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https://f1000research.com/articles/9-658/v1
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30 Jun 20
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{
"type": "Opinion Article",
"title": "Preliminary evidence for seasonality of Covid-19 due to ultraviolet radiation",
"authors": [
"Christos Karapiperis",
"Panos Kouklis",
"Stelios Papastratos",
"Anastasia Chasapi",
"Antoine Danchin",
"Christos A. Ouzounis",
"Christos Karapiperis",
"Panos Kouklis",
"Stelios Papastratos",
"Anastasia Chasapi",
"Antoine Danchin"
],
"abstract": "The Covid-19 pandemic has spread across the world during early 2020, with unforeseen consequences. Beyond social measures and biomedical research, it is important to assess the seasonality of the epidemic to inform strategies, with limited available data in the short period of time between the March equinox and the June solstice. While the effect of multiple factors is being investigated, little attention has been paid to ultraviolet (UV) radiation, a key parameter of seasonal forcing. We review the effects of UV radiation, proposing it as a potential element of seasonality, and provide evidence from the current literature and scant, yet revealing, observations. Explicit consideration should be given to UV radiation for the seasonality of Covid-19 at high latitudes and altitudes, based on the SARS and MERS epidemics and coronavirus diseases, and not just the ‘warmer days’ of summer.",
"keywords": [
"Covid-19",
"Seasonality",
"Ultraviolet radiation"
],
"content": "\n\nMuch attention has been paid by news reports, publications, press releases, preprints and white papers concerning containment measures for the Covid-19 pandemic, caused by the highly infectious and contagious virus SARS-CoV-2 in the human-to-human transmission chain (Anderson et al., 2020). From the onset of the epidemic, the naming of the new disease was not reminiscent of the previous, related disease SARS (Holmes, 2003), perhaps generating a certain degree of incertitude. The term Covid-19 has obfuscated its similarity and kinship to SARS for the public, with an initial tepid response in the Western world, as the disease started spreading globally. If Covid-19 had not been initially contrasted to influenza, responses might have been stronger and more efficient (Vetter et al., 2020). This is all history. Now, there is an acute need for swift, simple measures that can limit global contagion (Hunter, 2020), and ways to understand the epidemic response to climate factors.\n\nThere have been multiple reports and, admittedly, a hope that Covid-19 might exhibit a seasonal pattern, that will slow down the spread of the epidemic (Bukhari & Jameel, 2020). In fact, the initial confusion with flu-like symptoms might have contributed to this propitious hypothesis, obscuring the dangers for the epidemic in areas such as sub-Saharan Africa (Araújo & Naimi, 2020). A host of studies have examined seasonal fluctuations and regional climate parameters, including temperature (Auler et al., 2020; Shi et al., 2020), humidity (Luo et al., 2020), a combination of both (Mecenas et al., 2020; Şahin, 2020; Sajadi et al., 2020; Wu et al., 2020), along with wind speed (as an indicator for long-range airborne spread) (Chen et al., 2020), precipitation levels (Oliveiros et al., 2020), some in conjunction to population density (Liu et al., 2020; Pedrosa, 2020). Yet, only minor correlations with temperature, even controlling for a lag of detected cases, (Ma et al., 2020; Shi et al., 2020) or precipitation (Sobral et al., 2020) have been observed. Press releases reporting these studies state that seasonality can be a significant factor with summer days approaching. However, at the time of initial writing and review in parallel with the appearance of these reports, Northern and Southern hemispheres were at the March (‘Spring’ for the North) Equinox or just after it, confounding real-time analysis and derivation of solid conclusions that connect the epidemic with seasonal fluctuations to a certain extent (Bannister-Tyrrell et al., 2020), with little or no evidence found for local temperature effects, e.g. Spain (Briz-Redón & Serrano-Aroca, 2020) or Japan (Ujiie et al., 2020). Previous findings on the diminished presence of SARS-CoV on surfaces at higher temperatures, attenuating transmission, have been pointed out (Bannister-Tyrrell et al., 2020). Additional evidence for potential seasonality of Covid-19 arises from four other endemic coronaviruses, despite a significant uncertainty in epidemiological parameters, with the risk of a false impression of containment over the summer period (Neher et al., 2020).\n\nSunlight, as the surrogate and key element of seasonality, emits at infrared (IR), visible and ultraviolet (UV) wavelengths. While IR is responsible for heat transmission with low-energy electromagnetic (EM) waves, UV is responsible for high-energy EM waves – even at low temperatures, e.g. at high altitude or high latitudes1. Therefore, any assessment for the seasonality of Covid-19 should consider UV emission (typically UV-A or -B for sunlight), also hinting at the targeted deployment of artificial UV-C rays (naturally absorbed by the atmosphere and the ozone layer)2. In addition, it should be noted that extensive UV exposure can occur even in overcast skies, as this EM wavelength can penetrate water-droplet clouds that diminish UV radiation by as little as 15% (Calbó et al., 2005). Finally, there is also a correlation between vitamin D and UV, which implicates UV in human physiology (Asyary & Veruswati, 2020; Engelsen, 2010; Juzeniene et al., 2010; Tamerius et al., 2011). Statements concerning the absence of UV effects on Covid-19 seasonality over a short period of time and in an area with multiple confounding factors cannot be conclusive, as previously shown (Yao et al., 2020). More comprehensive studies now appearing suggest otherwise, suggesting a role for UV (Carleton et al., 2020) (Box 1). Consequently, there should be more focus on the effects of UV radiation, as a parameter that might slow virus transmission in open spaces under natural light and solar UV exposure as summer days are longer, in the period examined so far (Chiyomaru & Takemoto, 2020)3. Artificial sources of UV (-C) light for built environments that can be installed to eliminate the spread of infectious viral particles in public spaces can be deployed. Known examples include the use of UV to eradicate SARS-CoV-2 from banknotes, buses and hospitals. Indeed, studies on the related SARS-CoV report virion inactivation using 254nm UV light, heat or chemicals (Darnell et al., 2004).\n\n14-Apr: Chiyomaru & Takemoto, 2020\n\n16-Apr: Merow & Urban, 2020\n\n27-Apr: Asyary & Veruswati, 2020\n\n28-Apr: Carleton et al., 2020\n\n07-May: Yao et al., 2020\n\nIt is known that UV light (i.e. UV-C) affects nucleic acids in a detrimental fashion for organisms, including viruses and bacteria, with ssRNA viruses being more sensitive than other viral types by 2- to 3-fold (Tseng & Li, 2007). Germicidal UV is standard practice for cell culture and sterilization protocols (Li et al., 2005; Rae et al., 2008). Studies on the H1N1 influenza virus report inactivation with low doses of 222nm UV light, suggesting potential low dose-rate UV-C radiation solutions for reducing the spread of viral infections across indoor public spaces (Welch et al., 2018). Interestingly, a recent study on the effect of meteorological factors on influenza virus in Northern Europe has found some correlation between UV and spread of the epidemics during 2010–2018 (Ianevski et al., 2019), thus pinpointing one of the elements of seasonality, albeit for a different infectious disease. It is not known how resilient SARS-CoV-2 can be under strong, short-wave UV light, yet application of technologies, such as appropriate LED or UV-arrays, no matter how expensive, could in principle be deployed in crowded spots, such as mass transport systems, with a certain urgency.\n\nThere is limited information for non-human hosts of coronaviruses in the wild. It is remarkable, however, that a survey of populations for eight species of seabirds in the Southwestern Indian Ocean – including Réunion and Madagascar – did not detect any coronavirus presence in 338 samples, of which only a handful (39) were collected during the winter (Lebarbenchon et al., 2013). This serendipitous observation might suggest that the summer sun keeps these populations in the wild healthy and protects them from viral infections, however mild. More research into that direction will definitely be worthwhile, as the analogy of this finding cannot be readily translated for public health: neither the role of UV radiation nor seasonality can be inferred from these remarkable observations (C. Lebarbenchon, personal communication, University of Réunion). How wildlife responds to coronavirus infections with regard to seasons and climate change is not entirely understood at present.\n\nRegarding seasonality, our guide should be coronavirus epidemiology in general (Neher et al., 2020; Nickbakhsh et al., 2020), and the SARS/MERS outbreaks and their containment in particular – maybe more so than the flu. While models accept, simulate and interpret seasonality (Carleton & Meng, 2020; Kissler et al., 2020; Li et al., 2020), they do not usually refer to UV radiation in an explicit manner (rate, i.e. strong sunshine, or duration, i.e. day length). In the case of SARS and MERS, these epidemics did not spread widely, due to containment and mitigation strategies, including limited quarantine, which is not comparable to the scale we are experiencing today globally (Nickbakhsh et al., 2020). For instance, seasonal fluctuation for MERS was primarily limited at latitudes as low as 200 – in Saudi Arabia with high temperatures – although most infections occurred within hospitals (Zumla et al., 2015), and over a short period, at 400 latitude, e.g. in South Korea (Kim et al., 2017). We need to know how SARS-CoV-2 responds to UV and what its viability is on surfaces (van Doremalen et al., 2020), under simulated sunlight, which is currently unknown. Any chance of seasonal variation will provide valuable time in the North and ring an alarm in the South, as modelling the variation of UV radiation and temperature suggests a decline during summertime (Merow & Urban, 2020), with this study being the first following our initial hypothesis connecting UV radiation with Covid-19 growth rates (Karapiperis et al., 2020).\n\nSeasonal fluctuation can be a factor that will limit Covid-19, and the reason may not be due to high temperatures – as the media, but also a number of epidemiological studies, keep on reporting – but sustained UV radiation (Merow & Urban, 2020), as demonstrated in comparative studies (Lytle & Sagripanti, 2005). One piece of evidence is that, until the March Equinox 2020, Covid-19 was imported into the Southern Hemisphere but did not achieve epidemic status rapidly until recently (Figure 1). It is challenging to compare spread in real time for seasonality (Martinez, 2018), with 90% of the human population residing in the Northern Hemisphere. Using data from Peru, a country with the highest UV levels (Suarez Salas et al., 2017), there is a correlation between elevation and incidence, along population density (Figure 2), as latitude comparisons are not currently feasible. High-elevation countries such as Bhutan and Nepal exhibit low incidence rates for Covid-19, while exceptions due to travel patterns include countries as diverse as Andorra and China, all in the Northern hemisphere.\n\nData were imported from ECDC and refer to the period 31-Dec-2019 to 5-May-2020, © ECDC (2005–2019). Cross-checks with other datasets were performed, e.g. from HealthMap (Xu & Kraemer, 2020) (https://www.healthmap.org/covid-19/). Visualization was facilitated by Charticulator (https://charticulator.com/app/index.html, © 2018 Microsoft Corporation). Dates are shown on the x-axis, per day (vertical green line signifies the March equinox). Countries are classified in three groups on the y-axis (separated by horizontal lines, in orange) and listed alphabetically to avoid clashes over latitudes – top: Northern (73), middle: Southern (10) for contrast to North, bottom (70): equatorial (between 23N and 23S), 153 in total. Daily counts normalized per 100K population (shown in cells), to display spread (light: low values, dark: high values). It can be seen that at most countries in the North, there is some attenuation, except Russia, Turkey, the UK and the USA at present (dark-blue, at the top block). While many other factors contribute to the spread, it remains to be seen whether the pandemic will switch across North and South, indicating a seasonality pattern, with UV as a salient element compared to the more variable parameter of average daily temperature – and despite the fact of a large number of recorded cases. This will crucially depend on mitigation measures, travel restrictions and various other public health strategies around the world.\n\nA cursory view of the situation using elevation, as seasonality is currently difficult to track (see main text). We assess incidence in Peru, as the country with the highest UV radiation levels and high elevation contrasts (Suarez Salas et al., 2017). Reported Covid-19 cases in 25 departments (provinces) (from: https://covid19.minsa.gob.pe/sala_situacional.asp), 5-May-2020. Despite the fact that case reports have a resolution per province, there is a trend for high incidence in the low-lying East and coastal plains, while high altitudes with increased UV radiation levels (Suarez Salas et al., 2017) have lower counts. Left panel, generated by ClusVis (Metsalu & Vilo, 2015): Heatmap of PCA-based clusters, using correlation as a metric and complete linkage clustering for elevation, Covid-19 incidence (per thousand population) and population density, the latter used to capture crowding effects. Unit variance scaling applied to columns, scale shown (top right). Annotations: green for high, yellow for low – thresholds: high density >50/km2, high incidence >568pM (world average 13-May-2020), high elevation >200m. Annotations on the left block of the heatmap (also shown on the country map, right panel): 19/25 provinces consistent with high density/incidence and low elevation (6), and low density with low incidence/high elevation (10) or the reverse (3). There are just 6/25 provinces of low density and high incidence/elevation (inconsistent with UV-vs-incidence), four of which being coastal. Two clusters in rows and columns shown, delineating low-density/high-elevation provinces (bottom cluster, row-wise), and connecting Covid-19 incidence/density (right cluster, column-wise). Right panel: With no resolved data for UV per province, we used elevation of capital city as a UV estimate (i.e. altitude – selected from a number of other metrics). Annotation blocks (from left panel, see above) are shown per province; those consistent with UV-vs-incidence have a grey border (19), those inconsistent with UV-vs-incidence have a cyan border (6). The pattern provides, perhaps for the first time, some evidence of correlation between UV and Covid-19 incidence across altitudes and not latitudes – with UV being a strong candidate for an additional driver of seasonality at lower altitudes elsewhere (summer months).\n\nAs summer is approaching in the North, with increased UV radiation due to increased day length, there is a chance for a decelerating pace of transmission in the open. Artificial means to irradiate public spaces might be an option.\n\n\nData availability\n\nCovid-19 data from ECDC available from: : https://www.ecdc.europa.eu/en/publications-data/download-todays-data-geographic-distribution-covid-19-cases-worldwide (downloaded 5-May-2020).\n\nCovid-19 data from Peru available from: https://covid19.minsa.gob.pe/sala_situacional.asp (downloaded 12-May-2020).\n\n1https://www.ncbi.nlm.nih.gov/books/NBK304366/\n\n2https://www.who.int/uv/faq/whatisuv/en/index2.html\n\n3Preprint (https://osf.io/397yg/) was the first report on UV as a factor for Covid-19 seasonality (Box 1).",
"appendix": "Acknowledgements\n\nA previous version of this article was published on OSF Preprints: https://doi.org/10.31219/osf.io/397yg (Karapiperis et al., 2020).\n\nStelios Papastratos is an external member of BCPL.\n\n\nReferences\n\nAnderson RM, Heesterbeek H, Klinkenberg D, et al.: How will country-based mitigation measures influence the course of the COVID-19 epidemic? Lancet. 2020; 395(10228): 931–34. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAraújo MB, Naimi B: Spread of SARS-CoV-2 Coronavirus likely to be constrained by climate. medRxiv. 2020. Publisher Full Text\n\nAsyary A, Veruswati M: Sunlight exposure increased Covid-19 recovery rates: A study in the central pandemic area of Indonesia. Sci Total Environ. 2020; 729: 139016. 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PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "68930",
"date": "18 Aug 2020",
"name": "Gustavo Zubieta-Calleja",
"expertise": [
"Reviewer Expertise High altitude medicine",
"physiology",
"chronic hypoxia",
"chronic mountain sickness",
"U-V studies at high altitude",
"biophysics",
"COVID clinical aspects",
"silent hypoxemia",
"pneumolysis"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 \"Preliminary evidence for seasonality of Covid-19 due to ultraviolet radiation\" is of great interest in this COVID-19 Pandemic. The article is well written however a few points need to be addressed:\nThe first Figure is confusing and difficult to understand. The countries mentioned cannot be totally understood in the Y axis. The X-axis cannot be discerned, but it is assumed it is dates. The overall idea is good as it shows the seasonal variation.\n\nThe U-V radiation and high altitude has been discussed before in South America. The first mention as to the U-V radiation was in an article on it's effect on the eyes of high altitude residents:\n\nActa Ophthalmol. doi: 10.1111/j.1755-3768.2008.01488.x. Epub 2009 Mar 20. Lens autofluorescence is not increased at high altitude1.\n\nThe concept of the benefits of high altitude and the Ultra-Violet sterilizing effects were mentioned in an article on Longevity at high altitude in 2017 https://zuniv.net/pub/Longevity.pdf2.\n\nAt the beginning of the COVID-19 pandemia, several interviews, website publications and articles on the subject were published. Full references are available at:https://altitudeclinic.com/blog/2020/06/covid-19-pubs/3.\nSome should be included in the Timeline Box 1.\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? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes",
"responses": []
},
{
"id": "82697",
"date": "10 May 2021",
"name": "Matthew Baylis",
"expertise": [
"Reviewer Expertise Epidemiology",
"climate and health",
"One Health."
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper explores the possible impact of UV exposure on Covid-19. In my view it has a number of weaknesses.\nThe first paragraph is strange. It presents some arguably minor aspects of Covid-19 and then concludes we need to understand climate factors. This is a non-sequiteur. The paragraph should present a coherent argument for why we need to understand climate impacts on Covid-19.\n\nThe second paragraph considers some relevant literature. This is helpful, but not up to date [to today] - notably missing 1 (interest declared).\n\nThe reference to deploying artificial UV-C is strange and seems wrong in a paper about seasonality. As described, UV-C from artificial sources could be used to inactivate SARS-CoV-2 in e.g., hospitals, but it cannot influence seasonality of transmission. I suggest this is because it is in a paragraph taken from the pre-print.\n\nPage 4. The paragraph on seabirds is very weak and should be removed. The cited paper found no evidence of coronavirus [or other viruses] in any season in the seabirds; the lack of detection of coronaviruses is not evidence at all of an impact of season.\n\nPage 4. The discussion of SARS/MERS should consider the evidence that they had a seasonal cycle or were affected by climate or UV.\n\nPage 4. The suggestion that the southern hemisphere seems to have escaped the worst (supportive of seasonality etc) has been overtaken by events (e.g., Brasil) and the authors may wish to update the statement. Really, this was the danger from publishing analyses so early in the pandemic. This, and other examples, seem to be cherry picking examples to support their hypothesis.\n\nFigure 1. I have several concerns about this figure. The authors say the figure will be updated but it ends in May 2020. It is very hard to discern patterns in the figure It says in the title \"three countries\" but I think there are >140. The data are counts – of what? Cases, hospitalisations, deaths? Whichever, it is well known that different countries count these measures in different ways [e.g., UK, death from covid = death by any cause <28 days from a covid+ve test]. Further, many other factors will affect these counts and it is wrong to only look at climate factors.\n\nFigure 2 is very hard to follow. Nevertheless, I note that the right-hand panel makes no attempt to include population density; while in the left hand panel it is mentioned but not actually accounted for. In both cases, population density is strongly associated with altitude, and altitude with UV - so population density MUST be accounted for.\nAll in all, this is a weak paper. Its arguments are poor, it cherry picks examples, the data analysis is rudimentary and needs to include many other possible explanatory factors in addition to those related to UV.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Partly\n\nAre all factual statements correct and adequately supported by citations? Partly\n\nAre arguments sufficiently supported by evidence from the published literature? No\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? No",
"responses": []
}
] | 1
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https://f1000research.com/articles/9-658
|
https://f1000research.com/articles/9-656/v1
|
29 Jun 20
|
{
"type": "Research Article",
"title": "Isolation and characterization of a novel bacterial strain from a Tris-Acetate-Phosphate agar medium plate of the green micro-alga Chlamydomonas reinhardtii that can utilize common environmental pollutants as a carbon source",
"authors": [
"Mautusi Mitra",
"Kevin Manoap-Anh-Khoa Nguyen",
"Taylor Wayland Box",
"Jesse Scott Gilpin",
"Seth Ryan Hamby",
"Taylor Lynne Berry",
"Erin Harper Duckett",
"Kevin Manoap-Anh-Khoa Nguyen",
"Taylor Wayland Box",
"Jesse Scott Gilpin",
"Seth Ryan Hamby",
"Taylor Lynne Berry",
"Erin Harper Duckett"
],
"abstract": "Background: Chlamydomonas reinhardtii, a green micro-alga can be grown at the lab heterotrophically or photo-heterotrophically in Tris-Phosphate-Acetate (TAP) medium which contains acetate as the sole carbon source. When grown in TAP medium, Chlamydomonas can utilize the exogenous acetate in the medium for gluconeogenesis using the glyoxylate cycle, which is also present in many bacteria and higher plants. A novel bacterial strain, LMJ, was isolated from a contaminated TAP medium plate of Chlamydomonas. We present our work on the isolation and physiological and biochemical characterizations of LMJ. Methods: Several microbiological tests were conducted to characterize LMJ, including its sensitivity to four antibiotics. We amplified and sequenced partially the 16S rRNA gene of LMJ. We tested if LMJ can utilize cyclic alkanes, aromatic hydrocarbons, poly-hydroxyalkanoates, and fresh and combusted car motor oil as the sole carbon source on Tris-Phosphate (TP) agar medium plates for growth. Results: LMJ is a gram-negative rod, oxidase-positive, mesophilic, non-enteric, pigmented, salt-sensitive bacterium. LMJ can ferment glucose, is starch hydrolysis-negative, and is very sensitive to penicillin and chloramphenicol. Preliminary spectrophotometric analyses indicate LMJ produces pyomelanin. NCBI-BLAST analyses of the partial 16S rRNA gene sequence of LMJ showed that it matched to that of an uncultured bacterium clone LIB091_C05_1243. The nearest genus relative of LMJ is an Acidovorax sp. strain. LMJ was able to use alkane hydrocarbons, fresh and combusted car motor oil, poly-hydroxybutyrate, phenanthrene, naphthalene, benzoic acid and phenyl acetate as the sole carbon source for growth on TP-agar medium plates. Conclusions: LMJ has 99.14% sequence identity with the Acidovorax sp. strain A16OP12 whose genome has not been sequenced yet. LMJ’s ability to use chemicals that are common environmental pollutants makes it a promising candidate for further investigation for its use in bioremediation and, provides us with an incentive to sequence its genome.",
"keywords": [
"Chlamydomonas",
"Acidovorax sp.",
"TAP medium",
"LMJ",
"bioremediation",
"16S rRNA gene",
"penicillin-sensitivity",
"pyomelanin"
],
"content": "Introduction\n\nOur research laboratory primarily focuses on the functional genomics of eukaryotic oxygenic photosynthesis using the model green micro-alga, Chlamydomonas reinhardtii. The work in this article stemmed from a side research project of four undergraduates from the University of West Georgia and, one high school student from the Carrollton High School in Georgia. This undergraduate/high school student research project was centered on minimizing bacterial contamination of C. reinhardtii in our research lab.\n\nChlamydomonas can be grown at the lab heterotrophically or photo-heterotrophically at room temperature in Tris-Phosphate-Acetate (TAP) medium, which contains acetate as the sole carbon source1. When grown in TAP medium, Chlamydomonas can utilize the exogenous acetate to synthesize glucose using the glyoxylate cycle, without being dependent on photosynthesis for glucose biosynthesis2. Aerobic bacteria can also utilize glyoxylate cycle to metabolize fatty-acids or two carbon-compounds such as acetate to synthesize oxaloacetate, which can be used for gluconeogenesis3,4. The acetate in the TAP medium can be substituted with alternative carbon sources to test if bacteria can use the tested chemicals as the sole alternative carbon and energy source.\n\nWe isolated a bacterial strain from a contaminated Chlamydomonas TAP-agar medium culture plate. This bacterium was named LMJ, after the abbreviated name of the Chlamydomonas strain it contaminated: LMJ.SG0182. We performed growth analyses and several microbiological tests on LMJ. LMJ grows faster on TAP medium than on lysogeny broth (LB) medium, is a mesophilic, non-enteric, gram-negative bacillus. LMJ does not grow on Mannitol Salt Agar (MSA) and MacConkey agar (MAC) and is sensitive to 1% NaCl. LMJ is starch hydrolysis-negative, gamma hemolytic and is oxidase-positive.\n\nLMJ has a light pinkish-brown color on LB agar and creamy white color on TAP agar. We have found that on TAP + 1% tryptone medium plates in light, LMG colonies are pinkish brown pigmented, unlike that on the TAP-agar medium plates, indicating tryptone ingredients give the pink-brown color to LMJ on TAP+1% tryptone medium. Preliminary spectrophotometric analyses of the pigment/chemicals exuded by LMJ indicate that the light pink-brown pigment is pyomelanin. Pyomelanin is an auto-oxidized and self-polymerized product of homogentisate (HGA), which is an important intermediate in the tyrosine catabolic pathway5. Pyomelanin is one of the many forms of melanin that is produced by bacteria, fungus and other organisms5–10. Generation of pyomelanin can provide a survival advantage, scavenge free radicals, bind various drugs, offer protection against light and reactive oxygen species, and is involved in iron reduction and acquisition, and extracellular electron transfer11–17.\n\nWe have amplified and sequenced partially LMJ’s 16S rRNA gene. The best hit identified via the NCBI-BLAST analyses of the partial 16S rRNA gene sequence of LMJ is that of an uncultured bacterium clone LIB091_C05_1243 (Accession #: JX086489.1). We have submitted the partial 16S rRNA sequence of LMJ to the GenBank with the definition: Bacterium strain clone LIB091_C05_1243 variant 16S ribosomal RNA gene, partial sequence (Accession number: MN633292.1). The nearest relative of LMJ with a genus name is Acidovorax sp. strain A16OP12 (Accession #: MN519578.1).\n\nBetaproteobacteria are highly metabolically diverse and contain chemolithoautotrophs, photoautotrophs, generalist heterotrophs and opportunistic pathogens. Acidovorax is a genus of betaproteobacteria of the order Burkholderiales and family Comamonadaceae18,19. Strains classified as Acidovorax have been associated with soils, wastewater treatment plants, plants, and clinical samples20–28. Reflecting the diversity inherent in this widespread distribution, isolates within this diverse genus have been linked to a variety of phenotypes ranging from plant pathogenicity, to denitrification, to the biodegradation of contaminants29.\n\nAcidovorax sp. can degrade alkanes, polyhydroxyalkanoates (PHA) and polycyclic aromatic hydrocarbons (PAH)30–38. We tested the growth of LMJ on Tris-Phosphate (TP)-agar medium containing PAH, PHA, other aromatic compounds, cyclic alkanes and 10W30 car motor oil to determine if LMJ can use these compounds as the sole carbon source. LMJ was able to use cyclohexyl chloride, phenanthrene, naphthalene, benzoate, phenyl acetate and fresh and combusted 10W30 car motor oil. The abovementioned chemicals occur as common environmental pollutants in soil and water. In future, whole genome sequencing of LMJ and proper quantitative assays to determine LMJ’s ability to degrade (and remove) toxic chemicals from the TP-growth medium are needed to probe its full potential for usage in environmental bioremediation. In this primarily undergraduate and high school student-driven research project, we are presenting our research on the isolation and physiological and biochemical characterizations of the novel bacterial strain, LMJ.\n\n\nMethods\n\nWe used the Chlamydomonas wild type strain 4A+ (CC- 4051 4A+ mt+) strain in our antibiotic-sensitivity testing experiments. 4A+ strain was maintained on TAP agar medium in dim light intensities (15-20 µmol m-2s-1) at 22°C. Liquid 4A+ cultures were grown in 50 mL flasks in 15 mL of TAP in low light (50-80 µmol m-2s-1) for 3 days. LMJ bacterial strain was maintained in the lab under dim light at 22°C on either TAP or LB agar medium plates. Liquid cultures of LMJ were grown in culture tubes in 3 mL of TAP or LB medium. 4A+ and LMJ liquid cultures were shaken at 150 rpm for aeration on a MaxQ420HP incubator shaker (Thermo Fisher Scientific, Waltham, MA). Light intensities were measured using a LI-250A light meter (LI-COR, Inc., Lincoln, NE).\n\nThe detailed protocol utilized to produce the TAP and TP medium used in this article is available from Protocols.io: https://doi.org/10.17504/protocols.io.bgzujx6w.\n\na) Testing salt-sensitivity of LMJ: LMJ was streaked on LB, TAP, LB-1% NaCl and TAP +1% NaCl agar medium plates. Streaked LMJ media plates were incubated either 22°C or at 37ºC for 3 days and then imaged.\n\nb) Testing bile salts-/salt-sensitivity and ability to ferment lactose/mannitol: Growth assays on MAC and MSA were performed by streaking LMJ on MAC and MSA plates purchased from Carolina Biological (Burlington, NC). Media plates were incubated at room temperature for 4 days and then imaged to capture the LMJ growth and the pH change in the media. Escherichia coli and Staphylococcus aureus was used as a positive control for the MAC and the MSA experiment, respectively.\n\nc) Testing LMJ’s ability to use different sugars as a sole carbon source: LMJ was streaked on agar plates containing TP-phenol red medium (pH 7.2) supplemented with 1% glucose, 1% sucrose and 1% lactose, respectively. Media plates were imaged after 5 days of growth at 22°C to capture the LMJ growth and the pH change in the media.\n\nd) Testing LMJ’s ability to use different hydrocarbons and aromatic compounds as a sole carbon source: TP-agar plates were coated with different doses of cyclohexyl chloride, phenanthrene, naphthalene, benzoic acid, phenyl acetate and fresh and combusted 10W30 oil, using a modified technique39. 1% stock solutions of all chemicals (except 10W30 motor oil) were prepared by either dissolving or diluting the chemicals in chloroform. 10W30 fresh and used motor oils were diluted in chloroform to make a 2% (v/v) stock solution. 4 mL of 1% cyclohexyl chloride and 1% polyhydroxybutyrate (PHB) were coated on a TP plate. 2 mL and 4 mL of 1% phenanthrene stock solution were coated on TP plates. 0.5 mL, 1 mL and 2 mL of 1% napthalene, 1% benzoate and 1% phenyl acetate and 2% combusted and fresh 10W30 car motor oil were coated on TP plates. LMJ was streaked on the chemical-coated TP plates and incubated at room temperature. After 2 weeks, the plates were imaged using a Samsung Galaxy S5 camera. Images of all medium plates are available as Underlying data40.\n\na) Gram staining: LMJ from LB-agar plate and LB and TAP shaking liquid cultures were used for Gram staining. Gram staining was performed using standard Gram staining protocol using Gram stain reagents from VWR (Radnor, PA). Gram-stained cells were viewed under oil immersion lens with a X100 magnification. Images of gram-stained cells were taken by a Samsung Galaxy S5 camera using a cell phone adapter for the microscope eyepiece.\n\nb) Oxidase test: Oxidase test was performed using Difco DrySlide Oxidase Disposable Slide purchased from Carolina Biological (Burlington, NC). The dry slide contains a film coated with an oxidase reagent (tetramethyl-p-phenylenediamine dihydrochloride). LMJ and Microbacterium sp. cells were taken from tryptic soy agar medium plates which does not contain any fermentable sugar. Fermentable sugars generate acid which can drop the pH below 5.1 and can give a false negative oxidase test result (https://microbeonline.com/oxidase-test-principle-procedure-and-oxidase-positive-organisms/) (Carolina Biological, Burlington, NC). Cells were streaked on the dry slide in two separate reaction areas. After 10 seconds, the color of the streak was captured with a Samsung Galaxy S5 camera.\n\nc) Starch hydrolysis test: Starch hydrolysis test was performed on Mueller-Hinton-agar medium plates purchased from Carolina Biological (Burlington, NC). LMJ and E. coli were streaked on Mueller-Hinton agar medium plates and incubated for 48 hours at 37°C. After 48 hours of growth, plates were imaged. After imaging, Mueller-Hinton agar plates were flooded with Gram iodine and incubated for 10 minutes to test for the absence or presence of starch in the medium and the plate was re-imaged.\n\nWe modified the Kirby-Bauer (KB) disc diffusion method to perform the antibiotic susceptibility tests on TAP-agar plates (https://www.asm.org/Protocols/Kirby-Bauer-Disk-Diffusion-Susceptibility-Test-Pro). We grew LMJ in 3 mL of liquid TAP or in liquid LB and diluted the overnight culture 5-fold with liquid medium. The optical density of the 5-fold diluted culture was measured at 600 nm with a Beckman Coulter DU 730 Life science UV/Vis spectrophotometer (Brea, CA)41. The optical density of culture was adjusted with liquid medium to achieve an optical density of 0.08–13 which matches the 0.5 McFarland standard. Chlamydomonas strain 4A+ was grown in liquid TAP for three days and the optical density of the Chlamydomonas culture was measured at 750 nm with a Beckman Coulter DU 730 Life science UV/Vis spectrophotometer (Brea, CA)42. Measurement at 750 nm avoids the absorption of light by cellular pigments (chlorophyll and carotenoids) and is treated as a pure light scattering measurement42. The optical density of the Chlamydomonas culture was adjusted with TAP medium to achieve an optical density of 0.3–0.4.\n\nUsing a sterile swab, optical density-adjusted cultures of LMJ and Chlamydomonas were plated on the TAP plates. Two sterile filter paper discs were placed on each TAP plate with a separating distance of 4–5 cm. On one disc 50 µg or 100 µg of the antibiotic in a 20 µL total volume and on the other disc, 20 µL of sterile water was added (control). Antibiotics that were applied were: penicillin; neomycin, chloramphenicol and polymyxin B. Antibiotic plates were incubated at room temperature for 3–4 days. LMJ and Chlamydomonas plates were imaged after 3 and 4 days of incubation respectively, as Chlamydomonas grows slowly than bacteria. Diameters of zone of inhibitions were measured using a ruler. Standard deviations and means were calculated using Excel and, statistical analyses of the data from three biological replicates per experiment were performed using Microsoft Excels’ t-Test: Paired Two Sample for Means tool in the analysis ToolPak. Both One-Tailed and Two-Tailed Hypothesis Tests were performed. Statistical analyses43 and images of all antibiotic plates44 are available as part of the Underlying data.\n\nLMJ from TAP-agar plates were streaked on fresh TAP +1% tryptone-agar plates. One plate was incubated under light intensity of 350 µmol photons m-2s-1 and the other plate was kept in dark at 22ºC. Images of the plates were taken after 3 days of growth to monitor pigment production. LMJ cells were treated in three different ways for spectrophotometric analyses. 1) LMJ was grown in 2.5 mL of LB medium at 37°C for 3 days. 2 mL of the LB culture was centrifuged at 5000 X g for 2 minutes. The supernatant and the cell pellet were collected. Supernatant was alkalinized (20 µL of 6 M NaOH per ml of sample) and centrifuged at 16,000 X g for 2 minutes and the supernatant was collected. 2) LMJ cell pellet from treatment 1 was resuspended in 200 µL of 1% NaNO2 solution and 1mL of 0.1N-HC1 by vortexing the sample for few minutes and incubated for 5 minutes. 3) LMJ cells from a LB-agar medium plate was resuspended in 1 mL of 6M NaOH by vortexing and incubated for 5 minutes. Acidified and alkalinized tubes of cell pellet washes from treatments 2 and 3 were centrifuged at 16,000 X g for 2 minutes and the supernatants were collected separately.\n\nSupernatants obtained from the above stated three treatments were filtered using a 5 mL syringe fitted to a nylon membrane filters with a cut-off of 0.45 µm. Pigment formation in the alkalinized supernatant from the liquid LB culture and, the alkalinized- and acidified- supernatants from cell pellet washes were analyzed by using the wavelength scan program ranging 200 - 600 nm in a Beckman Coulter DU 730 Life science UV/Vis spectrophotometer (Brea, CA). To detect pyomelanin in the supernatant of the LB liquid culture, the absorption peak at the 400 - 405 nm region was monitored. To detect homogentisate (HGA) in the alkalinized supernatant from the cell pellet wash, the absorption peak at the 290 nm was monitored. To detect 1:4-benzoquinone-2-acetic acid (benzoquinone acetate; BQA), the absorption peak at the 250 nm was monitored in the acidified supernatant obtained from the cell pellet wash. Our protocol is a “quick and crude” version of the protocol available in the literature for preparation of samples for spectrophotometric assays of pyomelanin, BQA and HGA45–47.\n\nGenomic DNA was isolated using Qiagen’s blood and cell culture DNA mini kit (Qiagen, Valencia, CA) according to the protocol given in the technical manual. DNA concentration and purity of the isolated genomic DNA sample were measured using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA). Genomic DNA sample was separated on a 1% agarose gel and the quality of the sample was visualized by imaging the gel with a BioRad Molecular Imager Gel Doc XR+ (BioRad, Hercules, CA).\n\nForward and reverse 16S rRNA PCR primers were designed based on sequences in the literature for selecting primer pairs with the best overall coverage and phylum spectrum to reduce the bias in PCR-based microbial diversity studies48. The sequence of the forward primer 16SF is: 5’ CCTACGGGNGGCWGCAG 3’ and that of the reverse primer 16SR is: 5’ GACTACHVGGGTATCTAATCC 3’. HotStar Taq Plus DNA polymerase enzyme kit (Qiagen, Valencia, CA) was used for PCR following the given cycling conditions given in the Qiagen protocol booklet.\n\nThe PCR-amplified partial 16S rRNA genomic product was excised from the agarose gel and purified using the QIAquick Gel Extraction Kit (Qiagen, Valencia, CA). The purified PCR product was cloned using the TOPO TA cloning kit in the pCR4-TOPO TA vector (Thermo Fisher Scientific, Waltham, MA) according to the protocol given in the technical manual. One clone harboring the partial 16S rRNA gene of LMJ was sequenced by the UC Berkeley DNA Sequencing Facility. Chromas Lite and BLAST program were used to analyze DNA sequences. Raw electropherogram files and sequence text files are available as Underlying data49.\n\n\nResults\n\nWe found a creamy white bacterial contamination on a Chlamydomonas strain, LMJ.SG0182 TAP plate at our laboratory. (Figure 1A). We purified the bacterial strain using the streak plate method to isolate single bacterial colonies on fresh TAP medium plates. We picked ten single colonies and transferred them to a LB agar medium plate (Figure 1B). Colony # 10 was selected for our studies (Figure 1B). Colony # 10 was streaked on fresh LB and TAP-agar medium plates to maintain the purified bacterial strain in the lab (Figure 1C, D). We named this bacterial strain as LMJ after the abbreviated name of the Chlamydomonas strain LMJ.SG0182, as it was isolated from this Chlamydomonas strain’s culture plate. LMJ is an acid-devouring strain as it was able to grow on the TAP-agar medium (Figure 1D). LMJ culture on LB agar medium appears pigmented with a light pinkish-brown color (Figure 1C) but on TAP agar medium the culture is creamy white (Figure 1D).\n\n(A) TAP-agar medium plate showing bacterial contamination of a Chlamydomonas strain, LMJ.SG0182 at room temperature (22ºC). The bacterium is named LMJ, after the abbreviated Chlamydomonas LMJ.SG0182 strain. (B) Ten single colonies of LMJ on a lysogeny broth (LB)-agar medium plate. Colony # 10 outlined by the black circle was picked for culture stock maintenance and further analyses. (C) LB-agar medium plate of purified LMJ strain (D) TAP-agar medium plate of purified LMJ strain. Culture plates shown in (C) and (D) were imaged after 5 days of growth at 37ºC.\n\n\n\nTAP medium recipe can be found at the website of Chlamydomonas Resource Center. Hutner’s trace element solution is an ingredient in the TAP medium. Hutner’s trace element recipe can be found at https://www.chlamycollection.org/methods/media-recipes/hutners-trace-elements/. M9 medium is the standard minimal medium for growing bacteria. In our lab we use a slightly modified TAP medium recipe which has a final concentration of phosphate, nitrogen, magnesium and calcium approximately 10-fold, 2-fold, 2-fold and 2-fold higher than that in the TAP recipe found on the Chlamydomonas Resource Center website, respectively. Final concentrations of acetate and Hutner’s trace elements are the same in both TAP recipes. Table 1 compares the chemical ingredients in our lab’s TAP recipe with that present in the standard M9 medium described at http://www.thelabrat.com/protocols/m9minimal.shtml. M9 medium has a final concentration of phosphate, nitrogen, magnesium and carbon in the medium approximately 70-fold, 2.5-fold, 4-fold and 5-fold higher than that present in our lab’s TAP medium, respectively. Additionally, M9 contains 0.05% salt (8.56 mM) (Table 1). TAP has additional trace elements like iron, zinc, copper, manganese, cobalt, boron and molybdenum which are components of the Hutner’s trace element solution (Table 1). We have substituted the acetate in the TAP medium with alternative carbon sources to perform various experiments that are described below in the result section.\n\nTAP medium is used for growing Chlamydomonas heterotrophically or photo-heterotrophically. M9 medium is the standard minimal medium for growing bacteria. Note: Hutner’s trace element is an ingredient in the TAP medium. TAP trace elements and EDTA shown in the table are components in the Hutner’s trace element solution. pH of Hutner’s solution is adjusted to 6.5 using KOH (not NaOH) pellets before it is used to make the TAP medium. Both growth media have (pH 7-7.2). Acetate in the TAP medium can be substituted with alternative carbon sources. TAP medium minus acetate is the TP medium in our work and the media recipes can be found at https://doi.org/10.17504/protocols.io.bgzujx6w.\n\nGram staining of LMJ cells from a LB-agar medium plate revealed that LMJ cells are straight to slightly curved rods (Figure 2). Cells occur singly, in pairs or in short chains. LMJ is a gram-negative bacterium (Figure 2).\n\nLMJ cells from a LB-agar medium plate was used for gram-staining. Gram-stained cells were visualized and imaged under an oil immersion lens of a bright-field microscope.\n\n\n\nWe have monitored the growth of LMJ on LB-agar, TAP-agar and Mueller-Hinton agar medium over 5 days. LMJ has less dense growth on LB agar than on TAP agar or on Mueller-Hinton agar at 22°C (Figure 3A, B, E). LMJ has dense growth at 37°C than at 22°C on all three media tested (Figure 3C, D, F). It is to be noted that LMJ appears lightly pigmented on both LB-agar and Mueller-Hinton agar but not on TAP-agar (Figure 1C and Figure 3C, E, F).\n\n(A) Growth on LB-agar medium plate at room temperature (22ºC). (B) Growth on TAP-agar medium plate at room temperature. (C) Growth on LB-agar medium plate at 37ºC. (D) Growth on TAP-agar medium plate at 37ºC. (E) LMJ growth on Mueller-Hinton agar medium plate at 22ºC. (F) LMJ growth on Mueller-Hinton agar medium plate at 37ºC. Culture plates were imaged after 5 days of growth.\n\n\n\nLMJ grows on the nutrient-rich LB agar medium slowly compared to that on the TAP agar medium (Figure 3). TAP does not contain NaCl (Table 1) but LB medium has 1% NaCl (http://2018.igem.org/wiki/images/f/f2/T--Toronto--_LB_Medium_Preperation_Protocol.pdf). Gram-negative bacteria are usually sensitive to salt. Hence, we studied the growth of LMJ on LB, LB -1% NaCl, TAP, and TAP +1% NaCl-agar medium at 22°C and at 37°C. LMJ grew faster on LB-1% NaCl agar compared to that on the LB agar at both temperatures (Figure 4A-D). LMJ grew very slowly on TAP +1% NaCl agar compared to that on the TAP-agar at both temperatures (Figure 4E-H). The slow growth of LMJ was very pronounced at 22°C than at 37°C on both LB-agar and TAP +1% NaCl-agar medium (Figure 4A, C, E, G). Taken together, our results show that LMJ is sensitive to even 1% of NaCl in LB that is tolerated well by many Gram-negative bacteria.\n\n(A) LMJ growth on LB (contains 1% NaCl)-agar medium plate at 22ºC. (B) LMJ growth on LB (minus 1% NaCl)-agar medium plate at 22ºC. (C) LMJ growth on LB (contains 1% NaCl)-agar medium plate at 37ºC. (D) LMJ growth on LB (minus 1% NaCl)-agar medium plate at 37ºC. (E) LMJ growth on TAP+1% NaCl-agar medium plate at 22ºC. (F) LMJ growth on TAP-agar medium plate at 22ºC. (G) LMJ growth on TAP+1% NaCl-agar medium plate at 37ºC. (H) LMJ growth on TAP-agar medium plate at 37ºC. Culture plates were imaged after 3 days of growth.\n\n\n\nIn Figure 2, LMJ cells from a LB-agar medium plate was used for Gram staining. We performed Gram staining using cells from LMJ liquid TAP and liquid LB cultures that were shaking in an incubator shaker at 150 rpm at 37°C (Figure 5). Although the turbidity of the TAP or the LB liquid culture indicates dense growth (Figure 5A, C), Gram stain of LMJ liquid cultures revealed mainly cell lysis and membrane debris with very few rods and round intact cells (Figure 5B, D; Underlying data50). We have monitored the LMJ LB and liquid culture growth over a period 96 hours and we did not see any clearing of the TAP and LB liquid cultures (see Underlying data50). Cells from the liquid TAP culture were plated on a fresh TAP-agar medium plate and LMJ grew back within 4 days at room temperature (see Underlying data50). Cells in liquid culture were not dead although we saw mainly cell debris and very few intact cells after gram staining. Taken together, our results indicate that LMJ is probably a biofilm forming bacteria. It could not form biofilm in a shaking liquid culture and cells were prone to lysis in the shaking liquid culture (see Discussion).\n\n(A) A culture of LMJ grown for 24 h in liquid TAP and liquid TAP medium (control). (B) Gram-stained LMJ cells from the 24 hours-TAP liquid culture. (C) A 24 hours-grown culture of LMJ in liquid LB and liquid LB medium (control). (D) Gram-stained LMJ cells from the 24 hours-grown LMJ LB liquid culture. Mostly membrane debris, few rods and round shaped cells can be seen. Original gram-stained files can be found at 10.6084/m9.figshare.12420893.\n\n\n\nMany bacteria are known to produce a brown-black melanin pigment called pyomelanin5,17. Light and oxygen are needed for the formation of pyomelanin45. Aromatic amino acid tyrosine is needed for pyomelanin formation5. Phenylalanine can also contribute to the synthesis of pyomelanin as it can be can be converted to tyrosine by the enzyme phenylalanine-4-hydroxylase51. LB medium contains 1% tryptone which is a rich source of different amino acids including the aromatic amino acids (https://khimexpert.com/wp-content/uploads/2018/12/GCM23-Tryptone.pdf).\n\nWe tested if the light brown color of LMJ on LB medium is because of tryptone which provides aromatic amino acids. We grew LMJ on TAP +1% tryptone-agar medium plates in dark and in light. In the dark, on TAP + 1% tryptone medium plate LMJ did not appear to be pigmented, unlike that in the light (Figure 6). We found that 1% tryptone and light are needed for pigment production in LMJ (Figure 6).\n\n(A) LMJ on TAP +1% tryptone agar at room temperature. (B) LMJ on TAP +1% tryptone-agar under light intensity of 350 µmol photons m-2s-1 at room temperature. Images were taken after 3 days of growth.\n\n\n\nPyomelanin is a negatively charged extracellular pigment of high molecular weight, derived from the tyrosine catabolism pathway11(Figure 7). Pyomelanin is usually produced because of defects in the tyrosine catabolism pathway that leads to an accumulation of homogentisate (HGA) and occurs naturally in many pyomelanin over-producing strains5,52 (Figure 7). Accumulation of HGA can occur because of an inactivation of the enzyme homogentisate 1, 2 dioxygenase (HmgA)52,53 (Figure 7). Accumulated HGA is secreted out of the cells by an ABC transporter and is auto-oxidized to 1:4-benzoquinone-2-acetic acid (benzoquinone acetate; BQA) and then polymerized to form the brown pigment pyomelanin (Figure 7). We wanted to see if we can detect HGA, BQA and pyomelanin in the alkalinized LMJ LB liquid culture medium and in the acidified and alkalinized LMJ cell pellet-wash supernatants45–47.\n\nPhhC: Family I aminotransferase; Hpd: 4-hydroxyphenylpyruvate dioxygenase; HmgA: homogentisate 1,2-dioxygenase; MalA: maleylacetoacetate isomerase; FahA: fumarylacetoacetate hydrolase; HatABCDE: ABC transporter.\n\nPyomelanin absorption peak is between 400 and 405 nm45–47. We observed a 402 nm absorption peak in the absorption curve of alkalinized LMJ liquid LB culture medium (Figure 8A). BQA absorbs maximally in the UV region at 250 nm45. We observed an absorption peak at 252 nm in the absorption curve of acidified-cell pellet wash supernatant derived from LMJ liquid LB culture (Figure 8B). HGA absorbs maximally in the UV region at 290 nm45. We observed an absorption peak at 291 nm in the absorption curve of alkalinized cell pellet wash supernatant derived from LMJ cells from a LB-agar medium plate (Figure 8C). A slight 1-2 nm shift was observed in the HGA and BQA absorption peaks in our samples compared to the values found in literature (see Discussion)45. Taken together our results strongly indicate the LMJ produces pyomelanin and its production is stimulated under light by tryptone (Figure 6 and Figure 8). We did not have tyrosine at the time of submission of this work.\n\n(A) Absorption curve of the pigment, exuded out in the LMJ LB liquid culture at 37ºC and the corresponding absorbance reading and the absorption peak. (B) Absorption curve of the acidified LMJ cell pellet wash and the corresponding absorbance reading and the absorption peak. LMJ cells were harvested from the LB liquid culture (72 hours old) by centrifugation. (C) Absorption curve of the alkalinized LMJ cell pellet wash and the corresponding absorbance reading and the absorption peak. LMJ cells were harvested from a LB-agar medium plate. Absorption maxima of the alkalinized LB medium (A), acidified (B) and alkalinized (C) cell pellet washes were measured using the wavelength scan program in the UV-visible light wavelength range of 200–600 nm in a UV-Vis spectrophotometer. The absorbance readings at the absorption peaks are outlined by the red box. The black arrows and the black line points to the absorption maxima in the absorption curve.\n\nMAC is a selective and differential medium used for isolation and differentiation of enteric gram-negative bacteria based on their ability to ferment lactose. Bile salts and crystal violet in MAC inhibit the growth of gram-positive bacteria. Lactose in MAC is a source of fermentable carbohydrate. Neutral red is a pH indicator present in MAC that turns red at a pH below 6.8 and is colorless at a pH greater than 6.8. Bacteria that ferment lactose and thereby produce acid in the medium will appear pink because of the neutral red turning red while bacteria that are lactose non-fermenters will produce normal-colored or colorless colonies. LMJ was unable to grow on MAC (Figure 9A; right) unlike E. coli, a gram-negative enteric bacterium (Figure 9A; left). E. coli appeared pink on MAC plate, indicating it can ferment lactose (Figure 9A; left). Additionally, bile salts precipitated out of the MAC medium surrounding the growth of E. coli because of the acidic pH (Figure 9A; left). Our results indicate that LMJ is a non-enteric bacterium. Inability of LMJ to grow on MAC could be because: 1) LMJ cannot use lactose as a carbon/energy source; 2) because it is sensitive to bile salts and crystal violet in the MAC medium or 3) a combination of 1 and 2.\n\n(A) LMJ (on right) and Escherichia coli (left) on MacConkey Agar medium plate. LMJ fails to grow on MacConkey agar medium plate. E. coli appears pinkish because it ferments lactose to acid, which causes the neutral red pH indicator to turn red. The dark opaque pink haze on the medium around the E. coli growth is the bile precipitation in acidic environment (B) LMJ (on right) and Staphylococcus aureus (left) on Mannitol Salt Agar medium plate. LMJ fails to grow on Mannitol Salt Agar medium plate. Culture plates were imaged after 4 days of growth at room temperature.\n\nMSA is a selective and differential medium. MSA contains a high concentration (about 7.5–10%) of NaCl. This makes MSA selective for many high salt-tolerant gram-positive bacteria (Staphylococcus, Enterococcus and Micrococcus) as the high salt concentration is inhibitory for growth of most bacteria. MSA also contains the sugar mannitol and the pH indicator phenol red. If a bacterium can ferment mannitol to acid, the color of phenol red in the agar will change from red to yellow. LMJ being gram-negative and salt-sensitive (Figure 2 and Figure 4), fails to grow on MSA (Figure 9B, right) while Staphylococcus aureus, a mannitol-fermenter, grew on MSA and produced acid that changed the phenol red’s color to yellow (Figure 9B, left).\n\nWe grew LMJ on TP + 1% sugar agar which contained phenol red as the pH indicator. We replaced the carbon source acetate in the TAP medium with sugars like glucose, sucrose and lactose (Figure 10). TAP minus acetate medium is called TP medium in this work. Figure 10A, Figure 10C and Figure 10E represent control TP + 1% glucose, TP + 1% sucrose and TP + 1% lactose plates, respectively. LMJ grew very well on TP + 1% glucose agar and fermented glucose to produce acid (Figure 10B). It did grow to some extent on TP +1% sucrose agar but not as well as it did on the TP +1% glucose agar (Figure 10B, D). It was hard to confirm if LMJ fermented sucrose to any significant degree as there was no distinct color change of phenol red like that in Figure 10B (Figure 10D). But it is to be noted that the color of phenol red in the control TP +1% sucrose agar was light red (Figure 10C) and that on LMJ-TP +1% sucrose agar was light orange (Figure 10D), indicating there could have been some sugar fermentation. No visible growth of LMJ was observed on TP +1% lactose agar. Taken together with results from the LMJ growth on MAC medium (Figure 9A), LMJ cannot use the disaccharide lactose as a carbon source (Figure 9A and Figure 10F) but can use monosaccharide glucose and disaccharide sucrose as alternative carbon sources (Figure 10B, D).\n\n(A) Control TP + 1% glucose agar medium plate with phenol red as a pH indicator. (B) LMJ growth on TP +1% glucose agar medium plate containing phenol red. LMG can ferment glucose to produce acid. (C) Control TP +1% sucrose agar medium plate with phenol red as a pH indicator. (D) LMJ growth on TP +1% sucrose agar medium plate containing phenol red. Trace amount of sugar fermentation detected. (E) Control TP +1% lactose agar medium plate with phenol red as a pH indicator. (F) LMJ fails to grow on TP +1% lactose agar medium plate containing phenol red. Phenol red’s color turns yellow when sugars are fermented to produce acid. Culture plates were imaged after 5 days of growth at room temperature.\n\nStarch hydrolysis test is used to identify bacteria that can hydrolyze starch (amylose and amylopectin) using the enzymes α-amylase and oligo-1,6-glucosidase. Because of the large size, amylose and amylopectin molecules cannot cross the bacterial cell wall. To use starch as a carbon source, bacteria must secrete α-amylase and oligo-1,6-glucosidase into the extracellular space. These enzymes break the starch molecules into smaller glucose subunits which can be utilized by the cells. To interpret the results of the starch hydrolysis test, Gram iodine was added to the agar. Iodine reacts with the starch to form a dark brown/blue color. Hydrolysis of starch will create a clear zone around the bacterial growth. Both LMJ (Figure 11B) and E. coli (Figure 11D) tested negative in the starch hydrolysis test as there was no clear zone around the growth on the Mueller Hinton agar. We did not have an access to a starch-positive strain to use as a positive control in the starch hydrolysis test.\n\n(A) 48 hours-growth of LMJ on Mueller-Hinton medium which contains 0.15% starch. (B) LMJ Mueller-Hinton plate shown in (A) was treated with Gram iodine. (C) 48 hours-growth of E. coli on Mueller-Hinton medium which contains 0.15% starch. (D) E. coli Mueller-Hinton plate shown in (C) treated with Gram iodine. E. coli and LMJ fail to hydrolyze starch on Mueller-Hinton medium as there are no visible clear zones around the bacterial growth after gram iodine treatment. The brown-blue color of the medium upon Gram iodine treatment occurs because of the reaction of starch in the medium with iodine. Mueller-Hinton medium plates were incubated with Gram iodine for 10 minutes at room temperature and then imaged.\n\nThe oxidase test is used to identify aerobic, facultative anaerobic or microaerophilic bacteria that produce cytochrome c oxidase, an enzyme of the electron transport chain. When present, cytochrome c oxidase oxidizes the oxidase reagent (tetramethyl-p-phenylenediamine; TMPD) to indophenols (purple colored product) within 5–10 seconds. When the enzyme is not present, the reagent remains reduced and is colorless. Oxidase-positive strains take 5–10 seconds to form indophenols and the color change occurs within 30 seconds. Delayed oxidase-positive strains form indophenols within 60–90 seconds and oxidase-negative strains can either remain colorless or take more than 2 minutes to show the purple color because of slow spontaneous non-enzymatic oxidation of TMPD in air (https://www.asm.org/Protocols/Oxidase-Test-Protocol). Our oxidase test results show that LMJ is oxidase-positive (Figure 12; left) and Microbacterium sp. is oxidase-negative (Figure 12; right). Hence LMJ is an aerobic bacterium and uses cytochrome c in the electron transport chain (Figure 3–Figure 6, Figure 8 and Figure 12).\n\nCells of LMJ (on the left) and Microbacterium sp. (on the right) streaked on a disposable slide containing a film coated with oxidase reagent (tetramethyl-p-phenylenediamine dihydrochloride). Image of the slide was taken after 10 seconds of the application of the cells on the slide. LMJ is cytochrome c oxidase-positive as cytochrome c oxidase, if present, oxidizes the oxidase reagent on the film to form purple colored-indophenols. Microbacterium sp., a yellow-pigmented bacterium, is oxidase-negative and fails to form the purple-colored product. Cells were taken from strain specific-tryptic soy agar medium plates.\n\nWe tested four antibiotics to determine which of these four antibiotics and at what dose, will be effective in inhibiting growth of LMJ with no/minimal detrimental effect on Chlamydomonas growth. In the first set of experiments, we determined the antibiotic-sensitivity of LMJ and Chlamydomonas 4A+ strain separately to identify the effective antibiotic and, the required dose. In the second set of experiments, we streaked Chlamydomonas and LMJ together on the TAP-agar medium plate containing the antibiotic with the effective dose that we found to be potent against LMG (without hindering Chlamydomonas growth) from the first experiment. This was done to confirm that the selected antibiotic is effective in eliminating LMJ contamination on Chlamydomonas TAP-agar plates.\n\nWe modified the traditional KB disc diffusion method to test for antibiotic-sensitivity as described under Method section. We tested two different doses (50 µg and 100 µg) of penicillin, polymyxin B, neomycin and chloramphenicol. Table 2 shows the average diameter of the zone of inhibition for each antibiotic dose, with the standard deviations. Statistical analyses are available as part of the Underlying data43. Images of all antibiotic plates are available as Underlying data44.\n\nZones of inhibitions in the presence of four different antibiotics were studied for Chlamydomonas reinhardtii and the bacterial strain, LMJ. Grey and white rows represent 50 µg and 100 µg dose of each antibiotics applied on the filter paper discs, respectably. Three biological replicates were used to calculate the mean and standard deviations shown in the table (10.6084/m9.figshare.12407735; 10.6084/m9.figshare.12407741).\n\na) Penicillin-sensitivity testing: Penicillin affects peptidoglycan biosynthesis and compromises cell wall integrity in gram-positive bacteria. Chlamydomonas was resistant to both 50 µg (82.5 IU units) and 100 µg (165 IU units) as it did not show any zone of inhibitions (Table 2). LMJ was highly sensitive to penicillin (Table 2), despite being a gram-negative bacterium. p-values for the one-tailed and two-tailed hypothesis test for both doses were infinitely small and statistically significant. To ensure that the penicillin-sensitivity of LMJ is not because of the TAP-agar medium used in our experiments, we tested penicillin-sensitivity of LMJ and E. coli (control) on LB-agar medium and LB-1% NaCl agar medium using 50 µg and 100 µg of penicillin (Figure 13). We found that LMJ did not grow on LB-agar in the presence of penicillin (Figure 13A, B). LMJ grew on LB-1% NaCl + penicillin plates and showed a prominent zone of inhibition like it showed on TAP-agar penicillin plates (Figure 13C, D). E. coli was not sensitive to either of the two penicillin doses (Figure 13E, F). We have shown in Figure 4A, E that 1% NaCl inhibits growth of LMJ at 22°C. Hence penicillin in combination with 1% NaCl completely prevented growth of LMJ on LB-agar + penicillin plates (Figure 13A, B). We did not measure the diameter of the inhibition zones as this experiment was performed to compare penicillin sensitivity of two gram-negative bacteria, namely E. coli and LMJ on LB agar medium.\n\nOn the medium plates shown in (A–F), the filter paper disc on the right side is the water control disc and the one on the left side is the antibiotic disc containing either 50 µg (82.5 units) or 100 µg (165 units) of penicillin. Plates were imaged after incubation at 22ºC for 4 days. (A) LMJ on LB-agar medium plate and the antibiotic disc contains 50 µg of penicillin. (B) LMJ on LB-agar medium plate and the antibiotic disc contains 100 µg of penicillin. (C) LMJ on LB minus 1% NaCl-agar medium plate and the antibiotic disc contains 50 µg of penicillin. (D) LMJ on LB minus 1% NaCl-agar medium plate and the antibiotic disc contains 100 µg of penicillin. (E) E. coli on LB-agar medium plate and the antibiotic disc contains 50 µg of penicillin. (F) E. coli on LB-agar medium plate and the antibiotic disc contains 100 µg of penicillin. Antibiotic plates were imaged after 3 days of growth.\n\nb) Polymyxin B-sensitivity testing: Polymyxin B binds to the lipopolysaccharide of the outer membranes of gram-negative bacteria and increases the permeability of the bacterial outer membrane, which causes cell death. At 50 µg (500 IU) dose, LMJ is more sensitive to polymyxin B than Chlamydomonas (Table 2). The p-values for the one-tailed and two-tailed tests for the 50-µg dose were 0.4% and 0.09%, respectively, indicating that the polymyxin B sensitivity of LMJ is statistically significant. The p-values for the one-tailed and two-tailed tests for the 100-µg dose were 6.4% and 12.9%, respectively indicating that there was no significant difference in polymyxin b-sensitivity between Chlamydomonas and LMJ (Table 2).\n\nc) Neomycin-sensitivity testing: Neomycin inhibits protein translation by binding to the 30S subunit of bacterial ribosomes. At 50 µg dose, there was no statistically significant difference in neomycin-sensitivity between Chlamydomonas and LMJ as the p-values for the one-tailed and two-tailed tests for the 50 µg dose were 50% and 100%, respectively. At 100 µg dose, LMJ was more sensitive to neomycin than Chlamydomonas (Table 2). The p-values for the one-tailed and two-tailed tests for the 100-µg dose were 0.2% and 0.5 % respectively, indicating that there was a significant difference in neomycin-sensitivity between Chlamydomonas and LMJ (Table 2).\n\nd) Chloramphenicol-sensitivity testing: Chloramphenicol inhibits protein synthesis by binding to the 50S subunit of bacterial ribosomes. Chlamydomonas was resistant to both 50 µg and 100 µg of chloramphenicol as it did not show any zone of inhibitions (Table 2). LMJ was highly sensitive to chloramphenicol compared to Chlamydomonas (Table 2).\n\ne) Testing the potency of penicillin and chloramphenicol in minimizing LMJ contamination on Chlamydomonas culture plates: Our first set of experiments determined penicillin and chloramphenicol are the best antibiotic choices for minimizing LMG contamination. We tested combined growth of LMJ and the wild type Chlamydomonas strain 4A+ on TAP agar plate containing 50 µg penicillin/mL of the medium (Figure 14A) and on TAP-agar plate containing 50 µg chloramphenicol/mL of the medium (Figure 14B). LMJ growth was not visible on both penicillin and chloramphenicol TAP plates (Figure 14).\n\n(A) Chlamydomonas strain 4A+ and LMJ strain streaked on TAP-agar plate containing 50 µg of penicillin/mL of TAP medium. (B) Chlamydomonas strain 4A+ and LMJ strain streaked on TAP-agar plate containing 50 µg chloramphenicol/mL of TAP medium. TAP-agar antibiotic plates were incubated at room temperature for 2 weeks before they were imaged.\n\nFigure 15A shows a schematic diagram of a full-length 16S rRNA gene based on the E. coli 16S rRNA gene. The nine hypervariable regions, V1- V9, spanned nucleotides 69-99, 137-242, 433-497, 576-682, 822-879, 986-1043, 1117-1173, 1243-1294 and 1435-1465, respectively54–56. 11 nucleotides (788-798) within C4 are invariant in bacteria and is represented in the schematic as a black box within the C4 region57. The forward PCR primer, 16SF, spans nucleotides 340-356 in the C2 region and the reverse PCR primer, 16SR, spans nucleotides 784-804 within the C4 region of 16S rRNA gene (Figure 15A). Figure 15B shows that PCR amplification of the partial 16S rRNA gene generated an amplicon of approximately 460 bp in size (Figure 15A).\n\n(A) A schematic diagram showing the conserved and hypervariable regions in the 16S rRNA gene. The interspersed conserved regions (C1–C9) are shown in gray, and the hypervariable regions (V1–V9) are depicted in white. The black box within the C4 region represents 11 nucleotides (788 -798 base pairs) that are invariant in bacteria. PCR primers are shown in thick black arrows. Forward primer is in the C2 region and the reverse primer is in the C4 region. The figure is based on the 16S rRNA gene sequence of E. coli. (B) A DNA agarose gel showing the results of PCR with the primers shown in (A). Lane 1 represents PCR with water (zero DNA control) and Lane 2 showing the PCR product (approximately 460 bp in size) amplified by the PCR primers. 1kb plus DNA ladder was used as a DNA molecular size ladder.\n\nThe amplicon shown in Figure 15B was sequenced to determine the nearest relative of LMJ. NCBI-nucleotide BLAST analyses identified the best match to LMJ’s 16S rRNA gene partial sequence as that of an uncultured bacterium clone LIB091_C05_1243 16S ribosomal RNA gene partial sequence (Accession #: JX086489.1). This hit had a score of 843; 0 E-value and percent identity of 99.35%. Figure 16A shows three nucleotide substitutions (transitions) that are present in LMJ 16S rRNA gene sequence relative to uncultured bacterial clone hit (altered nucleotide in LMJ is shown in red font). Two of these nucleotide substitutions are in the conserved regions, C2 (nucleotide position 348) and, C3 (nucleotide position 542) and the third nucleotide substitution is in the V4 region (nucleotide position 607) (Figure 16A).\n\n(A) A schematic diagram showing the nucleotide changes in LMJ in the 16S rRNA region spanning the C2 and C4 regions in comparison to the best NCBI- BLAST hit (score of 843; E-value 0 and percent identity of 99.35%): Uncultured bacterium clone LIB091_C05_1243 16S ribosomal RNA gene partial sequence (Accession #: JX086489.1). (B) A schematic diagram showing the nucleotide changes in LMJ in the 16S rRNA region spanning the C2 and C4 regions in comparison to a second best BLAST hit with a genus name (score of 837; E-value 0 and percent identity of 99.14%): Acidovorax sp. strain A16OP12 16S ribosomal RNA gene, partial sequence (Accession #: MN519578.1). Conserved regions and the hypervariable regions are depicted in grey and white, respectively. The invariant 11 bp region (788 -798 base pairs) within the C4 region is shown by a black box within the gene. Black nucleotides show the native nucleotides in the BLAST hit that were substituted by the depicted red nucleotides in LMJ 16S rRNA gene sequence. The black bold numbers within the parenthesis beside the nucleotides show the specific nucleotide position where the nucleotide changes have occurred. Nucleotide positions shown in the figures have been assigned according to that of the 16S rRNA gene sequence of E. coli.\n\nWe found that the nearest relative of LMJ with a specific genus name is Acidovorax sp. strain A16OP12 16S (Accession #: MN519578.1). Acidovorax sp. hit had a score of 837, 0 E-value and 99.14% sequence identity. In addition to the above stated three nucleotide substitutions, a fourth nucleotide substitution (another transition at nucleotide position 798) within the 11 bp invariant region in the C4 region was observed, when LMJ’s partial 16S rRNA sequence was compared with that of the Acidovorax sp. strain A16OP12 (Accession #: MN519578.1) (Figure 16B). We have deposited the partial 16S rRNA sequence of LMJ in GenBank with the definition: Bacterium strain clone LIB091_C05_1243 variant 16S ribosomal RNA gene, partial sequence (Accession number: MN633292.1). Raw electropherogram files and sequence text files are available as Underlying data49.\n\nAcidovorax sp. utilize alkanes, PAH and PHA as alternative carbon sources for growth30–38. Since the nearest relative of LMJ is Acidovorax, we tested if LMJ can utilize the above mentioned chemical compounds as a carbon source for growth. TP medium (lacks a carbon source) was supplemented with the following three different categories of external carbon sources in separate experiments: 1) saturated alkane hydrocarbons, 2) aromatic compounds (includes PAH) and, 3) PHA: PHB. Images of all medium plates are available as Underlying data40.\n\na) Testing hydrocarbons (cycloalkanes and 10W30 oil) as the sole carbon source: LMJ was streaked on TP-agar plate as a control to show that LMJ does not grow on a TP medium plate (Figure 17A). 1% cyclohexyl chloride (Figure 17B), 2% (v/v) fresh 10W30 motor oil (Figure 17C) and 2% (v/v) combusted 10W30 motor oil (Figure 17D) were used as carbon sources. We tested one dose of 1% cyclohexyl chloride (4 mL) and three different doses (0.5, 1 and 2 mL) of the 2% (v/v) stock solutions of the fresh and combusted 10W30 motor oil. In Figure 17, we are presenting the results of use of only one dose (2 mL) per types of motor oil. LMJ can grow well on TP-agar media plates coated with 4 ml of 1% cyclohexyl chloride (Figure 17B), and 2 mL of 2% fresh and 2% combusted 10W30 motor oil as a carbon source (Figure 17C, D).\n\nTris-Phosphate (TP) agar medium plates shown in Figure 17B-17D were coated with different hydrocarbons. LMJ was streaked on the control TP-agar medium plate and on the hydrocarbon-coated TP-agar medium plates. After 2 weeks of growth at room temperature, medium plates were imaged. (A) TP-agar medium plate streaked with LMJ. LMJ does not grow on TP medium as it lacks a carbon source. (B) LMJ growth on TP-agar medium plate coated with 4 mL of 1% cyclohexyl chloride diluted with chloroform. (C) LMJ growth on TP-agar medium plate coated with 2 mL of 2% (v/v) fresh 10W-30 car motor oil. (D) LMJ growth on TP-agar medium plate coated with 2 mL of 2% (v/v) combusted 10W-30 car motor oil.\n\nb) Testing PAH and other aromatic compounds as the sole carbon source: Phenanthrene and napthalene are PAHs. Anthropogenic releases of benzoic acid and sodium benzoate into the environment are primarily from their uses as preservatives. Phenylacetate is found in the environment as a common carbon source and is a central intermediate in the degradation of many aromatic compounds such as phenylalanine, phenylacetaldehyde, 2-phenylethylamine and environmental contaminants like styrene and ethylbenzene58.\n\nLMJ was streaked on the TP plate (lacks a carbon source) (Figure 18A). 1% phenanthrene (Figure 18B), 1% napthalene (Figure 18C), 1% benzoic acid (Figure 18C) and 1% phenyl acetate (Figure 18D) were used as carbon sources in TP medium plates. We tested two doses of phenanthrene (2 and 4 mL) and three different doses (0.5, 1 and 2 mL) of napthalene, benzoic acid and phenyl acetate. LMJ can grow on TP-agar media plates coated with 4 mL doses of 1% phenanthrene (Figure 18B), 2 mL of 1% naphthalene (Figure 18C), 0.5 mL of 1% benzoic acid (Figure 18D) and 0.5 mL of 1% phenyl acetate as a sole carbon source (Figure 18E).\n\nTP medium plates shown in panels (B–E) were coated with different polycyclic and monocyclic aromatic compounds. LMJ was streaked on the control TP-agar medium plate and on the aromatic compound-coated TP-agar medium plates. After 2 weeks of growth at room temperature, medium plates were imaged. (A) TP plate streaked with LMJ. LMJ does not grow on TP plate as it lacks a carbon source. (B) LMJ growth on TP plate coated with 4 mL of 1% phenanthrene dissolved in chloroform. (C) LMJ growth on TP plate coated with 2 mL of 1% naphthalene dissolved in chloroform. (D) LMJ growth on TP plate coated with 0.5 mL of 1% benzoic dissolved in chloroform. (E) LMJ growth on TP plate coated with 0.5 mL of 1% phenyl acetate dissolved in chloroform.\n\nc) Testing PHB as the sole carbon source: PHB is a polymer belonging to the polyesters class (PHA) that are of interest as bio-derived and biodegradable plastics32. We tested one dose of 1% PHB (4 mL) on the TP-agar plate (Figure 19). LMG can grow on TP + PHB agar but it grows slowly (Figure 19).\n\n(A) LMJ growth on TP-agar medium plate coated with 4 mL of 1% polyhydroxybutyrate (PHB). (B) Zoom up of the TP + PHB plate shown in (A). TP-agar medium plate (lacking a carbon source) shown in Figure 18A served as the negative control for this experiment. Plate was imaged after two weeks of growth at room temperature.\n\n\nDiscussion\n\nLMJ is a mesophilic, acetate-loving, pigmented, gram-negative rod. It is a salt-sensitive, oxidase-positive, starch hydrolysis-negative, non-enteric bacterium (Figure 1–Figure 12). LMJ is gamma hemolytic (results available as Underlying data59). NCBI-BLAST analyses of the partial 16S rRNA gene sequence of LMJ revealed that its best match is the partial 16S rRNA gene sequence of an uncultured bacterium clone LIB091_C05_1243 16S (Accession #: JX086489.1). The isolation source for this NCBI match is from monochloramine-treated drinking water biofilms60. The second best match to the LMJ’s partial 16S rRNA gene sequence is that of Acidovorax sp. strain A16OP12. This Acidovorax strain is isolated from a lake sediment in China (NCBI, Accession #: MN519578.1).\n\nPseudomonas facilis, Pseudomonas delafieldii, E. Falsen (EF) Group 13, EF Group 16, and several clinical/environmental isolates have been assigned a new genus name, Acidovorax, and belongs to the acidovorans (acid-devouring bacteria) rRNA complex in rRNA superfamily III (betaproteobacteria) in the family Comamonadaceae19. Members of these genera are aerobic, mesophilic, gram-negative, oxidase-positive rods, starch-hydrolysis-negative and are commonly found in biofilm communities in soil, water and on plants61. Pyrosequencing analysis of bacterial biofilm communities in a water meter of a drinking water distribution system in Illinois revealed that 58% of betaproteobacteria in the biofilm on the water meter were Acidovorax sp.62. Biofilm formation is essential for the virulence of phytopathogenic strains of Acidovorax species24,25,27,63–65. Our results indicate that LMJ forms biofilm (Figure 5).\n\nMany bacteria produce extracellular and surface-associated components such as membrane vesicles, extracellular DNA and moonlighting cytosolic proteins for which the biogenesis and export pathways are not fully understood66. In Pseudomonas aeruginosa, cells undergo explosive cell lysis as a mechanism for the biogenesis of bacterial membrane vesicles and biofilm66. LMJ liquid cultures were inoculated with tooth picks (Figure 5). We have noticed LMJ growth on the tooth pick that is immersed in the shaking liquid media. In future, we will gram-stain cells from non-shaking liquid cultures to compare with that from the shaking cultures. We can isolate MVs from LMJ liquid cultures and stain it with FM4-64 fluorescent dye to monitor the red fluorescence. FM4-64 shows low fluorescence in water but fluoresces intensely on binding to the membrane66. We can detect extracellular DNA from LMJ liquid culture by staining with TOTO-1 iodide, which is a cell-impermeant, high-affinity nucleic acid stain66. These studies should confirm if LMJ undergoes explosive cell lysis in liquid cultures to form biofilm.\n\nDespite being a gram-negative bacterium, LMJ is very susceptible to penicillin compared to E. coli (Table 2; Figure 13). In a KB test if the diameter of the zone of inhibition is more than 29 mm, with a 10-µg dose (16.5 IU units) of penicillin, then the bacteria is sensitive to penicillin (see Methods). We used penicillin doses that were 5- to 10-fold higher than what is usually used in a KB test. There is one report of a multiple-β-lactam-antibiotic-resistant Acidovorax sp. strain MR-S767.\n\nIf the diameter of the zone of inhibition is more than 17 mm, with a 30-µg dose of chloramphenicol in a KB test, the bacterium is susceptible to chloramphenicol (https://www.flinnsci.com/antibiotic-sensitivity-disk-chloramphenicol-30-g/ab1400/). LMJ is sensitive to chloramphenicol but Chlamydomonas is resistant. If the diameter of the zone of inhibition is more than 16 mm, with a 30 µg dose of neomycin in a KB test, the bacterium is susceptible to neomycin (https://www.flinnsci.com/antibiotic-sensitivity-disks-neomycin-30-g/ab1403/). If the diameter of the zone of inhibition is more than 12 mm with the application of 300 IU of polymyxin B in a KB test, then the bacteria is sensitive to polymyxin B68. We used antibiotic doses of neomycin and polymyxin B were 1.7- to 3.3-fold higher than what is used for KB tests (Table 2). LMJ is resistant to neomycin and polymyxin B (Table 2). We have not found any studies to date that have tested the sensitivity of Acidovorax sp. to polymyxin B, neomycin and chloramphenicol.\n\nMany phytopathogenic Acidovorax strains are pigmented18. LMJ has light pink-brown pigmentation. Pseudomonas aeruginosa has water soluble pinkish red pyorubrin pigment and dark brown pyomelanin pigment69–71. Pyorubrin’s biosynthesis is enhanced by the addition of 1% DL-glutamate in the growth media69. Pyorubrin has a characteristic absorption peak in the visible light at 520 nm70. Pyomelanin is produced as a product of tyrosine catabolism via homogentisate (HGA) (Figure 7). Pyomelanin biosynthesis needs oxygen and light and can be enhanced by the addition of 0.36–1% tyrosine in the growth medium. Pyomelanin has a characteristic absorption peak in the visible light ranging from 400–405 nm46,47.\n\nWe used 1% tryptone as we did not have access to tyrosine at our lab. Commercial tryptone contains 1.86 grams of tyrosine/100 grams of tryptone (https://khimexpert.com/wp-content/uploads/2018/12/GCM23-Tryptone.pdf). The 1% tryptone contained 0.186 g of tyrosine/L of liquid medium which is approximately 19- to 54-fold lower than what researchers have used in inducing pyomelanin production in bacteria47,69. Although 1% tryptone induced pigment production in LMJ on TAP medium (Figure 6) it was not dark brown like that observed in other bacterial or fungal strains5,46,47. In future, we plan to test the enhancement of pigment production in LMJ with the addition of 1% tyrosine in the growth media.\n\nLMJ pigmentation on TAP+1% tryptone in light and in dark and spectrophotometric pigment analyses strongly indicate that the light pink-brown pigmentation in LMJ is due to light-induced HGA-based pyomelanin production (Figure 6 and Figure 8). In our experiments, we have a very small shift of 1–2 nm in the absorption peaks for BQA and HGA (Figure 8B, C) because we did not buffer our samples to a pH of 6.7–6.8 with phosphate buffer69. We performed this experiment using a “quick and crude” protocol to get preliminary data before we optimize our experimental protocol according to the protocols found in the literature45,47. We plan to optimize our protocols for preparing the samples for the spectrophotometric analyses using the method described in the literature69. We would also like to test if the addition of ascorbic acid in the buffered-acidified cell pellet washes can reduce BQA back to HGA via monitoring the shift in absorption maxima from 250 to 290 nm, as described previously69.\n\nPyomelanin protects cells against oxidative stress5,15. It would be interesting to probe pyomelanin’s role in oxidative stress protection in LMJ. LMJ growth can be monitored on TAP +1% tyrosine agar plate in the presence/absence of hydrogen peroxide and photo-sensitizers like Rose Bengal, that generates the reactive oxygen species, singlet oxygen, in the presence of light and oxygen72. Experiments can be performed in the absence and presence of inhibitors of 4-hydroxyphenylpyruvate dioxygenase (HPPD) which are herbicides like 2-[2-nitro-4-(trifluoromethyl) benzoyl]-1,3-cyclohexanedione (NTBC) (Figure 7)11,73. Additionally, effects of different light intensities, pH, UV light, heat and salt stress on pyomelanin production can be determined.\n\nTwo and three nucleotide substitutions in the conserved regions of the 16S rRNA gene were observed in LMJ when compared with that of the uncultured bacterial clone LIB091_C05_1243 and Acidovorax sp. strain A16OP12, respectively (Figure 16). These results show that conserved regions of the 16S rRNA are not really “conserved” and conserved regions of the 16S rRNA gene exhibit considerable variations that need to be considered, when using this gene as a biomarker74.\n\nDifferent environmental strains of Acidovorax sp. exist that can degrade polychlorinated-biphenyls, PAH, plastic films and saturated hydrocarbons (alkanes) which are major environmental pollutants30–38,75–79. In our first preliminary round of experiments, presented in this work, we have shown that at 22°C on TP medium, LMJ can use PAH, aromatic acids/esters, PHB, saturated chloro-alkane and motor oil as the sole carbon source (Figure 17–Figure 19). Our second round of experiments will focus on optimizing the process of uniformly coating the medium surface with chemicals as we had uneven deposition of chemical crystals on the plates. Uneven coating can affect LMJ’s utilization of aromatic chemicals.\n\nAcidovorax sp. DP5 displays a high extracellular depolymerase enzyme activity when grown in medium containing 0.25% (w/v) of PHB and 1 gram/L of urea as carbon and nitrogen source, respectively32. The depolymerase enzyme produced by strain Acidovorax sp. DP5 showed high percentage of degradation of PHB films in an alkaline condition at pH 9 under a temperature of 40°C32. Our third round of experiments would be to test different types of nitrogen sources, pH and temperature that would allow optimal growth on PHB and PAH-containing TP medium plates. It would be interesting to compare the growth of LMJ on M9 bacterial minimal medium against the growth on TP medium, when testing alternative carbon sources, as M9 medium has a higher phosphate and nitrogen content than the TAP medium, respectively (Table 1). We need to collaborate with a research lab that can test the concentration of these chemicals on the TP medium plates before and after the LMJ growth to get additional support that LMJ is removing PHB, PAH and other hydrocarbons in the TP medium plates.\n\nIn summary, LMJ shares morphological and many biochemical traits with Acidovorax sp. Currently in the NCBI database, there are 60 genome assemblies of 17 characterized Acidovorax species and 67 genome assemblies of uncharacterized Acidovorax environmental isolates. The genome of the nearest relative of LMJ, Acidovorax sp. strain A16OP12, an environmental isolate, has not been sequenced yet. Because of funding limitations, we could not sequence the whole genome of LMJ at the time of this study. But we will have funds in fall 2020 to sequence the LMJ’s genome using Pacific Biosciences technology. This will allow us to: 1) determine assign a specific scientific genus/species/strain name to LMJ and, 2) will identify the genes in LMJ that play a role in its metabolic diversity relevant to bioremediation of common environmental pollutants.\n\n\nData availability\n\nNCBI GenBank: Bacterium strain clone LIB091_C05_1243 variant 16S ribosomal RNA gene, partial sequence. Accession number MN633292.1.\n\nFigshare: Antibiotic sensitivity data for the bacterial strain LMJ (Bacterium strain clone LIB091_C05_1243 variant 16S ribosomal RNA; GenBank Accession # MN633292.1). https://doi.org/10.6084/m9.figshare.12407735.v143.\n\nThis project contains the following underlying data:\n\nData S1 LMJ (XLSX). (Means and standard deviations of the zones of inhibitions of the bacterial strain LMJ and Chlamydomonas, induced by 4 antibiotics.)\n\nData S2 LMJ (XLSX). (Statistical analyses of the zones of inhibitions of the bacterial strain LMJ and Chlamydomonas, induced by 4 antibiotics.)\n\nFigshare: Images of antibiotic plates of the bacterial strain LMJ (Bacterium strain clone LIB091_C05_1243 variant 16S ribosomal RNA; GenBank Accession # MN633292.1) and green micro-alga Chlamydomonas from the antibiotic susceptibility disc diffusion tests. https://doi.org/10.6084/m9.figshare.1240774144.\n\nThis project contains 16 images of antibiotic plates used for the antibiotic susceptibility tests using the disc diffusion method for Chlamydomonas and the bacterial strain, LMJ.\n\nFigshare: Tests using Tris-Phosphate medium (TP) to see if hydrocarbons, aromatic compounds and polyhydroxyalkanoates can be used by the bacterium LMJ (Bacterium strain clone LIB091_C05_1243 variant 16S ribosomal RNA; GenBank Accession # MN633292.1) as the sole carbon source. https://doi.org/10.6084/m9.figshare.1240782240.\n\nThis project contains 23 images of Tris-Phosphate medium plates containing different alternative carbon sources. Bacterium LMJ was streaked on these chemical plates to test if LMJ can utilize these chemicals as the sole carbon source for energy and growth.\n\nFigshare: 16S rRNA partial gene sequences of the bacterial strain LMJ (Bacterium strain clone LIB091_C05_1243 variant 16S ribosomal RNA; GenBank Accession # MN633292.1). https://doi.org/10.6084/m9.figshare.1241037249.\n\nThis project contains the following underlying data:\n\nABI extension files obtained from partial sequencing of the LMJ strain 16S rRNA.\n\nCorresponding text files of DNA sequences.\n\nFigshare: Growth of bacterial strain LMJ (Bacterium strain clone LIB091_C05_1243 variant 16S ribosomal RNA; GenBank Accession # MN633292.1) and Staphylococcus aureus on Tryptic Soy Agar medium plates containing 5% sheep blood. https://doi.org/10.6084/m9.figshare.1242088459.\n\nThis project contains five images of strain LMJ and Staphylococcus aureus grown on Tryptic Soy Agar medium plates containing 5% sheep blood.\n\nFigshare: TAP and LB liquid cultures of the bacterial strain LMJ (Bacterium strain clone LIB091_C05_1243 variant 16S ribosomal RNA; GenBank Accession # MN633292.1) grown for 4 days and Gram stains of LMJ from these liquid cultures. Growth of LMJ on TAP-agar from a 96 hours-grown TAP liquid culture is also shown. https://doi.org/10.6084/m9.figshare.1242089350.\n\nThis project contains images of bacterial strain LMJ grown in liquid TAP and liquid LB medium for 96 hours, taken at 24-h intervals, alongside images of Gram stains for strain LMJ grown in each medium.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
"appendix": "Acknowledgments\n\nWe would like to thank: Dr. Krishna K. Niyogi (UC Berkeley, Professor, Department of Plant & Microbial Biology, CA, USA) for giving us the Chlamydomonas 4A+ strain; Dr. Farooq A. Khan (Professor, University of West Georgia, Chemistry Department) for providing us with napthalene and, Mr. Joseph H. Douglas (Lab Coordinator, University of West Georgia, Biology Department) for providing us with 10W30 fresh and used car motor oil.\n\n\nReferences\n\nSueoka N: Mitotic replication of deoxyribonucleic acid in Chlamydomonas reinhardti. Proc Natl Acad Sci U S A. 1960; 46(1): 83–91. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKunze M, Pracharoenwattana I, Smith SM, et al.: A central role for the peroxisomal membrane in glyoxylate cycle function. Biochim Biophys Acta. Elsevier; 2006; 1763(12): 1441–52. PubMed Abstract | Publisher Full Text\n\nKornberg HL, Krebs HA: Synthesis of cell constituents from C2-units by a modified tricarboxylic acid cycle. 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PubMed Abstract | Publisher Full Text\n\nPalumbo SA: Role of iron and sulfur in pigment and slime formation by Pseudomonas aeruginosa. J Bacteriol. 1972; 111(2): 430–6. PubMed Abstract | Free Full Text\n\nKandela SA, al-Shibib AS, al-Khayat BH: A Study of Purified Pyorubrin Produced by Local Pseudomonas Aeruginosa. Acta Microbiol Pol. 1997; 46(1): 37–43. PubMed Abstract\n\nLedford HK, Chin BL, Niyogi KK: Acclimation to singlet oxygen stress in Chlamydomonas reinhardtii. Eukaryot Cell. 2007; 6(6): 919–30. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSiehl DL, Tao Y, Albert H, et al.: Broad 4-hydroxyphenylpyruvate dioxygenase inhibitor herbicide tolerance in soybean with an optimized enzyme and expression cassette. Plant Physiol. 2014; 166(3): 1162–76. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMartinez-Porchas M, Villalpando-Canchola E, Suarez LEO, et al.: How conserved are the conserved 16S-rRNA regions? PeerJ. 2017; 5: e3036. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHaigler BE, Wallace WH, Spain JC: Biodegradation of 2-nitrotoluene by Pseudomonas sp. strain JS42. Appl Environ Microbiol. 1994; 60(9): 3466–9. PubMed Abstract | Free Full Text\n\nLessner DJ, Parales RE, Narayan S, et al.: Expression of the nitroarene dioxygenase genes in Comamonas sp. strain JS765 and Acidovorax sp. strain JS42 is induced by multiple aromatic compounds. J Bacteriol. 2003; 185(13): 3895–904. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOhtsubo Y, Maruyama F, Mitsui H, et al.: Complete genome sequence of Acidovorax sp. strain KKS102, a polychlorinated-biphenyl degrader. J Bacteriol. 2012; 194(24): 6970–1. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKoma D, Sakashita Y, Kubota K, et al.: Degradation of Car Engine Base Oil by Rhodococcus sp. NDKK48 and Gordonia sp. NDKY76A. Biosci Biotechnol Biochem. 2003; 67(7): 1590–3. PubMed Abstract | Publisher Full Text\n\nIbrahim HMM: Biodegradation of used engine oil by novel strains of Ochrobactrum anthropi HM-1 and Citrobacter freundii HM-2 isolated from oil-contaminated soil. 3 Biotech. 2016; 6(2): 226. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "69019",
"date": "10 Aug 2020",
"name": "Dickson Aruhomukama",
"expertise": [
"Reviewer Expertise Microbiology",
"Microbial Genomics and Bioinformatics",
"Biotechnology"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nFirst, I would like to commend the authors for the good work. I find the work very interesting and comprehensive in several aspects. Second, I find every pursuit of identifying candidates that could be used in bioremediation of great importance. Third, I commend the authors for exploring to the dot a variety of methods in this work. I hope that the authors can sequence the bacterial strain and continue to pursue this work to further understand its role in bioremediation.\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": "65744",
"date": "12 Aug 2020",
"name": "Ruby A. Ynalvez",
"expertise": [
"Reviewer Expertise Molecular Biology and Biochemistry"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper gave a report on a novel bacterium that share morphological and many biochemical traits with Acidovorax sp. I find the paper interesting and worthy of publishing.\nThe isolation of this bacteria was highly interesting as the work in this article stemmed from a side research project of young investigators. There were four undergraduates and, one high school student.\n\nThe bacterium being reported is a promising candidate for further investigation for its use in bioremediation.\n\nThe bacterium produces pyomelanin. This finding opens another avenue for investigation - - to probe pyomelanin’s role in oxidative stress protection in LMJ.\n\nSufficient and scientifically sound methods have been done in order to characterize this novel bacterial strain. Results presented were adequate to give detailed characterization of the novel isolate. In addition, I appreciate that the authors provided sufficient and clear details of methods and analysis to allow replication by others.\n\nThis paper reported an interesting finding that has a potential for useful application in the future. I hope that the authors can continue to further characterize this novel strain, LMJ.\n\nI just have few and minor comments:\nThe optical density of 0.08–13 which matches the 0.5 McFarland standard seem to be a very wide range and 13 is too high. I think this is a typographical error.\n\nIs there a need to present the data from each replication? Consider presenting just the means and SDs and mentioned in the methods and/or underneath the table the number of replications done.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "69021",
"date": "19 Aug 2020",
"name": "Juan Campos Guillén",
"expertise": [
"Reviewer Expertise Molecular microbiology",
"RNA expression",
"tRNA methabolism."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 Mitra et al lays out a very interesting case for a connection between a novel bacterial strain (Acidovorax sp.) that was isolated from a contaminated TAP medium plate of Chlamydomonas and that can utilize common environmental pollutants as a carbon source. I also agree that the identification and characterization of Acidovorax sp., is absolutely required to reduce prevalence and prevent the further spread of contamination on TAP medium. The work is well performed and easy to follow. I think that this adds to the body of knowledge and recommend for acceptance with minor comments addressed below: Please see below for individual points:\nFigure 2 could be eliminated or improve the quality of the figure.\n\nI suggest changing the figure 8 for a graphic or table.\n\nIn page. 17, first line change LMG for LMJ.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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/9-656
|
https://f1000research.com/articles/9-315/v1
|
30 Apr 20
|
{
"type": "Research Article",
"title": "SARS-CoV-2 epidemic in India: epidemiological features and in silico analysis of the effect of interventions",
"authors": [
"Archisman Mazumder",
"Mehak Arora",
"Vishwesh Bharadiya",
"Parul Berry",
"Mudit Agarwal",
"Priyamadhaba Behera",
"Hemant Deepak Shewade",
"Ayush Lohiya",
"Mohak Gupta",
"Aditi Rao",
"Giridara Gopal Parameswaran",
"Archisman Mazumder",
"Mehak Arora",
"Vishwesh Bharadiya",
"Parul Berry",
"Mudit Agarwal",
"Hemant Deepak Shewade",
"Ayush Lohiya",
"Mohak Gupta",
"Aditi Rao",
"Giridara Gopal Parameswaran"
],
"abstract": "Background: After SARS-CoV-2 set foot in India, the Government took a number of steps to limit the spread of the virus in the country. This included restricted testing, isolation, contact tracing and quarantine, and enforcement of a nation-wide lockdown starting 25 March 2020. The objectives of this study were to i) describe the age,gender distribution and mortality among COVID-19 patients identified till 14 April 2020 and predict the range of contact rate; and ii) predict the number of active COVID-19 patients after 40 days of lockdown. Methods: We used a cross-sectional descriptive design for first objective and a susceptible-infected-removed model for in silico predictions. We collected data from government-controlled and crowdsourced websites. Results: Studying age and gender parameters of 1161 Indian COVID-19 patients, the median age was 38 years (IQR, 27-52) with 20-39 year-old males being the most affected group. The number of affected patients were 854 (73.6%) men and 307 (26.4%) women. If the current contact rate continues (0.25-27), India may have 110460 to 220575 infected persons at the end of 40 days lockdown. Conclusion: The disease is majorly affecting a younger age group in India. Interventions have been helpful in preventing the worst-case scenario in India, but will be unable to prevent the spike in number of cases.",
"keywords": [
"COVID-19",
"Coronavirus",
"SIR model",
"India"
],
"content": "Introduction\n\nSince December 2019, SARS-CoV-2, a novel virus of the Coronaviridae family of RNA viruses, has caused a widespread outbreak of the disease, now known as COVID-19, and was declared to be a pandemic by the World Health Organization (WHO) on March 11 20201–3. Human to human transmission occurs primarily through close-contact with the infected person, through fomites in the immediate surroundings of the infected person and via droplets of respiratory secretion, although there is limited evidence pointing to a possibility of airborne and faeco-oral transmission as well4–7. According to a few case studies, transmission may also occur via viral shedding in “pre-symptomatic” individuals during the incubation period8,9.\n\nThe incubation period for COVID-19 is thought to be within 14 days of exposure, with a median incubation period of 4–5 days4,10,11. Globally, the median age of patients affected by COVID-19 is 47 years with the most common clinical findings being fever and cough4,12. About 18% of patients develop shortness of breath4. Severe disease (including dyspnea defined as a respiratory rate of 30/min, blood oxygen saturation of 93%, a partial pressure of arterial oxygen to fraction of inspired oxygen ratio<300, and/or lung infiltrates>50% within 24 to 48 hours) has been reported to occur in 14% of elderly patients with pre-existing chronic diseases13. Critical disease requiring intensive care unit admission has been reported in 5% of patients, and overall case-fatality rate is 2.3%13. Currently, there are no approved treatments for COVID-19 and clinical trials, such as the WHO SOLIDARITY trial, are underway to evaluate the effectiveness of drugs like lopinavir-ritonavir, remdesivir, hydroxychloroquine and azithromycin3,14.\n\nIndia reported its first case of COVID-19 on 30 January 2020; a medical student who had travelled from Wuhan, China, the then epicentre of COVID-1915. On 15th March 2020, India sealed its borders and stopped all international flights, meaning all initial imported cases in India arrived before 15 March 2020. According to the data available in the public domain, as of 12 April 2020, India had 8606 cases (both imported cases and due to person-to-person transmission). According to the Indian Council of Medical Research (the apex national medical research body), as of 31st March 2020, community transmission had not started then and India was in category 2 of WHO classification for transmission patterns, i.e. sporadic cases without evidence of community transmission16. Recent evidence suggests 104 (1.8%) of the 5,911 patients with severe acute respiratory infection who tested positive for COVID-19, and 40 (39.2%) COVID-19 cases did not report any history of contact with a known case or international travel. This indicates imminent community transmission in the near future17.\n\nSince the beginning of the outbreak in India, there have been a number of interventions done by both state level and central level governments (Figure 1). These included restricting the inflow of international passengers, self-quarantine measures, directives on testing and management strategies, and in-country travel restrictions. During this period, the testing strategy also changed from being focused on foreign travel and contact history initially to including all individuals with severe acute respiratory illness and symptomatic health care providers. Gradually there were more social distancing measures in March, which were then followed by state-wise lockdowns, ultimately culminating in a nation-wide 21 day lockdown from 25 March 2020. As of 14th April, the lockdown had been extended until 3rd May 2020 (40 days).\n\nData Source: ICMR-NIE and MoHFW (www.mohfw.gov.in and www.covidindiaupdates.in). Orange, before lockdown; blue, after lockdown began.\n\nIn addition to the epidemiological parameters of COVID-19 in India, the other important question in the current scenario concerns the mathematical parameters of the initial spread of COVID-19 in India, and what are the epidemiological aspects that can predict this spread. We acknowledge there are certain difficulties in making precise calculations due to the rapidly changing dynamicity of the epidemic in the early stages, limited availability of data in the public domain, and limited testing capacity. Nonetheless, mathematical models with reasonable assumptions based on available information can help in analysis of the currently available data to provide important insights for guiding public health interventions18. The most basic of these models is the susceptible-infected-removed (SIR) model19,20, which we have used in the current Indian scenario to determine the range in which contact rate β lies, calculate the range of the current reproduction number, Rt, and predict the number of COVID-19 infections at the end of the 40 day lock down period.\n\n\nMethods\n\nThis was a cross-sectional descriptive analysis of the laboratory confirmed COVID-19 patient-wise data collected from a crowdsourced database (https://www.covid19india.org; includes data from state government and central government agencies). The data was taken for cases confirmed up to 14 April 2020, 7:20 PM Indian standard time or earlier.\n\nData analysis was done in regard to the age distribution, status of patients and gender distribution using Microsoft Office Excel 2007 (Microsoft, Redmond, WA, USA). Fatality rate in any category was found by dividing the number of deaths in the category by the number of affected individuals of that category.\n\nThe SIR model19,20 divides the (fixed) population of N individuals into three \"compartments\", which vary as a function of time (for purpose of this study, we have not included vital dynamics like birth and death rate):\n\nS(t) - S(t) are those susceptible but not yet infected with the disease (in a novel disease like COVID-19, the entire population is assumed to be susceptible as there is no pre-existing immunity);\n\nI(t) - I(t) is the number of infectious individuals;\n\nR(t) - R(t) are those individuals who have been removed from the infected population (includes those who have recovered from the disease and also deaths).\n\nThe SIR model describes the change in the population within each of these compartments in terms of two parameters, β and γ (Figure 2). β describes the effective contact rate of the disease: a susceptible individual comes into contact with an infectious individual and acquires the infection. This parameter considers both the number of people contacted per unit time, and the effectiveness of transmission in each contact. It reflects the force of infection of the disease and helps us understand at what rate the epidemic is progressing. γ is the mean removal rate: in our model, it is calculated using the removed cases against new cases on a daily basis.\n\nβ and γ are useful in the SIR model using the following differential equations:\n\nComparing the equation from the SIR model and the general equation of exponential growth,\n\nWe have used the new cases of COVID-19 daily data available from 24th March to 13th April 2020, to estimate the two parameters (assuming a lag period of 11 days), β and Rt (time varying reproductive number) with the help of SIR model (www.statista.com used for extraction of variables). We assumed that the recovery rate γ would remain constant for the population. The removal rate followed a normal distribution, and the mean was calculated with the data available from 1 March to April 4 202021. Since the effect of interventions would reflect in the contact rate β, we then took the value of γ to be constant equal to the mean (0.103) and ran the SIR model multiple times by varying the value of β, and comparing the trends with the real data.\n\nWe plotted the trend line for the real data using Microsoft Office Excel 2007, and used the equation of the curve to find out the trend of β in India in the present-day scenario by comparing it to equation 4. We then used the present trends of β to estimate the expected number of infections at the end of 40 days lockdown.\n\nAnonymized data available data in the public domain was used for analysis. Ethical approval was not required.\n\n\nResults\n\nThere were 10939 total patients recorded; age and gender data were available for 1161 (10.61%). Of 1161 patients, 854 (73.6%) were men and 307 (26.4%) were women. Median age was 38 years (IQR, 27–52). Nearly half of the patients (43.2%) were in the age group of 20–39 years. The median age of women was 40 years (IQR, 24–56.5) and men was 38 years (IQR, 27–51) (Table 1).\n\n*Normalized values have been calculated by dividing the percentage of patients in each category by the percentage of Indian population in that category.\n\nOut of 1161 patients, 1038 hospitalizations and 29 deaths were documented. The majority of them (65.5%) were in the 60–79 year age group. The median age of deceased patients was 67 years (IQR, 57–71). The mortality rate in male patients was 2.3% and for female patients was 2.9%. Overall case fatality was 2.5%. There was a 10.6% mortality in the 60–79 years age group and 16.7% in individuals aged equal to or above 80 years (Table 2).\n\n*Hospitalized include patients in facility isolation, in ICU or under medical care in hospital\n\nIn total, 94 patients had recovered. Among the recovered patients, 47 (50.0%) were in the 20–39 year age group, 22(23.4%) in the 40–59 year age group. Median age for recovered patients was 35 years (IQR, 24–53).\n\nAfter running multiple simulations using the SIR model, all assuming different values of β we found that the value of β in the current Indian scenario calculated from the trendline of real data lies around 0.272, which is also visible in the graph of real-time active cases lying between β=0.25 and β=0.28 curves (Figure 3).\n\nDatasource: https://www.statista.com/statistics/1104054/india-coronavirus-covid-19-daily-confirmed-recovered-death-cases/.\n\nAlso visible in the graph, is the real data line shifting from 0.24 to 0.28 from day 8. Also, as Rt= β/γ, the value of the reproductive number is found to be varying between 2.5–2.8 in the Indian scenario until 13th April 2020. Considering the present trend of β, the number of infected persons after the 40 day lockdown period may vary from 110,460 to 220,575.\n\n\nDiscussion\n\nTo the best of our knowledge, this is the first study to predict the number of COVID-19 infected persons in India after the 40 days of lockdown. We have used crowd sourced data due to lack of availability of official data. We could not include all the cases in the epidemiological analysis due to unavailability of the demographic details of all the patients. Limitations notwithstanding, there were three key findings.\n\nFirst, the median age of affected individuals is lower than reported in other countries. In study cohorts of Wuhan, the median age of affected patients ranges from 49–56 years4,22,23. In previous studies on COVID-19, it has been established that risk increases with age and comorbidities13,22,23. The broad based nature of India’s population pyramid means there are more people in the younger age group and very few people in the ≥80 years age group. Another reason is that as there is limited evidence of community spread in the epidemic in India so far; it has been reported to be driven by imported cases who mostly belong to the younger age group. In 2018, Indian residents between 35 and 49 years of age took the most holidays outside the nation24. On normalizing the percentage of patients in each age group with the corresponding percentage representation of the population, we observed that the highest number of patients is in the 60–79 years age group category. Interestingly, according to this analysis, men in the 60–79 years age group are affected more than the ≥80 years age group. This is something that has not been reported until now and it has to be seen whether this changes as the number of cases in India grows. This is new to the literature of COVID-19 and needs to be studied further.\n\nSecond, we found a case fatality rate of 2.5%, which is lower than countries like Italy25. The reason for this could be that Italy is in the later phases of the epidemic with widespread community transmission and high mortality due to overburdening of the health system. This has also led to focussing testing on severe patients and possible advise to suspected patients with mild symptoms to stay at home. In China, the case fatality rate was found as 2.3%, 14.8% in the above 80 years population, and 8.0% in the 70–79 years population13. Our estimate gives a slightly higher value, which may be due a smaller sample size or can also be because mild cases of COVID-19 have so far been missed due to restricted testing strategy during the initial stage.\n\nThird, from the SIR model, the values of β and the trend for Rt shows that the interventions which were put in place by the Indian government starting from mid-March were partially effective, preventing the scenario where Rt can reach even more than four26. The increase in β from day eight is probably due to the identification of a cluster in Delhi27. Additionally, Indian Council of Medical Research recently updated the testing strategy, which might have increased the detection of the positive cases significantly. According to a study in the early days of the epidemic, Wuhan city and Hubei province reported Rt between 1.85 and 4.46, which aligns with our study findings26. All over China, the Rt varied from 1.23 to 5.77. South Korea, which has high population density like India, had a decreasing trend of Rt from 9.72 on 20 February to 1.50 on 7 March. This indicates that the interventions have been helpful in preventing the worst case scenario in India but is unable to prevent the spike in number of cases27,28.\n\nAt the end of the 40 days lockdown, India is likely to have a significant number of infected persons (110,460 to 220,575) that would include both symptomatic and asymptomatic individuals. It may also enter the exponential growth phase and it would become very difficult to contain at that stage. The situation can still be controlled if Rt can be brought down close to one. This indicates the need for more effective strategies and ensuring optimum testing to avoid underestimation of danger.\n\nFuture research is needed to support the current findings that unlike other countries, in India, COVID-19 is mostly affecting the 20–39 years age group since our data analysis was restricted to individuals in whom data was available. The SIR model used does not account for age-structure or comorbidities. Hence we are addressing more dynamicity and realness by taking into account multiple compartments with robust mathematical assumptions in our future models to study the current pandemic. Comprehensive studies based on the clinical manifestations and laboratory parameters including genomic sequencing to detect mutations of the virus also needs to be done for the Indian population to see if the clinical features and the viral strains differ from other populations.\n\nTo conclude, the COVID-19 epidemic in India is more affecting younger age groups as compared to other countries. Our mathematical model predicts that India will have a significant number of infected persons after the 40 days lockdown. Hence, strict social distancing, and optimum testing followed by isolation and quarantine are vital elements to control the COVID-19 epidemic.\n\n\nData availability\n\nRaw data for daily cases of COVID-19 with gender, age and patient status obtained from: patientdb.covid19india.org\n\nRaw data for estimation of β obtained from: https://www.statista.com/statistics/1104054/india-coronavirus-covid-19-daily-confirmed-recovered-death-cases/\n\nFigshare:Mazumdaer A et al study - COVID 19 India data, https://doi.org/10.6084/m9.figshare.c.4940472.v229.\n\nThis project contains the following data:\n\n- Confirmed daily cases of COVID-19\n\n- Full patient dataset COVID-19\n\n- Indian population data\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).",
"appendix": "Acknowledgements\n\nWe thank Krushna Prasad Pradhan of National Institute of Technology, Rourkela, Odisha, India and members of the Indian COVID Apex Research Team (i-CART) for their valuable feedback and sharing their expertise. The authors thank the Department for International Development (DFID), UK, for funding the Global Operational Research Fellowship Programme at the International Union Against Tuberculosis and Lung Disease (The Union), Paris, France, in which H D Shewade works as a senior operational research fellow.\n\nDisclaimer: The views represented here are those of the authors and do not represent the institutions they are affiliated to.\n\n\nReferences\n\nZhu N, Zhang D, Wang W, et al.: A Novel Coronavirus from Patients with Pneumonia in China, 2019. N Engl J Med. 2020; 382(8): 727–733. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTang X, Wu C, Li X, et al.: On the origin and continuing evolution of SARS-CoV-2. Natl Sci Rev. 2020; nwaa036. Publisher Full Text | Free Full Text\n\nWorld Health Organization: WHO Director-General’s opening remarks at the media briefing on COVID-19 - 18 March 2020. 2020. Reference Source\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 | Free Full Text\n\nModes of transmission of virus causing COVID-19: implications for IPC precaution recommendations. 2020. Reference Source\n\nvan Doremalen N, Bushmaker T, Morris DH, et al.: Aerosol and Surface Stability of SARS-CoV-2 as Compared with SARS-CoV-1. N Engl J Med. 2020; 382(16): 1564–1567. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLu S, Lin J, Zhang Z, et al.: Alert for non-respiratory symptoms of Coronavirus Disease 2019 (COVID-19) patients in epidemic period: A case report of familial cluster with three asymptomatic COVID-19 patients. J Med Virol. 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 | Free Full Text\n\nQian G, Yang N, Ma AHY, et al.: A COVID-19 Transmission within a family cluster by presymptomatic infectors in China. Clin Infect Dis. 2020; pii: ciaa316. PubMed Abstract | Publisher Full Text\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 | Free 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 | Free Full Text\n\nYoung BE, Ong SWX, Kalimuddin S, et al.: Epidemiologic Features and Clinical Course of Patients Infected With SARS-CoV-2 in Singapore. JAMA. 2020. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWu Z, McGoogan JM: Characteristics of and Important Lessons From the Coronavirus Disease 2019 (COVID-19) Outbreak in China: Summary of a Report of 72 314 Cases From the Chinese Center for Disease Control and Prevention. JAMA. 2020. PubMed Abstract | Publisher Full Text\n\nAdaptive COVID-19 Treatment Trial (ACTT) - Full Text View - ClinicalTrials.gov. 2020. Reference Source\n\nCOVID 100casesIndia. 2020. Reference Source\n\nGlobal surveillance for COVID-19 caused by human infection with COVID-19 virus. Interim Guidance. WHO; 2020. Reference Source\n\nGupta N, Praharaj I, Bhatnagar T, et al.: Severe acute respiratory illness surveillance for coronavirus disease 2019, India, 2020. Indian J Med Res. 2020. PubMed Abstract | Publisher Full Text\n\nPredictive models for COVID-19-related deaths and infections. The Union. 2020. Reference Source\n\nKermack WO, McKendrick AG, Walker GT: A contribution to the mathematical theory of epidemics. Proc R SocLond Ser Contain Pap Math Phys Character. 1927; 115(722): 700–721. Publisher Full Text\n\nHuppert A, Katriel G: Mathematical modelling and prediction in infectious disease epidemiology. Clin Microbiol Infect. 2013; 19(11): 999–1005. PubMed Abstract | Publisher Full Text\n\nIndia - daily cumulative COVID-19 by type 2020.2020. Reference Source\n\nHuang C, Wang Y, Li X, et al.: Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet. 2020; 395(10223): 497–506. PubMed Abstract | Publisher Full Text | Free Full Text\n\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 | Free Full Text\n\nIndia Tourism Source (Domestic & Outbound) Market Insights 2019 - ResearchAndMarkets.com. 2019. Reference Source\n\nLivingston E, Bucher K: Coronavirus Disease 2019 (COVID-19) in Italy. JAMA. 2020; 323(14): 1335. Publisher Full Text\n\nHuang Y, Yang L, Dai H, et al.: Epidemic situation and forecasting of COVID-19 in and outside China. Bull World Health Organ, 2020. Publisher Full Text\n\nPhilip SA: How Tablighi Jamaat emerged as the 'largest known' Covid-19 source in South Asia. 2020. Reference Source\n\nHuge crowds as lockdown sparks mass migration. 2020. Reference Source\n\nShewade HD: Mazumdaer A et al study - COVID 19 India data. figshare. Collection. 2020. http://www.doi.org/10.6084/m9.figshare.c.4940472.v2"
}
|
[
{
"id": "63079",
"date": "22 May 2020",
"name": "Sarika Palepu",
"expertise": [
"Reviewer Expertise Public health and Epidemiology of infectious diseases"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe mathematical model used is \"SIR\", standing for \"Susceptible, Infectious, Recovered/dead\". This needs modification throughout the manuscript.\n\nIn COVID-19, re-infection is being seen in a few patients. This can be explained in limitation as to why \"SIRS\" model was not considered over \"SIR\". And also, not all susceptible individuals are infected. Why was the \"SEIR\" model not considered by authors?\n\nAlthough, the model mentions that the birth rate and death rate were not taken into account, its effect on outcome estimation has not been explicitly mentioned. The reason that the duration of infection is much lesser than the lifetime of an individual can be explanatory for non-inclusion of birth and death rate.\n\nClear mention of the force of infection with proper notation is required.\n\nReasons for the younger age group being affected more can be explained in greater detail.\n\nVarious studies have been published to date with different modelling techniques for the estimation of COVID-19 cases. They can be used for comparison in the discussion section.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "63081",
"date": "29 May 2020",
"name": "Soumya Swaroop Sahoo",
"expertise": [
"Reviewer Expertise Community Medicine and 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\nA very good attempt to present the Indian COVID-19 scenario by the authors. I congratulate them on their work. However a few queries:\nThe data analysis has been performed on 1161 patients. To project it for such a large population has limited scope.\n\nThe range of the projection of 1.10 lakh to 2.20 lakh is quite wide. How do we interpret it?\n\nIn COVID, most of the patients recover in due course. If possible, the SEIR model could have been used for a better picture.\n\nThe discussion section contains a comparison with the descriptive data of different countries. It could have been compared with the modeling data of different studies of other countries to highlight the similarity/difference of the Indian scenario.\n\nThe authors in the discussion state that \"we observed that the highest number of patients is in the 60–79 years age group category. The exact percentage projected needs to be mentioned.\n\nThe assumptions that the authors took for the modeling approach needs to be clearly stated in the methodology apart from birth and death 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?\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": "5607",
"date": "29 Jun 2020",
"name": "PRIYAMADHABA BEHERA",
"role": "Author Response",
"response": "Reply to Dr. Palepu’s (Reviewer 1) comments: Comment 1: The mathematical model used is \"SIR\", standing for \"Susceptible, Infectious, Recovered/dead\". This needs modification throughout the manuscript. Author Response 1: We have used the full form as Susceptible-Infectious-Removed model, where removed includes Deceased and Recovered. Thank you. Comment 2: In COVID-19, re-infection is being seen in a few patients. This can be explained in limitation as to why the \"SIRS\" model was not considered over \"SIR\". And also, not all susceptible individuals are infected. Why was the \"SEIR\" model not considered by authors? Author Response 2: We have added the reason for not using the SIRS model in the methods section. We didn’t consider the SEIR model because, at the time when this study was done, sufficient data was not available in the public domain for a reliable estimate of alpha for India which is the rate of transition from the exposed pool to the infected pool. In view of the limitations in this model, we have developed another model that is available at https://doi.org/10.1101/2020.05.13.20096826. Thank you. Comment 3: Although, the model mentions that the birth rate and death rate were not taken into account, its effect on outcome estimation has not been explicitly mentioned. The reason that the duration of infection is much lesser than the lifetime of an individual can be explanatory for non-inclusion of birth and death rate. Author Response 3: We have added the reason for the non-inclusion of vital dynamics in the methods section and how their inclusion would not significantly affect the results. Thank you. Comment 4: Clear mention of the force of infection with proper notation is required. Author Response 4: For the study, we have calculated gamma and tried to compare the real trends with trends for the varying value of beta. For this reason, we have not clearly mentioned the force of infection as it would not be reliably derived from our study. Also, because of varying Rt, it would be difficult to give a definite value for the force of infection in India. Thank you. Comment 5: Reasons for the younger age group being affected more can be explained in greater detail. Author Response 5: We have explained possible reasons like a high percentage of the population in the younger age group in India and possible younger age of imported cases in more detail in the discussion. Thank you. Comment 6: Various studies have been published to date with different modeling techniques for the estimation of COVID-19 cases. They can be used for comparison in the discussion section. Author Response 6: We have added discussions about other modeling studies of India in the discussion section. Thank you. When we were doing the study, the epidemic had progressed in other countries, so we compared our data with the actual data of the other countries. Also, the aim of our study was not to exactly predict the number of cases but to see the trend of the real data and get an idea about parameters like Rt, and contact rate. We extrapolated to give an idea of the number of cases India could have reached if the Rt had not been decreased by strict interventions. It must be noted that our study does not predict the future trend of beta, rather extrapolation has been done based on the range of beta in India till the time of the study."
},
{
"c_id": "5608",
"date": "29 Jun 2020",
"name": "PRIYAMADHABA BEHERA",
"role": "Author Response",
"response": "Reply to Dr. Sahoo’s (Reviewer 2) comments:Comment 1: The data analysis has been performed on 1161 patients. To project it for such a large population has limited scope.Author Response 1: Thanks for your valuable comments. The data for 1161 patients were used for the study of the epidemiological features of the affected people in India. For the model, the real data of the number of active cases was plotted. However, we agree that using data from the initial stage of the epidemic in India is one of our limitations, and this has been included in the discussion. This study was done to provide an overview of what India might be dealing with.Comment 2: The range of the projection of 1.10 lakh to 2.20 lakh is quite wide. How do we interpret it?Author Response 2: We have incorporated the explanation for this wide range of finding in the discussion. With the preliminary data available in the public domain, we obtained this interval based on the range of beta at the time of the study. Our study does not predict the beta for India in the future, rather we extrapolated based on the range at the time of the study. The range gives an idea of the number of infections if the contact rate wasn’t affected by lockdown and strict social distancing. Thank you.Comment 3: In COVID, most of the patients recover in due course. If possible, the SEIR model could have been used for a better picture.Author Response: As we have mentioned in the reply to Dr. Palepu’s 2nd comment, we didn’t consider SEIR model because, at the time when this study was done, sufficient data was not available in the public domain for a reliable estimate of alpha for India which is the rate of transition from the exposed pool to the infected pool. In view of the limitations in this model, we have developed another model that is available at https://doi.org/10.1101/2020.05.13.20096826. Thank you.Comment 4: The discussion section contains a comparison with the descriptive data of different countries. It could have been compared with the modeling data of different studies of other countries to highlight the similarity/difference of the Indian scenario.Author Response 4: When we were doing the study, the epidemic had progressed in other countries, so we found it better to compare our data with the actual data of the other countries. We have modified our discussion including modeling studies form the Indian scenario. Thank you.Comment 5: The authors in the discussion state that \"we observed that the highest number of patients is in the 60–79 years age group category. The exact percentage projected needs to be mentioned.Author Response 5: This observation is based on the normalized values calculated and tabulated in Table 1. The language has been modified for clarity. Thank you.Comment 6: The assumptions that the authors took for the modeling approach needs to be clearly stated in the methodology apart from birth and death rate.Author Response 6: Apart from the assumption regarding birth and death rate, we have assumed the value of removal rate gamma to be constant as we have mentioned in the text. We assume uniform transmission for all our extrapolations. These have been described in the method section. Thank you."
}
]
}
] | 1
|
https://f1000research.com/articles/9-315
|
https://f1000research.com/articles/9-583/v1
|
09 Jun 20
|
{
"type": "Brief Report",
"title": "Strengthening the core health research capacity of national health systems helps build country resilience to epidemics: a cross-sectional survey",
"authors": [
"Rony Zachariah",
"Dermot Maher",
"Abraham Aseffa",
"Mahnaz Vahedi",
"Pascal Launois",
"Mohammed Khogali",
"Garry Aslanyan",
"John C. Reeder",
"Dermot Maher",
"Abraham Aseffa",
"Mahnaz Vahedi",
"Pascal Launois",
"Mohammed Khogali",
"Garry Aslanyan",
"John C. Reeder"
],
"abstract": "Background: TDR, The Special Programme for Research and Training hosted at the World Health Organization, has long supported Low- and Middle-Income Countries in strengthening research capacity through three training programmes: the Postgraduate Training Scheme (PGTS), the Clinical Research and Development Fellowship (CRDF), and the Structured Operational Research Training InitiaTive (SORT IT). In the advent of the COVID-19 pandemic, we assessed whether those trained through these programmes were involved in the COVID-19 response and if so, in which area(s) of the emergency response they were applying their skills. Methods: From the records for each training programme, we identified the individuals who had completed training during the relevant timespan of each programme: 1999-2018 for the CRDF scheme, 2015-2020 for PGTS, and 2009-2019 for SORT-IT. Between March and April 2020, we sent trainees an online questionnaire by e-mail. Results: Out of 1254 trained, 1143 could be contacted and 699 responded to the survey. Of the latter, 411 were involved with the COVID-19 response, of whom 315 (77%) were applying their acquired skills in 85 countries. With some overlap between programmes, 84% of those trained through CRDF were applying their skills in 27 countries, 91% of those trained through PGTS were applying their skills in 19 countries, and through SORT IT, this was 73% in 62 countries. Skills were being applied in various areas of the emergency response, including: emergency preparedness, situation analysis/surveillance, infection control and clinical management, data generation, mitigating the effect of COVID on the health system, and research. Depending on the type of training programme, 26-74% were involved in implementation, operational or clinical research. Conclusion: Research training programmes build research capacity and equip health workers with transferable core competencies and skillsets prior to epidemics. This becomes invaluable in building health system resilience at a time of pandemics.",
"keywords": [
"COVID-19",
"Pandemic",
"Health systems",
"Training",
"Emergency preparedness"
],
"content": "Introduction\n\nOne of the lessons that should have been learned from the 2014–2015 Ebola epidemic in West Africa, the largest and longest Ebola outbreak in history, was “the need to be better prepared for the next epidemic”1. “The next epidemic” which is happening now in 2020 and is the COVID-19 pandemic. This pandemic, of unprecedented global scale and impact, has tested the preparedness and resilience of every country. Among the many factors that contribute to preparedness and resilience, the capacity to undertake health research is a vital component of the response to infectious disease outbreaks. As we have seen from the current pandemic, all countries are at risk of infectious disease outbreaks and need to strengthen their capacity for a timely and effective research response. Capacity to undertake research varies widely among countries, reflecting the extent of investment and efforts to build and retain that capacity, usually over a long period of time. TDR, The Special Programme for Research and Training hosted at the World Health Organization (WHO), has long supported Low- and Middle-Income Countries (LMICs) in strengthening research capacity, through the range of activities needed to develop the necessary institutional base, research infrastructure, training programmes, career development pathways, research portfolio, regulatory frameworks and networks.\n\nFortunately, most countries, most of the time, do not have such outbreaks, so the opportunities for developing capacity for research through “on the job” learning during an outbreak are limited. This has two key implications. Firstly, developing capacity for research on infectious diseases, including: outbreaks takes place to a large extent before an outbreak (or between outbreaks) and is blind to the next specific infectious agent. Secondly, developing adaptable capacity for research on other health problems contributes to generic research capacity, which becomes applicable during infectious disease outbreaks. TDR supports a number of long-term programmes to strengthen capacity for research on infectious diseases: the Postgraduate Training Scheme (PGTS) on implementation research, the Clinical Research and Development Fellowship (CRDF) scheme on clinical research, and the Structured Operational Research Training InitiaTive (SORT IT) on operational research, a partnership-based initiative led by TDR and implemented in collaboration with various partners (Box 1).\n\nPostgraduate Training Scheme: TDR strengthens individual and institutional capacity for implementation research by supporting a network of seven universities in disease-endemic regions to train Master’s students on courses relevant to implementation research.\n\nClinical Research and Development Fellowship (CRDF) scheme: TDR supports placements of researchers with relevant partners (including pharmaceutical companies, product development partnerships, and public research institutions involved in clinical research) to strengthen their skills in clinical trials. The scheme started in 1999 with a small number of fellows and was scaled up in 2008 and 2014, with the support of the Bill & Melinda Gates Foundation (Gates Foundation) and in partnership with the International Federation of Pharmaceutical Manufacturers & Associations (IFPMA).\n\nStructured Operational Research Training Programme (SORT IT): TDR coordinates a global partnership-based initiative to support countries and institutions to build sustainable operational research capacity. The target audience is front-line health workers from disease control programmes. The focus of training is on teaching practical skills for the generation of high quality, timely and disaggregated data for evidence-informed decision-making to improve public health.\n\nStimulated by examples of people who trained on these programmes and used the skills they gained to contribute to the COVID-19 response, we were interested to assess this more systematically. We therefore assessed whether those trained were involved in the COVID-19 response and if so, in which area(s) of the emergency response they were applying their skills.\n\n\nMethods\n\nWe used three online questionnaires in English (one per programme, see Extended data2; pre-tested on four selected trainees, following which minor changes were made to improve clarity) to gather information from the individuals who had been trained through the three programmes.\n\nFrom the records of each training programme, we identified individuals who had completed training during the relevant timespan of each programme: 1999–2018 for the CRDF scheme, 2015–2020 for the PGTS, and 2009–2019 for SORT-IT. For those people with available contact details, we sent online questionnaires by e-mail (in March 2020 for SORT-IT and in April 2020 for the CRDF and PGTS) asking if they were currently involved in the COVID-19 response. We asked about the nature of their involvement, and if they were applying their acquired skills in responding to the pandemic.\n\nThe survey data was exported to Microsoft Excel for data analysis.\n\nAs part of monitoring and evaluation of TDR supported training programmes, routine online surveys are conducted to gather information for improving the quality and performance of such trainings. Participation in this survey was voluntary and individual consent was obtained for use of anonymized data for reporting and dissemination, including through publications, via the use of a yes/no tick box question within the questionnaires. As this study was part of routine monitoring and evaluation of a training programme, and potential ethical concerns were addressed (responders were all adults, response was voluntary, data were anonymized, personal identifiers were removed and no sensitive personal questions were included that could risk psychological or social harm), this was thus considered a minimal risk study and specific ethical approval for sending questionnaires was not required.\n\n\nResults\n\nA total of 1143 individuals out of 1254 trained could be contacted; 699 responded to the survey. Table 1 shows the number of participants who reported involvement in the COVID-19 response, the number applying their acquired skills and the number of countries involved. Of 699 individuals who responded to the survey, 411 reported involvement in the COVID-19 response, with 315 (77%) applying their acquired skills in 85 countries around the globe. With some overlap between programmes, 84% of those trained through CRDF were applying their skills in 27 countries, 91% of those trained through PGTS were applying their skills in 47 countries, and through SORT IT, this was 73% in 62 countries.\n\n1 Percentage is calculated using the number who responded as the denominator\n\n2 Percentage is calculated using the number involved with COVID-19 as the denominator\n\n3 This figure represents numbers of individual countries without overlaps between programmes.\n\nTDR: UNICEF, UNDP, World Bank, WHO Special Programme for Research and Training in Tropical Diseases.\n\nTable 2 shows that trainees are applying their skills in a range of critical areas of the COVID-19 pandemic response. In terms of research, 74% of those trained through the CRDF scheme, were involved in clinical research, most commonly as a clinical trial manager. For PGTS, 45% were involved in implementation, operational research or clinical research, while 26% of trainees from the SORT IT programme were involved in implementation and/or clinical research.\n\n1 Participants may be involved in more than one area (so percentages do not add up to 100%).\n\nCRDF: Clinical Research and Development Fellowship; PGTS: Post Graduate Training Scheme; SORT IT: Structured Operational Research and Training InitiaTive\n\n\nDiscussion\n\nThe survey findings show that substantial numbers of health workers who were trained to improve their research capacity prior to the COVID-19 pandemic are currently involved in a wide range of emergency response activities.\n\nThis suggests that the respondents have used the specific skills they gained through trainings in combination with their abilities and knowledge as transferable competencies in responding to COVID-19 through a range of research and health system areas. This reinforces the value of TDR’s emphasis on developing core competencies (i.e. sets of skills combined with abilities and knowledge) through research training4,5. It also underscores the longer-term gains of investing in research capacity building programmes.\n\nRegarding contribution to the research response to COVID-19, the high involvement of those trained through the CRDF scheme in clinical research (74%) is a practical example of applying the recommendation in the 2018 World Bank report “Money and microbes: strengthening clinical research capacity to prevent epidemics” concerning leveraging capacity-building from the private sector6.\n\nThe significant involvement of trainees from PGTS and SORT IT in implementation and operational or clinical research shows that strengthening of core national research capacity before (or between) epidemics can make an important contribution to the timely mobilization of research resources during an epidemic.\n\nThe role played by TDR in supporting LMICs to strengthen capacity for clinical and implementation/operational research is in line with the WHO R&D blueprint1 (the global strategy and preparedness plan that allows the rapid activation of research and development activities during epidemics). The development of the R&D blueprint in the aftermath of the 2014–2015 Ebola epidemic in West Africa was a recognition of the need to galvanize research, with the aim “to fast-track the availability of effective tests, vaccines and medicines that can be used to save lives and avert large scale crisis”1. The focus on R&D needs to be complemented by efforts to promote implementation research, which helps to make sure that as new diagnostics, drugs and vaccines emerge from R&D pipelines they are evaluated in clinical trials and approved, they are made available to all who could benefit from them. Resources are needed to strengthen capacity for implementation/operational research, as well as for clinical research, in the LMICs where outbreaks are likely to occur7.\n\nRegarding contribution to the broad health system response to COVID-19, the survey results show that more than seven-in-ten of all trained prior to the COVID-19 pandemic are currently involved in a range of health system areas. These areas include: critical preparedness and response, situation analysis/surveillance, infection control and clinical management, data generation, analysis and reporting, and mitigating the effect of COVID-19 on other diseases. The research training has thus had wider benefits going beyond research, to provide generic skills that can be applied to a range of areas needed to tackle the pandemic.\n\nIn conclusion, the three TDR-supported training programmes have strengthened the health research capacity of health workers, thereby contributing not only to research but also to emergency preparedness and the broad health systems response to COVID-19. Such training programmes help build country resilience to epidemics.\n\n\nData availability\n\nOpen Science Framework: TDR_Training_COVID_Survey, https://doi.org/10.17605/OSF.IO/7YSZ22.\n\nThis project contains the following underlying data:\n\nDataset_1_CRDF_Survey_data_F1000.csv (Contains survey data on Clinical Research and development fellowships).\n\nDataset_2. PGTS_Survey_data_F1000.csv (Contains survey data on Post Graduate Training Scheme)\n\nDataset_3. SORT_IT_Survey_Data_F1000.csv (Contains survey data on Structured Operational Research and Training Initiative)\n\nOpen Science Framework: TDR_Training_COVID_Survey, https://doi.org/10.17605/OSF.IO/7YSZ22.\n\nThe project contains the following extended data:\n\nExtended_data_questionnaire_1_CRDF.docx (Survey questionnaire for Clinical Research and development fellowships)\n\nExtended_data_questionnaire_2_PGTS.docx (Survey questionnaire for Post Graduate Training Scheme)\n\nExtended_data_questionnaire_3_SORT_IT.docx (Survey questionnaire for Structured Operational Research and Training Initiative)\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 all collaborating partners involved with the Postgraduate Training Scheme and Clinical Research and Development Fellowship scheme, and all implementing partners of the global SORT IT partnership coordinated by TDR. (https://www.who.int/tdr/capacity/strengthening/sort/en/)\n\nTDR is able to conduct its work thanks to the commitment and support from a variety of funders. These include our long-term core contributors from national governments and international institutions, as well as designated funding for specific projects within our current priorities. A full list of TDR donors is available on our website at: https://www.who.int/tdr/about/funding/en/\n\n\nReferences\n\nWHO: An R&D blueprint for action to prevent epidemics. Plan of action. 2016. (accessed 6 May 2020). Reference Source\n\nZachariah R: TDR_Training_COVID_Survey.2020. http://www.doi.org/10.17605/OSF.IO/7YSZ2\n\nSpecial Programme for Research and Training in Tropical Diseases (TDR). (accessed 6 May 2020). Reference Source\n\nSpecial Programme for Research and Training in Tropical Diseases (TDR).Using the TDR global competency framework for clinical research. A set of tools to help develop clinical research. (accessed 11 May 2020). Reference Source\n\nAlonge O, Rao A, Kalbarczyk A, et al.: Developing a framework of core competencies in implementation research for low-and middle-income countries: a mixed methods study. BMJ Global Health. 2019; 4(5): e001747. Publisher Full Text\n\nWorld Bank: Money and microbes: strengthening clinical research capacity to prevent epidemics. Washington, DC: World Bank Group. 2018. (accessed 6 May 2020). Reference Source\n\nWHO: The world health report 2013: research for universal health coverage. Geneva: World Health Organization, 2013. (accessed 6 May 2020). Reference Source"
}
|
[
{
"id": "64567",
"date": "15 Jun 2020",
"name": "Bethany Hedt-Gauthier",
"expertise": [
"Reviewer Expertise Health systems research",
"research training and capacity building"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nOverall, this is a clear brief report with straight forward methods and justifiable conclusions. While it is possible that skills used were gained outside of the three training mechanisms, or that skills would have been gained elsewhere in the absence of these mechanisms, what is most important is that trainees identify that they are using skills in the COVID-19 response and the trainees attribute the skills to the specific training mechanisms described here.\nA few points to address:\nFor the specific question, “what percent of trainees are currently using skills from training in COVID-19 response?” The more accurate denominator for this is all reporting trainees. To say that 77% of trainees are using skills for COVID response, when only 315 out of 699 reported as such, is not accurate. The authors clarify in the table with a footnote, but either they need to add a clause of: Of the 59% working on COVID-19 response, 77% report using their skills as part of that response. Or they need to recalculate and say that 45% report using their skills for the COVID-19 response. This is true for overall and by training type reports and should be reflected in the abstract and main text.\n\nCan the authors include reporting by gender and a short discussion? There has been expressed concern about how COVID response has been gendered, specifically with working women being excluded from participating in response either because of systemic issues or through home demands. I would be interested to see if this is an issue with trainees.\n\nPlease add a statement about any influence of non-response and desirability bias.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "5638",
"date": "26 Jun 2020",
"name": "Rony Zachariah",
"role": "Author Response",
"response": "Point by point responses to peer reviewer comments Reviewer 1: Dr. Bethany Hedt Dear Dr Bethany, Thank you very much for taking time to review this paper and your useful comments and suggestions. We have revised the manuscript in line with your suggestions. We have provided a point-by-point response to your comments and suggestions using bold font. Reviewer: Overall, this is a clear brief report with straight forward methods and justifiable conclusions. While it is possible that skills used were gained outside of the three training mechanisms, or that skills would have been gained elsewhere in the absence of these mechanisms, what is most important is that trainees identify that they are using skills in the COVID-19 response and the trainees attribute the skills to the specific training mechanisms described here. Response: Thank you for this encouraging comment Reviewer: For the specific question, “what percent of trainees are currently using skills from training in COVID-19 response?” The more accurate denominator for this is all reporting trainees. To say that 77% of trainees are using skills for COVID response, when only 315 out of 699 reported as such, is not accurate. Response: We have now clearly indicated that for the 77% applying SORT IT skills, the denominator is “those involved with COVID-19” which is 411. Understandably, if one is not involved with COVID-19, it would be impossible to apply SORT IT skills. We thus prefer to keep the denominator of 411 and not 699. Reviewer: The authors clarify in the table with a footnote, but either they need to add a clause of: Of the 59% working on COVID-19 response, 77% report using their skills as part of that response. Or they need to recalculate and say that 45% report using their skills for the COVID-19 response. This is true for overall and by training type reports and should be reflected in the abstract and main text. Response. As suggested above, we have added the 59% (those involved with COVID-19) to the abstract and the results section. Reviewer: Can the authors include reporting by gender and a short discussion? There has been expressed concern about how COVID response has been gendered, specifically with working women being excluded from participating in response either because of systemic issues or through home demands. I would be interested to see if this is an issue with trainees. Response: Of the 411 involved with COVID, 152 (37%) were women. We have indicated this in the abstract and results section. As this proportion is more or less a reflection of the proportions trained by gender we have avoided further discussion. Reviewer: Please add a statement about any influence of non-response and desirability bias. Response: In the Discussion, we have stated that “Limitations of this study are that we had no comparison group and we are unable to know the influence of non-response and social desirability bias.”"
}
]
},
{
"id": "64563",
"date": "16 Jun 2020",
"name": "Stephen Graham",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThanks for the opportunity to review this brief report which provides interesting and novel data, well written and presented.\n\nSome limitations of interpretation of the data could be highlighted in a short paragraph in the discussion, such as:\nThere is no group for comparison that represents the numbers (%) of those not trained who are involved in some way in the COVID response.\n\nCould responders represent a biased sample, by nature of actively participating in this evaluation? The total numbers of past trainees involved may not be as high as “seven-in-ten”.\n\nThe extent and quality of contribution to the COVID response is not clearly defined\n\nSuggest in Introduction, the second sentence to delete “which” or “and”.\n\nIs this pandemic unprecedented in scale? Over 1.5 billion people infected (asymptomatic) with M.tb, around 10 million TB cases and 1.5 million TB-related deaths annually? What about the global death toll associated with the 1918 influenza pandemic?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? 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": "5639",
"date": "26 Jun 2020",
"name": "Rony Zachariah",
"role": "Author Response",
"response": "Reviewer 2: Dr. Steve Graham Dear Dr Steve Graham, Thank you very much indeed for having taken the time to review this paper and your useful comments and suggestions. We have revised the manuscript in line with your suggestions. We have provided a point-by-point response to your responses using bold font below Reviewer: Thanks for the opportunity to review this brief report which provides interesting and novel data, well written and presented. Response: Thank you Reviewer: Some limitations of interpretation of the data could be highlighted in a short paragraph in the discussion, such as: There is no group for comparison that represents the numbers (%) of those not trained who are involved in some way in the COVID response. Could responders represent a biased sample, by nature of actively participating in this evaluation? The total numbers of past trainees involved may not be as high as “seven-in-ten”. The extent and quality of contribution to the COVID response is not clearly defined Response: We agree and have added a para on limitations and have covered thee above points Reviewer: Suggest in Introduction, the second sentence to delete “which” or “and”. Response: done Reviewer: Is this pandemic unprecedented in scale? Over 1.5 billion people infected (asymptomatic) with M.tb, around 10 million TB cases and 1.5 million TB-related deaths annually? What about the global death toll associated with the 1918 influenza pandemic? Response: Since COVID-19 cases were first reported to WHO on 31 December 2019 over 8 million cases have now been reported and the numbers are climbing. We thus prefer to still regard this pandemic of a new human pathogen as unprecedented and we thank you for your kind understanding."
}
]
},
{
"id": "64566",
"date": "23 Jun 2020",
"name": "Desalegn Woldeyohannes",
"expertise": [
"Reviewer Expertise Public health",
"biomedical science"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe survey presents evidence of the usefulness of building up health research undertaking capacity to help contribute to strengthening the national health system resilience to epidemics. It addresses an important topic, given ongoing COVID-19 pandemic. The authors found that the research training programme, which was originally designed to strengthen the research capacity of health workers with the necessary competencies further contributing to health system resilience during COVID-19. It is great to see that researchers such as Rony and colleagues have evaluated the contribution of related training for strengthening the existing health system before or during epidemics. Such study findings will undoubtedly add new knowledge in the area, and could further be applied to reinforce health policy and practice.\n\nThe survey was well-designed, respective data analysis were performed largely with care. The presentation is excellent, with careful writing including tables along with the article help the follow-up to the reader. Below are more specific comments and suggestions by section:\nAbstract:\nA very informative abstract, although data collection and instrument tool used to collect the data could also be included in the method section as well as in the text.\n\nIntroduction: It was a nice overview and set the scene beautifully for the survey. Be there any similar initiatives in the past that could add to the statement of the problem? In this aspect, I would suggest the inclusion of a few statements from research findings on the 2014-2015 Ebola outbreak with respect to how it impacted the health system operation in affected countries in West Africa. By doing so, the necessity of carrying out the current survey would better be justified.\n\nMethods: The survey used a pre-tested questionnaire to collect data, and necessary changes were made to the questionnaire accordingly. Further to this, I would like the following points to be considered:\nStudy design, only mentioned in the title.\n\nA few statements on how items in each questionnaire were developed, for example, whether benchmarked/adopted/modified, or what ways had been followed otherwise in developing them.\n\nWere validity and reliability tests performed on the questionnaire in addition to pre-test so that assuring quality?\n\nHow data were summarized and analysed? Were there any missing data reported, given the low response rate (56%) obtained for the survey, how authors took control of these while analysing the data?\n\nResults:\nWell summarized using tables and text. What does ‘’other’’ refer to in Table 2? Good to mention it as a footnote as, for example the percentage of trainees applying skills in PGTS group for the same was significant, in fact second (67%) next to situation analysis/surveillance (81%), please list them all.\n\nDiscussion: While the dearth of literature/information in the area has largely prevented authors of making a robust scientific argument about the survey findings, their implications have clearly be presented towards practical application, and in national health policy enforcement for a similar pandemic. Further, would authors to expect similar results (percentage) if high response rate was obtained vis-à-vis the low response rate reported on the survey (56%)? This warrants discussion, perhaps as a limitation of the survey.\n\nConclusion: The conclusion was drawn adequately supported by the results.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? 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": "5656",
"date": "29 Jun 2020",
"name": "Rony Zachariah",
"role": "Author Response",
"response": "Point by point responses to peer reviewer commentsReviewer 1: Dr. Desalegn WoldeyohannesDear Dr Woldeyohannes,Thank you very much for reviewing this paper and your useful suggestions. We have revised the manuscript and have provided a point-by-point response using bold font.Reviewer: The survey presents evidence of the usefulness of building up health research undertaking capacity to help contribute to strengthening the national health system resilience to epidemics. It addresses an important topic, given ongoing COVID-19 pandemic. The authors found that the research training programme, which was originally designed to strengthen the research capacity of health workers with the necessary competencies further contributing to health system resilience during COVID-19. It is great to see that researchers such as Rony and colleagues have evaluated the contribution of related training for strengthening the existing health system before or during epidemics. Such study findings will undoubtedly add new knowledge in the area, and could further be applied to reinforce health policy and practice. The survey was well-designed, respective data analysis were performed largely with care. The presentation is excellent, with careful writing including tables along with the article help the follow-up to the reader. Below are more specific comments and suggestions by section:Response: Thank you for these encouraging comments.Reviewer: Abstract:A very informative abstract, although data collection and instrument tool used to collect the data could also be included in the method section as well as in the text.Response: We have mentioned that we used an on-line questionnaire form.Reviewer: Introduction:It was a nice overview and set the scene beautifully for the survey. Be there any similar initiatives in the past that could add to the statement of the problem? In this aspect, I would suggest the inclusion of a few statements from research findings on the 2014-2015 Ebola outbreak with respect to how it impacted the health system operation in affected countries in West Africa. By doing so, the necessity of carrying out the current survey would better be justified.Response: Unfortunately, there were no similar surveys done in relation to the 2014/2015 Ebola outbreak assessing the role of research training on building health system resilience in the affected countries. Reviewer: Methods:The survey used a pre-tested questionnaire to collect data, and necessary changes were made to the questionnaire accordingly. Further to this, I would like the following points to be considered:•Study design, only mentioned in the title.Response: We have added the study design to the Methods. Reviewer: •A few statements on how items in each questionnaire were developed, for example, whether benchmarked/adopted/modified, or what ways had been followed otherwise in developing them. Response: We have added some brief details to the methods. Reviewer: •Were validity and reliability tests performed on the questionnaire in addition to pre-test so that assuring quality?Response: No, but we will consider this in the future. Thank you.Reviewer: •How data were summarized and analysed? Were there any missing data reported, given the low response rate (56%) obtained for the survey, how authors took control of these while analysing the data?Response. 699 (61%) out of 1143 responded to the survey and there might indeed be desirability bias. We have mentioned this in the limitations of the study. This limitation not with-standing, the overall numbers involved with the COVID-19 pandemic and the numbers using their skills is still encouraging. Reviewer: Results:Well summarized using tables and text. What does ‘’other’’ refer to in Table 2? Good to mention it as a footnote as, for example the percentage of trainees applying skills in PGTS group for the same was significant, in fact second (67%) next to situation analysis/surveillance (81%), please list them all.Response: We have listed some of the items under ‘other’ in table 2Reviewer: Discussion:While the dearth of literature/information in the area has largely prevented authors of making a robust scientific argument about the survey findings, their implications have clearly be presented towards practical application, and in national health policy enforcement for a similar pandemic. Further, would authors to expect similar results (percentage) if high response rate was obtained vis-à-vis the low response rate reported on the survey (56%)? This warrants discussion, perhaps as a limitation of the survey.Response. We have already alluded to this point in the limitations. Reviewer: Conclusion:The conclusion was drawn adequately supported by the results.Response: Thank you."
}
]
}
] | 1
|
https://f1000research.com/articles/9-583
|
https://f1000research.com/articles/8-2059/v1
|
05 Dec 19
|
{
"type": "Study Protocol",
"title": "Clinical parameters and biomarkers predicting spontaneous operational tolerance after liver transplantation: a scoping review protocol",
"authors": [
"Christian Appenzeller-Herzog",
"Steffen Hartleif",
"Julien Vionnet",
"Steffen Hartleif",
"Julien Vionnet"
],
"abstract": "Objective: This scoping review aims at systematically identifying prognostic factors for spontaneous immunosuppression (IS) free allograft tolerance (operational tolerance, OT) in non-viral hepatitis and non-autoimmune disease liver transplant (LT) recipients who are undergoing immunosuppression withdrawal (ISW). The results may inform the subsequent conduct of a systematic review with a more specific review question. Background: LT is currently the most effective treatment for end-stage liver diseases. Whereas the short-term outcomes after LT have dramatically improved over the last decades, the long-term outcomes remain unsatisfactory, mainly because of side effects of lifelong IS, such as infections, cardiovascular diseases, malignancies, and nephrotoxicity. ISW studies have shown that OT can be achieved by a subset of LT recipients and recent research has identified biomarkers of OT in these patients. However, an evidence-based selection algorithm for patients that can predictably benefit from ISW is not available to date. The planned review will, therefore, map existing knowledge on prognostic clinical parameters and biomarkers for OT. Inclusion criteria: We will consider studies that record any clinical parameter or biomarker before the initiation of ISW in non-viral hepatitis and non-autoimmune disease LT recipients and analyse their possible association with ISW outcomes (OT or non-tolerance). Studies addressing the effectiveness of OT-inducing treatments will be excluded. Methods: Embase, MEDLINE, and Cochrane Library will be searched for relevant articles or conference abstracts. Full-texts of selected abstracts will be independently screened for inclusion by two reviewers. References and citing articles of included records will be screened for additional relevant records. Clinical trial registries will be searched for ongoing studies, and their investigators contacted for the sharing of unpublished data. Data from included records will be independently extracted by two reviewers using a prespecified data extraction table and presented in both tabular and narrative form.",
"keywords": [
"biomarker",
"clinical parameter",
"flow cytometry",
"gene expression profiling",
"immunosuppression",
"immunosuppression withdrawal",
"liver biopsy",
"liver transplantation",
"operational tolerance",
"regulatory lymphocytes",
"scoping review",
"Tregs"
],
"content": "Introduction\n\nLiver transplantation (LT) currently remains the only long-term treatment option for patients with end-stage liver failure. The success of LT was enabled by the introduction of effective pharmacological immunosuppressive strategies, which mostly target recipient T lymphocyte responses. The drugs that mediate immunosuppression (IS) in LT recipients exert their effects either by inhibiting intracellular T lymphocyte signalling or cellular proliferation. Calcineurin inhibitors (CNIs)1 and mammalian target of rapamycin inhibitors (mTOR-I)2 target the former, whereas corticosteroids3 or antimetabolites like mycophenolate mofetil4 or azathioprine5 impair the latter. Moreover, biologic agents blocking the anti-interleukin 2 receptor on activated T lymphocytes (e.g., basiliximab6) or inhibiting T lymphocyte costimulation (preliminary data in kidney transplantation: belatacept7) have been developed more recently to reduce CNI exposure.\n\nWhile providing effective protection against acute and chronic cellular rejection of the allograft, lifelong IS, particularly corticosteroids and CNIs, are known to cause significant side effects8. Common side effects include various malignancies, cardiovascular and metabolic diseases, renal toxicity, as well as susceptibility to infections9–14. These significant side effects account for chronic morbidity and impair quality of life of LT recipients. Therefore, efforts to minimise exposure to immunosuppressive drugs while preserving graft integrity are warranted.\n\nAmong all solid organ transplants (SOT), the transplanted liver exhibits unique immunoregulatory properties, which render liver allografts less dependent on IS15–17. The attributed mechanisms of liver allograft tolerance are complex and may include deficient antigen presentation, large antigen load, neutralisation of alloantibodies, regulatory T cell (Treg) generation, and long-term microchimerism18–22. Accordingly, LT recipients usually require less intensive IS treatment with lower levels and/or numbers of immunosuppressive drugs compared to other SOT recipients23. In addition, human leukocyte antigen (HLA) match requirements between donor and recipient are less stringent, and the incidence and severity of acute cellular rejection (ACR) episodes are lower and usually better tolerated in LT as compared to other SOT recipients24.\n\nBased on these particular features, clinical studies that examined IS minimization or even complete IS withdrawal (ISW) in LT recipients have been initiated already in the 1990s25–28. Most of these ISW studies (at least all of the recent ones) applied predefined eligibility criteria such as absence of recent rejection episodes or absence of significant histological lesions in a baseline biopsy29,30. In all studies, a significant subset of study participants exhibited stable allograft function and histological graft integrity despite complete ISW31. In agreement with the nomenclature used in the literature, we herein call this state of spontaneous immunological transplant tolerance operational tolerance (OT)32. However, the majority of study participants still would experience an ACR episode or develop abnormal liver function tests following ISW and eventually require the reinstitution of immunosuppressive drugs31 (ISW failure). The mechanisms underlying ISW success or failure in LT recipients are currently not completely elucidated. Likewise, whether ISW outcomes may be predictable at all (see below) or IS minimisation is a safer alternative to complete ISW is not yet known33.\n\nThe discovery of OT has promoted extensive research activity over the last two decades34. On the one hand, it is important to explore the factors that are associated with or enable the development of OT in a subset of transplant recipients35. More detailed knowledge on such predictors of spontaneous OT will help to refine the eligibility criteria for LT recipients to participate in ISW trials and hopefully increase the fraction of successful ISW attempts. On the other hand, researchers have started to address the question as to whether OT can be induced by immune manipulation prior to ISW. Thus, infusion of donor-derived hematopoietic stem cells36–40, Treg41, regulatory dendritic cells (DCreg)42 or mesenchymal stem cells43,44, as well as lymphodepletion protocols using T lymphocyte-directed antibodies45 have been or are being tested for their potential to induce tolerance31.\n\nRegarding the therapeutic dilemma of deleterious effects of chronic IS vs. the risk of ISW failure and graft injury after LT, there is a medical need to define clinical and biochemical markers to predict the success of ISW. Up to now, there is only one systematic review that addressed the benefits and harms of ISW in LT recipients46. It focused on CNI and included only randomized controlled trials (RCTs) comparing ISW and IS continuation after LT. The authors identified a single ongoing RCT, which has been published in the meantime47. In this RCT, the non-inferiority analysis of ISW vs. unchanged IS maintenance treatment on a composite morbidity/mortality endpoint was inconclusive. Based on these results and an unpublished scoping search in the literature that did not identify any new RCTs on this comparison, we concluded that there was not enough data for a new systematic review approach comparing ISW and IS continuation after LT.\n\nIn contrast, the number of publications that highlight predisposing factors or biomarkers for spontaneous OT in ISW cohorts is increasing35. We, therefore, reasoned that the systematic scoping for evidence on such factors would best inform the community regarding the therapeutic dilemma of IS after LT. Accordingly, this scoping review will for the first time systematically collect biomarkers and clinical parameters that are likely predictors of spontaneous OT. The anticipated results shall set the basis for subsequent evidence syntheses or clinical trials with a sharpened research focus. Any evidence that will help understand the spontaneous development of OT and increase the fraction of successful ISW by enabling an informed preselection of ISW candidates is of great value to the community, as it will provide valuable guidance in the therapeutic dilemma of IS after LT.\n\nThe objective of this scoping review will be to identify prognostic factors for spontaneous OT in non-viral hepatitis and non-autoimmune disease LT recipients who are undergoing ISW. The obtained results may inform the subsequent conduct of a systematic review with a more targeted review question.\n\nSpecifically, the review questions are:\n\ni) What are clinical parameters and biomarkers that predispose LT recipient ISW candidates to achieve spontaneous OT?\n\nii) What are the success rates of ISW and achievement of spontaneous OT in LT recipients?\n\niii) What are the rates of graft loss in LT recipients following ISW?\n\n\nProtocol\n\nEligibility criteria\n\nPopulation, Intervention, Outcomes\n\nThe primary eligibility criterion will be the assessment of spontaneous OT, i.e. rejection-free liver allograft survival for at least one year following ISW. LT recipients of any age or stage will be included, but recipients with underlying autoimmune diseases, replicative viral disease and/or multi-organ recipients will be excluded. Studies reporting on mixed populations will be included, if less than 20% of the study population has a viral or autoimmune liver disease aetiology. Studies that do not report the liver disease aetiologies for LT in their population will also be included. All pharmacological IS regimens including combination treatments being completely withdrawn will be eligible. However, studies addressing dose reduction of IS, withdrawal of a subset of drugs from IS combination treatments (e.g. withdrawal of corticosteroids in patients on CNI maintenance treatment), or conversion between IS regimens (e.g. CNI to mTOR-I conversion vs. CNI continuation) will be excluded.\n\nWe will include studies that assess an association of pre-ISW clinical parameters or biomarkers on the development of OT. Studies exclusively addressing the effectiveness of induction or immunomodulation therapies for development of OT (using lymphodepletion or infusion with regulatory immune cells) will be excluded. Prespecified pre-ISW clinical parameters potentially predicting OT are sex, recipient age at LT, time since LT, history of episodes of rejection, liver histology, pharmacologic IS regimen, living (LD) or deceased donor (DD) LT, degree of kinship (or HLA match) of the donor, lymphocytotoxic crossmatch, liver disease aetiology, and previous pregnancies, SOT, or blood transfusions. Prespecified pre-ISW biomarkers potentially predicting OT are any up- or downregulated immune cell subsets (detected by flow cytometry or mass cytometry), any up- or downregulated genes or micro RNAs in the liver allograft or peripheral blood (detected by gene microarray, quantitative PCR, or RNA-seq), epigenetic markers, and anti-HLA antibodies (detected by ELISA, single antigen bead assay, or complement-dependent cytotoxicity assay). Owing to the risk of confounding by interrupted IS in the OT cohort (i.e. featuring successful ISW), data on post-ISW biomarkers will be excluded unless the same biomarkers were measured in the same patients already before ISW.\n\nTypes of study to be included\n\nWe will include prospective, retrospective, randomised, and non-randomised studies irrespective of publication status and including case-control and cross-sectional designs. By reporting on those patients that did not achieve OT after ISW most relevant studies would include a “control cohort” by default. Principal investigators of ongoing studies and conference abstracts will be contacted twice by email for the sharing of their data. Conference abstracts where the data was subsequently published in a peer-reviewed article will be excluded. Animal studies, case reports, case series (i.e. publications where patient histories of exclusively tolerant or non-tolerant ISW-liver recipients are reported48), reviews, letters, and editorials will be excluded. No language or publication date restrictions will be applied.\n\nIdentification of relevant literature. An information specialist (CA-H) will develop the search strategies, which will be reviewed by a second information specialist. Database-specific subject headings and text words (synonyms and word variations) for liver transplantation, ISW, and OT, graft survival, or liver biopsy will be used. We will search the electronic databases Embase via Elsevier, Medline via Ovid, and the Cochrane Central Register of Controlled Trials (CENTRAL). The search string for Embase is provided in Box 1. We will also search the study registry clinicaltrials.gov as well as the World Health Organization’s International Clinical Trials Registry Platform (ICTRP) for ongoing studies. All retrieved references will be exported to EndNote X9 and deduplicated.\n\n(‘liver transplantation’/exp OR (OLT OR LTx):ab,ti OR ((‘liver’/de OR ‘liver lobe’/exp OR ‘liver disease’/exp OR ‘obstructive bile duct disease’/exp OR ‘bile duct atresia’/de OR (liver OR hepatic OR hepato* OR hepatis OR hepatitis OR intrahepatic OR extrahepatic OR cirrhosis OR cirrhotic OR ‘periportal fibrosis’ OR jaundice OR icterus OR bilirubinaemia OR cholestasis OR cholestatic OR ((bile OR biliary OR choledoch*) NEAR/3 (obstruction OR stasis OR occlusion OR stenosis OR stricture OR obliteration OR atresia OR agenesi*))):ab,ti) AND (‘transplantation’/de OR ‘organ transplantation’/de OR ‘allotransplantation’/de OR ‘orthotopic transplantation’/de OR ‘recipient’/exp OR (transplant* OR Tx OR allotransplant* OR graft* OR allograft* OR recipient*):ab,ti)))\n\nAND\n\n(((‘immunosuppressive agent’/exp OR ‘calcineurin inhibitor’/exp OR ‘mammalian target of rapamycin inhibitor’/de OR ‘immunosuppressive treatment’/de) AND (‘treatment withdrawal’/exp OR ‘weaning’/de)) OR ((((immunosuppress* OR immuno-suppress* OR immune-suppress* OR immunodepress* OR immuno-depress* OR immune-depress* OR anti-rejection OR antirejection OR ‘immune system-suppressing’ OR ‘transplantation reaction inhibition’ OR anti-metaboli* OR antimetaboli* OR azathioprine OR belatacept OR cyclophosphamide OR daclizumab OR ‘mycophenolate mofetil’ OR MMF OR ‘mycophenolic acid’ OR cellcept OR ‘calcineurin inhibitor*’ OR ‘protein phosphatase 2B inhibitor*’ OR cyclosporin* OR ciclosporin* OR neoral OR sandim* OR tacrolimus OR advagraf OR prograf* OR fk506 OR fk-506 OR ‘mammalian target of rapamycin inhibitor*’ OR ‘mammalian target of rapamycin kinase inhibitor*’ OR ‘mechanistic target of rapamycin inhibitor*’ OR ‘mechanistic target of rapamycin kinase inhibitor*’ OR ‘mTOR inhibitor*’ OR ‘mTOR kinase inhibitor*’ OR everolimus OR rad001* OR rad-001* OR rapamune OR rapamycin OR sirolimus) NEAR/4 (withdraw* OR taper* OR wean* OR minimization OR minimisation OR minimizing OR minimising OR sparing OR eliminat* OR reduction OR reducing OR lower* OR cessation OR discontinu* OR interrupt* OR abstinence OR avoid* OR stop* OR downgrad* OR diminish* OR free*)) OR is-withdraw* OR is-taper* OR is-wean* OR is-minimization OR is-minimisation OR is-minimizing OR is-minimising OR is-sparing OR is-eliminat* OR is-reduction OR is-reducing OR is-lower* OR is-cessation OR is-discontinu* OR is-interrupt* OR is-abstinence OR is-avoid* OR is-stop* OR is-downgrad* OR is-diminish* OR is-free):ab,ti))\n\nAND\n\n(‘transplantation tolerance’/de OR ‘immunological tolerance’/de OR ‘immunoregulation’/de OR ‘immunoreactivity’/de OR ‘graft survival’/de OR ‘liver biopsy’/de OR (tolerogen* OR ‘tolerant patient*’ OR ‘tolerant state’ OR ‘state of tolerance’ OR ‘sustained weaning’ OR ((transplant* OR posttransplant* OR operational* OR immune OR immunologic* OR alloimmune OR allograft* OR graft* OR alloantigen* OR antigen* OR chimerism OR donor-specific OR peripheral) NEAR/3 (tolerance OR tolerant OR tolerated OR tolerating OR acceptance OR protect* OR quiescen* OR unresponsive* OR nonresponsive* OR un-responsive* OR non-responsive*)) OR immunoregulat* OR immunosurveill* OR immunoreactiv* OR immunoactiv* OR ((immune OR immunologic*) NEXT (regulat* OR surveill* OR reactiv* OR activ*)) OR ((graft OR allograft OR transplant* OR liver OR hepatic) NEAR/3 (survival OR health OR function OR ‘resistance to rejection’)) OR ((inhibit* OR decrease OR abolish OR suppress* OR reduc* OR ameliorat* OR improve* OR absent OR avoid* OR prevent*) NEAR/3 (graft OR allograft OR transplant* OR liver OR hepatic) NEAR/3 (injury OR complication* OR dysfunction OR inflammation OR fibrosis OR infiltration)) OR ((inhibit* OR decrease OR abolish OR suppress* OR reduc* OR ameliorat* OR improve* OR absent OR avoid* OR prevent*) NEAR/3 (rejection OR ‘immune response*’ OR ‘alloimmune response*’ OR ‘T-cell response*’ OR ‘B-cell response*’ OR ‘antibody response*’ OR ‘humoral response*’)) OR ((liver OR hepatic) NEAR/3 (biopsy OR biopsies OR puncture*))):ab,ti)\n\nNOT\n\n((‘animal’/de OR ‘animal experiment’/exp OR ‘nonhuman’/de) NOT (‘human’/exp OR ‘human experiment’/de))\n\nNOTE: The subject heading \"graft rejection\" (and respective free text terms) was omitted from the third search block, because its tentative inclusion resulted in a non-manageable increase of hits.\n\nOne reviewer (CA-H) will screen the deduplicated references based on their titles and abstracts. All potentially relevant references will be retrieved in full-text and independently assessed by two reviewers (CA-H, JV). Any disagreements over eligibility will be resolved by consensus. Where necessary, a third review author (SH) will make a final judgement. All judgements at the full-text screening stage will be collected in a standardised MS Excel 2016 form. Articles in foreign languages that none of the review authors is familiar with will be checked for eligibility by other researchers before translation will be considered. Potentially relevant ongoing studies and conference abstracts will only be included if principal investigators will provide us with chartable data that relate to our primary outcomes (see below).\n\nTo identify possible additional studies that will escape our electronic database searches, we will screen the bibliographic references and the citations of all included articles that are indexed in Scopus or the Web of Science.\n\nQuality appraisal. Within the framework of this scoping review, no quality appraisal is planned.\n\nData charting. Next to reported prognostic and non-prognostic factors (clinical parameters and biomarkers) for OT, which will be the primary outcomes, we will also chart the percentage of successful ISW and achievement of sustained OT and the rate of graft loss in each trial as the secondary outcomes. Two reviewers (CA-H, JV) will independently chart the data from each eligible article using a jointly developed MS Excel 2016 charting form that will be pilot tested using four eligible full-text articles. If necessary, the charting form will be updated in an iterative process. Any disagreements will be solved by discussion. Data will be sought for the following variables:\n\nArticle characteristics such as first author, year of publication, country of origin, and bibliographic details\n\nFunder\n\nTrial ID\n\nMono- or multicenter study\n\nStudy design, IS maintenance control group yes/no\n\nDD and/or LD LT\n\nRecipient age at LT\n\nDonor age\n\nLiver disease aetiology\n\nTime from LT to ISW\n\nDuration of follow-up\n\nIS drug(s)\n\nISW schedule\n\nMethod(s) for assessing OT\n\nTotal number of patients that are included in the prognostic analyses\n\nPercentage of successful ISW (achievement of OT)\n\nPercentage of graft loss\n\nBiomarkers predicting OT\n\nClinical parameters predicting OT\n\nNumerical evidence for positive associations\n\nBiomarkers explicitly not predicting OT\n\nClinical parameters explicitly not predicting OT\n\nStrategy for data synthesis and presentation. For each included article, ongoing study, or conference abstract with data, we will present the charted data in a \"results of individual sources of evidence\" table. For the synthesis of collated prognostic factors (biomarkers and clinical parameters) for OT, we will use descriptive statistics showing the individual sources of evidence that support each factor. In addition to a tabular view, the results will be narratively synthesized in the review text. Together, these results will provide a comprehensive scope of past research activity on this topic and likely identify promising future research avenues.\n\nThis scoping review will be conducted along with the guidelines by the Joanna Briggs Institute49 and reported according to the “Preferred Reporting Items for Systematic reviews and Meta-Analyses extension for Scoping Reviews” (PRISMA-ScR) statement50.\n\nThe completed review will be published in a peer-reviewed journal.\n\n\nStudy status\n\nStart date of search: July 2019; anticipated completion date of review: May 2020.\n\nCurrent study status: preliminary searches, yes; piloting of the study selection process, yes; formal screening of search results against eligibility criteria, started; data extraction, no; data analysis, no.\n\n\nConclusions\n\nSince the first reports of spontaneous OT in LT25–28, numerous studies of ISW have been published (reviewed in 32). These studies were initially uncontrolled and heterogeneous in their design, rendering any comparison between them and any conclusions difficult to draw. Following the creation of international consortia (Immune Tolerance Network – ITN – in the US and Reprogramming the Immune System for the Establishment of Tolerance – RISET – in Europe), inclusion/exclusion criteria of ISW studies in LT have been harmonised, thus allowing cross-comparisons and cross-validations between studies. For instance, two ongoing multicenter trials (LIFT51 and OPTIMAL52) share the same inclusion/exclusion criteria.\n\nThese ISW trials have in parallel fuelled the need to find reliable biomarkers for the identification of those patients who are more likely to successfully stop IS, a problem that is most critical for the safety and future applicability of ISW. While clinically not available yet, several biomarkers have already been evaluated in LT recipients. In this scoping review, we will map all information on this body of literature. In addition to that, our searches in clinical trial registries will provide an overview of the current research activity in the field. The anticipated results will allow us to determine possible research gaps and whether any future systematic reviewing and meta-analysis efforts are warranted.\n\n\nData availability\n\nNo data are associated with this article.",
"appendix": "References\n\nHaddad EM, McAlister VC, Renouf E, et al.: Cyclosporin versus tacrolimus for liver transplanted patients. Cochrane Database Syst Rev. 2006; (4): CD005161. PubMed Abstract | Publisher Full Text\n\nTouzot M, Soulillou JP, Dantal J: Mechanistic target of rapamycin inhibitors in solid organ transplantation: from benchside to clinical use. Curr Opin Organ Transplant. 2012; 17(6): 626–33. PubMed Abstract | Publisher Full Text\n\nAl-Sinani S, Dhawan A: Corticosteroids usage in pediatric liver transplantation: To be or not to be! Pediatr Transplant. 2009; 13(2): 160–70. PubMed Abstract | Publisher Full Text\n\nKaltenborn A, Schrem H: Mycophenolate mofetil in liver transplantation: a review. Ann Transplant. 2013; 18(1): 685–96. PubMed Abstract | Publisher Full Text\n\nGermani G, Pleguezuelo M, Villamil F, et al.: Azathioprine in liver transplantation: a reevaluation of its use and a comparison with mycophenolate mofetil. Am J Transplant. 2009; 9(8): 1725–31. PubMed Abstract | Publisher Full Text\n\nZhang GQ, Zhang CS, Sun N, et al.: Basiliximab application on liver recipients: a meta-analysis of randomized controlled trials. Hepatobiliary Pancreat Dis Int. 2017; 16(2): 139–46. PubMed Abstract | Publisher Full Text\n\nPerez CP, Patel N, Mardis CR, et al.: Belatacept in Solid Organ Transplant: Review of Current Literature Across Transplant Types. Transplantation. 2018; 102(9): 1440–52. PubMed Abstract | Publisher Full Text\n\nGelson W, Hoare M, Dawwas MF, et al.: The pattern of late mortality in liver transplant recipients in the United Kingdom. Transplantation. 2011; 91(11): 1240–4. PubMed Abstract | Publisher Full Text\n\nCampbell KM, Yazigi N, Ryckman FC, et al.: High prevalence of renal dysfunction in long-term survivors after pediatric liver transplantation. J Pediatr. 2006; 148(4): 475–80. PubMed Abstract | Publisher Full Text\n\nGreen M, Michaels MG: Epstein-Barr virus infection and posttransplant lymphoproliferative disorder. Am J Transplant. 2013; 13 Suppl 3: 41–54; quiz. PubMed Abstract | Publisher Full Text\n\nHenchoz S, Fraga M, Saouli AC, et al.: [Outpatient follow-up of liver transplant recipients: the essential role of the general practitioner]. Rev Med Suisse. 2019; 15(660): 1488–95. PubMed Abstract\n\nJain A, Mazariegos G, Kashyap R, et al.: Comparative long-term evaluation of tacrolimus and cyclosporine in pediatric liver transplantation. Transplantation. 2000; 70(4): 617–25. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNg VL, Fecteau A, Shepherd R, et al.: Outcomes of 5-year survivors of pediatric liver transplantation: report on 461 children from a north american multicenter registry. Pediatrics. 2008; 122(6): e1128–35. PubMed Abstract | Publisher Full Text\n\nVenick RS: What is the future of pediatric liver transplantation? Optimal management of long-term recipients. Liver transpl. 2014; 20 Suppl 2: S19–21. PubMed Abstract | Publisher Full Text\n\nDemetris AJ, Bellamy CO, Gandhi CR, et al.: Functional Immune Anatomy of the Liver-As an Allograft. Am J Transplant. 2016; 16(6): 1653–80. PubMed Abstract | Publisher Full Text\n\nHeymann F, Tacke F: Immunology in the liver--from homeostasis to disease. Nat Rev Gastroenterol Hepatol. 2016; 13(2): 88–110. PubMed Abstract | Publisher Full Text\n\nLondoño MC, Rimola A, O'Grady J, et al.: Immunosuppression minimization vs. complete drug withdrawal in liver transplantation. J Hepatol. 2013; 59(4): 872–9. PubMed Abstract | Publisher Full Text\n\nPender MP: Activation-induced apoptosis of autoreactive and alloreactive T lymphocytes in the target organ as a major mechanism of tolerance. Immunol cell Biol. 1999; 77(3): 216–23. PubMed Abstract | Publisher Full Text\n\nThomson AW, Knolle PA: Antigen-presenting cell function in the tolerogenic liver environment. Nat Rev Immunol. 2010; 10(11): 753–66. PubMed Abstract | Publisher Full Text\n\nCrispe IN: Immune tolerance in liver disease. Hepatology. 2014; 60(6): 2109–17. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJenne CN, Kubes P: Immune surveillance by the liver. Nat Immunol. 2013; 14(10): 996–1006. PubMed Abstract | Publisher Full Text\n\nStarzl TE, Demetris AJ, Trucco M, et al.: Systemic chimerism in human female recipients of male livers. Lancet. 1992; 340(8824): 876–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCalne RY: Immunological tolerance--the liver effect. Immunol Rev. 2000; 174(1): 280–2. PubMed Abstract | Publisher Full Text\n\nNavarro V, Herrine S, Katopes C, et al.: The effect of HLA class I (A and B) and class II (DR) compatibility on liver transplantation outcomes: an analysis of the OPTN database. Liver Transpl. 2006; 12(4): 652–8. PubMed Abstract | Publisher Full Text\n\nStarzl TE, Demetris AJ, Trucco M, et al.: Cell migration and chimerism after whole-organ transplantation: the basis of graft acceptance. Hepatology. 1993; 17(6): 1127–52. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTzakis AG, Reyes J, Zeevi A, et al.: Early tolerance in pediatric liver allograft recipients. J Pediatr Surg. 1994; 29(6): 754–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRamos HC, Reyes J, Abu-Elmagd K, et al.: Weaning of immunosuppression in long-term liver transplant recipients. Transplantation. 1995; 59(2): 212–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMazariegos GV, Reyes J, Marino IR, et al.: Weaning of immunosuppression in liver transplant recipients. Transplantation. 1997; 63(2): 243–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFeng S, Ekong UD, Lobritto SJ, et al.: Complete immunosuppression withdrawal and subsequent allograft function among pediatric recipients of parental living donor liver transplants. JAMA. 2012; 307(3): 283–93. PubMed Abstract | Publisher Full Text\n\nBenítez C, Londoño MC, Miquel R, et al.: Prospective multicenter clinical trial of immunosuppressive drug withdrawal in stable adult liver transplant recipients. Hepatology. 2013; 58(5): 1824–35. PubMed Abstract | Publisher Full Text\n\nLevitsky J, Feng S: Tolerance in clinical liver transplantation. Hum Immunol. 2018; 79(5): 283–7. PubMed Abstract | Publisher Full Text\n\nFeng S, Bucuvalas J: Tolerance after liver transplantation: Where are we? Liver Transpl. 2017; 23(12): 1601–14. PubMed Abstract | Publisher Full Text\n\nPorrett P, Shaked A: The failure of immunosuppression withdrawal: patient benefit is not detectable, inducible, or reproducible. Liver Transpl. 2011; 17 Suppl 3: S66–8. PubMed Abstract | Publisher Full Text\n\nMathew JM, Leventhal JR: Clinical transplant tolerance: Coming of age. Hum Immunol. 2018; 79(5): 255–7. PubMed Abstract | Publisher Full Text\n\nVionnet J, Sánchez-Fueyo A: Biomarkers of immune tolerance in liver transplantation. Hum Immunol. 2018; 79(5): 388–94. PubMed Abstract | Publisher Full Text\n\nDonckier V, Troisi R, Toungouz M, et al.: Donor stem cell infusion after non-myeloablative conditioning for tolerance induction to HLA mismatched adult living-donor liver graft. Transpl Immunol. 2004; 13(2): 139–46. PubMed Abstract | Publisher Full Text\n\nTryphonopoulos P, Ruiz P, Weppler D, et al.: Long-term follow-up of 23 operational tolerant liver transplant recipients. Transplantation. 2010; 90(12): 1556–61. PubMed Abstract | Publisher Full Text\n\nDonckier V, Craciun L, Lucidi V, et al.: Acute liver transplant rejection upon immunosuppression withdrawal in a tolerance induction trial: potential role of IFN-gamma-secreting CD8+ T cells. Transplantation. 2009; 87(9 Suppl): S91–5. PubMed Abstract | Publisher Full Text\n\nTryphonopoulos P, Tzakis AG, Weppler D, et al.: The role of donor bone marrow infusions in withdrawal of immunosuppression in adult liver allotransplantation. Am J Transplant. 2005; 5(3): 608–13. PubMed Abstract | Publisher Full Text\n\nDonckier V, Troisi R, Le Moine A, et al.: Early immunosuppression withdrawal after living donor liver transplantation and donor stem cell infusion. Liver Transpl. 2006; 12(10): 1523–8. PubMed Abstract | Publisher Full Text\n\nTodo S, Yamashita K, Goto R, et al.: A pilot study of operational tolerance with a regulatory T-cell-based cell therapy in living donor liver transplantation. Hepatology. 2016; 64(2): 632–43. PubMed Abstract | Publisher Full Text\n\nThomson AW: DCreg in Living Donor Liver Transplantation. https://ClinicalTrials.gov; 2017 [updated August 30]. Reference Source\n\nDetry O, Vandermeulen M, Delbouille MH, et al.: Infusion of mesenchymal stromal cells after deceased liver transplantation: A phase I-II, open-label, clinical study. J Hepatol. 2017; 67(1): 47–55. PubMed Abstract | Publisher Full Text\n\nSturm E, Hartleif S: Safety and Tolerance of Immunomodulating Therapy With Donor-specific MSC in Pediatric Living-Donor Liver Transplantation. https://ClinicalTrials.gov; 2017 [updated March 10]. Reference Source\n\nBenítez CE, Puig-Pey I, López M, et al.: ATG-Fresenius treatment and low-dose tacrolimus: results of a randomized controlled trial in liver transplantation. Am J Transplant. 2010; 10(10): 2296–304. PubMed Abstract | Publisher Full Text\n\nPenninga L, Wettergren A, Chan AW, et al.: Calcineurin inhibitor minimisation versus continuation of calcineurin inhibitor treatment for liver transplant recipients. Cochrane Database Syst Rev. 2012; (3): CD008852. PubMed Abstract | Publisher Full Text\n\nShaked A, DesMarais MR, Kopetskie H, et al.: Outcomes of immunosuppression minimization and withdrawal early after liver transplantation. Am J Transplant. 2019; 19(5): 1397–409. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDekkers OM, Egger M, Altman DG, et al.: Distinguishing case series from cohort studies. Ann Intern Med. 2012; 156(1 Pt 1): 37–40. PubMed Abstract | Publisher Full Text\n\nPeters MD, Godfrey CM, Khalil H, et al.: Guidance for conducting systematic scoping reviews. Int J Evid Based Healthc. 2015; 13(3): 141–6. PubMed Abstract | Publisher Full Text\n\nTricco AC, Lillie E, Zarin W, et al.: PRISMA Extension for Scoping Reviews (PRISMA-ScR): Checklist and Explanation. Ann Intern Med. 2018; 169(7): 467–73. PubMed Abstract | Publisher Full Text\n\nSanchez-Fueyo A: Liver Immunosuppression Free Trial. https://ClinicalTrials.gov; 2015 [updated October]. Reference Source\n\nMarkmann JF: Evaluation of Donor Specific Immune Senescence and Exhaustion as Biomarkers of Tolerance Post Liver Transplantation. https://ClinicalTrials.gov; 2016 [updated January 6]. Reference Source"
}
|
[
{
"id": "57506",
"date": "13 Jan 2020",
"name": "Deirdre Kelly",
"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 scoping review systematically sets out how the Authors will consider studies that will record clinical details or biomarkers of relevance in the initiation of immunosuppression withdrawal. It is an ambitious review and although the Authors have laid out their strategy quite clearly by searching the relevant registries, databases and publications they do not specify whether they will include studies in children as well as adults. Most of the references are adult studies, so presumably this is the focus. I think the Authors will need to differentiate between IS minimisation or complete IS withdrawal and be clear which definitions they will include in their review for analysis..\nWith regard to the review questions:\nThe clinical parameters and biomarkers (Question 1) may be difficult to clearly identify whereas review questions 2 and 3 are more likely to be clearly defined in studies and databases.\n\nThe relevance of this review and its potential benefit to patients is obvious, I am concerned that it will require an extensive amount of work without identifying a clear outcome of value to Clinicians or Patients.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? No\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable",
"responses": [
{
"c_id": "5175",
"date": "29 Jan 2020",
"name": "Christian Appenzeller-Herzog",
"role": "Author Response",
"response": "Dear Prof. Kelly,Thank you very much for your constructive reviewer report and for drawing our attention to different issues in our protocol and possibilities for improvement. We are most happy with your judgements that this ambitious review has an obvious potential benefit to patients and that our protocol paper is of an acceptable scientific standard. With regard to your criticism, we would like to address the three points you raised: i) pediatric or adult patients?, ii) IS withdrawal and minimisation?, iii) difficulties to cope with review question 1: Pediatric/adult: This review will include all ISW studies irrespective of LT recipient age. Our protocol already states in the subsection “population, intervention, outcomes” that “LT recipients of any age or stage will be included”. To make this clearer to the reader we have now also added this information in the abstract, which now reads: “We will consider studies that record any clinical parameter or biomarker before the initiation of ISW in paediatric or adult non-viral hepatitis and non-autoimmune disease LT recipients …” The question whether or not OT-predicting factors differ in children and adults is interesting and important. Potentially, the planned systematic mapping of published predicting factors could provide some clues on this in the final review. IS withdrawal/minimisation: This review will only focus on full ISW studies and exclude studies on IS minimisation. Our protocol already holds the following statement in the subsection “population, intervention, outcomes”: “However, studies addressing dose reduction of IS, (…) will be excluded”. To clarify that also IS minimization studies will be considered ineligible we have now implemented the following extension: “However, studies addressing dose reduction of IS including IS minimisation, (…) will be excluded”. Review question 1: This is an important issue and we thank you for pointing this out. The objective of this scoping review is to collect and map all available evidence of OT-predicting factors. If no such factors can be identified, this result, albeit negative, will still be of importance and worth reporting. However, as you are pointing out, the formulations in our protocol are implying that our aim is to “identify” such factors in the literature. Instead, we should state that our objective is the mapping of all available evidence on such factors. In response to this important point of criticism, we have implemented the following revisions: Abstract: “This scoping review aims at systematically mapping reported prognostic factors for spontaneous immunosuppression (IS) free allograft tolerance (operational tolerance, OT) in non-viral hepatitis and non-autoimmune disease liver transplant (LT) recipients who are undergoing immunosuppression withdrawal (ISW).” Study aim and objectives/questions: “The objective of this scoping review will be to map all published prognostic factors for spontaneous OT in non-viral hepatitis and non-autoimmune disease LT recipients who are undergoing ISW.” We sincerely hope that these amendments to our protocol will help convince you to fully approve the revised version of our manuscript. Thank you again for your invaluable help in this matter. Christian Appenzeller-Herzog, Steffen Hartleif, Julien Vionnet"
}
]
}
] | 1
|
https://f1000research.com/articles/8-2059
|
https://f1000research.com/articles/9-652/v1
|
26 Jun 20
|
{
"type": "Research Article",
"title": "Comparative analysis of mechanical complications of emergency versus planned ultrasound-guided internal jugular venous (IJV) cannulation: data from the emergency room of a third-level hospital in Quito, Ecuador",
"authors": [
"Augusto Maldonado",
"Luis Vidal",
"Jorge Estrella Porter",
"Pablo Endara",
"Augusto Maldonado",
"Luis Vidal",
"Pablo Endara"
],
"abstract": "Background: Central venous catheters (CVC’s) are useful tools for the treatment of critically ill patients, especially in the emergency room, and are recognized for decreasing rates of failure and mechanical complications. Certain parameters can act as predictors to determine the likelihood of this type of complication. The aim of this study is to determine the incidence and predictors of mechanical complications using internal jugular venous (IJV) cannulation, especially when considering planned versus emergency cannulation. Methods: A prospective, observational study was performed at Hospital de los Valles, Quito, Ecuador, during a three-year period. All patients who presented to the emergency room with an indication for IJV cannulation and with no potential contraindications were included. Demographic, safety and procedure related data were collected. Variables were analyzed using STATA, and p-values <0.05 were considered significant. Results: A total of 142 patients were included. The majority of procedures (64%) were planned. The main indication for CVC placement via IJV cannulation was chronic renal failure (54%), all of which were planned procedures, followed by sepsis (15%), where most procedures (91%) were emergencies. The IJV was anatomically located lateral to the carotid artery in 38.73% of cases. Mechanical/technical complications were reported in 13 patients. Cannulation time greater than two minutes was found to be the only factor independently associated with a higher probability (12.4 times) of developing mechanical/technical complications. The vessel location did not affect the incidence of complications either in emergency or planned procedures when using ultrasound. Conclusions: Ultrasound-guided IJV cannulation is a safe technique that can be performed as an emergency or planned procedure without increased complication rates in the emergency room. Additionally, a puncture time of less than two minutes is associated with the safest profile in this patient population.",
"keywords": [
"Ultrasound",
"Central Venous Access",
"Internal Jugular Vein Cannulation",
"Mechanical complications",
"Puncture time"
],
"content": "Introduction\n\nCentral venous catheters (CVC’s) are a very useful tool for the treatment of critically ill patients1. They are used for the delivery of vasoactive agents, parenteral nutrition, long-term antibiotics, blood sampling, hemodynamic monitoring, and/or venous access for haemodialysis patients2. Historically, vein cannulation has been a procedure performed with a blind approach, following only anatomical landmarks for cannulation, thus the incidence of complications has been estimated to be as high as 40%3. Consequently, the use of ultrasound-guided techniques for vein cannulation has helped to reduce the rate of complications4, and its use has been standardized in many emergency rooms around the world5.\n\nUltrasound-guided techniques in the emergency room have long been documented as beneficial. In fact, in 2001, the Agency for Healthcare Research and Quality Evidence Report recognized bedside-ultrasonography for central venous access as one of the eleven practices with “strength of evidence for supporting more widespread implementation”2. The American College of Emergency Physicians recommends in its Ultrasound Guidelines Policy Statement (last approved in 2016) that emergency room physicians should be trained and proficient in these techniques6. However, there is evidence that even with the recommendations to use ultrasound-guided techniques for central venous access, up to 67% of physicians do not routinely use ultrasound to guide the procedure7.\n\nInsertion of ultrasound-guided CVCs with the conventional technique and with experienced personnel has allowed positive results with reduced costs8; nonetheless, it requires a thorough knowledge of the vascular anatomy and it is operator-dependent9. The technique is also limited in certain settings, like the emergency room, where elements such as previous vascular access, mechanical/ventilation and tracheostomy cannulas can affect access. Other factors affecting the success of the procedure are the presence of thrombosed veins, hematomas and anatomical variations10. These factors are associated with increased mechanical complications and failure rates11. This justifies the use of ultrasound to detect these possible anatomical variations before starting the procedure12.\n\nUltrasound-guided internal jugular venous (IJV) cannulation, one of the preferred methods for central venous access, is recognized for decreasing rates of failure and mechanical complications13. Main disadvantages of ultrasound-guided IJV cannulation access include the limited availability of the technology in low resource countries, a longer procedural time when the Doppler mode is used, and limited training from medical personnel regarding the ultrasound technique14.\n\nThe aim of this study is to determine the incidence of complications from ultrasound-guided jugular vein cannulation and compare the adverse outcomes in emergency versus planned cannulation, as well as to determine the factors that are associated with complications.\n\n\nMethods\n\nPatients’ written informed consent was obtained for the standard clinical procedure; patients were also notified through this document about the inclusion of their data in this study. All patients agreed. The Ethics Committee of Universidad San Francisco de Quito reviewed the study to the project.\n\nThis study was performed in Hospital de los Valles (HDLV), Quito, Ecuador. The emergency room department takes care of approximately 20,000 patients per year, of whom 2–4% are in critical condition. The study was designed as a prospective observational study, collecting data over a three-year period of time, between 2010 and 2013.\n\nAll patients who were admitted to the emergency room at HDLV and had an indication for CVC insertion, either emergency or planned, were eligible for the study. We excluded patients that did not consent to the CVC insertion, those with a visualized thrombus in the internal jugular vein and those who had trauma of the great vessels in the neck and thorax, according to recommendations based on previous studies11. A total of 142 consecutive patients were included in the study; informed consent for medical procedure was signed at the hospital just after the indication for CVC, either emergent or planned, was stablished. The procedure was explained to patients and/or relatives.\n\nIn order to guarantee the best possible technique for the study, physicians performing the IJV central line procedure had to fulfil the following inclusion criteria:\n\n1. Degree in Emergency Medicine\n\n2. A previous history of at least 300 CVC lines.\n\n3. An average of at least 80 CVC placements in the previous years.\n\n4. At least 100 IJV CVC placements using the same technique as used this study (see following section).\n\nThe technique used at the emergency room of our hospital (HDLV) starts with ultrasound-guided visualization of the internal jugular vein anatomy, using a high frequency superficial transducer (General Electric Logiq Book XP portable ultrasound). The CVC is placed with a high frequency 5–15 MHz electronic linear array. After this is done, the distance between skin to the centre of the vessel is measured using a measuring tape and it is corrected for the angulation factor that is going to be used. We mark the skin at the point of insertion. Using the Seldinger technique under sterile conditions and local anaesthesia (lidocaine 1% without epinephrine), the procedure starts with needle puncture (18–20 gauge) and visual guidance of the tip, direction and depth while advancing with continuous ultrasound monitoring. Venous blood is obtained, and the wire is introduced with visual confirmation of an intraluminal location. The dilator is positioned and the catheter is introduced. Finally, and to verify the position of the tip of the catheter, we use the saline flush test using the ultrasound subxiphoid window, to determine if the catheter is too close to the right atrium15.\n\nEmergency CVC via IJV placement was considered in any patient that fulfilled at least one the following criteria:\n\n1. Acute disease, with potential risk of death.\n\n2. Hemodynamic instability.\n\n3. Need for use of vasoactive drugs.\n\n4. Acute decompensation from a chronic illness that puts life at risk.\n\n5. Indication for emergent haemodialysis.\n\nPlanned CVC via IJV placement was considered in any of the following cases:\n\n1. Stable patients with diseases that require a planned procedure for:\n\na. Parenteral nutrition.\n\nb. Haemodialysis.\n\nc. Monitoring fluid administration.\n\n2. Chemotherapy.\n\n3. Inability to place a peripheral line.\n\nThe evaluation of which patients fulfilled emergency vs planned CVC criteria was done by the emergency physicians in charge of placing the IJV central line, with help from doctors from different specialties who were also evaluating each patient for baseline medical conditions.\n\nCVC complications are usually divided into two types: mechanical-technical and infectious. In this study, only mechanical and technical complications were evaluated since infectious require longer follow-ups and specialized studies to determine their origin. Mechanical complications were defined as either: haemothorax, pneumothorax, carotid puncture and/or hematoma at the puncture site16. Technical difficulties were defined as either: double or multiple puncture attempts, guide wire blockage and/or catheter obstruction17. All mechanical complications and/or technical difficulties were identified by the physician performing the procedures and confirmed by a second physician who was a trained observer and who was present during and after the procedure.\n\nThe data were collected prospectively, and a registry was made using a Microsoft Excel database, with access limited to the researchers. For each patient, one physician was assigned to the procedure while another one was in charge of registering the data, including demographics, type of procedure (planned vs emergent), time of the procedure, complications and technical difficulties. All the information related to the procedure was registered in the medical record of each patient.\n\nData is shown as number and percentage for categorical variables; for continuous variables, the mean, median and ranges were also considered. Categorical variables and proportions were compared using Fisher’s exact test, and continuous variables were compared using the Wilcoxon Rank sum test. To investigate risk factors associated with complications, a univariate and multivariate logistic regression analysis was performed in those with all the data available (N=133), and the odds ratios (ORs) with confidence intervals (CIs) and p-values were calculated. Those variables that were significant in the univariate analysis were included in the multivariate analysis. Patients who developed mechanical and technical complications were compared with those who did not present complications. STATA version 13 software was used for analysis and p-values <0.05 were considered statistically significant.\n\n\nResults\n\nThere was a total of 142 patients included in the study, among whom 55% were male and the mean age was 59 years18. Emergency procedures were performed in 36% of the cases. The main indication for CVC placement via IJV cannulation was chronic renal failure (54%), all of which were planned procedures, followed by sepsis (15%), where most procedures (91%) were done as emergencies. Approximately one third of the studied sample (35.2%) had an anatomical orientation of the internal jugular vein above the carotid artery. The median puncture time was two minutes. In most of the patients (66.2%), the time from needle puncture to the vein cannulation was less than two minutes (Table 1).\n\n§ Conditions must be adapted to your specific primers and or product length\n\nSD, standard deviation.\n\nOut of 133 patients for whom time data was available, 13 (9.8%; 95% CI: 5.3%–16.13%) had some type of complication: six patients (4.5%) had a mechanical complication, of which two (1,5%) were haematomas and four (3%) were arterial punctures, and seven patients (5.3%) had a technical complication, of which three (2.3%) were wire guide blockages and four (3%) were double punctures. Patients who developed mechanical complications were statistically younger than those who did not develop mechanical complications (40 years versus 60 years; p-value: 0.02). There were no age differences between those with and without technical complications.\n\nMedian puncture time was statistically longer in those with complications (19.9 minutes) compared to those without complications (3.3 minutes) (p-value: 0.0002). A similar pattern was found when considering mechanical or technical complications separately, where median puncture time was statistically higher in those with complications versus those without complications (Table 2).\n\nP-values are determined from non-parametric test-Wilcoxon Rank sum test.\n\nThere was no available data on procedure time for nine patients.\n\n** Referred to the time it took from skin puncture to vessel cannulation.\n\nAfter univariate analysis, a puncture time longer than two minutes was statistically associated with an 8.1 times increased risk of developing complications (CI: 2.1-31; p-value: 0.002), while there was no statistical increase in complications in emergency versus planned procedures. After taking into account the effect of age, gender, type of procedure (emergency or planned) and vessel anatomical location on the association, the only independent factor associated with an increased risk of complications was a puncture time higher than two minutes (OR: 7.8; CI: 2-31; p-value: 0.004) (Table 4). When the analysis was restricted to those with mechanical or technical complications, puncture time remains the only significant predictor of mechanical complications. Other variables such as the type of procedure (emergency versus planned) and vessel localization did not reveal any statistical association (Table 3).\n\nOR, odds ratio; CRF, chronic renal failure; CVC, central venous cannulation.\n\nAdjusted OR includes age and gender, other variables such as vessel location did not affect the results.\n\n* Refers to the time it took to vessel cannulation.\n\n\nDiscussion\n\nThe complication rates for central venous catheterization when using the landmark technique versus the ultrasound-guided technique have been clearly established in the literature, favoring the ultrasound-guided procedure4; nevertheless, there is no clear comparative analysis in the literature about complications during ultrasound-guided procedures in emergency versus planned situations and this is what this study tries to determine.\n\nWhen the puncture time exceeded two minutes, the chance of having a mechanical complication was 12.4 times higher compared to a puncture time of less than two minutes. All other variables such as the type of procedure (emergency versus planned) and vessel location did not show any statistical differences. This finding is important for clinical practice because it advices the operator that once the two minutes puncture time interval has been surpassed, the risk of complications has increased considerably, allowing them to be primed to look for and manage the possible associated mechanical complications. Additionally, it was demonstrated that clinicians can confidently perform a CVC insertion with ultrasound, either in emergency or planned situations, since there is no significant difference in the development of complications.\n\nThe rate of mechanical complications with ultrasound-guided central venous catheterization varies widely among literature. For example, a recent study published by Björkander et al. evaluated a total of 10,949 CVC insertions and found a mechanical complication rate of 1.1%16. On the other hand, the study published by Eisen et al., showed a mechanical complication rate of 14% during ultrasound-guided central venous catheterization19. Bleedings, pneumothorax, transient nerve injuries, and self-limiting arrhythmias are described as the most common mechanical complications16. The reason for a low rate of mechanical complications in our study (4%) is thought to be related to the level of confidence and expertise of the doctors performing the procedure while using ultrasound. Other complications such as pneumothorax, hematomas and incorrect positioning reported by our study are comparable to those of others20.\n\nRisk factors associated with mechanical complications have been described by literature: the need for help19, more than two failed punctures19, expertise of the operator21, history of previous catheterizations22, preprocedural coagulopathy and route of catheterization16. In our study, the most significant variable associated with complications was the time puncture. The need for help was not identified as a variable, and the rate of failed punctures was minimal.\n\nThe results of this study might be affected by the small number of patients chosen. However, the analysis provided was able to detect statistically significant differences between groups by complications. More patients should be recruited in future studies to confirm these findings and to guide future protocols focusing on puncture time as a predictor for mechanical complications during emergency or planned ultrasound-guided IJV cannulation.\n\n\nConclusions\n\nTraining in vascular access with ultrasound guidance has an enormous impact in patient safety and in the reduction of complications related to CVC. Some relevant findings were determined during the present study regarding the ultrasound-guided technique for IJV placement of a catheter. First, it was confirmed that ultrasound-guided jugular vein access is a safe strategy to perform in the emergency room. Second, it was demonstrated that the type of situation, emergency versus planned, has no influence on the outcome and complications related to the IJV access of the patient. Third, it was shown that puncture time (time from the skin puncture to the vessel cannulation) was the most predictive variable for the development of complications. The only and most important factor to predict procedural success, in this study, was a time of less than two minutes from skin puncture to IJV cannulation. A greater sample size might be necessary to confirm these findings.\n\n\nData availability\n\nHarvard Dataverse: Comparative analysis of mechanical complications of emergent versus planned ultrasound-guided internal jugular venous cannulation (IJV): data from the Emergency Room of a third-level hospital in Quito-Ecuador - RAW DATA SET ENGLISH VERSION. https://doi.org/10.7910/DVN/YAZQVA18\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\nBose N, Patel H, Kamat H: Evaluation of ultrasound for central venous access in ICU by an in experienced trainee. Indian J Crit Care Med. 2014; 18(1): 26–32. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLeibowitz A, Oren-Grinberg A, Matyal R: Ultrasound Guidance for Central Venous Access: Current Evidence and Clinical Recommendations. J Intensive Care Med. 2020; 35(3): 303–321. PubMed Abstract | Publisher Full Text\n\nPaul RG, Price S: Central venous cannulation. Medicine. 2014; 42(8): 473–474. Publisher Full Text\n\nSaugel B, Scheeren TWL, Teboul JL: Ultrasound-guided central venous catheter placement: a structured review and recommendations for clinical practice. Crit Care. 2017; 21(1): 225. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGottlieb M, Sundaram T, Holladay D, et al.: Ultrasound-Guided Peripheral Intravenous Line Placement: A Narrative Review of Evidence-based Best Practices. West J Emerg Med. 2017; 18(6): 1047–1054. PubMed Abstract | Publisher Full Text | Free Full Text\n\nUltrasound Guidelines: Emergency, Point-of-Care and Clinical Ultrasound Guidelines in Medicine. Ann Emerg Med. 2017; 69(5): e27–e54. PubMed Abstract | Publisher Full Text\n\nBailey PL, Glance LG, Eaton MP, et al.: A Survey of the Use of Ultrasound During Central Venous Catheterization. Anesth Analg. 2007; 104(3): 491–497. PubMed Abstract | Publisher Full Text\n\nSeleznova Y, Brass P, Hellmich M, et al.: Cost-effectiveness-analysis of Ultrasound Guidance for Central Venous Catheterization Compared With Landmark Method: A Decision-Analytic Model. BMC Anesthesiol. 2019; 19(1): 51. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRando K, Castelli J, Pratt JP, et al.: Ultrasound-guided internal jugular vein catheterization: a randomized controlled trial. Heart Lung Vessels. 2014; 6(1): 13–23. PubMed Abstract | Free Full Text\n\nGibson F, Bodenham A: Misplaced central venous catheters: applied anatomy and practical management. Br J Anaesth. 2013; 110(3): 333–346. PubMed Abstract | Publisher Full Text\n\nKornbau C, Lee KC, Hughes GD, et al.: Central line complications. Int J Crit Illn Inj Sci. 2015; 5(3): 170–178. PubMed Abstract | Publisher Full Text | Free Full Text\n\nde Almeida CES: Vascular access: the impact of ultrasonography. Einstein (Sao Paulo). 2016; 14(4): 561–566. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRay BR, Mohan VK, Kashyap L, et al.: Internal jugular vein cannulation: A comparison of three techniques. J Anaesthesiol Clin Pharmacol. 2013; 29(3): 367–371. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMaietta PM: Accidental carotid artery catheterization during attempted central venous catheter placement: a case report. AANA J. 2012; 80(4): 251–255. PubMed Abstract\n\nGekle R, Dubensky L, Haddad S, et al.: Saline Flush Test: Can Bedside Sonography Replace Conventional Radiography for Confirmation of Above-the-Diaphragm Central Venous Catheter Placement? J Ultrasound Med. 2015; 34(7): 1295–1299. PubMed Abstract | Publisher Full Text\n\nBjörkander M, Bentzer P, Schött U, et al.: Mechanical complications of central venous catheter insertions: A retrospective multicenter study of incidence and risks. Acta Anaesthesiol Scand. 2019; 63(1): 61–68. PubMed Abstract | Publisher Full Text\n\nMiguelena D, Pardo R, Morón-Duarte LS: Central venous catheter-related complications in critically ill children. Rev Salud Publica (Bogota). 2013; 15(6): 886–898. PubMed Abstract\n\nEstrella J, Maldonado A, Endara P, et al.: Comparative analysis of mechanical complications of emergent versus planned ultrasound-guided internal jugular venous cannulation (IJV): data from the Emergency Room of a third-level hospital in Quito-Ecuador - RAW DATA SET ENGLISH VERSION. Harvard Dataverse, V1. 2020. http://www.doi.org/10.7910/DVN/YAZQVA\n\nEisen LA, Narasimhan M, Berger JS, et al.: Mechanical complications of central venous catheters. J Intensive Care Med. 2006; 21(1): 40–46. Publisher Full Text\n\nSazdov D, Srceva MJ, Todorova ZN: Comparative Analysis of Ultrasound Guided Central Venous Catheterization Compared to Blind Catheterization. Pril Makedon Akad Na Nauk Umet Oddelenie Za Med Nauki. 2017; 38(2): 107–114. PubMed Abstract | Publisher Full Text\n\nOdendaal J, Kong VY, Sartorius B, et al.: Mechanical complications of central venous catheterisation in trauma patients. Ann R Coll Surg Engl. 2017; 99(5): 390–393. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDexheimer Neto FL, Roehrig C, Morandi P, et al.: Safety of a training program for ultrasound-guided internal jugular vein catheterization in critically ill patients. Rev Assoc Med Bras. 2011; 57(4): 394–7. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "75569",
"date": "14 Dec 2020",
"name": "Donaldo Arteta Arteta",
"expertise": [
"Reviewer Expertise Medicine",
"Critical Care",
"Internal Medicine",
"Nutritional Support."
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis work for me has several inconsistencies in the design, errors in the measures of central tendency from the statistical point of view, and it does not seem to me that the Wilcoxon test that was used was better than the two-tailed unpaired Student's t-test. Two-color tables confuse the reader and are not well organized or aligned. The value of \"p\" is repeated a lot. I believe that a rethinking should be done according to suggestions that I have sent by email before indexing it. Please see the attachment in Word.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
},
{
"id": "77072",
"date": "13 Jan 2021",
"name": "Muhammet Emin Naldan",
"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\nWriters said that ‘Patients who developed mechanical complications were statistically younger than those who did not develop mechanical complications (40 years versus 60 years; p-value: 0.02).’ What causes do they attribute these complications? Why these problems were seen in younger patients? What the writers think about this?\n\nThey said that 'The complication rates for central venous catheterization when using the landmark technique versus the ultrasound-guided technique have been clearly established in the literature, favoring the ultrasound-guided procedure4; nevertheless, there is no clear comparative analysis in the literature about complications during ultrasound-guided procedures in emergency versus planned situations and this is what this study tries to determine.' They should look at the literature and say the complication rates for central venous catheterization when using the landmark technique. Because writers say in this article ultrasound-guided procedures are very important and have less complicatıon from landmark techniques.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/9-652
|
https://f1000research.com/articles/9-651/v1
|
26 Jun 20
|
{
"type": "Research Article",
"title": "Pilot study for the evaluation and adaptation of a Four Item-Acne-Scar Risk Assessment Tool (4-ASRAT): a resource to estimate the risk of acne-induced scars",
"authors": [
"Jorge Estrella Porter",
"Mikaela Camacho",
"María Isabel Viteri",
"Katherine Aguilar",
"Drifa Belhadi",
"Vincenzo Bettoli",
"Anita del Rocío Buestán",
"Brigitte Dréno",
"Pablo Endara",
"Alison Layton",
"Nelly Machado",
"Rosa Mateus",
"Jerry Tan",
"Enrique Terán",
"Paola Yépez",
"Jonathan Guillemot",
"Mikaela Camacho",
"María Isabel Viteri",
"Katherine Aguilar",
"Drifa Belhadi",
"Vincenzo Bettoli",
"Anita del Rocío Buestán",
"Brigitte Dréno",
"Pablo Endara",
"Alison Layton",
"Nelly Machado",
"Rosa Mateus",
"Jerry Tan",
"Enrique Terán",
"Paola Yépez"
],
"abstract": "Background: Acne-induced scarring is associated with a similar burden as acne, i.e. diminished quality of life, and may be avoided if patients receive appropriate and timely acne treatment. In 2017, a four item-Acne-Scar Risk Assessment Tool (4-ASRAT) was designed by Tan et al. to categorise patients with acne into lower-risk or higher-risk for acne scarring. Its applicability outside the initial study population (France, Brazil and United States) remains to be determined. Methods: A study protocol was developed to create a systematic approach for validating and adapting 4-ASRAT to different populations, Ecuador in this case. The protocol was reviewed by 11 local and international dermatologists and pilot-tested in an Ecuadorian population using a sample of 10 participants who currently had or had had acne. Feedback from the pilot study was used to improve the study protocol. The results of the pilot study are included here, and the final study protocol is available as extended data. Results: The protocol proved to be applicable. Images taken of participants were a valuable resource for dermatological evaluation about the presence or absence of acne scars. Tangential light is necessary for this evaluation. Although dermatological assessments varied, we concluded that assessment by three local dermatologists for each participant was adequate for reaching a consensus on the presence or absence of acne scars.\n\nConclusions: Considering the morbidity related to acne and acne scars, tools designed as prevention that alert patients about risk of developing scarring are necessary. The proposed protocol shows a feasible way of validating and adapting 4-ASRAT to different populations.",
"keywords": [
"acne",
"acne scars",
"prevention tool",
"risk factors",
"scarring",
"scarring prevention"
],
"content": "Introduction\n\nAcne is among the most common dermatological conditions, with an estimated prevalence among the general population of 9.4%1. Prevalence among those aged 12 to 24 years can reach as high as 85%, with variations in different populations2. While acne can be perceived as a trivial condition due to its temporary nature, the emotional and psychological burden is high3. Patients with acne have diminished quality of life4, and are more likely to suffer from depression and anxiety5. The extent of these impacts are comparable to that of patients with chronic disability associated with asthma, epilepsy, diabetes or arthritis6,7.\n\nAcne often leads to the development of scars, which can be permanent8. Acne scarring is also associated with a psychosocial burden similar to that seen with acne9. Scarring may, however, be avoided if patients receive appropriate and timely acne treatment 10–12. Although risk factors for the development of acne scars are well-known, only one tool exists to predict the risk of acne scarring to support treatment initiation decision-making13.\n\nIn 2017, Tan et al.13 described the first, and to our knowledge only, tool which assesses the risk for acne scarring in patients with acne based on four major risk factors: “severity of acne”, “family history of acne scarring”, “squeezing and picking behaviours” and “duration of acne”. For simplicity, we named this tool the Four-item Acne Scar Risk Assessment Tool (4-ASRAT). The tool is a short questionnaire, which can be either self-administered or administered by a healthcare worker. Using a score associated with each item to the questionnaire and a score threshold, 4-ASRAT provides a binary outcome on the risk of acne scarring by categorising respondents as being at “lower” or “higher risk”. 4-ASRAT was calibrated and validated using cross-validation based on a pre-existing database containing a large sample of young adults from the United States, France and Brazil, and resulted in a sensitivity of 82% and specificity of 43%. However, its applicability to other populations is uncertain.\n\nThis study presents a protocol for validating and, if necessary, adapting 4-ASRAT to any population. The proposed protocol was tested via a pilot study in an Ecuadorian population to test its applicability and obtain feedback to develop a final version. With this study, we intend to develop and disseminate a protocol for the adaptation of 4-ASRAT to other populations, thereby promoting best practices for timely acne care and acne scar prevention.\n\n\nMethods\n\nThis study consisted of two phases: first, the development of a study protocol for the evaluation and adaptation of 4-ASRAT to different populations and, second, the pilot of the said protocol in a real-world scenario in Quito, Ecuador, to obtain feedback and improve the proposed protocol. A study using the final protocol in a large Ecuadorean population will be the subject of a subsequent publication.\n\nThe protocol aimed to provide a replicable process to evaluate and adapt 4-ASRAT to any population. It was conceived as a reviewed version of the initial study protocol proposed by Tan et al. To be effective, the protocol must provide a strategy to answer three questions: What set of risk factors should the adapted 4-ASRAT use? What score should be associated with each item? What score threshold should it use?\n\nIn addition to the nature of the information that it must lead to, the study protocol must address cost-effectiveness. The adaptation of 4-ASRAT must remain simple for it to be put in practice: the application of the protocol should be inexpensive, in terms of time, human resources and money. This means that the study protocol should also be readily available and provide adequate detail to ensure replicability.\n\nAs part of the protocol, a 22-question survey regarding acne scaring risk factors, epidemiology and self-perception of acne scars was developed (available as Extended data). Based on acne scar prevalence, the sample of respondent should be at least 250 participants14. This questionnaire, which was not formally assessed in this pilot study, is designed to establish the significant risk factors related to acne scars for the evaluation and possibly the adaptation of 4-ASRAT. Participants should go to a photobooth immediately after filling the questionnaire (characteristics of the photobooth details available as Extended data)15, where three pictures will be taken for each participant (front, and right and left profiles). A group of independent dermatologists then evaluate participants’ pictures to determine the presence or absence of acne scars. Their evaluation will be considered as the gold standard for data analysis.\n\nData will be computed and calibration and discrimination of the tool will be calculated to validate 4-ASRAT in the studied population. If 4-ASRAT proves invalid for the given population, the adaptation process then begins. Based on the questionnaire results and dermatologists’ evaluations, risk factors for acne scaring and their respective weight to determine the relevant list of risk factors, the scores associated with each response and the optimal score threshold. The complete version of the protocol can be consulted as Extended data16.\n\nThe pilot study was conducted to evaluate the feasibility of the aforementioned study protocol and improve its design before a full-scale research conduct. Besides the study of the data collected as part of the application of the standard study protocol, additional information was collected using observation of the data collection in real-world settings and by obtaining feedback from dermatologists.\n\nIn practice, the pilot consisted of a small-scale application of the study protocol with particular attention to the data collection process and a data analysis approach benefitting from expert input.\n\nA sample of 10 participants, which is among the range recommended for pilot studies17, was recruited by an open invitation through social media and flyers in Universidad San Francisco de Quito USFQ, during October 2018, using the same inclusion criteria as in the protocol:\n\nTo be a person aged 18–25 at the time of participation.\n\nTo have suffered acne at any time point, including having active acne at the time of the study.\n\nTo belong to [sample pool population]\n\nTo accept signing the informed consent, including consent for photographs to be captured.\n\nTo have no visible facial hair at the moment of the photograph\n\nTo have no facial make-up at the moment of the photograph\n\nParticipants answered the 22-question survey and were then taken to the photobooth for the three necessary photographs. Data collected as part of the pilot was recorded and tabulated as planned in the study protocol. Participants photographs were showed to a total of 11 dermatological professionals (seven local dermatologists and four international experts) for the evaluation of the presence or absence of acne scars in participants. This process was conceived to determine the minimum number of dermatologists needed to reach a consensus in the evaluation of the presence or absence of acne scars. The following inclusion criteria were used for dermatologists:\n\nTo be a medical doctor with a specialty degree in dermatology.\n\nTo have at least five years of experience in seeing patients with acne.\n\nTo have provided medical attention to at least 35 acne patients per year over the last 5 years.\n\nTo consent to the project participation an accept the workload proposed.\n\nPreferred: to have at least one publication related to acne in a scientific journal.\n\nDermatologists provided feedback on photography quality to improve the final study protocol. Besides elementary descriptive statistics, no statistical analysis was necessary at the stage of the pilot to evaluate the applicability of the protocol.\n\nThe study protocol and pilot study were approved by Universidad San Francisco de Quito’s Institutional Review Board (Comité de Ética de Investigación en Seres Humanos Universidad San Francisco de Quito) on September 25th 2018 (2018-193IN). Informed consent was obtained from each participant.\n\n\nResults\n\nThe pilot study was carried out in October 2018 with a sample of ten participants recruited from students of Universidad San Francisco de Quito USFQ (six women and four men, aged 18–25, mean age: 23 years). Results about the evaluation of the presence or absence of acne scars are shown in Table 1.\n\nD: dermatologist; P: acne scars are present, more than mild; p: Scars are present, but mild; A: acne scars are absent. This table presents the results of the protocol pilot with ten participants evaluated by 11 dermatologists, of whom seven were local dermatologists and four were international acne expert dermatologists (expert in table). For participant 01-P, the participant’s self-evaluation is that acne scars are present. Local dermatologist D1 evaluated that mild acne scares are present, local dermatologists D2 and D3 reported that acne scars beyond mild are present and local and expert dermatologists D4 to D11 reported that acne scars are absent. A simple majority showed to be equally effective in professional evaluation than more complex systems of determining consensus with compound majorities.\n\nParticipants and dermatologists were able to complete the study without issues. Scarring assessments varied widely: compared to the gold standard established by international experts – who generally agreed – local dermatologists had a little more difficulty agreeing, with consensus reached for 6 out of 10 cases vs. 9 out of 10 for international experts. When a consensus was reached among local dermatologists, the outcome always concurred with that of international experts. This suggests that local dermatologists are an adequate proxy for international experts provided a minimum number of participating local dermatologists, which we established at three.\n\nParticipant self-evaluations varied more widely from the expert gold standard: half the time self- and international expert assessments concurred. When they differed, participants were more likely to minimize the presence of scars (false negatives, 3/10) rather than exaggerate (false positives, 1/10).\n\nDermatologists generally validated the approach for photographing, with three main recommendations reached by consensus:\n\n- Tangential light is mandatory to assess scar volume and depth in photography.\n\n- Besides from front, left and right angle photographs, an oblique picture of the participants should be included.\n\n- Make-up, even if invisible, should not be allowed in participants.\n\nSurveys and photographs were easily collected.\n\nSimilar to Tan et al.’s study, the protocol proposes a methodology for studying at a single timepoint younger adults (18–25 years), and considers their history of acne and acne scarring in order to identify acne-scarring risk factors and their respective weight. Depending on the context, incentives may be used to promote participation. The study is designed to be completed in a six-month timeframe. All of the recommendations given by dermatologists were considered for its final version, and three dermatologists were established as the minimum number to reach consensus. No further adaptations to the protocol were required based on the pilot study.\n\n\nDiscussion\n\nThe proposed protocol showed applicability and ease of execution, while confirming its usefulness in obtaining the necessary data for the validation and adaptation of 4-ASRAT (epidemiologic data, self-evaluation about presence or absence of acne scars, associated risk factors, and images for dermatological evaluation). However, some limitations were found.\n\nAlthough preferable for accuracy, a prospective study including younger participants (children who have not developed acne scars yet) with follow-up evaluations was avoided for practical reasons: a study with these characteristics is impractical because of the time it implies in terms of follow-up, resulting in a low probability of applying the protocol in other populations.\n\nA single-site study was also chosen (a university) to take advantage of the concentration of eligible volunteers who were willing to participate. The study centre should be selected, if possible, based on the representativeness of the sample pool compared to the general population and should include individuals not only from a single institution (university) but rather more representative of the population’s context.\n\nRecruiting a representative population sample is a complex and costly exercise, which seems unrealistic in the context of acne-scar prevention. Although the lack of representativeness of the sample proposed in the study protocol may be perceived as a limitation, we prefer the local and cost-effective adaptation of 4-ASRAT to the use of the non-calibrated tool. Researchers should seek aggregated groups, such as universities, which are the best proxies to the general population for the adaption study.\n\nParticipant photography will never equal the quality of face-to-face evaluation, as bidimensional imaging does not yet allow volumetric assessment, a necessary element for determining the severity of a suspected scar18. Tangential light must be included in the final study, to obtain a more three-dimensional image of participants, as suggested by dermatologists during the pilot study. Again, we prefer the more cost-effective approach using photography, despite its limitations, rather than professional individual evaluations, which increase the study costs significantly.\n\nRegarding scar assessments, comparing self- to local professional assessments, the pilot suggests the need for dermatological assessments rather than reliance on self-assessment. As opposed to an earlier study, the pilot results hint towards a tendency of participants to minimise the presence of acne scars rather than overestimate, which was earlier found19. It was shown that a simple majority consensus is sufficient to determine the presence or absence of acne scars, so the use of three dermatologists is recommended for final evaluation (minimum number needed to reach a simple majority consensus).\n\n\nConclusions\n\nWe developed and piloted a readily available study protocol to evaluate and adapt 4-ASRAT to any population. We showed this protocol to be applicable in practice, provided that certain precautions were taken, including photography quality and local dermatologist support. Due to the scarcity of tools to assess the risk of acne scaring, the use of an adapted and validated tool for prediction of acne scar risk in a particular population is a valuable public health measure.\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\nHarvard Dataverse: Questionnaire for the validation and adaptation of a tool to estimate the risk of acne-induced scars in different population. https://doi.org/10.7910/DVN/WGDWB015.\n\nThis project contains the questionnaire to be given to participants.\n\nHarvard Dataverse: Protocol for the validation and adaptation of a tool to estimate the risk of acne-induced scars in different populations. https://doi.org/10.7910/DVN/NED0GS16.\n\nThis project contains the protocol to be conducted for the main study.\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\nThe authors of this manuscript would like to acknowledge the support of SIME (Sistemas Médicos) and their research and administration team, Dr. Jaime Ocampo, Dr. Andrés Caicedo and Sulema Romero.\n\n\nReferences\n\nTan JKL, Bhate K: A global perspective on the epidemiology of acne. Br J Dermatol. 2015; 172(Suppl 1): 3–12. PubMed Abstract | Publisher Full Text\n\nAsai Y, Baibergenova A, Dutil M, et al.: Management of acne: Canadian clinical practice guideline. CMAJ. 2016; 188(2): 118–126. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHazarika N, Archana M: The Psychosocial Impact of Acne Vulgaris. Indian J Dermatol. 2016; 61(5): 515–520. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZauli S, Caracciolo S, Borghi A, et al.: Which factors influence quality of life in acne patients? J Eur Acad Dermatol Venereol. 2014; 28(1): 46–50. PubMed Abstract | Publisher Full Text\n\nBarnes LE, Levender MM, Fleischer AB Jr, et al.: Quality of Life Measures for Acne Patients. Dermatol Clin. 2012; 30(2): 293–300. PubMed Abstract | Publisher Full Text\n\nTasoula E, Gregoriou S, Chalikias J, et al.: The impact of acne vulgaris on quality of life and psychic health in young adolescents in Greece. Results of a population survey. An Bras Dermatol. 2012; 876: 862–869. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMallon E, Newton JN, Klassen A, et al.: The quality of life in acne: a comparison with general medical conditions using generic questionnaires. Br J Dermatol. 1999; 1404: 672–676. PubMed Abstract | Publisher Full Text\n\nDréno B, Tan J, Kang S, et al.: How People with Facial Acne Scars are Perceived in Society: an Online Survey. Dermatol Ther (Heidelb). 2016; 6(2): 207–218. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChuah SY, Goh CL: The Impact of Post-Acne Scars on the Quality of Life Among Young Adults in Singapore. J Cutan Aesthetic Surg. 2015; 8(3): 153–158. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMadden WS, Landells ID, Poulin Y, et al.: Treatment of acne vulgaris and prevention of acne scarring: canadian consensus guidelines. J Cutan Med Surg. 2000; 4(Suppl 1): S2–13. PubMed Abstract\n\nTan J, Tanghetti E, Baldwin H, et al.: The Role of Topical Retinoids in Prevention and Treatment of Atrophic Acne Scarring: Understanding the Importance of Early Effective Treatment. J Drugs Dermatol 2019; 18(3): 255–260. PubMed Abstract\n\nWang JV, Saedi N: The utility of understanding atrophic acne scar formation for prevention and treatment. Br J Dermatol. 2018; 179(4): 819. PubMed Abstract | Publisher Full Text\n\nTan J, Thiboutot D, Gollnick H, et al.: Development of an atrophic acne scar risk assessment tool. J Eur Acad Dermatol Venereol. 2017; 31(9): 1547–1554. PubMed Abstract | Publisher Full Text\n\nJerry T, Sewon K, James L: Prevalence and Risk Factors of Acne Scarring Among Patients Consulting Dermatologists in the USA. J Drugs Dermatol. 2017; 16(2): 97–102. PubMed Abstract\n\nEstrella J, Camacho M, Viteri MI, et al.: Questionnaire for the validation and adaptation of a tool to estimate the risk of acne-induced scars in different population. Harvard Dataverse, V1. 2020. http://www.doi.org/10.7910/DVN/WGDWB0\n\nEstrella J, Camacho M, Viteri MI, et al.: Protocol for the validation and adaptation of a tool to estimate the risk of acne-induced scars in different populations. Harvard Dataverse, V1. 2020. http://www.doi.org/10.7910/DVN/NED0GS\n\nJohanson GA, Brooks GP: Initial Scale Development: Sample Size for Pilot Studies. Educ Psychol Meas. 2010; 70(3): 394–400. Publisher Full Text\n\nClark AK, Saric S, Sivamani RK: Acne Scars: How Do We Grade Them? Am J Clin Dermatol. 2018; 19(2): 139–144. PubMed Abstract | Publisher Full Text\n\nde Almeida H, Cecconi J, Duquia RP, et al.: Sensitivity and specificity of self-reported acne in 18-year-old adolescent males. Int J Dermatol. 2013; 52(8): 946–8. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "65623",
"date": "03 Aug 2020",
"name": "Maria Fernanda Torres-Calderón",
"expertise": [
"Reviewer Expertise Acne and Rosacea",
"Acne scars."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nRegarding the 4 ASRAT tool, I believe it can be good for screening the risk of developing scars, as tools with good sensitivity and moderate specificity are. It could be helpful as early intervention to prevent the detriment of their self esteem and mental health. The extended questionnaire, if applied to patients who already have acne, it could be helpful to intervene as soon as possible, under the premise that they are prone to develop acne scars or if they already have a few, so they do not develop any more scarring. This tool could translate into better adherence to treatment and less scarring, as well as knowing they are prone to develop them, for their future offspring.\n\nRegarding patients who already have acne scars, I think this tool will only show that if left untreated they will develop more scars.\nI believe the 4 ASRAT tool could incorporate questions from the extended questionnaire, but those specifically regarding risk factors for developing inflammatory acne, and the questionnaire should be applied by either a physician or a healthcare personnel, in order to standardize and reassure the questions are adequately interpreted by the patient. This could perhaps be done at the primary care physician office, with a referral to dermatology if the patient has a high risk of developing scars. Also, the full questionnaire would take around 20 minutes, so perhaps the most relevant questions should be kept to make it a 5-7 minute questionnaire or if most questions should remain the questionnaire should be made by trained personnel to make it objective and quicker.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "5823",
"date": "18 Aug 2020",
"name": "Jorge Estrella",
"role": "Author Response",
"response": "Dear Dr. Vicente Torres ad Dr. María Fernanda Torres-Calderón:As the lead author, we welcome with great joy your peer review. Like you, we believe that 4-ASRAT is a simple tool, easy to use and to understand, but with great potential for use as a primary prevention tool, which will allow the early identification of those patients at high risk of developing acne scars. Although the original questionnaire has more than 20 questions, the next phase of the study will allow us to determine which of these questions have greater relevance and weight as predictors of the risk of acne scars, with which we will be able to develop a questionnaire with fewer questions to be applied to the patients. We believe that its use in primary care, including by general practitioners, will allow the timely referral of patients with acne and high risk of scars, to dermatologists, so patients will be able to receive prompt and specialized care."
}
]
},
{
"id": "68895",
"date": "17 Aug 2020",
"name": "Zhou Bing-Rong",
"expertise": [
"Reviewer Expertise Phototherapy."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 4-ASRAT will be a useful and powerful tool for assessing whether acne scars occur in patients with acne lesions. As we all know, the treatment of these scars is still full of challenges. Therefore, if there is a simple and easy way to assess the risk of scar formation in acne patients and take early prevention measures, it may help to reduce the incidence of acne scars.\nThe current 4-ASRAT questionnaire is relatively suitable for rapid assessment of acne scar formation risk in a short period of time. Although it has good predictive sensitivity and specificity, I think the questionnaire still needs to add more specific indicators. It is better to be able to evaluate under the guidance of dermatologists. If the assessment items can include risk tools to predict the formation of acne atrophic scar, hypertrophic scar and keloid respectively, it will help doctors to deal with the corresponding problems. In addition, because in this study, according to the patient's facial lesions photos, in some cases, both dermatology experts and local dermatologists did not reach a high degree of consistency, which indicates that the definition of acne scar is still vague. Although it is very difficult to reach 100% consensus, it is important to make a more appropriate definition of acne scar before the assessment and to train the assessors for a certain period of time.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "5824",
"date": "18 Aug 2020",
"name": "Jorge Estrella",
"role": "Author Response",
"response": "Dear Dr Zhou Bing-Rong:As the lead author, we welcome with great pleasure your peer review. We definitely recognize that the management of acne scars still represents a challenge in dermatological clinical practice, so preventing the development of these scars should be a priority. According to our bibliographic research, one of the best ways to prevent the formation of acne scars is the timely identification of those patients with the greatest risk of developing them, so that they can receive timely and adequate treatment of their underlying disease. This is the importance of developing a tool like 4-ASRAT.The questions posed for 4-ASRAT are based on the identified risk factors for the development of acne scars. Because not all factors have the same relative risk, not all questions will be used in the final tool. A next study phase is being developed to identify which factors within those proposed are the ones that weigh the most and which consequently should be included in the final tool.Additionally, we believe that although 4-ASRAT should be used by dermatologists, it has a potential use also in primary medical care, where many patients with acne are treated but their risk of developing acne scars is not identified for their prompt referral to the service of dermatology.Finally, we do agree that defining acne scars is difficult and sometimes subjective. For subsequent studies with 4-ASRAT, we will clarify the definitions of acne scars to guarantee greater homogeneity among dermatologists in the evaluation of the same patient with acne."
}
]
}
] | 1
|
https://f1000research.com/articles/9-651
|
https://f1000research.com/articles/9-62/v2
|
04 Feb 20
|
{
"type": "Method Article",
"title": "Ensemble machine learning modeling for the prediction of artemisinin resistance in malaria",
"authors": [
"Colby T. Ford",
"Daniel Janies",
"Daniel Janies"
],
"abstract": "Resistance in malaria is a growing concern affecting many areas of Sub-Saharan Africa and Southeast Asia. Since the emergence of artemisinin resistance in the late 2000s in Cambodia, research into the underlying mechanisms has been underway. The 2019 Malaria Challenge posited the task of developing computational models that address important problems in advancing the fight against malaria. The first goal was to accurately predict artemisinin drug resistance levels of Plasmodium falciparum isolates, as quantified by the IC50. The second goal was to predict the parasite clearance rate of malaria parasite isolates based on in vitro transcriptional profiles. In this work, we develop machine learning models using novel methods for transforming isolate data and handling the tens of thousands of variables that result from these data transformation exercises. This is demonstrated by using massively parallel processing of the data vectorization for use in scalable machine learning. In addition, we show the utility of ensemble machine learning modeling for highly effective predictions of both goals of this challenge. This is demonstrated by the use of multiple machine learning algorithms combined with various scaling and normalization preprocessing steps. Then, using a voting ensemble, multiple models are combined to generate a final model prediction.",
"keywords": [
"malaria",
"Plasmodium falciparum",
"machine learning",
"parallel computing",
"Apache Spark",
"big data",
"artemisinin",
"bioinformatics",
"DREAM Competition"
],
"content": "Introduction\n\nMalaria is a serious disease caused by parasites belonging to the genus Plasmodium which are transmitted by Anopheles mosquitoes in the genus. The World Health Organization (WHO) reports that there were 219 million cases of malaria in 2017 across 87 countries1. Plasmodium falciparum poses one of greatest health threats in Southeast Asia, being responsible for 62.8% of malaria cases in the region in 20171.\n\nArtemisinin-based therapies are among the best treatment options for malaria caused by P. falciparum2. However, emergence of artemisinin resistance in Thailand and Cambodia in 2007 has been cause for research3. While there are polymorphisms in the kelch domain–carrying protein K13 in P. falciparum that are known to be associated with artemisinin resistance, the underlying molecular mechanism that confers resistance remains unknown4. The established pharmacodynamics benchmark for P. falciparum sensitivity to artemisinin-based therapy is the parasite clearance rate5,6. Resistance to artemisinin-based therapy is considered to be present with a parasite clearance rate greater than five hours7. By understanding the genetic factors that affect resistance in malaria, targeted development can occur in an effort to abate further resistance or infections of resistant strains.\n\n\nPrediction of artemisinin IC50\n\nFirst, we created a machine learning model to predict the IC50 of malaria parasites based on transcription profiles of experimentally-tested isolates. IC50, also known as the half maximal inhibitory concentration, is the drug concentration at which 50% of parasites die. This value indicates a population of parasites’ ability to withstand various doses of anti-malarial drugs, such as artemisinin.\n\nTraining data was obtained from the 2019 DREAM Malaria Challenge8,9. The training data consists of gene expression data of 5,540 genes of 30 isolates from the malaria parasite, Plasmodium falciparum. For each malaria parasite isolate, transcription data was collected at two time points [6 hours post invasion (hpi) and 24 hpi], with and without treatment of dihydroartemisinin (the metabolically active form of artemisinin), each with a biological replicate. This yields a total of at eight data points for each isolate. The initial form of the training dataset contains 272 rows and 5,546 columns, as shown in Table 1.\n\nThe transcription data was collected as described in Table 2. The transcription data set consists of 92 non-coding RNAs (denoted by gene IDs that begins with ’MAL’), while the rest are protein coding genes (denoted by gene IDs that start with ’PF3D7’). The feature to predict is DHA_IC50.\n\n(Adapted from Turnbull et al., (2017) PLoS One11).\n\nWe used Apache Spark10 to pivot the dataset such that each isolate was its own row and each of the transcription values for each gene and attributes (i.e. timepoint, treatment, biological replicate) combination was its own column. This exercise transformed the training dataset from 272 rows and 5,546 columns to 30 rows and 44,343 columns, as shown in Table 3. We completed this pivot by slicing the data by each of the eight combinations of timepoint, treatment, and biological replicate, dynamically renaming the variables (genes) for each slice, and then joining all eight slices back together.\n\nExample code shown below in the section labeled code 1. By using the massively parallel architecture of Spark, this transformation can be completed in a minimal amount of time on a relatively small cluster environment (e.g., <10 minutes using a 8-worker/36-core cluster with PySpark on Apache Spark 2.4.3).\n\n\n\nLastly, the dataset is then vectorized using the Spark VectorAssembler, and converted into a Numpy12-compatible array. Example code shown below in Code 1. Vectorization allows for highly scalable parallelization of the machine learning modeling in the next step.\n\n\n\nWe used the Microsoft Azure Machine Learning Service13 as the tracking platform for retaining model performance metrics as the various models were generated. For this use case, 498 machine learning models were trained using various scaling techniques and algorithms. We then created two ensemble models of the individual models using Stack Ensemble and Voting ensemble methods. Scaling and normalization methods are shown in Table 14.\n\nThe Microsoft AutoML package14 allows for the parallel creation and testing of various models, fitting based on a primary metric. For this use case, models were trained using Decision Tree, Elastic Net, Extreme Random Tree, Gradient Boosting, Lasso Lars, LightGBM, RandomForest, and Stochastic Gradient Decent algorithms along with various scaling methods from Maximum Absolute Scaler, Min/Max Scaler, Principal Component Analysis, Robust Scaler, Sparse Normalizer, Standard Scale Wrapper, Truncated Singular Value Decomposition Wrapper (as defined in Table 14). All of the machine learning algorithms are from the scikit-learn package15 except for LightGBM, which is from the LightGBM package16. The settings for the model sweep are defined in Table 4. The ‘Preprocess Data?’ parameter enables the scaling and imputation of the features in the data.\n\nOnce the 498 individual models were trained, two ensemble models (voting ensemble and stack ensemble) were then created and tested. The voting ensemble method makes a prediction based on the weighted average of the previous models’ predicted regression outputs whereas the stacking ensemble method combines the previous models and trains a meta-model using the elastic net algorithm based on the output from the previous models. The model selection method used was the Caruana ensemble selection algorithm17.\n\nThe voting ensemble model (using soft voting) was selected as the best model, having the lowest normalized Root Mean Squared Error (RMSE), as shown in Table 5. The top 10 models trained are reported in Table 6. Having a normalized RMSE of only 0.1228 and a Mean Absolute Percentage Error (MAPE) of 24.27%, this model is expected to accurately predict IC50 in malaria isolates. See Figure 1 for a visualization of the experiment runs and Figure 2 for the distribution of residuals on the best model.\n\nNote that the top performing model (VotingEnsemble) is the final IC50 model discussed in this paper.\n\nEach orange dot is an iteration with the blue line representing the minimum RMSE up to that iteration.\n\n\nPrediction of resistance status\n\nThe second task of this work was to create a machine learning model that can predict the parasite clearance rate (fast versus slow) of malaria isolates. When resistance rates change in a pathogen, it can be indicative of regulatory changes in the pathogen’s genome. These changes can be exploited for the prevention of further resistance spread. Thus, a goal of this work is to understand genes important in the prediction of artemisinin resistance.\n\nAn in vivo transcription data set from Mok et al., (2015) Science18 was used to predict the parasite clearance rate of malaria parasite isolates based on in vitro transcriptional profiles (see Table 8).\n\nThe training data consists of 1,043 isolates with 4,952 genes from the malaria parasite Plasmodium falciparum. For each malaria parasite isolate, transcription data was collected for various PF3D7 genes. The form of the training dataset contains 1,043 rows and 4,957 columns, as shown in Table 7. The feature to predict is ClearanceRate.\n\nThe training data for this use case did not require the same pivoting transformations as in the last use case as each record describes a single isolate. Thus, only the vectorization of the data was necessary, which was performed using the Spark VectorAssembler and then converted into a Numpy-compatible array12. Example code is shown below. Note that this vectorization only kept the numerical columns, which excludes the Country, Kmeans_Grp, and Asexual_stage__hpi_ attributes as they are either absent or contain non-matching factors (i.e. different set of countries) in the testing data.\n\n\n\nOnce the 98 individual models were trained, two ensemble models (voting ensemble and stack ensemble) were then created and tested as before. Model search parameters are shown in Table 9.\n\nThe voting ensemble model (using soft voting) was selected as the best model, having the highest area under the receiver operating characteristic curve (AUC), as shown in Table 11. The top 10 of the 100 models trained are reported in Table 10. Having a weighted AUC of 0.87 and a weighted F1 score of 0.80, this model is expected to accurately predict isolate clearance rates. A confusion matrix of the predicted results versus actuals is shown in Table 12. See Figure 3 for a visualization of the experiment runs and see Figure 4 and Figure 5 for the ROC and Precision-Recall curves on the best model.\n\nNote that the top performing model (VotingEnsemble) is the clearance rate model discussed in this paper.\n\nEach orange dot is an iteration with the blue line representing the maximum AUC up to that iteration.\n\nsubsectionFeature importance Feature importances were calculated using mimic-based model explanation of the ensemble model19. The mimic explainer works by training global surrogate models to mimic blackbox models. The surrogate model is an interpretable model, trained to approximate the predictions of a black box model as accurately as possible. See Figure 6 and Table 13.\n\n\nDiscussion\n\nBy using distributed processing of the data preparation, we can successfully shape and manage large malaria datasets. We efficiently transformed a matrix of over 40,000 genetic attributes for the IC50 use case and over 4,000 genetic attributes for the resistance rate use case. This was completed with scalable vectorization of the training data, which allowed for many machine learning models to be generated. By tracking the individual performance results of each machine learning model, we can determine which model is most useful. In addition, ensemble modeling of the various singular models proved effective for both tasks in this work.\n\nThe resulting model performance of both the IC50 model and the clearance rate model show relatively adequate fitting of the data for their respective predictions. While additional model tuning may provide a lift in model performance, we have demonstrated the utility of ensemble modeling in these predictive use cases in malaria.\n\nIn addition, this exercise helps to quantify the importance of genetic features, spotlighting potential genes that are significant in artemisinin resistance. The utility of these models will help in directing development of alternative treatment or coordination of combination therapies in resistant infections.\n\n\nPreprint\n\nAn earlier version of this article can be found on bioRxiv (doi: 10.1101/856922).\n\n\nData availability\n\nThe challenge datasets are available from Synapse (https://www.synapse.org/; Synapse ID: syn18089524). Access to the data requires registration and agreement to the conditions for use at: https://www.synapse.org/#!Synapse: syn18089524.\n\nChallenge documentation, including the detailed description of the Challenge design, data description, and overall results can be found at: https://www.synapse.org/#!Synapse:syn16924919/wiki/583955.\n\nWhole genome expression profiling of artemsinin-resistant Plasmodium falciparum field isolates, Accession number GSE59099: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE59099.\n\n\nSoftware availability\n\nSource code available from: https://github.com/colbyford/malaria_DREAM2019\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.359045921\n\nLicense: GPL-3.0",
"appendix": "References\n\nFact sheet about malaria. World Health Organization. 2019. Reference Source\n\nGuidelines for the treatment of malaria. World Health Organization. 2015. Reference Source\n\nDondorp AM, Nosten F, Yi P, et al.: Artemisinin resistance in Plasmodium falciparum malaria. N Engl J Med. 2009; 361(5): 455–467. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOuattara A, Kone A, Adams M, et al.: Polymorphisms in the K13-propeller gene in artemisinin-susceptible Plasmodium falciparum parasites from Bougoula-Hameau and Bandiagara, Mali. Am J Trop Med Hyg. 2015; 92(6): 1202–1206. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSaralamba S, Pan-Ngum W, Maude RJ, et al.: Intrahost modeling of artemisinin resistance in Plasmodium falciparum. Proc Natl Acad Sci U S A. 2011; 108(1): 397–402. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWhite NJ: The parasite clearance curve. In: Malar J. 2011; 10: 278. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAshley EA, Dhorda M, Fairhurst RM, et al.: Spread of artemisinin resistance in Plasmodium falciparum malaria. N Engl J Med. 2014; 371(5): 411–423. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDavis S, Button-Simons K, Bensellak T, et al.: Leveraging crowdsourcing to accelerate global health solutions. Nat Biotechnol. 2019; 37(8): 848–850. PubMed Abstract | Publisher Full Text\n\nGhouila A, Siwo GH, Entfellner JD, et al.: Hackathons as a means of accelerating scientific discoveries and knowledge transfer. Genome Res. 2018; 28(5): 759–765. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZaharia M, Xin RS, Wendell P, et al.: Apache spark: A unified engine for big data processing. Commun ACM. 2016; 59(11): 56–65. Publisher Full Text\n\nTurnbull LB, Siwo GH, Button-Simons KA, et al.: Simultaneous genome-wide gene expression and transcript isoform profiling in the human malaria parasite. PLoS One. 2017; 12(11): e0187595. PubMed Abstract | Publisher Full Text | Free Full Text\n\nvan der Walt S, Colbert SC, Varoquaux G: The numpy array: A structure for efficient numerical computation. Comput Sci Eng. 2011; 13(2): 22–30. Publisher Full Text\n\nMicrosoft Azure Machine Learning Service. 2019. Reference Source\n\nMicrosoft: Azure Machine Learning AutoML Core version 1.0.79. 2019. Reference Source\n\nPedregosa F, Varoquaux G, Gramfort A, et al.: Scikit-learn: Machine learning in Python. J Mach Learn Res. 2011; 12: 2825–2830. Reference Source\n\nKe G, Meng Q, Finley T, et al.: Lightgbm: A highly efficient gradient boosting decision tree. In: I. Guyon, U. V. Luxburg, S. Bengio, H. Wallach, R. Fergus, S. Vishwanathan, and R. Garnett, editors, Advances in Neural Information Processing Systems. Curran Associates, Inc. 2017; 30: 3146–3154. Reference Source\n\nCaruana R, Niculescu-Mizil A, Crew G, et al.: Ensemble selection from libraries of models. In: Proceedings of the Twenty-first International Conference on Machine Learning, ICML ’04, New York, NY, USA, 2004; 18. Publisher Full Text\n\nMok S, Ashley EA, Ferreira PE, et al.: Drug resistance. Population transcriptomics of human malaria parasites reveals the mechanism of artemisinin resistance. Science. 2015; 347(6220): 431–435. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLundberg SM, Lee S: A unified approach to interpreting model predictions. In: I. Guyon, U. V. Luxburg, S. Bengio, H. Wallach, R. Fergus, S. Vishwanathan, and R. Garnett, editors, Advances in Neural Information Processing Systems. Curran Associates, Inc., 2017; 30: 4765–4774. Reference Source\n\nMicrosoft: Microsoft Azure Machine Learning - AutoML Preprocessing. 2019. Reference Source\n\nFord C: colbyford/malaria_DREAM2019: Ensemble Machine Learning Modeling for the Prediction of Artemisinin Resistance in Malaria - Code Release (without data) for Research Publication (F1000) (Version v1.1). Zenodo. http://www.doi.org/10.5281/zenodo.3634793"
}
|
[
{
"id": "60584",
"date": "17 Mar 2020",
"name": "Stefan Jaeger",
"expertise": [
"Reviewer Expertise machine learning",
"artificial intelligence",
"data science",
"malaria screening"
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors present a machine learning approach for detecting malaria drug-resistance based on genetic attributes. To this end, they train many different models, which they combine with known ensemble methods like voting. The detection of malaria drug resistance is an important medical problem and the application of machine learning in this context deserves further exploration. However, the paper has several shortcomings that the authors need to address:\nThe author should provide a better description of the state-of-the-art and existing literature at the beginning of their paper.\n\nAlso demonstrate the need for such an approach. It is implicitly suggested, but greater clarity is needed on what gaps this approach fills. This can be addressed through previous bullet also.\n\nThe overall structure of the paper lacks clarity and concrete results. The authors claim that their exercise helps to “quantify the importance of genetic features, spotlighting potential genes that are significant in artemisinin resistance. The utility of these models will help in directing development of alternative treatment or coordination of combination therapies in resistant infections.” However, the experimental validation of these statements is insufficient, and the derived feature importance need to be discussed in more detail to convince the reader. In this context, Figure 6 need to be explained and discussed. What is the black block mimic model? Why has it been chosen by the authors for ranking features? In what way do other feature ranking schemes like PCA differ?\n\nThe paper describes two experiments: a regression experiment with the IC50 value as target, and a classification experiment with three different parasite clearance rates. However, both experiments need further justification. In the first experiment, the number of rows (patterns) seems to be very small compared to the number of features (genetic attributes), which makes over-training very likely. The authors need to comment on this and address the issue if possible. In the second experiment, it is unclear how the three different clearance rates relate to drug-resistance. What is the correlation between these classes and drug-resistance? Why have the authors trained many more models for the first experiment?\n\nListing of source code for formatting data is unnecessary and not suitable for a research paper. They have provided links to their code so including it in the paper seems superfluous, unless they want to make a point about it, which is absent. Further, that their example output after vectorization contains NaNs does not inspire confidence in the quality of the code; and, obviously needs further discussion.\n\nThe authors also cite that an earlier version of this article is available on bioRxiv. They should include discussion on what improvements are in this work that substantially improve over that.\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? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Partly",
"responses": [
{
"c_id": "5413",
"date": "29 Apr 2020",
"name": "Colby Ford",
"role": "Author Response",
"response": "We sincerely appreciate the reviewers' feedback on this work and have improved the article based on your recommendations. We have addressed each comment as follows in the article: Added additional examples of ML-based work in genomics, other tropical diseases, and in malaria. Added a brief explanation about the utility of this approach and its benefit over manual analysis. Addressed the reviewers' questions in the article around the explanability and black box methods and gave examples of the role of certain important genes identified here. Addressed the small observation size and the training of many models in the article and have better explained the relationship between drug resistance and parasite clearance rates. Further information on this data can be found in Mok et al., 2015. Also, the reason the second use case has fewer models trained was due to performance and risk of overfitting. However, with more observations, the machine learning modeling performance may increase with additional training and tuning time. The example code segments have been removed from the article. For the data quality concern, this is the data provided by the DREAM competition, thus isn't something we can control. The previous version on bioRxiv is nearly identical and was published there until the gateway was set up on F1000 and the publication embargo was lifted."
}
]
}
] | 2
|
https://f1000research.com/articles/9-62
|
https://f1000research.com/articles/9-647/v1
|
25 Jun 20
|
{
"type": "Case Report",
"title": "Case Report: A forty year-survivor of Tetralogy of Fallot with pulmonary atresia and chronic pediatric shunt thrombosis; findings from cardiac CT scan",
"authors": [
"Achour Asma",
"Walid Mnari",
"Mabrouk Abdelali",
"Ahmed Zrig",
"Ahmed Miladi",
"Mejdi Ben Messaoud",
"Badii Hmida",
"Mezri Maatouk",
"Walid Mnari",
"Mabrouk Abdelali",
"Ahmed Zrig",
"Ahmed Miladi",
"Mejdi Ben Messaoud",
"Badii Hmida",
"Mezri Maatouk"
],
"abstract": "Here we illustrate a rare case of Tetralogy of Fallot (TOF) with pulmonary atresia in a 40-year-old survivor patient, despite a chronic pediatric shunt thrombosis. The patient became symptomatic at 38-years-old with progressive dyspnea on exertion and short cyanosis spells. The clinical findings were unspecific except for sinus tachycardia. To reassess cardiac abnormalities and associated intrathoracic malformations, a cardiac CT-scan was performed. Here we explain the mechanism for prolonged survival as an expansion of several and huge major aortopulmonary collateral arteries. This case predicts an exceptional late outcome of untreated TOF.",
"keywords": [
"Computed tomography",
"cardiac imaging",
"Tetralogy of Fallot",
"Pulmonary atresia",
"Blalock Taussig shunt."
],
"content": "Introduction\n\nTetralogy of Fallot (TOF) is the most frequent form of cyanotic congenital heart disease. Only a few patients come of age without surgical intervention mainly in the extreme form with pulmonary atresia1. Here, we relate the case of a patient who survived until the age of 40 years without surgical curative intervention. The patient had an unusual association of chronic Blalock-Taussig shunt (BTS) thrombosis and huge major aortopulmonary collateral arteries (MAPCAs). This case provides the main role that a cardiac CT scan can lay in understanding the late outcome of these untreated cardiac abnormalities.\n\n\nCase report\n\nA 40-year-old man with “complex” cardiac disease and dyspnea was referred to our department for imaging. Old medical records with conventional angiographic studies inferred the diagnosis of TOF with pulmonary atresia, made at birth, treated by BTS. As the patient was asymptomatic, he had not consulted previously, and had been lost to follow-up. Two years ago, he began to present dyspnea on exertion, and also short cyanosis spells.\n\nClinical findings on presentation were unspecific except for sinus tachycardia. ECG showed biventricular hypertrophy and incomplete right bundle branch block. Echocardiography confirms the diagnosis of TOF with pulmonary atresia. To reassess cardiac abnormalities, primarily pulmonary trunk morphology and associated intrathoracic malformations, a 128-slice CT scan with ECG synchronization was performed. The scan revealed a large ventricle septal defect with overriding aorta (Figure 1, curved arrow) and right ventricle hypertrophy (Figure 1, asterisks). The pulmonary trunk was atretic, showing characteristic seagull pattern (Figure 2, arrows). The right pulmonary artery had a good diameter, while the left was significantly smaller (Figure 2). Pulmonary blood flow was given by MAPCA connecting blood vessels between the aorta and the pulmonary arteries (Figure 3, arrows). MAPCA was more numerous on the right side supplying the largest pulmonary artery. The BTS was completely thrombosed with a total heterogeneous filling defect and parietal calcifications (Figure 4, arrows); this is compatible with chronic thrombosis.\n\nThe mechanism for prolonged survival, despite the old shunt thrombosis, was the expansion of several and huge MAPCA allowing sufficient pulmonary blood flow. Two months after his hospitalization, the patient becom asymptomatic, receiving life-long conservative therapy including anticoagulation with a vitamin K antagonist (acenocoumarol 4 mg once a day) and a low dose of diuretic (Furosemide 40 mg once a day).\n\nCurved arrow shows the ventricular septal defect; arrows show the overriding Ao; and asterisks show the RV hypertrophy. LA, left atrium; RA, right atrium; LV, left ventricle; RV, right ventricle; Ao, aorta.\n\nArrows shows the atretic pulmonary trunk. LPA, left pulmonary artery; RPA, right pulmonary artery.\n\nArrows show major aortopulmonary collateral arteries.\n\nArrows show a total heterogeneous filling defect of the tube with parietal calcifications.\n\n\nDiscussion\n\nTOF is the most frequent cyanotic congenital heart disease. It includes four characteristic anatomical findings: sub-arterial ventricular septal defect, aorta straddling of the ventricles, a variable obstruction through the pulmonary outflow tract, and right ventricle hypertrophy2. The spectrum of severity within this lesion depends upon the anatomy of the pulmonary arteries. The different forms of TOF include TOF with a patent foramen oval/atrial septal defect, TOF with absent pulmonary valve, and TOF with pulmonary atresia, which is the extreme form characterized by absence of flow from the right ventricle to the pulmonary arteries1. In the latter event, pulmonary vascularization is derived from patent ductus arteriosus and aortopulmonary collaterals with significant variability from one patient to another2.\n\nMAPCAs are arteries that grow to irrigate lung circulation when native pulmonary arteries are underdeveloped. MAPCAs often arise from the descending aorta but also from the aortic arch and other systemic arteries like subclavian, the carotid, or rarely the coronary arteries. Pulmonary atresia-ventricular septal defects are classified into 3 types3: type A is associated with presence of the native pulmonary arteries with a pulmonary-aortic duct; type B has MAPCAs and native pulmonary arteries, such as in our case; type C has only MAPCAs, which are seen to provide the pulmonary blood without native pulmonary arteries.\n\nAdulthood clinical presentation, as in our case, is extremely rare. Without surgical intervention, most patients die at a young age with a rate of survival of 66% at one year of age, 11% at 20 years, and 3% at 40 years. In contrast, the survival rate after surgical treatment is over 90% at 40 years old2. The surgical option includes palliative forms, such as BTS, which consists in the creation of systemic to pulmonary shunt. Complete repair is the main option and associated with excellent outcome results4. In this case, despite a ductus arteriosus closure and a chronic pediatric shunt thrombosis, the patient survived at the age of 40 due to large and developed MAPCAs. The oldest survivor ever reported in the literature is 59 years old5.\n\nAfter a cardiac ultrasound, multidetector angiography CT scan is indicated to the assessment of TOF with pulmonary atresia mainly for associated intrathoracic malformations and to establish the precise MAPCA cartography before curative surgery6,7. Reformatted images allow a comprehensive analysis of pulmonary artery anatomy, measurements of the lumen of ascending aorta, analysis of the origins and course of coronary arteries, study location, the size, and flow in prior shunts8. The excellent spatial resolution and fast acquisition of multidetector CT make it a prime imaging tool to demonstrate even distal aortopulmonary collateral anatomy. Radiation should be reduced as low as possible in the pediatric population.\n\n\nConclusion\n\nUntreated pulmonary atresia with a ventricular septal defect is uncommon in adults. Most patients die from serious respiratory troubles or congestive cardiac failure very early. An angiography CT scan is the main imaging tool to delineate the pulmonary arterial supply, which is essential for an appropriate surgical approach.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and/or clinical images was obtained from the patient.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.",
"appendix": "References\n\nDobrocky T, Klink T, Weisstanner C, et al.: Imaging findings in uncorrected tetralogy of Fallot and pulmonary atresia with major aortopulmonary collateral arteries and septic embolism. Acta Radiol Short Rep. 2014; 3(1): 2047981613515211. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVaujois L, Gorincour G, Alison M, et al.: Imaging of postoperative tetralogy of Fallot repair. Diagn Interv Imaging. 2016; 97(5): 549–60. PubMed Abstract | Publisher Full Text\n\nTchervenkov CI, Roy N: Congenital heart surgery nomenclature and database project: pulmonary atresia-ventricular septal defect. Ann Thorac Surg. 2000; 69(4 Suppl): S97–105. PubMed Abstract | Publisher Full Text\n\nLapierre C, Dubois J, Rypens F, et al.: Tetralogy of Fallot: Preoperative assessment with MR and CT imaging. Diagn Interv Imaging. 2016; 97(5): 531–41. PubMed Abstract | Publisher Full Text\n\nFukui D, Kai H, Takeuchi T, et al.: Longest survivor of pulmonary atresia with ventricular septal defect: well-developed major aortopulmonary collateral arteries demonstrated by multidetector computed tomography. Circulation. 2011; 124(19): 2155–2157. PubMed Abstract | Publisher Full Text\n\nEnaba MM, Hasan DI, Alsowey AM, et al.: Multidetector Computed Tomography (CT) in Evaluation of Congenital Cyanotic Heart Diseases. Pol J Radiol. 2017; 82: 645–59. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHan BK, Rigsby CK, Hlavacek A, et al.: Computed tomography imaging in patients with congenital heart disease, part-I Rationale and utility. An expert consensus document of the Society of Cardiovascular Computed Tomography (SCCT). J Cardiovasc Comput Tomogr. 2015; 9(6): 475–92. PubMed Abstract | Publisher Full Text\n\nAhmed S, Johnson PT, Fishman EK, et al.: Role of Multidetector CT in Assessment of Repaired Tetralogy of Fallot. Radiographics. 2013; 33(4): 1023–36. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "65541",
"date": "13 Jul 2020",
"name": "Jean-Pierre Laissy",
"expertise": [
"Reviewer Expertise Cardiac imaging"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nGeneral Comments This case report describes CTA findings of a 40 year-old patient treated shortly after his birth by a Blalock-Taussig shunt for a tetralogy of Fallot, without use of any exam between the postoperative period and the actual CT. One of the main drawbacks of the BTS is the absence of growth and stretching, so thrombosis is very frequent. Fortunately, the patient developed large systemic to pulmonary arterial anastomoses, classifying the disease as a type B. The authors state that pulmonary atresia is untreated; this is partially true since BTS was used to compensate pulmonary artery flow from subclavian artery; this is mentioned in the Introduction (“without surgical curative intervention”) Some sentences are unclear and should be rewritten. In particular all the verbs should be in the preterit mode.\n\nSpecific comments Abstract Last sentence “This case predicts an exceptional late outcome of untreated TOF” is a wrong affirmation since BTS is one of the treatment options as described below.\n\nCase report Second para. In what way sinus tachycardia is specific? Line 8. “CT” should be preferred to “The scan” Line 9. What do the authors mean by “overriding aorta“? Line 10. “The right pulmonary artery had a good diameter should read “The right pulmonary artery had a good diameter”. Line 12. “MAPCA were more numerous” Line 16 “the patient becom” should read “the patient became” Parts of the following sentences should be reformulated such as: “pulmonary flow was given…”, “this is compatible with chronic thrombosis “ “receiving life-long conservative therapy”…\n\nDiscussion Line 4. “The different forms of TOF include TOF” should read “The different forms include TOF” Line 9. One should read “other systemic arteries like subclavian, carotid, or rarely coronary arteries. “. Line 18. The authors mention “In this case, despite a ductus arteriosus closure” Was it spontaneous or under surgery? The following sentence is difficult to understand and should be rewritten : “After cardiac ultrasound evaluation, multidetector CT angiography is useful to assess…” Line 26. What means “study location,”?\n\nReferences OK.\n\nIllustrations OK. Fig 2 should read “Multiplanar reconstruction image”; the following “from a cardiac CT scan with ECG synchronization”should be deleted.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? 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": "70020",
"date": "01 Sep 2020",
"name": "Hanne B Ravn",
"expertise": [
"Reviewer Expertise Critical care of patients with congenital heart 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\nImplications of MAPCAS detected this late after the initial event are missing. Data on vital values, including resting oxygen saturations, physical capacity, biventricular function and implications of a long-lasting systemic pressure in the pulmonary circulation are missing. Supposedly, the patient has an Eisenmenger equivalent condition. Apart from the fact that patient is a rarity, there is not much to be appreciated from the case-report. A reference for the survival data in untreated TOF patients is warranted.\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? No",
"responses": []
}
] | 1
|
https://f1000research.com/articles/9-647
|
https://f1000research.com/articles/9-178/v1
|
11 Mar 20
|
{
"type": "Brief Report",
"title": "What improvements do Cochrane systematic reviewers suggest for the design of trials?",
"authors": [
"Stefania Pirosca",
"Mike Clarke",
"Shaun Treweek",
"Stefania Pirosca",
"Mike Clarke"
],
"abstract": "Background: Many randomised trials have serious methodological flaws that fatally undermine their results, which makes the research wasteful. This is of concern for many, including those doing systematic reviews that include trials. Cochrane systematic reviews all have a section called ‘Implications for research’, which allows authors of the review to present their conclusions on how future research might be improved. Looking at these conclusions might highlight priority areas for improvement. Methods: We focused on the Cochrane Schizophrenia Review Group and the Multiple sclerosis and rare diseases of the central nervous system Review Group (the MS Review Group). Reviews with citations between 2009 and 2019 were identified and the recommendations given by review authors in ‘Implications for research’ were put into categories. Results: Between 2009 and 2019 we identified 162 reviews for the Schizophrenia Review Group and 43 reviews for the MS Review Group. We created 22 categories in total, of which 12 were common to both groups. The five most used categories were the same for both: better choice of outcomes; better choice of intervention/comparator; longer follow-up; larger sample size; use of validated scales. Better choice of outcomes and/or intervention/comparator was recommended in over 50% of reviews. Longer follow-up and larger sample size were recommended in over a third, with use of validated scales being suggested in around a fifth of reviews. There was no obvious pattern of improvement over time for trials included in systematic reviews published by both groups. Conclusions: We suggest that trialists working in these areas (and others too) ask themselves why they have chosen their particular outcomes, intervention and comparator, whether follow-up is long enough, if the sample size is big enough and whether the scales they choose to measure their outcomes are appropriate.",
"keywords": [
"Randomised controlled trial",
"Trial methodology",
"Systematic reviews",
"Schizophrenia",
"Multiple sclerosis."
],
"content": "Introduction\n\nRandomised trials are at the heart of evidence-informed healthcare systems. Not all trials, however, are created equal. Some are excellent, others have serious methodological flaws that fatally undermine their results and make the research wasteful1–3. This is of particular importance for the systematic reviews that should be used to underpin decision making in health care, and systematic reviewers often identify relevant issues and suggest ways to improve the quality of future trials. Cochrane is an international organisation that aims to provide high-quality information to support health decisions by systematically reviewing research, especially from randomised trials to investigate the effects of healthcare interventions. It is organized across more than 50 Cochrane Review Groups, each of which looks after a particular area of health.\n\nCochrane pays great attention to the methodological quality of both the reviews and the studies they include4. Cochrane systematic reviews all have a section called ‘Implications for research’, which allows the authors of the review to present their conclusions on how future research might be improved, for example by discussing the types of interventions that need more evaluation, or the outcomes which should, or should not, be measured and reported5.\n\nThe work described here looked at the ‘Implications for research’ sections of reviews published by two Cochrane Review Groups between 2009 and 2019 (2019 was incomplete at the time of the work). Our aims were to:\n\n1. Categorise the recommendations made in ‘Implications for research’.\n\n2. Explore whether there have been changes over time and between the two groups.\n\n\nMethods\n\nWe focused on the Schizophrenia Review Group and the Multiple sclerosis and rare diseases of the central nervous system Review Group (which we call the MS Review Group hereafter). The Schizophrenia Review Group was chosen because it has a long-standing interest in what is written in the ‘Implications for research’ section and we expected it to have good, consistent reporting. The MS group was selected because one of us (SP) had a special interest in this clinical area.\n\nThe work was split into two stages:\n\n1. Identify and extract the ‘Implications for research’ sections of all reviews in both groups with a citation date of 2009 to 2019. All reviews are available at the following URLs:\n\n1. Schizophrenia Review Group: https://schizophrenia.cochrane.org/topic-tree\n\n2. MS Review Group: https://msrdcns.cochrane.org/our-review\n\n2. Categorise the recommendations on trial design and methodology made by the review authors.\n\nStage 1 was straightforward because the ‘Implications for research’ sections of Cochrane reviews are easily identified. Stage 2 involved reading through the text and putting the issues raised by review authors into categories. We used a simple Excel spreadsheet data extraction form for this (see Data availability). We did not try to fit the data into pre-existing categories but were guided by what came from the data. The initial categorisation was done by SP before being discussed with ST until agreement was reached. No statistical analysis was necessary beyond counting.\n\n\nResults\n\nFor the period 2009 to 2019, we identified 162 reviews for the Schizophrenia Review Group and 43 reviews for the MS Review Group. A wide variety of interventions were covered by these reviews, including drugs, educational and behavioural interventions, and other therapies such as physical exercise and acupuncture. The median number of included studies in reviews from the Schizophrenia Group was seven (range 0 to 174); for the MS Group it was five (range 0 to 45).\n\nWe created 22 categories in total, of which 12 were common to both review groups (Table 1). Six categories were unique to the Schizophrenia Review Group and four were unique to the MS Review Group. The five most used categories were the same for both groups (better choice of outcomes, better choice of intervention/comparator, longer follow-up, larger sample size and use of validated scales). However, the use of these five categories varied over time for each group (Figure 1 and Figure 2).\n\nA total of 22 categories were identified, 12 of which were common to both the Schizophrenia and Multiple Sclerosis (MS) Review Groups. The five most frequently occurring implications for research (marked in grey) were the same for both review groups.\n\nThe percentages shown are the average over a two-year period, apart from 2019 which was incomplete at the time the work was done. While some categories are used in well over half of all review published in a given year, there is no clear pattern over time.\n\nMS=Multiple sclerosis and rare diseases of the central nervous system. The percentages shown are the average over a two-year period, apart from 2019 which was incomplete at the time the work was done. While some categories are used in well over half of all reviews published in a given year, there is no clear pattern over time. Use of ‘Better choice of outcomes’ may be increasing but review numbers are small so caution is needed in interpreting this increase.\n\n\nConclusions\n\nThere is substantial overlap in the types of recommendation made in the ‘Implications for research’ sections of systematic reviews done by the Cochrane Schizophrenia and MS Review Groups. These were clearer in the Schizophrenia Review Group’s reviews because of their structured approach to presenting implications for research in accordance with published guidance6. Their reviews also routinely include a suggested design for a future trial in this section.\n\nThe five most frequently made recommendations are the same for both groups with better choice of outcomes being top of the list and cited in over half the 205 reviews included in our study. Looking across the decade to 2019, there is no obvious pattern of decrease in the areas of methodology that need to be improved in trials included in systematic reviews published by both groups.\n\n\nLimitations\n\nIt is possible that different researchers would have categorised the implications for research differently, although we did use two reviewers working independently and there was little disagreement. Selecting other Cochrane review groups may have led us to conclude that different recommendations were most common but without doing that work we have no way of knowing this.\n\nDespite looking at just two Cochrane review groups, we believe that our findings are likely to be generalisable to other areas of health and health care but, at a minimum, a good start for the 2020s would be for researchers planning trials in schizophrenia and multiple sclerosis to ask themselves the following questions:\n\n1. Why did we choose these outcomes?\n\n2. Why is this the right intervention and comparator?\n\n3. Is follow-up long enough?\n\n4. Is the sample size large enough?\n\n5. Will validated scales be used to measure outcomes?\n\n\nData availability\n\nOpen Science Framework: Data extracted from the Implications for Research sections of reviews from two Cochrane Review Groups. https://doi.org/10.17605/OSF.IO/XCJ7R\n\nThis project contains the following underlying data:\n\n- Data from Implications for Research section of Cochrane MS Review Group 2009–2019.csv (Data extracted from Cochrane MS Review Group)\n\n- Data from Implications for Research section of Cochrane Schizophrenia Review Group 2009-2019.csv (Data extracted from Cochrane Schizophrenia Review Group)\n\nOpen Science Framework: Data extraction form used in 'What improvements do Cochrane systematic reviewers suggest for the design of trials?'. https://doi.org/10.17605/OSF.IO/FNJVZ\n\nThis project contains the following extended data:\n\n- Data extraction form.csv (Study data extraction form)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
"appendix": "References\n\nGlasziou P, Chalmers IC: Research waste is still a scandal—an essay by Paul Glasziou and Iain Chalmers. BMJ. 2018; 363: k4645. Publisher Full Text\n\nYordanov Y, Dechartres A, Porcher R, et al.: Avoidable waste of research related to inadequate methods in clinical trials. BMJ. 2015; 350: h809. PubMed Abstract | Publisher Full Text | Free Full Text\n\nIoannidis JP: Clinical trials: what a waste. BMJ. 2014; 349: g7089. PubMed Abstract | Publisher Full Text\n\nHiggins JPT, Thomas J, Chandler J, et al. (editors): Cochrane Handbook for Systematic Reviews of Interventions version 6.0. (updated July 2019). Cochrane. 2019. Reference Source\n\nClarke L, Clarke M, Clarke T: How useful are Cochrane reviews in identifying research needs? J Health Serv Res Policy. 2007; 12(2): 101–3. PubMed Abstract | Publisher Full Text\n\nBrown P, Brunnhuber K, Chalkidou K, et al.: How to formulate research recommendations. BMJ. 2006; 333(7572): 804–6. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "61192",
"date": "16 Mar 2020",
"name": "Livia Puljak",
"expertise": [
"Reviewer Expertise Methodological research",
"clinical 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\nI have reviewed with interest the manuscript titled “What improvements do Cochrane systematic reviewers suggest for the design of trials? [version 1; peer review: awaiting peer review]”. The authors report a study based on a very elegant idea, analysing content of the “Implications for research” section in selected Cochrane reviews. I would like to suggest the following minor revisions:\n\nTitle: I would suggest revising in a way to reflect the fact that these recommendations came from Cochrane reviews. When I first read the title, I initially thought that this manuscript reports results of a survey among Cochrane authors.\n\nAbstract\n“Reviews with citations between 2009 and 2019 were identified” – should this be “reviews published between 2009 and 2019”?\n\n“We created 22 categories” – I would suggest revising “categories” into “categories of recommendations”.\n\nConclusion: I am not sure that it is sufficient to recommend to trialists to “ask themselves”. The problems that were mentioned (i.e. recommendations) were consistent over the analysed period. I think it would be more meaningful to conclude that we need to analyse the effects of interventions that will force/motivate trialists to change their actions when designing clinical trials.\nMethods\nIt would be useful to have a section called Data extraction, to describe all the data that were extracted. For example, in the beginning of the Results, the authors have mentioned type of interventions, and number of studies included in analysed reviews, but this was not mentioned as extracted in the Methods.\nResults\nResults section is rather short. I would appreciate to read more text in this section, i.e. about categories specific only to each review group analyzed. However, I understand that this is brief report, so I would not insist on this, if this is not feasible for this type of article.\n\nIn the Discussion, the authors mentioned that some categories were “clearer”, but I am not really sure what this means. I also do not see anything mentioned about that in the Methods, and Results.\n\nIn the Abstract, the authors wrote “ There was no obvious pattern of improvement over time for trials included in systematic reviews published by both groups”, and in the Results, the authors wrote “However, the use of these five categories varied over time for each group”. Is this supposed to be the same? The expression “varied” is very non-specific.\nDiscussion\nI would prefer to see this explained in more detail; I am not sure I understand completely what it means: “These were clearer in the Schizophrenia Review Group’s reviews because of their structured approach to presenting implications for research in accordance with published guidance”. What kind of “structured approach” is this?\n\nIs this a formal requirement, is there a reference to be used for this sentence: “Their reviews also routinely include a suggested design for a future trial in this section.”\n\n“cited in over half” – I would suggest to revise “mentioned (or used) in over half”.\n\nConclusion statement at the end of the manuscript: the same comment as for the conclusion statement at the end of the Abstract.\n\nPerhaps the authors should mention some other resources that the trialists should use to improve some of these aspects, such as core outcome sets.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "5647",
"date": "25 Jun 2020",
"name": "Shaun P. Treweek",
"role": "Author Response",
"response": "Response to comments on F1KR00CDE F1R-VER24991-A22/6/2020 Thanks for the reviewers’ comments on our manuscript. Our responses are below. We have made the necessary changes to the manuscript using track changes so that they can be easily seen. We have also submitted a clean version with the changes accepted. Reviewer #1 Title: I would suggest revising in a way to reflect the fact that these recommendations came from Cochrane reviews. When I first read the title, I initially thought that this manuscript reports results of a survey among Cochrane authors. We have changed the title to “Learning from Cochrane systematic reviews: what improvements do these suggest for the design of trials?” “Reviews with citations between 2009 and 2019 were identified” – should this be “reviews published between 2009 and 2019”? In the first draft of the report we had ‘published’ but we have decided to change to ‘citations’ because of the way Cochrane makes its reviews available. Through ‘citation’ we mean that we examined the most recent version of any review that was first published or was updated in 2009 to 2019. This is important because reviews from before 2009 were still “published” during the decade, but were not updated in that time period. “We created 22 categories” – I would suggest revising “categories” into “categories of recommendations”. Done. Conclusion: I am not sure that it is sufficient to recommend to trialists to “ask themselves”. The problems that were mentioned (i.e. recommendations) were consistent over the analysed period. I think it would be more meaningful to conclude that we need to analyse the effects of interventions that will force/motivate trialists to change their actions when designing clinical trials. We have added a few sentences including the available resources for researchers to change their actions in the ‘Conclusions’ section: There are resources that can help with the above. For example, COMET (http://www.comet-initiative.org) can help with outcome choice and PRECIS-2 can help with design decisions around comparators and follow-up9. Discussions with staff at clinical trial units and other research support centres are also likely to improve designs. Change is slow at present and initiatives to encourage, or force, trialists to consider these questions would be welcome, especially from funders. In the abstract, we have also added a few words to address this comment (addition in italics, which is also included in the main Conclusions): We suggest that trialists working in these and other areas ask themselves, or are compelled to do so by others (e.g. funders),… It would be useful to have a section called Data extraction, to describe all the data that were extracted. For example, in the beginning of the Results, the authors have mentioned type of interventions, and number of studies included in analysed reviews, but this was not mentioned as extracted in the Methods. We have added a new file in the repository including all the data we extracted so that readers can see the data we extracted. We have added the following text to the ‘Methods’ section: Additionally we extracted information on the number of included participants and studies, the risk of bias etc. (See Data Availability for a file containing the full list). Results section is rather short. I would appreciate to read more text in this section, i.e. about categories specific only to each review group analyzed. However, I understand that this is brief report, so I would not insist on this, if this is not feasible for this type of article. As this is a Brief Report, we have decided to keep the ‘Results’ section to the point and we have not repeated in words information that is in the table. We have added some extra detail though, see response to Comment #8. In the Discussion, the authors mentioned that some categories were “clearer”, but I am not really sure what this means. I also do not see anything mentioned about that in the Methods, and Results. We have replaced this text with: However, the ranking of each category in that top five list varied from one year to the next; no category was consistently most used (Figures 1 and 2). There was no obvious pattern of improvement over time for trials included in systematic reviews published by both groups. In the Abstract, the authors wrote “ There was no obvious pattern of improvement over time for trials included in systematic reviews published by both groups”, and in the Results, the authors wrote “However, the use of these five categories varied over time for each group”. Is this supposed to be the same? The expression “varied” is very non-specific. The two sentences refer to different things: the one in the abstract to general improvement over time, the one in Results to the position of a given category in the top five from one year to the next. We have modified the text, see response to Comment #7. I would prefer to see this explained in more detail; I am not sure I understand completely what it means: “These were clearer in the Schizophrenia Review Group’s reviews because of their structured approach to presenting implications for research in accordance with published guidance”. What kind of “structured approach” is this? Consistency was the key advantage and we have changed the wording of this sentence accordingly: These were easier to identify in the Schizophrenia Review Group’s reviews because of their consistent approach to presenting implications for research in accordance with published guidance6 Is this a formal requirement, is there a reference to be used for this sentence: “Their reviews also routinely include a suggested design for a future trial in this section.” We are not aware of this being a formal requirement, or of a reference we can point readers to. We have modified the sentence so that it now comes across as an observation rather than review group policy: Many of their reviews also include a suggested design for a future trial in this section. “cited in over half” – I would suggest to revise “mentioned (or used) in over half”. We have changed to ‘used in over half’. Conclusion statement at the end of the manuscript: the same comment as for the conclusion statement at the end of the Abstract. We have addressed this issue. See response to comment 4. Perhaps the authors should mention some other resources that the trialists should use to improve some of these aspects, such as core outcome sets. See response to Comment #4."
}
]
},
{
"id": "62266",
"date": "29 Apr 2020",
"name": "Christian Gluud",
"expertise": [
"Reviewer Expertise Evidence based clinical practice."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nPirosca and colleagues have examined the 'Implications for research' section of 205 Cochrane reviews published between 2009 to 2019 from two editorial groups. The five most used categories were the same for both editorial groups: better choice of outcomes; better choice of intervention/comparator; longer follow-up; larger sample size; use of validated scales. Better choice of outcomes and/or better choice of intervention/comparator was recommended in over half of reviews; longer follow-up and larger sample size were recommended in over a third; and use of validated scales being suggested in around a fifth of reviews.\n\nI think the authors should also discus their interesting findings from the point of view: how good are the Cochrane authors? E.g. we know from numerous studies (e.g., Rebecca M Turner et al.) that 80% to 90% of all Cochrane reviews do not reach a fair meta-analytic sample size providing the necessary power to assess medium large and small (but still clinically relevant) intervention effects. How come then that almost 66% of reviews do not mention larger sample sizes?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "5646",
"date": "25 Jun 2020",
"name": "Shaun P. Treweek",
"role": "Author Response",
"response": "Response to comments on F1KR00CDE F1R-VER24991-A22/6/2020 Thanks for the reviewers’ comments on our manuscript. Our responses are below. We have made the necessary changes to the manuscript using track changes so that they can be easily seen. We have also submitted a clean version with the changes accepted. Reviewer #2 I think the authors should also discuss their interesting findings from the point of view: how good are the Cochrane authors? E.g. we know from numerous studies (e.g., Rebecca M Turner.) that 80% to 90% et al of all Cochrane reviews do not reach a fair meta analytic sample size providing the necessary power to assess medium large and small (but still clinically relevant) intervention effect. How come then that almost 66% of reviews do not mention larger sample sizes? We have added the following text to the ‘Conclusions’ section: Previous research found that small, underpowered studies make up the entirety of evidence in most meta-analyses reported by Cochrane reviews7. Only 35% of the reviews in our sample mentioned sample size in the Implication for research, which suggests that reviewers may be underestimating the size of this problem. The most frequent issue raised in our work was the choice of outcome, a persistent problem that led to the COMET initiative to facilitate the development of core outcome sets5,8. Cochrane reviewers need to continue to highlight trial design problems; indeed they could perhaps do so more often, especially around sample sizes."
}
]
}
] | 1
|
https://f1000research.com/articles/9-178
|
https://f1000research.com/articles/9-311/v1
|
29 Apr 20
|
{
"type": "Research Article",
"title": "Assessment of a demonstrator repository for individual clinical trial data built upon DSpace",
"authors": [
"Birol Tilki",
"Thomas Schulenberg",
"Steve Canham",
"Rita Banzi",
"Wolfgang Kuchinke",
"Christian Ohmann",
"Birol Tilki",
"Thomas Schulenberg",
"Steve Canham",
"Rita Banzi",
"Wolfgang Kuchinke"
],
"abstract": "Background: Given the increasing number and heterogeneity of data repositories, an improvement and harmonisation of practice within repositories for clinical trial data is urgently needed. The objective of the study was to develop and evaluate a demonstrator repository, using a widely used repository system (DSpace), and then explore its suitability for providing access to individual participant data (IPD) from clinical research. Methods: After a study of the available options, DSpace (version 6.3) was selected as the software for developing a demonstrator implementation of a repository for clinical trial data. In total, 19 quality criteria were defined, using previous work assessing clinical data repositories as a guide, and the demonstrator implementation was then assessed with respect to those criteria. Results: Generally, the performance of the DSpace demonstrator repository in supporting sensitive personal data such as that from clinical trials was strong, with 14 requirements demonstrated (74%), including the necessary support for metadata and identifiers. Two requirements could not be demonstrated (inability to incorporate de-identification tools in the submission workflow, lack of a self-attestation system) and three requirements were only partially demonstrated (ability to provide links to de-identification tools and requirements, incorporation of a data transfer agreement in system workflow, and capability to offer managed access through application on a case by case basis). Conclusions: Technically, the system was able to support most of the pre-defined requirements, though there are areas where support could be improved. Of course, in a productive repository, appropriate policies and procedures would be needed to direct the use of the available technical features. A technical evaluation should therefore be seen as indicating a system’s potential, rather than being a definite assessment of its suitability. DSpace clearly has considerable potential in this context and appears a suitable base for further exploration of the issues around storing sensitive data.",
"keywords": [
"Repository",
"clinical trial",
"individual participant data",
"data sharing",
"DSpace"
],
"content": "Introduction\n\nThe sharing of clinical trial data still occurs mainly with in a closed professional evironment through direct and personal sharing, rather than via accessible data repositories. A multi-stakeholder taskforce addressing this problem recommended that data and documents from clinical trials available for sharing should be transferred to a suitable data repository to help ensure that the data objects are properly prepared, are available in the longer term, are stored securely and are subject to rigorous governance1. A recent study has shown that an increasing number of such repositories are available for sharing of individual participant data (IPD) from clinical studies2. There are many different types of repositories, however, such as generic repositories for all kinds of life-science data, repositories exclusively for clinical research data and specialised repositories with a specific focus, e.g. a single disease area, and major heterogeneity exists with respect to data-upload, data-handling, and data-access processes. This heterogeneity of repository types and features, reflects both the different purposes and perspectives of repository founders, and the relative immaturity of repository data-sharing services. Given the lack of a consensus about the services required from a data repository, each organisation has implemented its own policies and systems to meet its own priorities. Greater harmonisation of practices within repositories, coupled with the implementation of quality criteria for repositories, may diminish the reluctance of many researchers to share the data from their studies, thus promoting data-sharing, discoverability, and re-use3,4.\n\nIn a consensus building exercise, the necessity for compliance of repositories for clinical trial data and related data objects with quality criteria was emphasised1. The services any repository provides should conform to specified quality standards, to give its users confidence that their data and documents will be stored securely and in accordance with the specific data transfer agreements they have agreed.\n\nThis paper explores the suitability of a widely used data repository system, DSpace, for supporting the long-term management of IPD generated from clinical research while conforming to defined quality criteria. Though DSpace is a repository system used for open data, it is increasingly used also for restricted data access because it provides several built-in features that make it adaptable for restricted data sharing. The work was carried out as part of a broader set of activities aimed at developing mechanisms for the sharing of IPD from clinical research (https://www.corbel-project.eu/home.htm). It builds on previous published papers describing principles and practical recommendations for IPD sharing1, offering a detailed analysis of the processes involved in depositing, managing and sharing IPD5, and evaluating existing repositories for their suitability for the deposition of IPD, specifically for researchers in the non-commercial sector2. In the latter analysis, repositories were assessed against a set of quality criteria, referring to the processes of data upload, storage, de-identification, and quality controls, metadata, identifiers, flexibility of access and long-term preservation. The aim of this paper is to describe the development of a demonstrator repository based on the DSpace system and assess it using a pre-defined set of quality criteria and requirements.\n\nThe reason for developing this repository was to explore further various technical and workflow issues around the long-term management of IPD, in practical terms, using a well-known repository system applied to IPD from clinical research. The demonstrator is intended as an illustrative example only and this paper deals only with technical aspects of the repository system, i.e. its evaluation as a suitable infrastructure. It is clear that many aspects of a repository’s suitability for IPD are linked to the procedures and processes implemented by the institution hosting the repository. In other words, a strong technical infrastructure is a necessary but not sufficient indicator of quality.\n\n\nMethods\n\nWriting a bespoke repository system from scratch was seen as unrealistic, given resource constraints, and in any case less useful than using an existing system – one that would also be available to potential repository managers. A variety of systems were considered as the possible base system for the demonstrator repository (e.g. Figshare, DSpace). These and other systems were assessed with respect to standardised criteria, e.g. cross-country use, type of activities to be supported (project, service, collaboration, platform, etc.), modules/architecture/components included, research use cases/projects/studies the tool is used, etc.6. DSpace was rated as the system with the greatest potential for a demonstrator repository, particularly in an academic context. The reasons for selection of DSpace as the repository system are discussed more fully in 6.\n\nHere only a short summary of the assessment is presented. DSpace was selected because it appears to be by far the most popular of the various repository systems, with almost 2884 users, 2204 of them listed as ‘academic’ (including the University of Cambridge, Yale, Duke University and the University of Edinburgh amongst many around the world; https://duraspace.org/registry/). Three of 25 repositories for IPD from clinical trial data, evaluated in a recent review, are built upon DSpace (Dryad, Drum, Edinburgh DataShare)2.\n\nDSpace is an open source system and can be modified and extended by users. It claims about 100 contributors to the code base, with the Dryad repository, which runs on DSpace, being an example of how the system can be extended. It is possible to download and run a pre-configured ‘out of the box’ solution, but DSpace also claims to be fully modifiable, even though many of the modifications listed are relatively superficial (e.g. themes, screen configurations, search parameters). The system also appeared compliant with most of the relevant standards (e.g. Open Archives Initiative Protocol for Metadata (OAI-PMH)), developed for harvesting metadata descriptions from records), runs on a variety of operating systems and can use either Oracle or PostgreSQL as the back-end database store (https://duraspace.org/dspace/). There also appeared to be an active user group and comprehensive documentation, including a Wiki (https://wiki.duraspace.org/display/DSPACE/). An alternative to DSpace would have been to use Invenio (https://invenio-software.org/), which delivers the repository units for Zenodo, OpenAIRE and CERN Open Data. Invenio was very focused on open data only and DSpace offered in contrast more possibilities to adjust to restricted data sharing.\n\nA data repository was established between October 2018 and June 2019 within the Coordination Centre for Clinical Trials at the University of Düsseldorf, by BT (first author) using version 6.3 of DSpace. Additional software was installed to supplement DSpace functioning and manage servers and common server functionality.\n\nFull list of the software and hardware used for the repository installations and details of the technical implementation of the demonstrator repository:\n\nDSpace is a framework of a considerable number of different software tools that must work together to achieve an efficient DSpace installation. Prerequisite software tools must be downloaded, installed, tested, configured and integrated with each other. In addition to DSpace itself, the following were installed:\n\nUbuntu 16 and Ubuntu 18 (Linux operating system)\n\nJava 8 (Java Development Kit)\n\nApache Maven 3.3.9 (Java build tool)\n\nApache Ant 1.9.13 (Java build tool)\n\nPostgreSQL 9.5 (with pgcrypto installed) as the relational database back end\n\nApache Tomcat 9.0.11 (Java Servlet, Server Pages, and Web Socket Engine)\n\nDSpace can be installed at different scales, allowing different amounts of data to be handled. In our usage scenario we assumed the storage of several hundred trials with a size of 10–100 MB per trial, uploaded over several years. We therefore decided to install a mid-range version of DSpace, able to accommodate a large number of clinical trials datasets. The virtual server was established with:\n\n6 GB RAM in total: approximately 2 GB for Ubuntu 16/18, 2 GB for PostgreSQL, 2 GB for Tomcat.\n\n200 GB system storage. Deducting 40 GB for system and application use this provides enough storage for 1600 datasets (at 100MB per dataset).\n\nThis mid-range system is capable of supporting an application with either a large number of items (roughly 50,000 files and associated metadata) or a large volume of activity (searches, accesses, downloads, etc.).\n\nFor testing, publicly available data and documents as from clinical trials were uploaded to the demonstrator repository. The data used are displayed on the welcome page of the DSpace demonstrator repository (http://90.147.75.211:8080/xmlui/).\n\nThe quality criteria for assessment were taken from an original collection of 34 attributes. These features were proposed and agreed among a group of researchers and were built on previous work and discussion within CORBEL and the IMPACT Observatory project2. They were meant to provide a general characterisation of a repository and included aspects used to help assess a repository’s relative maturity and its suitability for clinical research data. From these criteria set, 8 features were selected and applied in a review on evaluation of repositories, aimed to capture the most important features for clinical researchers to deposit data in a repository2.\n\nThese 8 criteria identified as being key to successful management of IPD are listed below2.\n\n1. Guidelines for data upload and storage\n\n2. Support for data de-identification\n\n3. Data quality controls\n\n4. Contracts for upload and storage\n\n5. Availability of metadata\n\n6. Application of identifiers\n\n7. Flexibility of access\n\n8. Repository long term preservation\n\nMore generic standards and criteria for trustworthy digital repositories have been developed and are being applied7–10; e.g., Data Seal of Approval, International Council for Science World Data Systems). The relationship as well as differences between the eight criteria and other standards and criteria available for repositories is explored further in the Discussion section (see also Table 3).\n\nTo promote interoperability and retain meaning within interpretation and analysis, shared data should be, as far as possible, structured, described and formatted using widely recognised data and metadata standards (e.g. Clinical Data Interchange Standards Consortium (CDISC), Core Outcome Measures in Effectiveness Trials (COMET), Medical Dictionary for Regulatory Activities (MedDRA))1. Although researchers should be encouraged to use these standards, this criterion was not included in our study. Here we focus on standards for metadata.\n\nIn order to make the assessment of the criteria more operational and to distinguish features of the system (technical features) from measures around the system (e.g. policies and procedures), the criteria were split into specific requirements. This was performed by the group of authors. Table 1 provides a detailed breakdown of the eight criteria in terms of their associated ‘requirements’ – i.e. the features one would normally expect to see implemented. ‘System’ features (i.e. repository system and its technical features), are distinguished from ‘Procedures’ (i.e. function of the repository’s policies and procedures).\n\nSystem: To be demonstrated by the repository system’s technical features. Procedures: Function of the repository’s governance, policies, procedures.\n\nFor example, to support ‘Guidelines for data upload and storage’, the requirements for the repository could include:\n\na) being able to support a wide variety of file and metadata types,\n\nb) providing easy to use mechanisms for the upload of files, including technical instructions,\n\nc) providing rules and guidelines for data upload and storage (e.g. which formats or metadata schema to use and when).\n\na), and b) are mainly aspects of the repository system and its technical features, whilst c) is more a function of the repository’s policies and procedures.\n\nIn the context of this study it is important to stress that only the requirements labelled as ‘system’ attributes in Table 1 were evaluated (19 of 29, or 66%). Each of these system features was assessed and its level of fulfilment within DSpace classified as:\n\ndemonstrated\n\npartially demonstrated\n\nnot demonstrated\n\nThe assessment of the requirements was performed by BT and based on publicly available information about DSpace (web pages, user manuals, Q&A pages, reports, etc.). DSpace was not contacted directly and but there was contact with the DSpace community. The Coordination Centre for Clinical Trials in Düsseldorf participated at a meeting of the German user community.\n\n\nResults\n\nThe results are summarised in this section and in Table 2.\n\nGrey: not considered by the Banzi criteria2.\n\nDSpace exhibits a flexible approach to file storage by supporting a range of file types and metadata schemas (1a demonstrated). With a variety of tools available, along with detailed technical guidance, it also provides mechanisms for upload of files, including instructions (1b demonstrated).\n\nThe DSpace system has no published requirements or guidelines relating to the de-identification of uploaded data. It is the submitter’s responsibility to ensure that documents are consistent with current standards, guidelines and policies from official bodies and scientific organisations. The submitter is, however, able to use links to requirements, guidelines and/or tools, if these are established by the system’s administrator (2a partially demonstrated). As far as we could tell, neither the DSpace repository system nor the user community have implemented de-identification tools or programs, able to perform and document de-identification on an existing dataset (2b not demonstrated).\n\nThe control of the quality of data is more a question of procedures and workflow around a repository than technical features available in a particular system. Nevertheless, there are some technical features that could facilitate a quality control workflow. Some of these features are available within DSpace, usually as optional and configurable additions to the data upload process but they are limited to a predefined review workflow. This covers a single reviewer workflow, collection’s workflow steps and a score review workflow. This is certainly an important feature but does not correspond to a full quality-controlled process, which needs additional features like monitoring and tracking uploads, rejections, edits; reports about reviews in process and performed, etc. (3a partially demonstrated).\n\nA formal data transfer contract signed by the data generator and the repository administrator should be a prerequisite for transferring clinical trial data to a repository, not least to clarify potential legal responsibilities under data protection legislation. At the end of the manual submission process in DSpace, the submitter (data generator) is asked to grant the repository service an appropriate distribution license (different licences can be made available to different user communities). Agreeing a distribution licence is not the same, however, as enforcing a data transfer agreement. Confirmation of the existence of a signed data transfer protocol can be required from the user, i.e. integrated within the distribution licence, if implemented. The demonstrator repository is not, however, able to provide support for constructing and editing such a document (4a partially demonstrated).\n\nDSpace can support multiple extended metadata schemas for describing an item. A qualified Dublin Core metadata is provided by default. Multiple schemas can be configured, and metadata fields selected from a mix of configured schemas to describe items (5a demonstrated). In addition, a new metadata schema can be created. In the demonstrator repository, the ECRIN Clinical Trial Metadata Schema was created11 (5b demonstrated). DSpace has several tools to help data generators export content and metadata, ingest content and metadata tools and batch edit metadata (5c demonstrated). DSpace offers OAI-MPH, a standard protocol for metadata harvesting. Metadata are public in DSpace. Communities, Collections and Items are discoverable in the browse and search systems regardless of read authorisation. Therefore, everyone can access metadata of items openly (5d demonstrated).\n\nDSpace uses the CNRI Handle System primarily to create a persistent identifier for every object (item, collection and community) stored in the system (6a demonstrated). DSpace also allows other persistent identifiers, such as a digital object identifier (DOI), to be applied to data sets to improve discoverability and to allow correct citation in DSpace. This is in parallel to the Handle System (6b demonstrated).\n\nDSpace has sophisticated embargo management as well as full open access. Embargo settings allow submitters to define embargoes linked to specific dates, that by default are applied to all anonymous (non-administrator) access requests. Advanced embargo settings can be used to apply (or exclude) embargo policies for particular user groups (7a demonstrated). The DSpace system supports several common authentication systems, but web based self-attestation, in which there is no active management of access, and where instead access is open once the user has identified themselves, is not supported (7b not demonstrated). Resources can be made available only to certain \"privileged\" users. With this functionality access through a group membership can be implemented (7c demonstrated). The ‘request a copy’ functionality exists in DSpace to facilitate access in cases when uploaded content is not openly shared. With this feature, the data submitter or owner interacts directly with the requester on a case-by-case basis. More complex request evaluation processes, for example involving a data access committee, are not directly supported in DSpace, though could in theory be integrated into any dialog between the requestor and the data submitter (7d demonstrated). The DSpace administrator can assign permissions to a privileged user at the item, community and collections level, allowing granular access to different parts of datasets collections (7e demonstrated).\n\nAlthough long term preservation of data is primarily a matter of resourcing and institutional commitment, together with a clear policy about what should happen to data if a repository is closed, a repository can, at a technical level, provide additional support for long term data management. DSpace does this in several ways: different preservation levels, a distributed architecture and checksums (8a, in so far it is a technical issue, demonstrated). Bit preservation assures that a file remains the same over time – not a single bit is changed. Functional preservation means that the file does change over time so that the material continues to be immediately usable in the same way it was originally while the digital formats (and physical media) evolve over time. DSpace has implemented a strategic plan for sustainability. Because it uses open technology, has a broad dissemination and usage, with a large user community and many diverse applications, the long-term availability and maintenance of the system is expected, if not guaranteed (8b demonstrated).\n\n\nDiscussion\n\nThe performance in supporting sensitive personal data such as that from clinical trials was strong, with 14 requirements demonstrated (74%). This included strong support for different file types and metadata systems, a range of access control systems, including embargoes and granular access management, an integrated persistent identifier scheme plus support for other identifiers like DOIs, and good support for data management in the long-term.\n\nOf the two areas that were not demonstrated at all, the first – the inability to incorporate de-identification tools in the submission workflow – is arguably an over ambitious requirement. Although general techniques certainly exist for de-identification this should normally be an exercise that is planned, documented and tested on a study-by-study basis, rather than an automatic process. Having links available to de-identification resources is probably a more realistic requirement.\n\nThe second missing requirement, the lack of a self-attestation system, is a feature that some data generators might want to use, as it requires much less administrative overhead then setting up access rights for groups and individuals. It would require an administrator to define the fields required for self-attestation and, like the current user registration process, it could be backed up by a system requiring confirmation of the email address given. Given the range of other access options available in DSpace it may not be a serious omission, but it is a missing feature that would be ‘nice to have’.\n\nOf the three areas that were partially demonstrated, the need for repository managers to establish links to de-identification and other tools, rather than have them built-in to the system, may represent an additional task but it is one that should be relatively easy to do. It can also be argued that this approach is more flexible, and easier to keep up to date, than a set of links integrated into the system.\n\nThe second partially demonstrated area related to quality control. The submission workflow allows for up to three review stages, which is good, but few other elements of quality control and monitoring seemed to be built into the system. For repository managers handling sensitive data, it would be useful to have reports on upload and access or access request activity, and the ability to integrate checklists of required features or information (such as de-identification status, metadata completeness, access types allowed or identifiers applied), as might be applied during the review process, to tag on the data itself (i.e. within internal system metadata). This would allow the status of the data in the repository to be better monitored and potential issues with data quality and/or legal issues to be more quickly identified.\n\nThe third partially demonstrated issue related to data transfer agreements, governing the terms of data upload and storage. Sensitive data requires more than a simple upload to a repository because, unless the data is fully anonymised, there are likely to be legal issues that need to be clarified, for instance exactly which institution is acting as the Data Controller, as that term is defined in the General Data Protection Regulation (GDPR). (At the very least, the legal status of the data needs to be clear, i.e. does it fall under data protection legislation, and if so which, or is it exempt from such consideration because of the way it has been prepared.) In addition, there may be questions about who is responsible for versioning data if it is changed, for paying any associated costs, about the access management required, and who needs to review access requests if access is managed (etc.). These considerations go well beyond any general agreement whereby data generators simply grant the repository the right to make their data available under a selected licence – and for sensitive data they may need to be considered on a study by study basis.\n\nIt would therefore be very useful if – as a configured option – the system could enforce a clear check that such a data transfer agreement was in place, preferably with the date of its application. (At the moment that seems possible, but a rather complex workaround is required.) It would be even better if the system could also indicate where the data transfer agreement was stored and link to it, or even display its provisions within the system. Ideally, a mature system would even allow the agreement to be drafted and agreed within the system, as part of a private interchange between the data uploader and the repository.\n\nA limitation of the study is that it is focusing only on the 8 repository features defined in Banzi et al.2. Other quality features not considered here may also be very important, for example good data security. This study should therefore be seen as a starting point, which will need further extension, perhaps using alternate approaches and systems (see next section).\n\nWe focused on attributes that we thought were particularly important for clinical trial and similar data. Aspects of quality for data repositories that have been cited by other authors, but which have not been explicitly considered in our approach include:\n\nTransparency and accountability\n\nTimely management\n\nMetadata repository\n\nData versioning\n\nAuditing of repositories\n\nAdequate funding and staff\n\nDiscoverability\n\nTechnical infrastructure\n\nTransparency and accountability have been referenced by Hrynaszkiewicz et al.7 and by Burton et al.8. Allowing access to data in a timely manner and including a proportionate review of the scientific rationale, without introducing unnecessary barriers has been formulated by 7. Science Europe supports the idea of a metadata repository, enabling referencing to related relevant information, such as other data and publications and asks for support of data versioning10. Effective audits are proposed by Burton et al.8. The ICSU World Data System requires that the repository has adequate funding and sufficient members of qualified staff managed through a clear system of governance to effectively carry out the mission and that the repository enables users to discover the data and refer to them in a persistent way through proper citation9. The ICSU World Data System9 requires that the repository functions on well-supported operating systems and other core infrastructural software and is using hardware and software technological appropriate to the services it provides to its designated community. In addition, the technical infrastructure of the repository should provide for protection of the facility and its data, product, services, and users8. The need to try and integrate these different approaches to assessing data repositories is discussed in the next section.\n\nAnother weakness of the study is that the assessment of the quality criteria is (necessarily) subjective – the criteria are not quantitative. In our approach, a rather simple scale based upon “demonstrated”, “partially demonstrated” and “not demonstrated” was used. The definition of the different categories may not have been precise enough to give an accurate representation of the repository’s functioning.\n\nFinally, there may be an issue related to the sources and completeness of the information used. We only took publicly available information about DSpace into consideration (web pages, user manuals, Q&A pages, reports, etc.). We did not contact DSpace directly and were not in contact with their developers. We did, however, participate at a meeting of the German user community.\n\nDifferent approaches have been used to assess the quality of repositories dedicated to data sharing, both of sensitive data and more generally, with different emphases laid upon different features. For instance, Hrynaszkiewicz at al.7 proposed additional features for data repositories to better accommodate non-public clinical datasets, including Data Use Agreements, whilst Burton et al.8 introduced the term “Data Safe Haven”, for sensitive data, and provided 12 criteria that characterised such a haven.\n\nThe Core Trustworthy Data Repositories Requirements9 are intended to reflect the characteristics of trustworthy repositories (for all types of data). All requirements are mandatory and are equally weighted, standalone items. Although some overlap is unavoidable, duplication of the evidence sought among requirements has been kept to a minimum where possible. The choices contained in the supplied checklists (e.g., repository type and curation level) are not considered to be comprehensive, and additional space is provided in all cases for the applicant to add ‘other’ (more idiosyncratic) information. This and any comments given may then be used to refine such lists in the future. The CoreTrustSeal Board offers all interested data repositories a core-level certification based on the DSA–WDS Core Trustworthy Data Repositories Requirements catalogue and procedures9.\n\nOne initiative of Science Europe10 was to develop a set of core requirements for data management plans (DMPs), as well as a list of criteria for the selection of trustworthy repositories where researchers can store their data for sharing. The different approaches are compared in Table 3. In light of the development of the European Open Science Cloud (EOSC) and the increasing pressure for data sharing, these requirements and criteria should help to harmonise rules on data management throughout Europe. This will aid researchers in complying with research data management requirements even when working with different research funders and research organisations.\n\nIn general, it may be necessary to better distinguish between criteria that are properties of the underlying infrastructure (e.g. staff preparation, physical security, logical security, appropriate technology) and those which are more tightly coupled to a specific repository system. In fact, we would suggest that there are three (overlapping) ‘layers’ of attributes that need to be considered – those associated with the underlying organisational infrastructure, those linked to the repository’s technical systems, and those derived from procedures and workflows. Future attempts to assess the quality of repositories should perhaps consider these layers more explicitly. In this study we were focused on the ‘system’ attributes, but a broader description and assessment of a demonstrator repository should examine all three aspects, perhaps across each of the three main functional areas of a data repository, i.e. data upload, data storage and data access.\n\nNone of the approaches described above is sufficient to classify the quality of repositories for clinical trial data, as pointed out by Banzi et al.2. It may be that we need to differentiate criteria that should apply to all or most data repositories from those that only apply, or become more significant, in the context of particular types of data, like IPD. A general assessment, and especially a general ‘score’, of repositories may therefore be less meaningful than an assessment for particular types of data or data usage. Despite these difficulties we believe that it would be useful to try and achieve a consensus about what ‘quality’ means in terms of data repositories, in different contexts, both to support repository managers and to help guide and promote their use by researchers.\n\n\nConclusion\n\nWe assessed the suitability of DSpace to support a repository of sensitive data, such as that from clinical trials, using quality criteria that we had previously identified as being critical to managing such data. Technically, the system was able to support most of the features required, including the necessary support for metadata and identifiers, though there are areas – for instance explicit support of data transfer agreements – where support could be improved. Of course, in a productive repository, appropriate policies and procedures would be needed to direct the use of the available technical features. A technical evaluation should therefore be seen as indicating a system’s potential, rather than being a definite assessment of its suitability. DSpace clearly has considerable potential in this context and appears a suitable base for further exploration of the issues around storing sensitive data.\n\nThis work should stimulate the discussion about quality assessment and certification of repositories. The discussion is of particular importance for repository managers as well as standardising organisations in the field (e.g. Data Seal of approval). Another target group are researchers willing to deposit data in a repository, who have an interest that definite quality criteria are fulfilled by the repository.\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\nThe ECRIN demonstrator repository for clinical trial data: http://90.147.75.211:8080/xmlui/\n\nAdditional information on the CORBEL project is available on the CORBEL website (https://www.corbel-project.eu/home.html).",
"appendix": "Acknowledgements\n\nThe authors wish to thank Serena Battaglia (ECRIN) for technical and organisational support of the study and Stefan Klein (Biomedical Imaging Group Rotterdam, Erasmus University, Netherlands) for support of handling images with XNAT.\n\n\nReferences\n\nOhmann C, Banzi R, Canham S, et al.: Sharing and reuse of individual participant data from clinical trials: principles and recommendations. BMJ Open. 2017; 7(12): e018647. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBanzi R, Canham S, Kuchinke W, et al.: Evaluation of repositories for sharing individual-participant data from clinical studies. Trials. 2019; 20(1): 169. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAustin CC, Brown S, Fong N, et al.: Research data repositories: Review of current features, gap analysis, and recommendations for minimum requirements. IASSIST Quarterly. Winter 2015; 39(4): 24. Publisher Full Text\n\nOECD Global Science Forum: Business models for sustainable research data repositories. OECD Science Technology and Industry Policy Paper No. 47, 2017. Reference Source\n\nOhmann C, Canham S, Banzi R, et al.: Classification of processes involved in sharing individual participant data from clinical trials [version 2; peer review: 3 approved]. F1000Res. 2018; 7: 138. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBanzi R, Canham S, Ohmann C, et al.: Report about a workshop on sensitive data: Repositories for sharing individual participant data from clinical trials and existing tools/services for storing clinical trial data (Version 1). Zenodo. 2018. Assessed 15 April 2020. Publisher Full Text\n\nHrynaszkiewicz I, Khodiyar V, Andrew L, et al.: Publishing descriptions of non-public clinical datasets: proposed guidance for researchers, repositories, editors and funding organisations. Res Integr Peer Rev. 2016; 61: 6. Publisher Full Text\n\nBurton PR, Murtagh MJ, Boyd A: Data Safe Havens in Health Research and Healthcare. Bioinformatics. 2015; 31(20): 3241–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nICSU, World Data System: Core Trustworthy Data Repositories Requirements. 2016. Reference Source\n\nScience Europe: Practical guide for the international alignment of research data management. 2018. Reference Source\n\nCanham S, Ohmann C: ECRIN Clinical Research Metadata Schema Version 2 (April 2018). Zenodo. 2018. Publisher Full Text"
}
|
[
{
"id": "62923",
"date": "20 May 2020",
"name": "Agustina Martinez-Garcia",
"expertise": [
"Reviewer Expertise Digital archiving",
"digital repository platforms",
"research data management"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSummary\n\nThis is the review of the research paper “Assessment of a demonstrator repository for individual clinical trial data built upon the DSpace open source platform”. The paper describes the assessment and implementation of a repository demonstrator, for the storage and dissemination of clinical trial data, with a particular focus on Individual Participant Data (IPD). The developed demonstrator is built upon the open source and community developed DSpace repository platform (https://duraspace.org/dspace/). This repository platform is data agnostic and can be used to both serve fully open content and content that requires some form of managed access. This paper will be very useful to those looking at evaluating repository platforms for archiving and disseminating research data more generally.\n\nThe paper focuses on describing the technical criteria used for assessing the suitability of this platform for the storage and dissemination of clinical data, although a good overview of other operational aspects such as the development of guidelines, data deposition rules and quality review in the context of repository submission workflows, is also described. It also includes a summary of the technical requirements (software dependencies and deployment infrastructure) which can be useful to others evaluating the use of this repository platform for the storage and dissemination of research data.\n\nResearch methodology\n\nOverall, the paper includes sufficient details about the methods and analysis undertaken. The authors have explored recent studies in the area, i.e. the suitability assessment presented builds upon a previous study looking at a range of existing repository platforms for sharing clinical trial data, and sensitive data more broadly. The results from this study are the basis for selecting the DSpace platform. In this respect, and although the authors include references to materials where the rationale for selecting this platform is presented, it would have been useful to include a summary table outlining key criteria and some details of the other platforms evaluated. The paper only mentions other platforms (e.g. Figshare, or Zenodo) in passing.\n\nOne strength of the paper is that the authors reflect on and present the perceived weaknesses of their study. However, and given the sensitive nature of the data underpinning clinical trials, I found it quite surprising that data security features were not included as part of the key criteria defined for this initial assessment, given that not meeting these criteria could impact the suitability of this platform for the archival of clinical data. The authors acknowledge this weakness of their study and state that criteria relating to data security should be considered in future extensions of the study. As part of future assessment, the authors should consider looking at robust security testing of the platform, such as performing penetration testing.\n\nAnother weakness of the study, even though the authors acknowledge it in the paper, is that they have only evaluated openly available documentation for the DSpace platform. Such documentation can often be incomplete in community-based projects, owing to potential lack of resources. More detail about why they took this approach would have been useful. Moreover, and given that DSpace is a very popular platform within the academic community as acknowledged in the paper, the authors could have informally contacted other institutions currently using the platform to find out more about their experiences of the platform when put to similar uses, and their opinion on the platform’s strengths and weaknesses.\n\nContent review\n\nThe paper reads very well, and the content structure is appropriate. The “Introduction” section sets the scene nicely and provides sufficient background information, with relevant and current literature references. One minor observation is that, when authors introduce the work of a dedicated taskforce addressing the problem of current forms of sharing clinical data, and propose to use data repositories, there is no mention of the importance of gaining consent for data archiving, sharing and re-use from research participants. This is a key barrier to data sharing, and one that we encounter as providers of Research Data Management Services, when researchers wish to deposit their data with our Institutional Repository.\n\nThe “Methods” section is well developed: the “Technical infrastructure for the demonstrator repository” section provides useful details for those seeking to use similar platforms; and sufficient information is provided so that a similar assessment can be performed on other platforms, or for study replication (even though the analysis is partially qualitative). As mentioned earlier, it would have been useful to include a summary table outlining key criteria and some details of the other platforms evaluated for completeness.\n\nIn the “Assessment of quality criteria for the reference implementation”, the paragraph beginning with “To promote interoperability …” is a bit unclear and contradictory. It mentions the importance of using metadata standards for describing, structuring and formatting content, which I agree is very important; but they have excluded them as part of the assessment criteria. In particular, the sentence “Here we focus on standards for metadata” is very confusing as the examples given earlier all refer to metadata standards. Is the sentence intended to mean that the study is only concerned with metadata standards and does not consider data format standards?\n\nThe “Results” section reads very well and is clear. The summary table together with the different criteria-based subsections include relevant, high-level information about the technical assessment that has been performed. With respect to requirement 2a around de-identification tools, perhaps it is worth mentioning that, although not specifically implemented by the community, the DSpace platform does have a mechanism / framework in place (i.e. curation system) that allows for easy integration of such tools within DSpace’s standard submission workflows (see https://wiki.lyrasis.org/display/DSDOC6x/Curation+System).\n\nIt is mentioned in the section “Formal contract regarding upload and storage” that the implemented demonstrator does not provide support for constructing and editing the distribution licence. However, the distribution licence text can be edited or customised, as we have done so in our Institutional DSpace repository instance. Perhaps, what the authors mean instead is that the platform does not provide a user interface to do this easily.\n\nThe section about repository long-term preservation could have incorporated more detailed information about the DSpace’s platform’s capabilities around content preservation and relevant references and links to relevant literature. For example, open source integrations of the DSpace platform with preservation systems exist, e.g. integration with Archivematica (https://figshare.com/articles/Automating_OAIS_compliant_digital_preservation_using_Archivematica_and_DSpace/11274143/1). The authors seem to mix the platform’s long-term availability based on a number of aspects such as technology sustainability plans, or wide use, with the platform’s capabilities for preservation of the repository content itself. The former is not directly related to preservation but to the long-term sustainability of the platform.\n\nLastly, a number of sections in the paper talk about self-attestation functions in the context of access to repository content (requirement 7b – web-based self-attestation of the user). I am not familiar with this term, and the general reader would benefit with a clearer definition of the term and such functions. I can only guess, based on context and my knowledge of repository platforms, that the authors mean the repository’s ability for user self-registration to be able to access repository content, or functions for only giving access to content once certain information about the user has been collected and verified. E.g. the repository allows to incorporate a form asking content requesters to supply information about what uses they will make of the data, purpose of their research, contact information and /or email address to be verified, etc. If this is the case, this should be made much more explicit in the paper.\n\nMinor edits and structure comments\n\nIn the “Results” subsection of the abstract, the sentence “Two requirements could not be demonstrated (inability to incorporate de-identification tools in the submission workflow, lack of a self-attestation system) …” is not clear. It needs to be rephrased, e.g. “ability to incorporate …” and “support for self-attestation …”. Otherwise it reads as though the things in parenthesis are actually the requirements.\n\nIn the “Conclusions” subsection of the abstract, “productive repository” should read “production ready repository” or similar.\n\nIn the “Introduction” section, first sentence, “evironment” should read “environment.\n\nTable 3, third row “Control of quality of data”, C6 should read “Quality assurance” instead of “insurance”. Also, Table 3 appears much earlier (page 7) than its reference within the paper (page 11). I found this quite confusing when reading the paper as it appeared straight after Table 2, and completely out of context. It would be much clearer if the table was moved closer to its reference in the text, towards the end of the paper.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? 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": "5642",
"date": "26 Jun 2020",
"name": "Christian Ohmann",
"role": "Author Response",
"response": "The process of selection of DSpace as platfom for the demonstrator repository has been better explained. A table outlining key criteria of other platforms was not included but the information can be extracted from a reference.given. The reasons why missing security features and encryption were not considered in the paper were explained. A statement was included to motivate the use mainly public information. The importance of consent was stressed in the introduction. . The confusion over metadata was clarified. In the section « De-identification practices », a line was added in response to the reviewers comment. In « Formal contract regarding upload and storage » an explanation reflecting the comment of the reviewer was given. The section about « Long term preservation and sustainabiltiy » has been renamed and rewritten. In the section « Flexibility of access » the meaning of the term self-attestation has been clarified. ."
}
]
},
{
"id": "63998",
"date": "02 Jun 2020",
"name": "Rob Baxter",
"expertise": [
"Reviewer Expertise Development & provision of large-scale data services for both open and sensitive data (the Edinburgh International Data Facility",
"the Scottish National Safe Haven)."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 useful experiment in trialling DSpace as a candidate repository for potentially sensitive clinical trial data. The assessment criteria used focussed on the “system” level, keeping the scope manageable, and map well onto more formal existing frameworks. The conclusions that DSpace is not a bad place to start — necessary but insufficient — are sound and offer a useful guide to people faced with similar challenges in enabling the sharing of sensitive data. I have a few specific observations around methods and analysis, noted below.\nWhile geared more towards open data, the FAIR principles (https://www.force11.org/fairprinciples) are an increasingly important set of criteria for research data repos and complement some of the approaches in Table 3. Perhaps they could be added to the mapping?\nThere is no mention of encryption in the 8 assessment criteria, but encryption is hinted at in the software config (\"PostgreSQL 9.5 (with pgcrypto installed) as the relational database back end”). For a repo system handling sensitive data, I’d like to see encryption at rest and encryption in flight as two additional criteria. Perhaps this is implicit in the experiment (the pgcrypto extension offers a tantalising hint!), and if so it’s worth making it explicit. If encryption wasn’t considered as a criterion, it’s worth adding an explanation: certainly encrypting archive data is controversial — what if you lose the keys? — but an Internet-accessible database of sensitive data is a worrying thing to have exposed unencrypted.\nGeneral, automatic de-identification of data is hard, as I’m sure the authors are fully aware! While they do cover de-identification support (or rather, the lack of it) in DSpace, I wonder if they would like to comment on whether they would regard some form of basic personally-identifiable data quality checking as a “must” for repository systems dealing with sensitive data? (Looking for names, addresses, email addresses, etc. in submissions.) How easy would it be for an absent-minded researcher to upload PII into DSpace and make it publicly readable by default? Should the assessment criteria be tighter here? Perhaps this is food for future work.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? 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": "5643",
"date": "26 Jun 2020",
"name": "Christian Ohmann",
"role": "Author Response",
"response": "A reference to the FAIR and TRUST principles was included in the paper.It was explained why security features end encryption were not considered in detail in the paper. The recommendation about exploring basic personally-identifiable data quality checking as a \"must\" for repository systems will be followed up in future work."
}
]
}
] | 1
|
https://f1000research.com/articles/9-311
|
https://f1000research.com/articles/9-644/v1
|
25 Jun 20
|
{
"type": "Study Protocol",
"title": "The impact of the COVID-19 pandemic on self-harm and suicidal behaviour: protocol for a living systematic review",
"authors": [
"Ann John",
"Emily Eyles",
"Luke A. McGuinness",
"Chukwudi Okolie",
"Babatunde K. Olorisade",
"Lena Schmidt",
"Roger T. Webb",
"Ella Arensman",
"Keith Hawton",
"Nav Kapur",
"Paul Moran",
"Rory C. O'Connor",
"Siobhan O’Neill",
"David Gunnell",
"Julian P.T. Higgins",
"Emily Eyles",
"Luke A. McGuinness",
"Chukwudi Okolie",
"Babatunde K. Olorisade",
"Lena Schmidt",
"Roger T. Webb",
"Ella Arensman",
"Keith Hawton",
"Nav Kapur",
"Paul Moran",
"Rory C. O'Connor",
"Siobhan O’Neill",
"David Gunnell",
"Julian P.T. Higgins"
],
"abstract": "Background: The COVID-19 pandemic has caused widespread morbidity and mortality as well as disruption to people’s lives and livelihoods around the world; this has occurred as a result of both infection with the virus itself and the health protection measures taken to curb its spread. There are concerns that rates of suicide, suicidal behaviours and self-harm may rise during and in the aftermath of the pandemic. Given the likely rapidly expanding research evidence base on the pandemic’s impact on rates of suicide, suicidal behaviours and self-harm and emerging evidence about how best to mitigate such effects, it is important that the best available knowledge is made readily available to policymakers, public health specialists and clinicians as soon as is possible. To facilitate this, we plan to undertake a living systematic review focusing on suicide prevention in relation to COVID-19. Method: Regular automated searches will feed into a web-based screening system which will also host the data extraction form for included articles. Our eligibility criteria are wide and include aspects of incidence and prevalence of suicidal behaviour, effects of exposures and effects of interventions in relation to the COVID-19 pandemic, with minimal restrictions on the types of study design to be included. The outcomes assessed will be death by suicide; self-harm or attempted suicide (including hospital attendance and/or admission for these reasons); and suicidal thoughts/ideation. There will be no restriction on study type, except for single case reports. There will be no restriction on language of publication. The review will be updated at three-monthly intervals if a sufficient volume of new evidence justifies doing so. Conclusions: Our living review will provide a regular synthesis of the most up-to-date research evidence to guide public health and clinical policy to mitigate the impact of COVID-19 on suicide. Protocol registration: PROSPERO CRD42020183326 01/05/2020",
"keywords": [
"COVID-19",
"Living systematic review",
"Suicide",
"Attempted suicide",
"Self-harm",
"Suicidal thoughts"
],
"content": "Introduction\n\nThe COVID-19 pandemic is causing widespread disruption and loss of life globally. By mid-May 2020 over 4.4 million people had been infected and 300,000 had died [https://www.worldometers.info/coronavirus/]. There are concerns about the impact of the pandemic on population mental health (Holmes et al., 2020). These stem from the impact of the virus itself on the people infected, as well as front line workers caring for them (Kisely et al., 2020), and from the public health measures to minimise spread of the virus – in particular physical distancing, leading to social isolation, disruption of work, businesses and education. Physical distancing measures have resulted in substantial rises in unemployment, falls in GDP and concerns that many nations will enter a period of deep economic recession.\n\nThere are concerns that suicide and self-harm rates may rise during and in the aftermath of the pandemic (Gunnell et al., 2020; Reger et al., 2020). Time-series modelling indicated that the 1918–20 Spanish Flu Pandemic, which caused well over 20 million deaths worldwide, led to a modest rise in the national suicide rate in the USA (Johnson & Mueller, 2002; Wasserman, 1992). Likewise, there is evidence that suicide rates increased briefly amongst people aged over 65 in Hong Kong during the 2003 SARS epidemic, predominantly amongst those with more severe physical illness and physical dependency (Cheung et al., 2008).\n\nThe current context is, however, very different from previous epidemics and pandemics. The 2003 SARS epidemic was restricted to relatively few countries. Furthermore, in the time since the 1918–20 influenza pandemic, global and national health systems have improved, international travel and the speed of communication of information (and disinformation) have increased, antibiotics are available to treat secondary infection, and national economies have become more inter-dependent. The availability of the internet and technology for communication has made it easier for people to communicate and engage in home working and home schooling. However, there are huge inequalities in relation to access to technology and ability to stay safe and continue to work, within and between countries. Public health policies and responses, and the degree of access to technology to facilitate online clinical assessments and treatments differ greatly between countries.\n\nKey concerns in relation to suicide prevention include: uncertainty regarding how best to assess and support people with suicidal thoughts and behaviours, whilst maintaining physical distancing; people who have attempted suicide may not attend hospitals because they are worried about contracting COVID-19 or being a burden on the healthcare system at this time; the uncertain impact on mental health and risk of suicide in the general population of physical distancing; an economic recession may adversely impact on suicide rates (Chang et al., 2013; Stuckler et al., 2009). There may be an increase in prevalence of suicide risk factors such as mental illness, domestic violence, alcohol misuse and bereavement (Turecki et al., 2019); the insensitive or irresponsible media reporting of suicide deaths associated with COVID-19 may be harmful; and in some countries access to highly lethal suicide methods such as firearms and pesticides may rise (Gunnell et al., 2020).\n\nIn the context of the COVID-19 pandemic there is likely to be a rapidly expanding research evidence base on its impact on suicide rates, and how best to mitigate such effects. It is therefore important that the best available knowledge is made rapidly available to policymakers, public health specialists and clinicians. To facilitate this, we plan to conduct a living systematic review focusing on suicide prevention in relation to COVID-19. Living systematic reviews are high-quality, up-to-date online summaries of research that are regularly updated, using efficient, often semi-automated, systems of production (Elliott et al., 2014).\n\nThe overarching aim of the review is to identify and appraise any newly published evidence from around the world that assesses the impact of the COVID-19 pandemic on suicide deaths, suicidal behaviours, self-harm and suicidal thoughts, or that assesses the effectiveness of strategies to reduce the risk of suicide deaths, suicidal behaviours, self-harm and suicidal thoughts, resulting from the COVID-19 pandemic.\n\n\nMethods\n\nWe will undertake a living systematic review (Figure 1) following published guidance for such reviews and for how expediated ‘living’ recommendations should be formed where relevant (Akl et al., 2017; Elliott et al., 2017). The review was prospectively registered (PROSPERO ID CRD42020183326; registered on 1 May 2020). This protocol is reported in line with the Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols guideline (Moher et al., 2015).\n\nStudy participants may be adults or children of any ethnicities living in any country. Outcomes of interest are:\n\n1. Deaths by suicide\n\n2. Self-harm (intentional self-injury or self-poisoning regardless of motivation and intent) or attempted suicide (including hospital attendance and/or admission for these reasons)\n\n3. Suicidal thoughts/ideation\n\nStudies must address one of the following research questions:\n\n(i) What is the prevalence/incidence?\n\nPrevalence/incidence of each outcome during pandemic (including modelling studies to predict incidence / prevalence)\n\n(ii) What is the comparative prevalence/incidence?\n\nPrevalence/incidence of each outcome during pandemic vs not during pandemic\n\n(iii) What are the effects of interventions?\n\nEffects of public health measures to combat COVID-19 (including physical distancing, school closures, interventions to address loss of income, interventions to tackle domestic violence) on each outcome\n\nEffects of changed and new approaches to clinical management of (perceived) elevated risk of self-harm or suicide risk on each outcome (any type of intervention is relevant)\n\n(iv) What are the effects of other exposures?\n\n• Impact of media portrayal of each outcome and misinformation attributed to the pandemic on each outcome\n\n• Impact of bereavement from COVID-19 on each outcome\n\n• Impact of any COVID-19 related behaviour changes (domestic violence, alcohol, cyberbullying) on each outcome\n\n• Impact of COVID-19-related workload on crisis lines on each outcome\n\n• Impact of infection with COVID-19 (self or family member) on each outcome\n\n• Impact of increased availability of analgesics, firearms and pesticides on each outcome (method-specific and overall suicide rates)\n\n• Impact of COVID-19 related socio-economic exposures (recession/depression: unemployment, debt, deprivation at the person-, family- or small-area level) on each outcome\n\n• Impact on health and social care professionals: the stigma of working with COVID-19 patients or the (perceived) risk of infection/being a ‘carrier’, as well as work-related stress on each outcome\n\n• Impact of changes in/reduced intensity of treatment for patients with mental health conditions, in particular those with severe psychiatric disorders.\n\n• Impact of any other relevant exposure on our outcomes of interest.\n\nQualitative research. We will include any qualitative research addressing perceptions or experiences around each outcome in relation to the COVID-19 pandemic (e.g. stigma of infection, isolation measures, complicated bereavement, media reporting, experience of remote methods of self-harm / suicide risk assessment or provision of treatment; experience of seeking help for individuals in suicidal crisis); narratives provided for precipitating factors for each outcome.\n\nNo restrictions will be placed on the types of study design eligible for inclusion, with the exception of single case reports. Pre-prints will be assessed at the time of publication. There will be no restriction on language of publication. We will draw on a combination of internet-based translation systems and network of colleagues to translate evidence in a language other than English.\n\nThe process will be supported using automation technology and in three-month intervals reviewers will decide on extracting new data and updating the review.\n\nWe will search the following electronic databases: PubMed; Scopus; medRxiv, bioRxiv; the COVID-19 Open Research Dataset (CORD-19) by Semantic Scholar and the Allen Institute for AI, includes relevant records from Microsoft Academic, Elsevier, arXiv and PMC); and the WHO COVID-19 database. A sample search strategy (for PubMed) appears in Box 1. We have developed a workflow that automates daily searches of these databases, and the code supporting this process can be found at https://github.com/mcguinlu/COVID_suicide_living (McGuinness & Schmidt, 2020)). Searches are conducted daily via PubMed and Scopus application program interface and the bioRxiv and medRxiv RSS feeds. Conversion scripts for the daily updated WHO and the weekly updated CORD-19 corpus are used to collect information from the remaining sources. The software includes a systematic search function based on regular expressions to search results retrieved from the WHO, CORD-19 and preprint repositories (search strategy available as extended data (John et al., 2020)). We will investigate the use of other databases to capture papers made available prior to peer review and assess eligibility and review internally.\n\nA two-stage screening process will be undertaken to identify studies meeting the eligibility criteria. First, two authors (either CO or EE) will assess citations from the searches and identify potentially relevant titles and abstracts. Second, either DG, AJ or RW will assess the full texts of potentially eligible studies to identify studies to be included in the review. This process was managed via a custom-built online platform (Shiny web app, supported by a MongoDB database). The platform also allows for data extraction via a built-in form (extended data (John et al., 2020)). At the time of writing we are processing new records virtually every working day. Regularity of this task may reduce over time though it will be done at least monthly. We have the resources to display the current state of the review and screening process publicly on a regular basis.\n\n((selfharm*[TIAB] OR self-harm*[TIAB] OR selfinjur*[TIAB] OR self-injur*[TIAB] OR selfmutilat*[TIAB] OR self-mutilat*[TIAB] OR suicid*[TIAB] OR parasuicid*[TIAB) OR (suicide[TIAB] OR suicidal ideation[TIAB] OR attempted suicide[TIAB]) OR (drug overdose[TIAB] OR self?poisoning[TIAB]) OR (self-injurious behavio?r[TIAB] OR self?mutilation[TIAB] OR automutilation[TIAB] OR suicidal behavio?r[TIAB] OR self?destructive behavio?r[TIAB] OR self?immolation[TIAB])) OR (cutt*[TIAB] OR head?bang[TIAB] OR overdose[TIAB] OR self?immolat*[TIAB] OR self?inflict*[TIAB]))) AND ((coronavirus disease?19[TIAB] OR sars?cov?2[TIAB] OR mers?cov[TIAB]) OR (19?ncov[TIAB] OR 2019?ncov[TIAB] OR n?cov[TIAB]) OR (\"severe acute respiratory syndrome coronavirus 2\" [Supplementary Concept] OR \"COVID-19\" [Supplementary Concept] OR COVID-19 [tw] OR coronavirus [tw] OR nCoV[TIAB] OR HCoV[TIAB] OR ((virus*[Title] OR coronavirus[Title] OR nCoV[Title] OR infectious[Title] OR HCoV[Title] OR novel[Title])AND (Wuhan[Title] OR China[Title] OR Chinese[Title] OR 2019[Title] OR 19[Title] OR COVID*[Title] OR SARS-Cov-2[Title] OR NCP*[Title]) OR “Coronavirus”[MeSH]))))\n\nOne author (DG, AJ or RW) will extract data from each included study using a piloted data extraction form (extended data (John et al., 2020)), and the extracted data will be checked by one other author. Disagreements will be resolved in the first instance through discussion, and where this fails, by referral to a third reviewer (KH or NK). Irrespective of study design, data source and outcome measure examined, the following basic data will be extracted: citation; study aims and objectives; country/setting; characteristics of participants; methods; outcome measures (related to suicidal behaviour and COVID-19); key findings; strengths and limitations; reviewer’s notes. For articles where causal inferences are made i.e. randomized or non-randomized studies examining the effects of interventions or aetiological epidemiological studies of the effects of exposures, we will use a suitable version of the ROBINS-I or ROBINS-E tool to assess risk of bias as appropriate based on the research question and study design (Morgan et al., 2017; Sterne et al., 2016).\n\nWe will synthesise studies according to themes based on research questions, using tables and narrative. Results will be synthesised separately for studies in the general population, in health and social care staff and other at-risk occupations, and in vulnerable populations (e.g. people of older age or those with underlying conditions that predispose them to becoming severely ill or dying after contracting Covid-19). Where multiple studies address the same research questions, we will assess whether meta-analysis is appropriate and conduct it where suitable following standard guidance available in the Cochrane Handbook (Deeks et al., 2019).\n\nWe plan to maintain the review in living mode for at least 12 months, from publication of the protocol. We will undertake monthly screening and consider full updates considered at least every 3 months, if sufficient relevant evidence is published, although these are likely to happen monthly in the first instance. We will extend the living mode at 6-monthly intervals if evidence is still being published regularly. We anticipate an end to the living phase of the review at most 24 months after initiation, at which point we plan to publish the cumulated evidence in the form of a standard systematic review. Our decision to update the review will depend on the likely impact of the new evidence on the conclusions of the review. Impactful evidence may be (i) evidence that affects policy and/ or (ii) substantial, high-quality research studies (e.g. a randomised trial or population-based observational cohort study).\n\nWhen a decision is made to move to from a living review to a static publication, two reviewers will independently assess 10% of all citations screened to determine whether any records were likely to have been missed, with further independent screening in the case of concerns. In addition, all extracted data will be checked against each relevant paper by an additional reviewer.\n\nIn the context of the COVID-19 pandemic there is a high level of uncertainty relating to its impact on suicide rates and how best to mitigate such effects and there is likely to be a rapidly emerging research evidence base to address this (Gunnell et al., 2020; Holmes et al., 2020; Reger et al., 2020). It is therefore important that the best available knowledge is made rapidly available to policymakers, public health specialists and clinicians to inform their practice.\n\nThis living review, along with further updates, will be published via F1000Research. This review was registered on PROSPERO, with ID CRD42020183326. All further data are publicly available via our Harvard Dataverse repository including all results of the continuous evidence surveillance and screening. Findings from the review will be widely disseminated through conference presentations, policy briefings, peer-reviewed publications, a project website, and traditional and social media outlets. Many of the co-authors are members of the International Covid-19 suicide prevention research collaboration (IPCSPRC), we will also use this network and its partnership with the International Association for Suicide Prevention to disseminate findings.\n\nWe are currently searching and screening on a daily basis.\n\n\nData availability\n\nNo data are associated with this article.\n\nHarvard Dataverse: Underlying data for: The impact of the Covid-19 pandemic on suicidal behaviour: a living systematic review protocol. https://doi.org/10.7910/DVN/9JYHLS (John et al., 2020)\n\nThis project contains the following extended data:\n\nSearch.docx (additional information about the searches, including full search strategies)\n\ndata_extraction_form.docx (Data extraction sheet/study report)\n\nlsr_fig1.png (Figure 1 from protocol)\n\nPROSPERO_registration.pdf (Prospero registration)\n\nHarvard Dataverse: PRISMA-P checklist for ‘The impact of the COVID-19 pandemic on self-harm and suicidal behaviour: protocol for a living systematic review’ https://doi.org/10.7910/DVN/9JYHLS (John et al., 2020)\n\n\nSoftware availability\n\nThe development version of the software for automated searching is available from Github: https://github.com/mcguinlu/COVID_suicide_living.\n\nArchived source code at time of publication: http://doi.org/10.5281/zenodo.3871366 (McGuinness & Schmidt, 2020)\n\nLicense: MIT",
"appendix": "References\n\nAkl EA, Meerpohl JJ, Elliott J, et al.: Living systematic reviews: 4. Living guideline recommendations. J Clin Epidemiol. 2017; 91: 47–53. PubMed Abstract | Publisher Full Text\n\nChang SS, Stuckler D, Yip P, et al.: Impact of 2008 global economic crisis on suicide: time trend study in 54 countries. BMJ. 2013; 347: f5239. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCheung Y, Chau PH, Yip PSF: A revisit on older adults suicides and Severe Acute Respiratory Syndrome (SARS) epidemic in Hong Kong. Int J Geriatr Psychiatry. 2008; 23(12): 1231–1238. PubMed Abstract | Publisher Full Text\n\nDeeks JJ, Higgins JP, Altman DG, et al.: Chapter 10: Analysing data and undertaking meta-analyses. Cochrane handbook for systematic reviews of interventions. 2nd ed. John Wiley & Sons. 2019. Reference Source\n\nElliott JH, Synnot A, Turner T, et al.: Living systematic review: 1. Introduction—the why, what, when, and how. J Clin Epidemiol. 2017; 91: 23–30. PubMed Abstract | Publisher Full Text\n\nElliott JH, Turner T, Clavisi O, et al.: Living systematic reviews: an emerging opportunity to narrow the evidence-practice gap. PLoS Med. 2014; 11(2): e1001603. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGunnell D, Appleby L, Arensman E, et al.: Suicide risk and prevention during the COVID-19 pandemic. Lancet Psychiatry. 2020; 7(6): 468–471. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHolmes EA, O'Connor RC, Perry VH, et al.: Multidisciplinary research priorities for the COVID-19 pandemic: a call for action for mental health science. Lancet Psychiatry. 2020; 7(6): 547–560. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJohn J, Catherine Eyles E, McGuinness LA, et al.: Underlying data for: The impact of the Covid-19 pandemic on suicidal behaviour: a living systematic review protocol. Harvard Dataverse, V2. 2020. http://wwww.doi.org/10.7910/DVN/9JYHLS\n\nJohnson NPAS, Mueller J: Updating the accounts: global mortality of the 1918-1920\" Spanish\" influenza pandemic. Bull Hist Med. 2002; 76(1): 105–115. PubMed Abstract | Publisher Full Text\n\nKisely S, Warren N, McMahon L, et al.: Occurrence, prevention, and management of the psychological effects of emerging virus outbreaks on healthcare workers: rapid review and meta-analysis. BMJ. 2020; 369: m1642. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMcGuinness L, Schmidt L: mcguinlu/COVID_suicide_living: Initial Release (Version v1.0.0). Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3871366\n\nMoher D, Shamseer L, Clarke M, et al.: Preferred reporting items for systematic review and meta-analysis protocols (PRISMA-P) 2015 statement. Syst Rev. 2015; 4(1): 1. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMorgan R, Sterne JA, Higgins JP, et al.: A new instrument to assess Risk of Bias in Non-randomised Studies of Exposures (ROBINS-E): Application to studies of environmental exposure Global Evidence Summit. Cape Town. 2017. Reference Source\n\nReger MA, Stanley IH, Joiner TE: Suicide Mortality and Coronavirus Disease 2019—A Perfect Storm? JAMA Psychiatry. 2020. PubMed Abstract | Publisher 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\nStuckler D, Basu S, Suhrcke M, et al.: The public health effect of economic crises and alternative policy responses in Europe: an empirical analysis. Lancet. 2009; 374(9686): 315–323. PubMed Abstract | Publisher Full Text\n\nTurecki G, Brent DA, Gunnell D, et al.: Suicide and suicide risk. Nat Rev Dis Primers. 2019; 5(1): 74. PubMed Abstract | Publisher Full Text\n\nWasserman IM: The impact of epidemic, war prohibition and media on suicide: United States, 1910–1920. Suicide Life Threat Behav. 1992; 22(2): 240–254. PubMed Abstract"
}
|
[
{
"id": "65517",
"date": "02 Jul 2020",
"name": "Tari Turner",
"expertise": [
"Reviewer Expertise Living evidence synthesis methods."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 clear and well written protocol for a living systematic review of evidence on the impact of the COVID-19 pandemic on self-harm and suicidal behaviour. This is an important topic, a living systematic review approach is clearly useful for this topic at this time, and the methods described are also appropriate.\nI note that the review questions are complex and this will both make the review more useful, but also more challenging to do and to describe.\nIs there any plan to review the search strategy to ensure the terms used remain appropriate? Will non-English studies be included? Have searches of trials registers been conducted to determine the likely workload over coming months?\nI would encourage the authors to provide more clarity on the approach and criteria they will use to make decisions about:\nchanging the frequency of screening\n\nwhether there is “sufficient relevant evidence” to justify an update.\nIdeally enough detail should be provided on these to allow another team to make the same decisions, using the same criteria; and I don’t think this is true of the descriptions as they currently stand.\nFor example, some other living review teams have committed to updating their reviews when new evidence is likely to have a clinically meaningful impact on the size of the effect, or change the direction or degree of certainty (GRADE) of the effect for key outcomes. I recognise that the complexity of the review questions and types of evidence being included in this review may make this challenging, but additional explanation would be helpful, and avoid potential future perceptions of bias based on these decisions. (These decisions may also require that some elements of data extraction and ROB assessment are taken before a decision to update is undertaken, and this should be considered for workflows.)\nAlso there is some confusion between the Figure and the text, with the figure appearing to suggest that update decisions are made 3 monthly, while the text suggests these decisions may initially be more frequent (monthly?); and the figure suggesting that screening is conducted daily (presumably 5/7?) but there is some ambiguity about commitment to this in the text.\nI recommend review of the article by a person with clinical expertise in the area, as I do not have these skills, and I look forward to reading the review.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable",
"responses": []
},
{
"id": "65519",
"date": "29 Jul 2020",
"name": "Edward A. S. Duncan",
"expertise": [
"Reviewer Expertise Mental Health and pre-hospital emergency care research. Currently undertaking 3 COVID-19 related studies",
"including two in mental health contexts."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper presents a protocol for a living systematic review on the impact of COVID-19 pandemic on self-harm and suicidal behaviour. This is an important and timely review.\nSuicidal and self-harm behaviours have increased during previous epidemics and pandemics and there are good theoretical reasons to believe that a similar patter will occur during the COVID-19 pandemic. The review aims to rapidly provide real-time evidence of the impact of COVID-19 pandemic on suicide deaths, suicidal behaviours, self-harm, and suicidal thoughts, and to assess the effectiveness of strategies that aim to reduce these.\nThe review is a mixed-method in design and will include both quantitative and qualitative (except single case studies in either method). The protocol is well described and presented in line with PRISMA guidelines.\nPsychInfo is not included as one of the listed search engines, which appears to be an omission. The link to medRxiv is broken.\nFactors for the authors to consider are as follows. How the authors will assess bias in quantitative research is clearly described. However, it is unclear how the authors will review the quality of qualitative studies. It is also unclear how the qualitative data will be synthesised with the quantitative data and inform the review of effectiveness study. The living systematic-review design lends itself to a greater integration of both quantitative and qualitative data as they are published.\nThe transition of the review to a static state and the dissemination strategy is well described.\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/9-644
|
https://f1000research.com/articles/9-643/v1
|
24 Jun 20
|
{
"type": "Research Article",
"title": "Association of food environment and cardiovascular mortality",
"authors": [
"Khushali Jhaveri",
"Anand Krishnan",
"Devika Kir",
"Kai Chen",
"Suveen Angraal",
"Khushali Jhaveri",
"Anand Krishnan",
"Devika Kir",
"Kai Chen"
],
"abstract": "Background: The association between healthy dietary habits and cardiovascular health is well established; however, it is unknown whether access to healthy and sustainable food sources correlates with better cardiovascular mortality. Methods: County-level data on Food Environment Index (FEI) for 2014 was retrieved from the County Health Rankings dataset. County-level data on cardiovascular diseases (CVD) mortality was obtained from the death registry files of the National Vital Statistics System. We used a linear regression model to assess the association between FEI and CVD mortality after adjusting for sex and race distributions, urban-rural distribution and median household income. Results: Data were available for 3069 counties. The mean FEI was 7.0 (SD, 1.2) and mean CVD mortality (per 100,000 population) was 252.7 (95% CI, 247.1-258.3) across the United States in 2014. There was a significant inverse association between FEI and adjusted CVD mortality (P <0.001). Every unit increase in FEI corresponded to a decline in CVD mortality of 19.1 per 100,000 population. Conclusions: Higher FEI correlates with reduced CVD mortality. Improving access to healthy sustainable food sources may help decrease cardiovascular mortality.",
"keywords": [
"Cardiovascular",
"mortality",
"food"
],
"content": "Introduction\n\nIn the United States, approximately 23.5 million Americans lack access to sustainable food sources like supermarkets1 . This disparity in food environment has been shown to markedly affect the cardiovascular diseases (CVD) risk factors in a population2. However, data regarding the association of the food environment with cardiovascular diseases mortality rate remains unknown. Previous studies have investigated the association between access to food and CVD risk2,3. However, these studies were not nationally representative and lacked the focus on the association between the food environment and CVD mortality. With an extremely high burden of cardiovascular mortality in the United States4, quantifying the association between food environment and CVD mortality rates at the national level can help appraise the disparity in food environment across the United States and can give critical information on how it relates to CVD mortality rates. Such an effort can assist policymakers to generate knowledge and allocate resources to mitigate the burden of cardiovascular diseases.\n\nWe sought to assess the association of food environment and cardiovascular mortality rate at the county level across the United States.\n\n\nMethods\n\nCounty-level data on the Food Environment Index (FEI) for the year 2014 was retrieved from the County Health Rankings and Roadmaps dataset5 . FEI is a standardized measure that calculates the food environment by accounting for limited access to a grocery store or supermarket, consistency of availability of food, and the burden of cost. FEI equally weighs 'limited access to food' and 'food insecurity' as the two indicators for the food environment. 'Limited access to food' is defined as the percentage of the population in a county that is low income and does not live close to a grocery store. Living in close proximity to a grocery store is defined as living less than 10 miles from a grocery store in rural areas and less than a mile in non-rural areas. This measure also includes low-income status, which is defined as having an annual family income of ≤200% of the federal poverty threshold for the family size6. The second indicator, 'food security,' represents a lack of constant access to the food supply and is defined as the percentage of the population who did not have access to a reliable source of food during the past year. We created a two-stage fixed-effects model to develop this measure using information from the Community Population Survey, Bureau of Labor Statistics, and the American Community Survey5. FEI ranges from 0 to 10, with 0 representing the worst and 10 the best possible food environment rating, respectively.\n\nData on county-level age-standardized CVD mortality rates were retrieved from National Vital Statistics System, the details of which have been previously published7. Counties were identified using their Federal Information Processing Standard (FIPS) codes, which are numerical values unique to every county. These unique identifiers were used to extract the county-level mortality data, matching for the corresponding FEI from the County Health Rankings dataset.\n\nA linear regression model was employed to determine the association of FEI with county-level age-standardized CVD mortality rates after adjusting for the following variables - sex, race, urban-rural distribution, educational level (defined as the percentage of the population who graduated some form of college), percentage of uninsured population under the age of 65 years, and percentage of adults smokers in the county. These data elements were obtained from 2010 US Census Bureau’s Census Population Estimates data and Behavioral Risk Factor Surveillance System data of 20148,9.\n\nFurther, we stratified the counties according to their FEI values and compared 100 counties with the highest FEI with 100 counties with the lowest FEI. Socio-economic demographic characteristics and surrogate markers for healthy dietary practices (prevalence of obesity and diabetes) were then compared amongst the two groups. These data elements were available in the County Health Rankings and Roadmaps dataset. Obesity was defined as the percentage of the population with a BMI ≥30kg/m2, and diabetes was assessed using the response to the question, \"Has a doctor ever told you that you have diabetes?\" Women with reported gestational diabetes mellitus were not included because the gestational diabetes mellitus is a distinct transient illness with different characteristics and entails different disease trajectories than diabetes. Analysis was conducted using R programming language version 3.6.010.\n\n\nResults\n\nData were available for 3069 counties. Mean FEI across the United States was 7.0 (SD, 1.2). Nationally, the mean CVD mortality rate (per 100,000 population) was 252.7 (95% CI, 247.1-258.3) in 2014. Per our analysis, FEI was inversely associated with CVD mortality rate, with a 19.1% decrease in CVD mortality rate for every unit increase in FEI (Figure 1). At the county level, FEI had a significant inverse association with the adjusted CVD mortality rate; adjusted for sex distribution, race distribution, urban-rural distribution, percentage of uninsured population under the age of 65 years, education attainment, and percentage of adult smokers in this analysis (P <0.001 and β = -3.6).\n\nCardiovascular mortality in deaths per 100,000 population.\n\nThe CVD mortality was 310.9 per 100,000 population (95% CI, 295.8-326.0) in counties with lowest FEI compared to 225.6 per 100,000 population (95% CI 218.7-232.6) in counties with the highest FEI. This difference accounted for a 27.4% relative risk reduction in CVD mortality rates between the two county groups. There was no significant difference seen in the mean age and sex distribution between the two groups. However, the counties with the highest FEI had a larger White population than counties with lower FEI (Table 1). Furthermore, the prevalence of obesity and diabetes was significantly higher in counties with lowest FEI when compared to the counties with the highest FEI; 34.4% vs. 28.1% for obesity, and 14.6%, and 8.7% for diabetes (P <0.001).\n\nCVD, cardiovascular diseases; FEI, food environment index; SD, standard deviation; CI, confidence interval; USD, United States Dollars.\n\n\nDiscussion\n\nOur study shows a significant association between FEI and CVD mortality rate. Higher FEI is associated with lower cardiovascular mortality rate. Furthermore, counties with the lowest FEI had a greater prevalence of obesity and diabetes compared to counties with the highest FEI. Our results provide an assessment of how disparities in access to nutritious food and food insecurity may affect population health outcomes. To our knowledge, this is the first study of its kind that evaluates the association between food environment and CVD mortality rate at the national level.\n\nThis study supplements existing literature in many ways. Prior studies have shown an association of healthy dietary habits with better cardiovascular health11–13. Further, Kelli et al. showed that people living in areas with low access to nutritious food are more likely to have an unfavorable cardiovascular risk profile than people living in areas with better access2. This argument has been explored by other studies as well, which found that access to poor quality food sources, such as convenience stores, is associated with lower quality diets3. However, the impact of the food environment on the cardiovascular mortality rates remained unexplored. In our analysis, we found an inverse association between access to food sources and cardiovascular mortality rates. These results underscore the potential benefit of targeting and improving FEI to improve the CVD mortality rates.\n\nThe food environment has been shown to be associated with cardiovascular health14,15. We found that counties with the lowest FEI had a greater prevalence of obesity and diabetes in comparison to counties with the highest FEI. This may, in part, be due to increased dependence on processed fast foods on which the populations with the lowest FEI rely on in the absence of a supermarket or other nutritious food sources16. Hence, decreasing barriers to access nutritious and reliable food sources may be an important step towards improving cardiovascular mortality. Future studies are needed to explore this association in a prospective design with longitudinal follow-up. Nevertheless, identifying areas with low food accessibility and diverting resources to these 'high risk' hotspots may aid in decreasing this disparity in the food environment and may help improve overall cardiovascular health. Furthermore, efforts should be directed in subsidizing nutritious food options as an incentive for our population to adopt better dietary practices and combat CVD.\n\nOur study has the following limitations. Firstly, due to the analysis design, we cannot ascertain a direct causal effect between FEI and CVD mortality. However, we accounted for sociodemographic factors across a large sample size and observed a significant correlation between FEI and CVD mortality rate. Secondly, our study is a socio-demographically adjusted population-based study. Hence, patient-level direct risk factors were not included in our analysis. However, sociodemographic factors play a critical role in assessing cardiovascular health at the population level17. Thirdly, while the food insecurity takes both proximities to nutritious foods and income into account, it does not account for access to or use of food assistance benefits among eligible families. Finally, we could not account for the health status data as an adjustment covariate in our model. Given the county-level health status data is not available, this represents a future prospect for studies investigating the food environment and CVD mortality.\n\nIn conclusion, higher FEI is associated with lower cardiovascular mortality rates. These findings support broad-based interventions to reduce existing disproportion in access to nutritious and reliable food sources in order to minimize the burden of cardiovascular mortality in the United States.\n\n\nData availability\n\nData can be accessed through the 2014 County Health Rankings and Roadmaps dataset (https://www.countyhealthrankings.org/), National Vital Statistics System (https://www.cdc.gov/nchs/nvss/index.htm), the 2010 US Census Bureau (https://www.census.gov) and 2014 Behavioral Risk Factor Surveillance System (https://www.cdc.gov/brfss/index.html).\n\nSource code is available from: https://github.com/suveenangraal/CountyHealth\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.387194710\n\nLicense: Creative Commons Attribution 4.0 International",
"appendix": "References\n\nThe Food Trust: The Grocery Gap: Who Has Access to Healthy Food and Why It Matters. Accessed 1 May, 2019. Reference Source\n\nKelli HM, Hammadah M, Ahmed H, et al.: Association Between Living in Food Deserts and Cardiovascular Risk. Circ Cardiovasc Qual Outcomes. 2017; 10(9): e003532. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRummo PE, Meyer KA, Boone-Heinonen J, et al.: Neighborhood availability of convenience stores and diet quality: findings from 20 years of follow-up in the coronary artery risk development in young adults study. Am J Public Health. 2015; 105(5): e65–73. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSidney S, Quesenberry CP Jr, Jaffe MG, et al.: Recent trends in cardiovascular mortality in the United States and public health goals. JAMA Cardiol. 2016; 1(5): 594–599. PubMed Abstract | Publisher Full Text\n\nCounty Health Rankings & Roadmaps Program: Food environment index. Accessed 1 May, 2019. Reference Source\n\nInstitute for Research on Poverty: What Are Poverty Thresholds And Poverty Guidelines? Accessed 1 May, 2019. Reference Source\n\nDwyer-Lindgren L, Bertozzi-Villa A, Stubbs RW, et al.: Us county-level trends in mortality rates for major causes of death, 1980-2014. JAMA. 2016; 316(22): 2385–2401. PubMed Abstract | Publisher Full Text | Free Full Text\n\nThe United States Census Bureau. Accessed 9 May, 2020. Reference Source\n\nCenters for Disease Control and Prevention: The Behavioral Risk Factor Surveillance System. Accessed 1 May, 2019. Reference Source\n\nAngraal S: CountyHealth. Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3871947\n\nMillen BE, Quatromoni PA: Nutritional research within the Framingham Heart Study. J Nutr Health Aging. 2001; 5(3): 139–43. PubMed Abstract\n\nRos E, Martínez-González MA, Estruch R, et al.: Mediterranean diet and cardiovascular health: Teachings of the PREDIMED study. Adv Nutr. 2014; 5(3): 330S–336S. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWang X, Ouyang Y, Liu J, et al.: Fruit and vegetable consumption and mortality from all causes, cardiovascular disease, and cancer: systematic review and dose-response meta-analysis of prospective cohort studies. BMJ. 2014; 349: g4490. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAdams EJ, Grummer-Strawn L, Chavez G: Food insecurity is associated with increased risk of obesity in California women. J Nutr. 2003; 133(4): 1070–1074. PubMed Abstract | Publisher Full Text\n\nLamichhane AP, Puett R, Porter DE, et al.: Associations of built food environment with body mass index and waist circumference among youth with diabetes. Int J Behav Nutr Phys Act. 2012; 9(1): 81. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAthens JK, Duncan DT, Elbel B: Proximity to Fast-Food Outlets and Supermarkets as Predictors of Fast-Food Dining Frequency. J Acad Nutr Diet. 2016; 116(8): 1266–1275. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWinkleby MA, Kraemer HC, Ahn DK, et al.: Ethnic and socioeconomic differences in cardiovascular disease risk factors: Findings for women from the third national health and nutrition examination survey, 1988-1994. JAMA. 1998; 280(4): 356–362. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "65650",
"date": "15 Jul 2020",
"name": "Javier Valero-Elizondo",
"expertise": [
"Reviewer Expertise Outcomes research",
"publich health",
"cardiovascular disease prevention and 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\nJhaveri & Krishnan et al. present a short but impactful manuscript, where they studied the relationship between food environment and cardiovascular mortality, using various national-level sources. The current manuscript is clearly written, with an appropriate analysis plan and with the conclusions being adequately supported by the presented results. Jhaveri & Krishnan et al. arrived at the conclusion that a higher (i.e. more favorable) food environment index was associated with lower cardiovascular mortality. Both ‘limited access to nutritious food sources’ and ‘food insecurity’ are becoming important topics both on public health policy and cardiovascular disease epidemiology research circles. This is an important contribution to the scientific literature, delivering a timely message that can potentially have a direct effect on individuals from lower income levels, who are already at a higher risk of cardiovascular disease. I do, however, have a few comments:\n\nMajor comments:\n\nIntroduction:\nLine 11: It appears that the authors are trying to convey the importance of their aim: study the relation of food environment and CVD mortality, which can certainly inform different stakeholders, from healthcare all the way to public health policy. The issue that I see is that this sentence is redundant, stating the intended goal, and repeating itself as it ends. I would recommend rephrasing this sentence, especially in light of the Introduction being as succinct.\nMethods:\n\nI am not quite sure I understand what ‘measure’ was created by the two-stage fixed-effects model. Could the authors elaborate on this section? I understand this is the method used by the County Health Rankings – did the authors create their own?\n\nCould the authors explain what methodology or criteria were used to identify/select the variables included in the linear regression model for FEI and CVD mortality rates? In addition, I understand that lack of insurance is especially important in the population younger than 65 years of age due to lack of Medicare – but does this mean that insurance status was collected for all ages, and “uninsured” (among those < 65 years of age) was the variable included in the model? Or was the study population only individuals < 65 years of age? This is not clear.\n\nSeeing as they are related to CVD mortality, were other CV risk factors available to inform the current analysis (hypertension, for example)? It would be interesting to see, especially for hypertension, since close to 50% of US adults have hypertension, and many treat it with lifestyle modifications, with diet and exercise at their center – which would be germane to the topic at hand and would add another level of relevance, without necessarily adding complexity to the analysis.\n\nRelated to the comment above: I notice that “leisure physical activity” is included in Table 1 of the results, but is not mentioned in the Methods or Results sections. Was this intended?\nDiscussion:\n\nIn the Limitations section specifically, the second limitation brings some confusion. On the one hand, I understand that the study was “socio-demographically adjusted, population-based”, but then the authors mention that patient-level direct risk factors were not included in the analysis. Could the authors expand on this area? I am not entirely clear which risk factors belong in this section, seeing that some were used (e.g. smoking, diabetes and obesity), and some where not (e.g. physical activity, or potentially, hypertension).\n\nMinor comments:\n\nAbstract:\nBackground: I wouldn’t utilize the phrasing “better cardiovascular mortality”. Perhaps “lower cardiovascular mortality” or “improved mortality outcomes”?\nKeywords:\n\nIt is standard practice that keywords be listed alphabetically.\nIntroduction:\n\n“United States” appears 3 times in a 1-paragraph Introduction. I would suggest using the “US” acronym after defined the first time, to avoid repetitiveness in such a small space (even beyond the Introduction, at the authors’ discretion).\n\nI notice that “cardiovascular mortality” and “CVD mortality” are used interchangeably at different times. For the sake of consistency, I would recommend choosing one and using throughout, which would reflect greatly on the authors in terms of their order, methodology, and attention to detail (once more, even beyond the Introduction, at the authors’ discretion).\nMethods:\n\nThe paper would benefit greatly from a short sentence depicting the study population, and exclusion/inclusion criteria.\n\nItems like food insecurity are often asked at a family level, and in the current manuscript, it seems like results are reported per capita. Is there any information that the authors could provide as to the actual measurement of the FEI individual components, and how they were estimated? If allowed by word count.\n\nTypo: a space is needed between the words “was” and “conducted” in the last line of the Methods section.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "86136",
"date": "28 Jun 2021",
"name": "Maria Gabriela M. Pinho",
"expertise": [
"Reviewer Expertise Epidemiology",
"Nutrition",
"Food environment"
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIntroduction: The introduction is too superficial and it seems to be lacking relevant references. In addition, some of cited literature is not accurate. For example, in the sentence “Previous studies have investigated the association between access to food and CVD risk”, the authors cite the work of Rummo et al. 2015. However, this work analysed the relationship between the food environment and diet quality, not CVD risk.\nThe rationale for the study is also not clearly elaborated. How exactly would the understanding on the association between food environment and CVD mortality help in the assessment of the disparity in food environment across the United States?\nMethods: The authors mention that the FEI was collected from the County Health Rankings and Roadmaps dataset. However, more details about this data source is needed. For example, what data was used to calculate ‘limited access to food’ and ‘food insecurity’?\nPlease describe this “two-stage fixed effects model” in more detail. And did you test if multilevel modelling was needed? - e.g. was there clustering at county level?\nIt is not clear how CVD mortality rate was measured. From figure 1, CVD mortality seems to range from 0 to 550, what does it mean?\nStratified analyses and comparison of FEI groups according to obesity and diabetes is a surprise here. There should be a rationale for this in the introduction, and this should be mentioned as additional aims of the study.\nFigure 1: This relationship does not seem to be linear. I wonder if the authors tested the assumptions for a linear regression before performing this type of model? This is not mentioned in the methods section. If assumptions are not met, other modelling methods should be performed.\nAlso, it is not clear from this figure that “there was a 19.1% decrease in CVD mortality rate for every unit increase in FEI (Figure 1)”.\nTable 1: Physical activity appears in this table, but no mention is made in the methods section. Was it account for in your models?\nConclusion: Conclusions seem to be supported by the results. However, I have concerns about the methodology and if statistical analyses are appropriated for this type of data.\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?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/9-643
|
https://f1000research.com/articles/9-642/v1
|
24 Jun 20
|
{
"type": "Research Article",
"title": "Effect of sodium hypochlorite solution and gel with/without passive ultrasonic irrigation on Enterococcus faecalis, Escherichia coli and their endotoxins",
"authors": [
"Amjad Abu Hasna",
"Laiana Pereira Da Silva",
"Fernanda Carvalho Pelegrini",
"Cláudia Luísa Ribeiro Ferreira",
"Luciane Dias de Oliveira",
"Cláudio Antonio Talge Carvalho",
"Laiana Pereira Da Silva",
"Fernanda Carvalho Pelegrini",
"Cláudia Luísa Ribeiro Ferreira",
"Luciane Dias de Oliveira",
"Cláudio Antonio Talge Carvalho"
],
"abstract": "Background: Sodium hypochlorite (NaOCl) is the most commonly used irrigant in endodontics. The purpose of this study was to evaluate the effect of NaOCl solution (2.5%) and gel (3%) with/without passive ultrasonic irrigation (PUI) on Enterococcus faecalis, Escherichia coli, and their endotoxins, lipopolysaccharide (LPS) and lipoteichoic acid (LTA). Methods: 40 human lower premolars were contaminated with E. coli (ATCC 25922) for 28 days and E. faecalis (ATCC 29212) for 21 days. Specimens were randomly divided into four groups: (1) 2.5% NaOCl irrigating the canals without PUI activation; (2) 2.5% NaOCl with PUI; (3) 3% NaOCl gel irrigating the canals without PUI; and (4) 3% NaOCl gel with PUI. 40 mL of irrigant was used for each group. PUI activation was carried out using E1-Irrisonic stainless-steel tip at 10% frequency. After treatment, all specimens were filled with 3mL of 17% ethylenediaminetetraacetic acid (EDTA) for 3min and then washed with nonpyrogenic saline solution. Three samples were collected from the canals: S1, at baseline to confirm biofilm formation; S2 after treatment; and S3 after EDTA. Samples were assessed for E. coli and E. faecalis colony forming units, and LPS and LTA were assessed using chromogenic kinetic LAL assay and ELISA, respectively. Data were analyzed by Kruskal-Wallis, Friedmann and Dunn tests with α≤0.05. Results: All groups were effective in reducing the microbial load of E. coli and E. faecalis after treatment without a significant difference among the groups. NaOCl and NaOCl gel groups had no significant difference in reducing LPS and LTA. Statistically increased reduction was seen for NaOCL + PUI and NaOCl gel + PUI compared for groups without PUI. Conclusions: NaOCl gel has the same antimicrobial action of NaOCl solution and can partially detoxify endotoxins. PUI improves NaOCl (gel or solution) action over E. faecalis and E. coli and their endotoxins.",
"keywords": [
"Sodium hypochlorite",
"Passive ultrasonic irrigation",
"Enterococcus faecalis",
"Escherichia coli",
"Endotoxins."
],
"content": "Introduction\n\nSodium hypochlorite (NaOCl) is the most commonly used irrigant in endodontics (Iqbal, 2012). It has been used since the second half of the 18th century (Sedgley, 2004) because it has antimicrobial action (Luebke, 1967) and dissolves necrotic tissues (Taylor & Austin, 1918).\n\nEnterococcus faecalis is a Gram-positive bacterium found in the root canal system (RCS) and can be disinfected by NaOCl (Siqueira et al., 2000). It may also be found in secondary infections of endodontically treated teeth (Machado et al., 2020). In addition, Escherichia coli, a Gram-negative bacterium, is also found in endodontic infections (Narayanan & Vaishnavi, 2010).\n\nBacteria have endotoxins in their outer membrane known as lipoteichoic acid (LTA) in Gram-positive bacteria (Ginsburg, 2002) and lipopolysaccharide (LPS) in Gram-negative bacteria (Mergenhagen & Varah, 1963). Endotoxins can be released during the duplication or death of these bacteria in infected RCS and this has a role in developing periapical lesions (Endo et al., 2012). Endodontic treatment using NaOCl can detoxify endotoxins, but not completely (Cavalli et al., 2017).\n\nNaOCl is a cytotoxic substance (Salazar-Mercado et al., 2019); overflow during endodontic treatment can cause diverse exacerbations (Goswami et al., 2014). Thus, NaOCl gel may be safer due to its minor apical extrusion tendency (Nesser & Bshara, 2019). Passive ultrasonic irrigation (PUI) improves RCS disinfection (Plotino et al., 2007), removing the smear layer and vapor lock during endodontic treatment (Bueno et al., 2019; Dioguardi et al., 2019), and permits greater penetration of irrigants to the dentinal tubules (Sáinz-Pardo et al., 2014).\n\nThe purpose of this study was to evaluate the effect of NaOCl solution (2.5%) and gel (3%) with/without PUI on E. faecalis, E. coli, and their endotoxins, LTA and LPS, respectively.\n\n\nMethods\n\nThis study was approved by the research ethics committee of São Paulo State University, Institute of Science and Technology (no1.504.995). The teeth used in this study were obtained from clinics where teeth are donated during routine procedures and following authorization of the patients. The research team presented the terms of donation by the clinics from which the teeth where obtained to the research ethics committee when submitting the study methodology. A total of 40 human lower premolars were collected (based on dimensional and morphological similarities).\n\nTo standardize root canal diameter, the teeth were initially instrumented with a #30 K-file (Maillefer, Ballaigues, Switzerland) and irrigated with 5 mL of NaOCl 1% for each file used. The canals were dried with sterile paper points (Dentsply Ind Com LTDA, RJ, Petrópolis, Brazil) and the apical region was sealed with light-cured resin composites (3M Dental Products, St Paul, MN). The outer surfaces of the specimens were covered with two layers of epoxy adhesive (Araldite - Brascola, Sao Paulo, Brazil), except the cervical opening (de Oliveira et al., 2007). Then they were fixed with chemically activated acrylic resin in 24-well plates and sterilized by gamma radiation with cobalt 60 (20 KGy for 6 hours) (Csako et al., 1983).\n\nSpecimens were contaminated with E. coli (ATCC 25922) for 28 days and E. faecalis (ATCC 29212) for 21 days and incubated at 37°±1°C, following the protocol of Maekawa et al. (2013).\n\nSpecimens were divided into four experimental groups (n=10/group) as follows: (1) 2.5% NaOCl (Asfer, São Caetano do Sul, São Paulo, Brazil) irrigating the canals without PUI activation; (2) 2.5% NaOCl irrigating the canals with PUI; (3) 3% NaOCl gel (Ultradent, South Jordan, UT, USA) irrigating the canals without PUI; and (4) 3% NaOCl gel irrigating the canals with PUI. All specimens were instrumented as a part of the biomechanical preparation by Reciproc R40 (VDW, Munich, Germany) following the protocol of each experimental group (Table 1).\n\nNaOCL, sodium hypochlorite; PUI, passive ultrasonic irrigation.\n\nPUI activation was performed using an E1-Irrisonic stainless-steel tip (Helse, Santa Rosa de Viterbo, Brazil) (0.10mm in diameter) at the working length using CVDente 100 ultrasound activator (CVDentus, São José dos Campos, Brazil) at 10% frequency.\n\nAfter treatment, all specimens were filled with 17% ethylenediaminetetraacetic acid (EDTA) (Biodinâmica, Ibiporã, PR, Brazil) for 3min and then washed with nonpyrogenic saline solution.\n\nThree samples were collected during the experiment, as in (Maekawa et al., 2013): S1, at baseline to confirm biofilm formation; S2, immediately after treatment; and S3, after EDTA application.\n\nSerial dilutions of all samples were performed with sterile saline solution and aliquots of 30µl of each sample were seeded in two different culture medias: Enterococcosel agar (Becton, Dickinson and Company Sparks, MD, USA) for E. faecalis; and MacConkey agar (Himedia Laboratories, Mumbai, India) for E. coli. The plates were kept at 37°C for 24h and then CFU/mL were counted.\n\nLPS levels in each sample was assessed as in Machado et al. (2020) using kinetic chromogenic limulus amebocyte lysate assay (Lonza, Walkersville, MD, USA). The plates were incubated at 37±1°C for 10 min in a KineticQCL reader, which was coupled to a computer with the WinKQCL software (Lonza). As soon as the kinetic test started, absorbance at 405 nm was read in each microplate well and automatically calculated the log/log linear correlation between reaction time of each standard solution and corresponding endotoxin concentration.\n\nLTA was assessed using enzyme-linked immunosorbent assay using ELISA 96-well plates (Nunc Thermo Scientific, Waltham, MA, USA) sensitized with anti-LTA monoclonal antibody (manufacturer) and kept overnight at relative humidity. Next day, the plates were washed with a wash buffer (PBS with 0.05% Tween 20) and incubated with a blocking buffer (PBS with 2% bovine serum albumin, BSA) for 1 h at room temperature. Then, they were washed with a wash buffer and received 100 μL of the samples collected and 100 μL of the LTA standard followed by serial 2-fold dilutions (standard curve) and maintained for 2 hours at room temperature. Afterwards, the plates were washed again and 100 µL of anti-LTA antibody was added for 1 hour at room temperature. The plates were washed again and 100 μl of horseradish peroxidase HRP conjugated rabbit IgG antibody was added for 1 hour at room temperature. Lastly, the plates were washed, and the reaction was developed using tetramethylbenzidine (TMB). After 20 min under the light, 50 μL of stop solution (2 N sulfuric acid) was added to each well of the plate and optical densities were read in the microtiter plate reader (BioTek Instruments, Inc., Winooski, VT, USA) at 450 nm absorbance (Machado et al., 2020).\n\nData were analyzed using Kruskal-Wallis, Friedmann and Dunn tests with α≤0.05 byGraphPad Prism 6 (La Jolla, CA, USA)\n\n\nResults\n\nAll experimental groups were effective in reducing the microbial load (CFU/mL) of E. coli (Figure 1) and E. faecalis (Figure 2) in S2 (from S1 levels). There was no significant difference among the experimental groups for S2 or S3 (Table 2).\n\nCFU, colony forming units; NaOCL, sodium hypochlorite; PUI, passive ultrasonic irrigation.\n\nCFU, colony forming units; NaOCL, sodium hypochlorite; PUI, passive ultrasonic irrigation.\n\nNaOCl and NaOCl gel groups had no significant difference in reducing LPS and LTA at S2. Groups with PUI showed a statistically increased reduction in LPS and LTA compared with groups without PUI (Figure 3). As a limitation of this study, the LPS and LTA levels were not assessed in S3.\n\nNaOCL, sodium hypochlorite; PUI, passive ultrasonic irrigation.\n\n*Different letters indicate statistically significant differences (p<0.05). Uppercase letters indicate difference in rows (Friedman test; intra-groups) and lowercase letters indicate difference in columns (Kruskal-Wallis test; inter-groups).\n\n\nDiscussion\n\nWell endodontically treated teeth fail mainly because of secondary intraradicular infection (Siqueira, 2001). E. faecalis and E. coli, among other bacteria, are the most detected microorganisms in periapical lesions (Geibel et al., 2005).\n\nThe use of NaOCl as an irrigant cannot be over-emphasized. In this study, it was effective in disinfecting E. faecalis and E. coli (Table 2). These results agree with the results of Siqueira et al. (2000), who showed that NaOCl was an effective irrigant over E. faecalis at different concentrations (1%, 2.5%, and 5.25%). In addition, Valera et al. (2009) showed that 1% NaOCl is effective in disinfecting E. faecalis inoculated in the RCS. There have also been more recent studies that have the same results (Carvalho et al., 2020; Plutzer et al., 2018; Valera et al., 2014). Similarly for E. coli, NaOCl has been reported in the literature as an effective irrigant in disinfecting microorganisms in the RCS lumen at 2.5% concentration (Maekawa et al., 2011) and in preventing E. coli regrowth (Valera et al., 2015).\n\nIn the present study, NaOCl was not effective in detoxifying LPS and LTA completely (Figure 3). In support of this, the literature has shown that NaOCl is not effective in reducing endotoxin levels in the RCS (de Oliveira et al., 2007), i.e. it reduces the endotoxin level but does not completely eliminate them (Neelakantan et al., 2019). However, adding chloride alkali electrolyte-stable anionic surfactant has been shown to improve NaOCl effectivity because it reduces superficial tension (Valera et al., 2015).\n\nNaOCL, sodium hypochlorite; PUI, passive ultrasonic irrigation; LPS, lipopolysaccharide; LTA, lipoteichoic acid.\n\nNaOCl gel has been suggested as an alternative endodontic irrigant because theoretically it has the same antimicrobial action of NaOCl solution, but with less apical extrusion and could thus be safer (Nesser & Bshara, 2019). It is effective in reducing the microbial load, but has been shown to be less effective when compared to NaOCl solution of a lower concentration (Poggio et al., 2010; Zand et al., 2016). In the present study, NaOCl gel was shown to be just as effective as NaOCl solution in reducing microbial load.\n\nTo the best of our knowledge, there are no studies in the literature evaluating the effect of NaOCl gel over endotoxins. In this study it was statistically as effective as the solution. But both were more effective when combined with PUI as it increases NaOCl penetration into dentinal tubules (Faria et al., 2019). The present study is novel as there are no studies evaluating how PUI can affect NaOCl solution or gel action on endotoxins.\n\nPUI is still being studied due to divergence of results in the literature. For example, Paiva et al. (2013) used PUI after instrumentation and showed it was ineffective in reducing microbial load; however, Mohmmed et al. (2018) showed that PUI is effective in biofilm removal from lateral canals. However, this activity may be influenced by the irrigation protocol (irrigation time; irrigant volume; instrument shape and material; and the irrigation frequency and intensity) (van der Sluis et al., 2007).\n\nIn conclusion, our study showed that NaOCl gel has the same antimicrobial action of NaOCl solution and can partially detoxify endotoxins. PUI improves NaOCl (gel or solution) action over E. faecalis and E. coli formation and their endotoxins (LPS and LTA).\n\n\nData availability\n\nHarvard Dataverse: Raw Data of NaOCl solution and gel, https://doi.org/10.7910/DVN/JNK3TH (Abu Hasna, 2020).\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).",
"appendix": "References\n\nAbu Hasna A: Raw Data of NaOCl solution and gel. Harvard Dataverse, V1, 2020. Publisher Full Text\n\nBueno CRE, Cury MTS, Vasques AMV, et al.: Cleaning effectiveness of a nickel-titanium ultrasonic tip in ultrasonically activated irrigation: a SEM study. Braz Oral Res. 2019; 33: e017. PubMed Abstract | Publisher Full Text\n\nCavalli D, Toia CC, Flores Orozco EI, et al.: Effectiveness in the removal of endotoxins and microbiological profile in primary endodontic infections using 3 different instrumentation systems: A randomized clinical study. J Endod. 2017; 43(8): 1237–1245. PubMed Abstract | Publisher Full Text\n\nCarvalho CAT, Hasna AA, Carvalho AS, et al.: Clinical Study of Sodium Hypochlorite, Polymyxin B And Limewater Effect on MMP-3,-8,-9 In Apical Periodontitis. Braz Dent J. 2020; 31(2): 116–121. Publisher Full Text\n\nCsako G, Elin RJ, Hochstein HD, et al.: Physical and biological properties of U.S. standard endotoxin EC after exposure to ionizing radiation. Infect Immun. 1983; 41(1): 190–196. PubMed Abstract | Free Full Text\n\nde Oliveira LD, Jorge AOC, Carvalho CAT, et al.: In vitro effects of endodontic irrigants on endotoxins in root canals. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2007; 104(1): 135–142. PubMed Abstract | Publisher Full Text\n\nDioguardi M, Di Gioia G, Illuzzi G, et al.: Passive Ultrasonic Irrigation Efficacy in the Vapor Lock Removal: Systematic Review and Meta-Analysis. ScientificWorldJournal. 2019; 2019: 6765349. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEndo MS, Martinho FC, Zaia AA, et al.: Quantification of cultivable bacteria and endotoxin in post-treatment apical periodontitis before and after chemo-mechanical preparation. Eur J Clin MicrobioI Infect Dis. 2012; 31(10): 2575–2583. PubMed Abstract | Publisher Full Text\n\nFaria G, Viola KS, Coaguila-Llerena H, et al.: Penetration of sodium hypochlorite into root canal dentine: effect of surfactants, gel form and passive ultrasonic irrigation. Int Endod J. 2019; 52(3): 385–392. PubMed Abstract | Publisher Full Text\n\nGeibel MA, Schu B, Callaway AS, et al.: Polymerase chain reaction-based simultaneous detection of selected bacterial species associated with closed periapical lesions. Eur J Med Res. 2005; 10(8): 333–338. PubMed Abstract\n\nGinsburg I: Role of lipoteichoic acid in infection and inflammation. Lancet Infect Dis. 2002; 2(3): 171–179. PubMed Abstract | Publisher Full Text\n\nGoswami M, Chhabra N, Kumar G, et al.: Sodium hypochlorite dental accidents. Paediatr Int Child Health. 2014; 34(1): 66–69. PubMed Abstract | Publisher Full Text\n\nIqbal A: Antimicrobial irrigants in the endodontic therapy. Int J Health Sci(Qassim). 2012; 6(2): 186–192. PubMed Abstract | Free Full Text\n\nLuebke RG: Pulp cavity debridement and disinfection. Dent Clin North Am. 1967; 603–613. PubMed Abstract\n\nMachado FP, Khoury RD, Toia CC, et al.: Primary versus post-treatment apical periodontitis: microbial composition, lipopolysaccharides and lipoteichoic acid levels, signs and symptoms. Clin Oral Investig. 2020. PubMed Abstract | Publisher Full Text\n\nMaekawa LE, Valera MC, deOliveira LD, et al.: Effect of Zingiber officinale and propolis on microorganisms and endotoxins in root canals. J Appl Oral Sci. 2013; 21(1): 25–31. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMaekawa LE, Valera MC, deOliveira LD, et al.: In vitro evaluation of the action of irrigating solutions associated with intracanal medications on Escherichia coli and its endotoxin in root canals. J Appl Oral Sci. 2011; 19(2): 106–112. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMergenhagen SE, Varah E: Serologically specific lipopolysaccharides from oral veillonella. Arch Oral Biol. 1963; 8: 31–36. PubMed Abstract | Publisher Full Text\n\nMohmmed SA, Vianna ME, Penny MR, et al.: Investigations into in situ Enterococcus faecalis biofilm removal by passive and active sodium hypochlorite irrigation delivered into the lateral canal of a simulated root canal model. Int Endod J. 2018; 51(6): 649–662. PubMed Abstract | Publisher Full Text\n\nNarayanan LL, Vaishnavi C: Endodontic microbiology. J Conserv Dent. 2010; 13(4): 233–239. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNeelakantan P, Herrera DR, Pecorari VGA, et al.: Endotoxin levels after chemomechanical preparation of root canals with sodium hypochlorite or chlorhexidine: a systematic review of clinical trials and meta-analysis. Int Endod J. 2019; 52(1): 19–27. PubMed Abstract | Publisher Full Text\n\nNesser SFA, Bshara NG: Evaluation of the apical extrusion of sodium hypochlorite gel in immature permanent teeth: An in vitro study. Dent Med Probl. 2019; 56(2): 149–153. PubMed Abstract | Publisher Full Text\n\nPaiva SSM, Siqueira JF, Rôças IN, et al.: Molecular microbiological evaluation of passive ultrasonic activation as a supplementary disinfecting step: a clinical study. J Endod. 2013; 39(2): 190–194. PubMed Abstract | Publisher Full Text\n\nPlotino G, Pameijer CH, Grande NM, et al.: Ultrasonics in endodontics: a review of the literature. J Endod. 2007; 33(2): 81–95. PubMed Abstract | Publisher Full Text\n\nPlutzer B, Zilm P, Ratnayake J, et al.: Comparative efficacy of endodontic medicaments and sodium hypochlorite against Enterococcus faecalis biofilms. Aust Dent J. 2018; 63(2): 208–216. PubMed Abstract | Publisher Full Text\n\nPoggio C, Arciola CR, Dagna A, et al.: Antimicrobial activity of sodium hypochlorite-based irrigating solutions. Int J Artif Organs. 2010; 33(9): 654–659. PubMed Abstract | Publisher Full Text\n\nSáinz-Pardo M, Estevez R, dePablo ÓV, et al.: Root canal penetration of a sodium hypochlorite mixture using sonic or ultrasonic activation. Braz Dent J. 2014; 25(6): 489–493. PubMed Abstract | Publisher Full Text\n\nSalazar-Mercado SA, Torres-León CA, Rojas-Suárez JP: Cytotoxic evaluation of sodium hypochlorite, using Pisum sativum L as effective bioindicator. Ecotoxicol Environ Saf. 2019; 173: 71–76. PubMed Abstract | Publisher Full Text\n\nSedgley C: Root canal irrigation--a historical perspective. J Hist Dent. 2004; 52(2): 61–65. PubMed Abstract\n\nSiqueira JF: Aetiology of root canal treatment failure: why well-treated teeth can fail. Int Endod J. 2001; 34(1): 1–10. PubMed Abstract | Publisher Full Text\n\nSiqueira JF, Rôças IN, Favieri A, et al.: Chemomechanical reduction of the bacterial population in the root canal after instrumentation and irrigation with 1%, 2.5%, and 5.25% sodium hypochlorite. J Endod. 2000; 26(6): 331–334. PubMed Abstract | Publisher Full Text\n\nTaylor HD, Austin JH: The solvent action of antiseptics on necrotic tissue. J Exp Med. 1918; 27(1): 155–164. PubMed Abstract | Publisher Full Text | Free Full Text\n\nValera MC, daCardoso FGR, Chung A, et al.: Comparison of Different Irrigants in the Removal of Endotoxins and Cultivable Microorganisms from Infected Root Canals. ScientificWorldJournal. 2015; 2015: 125636. PubMed Abstract | Publisher Full Text | Free Full Text\n\nValera MC, Maekawa LE, Chung A, et al.: The effect of sodium hypochlorite and ginger extract on microorganisms and endotoxins in endodontic treatment of infected root canals. Gen Dent. 2014; 62(3): 25–29. PubMed Abstract\n\nValera MC, daSilva KCG, Maekawa LE, et al.: Antimicrobial activity of sodium hypochlorite associated with intracanal medication for Candida albicans and Enterococcus faecalis inoculated in root canals. J Appl Oral Sci. 2009; 17(6): 555–559. PubMed Abstract | Publisher Full Text | Free Full Text\n\nvan der Sluis LWM, Versluis M, Wu MK, et al.: Passive ultrasonic irrigation of the root canal: a review of the literature. Int Endod J. 2007; 40(6): 415–426. PubMed Abstract | Publisher Full Text\n\nZand V, Lotfi M, Soroush MH, et al.: Antibacterial Efficacy of Different Concentrations of Sodium Hypochlorite Gel and Solution on Enterococcus faecalis Biofilm. Iran Endod J. 2016; 11(4): 315–319. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "65501",
"date": "22 Jul 2020",
"name": "Talal Al-Nahlawi",
"expertise": [
"Reviewer Expertise Endodontics and operative dentistry"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a well designed study that evaluated the effect of sodium hypochlorite as an irrigant solution and gel over one Gram-positive bacterium and another Gram-negative one, as well over their endotoxins with and without passive ultrasonic irrigation (PUI).\n\nThis study may be the first study that evaluated the effect of sodium hypochlorite gel over the endotoxins of endodontic pathogens and this has a great clinical relevance.\nThe introduction: It presented briefly the current literature and a historic background about the use of sodium hypochlorite. As a suggestion for the researchers in their future projects, I think that it is more appropriate to test another Gram-negative bacterium than e.coli. Even e.coli was found in the root canal system, however, I think that there are a variety of microorganisms that may be tested and have more relevance in the literature as example: Porphyromonas gingivalis (P. gingivalis) as in the study of Wang et al (2019)1 and others in the study of Lukic et al (2020)2.\nThe methods: The study design was carefully planned, I think that the study in its current version is accepted to be indexed, all the cited articles in the methodology section have more details about the execution of this study. I am just wondering why the researchers did not use the sodium hypochlorite in the same concentration in both solution and gel forms? It is not a big deal here as the concentration is almost the same (2.5 and 3 %), however, I think this should be explained in the discussion section, or at least mentioning a previous study that used different concentrations.\n\nThe discussion: Why did the authors not take advantage of the positive results obtained from using PUI in this study to be discussed furtherly in the discussion section or even in the introduction section? I think another paragraph will improve the discussion section. The conclusions of this paper drawn adequately supported by the results.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "69901",
"date": "19 Oct 2020",
"name": "Brenda P F A Gomes",
"expertise": [
"Reviewer Expertise Endodontics"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 evaluates the effect of NaOCl solution (2.5%) and gel (3%) with/without passive ultrasonic irrigation (PUI) on Enterococcus faecalis, Escherichia coli, and their endotoxins, lipopolysaccharide (LPS) and lipoteichoic acid (LTA). The article is very well written, responding adequately to the objectives proposed in this study. The language should be revised. The present study has a great clinical relevance.\nThe title of the work is clear, concise, short and objective. Furthermore, it summarizes the authors conclusions, which is a positive point.\nThe abstract proposed by the authors clearly summarizes the objective, materials and methods, statistical analysis, results and conclusions.\nThe introduction presents a background based on previous studies and at most recent literature. The authors could add a paragraph about the use of PUI in endodontics to address all of the research factors.\nThe authors did not report power calculation of the sample. Was the sample size based on previous studies? They should answer this question in the text.\nThe authors well described the specimens’ preparation, the tests performed and the statistical analyses used in the study.\nThe results section is well described and synthetizes the results obtained in the present study. The tables and figures are descriptive. Table 2 will be better viewed if it is before the discussion and the results present in this table could be better explored in the written presentation of the results.\nIn the discussion, the authors could add one more paragraph about the use of PUI, as suggested in the introduction.\nThe conclusions of this paper drawn adequately supported by the results.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/9-642
|
https://f1000research.com/articles/9-641/v1
|
24 Jun 20
|
{
"type": "Research Article",
"title": "Prevalence of Alzheimer's disease according to anticholinesterase drug usage from 2012 to 2017 in the state of Paraná, Brazil",
"authors": [
"Dayanna Hartmann Cambruzzi Mendes",
"Glaucia Renee Hilgemberg",
"Flavio Silva Posseti",
"Jéssica Wouk",
"Emerson Carraro",
"Glaucia Renee Hilgemberg",
"Flavio Silva Posseti",
"Jéssica Wouk",
"Emerson Carraro"
],
"abstract": "Introduction: Alzheimer's disease (AD) is a public health problem in Brazil due to the growing number of older adults in this population. Knowing the prevalence of AD in the Paraná state is essential to improve patients’ quality of life. The objective of the study was to estimate AD prevalence in the state of Paraná, based on the prescription of anticholinesterases, from 2012 to 2017. Methods: Patients diagnosed with AD, aged 60 years or over, who used Brazil’s Unified Public Health System (SUS) and received AD medication from the Department of Pharmaceutical Assistance of the state of Paraná, from 2012 to 2017, were the target population of the present study. The medication data were collected from the Computerized system management and Monitoring of Exceptional Drugs (SISMEDEX), and the population’s data were collected from the Brazilian Institute of Geography and Statistics (IBGE). Results: The sample consisted of 52,687 patients, and the median prevalence of AD in the established period for the state was 642.6/100,000 inhabitants (0.64%). In all ages and macro-regions, women showed the highest prevalence rates. The median prevalence for women in the established period was 755.4/100,000 inhabitants (0.75%). Among men, the median prevalence was 510.2/100,000 inhabitants (0.51%). In the temporal analysis of prevalence, between 2012 and 2017, a reduction of 23% was found in the state rate. The prevalence rate of Paraná for AD is nine times lower than the Brazilian average. Conclusions: Paraná’s prevalence rate for AD is lower than the rates of Brazil and the world, suggesting that AD is underdiagnosed in most municipalities of this state.",
"keywords": [
"Alzheimer's disease",
"Brazil",
"Prevalence",
"Pharmacoepidemiology",
"epidemiology"
],
"content": "Introduction\n\nThe growth of the elderly population is a worldwide phenomenon. As life expectancy rises, epidemiological changes cause risk factors for noncommunicable diseases (NCDs) to increase1. Among NCDs, neuropsychiatric disorders must be highlighted because, in 2013, they were responsible for more than a third, 34%, of all morbidity reported in Brazil2.\n\nAccording to the latest report from Alzheimer’s Disease International, dementia is the most common neurological disease in the elderly population. It affects 50 million people worldwide, with the number expected to triple by 2050. More than half of all people with dementia live in low- and middle-income countries, where only 10% of individuals receive adequate diagnosis3.\n\nAmong dementias, Alzheimer's disease (AD) is the most prevalent, accounting for 50–70% of cases. AD is a neurodegenerative disease that causes progressive deterioration of intellectual and routine skills, as well as cognitive impairment4. In Brazil, it is estimated that 6% of the 15 million people who are over the age of 60 suffer from AD4,5.\n\nThe literature describes a strongly positive relationship between age and dementia. After the age of 65, the prevalence rate of dementia doubles every five years6. Long-term care by both the patients’ family and the state becomes inevitable as the disease progresses, mainly due to the loss of patients’ functional capacity. Therefore, with the increase in the elderly population in Brazil, AD represents a significant public health problem7,8.\n\nThe global AD prevalence rate of people aged 60 is 3.9%, with a significant variation between continents, with a rate of 1.6% in Africa, 6.4% in North America, 7.1% in South America, 5.5% in Asia, and 9% in Europe9,10. Brazilian studies suggest a prevalence of 7.1% for dementia among people over the age of 652,11,12. Considering the dementia prevalence and the high number of older adults (15 million) in 2013, it is estimated that 1.1 million people lived with this condition in Brazil13.\n\nFew studies are available specifically on the prevalence and incidence of AD in Brazil. Most studies address all dementias, have a small sample size, or are developed in an institutionalized environment. Furthermore, a significant variation in the results is found, which hinders the identification of the true dementia prevalence among elderly Brazilians2,11,12.\n\nAD has no cure, and even though available medications alleviate the symptoms and delay the progression of the disease, current therapy is far from being satisfactory14. The drug treatments currently available for AD are acetylcholinesterase inhibitors (AChEIs), such as rivastigmine, galantamine and donepezil, and N-methyl-D-aspartate (NMDA) receptor antagonist, such as memantine15.\n\nAChEIs are the primary drugs used to treat AD. Since 2002, Brazil counts on the Assistance Program for Alzheimer's Disease Patients (PAPDA), which aims to address 50% of AD patients throughout the country16. The government offers, through Brazil’s Unified Public Health System (SUS), free treatment for patients with AD, according to the Clinical Protocol and Therapeutic Guidelines (PCDT) - Alzheimer's Disease. The PCDTs are made by technical-scientific consensus and aim at rationalizing medical prescription and dispensing, through previously established diagnostic criteria, with recommended treatments (drugs and their respective dosages) and mechanisms for monitoring effectiveness17.\n\nMedicines are highly relevant inputs to establish policies and priorities adjusted to the population's health needs but are rarely used in prevalence studies15. The information resulting from the use of specific drugs is predictably more reliable since drugs used in the treatment of cognitive symptoms follow specific protocols from the Brazilian Ministry of Health and can only be applied in the treatment of AD18.\n\nIn Brazil, AD is not part of the national list of notifiable diseases, therefore there is no national and official registry of patient cases in the country19. The only official references related to AD in the country are the registration of medicines dispensed for the disease, which is provided by SUS, and the AD death certificates that are registered in the Ministry of Health's Mortality Information System (SIM).\n\nData related to drugs dispensed by SUS may estimate cases of AD in the state since the control of patients in this dispensation program is individualized and rigorous. The patient is diagnosed based on ICD-10 (International Classification of Diseases, 10th Revision), which reduces diagnosis errors20.\n\nIn Brazil, there are several publications on AD; however, few are about the prevalence of the disease in a large sample of the population, and none are specific to the state of Paraná. The lack of national and state data triggers a deficiency in the understanding of the disease’s epidemiological profile in the country and the state, despite its well-known impact on public health.\n\nThe present study aimed to present the historical estimate of AD prevalence in the state of Paraná, based on the prescription of anticholinesterases among SUS users, from 2012 to 2017.\n\n\nMethods\n\nThe present study was approved by the Research Ethics Committee of Midwest State University of Paraná (UNICENTRO), under number 2.334.461/2017. The requirement for patient consent was dismissed by the ethics and research committee because this study uses secondary only data.\n\nA cross-sectional, retrospective, and descriptive study, with a quantitative approach, was performed in the state of Paraná from 2012 to 2017. The study population consisted of patients diagnosed with AD, users of SUS, in the state of Paraná and who received medication for AD in the studied period. Patients who were diagnosed based on PCDT for AD and had one of the following ICD 10 diagnostic codes were included; G30.0, G30.1, and G30.8.\n\nOnly patients who withdrew medication through the SISMEDEX program were included in the present study. Therefore, only data from the population over 60 years old who received their medications from SUS (free of charge) during the study period were analyzed. People with AD who privately purchased their drugs were not counted in this study. Also, patients under the age of 60 or with different ICD from those determined in the inclusion criteria who withdrew medication through the SISMEDEX program, were excluded from the study.\n\nA sample size calculation was not performed, since all 399 municipalities in Paraná were studied, as well as all patients over 60 years old, respecting the study inclusion criteria. The cut-off age, 60, was determined based on the majority of prevalence studies.\n\nThe following SISMEDEX system variables were collected; patient's gender, city and age, the medication that was interrupted and used for AD, and the health region to which the patient belongs. The data on this system are filled by pharmacists from the health region responsible for dispensing the drugs. Estimates of population by sex, age group (>60 years) and year used to calculate the prevalence coefficients were obtained from the Brazilian Institute of Geography and Statistics (IBGE), calculated on July 1st of each year.\n\nThe results of the study are separated by age groups and macro-regions of Paraná; the macro-regions comprise the 22 health regions that are governmental divisions. Regional health encompasses nearby municipalities that share as SUS health structures.\n\nInitially, a description of the main characteristics of the sample was performed for data analysis. The AD prevalence of each year was estimated by dividing the number of cases of AD in patients over 60 years old in Paraná (numerator) by the population over 60 years old residing in Paraná during the same period (denominator), multiplied by 100,000, with the result expressed per 100,000 inhabitants.\n\nThe descriptive analysis of the data included frequency distributions for categorical variables as well as measures of central tendency (mean, median) for continuous variables. The chi-square test was used to assess differences in proportions. Differences were considered statistically significant at p<0.05.\n\nThe data were analyzed using the IBM SPSS Statistics v.20.0 computer program. Armonk, New York: IBM Corp.\n\n\nResults\n\nDuring the study period, 65,494 patients registered in SISMEDEX who received IAChE drugs were identified. The final sample consisted of 52,687 patients diagnosed with AD using IAChE in the state of Paraná in the years 2012 to 201721. This sample served as a basis for determining the prevalence of the disease according to the year and patients’ gender and age group.\n\nThe mean age of the sample was 79.89 ± 8.1, in which female cases represented 63.5% (n = 33,465) and male cases, 36.5% (n = 19,222).\n\nAll patients received drugs dispensed by the health regions of Paraná. 12,807 patients were excluded from the study, 1,239 patients being under 60 years old, and 11,568 patients for having multiple records. SISMEDEX generates a new patient in the system every time a medication or its’ dose changes, or when the dispensing address is altered; therefore, some records are multiplied. This issue was corrected by selecting patients using their initial registration number in the system and not by the type of medication dispensed, change in dosage, or delivery address.\n\nRegarding the sample’s ICDs, the following percentages were found; 62% (n = 32,666) for G30.0 (early onset Alzheimer's disease), 33% (n = 17,386) for G30.1 (late onset Alzheimer's disease) and only 5% (n = 2,635) for 30.8 (other forms of Alzheimer's disease).\n\nThe median overall prevalence of AD among the elderly in the state of Paraná from 2012 to 2017 (Table 1) was 642.6/100,000 inhabitants (0.64%). Overall, the prevalence in females was 41% higher, at 755.4/100,000 inhabitants (0.75%) and 510.2/100,000 inhabitants (0.51%) among men (Table 1).\n\nWhen separated by age groups, the median results of AD prevalence in the state of Paraná showed a trend of progression, associated with age, regardless of gender. The median prevalence of AD in both genders aged 60–69 was lower than 1% (107.4/100,000 inhabitants). These results contrast with the median prevalence of AD in patients over the age of 80, in both genders, which was higher than 2%.\n\nWhen compared to the prevalence in males in the same age groups, the prevalence of AD in women aged 60 to 69, 70 to 79 and 80 or over was 29%, 28%, and 41% higher, respectively. Differing between genders, in men over 80 years old, the median prevalence was 2054.0/100,000 inhabitants (2.05%), and for women in the same age group, the prevalence was 2903.7/100,000 inhabitants (2.90%).\n\nThe median prevalence of AD among the elderly in the state of Paraná during the period studied vary according to the states’ macro-regions (Figure 1). Fewer cases were found in the east macro-region of the state: 401.6/100,000 inhabitants, which is 60% lower than the median of cases in the entire state in the same period. In contrast, when compared to the state’s median, the northwest macro-region showed a rate that is 96% higher, with 1235.5/100,000 inhabitants. West and north macro-regions presented a rate of 642.6/100,000 habitants and 614.5/100,000 inhabitants, respectively. The northwest macro-region has the highest number of cases in the state, showing a prevalence rate three times higher than the east macro-region, which presents the lowest number of cases in this period.\n\nAs shown in Figure 1, all macro-regions of Paraná presented the highest prevalence of AD in 2012, with 698.4 cases/100,000 inhabitants, with a decline in rates within the following years, showing a 23% reduction in 2017, with 556.1 cases/100,000 inhabitants. Variation was found in the north macro-region, showing stability for the female gender in the last three years analyzed. In the same macro-region, male patients showed prevalence stability during the first three years studied, presenting a decline in 2015 and, once again, stability in 2016.\n\nAD prevalence rates are shown in Figure 2, Figure 3, and Figure 4 (per 100,000 habitants), according to age group, year, patient’s gender, and Paraná macro-regions.\n\nThe prevalence of AD among 60–69 years old patients is shown in Figure 2. The northwest macro-region presented the highest number of cases for both genders, with a median prevalence of 287.8/100,000 inhabitants and 225.4/100,000 inhabitants for women and men, respectively. In general, rates declined for this age group over the years studied, showing occasional increases, as in 2016 in the northwest macro-region among women, and in the north macro-region in 2013 among women and in 2014 among men.\n\nThe prevalence of AD among 70–79-year-old patients is shown in Figure 3. Among this group age, the northwest macro-region demonstrated the highest number of cases in both genders during the period studied, with a median prevalence of 1801.9/100,000 inhabitants and 1218.1/100,000 inhabitants for women and men, respectively. A decline in AD prevalence from 2012 to 2017 was observed, with some exceptions in specific years and intensity variations according to the macro-region.\n\nIn the age group of patients aged 80 and over (Figure 4), the highest prevalence rates in the state were found for both genders and all macro-regions during the period studied. When compared to other age groups, the decline in AD prevalence was lower for patients aged 80 and over, with higher fluctuations over the years studied. For women, rates declined in the east and west macro-regions and fluctuated in the northwest and north macro-regions. Among men, rates declined over the years studied, but in the north macro-region, a decrease was observed in 2016, with a subsequent increase in 2017.\n\n\nDiscussion\n\nIn the present study, the prevalence of AD in the state of Paraná between the years 2012 and 2017 was assessed based on data collection of patients with a confirmed diagnosis who used the government's drug treatment program. Thus, the prevalence of AD described for patients undergoing treatment was 642.6/100,000 inhabitants (0.64%). When comparing the results of the present study and the prevalence rates of AD in other Brazilian studies, similarities were found. Nitrini et al. (2004) demonstrated a prevalence rate of 0.77% in Catanduva-SP12. However, other studies demonstrated higher AD prevalence rates. Three studies performed in the city of São Paulo showed a prevalence of 4.9%22, 5.1%23, and 6.8%24.\n\nA recent systematic review on the prevalence of dementia in Brazil showed a prevalence ranging from 5.1% to 17.5%, in which AD was the most prevalent and responsible for 60% of cases5. The variations in prevalence may be attributed to differences between the population, the number of samples and the methods applied in each study. It is also important to highlight that the Brazilian studies used for the comparison took place in the southeast region of the country, and the majority were based on institutionalized individuals.\n\nWorldwide prevalence data for AD, published in 2010 by the 10/66 Dementia Research Group, demonstrated the differences in the prevalence of dementia among developing countries. The rate of AD in Cuba was 11%, in Mexico 7.3%, in India 7.5%, in Africa 5.85%, and China 7.2%25.\n\nInternational studies also showed higher prevalence rates than those reported in the present study, which can be attributed to the lack of diagnosis and treatment for AD in Paraná. Only 14% of developing countries have reports containing the number of people with AD being diagnosed and treated. Studies suggest that up to 90% of people with dementia in low- and middle-income countries are unaware of their condition3,15,25.\n\nTable 1 reports the cases of AD by age group, and as age increases, the number of patients also increases. The age group of 80 years old and over showed 68% of AD cases found in the study. These data are congruent with Brazilian and international population studies in which it is verified that the prevalence of AD increases almost exponentially with age. Therefore, among the most advanced age groups, the most significant number of patients was found in the present study3,15. Literature reports that dementia prevalence doubles approximately every five years, matching the data from the present study in which AD prevalence increased by an average of 2.5 times every five years until the age of 7915,25,26. The demographic trends of the country, characterized by population aging, contribute to the increase of AD cases over the years, as age is the leading risk factor for its development27,28.\n\nThe results of the present study also suggest a tendency to a higher prevalence of AD among women in Paraná (Table 1), with a median coefficient of 755.4/100,000 inhabitants (0.75%) among women and 510.2/100,000 inhabitants (0.51%) among men. The median overall prevalence rate was 48% higher for women than for men. This result may be justified by women's higher life expectancy, the prevalence of older women in the sample, the feminization of aging, and the fact the majority of those who use health services are women29–31.\n\nWhen comparing men and women by age group (Table 1), once again, the results demonstrated the highest rates of AD among women regardless of age group. These results also match the data presented in the demographic and epidemiological profile of users of high-cost drugs in SUS, in which women showed higher rates of AD drug consumption, at 1.72% against 1.36% for men32.\n\nIn 2010, the population pyramid in Paraná had a barrel-like shape, characteristic of populations in the aging process, with a narrow shape on the base and a wider shape on the top, particularly among women due to over-mortality of men at advanced age33. Within this panorama, a phenomenon called the feminization of aging is observed. According to the World Health Organization (WHO), older women are living longer than men almost everywhere in the world34.\n\nMen's average life expectancy is 69.1 years old, while women’s is 73.8 years old, a difference of 4.7 years. In Brazil, this difference in favor of women averages seven years35. There are several explanations for greater female longevity. One of them is women’s attitude towards health/illness and health service, as they do more tests and routine medical consultations. There are also biological differences between the genders, such as protective factors related to female hormones. Also, there are differences in lifestyle; men are generally more exposed to risks at work, and alcohol and tobacco consumption36.\n\nThe prevalence of differences between men and women may vary regionally within the state, due to the heterogeneity of the age profile. Although women showed higher rates than men, differences between genders were not significant, and consequently inconclusive. Therefore, it is not inferred that being a woman implies a higher chance of developing AD11,12.\n\nWhen the results were separated by age groups, and macro-regions of Paraná, more considerable differences were found. The macro-regions comprise the 22 Health Regions of the state, and they were used in this study to facilitate comparisons. The highest median prevalence rate for AD was identified in the northwest, with 1235.5/100,000 inhabitants, and the lowest rate was found in the east, with 401.6/100,000 inhabitants. The ten largest cities in Paraná concentrate 41% of the entire population of the state; 51.6% of people live in municipalities with more than 100,000 inhabitants37. Despite the large population concentration in the east region of the state (Curitiba and the coast), the population of Paraná stands out for the existence of a significant number of medium-sized cities in the state’s countryside, a fact that regionalizes the demographic polarization37.\n\nThe intra-state analysis reveals heterogeneity among the municipalities of Paraná, which demonstrated a human development index (HDI) ranging from 0.546 in Doutor Ulysses city (lowest in the state) to 0.823 in Curitiba (highest in the state), both cities in the east macro-region38. A total of 39 municipalities in the state of Parana present low HDIs (<0.658) (10%). Among these municipalities, the majority (25) are in the east macro-region of the state. Associating the hypothesis reported in Table 1 with the socio-economic aspects of the state, the present study suggests that the prevalence of AD is lower in cities with a lower HDI23. The northwest macro-region has a large number of cities with the highest HDIs of the state, which generally provides a better education, health, and life quality - factors that protect against dementia. According to the literature review of Sharp and Gatz (2011), the relationship between schooling and risk of dementia was more significant in developed regions with a higher HDI than in developing regions, matching with the findings of the present study39.\n\nIn general, the largest municipalities with the highest HDI also have the best information management in health and medical care, consequently producing more accurate and reliable information40,41. Thus, the possible explanation for a higher prevalence of AD in municipalities with a higher HDI may be associated with better investigation and diagnosis of AD cases40. Mendonça et al. (2016) inferred that small cities, generally located in more isolated geographical areas, with greater social and economic vulnerability, are overlooked by doctors, which hinders AD diagnosis and treatment41.\n\nThe index of the older adults in Paraná state from 2016 to 2019 relates the population number of older adults (≥60 years old) to those of children and young people (≤15 years old). These two extreme segments of the disparate age pyramid allow a more precise measure of comparability among the municipalities of the state. The east macro-region has the most significant number of cities in Paraná with a low rate of older adults. In this macro-region, an average of less than 49 older adults per 100 children and young people is found, characterizing a young population38. This average correlates with the literature, in which the prevalence of AD increases with the increase of age1,42. The opposite occurs in the northwest macro-region, where most cities show proportions close to 70 older adults for every 100 children and young people, characterizing an aging population, contributing to a greater number of cases of AD in this region.\n\nRisk factors associated with diabetes mellitus (DM) have been linked, in several observational studies, to the development of dementia since the early 2000s43. A worldwide trend suggests that the increase in AD risk occurs by a direct link between glucose dysregulation and neurodegeneration. Authors consider AD as type 3 diabetes mellitus, due to its similarity to histopathological, molecular, and biochemical anomalies43–45.\n\nCorrelating the prevalence of AD with diabetes mortality, between 2004 and 2014 the rate of deaths by DM in the state was 32.0 deaths/100,000 inhabitants, with a higher incidence in women46. Twelve health regions from Paraná presented higher DM mortality rates than the state, and three of them are in the northwest macro-region. This is in contrast to the east macro-region, in which most of the health regions present AD mortality rates below the state average46. Epidemiological evidence links these two diseases, Shinohara and Sato (2017) reported that the risk of developing AD is higher among people with DM than in the general population, especially in the eastern populations47.\n\nSome limitations of the present study should be highlighted. The sample consists of patients linked to SUS, which impedes the generalization of the results for the entire population. Additionally, this retrospective study refers to secondary data, which was originated with an administrative purpose and made it difficult to obtain more detailed information. Some clinical information was not required while filling out the database, such as the starting date of medication usage; therefore, this data could not be collected and used in the present study. As an example of a lack of information, more than half (62%) of the patients studied were diagnosed with ICD 30.0, early-onset AD. This number of diagnoses conflicts with the literature since early-onset AD (before age 65) affects 6–7% of the population, usually with family background and an autosomal dominant transmission pattern12,48. Inaccurate AD diagnosis triggers a false epidemiological profile, due to incorrect or incomplete database, distorting and altering the epidemiological reality49. Therefore, the correct input of ICD codes in the SISMEDEX/PR database must be reviewed before such results can be inferred.\n\nAlso, the scarcity of scientific evidence and the difficulty of accessing information of this nature in Brazil is highlighted. Currently, no reliable information that addresses the dispensation of medication by the private sector is available, making it impossible to compare it with the public sector. Nevertheless, the present study shows that governmental information systems are essential secondary data sources, and they should be used mainly due to the low cost and availability of information over a long period. Data presented in this article is vital for prevalence studies; this information has never been published, and it guides further analysis.\n\n\nConclusions\n\nAs the population ages, the number of AD cases in Paraná increases during the studied period. Besides, Paraná’s prevalence rate for AD was nine times lower than the national rate average. This result suggests that AD is still underdiagnosed in most municipalities in Paraná.\n\nThe information presented was obtained from 52,687 patients, and it can provide subsidies for planning actions related to AD, and most important, collaborating to improve the quality of primary care. Also, an extended national assessment could be performed based in the present study, since the reality and needs of Brazil states are similar.\n\n\nData availability\n\nHarvard Dataverse: Prevalence of Alzheimer's disease according to anticholinesterase drugs usage from 2012 to 2017 in the state of Paraná, Brazil. https://doi.org/10.7910/DVN/FE2G6A21\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\nTerra NL, et al.: Envelhecimento, Memória e Doença de Alzheimer. 1. ed. Porto Alegre: EDIPUCRS. 2017; 1: 67.\n\nBurla C, Camarano AA, Kanso S, et al.: Panorama prospectivo das demências no Brasil: um enfoque demográfico. Ciência & Saúde Coletiva. 2013; 18(10): 2949–2956. Publisher Full Text\n\nAlzheimer’s Disease International (AID): Informe Mundial Sobre El Alzheimer 2018. Boletim Londres Set. 2018.\n\nOzello G, da Silva HS, Guimarães C, et al.: Impacto econômico da doença de Alzheimer no Brasil: é possível melhorar a assistência e reduzir custos?. Ciência &Saúde Coletiva. 2014; 19(11). Publisher Full Text\n\nBoff MS, Sekyia F, Bottino C: Revisão sistemática sobre prevalência de demência entre a população brasileira. Rev Med (São Paulo). 2015; 94(3): 154–161. Publisher Full Text\n\nForlenza OV: Tratamento farmacológico da doença de Alzheimer. Rev Psiq Clín. 2005; 32(3): 137–148. Publisher Full Text\n\nFerreira S, Massano J: Terapêutica farmacológica na doença de Alzheimer: progressos e esperanças futuras. Arq Med. 2013; 27(2): 65–86. Reference Source\n\nMaia CSC, et al.: Doença de Alzheimer: um perfil diagnóstico dentro da estratégia de saúde da família. Geriatrics, Gerontology and Aging. 2013; 7(4); 259–263. Reference Source\n\nCAHILL S, Pierce M, Werner P, et al.: A Systematic Review of the Public’s Knowledge and Understanding of Alzheimer’s Disease and Dementia. Alzheimer Dis Assoc Disord. 2015; 29(3): 255–275. PubMed Abstract | Publisher Full Text\n\nPrince M, Bryce R, Albanese E, et al.: The global prevalence of dementia: a systematic review and metaanalysis. Alzheimers Dement. 2013; 9(1): 63–75.e2. PubMed Abstract | Publisher Full Text\n\nHerrera JE, Paulo C, Ricardo N, et al.: Estudo epidemiológico populacional de demência na cidade de Catanduva - Estado de São Paulo - Brasil. Revista de Psiquiatria Clínica. 1997; 25(2): 70–73. Reference Source\n\nNitrini R, Caramelli P, Herrera E Jr, et al.: Incidence of Dementia in a Community-Dwelling Brazilian Population. Alzheimer Dis Assoc Disord. 2004; 18(4): 241–246. PubMed Abstract\n\ndaCosta RDF, Osorio-de-Castro CG, da Silva RM, et al.: Aquisição de medicamentos para a Doença de Alzheimer no Brasil: uma análise no sistema federal de compras, 2008 a 2013. Ciência & Saúde Coletiva. 2015; 20(12) 3827–3838. Publisher Full Text\n\nCintra MTG, et al.: Avaliação do Programa Público Brasileiro de Tratamento da Doença de Alzheimer no ano de 2008. Reference Source\n\nAlmeida-Brasil CC, de Oliveira Costa J, Aguiar VF, et al.: Acesso aos medicamentos para tratamento da doença de Alzheimer fornecidos pelo Sistema Único de Saúde em Minas Gerais, Brasil. Cadernos de Saúde Pública. 2016; 32(7): e00060615. Publisher Full Text\n\nBRASIL. Ministério da Saúde: Portaria n. 703 de 12 de Abril de 2002. Dispõe sobre a instituição, no âmbito do Sistema Único de Saúde, do Programa de Assistência aos Portadores da Doença de Alzheimer. 2002. Reference Source\n\nBRASIL. Ministério da Saúde: Portaria Conjunta n. 13 de 28 de Novembro de 2017. Aprova o Protocolo Clínico e Diretrizes Terapêuticas da Doença de Alzheimer. 2017. Reference Source\n\nBRASIL. Ministério da Saúde: Portaria n. 1.554 de 30 de Julho de 2013. Dispõe sobre as regras de financiamento e execução do Componente Especializado da Assistência Farmacêutica no âmbito do Sistema Único de Saúde (SUS). 2013. Reference Source\n\nBRASIL. Mistério da Saúde: Portaria n. 204 de 17 de Fevereiro de 2016. Define a Lista Nacional de Notificação Compulsória de doenças, agravos e eventos de saúde pública nos serviços de saúde públicos e privados em todo o território nacional, nos termos do anexo, e dá outras providências. 2016. Reference Source\n\nOMS, OrganizaÇÃo Mundial Da SaÚde: Classificação Estatística Internacional de Doenças e Problemas Relacionados à Saúde - CID-10. 10ª Edição. 2008. Reference Source\n\nMendes DH: Prevalence of Alzheimer's disease according to anticholinesterase drugs usage from 2012 to 2017 in the state of Paraná, Brazil. Harvard Dataverse, V1. 2020. http://www.doi.org/10.7910/DVN/FE2G6A\n\nHerrera E Jr, Caramelli P, Silveira AS, et al.: Epidemiologic survey of dementia in a community-dwelling Brazilian population. Alzheimer Dis Assoc Disord. 2002; 16(2): 103–108. PubMed Abstract | Publisher Full Text\n\nScazufca M, Menezes PR, Vallada HP, et al.: High prevalence of dementia among older adults from poor socioeconomic backgrounds in São Paulo, Brasil. Int Psychogeriatr. 2008; 20(2): 394–405. PubMed Abstract | Publisher Full Text\n\nBottino CM, Azevedo D Jr, Tatsch M, et al.: Estimate of Dementia Prevalence in a Community Sample from São Paulo, Brasil. Dement Geriatr Cogn Disord. 2008; 26(4): 291–299. PubMed Abstract | Publisher Full Text\n\nAlbanese E, Liu Z, Acosta D, et al.: Equity in the delivery of community healthcare to older people: findings from 10/66 Dementia Research Group cross-sectional surveys in Latin America, China, India and Nigeria. BMC Health Serv Res. 2011; 11: 153. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChaves ML, Camozzato AL, Godinho C, et al.: Incidence of Mild Cognitive Impairment and Alzheimer Disease in Southern Brazil. J Geriatr Psychiatry Neurol. 2009; 22(3): 181–187. PubMed Abstract | Publisher Full Text\n\nLebrÃo ML: O envelhecimento no Brasil: aspectos da transição demográfica e epidemiológica. Realic Saúde Coletiva. 2007; 4(17): 135–140. Reference Source\n\nAvila RI, Machado AM: Transição demográfica brasileira: desafios e oportunidades na educação, no mercado de trabalho e na produtividade. Revista Indicadores Econômicos FEE. 2016; 43(3). Reference Source\n\nAprahamian I, Martinelli JE, Yassuda MS: Doença de Alzheimer: revisão da epidemiologia e diagnóstico. Revista Brasileira de Clinica Médica. 2009; 7(27): 27–35. Reference Source\n\nLopes MA, Hototian SR, Reis GC, et al.: Systematic review of dementia prevalence 1994 to 2000. Dement Neuropsychol. 2007; 1(3): 230–240. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVallin J: Mortalidade, sexo e gênero. Séries Demográficas. 2015; 2(1): 15–54. Reference Source\n\nAcurcio FA, Brandão CM, Guerra AA Jr, et al.: Perfil demográfico e epidemiológico dos usuários de medicamentos de alto custo no Sistema Único de Saúde. Revista Brasileira de Estudos Populacionais. 2009; 26(2): 263–282. Publisher Full Text\n\nIBGE, Instituto Brasileiro de Geografia e Estatística: Pesquisa Nacional por Amostra de Domicílios. Um panorama da saúde no Brasil – Acesso e utilização dos serviços, condições de saúde e fatores de risco e proteção à saúde. PNAD, Rio de Janeiro. 2013; 33: 1–133. Reference Source\n\nOMS, Organização Pan-Americana da Saúde – Opas: Envelhecimento ativo: uma política de saúde. World Health Organization, Brasília, Tradução Suzana Gontijo. 2006; 60. Reference Source\n\nIBGE, Instituto Brasileiro de Geografia e Estatística: Resultado dos Dados Preliminares do Censo 2010. Boletim, Rio de Janeiro. 2011; 33: 1–133.\n\nGomes MM, Turra CM, Fígoli MG, et al.: Passado e presente: condições de vida na infância e mortalidade de idosos. Revista de Saúde Pública. 2015; 49: 2–11. Publisher Full Text\n\nMoura R, et al.: Os vários Paranás: diversidade, desigualdade e inserção diferenciada na divisão social do trabalho. Revista Paranaense de Desenvolvimento-RPD. 2011; 111: 145–150. Reference Source\n\nIPARDES, Instituto Paranaense de Desenvolvimento Econômico e Social: Caderno estatístico dos Municípios 2018.\n\nSharp ES, Gatz M: Relationship between education and dementia: an updated systematic review. Alzheimer Dis Assoc Disord. 2011; 25(4): 289–304. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPinto PALA: Um Estudo sobre a Distribuição Espacial dos Médicos no Brasil. Tese (Pós Graduação em Economia) - Universidade Federal de Pernambuco, 2015; 125. Reference Source\n\nMendonÇa FF, de Mattos LF, de Oliveira EB, et al.: Participation of small municipalities in the Mais Médicos para o Brasil (More Doctors to Brazil) Program in the macro-region of Northern Paraná. Ciência e Saúde Coletiva. 2016; 21(9): 2907–2915. Publisher Full Text\n\nAraujo AMGD, et al.: Linguagem Em Idosos Com DoenÇa De Alzheimer: Uma RevisÃo SistemÁtica. Revista CEFAC, João Pessoa, PB. 2015; 17(5): 1657–1663. Publisher Full Text\n\nChen C, Zhang J, Hua S, et al.: An updated Meta-Analysis of cohort studies: diabetes and risk of Alzheimer’s disease. Diabetes Res Clin Pract. 2017; 124: 41–47. PubMed Abstract | Publisher Full Text\n\nSchilling MA: Unraveling Alzheimer’s: Making Sense of the Relationship between Diabetes and Alzheimer’s Disease. J Alzheimers Dis. 2016; 51(4): 961–977. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSantos ALM, Fraga VG, Magalhães CA, et al.: Doença de alzheimer e diabetes mellitus tipo 2: qual a relação? Rev Bras Neurol. 2017; 53(4): 17–26. Reference Source\n\nPARANÁ, Governo do Estado do Paraná: Plano Estadual De SaÚde ParanÁ 2016 - 2019. 2016; 200. Reference Source\n\nShinohara M, Sato N: Bidirectional interactions between diabetes and Alzheimer's disease. Neurochem Int. 2017; 108: 296–302. PubMed Abstract | Publisher Full Text\n\nTruzzi A, Laks J: Doença de Alzheimer esporádica de início precoce. Rev Psiquiatr Clin. 2005; 32(1): 43–46. Publisher Full Text\n\nSantos AFA: Avaliação da qualidade da codificação atribuída aos diagnósticos nas internações em um hospital de pequeno porte no Vale do Paraíba. São Paulo. Dissertação (Saúde Pública) - Universidade De SÃo Paulo. 2014; 70. Publisher Full Text"
}
|
[
{
"id": "84519",
"date": "18 May 2021",
"name": "Aleksi Hamina",
"expertise": [
"Reviewer Expertise 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\nThis is a retrospective pharmacoepidemiological study on the use of acetylcholinesterase inhibitors (AChEI) among older residents of Paraná, Brazil. The topic is of importance due to Brazil’s heavily increasing older population and due to the apparent lack of studies concerning the subject. The authors have framed the research as a study on the prevalence of Alzheimer’s disease (AD) in the population, but I disagree with this framing for multiple reasons. However, despite obvious limitations, the study offers interesting data on drug utilization. Here are some of the issues I would wish the authors to comment on:\nAs I mentioned, the study is framed as a study on the prevalence of AD. The study suggests a low prevalence of AChEI in the population, which may be considered somewhat unsurprising. Could the authors explain why they suggest using drug use as a proxy for disease prevalence in this case? I would suggest reframing this as a drug utilization study throughout the manuscript.\n\nCould the authors describe the diagnosis process? The manuscript mention that the diagnosis criteria are based on international standards, but these standards are not referenced. In the discussion, the authors write that the diagnosis is confirmed, but there is no mention of how this confirmation is made earlier in the text.\n\nWhy is memantine not investigated in this study? If available, it should be included in the analyses. If not, it should be discussed as a limitation.\n\nDespite obvious pitfalls in using AChEI prevalence as a proxy for AD prevalence, the authors make detailed hypotheses on biological reasons for the findings. This is problematic since the analyzed sample of people with probable AD is likely to be very biased. I would suggest removing the discussion related to glucose deregulation from the discussion section completely. Moreover, I suggest the authors add to the limitation section that this sample is likely to be a small part of the people with AD in the Paraná’s population.\n\nRelated to the previous comment, the authors refer to the disproportionality of early AD as inaccurate diagnoses. Could the authors comment on why this theory would be preferable to sampling bias?\n\nAre there previously published estimates as to how large a part of the older population uses the SUS? If not, this should be discussed as a limitation in the discussion.\n\nMinor comments:\nPlease refer to AChEIs systematically with the same term throughout the manuscript.\n\nPlease refer to gender-related terminology systematically, i.e., use “gender” instead of “sex” and “men and women” instead of “males and females”.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "71304",
"date": "19 Jun 2024",
"name": "Peter K Panegyres",
"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\nThe authors describe Alzheimer's disease as a public health problem in the state of Parana in Brazil. They researched over 50,000 patients over 60 years of age who used Brazil's Unified Public Health System and found that the prevalence rate of Alzheimer's disease in Parana is lower than rates of Brazil and worldwide, indicating that Alzheimer's disease is underdiagnosed in the State.\nInteresting research and an excellent article which in my opinion should be indexed.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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/9-641
|
https://f1000research.com/articles/7-622/v2
|
07 Jun 18
|
{
"type": "Research Article",
"title": "Fibroblast growth factor-6 enhances CDK2 and MAKT expression in microvesicles derived from human stem cells extracted from exfoliated deciduous teeth",
"authors": [
"Ahmed Othman",
"Rabab Mubarak",
"Dina Sabry",
"Rabab Mubarak",
"Dina Sabry"
],
"abstract": "Background: Stem cells from human exfoliated deciduous teeth (SHEDs) are considered one of the most convenient sources of adult stem cells. This study aimed to examine the effect of fibroblast growth factor 6 (FGF-6) on SHEDs and evaluate CDK2 and MATK gene expression in SHED-derived microvesicles (MVs). SHEDs were cultured from deciduous teeth pulp. Methods: SHEDs were divided into two groups: the control group and test groups, with and without FGF-6 supplementation, respectively. After the third passage, SHED proliferation was assessed by MTT assay. MVs were purified and CDK2 and MATK gene expression was assessed by real time polymerase chain reaction. SHEDs were identified by their positivity for CD90 and CD73, and negativity for CD45 and CD34. Results: SHEDs proliferation in the test group was significantly higher than in the control group (P<0.001). mRNA from SHED-derived MVs from the test group exhibited a markedly elevated expression of CDK2 and MAKT, (P<0.002 and P<0.005, respectively) in comparison with those of the control group. FGF-6 enhanced the proliferation of SHEDs. Proliferation enhancement is favorable for the production of a large number of stem cells, which will then be beneficial for cell-based therapies. Conclusions: CDK2 and MAKT genes in SHED-derived MVs can be used as molecular biomarkers for SHED proliferation.",
"keywords": [
"Stem cells from human exfoliated deciduous teeth",
"fibroblast growth factor 6",
"microvesicles",
"CDK2",
"MAKT"
],
"content": "Introduction\n\nStem cells from human exfoliated deciduous teeth (SHEDs) are a type of adult stem cell acquired from the dental pulp of human exfoliated deciduous teeth. SHEDs stand out from other types of adult stem cells since they possess a remarkable growth and proliferation rate, providing an adequate stem cell source for any prospective clinical or laboratory use. The natural exfoliation of deciduous teeth provides a good chance to procure and isolate SHEDs without effort or complications, and with little or no trauma. SHEDs have the capacity to differentiate into many cell types, including chondrocytes, osteoblasts, adipocytes, cardiomyocytes and neurons1–3\n\nFibroblast growth factors (FGFs) are a family of secreted cytokine proteins that have a role in the regulation and direction of numerous cellular processes, including proliferation, differentiation, migration or metabolism4. FGF-6 is part of the FGF-4 subfamily of canonical FGFs. Like the other members of FGF-4 subfamily, FGF-6 is a secreted protein with a cleavable N-terminal signal peptide that binds and activates FGF receptors as an extracellular mediator4. Despite the fact that FGF-6 expression is almost completely limited to myogenesis5, it was found to exert a proliferating effect on human osteoblasts under specific conditions6.\n\nRecently, microvesicles (MVs) have been identified as an approach deployed by stem cells as a mean of mediating intercellular interactions7. These phospholipid membrane-bound MVs partake in intercellular interactions, such as proliferation, differentiation and gene expression alteration, via their content of mRNA, miRNA and transfer proteins7,8.\n\nHuman liver stem cell-derived MVs were found to have a role in hepatic regeneration, since they transfer proteins and mRNAs associated with the control of transcription, translation, proliferation, and apoptosis to hepatocytes9. Cyclin-dependent kinase 2 (CDK2) and megakaryocyte-associated tyrosine kinase (MATK) genes are associated with cellular proliferation as they were found in the mRNA of purified MVs at the time of microarray analysis and reverse transcription-quantitative PCR (RT-qPCR)9.\n\nCDK2 is the catalytic subunit of the cyclin-dependent protein kinase complex, which controls advancement through the cell cycle via its involvement in the G1 to S phase transition10,11. MATK has been identified by Avraham et al. as an intracellular tyrosine kinase that participates in the proliferation and survival of megakaryocyte progenitors12. Furthermore, Findings by Herrera et al. demonstrated that MATK conveyed by MVs was one of the genes responsible of liver stem cell proliferation9.\n\nThe current study was performed to evaluate SHED proliferation upon supplementation with FGF-6 in comparison with SHEDs in regular culture medium without FGF-6 supplementation; it also aimed to assess the effect of FGF-6 supplementation on CDK2 and MAKT gene expression in purified proliferated stem cell MVs.\n\n\nMethods\n\nA total of 28 deciduous teeth indicated for extraction were collected from 25 patients at the Pediatric Dentistry Department in Faculty of Dental Medicine, Cairo University. Patient age ranged from 7 to 12 years. Collection was done at the pediatric clinic over 3 days, we looked for deciduous teeth indicated for extraction due to their natural shedding time in order to make room for their permanent successors, so no ethical concerns would arise. Deciduous tooth collection was conducted after obtainment of the guardians’ written informed consent at Pediatric Dentistry Department in the Faculty of Dental Medicine Cairo University, with the approval of the Ethics Committee of the Faculty of Oral and Dental Medicine, Cairo University. Subjects were identified by their treating physician, following which we contacted the guardians of the subjects for consent to use the extracted teeth. Stem cell propagation (at the Medical Biochemistry Department in the Faculty of Medicine Cairo University) was performed in accordance with recommendations and with the approval of the Ethics Committee of the Faculty of Oral and Dental Medicine, Cairo University.\n\nDeciduous tooth surfaces were washed several times with Dulbecco’s PBS (Biowest, USA). Dental pulp was extracted delicately from teeth using a sterile endodontic barbed broach and placed in falcon tube containing PBS (Biowest, USA).\n\nSHEDs culture and characterization were done after taking established procedures into account13. A total of 3 mg collagenase type II (Sigma Aldrich, USA) was dissolved in PBS to digest the extracted dental pulp tissues for 1 h at 37°C in a 5% CO2 incubator and shaken well at 10 min intervals until the tissues were fully digested. The samples were strained using a cell strainer (40 µm nylon PP) (Bio Basic, Inc., Canada) to remove tissue debris and then centrifuged for 10 min at 3000 rpm at 5°C to obtain pellets of isolated cells. The supernatant fluid was discarded and cell suspension was obtained by pipetting cells in RPMI 1640 culture medium (Biowest, USA). Next, the isolated cell pellets were seeded in 75 cm3 tissue culture flasks for cell culture propagation. Culture medium (RPMI 1640) (was supplemented with 1% Pen/Strep solution (Lonza, USA) and 10% fetal bovine serum (FBS) (Lonza, USA) were supplemented to the culture media to achieve cell propagation at 37°C in humidified CO2 incubator for 7–10 days, with medium changes every 3 days.\n\nCells were identified as being mesenchymal stem cells (MSCs) by their morphology and adherence to the plastic flask. In addition, quantification of several expressed MSCs markers was conducted using flow cytometry analysis. Adherent cells were trypsinized and subjected to centrifugation to form cell pellet. Next, 1×105 cells were incubated with 10 μl monoclonal CD90 PE (catalog number FAB2067A; R&D Systems), CD73PE (catalogue number FAB5795P; R&D systems) CD34 PE (catalogue number FAB72271P; R&D Systems) and CD45 PE (catalog number DAB1430P; R&D Systems) antibodies, at 4°C in the dark. Same species isotypes served as a negative control, Mouse IgG1 PE conjugated antibody (catalog number IC002P; R&D Systems). After a 20 min incubation, 2 ml PBS containing 2% FBS was added to a tube of monoclonal treated cells. The mixtures were then centrifuged for 5 min at 2500 rpm, followed by discarding the supernatant and re-suspending cells in 500 μl PBS containing 2% FBS. Cell analysis was performed using a CYTOMICS FC 500 Flow Cytometer and analyzed using CXP Software version 2.2.\n\nPassaging of SHEDs was done according to established protocols14, with modifications for this experiment. Sub-culturing and passaging was done when adherent cells primary culture (passage zero) have reached 80% confluence. 103–105 cells were seeded into 24-well plates prior to grouping and subsequent passaging till reached third passages. Seeded cells were divided into two groups: control group (SHEDs untreated with FGF-6) and test group (SHEDs treated with FGF6). FGF-6 was added at concentration 20 ng/ml for test group.\n\nMTT reagent, supplied ready for use after the third passage of the SHEDs, was obtained from Tacs Trevigen (Gaithersburg, USA). For the cell viability assay, the two cell groups were seeded in three 96-well tissue culture plates each, at 103 cells/ml per well. The MTT reagent was added and the plate was incubated in the dark for 2–4 h. Detergent reagent (catalog number # 4890-25-02, TACS) was added to each well to solubilize formazan dye prior to absorbance measurement. The absorbance in each well was measured at a range from 490 to 630 nm using an enzyme-linked immunosorbent assay plate reader (Stat Fax 2200, Awareness Technologies, Florida, USA)15.\n\nMVs were obtained from supernatants of third-passage MSCs (5×106 cells/ml) cultured in RPMI-1640 deprived of FBS and supplemented with 0.5% of bovine serum albumin (BSA) (Sigma Aldrich, USA). After centrifugation at 2000g for 20 min to remove debris, cell-free supernatants were centrifuged at 100,000g for 1 h at 4°C, washed in serum-free medium 199 containing 25 mM HEPES (Sigma) and submitted to a second ultracentrifugation under the same conditions16. MVs were then prepared for electron microscopy characterization. Briefly MVs were diluted in 145 µL PBS containing 0.2% paraformaldehyde (w/v). 10 µl was administered to a formvar-carbon-coated 300 mesh grid (Electron Microscopy Sciences, Hatfield, USA) for 7 min, followed by staining with 1.75% uranyl acetate (w/v). Samples were left to dry at room temperature for 2 h and imaged by transmission electron microscopy (TEM) (CM-10, Philips, Eindhoven, The Netherlands) at 100 kV afterwards17.\n\nTotal RNA was isolated from MVs using an RNA purification kit (Gene JET, Kit, #K0731, Thermo Fisher Scientific, Inc.). RNA quantification using RT-qPCR was achieved using a one-step reaction (SensiFAST™ SYBR® Hi-ROX One-Step Kit, catalog no. PI-50217 V; Bioline, UK). Sequence-specific primers (Bio Basic, USA) for the studied target genes (CDK2 and MATK) and reference housekeeping gene (β-actin) were used. The prepared reaction mix samples were applied in real time PCR (StepOne Applied Biosystem, Foster city, USA). The cDNA was subsequently amplified using a SYBRGreen I PCR Master kit (Fermentas) in a 48-well plate as follows: 10 min at 95°C for enzyme activation, followed by 40 cycles of 15 s at 95°C, 20 s at 60°C and 30 s at 72°C for the amplification step. Changes in the expression of each target were normalized relative to the mean Cq values of β-actin as housekeeping gene by the 2−∆∆Cq method. We used 1 µM of both primers specific for each target gene. Primers sequences were as follows: CDK2 sense, 5'-AATCCGCCTGGACACTGAGA-3' and antisense, 5'-CCAGCAGCTTGACAATATTAGGA-3' (Genbank accession number XM011537732.1); MATK sense, 5'-CCGCGACGTCATCCACTAC-3' and antisense, 5'-TTGTAATGCTCCACCATGTCCAT-3' (Genbank accession number AH006874.3); and GAPDH sense, 5'-GGCGGCACCACCATGTACCCT-3' and antisense, 5'-AGGGGCCGGACTCGTCATACT-3' (Genbank accession number AAG01996.1).\n\nData were coded and entered using SPSS version 23. Data are presented as the median and interquartile range for quantitative data Comparisons between quantitative variables were done using the non-parametric Mann-Whitney test. Correlations between quantitative variables were done using Spearman’s correlation coefficient. P-values less than 0.05 were considered as statistically significant.\n\n\nResults\n\nThe SHEDs in culture were identified morphologically by their fusiform fibroblast like form for both groups. SHEDs in the test group exhibited an increase in proliferation during culture and passaging, when compared with the control group (Figure 1). Flow cytometric analysis SHEDs were identified as they were negative for CD34 and CD45 and positive for CD90 and CD73 (Figure 2A).\n\n(A) Passage one shows stem cells with spindle-like morphology as grow from human exfoliated deciduous teeth pulp in few number. (B and C) Passages two (B) and three (C) show an increase in number of stem cells with spindle-like morphology. Isolation, morphological observation of stem cells from human exfoliated deciduous teeth in the test group through phase contrast microscopy. (D) Passage one shows a marked increase in number and confluency of stem cells with spindle-like morphology in comparison with control group in passage one. (E and F) Passages two (E) and three (F) show a pronounced, confluent and expanded SHED with fibroblast like morphology in relation to control groups of second and third passages.\n\n(A) Flow cytometry analysis of CD90, CD73, CD34 and CD45 for stem cell characterization. (B) Electron microscopy ultrastructure of released microvesicles (black arrow) from the mesenchymal stem cells of dental pulp.\n\nThe proliferated SHEDs were assessed using MTT assay. The viability of the cells in the test group was significantly higher (P<0.001) in comparison with that of the control group (Table 1).\n\nData presented as median (IQR).\n\nSHEDs, stem cells from human exfoliated deciduous teeth; FGF-6, fibroblast growth factor-6.\n\nTEM detected MVs purified from SHED after ultracentrifugation (Figure 2B). MVs were characterized by their size (500 nm), as detected by TEM.\n\nPurified MVs demonstrated a significant positive expression intensity of CDK2 (P=0.002) and MATK (P=0.005) in the test group in relation with the control group. A box plot (Figure 3) shows that expression of CDK2 and MATK is higher in the test group than the control group, as they display a higher interquartile range (IQR) and medium. The correlation coefficients of CDK2 and MATK expression to cell viability were positive for both genes in the test group (Figure 4).\n\n(A and B) Expression of each gene is higher in the FGF-6-supplemented group than in the control group, since higher interquartile range (IQR) and median values are observed.\n\n(A) Expression of CDK2 is positively correlated with cell proliferation in the test group. (B) Expression of MATK is positively correlated with cell proliferation in the test group.\n\n\nDiscussion\n\nThe present study was developed to deal with difficulties sourcing stem cells and a lack of sufficient stem cells for reliable tissue formation. The study focused on stem cells isolated from human deciduous teeth (SHEDs) and tissue-inducing substances, which in this case is FGF-6. The reason we chose SHEDs for the isolated stem cells is that they present an opportune source of adult stem cells; the deciduous teeth are naturally exfoliating so there are no ethical problems surrounding their use, and the isolation of cells is simple, painless, convenient and time-efficient19.\n\nThere are several criteria for SHED identification; they were identified as stem cells by their morphology under the inverted microscope, as they appeared as fibroblast-like cells. Another feature exhibited was that they have a plastic adherence feature under our normal culture conditions1. SHEDs were also identified as ectomesenchymal stem cells through the quantification of several expressed mesenchymal stem cell markers using flow cytometry; they were shown to be positive for CD90 and CD73, and negative for CD45 and CD3420.\n\nIn this case, the stem cells are quiescent21, unlike progenitor cells, meaning growth factor treatment is required to produce a large amount of cells. In our experiment, FGF-6 was chosen as it has, to our knowledge, never used on SHEDs before, and would avoid the conflicting reports of the effects of bFGF on SHEDs22,23.\n\nMVs were isolated from the proliferated SHEDs for three reasons. The first reason was to assess the two groups of proliferated SHEDs (i.e. the control group and the group supplemented with FGF-6) via the expression of CDK2 and MATK. The second reason was to explore the MVs RNA since the physiological functions of MVs in many tissues remain mostly unknown; on the other hand, their potential roles in pathological settings have been studied largely in the fields of oncology and immunology. The third reason was that the MVs have great potential for use in research24. It was demonstrated that the cell-derived MVs are found to be able to change phenotypes of different cells to become stem cells via epigenetic reprograming or infectious particle transfer24.\n\nIn this study, the CDK2 gene was used to obverse SHEDs proliferation since it has been used to monitor proliferation in many types of stem cells, such as neural progenitor stem cells25 and liver stem cells9. CDK2 also encodes a serine/threonine protein kinase family member, with receptors in this family having a role in the regulation of cell proliferation, programmed cell death, cell differentiation, and embryonic development26.\n\nMegakaryocyte-associated tyrosine kinase is the enzyme which is encoded by MATK in humans. This enzyme possesses a similar amino acid sequence to tyrosine-protein kinase CSK. It was chosen for our experiment as it is not frequently used for the assessment of SHED proliferation, to evaluate whether this ambiguous gene can be studied in further research to assess the proliferation rate of SHEDs and other types of stem cells it is known to be capable of phosphorylating and inactivating Src family kinases, and may inhibit T-cell proliferation12.\n\n\nConclusion\n\nFrom the present study, it can be concluded that SHED proliferation was significantly higher in culture medium with FGF-6 present than in culture medium with no FGF-6; there was a corresponding increase in positive expression of CDK2 and MATK mRNA from extracted MVs. The intensity of CDK2 and MATK gene expression in SHEDs supplemented with FGF-6 was significantly higher than SHEDs not supplemented with FGF-6.\n\n\nData availability\n\nDataset 1. Raw data for the MTT cell viability assay and for reverse transcription-quantitative PCR. DOI: 10.5256/f1000research.14900.d20379618.",
"appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nMiura M, Gronthos S, Zhao M, et al.: SHED: stem cells from human exfoliated deciduous teeth. Proc Natl Acad Sci U S A. 2003; 100(10): 5807–5812. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRai S, Kaur M, Kaur S: Applications of stem cells in interdisciplinary dentistry and beyond: an overview. Ann Med Health Sci Res. 2013; 3(2): 245–254. PubMed Abstract | Free Full Text\n\nYin Z, Wang Q, Li Y, et al.: A novel method for banking stem cells from human exfoliated deciduous teeth: lentiviral TERT immortalization and phenotypical analysis. Stem Cell Res Ther. 2016; 7: 50. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOrnitz DM, Itoh N: The Fibroblast Growth Factor signaling pathway. Wiley Interdiscip Rev Dev Biol. 2015; 4(3): 215–266. PubMed Abstract | Publisher Full Text | Free Full Text\n\nArmand AS, Laziz I, Chanoine C: FGF6 in myogenesis. Biochim Biophys Acta. 2006; 1763(8): 773–8. PubMed Abstract | Publisher Full Text\n\nBosetti M, Leigheb M, Brooks RA, et al.: Regulation of osteoblast and osteoclast functions by FGF-6. J Cell Physiol. 2010; 225(2): 466–471. PubMed Abstract | Publisher Full Text\n\nSabin K, Kikyo N: Microvesicles as mediators of tissue regeneration. Transl Res. 2014; 163(4): 286–295. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEttelaie C, Collier ME, Maraveyas A, et al.: Characterization of physical properties of tissue factor containing microvesicles and a comparison of ultracentrifuge-based recovery procedures. J Extracell Vesicles. 2014; 3(1): 23592. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHerrera MB, Fonsato V, Gatti S, et al.: Human liver stem cell-derived microvesicles accelerate hepatic regeneration in hepatectomized rats. J Cell Mol Med. 2010; 14(6 B): 1605–1618. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJulian LM, Carpenedo RL, Rothberg JL, et al.: Formula G1: Cell cycle in the driver’s seat of stem cell fate determination. BioEssays. 2016; 38(4): 325–332. PubMed Abstract | Publisher Full Text\n\nWierød L, Rosseland CM, Lindeman B, et al.: CDK2 regulation through PI3K and CDK4 is necessary for cell cycle progression of primary rat hepatocytes. Cell Prolif. 2007; 40(4): 475–487. PubMed Abstract | Publisher Full Text\n\nAvraham S, Jiang S, Ota S, et al.: Structural and functional studies of the intracellular tyrosine kinase MATK gene and its translated product. J Biol Chem. 1995; 270(4): 1833–1842. PubMed Abstract | Publisher Full Text\n\nHuang GT, Gronthos S, Shi S: Mesenchymal stem cells derived from dental tissues vs. those from other sources: their biology and role in regenerative medicine. J Dent Res. 2009; 88(9): 792–806. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhang N, Chen B, Wang W, et al.: Isolation, characterization and multi-lineage differentiation of stem cells from human exfoliated deciduous teeth. Mol Med Rep. 2016; 14(1): 95–102. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShabbir A, Cox A, Rodriguez-Menocal L, et al.: Mesenchymal Stem Cell Exosomes Induce Proliferation and Migration of Normal and Chronic Wound Fibroblasts, and Enhance Angiogenesis In Vitro. Stem Cells Dev. 2015; 24(14): 1635–1647. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCollino F, Deregibus MC, Bruno S, et al.: Microvesicles derived from adult human bone marrow and tissue specific mesenchymal stem cells shuttle selected pattern of miRNAs. PLoS One. 2010; 5(7): e11803. PubMed Abstract | Publisher Full Text | Free Full Text\n\nvan der Pol E, Coumans FA, Grootemaat AE, et al.: Particle size distribution of exosomes and microvesicles determined by transmission electron microscopy, flow cytometry, nanoparticle tracking analysis, and resistive pulse sensing. J Thromb Haemost. 2014; 12(7): 1182–1192. PubMed Abstract | Publisher Full Text\n\nOthman A, Mubarak R, Sameer M, et al.: Dataset 1 in: Fibroblast growth factor-6 enhances CDK2 and MAKT expression in microvesicles derived from human stem cells extracted from exfoliated deciduous teeth. F1000Research. 2018. Data Source\n\nMa L, Makino Y, Yamaza H, et al.: Cryopreserved Dental Pulp Tissues of Exfoliated Deciduous Teeth Is a Feasible Stem Cell Resource for Regenerative Medicine. Câmara NOS ed. PLoS One. 2012; 7(12): e51777. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVishwanath VR, Nadig RR, Nadig R, et al.: Differentiation of isolated and characterized human dental pulp stem cells and stem cells from human exfoliated deciduous teeth: An in vitro study. J Conserv Dent. 2013; 16(5): 423–428. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCheng T, Scadden D: Handbook of Stem Cells, Two-Volume Set: Volume 1-Embryonic Stem Cells... Chapter 8-Google Books. 2014.\n\nSukarawan W, Nowwarote N, Kerdpon P, et al.: Effect of basic fibroblast growth factor on pluripotent marker expression and colony forming unit capacity of stem cells isolated from human exfoliated deciduous teeth. Odontology. 2014; 102(2): 160–166. PubMed Abstract | Publisher Full Text\n\nNowwarote N, Pavasant P, Osathanon T: Role of endogenous basic fibroblast growth factor in stem cells isolated from human exfoliated deciduous teeth. Arch Oral Biol. 2015; 60(3): 408–415. PubMed Abstract | Publisher Full Text\n\nQuesenberry PJ, Aliotta JM: Cellular phenotype switching and microvesicles. Adv Drug Deliv Rev. 2010; 62(12): 1141–1148. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJablonska B, Aguirre A, Vandenbosch R, et al.: Cdk2 is critical for proliferation and self-renewal of neural progenitor cells in the adult subventricular zone. J Cell Biol. 2007; 179(6): 1231–1245. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDe Bondt HL, Rosenblatt J, Jancarik J, et al.: Crystal structure of cyclin-dependent kinase 2. Nature. 1993; 363(6430): 595–602. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "35756",
"date": "16 Jul 2018",
"name": "Asma Abdullah Nurul",
"expertise": [
"Reviewer Expertise stem cell",
"regenerative medicine",
"molecular immunology"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors have presented a work on the effects of fibroblast growth factor-6 on the expression of CDK2 and MATK in microvesicles derived from stem cells from exfoliated deciduous teeth (SHED). In the study, the authors have shown that treatment with FBF-6 enhances the proliferative activity as well as the expressions of CDK2 and MATK in MV derived from SHED. This study is somewhat preliminary and the manuscript requires modifications to further improve it.\nThere is inconsistency in the usage of terminology for example MATK was misspelled with MAKT many times (also in the title).\n\nFigure 2 should include population gating and isotype control.\n\nThe authors should provide r value of the correlation coefficients of the CDK2 and MATK expressions to cell proliferative activity as shown in Figure 4.\n\nConclusion need to be rephrased.\n\nThe language use in the manuscript is unsuitable for publication. I would like to suggest submitting the manuscript for professional English editing.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
}
] | 2
|
https://f1000research.com/articles/7-622
|
https://f1000research.com/articles/9-636/v1
|
23 Jun 20
|
{
"type": "Review",
"title": "Epidemiology of mental health problems in COVID-19: a review",
"authors": [
"Md. Mahbub Hossain",
"Samia Tasnim",
"Abida Sultana",
"Farah Faizah",
"Hoimonty Mazumder",
"Liye Zou",
"E. Lisako J. McKyer",
"Helal Uddin Ahmed",
"Ping Ma",
"Samia Tasnim",
"Abida Sultana",
"Farah Faizah",
"Hoimonty Mazumder",
"Liye Zou",
"E. Lisako J. McKyer",
"Helal Uddin Ahmed",
"Ping Ma"
],
"abstract": "The novel coronavirus disease 2019 (COVID-19) has become a pandemic affecting health and wellbeing globally. In addition to the physical health, economic, and social implications, the psychological impacts of this pandemic are increasingly being reported in the scientific literature. This narrative review reflected on scholarly articles on the epidemiology of mental health problems in COVID-19. The current literature suggests that people affected by COVID-19 may have a high burden of mental health problems, including depression, anxiety disorders, stress, panic attack, irrational anger, impulsivity, somatization disorder, sleep disorders, emotional disturbance, posttraumatic stress symptoms, and suicidal behavior. Moreover, several factors associated with mental health problems in COVID-19 are found, which include age, gender, marital status, education, occupation, income, place of living, close contact with people with COVID-19, comorbid physical and mental health problems, exposure to COVID-19 related news and social media, coping styles, stigma, psychosocial support, health communication, confidence in health services, personal protective measures, risk of contracting COVID-19, and perceived likelihood of survival. Furthermore, the epidemiological distribution of mental health problems and associated factors were heterogeneous among the general public, COVID-19 patients, and healthcare providers. The current evidence suggests that a psychiatric epidemic is cooccurring with the COVID-19 pandemic, which necessitates the attention of the global health community. Future epidemiological studies should emphasize on psychopathological variations and temporality of mental health problems in different populations. Nonetheless, multipronged interventions should be developed and adopted to address the existing psychosocial challenges and promote mental health amid the COVID-19 pandemic.",
"keywords": [
"COVID-19",
"Coronavirus",
"Mental Health",
"Mental Disorders",
"Epidemiology",
"Review"
],
"content": "Introduction\n\nThe novel coronavirus disease 2019 (COVID-19) caused by the SARS-CoV-2 virus has become a pandemic with a growing number of cases globally1. With the rapid spread of the COVID-19, global health systems are experiencing critical challenges in preventing infections, identifying and managing COVID-19 cases, and ensuring effective strategies to protect public health2,3. These challenges, although primarily emerging from an infectious disease with physical health implications, may also affect mental health and wellbeing profoundly4,5. People around the world are grappled with fear and worry about their personal safety, a lack of effective vaccine or treatment, and adverse socioeconomic consequences like unemployment and lack of access to necessary commodities resulting from quarantine and lockdown measures in different contexts6,7. These issues may have multiple impacts on mental health across populations, which necessitates the attention of global health researchers and practitioners.\n\nPrevious studies suggest that depression, anxiety disorders, substance abuse, increased suicidal tendencies, and PTSD commonly follow major economic crises or natural disasters8–10. If similar patterns hold for the COVID-19 pandemic, the psychological effects of persistence stress among the general population and exacerbation of several mental health disorders among the vulnerable individuals will further strain the current health care system. It may also prevent resumption to normal life for many people when the physical threat to viral infection eventually subsides. The disruption of a normal life as a result of a government-imposed lockdown or stay home orders has significantly impacted the mental health of the affected individuals6,7. A recent umbrella review of mental health outcomes of quarantine and similar prevention strategies has found that depression, anxiety disorders, mood disorders, posttraumatic stress symptoms, sleep disorders, panic, stigmatization, low self-esteem, lack of self-control are highly prevalent among individuals impacted with physical isolation11. Another rapid review suggested that stressors like prolonged quarantine, fear of infection, frustration, boredom, inadequate supplies, inadequate information, financial loss, and stigma have resulted in long-lasting posttraumatic stress symptoms, confusion, and anger in the mass population12.\n\nThe psychological impact of COVID-19 among individuals who are tested positive is another global health concern13. Although the psychological dimensions of COVID-19 are yet to be understood, previous studies suggest that infectious outbreaks critically affect the mental health of the patients who may experience anxiety symptoms, fear, and a lack of hope regarding the uncertainties in treatment and health outcomes11. Several factors influence mental health in this vulnerable population, which include isolation after being diagnosed with the disease, stigma and discrimination, prolonged hospitalization, and a lack of social support7,11,14. In COVID-19, these challenges may become more prevalent alongside psychosocial stressors experienced by people in general. Moreover, patients with limited access to healthcare services or those who have preexisting conditions are likely to have higher psychological stress during this pandemic15,16. Furthermore, multiple mental health problems may have co-existed among individuals and populations even before the onset of the pandemic17,18, which may increase their susceptibility to adverse mental health outcomes following a COVID-19 diagnosis. Increasing reports and studies inform that patients with COVID-19 may have depression, anxiety disorders, psychological distress, and suicidal behavior14,19,20, which necessitates an in-depth understanding of the mental health epidemiology during this pandemic.\n\nCOVID-19 may also affect the mental health and wellbeing among healthcare professionals, especially among those who are working as frontline providers21,22. As COVID-19 cases are impacting the capacities of health systems globally, many of the healthcare providers are working beyond their regular schedules to meet the increased demand for critical care. This makes those healthcare providers susceptible to anxiety, depression, burnout, and insomnia22,23. A review suggests that physicians face frequent challenges in providing care to their patients, whereas the healthcare systems often mandate the physicians to keep records of their physician-patient encounters along with several clerical responsibilities24, which is likely to increase during this pandemic. Moreover, working without adequate personal protective equipment and other preventive measures increases the fear of contracting the infection, which is becoming a growing concern as a high prevalence of infection among healthcare providers is reported in China, Italy, and the USA25–27. Furthermore, a lack of social support, working under stress, guilt about suboptimal care to the patient or leaving hospitals understaffed, or and worrying about their families may result in critical mental health challenges among healthcare providers amid COVID-1923,28–30.\n\nIn this narrative literature review, we aimed to describe the epidemiology of mental health problems in COVID-19 from available articles retrieved from Medline and Google Scholar using a non-systematic approach. The epidemiological burden of mental health problems is primarily discussed as the prevalence, proportion, or rate of mental health problems from studies that focused on individuals or populations affected by COVID-19. Secondarily, key factors associated with those problems are summarized to inform the risk and protective factors as well as highlight the distributions of epidemiological burden based on variations in those factors. Lastly, we discussed how the current epidemiological evidence might inform future research, policy development, and practice for improving global mental health amid COVID-19 pandemic and future public health emergencies.\n\n\nLiterature review on the epidemiology of mental health problems in COVID-19\n\nGeneral population. Several studies have reported the burden of mental health problems among the general population during COVID-1913,19,31–42. For example, a study by Lei and colleagues used self-rating anxiety scale (SAS) and the self-rating depression scale (SDS) to evaluate mental health status among 1593 respondents aged 18 years and above in Southern China35. In this study, the prevalence of anxiety and depression was 8.3% and 14.6%, respectively. Moreover, the prevalence was much higher (12.9%, 22.4%) among individuals who had someone in their social network who had been quarantined compared to the remaining respondents (6.7%, 11.9%). Another cross-sectional study by Liang and colleagues assessed mental health among 584 youths using the General Health Questionnaire (GHQ-12), Negative coping styles scale, and PTSD Checklist-Civilian Version (PCL-C)36. In this study, nearly 40.4% of participants had psychological problems, whereas 14.4% of participants had posttraumatic stress disorder (PTSD) symptoms. A similar study assessed depression and anxiety among 8079 Chinese students using Patient Health Questionnaire (PHQ-9) and the Generalized Anxiety Disorder (GAD-7) questionnaire, respectively13. This study found that the prevalence of depression, anxiety, and a combination of both was 43.7%, 37.4%, and 31.3%, respectively.\n\nPublic health interventions like quarantine and isolation may have impacted mental health during COVID-19, which was assessed in several studies. Tang and colleagues assessed PTSD and depressive symptoms in 2485 home quarantined participants from 6 universities using PTSD Checklist Civilian Version and PHQ-9, and data on sleep duration37. This study found that the prevalence of PTSD and depression was 2.7% and 9.0%, respectively. Other mental health problems included feeling extreme fear and short sleep durations.\n\nMental health at the population level was evaluated using diverse methods. For example, Li and colleagues analyzed Weibo posts from 17,865 active Weibo users using several machine-learning predictive models38. They calculated word frequency and scores of emotional (anxiety, depression, and indignation, for example)) and cognitive (social risk judgment and life satisfaction) indicators before and after the declaration of COVID-19 outbreak on 20 January 2020. This study found that negative emotions like anxiety, depression, indignation, and sensitivity to social risks increased over time, whereas the scores of positive emotions like Oxford happiness and life satisfaction decreased. Another study using the WeChat-based survey among 1577 community-based adults Wuhan, China, used validated GAD-2 (cutoff ≥3) and PHQ-2 (cutoff ≥3) to assess anxiety and depression, respectively39. The prevalence of probable anxiety and depression was 23.84% (95% Confidence Interval [CI]: 21.8–26.0) and 19.21% (95% CI: 17.3–21.2), respectively.\n\nIn addition to these cross-sectional studies, a longitudinal study surveyed a total of 1738 respondents from 190 Chinese cities, 333 respondents participated in both sessions of data collection40. This study assessed mental health in the general population two times: first at the beginning of the outbreak and four weeks later, when the outbreak became more widespread. This study found that there was a statistically significant longitudinal reduction in mean Impact of Event Scale-Revised (IES-R) scores (from 32.98 to 30.76, p < 0.01) after four weeks, which was not clinically significant as both scores were above the cut-off for PTSD symptoms. Moreover, the participants had moderate-to-severe stress, anxiety, and depression at the beginning at 8.1%, 28.8%, and 16.5%, respectively, which did not change significantly after four weeks.\n\nWhile most studies have assessed the mental health impacts during COVID-19 in Chinese populations, fewer studies reported how this pandemic impacted mental health and wellbeing at the population level in other countries. For example, a study conducted in Turkey administered Hospital Anxiety and Depression Scale (HADS) and the Health Anxiety Inventory (HAI) among 343 participants and found that 23.6% of the participants scored above the depression cut-off point whereas 45.1% scored higher than the cut-off point for anxiety41. Furthermore, a study conducted among 662 Indian adults found that more than 80% of the participants were preoccupied with COVID-19 related thoughts42. Moreover, sleep difficulties, paranoia about having COVID-19 infection and distress were reported by 12.5%, 37.8%, and 36.4% participants, respectively. Furthermore, more than 80% of participants in this study perceived a need for mental health services.\n\nCOVID-19 has also influenced suicidal behavior among vulnerable individuals in different contexts. Several case studies based on news media reports are found, where individuals committed suicide in fear of COVID-1931–33,43. These cases from India, Pakistan, and Bangladesh have shown how a perceived susceptibility and fear of getting infected has resulted in suicidal attempts among individuals, highlighting a high mental health burden of this pandemic.\n\nPatients with COVID-19. Empirical studies suggest that patients who were tested positive for COVID-19 had experienced adverse mental health outcomes14,19,20,29,44,45. A mixed-method study by Guo and colleagues evaluated and compared the mental status and inflammatory markers among 103 patients tested positive with COVID-19 and recruited 103 matched controls that were COVID-19 negative14. This study revealed that COVID-19 patients had higher levels of depression (p < 0.001), anxiety (p < 0.001), and post-traumatic stress symptoms (p < 0.001) compared to non-COVID controls. Moreover, the levels of C-reactive protein (CRP), which is a peripheral inflammatory indicator, positively correlated with the PHQ-9 total score (R = .37, p = 0.003, Spearman's correlation) of COVID-19 patients with symptoms of depression. Moreover, a web-based cross-sectional survey among 7,236 Chinese individuals identified the overall prevalence of GAD, depressive symptoms, and deteriorated sleep quality as 35.1%, 20.1%, and 18.2%, respectively29. Furthermore, suicidal behavior among individuals who were tested positive for COVID-19 is another mental health concern. A case from India suggests that COVID-19 may critically impact psychosocial wellbeing and influence suicidal attempts among the affected individuals, which may also aggravate if the patient has other comorbid diseases19.\n\nCOVID-19 may have impacted psychological health among COVID-19 positive individuals with preexisting mental health problems differently than patients with no such history. This was assessed in a study by Hao and colleagues, who evaluated mental health among 76 psychiatric patients and 109 healthy controls from Chongqing, China using the IES-R for PTSD symptoms, Depression, Anxiety and Stress Scale (DASS-21) for anxiety, depression, distress, and Insomnia Severity Index (ISI) for sleep disorders44. In this study, the mean scores in IES-R, DASS-21 subscales, and ISI were significantly higher among psychiatric patients than healthy controls (p < 0.001). Moreover, worries about their health, anger issues, impulsivity, and suicidal ideation were higher in psychiatric patients compared to the healthy controls (p < 0.05). About one-third of psychiatric patients fulfilled the diagnostic criteria for PTSD, and more than one-quarter of the recruited psychiatric patients had moderately severe to severe insomnia. This study informs that the severity of adverse psychological effects on psychiatric patients during the COVID-19, which similar to another study by Bo and colleagues45.\n\nMultiple primary studies synthesized in reviews provide collective insights on the epidemiological burden. A recent meta-analytic review of 12 studies assessed mental health among 976 patients with COVID-1920. More of the cases were from Wuhan and other cities in China. These studies provided evidence for neuropsychiatric problems, including delirium. For example, confusion was observed in 65% of the intensive care unit patients, whereas agitation was found in 69% of the intensive care unit patients in one study. Moreover, altered consciousness in 21% of patients who subsequently died as reported in another study. During discharging from the hospital, 33% of COVID-19 patients in a study were experiencing the dysexecutive syndrome.\n\nHealthcare providers. Several studies have evaluated the burden of mental health problems among healthcare providers during COVID-1921,28,46–55. In a single-center cross-sectional survey using a numeric rating scale (NRS) on fear, Hamilton Anxiety Scale (HAMA), and Hamilton Depression Scale (HAMD), 2299 participants were enrolled, including 2042 healthcare providers and 257 administrative staff from the same institution48. In this study, significant differences in the severity of fear, anxiety, and depression were observed between the two groups. In addition, frontline healthcare providers with close contact with COVID-19 patients were 1.4 times more likely to experience fear and nearly twice more likely to experience anxiety and depression compared to the non-clinical staff. Another study by Cao and colleagues found 6.3% of the participating doctors felt nervous after listening to the news on mass media that some doctors were positive for COVID-19, whereas 52.6% of the participating nurses reported negative emotions, worrying about family, fear of infection, and stress about heavy workload49. Moreover, a study from Wuhan assessed mental health in 105 healthcare providers who were positive for COVID-19, which found that 88.3% of the participants experienced either psychological stress or emotional changes during the isolation period50. Furthermore, a comparative assessment by Chen and colleagues found that the mean SDS and SAS scores were significantly higher among Chinese healthcare providers than that of the general Chinese population (47.1 ± 10.5 vs. 41.9 ± 10.6 for SDS; 40.3 ± 11.5 vs. 29.8 ± 10.1 for SAS; Ps < 0.001)51.\n\nSimilar studies in different institutions provide a detailed overview of how COVID-19 has impacted mental health among healthcare providers. A study conducted among 120 healthcare provider medical staff used Symptom Checklist 90 (SCL-90), SAS, SDS, and PCL-C to assess their mental health52. Among the study participants, 60 staff from COVID-19 designated hospitals were assigned to the experimental group, and 60 staff from the non-designated hospital were assigned to the control group. This study found that the SCL-90 scores of somatization, depression, anxiety, and terror were much higher among the staff at the COVID-19 designated hospital. Moreover, the SAS (45.89±1.117), SDS (50.13±1.813), and PCL-C (50.13±1.813) scores were significantly higher in the experimental group than the control participants. Furthermore, the average PSQI score in the experimental group was 16.07±3.761, which informed that the sleep quality was poor among those participants, whereas 61.67% and 26.67% have moderate and severe insomnia, respectively. Another study by Wu and colleagues, the frequency of burnout was assessed and compared between 220 physicians and nurses on the frontline wards and those working in the usual wards using the Maslach Burnout Inventory-medical personnel53. The findings of this study suggest that the frontline participants had a lower frequency of burnout (13% vs. 39%; p < 0.0001) and were less worried about being infected with coronavirus compared with participants from usual wards. This suggests that working as a frontline provider may not be an independent risk factor for mental health problems. Rather, healthcare providers from both frontline and usual wards may have unique mental health needs and both groups should be considered for preventive mental health measures amid this pandemic.\n\nSimilar studies have shown a high prevalence of multiple mental health problems across contexts. For example, a cross-sectional survey in Gansu, China, assessed mental health among 79 doctors and 86 nurses with a questionnaire set consisted of the SAS, SDS, and the simplified coping style questionnaire (SCSQ)54. The prevalence rates of anxiety and depression symptoms among doctors were 11.4% and 45.6%, respectively, whereas the prevalence among nurses was 27.9% and 43.0%, respectively. Moreover, Li and colleagues conducted a cross-sectional study in 948 medical staff in Wuhan and Ningbo, China55. In this study, the medical staff in Wuhan had higher insomnia than in Ningbo (58.90 vs. 24.97%; p = 0.001) and had higher general psychological symptoms (13.24 vs. 8.64%; p = 0.044). Moreover, Li and colleagues conducted a study among 4369 female healthcare providers. The prevalence of depression, anxiety, and acute stress symptoms among the participants was 14.2%, 25.2%, and 31.6%, respectively21. Furthermore, Tan and colleagues assessed psychological effects on the workforce returning to work during the COVID-19 using the IES-R, DASS-21, and ISI scales46. Among the study participants, 10.8% had PTSD symptoms after returning to work. Furthermore, participants in this study had a prevalence of anxiety disorders (3.8%), depression (3.7%), stress (1.5%), and insomnia (2.3%).\n\nAnother study by Kang and colleagues evaluated the mental health status of 994 medical and nursing staff in Wuhan28. In this study, 36.9% of the participants had subthreshold mental health disturbances (mean PHQ-9: 2.4), 34.4% had mild disturbances (mean PHQ-9: 5.4), 22.4% had moderate disturbances (mean PHQ-9: 9.0), and 6.2% had severe disturbance (mean PHQ-9: 15.1) in the immediate wake of the viral epidemic. Studies outside China have shown a similar burden of mental health problems among the healthcare providers during COVID-19. For example, Chew and colleagues assessed mental health among 906 healthcare workers in Singapore and India47. Among the study participants, 48 (5.3%) were screened positive for moderate to very-severe depressive symptoms, 79 (8.7%) had moderate to extremely severe anxiety symptoms, 20 (2.2%) had moderate to extremely severe stress, and 34 (3.8%) had moderate to severe levels of psychological distress. Moreover, a headache was the commonest reported symptom (32.3%), whereas 33.4% of participants reported having more than four neuropsychiatric symptoms.\n\nAge. Several studies reported younger age as a risk factor for mental health problems amid COVID-1928,29,56. For example, Huang and colleagues found that younger people had a significantly higher prevalence of generalized anxiety and depressive symptoms compared to older people29. Moreover, Wang and colleagues reported the anxiety risk of people above 40 years old was 0.40 times (95% CI: 0.16–0.99) than participants aged below 40 years, suggesting that younger individuals had a higher risk of anxiety56. However, Chew and colleagues found mental health problems as more prevalent among older adults, contradicting studies discussed earlier47.\n\nGender. Female gender was reported as a common risk factor for mental health problems in several studies13,21,28,41,55–57. For example, Li and colleagues reported that female patients were more likely to have anxiety (OR = 3.206, 95% CI: 1.073–9.583, p < 0.05) and depression (OR = 9.111, 95% CI: 2.143–38.729, p < 0.01) compared to male patients57. In another study by Guo and colleagues, female patients with COVID-19 had a higher perceived helplessness compared to male patients (Z = 2.56, p = 0.010), and controls of both female (Z = 2.37, p = 0.018) and male genders (Z = 2.87, p = 0.004)14. Another study among medical staffs in Wuhan conducted by Li and colleagues found that the symptoms of insomnia were significantly related to female gender (OR = 1.379, p = 0.042, 95% CI: 0.65–2.17)55.\n\nMarital status. Marital status was associated with mental health status among individuals experiencing mental health problems during COVID-19. Li and colleagues found that insomnia was related to marital status (OR = 0.57, p = 0.046, 95% CI: 0.33–0.99) among medical staff in Ningbo, China55. Another study by Tan and colleagues reported that the severity of psychiatric symptoms in the workforce returning to the workplace was significantly associated with marital status (p < 0.05)46.\n\nEducation. Several studies have reported education as an associated factor of mental health status and problems during the COVID-19 pandemic13,35–37,55,56. For example, Liang and colleagues found that mental health among young participants was significantly related to being less educated (OR = 8.71, 95% CI: 1.97–38.43)36. Also, Lei and colleagues reported that low education was associated with poor mental health outcomes35. In contrast, Zhou and colleagues found that students in senior high school and those with higher grades had a greater prevalence of depressive and anxiety symptoms13. Similarly, Wang and colleagues reported that those with a bachelor's degree group had a depression risk of 0.39 times (95% CI: 0.17-0.87) compared to participants with a master's degree or above56. This evidence highlights that education may have some protective roles as seen in the study by Lei or Liang and colleagues, but later studies emphasized that the added academic stress may affect mental health amid this pandemic.\n\nOccupation and income. Empirical studies reported associations of occupation, income, and economic conditions with susceptibility to mental health problems21,29,35,36,56. For example, Huang and colleagues found that healthcare workers were more likely to have poor sleep quality compared to other occupational groups29. Another study by Liang and colleagues found that participants working as an employee in the local enterprises (OR = 2.36, 95% CI: 1.09-5.09) had an elevated risk of poor mental health outcomes compared to other occupational groups36. Also, lost economic opportunities due to lockdown were reported in a suicide case of a farmer in India, which informs socioeconomic challenges that may critically impact mental health among marginalized individuals43. Moreover, people with property loss and adverse economic conditions are more vulnerable to mental health problems35. These highlight the impacts of occupational stress as well as economic instability or challenges in mental health during COVID-19.\n\nPlace of living and close contact with people with COVID-19. In several studies, the location or place of living was associated with mental health problems among study participants35,41,55. For example, Özdin and colleagues reported that living in urban areas was associated with depression41. Moreover, Lei and colleagues found that individuals living in Chongqing had higher anxiety and depression scores than those living in Yunnan Province35, highlighting two cities in the same country may have different levels of associations with mental health among residents living in those cities. Another study by Li and colleagues found that patients with contact history and people in the epidemic area were more likely to experience depression (OR = 3.267, 95% CI: 1.082-9.597, p <0.05)55. Moreover, close contact with COVID-19 patients impacted mental health and wellbeing. For example, suspected or confirmed cases among family members or relatives were significantly associated with depressive symptoms (OR = 1.93; 95% CI: 1.29-2.86; p = 0.001) among women healthcare providers21, which added psychosocial burden in addition to their occupational stress in hospitals where they spent the most time during COVID-19.\n\nComorbid physical health problems. The presence of comorbid physical health problems like diabetes, cerebrovascular diseases, heart diseases and other chronic conditions as a risk factor associated with mental health problems amid COVID-19 in several studies21,41,44,46,47. For example, accompanying chronic diseases were identified as risk factors for anxiety during COVID-19 in a study by Özdin and colleagues41. Another study by Chew and colleagues found that comorbid physical symptoms experienced in the preceding month were significantly associated with depression (OR = 2.79, 95% CI: 1.54-5.07, p = 0.001), anxiety (OR = 2.18, 95% CI: 1.36-3.48, p = 0.001), stress (OR = 3.06, 95% CI: 1.27-7.41, p = 0.13), and PTSD (OR = 2.20, 95% CI: 1.12-4.35, p = 0.023) among the study participants47.\n\nComorbid mental health problems. People with preexisting mental health problems are highly vulnerable to experience psychological impacts of COVID-1921,35–37,41,44,47,54,55. For example, Hao and colleagues found that comorbid psychiatric illness was significantly associated with higher mean IES-R, DASS depression, anxiety, and stress subscale scores and ISI scores (p < 0.05). The findings of this study inform that the severity of negative mental health impacts was higher among individuals with a clinical history of psychiatric comorbidity44. Also, people with substance use disorders are susceptible to COVID-1958, which may also increase their vulnerability to subsequent mental health problems. In another study, Liang and colleagues found that suffering from PTSD symptoms was significantly associated with mental health (OR = 1.05, 95% CI: 1.03-1.07) among young participants36. Moreover, Zhu and colleagues reported that a history of depression or anxiety was a significant risk factor for doctors (T = -2.644, p = 0.010, 95% CI: -10.514~-1.481), which was also associated with an increased risk of anxiety symptoms (T = -3.635, p = 0.000, 95% CI: -16.360~-4.789) and depression symptoms (T = -2.835, p = 0.005, 95% CI: -18.238~-3.254) among the participating nurses54.\n\nExposure to COVID-19 related news and social media. Several studies have reported that exposure to COVID-19 related social media contents or mass media news was associated with adverse mental health outcomes amid COVID-1929,39. Ni and colleagues found that spending more than 2 hours per day on COVID-19 related news via social media was associated with anxiety and depression among community-based adults39. Another study by Huang and colleagues reported that time spent focusing on the COVID-19 for equal or more than 3 hours per day was associated with generalized anxiety disorder29.\n\nCoping styles. In COVID-19, mental health outcomes among individuals were associated with their coping skills. For example, Liang and colleagues assessed the magnitude and predictors of mental health problems among youth and found that negative coping styles were significantly associated (OR = 1.03, 95% CI: 1.00–1.07) with adverse mental health outcomes36. Similar findings from Zhu and colleagues suggest that the total score of positive coping was negatively correlated with the total score of anxiety (r = -0.182, p = 0.002) and depression (r = -0.253, p = 0.001) among individuals affected by the COVID-19 pandemic54.\n\nPsychosocial support. Psychosocial stressors during COVID-19 had low impacts on individuals who had better psychological and social support from their family and social networks35,39. Lei and colleagues found that individuals with no psychosocial support were highly vulnerable to anxiety and depression during this pandemic35. Moreover, Ni and colleagues reported that social support was associated with a lower risk of anxiety and depression among both health professionals and community-based adults in their study sample39.\n\nOther factors associated with mental health impacts during COVID-19. Several other factors associated with the mental health impacts of COVID-19 has been reported. For example, Wang and colleagues reported that a high level of confidence in doctors, perceived likelihood of survival, personal protective measures, satisfaction with health communication, and a low risk of contracting COVID-19 minimized the risks of adverse mental health outcomes during COVID-1940. Similarly, Tan and colleagues found that personal prevention measures including hand hygiene and wearing face masks and organizational measures, including improvement of workplace hygiene and proactive approaches from the company, were associated with a significantly lower risk of mental health problems (p < 0.05) among healthcare providers46. In addition, stigma to COVID-19 was a major challenge associated with adverse mental health outcomes and events, including suicidal behavior14,59. Moreover, exercise habit was found as a common protective factor of depression, anxiety, and acute stress symptoms in female healthcare providers in a study by Li and colleagues21. Furthermore, participants in this study reported that working in other departments (OR = 0.78; 95% CI: 0.65 to 0.94; p = 0.009) was a protective factor of mental health during COVID-19. Among the study participants, those who worked in the isolation wards had more resigning thoughts (16.9% vs. 7.8% in other departments; p < 0.001) and life-threatening thoughts once infected (67.7% vs. 56.4% in other departments; p < 0.001), which suggests that the placement within workplace had significantly impacted mental health outcomes among the participants.\n\n\nDiscussion\n\nThis review evaluated and narratively synthesized available scholarly articles on the impacts of COVID-19 on mental health in different populations alongside factors associated with mental health conditions in those studies. The available literature suggests that people have a wide range of mental health problems, including depression, anxiety disorders, stress, panic, anger, impulsivity, somatization disorder, sleep disorders, emotional disturbance, PTSD, suicidal behavior, and so on14,21,28,35,37,41,44,46,47,60. Studies have shown that the general population who had been experiencing different levels of psychosocial stressors amid the COVID-19 pandemic had developed mental health problems13,31–42. In addition to general stressors, a fear of ongoing outbreak, susceptibility to infection, exposure, or close contact with someone with COVID-19 affected mental health and wellbeing among the general population35,41. Moreover, individuals with COVID-19 diagnosis had profound psychological distress, anxiety, depression, and other mental health problems compared to those who were not infected14,19,20,29,44. This illustrates the fear of adverse health outcomes due to COVID-19 may have affected mental health, which also highlights the mental health aspect of a physical health problem among those individuals. Furthermore, healthcare providers working as frontline workers or other departments had varying levels of psychosocial burden amid this pandemic28,46–55. For those workers, prolonged exposure or contact to persons with COVID-19, preexisting psychosocial challenges, and low institutional and social support contributed to the deterioration of mental health status28,46,47,50,52. This review also found several socio-demographic and psychosocial factors associated with mental health problems in the COVID-19 pandemic. People of younger and older age had different risks of developing mental health problems28,56, whereas women were at a higher risk of such problems compared to men13,41,56,57. Moreover, marital status, education, and economic challenges, including unemployment, loss of income, or economic opportunities due to lockdown or other social measures were associated with mental health outcomes36,41,46,60. Furthermore, the place of residence or living near outbreak areas impacted mental wellbeing41,55, whereas comorbid physical and mental health problems made individuals highly susceptible to mental health problems during this pandemic35–37,41,44,54,55. In addition, spending more time on social media content or news related to COVID-19, lack of social support, stigma, inadequate personal preventive measures, and working in COVID-designated departments were associated with a high risk of mental health problems amid this pandemic14,29,39,46,61. These findings on mental health burden and associated factors inform several critical insights on the psychosocial epidemiology of this pandemic alongside unique perspectives on how to mitigate mental health problems through future research, policymaking, and practice.\n\nUnderstanding the psychopathological variations in COVID-19. Empirical studies evaluating mental health amid COVID-19 have shown how different factors associated with mental health problems impacted individuals and populations. However, those factors may not affect everyone in a similar way. For example, people with existing mental disorders or poor socioeconomic challenges may have suboptimal mental health status15,17, which highlights the need for a comprehensive assessment of risk factors for specific mental health problems alongside a holistic assessment of overall mental health and wellbeing in those individuals and populations. Moreover, individuals may have different levels of psychological resilience and coping mechanisms to stressful events, which may result in different mental health outcomes among individuals despite having similar exposure to psychosocial stressors62. These unique variations are evident in the existing literature on COVID-19 and mental health, which necessitates more research on psychopathological processes and outcomes in individuals and populations globally.\n\nEvaluating the temporality of mental health impacts of COVID-19. Most studies available on COVID-19 and mental health are cross-sectional in nature, which may not inform the incremental changes in mental health outcomes among the affected individuals. Although some studies recruited matched controls or compared with the earlier mental health status of the individuals14,52, there is a critical need to evaluate such changes using longitudinal study designs. Moreover, such studies should evaluate how different risk factors contribute to psycho-pathogenesis in diverse populations in different contexts over time.\n\nImproving measurements and methodological approaches. Studies in the review have used a wide range of methods and instruments for measuring mental health, which included SAS, SDS, GHQ, PHQ, GAD-7, IES-R, HADS, HAMA, and HAMD. Many of these are widely used across mental health studies63; however, the reliability and validity of these instruments in the context of a pandemic affecting almost every aspect of human life remain unknown. In addition, the meanings of sub-scales or questions in each instrument may appear to be different from the individuals who experienced profound psychosocial stress during this pandemic. One consideration for future research can be focused on the development and adoption of instruments that may provide more accurate measurements of mental health and psychosocial constructs in this pandemic as well as other public health emergencies.\n\nPromoting multi-disciplinary mental health research. COVID-19 has brought unique challenges to all aspects of health and wellbeing, which include social, cultural, educational, occupational, economic, political, and other dimensions of human lives7. This phenomenon is evident in the findings of this review as well; a wide range of factors associated with mental health problems suggest that those should be examined using multi-disciplinary approaches enabling researchers from different disciplines to bring their unique insights and resources to better understand the social determinants of mental health in the era of a pandemic. Although health systems remain on the focus in such research efforts, other social systems and stakeholders should be engaged in future mental health research in COVID-19 and similar disruptive global events64,65.\n\nEmphasizing on research and evidence mapping on mental health. This review found a high burden of different mental health problems reported in empirical studies, case studies, and reviews. However, it cannot be concluded that the current evidence illustrates the true burden of mental health problems for several reasons. One such reason is a critical lack of studies from Africa, the Americas, and South Asia, which informs a lack of mental health research during COVID-19 in those contexts. Perhaps, those countries started having COVID-19 cases after China and European countries, which could be a reason for delayed research and scholarly publications on mental health during COVID-19. Another challenge is the rapidly evolving literature on COVID-19 that provides early stage of research evidence66. As more primary studies become available, it will be necessary to perform a systematic appraisal and synthesis of the evidence of those studies for effective decision-making. These issues should be addressed through strengthening research capacities in resource-constrained contexts and facilitating rigorous evidence synthesis in global mental health.\n\nDeveloping effective mental health interventions and strategies. The current evidence on the epidemiological burden of mental health problems in COVID-19 necessitates the development and implementation of multipronged interventions and strategies addressing the same. In this discourse, the mental health needs in vulnerable populations, including those with preexisting physical and mental health problems, should be assessed and managed carefully. Moreover, as in-person mental health services are most disrupted, psychosocial interventions offered through digital platforms like the internet, social media, mobile phones, and apps are increasingly being adopted67. However, the effectiveness, safety, and quality of such digital mental health interventions should be assessed prior to recommending those in practice68. Moreover, the digital divide in different population groups like those who have low education, old age, or living in a rural area with limited access to technologies may not use these services69. Therefore, context-specific strategies allowing multiple ways for access to mental health should be ensured. In this regard, mental health policies and programs should be revisited and strengthened, considering the operational challenges amid COVID-19.\n\nPrioritizing and integrating mental health in existing systems of care. A high prevalence of mental health problems inform a widespread need for mental health services, whereas most countries lack adequate infrastructure and human resources to provide the same. Earlier, scholars and practitioners have argued about integrating mental health services in primary care70, which may substantially improve access to mental health services globally. Another way to improve mental health care can be strengthening community-based and social health programs and incorporating mental health components with the provision of referral care whenever required71. The appropriateness of such approaches should be assessed for each context and adopted, ensuring infection prevention strategies complementing those approaches.\n\nImproving access to information and mental health resources. Many studies highlighted the fact that access to accurate information was associated with a lower risk of mental health problems. Since the beginning of the pandemic, rumors or misinformation had emerged in mass and social media platforms72. This highlights inadequate health communication amid this pandemic, which might have induced fear and affected mental health globally. It is essential to ensure effective health communication regarding the facts and preventive measures in a timely way to prevent public anxiety and fear of COVID-1972. Moreover, access to resources promoting positive mental health can substantially help in addressing and self-managing mental health challenges among individuals73. Online resources like self-help meditation, mental health education, information on early signs, and caregiving could be helpful, which should be considered for preventing COVID-19 and associated mental health problems.\n\nAddressing mental health inequities. Several social determinants of mental health are identified in the current literature, which emphasizes the roles of the structural inequities in different contexts that influence psychosocial stressors and overall mental health outcomes among the affected individuals. Therefore, addressing inequities in the social determinants of mental health should be a long-term goal of future policymaking and mental health practice74. Such efforts, if adopted, might help in developing resilience to mental health challenges, preventing mental disorders, and promoting positive mental health at the population level.\n\nMobilizing social and community resources and organizations. Public health challenges necessitate mobilizing resources available within and between contexts. In COVID-19, psychosocial challenges experienced by individuals may be addressed by local and regional organizations through enhancing collaboration and extending social capital. Mobilizing two types of social capital, i.e., bonding capital within a context and bridging capital from another context, may help communities to improve their psychosocial wellbeing and other factors associated with mental health75, which can be considered as a strategy amid the COVID-19 pandemic.\n\nStrengthening mental health systems for COVID-19 and future public health emergencies. Health systems around the world have experienced a critical lack of preparedness in fighting COVID-19, which includes the mental health impacts of this pandemic as well. Most nations do not have strong mental health systems that may ensure a continuum of mental health care from prevention to institutional care for severe mental disorders; therefore, those systems may not address the added burden of mental health problems in this pandemic. Perhaps, one of the greatest lessons from COVID-19 would be strengthening mental health systems ensuring resilience to systematic shocks as seen during public health emergencies11. Potential strategies to achieve such resilience may include establishing mental health policies, developing population-based programs, enhancing institutional capacities to develop mental health workforce, revising health systems financing for mental health, engaging communities, and institutions to address barriers to access to mental health and promote positive mental health across populations.\n\nThere are several limitations of this review, which should be acknowledged. First, this is not a systematic review, which makes this review subject to selection bias while searching for databases without a structured format. For the same reason, a rigorous appraisal and synthesis of the global evidence are not done in this review that affects the quality of evidence presented in this study. Second, this review did not focus on a specific mental health problem, which could have provided a more in-depth analysis of the literature on that issue. Third, this review did not focus on context-specific correlates of mental health problems separately, which could have facilitated local-level decision making. Despite these challenges, this narrative review provides a comprehensive overview of mental health epidemiology in COVID-19 based on the early-stage research findings. The limitations of this review should be addressed in future reviews adopting a more systematic approach with the emergence of more epidemiological studies, which would inform substantially better evidence for policymaking and practice.\n\n\nConclusion\n\nCOVID-19 is a global public health emergency with enormous impacts on mental health. This narrative review found that individuals affected in the pandemic may have a high epidemiological burden of depression, anxiety disorders, stress, panic attack, somatization disorder, sleep disorders, emotional disturbance, PTSD symptoms, suicidal behavior, and many more mental health problems. Moreover, a wide range of demographic and psychosocial factors are associated with mental health problems during this pandemic that highlights some people who are especially vulnerable to those adverse outcomes. Taken together, the epidemiological distribution of mental health problems informs a psychiatric epidemic is cooccurring with the COVID-19 pandemic, which is evidently becoming a global health challenge. This evidence should be widely communicated with the general public and global health community to prevent the psychological consequences of COVID-19 in different population groups. Nonetheless, it is essential to identify highly vulnerable individuals and connect them to required care, whereas access to mental health services and resources should be promoted, aiming positive mental health outcomes and psychosocial resilience across populations. This review provides an overview of early research on mental health epidemiology and encourages extensive research and evidence synthesis in the future, exploring the severity and causality of mental health problems. Lastly, evidence-based policymaking and practice should be adopted to guide how those mental health challenges can be mitigated in different contexts amid COVID-19 pandemic and future public health emergencies.\n\nPre-print: A previous version of this article is available as a preprint from PsyArXiv: https://doi.org/10.31234/osf.io/q8e5u76\n\n\nData availability\n\nNo data are associated with this article.",
"appendix": "References\n\nWorld Health Organization: Coronavirus disease (COVID-19) pandemic. 2020; (accessed May 25, 2020). Reference Source\n\nLegido-Quigley H, Asgari N, Teo YY, et al.: Are high-performing health systems resilient against the COVID-19 epidemic? Lancet. 2020; 395(10227): 848–50. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShamasunder S, Holmes SM, Goronga T, et al.: COVID-19 reveals weak health systems by design: Why we must re-make global health in this historic moment. Glob Public Health. 2020; 1–7. PubMed Abstract | Publisher Full Text\n\nPfefferbaum B, North CS: Mental Health and the Covid-19 Pandemic. N Engl J Med. 2020. Publisher Full Text\n\nUnited Nations: COVID-19 and the Need for Action on Mental Health. 2020; (accessed May 25, 2020). Reference Source\n\nNicola M, Alsafi Z, Sohrabi C, et al.: The Socio-Economic Implications of the Coronavirus and COVID-19 Pandemic: A Review. Int J Surg. 2020; 78: 185–193. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHolmes EA, O’Connor RC, Perry VH, et al.: Multidisciplinary research priorities for the COVID-19 pandemic: a call for action for mental health science. Lancet Psychiatry. 2020; 7(6): 547–560. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChaves C, Castellanos T, Abrams M, et al.: The impact of economic recessions on depression and individual and social well-being: the case of Spain (2006-2013). Soc Psychiatry Psychiatr Epidemiol. 2018; 53(9): 977–86. PubMed Abstract | Publisher Full Text\n\nGranados JAT, Christine PJ, Ionides EL, et al.: Cardiovascular Risk Factors, Depression, and Alcohol Consumption During Joblessness and During Recessions Among Young Adults in CARDIA. Am J Epidemiol. 2018; 187(11): 2339–45. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBeaglehole B, Mulder RT, Frampton CM, et al.: Psychological distress and psychiatric disorder after natural disasters: Systematic review and meta-analysis. Br J Psychiatry. 2018; 213(6): 716–22. PubMed Abstract | Publisher Full Text\n\nHossain MM, Sultana A, Purohit N: Mental Health Outcomes of Quarantine and Isolation for Infection Prevention: A Systematic Umbrella Review of the Global Evidence. SSRN Electron J. 2020. Publisher Full Text\n\nBrooks SK, Webster RK, Smith LE, et al.: The psychological impact of quarantine and how to reduce it: rapid review of the evidence. Lancet. 2020; 395(10227): 912–20. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhou SJ, Zhang LG, Wang LL, et al.: Prevalence and socio-demographic correlates of psychological health problems in Chinese adolescents during the outbreak of COVID-19. Eur Child Adolesc Psychiatry. 2020; 1–10. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGuo Q, Zheng Y, Shi J, et al.: Immediate psychological distress in quarantined patients with COVID-19 and its association with peripheral inflammation: a mixed-method study. Brain Behav Immun. 2020; S0889-1591(20)30618-8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKavoor AR: COVID-19 in People with Mental Illness: Challenges and Vulnerabilities. Asian J Psychiatr. 2020; 51: 102051. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGuan WJ, Liang WH, Zhao Y, et al.: Comorbidity and its impact on 1590 patients with Covid-19 in China: A Nationwide Analysis. Eur Respir J. 2020; 55(5): 2000547. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHossain MM, Khan N, Sultana A, et al.: Prevalence of comorbid psychiatric disorders among people with autism spectrum disorder: An umbrella review of systematic reviews and meta-analyses. Psychiatry Res. 2020; 287: 112922. PubMed Abstract | Publisher Full Text\n\nHossain MM, Purohit N, Sultana A, et al.: Prevalence of mental disorders in South Asia: An umbrella review of systematic reviews and meta-analyses. Asian J Psychiatr. 2020; 51: 102041. PubMed Abstract | Publisher Full Text\n\nHossain MM, Tasnim S, Sultana A, et al.: COVID-19 and suicide of an army soldier in India: perspectives on psychosocial epidemiology of suicidal behavior. SocArXiv. 2020. Publisher Full Text\n\nRogers JP, Chesney E, Oliver D, et al.: Psychiatric and neuropsychiatric presentations associated with severe coronavirus infections : a systematic review and meta-analysis with comparison to the COVID-19 pandemic. Lancet Psychiatry. 2020; 1–17. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLi G, Miao J, Wang H, et al.: Psychological impact on women health workers involved in COVID-19 outbreak in Wuhan: a cross-sectional study. J Neurol Neurosurg Psychiatry. 2020; jnnp-2020-323134. PubMed Abstract\n\nSultana A, Sharma R, Hossain M: Burnout Among Healthcare Providers During COVID-19 Pandemic : Challenges and Evidence-based Interventions. SocArXiv Pap. 2020. Publisher Full Text\n\nPappa S, Ntella V, Giannakas T, et al.: Prevalence of depression, anxiety, and insomnia among healthcare workers during the COVID-19 pandemic: A systematic review and meta-analysis. Brain Behav Immun. 2020; S0889-1591(20)30845-X. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPatel RS, Bachu R, Adikey A, et al.: Factors related to physician burnout and its consequences: A review. Behav Sci (Basel). 2018; 8(11): 98. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEhrlich H, McKenney M, Elkbuli A: Protecting our healthcare workers during the COVID-19 pandemic. Am J Emerg Med. 2020. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWang J, Zhou M, Liu F: Reasons for healthcare workers becoming infected with novel coronavirus disease 2019 (COVID-19) in China. J Hosp Infect. 2020; 105(1): 100–101. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChirico F, Nucera G, Magnavita N: COVID-19: Protecting Healthcare Workers is a priority. Infect Control Hosp Epidemiol. 2020; 1. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKang L, Ma S, Chen M, et al.: Impact on mental health and perceptions of psychological care among medical and nursing staff in Wuhan during the 2019 novel coronavirus disease outbreak: A cross-sectional study. Brain Behav Immun. 2020. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHuang Y, Zhao N: Generalized anxiety disorder, depressive symptoms and sleep quality during COVID-19 outbreak in China: a web-based cross-sectional survey. Psychiatry Res. 2020; 288: 112954. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWalton M, Murray E, Christian MD: Mental health care for medical staff and affiliated healthcare workers during the COVID-19 pandemic. Eur Hear J Acute Cardiovasc Care. 2020; 9(3): 241–247. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGoyal K, Chauhan P, Chhikara K, et al.: Fear of COVID 2019: First suicidal case in India ! Asian J Psychiatr. 2020; 49: 101989. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMamun MA, Griffiths MD: First COVID-19 suicide case in Bangladesh due to fear of COVID-19 and xenophobia: Possible suicide prevention strategies. Asian J Psychiatr. 2020; 51: 102073. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMamun MA, Ullah I: COVID-19 suicides in Pakistan, dying off not COVID-19 fear but poverty? – The forthcoming economic challenges for a developing country. Brain Behav Immun. 2020. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWang C, Pan R, Wan X, et al.: Immediate psychological responses and associated factors during the initial stage of the 2019 coronavirus disease (COVID-19) epidemic among the general population in China. Int J Environ Res Public Health. 2020; 17(5): 1729. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLei L, Huang X, Zhang S, et al.: Comparison of Prevalence and Associated Factors of Anxiety and Depression Among People Affected by versus People Unaffected by Quarantine During the COVID-19 Epidemic in Southwestern China. Med Sci Monit. 2020; 26: e924609. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLiang L, Ren H, Cao R, et al.: The Effect of COVID-19 on Youth Mental Health. Psychiatr Q. 2020; 1–12. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTang W, Hu T, Hu B, et al.: Prevalence and correlates of PTSD and depressive symptoms one month after the outbreak of the COVID-19 epidemic in a sample of home-quarantined Chinese university students. J Affect Disord. 2020; 274: 1–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLi S, Wang Y, Xue J, et al.: The Impact of COVID-19 Epidemic Declaration on Psychological Consequences: A Study on Active Weibo Users. Int J Environ Res Public Health. 2020; 17(6): 2032. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNi MY, Yang L, Leung CMC, et al.: Mental Health, Risk Factors, and Social Media Use During the COVID-19 Epidemic and Cordon Sanitaire Among the Community and Health Professionals in Wuhan, China: Cross-Sectional Survey. JMIR Ment Heal. 2020; 7(5): e19009. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWang C, Pan R, Wan X, et al.: A longitudinal study on the mental health of general population during the COVID-19 epidemic in China. Brain Behav Immun. 2020. PubMed Abstract | Publisher Full Text | Free Full Text\n\nÖzdin S, Bayrak Özdin Ş: Levels and predictors of anxiety, depression and health anxiety during COVID-19 pandemic in Turkish society: The importance of gender. Int J Soc Psychiatry. 2020; 20764020927051. PubMed Abstract | Publisher Full Text\n\nRoy D, Tripathy S, Kar SK, et al.: Study of knowledge, attitude, anxiety & perceived mental healthcare need in Indian population during COVID-19 pandemic. Asian J Psychiatr. 2020; 51: 102083. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHossain MM, Purohit N, Sharma R, et al.: Suicide of a farmer amid COVID-19 in India: Perspectives on social determinants of suicidal behavior and prevention strategies. SocArXiv. 2020. Publisher Full Text\n\nHao F, Tan W, Jiang L, et al.: Do psychiatric patients experience more psychiatric symptoms during COVID-19 pandemic and lockdown? A case-control study with service and research implications for immunopsychiatry. Brain Behav Immun. 2020; S0889-1591(20)30626–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBo HX, Li W, Yang Y, et al.: Posttraumatic stress symptoms and attitude toward crisis mental health services among clinically stable patients with COVID-19 in China. Psychol Med. 2020; 1–2. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTan W, Hao F, McIntyre RS, et al.: Is returning to work during the COVID-19 pandemic stressful? A study on immediate mental health status and psychoneuroimmunity prevention measures of Chinese workforce. Brain Behav Immun. 2020; S0889-1591(20)30603–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChew NWS, Lee GKH, Tan BYQ, et al.: A multinational, multicentre study on the psychological outcomes and associated physical symptoms amongst healthcare workers during COVID-19 outbreak. Brain Behav Immun. 2020; S0889-1591(20)30523–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLu W, Wang H, Lin Y, et al.: Psychological status of medical workforce during the COVID-19 pandemic: A cross-sectional study. Psychiatry Res. 2020; 288: 112936. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCao J, Wei J, Zhu H, et al.: A Study of Basic Needs and Psychological Wellbeing of Medical Workers in the Fever Clinic of a Tertiary General Hospital in Beijing during the COVID-19 Outbreak. Psychother Psychosom. 2020; 1–3. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJin Y-H, Huang Q, Wang Y-Y, et al.: Perceived infection transmission routes, infection control practices, psychosocial changes, and management of COVID-19 infected healthcare workers in a tertiary acute care hospital in Wuhan: a cross-sectional survey. Mil Med Res. 2020; 7(1): 24. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChen Y, Zhou H, Zhou Y, et al.: Prevalence of self-reported depression and anxiety among pediatric medical staff members during the COVID-19 outbreak in Guiyang, China. Psychiatry Res. 2020; 288: 113005. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWu K, Wei X: Analysis of Psychological and Sleep Status and Exercise Rehabilitation of Front-Line Clinical Staff in the Fight Against COVID-19 in China. Med Sci Monit Basic Res. 2020; 26: e924085. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWu Y, Wang J, Luo C, et al.: A Comparison of Burnout Frequency Among Oncology Physicians and Nurses Working on the Frontline and Usual Wards During the COVID-19 Epidemic in Wuhan, China. J Pain Symptom Manage. 2020; published online April 10. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhu J, Sun L, Zhang L, et al.: Prevalence and Influencing Factors of Anxiety and Depression Symptoms in the First-Line Medical Staff Fighting Against COVID-19 in Gansu. Front Psychiatry. 2020; 11: 386. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLi X, Yu H, Bian G, et al.: Prevalence, risk factors, and clinical correlates of insomnia in volunteer and at home medical staff during the COVID-19. Brain Behav Immun. 2020; published online May 5. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWang Y, Di Y, Ye J, et al.: Study on the public psychological states and its related factors during the outbreak of coronavirus disease 2019 (COVID-19) in some regions of China. Psychol Health Med. 2020:1–10. PubMed Abstract | Publisher Full Text\n\nLi X, Dai T, Wang H, et al.: [Clinical analysis of suspected COVID-19 patients with anxiety and depression]. Zhejiang Da Xue Xue Bao Yi Xue Ban. 2020; 49(2): 203–8. PubMed Abstract | Publisher Full Text\n\nVolkow ND: Collision of the COVID-19 and Addiction Epidemics. Ann Intern Med. 2020; M20–1212. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGriffiths MD, Mamun MA: COVID-19 suicidal behavior among couples and suicide pacts: Case study evidence from press reports. Psychiatry Res. 2020; 289: 113105. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhang C, Yang L, Liu S, et al.: Survey of Insomnia and Related Social Psychological Factors Among Medical Staff Involved in the 2019 Novel Coronavirus Disease Outbreak. Front Psychiatry. 2020; 11: 306. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhang Y, Ma ZF: Impact of the COVID-19 Pandemic on Mental Health and Quality of Life among Local Residents in Liaoning Province, China: A Cross-Sectional Study. Int J Environ Res Public Health. 2020; 17(7): 2381. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTugade MM, Fredrickson BL, Barrett LF: Psychological resilience and positive emotional granularity: Examining the benefits of positive emotions on coping and health. J Pers. 2004; 72(6): 1161–90. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCameron IM, Crawford JR, Lawton K, et al.: Psychometric comparison of PHQ-9 and HADS for measuring depression severity in primary care. Br J Gen Pract. 2008; 58(546): 32–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMazumder H, Hossain MM, Das A: Geriatric Care during Public Health Emergencies: Lessons Learned from Novel Corona Virus Disease (COVID-19) Pandemic. J Gerontol Soc Work. 2020; 63(4): 257–258. PubMed Abstract | Publisher Full Text\n\nHossain MM, Mazumder H, Tasnim S, et al.: Geriatric health in Bangladesh during COVID-19: challenges and recommendations. SocArXiv. 2020. Publisher Full Text\n\nHossain MM: Current status of global research on novel coronavirus disease (COVID-19): a bibliometric analysis and knowledge mapping. F1000Res. 2020; 9: 374. Publisher Full Text\n\nLiu S, Yang L, Zhang C, et al.: Online mental health services in China during the COVID-19 outbreak. Lancet Psychiatry. 2020; 7(4): e17–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYao H, Chen JH, Xu YF: Rethinking online mental health services in China during the COVID-19 epidemic. Asian J Psychiatr. 2020; 50: 102015. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChang BL, Bakken S, Brown SS, et al.: Bridging the digital divide: Reaching vulnerable populations. J Am Med Informatics Assoc. 2004; 11(6): 448–57. PubMed Abstract | Publisher Full Text | Free Full Text\n\nThielke S, Vannoy S, Unützer J: Integrating Mental Health and Primary Care. Prim Care. 2007; 34(3): 571–92. 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}
|
[
{
"id": "71938",
"date": "07 Oct 2020",
"name": "Sarah Alonzi",
"expertise": [
"Reviewer Expertise Mental health during COVID-19",
"PTSD",
"health psych"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nReview of Epidemiology of mental health problems in COVID-19: a review.\nThank you for the opportunity to review this manuscript reporting the prevalence of depression, anxiety, and other adverse mental health effects of the COVID-19 pandemic. This is an interesting narrative review, with useful recommendations for researchers and clinicians. Minor revisions are needed to make findings and implications clearer to readers. I hope these suggestions can be of help!\n\nAbstract The abstract contained minor grammatical and sentence structure issues, which could be remedied as suggested.\nTwo sentences in the abstract, (1) “the current literature” through “behavior” and (2) “moreover” through “survival”, are very long. These sentences can be broken up, or focus on a few of these factors (that authors deem most important for research and practice) to make the focus of the paper clearer to readers.\n\nRemove “on” from “future epidemiological studies should emphasize on psychopathological variations”\nIntroduction\nParagraph 2, sentence 2. I found the sentence hard to read. It could be improved by changing persistence stress to persistent stress.\n\nParagraph 2, final sentence. The fact that the review was “rapid” does not need to be mentioned and playing up the rapid peer review may undermine readers’ confidence in the validity of those findings.\n\nParagraph 3, sentence 1. Change individuals who are tested positive to individuals who have tested positive.\n\nParagraph 4, sentence 2. “As COVID-19 cases are impacting the capacities of health systems” is too vague. Include the way that they are impacting the capacities (e.g., adversely).\nLiterature review on the epidemiology of mental health problems in COVID-19\n\nGeneral population. In this section, it is unclear to me whether you are referring to the “general population” of a specific region, country, etc., or just summarizing existing population-level studies across locations and cultures. I would assume there is some variation in population-level stress across locations, so the term general population might be too broad. In addition to the heading, the body would benefit from more description of the countries and populations these reviews are focused on.\n\nParagraph 5, final sentence. The sentence “furthermore, more than 80% of participants in this study perceived a need for mental health services,” is unclear. Did 80% perceive that they needed MH services, or that there should be MH services made available for the general population to cope with COVID-19?\n\nPatients with COVID-19. Removed were from “Furthermore, suicidal behavior among individuals who were tested positive”.\n\nParagraph 9, sentence 5. The sentence, “moreover, altered consciousness in 21% of patients who subsequently died as reported in another study,” is confusing and needs revision.\nFactors associated with mental health problems during COVID-19\n\nMarital status. As you did in the previous paragraphs, it is necessary to state what marital status is associated with better or worse mental health outcomes.\n\nComorbid mental health problems. The statement, “also, people with substance use disorders are susceptible to COVID-19, which may also increase their vulnerability to subsequent mental health problems” was quite confusing to me. The phrasing makes it seem as if people with substance use disorders are more susceptible than others, which id not supported by the literature.\nDiscussion\nEmphasizing on research and evidence mapping on mental health. The sentence starting with “perhaps, those countries” and ending with “on mental health during COVID-19” should be rephrased. Perhaps is unnecessary in this context, as data show that the countries listed in the previous section did, in fact, begin seeing COVID-19 cases later than China and Europe.\nLimitations\n\nI would recommend adding that most of the literature cited in your review emerged from studies in China at the beginning of what became a global pandemic. As such, these findings might not be consistent across other affected countries and it is unclear how these mental health trends are developing as the pandemic continues.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Partly\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes",
"responses": []
},
{
"id": "69817",
"date": "09 Oct 2020",
"name": "Reindolf Anokye",
"expertise": [
"Reviewer Expertise health psychology",
"mental health",
"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\nThank you for giving me the opportunity to review this article reporting the epidemiology of mental health problems in COVID-19. This is an important well-written article that will contribute to the literature in public health. The authors have done a great job putting this together and I would recommend that this review is published.\nI would also recommend that authors take their time to go through the article and correct any grammatical errors in the manuscript.\nAlso, words such as psychological impact, psychological dimensions and psychological stress should not be used interchangeably with mental health if it is not probably a direct quote. For example, the sentence “psychological dimensions of COVID-19 are yet to be understood” could be rephrased as the mental health burden of COVID-19. Another statement “COVID-19 may have impacted psychological health among COVID-19 positive individuals” could also be rephrased. The consistency in the usage of mental health throughout the manuscript will make it easier for readers who are not expertise in this area to understand what the article is reporting.\nFurthermore, please indicate in the limitations section that most of the included studies were conducted in countries/places that reported most of the early cases of COVID-19.\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/9-636
|
https://f1000research.com/articles/8-2099/v1
|
13 Dec 19
|
{
"type": "Research Article",
"title": "E-cigarette use and onset of first cigarette smoking among adolescents: An empirical test of the ‘common liability’ theory",
"authors": [
"Hui G. Cheng",
"Edward G. Largo",
"Maria Gogova",
"Edward G. Largo",
"Maria Gogova"
],
"abstract": "Background: E-cigarettes have become the most commonly used tobacco products among youth in the United States (US) recently. It is not clear whether there is a causal relationship between e-cigarette use and the onset of cigarette smoking. The “common liability” theory postulates that the association between e-cigarette use and cigarette smoking can be attributed to a common risk construct of using tobacco products. This study aims to investigate the relationship between ever e-cigarette use and cigarette smoking onset in the US using a structural equation modeling approach guided by the “common liability” theory. Methods: The study population is non-institutionalized civilian adolescents living in the US, sampled in the longitudinal Population Assessment of Tobacco and Health study. Information about tobacco product use was obtained via confidential self-report. A structural equation modeling approach was used to estimate the relationship between e-cigarette use at wave 1 and the onset of cigarette smoking at wave 2 after controlling for a latent construct representing a “common liability to use tobacco products.” Results: After controlling for a latent construct representing a “common liability to use tobacco products”, ever e-cigarette use does not predict the onset of cigarette smoking (β=0.10, 95% CI= -0.09, 0.29, p=0.299). The latent “common liability to use tobacco products” is a robust predictor for the onset of cigarette smoking (β=0.42; 95% CI=0.08, 0.76; p=0.015). Conclusions: Findings from this study provide supportive evidence for the ‘common liability’ underlying observed associations between e-cigarette use and smoking onset.",
"keywords": [
"Adolescents",
"e-cigarette",
"cigarette smoking",
"common liability theory"
],
"content": "Introduction\n\nThe prevention of underage cigarette smoking is an essential component to curtail the substantial disease burden associated with cigarette smoking1. The prevalence of underage cigarette smoking has been declining in the US over the past two decades2. E-cigarettes (or e-vapor products) were introduced to the market as potentially reduced-harm alternatives to combustible cigarettes and have gained popularity since they became generally accessible around 20093,4. In the US, the prevalence of youth e-cigarette use increased sharply between 2011 and 2015 and, after a brief decline, increased again in 20183,4. Multiple national surveys have shown that the majority of e-cigarette use is infrequent or experimental4–6. Despite this, the existence of exclusive e-cigarette users who do not use other tobacco products raises concerns about whether e-cigarettes may play a causal role in the uptake of cigarette use and, therefore, offset the decline in underage cigarette use.\n\nA review of existing literature revealed some inconsistencies in evidence about the relationship between e-cigarette use and cigarette smoking. To infer causality, studies with an experimental design arguably provide the best evidence. Nonetheless, a randomized controlled trial of e-cigarette use among nonusers is not feasible7. In this context, Vasiljevic and colleagues sought to evaluate the impact of exposure to advertisements promoting e-cigarettes on appeal and susceptibility of tobacco smoking using an experimental design. Youth (11–16 years of age) nonusers of e cigarettes and combustible cigarettes were randomized to e-cigarette advertisement groups (a candy-flavored e-cigarette advertisement group and a non-flavored e-cigarette advertisement group), or to a no advertisement group (control). This study found no effect of exposure to e-cigarette advertisements on appeal or susceptibility of cigarette smoking. Moreover, it had no effect on the perceived harm of cigarette smoking, another robust predictor for cigarette smoking onset8. Despite the lack of direct behavioral measures, this study suggests that the exposure to e-cigarette advertisement had no acute effect on the measures related to cigarette smoking.\n\nThe largest body of literature comes from individual-level observational studies showing a consistent positive association linking e-cigarette use with a higher risk of cigarette smoking onset, even after adjusting for a range of variables known to be associated with cigarette smoking9–12. In a systematic review and meta-analysis, Soneji and colleagues (2017) concluded that e-cigarette ever use was associated with greater risk for subsequent cigarette initiation and past 30-day use13. Nonetheless, in the context of observational studies, it is almost impossible to control for an exhaustive list of confounders; the positive relationship can be attributed to unmeasured confounders associated with e-cigarette use, which would produce a spurious relationship between e-cigarette use and cigarette smoking without the presence of a causal relationship7,14.\n\nEcological prevalence trends are inconsistent with the notion that e-vapor use increases cigarette smoking. At the country level, e-cigarettes emerged and gained popularity during the period when the sharpest decline in adolescent cigarette smoking was observed in the US and other countries5,15,16. To further quantify the potential change over time, Dutra and Glantz applied interrupted time series analysis using data from National Youth Tobacco Surveys, 2004–2014, and found no difference in the rate of decline in ever (p=0.57) or current (p=0.23) cigarette smoking before and after 2009, when e-cigarette became available in the US market2. That is, the rate of decline in youth cigarette smoking did not change after the emergence of e-cigarette. In addition to country-level analysis, Dutra and Glantz used individual-level data to build a prediction model based upon data from the pre-e-cigarette era (2004–2009). This model correctly predicted ~70% of ever smokers and ~75% of current smokers for the post-e-cigarette era (i.e., 2010 to 2014). These predictions are similar to the predictions for the pre-e-cigarette era (i.e., 2004 to 2009). This finding suggests that smokers’ characteristics did not change before and after the introduction of e-cigarettes. Although e-cigarettes became available in the US in 2009, e-cigarette use was uncommon between 2009 and 2013. Levy and colleagues specified 2014, when e-cigarette use became prominent among youth in the US, as the reference time point and assessed the relationship between e-cigarette prevalence and cigarette smoking prevalence at an ecological level17. In this study, they found strong inverse associations between the prevalence of e-cigarette use and the prevalence of cigarette smoking among adolescents and young adults. Moreover, the inverse associations were consistently shown in all US national surveys identified via a systematic literature search17. Also at the ecological level, a few studies compared youth cigarette smoking prevalence between states with and without regulations to prohibit sales of e-cigarettes to minors, with the assumption that cigarette smoking prevalence should be higher in states that are less-restrictive toward underage access to e-cigarettes if e-cigarette use increases the risk of cigarette smoking. These studies have produced inconsistent findings with two studies documenting lower youth cigarette smoking in states without an e-cigarette age restriction18,19, and one study documenting the opposite20. Nonetheless, it is well known that ecological studies are prone to “ecological fallacy”, which may introduce inconsistent findings for causal inference.\n\nOne potential explanation of the consistently observed association between e-cigarette use and cigarette smoking from individual observational studies is the “common liability” theory, which postulates that any observed relationships between e-cigarettes and combustible tobacco cigarettes is attributed to the “liability” to use tobacco products of the individual; once this “common liability” is controlled for, there is no causal relationship between e-cigarette use and cigarette smoking21–24. Several lines of evidence align with the “common liability” theory. First, the relationship is not unique to e-cigarettes. Various studies showed that the use of any type of tobacco products (e.g., cigar, hookah, etc.) and/or other substances (e.g., alcohol and cannabis) predicted cigarette smoking onset (e.g., 11,12,24,25). Moreover, the risk of cigarette smoking onset increases with the number of types of tobacco product used11. Findings from a recent study using a propensity scoring approach found supportive evidence that the frequently observed association between e-cigarette use and smoking is largely attributed to shared risk factors, using data from Monitoring the Future, a national school survey of 8th, 10th, and 12th graders26. Second, the relationships between cigarette smoking and e-cigarette use are often reciprocal. For example, Leventhal and colleagues (2015) found that high school never triers of combustible tobacco products who had ever used e-cigarettes were more likely to start using any combustible tobacco product at 6- and 12-month follow-up than those who had never used e-cigarettes at baseline. Likewise, among never triers of e-cigarettes at baseline, those who had ever used combustible tobacco products were more likely to start e-cigarette use at follow-ups than those who had never used combustible tobacco products27. This bidirectional relationship has been replicated in some more recent studies12,28,29. Third, e-cigarette use predicts the onset of a range of other substance use behaviors, not limited to cigarette smoking10,25. These lines of evidence support the notion that the relationship is not limited or specific to e-cigarettes, and common risk factors may underlie the use of various tobacco products30.\n\nPoorer inhibitory control and elevated impulsivity are phenotypes of externalizing problems, which has been linked to precocious use of various substances31–34. A study found similar levels of inhibitory control and impulsivity between youth e-cigarette users who never smoked a cigarette and youth smokers who never used an e-cigarette. Both groups showed poorer inhibitory control and elevated impulsivity compared to youth not using any tobacco products35.\n\nGiven this background and guided by the ‘common liability’ theory, the aim of this study is to estimate the prospective relationship between e-cigarette ever use and the onset of cigarette smoking after controlling for a “common liability” to use tobacco in US adolescents using a structural equation modeling approach. In essence, we first created a unidimensional construct to represent the ‘liability’ to use tobacco products, and then regressed the onset of cigarette use on antecedent e-cigarette use taking into account the latent ‘liability’ to use tobacco products.\n\n\nMethods\n\nThe study population consisted of non-institutionalized civilian adolescents 12–17 years of age living in the US, sampled in the longitudinal Population Assessment of Tobacco and Health (PATH) study. A multi-stage sampling method was used to draw nationally representative samples after Institutional-Review-Board-approved parent consent and youth assent36. In contrast to school surveys of adolescents, the PATH sample includes young people irrespective of school attendance, and its sampling frame includes college dormitories and children of active-duty military living in the US. More details about the PATH methodology is provided elsewhere36.\n\nIn this study, PATH wave 1 (2013–2014) and wave 2 (2014–2015) data were used. Participation levels were 54% at the household level and 78% at the individual level for the youth survey at wave 1. Wave 2 follow-up rate was 88% for youth. Because the outcome in this study is the onset of cigarette smoking, the study sample consists of wave 1 never smokers who were followed up at wave 2. We did not include “aged-up” adolescents (i.e., those who became 18 at wave 2) to retain a sample relevant for underage smoking (n=9,045).\n\nPATH confidential assessments were audio computer assisted self-interviews (ACASI), with standardized multi-item modules on use of various tobacco products, including cigarettes, e-cigarettes, cigars (including traditional cigars, cigarillos, and filtered cigars), smokeless tobacco, snus, hookah, pipe, dissolvable tobacco, bidis, and kretek. Survey questions about ever use of these tobacco products are typically in the format of “Have you ever smoked/used …, even one or two puffs/times?” In this study, the outcome is the onset of ever cigarette smoking at wave 2, which is defined as smoking cigarettes (even one or two puffs) for the first time between wave 1 and wave 2 assessments among adolescents who had never smoked cigarettes at wave 1. PATH also assessed lifetime history (i.e., ever use) of alcohol, cannabis, Ritalin® or Adderall®, Painkillers/sedatives/tranquilizers, cocaine, stimulants, and other drugs, respectively.\n\nSex (male or female) and age categories (12–14 or 15–17 years of age at baseline) were included as covariates. (The PATH Public Use File only included a binary variable for age.) Information about these covariates is from survey items in the Demographics module. When these items are missing, information from the household screening roster is drawn.\n\nGuided by the “common liability” theory, we used a structural equation modeling approach to test whether there is a specific association between e-cigarette ever use at wave 1 and the onset of first cigarette smoking at wave 2 holding constant a latent construct for the “liability to use tobacco products.”\n\nFirst, we built a latent construct for the common liability to use tobacco products using confirmatory factor analysis methods. The observed variables were lifetime ever use of specific tobacco products. All tobacco products assessed in PATH wave 1 were included to create the latent construct. Snuff and chewing tobacco, snus, and dissolvable tobacco products were combined to create a “smokeless tobacco products” variable due to the considerations that a) youth participants may not differentiate these oral tobacco products well, and b) low occurrence of dissolvable tobacco use (n=9) and high correlation between smokeless tobacco use and snus use (i.e., 69% of snus users had also used smokeless tobacco). All observed variables were treated as categorical variables. The variance of the latent construct was fixed to one in order to obtain factor loading and threshold estimates for each observed variable. After ensuring a good fit of the measurement model (as described in the next paragraph), we built the structural portion of the model to assess the relationship between e-cigarette ever use at wave 1 and the onset of first cigarette smoking at wave 2. Specifically, we drew a path from the latent construct to the onset of first cigarette smoking as well as a direct path from e-cigarette ever use to the onset of first cigarette smoking. Figure 1 provides a conceptual description of the model. If the direct path from e-cigarette to cigarette smoking is statistically robust, it provides evidence that e-cigarettes plays a role for cigarette smoking onset over and beyond the common liability to use tobacco products. If not, it supports the notion that the frequently observed association between e-cigarettes ever use and smoking is attributed to a common liability to use tobacco products.\n\nSeveral model fit indices were used to assess the goodness of fit of the measurement and the final structural models. These fit indices include root mean square of approximation (RMSEA)37, comparative fit index (CFI)38, and Tucker-Lewis index (TLI) . A RMSEA<0.08 and CFI/TLI > 0.90 are considered as indications of reasonably good model fit39,40.\n\nAnalysis weights were used to adjust for selection probability, nonresponse patterns, possible deficiencies in the sampling frame, and attrition. The PATH User Guide provides details about the calculation of weights36. Balanced repeated replication method was used to generate standard errors and 95% confidence intervals (CI). Analyses were conducted using Stata 15.0 (StataCorp, College Station, Texas, USA) and Mplus 8.1 (Muthén & Muthén, Los Angeles, CA, USA).\n\n\nResults\n\nTable 1 presents the estimated ever use of tobacco products as well as factor loadings and thresholds from confirmatory factor analysis among youths who had never tried a cigarette at wave 1 (n=9,045). Among the sample of youth never smokers, e-cigarette was the most common tobacco product ever tried (3.7%), followed by hookah (2.5%), with bidi and kretek being the least common tobacco products ever tried (0.1%). The measurement model fits reasonably well (RMSEA=0.019, 90% CI=0.014, 0.024; CFI=0.963; TLI=0.945); all factor loadings were greater than 0.4 and statistically significant (p<0.001). Each individual’s factor score, a score reflecting the latent liability to use tobacco products, was calculated based on both the threshold and the loading of each item. Therefore, an individual who used bidi, kretek, or pipe (items with greater thresholds) would have a higher factor score compared to an individual who used e-cigarette or hookah only (items with lower thresholds). An individual who used multiple products would generally have a higher factor score than an individual who used a single product. This factor score represents an individual’s liability (unobserved) to use tobacco products.\n\nData from Population Assessment of Tobacco and Health wave 1 and 2, 2013–201541. (Unweighted n=9,045 12–17 Year Oldsa).\n\na Analytical sample consists of youths who had never smoked a puff of cigarette at wave 1 and followed up and remained youths at wave 2.\n\nb smokeless tobacco includes smokeless tobacco, snus, and dissolvable tobacco products.\n\nOf the 9,045 never cigarette smokers at wave 1 assessment, 338 adolescents smoked a cigarette for the first time between wave 1 and wave 2 assessments (weighted incidence= 3.8%). The structural equation model shown in Figure 1 fits data well (RMSEA=0.015, 90% CI=0.011, 0.020; CFI=0.969; TLI=0.954). The model shows that after accounting for the general liability to use tobacco products, e-cigarette ever use at wave 1 is not associated with cigarette onset at wave 2 (β=0.13; 95% CI= -0.07, 0.32; p=0.204). The latent general liability variable is positively associated with wave 2 onset of ever cigarette smoking (β=0.38; 95% CI=0.07, 0.69; p=0.015). Because sex and age are the two most important exogenous variables that are related to tobacco use behaviors, they were introduced as covariates in the next step to assess any potential changes in estimates and statistical inference. Including sex and age as covariates introduced little change in estimates, and statistical inference remained the same (β=0.16; 95% CI= -0.03, 0.35; p=0.095; for the e-cigarette to cigarette onset path; β=0.33; 95% CI= 0.04, 0.61; p=0.025; for the ‘liability to use tobacco’ latent construct to cigarette onset path).\n\nIn order to further test the model with a latent construct for the liability for use of psychoactive substances (as was originally proposed by the ‘common liability theory’), we expanded the measurement model to include other substances measured in PATH (i.e., alcohol, cannabis, prescription Ritalin and Adderall, cocaine/crack, stimulants, and other drugs (heroin, inhalants, solvents, and hallucinogens). The expanded measurement and structural equation models both have good fit (RSMEA<0.02, CFI and TLI>0.90; see Table 2 for estimated ever use, factor loadings, and thresholds). Similar to results from the tobacco liability model shown above, the e-cigarette-specific path is not statistically significant (β=0.13; 95% CI= -0.002, 0.27; p=0.053 without adjusting for sex and age; β=0.14; 95% CI= -0.002, 0.29; p=0.054 with adjusting for sex and age), and the latent ‘liability’ construct is a robust predictor of cigarette smoking onset (β=0.44; 95% CI=0.28, 0.61; p<0.001 without adjusting for sex and age; β=0.43; 95% CI=0.25, 0.60; p<0.001 with adjusting for sex and age).\n\nData from Population Assessment of Tobacco and Health wave 1 and 2, 2013–201541. (Unweighted n=9,045 12– 17 Year Oldsa).\n\na Analytical sample consists of youths who had never smoked a puff of cigarette at wave 1 and followed up and remained youths at wave 2.\n\nb Smokeless tobacco products includes snuff and chew, snus, and dissolvable tobacco products.\n\nc Other drugs include heroin, inhalants, solvents, and hallucinogens.\n\n\nDiscussion\n\nIn this study, we found that after accounting for the latent ‘common liability to use tobacco products’ construct, ever e-cigarette use does not predict the onset of smoking among US adolescents.\n\nThis study’s findings should be interpreted in light of the following limitations.\n\nFirst, this study is observational in nature. Unmeasured heterogeneity is possible (variables not accounted for by the latent ‘liability’ construct), and no definitive evidence for a causal relationship is warranted. Second, the assessment was based on self-report information. There is possible under-reporting of tobacco and other drug use due to socially desirable responding42. The use of ACASI can help ameliorate this limitation. Measures for ever use of tobacco products have been shown to have good validity based on self-reported information43. Third, the response level at the household screening is moderate. Nonetheless, it is comparable to other modern national household surveys in the US44, and post-stratification was applied to bring the sample into balance with the US adolescent population.\n\nStrengths of this study include a) the prospective design provides clear temporal relationship, and is less prone to differential recall as compared to cross-sectional surveys; b) compared to prevalence-based measures, newly incident cigarette use focuses on the onset process without any interference of the persistence process45; c) by using nationally representative data, our results are generalizable to the general US adolescent population; and d) the use of ACASI and relatively low attrition in PATH enhances internal validity by reducing potential socially desirable responding and bias associated with attrition.\n\nOur approach is driven by the “common liability” theory. To the best of our knowledge, this is the first study to use a structural equation modeling approach to create a latent unidimensional ‘liability’ variable, which is consistent with the conceptualization of “liability”, which denotes “a latent (unobservable) quantitative trait that, when measured, ‘would give us a graded scale of the degree of affectedness or of normality’24,46.”\n\nTobacco products use is the result of complex genetic-environment interplay. Under the “common liability” conceptualization, the sequence of tobacco product use is opportunistic and depends upon various environmental factors including accessibility, local policies, and social norms. Therefore, the threshold for each product may change from one culture to other. For example, in a cigar-prone culture, the threshold for cigar use may be lower than that for e-cigarettes. The latent ‘liability’ construct is capable of accommodating these types of environmental variations.\n\nA few hypotheses have been developed to provide theoretical grounds and underlying mechanisms for the relationship between e-cigarette use and subsequent cigarette smoking among youth. Here, we highlight three main theories as cited by the Academies of Sciences Engineering Medicine: the ‘diversion theory’, ‘catalyst theory’, and ‘common liability theory’47. In brief, the ‘diversion theory’ hypothesizes that e-cigarette deters tobacco cigarette use by diverting ‘high-risk’ individuals to e-cigarette from combustible cigarettes22,48. The ‘catalyst theory’ postulates that e-cigarette use increases the risk of combustible cigarette use by first attracting ‘low-risk’ individuals to e-cigarettes, as a reduced-harm product, and then increasing proclivity to try combustible tobacco cigarettes49. The ‘common liability theory’ proposes that any observed relationships between e-cigarettes and combustible tobacco cigarettes is completely attributed to shared risk factors such as impulsivity and novelty-seeking (e.g., common liability to use tobacco products;21–24). If the ‘diversion theory’ is true, e-cigarette use should decrease the onset of cigarette smoking (or accelerate the declining trend of smoking); if the ‘catalyst theory’ is true, e-cigarette use should increase the onset of cigarette smoking; if the ‘common liability’ theory is true, a null relationship should be observed. Our findings provide supporting evidence for the ‘common liability’ theory for the US youth population as a whole. Our finding is in line with findings from a recent study using propensity scoring methods, which found supportive evidence that the observed association between e-cigarette use and cigarette smoking was attributed to shared risk factors for tobacco use26. Nonetheless, it is possible that different hypotheses apply to heterogeneous groups of individuals or contexts. For example, e-cigarettes may provide an alternative to cigarette smoking for some adolescents, whereas they may expose some other adolescents to a more smoking-prone environment. The onset of cigarette smoking is a complex interplay between micro-, meso-, and macro-level factors. Future studies with assessments of a breadth of these relevant factors and due attention to potential heterogeneities may provide more insights about the role of e-cigarette in smoking onset to guide targeted prevention and intervention efforts among youth. Applications of similar approaches in other cultures will help assess the reproducibility and consistency of the evidence, which is required for causal inference. It is noteworthy in this context that tobacco use behaviors in youth are often experimental and highly dependent on the availability of tobacco products6,50–52, and an infrequent use pattern (1–2 days during the past 30 day) is more pronounced among exclusive e-cigarette users compared to youths who use other tobacco products concurrently51. In this context, future studies incorporating the frequency of e-cigarette use and examining the relationship between e-cigarette use and the progression to more established cigarette smoking will provide useful insights about the e-cigarette-use-and-smoking relationship.\n\n\nData availability\n\nNational Addiction & HIV Data Archive Program: Population Assessment of Tobacco and Health (PATH) Study [United States] Public-Use Files (ICPSR 36498), https://doi.org/10.3886/ICPSR36498.v941.\n\nAll data are publicly available. In order to download the data, users must first agree with the ICPSR Terms of Use, specific to each dataset, which can be viewed by clicking the download button. Data used in this study were downloaded on May 14, 2018.",
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Publisher Full Text\n\nNational Academies of Sciences, Engineering, and Medicine; Health and Medicine Division; Board on Population Health and Public Health Practice; et al.: Public health consequences of e-cigarettes. Washington, DC: The National Academies Press; 2018. PubMed Abstract | Publisher Full Text\n\nEtter JF: Gateway effects and electronic cigarettes. Addiction. 2018; 113(10): 1776–1783. PubMed Abstract | Publisher Full Text\n\nSchneider S, Diehl K: Vaping as a Catalyst for Smoking? An Initial Model on the Initiation of Electronic Cigarette Use and the Transition to Tobacco Smoking Among Adolescents. Nicotine Tob Res. 2016; 18(5): 647–653. PubMed Abstract | Publisher Full Text\n\nAbrams DB, Glasser AM, Pearson JL, et al.: Harm Minimization and Tobacco Control: Reframing Societal Views of Nicotine Use to Rapidly Save Lives. Annu Rev Public Health. 2018; 39: 193–213. PubMed Abstract | Publisher Full Text\n\nVillanti AC, Pearson JL, Glasser AM, et al.: Frequency of Youth E-Cigarette and Tobacco Use Patterns in the United States: Measurement Precision Is Critical to Inform Public Health. Nicotine Tob Res. 2017; 19(11): 1345–1350. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHair EC, Romberg AR, Niaura R, et al.: Longitudinal Tobacco Use Transitions Among Adolescents and Young Adults: 2014-2016. Nicotine Tob Res. 2019; 21(4): 458–468. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "57839",
"date": "08 Jan 2020",
"name": "Arielle S. Selya",
"expertise": [
"Reviewer Expertise Behavioral science",
"esp. nicotine use and dependence among adolescents"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 focuses on the controversial question of whether e-cigarettes causally lead to later cigarette smoking among adolescents, or whether the use of both conventional cigarettes and e-cigarettes are due to a common liability. The paper uses structural equation modeling to model the pre-existing likelihood of e-cigarette use as a latent factor, finds that e-cigarettes do *not* have a causal effect on later conventional smoking over and above the latent factor. This paper uses an innovative method to address a complicated statistical question. The paper is well-written, and the implications are important.\n\nMajor comments:\nThe introduction has an excellent literature review that makes a convincing case for the common liability hypothesis, even using papers that at face value support the “gateway” hypothesis of e-cigarettes but identifying interesting counterpoints in the analyses of these papers.\n\nMy main concern is about the choice to include only substance use variables in the latent factor representing liability to use tobacco products. Propensity to use tobacco products includes a variety of other factors as well (socioeconomic, personality characteristics, risk perception, peer group and family substance use, mental health, etc). Are these other variables available in the PATH study, and if so what was the rationale to restrict only to behavioral tobacco use measures? What would the results look like if other covariates were also included?\n\nI would recommend a re-organization of the Discussion, to discuss interpretation of findings, implications, and relation to existing literature first, followed by limitations and strengths.\nMinor comments:\nI think a brief description of “standardized threshold” (which appears first in Table 1) would be helpful.\n\nOn p. 6, last paragraph, why are exact values not reported for the fit measures (“RMSEA <0.02, CFI and TLI > 0.90”) as they are elsewhere?\n\nWhat does the acronym ACASI mean (p.7)?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "5441",
"date": "04 May 2020",
"name": "Hui Cheng",
"role": "Author Response",
"response": "Dr. Selya’s commentsGeneral comments: This manuscript focuses on the controversial question of whether e-cigarettes causally lead to later cigarette smoking among adolescents, or whether the use of both conventional cigarettes and e-cigarettes are due to a common liability. The paper uses structural equation modeling to model the pre-existing likelihood of e-cigarette use as a latent factor, finds that e-cigarettes do *not* have a causal effect on later conventional smoking over and above the latent factor. This paper uses an innovative method to address a complicated statistical question. The paper is well-written, and the implications are important. Authors response: We thank the reviewer for recognizing the significance and novelty of the work and the helpful comments. We have revised the original manuscript based on these comments as detailed below. Major comments: 1. The introduction has an excellent literature review that makes a convincing case for the common liability hypothesis, even using papers that at face value support the “gateway” hypothesis of e-cigarettes but identifying interesting counterpoints in the analyses of these papers. Authors response: We thank the reviewer for recognizing the value of the research question. 2. My main concern is about the choice to include only substance use variables in the latent factor representing liability to use tobacco products. Propensity to use tobacco products includes a variety of other factors as well (socioeconomic, personality characteristics, risk perception, peer group and family substance use, mental health, etc). Are these other variables available in the PATH study, and if so what was the rationale to restrict only to behavioral tobacco use measures? What would the results look like if other covariates were also included? Authors response: We agree with the reviewer that the propensity to use tobacco products is multifaceted, including important factors mentioned by the reviewers.In this study, we were guided by the ‘common-liability’ theory and hypothesized that the use of various tobacco products is driven by an unobserved latent liability to use tobacco products. That is, a single-dimension latent variable (i.e., the liability to use tobacco products) gives rise to the use of different tobacco products. This latent liability to use tobacco products likely varies by socioeconomic, personality characteristics, risk perception, peer influence, and familial history of substance use, mental health etc., which can be precedents, consequences, or both (feedback loops) of tobacco use. That is, our expectation is that the latent variable, which represents an individual’s liability to use tobacco products, is correlated with these other variables. Practically, one complexity to include various other factors in the structural equation framework is that these variables are not necessarily manifestations of a single dimension, which complicates the structure of the measurement model. Due to the above theoretical and practical reasons, we chose to take a simpler theory-driven approach to define the latent construct by tobacco use behaviors and only included sex and age as covariates because they are exogenous to the liability-and-smoking-onset relationship. In an exploratory step, we further expanded the model to include other substances because it has been shown that adolescent substance use is highly opportunistic and tobacco use is highly correlated with other substance use among adolescents in the original submission.That said, the reviewer’s concern is well taken. We have expanded the model to include aforementioned variables as covariates to the extent that they were assessed in PATH wave 1 survey. Specifically, we took a two-step approach to include additional covariates. First, we added race/ethnicity (non-Hispanic White, non-Hispanic Black, Hispanics, and non-Hispanic others), availability of tobacco products in the household (yes/no), and a measure of novelty seeking (“agree” or “strongly agree” to the question “I like new and exciting experiences, even if I have to break the rules. Do you…”). In the second step, we additionally included participant’s self-rated health (excellent/non-excellent), harm perception of cigarettes (“a lot of harm” to the question “How much do you think people harm themselves when they smoke cigarettes”), and participant’s grade (mostly A’s or A’s and B’s vs. others) because these variables are more likely to have feedback loops with tobacco use. These covariates were modeled to correlate with the latent liability construct and to predict the onset of cigarette smoking at wave 2.The inclusion of these variables induced little change in estimates, although the latent-liability-to-smoking-onset path became marginally significant statistically, possibly due to the loss of precision associated with the additional covariates. Nonetheless, e-cigarette did not predict smoking onset in either model (Additional model 1: β=0.16; 95% CI= -0.04, 0.35; p=0.109; Additional model 2: β=0.16; 95% CI= -0.03, 0.35; p=0.102), and the latent liability of tobacco use predicted smoking onset in the first additional model (β=0.31; 95% CI= 0.01, 0.61; p=0.047) as well as in additional model 2 (β=0.31; 95% CI= 0.01, 0.61; p=0.047).We observed little change in estimates for the e-cigarette-to-smoking relationship as well as the liability-to-smoking relationship across the four models (no covariates, sex-and-age as covariates, additional model 1, and additional model 2 described above), which suggests that the model is robust to variations of covariates.In the revised manuscript, we have added this additional information in the Results section and revised the Discussion section accordingly. 3. I would recommend a re-organization of the Discussion, to discuss interpretation of findings, implications, and relation to existing literature first, followed by limitations and strengths. Authors response: We thank the reviewer for the suggestion. We have moved the ‘Limitation and strengths’ section to the end of the manuscript. Minor comments: 1. I think a brief description of “standardized threshold” (which appears first in Table 1) would be helpful. Authors response: We thank the reviewer for bringing it to our attention. After further consideration, we think the unstandardized thresholds are more meaningful than the standardized threshold. In the revised version, we have changed the standardized thresholds to unstandardized thresholds, which represents the ‘difficulty parameter’ of items. We have also added the following footnote for “standardized loading” in Table 1:“Factor loadings are standardized based on the variances of the continuous latent variables as well as the variances of the outcome variables.” 2. On p. 6, last paragraph, why are exact values not reported for the fit measures (“RMSEA <0.02, CFI and TLI > 0.90”) as they are elsewhere? Authors response: In the revised version, we have added exact values for these fit indices in the revised version as follows,“RSMEA=0.015, 90% CI=0.012, 0.017; CFI=0.948, TLI=0.938 for the model without sex and age; RSMEA=0.016, 90% CI=0.015, 0.018; CFI=0.922, TLI=0.905 for the model out sex and age as covariates”We did not report the exact values in the previous version because we considered the expanded model a post-estimation exploratory step and the results are similar to those of the main model. 3. What does the acronym ACASI mean (p.7)? Authors response: ACASI stands for audio computer assisted self-interviews. It was defined at first appearance under “Assessment.” Since it only appeared three times in the manuscript, we have replaced the acronyms with its full name in the revised version."
}
]
},
{
"id": "57841",
"date": "28 Jan 2020",
"name": "Alexa Gallagher",
"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\nCheng et al. (2019) examines the association between e-cigarette use and onset of cigarette smoking in youth between the ages of 12 and 17 years in the U.S. They utilize nationally representative and publicly available data from the PATH study. The research was guided by “common liability” theory, which “postulates that any observed relationship between e-cigarettes and combustible tobacco products is attributed to the liability to use tobacco products of the individual; once this common liability is controlled for, there is no causal relationship between e-cigarette use and cigarette smoking.” The authors report that they are the first study to use structural equation modeling to answer this question.\n\nIntroduction\nCheng and colleagues ask an interesting and timely research question. They include a series of recent publications in their background as they lay out their justification. The introduction, however, is rather long and somewhat unfocused.\n\nI would lead the discussion of the existing literature with the paragraph starting, “The largest body of literature comes from individual-level observational studies showing a consistent positive association…” The Vasiljevic study looking at exposure to e-cigarette advertisements does not seem directly relevant. Although I understand that it utilizes an experimental design, it could be cut to shorten the rather long introduction.\n\nThe authors’ reference #26 should be discussed more fully in the introduction and discussion sections, as it identifies the components of common liability to use tobacco products and could shed light on the proper covariates to include in the SEM.\n\nThe discussion of ecological studies is lengthy and could be shortened.\n\nCommon liability theory should be introduced and described in the introduction, as it underlies the whole project. What is it, how is it measured, why is it appropriate for this project?\n\nMethods\nSEM is not that commonly used by epidemiologists. The paper would be more accessible if it included a brief introduction to SEM, including model selection, model evaluation, and how to interpret the results. The authors should explain why SEM is the right way to analyze a hypothesis based in common liability theory.\n\nThe authors used data from Wave 1 (2013-2014) and Wave 2 (2014-2015) of the PATH study. Were PATH Wave 3 (2015-2016) data available and considered? If available but not included, why not?\n\nAre the questions the same in Wave 2 for those who are 18? If so, have you considered running a sensitivity model that includes the 18-year-olds? Have other authors made this same decision to exclude them? How many Wave 1 youth were excluded because they had aged up by Wave 2? It may make less sense for future studies to make this cut-off considering changes in the minimum legal age to purchase tobacco products.\n\nThe authors ran two sets of models, one limited to tobacco products and a second, which they call the ‘expanded liability model’, that includes tobacco products and psychoactive substances. This expanded liability model is not introduced until the results section. It should be described in the methods.\n\nSex and age were included as covariates. Were any of the other risk factors for smoking available in PATH, such as were identified in authors’ reference 26? If so, why not use those?\n\nPlease provide additional information on missing information. The only mention of it is as it relates to the covariates of age and sex. The authors report that this information was obtained from the Demographics module, which if missing was taken from the household screening roster. Was there missing information for the questions related to tobacco products, alcohol, cannabis, and other psychoactive drugs. If so, provide numbers and describe how it was handled. If there was limited missing information, that could be noted as a strength. Also, was there evidence that the data were, or were not, missing systematically? This can be assessed by comparing the available information for respondents who answered the substance use questions vs. those who did not.\n\nResults\nPlease provide the number of eligible participants at Wave 1 and Wave 2 and report numbers and reasons for any that were excluded. This could be done in the form of a figure ending with the final number included in your analysis.\n\nAlso, please include a baseline table that provides basic demographic information. This could also be described in text.\n\nThe titles for tables 1 and 2 indicate that that data is from Wave 1 and Wave 2. We are interested in ever use of the products listed in the table at Wave 1 and ever use of cigarettes at Wave 2. Please clarify what is provided in the table and update the table titles/descriptions.\n\nIn fact, perhaps tables 1 and 2 could be combined to include demographic data from Wave 1; ever use of e-cigarette, cigar, pipe, hookah, SLT, bidi, alcohol, cannabis, prescription Ritalin and Adderall, cocaine/crack, stimulants, and other drugs at Wave 1; and ever cigarette use at Wave 3.\n\nI’d suggest placing the model results described in the text in a table.\nDiscussion\nThe authors conclude that “after accounting for the latent ‘common liability to use tobacco products’ construct, ever e-cigarette use does not predict the onset of smoking among US adolescents.” This statement is supported by the results provided, assuming that the variables considered by the authors reasonably captured the common liability they hypothesized exists. This point – whether or not the covariates included were sufficient – needs further explanation and justification.\n\nThe authors identify several appropriate limitations and strengths. However, the limitations should address more explicitly the availability of data to describe smoking propensity, as defined in reference 26 or elsewhere. The authors identify lack of recall bias as a strength of the study, but the interval between waves in PATH is quite short, so recall bias is not a major concern.\n\nThe discussion section should place the current results into context of what is already known about smoking initiation in youth. The authors note that their findings are in line with a study by Kim et al. (2019) and they should discuss this reference more fully. Several other observational studies are referenced in the introduction and further description of these observational studies might be useful here as well.\n\nThank you for the opportunity to be a reviewer of this study. It is my recommendation that this paper be approved with reservations.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "5442",
"date": "04 May 2020",
"name": "Hui Cheng",
"role": "Author Response",
"response": "Dr. Gallagher and Dr. Sulsky comments Cheng et al. (2019) examines the association between e-cigarette use and onset of cigarette smoking in youth between the ages of 12 and 17 years in the U.S. They utilize nationally representative and publicly available data from the PATH study. The research was guided by “common liability” theory, which “postulates that any observed relationship between e-cigarettes and combustible tobacco products is attributed to the liability to use tobacco products of the individual; once this common liability is controlled for, there is no causal relationship between e-cigarette use and cigarette smoking.” The authors report that they are the first study to use structural equation modeling to answer this question. 1. Introduction 1.1 Cheng and colleagues ask an interesting and timely research question. They include a series of recent publications in their background as they lay out their justification. The introduction, however, is rather long and somewhat unfocused. Authors response: We thank the reviewer for recognizing the value of the research question. We have amended the introduction to make it more focused based on reviewers’ helpful comments as follows. 1.2 I would lead the discussion of the existing literature with the paragraph starting, “The largest body of literature comes from individual-level observational studies showing a consistent positive association…” The Vasiljevic study looking at exposure to e-cigarette advertisements does not seem directly relevant. Although I understand that it utilizes an experimental design, it could be cut to shorten the rather long introduction. Authors response: We have reorganized the discussion of existing literature in the revised manuscript. It now begins with observational studies, followed by the experimental study by Vasiljevic and colleagues, and then ecological evidence. The description of the Vasiljevic study has been shortened. 1.3 The authors’ reference #26 should be discussed more fully in the introduction and discussion sections, as it identifies the components of common liability to use tobacco products and could shed light on the proper covariates to include in the SEM. Authors response: We have added more details of reference #26 (now reference # 15) in the revised manuscript (the middle of the 5th paragraph in the Introduction and the middle of the second paragraph in Discussion). During the revision, another study using similar method has been published. In this study, an inverse association was found between e-cigarette use and subsequent cigarette smoking using data from NYTS. We have added this study in the revised manuscript (ref. #16). 1.4 The discussion of ecological studies is lengthy and could be shortened. Authors response: We have shortened the discussion about ecological studies in the revised manuscript. 1.5 Common liability theory should be introduced and described in the introduction, as it underlies the whole project. What is it, how is it measured, why is it appropriate for this project? Authors response: In the revised manuscript, we have added more details about the common liability theory (the beginning of the 5th paragraph of the Introduction). 2. Methods 2.1 SEM is not that commonly used by epidemiologists. The paper would be more accessible if it included a brief introduction to SEM, including model selection, model evaluation, and how to interpret the results. The authors should explain why SEM is the right way to analyze a hypothesis based in common liability theory. Authors response: We have added a brief introduction of SEM and why we think SEM is appropriate for this analysis based on the common liability theory (2nd paragraph under Analysis Approach). 2.2 The authors used data from Wave 1 (2013-2014) and Wave 2 (2014-2015) of the PATH study. Were PATH Wave 3 (2015-2016) data available and considered? If available but not included, why not? Authors response: PATH wave 3 data were not available when we started this project. During the course of the project, they became available. We did repeat the analysis using wave 2 and 3 data, and the results are similar. We have added estimates using wave 2 and 3 data in the Results section of the revised manuscript and in the new Table 3. Nonetheless, we consider wave 1 and 2 data our primary focus because it is what we set out to do. Also, PATH wave 1 is the true baseline cohort, which is a representative sample of youth. Wave 2 is a follow-up of the wave 1 cohort with ‘shadow’ youth (those younger than 12) sampled at wave 1. In addition, for longitudinal studies like PATH, attrition occurs at each follow-up. When using wave 2 and 3 data, attrition may play a larger role. 2.3 Are the questions the same in Wave 2 for those who are 18? If so, have you considered running a sensitivity model that includes the 18-year-olds? Have other authors made this same decision to exclude them? How many Wave 1 youth were excluded because they had aged up by Wave 2? It may make less sense for future studies to make this cut-off considering changes in the minimum legal age to purchase tobacco products. Authors response: We did discuss whether we should include aged-up youth in the analysis, and we decided not to because the focus of this study is on youth smoking onset. During wave 1 and 2 data collection (2013-2015), cigarettes were legal for 18-year-olds to purchase in most places. Risk profiles and mechanisms may differ for underage smoking from of-age smoking. We wanted to retain a focus on youth smoking onset and, therefore, did not include 18 year olds in the current study. 2.4 The authors ran two sets of models, one limited to tobacco products and a second, which they call the ‘expanded liability model’, that includes tobacco products and psychoactive substances. This expanded liability model is not introduced until the results section. It should be described in the methods. Authors response: We have added a paragraph in the Method section to describe a series of models to explore the sensitivity of the main model results, including the model with additional psychosocial covariates and the model with wave 2 and wave 3 data, as suggested by the reviewers (fourth paragraph in the Analysis section). 2.5 Sex and age were included as covariates. Were any of the other risk factors for smoking available in PATH, such as were identified in authors’ reference 26? If so, why not use those? Authors response: We have added additional covariates in the series of exploratory analysis described in the revised manuscript. Please see our response to reviewer #1’s comment #2 for a detailed explanation. 2.6 Please provide additional information on missing information. The only mention of it is as it relates to the covariates of age and sex. The authors report that this information was obtained from the Demographics module, which if missing was taken from the household screening roster. Was there missing information for the questions related to tobacco products, alcohol, cannabis, and other psychoactive drugs. If so, provide numbers and describe how it was handled. If there was limited missing information, that could be noted as a strength. Also, was there evidence that the data were, or were not, missing systematically? This can be assessed by comparing the available information for respondents who answered the substance use questions vs. those who did not. Authors response: We have added a sentence in the revised manuscript to indicate that for all tobacco use variables, there were <3% missing values. In addition, we specified a robust weighted least square mean and variance adjusted estimator which uses a full weight matrix. Therefore, a participant is included in the model unless the participant has missing values in all indicator variables (i.e., tobacco use and substance use variables). None of wave 1 never smokers had missing values in all of these variables, so we are able to include all never smokers in our model. We have added this information in the last paragraph of the Methods section in the revised manuscript. 3. Results 3.1 Please provide the number of eligible participants at Wave 1 and Wave 2 and report numbers and reasons for any that were excluded. This could be done in the form of a figure ending with the final number included in your analysis. Authors response: In the revised manuscript, we added a sentence about the number of participants included in the current study: Among 13,651 participants at wave 1, 11,792 were never smokers, among whom 9,045 remained youth (12-17 years of age) and were followed up for wave 2 assessment. The sampling procedure of PATH is described in detail in PATH User’s Guide (ref. #39) 3.2 Also, please include a baseline table that provides basic demographic information. This could also be described in text. Authors response: In the revised manuscript, we have added basic demographics of the sample in Table 1 and a sentence in the first paragraph of the Results section: Of the 9,045 participants, 51% were males; 64% were 12-14 year olds; and 54% were non-Hispanic whites. 3.3 The titles for tables 1 and 2 indicate that that data is from Wave 1 and Wave 2. We are interested in ever use of the products listed in the table at Wave 1 and ever use of cigarettes at Wave 2. Please clarify what is provided in the table and update the table titles/descriptions. Authors response: We have adjusted the titles of Table 1 and Table 2 to reflect the estimates are among wave 1 never smokers. 3.4 In fact, perhaps tables 1 and 2 could be combined to include demographic data from Wave 1; ever use of e-cigarette, cigar, pipe, hookah, SLT, bidi, alcohol, cannabis, prescription Ritalin and Adderall, cocaine/crack, stimulants, and other drugs at Wave 1; and ever cigarette use at Wave 3. Authors response: In the revised manuscript, we have combined the previous Table 1 and 2 into one table (new Table 1). 3.5 I’d suggest placing the model results described in the text in a table. Authors response: We have added Table 2 which contains model results. 4. Discussion 4.1 The authors conclude that “after accounting for the latent ‘common liability to use tobacco products’ construct, ever e-cigarette use does not predict the onset of smoking among US adolescents.” This statement is supported by the results provided, assuming that the variables considered by the authors reasonably captured the common liability they hypothesized exists. This point – whether or not the covariates included were sufficient – needs further explanation and justification. Authors response: As we indicated above, we have added additional covariates in the model in our exploratory analyses, and the estimates were very similar to the one from the main model. We think these additional results suggest a certain degree of robustness of the latent ‘common liability’ construct and the main model overall. We have added this information and related discussion in the revised manuscript. Please also see our response to comment 2.5 and to reviewer #1’s comment #2 for more details. 4.2 The authors identify several appropriate limitations and strengths. However, the limitations should address more explicitly the availability of data to describe smoking propensity, as defined in reference 26 or elsewhere. The authors identify lack of recall bias as a strength of the study, but the interval between waves in PATH is quite short, so recall bias is not a major concern. Authors response: Regarding concerns about other covariates, we have conducted additional exploratory analyses to incorporate additional covariates related to smoking onset based on previous literature. Please also see our response to comment 2.5 and to reviewer #1’s comment #2. Regarding recall bias, we understand that the follow-up interval is only a year; however, there has been literature showing inconsistencies in the reporting of tobacco use as well as alcohol drinking behaviors in the youth population. In a cross-sectional setting, a smoker may be more likely to recall other tobacco use and vice versa because they can be correlated behaviors. Therefore, we think it is a noteworthy strength of the current study. 4.3 The discussion section should place the current results into context of what is already known about smoking initiation in youth. The authors note that their findings are in line with a study by Kim et al. (2019) and they should discuss this reference more fully. Several other observational studies are referenced in the introduction and further description of these observational studies might be useful here as well. Authors response: We have revised both the Introduction and Discussion to provide fuller details of previous studies and their relevance to the current study to help put findings from the current study into context. In closing, we thank all three reviewers for their helpful and constructive comments. We hope we have provided sufficient information and revision to make the work acceptable for publication."
}
]
}
] | 1
|
https://f1000research.com/articles/8-2099
|
https://f1000research.com/articles/8-613/v1
|
02 May 19
|
{
"type": "Research Article",
"title": "Mutation profiling of anaplastic ependymoma grade III by Ion Proton next generation DNA sequencing",
"authors": [
"Ejaz Butt",
"Sabra Alyami",
"Tahani Nageeti",
"Muhammad Saeed",
"Khalid AlQuthami",
"Abdellatif Bouazzaoui",
"Mohammad Athar",
"Zainularifeen Abduljaleel",
"Faisal Al-Allaf",
"Mohiuddin Taher",
"Ejaz Butt",
"Sabra Alyami",
"Tahani Nageeti",
"Muhammad Saeed",
"Khalid AlQuthami",
"Abdellatif Bouazzaoui",
"Mohammad Athar",
"Zainularifeen Abduljaleel",
"Faisal Al-Allaf"
],
"abstract": "Background: Ependymomas are glial tumors derived from differentiated ependymal cells. In contrast to other types of brain tumors, histological grading is not a good prognostic marker for these tumors. In order to determine genomic changes in an anaplastic ependymoma, we analyzed its mutation patterns by next generation sequencing (NGS). Methods: Tumor DNA was sequenced using an Ion PI v3 chip on Ion Proton instrument and the data were analyzed by Ion Reporter 5.6. Results: NGS analysis identified 19 variants, of which four were previously reported missense variants; c.395G>A in IDH1, c.1173A>G in PIK3CA, c.1416A>T in KDR and c.215C>G in TP53. The frequencies of the three missense mutations (PIK3CA c.1173A>G, KDR c.1416A>T, TP53, c.215C>G) were high, suggesting that these are germline variants, whereas the IDH1 variant frequency was low (4.81%). However, based on its FATHMM score of 0.94, only the IDH1 variant is pathogenic; other variants TP53, PIK3CA and KDR had FATHMM scores of 0.22, 0.56 and 0.07, respectively. Eight synonymous mutations were found in FGFR3, PDGFRA, EGFR, RET, HRAS, FLT3, APC and SMAD4 genes. The mutation in FLT3 p.(Val592Val) was the only novel variant found. Additionally, two known intronic variants in KDR were found and intronic variants were also found in ERBB4 and PIK3CA. A known splice site mutation at an acceptor site in FLT3, a 3’-UTR variant in the CSF1R gene and a 5’_UTR variant in the SMARCB1 gene were also identified. The p-values were below 0.00001 for all variants and the average coverage for all variants was around 2000x. Conclusions: In this grade III ependymoma, one novel synonymous mutation and one deleterious missense mutation is reported. Many of the variants reported here have not been detected in ependymal tumors by NGS analysis previously and we therefore report these variants in brain tissue for the first time.",
"keywords": [
"Ependymoma",
"Palisading necrosis",
"Perivascular psuedorossettes",
"Ion Proton",
"Next Generation DNA sequencing",
"Glioma",
"pediatric brain tumors",
"Anaplastic Ependymoma"
],
"content": "Introduction\n\nEpendymal cells are macroglial cells which line the ventricles, the central canal of the spinal cord and form the blood-cerebrospinal fluid barrier, being involved in producing the cerebrospinal fluid1,2. These tumors account for only 4–8% of gliomas and, after astrocytomas and oligodendrogliomas, ependymomas are the least common3. Nearly one-third of brain tumors in patients younger than three years old are ependymomas and constitute around 5%–9% of all neuroepithelial malignancies1,4. These tumors are also found in the choroid plexus and may occur at any age, from one month to 81 years and without any gender preference5. In pediatric cases, the location of the tumor is intracranial, while adult ependymal tumors can have either an intracranial or a spinal localization6,7. The prognosis is better in older children as compared to young infants but nonetheless, in children with intracranial ependymomas, event-free survival after five years is less than 50%8. In adults, about 50% to 60% intracranial ependymomas are supratentorial; however, pediatric supratentorial ependymomas account for 25% to 35% of all ependymomas5,9. Adults present better prognosis with a 5-year survival of around 90%, while in the pediatric population it is around 60%. The five-year survival rate for supratentorial, infratentorial, and spinal cord ependymomas is 62%, 85%, and 97%, respectively, and for grade I, II, and III spinal cord ependymomas the five-year overall survival rate is 92%, 97% and 58%, respectively10–12.\n\nEpendymoma tumors are well circumscribed, soft, tan-red masses and may be associated with hemorrhage. Their microscopic appearance shows hypercellularity and distinct infiltrative margins with surrounding parenchyma, consisting of monomorphic cells with nuclear atypia and brisk mitotic activity. They may also have intramural or glomeruliod vascular proliferation, pseudopalisading necrosis, perivascular pseudo rosettes (5–10% cases), calcifications and hyalinized vessels1. Other diagnostic hallmarks include areas of fibrillary and regressive changes such as myxoid degeneration, palisading necrotic areas and the formation of true rosettes, composed of columnar cells arranged around a central lumen1,6. Immunologically, they are positive for epithelial membrane antigen (EMA), glial fibrillary acidic protein (GFAP) and S-100. According to the 2016 updated World Health Organization (WHO) classification of brain tumors, ependymomas are divided into 4 types on the basis of histologic appearance: (1) grade I subependymomas, (2) grade I myxopapillary ependymomas, (3) grade II ependymomas, (4) grade II or III RELA fusion-positive ependymomas and grade III anaplastic ependymomas13,14.\n\nPrevious studies have shown the use of comparative genomic hybridization (CGH) arrays to distinguish intracranial ependymomas from spinal ependymomas15. In contrast to other types of brain tumors, histological grading is not a good prognostic marker for outcome for ependymomas16,17. Several gene expression studies have been helpful in differentiating between intracranial and extra cranial ependymomas, but have not had clinical significance in directing therapy and their role in tumor origin and prognosis is not clear18,19. Studies using cDNA micro-arrays have shown that gene expression patterns in ependymomas correlate with tumor location, grade and patient age20. Cytogenetic studies have shown that chromosomal abnormalities are relatively common in ependymomas21. Loss of 22q has been the commonest abnormality found in ependymoma and, in some other tumors, gain of 1q or loss of 6q was observed21,22.\n\nTo date, there is a lack of information regarding the mutational signatures which distinguish the various subgroups of ependymomas. Another ependymoma cohort study found very few mutations and gene amplifications but a high expression of multi-drug resistance, DNA repair and synthesis enzymes23. Intracranial ependymomas differ from spinal ependymomas in the expression of these proteins, and protein expression is also dependent on the ependymoma grade23. For both intracranial and spinal ependymomas, very few mutations were reported by using whole exome sequencing24. In another study, profiling of NGS mutations was carried out for one case of grade II ependymoma25 using a GlioSeq panel, which contains a total of 30 genes. In order to determine the mutational patterns of grade III anaplastic ependymoma, we have sequenced DNA from this ependymoma tumor using the Ion Proton system for next generation DNA sequencing with the Ion Torrent’s AmpliSeq cancer HotSpot panel. This panel contains 50 genes, only 15 of which also appear in the GlioSeq panel used in previous research. These data provided an evaluation of mutational signatures of this anaplastic ependymoma which differs from the previous two studies, but confirms their conclusions about finding very few mutations in cancer driver genes, helping to direct diagnosis and therapy for ependymomal tumors.\n\n\nMethods\n\nThis study was performed in accordance with the principles of the Declaration of Helsinki. This study was approved by the Institutional Review Board (IRB) bioethics committee of King Abdullah Medical City (KAMC), Makkah, Kingdom of Saudi Arabia (IRB number 14-140). A written informed consent was obtained from the parent of this patient before starting the study.\n\nSpecimens from all patients willing to give written informed consent and diagnosed with gliomas were eligible to be included in this study. Specimens that cannot be unambiguously identified (no label or specimen number, and/or sample and requisition do not match), specimens with hematomas and blood clots, or specimens from patients who refused inclusion in the study were not eligible. The single patient’s tumor tissue (FFPE sections in PCR tubes) used in this NGS analysis was obtained from the histopathology laboratory of Al-Noor Specialty Hospital Makkah, after tumor excision and left frontal craniotomy in the neurosurgery department. The tumor was classified based upon similarity to the constituent cells of the central nervous system, such as astrocytes, oligodendrocytes and ependymal, glial cells, mitosis and cell cycle-specific antigens, used as markers to evaluate proliferation activity and biological behavior (the WHO grading system)13. The final diagnosis was made following radiological, histopathological and immunological examinations.\n\nA CT scan of the brain was performed by a multi-slice CT (MSCT), using a 64-detector-row scanner. The use of computed tomography (CT) allowed visualization of detailed images of the soft tissues in the body in 3D as well as in multiplanar reconstructions. Images were acquired with 5mm slice thickness throughout on a GE Medical Systems, light speed VCT, 64-slice multidetector CT (MDCT). High quality images were processed at low dose performance on Volara™ digital DAS (Data Acquisition System).\n\nThe excised tumor was fixed in 4% buffered formaldehyde, routinely processed and paraffin embedded. Four-micrometer-thick sections were prepared on clear ground glass microscope slides with ground edges and routinely stained using Dako Reagent Management System (DakoRMS) with hematoxylin and eosin (H and E) on a Dako Coverstainer (Agilent). For immunohistochemistry, sections were collected on Citoglas adhesion microscope slides (Citotest). Mouse monoclonal beta-catenin (14) (Sigma-Aldrich, cat. no. 224M-1), mouse monoclonal EMA (E29) (Sigma-Aldrich, cat. no. 247M-9), rabbit monoclonal EGFR (SP84) (Cell Marque, cat. no. 414R-16-ASR), mouse monoclonal Vimentin (vim 3B4) (Ventana-Roche, cat. no. 760-2512), GFAP EP672Y rabbit monoclonal (Ventana-Roche, cat. no. 760-4345) and E-cadherin (36) mouse monoclonal (Ventana-Roche, cat. no. 790-4497) and mouse monoclonal anti-Ki-67 (Leica Biosystems, cat. no. KI67-MM1-L-CE) antibodies were used for immunohistochemistry. Briefly, the tissue sections were deparaffinized with EZ Prep (Ventana, cat. no. 950-102) at 60°C for 1 hr. Immunohistochemistry was performed with the Ventana BenchMark XT automated stainer (Ventana, Tucson, AZ). After inactivation of the endogenous peroxidase using a UV-inhibitor for 4 min at 37°C, the primary antibody was added for 16 min at 37°C, followed by the application of HRP Universal Multimer for 8 min, and detected using the ultraView Universal DAB Detection Kit (cat. no. 760-500) for 38 min. Slides were counterstained with hematoxylin for 8 min and bluing reagent for 4 min before mounting with cover slips. Following staining, images were acquired using NIKON Digital Microscope Camera - DS-Ri1, with image software NIS Elements v.4.0. Appropriate positive controls for all of the studied antibodies were used.\n\nDNA isolation was carried out using the QIAamp DNA FFPE Kit (50), Cat. No. 56404. 5-10 Formalin-Fixed Paraffin-Embedded sections of 5 microns were deparaffinized using xylene, treated with ethanol to remove the xylene, and the pellet was dried at 65°C for 5 mins. The pellets were resuspended in ATL buffer then treated with proteinase K. The remaining steps were carried out according to the user manuals. DNA concentration was measured using Nanodrop2000C and 10 ng of DNA was used for NGS analysis. DNA was sequenced using the Ion PI v3 Chip Kit (Cat no. A25771, Thermo Fisher Scientific, USA) with the Ion Proton System (Cat no. 4476610, Thermo Fisher Scientific, USA)26. Libraries were prepared using Ion AmpliSeq cancer HotSpot Panel v1 (Cat no. 4471262, Thermo Fisher Scientific, USA) primer pools. The Ion AmpliSeq Library Kit 2.0 (Cat no. 4475345, Thermo Fisher Scientific, USA) and Ion PI Hi-Q OT2 200 Kit (Cat no. A26434, Thermo Fisher Scientific, USA) was used for library and template preparation respectively. Sequencing was carried out using Ion PI Hi-Q Sequencing 200 Kit (Cat no. A26433, Thermo Fisher Scientific, USA) reagents and libraries were tagged with Ion Express Barcode Adapters 1-16, Cat. No. 4471250 (Thermo Fisher Scientific, USA). After sequencing, amplicon sequences were aligned to the human reference genome GRCh37 (hg19) (Accession no. GCA_000001405.1) in the target region of the cancer HotSpot panel using the Torrent Suite Software v.5.0.2 (Thermo Fisher Scientific, USA). Variant call format files (vcf files) were generated by running the Torrent Variant Caller Plugin v5.2. Variant calling and creation of vcf files can also be carried out using non-proprietary software such as SAMtools66 or VarScan267, which also provide coverage analysis. The vcf file data were analyzed using Ion Reporter v5.6 (ThermoFisher Scientific, USA), which calculated allele coverage, allele frequency, allele ratio, variant impact, clinical significance, PolyPhen 2 scores, Phred scored, SIFT scores, Grantham scores and FATHMM scores. This vcf file analysis was also carried out by Advaita Bioinformatics’ iVariantGuide. PolyPhen2, SIFT, variant impact and clinical significance can be calculated using non-proprietary software SnpEff29 and SnpSift30. FATHMM scores can also be predicted using fathmm31 and Grantham scores according to the formula as described in Grantham, 197432. The heat map was generated by the clustering of predicted variant impact scores by Ion Reporter v5.6. The most deleterious score was picked for every gene to generate the heat map; thereafter, hierarchical clustering was conducted. The color codes indicate the following variant impacts using score values 0-8: (0) unknown; (1) synonymous; (2) missense; (3) non-frameshift block substitution; (4) non-frameshift indel; (5) nonsense; (6) stop-loss; (7) frameshift block substitution or indel; (8) splice variant.\n\n\nResults\n\nA six-year-old female patient presented with a history of right facial palsy for few months with ataxia and right-sided weakness. The patient had a chronic headache, vomiting and had repeatedly been treated for sinusitis. Unenhanced computed tomography (CT) of the brain was performed (Figure 1, panels A, B and C). A large lesion (5.4 x 7.5cm) was noticed in the left cerebral frontoparietal region. There was an indication of a predominant cystic component and large, eccentric clump of coarse calcification. Additionally, mass effect resulting in midline shift, along with mild scalloping of the internal cortex of the parietal bone, was noted. No hydrocephalic changes or intrinsic hemorrhagic focus were seen (Figure 1).\n\nA large lesion (5.4 x 7.5cm) in the left cerebral frontoparietal location with predominantly cystic components (panel A, green arrow), and a large, eccentric clump of coarse calcification (panel B; yellow arrow). Mass effect and mid line shift (panel C, red arrow) can also be seen. No hydrocephalic changes or intrinsic active hemorrhagic focus were observed.\n\nHistopathological examination revealed sheets of neoplastic cells with round to oval nuclei and abundant granular chromatin. A variable dense fibrillary background and endothelial proliferation was also noted. Hematoxylin and eosin (H&E) staining results are shown in Figure 2 and Figure 3. Panels A and B of Figure 2 show the tumor exhibiting delicate cytoplasmic processes, perivascular rosettes characteristic of ependymoma, focal calcification areas and pseudo palisading necrosis, characterized by a garland-like structure of hypercellular tumor nuclei lining up around irregular foci of tumor necrosis. Panel C shows glomeruloid vascular proliferation and panel D shows extensive palisading necrosis and true rosette formation. The exhibition of a true rosette with a central lumen and the formation of pseudo-palisading necrotic areas is also clear from Figure 3 (panel A). Panel B shows focal areas with numerous tumor giant cells and the presence of brisk mitotic activity, vascular formation and pseudo-palisading necrotic areas. Formation of true rosettes surrounding the microvascular proliferation within ependymal tumors usually signifies anaplastic transformation, which is characteristic of grade III ependymoma (panels C and D). Immunostaining is shown in Figure 4: (A) Ki-67 stain shows a high proliferation index, (B) vimentin positive, (C) GFAP positive, (D) EMA showing punctate cytoplasmic (perinuclear dot-like positivity) staining which is fairly diagnostic of ependymal tumor cells. Figure 5 shows beta-catenin positive (panels A and B) and E-cadherin positive (panels C and D) immunostaining, with both membranous and true rosette-like structures clearly visible in this staining. EGFR staining was negative (see Underlying data)33.\n\n(A) Focal calcification areas (blue arrow), palisading necrosis (yellow arrow) and perivascular pseudo-rosettes (white arrow). (B) Pseudo palisading necrosis, characterized by a garland-like structure of hypercellular tumor nuclei (black arrow) lining up around irregular foci of tumor necrosis (blue arrow). (C) The cellular tumor exhibiting glomeruloid vascular proliferation (black arrow). (D) Extensive palisading necrosis (green arrow) and true rosettes (yellow arrow).\n\n(A) Pseudo palisading necrotic areas, exhibiting true rosettes with central lumen (yellow arrow). (B) Focal areas with numerous tumor giant cells and the presence of a brisk mitotic activity (green arrows). (C) Tumor with vascular formation (yellow arrows) and pseudo palisading necrotic areas. (D) Formation of true rosettes (green arrows) surrounding the microvascular proliferation within ependymal tumors, usually signifies anaplastic transformation which is characteristic of ependymomas.\n\n(A) Ki-67 immunostaining indicates a high proliferation index in the tumor (70%). (B) Vimentin stain is positive. (C) GFAP stain is positive. (D) EMA stain is positive and shows punctate cytoplasmic (perinuclear dot-like) staining, fairly diagnostic of the ependymal nature of the tumor cells.\n\nImmunostaining is strongly positive for beta-Catenin (panel A 20x, panel B 40x) and true rosettes (red arrows) and palisading cells (blue arrow) are clearly visible. E-Cadherin stain is also positive in this tumor. Red and blue arrows indicate tumor cells arranged in true rossettes and formation of palisading structures, respectively (panel C 20x, panel D 40x).\n\nAlignment to the target regions (CHP2. 20131001.designed) of the reference genome (hg 19) was performed by the Ion Torrent Suite software v.5.0.2. For this tumor, NGS generated 6,252,341 mapped reads using the Ion PI v3 Chip, with more than 90% reads on target. Amplicon and target base read coverages for the sequencing are shown in Table 1. All 207 amplicons were sequenced with Ion AmpliSeq Cancer HotSpot Panel primer pool. As shown in Table 1, for this sample sequencing the uniformity of amplicon coverage was 95.17%, and the uniformity of base coverage on target was 94.81%. The average reads per amplicon was 34, 179, and the average target base coverage depth was 31,771. 100% of amplicons had at least 500 reads and the percentage of amplicons read end-to-end was 89.37% (Table 1). Initial analysis by the Ion Reporter 5.6 program found that a total of 1652 variants passed all filters. Initial analysis by Advaita’s iVariantGuide software showed 100% (1633) of variants passed all filters (see Extended data)33. The filter flags signify variants which do not meet certain criteria during variant calling. The flags refer to the quality or confidence of the variant call. The parameters of flags were read in from the input vcf file. If a variant passes all filters, it is marked as having passed. Six hundred and fifteen variants were identified using a filter for clinical significance that identifies drug response, likely to be pathogenic and pathogenic variants. The distribution of these variants, based on chromosomal position, region within the gene, variant class, functional class, variant impact and clinical significance, are shown in doughnut charts A – F (Figure 6). As shown in doughnut chart A, chromosome 17 has the highest number of variants (26%) and chromosome 8 has lowest number of variants (0.8%). 98.7% of variants are exonic and, according to variant class distribution, 73.8% are SNPs, 70.2% are missense variants, 25.4% are high impact variants and 46.8% are pathogenic. We have considered true mutations to be those with a Phred score above 20 and significant mutations called by Ion Reporter software were those with a p-value below 0.05.\n\nDistribution of variants according to filters, showing characteristics including the relative number of variants located on each chromosome, variant class, substitution type and the functional consequences of each variant, in order to interpret and score the severity and impact of variants and therefore predict the severity of the disease. Doughnut charts in panels shows variants passed for each individual filter for (A) Chromosomal distribution, (B) Region in the gene, (C) Variant class, (D) Variant effect on the protein structure, (E) Variant impact on the protein function and (F) Clinical significance of the variants as annotated on the ClinVar database.\n\nA summary of the all missense mutations found in the grade III tumor is shown in Table 2. In this tumor, NGS data analysis identified 19 variants, of which four were missense mutations, eight were synonymous mutations and seven were intronic variants. Known missense mutation c.395G>A; p.(Arg132His) in exon 4 of the IDH1 gene, c.1173A>G; p.(Ile391Met) in exon 7 of the PIK3CA gene, c.1416A>T; p.(Gln472His) in exon 11 of the KDR gene and c.215C>G; p.(Pro72Arg) in exon 4 of the TP53 gene were found in this tumor. The frequency, allele coverage, allele ratio, p-value and Phred score for these mutations is shown in Table 3. The p-values and Phred scores were significant for all of these mutations. The frequencies of the three missense mutations, namely PIK3CA c.1173A>G, KDR c.1416A>T and TP53 c.215C>G, were high, suggesting that these are germ line variants, whereas the IDH1 variant frequency was low (4.81%). As shown in Table 2, eight synonymous mutations were found in this tumor, in exon 14 of FGFR3 p.(Thr651Thr), exon 12 of PDGFRA p.(Pro566Pro), exon 20 of EGFR p.(Gln787Gln), exon 13 of RET p.(Leu769Leu), exon 2 of HRAS p.(His27His), exon 14 of FLT3 p.(Val592Val), exon 16 of APC p.(Thr1493Thr) and exon 9 of SMAD4 p.(Phe362Phe). The synonymous mutation in FLT3 (c.1776T>C; p.(Val592Val) detected in this tumor was a novel variant, while the other variants were previously reported. Additionally, two known intronic variants were identified in KDR (c.798+54G>A and c.2615-36A>CA) (Table 2). A known splice site mutation (c.1310-3T>C) at an acceptor site in FLT3 (rs2491231) and a single nucleotide variant in the 3’-UTR of the CSF1R gene (rs2066934) were also identified. Additionally, in SMARCB1 a 5’-UTR variant, and an intronic variant in ERBB4 and PIK3CA respectively were found. In Figure 7, the heat map of the variant impact for each gene is presented. The color gradation from green to red indicates unknown, synonymous, missense, nonsense, and splice variants, based upon their SIFT, PolyPhen2 and Grantham scores. Only variants in four genes had a positive PolyPhen2 score (variants in TP53, PIK3CA, IDH1 and KDR genes had a PolyPhen2 score of 0.083, 0.011, 0127 and 0.003, respectively). However, FATHMM scores for the prediction of the functional consequences of a variant suggest that only the IDH1 variant is pathogenic, with a score of 0.94. As described in the COSMIC data base, FATHMM scores above 0.5 are deleterious, but only scores ≥ 0.7 are classified as pathogenic.\n\nVariant impact takes into account the type of mutation (such as insertion, deletion or frame shift) and considers the location of the variant (intronic or exonic). The color gradation from green to red indicates unknown, synonymous, missense, nonsense and splice variants, calculated based upon their SIFT, PolyPhen2 and Grantham scores.\n\n\nDiscussion\n\nEpendymomas are brain tumors that arise throughout the central nervous system, within the supratentorial areas, the posterior fossa and the spinal cord. Histologic low-grade (WHO grade I) tumors, such as subependymomas and myxopapillary ependymomas, are usually slow progressing variants of ependymomas. In contrast, grade III ependymomas display anaplastic features like hypercellularity, high mitosis, proliferation of endothelial cells and palisading necrosis34. Histopathological evaluation of ependymoma tissue reveals pseudo-rosette formation, high mitotic activity, vascular proliferation and necrosis, EMA staining with perinuclear dot-like structures and with diffuse GFAP immunoreactivity35. Immunological staining with GFAP and vimentin is very helpful for the differential diagnosis of ependymomas from other non-ependymal tumors, such as astrocytic and choroid plexus tumors, and also in differentiating between the various grades of ependymomas13,14,36. It has been reported that the GFAP expression correlates with a loss of E-cadherin expression in anaplastic ependymomas, although in this case there was E-cadherin expression36. Changes in E-cadherin expression promote tumor invasion and metastasis37. Overexpression of EGFR is known to correlate with tumor grades in ependymomas (100%, 50%, and 0% in grade I, II and III, respectively)23. The tumor in our case is grade III anaplastic ependymoma and it stained negatively for EGFR, confirming this observation. Based upon the expression profiles of numerous angiogenesis genes (HIF-1a signaling, VEGF signaling, cell migration) and signaling pathway genes (PDGF signaling, MAPK signaling, EGFR signaling), posterior fossa ependymomas are subdivided into two groups19. In this case, a diagnosis of anaplastic ependymoma (WHO grade III) was made upon the observation of the above characteristics for the tumor. The pathology of the resected tissue demonstrated a hypercellular tumor with areas of perivascular pseudo rosettes, consistent with a diagnosis of ependymoma.\n\nDespite several investigations, the correlation between histological grading of ependymoma tumors and their prognosis is unclear8,34,38. Apart from histopathological grading, previous studies have focused on gross deletions and chromosomal abnormalities through cytogenetic studies and array-CGH profiling of ependymomas39,40. These studies helped to distinguish between intracranial and spinal cord ependymomas. Around 70% of supratentorial ependymas are known to carry a fusion gene which produces the C11orf95/RELA fusion transcript and the prognosis is poor for this tumor41. Ion Torrent PGM sequencing of a grade II ependymoma demonstrated MET and ATRX copy number gain25. Overexpression of L1 cell adhesion molecule (L1CAM), 1q25 copy number gain and a homozygous deletion in CDKN2A was also reported in some aggressive supratentorial ependymomas42. However, in the present case we did not detect any MET or CDKN2A mutations using the Ion AmpliSeq Cancer HotSpot panel.\n\nPatients with neurofibromatosis type 2 are predisposed to the development of ependymomas, and the gene for neurofibromatosis type 2 (NF2) maps to chromosome 22 (q1216,17). Mutations in the NF2 gene are uncommon in sporadic ependymomas and appear to be restricted to spinal tumors43. For spinal cord ependymomas, four out of eight tumors were found to have an NF2 mutation and all eight tumors had loss of heterozygosity (LOH) of chromosome 22, where the NF2 locus is found. However, five out of eight intracranial tumors exhibited LOH of chromosome 22 but no NF2 mutations24. A high rate of truncating mutations such as nonsense and frameshift mutations in the NF2 gene were also reported previously in spinal ependymomas44,45. Unfortunately, in the Ion AmpliSeq cancer HotSpot panel primer pool used in the present study NF2 gene was not included.\n\nWe have verified all mutations in various databases (COSMIC, ExAc and dbSNP) to confirm whether variants are novel. Only one detected in our case, a synonymous variant found in FLT3 (c.1776T>C; p.(Val592Val), is a novel variant. The IDH1 mutation c.395G>A; p.(Arg132His) we detected in this tumor is a substitution missense mutation which has been reported previously (COSM28746) in glioma tumors46. In this codon, another missense G>T mutation (COSM28750), and a compound substitution c.394_395CG>GT (COSM28751) are also known. Somatic IDH1 mutations in this codon have been found with greater frequency in diffuse astrocytomas, oligodendrogliomas, oligoastrocytomas and secondary glioblastomas. However, several grade II and grade III ependymal tumors tested did not show this mutation in the IDH1 gene47. For astrocytic tumors, the presence of this mutation is known to be associated with younger patients48. This observation supports our findings for this ependymoma tumor as the patient is six years-old. This mutation is pathogenic, having a FATHMM score of 0.94. Other variants detected in this tumor, such as those in FGFR3, PDGFRA, KDR (c.1416A>T), CSF1R, EGFR, RET, HRAS, PIK3CA, FLT3 (c.1310-3T>C), and SMAD4, are benign. Variants detected in this tumor have also been reported in other cancers: PDGFRA mutations in cervical adeno-squamous carcinomas; ERBB4 mutations in lung adenocarcinomas; FGFR3 mutations in breast, endometrial and ovarian cancers; CSF1R mutations in prostate cancer; EGFR mutations in lung adenocarcinomas; RET mutations in thyroid carcinomas; HRAS mutations in melanomas; and SMAD4 mutations in breast cancer. However, with the exception of the KDR variant c.1416A>T, this is the first time the above variants are reported in a brain tumor49–57.\n\nWe found an intronic variant in PIK3CA and one missense mutation in this gene. This missense mutation was also reported previously in hemangioblastoma and in colon adenocarcinoma58,59. Missense mutations in PIK3CA are known to promote glioblastoma tumor progression60. Mutations of the PTEN gene are rare in ependymomas and we have also not detected any PTEN mutations in this tumor61.\n\nMutations in cancer driver genes such as TP53, CDKN2A, and EGFR, which are frequently affected in gliomas, have been shown to be rare in ependymomas43,61,62. We have detected a TP53 mutation (c.215C>G, p.Pro72Arg, rs1042522) in this tumor with a frequency of 47.94%. This mutation p.(Pro72Arg) has also been reported previously in a medulloblastoma tumor in a young patient63. Previous studies have shown that out of 15 ependymoma tumors tested, only one case, a patient with a malignant ependymoma of the posterior fossa, had a mutation in exon 6 of the TP53 gene, which was silent, and in another study only one out of 31 ependymoma tumors tested contained a mutation in the TP53 gene64,65. However, in another study, out of 15 ependymoma tumors, none had a mutation in the TP53 gene, suggesting that this gene does not play an important role in the pathogenesis and development of ependymomas, unlike other brain tumor types61,65,66. Miller et al., (2018) through whole-exome sequencing of an anaplastic ependymoma tumor, have shown mutations in several cancer-related genes, as well as genes related to metabolism, neuro-developmental disorder, epigenetic modifiers and intracellular signaling67. These authors have shown resistance-promoting variant expression in a single ependymoma case at different stages of recurrence. However, these genes were not present in the cancer panel we used in this study. Using the human exome capture on Illumina, Bettegowda et al., (2013) have reported that in one out of eight grade III intracranial ependymomas, tumors have mutations in PTEN and TP53, and one tumor with HIST1H3C mutations24. The HIST1H3C p.(Lys27Met) mutation has also been reported previously in posterior fossa ependymomas68. Ependymomas may in fact represent a very heterogeneous class of tumors, each with distinct molecular profiles and, even within posterior fossa ependymomas, there are at least two distinct gene expression patterns, as demonstrated by Witt et al., (2011)19,69. Overall, in previous studies, a very low frequency of mutations was observed in both intracranial and spinal ependymomas and our findings also supports this observation19,24,25,41.\n\nThe Ion AmpliSeq Cancer HotSpot Panel consists of 207 primers in 1 tube, targeting 50 oncogenes and tumor suppressor genes that are frequently mutated in several types of cancers. The detected mutations were found to have high accuracy; 100% amplicons had at least 500 reads and 500x target base coverage was also 100%. This high level of accuracy and the high depth of coverage achieved with the Ion Proton system allowed us to reliably detect low frequency mutations with high confidence. Allele coverage in most of the variants is around 2000x, the p-value was 0.00001 and the Phred score was very high for all the variants, indicating high confidence in the variants found in this tumor. Apart from its use in whole-exome sequencing, cancer panel analysis has also become common practice for Ion Proton26. The Ion Proton instrument has the advantage of pooling samples using barcodes and the Ion PI chip. For pooled samples, sequencing enables a high throughput up to 15 Gb of data, with more than 60–80 million reads passing read filtering. The purpose of read filtering is to discard the reads that contain low quality sequences, to remove polyclonal reads, remove reads with an off-scale signal, remove reads lacking a sequencing key, remove adapter dimers, and remove short reads etc. If the computed mean read length from all the reads and the minimum total mapped reads in the sample is less than the specified threshold, that sample does not pass the quality control.\n\nRecent molecular diagnostics research had helped in subdividing glioblastomas, oligodendrogliomas and oligoastrocytomas into genetically diverse groups of tumors, and these mutational markers may help in predicting the prognosis and response to therapy70. However, such a strategy for the molecular subdivision of ependymomas has been not successful so far using mutational profiling. Epigenetic markers and fusion protein analysis have also helped in identifying new groups of supratentorial ependymoma tumors and in spite of the histopathological signs of malignancy, a small set of ependymomas had a very good prognosis, suggesting that this subgroup of tumors should not be diagnosed as classic ependymomas71. However, another study showed that methylation profiling did not identify a consistent molecular class within the supratentorial tumors, but successfully sub-classified posterior fossa ependymoma into two subgroups72.\n\nIn conclusion, we have identified four known missense mutations, eight synonymous and seven intronic, in this grade III ependymoma. Out of these, only one mutation in FLT3 (c.1776T>C, synonymous) is novel, and only one mutation in IDH1 (c.395G>A, missense) is deleterious, with all other mutations benign. Many of the variants we reported here were not detected in the ependymal tumors analyzed by NGS previously. HRAS c.81T>C, PIK3CA c.1173A>G, RET c.2307G>T, KDR c.1416A>T, APC c.4479G>A, EGFR c.2361G>A and FLT3 c.1310-3T>C variants have not been previously reported in brain tissue, as verified in COSMIC data base, although they have been reported in other tissues like lung and breast. Further studies are warranted, using NGS methods in all three grades of intracranial ependymomas to identify the genetic signatures that may distinguish between these tumors at the molecular genetic level.\n\n\nData availability\n\nRaw sequence reads for this tumor on Sequence Read Archive, Accession number SRP192752: https://identifiers.org/insdc.sra/SRP192752\n\nOpen Science Framework: Mutation profiling of anaplastic ependymoma grade III by Ion Proton next generation DNA sequencing. https://doi.org/10.17605/osf.io/y9sfg33\n\nThis project contains the following underlying data:\n\n- All variants before filteration.xlsx (spreadsheet of all annotated variants)\n\n- Final Variant Calls using HotSpot filter.xlsx (spreadsheet of annotated variants called using HotSpot filter)\n\n- TSVC_variants_IonXpress_013.vcf (file containing all variants (un-annotated) in vcf format)\n\n- TSVC_variants_IonXpress_013.vcf.gz.tbi (file containing all variants (un-annotated) in vcf.gz.tbi format)\n\n- heatmap-gr3 ion rep.csv (spreadsheet containing impact scores used to generate heat map)\n\n- EGFR Figure neg.jpg (images for EGFR staining)\n\n- Fig2A.jpg – Figur4D.jpg (raw image files used in Figure 2–Figure 5)\n\n- Radiology Fig.1.jpg – Radiology Figure 1 (2).jpg (raw image files used in Figure 1)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nOpen Science Framework: Mutation profiling of anaplastic ependymoma grade III by Ion Proton next generation DNA sequencing. https://doi.org/10.17605/osf.io/y9sfg33\n\nThis project contains the following extended data:\n\n- Gr-3 Advaiata Final report.pdf (Advaiata iVariant analysis report)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
"appendix": "Grant information\n\nThis study is supported by a grant from The Deanship of Scientific Research, Umm-Al-Qura University, Makkah to Dr. MM. Taher (Code. No. 43509008).\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 appreciate the technical help of Mrs. Rowa Abbas Bakhsh, Histopathology Division, Al-Noor Hospital, Makkah. The authors would like to thank the staff of Science and Technology Unit at Umm Al-Qura University for the continuous support.\n\n\nReferences\n\nDuff DJ, Miller DC: Ependymomas (Review). AJSP: Reviews & Reports. 2013; 18(5): 221–230. Publisher Full Text\n\nWu J, Armstrong TS, Gilbert MR: Biology and management of ependymomas. Neuro Oncol. 2016; 18(7): 902–913. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWesseling P, Capper D: WHO 2016 Classification of gliomas. Neuropathol Appl Neurobiol. 2018; 44(2): 139–150. 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}
|
[
{
"id": "53778",
"date": "17 Oct 2019",
"name": "Luni Emdad",
"expertise": [
"Reviewer Expertise Cancer Biology and Molecular Oncology",
"Genetics",
"Targeted experimental therapeutics development"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this study authors analyzed mutation patterns by next generation sequencing (NGS) in order to determine genomic changes in an anaplastic ependymoma. Authors identified one novel synonymous mutation and one deleterious missense mutation in this grade III ependymoma.\n\nComments:\nIn the clinical specimen section authors state “Specimens from all patients willing to give written informed consent and diagnosed with gliomas were eligible to be included in this study.” However, the specimen used in this study for NGS analysis was obtained from a single patient’s tumor tissue. What are the other samples and in what study they were used?\n\nFigure 2A: authors indicate yellow arrow showing palisading necrosis; is this the correct location?\n\nAuthors conclude they identified four known missense mutations, eight synonymous and seven intronic, in this grade III ependymoma. How global these signature molecules in the context of grade III ependymoma? Can the authors add additional correlation analysis by analyzing TCGA or other bioinformatics based data?\n\nAuthors indicate FLT3 (c.1776T>C, synonymous) is novel, which is interesting; is this mutation reported in other cancer than ependymoma? What is the clinical and functional significance of this mutation in ependymoma progression?\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": [
{
"c_id": "5594",
"date": "09 Jun 2020",
"name": "Mohiuddin Taher",
"role": "Author Response",
"response": "We are thankful to the reviewer-1 (Dr. Luni Emdad, Assistant Professor, Human and Molecular Genetics Department, Virginia Commonwealth University, Richmond, USA) for the helpful suggestions and comments. We have made the necessary changes in the revised manuscript following these suggestions. Our answers to the comments are listed below.Comment:1. In the clinical specimen, section authors state “Specimens from all patients willing to give written informed consent and diagnosed with gliomas were eligible to be included in this study.” However, the specimen used in this study for NGS analysis was obtained from a single patient’s tumor tissue. What are the other samples and in what study they were used?Answer:The present study is a part of the project on mutation profiling of brain tumors in the Saudi population which is supported by a grant from Umm-Al-Qura University (Code. No. 43509008). For this project, various gliomas such as GBM, astrocytomas, oligodendrogliomas, and ependymoma tumors (all grades) were collected. The NGS analysis is ongoing for many other tumors. The NGS analysis reported here is for a single case, as suggested by this referee, we have modified this statement in the revised manuscript in the ‘Methods’ section. Comment: 2. Figure 2A: Authors indicate yellow arrow showing palisading necrosis; is this the correct location?Answer: As suggested by the referee the corrections are made in this figure, and a new figure-2 and a new figure legend is added in the revised manuscript. Comment:3. Authors conclude they identified four known missense mutations, eight synonymous and seven intronic, in this grade III ependymoma. How global these signature molecules in the context of grade III ependymoma? Can the authors add additional correlation analysis by analyzing TCGA or other bioinformatics-based data?Answer:As suggested by the reviewer we have searched various databases including The Cancer Genome Atlas (TCGA) www.tcga.org, and summarize our answer to the reviewer’s comments below. A relevant section of this summary is included in the revised manuscript. The tumor type we described in this study is a rare tumor, and specifically, in many databases, it is not listed separately except in the ‘Integrative onco-genomics’ database (https://www.intogen.org). In the integrative onco-genomics database (https://www.intogen.org/search?cancer) high-grade glioma and low-grade glioma, and GBM projects are listed. Genes mutated such as TP53, and PDGFRA are in high-grade glioma data from St. Jude children's research hospital (HGG_D_STJUDE), and IDH1, TP53, PIK3CA, EGFR, are most recurrently mutated cancer driver genes in GBM_TCGA dataset (https://www.intogen.org). Also, under ‘ependymoma’ only, two cohorts are found, with a total of 94 samples, 10,607 total mutations, and one driver gene is mutated in this. In the ependymoma – DKFZ cohort, (EPD_PRY_DKFZ_2017), a total of 55 Samples, mutations with driver genes found are nil, total mutations found are 534, (Grobner et al., 2018). Ependymoma data from St. Jude children's research hospital cloud, 39 samples are found, with 1 driver gene mutated, mutations-10,073. By WGS, PIK3CA is detected as a mutational cancer driver in the ependymoma cohort. 2 out of 39 (5.13%) samples (3:179203765: T>A; AA, 345, 3:179221147: A>C, AA726), were found to have a mutation in this driver gene. (Parker et al., 2014). We have analyzed the TCGA (https://www.intogen.org/search?cancer) release 2020-02-01, dataset. Tumor types such as medulloblastoma (636 samples), pilocytic astrocytoma (621 samples), GBM (571 samples), and LGG (565 samples) are included in this but not ependymoma. Glioblastoma was the first cancer studied by TCGA in a pilot study. In the TCGA projects top mutated cancer genes in IDH1, TP53, EGFR, PIK3CA, and PDGFRA. In the TCGA database the missense IDH1 mutation p.(R132H) affected cases are 90.07%, and VEP impact (Ensembl Variant Effect Predictor) is moderate for this, and SIFT impact is deleterious low confidence (score 0.01), and the PolyPhen impact is also possibly damaging (score 0.813). Poor prognostic markers included genetic changes in the EGFR and PI3-kinase mutations in this group. Among most driver mutations IDH1/2 and TP53 tumor grade remain a prognostic factor in diffuse gliomas (Draaisma et al., 2015). The variant we found in the present ependymoma case such as in TP53, HRAS, SMAD4, PIK3CA not in TCGA. However, in ClinVar database SMAD4 synonymous variant is reported (Accession: VCV000132693.2, Variation ID: 132693). In cBioportal database (https://www.cbioportal.org) of whole-exome sequencing (WES) under diffuse glioma project for CNS/Brain tumors, only diffuse glioma, low-grade glioma, glioblastoma, medulloblastoma, and pilocytic astrocytoma and other brain tumors are classified, but ependymoma is not listed separately to apply the filter. As stated by the reviewer we can apply the search only globally under low-grade glioma (LGG) and glioblastoma (GBM World Health Organization grade IV)) projects, and for global mutation profiling not related to the variants, we described in our manuscript. On this portal under CNS/brain studies merged cohort of LGG and GBM; TCGA, (Ceccarelli et al., 2016) a total of 1102 samples are present in that 812 (72.4%) cases are with mutations. In IDH1 gene somatic mutations found in 411 samples (50.6%). More than 90% of them are missense mutations. There is promising clinical data in patients with IDH1 codon R132-mutant glioma treated with the IDH1-targeted inhibitor Ivosidenib an FDA-approved drug in another indication. This mutated amino acid was identified as a recurrent hotspot (statistically significant) in a population-scale cohort of tumor samples of various cancer types (Chang et al., 2016). Samples with TP53 mutations are 322 (39.7%), there are no FDA-approved or NCCN-compendium listed treatments specifically for patients with c.215C>G mutant diffuse glioma. Mutations in PIK3CA cases are 63 (7.8%). While Alpelisib in combination with Fulvestrant is FDA-approved for the treatment of patients with PIK3CA-mutant ER+/HER2- breast cancer, its clinical utility in patients with PIK3CA mutations in diffuse glioma is unknown. Mutations in EGFR cases are 87, (10.7%), laboratory data suggest that glioma cells with EGFR mutations may be sensitive to Lapatinib, an EGFR/HER2 tyrosine kinase inhibitor. While the EGFR tyrosine kinase inhibitor Afatinib is FDA-approved for the treatment of patients with EGFR G719-mutant non-small cell lung cancer, its clinical utility in diffuse glioma patients with EGFR mutations is unknown. PDGFRA mutations cases are 12 (1.5%), while Imatinib is NCCN-compendium listed for the treatment of patients with gastrointestinal stromal tumors (GISTs) harboring oncogenic PDGFRA alterations, its clinical utility in diffuse glioma patients with PDGFRA mutation is unknown. KDR case with mutations are 1%, there are no FDA-approved or NCCN-compendium listed treatments specifically for patients with KDR mutant diffuse glioma. FLT3 cases are 0.6%, while the multi-kinase inhibitor Midostaurin in combination with intensive chemotherapy is FDA-approved for the treatment of patients with FLT3 mutant acute myeloid leukemia (AML), its clinical utility in patients with FLT3 mutant diffuse glioma is unknown. RET and MET cases are 0.6%, APC cases are 0.2%, ERBB4 cases are 0.4%, no clinical data is available for these genes. FGFR3 mutation cases are 0.2%, while there is promising clinical data in patients with urothelial cancer harboring functionally characterized hotspot FGFR3 mutations treated with pan-FGFR-targeted inhibitors such as AZD4547, BGJ398, Debio1347 and Erdafitinib, their clinical utility in patients with FGFR3 mutant diffuse glioma is unknown. None of the cases found to have SMARCB1, CSF1R, SMAD4, and HRAS, mutations. Whereas when we searched in a glioma cohort of 1004 samples (https://www.cbioportal.org/MSKCC, Jonsson et al., 2019) on this portal cases found to have SMARCB1, CSF1R, SMAD4, and HRAS mutations are 1.3%, 1.4%, 0.6%, and 0.2% respectively. We have searched the database “Genomics of drug sensitivity in cancer” (cancerrxgene.org) to verify the studies targeting the signaling pathways of the four missense variants found in our studies. Compounds such as AGI-5198, GSK690693, Foretinib, and MIRA-1 shown to sensitize in GBM and LGG cell lines in vitro, targeting IDH1, PIK3CA, KDR, and TP53 pathways respectively (Iorio et al., 2016). ICGC (https://dcc.icgc.org) data portal 2504 cases in 6 projects were reported; 3 projects from the US, 1 each from Canada, Germany, and China respectively. GBM-TCGA US-595 case, pediatric brain cancer-DE, -554 cases, LGG-TCGA, US, 514 cases were reported. In top 20 mutated cancer genes with high functional impact somatic mutations in 393 cases of LGG affected in IDH1, 24 cases of GBM affected in IDH1, 11 cases of pediatric brain cancer in IDH1, 229 cases of LGG in TP53, 105 cases of GBM in TP53, 42 cases of pediatric brain cancer in TP53, 10 cases of pediatric brain cancer in TP53. 13 cases of LGG in EGFR, 19 cases of GBM in EGFR. Other genes were not in Top 20 genes. In the NCI's Genomic Data Commons (GDC) portal (https://portal.gdc.cancer.gov/) cases TCGA -GBM and TCGA -LGG are projects with 617, and 516 cases are reported, with mutations in 396, and 513 cases reported. IDH1 mutations were found in 394 out of 903 cases, cases were affected with mutations in TP53 are 233, 38 cases were affected in PIK3CA; EGFR, 95 cases, PDGFR 20 cases, in 2 projects. IDH1 c.395G>A p.(R132H) mutation (rs121913500), are reported in GBM, astrocytoma, and oligodendrogliomas in Clin variant database, #VCV000156444. The frequency of this variant is highest (64.3%) in GBM WHO grade IV, compared with astrocytoma WHO grade II (7.14%), anaplastic oligodendroglioma WHO grade III (14.3%) and anaplastic ependymoma WHO grade III (14.3%) (Yusoff et al., 2016). This variant also reported in AML as an adverse prognostic factor (Wagner et al., 2010). The NCI's genomic data commons (GDC) have reported 28 mutations in 102 affected cases with KDR mutations in all TCGA GBM projects. The expression KDR gene (vascular endothelial growth factor receptor 2 or VEGFR2) is increased in endothelial cells during tumor angiogenesis, and missense mutations cause constitutive activation of VEGFR2 in hemangioma. Patients with infantile capillary hemangioma are known to have constitutive activation of VEGFR2 signaling and carry a germline mutation (C482R) in the KDR gene (Jinnin, et al., 2008). TP53 c.215C>G, PIK3CA c.1173A>G, c.352+40A>G, EGFR c.2361G>A not reported in GDC. SMAD4 mutation in c.1086T>C (rs1801250) reported in breast cancer (Tram et al., 2011); HRAS mutation in c.81T>C rs12628, reported in chronic myeloid leukemia (Mir et al., 2015), PIK3CA mutation in c.352+40A>G, rs3729674 in breast cancer (Arsenic et al., 2015) ; ERBB4 mutation in c.421+58A>G, rs839541 in brain tissue (Mothersill et al., 2012), TP53 mutations in GBM mostly point mutations that lead to a gain of function (GOF) of the oncogenic variants of the p53 protein (Zhang et al., 2018). The TP53 variant rs1042522 we reported in the present case [(c.215C>G, p. (Pro72Arg)] was also reported in anaplastic astrocytoma grade-III (Pessoa et al., 2019), but not in ependymoma cases. In COSMIC database this variant is reported in several types of cancers. A mutation in Exon 5 of the TP53 gene was reported in one anaplastic ependymoma out of three cases (Tominaga et al., 1995). Whereas in our case the mutation was found in exon 4. Targeted therapy is being studied for the treatment of childhood ependymoma and other brain tumors utilizing the genomic data. NCI supported Clinical Trials for Ependymomal brain tumors 4 clinical trials are listed (https://www.cancer.gov/types/brain/patient/child-ependymoma-treatment-pdq). One study is enrolling the patients for drug targets (Carboplatin and Bevacizumab for Recurrent Ependymoma) that inhibit VEGF-promoted angiogenesis. Based on the interesting results observed in the reported small series of patients with recurrent ependymomas treated with bevacizumab, as well as on the evidence of VEGF-promoted angiogenesis in these tumors, we designed a phase II study to test the efficacy of bevacizumab in patients with recurrent ependymoma. Cabozantinib, a multi-kinase inhibitor of FLT3, MET, VEGFR2, and KIT, respectively, and clinical trials are undergoing for Non-Small Cell Lung Cancer, thyroid cancer, AML, and GBM treatment. References: Arsenic R, Treue D, Lehmann A, et al., Comparison of targeted next-generation sequencing and Sanger sequencing for the detection of PIK3CA mutations in breast cancer. BMC Clin Pathol. 2015; 15:20. doi: 10.1186/s12907-015-0020-6. eCollection 2015. Chang MT, Asthana S, Gao SP, et al., Identifying Recurrent Mutations in Cancer Reveals Widespread Lineage Diversity and Mutational Specificity. Nat Biotechnol. 2016; 34: 155-163. doi: 10.1038/nbt.3391. Ceccarelli M, Barthel FP, Malta TM, et al., TCGA Research Network; Noushmehr H, Iavarone A, Verhaak RGW. Molecular Profiling Reveals Biologically Discrete Subsets and Pathways of Progression in Diffuse Glioma (Ceccarelli et al., 2016). Draaisma K, Wijnenga MMJ, Weenink B, et al., PI3 kinase mutations and mutational load as poor prognostic markers in diffuse glioma patients. Acta Neuropathologica Communications. 2015; 3:88 DOI 10.1186/s40478-015-0265-4. Grobner SN, Worst BC, Weischenfeldt J, et al., ICGC PedBrain-Seq Project; ICGC MMML-Seq Project. The Landscape of Genomic Alterations Across Childhood Cancers. Nature. 2018; 555 :321-327. doi: 10.1038/nature25480. Iorio F, Knijnenburg TA, Vis DJ, et al., A landscape of pharmacogenomic interactions in cancer. Cell. 2016; 166: 740 – 754. doi: 10.1016/j.cell.2016.06.017. Jinnin, M, et al., Suppressed NFAT-dependent VEGFR1 expression and constitutive VEGFR2 signaling in infantile hemangioma. Nature Med. 14: 1236-1246, 2008. Jonsson P, Lin AL, Young RJ, et al., Genomic Correlates of Disease Progression and Treatment Response in Prospectively Characterized Gliomas. Clin Cancer Res. 2019; 25: 5537-5547. doi: 10.1158/1078-0432.CCR-19-0032. Mir R, Ah I, Javid J, et al., Polymorphism T81C in H-RAS Oncogene Is Associated with Disease Progression in Imatinib (TKI) Treated Chronic Myeloid Leukemia Patients. World J Oncol. 2015; 6: 321-328. DOI: 10.14740/wjon912e. Mothersill O, Kelly S, Rose EJ, Donohoe G. The effects of psychosis risk variants on brain connectivity: a review. Front Psychiatry. 2012; 3:18. doi: 10.3389/fpsyt.2012.00018. Parker M, Mohankumar KM, Punchihewa C, et al., C11orf95–RELA fusions drive oncogenic NF-κB signalling in ependymoma. Nature. 2014; 506: 451–455. Pessoa IA, Amorim CK, Ferreira WAS, et al., Detection and Correlation of Single and Concomitant TP53, PTEN, and CDKN2A Alterations in Gliomas. Int J Mol Sci. 2019; 20 (11). pii: E2658. doi: 10.3390/ijms20112658. Tominaga T, Kayama T, Kumabe T, et al., Anaplastic ependymomas: clinical features and tumour suppressor gene p53 analysis. Acta Neurochir (Wien). 1995; 135(3-4):163-70. https://doi.org/10.1007/BF02187763. Tram E, Ibrahim-Zada I, Briollais L, et al., Identification of germline alterations of the mad homology 2 domain of SMAD3 and SMAD4 from the Ontario site of the breast cancer family registry (CFR). Breast Cancer Res. 2011 ;13(4): R77. doi: 10.1186/bcr2926. Wagner K, Damm F, Göhring G, et al., Impact of IDH1 R132 Mutations and an IDH1 Single Nucleotide Polymorphism in Cytogenetically Normal Acute Myeloid Leukemia: SNP rs11554137 Is an Adverse Prognostic Factor. J Clin Oncol. 2010; 28: 2356-2364. doi: 10.1200/JCO.2009.27.6899. Yusoff AM, Zulfakhar FN, Sul’ain MD, et al., Association of The IDH1 C.395G>A (R132H) Mutation with Histological Type in Malay Brain Tumors. Asian Pac J Cancer Prev. 2016 ;17: 5195-5201. doi:10.22034/APJCP.2016.17.12.5195. Zhang Y, Dube C, Gibert M Jr, et al., The p53 Pathway in Glioblastoma. Cancers (Basel). 2018 Sep 1;10(9). pii: E297. doi: 10.3390/cancers10090297. Comment:4. Authors indicate FLT3 (c.1776T>C, synonymous) is novel, which is interesting; is this mutation reported in other cancer than ependymoma? What is the clinical and functional significance of this mutation in ependymoma progression?Answer:A relevant section is added to the revised manuscript regarding this comment. This mutation is not reported in the merged cohort of LGG and GBM-TCGA project (Ceccarelli et al., 2016). This driver gene FLT3 down-regulation is related to favorable clinical outcome in glioma patients (Liang et al., 2017). Interestingly, several pathogenic mutations were reported at Val592 coding positions c.1774 and c.1775 in the COSMIC database but all in acute myeloid leukemia (AML). However, the FLT3 synonymous mutation found in our ependymoma case (c.1776T>C) is within the juxta-membrane domain in exon 14, which codes the same amino acid valine p. (Val592Val), hence we don’t anticipate having any functional and clinical impact of this mutation. This variant is not reported in the COSMIC database or dbSNP also. The most common form of FLT3 mutation is in the internal tandem duplication (ITD) region of the juxta-membrane domain (AA 572-609), which occurs in 15–35% of patients with AML (Staudt et al., 2018). The presence of either a FLT3 ITD or TKD mutation may be associated with a response to the tyrosine kinase inhibitor, Midostaurin (Stone et al., 2017). FLT3 activating mutations were predominant in AML, and types of leukemias and lymphomas also (Metzeler et al., 2016; The AACR Project GENIE Consortium). In the database of Genomics of Drug Sensitivity in Cancer, https://www.cancerrxgene.org/compound, mutations in FLT3 are associated with altered sensitivity to several drugs tested in vitro, also several clinical trials are ongoing using these compounds (Daver et al., 2019). References: Staudt D, Murray H, McLachlan T, et al., Targeting Oncogenic Signaling in Mutant FLT3 Acute Myeloid Leukemia: The Path to Least Resistance. Int. J. Mol. Sci. 2018, 19, 3198; doi:10.3390/ijms19103198Liang, A., Zhou, B. and Sun, W. Integrated genomic characterization of cancer genes in glioma. Cancer Cell Int 2017; 17: 90. https://doi.org/10.1186/s12935-017-0458-y.Daver N, Schlenk RF, Russell NH and Levis MJ. Molecular targets for therapy Targeting FLT3 mutations in AML: review of current knowledge and evidence. Leukemia. 2019; 33: 299–312. doi: 10.1038/s41375-018-0357-9. Metzeler KH, Herold T, Rothenberg-Thurley M, et al., AMLCG Study Group. Spectrum and prognostic relevance of driver gene mutations in acute myeloid leukemia. Blood. 2016; 128: 686-698. doi: 10.1182/blood-2016-01-693879.Stone RM, Mandrekar SJ, Sanford BL, et al., Midostaurin Plus Chemotherapy for Acute Myeloid Leukemia with a FLT3 Mutation. N Engl J Med. 2017; 377: 454-464. doi: 10.1056/NEJMoa1614359.The AACR Project GENIE Consortium. AACR Project GENIE: powering precision medicine through an international consortium. Cancer Discovery. 2017; 7: 818-831. DOI: 10.1158/2159-8290.CD-17-0151."
}
]
},
{
"id": "58035",
"date": "26 Feb 2020",
"name": "Firoz Ahmad",
"expertise": [
"Reviewer Expertise Molecular Diagnostics",
"Cancer Genetics",
"Next Generation Sequencing."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors presented report on their NGS study on anaplastic ependymoma grade III by Ion Proton next generation DNA sequencing.\n\nThe article looks interesting, and is well written. Since this report is from a single case, a necessary correction has to be made in the material section. Also, the authors need to mention the tumor content of the FFPE block which was taken for the NGS analysis.\nI feel, the author need to cut down on the text significantly while explaining NGS data analysis and variant identification statistics. There is too much of information which is not required. The authors have already shared the coverage analysis report which is sufficient.\n\nExcept for IDH1 mutation, other variants are non significant and hence do not add much value clinically. Identification of FLT3 p.(Val592Val) does not carry any meaning unless and until the authors show the functional impact of this variant.\nOverall, good article but the text needs to be significantly cut down considering this to be a single case report.\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": [
{
"c_id": "5595",
"date": "09 Jun 2020",
"name": "Mohiuddin Taher",
"role": "Author Response",
"response": "Response to 2nd Reviewer’s Comments: We are thankful to this reviewer (Dr. Firoz Ahmed, Research Scientist, and Senior Manager-R&D, SRL Diagnostics, SRL Limited, Goregaon (West), Mumbai, India) for the helpful suggestions and comments. We have made the necessary changes in the manuscript following these suggestions. Our answers to the comments are listed below.Comment:The article looks interesting and is well written. Since this report is from a single case, a necessary correction has to be made in the material section. Also, the authors need to mention the tumor content of the FFPE block which was taken for the NGS analysis. Answer: We agree with the reviewer’s suggestion about the sample size. Regarding the single case, a correction is made in the manuscript in the Methods section. As suggested by the reviewer, we have indicated tumor tissue content of the FFPE block used in this investigation (Methods section). Comment:I feel the author needs to cut down on the text significantly while explaining NGS data analysis and variant identification statistics. There is too much of information which is not required. The authors have already shared the coverage analysis report which is sufficient. Answer: We agree with the reviewer’s suggestion, however, according to the journal's policy, we have described in detail manner several techniques used in this study and the results also in detail. The NGS data analysis such as variant identification and coverage analysis will be helpful to reproduce this work and help others to understand the data who are not familiar with NGS analysis.Comment:Except for IDH1 mutation, other variants are non-significant and hence do not add much value clinically. Identification of FLT3 p. (Val592Val) does not carry any meaning unless and until the authors show the functional impact of this variant. Answer: Referee-1 also made a similar comment, and we agree with this reviewer’s suggestion. We have made the necessary changes in the revised manuscript. We have identified in this study four missense variants; c.395G>A in IDH1, in c.1173A>G in PIK3CA in c.1416A>T in KDR, in c.215C>G in TP53. Other variants are synonymous and intronic ones. We agree with the reviewer’s comments that “Identification of FLT3 mutation p. (Val592Val) in exon 14 does not have any structural-functional impact as this is a synonymous variant coding for the same amino acid.” However, this variant is not reported in the COSMIC database or dbSNP also. It is a novel variant of FLT3, so it is important to report this variant. In the LOVD database 5 more synonymous mutations were reported in exon 14 (amino acids 572-609, in the juxtamembrane domain); in c.1746, c.1770, c.1773, c.1803 and in codon 1805; http://databases.lovd.nl/whole_genome/variants/FLT3. Internal tandem duplications of FLT3 (FLT3-ITD) are the most common mutations (in the juxtamembrane domain) associated with acute myelogenous leukemia (AML) and are a prognostic indicator associated with adverse disease outcome. Activating internal tandem duplication (ITD) mutations in FLT3 (FLT3-ITD) are detected in approximately 20% of acute myeloid leukemia (AML)."
}
]
}
] | 1
|
https://f1000research.com/articles/8-613
|
https://f1000research.com/articles/9-112/v1
|
13 Feb 20
|
{
"type": "Correspondence",
"title": "The deep(er) roots of Eukaryotes and Akaryotes",
"authors": [
"Ajith Harish",
"David Morrison",
"David Morrison"
],
"abstract": "Background: Locating the root node of the “tree of life” (ToL) is one of the hardest problems in phylogenetics. The root-node or the universal common ancestor (UCA) divides descendants into organismal domains. Two notable variants of the two-domains ToL (2D-ToL) have gained support recently. One 2D-ToL posits that eukaryotes (organisms with nuclei) and akaryotes (organisms without nuclei) are sister clades that diverged from the UCA and that Asgard archaea are sister to other archaea, whereas the other proposes that eukaryotes emerged within archaea and places Asgard archaea sister to eukaryotes. Williams et al. (Nature Ecol. Evol. 4: 138–147; 2020) re-evaluated the data and methods that support the competing two-domains proposals and concluded that eukaryotes are the closest relatives of Asgard archaea. Critique: We argue that important aspects of estimating evolutionary relatedness and assessing phylogenetic signal in empirical data were overlooked. We focus on phylogenetic character reconstructions necessary to describe the UCA or its closest descendants in the absence of reliable fossils. It is well known that different character types present different perspectives on evolutionary history that relate to different phylogenetic depths. Which 2D-ToL is better supported depends on which kind of molecular features – protein-domains or their component amino acids – are better for resolving common ancestors at the roots of clades. In practice, this involves reconstructing character compositions of the ancestral nodes all the way back to the UCA. We believe the criticisms of 2D-ToL focus on superficial aspects of the data and reflects common misunderstandings of phylogenetic reconstructions using protein domains (folds).\n\nClarifications: Models of protein domain evolution support more reliable phylogenetic reconstructions. In contrast, even the best available amino acid substitution models fail to resolve the archaeal radiation, despite employing thousands of genes. Therefore, the primary domains Eukaryotes and Akaryotes are better supported in a 2D-ToL.",
"keywords": [
"Asgard archaea",
"2D",
"tree of life",
"LUCA",
"phylogenomics",
"nonstationary",
"rooting",
"eukaryogenesis"
],
"content": "Background\n\nModels of character evolution are essential to determine the evolutionary relationships of organisms. Phylogenetic models that employ protein structural domains as characters place Asgards as sister to other archaea (Figure 1a), and archaea sister to bacteria in the “tree of life” (ToL)1–3. Whereas several analyses that employ amino-acids as characters fail to resolve the archaeal radiation (Figure 1b) or to identify a distinct ancestor of archaea4. Conflicts between different studies that employ different character types are often due to incompatible assumptions about the character-evolution processes5–7. In a recent study, Williams et al.4 compared the performance of several character-evolution models to evaluate which one of the ToL hypotheses is better supported. The authors tested the performance of several substitution models for amino acid characters using empirical data, but models for protein-domain characters with simulated data4.\n\n(a) The rooted tree (phylogeny) inferred by estimating the evolution of species-specific changes in protein domain composition. Directional character-evolution models place the root between eukaryotes and akaryotes. Named groups of organisms, including Asgardarchaeota are resolved into clades (i.e. a single ancestor). The Asgard archaea are sister to all other archaea, with euryarchaea being the closest relatives. The phylogeny shown is a condensed form obtained after collapsing the clades of the full tree shown previously1. (b) The unrooted tree inferred by estimating the evolution of protein/gene-specific changes in amino acid composition. The unrooted-tree is the same as in Figure S8d in the article by Williams et al.4. The group archaea, and Asgard archaea are unresolved; and a distinct archaeal ancestor is absent. Time-reversible character evolution models cannot identify the root (the universal common ancestor (UCA)) as well. Alternative rootings polarize the branching order in opposite directions implying incompatible relationships among the major organismal clades. Regardless of the rooting, neither Asgard archaea nor archaea as a whole can be resolved as a monophyletic group. Further, Argards do not share a unique common ancestor with other archaea. Even the best-fitting amino acid evolution models cannot resolve the archaeal radiation despite employing thousands of genes4. The poor resolution of archaea is seen in virtually all trees, with or without inclusion of long branches of bacteria. In such ambiguous cases, “character polarization” as in (a) is likely to be efficient, rather than the more commonly used “graphical polarization” of unrooted trees. Clade support is indicated for key groups as (a) Bayesian posterior probability, (b) bootstrap percentage.\n\nThe authors present a comprehensive analysis of protein sequence data and lucid arguments about the fit of the amino acid substitution models to the relevant datasets examined. However, description of the protein domain characters (which they refer to as protein folds) and relevant published analyses were not adequately explained (see references 2 and 3) or were overlooked (see references 1 and 8). Further, based on simple frequency distributions they suspect that identification of UCA and character compositions at the root node could be biased. Such simple frequency distributions can be misleading. Careful and rigorous analyses of empirical datasets1–3,8 that demonstrate the robustness of rooting and tree topology against many potential biases were ignored4. Here, we would like to clarify certain aspects of the published protein domain-based phylogenies so as to avoid further misunderstandings and to highlight their advantages for phylogenetic reconstruction.\n\nWilliams et al.4 rely on (i) simulated data to reject a robust phylogeny inferred from empirical data (Figure 1a) that supports the evolutionary kinship of eukaryotes and akaryotes (akaryote 2D-ToL)1–3; and (ii) the so-called bacterial rooting to interpret a partially resolved, unrooted-ToL (Figure 1b), asserting that Asgard archaea are the closest relatives of eukaryotes (eocyte 2D-ToL)4. Both assertions are questionable, since (i) simulated data neither reproduce nor represent empirical distributions, and (ii) poorly resolved trees obscure evolutionary relationships. We argue that Williams et al.4 have overlooked important aspects of assessing phylogenetic signal in empirical data, and that it may be premature to reject a well-supported phylogeny1–3 based on simulated data4.\n\n\nWhich molecular feature is a better phylogenetic character?\n\nReversibility of amino-acid replacements due to biochemical redundancy makes determining character compositions of ancestral nodes ambiguous, as character polarity is ambiguous. This has been a sticking point for locating a distinct archaeal common ancestor (CA) to resolve the archaeal radiation. This is routinely seen as a conspicuous absence of the archaeal CA as well as the universal CA (UCA) in unrooted trees (e.g. Figure 1b), inferred using time-reversible models of character evolution4,9,10. Without a distinct node to unite the archaeal branches, the archaea are unresolved, whereas eukaryotes and bacteria are resolved so that their CA nodes are discernable.\n\nProtein structural domains, unlike amino acids, are biochemically non-redundant (see below) and have proven to be excellent “genomic characters”1,2 that support a robust akaryote 2D-ToL (Figure 1a). Though undervalued, they afford many conceptual and technical advantages over amino acids for reliable phylogenetic modeling1,7,11 and estimating ancestral compositions2,3,12:\n\nSubstitutions between structural domains do not occur, unlike amino acid replacements, since each domain defines a distinctive biochemical function1 (Figure 2a).\n\nThe natural bias in gain/loss rates, arising from the difficulty of parallel gains and the relative ease of parallel losses, is useful for implementing directional (rooted) character-evolution models3,12,13.\n\n(a) Protein-domains are considered to be independent evolutionary units with a distinct tertiary fold, amino acid sequence and biochemical function. The majority of proteins are multi-domain proteins formed by duplication and recombination of domain units. Covariation of protein-domain composition among the 125 species sampled by Williams et al.4 (top) was compared by principal component analysis (PCA). Each circle in the PCA projection (top left) is a distinct species, defined by a species-specific domain cohort. Asgards are highlighted as filled circles. The frequency distribution (top right) shows the number of distinct protein-domains per species. Vertical intersecting lines in the histograms are the median numbers of protein-domains. Protein domain composition is characteristic of clades of species (top left). In contrast, covariation of amino acid composition (bottom) in a single-domain (super)family is not clade-specific, but gene family-specific. Multiple sequence alignments of a single domain (c.37.1) shared by 5/50 concatenated orthologous gene families from 125 species were sampled for the PCA projection. (b) Effects of severe perturbation of the domain composition in recovering clade-specific distributions was tested in a sample of 141 species. Although it is common to suspect that the rooting between akaryotes and eukaryotes could be biased due to a larger domain cohort in eukaryotes4, it is not the case2,3,12. Diversity of clade-specific domain composition (top right) measured simply as the number of protein domains4 is a poor descriptor of heterogeneity, and can be misleading. Clades are grouped by covarying “protein-domain types”, but not by numbers alone. The rooting is stable and the tree topology is virtually identical even after reducing the eukaryote cohort by 1/3rds (middle) or 2/3rds (bottom)8 of the original composition2. Description of the PCA projections and frequencies are the same as in (a).\n\nA key advantage of non-redundant characters is that estimating ancestral compositions and evolutionary paths of individual characters is much less ambiguous. In addition to identifying the root nodes, an added benefit of the built-in directionality is that mutually exclusive evolutionary fates of individual features – inheritance, loss or transfer – can be resolved efficiently using directional-evolution models1,8,13. Harish et al.1–3 demonstrated that difficult phylogenetic problems can be resolved efficiently by employing protein domain characters and directional evolution models.\n\nTo be clear, unrooted trees are not phylogenies per se, since the absence of root-ancestor(s) obscures ancestor-descendant polarity and phylogenetic relatedness14,15. Since identifying the closest relatives of extant groups is the same as determining the closeness of their common ancestors, time-reversible models and unrooted trees remain ineffective tools (Figure 1b). Thus, regardless of the gene-aggregation and tree-reconciliation method used for estimating a consensus unrooted tree4, the location of the archaeal CA or UCA remains ambiguous (Figure 1b). Support from fossils or other sources are not reliable, despite claims to the contrary4. Likewise, predicting the origins of single domains or single genes by estimating amino acid (or nucleotide) compositions also remains ambiguous (reviewed in refs 1,6,7). A sobering revelation is that some datasets/models may be of little use or relevance to resolve questions of deep time evolution – this is sad but true.\n\n\nWill more complex models minimize uncertainties?\n\nWilliams et al.4 argue that (i) directional-evolution models12,13 may be unsuitable to predict the unique origin of homologous protein domains; and (ii) the akaryote 2D-ToL1–3 is an unsatisfactory explanation of the evolution of clade-specific compositions of protein domains (Figure 2). Their arguments seem to imply that phylogenetic signal can be recovered only by modeling evolution of amino acid composition. However, the fact that even the best-fitting substitution models are inadequate4, despite ever increasing model complexity to resolve conflicting signals (Figure 1b), suggests that different protein domain-families may require different but incompatible substitution models (Figure 2a). Further, such incompatibilities are likely to make estimating the absolute origins of single-domain families and single genes difficult, since a majority of genes are formed by duplication and recombination of distinct domains1. As a result, distinguishing between gene duplication and horizontal gene transfer, as well as quantifying the extent of duplications and transfers using primary sequences, is highly ambiguous1–3.\n\nThe KVR13 model for protein domain data1,2 is an extension of the Markov k states (Mk) model16, a generic probability model for discrete-state characters. A variant at k ≥20 is suitable for modeling evolution of amino acids or copy numbers of gene or protein domain families. While time-reversible variants produce unrooted trees, such directional models consistently recover a 2D phylogeny (Figure 1a) in which akaryotes are the closest relatives of eukaryotes1,2,8. The KVR model assumes that the root ancestor has a different character composition than the rest of the tree, which is essentially an irreversible acyclic process. This is fully consistent with the idea that, on a grand scale, the “tree of life” describes broad generalizations of singular events and major transitions underlying striking sister clade differences. Since parallel evolution of homologous protein-domains or distinct domain permutations is very rare, the KVR model adequately captures the evolution of unique features.\n\nThe assumptions of the KVR model are also consistent with the idea that the idiosyncratic compositions of homologous protein-domains (Figure 2) is a characteristic of the clades1–3. In contrast, amino acid compositions in single-domain families are not (Figure 2a). That is, patterns of covariation of species-specific protein-domain compositions clearly distinguish eukaryotes from akaryotes (and archaebacteria from eubacteria). The systematic covariation of homologous domains among the clades is best explained as phylogenetic effect. Consequently, the akaryote 2D-ToL (Figure1a) was consistently recovered with robust support for the major clades regardless of the taxonomic/protein domain diversity sampled (Figure 2b), and regardless of the model complexity1–3,8,12. By contrast, patterns of amino acid covariation are indiscriminate with regard to organismal families, although gene families can be efficiently identified.\n\nThe KVR model is an optimal explanation of the evolution of clade-specific composition of homologous features. Complex variants of the KVR model that account for rate variation among both characters and branches also consistently recovered the akaryote 2D-ToL (Figure 1a) despite significantly different model fits1. More complex models are available, such as the no-common-mechanism model17, an extremely parameter-rich model that allows each character to have its own rate, branch length and topology parameters. Even more complex models can be implemented, which assume that the tempo and mode of evolution changes at each internal node along the phylogeny4. However, such over-specified models may not be optimal for generalizing the evolutionary process and may over-fit observed patterns – a form of model misspecification. For instance, empirical datasets are limited to a finite set of homologous protein domains that range between 2,000 and 10,000 characters depending on the protein structure classification scheme1. By contrast, Williams et al.4 use 1,000,000 characters in their simulations to estimate the fit between the simulated data and over-complex models4. That said, it remains to be seen whether more complex models perform better with empirical datasets.\n\n\nData and methods\n\nProteome sequences (predicted protein cohorts from genome sequences) were obtained from recently published studies4,8. Homologous protein structural domains were identified using the homology assignment tools provided by the SUPERFAMILY database as in previous studies1–3. Briefly, each proteome was queried against the hidden Markov model (HMM) library of homologous protein-domains defined at the Superfamily level in the SCOP (Structural Classification of Proteins) hierarchy. The taxonomic diversity of sequenced genomes and the number of unique protein domains identified for each species is shown in Table 1.\n\nDescriptive statistics of protein-domain compositions for each taxonomic sampling, including the frequency distribution and median number of protein domains for each clade (Archaea, Bacteria and Eukarya), were estimated and visualized using the ggplot2 package (v 3.2.1) in R (v3.6.2). Covariation of clade-specific protein-domain composition, as well as domain-specific amino acid composition, was compared using principal component analysis (PCA). Components were generated by an eigenvector decomposition of the character matrix. PCA scores were based on percentage identity of character compositions.\n\n\nData availability\n\nThe predicted protein cohorts from genome sequences taken from Williams et al.4 and Harish and Kurland8 were assessed.",
"appendix": "Acknowledgements\n\nWe thank Tom Williams for kindly providing the proteome sequences used in their study and for answering our questions. Tanai Cardona for comments on an earlier version of the article. Måns Ehrenberg for supporting the APC.\n\nAn earlier version of this article can be found on bioRxiv (DOI: https://doi.org/10.1101/2020.01.17.907717).\n\n\nReferences\n\nHarish A: What is an archaeon and are the Archaea really unique? PeerJ. 2018; 6: e5770. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHarish A, Kurland CG: Akaryotes and Eukaryotes are independent descendants of a universal common ancestor. Biochimie. 2017; 138: 168–183. PubMed Abstract | Publisher Full Text\n\nHarish A, Tunlid A, Kurland CG: Rooted phylogeny of the three superkingdoms. Biochimie. 2013; 95(8): 1593–1604. PubMed Abstract | Publisher Full Text\n\nWilliams TA, Cox CJ, Foster PG, et al.: Phylogenomics provides robust support for a two-domains tree of life. Nat Ecol Evol. 2020; 4(1): 138–147. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHillis DM: Molecular versus morphological approaches to systematics. Annu Rev Ecol Syst. 1987; 18(1): 23–42. Publisher Full Text\n\nMorrison DA, Morgan MJ, Kelchner SA: Molecular homology and multiple-sequence alignment: an analysis of concepts and practice. Aust Syst Bot. 2015; 28: 46–62. Publisher Full Text\n\nKurland CG, Harish A: The phylogenomics of protein structures: The backstory. Biochimie. 2015; 119: 284–302. PubMed Abstract | Publisher Full Text\n\nHarish A, Kurland CG: Mitochondria are not captive bacteria. J Theor Biol. 2017; 434: 88–98. PubMed Abstract | Publisher Full Text\n\nZaremba-Niedzwiedzka K, Caceres EF, Saw JH, et al.: Asgard archaea illuminate the origin of eukaryotic cellular complexity. Nature. 2017; 541(7637): 353–358. PubMed Abstract | Publisher Full Text\n\nSpang A, Saw JH, Jørgensen SL, et al.: Complex archaea that bridge the gap between prokaryotes and eukaryotes. Nature. 2015; 521(7551): 173–179. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMorrison DA: Multiple Sequence Alignment is not a Solved Problem. arXiv:1808.07717 [q-bio], 2018. Reference Source\n\nHarish A, Kurland CG: Empirical genome evolution models root the tree of life. Biochimie. 2017; 138: 137–155. PubMed Abstract | Publisher Full Text\n\nKlopfstein S, Vilhelmsen L, Ronquist F: A Nonstationary Markov Model Detects Directional Evolution in Hymenopteran Morphology. Syst Biol. 2015; 64(6): 1089–1103. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWilkinson M, McInerney JO, Hirt RP, et al.: Of clades and clans: terms for phylogenetic relationships in unrooted trees. Trends Ecol Evol. 2007; 22(3): 114–115. PubMed Abstract | Publisher Full Text\n\nMorrison DA: Phylogenetic analyses of parasites in the new millennium. Adv Parasitol. 2006; 631–124. PubMed Abstract | Publisher Full Text\n\nLewis PO: A likelihood approach to estimating phylogeny from discrete morphological character data. Syst Biol. 2001; 50(6): 913–925. PubMed Abstract | Publisher Full Text\n\nHuelsenbeck JP, Alfaro ME, Suchard MA: Biologically inspired phylogenetic models strongly outperform the no common mechanism model. Syst Biol. 2011; 60(2): 225–32. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "60618",
"date": "31 Mar 2020",
"name": "Edward Braun",
"expertise": [
"Reviewer Expertise Evolutionary genomics and computational biology"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIt is challenging to review a reply without considering the original paper carefully. In this case it is doubly challenging because the most relevant portion of Williams et al. (2020)1 is itself a reply to Harish and Kurland (2017)2. This prompted me reread Harish and Kurland (2017) and Williams et al. (2020) in detail and evaluate the Harish and Morrison manuscript (which I will call HM-F1000 hereafter) in light of those publications. Thus, I have decided to provide a review of HM-F1000 along with a limited post-publication review of Harish and Kurland (2017) and Williams et al. (2020). I will divide this review into two sections: 1) a discussion of the larger philosophical questions and 2) a description of the changes to HM-F1000 that I believe to be necessary as well as a few minor issues with HM-F1000.\n\nBefore I provide that combined review, I want to answer two questions: 1) does HM-F1000 warrant publication as a peer-reviewed publication? and 2) did HM-F1000 convince me that the Eukaryote/Akaryote* two-domain tree of life (2D-ToL) represents an accurate placement of the root of the tree of life (ToL)? My answer to the first question is “yes” and my answer to the second question is “not at this time.”\n\n* NOTE: Throughout this review I will use “akaryote” in because it is the terminology used in HM-F1000; however, I am agnostic regarding benefits of that term relative to “prokaryote.”\n\nI have answered the first question in the affirmative despite expressing substantially greater caution when I answer the second because identifying the position for the root of the ToL is arguably one of the most difficult problems in evolutionary biology. The field needs more ideas regarding the best way to estimate a robust topology for the ToL and place the root, not fewer. Excluding the HM-F1000 from the peer-reviewed literature would exclude their concise defense of the idea that the KVR (Klopfstein et al. 2015)3 model of evolution can be used with protein fold presence/absence data to place the root of the ToL.\n\n***** Section 1:\n\nWilliams et al. (2020) argued that using the KVR model with protein domain composition data was inappropriate based upon their simulations; they chose instead to focus on analyses using models of protein sequence evolution. It is certainly true that the KVR model is a simplistic model of evolution. However, the fact that the KVR model is imperfect does not invalidate the use of the model; as Box (1979) famously stated “all models are wrong but some are useful.”\n\nSimply showing that that a model has imperfect fit to empirical data (as Williams et al. 2020 did) does not mean the model is useless for inference. Indeed, it is very likely that the models of protein evolution, like the CAT, C60, and NDCH2 models, which Williams et al. (2020) used in their other analyses, also have an imperfect fit to the true underlying process of evolution. The central issue is whether analyses of protein fold data using the KVR model are more likely to recover true historical signal than analyses of aligned proteins using various models of protein sequence evolution.\n\nHM-F1000 highlights a corollary of this fundamental issue in its abstract when they state that “(i)t is well known that different character types present different perspectives on evolutionary history that relate to different phylogenetic depths.” With that said, is clear that the models of protein sequence evolution that Williams et al. (2020) used are much more sophisticated than the KVR model. So why should we embrace the results of the KVR model over the results of analyses using those sophisticated models of protein evolution? Obviously, the purpose of HM-F1000 is to convince readers to accept the results of the KVR model (applied to protein fold presence/absence data) as more likely to be correct (in this context I will use “correct” to mean “closer to the truth”) than the results of the models of protein evolution used by Williams et al. (2020). Why might that be the case? I can think of two reasons that I will discuss below:\n\n--- I. Historical signal might decay more rapidly in aligned protein sequences than in protein fold content data.\n\nThe simplest explanation for preferring the results of analyses using the protein fold data to those obtained using aligned proteins is the possibility that historical signal might have decayed in the latter. Mossel (2003)4 proved that “…it is impossible to reconstruct the topology of ‘deep’ trees with high mutation rates…” More accurately, Mossel (2003) identified a bound on the number of characters necessary for tree reconstruction, but that bound implies that impossible to reconstruct some past events (also see Sober and Steel 2002)5. Perhaps protein sequence alignments cannot provide accurate information about the deepest branches in the tree of life and we have to look to other data types, like protein fold content, to estimate the topology of the deepest branches in the tree of life.\n\nIn my opinion, two arguments are necessary to establish that data type is more important that model fit for reconstructing the deepest branches in the tree of life. HM-F1000 states that “(p)rotein structural domains, unlike amino acids, are biochemically non-redundant (see below) and have proven to be excellent ‘genomic characters’…” as a defense of the idea that protein fold data might be superior to aligned amino acids. I was expecting an explicit statement that it might be appropriate to view changes in the protein fold repertoire as rare genomic changes (RGCs; Rokas and Holland 2000; Bleidorn 2017)6,7. I think that linking protein folds to RGCs is important because it provides an explicit link between protein fold data and the body of theory surrounding RGCs. Specifically, the fact that analyses using the maximum parsimony criterion are expected yield the correct tree when applied to RGC data (Steel and Penny 2004; 2005)8,9. I believe this has implications for the idea that the relatively simple KVR model might be useful for rooting the tree of life.\n\nWhether the maximum parsimony criterion should be viewed as a simple model (or any sort of model) has been a topic of philosophical debate in phylogenetics (Goloboff 2003’ Huelsenbeck et al. 2008)10,11; I will accept the idea that maximum parsimony is “simple” for the sake of this argument (also see Yang 1995)12. However, if we accept that a “perfect RGC” model (which I define as a process that results in some binary character that can only undergo a single transition on one edge in the gene tree associated with that genomic character) it allows us to pose a question about the KVR model: is the KVR model consistent for characters generated by a hypothetical “asymmetric perfect RGC” model? The asymmetric perfect RGC model modifies the perfect model so the ensemble state frequencies at the root differ from the tip frequencies. I recognize that, in addition to the treatment of the root state frequencies, the KVR model differs from parsimony in an important way (specifically, the treatment of branch lengths). However, this conjecture regarding the behavior of the KVR model might point the way toward a falsifiable hypothesis because it lends itself to testing by simulation.\n\nThe question of whether the KVR model is consistent given the asymmetric perfect RGC model is interesting from theoretical standpoint but there is a second (and more important question) that should be answered: is whether the true underlying model of fold content is sufficiently close to the asymmetric perfect RGC model for that model to be useful? The true underlying model of fold evolution includes fold origination (which is almost certainly a very rare event) and horizontal transfer (likely to be much more common). Williams et al. (2020) discuss this in their supplementary materials, where they state “…a change from 0 to 1 might indicate de novo origin of an existing fold by convergent evolution (which is likely to be rare), or the gain of an existing fold by [horizontal gene transfer]; if the latter, then the pattern of presences and absences for that fold cannot be reliably used to infer the underlying tree.” I agree with the first part of that sentence (which is a reason why I have invoked the idea of RGCs) but I disagree with the second; even when there is horizontal gene transfer novel fold acquisition might be sufficiently rare for that type of event to be considered an RGC.\n\nAnswering those questions will be challenging and outside the scope of a short note like HM-F1000. In that context, I think it would be good for HM-F1000 to express a little more caution. Statements like “[t]he KVR model is an optimal explanation of the evolution of clade-specific composition of homologous features” (first full paragraph on page 5 of HM-F1000). The point of my arguments above is that it might be reasonable to view the KVR model as an excellent approximating model for protein fold evolution. The first author has written multiple papers dealing with patterns of protein fold evolution over deep evolutionary time and I do not want to disrespect those efforts, but I do not think this is a settled issue at this time.\n\nII. The best interpretation of the unrooted trees in Williams et al. (2020) is unclear.\n\nThe Harish and Kurland (2017) trees are the only intrinsically rooted trees under discussion in Williams et al. (2020). Since the position of the root of the 2D-ToL in Williams et al. (2020) ultimately represents the imposition of a root on an otherwise unrooted tree. Strictly from a logical standpoint there are four interpretations of the results of Harish and Kurland (2017) and Williams et al. (2020).\n\nBoth trees are inaccurate estimates of the ToL.\n\nThe Harish and Kurland (2017) topology is correct.\n\nThe unrooted Williams et al. (2020) topology is correct and the Harish and Kurland (2017) root is also correct (i.e., the root lies between eukaryotes and akaryotes).\n\nThe unrooted Williams et al. (2020) topology is correct and the root is not between eukaryotes and akaryotes.\n\nPossibility 3 is important, and it would seem to be implicit in Figure 1b of HM-1000. The authors should make this point more explicitly; they use Figure 1b to make another point, using it to stress that “Williams et al. have overlooked important aspects of assessing phylogenetic signal in empirical data, and that it may be premature to reject a well-supported phylogeny based on simulated data.” I think it would be valuable to make the point that, at least in principle, the Williams et al. (2020) results would be consistent with a tree rooted between eukaryotes and akaryotes that also places the root of akaryotes within the Asgard archaea.\n\nObviously, embracing the unrooted Williams et al. (2020) topology would require rejecting the akaryote topology of the Harish and Kurland (2017). Specifically, the fact that the Harish and Kurland (2017) tree is consistent with archaeal monophyly (as long as one assumes the root of the tree of life is not within archaea) makes it fundamentally inconsistent with the Williams et al. (2020) topology. However, the possibility of archaeal non-monophyly would seem to be consistent with Figures 1 and 2 in Harish (2018)13, both of which show substantial uncertainty at the base of Archaea. Figure 2 in Harish (2018) is based on distances calculated using protein fold data, raising some questions regarding the strength of support for monophyly of Archaea.\n\nOne aspect of the Harish and Kurland (2017) tree that HM-F1000 should acknowledge is the fact plants are not monophyletic. Specifically, the root of the eukaryotic sub-tree of Figure 3 in that paper was placed between rice and all other eukaryotes. Obviously, this is troubling given that the Harish and Kurland (2017) tree includes other angiosperms. In fact, the Harish and Kurland (2017) dataset includes two other grasses; non-monophyly of both angiosperms and grasses is unreasonable. Even placing the eukaryotic root between the green plants and other eukaryotes seems unlikely given the best available information about the eukaryotic tree (reviewed by Burki et al. 2020)14.\n\nOne might wonder whether the root position for the ToL should be viewed as accurate given the unexpected position of the eukaryotic root. However, it is reasonable to postulate that the rice data were problematic in some way. Alternatively, it could reflect the observation that different data perform differently at different levels in the tree (Chen et al. 2015) (HM-F1000 already alluded to this). If I had reviewed Harish and Kurland (2017) I would have asked the authors to conduct a second set of analyses after excluding rice to see if that changed the root of the eukaryotic sub-tree. I do not think it would be reasonable to ask HM-F1000 to add a reanalysis of the Harish and Kurland (2017) after excluding rice, but it would be nice for HM-F1000 to acknowledge this issue. ---\n\nLooking back, I realize that I have written this review as an advocate for the position articulated by HM-F1000. Given that tone it would be fair to ask why I not convince that their placement of the root between eukaryotes is accurate? I would answer that question I am not convinced that the tools exist to place of the root of the ToL is accurate exist at this point. As I discussed above, I have made an argument can be made that the KVR model might be able to yield an accurate estimate of the ToL. However, my reasons for that assertion rest on assumptions regarding the nature of the protein fold data. I believe that the Williams et al. (2020) analyses do show that the KVR model is imperfect; I simply disagree with their conclusion that this imperfect fit means that we should dismiss the Harish and Kurland (2017) result. Fundamentally, I view the use of the KVR model with protein fold data as similar to the Jukes-Cantor model. Felsenstein (2001)15 shared an anecdote regarding that model, stating that: “Tom Jukes once told me that the reason the Jukes–Cantor model was buried in the midst of a large empirical paper was that this was the only way to get it published. He felt that if he had attempted to publish it on its own, it would have been rejected by editors as idle and oversimplified speculation.” However, without the pioneering work of Jukes and Cantor (1969)16 and Neyman (1971)17 (or Felsenstein 1981)18 it is difficult to envision the development of the more sophisticated models of sequence evolution that developed over the subsequent five decades. Dismissing the use of protein fold data at this point will slow the development of those models. Will further model development support the Eukaryote/Akaryote 2D-ToL? I am uncertain whether it will, but I am interested to find out.\n\nI would like to add a final discussion regarding model fit. Although there is a long history of model development for protein sequences and the models are now quite sophisticated, there is still much that we do not know about protein evolution. Williams et al. (2020) used the LG+C60 model. Presumably this is the C60 profile mixture from Le et al. (2008) (although I was unable to find Le et al. 2008 cited in Williams et al. 2020) combined with the LG (Le and Gascuel 2008)19 matrix. However, Pandey and Braun (2020)20 reported that the Le et al. (2008)21 profile mixtures can exhibit surprising (and disturbing) behavior for at least one phylogenetic problem. The Le et al. (2008) mixture models are very similar to the CAT model (Lartillot and Philippe 2004); it is unclear whether these issues Pandey and Braun (2020) noted for the Le et al. (2008) models are general features of CAT-type models (or even more widespread than the particular case studied by Pandey and Braun 2020) but it is important to be careful regarding the use of any models of evolution so deep in the tree.\n\nIt is tempting to look at the sophistication of existing models of protein sequence evolution and conclude that the results obtained using those models trump other sources of information. Although I do not want to be overly dismissive of the Williams et al. (2020) analyses, which are state of the art, I do want to emphasize that I believe data type matters and that we should be looking at other sources of information. In my opinion, that is the message that HM-F1000 should convey; that is why I think some statements in HM-F1000, like the statement that the KVR model provides an optimal explanation for protein fold evolution, actually undercut the case. In my opinion, obtaining a strongly corroborated estimate of the deepest branches in the ToL, if it is possible, will require us to examine multiple sources of information and to be very careful regarding the models we use for analyses. That applies to analyses of aligned protein sequences and to analyses of protein fold content.\n\n***** Section 2: Minor issues and description of necessary revisions:\n\nI have written a fairly long review, but I feel the changes to HM-F1000 that are necessary are actually fairly minimal. I think HM-F1000 needs to walk back the claims that the KVR model is an optimal explanation for protein fold distribution and simply point out that it is likely to be a reasonable approximating model. I think HM-F1000 also needs to acknowledge that “both trees could be telling us part of the truth” (i.e., that a tree rooted between eukaryotes and akaryotes with a paraphyletic archaea might be a way to reconcile Harish and Kurland 2017 with Williams et al. 2020). HM-F1000 should also acknowledge the unexpected (and incorrect) rooting of the eukaryotic sub-tree. Finally, I hope the minor comments that follow are carefully considered.\n\nI was surprised that the work of Poole et al. (1998; 1999)22,23 was not cited. It provides another line of evidence supporting the placement of the root on the eukaryotic branch (i.e., it supports the Eukaryote/Akaryote 2D-ToL).\n\nThe first full line of the second column of the fourth page of HM-F1000 states “Support from fossils or other sources are not reliable, despite claims to the contrary [Williams et al. 2020].” I could not find an explicit statement in Williams et al. (2020) that makes this assertion. I agree with the basic point that the fossil record to establish the deep topology for the ToL provides, at most, limited information. However, HM-F1000 should be careful regarding the attribution of statements like this. I hope that I did not miss any such statement in Williams et al. (2020); if I have missed it, Harish and Morrison should point to the statement.\n\nThe legend of Figure 2 states “[t]he majority of proteins are multi-domain proteins formed by duplication and recombination of domain units.” However, Ekman et al. (2005)24 reports that fewer than 50% of prokaryotic (akaryotic) proteins are multidomain. I was unable to find an explicit survey of proteins showing that the numbers of multidomain proteins is generally >50% in the literature. This statement should have an associated citation and, if the number is <50% in some lineage be a bit more cautious. Perhaps something like “a large proportion” would be a better statement.\n\nThe legend of Figure 2 also states that “[a]lthough it is common to suspect that the rooting between akaryotes and eukaryotes could be biased due to a larger domain cohort in eukaryotes [Williams et al. 2020], it is not the case.” Since the statement that the large domain cohort of eukaryotes is a source of bias only cites Williams et al. (2020) I don’t think it is valid to state that “it is common to suspect” unless there are additional citations. The statement “…it is not the case” cites three papers with Harish as an author, but the evidence that a large domain cohort cannot be a source of bias was not clear to me. The explanation should be expanded a bit and moved to the main text.\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? Partly",
"responses": [
{
"c_id": "5618",
"date": "22 Jun 2020",
"name": "Ajith Harish",
"role": "Author Response",
"response": "Response to reviewerWe thank the reviewer for their detailed review and thoughtful comments. We agree with the issues raised by the reviewer. We revised the text accordingly. Suggestion: I have written a fairly long review, but I feel the changes to HM-F1000 that are necessary are actually fairly minimal. I think HM-F1000 needs to walk back the claims that the KVR model is an optimal explanation for protein fold distribution and simply point out that it is likely to be a reasonable approximating model. I think HM-F1000 also needs to acknowledge that “both trees could be telling us part of the truth” (i.e., that a tree rooted between eukaryotes and akaryotes with a paraphyletic archaea might be a way to reconcile Harish and Kurland 2017 with Williams et al. 2020). Response: We have included the following in the second to last paragraph of the manuscript,“Even if the archaeal radiation remains poorly resolved with more data, the better supported rooting between eukaryotes and akaryotes is consistent with a Eukaryote-Akaryote 2D-ToL (Figure 1b). That is, diversification of eukaryotes and akaryotes from the UCA is a better supported hypothesis rather than a prokaryote-to-eukaryote transition being assumed to interpret poorly resolved trees.” Suggestion: I was surprised that the work of Poole et al. (1998; 1999)22,23 was not cited. It provides another line of evidence supporting the placement of the root on the eukaryotic branch (i.e., it supports the Eukaryote/Akaryote 2D-ToL). Response: We have now cited the suggested article in our discussion about the relative age of eukaryotes and akaryotes in the last paragraph of section 1. Suggestion: The first full line of the second column of the fourth page of HM-F1000 states “Support from fossils or other sources are not reliable, despite claims to the contrary [Williams et al. 2020].” I could not find an explicit statement in Williams et al. (2020) that makes this assertion. I agree with the basic point that the fossil record to establish the deep topology for the ToL provides, at most, limited information. However, HM-F1000 should be careful regarding the attribution of statements like this. I hope that I did not miss any such statement in Williams et al. (2020); if I have missed it, Harish and Morrison should point to the statement. Response: The reference was to the Williams et al (2020) statement “At present, the best-supported root is on the branch separating bacteria and archaea67,68,80,81 or among the bacteria70,72, and the hypothesis that eukaryotes are younger than prokaryotes is supported by a range of phylogenetic, cell biological2,3 and palaeontological61,82–84 evidence.” We have revised our statement for clarity, also related to the discussion of the relative age of eukaryotes and akaryotes as: “Thus, the location of the archaeal-CA or UCA remains ambiguous at best (Figure 1b), regardless of the gene-aggregation and tree-reconciliation method used for estimating a consensus unrooted tree… Despite claims to the contrary, that the best-supported root is on the branch separating bacteria and archaea or that eukaryotes are younger than akaryotes7, support from fossils is not reliable either, since assigning fossils to extinct archaea/bacteria or UCA is even more ambiguous.” Suggestion: The legend of Figure 2 states “[t]he majority of proteins are multi-domain proteins formed by duplication and recombination of domain units.” However, Ekman et al. (2005)24 reports that fewer than 50% of prokaryotic (akaryotic) proteins are multidomain. I was unable to find an explicit survey of proteins showing that the numbers of multidomain proteins is generally >50% in the literature. This statement should have an associated citation and, if the number is <50% in some lineage be a bit more cautious. Perhaps something like “a large proportion” would be a better statement. Response: The statement now reads “a large proportion” as suggested. Suggestion: The legend of Figure 2 also states that “[a]lthough it is common to suspect that the rooting between akaryotes and eukaryotes could be biased due to a larger domain cohort in eukaryotes [Williams et al. 2020], it is not the case.” Since the statement that the large domain cohort of eukaryotes is a source of bias only cites Williams et al. (2020) I don’t think it is valid to state that “it is common to suspect” unless there are additional citations. Response: We have revised the statement as “Despite the suspicion that the rooting between akaryotes and eukaryotes could be biased due to a larger domain cohort in eukaryotes …. ” Suggestion: The statement “…it is not the case” cites three papers with Harish as an author, but the evidence that a large domain cohort cannot be a source of bias was not clear to me. The explanation should be expanded a bit and moved to the main text. Response: We included a short discussion in the third to last paragraph of section 1 as: “. In contrast, the separation of eukaryotes and akaryotes (and of archaea and bacteria) is unperturbed even after extreme perturbation of the domain composition in eukaryotes (e.g. by excluding up to two-thirds of the domain cohort, Figure 2b). The clades within eukaryotes and akaryotes are unperturbed as well11\"."
}
]
},
{
"id": "60012",
"date": "04 May 2020",
"name": "Jacob S. Berv",
"expertise": [
"Reviewer Expertise molecular systematics and phylogenetics"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nReview of: The deep(er) roots of Eukaryotes and Akaryotes\n\nJacob S. Berv and Stephen A. Smith\nIntroduction In the present article (Harish and Morrison, 2020), Harish and Morrison argue that prior work (Williams et al., 20201) to elucidate the structure of the deepest branches of the tree of life, was misled by reliance on particular data types and models which are unsuited to the task. In general, we agree that Harish and Morrison has merit as a scientific contribution, and represents a valid perspective. However, we are nonetheless cautious as to their conclusions.\n\nThe debate at issue concerns several hypotheses concerning the monophyly and placement of archaea, bacteria, and eukaryotes. Williams et al.’s rigorous phylogenetic analysis of “redundant” characters, like nucleotide or amino acid sequences, converges on a solution that places eukaryotes within the archaea, and as sister to bacteria – in a two-domain tree of life. This paper garnered significant attention at the time of publication earlier this year, and given the implications of their result, it is fair to subject it to additional scrutiny. The present article by Harish and Morrison, therefore, constitutes a rebuttal to Williams et al., which itself is in part a response to earlier work by Harish et al. (Harish, 2018; Harish and Kurland, 2017a, 2017b, 2017c)2,3,4,5.\n\nBefore we can have confidence in understanding the branching pattern reflecting at the root of the tree of life, it seems important to acknowledge that there are several key questions at play that are frequently confounded, as rightly emphasized by this and prior work by Harish et al.: 1) What is a domain? 2) How many domains are there? 3) What are the relationships of these domains to each other? Clearly articulating and answering these questions will require addressing two issues that have plagued prior studies of the origins of crown-group life. The first involves the information content of particular data types and identifying which data types are most likely to contain information relevant for discriminating between particular phylogenetic hypotheses. There is significant literature on this front, both from theoretical and empirical perspectives (e.g Dornburg et al., 2019; Reddy et al., 2017; Townsend and Leuenberger, 20116,7,8). The second key issue concerns the rooting of the tree of life, which presents several difficult challenges that may require addressing fundamental epistemological choices (below). In our review here, we will briefly outline these two issues and discuss the arguments presented by the article by Harish and Morrison.\nInformation content Harish and Morrison argue for a two-domain tree of life that places a clade of archaea and bacteria (together called Akaryotes) as sister to Eukaryotes. In particular, Harish and Morrison argue that phylogenetic characters derived from the presence/absence of protein domains are more suited to the task of elucidating the deepest roots of the tree of life than more traditional phylogenetic characters advocated for by Williams et al. Protein structural domains, which are ~200 amino acid or ~600 nucleotides long, each with unique structure and function (Harish, 2018), have been the focus of prior work by the authors, and we find the authors’ arguments in favor of their application to be justifiable. These data types, at the very least, serve as complementary to other data types used for phylogenetic reconstruction and offer some compelling properties relevant for deep phylogenetic reconstruction.\n\nHarish and Morrison point out that unlike traditional nucleotides and amino acid characters, structural domains may be relatively homoplasy free and therefore useful for clarifying the extremely difficult problem of the root of the tree of life. Using such characters for phylogenetic inference recognizes homology of structure that may be lost at the sequence level. Structural domains also exhibit compositional variance that isolates species into taxonomic clusters, whereas clustering of amino acid data generates clusters that reflect gene families, and not clades (Figure 2a). While there may be lineage specific compositional heterogeneity of amino acids within gene families, models that assume amino acid composition to be consistent across gene regions may be a poor fit to data.\n\nOne issue not addressed by Harish and Morrison but that we feel warrants comment regards branch lengths. A qualitative comparison of the branch lengths reported from prior work by Harish et al., (relying on protein domain characters), and the branch lengths reported in Williams et al. (relying on amino acid sequences), is strongly suggestive of the hypothesis that the trees reported in Williams et al. may be influenced by saturation artifacts. In Figure 2a and of Williams et al, the branch lengths connecting bacteria and eukaryotes to their respective positions in the unrooted tree of life are much greater than 1 substitution per site. This suggests that there are few to no sites in the alignment that are not variable at edges separating major putative clusters in these data. In other words, there may be no detectable homology between these clades and the rest of the data. This is a very high rate of evolutionary change in the context of divergences that occurred billions of years ago. Williams et al. discuss that the reason these branches may be so long is that the CAT+GTR+G4 model is better able to identify convergent substitutions on these branches (as validated by posterior predictive simulation). While this may be true, an alternative interpretation is that there is simply no information in those amino acid data directly relevant to this question, and different models are reflecting statistical differences, and not necessarily different signals in the data. In comparison, the branch lengths reported by Harish (2018) (for instance) seem to indicate much more realistic values (Figure 6 in Harish, 2018) indicates all branches are much shorter than 1 substitution/site). These observations, at the very least, suggest caution in interpreting the Williams et al. result without further analysis of the adequacy of the model to fit these data with so few shared characters, and would seem to argue in favor of more slowly evolving (but nonetheless apparently very informative) characters like protein domains employed by Harish et al. The rate of evolution across characters is an important consideration in addition to the rate of evolution of the locus when examining the utility of a particular data type for resolving a phylogenetic question (Dornburg et al., 2019).\nWhile the branch lengths of Williams et al. may give one pause, some relationships in Harish and Kurland (2017b) also warrant discussion. The rooted analyses that place Eukaryotes sister to Akaryotes result in several relationships that would be considered unusual as compared to most other analyses that focus on the resolution of early Eukaryotes. For example, the resulting analyses have plants as paraphyletic with strong support. Other relationships, while perhaps less egregiously different than other analyses are still uncommon. These results might call into question the quality of these data and analyses for resolving other relationships.\nRooting Harish and Morrison argue that “non-redundant” protein domain characters provide a key advantage which allows them to aid in directly estimating the position of the root of the tree of life, using non-reversible models. The issue of rooting cannot be overstated, and its lack of consideration by Williams et al. is an oversight. An unrooted tree does not describe phylogenetic relationships including monophyly. While typical phylogenetic analyses include an outgroup on which the tree can be rooted, the root of the tree of life presents distinct challenges. Harish (2018) discusses why rooting the tree of life is perhaps the most difficult phylogenetic problem: in the absence of outgroups or fossils, the typical approach has been to root the tree on bacteria, but of course, “the nearest neighbor in an unrooted tree need not necessarily be the closest relative” Harish (2018). We reiterate these sentiments – identifying support for the existence of a particular monophyletic clade does not constitute evidence of its relationships with other monophyletic groups—indeed, with an unknown root position, many alternative topological optimizations can be generated by attaching the root to different branches among the three domains a posteriori, which each fundamentally altering our understanding of the origins of crown-group life.\n\nThe common practice of rooting the ToL with bacteria is unsatisfying as it enforces a strong assumption, and we, therefore, agree with Harish and Morrison that directional models of character evolution may be a useful way forward. The KVR model (Klopfstein et al., 2015)9 Haris and Morrison advocate for assumes that the root possesses a different character composition than descendants, and since independent convergence of protein domains may be very rare, the KVR model may be informative in optimizing the position of an unknown root. Williams et al. investigated this proposal by Harish et al. through simulations and found that with simulated datasets that allowed protein fold compositions to vary over the tree, the KVR model often fails to find the correct root. They note that the root position appears to systematically converge toward branches that represent a majority of the compositional variance, which can be controlled by including or excluding taxa from within subclades. Harish and Morrison argue that the simulations employed by Williams et al. do not accurately capture important features of the empirical data, and so are of limited relevance. They note that their own experiments, which reduce the eukaryote sample by ½ or ⅔ still recover a stable root position between Akaryotes and Eukaryotes. These perspectives suggest a strong disagreement over what may be the most critical aspect of a larger problem. It seems to us therefore that continued development of approaches to objectively identify the position of the root may be helpful in making progress, whether or not the topology advocated for by Harish and Morrison is correct.\nIn sum, the present work by Harish and Morrison serves to emphasize that there are still a number of unresolved challenges in understanding the deepest roots of the tree of life. The development of tools like posterior predictive simulation may help us understand how well our models capture specific aspects of our data (e.g. Brown, 2014; Foster, 2004)10,11, but it will also be important to consider the likely signal of homology that may be present in different data types, as well as how best to objectively identify the position of the root of the tree of life.\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? Yes",
"responses": [
{
"c_id": "5619",
"date": "22 Jun 2020",
"name": "Ajith Harish",
"role": "Author Response",
"response": "Response to reviewersWe thank the reviewers for their detailed review and suggestions.Suggestion: One issue not addressed by Harish and Morrison but that we feel warrants comment regards branch lengths. Response: We have now included a discussion of branch lengths, and how branch lengths are not reliable proxies for assessing homology of clades, in the second to last paragraph of section 1, as follows: “Furthermore, branch-length estimation from sequences alignments is not a reliable proxy for assessing homology of clades, since it appears to be extremely sensitive to character composition. The latter depends on the inclusion/exclusion of characters, either the choice of: (1) alignable genes, or (2) aligned amino acids (alignment trimming). Both are dependent on the degree of sequence similarity, which can vary wildly in highly divergent taxa and affect the choice of characters. In contrast, the separation of eukaryotes and akaryotes (and of archaea and bacteria) is unperturbed even after extreme perturbation of the domain composition in eukaryotes (e.g. by excluding up to two-thirds of the domain cohort, Figure 2b). The clades within eukaryotes and akaryotes are unperturbed as well11.”."
}
]
},
{
"id": "63265",
"date": "29 May 2020",
"name": "John Gatesy",
"expertise": [
"Reviewer Expertise phylogenetics"
],
"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\nHarish and Morrison explore rooting the tree of Life given recently proposed hypotheses. They might consider the following in editing/improving their manuscript:\n\nFirst sentence of the background. Not sure I agree; it depends on what the authors think a 'model' is in this context. Are they referring to an explicit substitution or transition rate matrix model, or some more general concept?\n\nDefinition of 'domains' would seem to be more ambiguous than defining particular amino acids that are discrete subunits with a very simple genetic basis. Admittedly, the alignment of such amino acids can entail ambiguity, especially at these divergences, so I suppose all inference at this level is a challenge. But, determining whether a particular 'domain' is even a 'domain' (or not two domains or one and a half domains or a different domain) is in my view squishy.\n\nRooting using asymmetrical abstractions (models) is squishy too. This is basically never done except for when there is no outgroup. In this case, there is none, but I am disturbed by the authors confidence (e.g., Fig. 1a with maximum support) in rooting an ancient tree based on this model or that.\n\nPage 4 left column. I think that the following is an assertion, not a fact, \"Substitutions between structural domains do not occur, unlike amino acid replacements, since each domain defines a distinctive biochemical function\". Who says that one domain cannot transform into another? I do not understand this assertion. The authors have extreme confidence in this statement it seems, but perhaps this is the problem? Were they there, back 100s of millions of years ago to observe that one domain could not have evolved into another or that similar domains did not evolve convergently into what the authors assume are the same domain (even though it might not be the same 'domain')?\n\nPage 4 left column. I am assuming that the authors' preferred models, \"The natural bias in gain/loss rates, arising from the difficulty of parallel gains and the relative ease of parallel losses, is useful for implementing directional (rooted) character-evolution models\". The idea that there is some general rate across all domains for gain and loss and convergent gain seems naive to me, or at least, a poor criterion for rooting a tree with awesome confidence and high probability.\n\nI am not buying the idea that these domains are 'non-redundant'. I believe that the authors believe this, but that is about it. So, I do not think there is \"built in directionality\" if the authors' initial assumptions/assertions are accepted. It is true that one can root a tree if one assumes one can identify homologous domains accurately and apply a very specific general model to a situation that is not specific and surely not general in terms of rate. Rooting the tree of Life will always be depedent on some sort of model that assumes this or that about gains and losses and convergent gains as there is no outgroup (whether gains or losses of domains or genes or nucleotides or amino acids), but this just reinforces the authors' initial assertion in the paper that models that people imagine (which are poorly understood in terms of process) will drive results. The fact that the amino acid trees (unrooted) are completely in conflict with the domain-based tree is not a good sign as no congruence among different data, even in an unrooted context. I suppose it is okay for the authors to assert their tree is better, but I think many people will not agree or be convinced by trust in some general asymmetry model and domains that may or may not be the same thing in very divergent taxa. From the amino acid analysis side of the debate, their tree seems to refute the domain tree, even though it is unrooted (and vice versa I guess), so as an outside viewer of this debate, there seems to be a lot of work to do on an admittedly challenging problem. But, that was likely known before this contribution.\n\nDo the authors' asymmetry models take lateral transfer of domains into account as well? Since the authors admit that, \"Further, such incompatibilities are likely to make estimating the absolute origins of single-domain families and single genes difficult, since a majority of genes are formed by duplication and recombination of distinct domains\", if their asymmetry domain models for rooting do not take lateral transfer into account, this would seem problematic to me, given that they argue for the importance of lateral transfer of entire genes (and genes include 'domains').\n\nI think the following from the authors is an assertion, not a fact, \"Since parallel evolution of homologous protein-domains or distinct domain permutations is very rare, the KVR model adequately captures the evolution of unique features.\" If not a necessarily true, nothing the authors argue is either?\n\nThe authors note that, \"The systematic covariation of homologous domains among the clades is best explained as phylogenetic effect\". I have studied phylogenetics for 30 years, yet I still have not seen any compelling or useful definition of this term that makes any sense. This just seems like a vague explanation for an observed pattern of covariation. Many of the things that the authors seem to consider 'clade-specific' could just be 'grade-specific'. For example, 'fish' have lots of common features that sort of make sense together as all primitively swim around in water, have tails for propulsion underwater, and breath oxygen and feed underwater. Similarly, 'domains' characteristic of eukaryotes or archaeans might not define monophyletic groups, but might instead be characteristic of paraphyletic groups (e.g., Asgards or 'Others' in Fig. 1b). So, one wonders whether the robustly rooted tree of Life based on specifics of a particular model mean that much, or not. The fact that the tree strongly contradicts an unrooted tree based on independent data (Fig. 1b) does not give me much confidence as an outsider to the debate.\n\nThis has to be a gross overstatement? \"The KVR model is an optimal explanation of the evolution of clade-specific composition of homologous features.\" For example, the authors note that, \"More complex models are available, such as the no- common-mechanism model, an extremely parameter-rich model that allows each character to have its own rate, branch length and topology parameters.\" For this model, surely there would be fewer evolutionary steps; wouldn't that be more 'optimal'? What is the result for this model that could be interpreted as a better (more optimal?) fit to the data - certainly not optimal in terms of minimizing evolutionary steps, unless the tree topology for the model based analysis and parsimony are identical.\n\nIs the rationale for commenting on the previous publication clearly described? Yes\n\nAre any opinions stated well-argued, clear and cogent? Partly\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": "5620",
"date": "22 Jun 2020",
"name": "Ajith Harish",
"role": "Author Response",
"response": "Response to reviewerWe thank the reviewer for their suggestions. The comments helped us improve the clarity of the presentation. We revised the text extensively to address the issues raised.Suggestion:1. First sentence of the background. Not sure I agree; it depends on what the authors think a 'model' is in this context. Are they referring to an explicit substitution or transition rate matrix model, or some more general concept?Response: We revised it as: “Models of character evolution that specify assumptions about the frequency and propensity of character changes”Suggestion: 2. Definition of 'domains' would seem to be more ambiguous than defining particular amino acids that are discrete subunits with a very simple genetic basis. Admittedly, the alignment of such amino acids can entail ambiguity, especially at these divergences, so I suppose all inference at this level is a challenge. But, determining whether a particular 'domain' is even a 'domain' (or not two domains or one and a half domains or a different domain) is in my view squishy. Response: We use domain definitions from experimentally determined structures according to the SCOP (Structural Classification of Protein) scheme. In general, an independently folding unit is considered to be a domain. There is a large body of experimental work and literature on delimiting domains based on 3D structure and function as well as assigning domains and determining homology using computational tools. Arguably, assessing homology of a domain (or a protein) is more reliable than determining the homology of amino acids/nucleotides even in the absence of alignment ambiguities.Suggestion:3. Rooting using asymmetrical abstractions (models) is squishy too. This is basically never done except for when there is no outgroup. In this case, there is none, but I am disturbed by the authors confidence (e.g., Fig. 1a with maximum support) in rooting an ancient tree based on this model or that.Response: Our confidence and maximum support are based on the consistency of the rooting in all sampled “rooted trees” (> 50,000 after burnin). In addition, the reason for our confidence is that the alternative rootings have negligible support based on Bayes Factor estimates as shown in our earlier studies (Refs 2, 4 & 11).Agreed that most phylogeny software are designed to output/read unrooted trees, and so the support value for the root split is not usually reported, because it is not calculated.Suggestion:4. Page 4 left column. I think that the following is an assertion, not a fact, \"Substitutions between structural domains do not occur, unlike amino acid replacements, since each domain defines a distinctive biochemical function\". Who says that one domain cannot transform into another? I do not understand this assertion. The authors have extreme confidence in this statement it seems, but perhaps this is the problem? Were they there, back 100s of millions of years ago to observe that one domain could not have evolved into another or that similar domains did not evolve convergently into what the authors assume are the same domain (even though it might not be the same 'domain')?Response: Substitutions between structural-domains are not known to occur, unlike amino acid replacements, though, domain recombinations that generate new proteins and functions are frequent2,18. This is because each domain is associated with a distinctive biochemical function.We also included a reference (Bashton, M. & Chothia, C. The Generation of New Protein Functions by the Combination of Domains. Structure 15, 85–99 (2007).). We hope this will be useful to the readers as to why substitutions of domains are not known, or why they may not be possible. It could be useful to answer the questions raised in the previous comment.Suggestion:5. Page 4 left column. I am assuming that the authors' preferred models, \"The natural bias in gain/loss rates, arising from the difficulty of parallel gains and the relative ease of parallel losses, is useful for implementing directional (rooted) character-evolution models\". The idea that there is some general rate across all domains for gain and loss and convergent gain seems naive to me, or at least, a poor criterion for rooting a tree with awesome confidence and high probability. 6. I am not buying the idea that these domains are 'non-redundant'. I believe that the authors believe this, but that is about it. So, I do not think there is \"built in directionality\" if the authors' initial assumptions/assertions are accepted. It is true that one can root a tree if one assumes one can identify homologous domains accurately and apply a very specific general model to a situation that is not specific and surely not general in terms of rate. Rooting the tree of Life will always be depedent on some sort of model that assumes this or that about gains and losses and convergent gains as there is no outgroup (whether gains or losses of domains or genes or nucleotides or amino acids), but this just reinforces the authors' initial assertion in the paper that models that people imagine (which are poorly understood in terms of process) will drive results. The fact that the amino acid trees (unrooted) are completely in conflict with the domain-based tree is not a good sign as no congruence among different data, even in an unrooted context. I suppose it is okay for the authors to assert their tree is better, but I think many people will not agree or be convinced by trust in some general asymmetry model and domains that may or may not be the same thing in very divergent taxa. From the amino acid analysis side of the debate, their tree seems to refute the domain tree, even though it is unrooted (and vice versa I guess), so as an outside viewer of this debate, there seems to be a lot of work to do on an admittedly challenging problem. But, that was likely known before this contribution. 7. Do the authors' asymmetry models take lateral transfer of domains into account as well? Since the authors admit that, \"Further, such incompatibilities are likely to make estimating the absolute origins of single-domain families and single genes difficult, since a majority of genes are formed by duplication and recombination of distinct domains\", if their asymmetry domain models for rooting do not take lateral transfer into account, this would seem problematic to me, given that they argue for the importance of lateral transfer of entire genes (and genes include 'domains').8. I think the following from the authors is an assertion, not a fact, \"Since parallel evolution of homologous protein-domains or distinct domain permutations is very rare, the KVR model adequately captures the evolution of unique features.\" If not a necessarily true, nothing the authors argue is either? Response: We edited the text and re-wrote parts of the text to address issues raised in points 5-8. For a more detailed discussion of these matters, we recommend references 2-4, in addition to the ones mentioned below. But in brief, (a) The directional models do not assume a general rate of gain/loss. The relative rates were estimated using a Gamma distribution, up to 12 rate categories, and did not affect the rooting or tree topology (see refs 2, 4). (b) Convergent evolution of the same 3D structure is highly improbable given the what we know about the sequence-structure relationships. We have now included a reference that shows the improbability of the convergent evolution with an elegant experiment (Mackin, K. A., Roy, R. A. & Theobald, D. L. An empirical test of convergent evolution in rhodopsins. Mol. Biol. Evol. 31, 85–95 (2014)). (c) The skewed rates of losses over gains of unique gene families have been reported in other studies using presence/absence of genes (e.g. Zamani-Dahaj SA, Okasha M, Kosakowski J, Higgs PG. Estimating the frequency of horizontal gene transfer using phylogenetic models of gene gain and loss. Molecular biology and evolution. 2016 Jul 1;33(7):1843-57.). (d) In practice, it is not easy to distinguish convergent evolution from horizontal transfer (HT) using presence/absence patterns by itself, but given (b) and (c), HTs are a minority. Suggestion:9. The authors note that, \"The systematic covariation of homologous domains among the clades is best explained as phylogenetic effect\". I have studied phylogenetics for 30 years, yet I still have not seen any compelling or useful definition of this term that makes any sense. This just seems like a vague explanation for an observed pattern of covariation. Many of the things that the authors seem to consider 'clade-specific' could just be 'grade-specific'. For example, 'fish' have lots of common features that sort of make sense together as all primitively swim around in water, have tails for propulsion underwater, and breath oxygen and feed underwater. Similarly, 'domains' characteristic of eukaryotes or archaeans might not define monophyletic groups, but might instead be characteristic of paraphyletic groups (e.g., Asgards or 'Others' in Fig. 1b). So, one wonders whether the robustly rooted tree of Life based on specifics of a particular model mean that much, or not. The fact that the tree strongly contradicts an unrooted tree based on independent data (Fig. 1b) does not give me much confidence as an outsider to the debate.Response: We revised the sentence as “The non-random similarity of domain composition within clades and the systematic covariation of homologous domains among the clades is referred to as phylogenetic effect to imply shared ancestry of the members of a clade”. Moreover, as we have now clarified, we hope we can agree that homology assessment is key to assessing phylogenies. If one agrees that protein domains are better characters to assess homology than nucleotides/amino acids, then the phylogenies estimated with protein domains are indeed better to assess the relatedness of eukaryotes and akaryotes.Suggestion:10. This has to be a gross overstatement? \"The KVR model is an optimal explanation of the evolution of clade-specific composition of homologous features.\" For example, the authors note that, \"More complex models are available, such as the no- common-mechanism model, an extremely parameter-rich model that allows each character to have its own rate, branch length and topology parameters.\" For this model, surely there would be fewer evolutionary steps; wouldn't that be more 'optimal'? What is the result for this model that could be interpreted as a better (more optimal?) fit to the data - certainly not optimal in terms of minimizing evolutionary steps, unless the tree topology for the model based analysis and parsimony are identical. Response: We revised this statement and expanded the discussion in the penultimate paragraph of section 2. We clarify why the relatively simpler directional-evolution models such as the parametric KVR model and its non-parametric (parsimony) analog HK model are adequate for empirical data (1,800 characters) as opposed to the 1,000,000 simulated characters required to estimate the fit of data to the more complex models. The KVR and HK models do recover congruent phylogenies."
}
]
}
] | 1
|
https://f1000research.com/articles/9-112
|
https://f1000research.com/articles/9-631/v1
|
19 Jun 20
|
{
"type": "Research Article",
"title": "Lessons learned from recent randomized controlled trials comparing the immunogenicity of different infant vaccination schedules of pneumococcal conjugate vaccine",
"authors": [
"Rachel C. Pieciak",
"Christopher J. Gill",
"Christopher J. Gill"
],
"abstract": "Background: The technically complex pneumococcal conjugate vaccine (PCV) is arguably one of the most important and widely studied vaccines since the Hib vaccine. Given the complexity of its design, the cost of administering the PCV is tremendous. While we cannot make adjustments to the vaccine itself post licensure, we can manipulate the dosing schedule. And yet little work has been done to understand the differences in immune responses across different dosing schedules. Methods: Accordingly, we conducted a review of three recently published randomized control trials that compared immune responses across commonly used vaccine schedules in both high- and low-income countries. Results: Each of these studies assessed how changes to the number of doses, spacing between doses and the use/timing of a booster dose affected ELISA geometric mean concentrations post-primary and post-booster dose. If the goal is to administer vaccinations in the most immunologically efficient manner as possible, evidence from these studies would suggest that several commonly used vaccine schedules are missing the mark. Conclusions: In order to deliver the most “bang for its buck”, PCV dosing schedules should not only leverage convenience but also immunological data. Without the reexamination of PCV schedules the status quo will remain inefficient, ineffective and needlessly expensive, threatening the sustainability of its implementation long-term.",
"keywords": [
"pneumococcal conjugate vaccine",
"dosing schedules",
"expanded programme on immunization"
],
"content": "\n\n\n\nWe examined three studies comparing immune responses across PCV dosing schedules.\n\nCurrent dosing schedules are immunologically ineffective and inefficient.\n\nWe summarize evidence-based strategies for manipulating PCV dosing schedules.\n\nPCV dosing schedules should leverage immunological data rather than convenience.\n\n\nIntroduction\n\nThe pneumococcal conjugate vaccine (PCV) is arguably the most important and intensely studied vaccine since the Hib conjugate vaccine in the 1980s. It is an extremely safe and effective vaccine, yet complicated both in terms of its immunology and construction1. The technical complexity of its design also makes it very expensive to manufacture. Immunologically, it is not accurate to think of it as one vaccine, but rather 10 or 13 monovalent conjugated vaccines in one syringe, depending on which of the two dominant marketed vaccines one is talking about (PCV10 (Synflorix, Glaxo Smith Klein) or PCV13 (Pfizer), respectively). Logically, such a valuable and costly commodity should be used in the most immunologically astute way possible to yield maximum benefit for its cost. For licensed vaccines, one no longer has the option of changing the vaccine’s composition (choice of carrier protein, adjuvants, antigen content). Instead, the parameters for optimizing the immunogenicity of any licensed vaccine are rooted in details of the dosing schedule2: How early in life can vaccines be started? How many priming doses are optimal? How does the age of infants affect immunogenicity? How far apart should doses be spaced in time? How important is a booster dose? And how soon in life can booster doses be administered?\n\nUntil recently, the answers to these pragmatic questions have been elusive due to the near absence of comparative studies exploring the immunogenicity of different dosing regimens. However, in the past few years, a series of pivotal randomized controlled trials focused on dose schedules have been published and shed new light on each of these questions.\n\nIn this synthetic analysis, we seek to summarize the key learnings from each of these three seminal investigations, two in high-income settings (The Netherlands and United Kingdom) and one in a low-income setting (Nepal)3–5. It is our contention that these new data suggest that current vaccine schedules used specifically in the US (priming doses at 2, 4, 6 months with a booster at 12 months) and widely in low-income settings through the WHO’s Extended Program of Immunization (EPI) schedule (doses at 6, 10, and 14 weeks, with no booster dose) are immunologically inefficient and either wasteful in terms of administering too many doses of vaccine, or yield suboptimal immune responses due to schedules constructed around expediency rather than based on immunologic data. These learnings present an opportunity to reexamine PCV dosing schedules globally and to align these schedules with immunologic data such that these schedules deliver the greatest long-term cost-effectiveness.\n\n\nMethods\n\nFor the purposes of this analysis, our review centers on three recently published randomized controlled trials (Table 1). Given our interest in comparative immunogenicity, we focus on geometric mean concentrations (GMCs) as our outcome of interest. An advantage to GMCs is that there is no ceiling effect in measuring immune responses, as is the case with the proportion of study participants meeting or exceeding some threshold concentration of IgG, and this allows for greater separation of the intrinsic immunogenicity of different dosing strategies. While threshold measures are useful for inferring the proportion of a population that will be seroprotected at a given time, this simplification is distinct from the net magnitude of the immune response. Moreover, GMCs offer some clues as to the likely persistence of protection over time. Moreover, in the case of pneumococci, the commonly used threshold of protection for type specific capsular IgG of 0.35 μg/mL has only been validated for certain serotypes, with the protective relationship assumed to apply to the others6–8. Recent data has made clear that the relationship between IgG GMC and nasopharyngeal carriage varies substantially between serotypes and between high and low incidence settings9–12.\n\nWe also note that none of the three studies we reviewed were efficacy studies focused on prevention of clinical disease, so we cannot comment on how these data would translate into morbidity and mortality rates in clinical settings. However, this is an acceptable limitation since field efficacy is already established for PCV, and obviously this is mediated by immunogenicity.\n\n\nResults\n\nUsing a cohort of 400 healthy, term, Dutch infants, Spijkerman et al.3 conducted a large randomized control trial to test the immunogenicity of four different dosing schedules. In this study, infants were randomized (1:1:1:1) to receive three doses of PCV13 either at 2, 4, and 6 months (regimen 1); 2, 3 and 4 months (regimen 2); or to receive just 2 doses of PCV13 at 3 and 5 months (regimen 3) or at 2 and 4 months (regimen 4). Every group received a booster dose at age 11.5 months. To compare differences in immunogenicity across these four schedules, they used observed immunoglobin G (IgG) concentration values (GMC in μg/mL) one month after the primary series and one month after the booster dose.\n\nUpon observation one month post primary series, IgG GMCs for the 2-4-6 schedule were higher for three serotypes than the 2-3-4 and 3-5 schedules. The 3-5 schedule was superior to the 2-4-6 and 2-3-4 schedules for one serotype and the 2-3-4 schedule was higher than the 2-4 schedule for 5 serotypes (Figure 1).\n\nHowever, these high-level comparisons, in which the statistics sought to measure superiority of one schedule compared to all other schedules, may have significantly understated the observed differences in immunogenicity. When assessing this instead as pairwise comparisons by serotype across schedules, the differences in immune responses to the primary series were more striking. Unsurprisingly, 2-4-6 was statistically superior to 2-4 for 11/13 serotypes. Yet it was surprising that 2-4-6 was superior to 3-5 only for 4/13 serotypes (Table 2). The former is an intuitive finding, but the latter is somewhat surprising and quite interesting: delaying the onset of the primary series (as observed in the 3-5 dosing schedule) seemed to yield better immune responses despite the omission of one of the priming doses (as observed in the 2-4-6 and 2-3-4 schedules). This argues that the timing of doses may be more relevant than the total number of doses in the priming series.\n\nEmphasizing this same point, while also testing the question of dose spacing, the 2-4-6 schedule outcompeted 2-3-4 for 10/13 serotypes, indicating that the increased spacing between priming doses (2 months apart instead of 1) elicited significantly better immune responses. Delayed onset of dosing also appeared immunologically superior given that the 3-5 schedule yielded significantly higher GMCs than the 2-4 schedule for 11/13 serotypes. A surprising and counterintuitive finding was that two doses at 3-5 months yielded superior GMCs than did three doses at 2-3-4 months for 5/13 serotypes. This further demonstrates that the total number of doses given may be less important than the age of the child at the time of vaccine administration.\n\nThey also assessed persistence of immune responses following each schedule, testing GMCs again at 8 months (Figure 2) and before the booster dose at 11.5 months (Figure 3). Overall, the 2-4-6 schedule had better persistence at eight months, followed by the 3-5 schedule and both schedules were superior to the 2-4 and 2-3-4. These observations suggest that delaying the onset of doses coupled with the longer intervals between doses yield higher immune responses than dosing schedules where doses are administered earlier in life and closely together. If the goal of dosing schedules is to provide protection early in life, then the 2-4-6 or 3-5 schedules appear to be the most desirable of the four regimens. If we are thinking about this in terms of cost comparisons of these schedules in relation to the total number of doses given, the 3-5 schedule delivers comparable immune responses to the 2-4-6 schedule at a lower price. While the immune responses across all four regimes wane pre-booster, the 2-4-6 and the 3-5 schedules continue to offer the highest residual GMCs.\n\nDespite these differences in post primary and pre-boost GMCs, the immune responses following a booster at 11.5 months resulted in significant increases in GMCs, with little variation based on primary schedule (Figure 4). Here, the booster dose acts as the great equalizer between dosing schedules and emphasizes its importance for achieving high immune responses.\n\nIn summary, this simple yet elegant experiment yielded a wealth of important and in some cases surprising findings.\n\nFirst, the absolute number of doses given appeared to be less important than when doses were given. Delaying the onset of PCV administration until later in life (3 months instead of 2 months) may not be a prudent decision for settings that have not reached herd immunity since it leaves infants vulnerable to pneumococcus infection longer. However, in settings with established herd immunity, this herd immunity should provide critical, indirect protection to these infants and allow for flexibility. In these settings, moving the onset of dosing schedules to later in life may not add any undue risk to the infant.\n\nSecond, a larger spacing between doses (2 months apart vs. 1 month apart) appeared particularly important, as demonstrated by the fact that 3-5 and 2-4 generally outperformed the 2-3-4- schedule.\n\nLastly, the booster dose acted as the great equalizer, such that the IgG GMCs were essentially indistinguishable regardless of which priming series had been used.\n\nAlso notable was that serotype 3 appeared far less immunogenic than the others, a finding that has been noted elsewhere. Each of these findings has obvious practical implications, as will be discussed subsequently.\n\nFive years later in the UK, Goldblatt et al.4 conducted a similar study to observe how changing the number of priming doses in the PCV dosing schedules would impact serotype-specific IgG GMCs. Unlike the United States, which launched PCV7 using a 2-4-6 priming schedule with a booster at 12 months, the UK instead chose a 2+1 schedule (two priming doses plus a booster in the second year) based on earlier field immunogenicity studies suggesting that this approach would be effective. In the current study, Goldblatt pushed this concept to the next logical point, testing whether a further reduction in priming doses to a 1+1 schedule would be feasible and comparable to the current standard of care. Here, infants were randomized (1:1) to two different dosing schedules, a 2+1 dosing schedule (current standard of care) and an experimental 1+1 dosing schedule. In the 2+1 schedule, infants received two priming doses at 2 and 4 months with a booster dose at 12 months and in the 1+1 schedule, infants received a single priming dose at 3 months and a booster dose at 12 months.\n\nAs shown in Figure 5, consistent with expectations, the 2-4 schedule yielded higher GMCs than a single dose at 3 months for 12/13 serotypes. Yet, similar to what was observed in the experiment by Spijkerman et al.3, following the boost at 12 months, GMCs were quite similar between the two schedules, with observed GMC ratios close to 1.0 for most serotypes. Interestingly, while the 2-4 schedule had yielded statistically significantly higher post boost GMC ratios for 4/13 serotypes, a single dose at 3 months yielded significantly superior GMC ratios for 4 of the serotypes. In other words, neither schedule offered a clear advantage over the other. The lack of a clear difference between infants regardless of whether they received one or two priming doses, suggests that a 1+1 dosing schedule could be a less costly but similarly effective alternative to the 2+1 dosing schedule, provided that this is done in a setting where high levels of herd immunity had already been achieved. This finding has obvious practical implications.\n\nMoreover, when considering the post boost responses in terms of the proportion of infants achieving 0.35 μG of serotype specific IgG (the putative, though somewhat controversial, threshold for protection), 100% of participants were seroprotected against 12/13 serotypes6–8. The exception was again serotype 3, where only 75.9% and 78.8% of the infants randomized to the 2–4 month and 3 month priming schedules, respectively, met or exceeded that threshold.\n\nThe studies conducted by Spijkerman et al.3 and Goldblatt et al.4 were both conducted in high-income/low disease burden settings. It is safe to assume that these same issues in terms of dose timing, spacing, and dose number would be at least as important, if not more, in low-income/high disease burden settings. Accordingly, Kandasamy et al.5 conducted a PCV dose schedule study amongst a cohort of Nepalese infants to compare two different dosing schedules, this time using the PCV10 vaccine. Working within the WHO’s EPI schedule of 6-10-14 weeks, infants were randomized (1:1) to receive two priming doses of PCV10 at 6 and 10 weeks (6–10) or at 6 and 14 weeks (6–14), with both groups receiving a booster dose at 9 months of age to complete a 2+1 schedule. The rationale for moving the boost earlier in life to 12 months was to take advantage of the scheduled administration of the measles vaccine at that age, without which a fifth vaccination visit would have to have been created de novo.\n\nTo note, the post primary responses for the 6–10 and 6–14 groups were both assessed at 18 weeks. Theoretically, this strategy could penalize the 6-10 regimen to some degree, since it introduces another month for initial antibody concentrations to wane.\n\nWith that caveat, as shown in Figure 6, the post primary responses were quite similar between the 6–10 and 6–14 groups, though in general, the 6-14 group GMCs trended higher for most serotypes. These differences were less obvious when reassessed at 9 months, just prior to the boost dose, basically because GMCs had fallen to baseline for most serotypes. However, one month after the 9-month boost, there were robust immune responses to all serogroups (Figure 7). The GMCs were nearly identical for all PCV10 serotypes, irrespective of priming schedule. As seen in the papers by Spijkerman et al.3 and Goldblatt et al.4, once again, the booster dose acted as a grand equalizer, largely erasing the subtle differences following different priming series.\n\nSome exceptions were observed: post-booster GMCs for serotypes 18C and 19F were somewhat higher among infants who received doses at 6-14 than for 6-10. And again, serotype 3 was an immunological dud compared with all other serotypes. Apparently, the comparative underperformance of serotype 3 is not limited to PCV13, but also occurs with other PCV formulations, arguing that the technical construction of the vaccine seems less likely at fault13. Rather, there may be intrinsic limitations in how the immune system engages serotype 3 antigens specifically14.\n\n\nDiscussion\n\nFrom this body of evidence, several important messages emerge:\n\nFirst, that the number of doses in the primary series is less important than at what age doses are given.\n\nSecond, in order to maintain optimal levels of antibodies between doses it is important to administer these doses spaced apart by longer intervals.\n\nThird, deferring the start of vaccinations until infants are older, yields higher immune responses. Even a single month’s delay from 2 to 3 months of age yielded markedly higher GMCs for most serotypes.\n\nFourth, even when infants are dosed on different immunization schedules, the observed differences across these groups largely disappear after the administration of the booster dose. In order to harvest the full benefit of PCV, the booster dose is critically important and simultaneously reassures us that flexibility within the primary dosing schedule is possible. In low disease burden settings, dosing schedules could be reduced or started later in life without compromising protection from infection so long as the booster dose is administered. Any reduction to the number of doses could offer tremendous cost savings15. Being able to align modified vaccine schedules with other essential earlier-life vaccines (e.g. measles vaccine) could also indirectly improve adherence to these schedules.\n\nFifth, it is not necessary to wait until the second year of life to administer a booster dose. In fact, a booster dose given at 9 months also yielded excellent immunologic responses. While one must be cautious in comparing immune responses between different cohorts (the United Kingdom vs. Nepal, for example), the GMCs seen after a boost at 9 months were remarkably similar to those in the UK infants after a boost at 12 months. This finding has significant practical implications for countries adhering to the EPI schedule where measles vaccine is given at 9 months.\n\nWhile these messages emerge consistently throughout this body of work, their implications for change are vastly different depending on where you are in the world. For instance, in high-income countries, where herd immunity and disease surveillance programs are well-established, creating a dosing schedule with as few as two doses (one priming dose and a booster dose) may be an effective and cheaper alternative to commonly used dosing schedules. However, in low- and middle-income countries the need for early protection is crucial to child survival and the strategy for adapting the immunization schedule must take this into account.\n\nIn conclusion, dosing schedules need not and cannot be designed as “one size fits all”. The lack of scrutiny towards the current PCV schedules has left us with inefficient and ineffective dosing schedules as the status quo. These inefficiencies exist in high- and low-income settings alike. Given that the UK has achieved similar success to the US using a 2+1 vs. 3+1 schedule, and evidence here that a 1+1 schedule is also justifiable, one may conclude that the US schedule is wasteful. Similarly, given the criticality of the booster dose, the EPI schedule of 6, 10 and 14 weeks, with no booster, may be spending more than necessary on doses of vaccine, while using them in a way that immunologically inefficient. Key learnings from this synthesis suggest several strategies for adapting dosing schedules to deliver robust immune responses, including longer dose spacing, fewer doses and the possibility for earlier booster doses. These changes could provide countries with cheaper alternatives to commonly used dosing schedules without compromising protection against pneumococcal disease.",
"appendix": "References\n\nObaro SK: The new pneumococcal vaccine. Clin Microbiol Infect. 2002; 8(10): 623–33. PubMed Abstract | Publisher Full Text\n\nTrotter CL, McVernon J, Ramsay ME, et al.: Optimising the use of conjugate vaccines to prevent disease caused by Haemophilus influenzae type b, Neisseria meningitidis and Streptococcus pneumoniae. Vaccine. 2008; 26(35): 4434–45. PubMed Abstract | Publisher Full Text\n\nSpijkerman J, Veenhoven RH, Wijmenga-Monsuur AJ, et al.: Immunogenicity of 13-valent pneumococcal conjugate vaccine administered according to 4 different primary immunization schedules in infants: a randomized clinical trial. JAMA. 2013; 310(9): 930–7. PubMed Abstract | Publisher Full Text\n\nGoldblatt D, Southern J, Andrews NJ, et al.: Pneumococcal conjugate vaccine 13 delivered as one primary and one booster dose (1 + 1) compared with two primary doses and a booster (2 + 1) in UK infants: a multicentre, parallel group randomised controlled trial. Lancet Infect Dis. 2018; 18(2): 171–179. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKandasamy R, Gurung M, Thorson S, et al.: Comparison of two schedules of two-dose priming with the ten-valent pneumococcal conjugate vaccine in Nepalese children: an open-label, randomised non-inferiority controlled trial. Lancet Infect Dis. 2019; 19(2): 156–164. PubMed Abstract | Publisher Full Text\n\nAndrews NJ, Waight PA, Burbidge P, et al.: Serotype-specific effectiveness and correlates of protection for the 13-valent pneumococcal conjugate vaccine: a postlicensure indirect cohort study. Lancet Infect Dis. 2014; 14(9): 839–46. PubMed Abstract | Publisher Full Text\n\nSiber GR, Chang I, Baker S, et al.: Estimating the protective concentration of anti-pneumococcal capsular polysaccharide antibodies. Vaccine. 2007; 25(19): 3816–26. PubMed Abstract | Publisher Full Text\n\nKaur R, Pichichero M: Comparison of anti-capsular antibody quantity and functionality in children after different primary dose and booster schedules of 13 valent-pneumococcal conjugate vaccine. Vaccine. 2020; 38(28): 4423–4431. PubMed Abstract | Publisher Full Text\n\nDagan R, Givon-Lavi N, Fraser D, et al.: Serum serotype-specific pneumococcal anticapsular immunoglobulin g concentrations after immunization with a 9-valent conjugate pneumococcal vaccine correlate with nasopharyngeal acquisition of pneumococcus. J Infect Dis. 2005; 192(3): 367–76. PubMed Abstract | Publisher Full Text\n\nGoldblatt D, Ramakrishnan M, O'Brien K: Using the impact of pneumococcal vaccines on nasopharyngeal carriage to aid licensing and vaccine implementation; a PneumoCarr meeting report March 27-28, 2012, Geneva. Vaccine. 2013; 32(1): 146–52. PubMed Abstract | Publisher Full Text\n\nSimell B, Nurkka A, Lahdenkari M, et al.: Association of serotype-specific antibody concentrations and functional antibody titers with subsequent pneumococcal carriage in toddlers immunized with a 9-valent pneumococcal conjugate vaccine. Clin Vaccine Immunol. 2012; 19(1): 96–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVoysey M, Fanshawe TR, Kelly DF, et al.: Serotype-Specific Correlates of Protection for Pneumococcal Carriage: An Analysis of Immunity in 19 Countries. Clin Infect Dis. 2018; 66(6): 913–20. PubMed Abstract | Publisher Full Text\n\nLinley E, Bell A, Gritzfeld JF, et al.: Should Pneumococcal Serotype 3 Be Included in Serotype-Specific Immunoassays? Vaccines (Basel). 2019; 7(1). PubMed Abstract | Publisher Full Text | Free Full Text\n\nPoolman J, Kriz P, Feron C, et al.: Pneumococcal serotype 3 otitis media, limited effect of polysaccharide conjugate immunisation and strain characteristics. Vaccine. 2009; 27(24): 3213–22. PubMed Abstract | Publisher Full Text\n\nO'Brien KL: When less is more: how many doses of PCV are enough? Lancet Infect Dis. 2018; 18(2): 127–8. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "277333",
"date": "14 May 2024",
"name": "Anand Kawade",
"expertise": [
"Reviewer Expertise Vaccine clinical development",
"Infectious diseases",
"Pediatric community based research"
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nDate : 14 MAY 2024 Thank you very much for inviting me to review this article. Below are the detailed answer to above review questions: 1) This article was first published on 19 JUL 2020. The purpose of this review \" to understand the differences in immune responses across different dosing schedules of PCV \" might be important and contextual at the time of writing the first draft of the manuscript. Lot of significant data were generated since then till date to conclude on not only the effects of schedules on immunogenicity but on nasopharyngeal carriage which is now considered as proxy marker of public health impact of PCV. RCTs conducted in countries South Africa, UK, India, Vietnam and Gambia contributed significantly towards reduced dose schedules. Therefore, it is highly recommended to rewrite completely using current data. 2)It is recommended to use systematic review approach and guidelines like QUOROM 3) & 4) Request to use well defined statistical methods for meta-analysis 5) Will require recent data additionally 6) The conclusion is based of small data. The pneumococcal carriage and immunogenicity after PCV is very complex and is influenced by multiple factors. The dynamics of pneumococcal colonization (acquisition, colonization, removal & replacement) must be considered while interpreting the RCT data which was generated at certain time-points only. Overall decision Rejected in current status and Suggested to revise completely taking recent data in consideration\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
},
{
"id": "264515",
"date": "17 May 2024",
"name": "Paul Licciardi",
"expertise": [
"Reviewer Expertise immunology",
"pneumococcal vaccines"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript by Pieciak and Gill provides a review of the current evidence from RCTs relating to different PCV vaccines and schedules in infants. They provide insights into the how the best to use PCVs for infant vaccination based on vaccine immunogenicity studies, highlighting that there may be more efficient ways in which PCVs could be used to combat disease burden that take into consideration number of doses, timing of doses, use of a booster dose and interval between doses. These are all very critical issues in the field, and so I believe this manuscript is timely and would be a useful contribution to the field.\nHowever, as written, I do believe the manuscript needs significant changes. The major concern I have is that the authors have based their conclusions on three RCTs that have been published in the last 10 years or so. What was the justification for including just these three studies? While I believe their conclusions to be mostly valid, there needs to be a more thorough review of the literature, as there are a number of other RCTs conducted in different settings that would add value and are necessary. For example, there are studies that have been conducted in South Africa, Vietnam, The Gambia and India with different PCV products (the higher valency PCVs) and schedules and these should be included in any discussion of current PCV scheduling issues. Indeed, some important lines of evidence can also be gleaned from some of the original PCV7 studies, such as those from Fiji and elsewhere.\nOther points for consideration include:\n\nIntroduction – it is important to note that WHO recommends either a 2+1 or 3+0 schedule. Many countries are now looking to reduce the number of doses further to 1+1 but data is still lacking, especially for carriage and disease outcomes. The data in the Figures have been recreated from the papers in most instances – has there been appropriate authorization for the use of this data from the original study authors? In the methods, the authors state that they focus on IgG GMCs rather than the proportion of infants above the seroprotective 0.35ug/ml cutoff however this has been included in their results. This needs to be amended. Also, have the authors thought have including OPA data where these data are available? Results p9 – the authors mention that serotype 3 is not very immunogenic, which is true, but that it occurs with over vaccine formulations in addition to PCV13. This needs some correction, since PCV13 is the only licensed vaccine containing serotype 3. If the authors are referring to the PCV10 predecessor that contained serotype 3 (but was later removed) this needs to be clear. Discussion p10 – states that booster doses are important. While I agree with this argument, there is data from some trials suggesting no added value of a booster dose (in a 2+1 schedule) compared to 3+0, in terms of carriage. This is an important point, and requires some further discussion. Some additional discussion around the future of PCV use would be beneficial as there are many issues facing the field. For example, the use of reduced dose (1+1) schedules, higher-valency vaccines and impact on immunogenicity and where and timing of their use. This would provide some context in terms of where the field is heading towards.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "170341",
"date": "29 May 2024",
"name": "Amanda Leach",
"expertise": [
"Reviewer Expertise RCTs of interventions to prevent and treat otitis media in Australian First Nations children."
],
"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\nComments Whilst scheduling is an important question and issue for pneumococcal conjugate vaccine policy, this review uses data from just three studies to address the many complex questions posed. The authors give no indication as to how or why these three studies were selected, no search strategy or date of data retrieval is given (although the journal header says \"Last updated: 26 May 2022\", there is no explanation as to whether this applied to analysis), there are no study inclusion or exclusion criteria (a decision tree/flow diagram could be provided). There is no reference to several systematic reviews, reviews or key RCTs on this topic, nor why these were excluded (a few for example: Silferdel et al. (2009)1, Rodgers et al. (2013)2, Knoll et al. (2014) 3, Whitney et al. (2014)4, Madhi et al. (2017)5…). Of the total of 15 citations, the authors cite 4 studies of nasopharyngeal carriage, which is not an outcome incorporated into this analysis. I find this odd.\nGiven the paper was first published 19 June 2020, almost 3 years ago, the authors should explain whether there have been any further data published that would/should be included in this review.\nThe reporting could be improved by using appropriate guidelines for a review, such as PRISMA.\nHighlights\nPoints one and three could be combined.\n\nThe second dot point should be re-written: \"Current dosing schedules can be manipulated to become immunologically more effective and efficient.\"\n\nThe final dot point should be altered to “...immunological data in addition to convenience”. I would add “and local epidemiology”.\n\nTable 2 Includes serotype 2 which is not a PCV13 serotype and no data are reported by Spijkeman et al. for serotype 2. Please explain inclusion of serotype 2.\nPage 5 \"While the immune responses across all four regimes wane pre-booster, the 2-4-6 and the 3-5 schedules continue to offer the highest residual GMCs.\" - change \"regimes\" to \"regimens\".\nPage 9 In discussion of Kandasamy et al.'s Lancet paper on PCV10, firstly the comment about 18C and 19F is made, yet the authors make no reference to the carrier protein of these serotypes not being protein D, which could well be influencing the immune response. The authors could review the data on 18C and 19F. Also in this paragraph, the authors state “And again, serotype 3 was an immunological dud compared with all other serotypes. Apparently, the comparative underperformance of serotype 3 is not limited to PCV13, but also occurs with other PCV formulations…” PCV10 does not contain serotype 3 so I am unclear what data they are referring to as there are none reported by Kandasamy. Could the authors clarify this statement please?\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No",
"responses": []
}
] | 1
|
https://f1000research.com/articles/9-631
|
https://f1000research.com/articles/9-622/v1
|
17 Jun 20
|
{
"type": "Research Article",
"title": "Validating the Developmental and Well-Being Assessment (DAWBA) in a clinical population with high-functioning autism",
"authors": [
"Nadia Coscini",
"Ramya Srinivasan",
"David Skuse",
"Ramya Srinivasan",
"David Skuse"
],
"abstract": "Background: With increasing numbers of referrals to health services for assessment of Autism Spectrum Disorder (ASD), the Developmental and Well-Being Assessment (DAWBA) has been suggested as a useful screening instrument to assist in prioritising patients for review. It is an online interview for parents that has been previously validated for ASD in a non-clinical community sample of twins. Our study aimed to evaluate its predictive validity in a complex clinically-referred sample of children with suspected high-functioning autism. Methods: The sample comprised 136 children (females = 53; males = 83) who were referred for ASD assessment at the Social Communication Disorder Clinic (SCDC) at Great Ormond Street Hospital. Parents completed the DAWBA online prior to undergoing a multi-disciplinary team (MDT) assessment. This included completing the Developmental, Dimensional and Diagnostic Interview (3di) and the Autism Diagnostic Observation Schedule (ADOS). Two clinicians independently rated the DAWBA using DSM-5 diagnostic criteria and compared results to the MDT outcome, which was considered gold standard. Results: Compared with an MDT assessment, the DAWBA interview demonstrated good sensitivity (0.91) but poor specificity (0.12). Overall, 64% of cases were accurately assigned as case/non-case. Estimates of positive (0.66) and negative (0.43) predictive validity were influenced by the relatively high prevalence of ASD in the study sample (65%). Conclusion: The DAWBA online interview has excellent sensitivity in a clinical population of complex neurodevelopmental disorders, containing a high prevalence of ASD, but specificity was poor. As the SCDC offers tertiary opinions on disputed cases of suspected ASD, the population cohort limits the generalisability of these results. Further evaluation is required in community child mental health or paediatric services.",
"keywords": [
"Autism spectrum disorder",
"assessment",
"validity",
"sensitivity and specificity",
"social communication disorder",
"screening instrument."
],
"content": "Introduction\n\nThe Diagnostic and Statistical Manual (5th edition) describes Autism Spectrum Disorder (ASD) as a condition with social communication problems and patterns of repetitive behaviour or restricted interests (American Psychiatric Association, 2013). The prevalence of autism in England is 1.3% based on figures from the United Kingdom Department for Education (Department for Education (UK), 2017). There have been increasing numbers of referrals for autism, with services receiving twice the previous number, but without the required resources with which to make assessments (Dreaper, 2017). At an estimated cost of £800 per child per diagnostic assessment, service providers are under increasing pressure to assess and prioritise patients (Galliver et al., 2017). With recognition of the benefits of earlier diagnosis, there has also been a focus on earlier recognition of ASD. A recent study has shown that in the UK, the median age for being diagnosed with ASD is 4.5 years, but despite strategies to try and reduce this, it has remained the same between 2004 and 2014 (Brett et al., 2016). Therefore, there may be a benefit in screening potential cases of ASD in order to triage the need for further assessment. Application of a diagnostic tool prior to a multidisciplinary assessment in order to obtain a clinical history could also inform the diagnostic process and expedite the clinical evaluation.\n\nThere is a high prevalence of comorbid psychiatric disorders in children with ASD, with one study estimating this as up to 70% (Simonoff et al., 2008). Whilst there is a focus on earlier diagnosis of ASD, there is no corresponding emphasis on the diagnosis of comorbid disorders (Mannion & Leader, 2013). It is particularly important to recognise comorbidities because of the increased impact on functioning and quality of life for children diagnosed with ASD (Soke et al., 2018). Understanding comorbid disorders also has the potential to make the ASD diagnosis more accurate.\n\nThe best practice guidelines for diagnosing ASD recommend that a multi-disciplinary team (MDT) should undertake a thorough medical and developmental history, clinical examination and a semi-structured period of observation (NICE, 2011). The guidelines recommend the MDT should involve a paediatrician or psychiatrist, psychologist and speech pathologist and also suggest an autism assessment is helpful, but do not recommend any specific one (NICE, 2011). Frequently used tools include the Developmental, Dimensional and Diagnostic Interview (3di) (Skuse et al., 2004), the Autism Diagnostic Interview-Revised (ADI-R) (Lord et al., 1994a), and the Autism Diagnosis Observation Schedule (ADOS) (Falkmer et al., 2013). These interviews can take between 1.5–3 hours to complete and can only be implemented with trained personnel (McEwan & Brinkmann, 2000). Due to the length of these assessments, they are useful as part of the MDT but not as potential screening instruments to determine which children are most at risk and should be prioritised for review.\n\nBefore evaluating whether a screening tool may also have utility as a diagnostic instrument, it is useful to review its nature and function. The United Kingdom National Screening Committee defines it as a test that is applied in a systematic way to a specific population to identify people at risk in order to investigate further or prevent the disease in an individual who would not otherwise have looked for the disorder (The UK National Screening Committee, 1998, in Gilbert et al., 2001). In this situation, screening would not be for all children in the general population, but only for those about whom concerns have been raised about possible developmental problems, who are then subsequently referred for paediatric or psychiatric assessment. Fundamental values of a screening test were introduced by Wilson and Jungner in their seminal 1968 paper, which highlighted that it should be cheap, simple to administer, acceptable, reliable, valid and be able to be followed up appropriately (Wilson & Jungner, 1968). There is a need for an assessment that is inexpensive to administer prior to being seen in clinic that can help clinicians identify individuals most at risk of ASD.\n\nSince a screening test of ASD is likely to collect a large amount of data, this could also be utilised to its maximum capacity by integrating the results into a subsequent multi-disciplinary assessment. This would mean that a screening test could also be used as a diagnostic adjunct or instrument providing information to clinicians to assist them reaching a conclusion about whether an individual has autism. It may improve the efficiency of diagnosis by speeding up information-gathering since much of the relevant information would be collected prior to the diagnostic process. It may also support decision-making in difficult or unclear cases, and assist in gathering broader information about emotional and behavioural problems, helping to clarify the diagnosis when a specific presentation may be caused by different conditions.\n\nThe Developmental and Well-Being Assessment (DAWBA) may be useful in both these ways and its development was envisioned as such by its authors (Goodman et al., 2000). It has already been used in epidemiological mental health surveys in the UK for young people (Meltzer et al., 2000).\n\nThe Developmental and Well-Being Assessment (DAWBA) is an instrument for caregivers to complete that helps identify possible psychiatric disorders (Goodman et al., 2000). It is an interview that is completed online, which has been used in many research studies and all national UK studies looking at psychopathology in children and adolescents. The information gathered helps guide clinicians on areas of risk before the child attends clinic. It can be completed by multiple different informants including children over 11 years of age, their parents or main caregivers, and school teachers. The DAWBA has an incorporated diagnostic algorithm that can predict risk of psychiatric morbidity, and clinical raters can analyse the unstructured answers and synthesise information from the structured questions across the different disorder modules to refine the DAWBA-generated diagnoses.\n\nAny screening or diagnostic tool should be able to be applied to cases that are difficult to diagnose in ASD, due to the spectrum of symptoms or the presence of other comorbidities. There are other questionnaires used by some services as a screening tool, but they focus only on ASD symptoms. The advantage of using the DAWBA, is that it covers a wide range of psychopathologies and can be completed online in the parents’ own time. Furthermore, its need for review by a clinician means that it is not just a questionnaire relying on a statistical cut off, and context can be applied to interpreting the symptoms reported.\n\nThe initial validity study for the DAWBA was performed in both clinical and community samples. In the clinical sample of patients in a child and adolescent mental health clinic, there was strong agreement between the primary diagnosis yielded by the DAWBA and that of the clinical case notes. There was however, an increase in comorbid diagnoses made that were either only possible (not definitive) or completely absent in the case notes (Goodman et al., 2000). Within the community sample, the respondents reflected the expected diversity of clinical characteristics, and the DAWBA also found the expected difference in rates of diagnosis between a clinical and community sample (Goodman et al., 2000). There have been further studies that have validated the DAWBA, and it has also been used and validated in epidemiological studies (Ford et al., 2003; Heiervang et al., 2007). Two studies found high values of agreement between the DAWBA and clinical diagnosis (Alyahri & Goodman, 2006; Mullick & Goodman, 2005). Alyahri and Goodman compared the DAWBA and Strengths and Difficulties Questionnaire (SDQ) in a Yemeni population, including children in community psychiatric clinics, and found good agreement for internalising and externalising disorders (Alyahri & Goodman, 2006). The other validation study used the Bangladeshi translation of the DAWBA in a cohort of 100 children who had mental health referrals. The DAWBA diagnoses demonstrated a high value of agreement compared with clinical diagnoses (Mullick & Goodman, 2005). Another study looked at how the DAWBA performed when used as an adjunct to clinical diagnosis in a Swiss population of children referred to an outpatient mental health service (Aebi et al., 2012). Of 270 consecutive new referrals, half of the clinicians were randomised to receive data from the DAWBA as well as the clinical rater diagnosis, while the other half did not (Aebi et al., 2012). Overall there was fair to moderate agreement for all diagnoses, whether clinicians received data or not.\n\nThroughout these studies, inter-rater reliability between clinical raters has been reported to be consistently high (Aebi et al., 2012; Fleitlich-Bilyk & Goodman, 2004; Ford et al., 2003; Heiervang et al., 2007).\n\nIn a 2016 study looking at participants from a national twin study, the DAWBA was used to assess possible ASD and demonstrated high sensitivity (0.86) and specificity (0.87) (McEwen et al., 2016). This large population of twins was screened for ASD and parents completed the DAWBA interview. A small proportion of this group were then allocated into at-risk groups for ASD based on their DAWBA scores; high risk or low risk. Within these groups of predicted ASD and non-ASD, a subsample was followed up by an at-home visit by researchers completing a gold standard MDT ASD assessment, including the ADOS. As the sample in this study was community-based, and assessed based on at-risk screening, the utility for the DAWBA in a clinical setting needs to be tested. Its ability to predict an ASD diagnosis needs to be validated in a clinically-referred sample.\n\nWe propose comparing the outcomes of DAWBA- and MDT-diagnostic assessments for children who have been referred to a developmental clinic for concerns about ASD. If the DAWBA proves to have good predictive validity, then it may be useful as an efficient screening measure not only for ASD, but also for comorbid psychiatric disorders. This would allow clinicians and researchers to utilise the DAWBA with confidence. We expect, given the excellent history of inter-rater reliability, that the DAWBA will have good inter-rater reliability between clinical raters. Further to McEwan’s study, we are keen to find out how sensitive and specific the DAWBA is in diagnosing ASD in a clinically complex referral group which in this study was the Social and Communication Disorder Clinic, which is discussed below. The DAWBA may be useful as a screening test before clinical assessment to highlight a possible risk of diagnosis, which could be used as part of diagnosing ASD. If the DAWBA is validated for ASD, it is also possible that service providers could utilise the interview as part of a triaging system to prioritise referrals for assessment.\n\n\nMethods\n\nThe Social Communication Disorder Clinic (SCDC) and the multi-disciplinary team (MDT) assessment. The sample in this study included all individuals aged 4–18 years who were referred to the Social Communication Disorder Clinic (SCDC) at Great Ormond Street Hospital, and completed a DAWBA before a gold standard MDT ASD diagnostic assessment (n=136) between June 2014 and August 2017. Parents of children seen in the SCDC were asked to whether they were interested in participating in the Autism Families Study, using data collected for future research on autism spectrum disorders (Great Ormond Street Hospital for Children NHS, 2020).\n\nThe SCDC is a tertiary specialist clinic, which focusses on providing an extensive and evidence-based multi-disciplinary assessment for children referred from local community clinics who are uncertain about a child’s diagnosis of possible ASD. The SCDC implements the NICE guidelines for a gold standard MDT assessment for ASD, by incorporating a thorough patient medical history and examination, followed by a structured developmental interview (the 3di) and standardised observation (the ADOS). All prior assessments and reports are collated and reviewed by the MDT, and members of the team may also observe the child in school as part of the wrap-around approach to assess the child in all their environments. Parents of children referred to the clinic were asked to complete the DAWBA before their first appointment at the SCDC.\n\nAs a tertiary referral clinic, the children referred to the SCDC are not a typical population of children referred for assessment of autism in general. All the children were referred for second opinions on previous autism assessments. Many of the children had been referred because health professionals were unsure about whether they met the full criteria for ASD due to a complex clinical presentation.\n\nAll cases were reviewed and data collected on the results of ADOS scores (overall and modules), 3di subscale scores, Strengths and Difficulties Questionnaire (SDQ) scores, DAWBA computer predicted probabilities, DAWBA consensus ratings, and final MDT diagnosis. We also collected demographic data including parental level of education and pathway of referral. Pathways to referral were divided into two categories: via a paediatrician or by local Child and Adolescent Mental Health Services (CAMHS). Information about parental education was categorised both by whether parents had completed high school or not (defined as A levels at 17–18 years of age in this United Kingdom cohort).\n\nConfidentiality and consent. The standard protocol for confidentiality was followed according to the Data Protection Act 1998 (DPA) adopted by University College London and Great Ormond Street Hospital. Each child seen in clinic has details stored in a secure database that is only available to staff involved either with the clinic or approved research. Each DAWBA record has a unique de-identified number and records are not identifiable based on patient information. All families who were reviewed in the SCDC provided written informed consent to participate in future research through the Autism Families Study (Great Ormond Street Hospital for Children NHS, 2020). Ethics approval was already in place as per the Autism Families Study (REC reference number is 06/Q0508/60 and R&D number is 08BS06).\n\nThe DAWBA was completed by parents online or by trained research students who conducted the interview via phone and completed the online form. The DAWBA consists of a computer program using a structured interview format with extended answers that then uses an algorithm to predict psychiatric diagnoses. The first part of the DAWBA is the Strengths and Difficulties Questionnaire, which is further discussed in detail below. Respondents are then asked about symptoms of up to seventeen types of psychiatric diagnoses, including ASD. With these answers, the DAWBA’s algorithm uses its calculated scores to generate a probability of diagnosis for each disorder. These computer-predicted probabilities are termed the “DAWBA bands”.\n\nDAWBA ‘skip rules’ were employed in order to gather more focused data in areas where the respondent gives a set number of positive responses. Respondents also provided full text answers to open-ended questions, providing further description to assist clinical raters in their interpretation.\n\nThe Strengths and Difficulties Questionnaire. An overall understanding of the social and emotional wellbeing of each subject was measured using the Strengths and Difficulties Questionnaire (SDQ) section of the DAWBA. The SDQ is an interview that measures social and emotional difficulties by assessing the severity and impact of problems in five domains listed in order of assessment: emotional problems, conduct issues, hyperactivity, problems with peers, and prosocial behaviour. There are 20 questions on difficulties, and 5 questions on prosocial behaviour. Total difficulty scores were calculated by adding up the scores on the 20 items relating to difficulties. Respondents were able to select from 3 possibilities ranging from: “certainly true”, “somewhat true” and “not true”.\n\nTotal scores were reported in comparison to population norms generated by a survey of mental health in 5–15-year-old children in the United Kingdom (Meltzer et al., 2003). These overall reports were classified as increasing in severity from “close to average”, “slightly raised”, “high” and “very high” for domains that looked at difficulties. Reverse order classification was used for prosocial problems with categories referred to as “close to average”, “slightly lowered”, “low” and “very low”.\n\nThe DAWBA predictability bands. The algorithm within the DAWBA analysis tool was used to provide probabilities of various conditions including ASD. The probability was presented as one of six levels (numbered 0–5). Level 0 corresponds to less than 0.1% probability of the subject having the disorder. This continues with level 1 representing approximately 0.5% probability, level 2 being 3% probability, level 3 being 15%, level 4 being 50%, and level 5, greater than 70%.\n\nThe DAWBA clinical-rater consensus diagnosis. Using both the DAWBA-generated predictions and free text information provided by responders as part of their decision-making process, two independent clinical raters assessed the records for either a positive or negative ASD diagnosis. Diagnosis was informed by criteria as set out by the Diagnostic and Statistical Manual of Mental Disorders (5th edition; (American Psychiatric Association, 2013)). One assessor was a child psychiatrist who had previous experience as a DAWBA clinical rater. The second assessor was a paediatric clinician. Both the raters only had access to de-identified DAWBA records and were blind to the outcome of the MDT assessment. The clinical raters then met to discuss and compare the results and reached a Consensus diagnosis if there were any disagreements. In 15 cases where there was uncertainty about the Consensus diagnosis, a third senior clinical rater reviewed the cases to make a final diagnosis. The final DAWBA clinical rater diagnoses will be referred to as the Consensus diagnosis throughout the rest of this document.\n\nAs part of the gold standard MDT assessment completed by the SCDC, the cohort was assessed using the Developmental, Dimensional and Diagnostic Interview (3di) and the Autism Diagnostic Observation Schedule (ADOS).\n\nThe Developmental, Dimensional and Diagnostic Interview (3di). The 3di was administered by trained psychologists working as part of the SCDC team. The 3di is a interview based on parent-report which measures symptoms of ASD and associated comorbidities (Skuse et al., 2004). The 3di asks questions about the child’s sociodemographic information, developmental history and motor coordination which is entered into a computer program. Once the semi-structured interview is completed, the computer algorithm is able to immediately generate a short report. In total, there are 183 questions on sociodemographic details, motor and development history, 266 questions associated with ASD and 291 questions based on other mental disorders. Scores are generated based on the responses: 0 is given for no record of any symptoms, 1 is for small amount of evidence of asked-about behaviour, and 2 is for known or ongoing presence of the behaviour. Once the interviews were completed, the final scores were divided into three components representative of ASD: Reciprocal social interaction, Communication, and Repetitive and stereotyped patterns of behaviour. These scores were then used as equivalent ADI-Algorithm scores as discussed in the next section.\n\nThe 3di scores and ADI-Algorithm. The Autism Diagnostic Interview, Revised (ADI-R) is an interview that assesses the three domains impaired by ASD (social reciprocity, communication and restricted behaviours) and makes a diagnosis based on these three domains. The 3di was also modelled on these three domains and therefore subscale scores from the 3di can be used to calculate scores which are equivalent to the ADI-R (Santosh et al., 2009). When these scores are derived from the 3di they are termed the ADI-Algorithm scores.\n\nThe 3di conducted at the SCDC produces scores that reflect possible impairment in one of the following domains; Social reciprocity, Communication and Restricted behaviours. As the 3di output score is consistent with an ADI-Algorithm score, all results from the 3di are henceforth referred to as the ADI-Algorithm. The ADI-Algorithm scores were analysed based on the three separate domains, and they were also grouped together to look at Social reciprocity and Communication and all three combined.\n\nThe Autism Diagnostic Observation Schedule (ADOS). The ADOS was administered by trained psychologists and allied health specialists working as part of the SCDC team. The ADOS is a semi-structured instrument that measures the observed behaviour of children for possible ASD, and two currently available and validated versions were used (ADOS and ADOS-2). The assessment uses four modules that look at the domains of play, imagination, social interaction and communication. There are four modules that can be used based on a child’s level and each takes approximately 30 minutes to complete (Lord et al., 2000). There is a total score at the completion of the modules and these are based on the individual scores of the social and communication domains, and the sum of the play and imagination domains (Oosterling et al., 2010). We identified a positive result on the ADOS if it diagnosed Autism. If the threshold showed a category of Autism Spectrum or no diagnostic features, we classified it as a negative result.\n\nAnalysis was completed using data stored on Microsoft Excel and analysed with IBM SPSS Statistics 25.0 (IBM Corp, 2017). We investigated differences between groups using chi-square tests or two-sample t-tests and set levels of significance at 0.05. Characteristics of the sample considered the background demographics of the participants by generating a table looking at the number of children who received a diagnosis of ASD, their gender, level of maternal and paternal education, the referral pathway and the child’s age in years.\n\nThe Cohen’s kappa statistic was used to calculate the inter-rater reliability of the independent DAWBA clinical raters. The percentage of agreement was also measured for comparison.\n\nThe mean SDQ scores of the ASD and non-ASD groups were compared. These scores were then compared to mean scores in the general population. The mean score results for the ADOS and ADI-Algorithm were also compared between ASD and non-ASD groups. The ADI-Algorithm mean scores were divided into: Social reciprocity; Communication; Combined Social reciprocity and Communication scores; Restricted behaviours; and, Combined Social reciprocity, Communication and Restricted behaviours scores. Comparison between all the scores listed in the SDQ, ADOS and ADI-Algorithm were checked for significance using 2-sample t-tests.\n\nThe sensitivity, specificity and positive and negative predictive values of the Consensus diagnosis compared to the MDT diagnosis were calculated. The diagnostic results from the ADOS and ADI-Algorithm were also compared to the MDT diagnoses. We further compared the result of the Consensus diagnosis directly with the diagnostic results of the ADOS and ADI-Algorithm.\n\nFinally, we were interested to examine the predictive validity of the DAWBA algorithm relative to the Consensus diagnoses. Therefore, we calculated the sensitivity, specificity, PPV and NPV of five predictability bands of the DAWBA for ASD diagnosis compared with the MDT results. We measured the accuracy of the DAWBA probability bands in predicting ASD against the MDT diagnosis using a receiver operating characteristic (ROC) curve.\n\n\nResults\n\nA total of 149 participants completed the DAWBA before attending the Social Communication Disorder Clinic at GOSH. Of those that completed the DAWBA and underwent a multidisciplinary team assessment, 13 were excluded due to an incomplete DAWBA. This is demonstrated in Figure 1.\n\nA final total of 136 participant records were analysed. Of the total participants, 64% (87) were diagnosed by the MDT as having ASD. The mean age of the cohort was similar in both subgroups; 10.5 years for those with ASD compared to 10.4 for the non-ASD group. There was a similar rate of males diagnosed with ASD (59%) compared to those who were not diagnosed (65%). Table 1 lists the sample characteristics of the cohort. There was no evidence of any difference between the groups.\n\nASD, Autism Spectrum Disorder.\n\nParents of non-ASD children had a trend towards slightly higher level of education compared to parents of children diagnosed with ASD. 85% of those without ASD had a mother who competed higher education compared to 75% those with ASD. Similarly, 82% of those assessed as non-ASD had fathers who completed A levels compared to 72% who were diagnosed with ASD. The referral pathway for both groups was also quite similar. 63% of children with ASD were referred to the SCDC by their paediatrician compared to 67% in the non-ASD group. The rest were referred by a Child and Adolescent Mental Health Services (CAMHS) clinician.\n\nThe SDQ mean scores demonstrate the difference between the populations. As seen in Table 2, the mean scores for each section between the ASD and non-ASD groups are similar. There was evidence for a difference in degree of conduct problems (CP) between those who were diagnosed with ASD and those who were not (Mean CP subscale score – ASD: 3.8, SD 2.0, 95% CI 3.3–4.2; Non-ASD: 5.0, SD 2.6, 95% CI 4.3–5.8; p = <0.01). There was no evidence of difference on any of the other SDQ subscales between the two groups.\n\nNorms provided by (Meltzer et al., 2000). ASD, Autism Spectrum Disorder.\n\nSDQ scores can be represented categorically based on previously published cut-offs (Goodman, 2018). Table 3 visually represents the categorical values of the mean scores in both groups compared to the population norm.\n\nBlue, close to average; yellow, slightly raised/slightly lowered; orange, high/low; red, very high/very low. ASD, Autism Spectrum Disorder.\n\nTable 4 demonstrates the mean score for the ADOS between the ASD and non-ASD groups. There was strong evidence for a difference in the total ADOS score between those who were diagnosed with ASD and those who were not (Mean ADOS score – ASD: 12.1, SD 6.2, 95% CI 10.8–13.5; Non-ASD: 6.0, SD 4.5, 95% CI 4.5–7.4; p= <0.01).\n\nTable 5 demonstrates the mean scores for the ADI-Algorithm between the ASD and non-ASD groups. There was strong evidence for a difference in the Restricted behaviours scale score between those who were diagnosed with ASD and those who were not (Mean Restricted behaviours score – ASD: 4.8, SD 2.4, 95% CI 4.3–5.3; Non-ASD: 3.5, SD 2.1, 95% CI 2.9–4.2 p= 0.003). There was no evidence of difference on the other domain scores between the two groups.\n\nOut of 136 clinical ratings of the DAWBA, 119 (87.5%) had initial agreement. A further two reached Consensus between the two clinical raters. The remaining 15 reached Consensus together with a third clinical rater.\n\nAs discussed above, there was an 87.5% rate of agreement between diagnoses. The inter-rater reliability for the DAWBAs assessed by clinical raters in the SCDC cohort was poor (Cohen’s kappa = 0.43). Before completing the SCDC clinical-rating of the DAWBAs, the authors independently reviewed 308 DAWBA records of a different clinical group with excellent results for inter-rater reliability for ASD (Cohen’s kappa = 0.83) (IMAGINE ID Study, 2018).\n\nWhen comparing the Consensus diagnoses with the MDT diagnoses, the DAWBA showed a sensitivity of 0.91 and a specificity of 0.12. The Positive Predictive Value (PPV) was 0.66 while the Negative Predictive Value (NPV) of the Consensus diagnoses was 0.43 (Table 6).\n\nASD, Autism Spectrum Disorder.\n\nTable 7 also shows the results for the face-to-face assessments. This result for the ADOS assessment showed a sensitivity of 0.67 and specificity of 0.78, while the PPV was 0.86 and the NPV 0.53. The ADI-Algorithm showed a sensitivity of 0.53 and specificity of 0.54 when compared with the MDT diagnoses. PPV was 0.69 and NPV 0.38. We also compared it with where just the Social reciprocity and Communication scales met ADI-Algorithm criteria for ASD, which showed a sensitivity of 0.34 and a specificity of 0.63. The PPV was 0.63 and NPV 0.34.\n\nWe then assessed the validity of the Consensus diagnosis when using the ADOS and ADI-R assessments as the reference standard for diagnosis. The results show a similar pattern to when Consensus is compared to MDT diagnosis with sensitivity and low specificity as per Table 8.\n\nTable 9 demonstrates the sensitivity and specificity of the DAWBA prediction bands for an ASD diagnosis relative to MDT diagnosis. There were five possible cut-off levels for a positive result. As expected, sensitivity increased as the cut-off was reduced but was never higher than 0.76. The specificity decreased from 0.94 at the highest cut-off to 0.29 at the lowest. Figure 2 demonstrates the distribution of ASD and non-ASD cases within each prediction band.\n\n* according to MDT diagnosis. ASD, Autism Spectrum Disorder.\n\nMDT, multi-disciplinary team; ASD, Autism Spectrum Disorder.\n\nAn ROC curve was generated to compare the DAWBA probability bands with the MDT ASD diagnosis. Figure 3 demonstrates that the area under the curve was not significant (AUC = 0.58, 95% CI = 0.5–0.7).\n\nArea Under the Curve = 0.58 (p = 0.13 95% CI = 0.5 – 0.7).\n\n\nDiscussion\n\nThis study found that the DAWBA Consensus diagnosis was accurate in 64% of diagnoses compared to the gold standard MDT. It had good sensitivity (91%) but poor specificity (12%). These findings were unexpected. Based on previous studies, we had anticipated that there would be high sensitivity and specificity in diagnosing ASD. We suspect that the difference between the previous studies and these results relates to the unique quality of our sample population. Due to the DAWBA’s previously documented ability to differentiate autism from non-autism in a community twin sample, we expected that it would perform similarly well in discriminating autism from non-autism in a more complex clinical group referred for assessment. Unfortunately, the complexity of our sample has resulted in the DAWBA performing with poorer specificity when using Consensus rating, and poor sensitivity when using computer prediction. There are several interesting findings from this study that may go towards explaining this phenomenon, to help understand the possible role for the DAWBA in future studies and/or clinical practice.\n\nOne of the main factors to consider when interpreting the results of this study is the unique nature of the children who were referred to our clinic. The mixed sensitivity and specificity results appear disappointing in this sample, but the nature of this population means that the results cannot be generalised. It is possible that there was ascertainment bias in the DAWBA interview as many of the families attending this service were seeking a second opinion (and in some cases, a third opinion). This extended contact with services may have resulted in increased mental health literacy, which may have subsequently affected their answers. This could be suspected due to the very high number of positive Consensus clinical rater diagnoses that are based on reading open-text responses and the high SDQ scores in the cohort. In addition, the ADOS (a purely observational tool) was more differentiating between ASD and non-ASD than the 3di and DAWBA which rely, in part, on parent-report.\n\nA study by Salisbury et al. has highlighted that parents may already be ‘sensitised’ to ASD behaviours in children referred to a subspecialty developmental clinic compared to children in a primary care clinic. This may result in increasing the sensitivity of the interview, but not reflect its true specificity (Salisbury et al., 2018). The study looked at how two screening tests performed in children between 16–48 months of age who were assessed in a subspecialty developmental clinic. The two screening tests were the Parent’s Observations of Social Interactions (POSI) and the Modified Checklist for Autism in Toddlers (M-CHAT). This clinic had a high prevalence of ASD (61%) and because of the referral process, it was suggested that parents may then be more aware of the ASD symptoms and their significance, compared to parents in the general population (Salisbury et al., 2018). The high prevalence of ASD in our population with some generally very complex cases and parents’ frequent encounter with clinicians are similar. It is possible that in a more general community sample referred for first assessment of suspected autism that the findings may differ.\n\nAnother feature of our sample was that many families were seeking a second opinion in looking for answers, which suggests a raised level of concern for autism. A study in Norway found that parents concerned about ASD are more likely to report behaviour that is consistent with ASD (Havdahl et al., 2017). The authors were concerned about the diagnostic validity of the ADI-R and ADOS in Europe. Despite multiple studies and a systematic review showing high agreement of combined ADOS and ADI-R for autism diagnosis (Kim & Lord 2012, and Falkmer et al., 2013, cited in Havdahl et al., 2017), they were concerned the validity for both assessments were based on samples from subspecialty developmental clinics in the USA. Some studies had shown the ADI-R did not perform as well as the ADOS in a European population (Zander et al., 2015, and de Bildt et al., 2015, cited in Havdahl et al., 2017), and the authors wondered whether factors such differences in culture and parental awareness and reporting behaviour may be impacting this (Havdahl et al., 2017). Therefore, they investigated a subsample of Norwegian children that were part of a larger birth cohort study who were referred to a developmental assessment clinic. They looked at how the ADI-R and ADOS performed, with a secondary measure to see whether parental concern and reporting-behaviour contributed to the assessment outcomes (Havdahl et al., 2017). This was done by dividing the sample into two groups: those children referred for possible delay, and those referred because parents were specifically concerned about possible ASD symptoms. The study demonstrated that there were higher scores on the ADI-R parent-report assessment when parents were worried about possible ASD, even independently of having a subsequent diagnosis of ASD. However, this did not affect the ADOS scores. While parents who were concerned about possible ASD were able to give examples of concerning features and symptoms (Havdahl et al., 2017), this raises the question about whether they answer parent-report questionnaires in a specific way. This could be either subconsciously or consciously, but may impact on the instrument’s lower validity. By the time of referral to the SCDC, the parents of our sample population have already had one MDT assessment after referral from concerned primary care health professionals who may have already raised ASD concerns. Given the above findings, it is possible that this may be implicitly affecting the way in which the DAWBA interview was completed by parents in this cohort.\n\nThe high level of parental education in our cohort may also impact the health literacy of the SCDC population. There was a very high level of high school completion in both maternal and paternal education (>70% of parents in the ASD group, >80% of parents in the non-ASD group). Studies have shown that higher socioeconomic status increases the likelihood of ASD diagnosis (King & Bearman, 2011) and that highly educated parents increases the likelihood of ASD being diagnosed earlier (Tek & Landa, 2012; Windham et al., 2011). This could be associated with better access to resources, but also more health literacy or increased recognition of symptoms. This tendency to recognise symptoms and access services may have influenced the way the DAWBAs were answered and interpreted in our cohort.\n\nThe majority of referrals in this cohort came from paediatricians (64% in those with ASD and 67% in those without). Again, this highlights that paediatricians found these particular cases to be complex. Specifically, these children were seen by experienced paediatricians and MDTs who were uncertain and seeking clarification of diagnosis.\n\nThe poor inter-rater reliability from this study compared to the previously excellent inter-rater reliability calculated, is quite a stark contrast. The two unique aspects of the DAWBA in this cohort are the clinical complexity of the sample as previously discussed, and the reduction in background information provided by this version of the DAWBA. The previously reported high inter-rater reliability was based on a research project called the IMAGINE ID (Intellectual Disability and Mental Health: Assessing the Genomic Impact on Neurodevelopment). It is a research study creating a database of a children with intellectual disability, and their genotypic and phenotypic information (IMAGINE ID Study, 2018). When rating the information provided in the DAWBA by the IMAGINE ID cohort, the background risk provided significant additional information to the clinical raters, which helped contextualise the DAWBA results. This information would also have been available to the clinicians completing the MDT assessment in the clinic but was not completed by our DAWBA participants. In their initial validation study, Goodman et al. highlighted that clinically rated assessments enhance validity. Open-text answers allow responders to clarify their answers, and any misunderstandings around the questions or any inappropriate assumptions on typical or normative behaviour can be recognised later through the lens of a clinical rater (Goodman et al., 2000). This is supported by Breslau who has argued that the whole picture of a child’s situation needs to be contextualised in order to better make a diagnosis (Breslau, 1987). Future studies evaluating the DAWBA against clinical diagnosis should ensure that the background information is available to provide context and ensure validity of rating. This is particularly important in complex clinical populations.\n\nThe results of the Strengths and Difficulties Questionnaire show high scores and overlapping symptoms of emotional and behavioural problems between the two groups of children. The total difficulties score for both the ASD and non-ASD groups were three times higher than for the general population in the same age bracket, and children in the SCDC cohort were also nearly half as likely to have social skills. In addition, there was a significant level of conduct scores between the two groups, with levels at least three times higher than in the general population. These levels of difference raise questions about the profile of the children we assessed. It is possible that the challenge of conduct problems may be the reason for referral to this specialised clinic. It is also possible that increased conduct problems could be associated with undiagnosed psychiatric disorder and difficult-to-manage behaviour. In addition, frustrations associated with communication problems and being misunderstood in the context of a challenging ASD diagnosis might also influence the type of population that completed the DAWBA. This is a patient group where other MDTs have had difficulty discriminating whether there was a positive ASD diagnosis, so in retrospect, expecting the DAWBA to successfully discriminate for ASD may have been an unreasonable expectation.\n\nThe results of this study in comparison to previous validation studies of the 3di and ADOS, highlight how the performance of both these assessments reflect the complexity of the SCDC population. The ADI-Algorithm and ADOS are well-validated and have been used consistently for their reliable performance. The validity study for the 3di found high sensitivity (100%) and high specificity (98%) (Skuse et al., 2004) while the shortened version showed a high sensitivity (90–96%) and high specificity (85–96%) (Santosh et al., 2009). Similarly, the ADOS has been demonstrated to have high validity with high sensitivity (94–100%) and moderate specificity (67–94%) (Lord et al., 1994b). Charman and Gotham have discussed this in their review that results of specific screening tests are very much dependent on the sample. Factors affecting the sample include autism prevalence, the distinct features of the population, such as their age and intellect, socioeconomic and family characteristics, such as parental education, and if the assessment occurs before or after a diagnostic encounter (Charman & Gotham, 2013). They also highlight that the characteristics of the sample and the reason for screening are the two issues that affect the performance of screening assessments and the scores used to identify positive cases (Charman & Gotham, 2013). This is discussed further in the limitations section below.\n\nThe results from this study confirm that in a complex clinical group, even previously well-validated diagnostic tools can be inaccurate unless used as part of a broader MDT assessment. This further reinforces the need for rigorous assessments despite the estimated costs, and pressures faced by service providers receiving increased referrals (Galliver et al., 2017).\n\nThe DAWBA prediction bands function as a guide for clinicians about the risk of conditions for each participant and are helpful if clinical raters have not assessed the DAWBA. In the study used to validate the DAWBA bands, researchers found that while there were similar numbers of suggested diagnoses between the DAWBA band algorithm group and of the clinical raters, there was disagreement between the raters and computer-generated diagnoses, which were not always closely associated (Goodman et al., 2010). The current study showed that no computer cut-off level had a good balance of sensitivity and specificity. The highest cut-off level had good specificity but poor sensitivity, but no level had particularly good sensitivity. The ROC curve has been used for diagnostic tests, with the area under the curve representing how accurate the test is in differentiating between those with and without the investigated disorder (Hajian-Tilaki, 2013). Figure 3 showed that the DAWBA probability bands were not accurate in predicting ASD when compared with the gold standard in this population. Therefore, the results represented in the ROC encapsulate the overall performance of the DAWBA that was confounded by the baseline high levels of ASD and atypical presentations of the sample population. It is possible however, that in other settings, using DAWBA prediction bands may have specific utility. For example, if wanting to screen for ASD, it is possible that the lowest cut-off may provide a sensitive enough screening tool in other samples. Similarly, if there is uncertainty about a diagnosis, meeting the highest cut-off level may be considered sufficiently specific to aid in confirming a diagnosis.\n\nIt has been estimated that developmental assessments should have a sensitivity and specificity between 70–80% in order to be a good screening tool (American Academy of Pediatrics (Committee on Children with Disabilities), 2001). Charman and Gotham highlight that a child who is not picked up on screening in the first assessment will be picked up on any further screening if it is done intermittently (Charman & Gotham, 2013). Evidence for this is highlighted by the previously mentioned study of UK children referred to a developmental clinic, with assessments showing high sensitivity and low specificity for POSI and M-CHAT (Salisbury et al., 2018). With a similar prevalence rate as our study sample (61%), the authors concluded that the low specificity was due to the clinic population, and that the tools need to be looked at in more primary care clinics (Salisbury et al., 2018). However, they also highlight that clinics need to prioritise whether to use high specificity or high sensitivity to capture those at risk, which is also influenced by the amount of follow-up available (Salisbury et al., 2018). Service providers may need to consider this if using the DAWBA as a screening instrument and it would depend on the amount of resources that service providers have to provide follow up assessments.\n\nIn the initial validation study for the DAWBA, Goodman et al. predicted that the clinical rater diagnoses would be the most useful way to implement the DAWBA as a screening test to help services plan (Goodman et al., 2000). Already the DAWBA has been used in a study to confirm clinical diagnoses of ASD (Bedford et al., 2017) and has been shown to have a high sensitivity and specificity of diagnosing ASD in a twin study population (McEwan & Brinkmann, 2000). Its high sensitivity in what has been proven to be a highly complex and difficult-to-diagnose clinical group, suggests that its performance in a general referral clinic could be feasible.\n\nCharman and Gotham completed a review of screening assessments for prospective ASD diagnosis and found that these assessments are least helpful in tricky populations, where health providers need most assistance because the screening tests give less accurate results in these cases (Charman & Gotham, 2013). They do recommend the use of screening instruments in clinics, but with specific attention to the sample characteristics. This supports the findings in this cohort that appears to contain many difficult cases, and therefore less likely to have accurate results in screening tests.\n\nWhile the DAWBA as a screening test in a complex clinical population may be not be helpful, it may have a role as a diagnostic adjunct. As in previous validity studies showing that the DAWBA increased the diagnosis of comorbid disorders (Goodman et al., 2000; Aebi et al., 2012), it is possible that the DAWBA assisted the SCDC MDT in diagnosing comorbidities. Or in differentiating ASD diagnosis from alternative diagnoses such ADHD and anxiety. This information was not available within the scope of the study and is unfortunately a limitation. However, the data collected as part of the DAWBA, could be useful for clinicians in making decisions about children referred for further assessment in future.\n\nLimitations of this study include a small sample size, retrospective nature of the study, lack of follow-up, sample participant characteristics, possible clinical rater bias and limited research in the area. A larger sample size in this study may reveal more subtle differences between the DAWBA results of the ASD and non-ASD groups, or more significant evidence of difference between the two groups. A larger sample size would also allow for subgroup analysis such as looking at whether levels of parental education or referral pathway contributed to any differences.\n\nThe unique characteristics of this sample mean that it is not a diverse population. There was a high prevalence of ASD in the sample. Also, a majority had high-functioning ASD and many were difficult to diagnose and requiring a second opinion. With a more diverse sample, the results could be more generalisable. The retrospective nature of the study is also another limitation as it may have introduced selection bias, and meant a lack of follow-up. Neurodevelopmental disorders evolve with time and diagnoses can become clearer as length of follow up time is increased. Follow-up of cases may have made diagnoses more accurate or certain and confirmed the presence or absence of comorbid disorders.\n\nLack of research in this area may also have impacted on the findings. The DAWBA has been validated across 17 psychopathologies but research has only been targeted at distinguishing between internalised and externalised disorders. There is a lack of focus on individual psychiatric diagnoses, with only one study to date considering ASD specifically (McEwen et al., 2016). Finally, there may also be an element of bias in clinical raters. Other clinical raters might have different experiences and individual clinical biases that might make it harder to generalise the results of this study.\n\n\nConclusions\n\nThe DAWBA Consensus rating had excellent sensitivity and poor specificity in diagnosing ASD in a clinically complex referral population. These results, in such a unique population, may not be generalised to typical referral clinics. Therefore, the DAWBA may still have a role as a potential screening tool and diagnostic adjunct. Further study is required in a more general ASD referral community clinic.\n\n\nData availability\n\nThis validation study was conducted using data from patients who presented to the Social Communication Disorder Clinic at Great Ormond Street Hospital. While parents of patients gave permission for their data to be used for research, they did not consent to share this information publicly. This is in line with the recommendations by the Institutional Review Board and principle 9 of the Declaration of Helsinki. Those seeking access to the data should apply jointly to Prof. David Skuse of the Autism Families Study (d.skuse@ucl.ac.uk) and the GOSH/ICH Human Research Ethics Committee (Research.Governance@gosh.nhs.uk). Following application, consideration by these bodies will be made for allowing access to a de-identified data file.",
"appendix": "References\n\nAebi M, Kuhn C, Winkler Metzke C, et al.: The use of the development and well-being assessment (DAWBA) in clinical practice: a randomized trial. Eur Child Adolesc Psychiatry. 2012; 21(10): 559–67. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAlyahri A, Goodman R: The validation of the Arabic SDQ and DAWBA. East Mediterr Health J. 2006; 12(Suppl 2): S138–S146. PubMed Abstract\n\nAmerican Academy of Pediatrics (Committee on Children with Disabilities): Developmental surveillance and screening of infants and young children. Pediatrics. 2001; 108(1): 192–196. PubMed Abstract | Publisher Full Text\n\nAmerican Psychiatric Association: Diagnostic and Statistical Manual of Mental Disorders - Fifth Edition. Washington DC, American Psychiatric Association. 2013. Reference Source\n\nBedford R, Gliga T, Shephard E, et al.: Neurocognitive and observational markers: prediction of autism spectrum disorder from infancy to mid-childhood. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nde Bildt A, Sytema S, Zander E, et al.: Autism Diagnostic Interview-Revised (ADI-R) algorithms for toddlers and young preschoolers: Application in a non-US sample of 1,104 children. J Autism Dev Disord. 2015; 45(7): 2076–2091. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDepartment for Education (UK): Special educational needs in England: January 2017. Additional Tables: SFR37/2017. 2017. Reference Source\n\nDreaper J: Autism diagnoses 'could be reduced under NHS plan'. 2017. Reference Source\n\nFalkmer T, Anderson K, Falkmer M, et al.: Diagnostic procedures in autism spectrum disorders: a systematic literature review. Eur Child Adolesc Psychiatry. 2013; 22(6): 329–340. PubMed Abstract | Publisher Full Text\n\nFleitlich-Bilyk B, Goodman R: Prevalence of child and adolescent psychiatric disorders in southeast Brazil. J Am Acad Child Adolesc Psychiatry. 2004; 43(6): 727–34. PubMed Abstract | Publisher Full Text\n\nFord T, Goodman A, Meltzer H: The British Child and Adolescent Mental Health Survey 1999: the prevalence of DSM IV disorders. J Am Acad Child Adolesc Psychiatry. 2003; 42(10): 1203–1211. PubMed Abstract | Publisher Full Text\n\nGalliver M, Gowling E, Farr W, et al.: Cost of assessing a child for possible autism spectrum disorder? An observational study of current practice in child development centres in the UK. BMJ Paediatrics Open. 2017; 1(1): e000052. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGilbert R, Logan S, Moyer V, et al.: Assessing diagnostic and screening tests: Part 1. Concepts. West J Med. 2001; 174(6): 405–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGoodman A, Heiervang E, Collishaw S, et al.: The ‘DAWBA bands’ as an ordered-categorical measure of child mental health: description and validation in British and Norwegian samples. Soc Psychiatry Psychiatric Epidemiology. 2010; 46(6): 521–532. 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PubMed Abstract | Publisher Full Text\n\nHeiervang E, Stormark KM, Lundervold AJ, et al.: Psychiatric disorders in Norwegian 8- to 10-year-olds: an epidemiological survey of prevalence, risk factors, and service use. J Am Acad Child Adolesc Psychiatry. 2007; 46(4): 438–447. PubMed Abstract | Publisher Full Text\n\nIBM Corp: IBM SPSS Statistics for Windows. 25.0 ed. Armonl, NY: IBM Corp. 2017. Reference Source\n\nImagine id study: Intellectual Disability and Mental Health: Assessing Genomic Impact on Neurodevelopment - Report on Progress: February 2017 to February 2018. London, United Kingdom: Institute of Child Health: University College London. 2018Reference Source\n\nKim SH, Lord C: Combining information from multiple sources fot he diagnosis of autism spectrum disorders for toddlers and preschoolers from 12 to 47 months of age. J Child Psychol Psychiatry. 2012; 53(2): 143–151. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKing M, Bearman P: Socioeconomic Status and the Increased Prevalence of Autism in California. Am Sociol Rev. 2011; 76(2): 320–346. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLord C, Risi S, Lambrecht L, et al.: The Autism Diagnostic Observation Schedule-Generic: A standard measure of social and communication deficitis associate with the spectrum of autism. Journal of Autism and Developmental Disorders. 2000; 30(3): 205–223. PubMed Abstract | Publisher Full Text\n\nLord C, Rutter M, Le Couteur A: Autism diagnostic interview-revised: A revised version of a diagnostic interview for caregivers of individuals with possible pervasive developmental disorders. J Autism Dev Disord. 1994a; 24(5): 659–685. PubMed Abstract | Publisher Full Text\n\nLord C, Rutter M, Le Couteur A: The Autism Diagnostic Interview-Revised: A Revised version of a diagnostic interview for caregivers of individuals with possible pervasive developmental disorders. J Autism Dev Disord. 1994b; 24(5): 659–685. PubMed Abstract | Publisher Full Text\n\nMannion A, Leader G: Comorbidity in autism spectrum disorder: A literature review. Research in Autism Spectrum Disorders. 2013; 7(12): 1595–1616. Publisher Full Text\n\nMcewan IJ, Brinkmann AO: Androgen Physiology: Receptor and Metabolic Disorders. De Groot LJ, Chrousos G, Dungan K, Grossman A, Hershman JM, Koch C, Korbonits M, Mclachlan R, New M, Purnell J, Rebar R, Singer F, Vinik A. (eds.) Endotext. South Dartmouth MA: MDText.com, Inc. 2000. PubMed Abstract\n\nMcewen FS, Stewart CS, Colvert E, et al.: Diagnosing autism spectrum disorder in community settings using the Development and Well-Being Assessment: validation in a UK population-based twin sample. J Child Psychol Psychiatry. 2016; 57(2): 161–170. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMeltzer H, Gatward R, Goodman R, et al.: Mental health of children and adolescents in Great Britain. London: The Stationery Office: Social Survey Division of the Office for National Statistics on behalf of the Department of Health, the Scottish Health Executive and the National Assembly for Wales. 2000.Publisher Full Text\n\nMeltzer H, Gatward R, Goodman R, et al.: Mental health of children and adolescents in Great Britain. Int Rev Psychiatry. 2003; 15(1–2): 185–187. PubMed Abstract | Publisher Full Text\n\nMullick M, Goodman R: The prevalence of psychiatric disorders among 5-10 year olds in rural, urban and slum areas in Bangladesh: an exploratory study. Soc Psychiatry Psychiatr Epidemiol. 2005; 40(8): 663–71. PubMed Abstract | Publisher Full Text\n\nNICE: Autism Diagnosis in children and young people: Recognition, referral and diagnosis of children and young people on the autism spectrum.2011. Clinical Guidance 128. London: National Institute for Health and Care Excellence. PubMed Abstract\n\nOosterling I, Roos S, de Bildt A, et al.: Improved Diagnostic Validity of the ADOS Revised Algorithms: A Replication Study in an Independent Sample. J Autism Dev Disord. 2010; 40(6): 689–703. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSalisbury L, Nyce J, Charles D, et al.: Sensitivity and Specificity of 2 Autism Screeners Among Referred Children Between 16 and 48 Months of Age. J Dev Behav Pediatr. 2018; 39(3): 254–258. PubMed Abstract | Publisher Full Text\n\nSantosh P, Mandy W, Puura K, et al.: The construction and validation of a short form of the developmental, diagnostic and dimensional interview. European Child and Adolescent Psychiatry. 2009; 18(8): 521–524. PubMed Abstract | Publisher Full Text\n\nSimonoff E, Pickles A, Charman T, et al.: Psychiatric disorders in children with autism spectrum disorders: prevalence, comorbidity, and associated factors in a population-derived sample. J Am Acad Child Adolesc Psychiatry. 2008; 47(8): 921–929. PubMed Abstract | Publisher Full Text\n\nSkuse D, Warrington R, Bishop D, et al.: The developmental, dimensional and diagnostic interview (3di): A novel computerized assessment for autism spectrum disorders. J Am Acad Child Adolesc Psychiatry. 2004; 43(5): 548–558. PubMed Abstract | Publisher Full Text\n\nSoke G, Maenner M, Christensen D, et al.: Prevalence of Co-occurring Medical and Behavioral Conditions/Symptoms Among 4- and 8-Year-)ld Children with Autism Spectrum Disorder in Selected Areas of the United States in 2010. J Autism Dev Disord. 2018; 48(8): 2663–2676. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTek S, Landa R: Differences in Autism Symptoms between Minority and Non-Minority Todders. J Autism Dev Disord. 2012; 42(9): 1967–1973. PubMed Abstract | Publisher Full Text | Free Full Text\n\nThe UK National Screening Committee: Two: Definitions and Classifications of Population Screening Programmes. First Report of the National Screening Committee. London: Department of Health. 1998. Reference Source\n\nWilson J, Jungner G: Principles and practice of screening for disease. 1968. Reference Source\n\nWindham G, Anderson M, Croen L, et al.: Birth Prevalence of Autism Spectrum Disorders in the San Francisco Bay Area by Demographic and Ascertainment Source Characteristics. J Autism Dev Disord. 2011; 41(10): 1362–72. PubMed Abstract | Publisher Full Text\n\nZander E, Sturm H, Bolte S: The added value of the combined use of the Autism Diagnostic Interview-Revised and the Autism Diagnostic Observation Schedule: Diagnostic validity in a clinical Swedish sample of toddlers and young preschoolers. Autism. 2015; 19(2): 187–199. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "65067",
"date": "08 Jul 2020",
"name": "David Coghill",
"expertise": [
"Reviewer Expertise Child and adolescent mental health and neurodevelopmental disorders"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for the opportunity to review this interesting manuscript. The authors describe an evaluation of the predictive validity of the Developmental and Well-being Assessment (DAWBA) for ASD in a complex clinical sample. They conclude that, compared to the gold standard multi-disciplinary team assessment, the DAWBA had high sensitivity but low specificity.\nStrengths of the study include a very clear and transparent methodology and a very clear diagnostic process. I do however have several concerns with the way that the data has been analysed and reported. Looking at the results, particularly those shown in table 7, it is clear that whilst the authors comments on the sensitivity and specificity of the DAWBA assessment are clearly supported by the date, there are other interesting findings that have not been discussed. For example when compared to MDT assessment although the DAWBA has the poorest specificty, which is very important, it had a better classification accuracy that either the ADI-Algorithm for either social reciprocity and communication or the ADI when repetetive behaviour was added to this. Does this mean that if the DAWBA was substituted for the ADI (actually the 3 di - see below) in the MDT assessment there would be no loss of accuracy and perhaps an improvement for this type of complex case?\nIn this respect Table 5 is interesting as it suggests that the ADI algorithm scores did not differentiate between ASD and no ASD in this sample. I guess that this may be due to the complex nature and previous diagnostic uncertainty around these cases but this is not a line of reasoning that has been explored here.\nI therefore think that the authors could have addressed this data in a broader way rather than just focusing on the DAWBA aspects. It would have been interesting to see the 2x2 table for the ADI findings and the ADOS also so that we could have explored this further.\nThe negative comments about success of the DAWBA to discriminate ASD made in the discussion could equally be applied to the 3DI/ADI data. I know this is mentioned in the discussion but I think there could be more balance to this.\nI also found table 8 confusing. At first look it seems similar to table 7 but in fact it is 'the other way round' I think showing that the consensus diagnosis behaved similarly when compared to the separate ASD specific assessments as it did compared to the MDT diagnosis.\nWhilst on tables I think that tables 2 and 3 could probably be combined as could tables 4 and 5.\nI also found the data in the first row of table 9 confusing as it looks like there were 11 true positives and 3 false positives (but perhaps an reading incorrectly in which case the table is confusing). If this is so I cant quite see how only 42% are correctly classified and how sensitivity is so low.\nWhilst there is a lot of data presented I missed not seeing the confidence intervals around the Sn, Sp, PPV, NPV.\nI was not clear why the 3 DI results were translated into ADI and not reported for what they are. I understand that the process has been validated but any transformation like this will increase error and reduce accuracy. This may be part of the explanation why the ADI did so poorly as discussed above\nSimilarly the potential parental biases discussed for the DAWBA in the discussion could equally apply to the ADI.\nThe authors spend quite some time in the introduction pointing out that a potential benefit of the DAWBA is that it assesses a broad range of mental health disorders and symptoms. Indeed this is a criticism of many clinical assessments for ASD that start and finish with the question of whether someone does or does not have ASD and fails to make the comprehensive assessment that identifies either comorbid problems or alternative diagnoses. I was looking forward to reading about how the DAWBA performed in this respect and whether it allowed a more complete assessment. But there is not mention of this past the introduction which was disappointing for me at least. Was this data gathered and if so could it be presented?\nMinor issues\nThere are quite a lot of grammatical and stylistic issues that need to be addressed.\nIt is true that the sample are different to a regular population and many clinical samples but they are certainly not (at least as a group) unique. If they were there would be no reason to publish as the data and findings would not inform any other service providers. As it is there are many other specialist clinics who see similarly complex cases who will benefit from these findings\nwe are told in the discussion that a high proportion of the consensus diagnoses were based on free text responses. this has not been mentioned before. can it be quantified?\nIn the introduction is the prevalence of 1.3% for ASD in the UK an epidemiological prevalence or the current rate of diagnosis (administrative prevalence)?\nFor me the discussion is too long and describes other studies in too much detail for a paper. I think it could be edited down considerably to make sure it addresses the main issues in a more focused clear and concise manner.\n\nIs the work 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? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
},
{
"id": "71140",
"date": "21 Sep 2020",
"name": "Alexander C Wilson",
"expertise": [
"Reviewer Expertise Neurodevelopmental disabilities"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI enjoyed reading this article. The authors present psychometric analysis of the Developmental and Wellbeing Assessment (DAWBA; Goodman et al., 20001) in identifying autism in young people aged 4 to 18 referred to a specialist clinic for multidisciplinary assessment. Results revealed that the DAWBA autism module had high sensitivity but poor specificity for autism, as well as weak inter-rater reliability. The authors highlight the complexity of the sample as an important factor in accounting for the poor specificity of the instrument.\nStrengths of the paper include the rigorous assessment for autism and the detailed reporting. I do have a few questions and comments for the authors that I hope may be useful, and list those below.\nFirst, could you confirm whether DAWBA and MDT assessments were entirely distinct? This is important in interpreting the psychometric results. I appreciate that DAWBA raters were blinded to the MDT outcomes, but it is not clear whether DAWBA responses were reviewed as part of the MDT assessment. At one point, the authors state “All cases were reviewed and data collected on the results of ADOS scores (overall and modules), 3di subscale scores, Strengths and Difficulties Questionnaire (SDQ) scores, DAWBA computer predicted probabilities, DAWBA consensus ratings, and final MDT diagnosis” which I found somewhat confusing.\nSecond, I wonder if comparing the ability of the DAWBA to discriminate between MDT cases and non-cases to that of the ADOS and 3di (Table 7) is potentially misleading, given that the ADOS and 3Di both informed the MDT decision. It wouldn’t really be surprising that these other instruments would show better specificity, as they provided information to clinicians in making differential diagnoses. It is interesting that the ADOS tended to perform better than the 3Di, which suggests to me that the MDT decision was most strongly influenced by the clinical observation.\nThird, it would be really helpful in contextualising the poor specificity of the DAWBA to know more about the MDT non-cases. The nature of the sample was complexity and ambiguity in presentation, so I am left wondering whether these young people were ultimately diagnosed with other neurodevelopmental conditions that might closely mimic autism?\nFourth, could we see more about outcomes on other DAWBA modules? The introduction raises issues of differential diagnosis and co-occurring conditions, and the broad assessment that the DAWBA allows would seem to give excellent opportunity for addressing some of these questions. Related to my third point above: if some of the young people not diagnosed with autism received other diagnoses, were these predicted by outcomes on, say, the anxiety, ADHD and externalising modules? And were the autistic young people receiving multiple diagnoses?\nFifth, I feel there are some labelling decisions that may lead to confusion. Were all the 3Di scores used in analysis outputs of the 3Di-sv (Santosh et al, 20092)? If so, would it not make sense to refer to them as such? At present, I find it confusing that these are referred to as ADI-R scores, when the ADI-R was not actually administered. And could you confirm whether the full 3Di interview or the shorter version was administered? Relatedly, where you refer to “consensus diagnosis”, I wonder if this is potentially confusable with “MDT diagnosis”. I think I would prefer “DAWBA clinician diagnosis” or “DAWBA consensus diagnosis” for clarity.\nMore broadly, I think there were occasional lapses in clarity or sections that were overly wordy. While I really appreciate the detail in the article, I also feel it is verbose at times, and might benefit from some editing down. One specific point on which I became unclear, as I read the discussion, was exactly how much of the DAWBA assessment had been completed by participants when you mentioned that there was a “reduction in background information provided by this version of the DAWBA”.\nThese are the main points that come up for me in reading the article. Here are a couple of additional comments on individual parts:\n“The sample in this study included all individuals aged 4–18 years who were referred to the Social Communication Disorder Clinic (SCDC) at Great Ormond Street Hospital, and completed a DAWBA before a gold standard MDT ASD diagnostic assessment (n=136) between June 2014 and August 2017.” Does this mean that DAWBA data existed for all consecutive referrals between those dates? If not, could we see the proportion of families who completed this assessment?\n“The DAWBA was completed by parents online or by trained research students who conducted the interview via phone and completed the online form.” It would be useful to know a breakdown of cases with parent-completed forms vs interview-based assessments, as this might have affected the level of information on which clinical raters were relying for making diagnoses. I note the comment that free responses were particularly useful, and these might have differed based on whether online forms/interviews were completed.\n“If the threshold showed a category of Autism Spectrum or no diagnostic features, we classified it as a negative result.” Why were ADOS Autism Spectrum outcomes classified as non-autism?\n“As discussed above, there was an 87.5% rate of agreement between diagnoses. The inter-rater reliability for the DAWBAs assessed by clinical raters in the SCDC cohort was poor (Cohen’s kappa = 0.43).” I think this is potentially ambiguous, as 87.5% agreement sounds respectable, but then we move on to learn that this equates to a rather low Cohen’s kappa. Maybe rephrase along the lines of as “there was an 87.5% rate of agreement between diagnoses, which translates to poor inter-rater reliability (Cohen’s kappa = 0.43)”.\nThank you for the opportunity to read your work.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/9-622
|
https://f1000research.com/articles/9-615/v1
|
16 Jun 20
|
{
"type": "Software Tool Article",
"title": "Chanjo: Clincal grade sequence coverage analysis",
"authors": [
"Robin Andeer",
"Måns Magnusson",
"Anna Wedell",
"Henrik Stranneheim",
"Robin Andeer",
"Anna Wedell",
"Henrik Stranneheim"
],
"abstract": "Coverage analysis is essential when analysing massive parallel sequencing (MPS) data. The analysis indicates existence of false negatives or positives in a region of interest or poorly covered genomic regions. There are several tools that have excellent performance when doing coverage analysis on a few samples with predefined regions. However, there is no current tool for collecting samples over a longer period of time for aggregated coverage analysis of multiple samples or sequencing methods. Furthermore, current coverage analysis tools do not generate customized coverage reports or enable exploratory coverage analysis without extensive bioinformatic skill and access to the original alignment files. We present Chanjo, a user friendly coverage analysis tool for persistent storage of coverage data, that, accompanied with Chanjo Report, produces coverage reports that summarize coverage data for predefined regions in an elegant manner. Chanjo Report can produce both structured coverage reports and dynamic reports tailored to a subset of genomic regions, coverage cut-offs or samples. Chanjo stores data in an SQL database where thousands of samples can be added over time, which allows for aggregate queries to discover problematic regions. Chanjo is well tested, supports whole exome and genome sequencing, and follows common UNIX standards, allowing for easy integration into existing pipelines. Chanjo is easy to install and operate, and provides a solution for persistent coverage analysis and clinical-grade reporting. It makes it easy to set up a local database and automate the addition of multiple samples and report generation. To our knowledge there is no other tool with matching capabilities. Chanjo handles the common file formats in genetics, such as BED and BAM, and makes it easy to produce PDF coverage reports that are highly valuable for individuals with limited bioinformatic expertise. We believe Chanjo to be a vital tool for clinicians and researchers performing MPS analysis.",
"keywords": [
"Genomics",
"QC",
"MPS",
"Coverage analysis",
"Clinical analysis"
],
"content": "Introduction\n\nCompared to extensive serial Sanger sequencing, exome sequencing can be done at a small fraction of the cost per sample (the same order of magnitude as one average-sized gene) and whole exome sequencing (WES) has been more or less established in the clinic for a few years1. Sequencing technologies are continuing to improve at a rapid pace. The Illumina HiSeq X system, and the more recent Illumina Novaseq 6000, reduce the cost of whole genome sequencing (WGS) to slightly more than $1,000 for sequencing a human genome to 30x coverage2. For the first time it is now possible to analyze complete human genomes within reasonable time and cost. This will further increase the pace of implementation of massively parallel sequencing (MPS) in new areas, such as diagnostics of inherited genetic disease. When analyzing the enormous data volume from WES and WGS, it is important to identify underrepresented genomic regions by calculating and tracking coverage quality control metrics. This is particularly true if the sequence data is used in diagnostics, since low or not exposed regions can lead to false positive or false negative results3,4. There are a number of tools that provide basic coverage and overlap annotation functionalities: PicardTools5, BEDTools6, Sambamba7 and GATK8. These tools are excellent for comparing the overlap of two feature sets using single operations. However, they do not offer a solution for coverage analysis with different thresholds and different genomic and biological features. Moreover there are many laboratories around the world that work in a production setting where new samples are sequenced and analyzed every week. There is no present solution for persistent storage of coverage data that makes comparisons of hundreds or thousands of samples possible. This is essential to locate genomic regions that are hard to sequence and where the local sequencing pipeline gives insufficient information.\n\nFurthermore, to our knowledge there are no tools that support dynamic report generation. To address these needs, we have developed Chanjo, a fast and flexible toolkit for seamless coverage analysis of genomic and biological features across multiple samples. Chanjo has been incorporated into the clinical analysis pipeline at Clinical Genomics Science for Life Laboratory and analysed more than 4500 rare disease WES and WGS samples to date. We believe Chanjo to be a vital tool for clinicians and researchers performing MPS analysis.\n\n\nMethods\n\nChanjo is written in Python (3.2+). It follows UNIX conventions and is built around text streams that can be incorporated into pipelines (Figure 1). Chanjo is distributed via GitHub, installation is simple and robust thanks to extensive tests. Chanjo loads output files from Sambamba depth7 and stores coverage related statistics, e.g. average coverage and completeness in a SQL database. Completeness is defined as the percentage of bases meeting a user-defined coverage threshold for each genomic interval (Figure 2). Chanjo does not aim to analyze the whole genome, but will limit the analyses to predefined genomic regions of interest defined in BED format.\n\nChanjo supports any genomic intervals as long as they adhere to the BED format with two optional columns for linking exons to transcripts and genes. Hence, it is easy to set-up independent databases using different gene and transcripts definitions, e.g. ccds, refseq or ensembl. When the genomic intervals are defined and added to the database, it is simple to add additional samples.\n\nChanjo uses a predefined database schema where exons are organized into transcripts and genes (Figure 3). Extracting basic coverage metrics such as “average coverage”, “overall completeness”, etc. for different transcripts and genes is easily done through the Python application programing interface (API). Setting up a database with the “init” subcommand only needs to be done once. After the basic structure of the database is in place, the user can add an arbitrary number of samples to the database and include or exclude samples, as preferred from the downstream analysis. The SQL schema has been designed to be a powerful tool on its own for studying coverage. It allows for quick aggregate metrics across multiple samples and can be used as a general coverage API for accompanying tools. One example of such a tool is Chanjo-Report, a clinical-grade coverage report generator for Chanjo output developed to be used in a clinical setting. The report can be tailored to include any number of samples, genes or transcripts (Figure 4). The report can be exported as a PDF and is assembled via a web interface using Chanjo-Report together with a Chanjo database. This works as a powerful bridge between bioinformaticians and professionals that work with analysis who often lack programming skills.\n\nChanjo requires a installation of Python version 3.2 or above. In a production setting, a mySQL database is preferred, however it is possible to use sqlite, which comes with the python installation. Chanjo has been installed and tested on Linux and Mac OS environments while there should not be any problems to install on Windows. Performance wise, a standard computer will be sufficient to run Chanjo.\n\n\nUse cases\n\nIt is straight forward to setup a working demo of Chanjo to test how to use the tool:\n\n\n\nThe first command initializes a SQL database with tables, it will also use a reduced bed file to link exons, transcripts, and genes according to the definitions in “hgnc.min.bed”. Chanjo uses output generated by “sambamba depth” which includes average coverage and completeness data for each exon defined in “hgnc.min.bed”. The for loop loads data for 3 samples into the database for persistent storage. Finally, the CLI can be used to execute simple queries and output the results in JSON format.\n\n\n\nChanjo is intended to be used with a central database where samples are continuously added over time. This facilitates aggregate statistics, e.g. trending coverage metrics, poorly covered regions of the genome and comparing gene panel coverage across samples.\n\nIn many settings the workflow for a sample starts in the lab where DNA is prepared and sequenced. After that bioinformaticians prepare the output for analysis and hand it over to a researcher or clinician. Chanjo-Report is developed as a tool to present data generated by the bioinformaticians to the end-user. Here one can specify a subset of regions, in many cases a gene panel that is specific for a disease group, and get a well structured report of the fraction of transcripts/genes that are fully covered and which are not. This has proven essential when performing clinical tests with MPS data.\n\n\nSummary\n\nWe have developed a novel tool, Chanjo, for continuous and accurate coverage analysis for multiple samples, ideal for WES as well as WGS. We believe Chanjo will be useful for sequencing facilities in general and clinical facilities in particular, where stringent quality control is required. The user only needs to initialize a database once by using a definition of regions and links, e.g. transcripts and genes, in the BED format. Samples are then added by loading data from sambamba depth into the database. Chanjo has been implemented to be easily included in an existing workflow of sequencing analysis. Furthermore, we introduce Chanjo Report to present coverage reports that are easy to generate and to interpret for non-bioinformaticians. This enables, e.g. estimation of accuracy of negative analyses, indicating regions that may require resequencing or investigation using alternative technology.\n\nTo our knowledge there are no software freely available today capable of continuous coverage analysis across multiple samples with dynamic report generation like Chanjo.\n\n\nSoftware availability\n\n1. Source code available from: https://github.com/Clinical-Genomics/chanjo\n\n2. Archived source code as at time of publication: http://doi.org/10.5281/zenodo.326649\n\n3. Software license: MIT",
"appendix": "Acknowledgements\n\nThanks to Valtteri Wirta at Clinical Genomics for allowing this project to develop.\n\n\nReferences\n\nYang Y, Muzny DM, Reid JG, et al.: Clinical Whole-Exome Sequencing for the Diagnosis of Mendelian Disorders. N Engl J Med. 2013; 369(16): 1502–11. PubMed Abstract | Publisher Full Text | Free Full Text\n\nErika Check Hayden: Technology: The $1,000 genome. Nature News. 2014; 507(7492): 294. Publisher Full Text\n\nBrownstein CA, Beggs AH, Homer N, et al.: An international effort towards developing standards for best practices in analysis, interpretation and reporting of clinical genome sequencing results in the CLARITY Challenge. Genome Biol. 2014; 15(3): R53. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVrijenhoek T, Kraaijeveld K, Elferink M, et al.: Next-generation sequencing-based genome diagnostics across clinical genetics centers: Implementation choices and their effects. Eur J Hum Genet. 2015; 23(9): 1142–1150. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBroad Institute: Picard tools. Reference Source\n\nQuinlan AR, Hall IM: BEDTools: A flexible suite of utilities for comparing genomic features. Bioinformatics. 2010; 26(6): 841–842. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTarasov A, Vilella AJ, Cuppen E, et al.: Sambamba: Fast processing of NGS alignment formats. Bioinformatics. 2015; 31(12): 2032–2034. 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): 491–498. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAndeer R, Magnusson M, Kern J, et al.: chanjo: Chanjo 3.0.0. (Version v3.0.0) Zenodo. 2015. http://www.doi.org/10.5281/zenodo.32664"
}
|
[
{
"id": "92794",
"date": "20 Sep 2021",
"name": "Ryan M. Layer",
"expertise": [
"Reviewer Expertise bioinformatics",
"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\nChanjo is a tool that allows for the tracking and review of sequencing coverage statistics over time and across many samples and groups. While numerous tools exist to measure such statistics in a single batch, there are not any that enable tracking these over time, which can be very important for a diagnostics laboratory.\nCommentary on the manuscript:\nThe manuscript is by and large well written and clear.\n\nAll figures are missing descriptions, these need to be added.\nFigure 1:\nWhat do the shapes and colors mean?\n\nWhat does load do?\n\nWhat are you calculating?\n\nFigure 2:\nIs this something that appears on one of the reports or just a figure for describing what completeness is?\n\nWhat do the different parts of the figures represent?\n\nWhat is the actual % completeness of this example?\n\nFigure 3:\nThis is the one figure that might be fine without a description, but it might be worth explaining for those not familiar with relational databases that there are 3 tables used in Chanjo (table1, table2, and table3) and that name1 references information found in table2 and table3.\n\nFigure 4:\nHow was this generated? Is it a stock report or a customized one? What is this screenshot of a report supposed to be showing me?\n\nIn the manuscript it is described how Chanjo has been extensively tested, on the GitHubs for Chanjo and Chanjo Report, they have some automated checks, but the most recent commit in both of these reports is causing the automated test to fail. It would be worth fixing whatever is causing these tools to fail their own test cases.\nWhen you say “dynamic report generation” what exactly does this mean? Consider rewording part of this sentence: “This is particularly true if the sequence data is used in diagnostics, since low or not exposed regions can lead to false-positive or false-negative results.”\nCommentary on the documentation and tool:\nThe tool is pip-installable, thank you, that is phenomenally user-friendly.\n\nThe documentation of how to use the tool is in need of serious improvement. What is on the Github README vs what is on the https://clinical-genomics.github.io/chanjo/ do not match.\n\nThe documenting of this tool does not appear to be complete, or mostly complete. Documentation needs to be consistent and complete prior to sharing Chanjo publicly.\n\nThere is a nice demo of how to use Chanjo but it doesn’t fully explain what is being done or why.\n\nWhat does the link command do? Why do we need that?\n\nThe file “chanjo.yaml” is required to use the tool, but is not a required parameter. It is mentioned that Chanjo will just look for that file in the current working directory if not otherwise specified, but Chanjo is installed as a globally accessible tool, to be used anywhere so this feature doesn't make great sense.\n\n“chanjo load”, in the demo it is shown how to use the “--group” option, but in the introduction of the documentation, you show that this is not a required option in order to load a sample into Chanjo. But in order to use chanjo report a group is required. There seems to be a disconnect here in what is required for the functionality of the tools.\n\nReading through the Github issue for Chanjo I learned there is a “chanjo remove” command. Which for me as well as the issue creator could have been very useful during the setup stages after accidentally adding numerous samples without groups. The remove command no longer appears to be a command in chanjo, but could be very useful. Samples cannot be re-loaded twice and cannot be removed so it seems the only option here is to delete the database, which seems like an extreme example.\n\nIn the manuscript, it is described how Chanjo can be used with an SQL database and server or with SQLite. The demo shows how to use the SQLite version but I cannot find a description of how to use a full SQL database.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Partly\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? No\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Partly\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes",
"responses": []
},
{
"id": "127509",
"date": "24 Mar 2022",
"name": "Ksenia Lavrichenko",
"expertise": [
"Reviewer Expertise Bioinformatics",
"genomics",
"structural variants",
"short and long read sequencing"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAndeer and colleagues present a novel software tool Chanjo that aims at DNA variant quality control via coverage analyse in the context of high-throughput sequencing in diagnostics. Streamlining of the software promises easy incorporation into genomic diagnostic pipelines and accessibility for non-bioinformatic personnel.\n\nThe paper is well-articulated and easy to follow overall. The software is very well documented and distributed via package repositories which makes it easy to adapt. I have a few minor comments, aimed at making the advantages of using the tool more transparent:\n\nThe main one is that I am missing an actual use case and demonstration on why the tool is \"highly valuable\", even \"vital\", in the clinic. The current 'Use case' section belongs to the technical tutorial while in the paper I expect to see an actual example - perhaps one or a couple of genes for which among the 4500 cases (or respective reference samples) analysed the tool helped indicate a false positive or a false negative, or preferably both scenarios.\n\nIn the aforementioned use case, it could be relevant (and highly recommended) to compare Chanjo performance with other mentioned \"state of art\" tools in the chosen loci.\n\nAll the figures should be accessible outside of the context of the main text of the paper, therefore they need a caption with a respective description (and not just a figure title).\n\nPlease, do add the annotation of what the colors mean (as graphical legend or in the caption of the figure) or drop the colors if they do not convey a specific meaning, to reduce information noise.\n\nIf you do not mention the elements of a figure in the paper text, please remove them from the figure (see Figure 2, exon-intron)\n\nSentence 6 in the Introduction \"This is particularly true if the sequence data is used in diagnostics, since low or not exposed regions can lead to false positive or false negative results\": you mean \"low-covered\" regions? and \"not exposed\" as in \"heterochromatin\"?\n\nCould you explicitly mention some assumptions made or existing potential biases, and future improvements?\n\nCan this tool also be useful for newer technologies, e.g. long-read sequencing?\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/9-615
|
https://f1000research.com/articles/9-47/v1
|
27 Jan 20
|
{
"type": "Software Tool Article",
"title": "scRepertoire: An R-based toolkit for single-cell immune receptor analysis",
"authors": [
"Nicholas Borcherding",
"Nicholas L. Bormann",
"Nicholas L. Bormann"
],
"abstract": "Single-cell sequencing is an emerging technology in the field of immunology and oncology that allows researchers to couple RNA quantification and other modalities, like immune cell receptor profiling at the level of an individual cell. A number of workflows and software packages have been created to process and analyze single-cell transcriptomic data. These packages allow users to take the vast dimensionality of the data generated in single-cell-based experiments and distill the data into novel insights. Unlike the transcriptomic field, there is a lack of options for software that allow for single-cell immune receptor profiling. Enabling users to easily combine mRNA and immune profiling, scRepertoire was built to process data derived from 10x Genomics Chromium Immune Profiling for both T-cell receptor (TCR) and immunoglobulin (Ig) enrichment workflows and subsequently interacts with the popular Seurat R package. The scRepertoire R package and processed data are open source and available on GitHub and provides in-depth tutorials on the capability of the package.",
"keywords": [
"Single-cell RNA sequencing",
"immune receptor profiling",
"R",
"clonotypic analysis"
],
"content": "Introduction\n\nThe molecular resolution offered by single-cell sequencing (SCS) technologies has led to extensive investigations in the realms of developmental biology, oncology, and immunology. In terms of the latter field, SCS offers the ability to couple the exploration of transcriptomic heterogeneity in immune cells along a disease process with clonality1. A number of methods exist for dimensional reduction of mRNA data, reviewed by Chen et al.2 that have been implemented into R packages to assist in processing and analysis of SCS experiments. However, a gap exists in the processing of V(D)J sequencing, descriptive statistics, clonal comparisons, and repertoire diversity with the current SCS R packages.\n\nWith these limitations in mind, scRepertoire3 was generated (Figure 1). Built using R, scRepertoire is a toolkit to assist in the analysis of immune profiles for both B and T cells, while interacting with the popular Seurat pipeline4–6. scRepertoire also includes processed single-cell mRNA and V(D)J sequencing data of 12,911 tumor-infiltrating and peripheral-blood T cells derived from three renal clear cell carcinoma patient, which is characterized below to demonstrate the capabilities of the package.\n\nThe analysis starts with the single-cell immune and mRNA sequencing and Cell Ranger-based alignment with the 10x Genomics pipeline. With the TCR or Ig sequencing, scRepertoire can import the filtered overlapping DNA segments, or contigs. The alignments are filtered by cell type of interest and combined using the individual cell barcodes. Clonotypes can be called using the gene sequence of the immune receptor loci, CDR3 nucleotide sequence or CDR3 amino acid sequence. After clonotype assignment, more extensive clonotypic analysis can be performed at the individual sample level or across all samples. General outputs from scRepertoire can be imported into Seurat objects to visualize clonotype data overlaid onto the cell clustering. Likewise, metadata from the Seurat objects can be imported into scRepertoire to analyze clonotypes by assigned clusters.\n\n\nMethods\n\nSystem requirements for running scRepertoire3 include the installation of R v3.5.1 and the the Seurat R package (v3.1.2). Utilization of scRepertoire is dependent on the total number of single-cells being processed, with a base estimate of 1 Gb of random-access memory and a modern CPU.\n\nThe isolation and processing of the 10x-Genomics-based single-cell mRNA and V(D)J Chromium sequencing data for immune cells has previously been described7,8. In addition, T cells were identified using expression values for canonical T cell markers: CD3D, CD4, CD8A, CD8B1 and previous clustering. T cells were isolated and reclustered using the integration method from the Seurat R package (v3.1.2) with 20 principal components and a resolution of 0.54. All code used to generate the figures appearing in the manuscript is available at https://github.com/ncborcherding/scRepertoire.\n\nThe scRepertoire was built and tested in R v3.5.1. Analysis for scRepertoire was inspired from the bulk immune profiling tcR (v2.2.4) R package without derivations in code9. Clonotypes can be called using the combination of immune loci genes, a more sensitive approach, or the nucleotide/amino acid sequence of the complementary-determining region 3 (CDR3). In addition to the base functions in R, data processing was performed using the dplyr (v0.8.3) and reshape2 (v1.4.3) R packages. Visualizations are generated using the ggplot2 (v3.2.1) and ggalluvial (v0.11.1) R packages with color pallets derived from the use of colorRamps (v2.3) and RColorBrewer (v1.1.2) R packages. Diversity metrics are calculated using the vegan (v2.5-6) R package. Visual outputs of functions are stored as layers of geometric or statistical ggplot layering, allowing users to easily modify presentation.\n\n\nResults\n\nscRepertoire3 can be used to call clonotypes using the CDR3 amino acid/nucleotide sequences, by gene usage, or by the combination of CDR3 nucleotide sequences and genes. Using the quantContig function, unique clonotypes can be visualized as raw values or scaled to the size of the library for samples or by type (Figure 2A). The total abundance of clonotypes can also be visualized calling abundanceContig (Figure 2B) or relative abundance of clonotypes (Figure 2C). Additionally, the distribution of CDR3 nucleotide or amino acid sequences for clonotypes can be visualized with lengthContig (Figure 2D).\n\n(A) Scaled unique clonotypes by total number of TCRs sequenced by patient and type of sample (peripheral, P; tumor, T), using the quantContig function. (B) Total abundance of clonotypes by sample and type using the abundanceContig function. (C) Relative abundance of clonotypes using density comparing peripheral blood to tumor samples. (D) CDR3 nucleotide length analysis by sample using the lengthContig function. The bimodal nature of the curve is a function of calling clonotypes for cells with both one and two immune receptors sequenced.\n\nMore in depth analysis of clonal architecture is available. Within the framework of scRepertoire, analysis of clonal homeostasis, or the clonal space occupied by clonotypes of specific proportions, can be visualized by clonalHomeostasis function (Figure 3A). Similarly, clonalProportion can be called to look at the proportion of clonal space occupied by specific clonotypes (Figure 3B). Overlap between the samples can be calculated and visualized with clonalOverlap, using either the overlap coefficient or Morisita index methods (Figure 3C). Measured of diversity across samples or groups can be quantified with the clonalDiversity function, demonstrating an overall reduction in clonal diversity in tumor samples (Figure 3D).\n\n(A) Clonal homeostatic space representations across all six samples using the gene and CDR3 AA sequence for clonotype calling. (B) Relative proportional space occupied by specific clonotypes across all six samples using the gene and CDR3 AA sequence for clonotype calling. (C) Morisita overlap quantifications for clonotypes across all six samples. (D) Diversity measures based on clonotypes by sample type using Shannon, Inverse Simpson, Chao, and abundance-based coverage estimator (ACE) indices.\n\nAfter the processing and analysis of the TCR repertoire with the base features, the next step is using scRepertoire to interact with the single-cell mRNA data. The expression data for the 12,911 cells built into the package have already been clusters (Figure 4A), with a clear distribution of the clusters into peripheral-blood- versus tumor-predominant (Figure 4B). Using the combineSeurat function in scRepertoire, we can look at the clonotypic frequencies of cells that comprise the UMAP-based clusters (Figure 4C), with notable expansion in the C2, C3, and C6 clusters (Figure 4D). The C7 and C8 clusters also have a relatively high frequency. In addition to clonal distribution, we can also use highlightClonotypes to set specific sequences of clonotypes to be visualized (Figure 4E), with clonotype 1 referring to the amino acid sequence “CAVNGGSQGNLIF_CSAEREDTDTQYF” and clonotype 2 for the amino acid sequence \"NA_CATSATLRVVAEKLFF\". Interesting clonotype 2 is restricted to a subcluster of the C6 cluster (Figure 4E). After combining both the clonotype and expression data, interaction between categories, such as cluster label and clonotype frequency can be visualized with the alluvialGraph function.\n\n(A) UMAP projection of the ccRCC T cells (n=12,911) into 12 distinct clusters. (B) UMAP projection with peripheral blood (red) and tumor (blued) populations highlighted and an accompanying relative proportion composition of each cluster, scaled by the total number of peripheral blood and tumor cells, respectively. (C) Using the combineSeurat function places individual cells into groups by the number of clonotypes, which then can be displayed overlaid with the UMAP projection. (D) combineSeurat also calculates the frequency of clonotypes, which can be used to more closely examine cluster composition, such as the indicate boxplot. (E) After combing the clonotype information with the Seurat object, highlightClonotypes can be used to specifically highlight the individual clonotypes of interest using the sequence information. (F) Interaction of clonotypes between multiple categories can be examined using the alluvialGraph function.\n\n\nConclusions\n\nscRepertoire (9) is a R-based toolkit for the analysis of single-cell immune receptor profiling. The package is able to take the annotated filtered outputs from the 10x Genomics Cell Ranger platform and provide analysis a number of modalities, including calling clonotypes, clonal space/homeostasis, clonal diversity, and repertoire overlap between samples. Outputs from scRepertoire can combined with dimensional reduction strategies for single-cell RNA quantifications, allowing users to analyze mRNA and immune profiles together. Under the creative commons v4.0 license, the scRepertoire package is freely available from the GitHub repository and is extensively annotated to assist in implementation and modification.\n\n\nData availability\n\nZenodo: scRepertoire. https://doi.org/10.5281/zenodo.36122163.\n\nFolder ‘Data’ contains all data required to run the vignettes described in the Results. This is also available on GitHub.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nSoftware availability\n\nSource code is available from GitHub: https://github.com/ncborcherding/scRepertoire.\n\nArchived source code at the time of publication: https://doi.org/10.5281/zenodo.36122163.\n\nLicense: Creative Commons Attribution 4.0 International.",
"appendix": "References\n\nPapalexi E, Satija R: Single-cell RNA sequencing to explore immune cell heterogeneity. Nat Rev Immunol. 2018; 18(1): 35–45. PubMed Abstract | Publisher Full Text\n\nChen G, Ning B, Shi T: Single-Cell RNA-Seq Technologies and Related Computational Data Analysis. Front Genet. 2019; 10: 317. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBorcherding N, Bormann NL: scRepertoire (Version 0.0.1). Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3612216\n\nStuart T, Butler A, Hoffman P, et al.: Comprehensive Integration of Single-Cell Data. Cell. 2019; 177(7): 1888–1902.e21. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMacosko EZ, Basu A, Satija R, et al.: Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets. Cell. 2015; 161(5): 1202–14. PubMed Abstract | Publisher Full Text | Free Full Text\n\nButler A, Hoffman P, Smibert P, et al.: Integrating single-cell transcriptomic data across different conditions, technologies, and species. Nat Biotechnol. 2018; 36(5): 411–420. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBorcherding N, Ahmed KK, Voigt AP, et al.: Transcriptional heterogeneity in cancer-associated regulatory T cells is predictive of survival. bioRxiv. 2018; 478628. Publisher Full Text\n\nVishwakarma A, Bocherding N, Chimenti MS, et al.: Mapping the Immune Landscape of Clear Cell Renal Cell Carcinoma by Single-Cell RNA-seq. bioRxiv. 2019; 824482. Publisher Full Text\n\nNazarov VI, Pogorelyy MV, Komech EA, et al.: tcR: an R package for T cell receptor repertoire advanced data analysis. BMC Bioinformatics. 2015; 16: 175. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "59209",
"date": "03 Mar 2020",
"name": "Lorenzo Drufuca",
"expertise": [
"Reviewer Expertise Integrative Biology",
"Cancer Immunology"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper describes a new package (scRepertoire) of functions for the analysis of clonality in single cell experiments in the R analytical environment.\nWhile it is true, as it is claimed, that 'a gap exists in the processing of V(D)J sequencing, descriptive statistics, clonal comparisons, and repertoire diversity with the current SCS R packages', nonetheless few packages already exist for the analysis of clonality in bulk samples (such as the inspiring package tcR). From the text it is not immediately clear why there is the need for a brand new package rather than a simpler 'wrapper' to bridge the gap between single cell based data and existing analytical packages.\nAlthough package functionalities are clearly illustrated in the paper, software documentation could be improved: behaviour of some functions is not immediately clear without studying the code directly (e.g. the way multi-chains clonotypes are treated by combineContigs()).\nSimilar to its inspiring package (tcR) and leveraging on the vegan package, scRepertoire implements different measures for diversity. Nonetheless, it has been suggested that conventional diversity measures fail to describe properly clonotype distributions and several complementary methods have been proposed like the Recon package by Kaplinsky1 and Arnaout1 or Startrac package by Zhang and colleagues2. Interaction with such packages would greatly increase scRepertoire analytical effectiveness.\nSimilarly, the function abundanceContig(), especially in the unscaled form, is of little use per se. Interestingly, recent methods have been proposed to for the comparative analysis of clone size distributions3 and could be easily incorporated into scRepertoire adding considerable power to it (though assessment of required numerosity should be introduced).\nThe package claims to be designed both for TCR and BCR analysis but definition of clonality in B cells is slightly different than in T cells due to isotype switch and somatic hypermutation phenomena following activation. Therefore clonotype identity between two cells should be defined differently between BCR and TCR analysis.\nConcerning overlap, beside the nice representation as a heatmap, it could be useful to have the chance to output the matrix itself rather than the plot only.\n10x vdj methods occasionally fails to reconstruct complete clonotypes or it reconstructs putatively aberrant clonotypes (clonotypes with multiple beta chains). Currently scRepertoire does not allow to filter for specific chain compositions but such feature would be worth adding, together with a graphical visualization of relative frequencies of chain composition across clonotypes.\nPaired 10x gene expression profile and vdj scoped analysis are not guaranteed to reconstruct the information for the exact same pool of barcodes, thus the combineSeurat() function could be improved by allowing to specify whether an inner or Seurat-sided joining is to be performed and ensuring that the joining is performed correctly.\n\nIs the rationale for developing the new software tool clearly explained? Partly\n\nIs the description of the software tool technically sound? Partly\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes",
"responses": [
{
"c_id": "5592",
"date": "15 Jun 2020",
"name": "Nicholas Borcherding",
"role": "Author Response",
"response": "The paper describes a new package (scRepertoire) of functions for the analysis of clonality in single cell experiments in the R analytical environment. While it is true, as it is claimed, that 'a gap exists in the processing of V(D)J sequencing, descriptive statistics, clonal comparisons, and repertoire diversity with the current SCS R packages', nonetheless few packages already exist for the analysis of clonality in bulk samples (such as the inspiring package tcR). From the text it is not immediately clear why there is the need for a brand new package rather than a simpler 'wrapper' to bridge the gap between single cell based data and existing analytical packages. Thank you for your input. To clarify, indeed a gap exists. The vast majority of packages available for V(D)J single-cell analysis is python-based reconstructions of TCR/BCR sequences (see, scTCRseq , TRAPeS and TraCeR). The tcR package that the reviewer indicates is in fact deprecated and only functional for bulk sequencing, there is no barcoded-based method. However, the authors of tcR have a newer R package, immunarch, that is freely available and produced by the company immunomind. This newer package offers single-cell support, but the workflow within the package does not allow for the combination of both TCR/BCR chains, and will call clonotypes with a single chain. In addition, as the reviewers keenly mention in the accompanying comments, single-cell technologies have unique pitfalls that need to be addressed as opposed to just creating wrapper functions. These concepts have been added to a newer version of the manuscript. Although package functionalities are clearly illustrated in the paper, software documentation could be improved: behaviour of some functions is not immediately clear without studying the code directly (e.g. the way multi-chains clonotypes are treated by combineContigs()). We have added extensive documentation to the package. Similar to its inspiring package (tcR) and leveraging on the vegan package, scRepertoire implements different measures for diversity. Nonetheless, it has been suggested that conventional diversity measures fail to describe properly clonotype distributions and several complementary methods have been proposed like the Recon package by Kaplinsky and Arnaout or Startrac package by Zhang and colleagues. Interaction with such packages would greatly increase scRepertoire analytical effectiveness. We have added the function StartracDiversity() to the development version of the package to allow for the migr, exp, and tans indices to be calculated from the Seurat object after the addition of the clonotype information. This function is detailed in the vignette due to limited word count of this article. Similarly, the function abundanceContig(), especially in the unscaled form, is of little use per se. Interestingly, recent methods have been proposed to for the comparative analysis of clone size distributions and could be easily incorporated into scRepertoire adding considerable power to it (though assessment of required numerosity should be introduced). We have added the function clonesizeDistribution() to allow users to examine the hierarchical clustering based on clone size distributions of samples to the scRepertoire package along with a comprehensive description and covered the usage in the vignette accompanying the package. Due to the length limitation for the manuscript, we only reference the function in the manuscript with the appropriate citation. The package claims to be designed both for TCR and BCR analysis but definition of clonality in B cells is slightly different than in T cells due to isotype switch and somatic hypermutation phenomena following activation. Therefore clonotype identity between two cells should be defined differently between BCR and TCR analysis. We agree with the need to add thresholding for clonotype assignment for the BCR in the context of somatic hypermutation. We have separated the combineContig() function into combineTCR() and combineBCR() functions, the latter organizes the data similarly, but defines nucleotide sequences by the normalized Hamming Distance, with sequences greater than >0.85 assigned to the same group. In the strict definition of the clonotypes in the combineBCR() the vgene is also added to both the light and heavy chains. We have kept the ability to visualize and analyze clonotypes at the amino acid, nucleotide sequence and gene level as well, to all users more choices. Concerning overlap, beside the nice representation as a heatmap, it could be useful to have the chance to output the matrix itself rather than the plot only. When appropriate, the visualization functions have the exportTable variable that allows a user to get the table or matrix that enables the visualization. Although this feature is already present, we will add discussion to the manuscript and accompanying vignette. 10x vdj methods occasionally fails to reconstruct complete clonotypes or it reconstructs putatively aberrant clonotypes (clonotypes with multiple beta chains). Currently scRepertoire does not allow to filter for specific chain compositions but such feature would be worth adding, together with a graphical visualization of relative frequencies of chain composition across clonotypes. If the authors understand the suggestion, the reviewers are asking for additional filtering options for combineTCR() and combineBCR(), two separate functions created from the reviewers earlier points. The function has the ability to remove barcodes with NA values or with multiple chains. More specifically to the combineTCR() function we have added removeMulti (removes all barcodes with >= 3 chains) and filterMulti (selects the 2 highest expressing chains). In terms of the chain filtering and visualizations, we are more than happy to add additional filtering and visualization tools for users. If the reviewer could clarify more on the how they envisioned the chain composition visualization, we would appreciate more insight. Paired 10x gene expression profile and vdj scoped analysis are not guaranteed to reconstruct the information for the exact same pool of barcodes, thus the combineSeurat() function could be improved by allowing to specify whether an inner or Seurat-sided joining is to be performed and ensuring that the joining is performed correctly. We have added the variable NA into the new combineBCR() and combineTCR(), which functions by removing cells without clonotype information. If we understand the reviewer correctly, the differential joining function would also need to address the seurat object as a whole instead of just merging the meta.data."
}
]
},
{
"id": "60873",
"date": "09 Mar 2020",
"name": "Tim Stuart",
"expertise": [
"Reviewer Expertise Single-cell genomics",
"bioinformatics",
"epigenomics"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper presents a new R package for the analysis of T-cell and B-cell clonotypes inferred from V(D)J recombination events. The package is designed for the analysis of data generated using the 10x Genomics V(D)J kit that allows clonotypes and transcriptomes to be co-assayed in the same individual cells.\nOverall the package appears to fill an unmet need in the analysis of single-cell V(D)J data, and works nicely with the existing Seurat package for single-cell analysis.\nWhile the focus of the paper is on the software, a more detailed explanation of the biology of V(D)J recombination and how this information is detected in single cells would improve the clarity of the manuscript. A description of the major existing challenges and goals of immune clonotype analysis could also be added.\nI do not have extensive expertise in the immunology and so my review focuses on the software implementation. I leave discussion of the value and impact of the software to other reviewers. However I will reiterate comments from other reviewers in saying that there should be a distinction made between T and B cell clonotypes, due to the Ig class switching that occurs in B cells.\n\nThe paper also states that “The expression data for the 12,911 cells built into the package”. I believe the clonotype information is included in the package in the data directory, but I don’t think the expression data is included. Including large gene expression matrix within the R package is not advisable anyway, and the authors should make it available through other means (NCBI GEO, for example).\nCode review\nI would encourage the authors to submit the package to CRAN or another R repository. This assist in the distribution of the package, and the process of getting the package accepted on CRAN will likely greatly improve the code quality. At a minimum, the authors should try to get the package to pass R CMD check with no notes, warnings, or errors.\nCurrently the package includes all rendered figures in the vignettes folder. I would recommend removing these files, and also removing the `ggsave` function calls within the vignette to prevent these files being written while compiling the vignette. This will reduce the size of the package.\nSeveral functions in the package use scoping assignment to assign variables in the global environment. This is considered bad practice and should be avoided in all cases.\nExtra files such as .DS_Store should be removed from the git repository and from the package. Use the .gitignore and .Rbuildignore files for this.\nAvoid importing code within R functions, for example `require(ggplot2)` calls. Instead, document the dependencies using roxygen2, for example `@importFrom ggplot2 ggplot`.\n\nTo access data in a Seurat object, I highly recommend using the functions defined in Seurat for this purpose rather than accessing the slots directly. For example, use obj[[]] to access metadata rather than obj@meta.data and Idents(obj) rather than obj@active.ident\nIn general the documentation of functions can be greatly improved. Try to include a text description of each function, a detailed description of the parameters, document the returned values, and include an executable example.\nIt is generally not advisable to overwrite functions in base R or other packages with variable names, for example the `call` variable in `clonalDiversity` overwrites the base R `call` function.\nReplace code like `class(df)[1] == “Seurat”` with `inherits(x = df, what = \"Seurat”)`\nIn plotting functions such as clonalOverlap, consider returning the ggplot object rather than printing the object. For example, replace `suppressWarnings(print(plot))` with `return(plot)`. This will allow users to modify the plot that is generated.\n\nSome code sections are duplicated, for example L91:104 and L131:144 in seuratFunctions.R. Consider putting duplicated code into functions.\nImported functions should be added to the namespace. Documenting the imports using roxygen2 (as has been done for parameters and exports) will take care of this.\nThe highlightClonotypes function is a bit redundant with existing functions in Seurat, ie DimPlot function with the cells.highlight parameter.\n\nIs the rationale for developing the new software tool clearly explained? Partly\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes",
"responses": [
{
"c_id": "5593",
"date": "15 Jun 2020",
"name": "Nicholas Borcherding",
"role": "Author Response",
"response": "This paper presents a new R package for the analysis of T-cell and B-cell clonotypes inferred from V(D)J recombination events. The package is designed for the analysis of data generated using the 10x Genomics V(D)J kit that allows clonotypes and transcriptomes to be co-assayed in the same individual cells. Overall the package appears to fill an unmet need in the analysis of single-cell V(D)J data, and works nicely with the existing Seurat package for single-cell analysis. While the focus of the paper is on the software, a more detailed explanation of the biology of V(D)J recombination and how this information is detected in single cells would improve the clarity of the manuscript. A description of the major existing challenges and goals of immune clonotype analysis could also be added. The authors agree that a more extensive explanation of VDJ biology would be a benefit, however, due to the limitations of less than 1000 words and 10 citations, we are limited by the structure of the F1000Research. We have added citations to the readme file to help users. I do not have extensive expertise in the immunology and so my review focuses on the software implementation. I leave discussion of the value and impact of the software to other reviewers. However I will reiterate comments from other reviewers in saying that there should be a distinction made between T and B cell clonotypes, due to the Ig class switching that occurs in B cells. The paper also states that “The expression data for the 12,911 cells built into the package”. I believe the clonotype information is included in the package in the data directory, but I don’t think the expression data is included. Including large gene expression matrix within the R package is not advisable anyway, and the authors should make it available through other means (NCBI GEO, for example). The seurat object with the package is available in the zenodo repository and linked in the github repository. The raw data has been deposited in GEO and more information can be found in the two preprints cited. Code review I would encourage the authors to submit the package to CRAN or another R repository. This assist in the distribution of the package, and the process of getting the package accepted on CRAN will likely greatly improve the code quality. At a minimum, the authors should try to get the package to pass R CMD check with no notes, warnings, or errors. Thank you for the suggestions, the authors believe this point helped the structure of the package immensely. We have used the devtools check function and as of the version in the github repository and zenodo at resubmission, there is no warnings, errors, or notes. We have begun the process of submitting the package to Bioconductor and have passed the build phase of the process. Currently the package includes all rendered figures in the vignettes folder. I would recommend removing these files, and also removing the `ggsave` function calls within the vignette to prevent these files being written while compiling the vignette. This will reduce the size of the package. Thank you for the suggestion, this has been completed. Several functions in the package use scoping assignment to assign variables in the global environment. This is considered bad practice and should be avoided in all cases. Scoping assignments in these instances are to give users the options of exporting the tables or matrices that form the visualization. We have modified the activity of the exportTable parameter to return a data frame used if TRUE or visualization if FALSE (default). Extra files such as .DS_Store should be removed from the git repository and from the package. Use the .gitignore and .Rbuildignore files for this. Thank you for the suggestion, we have removed .DS_Store. Avoid importing code within R functions, for example `require(ggplot2)` calls. Instead, document the dependencies using roxygen2, for example `@importFrom ggplot2 ggplot`. Thank you for the suggestion, we have made this change. To access data in a Seurat object, I highly recommend using the functions defined in Seurat for this purpose rather than accessing the slots directly. For example, use obj[[]] to access metadata rather than obj@meta.data and Idents(obj) rather than obj@active.ident Thanks for the suggestion, we have changed this in the new version of the R package. In general the documentation of functions can be greatly improved. Try to include a text description of each function, a detailed description of the parameters, document the returned values, and include an executable example. Both reviewers have made this excellent suggestion, the new version of scRepertoire has more comprehensive documentation, including descriptions and working examples. It is generally not advisable to overwrite functions in base R or other packages with variable names, for example the `call` variable in `clonalDiversity` overwrites the base R `call` function. Thank you for the suggestion, we have modified this for all functions. Replace code like `class(df)[1] == “Seurat”` with `inherits(x = df, what = \"Seurat”)` We have made this change and also replace all class() evaluations. In plotting functions such as clonalOverlap, consider returning the ggplot object rather than printing the object. For example, replace `suppressWarnings(print(plot))` with `return(plot)`. This will allow users to modify the plot that is generated. Excellent suggestion and this has been fixed. Some code sections are duplicated, for example L91:104 and L131:144 in seuratFunctions.R. Consider putting duplicated code into functions. Thank you for the suggestion, part of the change in the “call” to cloneCall variable in the functions also including creating a function to deal with the user selection. These lines were consistently repeated across multiple functions, but now have been reduced. We have also extensively modularized the code and added these to utils.R . Imported functions should be added to the namespace. Documenting the imports using roxygen2 (as has been done for parameters and exports) will take care of this. This has been completed. The highlightClonotypes function is a bit redundant with existing functions in Seurat, ie DimPlot function with the cells.highlight parameter. Agreed, although redundant, this was a request from an early beta tester, we plan to keep the function in."
}
]
}
] | 1
|
https://f1000research.com/articles/9-47
|
https://f1000research.com/articles/8-2018/v1
|
28 Nov 19
|
{
"type": "Study Protocol",
"title": "Learning to make informed health choices: Protocol for a pilot study in schools in Barcelona",
"authors": [
"Laura Martínez García",
"Pablo Alonso-Coello",
"Laia Asso Ministral",
"Clara Ballesté-Delpierre",
"Carlos Canelo Aybar",
"Carol de Britos",
"Ana Fernández Rodríguez",
"Ana Gallego Iborra",
"Victoria Leo Rosas",
"Paloma Llaquet",
"Ena Pery Niño de Guzmán Quispe",
"Giordano Pérez-Gaxiola",
"Carolina Requeijo",
"Karla Salas-Gama",
"Laura Samsó Jofra",
"Jordi Terres",
"Iratxe Urreta",
"Sarah Rosenbaum",
"Pablo Alonso-Coello",
"Laia Asso Ministral",
"Clara Ballesté-Delpierre",
"Carlos Canelo Aybar",
"Carol de Britos",
"Ana Fernández Rodríguez",
"Ana Gallego Iborra",
"Victoria Leo Rosas",
"Paloma Llaquet",
"Ena Pery Niño de Guzmán Quispe",
"Giordano Pérez-Gaxiola",
"Carolina Requeijo",
"Karla Salas-Gama",
"Laura Samsó Jofra",
"Jordi Terres",
"Iratxe Urreta",
"Sarah Rosenbaum"
],
"abstract": "Introduction: The Informed Health Choices (IHC) project has developed learning resources to teach primary school children (10 to 12-year-olds) to assess treatment claims and make informed health choices. The aim of our study is to explore both the students’ and teachers’ experience when using these resources in the context of Barcelona (Spain). Methods: During the 2019-2020 school year, we will conduct a pilot study with 4th and 5th-year primary school students (9 to 11-year-olds) from three schools in Barcelona. The intervention in the schools will include: 1) a workshop with the teachers, and 2) lessons to the students. The data collection will include: 1) initial assessment of the resources by the teachers, 2) non-participatory observations during the lessons, 3) semi-structured interviews with the students after a lesson, 4) assessment of the lessons by the teachers, 5) treatment claim assessment by the students, and 6) final assessment of the resources by the teachers. We will use ad hoc questionnaires and guides to register the data. We will perform a quantitative and qualitative analysis of the data to explore understandability, desirability, suitability, usefulness, facilitators and barriers of the resources. The most relevant results will be discussed and some recommendations on how to use, how to adapt (if needed), and how to implement the IHC resources to this context will be agreed. We will publish the study results in a peer-reviewed journal and in several Internet resources (web pages, electronic bulletins, and social media), and we will present them to the different users of interest in conferences, workshops, and meetings. Ethical considerations: The study protocol has obtained an approval exemption from the Ethics Committee of the Hospital de la Santa Creu i Sant Pau (Barcelona, Spain).",
"keywords": [
"Children’s health",
"critical thinking",
"evidence-based medicine",
"health education",
"health promotion",
"public health."
],
"content": "Introduction\n\nIn our day-to-day, we hear and make claims about treatments that can improve or worsen our health (“treatment” can be defined broadly as any action to improve or maintain the health of individuals). Claims we make, or are exposed to, may be about therapeutic interventions (take drugs, undergo surgery or use medical devices), changes in lifestyle (follow dietary guidelines, do exercise), interventions involving alternative medicine (use medicinal herbs), public health or environmental interventions, or changes in how health care is provided, funded or managed1,2.\n\nMany of these claims, regardless of whether they are well-intentioned or driven by various interests, can be wrong, inadequate or untrustworthy3. When people make decisions based on untrustworthy treatment claims, or when they ignore trustworthy claims, they may harm their health and use resources inadequately3.\n\nIn order for people to make informed health choices, they need to be able to obtain, process and understand the relevant health information (health literacy) and use that information from a critical perspective (critical thinking)4–6. Unfortunately, many people lack that ability. A European survey showed that 58.3% of the Spanish population has a limited level of health literacy7.\n\nThe main objective of the Informed Health Choices (IHC) project is to teach people to assess treatment claims and make informed health choices.\n\nThe IHC project has a focus on enabling people to learn these skills at a young age and began their first work in developing learning resources for primary school children (10 to 12-year-olds) from low-income countries (Uganda)8. There are several reasons the IHC project started with primary school children: 1) children can learn about fair comparisons (controlled research) and critical appraisal (in some countries, teaching these basic capabilities is already part of the curriculum); 2) primary school interventions can reach a large population group, before many of them leave school; 3) compared to adults, children have more time to learn and show less resistance to change with regard to their beliefs, attitudes or behaviours; 4) teaching children to think critically improves their academic performance; and 5) learning how to think critically about claims about treatment effects can help them, once they become adults, to make decisions about their health and to contribute, as citizens or as health decision-makers, to develop and implement health policies9. In addition, the IHC project focused on the child population of low-income countries because making informed health choices can contribute to a more efficient use of resources in contexts with higher social and economic inequality8.\n\nThe IHC Working Group has developed several resources to help people understand the differences between trustworthy and untrustworthy health claims, and how to use reliable information to make informed health choices8. The main resources are: 1) key concepts, 2) learning resources, and 3) a tool to evaluate the ability to assess treatment claims.\n\nIHC key concepts. Using the principles of a spiral curriculum, the IHC Working Group has compiled a list of concepts that individuals need to understand and apply when assessing claims about treatment effects and making health choices3,10.\n\nThe list of concepts is reviewed and updated periodically. The list currently includes 44 concepts divided into three capability groups: 1) identify when the treatment claim has an untrustworthy basis, 2) recognise when evidence from comparisons of treatments is trustworthy and when it is not, and 3) make well-informed choices about treatments. Table 1 shows the list of key concepts11.\n\nIHC learning resources. Using a human-centred design approach12–14, the IHC Working Group has produced various learning resources (IHC resources) to teach children and their families to understand and apply some of the key concepts8.\n\n* The 12 concepts included in the learning resources of the IHC project for primary school children. The IHC Key Concepts' explanations are available from the “That’s a Claim” website. This table has been reproduced with permission from Oxman et al. (Box 3)11.\n\nThe following resources were produced for primary school children (10 to 12-year-olds): a book (that includes and explains 12 key concepts), an exercise book, a teachers’ guide, some activity cards, a poster and a song (Figure 1)8,15. The book tells a story, narrated as a comic, about a brother and a sister, John and Julie, who know two teachers and health researchers, professor Compare and professor Fair. The professors teach John and Julie: 1) what questions they should ask when someone says something about a treatment; 2) what questions health researchers ask to find out more about treatment effects; and 3) what questions they should ask when deciding to use a treatment or not15.\n\nThe effect of the resources was assessed in a cluster randomised trial conducted in Uganda9. In the trial, 120 schools were assigned randomly to receive the intervention with the resources (60 schools, 76 teachers and 6,383 children) or not receive it (60 schools, 67 teachers and 4,430 children)9. The study showed that the children who used the resources improved their ability to assess treatment claims in comparison with the group without resources (69% of the children who use the learning resources got a passing score vs. 27% of children in the control group)9. A follow-up study one year later showed that children retained this knowledge and, in fact, the proportion of children with a passing score increased from 69% to 80%16.\n\nAdditionally, the IHC project team developed and evaluated a podcast with several episodes for parents (that introduce and explain nine key concepts)8,14,17,18.\n\nTool to evaluate the ability to assess treatment claims. The IHC Working Group has created a database with questions to assess people’s understanding and ability to apply the key concepts; the CLAIM Evaluation Tools19. Each question is based on a scenario that involves a claim about a treatment. There are two types of questions: 1) individual multiple-choice questions and 2) several true or false statements19.\n\nThis tool is a flexible resource, since people may design a questionnaire according to the key concepts that they wish to evaluate, selecting the questions that are most relevant for their objectives19. For example, teachers can design questionnaires to assess children, and researchers can design questionnaires to assess interventions or to describe a population’s ability to make informed health choices19. All the questions have been designed to be answered by children over 10 years of age as well as by adults19. The CLAIM Evaluation Tools can be found on the Testing Treatments international website.\n\nIn the previously cited trials that assessed the effect of the IHC resources, the researchers used questions from this database9,18.\n\nThe IHC resources have proven to be effective in the Ugandan trial, but it is still unknown whether they may be useful in other contexts20. Different working groups from more than 25 countries are adapting, or planning adaptation, of the IHC resources to their context21.\n\nThe IHC Working Group has proposed the following contextualization activities to explore how these resources can be used in a context different from the one that they were originally designed for: 1) context analysis, 2) translation of the resources, 3) pilot study, 3) content adaptation, 5) resource production, and 6) validation of the tool to assess treatment claims20.\n\nCurrently, we do not have any specific learning resources to teach primary school children to think critically about their health in the context of Barcelona (Spain). The working group from the Iberoamerican Cochrane Centre (CCIb) - Sant Pau Biomedical Research Institute (IIB Sant Pau) has translated the IHC resources into Spanish and included them on the IHC website. The next step is to explore how to use and, if needed, how to adapt the IHC resources in this context.\n\n\nObjectives\n\nExplore the students’ and teachers’ experience when using the learning resources of the IHC project in the context of Barcelona (Spain).\n\nExplore potential changes to the IHC resources to adapt them to this context.\n\nExplore the feasibility of implementing the IHC resources in this context.\n\nEvaluate the ability of the students to assess treatment claims and make informed health choices after using the IHC resources in this context.\n\n\nMethods\n\nDuring the 2019–2020 school year, we will conduct a pilot study with 4th and 5th-year primary school students (9 to 11-year-olds) from three schools in Barcelona, based on methods used in the IHC project22. Table 2 shows and describes the different steps of the study.\n\nEstablishment of the IHC-Barcelona Working Group. We will establish a “coordination group” to lead and coordinate the pilot study and to ensure it is completed according to the established work plan. We will establish a multidisciplinary “advisory group” (researchers, teachers, paediatricians, student representatives, family representatives, education and health stakeholders, and translators) to review and advise on the development of the different steps of the pilot study.\n\nWe will aim for profile representativeness of the IHC-Barcelona Working Group members. We will identify researchers from CIBER of Epidemiology and Public Health (CIBERESP) and expert colleagues; teachers, student representatives, and family representatives from selected schools; paediatricians from Asociación Española de Pediatría de Atención Primaria (AEPap); education and health stakeholders from Catalan Education and Health Departments; and translators who participated in the IHC resources translation into Spanish. Potential members will be contact and invite to participate by email. We will request and register the conflicts of interest of all the members of the IHC-Barcelona Working Group.\n\nSelection of the schools. We will select a convenience sample of three schools in Barcelona. We will use the following eligibility criteria: 1) schools included in the school directory from the Department of Education from the Government of Catalonia (2018–2019); 2) schools that have participated in a health promotion programme (2016–2017)23; and 3) schools that take part in the initiative Escola Nova 21. We will also take into consideration whether the schools include students that are representative of the neighbourhood, if they are in different neighbourhoods of the city, and their type of funding (two public schools and one private or charter school).\n\nSelection of the students and teachers. We will select 4th and 5th-year primary school students (9 to 11-year-olds) in all the lines from the selected schools (in this context, the number of lines means the number of student groups per academic level). We expect to include a convenience sample of approximately 150 students (25 students per class * two lines per school * three schools). We will request written informed consent from the families (Extended data 1 and 2)24,25.\n\nWe will select one teacher from every 4th or 5th year class in the selected schools. We expect to include six teachers (one teacher per class * two lines per school * three schools). The profile of the participatory teachers, as well as the subject where the lessons will be included (for example, in Science, Ethics or even Spanish) will depend on the education plan and the availability of the resources in each school. We will request informed consent from the teachers (Extended data 1 and 3)24,26.\n\nThe intervention in the schools will include: 1) a workshop with the teachers, and 2) lessons to the students. Each of the activities is described below:\n\n1. Workshop with the teachers\n\nThe objective is to introduce and review the IHC project, the pilot study, and the IHC resources with the teachers.\n\nBefore the workshop, a paper copy of the resources translated into Spanish will be sent to the teachers for their review. During the workshop, a researcher from the IHC-Barcelona Working Group will introduce the IHC project and the pilot study. In addition, a mock lesson will be taught as an example (previously selected by the teachers). Finally, a teacher from each school will explain the plan to teach the lessons to the student body. The workshop will last approximately five and a half hours (Table 3; Extended data 4)27.\n\n2. Lessons to the students\n\nThe objective is to teach students to assess treatment claims and make informed health choices using the IHC resources.\n\nDuring the lessons, the story of each lesson must be read and discussed. The teacher will be able to adapt the lessons to their students depending on the education plan of each school. The criteria that the teachers must take into consideration are:\n\nContinuity of lessons (initial plan is one lesson/week for nine weeks)\n\nDuration of lessons (initial plan is 60 minutes approximately)\n\nCompletion of some or all activities and/or exercises proposed in the lessons\n\nResource format (in Spanish and/or English, printed and/or digital)\n\nCompletion of extra activities\n\nThe teachers will reach an agreement with the IHC-Barcelona Working Group regarding their proposal for adaptation.\n\nThe data collection will include: 1) initial assessment of the resources by the teachers, 2) non-participatory observations during the lessons, 3) semi-structured interviews with the students after a lesson, 4) assessment of the lessons by the teachers, 5) treatment claim assessment by the students (CLAIM questionnaire test), and 6) final assessment of the resources by the teachers. Each of the activities is described below:\n\n1. Initial assessment of the resources by the teachers\n\nThe objective is to explore the teachers’ initial perception of the IHC resources.\n\nWe will explore the teachers’ initial perception of the IHC resources using an ad hoc self-administered questionnaire after the workshop. The questionnaire will include: teacher’s impression of the students’ expected experience with the IHC resources (understandability, desirability, suitability, and usefulness), the teachers’ experience with the IHC resources (understandability, desirability, suitability, and usefulness), examples of treatment claims, and comments (Table 3; Extended data 5)28.\n\n2. Non-participatory observations during the lessons\n\nThe objectives are to assess (objectively) the degree of implementation of the IHC resources and explore the students’ experience when using the IHC resources.\n\nA researcher from the IHC-Barcelona Working Group will make the non-participatory observations during the lessons. For convenience, each lesson will be observed in two classes (18 observations). Which lesson is going to be observed in each class will be assigned randomly. The researcher will record his or her observations in an ad hoc guide that will include: researcher’s impression of the students’ and teachers’ experience with the IHC resources (understandability, desirability, suitability, and usefulness), technique used to teach the lesson, the facilitators and barriers to teach the lesson, examples of treatment claims, questions, and comments (Table 3; Extended data 6)29.\n\n3. Semi-structured interviews with the students after a lesson\n\nThe objective is to explore the students’ experience when using the IHC resources.\n\nA researcher from the IHC-Barcelona Working Group will hold, with the support of a teacher, semi-structured individual interviews with a selection of students after a lesson. For convenience, two interviews will be held per lesson (18 interviews). Which student is going to be interviewed in each class will be assigned randomly (using the alphabetical attendance sheet). In the event that any of the selected students does not wish to participate, the next student will be selected from the list. Each interview will last approximately 30 minutes, and its audio will be recorded and transcribed. The researcher will hold the semi-structured interview using an ad hoc guide that will include: the students’ experience with the IHC resources (understandability, desirability, suitability, and usefulness), examples of treatment claims, suggestions to improve the lesson, questions, and comments (Table 3, Extended data 7)30.\n\n4. Assessment of the lessons by the teachers\n\nThe objectives are to assess (self-reportedly) the degree of implementation of the IHC resources and explore the teachers’ experience when using the resources.\n\nAfter teaching each lesson, the teachers will assess it in an ad hoc self-administered questionnaire. The questionnaire will include: teacher’s impression of the students’ experience with the lesson (understandability, desirability, suitability, and usefulness), the teachers’ experience with the lesson (understandability, desirability, suitability, and usefulness), the technique used to teach the lesson, the facilitators and barriers to teach the lesson, suggestions to improve the lesson, questions, and comments (Table 3, Extended data 8)31.\n\n5. Treatment claim assessment by the students (CLAIM questionnaire test)\n\nThe objective is to evaluate the ability of the students to assess treatment claims and make informed health choices after using the IHC resources in this context.\n\nAfter completing all the lessons, the students will take a self-administered test to evaluate their ability to apply the concepts discussed during the lessons. The test will include 24 questions (15 multiple-choice questions and nine true or false statements) from the CLAIM Evaluation Tools (Table 3; the questionnaire is accessible upon request from the Testing Treatments website to preserve the validity of the questions). The evaluation will be in Spanish (even if the resources were used in English), on a paper copy, and with a duration of approximately 60 minutes.\n\n6. Final assessment of the resources by the teachers\n\nThe objectives are to explore the teachers’ final experience when using the IHC resources and compare their initial perception with the final experience.\n\nAfter completing all the lessons, we will explore the teachers’ final experience with the IHC resources using an ad hoc self-administered questionnaire. The questionnaire will include: teacher’s impression of the students’ experience with the IHC resources (understandability, desirability, suitability, and usefulness), the teachers’ experience with the IHC resources (understandability, desirability, suitability, and usefulness), and comments (Table 3; Extended data 9)32.\n\nQuantitative analysis. We will perform a descriptive analysis of the qualitative variables (absolute and relative frequencies) and the quantitative variables (mean and standard deviation or median and range).\n\nWith regard to the treatment claim assessment by the students, we will show the mean score and the standard deviation, the proportion of the students with a passing score (basic knowledge of the concepts and how to apply them, 13 points or more over 24), and the proportion of the students with a high score (clear knowledge of the concepts and how to apply them, 20 points or more over 24)33.\n\nQualitative analysis. We will register in an excel sheet the feedback from: 1) the initial assessments by the teachers, 2) the non-participatory observations, 3) the semi-structured interviews with the students, 4) the assessment of the lessons by the teachers, and 5) the final assessments by the teachers.\n\nWe will perform a thematic analysis based on the categories previously used in the IHC project (seriousness, user experience, facilitators and barriers, and potential changes) (Table 4)22,34. One researcher will identify, codify, and summarise the feedback using these categories and search for emerging categories; another researcher will check the codification. They will discuss and review the definitions and limits of each category. Finally, using the summarised data, they will explore the nature of the phenomena (understandability, desirability, suitability, usefulness, facilitators and barriers) and the possible explanations of the results.\n\nThe ‘Facilitators and barriers’ section of this table has been reproduced with permission from Nsangi et al. (Table 1)34.\n\nThe IHC-Barcelona Working Group will discuss most relevant results and ideas to improve users’ experience when using the IHC resources in the context of Barcelona (Spain). They will consensus the potential changes of IHC resources (Table 4). Finally, they will suggested and agreed recommendations - both practice and research purposes - on how to use, how to adapt (if needed), and how to implement the IHC resources to this context.\n\nThe dissemination activities of the pilot study results will include: 1) publication in a peer-reviewed journal, 2) publication in several Internet resources (for example, related web pages, electronic bulletins and social media), and 3) introduction to the different users of interest (researchers, teachers, paediatricians, student representatives, family representatives, education and health stakeholders, and translators) in conferences, workshops and meetings. The implementation activities will include: 1) offering support to the schools that have participated in the pilot study and that are interested in including the IHC resources in the following school years, 2) giving support to other schools that are interested in including the IHC resources in their education plan.\n\nRepresentatives from all the different areas of interest (researchers, teachers, paediatricians, student representatives, family representatives, education and health stakeholders, and translators) will be invited to be members of the IHC-Barcelona Working Group.\n\nThe study protocol has obtained an approval exemption (does not include patients, biological specimens or clinical data) from the Ethics Committee of the Hospital de la Santa Creu i Sant Pau (Barcelona, Spain). The participants will be informed about the study, their written informed consent will be requested, and the data will be anonymised.\n\nFigure 2 shows the work plan. Currently, we have started the intervention in schools with the teachers’ workshop.\n\n\nDiscussion\n\nIt is important that people learn how to think critically about their health and how to make informed choices. The IHC project tackles this challenge from an innovative perspective because: 1) it focuses in children and 2) uses learning resources designed and assessed to facilitate the teaching and learning process. By introducing the IHC resources in a new context, we hope to contribute to the global effort to help people make informed choices regarding their health.\n\nIntroducing the IHC resources in schools can be considered as a health promotion and education intervention35. According to the World Health Organization (WHO), the concept of health promotion comprises “the process of enabling people to increase control over, and to improve their health”36. Additionally, health education comprises “consciously constructed opportunities for learning involving some form of communication designed to improve health literacy, including improving knowledge, and developing life skills which are conducive to individual and community health”36. Health education and promotion interventions in schools have proven to be beneficial for the health of the population37,38.\n\nThere are several definitions of critical thinking, as well as several strategies to teach how to think critically39,40. In 1990, a Delphi panel of experts defined this ability as a “purposeful, self-regulatory judgment which results in interpretation, analysis, evaluation, and inference, as well as explanation of the evidential, conceptual, methodological, criteriological, or contextual considerations upon which that judgment is based”41. Therefore, promoting critical thinking at schools can be not only useful in the health area18, but also in other curricular areas (e.g., Mathematics, Science, Literacy)42.\n\nThe IHC project offers several learning resources that were created accurately and explicitly, and have been assessed in a cluster randomised trial16,18. Thus far, there are few studies that assess the effect of the learning resources when acquiring competences43. Moreover, the available studies show that the evaluated textbooks provide little learning support44–46. We must start demanding the same standards for evaluating educational interventions that are used for evaluating health interventions47.\n\nOur proposal has several strengths. Firstly, before the study, we have translated the IHC resources into Spanish. Different users of interest (translator, researchers, students, teachers, and medical doctors) participated in the translation process and fit the text of the IHC resources to this context. Secondly, we have expanded the profile of the users of interest (researchers, teachers, paediatricians, student representatives, family representatives, education and health stakeholders, and translators) to establish the study's multidisciplinary working group. Thirdly and lastly, we will pilot an intervention in the schools that has already been shown to be effective in a cluster randomised trial in Uganda, where over 100 schools participated (100 teachers and 10,000 children).\n\nOur proposal also has some limitations. The main limitation is using a convenience sample (small and geographically limited sample). However, an exploratory approach with few schools has some advantages: 1) assessing the feasibility of introducing the IHC resources into this education system, 2) familiarising ourselves with the intervention, and 3) adapting the methods to apply them to broader future studies in this context. It is also worth noting that we will not be assessing the impact of the IHC resources in this study; due to this, we will not include a control group and we will not have a questionnaire validated to assess treatment claims for students.\n\nThe next contextualization activities will be: 1) content adaptation - if needed, 2) context analysis, and 3) validation of the CLAIM questionnaire test into Spanish for use in this context. Finally, a cluster randomised trial with the adapted IHC resources could be conducted to evaluate their effect in the students’ ability to assess treatment claims in this context.\n\n\nData availability\n\nNo data are associated with this article.\n\nFigshare: IHC@BCN_01Information.pdf. https://doi.org/10.6084/m9.figshare.10272173.v124\n\nFigshare: IHC@BCN_02InformedConsentFamilies.pdf https://doi.org/10.6084/m9.figshare.10272215.v125\n\nFigshare: IHC@BCN_03InformedConsentTeachers.pdf https://doi.org/10.6084/m9.figshare.10272242.v126\n\nFigshare: IHC@BCN_04Workshop.pdf https://doi.org/10.6084/m9.figshare.10272275.v127\n\nFigshare: IHC@BCN_05InitialAssessment https://doi.org/10.6084/m9.figshare.10272278.v128\n\nFigshare: IHC@BCN_06NonParticipatoryObservations https://doi.org/10.6084/m9.figshare.10272281.v129\n\nFigshare: IHC@BCN_07SemiStructuredInterviews https://doi.org/10.6084/m9.figshare.10272287.v130\n\nFigshare: IHC@BCN_08AssessmentLessons https://doi.org/10.6084/m9.figshare.10272305.v131\n\nFigshare: IHC@BCN_09FinalAssessment https://doi.org/10.6084/m9.figshare.10272308.v132\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
"appendix": "Acknowledgements\n\nLaura Samsó Jofra is a doctoral candidate at the Paediatrics, Obstetrics and Gynaecology and Preventive Medicine Department, Universitat Autònoma de Barcelona, Barcelona, Spain.\n\nWe would like to thank the three schools in Barcelona (Escola Sant Martí, Escola Virolai and Institut Escola Antaviana) for their participation in the pilot study. Additionally, we would like to thank Escola Virolai for their participation in the translation of the IHC resources into Spanish.\n\nWe would like to thank Dr Andrew Oxman (Centre for Informed Health Choices, Norwegian Institute of Public Health, Oslo, Norway) for his advice and feedback on an earlier version of this protocol.\n\n\nReferences\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\nSchwitzer G: A guide to reading health care news stories. JAMA Intern Med. 2014; 174(7): 1183–6. PubMed Abstract | Publisher Full Text\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\nMoseley D, Baumfield V, Elliott JG, et al.: Frameworks for Thinking: A Handbook for Teaching and Learning. Cambridge: Cambridge University Press, 2005. Publisher Full Text\n\nSørensen K, Van den Broucke S, Fullam J, et al.: Health literacy and public health: a systematic review and integration of definitions and models. BMC Public Health. 2012; 12: 80. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWorld Health Organization: Health literacy. The solid facts. World Health Organization: Geneva, 2013. Reference Source\n\nSørensen K, Pelikan JM, Röthlin F, et al.: Health literacy in Europe: comparative results of the European health literacy survey (HLS-EU). Eur J Public Health. 2015; 25(6): 1053–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nThe Informed Healthcare Choices Group: Supporting informed healthcare choices in low-income countries - final report. IHC Working Paper, 2018. Reference Source\n\nNsangi A, Semakula D, Oxman AD, et al.: Effects of the Informed Health Choices primary school intervention on the ability of children in Uganda to assess the reliability of claims about treatment effects: a cluster-randomised controlled trial. Lancet. 2017; 390(10092): 374–388. PubMed Abstract | Publisher Full Text\n\nThe Informed Health Choices Group: Spiral curriculum. [Accessed November 7, 2019]. Reference Source\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. 2018; 7: 1784. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGiacomin J: What is human centred design? Design Journal. 2014; 17(4): 606–623. Publisher Full Text\n\nNsangi A, Semakula D, Rosenbaum S, et al.: Development of the Informed Health Choices resources to teach primary school children to assess claims about treatment effects in four countries. IHC Working Paper. 2017. Reference Source\n\nSemakula D, Nsangi A, Oxman M, et al.: Development of mass media resources to improve the ability of parents of primary school children in Uganda to assess the trustworthiness of claims about the benefits and harms of treatments. IHC Working Paper, 2018.\n\nInformed Health Choices Group: The Health Choices Book: Learning to think carefully about treatments. A health science book for primary school children. IHC Working Paper. 2016. Reference Source\n\nNsangi A, Semakula D, Oxman AD, et al.: Effects of the Informed Health Choices primary school intervention on the ability of children in Uganda to assess the reliability of claims about treatment effects, one-year follow-up: a cluster-randomised trial. Trials. 2019; (in review). Publisher Full Text\n\nThe Informed Healthcare Choices Group: The Health Choices programme podcast Kampala. Makerere University. 2016.\n\nSemakula D, Nsangi A, Oxman AD, et al.: Effects of the Informed Health Choices podcast on the ability of parents of primary school children in Uganda to assess claims about treatment effects: a randomised controlled trial. Lancet. 2017; 390(10092): 389–398. PubMed Abstract | Publisher Full Text\n\nAustvoll-Dahlgren A, Semakula D, Nsangi A, et al.: Measuring ability to assess claims about treatment effects: the development of the 'Claim Evaluation Tools'. BMJ Open. 2017; 7(5): e013184. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMartínez García L, Rosenbaum S, Informed Health Choices Group: Contextualising Informed Health Choices primary school resources for use in different countries. 25th Cochrane Colloquium; Edinburgh, UK. 2018. Reference Source\n\nThe Informed Health Choices Group: Informed Health Choices Newsletter 2019. [Accessed November 7, 2019]. Reference Source\n\nThe Informed Health Choices group: Guide for piloting the Informed Health Choices (IHC) learning resources. IHC Working Paper. 2017.\n\nSalvador M, Bastida A, Martínez SN, et al.: Programes de promoció de la salut en centres d’educació primària i secundària de Barcelona. Avaluació del curs 2016-2017. Barcelona: Agència de Salut Pública de Barcelona. 2018. Reference Source\n\nMartínez García L: IHC@BCN_01Information.pdf. figshare. Online resource. 2019. http://www.doi.org/10.6084/m9.figshare.10272173.v1\n\nMartínez García L: IHC@BCN_02InformedConsentFamilies. figshare. Online resource. 2019. http://www.doi.org/10.6084/m9.figshare.10272215.v1\n\nMartínez García L: IHC@BCN_03InformedConsentTeachers. figshare. Online resource. 2019. http://www.doi.org/10.6084/m9.figshare.10272242.v1\n\nMartínez García L: IHC@BCN_04Workshop. figshare. Online resource. 2019. http://www.doi.org/10.6084/m9.figshare.10272275.v1\n\nMartínez García L: IHC@BCN_05InitialAssessment. figshare. Online resource. 2019. http://www.doi.org/10.6084/m9.figshare.10272278.v1\n\nMartínez García L: IHC@BCN_06NonParticipatoryObservations. figshare. Online resource. 2019. http://www.doi.org/10.6084/m9.figshare.10272281.v1\n\nMartínez García L: IHC@BCN_07SemiStructuredInterviews. figshare. Online resource. 2019. http://www.doi.org/10.6084/m9.figshare.10272287.v1\n\nMartínez García L: IHC@BCN_08AssessmentLessons. figshare. Online resource. 2019. http://www.doi.org/10.6084/m9.figshare.10272305.v1\n\nMartínez García L: IHC@BCN_09FinalAssessment. figshare. Online resource. 2019. http://www.doi.org/10.6084/m9.figshare.10272308.v1\n\nDavies A, Gerrity M, Nordheim LV, et al.: Measuring ability to assess claims about treatment effects: establishment of a standard for passing and mastery. IHC Working Paper, 2017. Reference Source\n\nNsangi A, Semakula D, Glenton C, et al.: Informed health choices intervention to teach primary school children in low-income countries to assess claims about treatment effects: process evaluation. BMJ Open. 2019; 9(9): e030787. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSharples JM, Oxman AD, Mahtani KR, et al.: Critical thinking in healthcare and education. BMJ. 2017; 357: j2234. PubMed Abstract | Publisher Full Text\n\nWorld Health Organization: Health Promotion Glossary. World Health Organization: Geneva, 1998. Reference Source\n\nCaan W, Cassidy J, Coverdale G, et al.: The value of using schools as community assets for health. Public Health. 2015; 129(1): 3–16. PubMed Abstract | Publisher Full Text\n\nLangford R, Bonell CP, Jones HE, et al.: The WHO Health Promoting School framework for improving the health and well-being of students and their academic achievement. Cochrane Database Syst Rev. 2014; (4): CD008958. PubMed Abstract | Publisher Full Text\n\nAbrami PC, Bernard RM, Borokhovski E, et al.: Strategies for teaching students to think critically a meta-analysis. Rev Educ Res. 2015; 85: 275–314. Publisher Full Text\n\nMartínez García L, Oxman A: Frameworks for critical thinking about treatment claims, comparisons, and choices: a mapping review. 26th Cochrane Colloquium; Santiago de Chile, Chile. 2019. Reference Source\n\nFacione PA: Critical thinking: A statement of expert consensus for purposes of educational assessment and instruction. Research findings and recommendations. Newark, DE: American Philosophical Association. 1990. Reference Source\n\nRuddock G, Sainsbury M: Comparison of the core primary curriculum in England to those of other high performing countries. Research Report DCSF-RW048. London: Department for Children, Schools and Families, 2008. Reference Source\n\nBehnke Y: Textbook Effects and Efficacy. The Palgrave handbook of textbook studies. New York: Palgrave Macmillan, 2018. Publisher Full Text\n\nStern L, Roseman JE: Can Middle-School Science Textbooks Help Students Learn Important Ideas? Findings from Project 2061’s Curriculum Evaluation Study: Life Science. J Res Sci Teach. 2004; 41(6): 538–68. Publisher Full Text\n\nPudas AK: Investigating Possibilities to Develop Textbooks to Implement Global Education in Basic Education Instruction. IARTEM e-Journal. 2013; 5(2): 1–22. Reference Source\n\nVojir K, Rusek M: Science education textbook research trends: a systematic literature review. Int J Sci Educ. 2019; 41(11): 1496–1516. Publisher Full Text\n\nFerrer S: Educación basada en la evidencia: ¿qué pedagogías han probado que funcionan? Agencia SINC, la ciencia es noticia. 2018. Reference Source"
}
|
[
{
"id": "58609",
"date": "10 Feb 2020",
"name": "Loai Albarqouni",
"expertise": [
"Reviewer Expertise Evidence-based practice"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThanks for inviting me to review this protocol. The author proposes a pilot study to evaluate the IHC learning resources in 3 primary schools in Barcelona. the protocol is well -designed and -written. Few comments below:\n\nIntroduction\n\nI would cite a few references to support each of the 5 arguments for starting the IHC project in primary school.\nMethods\nI would justify the sampling methods & eligibility criteria of the schools and describe if these 3 schools are representatives to the average school in Barcelona (e.g. a number of students).\n\nI would consider TIDier Checklist to better describe intervention details.\n\nI would describe the time points for each of the outcome measures.\n\nData analysis – ‘qualitative’ variables should be replaced by ‘categorical’ and ‘quantitative’ variables by ‘continuous’ – Both quantitative measures.\n\nAuthors should describe whether the resources have been translated and validated into Spanish.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable",
"responses": [
{
"c_id": "5473",
"date": "12 May 2020",
"name": "Laura Martínez García",
"role": "Author Response",
"response": "Comment 1 - References in the introduction Response 1 We have included relevant references to support the reasons for starting the IHC project in primary school children. The text now reads: “There are several reasons the IHC project started with primary school children: 1) children can learn about fair comparisons (controlled research) and critical appraisal (in some countries, teaching these basic capabilities is already part of the curriculum) [Oxman 2020, START 2020]; 2) primary school interventions can reach a large population group, before many of them leave school [UNESCO 2019]; 3) compared to adults, children have more time to learn and show less resistance to change with regard to their beliefs, attitudes or behaviours [Vosniadou 2013]; 4) teaching children to think critically improves their academic performance [Oxman 2019]; and 5) learning how to think critically about claims about treatment effects can help them, once they become adults, to make decisions about their health and to contribute, as citizens or as health decision-makers, to develop and implement health policies [Nsangi 2017].” Comment 2 - Sample, eligibility criteria, and description of the schools Response 2 Sample We have included a relevant reference to justify the sampling. The text now reads: “To achieve the objective, we will select a convenience sample of three schools in Barcelona [Etikan 2016].” On the other hand, we already highlight this limitation in the “Discussion - Study strengths and limitations” section, the text reads: “The main limitation is using a convenience sample (small, geographically limited, and non-representative sample).” Eligibility criteria We have clarified that the IHC-Barcelona Working Group made a consensus to establish the eligibility criteria of the sample. The text now reads: “The IHC-Barcelona Working Group reach a consensus on eligibility criteria of the schools: 1) schools included in the school directory from the Department of Education from the Government of Catalonia (2018-2019); 2) schools that have participated in a health promotion programme (2016-2017) [Salvador 2018]; and 3) schools that take part in the initiative Escola Nova 21 (alliance of schools and civil society institutions for an advanced education system, carried out between 2016 - 2019, and responding to United Nations and UNESCO’s call for the participation of all sectors in an inclusive process to make possible the education paradigm shift). We will also take into consideration whether the schools include students that are representative of the neighbourhood, if they are in different neighbourhoods of the city, and their type of funding (two public schools and one private or charter school). Description of the schools We will describe the included schools and discuss their representativeness in the publication of the results of the pilot study. Comment 3 - TIDieR Checklist Response 3 We have included, as extended data, the description of the intervention in the schools using TIDieR checklist. The text now reads “The intervention in the schools will include: 1) a workshop with the teachers, and 2) lessons to the students (Extended data 4 provides a description of the intervention using the TIDieR checklist) [Hoffmann 2014, Martínez García 2020]. Each of the activities is summarised below:” Also, we have combined all extended data in a single document to facilitate its availability to readers. The text in the \"Data availability\" section now reads: “Extended data is available at: https://figshare.com/articles/IHC_BCNPilotStudy/12167622 [Martínez García 2020].” We have modified the reference according “Martínez García L. IHC@BCNPilotStudy [Internet]. figshare; 2020. Available at: https://figshare.com/articles/IHC_BCNPilotStudy/12221189/1 [Accessed April 30, 2020].” Comment 4 - Time points of outcomes measures Response 4 We have clarified the time points for data collection throughout the manuscript. The activities now read: Assessment of the IHC resources by the teachers before the lessons Non-participatory observations during the lessons Semi-structured interviews with the students after a lesson Assessment of the lessons by the teachers after a lesson Treatment claim assessment by the students at the end of the lessons Assessment of the IHC resources by the teachers at the end of the lessons Comment 5 - Data analysis Response 5 We have amended the text, now it reads: “We will perform a descriptive analysis of the categorical variables (absolute and relative frequencies), and the continuous variables (mean and standard deviation or median and range).” Comment 6 - Translation and validation into Spanish of IHC resources Response 6 We have included a relevant reference to support the method used to translate into Spanish the IHC resources. The text now reads “The working group from the Sant Pau Biomedical Research Institute (IIB Sant Pau) - Iberoamerican Cochrane Centre (CCIb) has translated the IHC resources into Spanish based on methods proposed by the IHC Working Group [IHC 2019].” On the other hand, we already highlight the strengths and limitations of translation process in the “Discussion - Study strengths and limitations” section, the text reads: Strength: “Firstly, before this study, we have translated the IHC resources into Spanish. A translator, researchers, students, teachers, and medical doctors participated in the translation process and fit the text of the IHC resources to this context.” Limitation: “It is also worth noting that we will not be assessing the impact of the IHC resources in this study; due to this, we will not include a control group and we will not have a questionnaire validated to assess treatment claims for students.” References [Etikan 2016] Etikan I, Abubakar Musa S, Sunusi Alkassim R. Comparison of Convenience Sampling and Purposive Sampling. American Journal of Theoretical and Applied Statistics. 2016;5(1):1-4. [Hoffmann 2014] Hoffmann TC, Glasziou PP, Boutron I, Milne R, Perera R, Moher D, et al. Better reporting of interventions: template for intervention description and replication (TIDieR) checklist and guide. BMJ. 2014;348:g1687. [IHC 2019] The Informed Health Choices group. Guide for translating the Informed Health Choices school resources. Informed Health Choices Working Paper, Norwegian Institute of Public Health, 2019. [Martínez García 2020] Martínez García L. IHC@BCNPilotStudy [Internet]. figshare; 2020. Available at: https://figshare.com/articles/IHC_BCNPilotStudy/12221189/1 [Accessed April 30, 2020]. [Nsangi 2017] Nsangi A, Semakula D, Oxman AD, Austvoll-Dahlgren A, Oxman M, Rosenbaum S, et al. Effects of the Informed Health Choices primary school intervention on the ability of children in Uganda to assess the reliability of claims about treatment effects: a cluster-randomised controlled trial. Lancet. 2017;390(10092):374-388. [Oxman 2020] Oxman A, Martínez García L. 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 [version 1; peer review: awaiting peer review]. F1000Research. 2020. [Salvador 2018] Salvador M, Bastida A, Martínez SN, Juárez O, Artazcoz L. Programes de promoció de la salut en centres d’educació primària i secundària de Barcelona. Avaluació del curs 2016-2017. Barcelona: Agència de Salut Pública de Barcelona; 2018. [START 2020] Health Research Board – Trials Methodology Research network (HRB-TMRN), National University of Ireland Galway. START – Schools Teaching Awareness of Randomised Trials [online]. Available at: https://www.hrb-tmrn.ie/public-engagement/start-competition [Accessed April 16, 2020] [UNESCO 2019] UNESCO Institute for Statistics. New Methodology Shows that 258 Million Children, Adolescents and Youth Are Out of School [online]. Available at: http://uis.unesco.org/sites/default/files/documents/new-methodology-shows-258-million-children-adolescents-and-youth-are-out-school.pdf [Accessed April 16, 2020] [Vosniadou 2013] Vosniadou S. International handbook of research on conceptual change. 2nd edition. Oxford: Routledge, 2013."
}
]
},
{
"id": "61277",
"date": "26 Mar 2020",
"name": "Luis Gabriel Cuervo",
"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\nWe congratulate the authors for developing this protocol for a pilot study to adapt a tested intervention with a lasting effect in education of benefit to children, teachers and families. This is especially important at a time when pseudoscience and false statements frequently confuse people. There is great potential and value in people being able to critically assess information and distinguish valid information from unreliable one. Well done. We were pleased to review this protocol and to provide recommendations to enhance it ahead of the study.\nIs the rationale for, and objectives of, the study clearly described?\nThe manuscript provides a detailed description of the study previously done in Uganda1,2,3 and needs to provide a similar level of detail of the protocol to be implemented in Barcelona. The rationale and objectives are described under \"Contextualization of the Informed Health Choices project\" yet the descriptions need to provide in-depth detail of how the pilot is to be prepared, implemented, analysed, reported and used to inform future developments. Although it is a pleasure to read the description they do of the above cited research conducted in Uganda, if length were an issue, the authors can summarize some details by citing the studies and instead offering more details from their own study.\nThe objectives need to explicitly state their intent to to assess the effects of the intervention and replicability of the study in a IHC setting in a Spanish speaking country, or to prepare the IHC project for its scaling up in Barcelona, Spain. This appears to be a central objective to the study. Also describe their aims regarding the scaling-up of the intervention beyond the pilot schools.\nThe information in the abstract needs to be matched to the key elements of the study, thus highlighting the ultimate purpose of the pilot and subsequent studies.\nIs the study design appropriate for the research question?\nPlease describe the target and study population in detail elaborating on the characteristics of the school districts and system in Barcelona and provide descriptive details of the target population (children, teachers, parents) disaggregating the information where possible by gender and other relevant variables (e.g. socioeconomic stratum, ethnicity, religion, etc.)4,5,6. This way it will be clear if the pilot relates to the target population and/or elicit any limitations that need to be considered in the analysis or conclusions, as well as considerations for subsequent research.\nElaborate on the criteria they will be using to select the participating schools in the convenience sample. The sample of teachers will be small thus it is important to illustrate any efforts to determine if these are representative of the broader population of teachers, to deal with imprecision or bias arising from the small sample, and to elaborate on considerations for their subsequent implementation research. How will they control if the selected teachers are over or under performers?\nWe believe there are important conceptual issues to address under Ethical considerations. First, the decision to waive an ethical review falls with the Ethics Committee; apparently this was the case. However, we have some concerns with the rationale presented:\nDescribe what review process is being undertaken to ensure that their is compliance with local laws, with the assent of children (if appropriate), and whether the informed consent process (and corresponding documents) are appropriate for each of the groups of participants so that there is an informed decision;\n\nElaborate on the processes to protect the privacy of participants considering that this is a small sample size making feasible that identities can be exposed;\n\nThe rationale for the exemption is flawed because this is research in human subjects. If there was a reason for conducting an expedited review (ideal) or waiving it would be this being a “less than minimal risk” or “minimal risk study with the prospect of direct benefit to individual subjects”. Definitions are provided and surely there is equivalent European regulation that applies.4,5.\nClarify what the management will be if a student, teacher or parent decline to participate, considering the whole grade is participating. Will informed consent be managed at the individual or class level?\n\nAre sufficient details of the methods provided to allow replication by others?\nThere are aspects that need more detail or perhaps a visual aid, such as:\nSpecify the times when measurements will be done illustrating them with a PERT Chart complementing their timetable (or GANTT). This will make the information clear and also help to determine if the evaluations will be done at an appropriate time, avoiding for example these extending into the holiday season.\n\nDescribe how the sample was chosen for the pilot and what implications this may have in the preparation of the larger implementation study.\n\nDetails of the specific skills sought in the IHC-Barcelona Working Group and the Advisory Group are missing. It is of relevance to know what skills and attitudes are sought in the advisory group members to perform a proper job, and how they will be assessed. What criteria will be used in selecting the researchers and experts from the institutions listed? Will education policy makers be involved from the outset (advisable) and what positions do they have in Barcelona’s education system? Kindly describe what is the Escola Nova 21 initiative and how it relates to Barcelona’s education structure and standards. Elaborate on the implications -including bias that can result from the selection, and how this will be controlled.\n\nDescribe what efforts will be made to determine the replicability and adaptations needed for the study in different settings in Barcelona and beyond.\n\nClarify the criteria that will be used to select the convenience sample as well the the justification for the sample size. Describe the process to be followed in selecting the schools meeting criteria 1, 2 and 3, and how these schools represent the broader education district of Barcelona, and the population of students. Elaborate how you will analyse representation.\n\nProvide specific dates for data collection and their place within the school calendar.\n\nClarify if the selected classes will be co-ed (see note on PROGRESS) and if groups turn out to have >25 students, how will this be managed.\nAre the data sets clearly presented in a usable and accessible format?\nThe data analysis section would benefit from a table with the variables that will be analysed, and the analysis strategies (e.g. summary statistics, dispersion assessment, etc.) for each variable.\n\nConsider ways to minimize the variability of the assessments from teachers.\n\nConsider having double data capturing and whether and the thought process behind having one researcher identifying, codifying and summarizing feedback instead of having a duplicate data-capturing with a third person as tie breaker, or other methods to enhance precision and assess consistency (page 10.)\n\nIn section “Dissemination of the results” elaborate on the engagement and appropriation by education and science authorities as a strategy to enhance implementation and sustainability in Barcelona and beyond. Elaborate on the participation (if any) of science communicators and journalists, and potential advocates for education.\n\nElaborate on the inclusion and exclusion criteria under “User participation” and describe the characteristics and more detailed relevant information of the group.\n\nIn section “Discussion” highlight any preparations to adjust to the COVID19 (and if this may provide suitable material) and if possible frame the study on relevant policies that promote critical appraisal learning and partnerships with civil society to increase the value and use of research for health. We list a couple of policy documents and surely the authors can illustrate some local and European policies that apply as well6,7\nWe were delighted to read this study and how it will build on the success of a previous study to facilitate the implementation of an effective intervention in a different setting. Congratulations to the authors! It is important for these new studies to uphold the methodological rigour of the original studies and we have made some recommendations towards this end. A significant part of the protocol elaborates on the earlier studies referenced and some additional enhancements and details are needed to level this protocol with previous studies. We have provided actionable recommendations and we look forward to a revised version. This study has a tremendous potential to address with a key population, the current crisis that pseudo-science, false news, and misleading claims have brought upon societies, and we believe it needs to be advanced reported and developed in collaboration with education and science and technology leaders, seeking to strengthen appropriation and uptake. We also believe that equity considerations need to be reflected in every aspect of this study, and we have made some recommendations towards this. Well done, we look forward to a revised version!\n\nIs the rationale for, and objectives of, the study clearly described? Partly\n\nIs the study design appropriate for the research question? Partly\n\nAre sufficient details of the methods provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable",
"responses": [
{
"c_id": "5474",
"date": "12 May 2020",
"name": "Laura Martínez García",
"role": "Author Response",
"response": "Comment 1.1 - Detailed description of the study Response 1.1 We assume that the details requested by the reviewers in this comment are disaggregated below. We describe our responses to each of the comments below. Comment 1.2 - Objectives Response 1.2 The primary objective of the pilot study is to “explore the students’ and teachers’ experience when using the learning resources of the IHC project in the context of Barcelona (Spain).” To achieve this objective, we will conduct a pilot study in a convenience sample of three schools in Barcelona. Our principal results will be recommendations - both for practice and research - on how to use, how to adapt (if needed), and how to implement the IHC resources in this context. To evaluate the effect of the intervention, we would have to conduct a cluster-randomised trial [Nsangi 2017]. We already described this limitation in the “Discussion - Study strengths and limitations” section; the text reads “It is also worth noting that we will not be assessing the impact of the IHC resources in this study; due to this, we will not include a control group and we will not have a questionnaire validated to assess treatment claims for students.” Comment 1.3 - Scaling-up the intervention Response 1.3 We have modified the text in the “Discussion - Implications for practice and research” section according to the suggestion of the reviewers. The text reads: “The next contextualization activities will be: 1) content adaptation - if needed, 2) context analysis (exploring factors that can impact scaling up), and 3) validation of the CLAIM questionnaire test into Spanish for use in this context. The findings of the contextualization activities could inform the design of a cluster-randomised trial, to determine the effectiveness of the IHC resources in this context prior to scaling-up its use.”. Comment 1.4 - Abstract Response 1.4 We have modified the text in the “Abstract” section according to the suggestion of the reviewers. The text reads: “The findings of the contextualization activities could inform the design of a cluster-randomised trial, to determine the effectiveness of the IHC resources in this context prior to scaling-up its use.”. Comment 2.1 - Study population Response 2.1 We have included, as extended data, the description of the intervention in the schools using TIDieR checklist. The text now reads “The intervention in the schools will include: 1) a workshop with the teachers, and 2) lessons to the students (Extended data 4 provides a description of the intervention using the TIDieR checklist) [Hoffmann 2014, Martínez García 2020].”. We will describe the study population (children, teachers, parents) and relevant related variables (e.g. gender, socioeconomic stratum, ethnicity, or religion, etc.) in the publication of the results of the pilot study. Comment 2.2 - Eligibility criteria of the sample Response 2.2 Relating the eligibility criteria of the sample, please see “Response 2” for Reviewer 1. Comment 2.3 - Generalisation and implementation considerations Response 2.3 The primary objective of the pilot study is to “explore the students’ and teachers’ experience when using the learning resources of the IHC project in in the context of Barcelona (Spain).” To achieve this objective, we will conduct a pilot study in a convenience sample of three schools in Barcelona. Our principal results will be recommendations - both for practice and research - on how to use, how to adapt (if needed), and how to implement the IHC resources in this context. To consider to generalise the results, we would have to include a representative sample [Nsangi 2017]. We have included this limitation in the “Discussion - Study strengths and limitations” section; the text reads “The main limitation is using a convenience sample (small, geographically limited, and non-representative sample). However, we will provide a detailed description of our data collection and research context to help other stakeholders consider transferring our results to their settings.”. To evaluate implementation considerations, we would have to conduct a context analysis [IHC 2020]. The mail objectives of a context analysis would be: 1) explore what demand there is for learning resources for teaching critical thinking about health in schools, 2) map where teaching critical thinking about health best fits in the curriculum, 3) identify and examine relevant resources already in use, and 4) explore conditions for introducing new learning resources [IHC 2020]. The development of context analysis in our setting is already considered in the “Discussion - Implications for practice and research” section, the text reads: “The next contextualization activities will be: 1) content adaptation - if needed, 2) context analysis, and 3) validation of the CLAIM questionnaire test into Spanish for use in this context. Finally, a cluster randomised trial with the adapted IHC resources could be conducted to evaluate their effect on the students’ ability to assess treatment claims in this context.”. Comment 2.4- Ethical considerations Response 2.4 We have modified the text in the “Ethical considerations” section according to the suggestion of the reviewers. The text now reads: “The study protocol has obtained an approval exemption (does not include patients, biological specimens or clinical data) from the Ethics Committee of the Hospital de la Santa Creu i Sant Pau (Barcelona, Spain) [CEIm 2020]. We will inform participants about the pilot study and will request their written informed consent (Extended data 1-3) [Martínez García 2020]. If a family does not want to participate, the student will participate in the lessons as a curricular activity but will not participate in any of the study data collection activities. We will anonymise the data removing participant and school name.” Comment 3.1 - Gantt chart Response 3.1 We have modified the text and Figure 2 according to the suggestion of the reviewers. The text now reads “Figure 2 is a Gantt chart illustrating the schedule of the pilot study.” Comment 3.2 - Eligibility criteria of the sample Response 3.2 Relating the eligibility criteria of the sample, please see “Response 2” for Reviewer 1. Relating the implementation considerations, please see “Response 2.3” above. Comment 3.3 - IHC-Barcelona Working Group Response 3.3 To establish the IHC-Barcelona Working Group, we only considered profession criteria (researchers, teachers, paediatricians, education and health stakeholders, and translators) or family-related criteria (student and family representatives). We will describe detail about IHC-Barcelona Working Group in the publication of the results of the pilot study. Comment 3.4 - Escola Nova 2 Response 3.4 Relating the eligibility criteria of the sample, please see “Response 2” for Reviewer 1. Comment 3.5 - Contextualising IHC resources Response 3.5 The primary objective of the pilot study is to “explore the students’ and teachers’ experience when using the learning resources of the IHC project in the context of Barcelona (Spain).” To achieve this objective, we will conduct a pilot study in a convenience sample of three schools in Barcelona. Our principal results will be recommendations - both for practice and research - on how to use, how to adapt (if needed), and how to implement the IHC resources in this context. The IHC project team has developed several guidelines for contextualising IHC resources [IHC 2019a, IHC 2019b, IHC 2017a, IHC 2017b]. Different working groups from more than 20 countries are adapting, or planning adaptation, of the IHC resources to their context [IHC Newsletter 2019, IHC Newsletter 2020]. Comment 3.6 - Sample and eligibility criteria Response 3.6 Relating the sample and eligibility criteria, please see “Response 2” for Reviewer 1. Comment 3.7 - Gantt chart Response 3.7 The schedule of the pilot study is illustrated in Figure 2. We will describe detail about data collection in the publication of the results of the pilot study. Comment 3.8 - Study population Response 3.8 We will describe detail about co-educational classes or the number of students in the publication of the results of the pilot study. Comment 4.1 - Study variables Response 4.1 The variables of the pilot study are already listed in “Table 3. Pilot study variables”. The reviewers' proposal “to include a data analysis plan” [Simpson 2015] is not a request of the F1000Research journal [https://f1000research.com/for-authors/article-guidelines/study-protocols]. Comment 4.2 - Variability of the assessments Response 4.2 We will use ad hoc questionnaires and guides to collect the data to minimize the variability of the assessments. We included questionnaires and guides as extended data, available at: https://figshare.com/articles/IHC_BCNPilotStudy/12221189/1. Comment 4.3 - Data collection Response 4.3 We have included a review process to improve the precision of the data that we will collect from “Non-participatory observations during the lessons” and “Semi-structured interviews with the students after a lesson”. The included text reads: “Another researcher will check the notes with the recorded audios. The two researchers will resolve potential disagreements by discussion, and if necessary, by consulting a third researcher.” Comment 4.4 - Dissemination of the results Response 4.4 We already included education and science authorities in the “Dissemination of the results” section; the text reads: “The dissemination activities of the pilot study results will include: 1) publication in a peer-reviewed journal, 2) publication in several Internet resources (for example, related web pages, electronic bulletins and social media), and 3) introduction to the different users of interest (researchers, teachers, paediatricians, student representatives, family representatives, education and health stakeholders, and translators) in conferences, workshops and meetings.”. Relating the implementation considerations, please see “Response 2.3” above. Comment 4.5 - IHC-Barcelona Working Group Response 4.5 Relating the description of IHC-Barcelona Working Group, please see “Response 3.3” above. Comment 4.6 - COVID19 Response 4.6 The first version of the “Learning to make informed health choices: Protocol for a pilot study in schools in Barcelona” was published on 28 Nov 2019 [Martínez García 2019]. We will describe detail about preparations to adjust to the COVID19 in the publication of the results of the pilot study. References [CEIm 2020] Ethics Committee for research with medicinal products (CEIm). 2020 [online]. Available at: http://www.recercasantpau.cat/en/clinical-research/ceic/. [Accessed April 16, 2020]. [Hoffmann 2014] Hoffmann TC, Glasziou PP, Boutron I, Milne R, Perera R, Moher D, et al. Better reporting of interventions: template for intervention description and replication (TIDieR) checklist and guide. BMJ. 2014;348:g1687. [IHC 2017a] The Informed Health Choices group. Guide for piloting the Informed Health Choices (IHC) learning resources. IHC Working Paper, Norwegian Institute of Public Health, 2017. [IHC 2017b] The Informed Health Choices group. Guide for translating and adapting the Informed Health Choices (IHC) podcast. Informed Health Choices Working Paper, Norwegian Institute of Public Health, 2017. [IHC 2019a] The Informed Health Choices group. Guide for translating the Informed Health Choices school resources. Informed Health Choices Working Paper, Norwegian Institute of Public Health, 2019. [IHC 2019b] The Informed Health Choices group. Resource production guide for translating and adapting Informed Health Choices learning resources. Informed Health Choices Working Paper, Norwegian Institute of Public Health, 2019. [IHC 2020] IHC CHOICE team. Protocol for a context analysis: Exploring the considerations for introducing digital learning resources for critical thinking about health in secondary schools. Informed Health Choices Working Paper, Norwegian Institute of Public Health, 2020. [IHC Newsletter 2019] The Informed Health Choices Group. Informed Health Choices Newsletter 2019 Norwegian Institute of Public Health. 2019 [online]. Available at: https://www.informedhealthchoices.org/wp-content/uploads/2019/03/IHC-Newsletter-2019.pdf [Accessed April 16, 2020]. [IHC Newsletter 2020] The Informed Health Choices Group. Informed Health Choices Newsletter 2020. Norwegian Institute of Public Health. 2020 [online]. Available at: https://www.informedhealthchoices.org/wp-content/uploads/2020/02/2020-Informed-Health-Choices-newsletter-1.pdf. [Accessed April 16, 2020]. [Martínez García 2019] Martínez García L, Alonso-Coello P, Asso Ministral L, Ballesté-Delpierre C, Canelo Aybar C, de Britos C, et al. Learning to make informed health choices: Protocol for a pilot study in schools in Barcelona [version 1; peer review: awaiting peer review]. F1000Research. 2020 (https://f1000research.com/articles/8-2018). [Martínez García 2020] Martínez García L. IHC@BCNPilotStudy [Internet]. figshare; 2020. Available at: https://figshare.com/articles/IHC_BCNPilotStudy/12221189/1 [Accessed April 30, 2020]. [Nsangi 2017] Nsangi A, Semakula D, Oxman AD, Austvoll-Dahlgren A, Oxman M, Rosenbaum S, et al. Effects of the Informed Health Choices primary school intervention on the ability of children in Uganda to assess the reliability of claims about treatment effects: a cluster-randomised controlled trial. Lancet. 2017;390(10092):374-388. [Simpson 2015] Simpson SH. Creating a Data Analysis Plan: What to Consider When Choosing Statistics for a Study. Can J Hosp Pharm. 2015; 68(4): 311–317."
}
]
}
] | 1
|
https://f1000research.com/articles/8-2018
|
https://f1000research.com/articles/8-1521/v1
|
28 Aug 19
|
{
"type": "Software Tool Article",
"title": "Pathfinder: open source software for analyzing spatial navigation search strategies",
"authors": [
"Matthew B. Cooke",
"Timothy P. O'Leary",
"Phelan Harris",
"Richard E. Brown",
"Jason S. Snyder",
"Timothy P. O'Leary",
"Phelan Harris",
"Richard E. Brown"
],
"abstract": "Spatial navigation is a universal behavior that varies depending on goals, experience and available sensory stimuli. Spatial navigational tasks are routinely used to study learning, memory and goal-directed behavior, in both animals and humans. One popular paradigm for testing spatial memory is the Morris water maze, where subjects learn the location of a hidden platform that offers escape from a pool of water. Researchers typically express learning as a function of the latency to escape, though this reveals little about the underlying navigational strategies. Recently, a number of studies have begun to classify water maze search strategies in order to clarify the precise spatial and mnemonic functions of different brain regions, and to identify which aspects of spatial memory are disrupted in disease models. However, despite their usefulness, strategy analyses have not been widely adopted due to the lack of software to automate analyses. To address this need we developed Pathfinder, an open source application for analyzing spatial navigation behaviors. In a representative dataset, we show that Pathfinder effectively characterizes the development of highly-specific spatial search strategies as male and female mice learn a standard spatial water maze. Pathfinder can read data files from commercially- and freely-available software packages, is optimized for classifying search strategies in water maze paradigms, and can also be used to analyze 2D navigation by other species, and in other tasks, as long as timestamped xy coordinates are available. Pathfinder is simple to use, can automatically determine pool and platform geometry, generates heat maps, analyzes navigation with respect to multiple goal locations, and can be updated to accommodate future developments in spatial behavioral analyses. Given these features, Pathfinder may be a useful tool for studying how navigational strategies are regulated by the environment, depend on specific neural circuits, and are altered by pathology.",
"keywords": [
"search strategy",
"water maze",
"learning",
"memory",
"rodent",
"reversal",
"goal"
],
"content": "Introduction\n\nAll living organisms move throughout space to survive. Amongst mammals, there is a diversity of spatial behaviors that depend on numerous factors such as anxiety1,2, learning3, and the nature and pattern of stimuli that predict goals4–6. Given rodents’ natural propensity to explore stimuli and environments, an array of rodent navigational tasks have been developed to investigate how various brain regions interact to control goal-direct behavior7. This has routinely been conducted using fixed-trajectory mazes such as the T-maze or radial maze. While these dry maze paradigms offer the convenience of fixed choice points that reduce ambiguity associated with classifying decisions and navigational responses, they cannot be used to study patterns of exploration in open environments.\n\nA popular approach for studying free navigation in animals has been the water maze, where rodents learn the location of a hidden escape platform in a pool of water based on distal and/or local cue configurations3 Early studies validated the usefulness of the water maze for studying spatial processing and described progressive stages of learning where a rodent searches for the platform with increasing spatial specificity8,9. The vast majority of studies have since used escape latency or path length as primary measures of spatial learning. However, water maze navigation is unconstrained and animals can solve the task using different strategies that may not always differ in terms of the time it takes to reach the platform8,9. Thus, while latency and path length measures are convenient, they discard a rich amount of behavioral data.\n\nOver the years, a number of groups have described manual and automated methods for classifying search strategies used by animals and humans in water maze experiments8,10–19. By mathematically relating the swim path to features of the maze environment one can identify and quantify the types of search strategies employed. Search strategy analyses have revealed that the ventral hippocampus is involved in coarse spatial goal-directed search16, that adult neurogenesis promotes spatially precise search20, and that spatially accurate search is reduced in humans with, and/or animal models of, Alzheimer’s disease21,22, autism23, traumatic brain injury22,24 and aging14,25. Despite the utility of these analyses they have been relatively uncommon to date, likely because commercially-available software packages often do not perform these analyses and the analytic methods used in previous work are not typically available in the form of an easy-to-use software package.\n\nTo facilitate the study of navigational search strategies, whether in the water maze or other 2-dimensional navigational paradigms, we created a new software application called Pathfinder. Pathfinder is a Python-based, open source tool with an intuitive graphical user interface and adjustable parameters for conducting detailed analyses of spatial search patterns. We validate Pathfinder with a mouse water maze dataset, where we find that male and female mice develop increasingly specific and direct spatial search strategies with additional days of training.\n\n\nMethods\n\nPathfinder is freely available under the GNU General Public License version 3.0.\n\nDetailed instructions on use and installation of the program can be found on Github at github.com/MatthewBCooke/Pathfinder. We recommend installing Anaconda for Python 3, as it includes all of the following packages that are needed to run Pathfinder: PIL (https://pillow.readthedocs.io/en/latest/), xlrd (https://xlrd.readthedocs.io/en/latest/), numpy (https://www.numpy.org), pickle (https://docs.python.org/3/library/pickle.html), scipy (https://www.scipy.org), matplotlib (https://matplotlib.org), and tkinter (https://wiki.python.org/moin/TkInter). The MATLAB engine is optional and needs to be installed separately for entropy calculations (MATLAB and Statistics Toolbox Release 2018b, The MathWorks, Inc., Natick, Massachusetts, United States). Once Anaconda is installed, Pathfinder can be downloaded via Github or by typing “pip install pathfinder” in a shell window (i.e. Mac terminal or Windows command line). Pathfinder is then opened by typing “pathfinder” into the shell window and pressing return.\n\nPathfinder has a simple, user-friendly interface for extracting information from spatial navigation tracking files that contain xy coordinates over time (Figure 1). While it can be used to analyze multiple types of 2D navigational data, it is optimized for rodent spatial water maze experiments and accepts inputs from commonly-used commercial tracking software, including Ethovison (Noldus), Anymaze (Stoelting) and WaterMaze (Actimetrics). Pathfinder can also open files exported from the open source tracking software, ezTrack26, enabling a cost-effective and fully open source workflow for detailed water maze behavioral analyses. Trial information from these programs are outputted in CSV or Excel format, which can then be inputted into Pathfinder through the File menu. The experimental setup is specified in the main window (Figure 1a). Pathfinder can automatically calculate the position and size of the maze and the goal location (provided they are constant across trials), or these parameters can be entered manually.\n\na) Screenshot of the main application window, where maze geometry is defined and input and output settings are established. b) Maze schematic and geometry for defining variables. The chaining corridor is centered on the goal platform and extends throughout all 4 quadrants; its width is specified in the main window. The larger thigmotaxis zone is specified in the main window; Pathfinder calculates the smaller thigmotaxis zone as half the width. Heading error is the angular distance between the actual path direction and a straight line to the goal (Pathfinder calculates average heading error at all points; only a single example shown). The angular corridor is used to define the directed search strategy, which depends on the accuracy of the animal’s trajectory as it approaches the platform. The width of the corridor (in degrees) is specified in the main window and is centered on the goal. c) Schematic of the Ideal Path Error (IPE) metric. The distance from the platform is measured at each timepoint provided by the tracking software (actual path; only a fraction of distances shown for clarity) to provide a cumulative distance measure. Assuming the same swim speed as the actual path, distances are similarly summed from the ideal path, to provide a cumulative ideal path measure. The ideal cumulative distance is subtracted from the actual cumulative distance to generate the IPE. d) Parameter bounds are entered in the settings window. e) The manual categorization window, for viewing trial paths and manually categorizing strategies.\n\nPathfinder relies on several variables that describe navigation relative to the pool and platform geometry: 1) Ideal Path Error (IPE): the summed error of the search path (Figure 1c). It is conceptually similar to the Cumulative Search Error (CSE) since it also measures proximity to the goal throughout the trial9,27. An advantage of proximity measures is that they can distinguish two trials that have equivalent latencies/path lengths but differ in average distance to the platform. When calculating the IPE, the distance from the goal is measured at each time point in the trial and summed to generate a cumulative distance measure of the actual path (similar to CSE). In contrast to the CSE, the IPE is calculated by subtracting the cumulative ideal path distance from the cumulative actual path distance. The cumulative ideal path is simply the sum of all of the distances between the goal and the position of the animal if it swam along a straight line to escape, using the average velocity from the trial. 2) Heading error: the angular distance between the current path and a straight line to the goal location. The current path direction is defined by a line connecting two temporally-adjacent xy coordinates. The average heading error is an average of all of the heading error values for the trial and the initial heading error is the average of the heading error values for the first second of the trial.\n\nAdditional variables are user-defined on the main window: 3) Angular Corridor Width: the size of the angular navigational corridor (in degrees) that extends from the start location and widens towards the goal, centered on the goal location. 4) Chaining Annulus Width: the width of the chaining annulus, a donut-shaped zone that is centered on the goal and spans all areas of the maze at a fixed distance from the maze wall. 5) Thigmotaxis zone size: the width of a zone that spans the perimeter of the maze and extends inward from the maze wall. Pathfinder also defines a “small” thigmotaxic zone that is half the width of this value. 6) Add goal: Pathfinder will perform all calculations and strategy analyses with respect to an unlimited number of goal locations. This can be used to measure performance and characterize strategies with respect to multiple goal locations (e.g. during spatial reversal, spatial choice). Selecting “truncate trials” will artificially end the trials if/when the subject reaches the additional goal locations. This is necessary, for example, to measure direct trajectories to a former goal location in a reversal paradigm (since the strategy will no longer meet direct path criteria if the former location in contacted and search continues elsewhere in the maze).\n\nOnce the variables are defined, boundaries must be set to establish the criteria for strategy categorization. Clicking “settings” will open up an additional window where strategy options can be selected and parameter bounds can be set (Figure 1d). Upon clicking “calculate”, Pathfinder categorizes trials into one of eight search strategies that are ordered according to the degree of spatial specificity (high to low): 1) direct path, 2) focal search, 3) directed search, 4) indirect search, 5) chaining, 6) scanning, 7) random search, and 8) thigmotaxis. These categories are mutually exclusive and follow a defined order (1 to 8), but the user can opt to exclude strategies from the analysis. Thus, Pathfinder determines, in a stepwise fashion, whether a given trial fulfills the criteria for direct swim. If so, it moves on to categorize the next trial. If not, it determines whether the trial fits the subsequent strategy, and so on. The strategies and their parameters are shown in Figure 2. In the output file (.csv), each trial is categorized and the following additional metrics are provided: latency and distance travelled to reach the goal, average distance from the goal, percent of maze traversed, velocity, initial and average heading error and IPE. Pathfinder also has the ability to calculate the entropy for each trial, a measure of disorder in the path, relative to the goal location. The entropy calculation calls the MATLAB engine and requires a MATLAB license. Entropy measures the performance by looking at a shift from more disordered swimming (high entropy) to more spatially strategic paths (low entropy), and has been previously found to be highly sensitive to water maze search performance28. Due to the manipulation of large matrices, calculating the entropy of trials is very slow.\n\nPathfinder categorizes each trial according to 1 of 8 possible strategies. Categorization proceeds sequentially in the order shown (unless some strategies are excluded from the analysis). For example, for a trial to be classified as Random Search, the path must cover a minimum proportion of the maze and not fit any of the criteria for strategies 1–6. In the examples shown, the blue square indicates the start point and the green circle indicates the middle of the pool. Parameter settings are those used in the present study and should be adjusted depending on changes to testing procedures and maze geometry.\n\nOccasionally, some trials cannot be categorized. The user therefore has the option to manually categorize uncategorized trials, by selecting this option on the main window. Additionally, there is an option to manually categorize all trials. Here, Pathfinder provides an image of the trial as well as shortcut keys to select the appropriate strategy. The software will also display the strategy it had automatically categorized for the displayed trial. Manual categorization will not overwrite the automatic categorization but will be displayed separately in the output file. This allows for comparison between the automatically calculated and user-selected strategy.\n\nIn addition to strategy categorization, Pathfinder will also create heatmaps as a useful visual representation of groups of trials. This is accomplished by counting the number of times animal(s) visit each bin in a hexagonal array that is overlaid on the maze (bin size is user-defined). The range of colors (cool to warm) can be automatically set to occupy the full scale. Alternatively, the user can manually set the maximum, above which all bins will read the hottest.\n\nAnimals. A group of 35 C57BL/6J mice (18 male, 17 female) were used in this experiment (Jackson Laboratories, Bar Harbor, Maine). Relative to commonly-used samples sizes of 8–10 mice/group, a large cohort was used to maximize reliability and detect potentially infrequent strategies. Mice were housed in same-sex groups (2–4/cage) in polyethylene cages (30 × 19 × 13cm) with pine chip bedding and a small tube and food and water available ad-libitum. Mice were housed under a reversed light-dark cycle (lights off 8:00am–8:00pm) and completed water-maze testing in the dark phase. Mice were first tested on the Barnes maze29 and were 18 weeks old when tested on the water maze for the current experiment. All efforts were made to minimize animal suffering, and all procedures adhered to guidelines from the Canadian Council on Animal Care and were approved by the Dalhousie University Committee on Laboratory Animals.\n\nSpatial water maze training. The water maze consisted of a plastic circular pool (110 cm diameter) painted black. The pool was filled with water (21–23°C), which was made opaque with the addition of non-toxic white tempera paint (Schola). A circular escape platform (14 cm height, 9 cm diameter) was positioned 1 cm below the water. The water maze was placed in a diffusely lit room with many extra-maze visual cues (posters on walls, a desk, the experimenter, geometric layout of testing room etc.).\n\nAnimals were tested over a total of 15 days. They first completed 8 days of acquisition training (A1-A8) with a hidden escape platform (4 trials/day). Across trials, mice were released into the pool from four different locations, with the order differing across mice. They were given a maximum of 60 sec to locate the escape platform, after which they were guided to the platform by the experimenter. Mice remained on the platform for 15–20 seconds before being removed from the pool. During daily test sessions, mice were tested in squads of 4 and each mouse was held in separate cages filled with a bedding of paper towel. The inter-trial interval ranged from 2–8 minutes. The day following acquisition training, memory was assessed with a single 60-sec probe trial with no escape platform present. Mice then completed a single day of re-training (Retrain) to reduce extinction that may occur during the probe trial. During re-training the escape platform is returned to the same location used in acquisition training.\n\nAfter acquisition re-training, reversal learning was assessed over 3 days (R1-R3) with the escape platform moved to the opposite side of the maze. A reversal probe trial (R probe) was then completed to assess memory for the location of the new escape platform location. Finally, a single day of visible platform training (Visible platform; 4 trials) was completed, where the escape platform was moved to a new location and made visible with the addition of a striped flag.\n\nBehavior was recorded with the WaterMaze (Actimetrics) video tracking system (5 samples per second), via a camera placed directly above the pool.\n\n\nResults\n\nTo validate Pathfinder, we trained mice for 8 days on a spatial water maze such that they achieved asymptotic performance according to standard metrics and should therefore have adopted distinct navigational strategies as they learned the procedural and spatial task demands. Following acquisition, mice received an unreinforced probe trial, 1 day of retraining, 3 days of reversal training (platform in opposite side of pool), another probe trial, and one day of visible platform training (outlined in Figure 3a).\n\na) Schematic outline of full behavioral paradigm. Individual performance metrics were analyzed for acquisition (b–f) and reversal (g–k) stages of testing. b) Latency to reach the platform decreased across days (day effect F7,231=75, P<0.0001; sex effect F1,33=0.3, P=0.6; interaction F7,231=0.9, P=0.5). Asterisks denote statistically significant differences from the subsequent days that are indicated by the numbers. c) Initial heading error decreased over days (day effect F7,231=39, P<0.0001; sex effect F1,33=0.4, P=0.6; interaction F7,231=0.7, P=0.6). d) Average heading error decreased over days (day effect F7,231=48, P<0.0001; sex effect F1,33=2.5, P=0.12; interaction F7,231=0.6, P=0.8). e) Idea path error decreased over days (day effect F7,231=79, P<0.0001; sex effect F1,33=0.3, P=0.6; interaction F7,231=1.0, P=0.4). f) Entropy decreased over days (day effect F7,231=75, P<0.0001; sex effect F1,33=1.3, P=0.3; interaction F7,231=0.5, P=0.8). g) Latency decreased over days (day effect F2,66=69, P<0.0001; sex effect F1,33=0.5, P=0.5; interaction F2,66=0.7, P=0.5). h) Initial heading error decreased over days and was greater in females on day 1 (day effect F2,66=9, P<0.001; sex effect F1,33=1.0, P=0.3; interaction F2,66=4.7, P=0.01). i) Average heading error decreased over days (day effect F2,66=21, P<0.0001; sex effect F1,33=0.2, P=0.7; interaction F2,66=0.9, P=0.4). j) Ideal path error decreased over days (day effect F2,66=98, P<0.0001; sex effect F1,33=0.5, P=0.5; interaction F2,66=0.7, P=0.5). k) Entropy decreased over days (day effect F2,66=39, P<0.0001; sex effect F1,33=0.6, P=0.4; interaction F2,66=2.6, P=0.08). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, †P<0.05 within day, male vs female comparison. Symbols = mean ± standard error.\n\nTo confirm that mice learned the task, we first analyzed performance using several metrics that indicate learning but do not reveal details about navigational strategies (Figure 3). We focused on acquisition and reversal phases since they are the main focus of our subsequent strategy analyses. Over the 8 days of acquisition, mice reached the platform faster, increasingly swam in the direction of the platform as measured by heading angle error and had lower IPE and entropy scores. The greatest performance improvements occurred during the first 4 days and, while all measures revealed improvements beyond day 4, only average heading error and entropy analyses revealed improvements beyond day 5. There were no sex differences in acquisition performance.\n\nReversal learning performance improvements were mostly apparent after the first day of training, likely because mice had learned the procedural aspects of the task and the spatial environment, and only had to learn a new platform location (Figure 3g–k)30. Path entropy decreased from days 2–3, indicating continued learning. Females and males were equivalent in all performance measures except males had a lower initial heading error on day 1 of reversal training (Figure 3h).\n\nPathfinder revealed clear differences in search strategies over days of training (Figure 4). Over the first two days of acquisition, mice were initially thigmotaxic. After learning that the pool wall did not afford escape, they then transitioned to chaining, random and scanning search patterns, all of which indicate spatially non-specific search away from the pool wall. Over days 2–3 mice transitioned to spatially-specific forms of search, with ~30% performing indirect searches to locate the platform. A similar proportion of trials were indirect searches over days 2–8 of training. Mice increasingly displayed directed searches, focal searches and direct paths such that, by the end of training, search was spatially specific on over 80% of trials. There were no major sex differences in strategy. The usefulness of strategy analyses (at least with default settings) for long probe trials is limited since spatially-specific strategies rely on IPE, which rapidly increases with trial duration. Additionally, animals will change strategies as they learn that the escape platform is not available in the expected location. Indeed, when the probe trial analysis was restricted to the first 10s, mice displayed focal and directed search strategies, indicating perseveration at the former platform location. When the analysis was conducted on longer segments, chaining was common, indicating that mice adopted a procedural strategy of searching in similar regions throughout the pool. Finally, when examining the entire probe trial, scanning and random searches dominated, indicating that mice eventually abandoned strategies that were no longer successful. During reversal, spatial specificity was initially very poor; mice primarily scanned, indicating preserved knowledge of the procedural requirements but no knowledge of the platform location. By the end of day 2 mice displayed levels of spatially-specific search strategies that were comparable to those at the end of the acquisition phase. Using the “add goal” feature, we also analyzed reversal strategies with respect to the original goal location (Figure 4b). This revealed a number of direct paths to the goal on the first day that quickly dissipated with additional trials as mice learning the new platform location.\n\na) Search strategies for male and female mice. Each set of stacked bars indicates strategies used for the 4 acquisition and reversal trials for each day. Probe strategies are shown for the entire trial (0–60s) and for the first 10, 20 and 30s. b) Reversal strategies relative to the original platform location (indirect search excluded from analyses, since short swims that bypass the old location but quickly go to the new location become incorrectly classified as indirect searches with current settings). c) Escape latencies for all 1888 trials varied by strategy. Symbols indicate individual trials, bars indicate means (Kruskal Wallis test, P<0.0001; Dunn’s tests: direct path vs all others except focal search, P<0.0001; focal search vs all others except direct path, P<0.0001; directed search vs all others except indirect search, P<0.0001; indirect search vs all others except directed search, P<0.0001; chaining vs all others except scanning and random, P<0.01; scanning vs all others except chaining and thigmotaxis, P<0.01; random vs all others except chaining, P<0.05; thigmotaxis vs all others except random search, P<0.05). d) Manual vs automatic categorization. For each strategy assigned by Pathfinder, the proportion that received the same classification (manually) by 2 raters is shown. “Raters 1+2” indicates the percentage of Pathfinder classified-trials that also received the same classification by both raters. e) Search strategy classification by rater 1 for each day of testing. f) Search strategy classification by rater 2 for each day of testing. g) Search strategy classification by Pathfinder for each day of testing.\n\nTo investigate possible relationships between strategy and conventional measures of water maze performance, we examined escape latencies for each strategy type, over all trials (1888 trials from all 15 days of testing; Figure 4c). Direct swim trials had the lowest latencies (2.9s on average) and was followed by the other spatially-specific strategies (focal search, 3.6s; directed search, 6.7s; indirect search, 5.8s). Non-specific strategies that avoided the pool wall were all significantly worse than the spatially specific strategies (chaining, 29s; scanning, 32s; random, 38s), and thigmotaxic trials were significantly worse than all other trial types (58s).\n\nTo determine how Pathfinder compared to subjective assessment of strategy, we compared Pathfinder categorization to manual scores generated by 2 independent raters (all trials). Rater 1 had experience in mouse behavior testing, but only brief training on water maze strategy classification. Rater 2 developed Pathfinder (MBC) and had extensive experience with strategy classification. Figure 4d shows the proportion of Pathfinder-categorized trials that received the same strategy classification via the manual raters. The greatest correspondence between automatic and manual categorization was seen for direct swims and thigmotaxis (~80% for both). Automatic-manual consistency was much lower for the other strategies, ranged from 25–75% and differed for the 2 raters. Overall consistency between the 2 manual raters was 65%. These data highlight the difficulty of intuitively differentiating complex search paths. Interestingly, when we averaged strategy analyses over all 15 days of testing, automatic and manual categorization resulted in similar patterns (Figure 4e–g). Thus, manual scoring is unreliable at the level of an individual trial, and human error can be masked when data are averaged.\n\nTo provide an intuitive visual inspection of search performance, we used Pathfinder to generate heatmaps of spatial occupancy at stages of testing that differed in spatial search patterns (Figure 5). Averaged over all trials and across sexes, mice swam in close proximity to the pool wall on day 1 of initial acquisition. By days 3 and 8 search was increasingly focused near the goal. Spatial preference was clearest on the probe trial, since these trials provided a longer temporal window to accumulate spatial occupancy samples. Day 1 of reversal testing resembled the probe trial, since mice spent the majority of time in the former platform location. By day 3, and on the probe trial, their spatial preference had shifted to the new, correct location. One set of heatmaps are presented using Pathfinder’s auto scale feature, which maximizes the color range within a trial and can be useful for visualizing within-trial details since it avoids saturation. However, by differentially scaling, it can also obscure or inflate differences across trials. We therefore include a second set of heatmaps that are all scaled equivalently.\n\nExamples of heatmaps for various testing days (all trials from both sexes combined). Top rows: heatmaps were automatically scaled by Pathfinder, to occupy the full color spectrum and facilitate visualization of spatial occupancy within a given day. Bottom rows: heatmaps were set to a common scale, to facilitate comparison across days. Scale indicates number of samples within a spatial bin.\n\n\nDiscussion\n\nHere we describe Pathfinder, an easy-to-use software package for analyzing patterns of spatial navigation. Pathfinder performs automatic classification of multiple search strategies that have been previously described in the rodent water maze, but it can also be used for analyzing navigational behavior in dry mazes, virtual mazes or any other environment where xy coordinates are provided. Currently, Pathfinder accepts inputs from three commonly-used, commercially-available tracking programs (Ethovision, Anymaze, Watermaze) and also the freely-available tracking software, ezTrack26. It requires no programming knowledge, but is open source and can be expanded by developers in the future. Using a mouse water maze dataset, we validated Pathfinder’s performance and found that mice progressed through a series of search strategies that had increasing levels of spatial search specificity, consistent with earlier reports16–18,20,21,25,31. Mice initially displayed thigmotaxic, random and chaining search strategies as they learned the procedural components of the task. Pathfinder effectively demonstrated that mice transitioned to spatially-specific, presumably hippocampal-dependent, strategies during the later stages of training. Pathfinder also revealed the reverse transition from spatial to procedural to random strategies in the probe trial. By analyzing reversal performance with respect to multiple goal locations, Pathfinder showed that mice redirect their spatial search from the previously-reinforced platform location to the new location. Mice displayed a variety of search strategies on any given day, even after escape latency performance had plateaued. Since manual classification based on static images of swim paths was slow and inconsistent, Pathfinder may therefore be a useful tool for objectively characterizing swim strategies in the rodent water maze and 2D spatial navigation in other behavioral paradigms.\n\nThe water maze was initially described nearly 40 years ago and quickly became popular due to the ease of training, strong motivation for escape, and consistent reliance on hippocampal function3,32. Escape latency and path length were quickly adopted as the primary measures of learning and, due to their simplicity and sufficiency for many experimental situations, they remain the most commonly-used metrics. However, they cannot always differentiate between behaviors that vary in the degree of spatial bias. For example, animals that employ a chaining strategy search nonspecifically in some cases can reach the platform as fast as animals that perform a directed spatial search (Figure 4c). Latency and path length are also less capable of detecting age-related impairments in spatial learning, prompting development of measures of proximity to the goal location, which has proven to be highly sensitive to group differences in both training and probe trial performance9,27,33. Our IPE proximity measure is similar to previous proximity measures with the exception that the cumulative ideal path distance is subtracted from the cumulative actual path distance to generate a path error measure. Finally, another recent metric that has been reported to be even more sensitive to group differences in spatial probe trial performance is entropy28. Entropy is originally a measure of thermodynamic disorder in a system but, when applied to the distribution of sampled sites in a maze, can also be used to measure the transition from high to low disorder in navigation, as animals focus their search on the precise goal location. By default, Pathfinder applies equal weighting to the path and goal components of the entropy measure and, compared to standard metrics, entropy was slightly better at detecting performance changes during the later stages of water maze acquisition.\n\nDespite their convenience, even the most precise individual measures cannot distinguish between multiple possible strategies that an animal might employ to reach a goal location. Thus, strategy analyses may be valuable for identifying the role that different circuits play in guiding behavior. Consistent with the lower spatial resolution of ventral hippocampal place cells34, strategy analyses have found that the ventral hippocampus is particularly important for developing coarse, non-specific search patterns in the water maze and that increasingly spatially localized search depends on sequential recruitment of intermediate and then dorsal hippocampus16. Adult-born neurons are believed to promote memory precision and, indeed, blocking neurogenesis greatly reduced the adoption of spatially-specific search strategies20. Strategy analyses in animals have revealed spatial precision-related deficits in models of aging25, stroke35, traumatic brain injury22,24, autism23 and Alzheimer’s pathology21,22. With the advent of virtual reality, it has also become possible to test whether rodent water maze findings generalize to humans15. Indeed, hippocampal damage and CA1-specific lesions impair human water maze performance according to standard measures such as latency to reach the platform36,37. Human water maze experiments have also revealed superior spatial memory and greater spatial strategy use in younger individuals, and in males compared to females14. Here, we did not observe any sex differences, consistent with a recent meta analysis that revealed that male and female mice display broadly comparable performance in the spatial water maze38.\n\nGiven the apparent utility of strategy classification, the question arises as to why it has not been used more extensively. One likely explanation is that it is not a standard feature of commercially-available software packages, therefore requiring time and programming experience to execute. Groups that have performed strategy analyses have developed their own software, using either a predefined parameter-based approach, like ours, or machine learning algorithms that classify based on user input8,11,12,15–20,25,39. Since most previous approaches have not been developed into freely-available software packages, Pathfinder may enable more widespread adoption of strategy analyses. Moreover, in conjunction with freely-available tracking programs such as ezTrack (which is already supported) or others39, users should be able to easily perform advanced navigation analyses at little cost.\n\nIt is worth noting that, with respect to water maze analyses, some behaviors (e.g. chaining and thigmotaxis) have been relatively well-described. In contrast, differences between spatially-specific search patterns (direct swim, directed search, focal search, indirect search) may be intuitive and quantifiable but the extent to which they are meaningful and result from distinct neural processes is less clear. Certainly, the fact that search strategies can now be easily quantified opens the door to future studies of the biology of complex navigation strategies. However, to some extent, strategy definitions are arbitrary, and it is therefore incumbent upon the user to determine which behaviors are relevant for their experimental paradigm.\n\nOne area where Pathfinder could be useful is for assessing spatial bias and choice behavior when there are multiple goal locations. Indeed, the water maze has been effectively used to study visuospatial goal discrimination40,41 and cue vs place-related choice behavior42,43. We have recently used Pathfinder to show that neurogenesis promotes spatial platform preference in a spatial alternation water maze, which was detected by a greater number of direct swims when the platform was in the rat’s preferred location than when it was in the non-preferred location44. Neurogenesis-deficient rats often vacillated between the two platform locations, similar to vicarious trial and error behavior that has been described at choice points in dry mazes45. Future software developments could possibly incorporate these types of movements between competing goal options to detect indecisiveness as animals refine goal-directed navigation behavior. Swim speeds are also currently not factored into Pathfinder’s classification scheme, but could provide useful additional information for strategies that incorporate goal expectancy46,47 or a transition between place- and cue-directed navigation5. In the water maze, multiple platform locations (> 2) are typically only used in matching-to-place variants where it is expected that subjects quickly forget previous goal locations30. However, there is evidence that search patterns can reflect memory for many individual goal locations as well as the overall distribution of goals, at recent and remote post-training intervals, respectively6. Since Pathfinder can analyze navigation with respect to an unlimited number of goal locations, it may be useful for future investigations of how multiple spatial goals interact to guide search.\n\nSpatial navigation and exploration have been studied in many paradigms and so it is worth reiterating that Pathfinder could be applied to study navigation by any species, in any open 2D environment, and not just the water maze. For example, it could be used to measure the spatial precision of homing behavior48,49, spatial preferences of mammals or invertebrates in novel environments50–52, or navigation with respect to other environmental features that are known to drive firing of select populations of neurons, such as local and distal cues53, objects54,55 and environmental borders56. An array of virtual environments also opens the door to similar analyses of spatial navigation in humans36,37,57,58. Finally, eye tracking data, as humans and nonhuman primates explore 2D scenes, provides a measure of navigation that is analogous to rodent spatial exploration59. Indeed, hippocampal-damaged subjects display disorganized, inefficient search in a scene exploration task and are impaired according to several water maze-inspired metrics such as cumulative search error and heading angle error60. As a user-friendly application that can be further developed to accommodate differences between these various paradigms, Pathfinder may be a useful tool for characterizing complex spatial behavior and bridging findings across humans and animal models.\n\n\nData availability\n\nFigshare: Raw data for \"Pathfinder: open source software for analyzing spatial navigation search strategies\", https://doi.org/10.6084/m9.figshare.9686987.v161\n\nThis project contains the following underlying data:\n\n- Water maze output\n\n- Pathfinder output\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\nSoftware source code available from: https://github.com/MatthewBCooke/Pathfinder\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.337276262\n\nLicense: GNU General Public License 3.0.",
"appendix": "Grant information\n\nThis work was funded by the Canadian Institutes of Health Research (JSS), the Natural Sciences and Engineering Research Council (JSS, REB), and the Michael Smith Foundation for Health Research (JSS, TPO).\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 Sabri Snyder for contributing the name, Pathfinder, Kurt Stover for contributing initial work towards automatic strategy classification and Tian Rui Zhang for assistance with manual strategy classification. A previous version of this work is available from: https://doi.org/10.1101/715961\n\n\nReferences\n\nKjelstrup KG, Tuvnes FA, Steffenach HA, et al.: Reduced fear expression after lesions of the ventral hippocampus. Proc Natl Acad Sci U S A. 2002; 99(16): 10825–10830. 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PubMed Abstract | Publisher Full Text\n\nBannerman DM, Bus T, Taylor A, et al.: Dissecting spatial knowledge from spatial choice by hippocampal NMDA receptor deletion. Nat Neurosci. 2012: 15(8): 1153–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nArruda-Carvalho M, Sakaguchi M, Akers KG, et al.: Posttraining ablation of adult-generated neurons degrades previously acquired memories. J Neurosci. 2011; 31(42): 15113–15127. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMcDonald RJ, White NM: Parallel information processing in the water maze: evidence for independent memory systems involving dorsal striatum and hippocampus. Behav Neural Biol. 1994; 61(3): 260–270. PubMed Abstract | Publisher Full Text\n\nSnyder JS, Cahill SP, Frankland PW: Running promotes spatial bias independently of adult neurogenesis. Hippocampus. 2017; 27(8): 871–882. PubMed Abstract | Publisher Full Text\n\nYu RQ, Cooke M, Zhao J, et al.: Adult neurogenesis promotes efficient, nonspecific search strategies in a spatial alternation water maze task. Behav Brain Res. 2019. PubMed Abstract | Publisher Full Text\n\nRedish AD: Vicarious trial and error. Nat Rev Neurosci. 2016; 17(3): 147–159. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHollup SA, Kjelstrup KG, Hoff J, et al.: Impaired recognition of the goal location during spatial navigation in rats with hippocampal lesions. J Neurosci. 2001; 21(12): 4505–4513. PubMed Abstract | Publisher Full Text\n\nWhishaw IQ, Cassel JC, Jarrad LE: Rats with fimbria-fornix lesions display a place response in a swimming pool: a dissociation between getting there and knowing where. J Neurosci. 1995; 15(8): 5779–5788. PubMed Abstract | Publisher Full Text\n\nSchenk F: A homing procedure for studying spatial memory in immature and adult rodents. J Neurosci Methods. 1989; 26(3): 249–258. PubMed Abstract | Publisher Full Text\n\nMaaswinkel H, Jarrard LE, Whishaw IQ: Hippocampectomized rats are impaired in homing by path integration. Hippocampus. 1999; 9(5): 553–561. PubMed Abstract | Publisher Full Text\n\nValente D, Golani I, Mitra PP: Analysis of the trajectory of Drosophila melanogaster in a circular open field arena. PLoS One. 2007; 2(10): e1083. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEilam D, Golani I: Home base behavior of rats (Rattus norvegicus) exploring a novel environment. Behav Brain Res. 1989; 34(3): 199–211. PubMed Abstract | Publisher Full Text\n\nDvorkin A, Szechtman H, Golani I: Knots: attractive places with high path tortuosity in mouse open field exploration. PLoS Comput Biol. 2010; 6(1): e1000638. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLeutgeb S, Leutgeb JK, Barnes CA, et al.: Independent Codes for Spatial and Episodic Memory in Hippocampal Neuronal Ensembles. Science. 2005; 309(5734): 619–623. PubMed Abstract | Publisher Full Text\n\nTsao A, Moser MB, Moser EI: Traces of Experiencein the Lateral Entorhinal Cortex. Curr Biol. 2013; 23(5): 399–405. PubMed Abstract | Publisher Full Text\n\nDeshmukh SS, Knierim JJ: Representation of non-spatial and spatial information in the lateral entorhinal cortex. Front Behav Neurosci. 2011; 5: 69. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSolstad T, Boccara CN, Kropff E, et al.: Representation of geometric borders in the entorhinal cortex. Science. 2008; 322(5909): 1865–1868. PubMed Abstract | Publisher Full Text\n\nCoutrot A, Silva R, Manley E, et al.: Global Determinants of Navigation Ability. Curr Biol. 2018; 28(17): 2861–2866.e4. PubMed Abstract | Publisher Full Text\n\nMaguire EA, Nannery R, Spiers HJ: Navigation around London by a taxi driver with bilateral hippocampal lesions. Brain. 2006; 129(Pt 11): 2894–2907. PubMed Abstract | Publisher Full Text\n\nMeister MLR, Buffalo EA: Getting directions from the hippocampus: The neural connection between looking and memory. Neurobiol Learn Mem. 2016; 134 Pt A: 135–144. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYee LTS, Warren DE, Voss JL, et al.: The hippocampus uses information just encountered to guide efficient ongoing behavior. Hippocampus. 2014; 24(2): 154–164. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCooke M, O'Leary T, Harris P, et al.: Raw data for \"Pathfinder: open source software for analyzing spatial navigation search strategies\". figshare. Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.9686987.v1\n\nCooke M, Harris P: MatthewBCooke/Pathfinder: Zenodo Release for DOI (Version 1.1.6). Zenodo. 2019. http://www.doi.org/10.5281/zenodo.3372762"
}
|
[
{
"id": "56738",
"date": "02 Dec 2019",
"name": "Matthew Holahan",
"expertise": [
"Reviewer Expertise Spatial learning and memory",
"Parkinson's disease",
"concussions",
"development",
"hippocampus",
"addiction"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 agree with the authors that the assessment of spatial information processing in rodents with automatized tracking systems aids in the reliability of findings across studies and enhances efficiency in which one can make conclusions about specific manipulations. The current work, which would have been nice to include, would be more complete with:\nA direct comparison to commercially available software - e.g., HVS Image provides similar tracking information so in this case, how do these two tracking systems fair head to head?\n\nA group of mice with hippocampal lesions.\n\nFor point 2, does the tracking software pick out well-established deficits in spatial behavior following hippocampal lesions and does the new software provide any additional information on the deficits in spatial navigation produced by hippocampal lesions. I wonder also if the additional measures provided by this software will give more insight into spatial learning deficits in models where deficits were not noted; that is to say, conventional tracking software or output measures might not revealed a deficit when a deficit was predicted. Would this new software provide more robust measures to indicate deficits? Further work is required to more fully test the software to demonstrate its reliability.\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": "5587",
"date": "15 Jun 2020",
"name": "Jason Snyder",
"role": "Author Response",
"response": "Matthew – thank you for the feedback. Our response is a little delayed, because we have timed it to coincide with a software/manuscript update, which took longer than anticipated. With respect to your points, we were unaware that any commercial software packages offer strategy-based analyses. There are some other free programs created by researchers, and it would definitely be interesting to do a comparison. It certainly would have been nice to validate it using an established model of hippocampal deficits, like a lesion. I do think it would pick up deficits that have been described, since hippocampal-lesioned rodents are typically thigmotactic and less spatially accurate, which Pathfinder clearly captures in the early stages of training in our experiment. We have validated it with neurogenesis-deficient rats (Yu, Behav Brain Res, 2019), where these rats displayed a strategy phenotype that was probably relatively subtle compared to hippocampal-lesioned animals. Finally, I do think it could be used to detect behavioral changes that other programs cannot. For this to work, it will be important for the user to understand their subject’s behavior and adjust the parameters accordingly such that the strategies are captured. This will increase strategy detection accuracy and sensitivity but users always need to be cautious when interpreting the output, and assess whether finely-tuned parameters are detecting meaningful differences or not. In any case, there should be lots of existing navigational datasets out there that could be run with Pathfinder to address these exact questions. We look forward to seeing what people find."
}
]
},
{
"id": "56564",
"date": "21 Jan 2020",
"name": "Robert McDonald",
"expertise": [
"Reviewer Expertise Neural basis of learning and memory."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 contribution: A recent paper submitted by Snyder and colleagues to F1000Research concerns the use of the Morris water task as an assay of learning and memory functions in the mammal. As noted 10 years ago, this is probably the most widely used assay of both spatial navigational processes and cognitive functions, in the rodent, in the history of psychology and behavioural neuroscience1. This paper introduces an open source analysis program, “Pathfinder”, for assessing the traditional measures of spatial learning and memory, and also an evaluation of swim patterns/strategies that change in a systematic way during different stages of learning. The paper provides a brief historical account of the discovery and use of the Morris water task (MWT) suggesting that the earlier work lacked sophistication in their analysis of this complex cognitive/navigational behaviour, relying mostly on latency and path length. This is followed by a description of the new software including that it easily interfaces with commonly used tracking and behavioural analysis systems, and that it will be available for free. Both of these features will ensure that it can be used widely by scientists with varying ideas and views. Importantly, the authors provide “proof of principle” experiments highlighting its strengths at assessing the classic measures of place learning and memory, as well as the newer assessment of swim patterns during the different stages of acquisition and retention. The graphical user interface is excellent and allows the experimenter to determine the particular parameters of interest. A nice addition to this part of the paper was the verification of the data analysis completed by the new system as compared to several human observers, showing a high level of agreement. We also believe that having this software available to anyone interested in learning and memory processes in a variety of less constrained open-field situations will be very beneficial.\n\nWe would like to raise some issues for the readers and authors to think about relating to historical context, including early uses of swim pattern analysis, potential benefits and pitfalls of the swim pattern analysis, the nature of the representation acquired during task acquisition, and the possibility of future advances that might prove impactful.\n\nHistorical context: It is important for the reader interested in using the MWT as a measure of learning and memory processes to get a better view of the history and development of the task which will place the new “Pathfinder” system in historical context. This exposition points to various empirical issues and theoretical questions that could be solved with future developments of the program. For example, the contributions of the “Lethbridge group” should be noted. In particular, Sutherland along with Whishaw and Kolb published some of the earliest lesion work using this task2,3. During this time, there was probably a significant amount of communication (see earliest papers and conference abstracts) between Morris and the Lethbridge group concerning the effects of fornix lesions, hippocampal ablations, and ultimately neurotoxins on the place learning version of this task. Sutherland went on to do important work using this paradigm investigating the subcortical and neocortical circuits implicated in this complex form of spatial navigation4,5. The Lethbridge group also contributed significantly to our understanding of the contributions of different neurotransmitters6, the important distinction between motor versus cognitive impairments7, the nature of the representation acquired during training8,9, etc.\n\nThe role of the HVS tracking system in making the MWT such an important tool for behavioural neuroscience and biomedical research should also not be forgotten. The two key labs (Morris and Sutherland) that contributed so much to the development of this task and understanding the brain systems and mechanisms involved used the HVS tracking system and then wrote their own data collection and analysis programs for use in their respective labs.\n\nWe also found in the paper, the general tendency to suggest that the early work was quite primitive and focused on latency and path length. I do not think this is accurate. If one looks at the early work by both groups, there was clear use of multiple parameters (latency, path length, heading angle, quadrant preference, thigmotaxia, rearing on the platform, wall and start-point revisits, and annulus crossings). This work was followed by assessment of different strategies used by rats following brain manipulations while acquiring the MWT. For example, rats with HPC lesions can show one successful strategy in which they learn to circumnavigate a certain distance away from the pool wall and run into the escape platform10. As well, other strategy changes have also been described following specific neuropharmacological manipulations11. The annulus crossings measure is interesting because it shows an early interest in spatial specificity of the place responses acquired following training on the MWT. The importance of this measure for understanding the neural basis of learning on this task was not clear at the time, but became evident when we were involved in addressing controversies over whether the HPC was important for learning this task or not12,13. The spatial specificity measure was an important part of our rebuttals to these claims14. Accordingly, we take significant credit for the idea that rats with HPC dysfunction can show a “place response” under some training conditions, but they do not show spatial specificity (but see Kolarik et al., 201815).\n\nDevan and colleagues16,17 also used a swim path analysis to counter claims18 that the cause of impairments on the standard, spatial version of the water task, induced by brain manipulations (lesions, pharmacology, etc) were due to partial reinforcement effects and not because the subjects were unable to learn and remember the location of the escape platform. According to Gonzalez and colleagues18, the deficits in these experimental subjects were caused by the brain manipulations disrupting the supports of learning and memory (sensory, motor, and motivation) that resulted in the subjects finding the platform fewer times during training than a normal subject. We assessed these serious claims and showed that normal rats that received partial reinforcement on the spatial version of the water task showed impaired escape latencies. However, using a combination of time bin and zone analysis, we showed that the partially reinforced group went directly to the correct spatial location early in the trial and when they did not find it, swam to the pool wall. Note that the pool wall is also a source of reinforcement because the subject is removed by the experimenter at this barrier when a trial ends. Based on our analysis we concluded that, even under partial reinforcement training conditions, control rats learn the precise location of the escape platform, but other reinforcement contingencies also have an influence on their subsequent behaviour as the experimenter is also associated with escape from the pool.\nWe have also shown that rats can sequentially learn two different escape locations and during a probe test visit the most recent location first and then the old location19. It would be interesting if the authors discussed how the “Pathfinder” system could be used to capture these complex sets of swim strategies, in the different experiments described, and how bin analyses could be incorporated.\n\nThe authors of the current paper also describe vicarious trial and error (VTE) like behaviours exhibited when multiple locations are in play at the same time. In our experience, the most compelling demonstration of these kinds of competition-like behaviours is during the final test day following 12 days of cue/place learning20. When the cued platform was moved to a new spatial location and the subjects were started from an equidistant point between the old spatial location and the new cue location, some of the rats went to the new cue position and the others went to the old spatial location first. Interestingly, watching videos of the subjects during this competition test revealed some interesting patterns of behaviour. Some went directly to the cue, some went directly to the place, others started heading to one platform position and then switched direction, and some would change course several times of move their head back and forth to go back and forth before making a decision. We imagine this emerging when representations acquired by two parallel learning and memory networks are “flickering” back and forth in controlling the subject’s behaviour21. Again, it is not clear from the manuscript how “Pathfinder” would pick up these kinds of micro-behaviours or the nature of further advancements in the program that might accommodate these more complex learning situations. Maybe a further expansion of this would be helpful in the manuscript.\nPotential benefits and pitfalls of the automated swim path analysis approach: As noted by the authors, it is not clear what the potential benefits of the swim path analysis produced by the “Pathfinder” system might be, and they provide some possible lines of research that might be fruitful. In our view, one area of enquiry that might be particularly fruitful is a reassessment of the learning and memory network centered on the HPC and the role of each of these areas (HPC, fornix, subiculum, thalamus, nucleus accumbens, mammillary bodies, prefrontal cortex, cingulate cortex, etc) to the different components of place navigation (strategies and strategy transitions). Another potentially interesting area of enquiry would be to revisit the contributions of various neurotransmitters including the ascending modulator systems as well as neurotransmitter systems localized in each sub-region. These lines of enquiry could be developed using both traditional as well as “state of the art” techniques including neurotoxic lesions, inactivation techniques, behavioural pharmacology, optogenetics, imaging, etc.\nOur experience with swim path analysis by human observers and comparing it to those generated by automated versions have been interesting. In a recent paper22, we assessed swim path strategies and transitions during the course of training on the spatial version of the water task based on early classic water task experiments and the approach taken by Ruediger and colleagues23. We assessed the navigation response strategy used by rats during all training trials by viewing each swim and categorizing the behaviour. We replicated their findings that mice with ventral HPC lesions are impaired early in training and not later in training. Importantly, we also discovered some clear differences that are relevant to the “Pathfinder” program. In our experiment, the VHPC lesioned rats used general random search strategies in the early and middle stages of training, while the sham rats switched to direct search strategies towards the escape platform earlier. This switch to direct search strategies by control rats was earlier than control mice in the Ruediger experiments. As we have argued before, these kinds of species and potentially strain differences are important to be aware of when using these kinds of automated analysis systems22.\nWe found other potential caveats associated with these automated approaches. For example, for some individual swims, the criterion for a specific strategy either: did not categorize the subject’s behaviour accurately; some swims could be categorized incorrectly; or no categorization fit the swim pattern exhibited. One of the most interesting examples we came across was late in training in which the swim strategies would be described as hybrid strategies. These include: thigmotaxia early in the swim and then a quick turn to the platform when the subject was close to the correct area of the escape location; circumnavigation but in zones A and B (HVS image); looping but with most time in zone A; single short looping behaviours to escape platform. The point of this simple analysis is that we understand the advantages of using automated algorithms for categorizing swim navigational strategies, but they have some disadvantages including missed strategies or swims categorized incorrectly. We strongly advocate that anytime a lab employs a new experimental manipulation (brain perturbation, strain, sex, age, etc) that swim navigational strategies are assessed with both automated and human observers.\n\nNature of the representations used to navigate in the Morris water task: One final issue that is relevant to the “Pathfinder” analysis program is that it remains a bit of a mystery as to what the actual representation(s) and associated networks that guide behaviour during acquisition and retention of the spatial version of the water task.\n\nBased on some experiments using the MWT9, it appears, somewhat counterintuitively that rats use the shape of the testing apparatus and directional information to locate the escape location. For these experiments, rats were trained on the standard, spatial version of the water task and then a probe trial was completed in which the maze was moved in the training context in a way that allowed the rats to either try to locate the escape platform via directional information or absolute spatial location information. The results clearly showed that a majority of the rats used a directional representation and not a representation of the relationships among the cues in the testing room. Our follow-up experiments assessing the implications of this work extended these results and showed that this directional representation seems to be based on pool shape information combined with head direction information encoded by the post-subiculum and related head direction network23. Even more striking, this directional representation transfers to new contexts, but the behaviours supported by this representation would be deleterious to performance unless the experimenter places the platform in the location based on directional information regardless of which context they are navigating. This is important to be aware of for various reasons, but particularly relevant in the current review because transferring to a new room will not result in random searches of the pool, as generally assumed. Rather, early on in training they will be swimming to a location that is now probably incorrect.\nFinally, we implore researchers to ensure that during crucial probe (transfer) tests that the experimenter stir the pool water after each swim to ensure that olfactory cues are not supporting the place response13.\nConclusions The Pathfinder program should prove to be a welcome addition to the toolbox available to researchers interested in the learning and memory processes involved in place navigation and the neural networks and mechanisms supporting these complex functions. Making it more accessible for scientists to look at various swim patterns during different stages of training will inevitably make it more mainstream to do so. In our view, further advancements to this new approach will increase its impact substantially.\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": "5588",
"date": "15 Jun 2020",
"name": "Jason Snyder",
"role": "Author Response",
"response": "Robert and Nancy – thank you for your detailed and thoughtful review. The additional background information and interpretations will be useful for those wishing to use Pathfinder when designing and analyzing their water maze experiments. We generally agree with the points you raise and would like to comment on a few of them specifically. We understand that it may have sounded like we imply that early water maze work was primitive. We would like to clarify that we in fact tried to emphasize the opposite! Our take is similar to yours, which is that the early work by Morris, Sutherland, Whishaw etc performed quite extensive analyses which were essential to validate the water maze. Ultimately, as behavioral testing has become much more common, and since latency and path length are decent measures of basic performance, they soon dominated and detailed analyses became less common. Of course, even the early work did not conduct strategy analyses that are comparable to those offered by Pathfinder, but certainly the quality of those original papers cannot be understated. We have made some minor adjustments to the text to clarify our view. You also raise some questions about how Pathfinder could be employed to detect strategies related to competing goals. We attempted to describe a few situations where rodents are tasked with deciding between spatial vs cued goals and multiple possible spatial goals. We cite these papers for the readers’ reference and feel that these provide a good illustration of how the water maze can be used to study decision-making , conflict between multiple strategies/brain regions and the like. But the possibilities are endless and so ultimately it will depend on the creativity of the users. We appreciate your comments about the importance of validating automated software with manual analyses. We agree that this is critical at the outset. Only when the user trusts their parameter settings should they proceed with the full analysis. Finally, the issue of hybrid strategies is one that Pathfinder currently cannot address, unless one is willing to bin their trials prior to importing into Pathfinder. For a hybrid approach one may want to try the software described in Vourous, 2018, Scientific Reports since it can detect multiple strategies in a single trial."
}
]
}
] | 1
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https://f1000research.com/articles/8-1521
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https://f1000research.com/articles/9-263/v1
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16 Apr 20
|
{
"type": "Research Article",
"title": "Evaluation of dental arches in orthodontic patients with condylar hyperplasia in a North Sumatra subpopulation: a cross-sectional study",
"authors": [
"Ervina Sofyanti",
"Trelia Boel",
"Denny Satria",
"Zuriyah Fionita Ritonga",
"Indah Hafniar Hasibuan",
"Trelia Boel",
"Denny Satria",
"Zuriyah Fionita Ritonga",
"Indah Hafniar Hasibuan"
],
"abstract": "Background: Condylar Hyperplasia (CH) is a self-limiting mandibular condyle disorder that shows asymmetry progress conjunction with associated occlusal changes as long as condylar growth is still active and leads to facial asymmetry. This study aimed to evaluate dental arches by analyzing dental arch asymmetry and form in orthodontic patients with CH in a North Sumatra subpopulation. Methods: This is a retrospective study of suspected CH patient’s clinical records who sought for the initial orthodontic treatment between January 2015 to March 2019. Patient with facial asymmetry (based on photography, posterior cross bite and midline deviation), positive temporomandibular joint disorder in functional analysis, and no history of facial trauma were included in the study. Dental arch asymmetry was based on the measurement of dental midline deviation, canine tip in the dental arch, distance of the upper canines from the palatal suture, and inter canine distance. The evaluation of dental arch was achieved by comparing arch width and length. Results: There was a significant difference (p<0.05) of upper canine distance from the palatal suture in female patients when evaluating upper dental arch asymmetry. There was a moderate correlation (r=0.379) in midline deviation between upper and lower dental arch. The dimension and dental arch form was mid and flat, and there was moderate correlation (r=0.448) between the upper and lower dental arch form in these CH patients. Conclusion: The evaluation of dental arch symmetry and arch form showed asymmetric occlusal characteristics in orthodontics patient with CH in North Sumatera subpopulation. In treating these patients, we recommend the plaster cast evaluation as essential and routine procedure in order to understand the complexity of occlusal change due to active growth of condylar and limitation in radiography evaluation.",
"keywords": [
"dental arch",
"condylar hyperplasia",
"asymmetry",
"form"
],
"content": "Introduction\n\nThe unilateral non-neoplastic overgrowth of mandibular condyle that leads to occlusal interferences and joint dysfunction or pain, is identified as condylar hyperplasia (CH). The elongation of condylar head, neck, and lower arch corpus leads to the development of occlusal disharmony, dental compensation, and asymmetry due to continued abnormal growth (Bharathi et al., 2014; Wolford et al., 2014). This deformity requires assessment in condylar growth and sometimes requires high condylectomy combined with orthognathic surgery and articular disc repositioning to achieve treatment stability (Lippold et al., 2007; Wolford et al., 2009). Sequential assessments (six to twelve month intervals) of CH aim to prevent worsening functional, esthetic, skeletal, and occlusal changes during orthodontic treatment. These examinations include clinical examinations, cast analysis, and radiographic evaluations in the abnormal condylar lower arch growth. Since CH is a rare disorder, previous studies explore the multiple factors of mandibular growth asymmetry and development. The characteristics of CH are as follows: excessive or persistent growth of condyle leads to facial asymmetry with occlusal discrepancies and temporomandibular joint disorder as concurrent symptoms. It has severe asymmetry over a long-time period and can grow past the growth period gradually (Almeida et al., 2015; Goulart et al., 2018; Raijmakers et al., 2012; Wolford et al., 2014).\n\nCH usually occurs during puberty and ceases at the same time as the completion of healthy growth (Raijmakers et al., 2012; Wolford et al., 2014). CH affects women more than men with a 3:1 ratio (Nitzan et al., 2008). It can also continue growing past the growth period (Almeida et al. 2015; Obwegeser & Makek, 1986; Pacheco et al., 2010). Finishing treatment in the soft tissues usually requires correction, especially in patients with longstanding condylar hyperplasia, and soft tissue procedures include facelifts, augmentation using medpor implants or fat grafts as alternative treatment (Alyamani & Abuzinada, 2012). The investigation of facial asymmetry with CH complexity should be integrally planned during orthodontic treatment (Olate et al., 2013).\n\nPrevious studies report that CH adversely affects morphology and size of the mandible, it also alters the occlusion development along with dental arches (Goulart et al., 2018; Mehrotra et al., 2011; Wolford et al., 2014). Genetic and environmental factors influence occlusal development and dental arch form variations. In planning orthodontics treatment, changing the dental arch form can also influence treatment stability. In patients with facial asymmetry, dental alveolar compensation is one treatment choice when treating malocclusion with dental arch asymmetry. Thus, this study aims to evaluate dental arches by analyzing the dental arch asymmetry and form in orthodontic patients with CH in a subpopulation of North Sumatra, Indonesia.\n\n\nMethods\n\nThis is a retrospective study of the clinical records of suspected CH patients that sought for the initial orthodontic treatment at the Dental Hospital, Universitas Sumatra Utara between January 2015 and March 2019. The Research Ethics Committee of the Universitas Sumatera Utara Medical Faculty approved this study (378/TGL/KEPK FK USU-RSUP HAM/2019). Those patients who were treated with fixed orthodontics at the Dental Hospital, University of Sumatra have signed informed consent, which includes data collection for research purposes.\n\nCH suspected subjects fulfilled the following characteristics: facial asymmetry based on photography, posterior cross bite and midline deviation in plaster casts analysis, and positive temporomandibular joint disorder in functional analysis. This study excluded patients with a history of facial trauma and hereditary disorder based on their medical records, and patients whose records had unclear morphology of condylar and lower arch features in pretreatment panoramic radiographs so that cannot be analyzed with asymmetry vertical mandibular with Kjellberg’s technique (Hirpara et al., 2016).\n\nEvaluation of dental arch symmetry was done using plaster casts taken from the patients’ medical records.\n\nInitial measurements. The well-trimmed plaster casts were positioned on millimeter rule paper and measurements were taken manually with a cephalometric and metal protractor. To determine the upper arch midline, a mark was located along the mid palatal suture. The symmetry axis of the upper arch midline was made by connecting the incisive papilla (As=Anterior superior) passing second palatal rugae to the most visible posterior upper arch landmark (Ps=Posterior superior) over the mid palatal suture. Once the Ps was marked, the end reference point was located on the border between the hard and soft palate. Then Ps mark of the upper arch was transferred to the lower arch (Pi=Posterior inferior) using a ruler positioned perpendicular and occluded upper and lower plaster casts. To determine the anterior lower arch point (Ai=Anterior inferior), the upper arch As was transferred to the lower arch using a ruler that occluded upper and lower plaster casts. The symmetry axis of the lower arch midline was obtained by connecting the Ai to the most visible posterior landmark (Pi) over the lingual frenulum to the posterior border of lower arch. After marking the midline symmetry axis, we can evaluate the dental arch asymmetry by measuring dental midline deviation (MD), canine tip in the dental arch (PC), distance of the upper canines from the palatal suture (DC) and inter canine distance (ICD) in the upper and lower arches (Scanavini et al., 2012).\n\nAngular and linear measurement of the dental arch asymmetry. Angular arch asymmetry was performed by measuring the right and left canines to the upper arch symmetry axis using a cephalometric protractor in the upper arch (this is known as midline deviation (MD) (Figure 1 and Figure 2). The position of the canine (PC) is the distance from the cusp tip on each side that were projected perpendicular to the symmetry axis using a metal protractor. Distance of canines (DC) is the linear measurement from right to left canine tips (Figure 3). This angular and linear measurement of upper and lower arch dental asymmetry has predictive accuracy and is marked as negative if shifted to the left (Scanavini et al., 2012).\n\nThe plaster cast model is positioned with a protractor at the cusp of the right upper arch canine (A); the protractor shows that PC of right upper arch canine is 46° (B).\n\nThe plaster cast model is positioned with a protractor at the cusp of the right lower canine (A); the protractor shows that PC of right lower arch canine is 39° (B).\n\nEvaluation of dental arch form. The evaluation of dental arch form by comparing arch width and length. Dental arch dimensions were as follows: L33 (inter canine width), from right canine cusp to left canine cusp; L66 (intermolar width), from right first molar cusp to left first molar cusp; L77 (posterior intermolar width), from right second molar cusp to left second molar cusp; L31 (sagittal dental arch), from the “arrow” of the anterior curve; L61, from the incisal edge to the line joining the mesiobuccal canines of the first molar; L71, the incisal edge to the line joining the distobuccal cusps of the second molar. These six factors characterized both the arch form and the dimension that will determine the dental arch ratio: L31/L33, L61/L66, L71/L77, L33/L66, L61/L71 (Figure 4 and Figure 5). The arch is classified as narrow if three dental arch ratios (L31/L33, L61/L66, L71/L77) are positive, the arch form is classified as wide if they are negative. The arch is classified as mid if none of those dental arch ratios significantly deviates from the average. The arch form is pointed if only the L31/L33 has a noticeableintensity significantly higher value than all the comparisons (L61/L66, L71/L77, L33/L66, L61/L71). The arch is classifiedas flat only if the L31/L33 has significantly lower value than all comparisons (L61/L66, L71/L77, L33/L66, L61/L71) (Raberin et al., 1993). Those measurements were repeated in 15 days after obtaining the initial data to assess the reliability with intra-rater measurement.\n\nThe sagittal dental arch from the arrow of the anterior curve (L31) (A); from right canine cusp to left canine cusp is identified as intercanine width (L33) (B); the incisal edge to the line joining the mesiobuccal canines of the first molar (L61) (C); the dental arch dimension from right first molar cusp to the left first molar cusp is intermolar width (L66) (D); the incisal edge to the line joining the distobuccal cusp of the second molar (L71) (E); from right second molar cusp to the left second molar cusp is the posterior intermolar width (L77) (F).\n\nThe sagittal dental arch from the arrow of the anterior curve (L31) (A); from right canine cusp to left canine cusp is identified as intercanine width (L33) (B); the incisal edge to the line joining the mesiobuccal canines of the first molar (L61) (C); the dental arch dimension from right first molar cusp to the left first molar cusp is intermolar width (L66) (D); the incisal edge to the line joining the distobuccal cusp of the second molar (L71) (E); from right second molar cusp to the left second molar cusp is the posterior intermolar width (L77) (F).\n\nStatistical analysis. Analyzing the significant difference of symmetrical angular and linear measurement of the dental arches based on gender was done using paired independent pair t-test (p<0.05). This study used the second measurement in analyzing symmetrical dental and dental arch form with chi-square and Cramer’s V correlation. SPSS version 17.0 was used for data analysis.\n\n\nResults\n\nThere were total of 14 male and 20 female patients who were suspected as CH patients between 10 to 30 years old based on their clinical records. Those subjects had adequate pre treatment plaster casts and had posterior cross bite and mid line deviation in upper and lower arch. There were facial asymmetry based on photography analysis. There were positive temporomandibular joint disorder based on functional analysis. There were vertical mandibular asymmetry based on pre treatment panoramic radiographs.\n\nTable 1 shows the distribution of upper and lower arch dental symmetry in this study. Based on gender, there was no significant difference in upper and lower dental symmetry between male and female (Table 2). There was a significant difference (p<0.05) of upper canine distance from the palatal suture in female patients, but no significant difference in other symmetry variables between male and female (Table 3), There was also no significant difference of midline deviation between male and female in this study (Table 4). However, there was a moderate correlation (r=0.379) of midline deviation in upper and lower dental arches of this studied subjects (Table 5). In these CH patients, the dental arch form in upper and lower dental arch were mid and flat. There was also a moderate correlation (r=0.448) between upper and lower dental arch form (Table 6).\n\nMD, dental midline deviation; PC, canine tip in the dental arch; DC, distance of the upper canines from the palatal suture.\n\n*Negative, the midline deviates to the left-side; positive, deviates to the right-side.\n\nMD, dental midline deviation; PC, canine tip in the dental arch; DC, distance of the upper canines from the palatal suture.\n\n*Negative, the midline deviates to the left-side; positive, deviates to the right-side. Statistically significant difference (p<0.05).\n\nPC, canine tip in the dental arch; DC, distance of the upper canines from the palatal suture.\n\nStatistically significant difference (p<0.05).\n\nStatistically significant difference (p<0.05).\n\nStatistically significant difference (p<0.05).\n\nStatistically significant difference (p<0.05).\n\n\nDiscussion\n\nThe dental arch evaluation is an essential issue in orthodontic treatment. We performed a retrospective study of clinical records of suspected CH patients in order to evaluate dental arch symmetry. The failure in early identification of CH can lead to unfavorable esthetic and functional orthodontic treatment resulting in the development of asymmetry. Identifying the growth pattern occurring in CH and deciding the activity of condylar growth are important in treating any malocclusion with lower arch asymmetry. Wolford et al. introduced an updated classification system based on growth, clinical, histological, and imaging characteristics (Wolford et al., 2014). Progressive unilateral growth expansion of condyle, leads to facial asymmetry, malocclusion, and indirectly affects the upper arch development, also shifting off the chin towards the unaffected side (Goulart et al., 2018; Wolford et al., 2014). A study by Mehrotra et al. in 2011 on five Indian adult active CH patients reported that facial asymmetry was the most common clinical finding, followed by midline shifting, protruded chin, contralateral cross bite, and upper arch occlusal canting (Mehrotra et al., 2011). CH clinical findings show midline shifting along with chin deviation to contralateral side with related posterior cross bite (Almeida et al., 2015). Generally, patients with active CH present facial asymmetry transversally, vertically or combined (Nitzan et al., 2008). There was a significant difference in soft-tissue asymmetry between CH subjects and controls as observed in three-dimensional photographs. Therefore, this method is a valid and suitable tool for the evaluation of progressive worsening occlusion, which is a limitation of radiographs (Verhoeven et al., 2013). In some emerging countries, panoramic radiographs were suitable for screening CH, although the diagnostic gold standard was to assess condylar growth activity using bone scintigraphy (Nolte et al., 2015). Previous studies have also reported that there were no significant association between vertical mandibular asymmetry and temporomandibular disorder symptom (Mendoza-García et al., 2019; Sofyanti et al., 2018). Therefore, we used photography, positive posterior cross bite from plaster cast and positive temporomandibular disorder in a cohort of 10–30 year old patients based on their initial dental record in this study.\n\nTable 1 shows that in our patients the midline shifted to the left on the upper and lower arch. This result might be supported by Haraguchi et al’s study that reported a higher proportion of wider left hemiface in the post pubertal stage than prepubertal stage in 1800 Japanese subjects (Haraguchi et al., 2008). However, this study result was different to a clinical study in Brazilian twin girls with CH who presented with facial asymmetry and skeletal class III whose chin and lower arch midline tend to deviate to the right side (Goulart et al., 2018).\n\nEven though previous study reported that CH affects more women than men, with a 3:1 ratio (Nitzan et al., 2008); however, there was no significant difference of angular and linear measurement for symmetrical analysis in upper and lower dental arch based on gender in this study (Table 2). There was also no significant difference of symmetry variables in upper and lower arch dental arch of CH patients based on gender, except the upper canines distance from the palatal suture of female patients (Table 3). The outcome of this study was also suggested by Al-Zubair, which reported that the location of the central incisor and canines to each other and to other teeth is the most potent factor in determining dental arch asymmetry (Al-Zubair, 2019).\n\nAccording to Scanavini et al, the differences of midline deviation in normal and class II malocclusion, is midline deviation dominant toward the left in the upper arch in each group (Scanavini et al., 2012). In this study, the different PC between the upper canines distance from the palatal suture were higher in the lower arch than the upper arch. As with the analysis of midline deviation, the lower arch showed a higher degree of asymmetry than its counterpart upper arch. In the current study, the average values of midline deviation were greater than in the previous study regardless of the type of the malocclusion. Since this study focused on CH patients, the variance of ssymmetry showed a moderate correlation in midline deviation of dental arch symmetry (Table 4) and dental arch form (Table 5). The presence of condylar hyperactivity influenced the degree of malocclusion with facial asymmetry and diversity of CH (Alyamani & Abuzinada, 2012; Pacheco et al. 2010; Pinto et al., 2016).\n\nPrasad et al. reported that dental arch width of untreated South Indian adults which had minimal crowding and spacing, is associated with gender, race and vertical facial morphology. Then, it was important to consider the individualized arch wires (Prasad et al., 2013) In Saudi normal population, it is suggested using a nickel titanium arch wire that will produce the lowest mean absolute error, cause minimal change in the dental arch form, and less customization of stainless steel wires necessary (Al-Barakati et al., 2016). If there was a failure to preserve the arch form, it might increase the probability of relapse (Bayome et al., 2011). Relapse related to improper arch wire changes can affect periodontal breakdown, recurrence of crowding of buccal segments, or increased crowding of labial segments particularly while inter-canine width and inter-molar width have been expanded. In the present study, relatively stocky arch guide showed mid and flat arch form for both upper and lower dental arch in the studied patients. This is also similar to the lower dental arch form of a Papuan population, which showed that prevalence of mid arch form was highest whilst the pointed lower arch form was the least in lower arch analysis of 18 to 25 year old Papuan students in Manado (Saputra et al., 2016). Another Indonesian sub-population study also reported that there was only significant difference of upper dental arch for full dentition of Buginese, Makassarese, and Torajanese individuals, which was null in the lower dental arch (Rieuwpassa et al., 2012).\n\nThe variance of upper and lower dental arch relationship in each malocclusion group appeared to have a specific etiology and different treatment modality especially in adult orthodontic treatment with facial asymmetry, such as: camouflage orthodontic treatment or orthognathic surgery. In interceptive orthodontic treatment, dental arch coordination should be evaluated during occlusal changing during mandibular growth. The development asymmetry of the lower arch might be due to the condylar growth activity and joint function and it is common that a growth period elapses after the patient’s asymmetric growth is completed (Pacheco et al., 2010). The asymmetry upper dental arch showed highly significant difference between right and left posterior arch segment whilst anterior segment showed no significant difference in class I occlusion of Iraqi young adult (Allabban et al., 2017). Further study, such as genetic investigation and follow-up of the patients, may help clinicians to understand the multi-characteristics of CH based on histological findings in this North Sumatra sub-population. Genetic and environmental factors can influence the development of left-right patterning during embryogenesis and remain in the masseter muscle in adults, including the PITX2 promoter (Sofyanti et al., 2018). Ethnicity also influences the development of dental arch form related with symmetry point. In the future, evaluation of plaster casts during orthodontic treatment is a requirement especially in treating malocclusion with mandibular asymmetry in order to anticipate the biomechanics of certain arch wire type. The clinicians should be careful in using arch wire coordination to obtain optimal occlusal relationship in order to maintain treatment stability.\n\n\nConclusion\n\nThe evaluation of dental arch symmetry and arch form showed asymmetric occlusal changes of orthodontic patients with CH in this North Sumatera subpopulation. In treating these patients, plaster cast evaluation is recommended as essential and routine procedure in order to understand the complexity of occlusal characteristics due to active growth of condylar and limitations in radiography evaluation during treatment. This phenomenon is marked as one obstacle in treating growing patients if there was no early detection and proper treatment of malocclusion with facial asymmetry.\n\n\nData availability\n\nHarvard Dataverse: Replication Data for: Angular and linear measurement of the dental arch asymmetry and dental arch form, https://doi.org/10.7910/DVN/GONORA (Sofyanti et al, 2020).\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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}
|
[
{
"id": "62453",
"date": "12 May 2020",
"name": "Maarten J. Koudstaal",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study aimed to evaluate dental arches by analyzing dental arch asymmetry and form in orthodontic patients with CH in a North Sumatra subpopulation. Retrospective study design in 34 patients, 10-30 years old.\nAbstract:\nAdequate.\n\nIntroduction:\nAdequate.\n\nMethods:\nParticipants Inclusion:\nCH suspected subjects fulfilled the following characteristics: facial asymmetry based on photography, posterior cross bite and midline deviation in plaster casts analysis, and positive temporomandibular joint disorder in functional analysis.\n\nIt is unclear if patients needed to have all of these? The reason for asking, a fair amount of patients with CH do not have any functional issues with the TMJ joint.\n\nAt what time points were the records taken and studied? The same protocol for all patients? And at what ages?\n\nResults:\nAge of inclusion? Would the authors expect a difference in the still active versus the non-active growth patients? If so please show the actual ages at inclusion and follow-up. And add a paragraph to the results about this.\n\nI do not see the patients classified as being either left or right side CH? Were they grouped? That does not make a lot of sense to me, especially when looking at the deviation of midline. Please elaborate.\n\nDiscussion:\nAdequate\n\nTables:\nThe legend should include explanation of the abbreviations used in the 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?\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": "5575",
"date": "12 Jun 2020",
"name": "Ervina Sofyanti",
"role": "Author Response",
"response": "Dear Dr. Maarten J. Koudstaal,We want to thank you for providing us with valuable advice for the manuscript. CH suspected subjects fulfilled the following characteristics: facial asymmetry based on photography, posterior crossbite and midline deviation in plaster casts analysis, and positive temporomandibular joint disorder in functional analysis. We thank the reviewer for the suggestion. Since this is a study from a retrospective study of suspected CH patient's clinical records, we assessed that any pain, clicking, crepitation, pain, and other sign or symptom of positive temporomandibular joint disorder from the extraoral analysis will be included. It is unclear if patients needed to have all of these? The reason for asking, a fair amount of patients with CH do not have any functional issues with the TMJ joint. We thank you for the comments. We include all these criteria in select cases based on patients' information medical records. We include patients with symptoms and/or TMD signs based on dental records. At what time points were the records taken and studied? The same protocol for all patients? And at what ages? We thank you for kindly advising. The orthodontics patient's data medical records was collected and selected during January 2015 and March 2019. However, this information was collected by different orthodontic clinicians and the age of patients based on the date of birth stated in the medical record. We have also revised the resources data by adding the sample’s age. (Sofyanti E, Boel T, Satria D, et al.: Replication Data for Angular and linear measurement of the dental arch asymmetry and dental arch form. Harvard Dataverse, V1, UNF:6:GiTmDSqATBmhdCM366FFoQ== [fileUNF],2020. http://www.doi.org/10.7910/DVN/GONORA). Results: Age of inclusion? Would the authors expect a difference in the still-active versus the non-active growth patients? If so please show the actual ages at inclusion and follow-up. And add a paragraph to the results about this. We thank you for the reviewer's advice. Based on the date of birth, the samples were selected between 10 to 30 years old in this study according to the previous study that reports about the age of condylar hyperplasia patients in the discussion phase. (Pacheco et al., 2010; Bharathi et al., 2014; Lopez & Herrera-Guadiola., 2016; Lopez et al., 2017; Goulart et al., 2018). The limitation of this study was less information about the activity of condylar growth due to the dental hospital didn/t have 3D CBCT in that time to evaluate the condylar precisely. I do not see the patients classified as being either left or right side CH? Were they grouped? That does not make a lot of sense to me, especially when looking at the deviation of the midline. Please elaborate. Based on previous medical records, we include samples with a deviation of midline in occlusion transversally. In this study, we analyzed the upper and lower arch based on Scanavini and Raberin method in the plaster cast of suspected CH as mentioned in the material and methodbased on previous studies about CH clinical findings."
}
]
}
] | 1
|
https://f1000research.com/articles/9-263
|
https://f1000research.com/articles/9-600/v1
|
12 Jun 20
|
{
"type": "Research Article",
"title": "Modeling the trend of reported malaria cases in Kisumu county, Kenya",
"authors": [
"Elsen Achieng",
"Vincent Otieno",
"Joseph Mung'atu",
"Vincent Otieno",
"Joseph Mung'atu"
],
"abstract": "Background: Although there has been an extensive scale-up of malaria interventions in Kenya, malaria infections persist at unacceptably high levels in some of the regions. Even with renewed calls to eradicate the disease through increased international donor assistance and country-specific government involvement, malaria is still a cause of worry in endemic regions. The objective of this study was to determine the factors associated with the incidence of malaria in Kisumu County over time. Methods: The study conducted secondary analysis of data from a cross-sectional survey of routinely reported malaria cases. The population of interest were patients confirmed to have malaria by laboratory test. A sample size of 384 was randomly selected from all laboratory-confirmed malaria cases as reported by health facilities in Kisumu County from January 2014 to December 2017. The analysis involved descriptive, trend analysis and time series analysis (ARIMA). A negative binomial regression model was used to measure the effect of each of the selected predictor variables on incidence of malaria and the incidence rate ratio, was reported. Frequency distribution of each of the categorical variables was calculated. Results: The overall pattern of the reported malaria cases had seasonal variations for weekly cases. The best-fitting time series model developed for predicting the number of weekly reported cases of malaria was ARIMA (2, 0, 1). It was observed that the negative binomial was actually the best model to fit the incidences of malaria because the dispersion parameter given by Poisson regression model had been reduced from 70.292 to 1.103. Conclusion: There is a need to encourage health professionals to regularly review and report cases of malaria in their facilities. This is because reporting rates, completeness and the consistency of malaria reported cases remain extremely low.",
"keywords": [
"Malaria",
"Trend Analysis",
"Modeling",
"Reported cases",
"ARIMA model",
"ACF and PACF plot",
"Negative Binomial",
"Poisson regression"
],
"content": "Introduction\n\nMalaria is among the major killer diseases within tropical regions. Sub-Saharan Africa bears its burden than any other region across the globe. The parasites are spread to humans through bites from infected Anopheles mosquitoes, introducing the protozoans into the human body. Even with renewed calls to eradicate the disease through increased international donor assistance and country specific government involvement, malaria is still a cause of worry in the endemic regions.\n\nAccording to the world malaria report, anti-malaria progress has slowed down in many parts of the world and it is unlikely that the world will achieve targets for 2020 set by the World Health organization (WHO) Global Technical Strategy in reducing malaria incidences. A year on from this recognition, still there is no significant progress in global malaria control (WHO, 2018). Globally, 219 million cases of malaria were estimated to have occurred in 2017 as compared to 239 million cases that occurred in 2010. Even though there were 20 million fewer cases by in 2017 than in the year 2010 worldwide, from 2015 to 2017 there was a slight upward change in trend, suggesting that progress had generally stalled (World Malaria Report, 2018). The WHO statistics of 2017 reveal that Sub-Saharan Africa is still suffering greatly from malaria deaths, with 200 million cases comprising of 92% of the global burden. Malaria has impacted the health and economic development of Sub-Saharan African countries negatively and is considered a major impediment to sustainable development in the world’s poorest regions (Gallup & Sachs, 2001).\n\nThe 2030 Agenda adopted in September 2015 by world leaders was to ensure sustainable development globally. The aim is to eradicate poverty, improving health, reduce inequity and address challenges related to climate change by the year 2030. In order to track progress, they created a number of Sustainable Development Goals (SDGs), each having a specific target to be realized in the next 15 years. One of the set targets is to end epidemics of neglected tropical diseases and malaria by 2030. A set of indicators to track success on malaria eradication is, the number of malaria incidence experienced in a population and the number of people affected by neglected tropical diseases and are seeking interventions against these diseases (“SDG Indicators,” 2018)\n\nThe inclusion of universal health coverage (UHC) reaffirms the position of the SDGs on global health priorities (Barasa et al., 2018). The purpose of UHC is to make sure that all citizens of a state are able to access curative, rehabilitative and preventive health services at minimum costs, thereby eliminating disparities in access to health care. This also includes free consultation, diagnosis services for normal ailments and getting drugs for free. The Kenyan UHC model of dispensation by the current government calls for the enhancement of HIV, tuberculosis and malaria treatment in the country, with the aim of eradication of these diseases.\n\nMore than 70% of the entire population in Kenya is at risk of malaria which is the major cause of ill health (KNBS, 2015). In 2017, confirmed malaria incidences were 2,783,846 as reported by the public health facilities. The diagnosed cases contribute correspondingly to the high number of malaria deaths in the Kenyan population, which increased from 2016 to 2017 as a result of stalled progress in malaria prevention.\n\nWith the aim of achieving the largest reduction rate in morbidity and mortality, the President’s Malaria Initiative (PMI) has always considered the regions of Kenya experiencing the high incidences of malaria since 2013. The counties of Kisumu, Vihiga, Migori, Bungoma, Homa Bay, Kakamega, Busia and Siaya all together have a projected population of about 9.6 million in 2018 and bear the highest incidence of malaria in Kenya (President’s Malaria Initiative, 2018a). The quarterly surveillance bulletin released in December 2016 revealed that, 73% of all confirmed malaria cases were reported from these counties (President’s Malaria Initiative, 2018b). Execution of malaria control interventions focuses on endemic areas and are usually guided by the epidemiological zones. They include community managing the cases as they occur and intermittent preventive treatment in pregnancy (Musuva et al., 2017)\n\nIt is in the light of this discourse that the Kenyan government and other developing partners in the health sector have remained dedicated in improving the delivery of health service in various parts of the country, with high priority being placed on prevention and control measures of malaria in endemic regions with eventual elimination. This being one of the objectives that were to be realized by 2018, together they aimed at reducing malaria burden by two-thirds (US President’s Malaria Initiative, 2019).\n\nMalaria situation with regard to facility reported cases and distribution of malaria remains unclear within the recent past in the study area, despite the above-mentioned studies in various parts of the country. Therefore, this study aimed at mapping the corresponding factors contributing to the incidence of malaria with the trend data on malaria cases across the region.\n\n\nMethods\n\nA cross-sectional survey using routinely reported national Programme data on malaria cases in the DHIS2 platform, as reported by health facilities in Kisumu County was conducted from January 2014 to December 2017.\n\nKisumu County is located in the former Nyanza Province and its headquarters are in Kisumu City, which is situated approximately 370 km west of the Kenyan capital, Nairobi. According to 2009 census, Kisumu County had a population of 968,879 people and covers an area landmass of 2085.9 km2 and 567 km2 covered by water.\n\nThe study population included individuals at risk of malaria in Kisumu County.\n\nAll patients confirmed to have malaria by laboratory test were recruited in the study and patients diagnosed with ailments other than malaria were excluded.\n\nThe Fisher et al. (1998) formula was used to determine the sample size for the study as follows:\n\n\n\nWhereby:\n\nn = is the desired sample size (if the target population is over 10,000) and Z = 1.96 which corresponds to 95% confidence level. p = is proportion of patients confirmed to have malaria by laboratory test while q = is (1-p) is proportion of patients confirmed not to have malaria by laboratory test. d = standard error at 95% confidence limit (0.05). Since the proportion of patients confirmed to have malaria by laboratory test (P) was not known, it was estimated to be 50% (Mugenda & Mugenda, 2013)\n\nTherefore, based on the given figures\n\n\n\nn = 384 laboratory-confirmed malaria patients\n\nAny patient who was referred to the laboratory by the clinical officer/nurse after presumptive diagnosis of malaria and fulfilled the criteria for inclusion was enrolled in the study.\n\nSecondary data of all laboratory-confirmed malaria cases as reported by all Health Facilities was downloaded from the District Health Information System (DHIS2) using the standard application programming interface provided. The data included weekly number of malaria cases. Patients testing Positive for malaria was the dependent variable and the predictors were locality, Time in years and Total number of patients tested for malaria. The predictors were assessed as given in Table 1. A conceptual framework depicting the predictor-outcome relationship is displayed in Figure 1.\n\nConsidering that secondary data was used, there was no interaction with human participants in this study. However, all the personal information generated from this study was treated with a lot of confidentiality. Approval to carry out the study was granted by the Ethics Research Committee at Kenyatta National Hospital/University of Nairobi (P511/05/2019). Permission to use the DHIS2 data was also granted Health Management Information Systems (HMIS) at the Ministry of Health Kenya (Ref. No.MOH/F/DHSMEI/01 Vol.I(17)).\n\nData was downloaded from the DHIS2 using the standard application programming interface provided. Each facility’s data was visually cross-checked subjected to range and limit tests to confirm obvious outliers or errors in transcription.\n\nWeekly facility-level data on malaria parasitological testing were extracted from the Integrated Disease Surveillance and Response (IDSR) registry through the DHIS2 platform. The analysis involved checking for completeness, consistency and accuracy of the data. This include descriptive, trend analysis and time series analysis (ARIMA). The ARIMA model was used and applied to time series data of malaria reported cases in Kisumu County.\n\nBased on the Box-Jenkins approach, model building involved ascertaining the order of the AR and MA components which was guided by the Auto Correlation Function and partial correlation coefficients plots. Dicker Fuller and Shapiro-Wilk test were used to test for stationarity and normality respectively. Additionally, Pearson’s Chi-square was used to test the goodness of fit of both Poisson and negative binomial regression models.\n\nThe influence of time in years, total number of patients tested and locality on the number of malaria cases was evaluated. Frequency distribution of each variable and each category for each of the categorical variables was calculated and presented using R statistical software version 3.5.1. A significant count model negative binomial regression model was used to measure the effect of each of the predictor variables on the number of malaria cases and the incidence rate ratio (IRR), was reported. The statistical significance was measured using the 95% confidence interval and P-values. Missing data was addressed using single imputation method.\n\n\nResults\n\nThe study included 384 reported malaria cases in the final analysis, for the period January 2014 to December 2017. A line graph illustrating reported malaria cases from 2014–2017 is shown in Figure 2. Figure 3 shows the trend in weekly reported malaria cases.\n\nThe procedure for model building involved ascertaining the order of the AR and MA components. This was guided by the auto correlation function and partial correlation coefficient plots based on the Box-Jenkins approach.\n\nThe autocorrelation function in Figure 4 of malaria reported cases from 2014 to 2017 at 95% confidence level spikes significantly at lag 1, 2, 3 and 4. After lag 4 it cuts off, implying that the moving average component of the ARIMA model of order 4 (MA= 4) would be needed to describe this data set.\n\nThe partial autocorrelation function in Figure 5 at 95% confidence level significantly spikes at lag 1 and then quickly cut off, implying that the autoregressive component of the ARIMA model is of order 1 (AR=1).\n\nThe ACF and PACF plots in Figure 4 and Figure 5 respectively suggest that a MA = 4 and AR =1 would be needed in describing this data set as coming from a moving average and autoregressive process, respectively.\n\nA constant mean, constant variance and a constant autocorrelation structure signifies a stationary series. The test for stationarity using augmented Dicker Fuller test as shown in Table 2 depicts that the time series is stationary with a significant p-value of 0.01.\n\nIn comparing the Akaike information criterion (AIC) values of the five likely ARIMA models depicted in Table 3, it was deduced that model ARIMA (2, 0, 1) is the best model for the data since it has the lowest AIC value of 5581.917.\n\nShapiro-Wilk normality test results (p-value ≤ 0.0001) in Table 4, depicts that the residuals are normally distributed. Consequently, it can be inferred that the true mean of the residuals is approximately equal to zero and there is a constant variance among residuals of the selected model.\n\nTable 5 shows the residual deviance for the fitted Poisson regression was given as 26219 on 373 degrees of freedom. To check the fit of the fitted Poisson model, the value of the residual deviance 26219 on 373 degrees of freedom was considered as observed in Table 5 26219373=70.29223, a dispersion parameter of 70.29223 is an indication that the data is over-dispersed as the value is far greater than 1. This implies that the model is not fit because the mean and variance of the response variable are not equal. If the mean and variance were equal, the residual deviance should be approximately equal to the degrees of freedom.\n\nSignificance level <0.05.\n\nIn this case, the assumption of mean equal to variance of the Poisson random variable was violated and so, a negative binomial regression model was deemed suitable and practical as they cater for over-dispersion. Also, they allow the likelihood ratio and other standard maximum likelihood tests to be applied. The fitted Poisson model had an AIC value of 29318 and a null deviance of 115777 on 383 degrees of freedom. Table 6 represents parameter estimates after validating the Poisson regression model using negative binomial regression model since the assumptions of Poisson regression was not met.\n\nSignificance level <0.05.\n\nThe AIC of this model is 5345.2, a deviance of 411.59 on 373 degrees of freedom also following the chi-square distribution D~χ2(n-p) degrees of freedom. The dispersion parameter was found to be 1.10346 as shown in Table 7. It was observed that the negative binomial was actually the best model which fit the incidences of malaria as the dispersion parameter given by Poisson regression model had been reduced from 70.29223 to 1.10346, an indication that the assumptions of Poisson regression were not met.\n\nln (Incidence) = 5.185 – 0.1155(Nyakach) – 0.4613(Kisumu East) – 0.0829(Muhoroni) + 0.3357(Seme) + 0.3001(Nyando)– 0.375(Kisumu Central) – 0.1022(Year2015) – 0.09154(Year2016) − 0.3084(Year2017) + 0.0008181(Total Tested)\n\nTable 5 below shows the goodness of fit results, which clearly indicates that negative binomial regression model is a better fit for the incidence of malaria compared to the Poisson model. To begin with, the ratios of deviance and Pearson chi-square to degree of freedom DDF∼χ2(n−p) for the Poisson model are far greater than 1, which is an indication of over-dispersion and thus Poisson is not adequate for modeling such data.\n\nSubsequently, DDF∼χ2(n−p) negative binomial model ratios are both close to one which exhibits a good fit to the data. Additionally, Pearson chi-square, the lower deviance, and the larger log likelihood values of negative binomial are against those of Poisson. Together all of them share a similar conclusion of favoring the negative binomial model.\n\nFrom the Table 4, Adjusting for Kisumu East, Muhoroni, Seme, Nyando, Kisumu Central, years 2015, 2016, 2017 and the total number of patients tested for malaria, the average number of reported malaria cases in Nyakach is e-0.1155(0.8909), 11% less than those in Kisumu West Sub-County.\n\nAdjusting for Nyakach, Muhoroni, Seme, Nyando, Kisumu Central, years 2015, 2016, 2017 and the total number of patients tested for malaria, the average number of reported malaria cases in Kisumu East is e-0.4613(0.6305), 37% less than those in Kisumu West Sub-County.\n\nAdjusting for Kisumu East, Nyakach, Seme, Nyando, Kisumu Central, years 2015, 2016, 2017 and the total number of patients tested for malaria, the average number of reported malaria cases in Muhoroni is e-0.0829(0.9204), 8% less than those in Kisumu West Sub-County.\n\nAdjusting for Muhoroni Kisumu East, Nyakach, Nyando, Kisumu Central, years 2015, 2016, 2017 and the total number of patients tested for malaria, the average number of reported malaria cases in Seme is e0.3357(1.3989), 40% more than those in Kisumu West Sub-County.\n\nAdjusting for Seme, Muhoroni Kisumu East, Nyakach, Kisumu Central, years 2015, 2016, 2017 and the total number of patients tested for malaria, the average number of reported malaria cases in Nyando is e0.3001(1.3499), 35% more than those in Kisumu West Sub-County.\n\nAdjusting for Nyando, Seme, Muhoroni Kisumu East, Nyakach, years 2015, 2016, 2017 and the total number of patients tested for malaria, the average number of reported malaria cases in Kisumu Central is e-0.375(0.6873), 31% less than those in Kisumu West Sub-County.\n\nAdjusting for Kisumu Central, Nyando, Seme, Muhoroni Kisumu East, Nyakach, years 2016,2017 and the total number of patients tested for malaria, the average number of reported malaria cases in the year 2015 is e-0.1022(0.9028), 10% less than the year 2014.\n\nAdjusting for Kisumu Central, Nyando, Seme, Muhoroni Kisumu East, Nyakach, years 2015,2017 and the total number of patients tested for malaria, the average number of reported malaria cases in the year 2016 is e-0.09154(0.9125), 9% less than the year 2014.\n\nAdjusting for Kisumu Central, Nyando, Seme, Muhoroni Kisumu East, Nyakach, years 2015, 2016 and the Total number of patients Tested for malaria, the average number of reported malaria cases in the year 2017 is e-0.3084(0.7346), 27% less than the year 2014.\n\nAdjusting for Kisumu Central, Nyando, Seme, Muhoroni Kisumu East, Nyakach, years 2015, 2016 and 2017, the average number of reported malaria cases e0.0008181(1.0008) increases by 0.08% for every increase in the number of patients tested for malaria.\n\nKisumu East Sub-county is statistically associated with the incidence of malaria while adjusting for Nyakach, Muhoroni, Seme, Nyando, Kisumu Central, Years 2015, 2016, 2017 and the Total number of patients Tested for malaria (p-value ≤0.0001)\n\nSeme Sub-county is statistically associated with the incidence of malaria while adjusting for Kisumu East, Nyakach, Muhoroni, Nyando, Kisumu Central, Years 2015, 2016, 2017 and the Total number of patients Tested for malaria (p-value = 0.0001)\n\nNyando Sub-county is statistically associated with the incidence of malaria while adjusting for Kisumu East, Nyakach, Muhoroni ,Seme, Kisumu Central, years 2015, 2016, 2017 and the total number of patients tested for malaria (p-value = 0.0006)\n\nKisumu Central Sub-county is statistically associated with the incidence of malaria while adjusting for Kisumu East, Nyakach, Muhoroni, Nyando, Seme, years 2015, 2016, 2017 and the Total number of patients Tested for malaria (p-value = 0.0024)\n\nControlling for all other covariates, Nyakach and Muhoroni localities do not show any significant association with the incidence of malaria in Kisumu County (p-value = 0.205, 0.339) respectively.\n\nThe year 2017 is statistically associated with the incidence of malaria while adjusting for Kisumu East, Nyakach, Muhoroni, Nyando, Seme, Kisumu Central, years 2015, 2016 and the total number of patients tested for malaria (p-value = < 0.0001)\n\nControlling for all other covariates, the years 2015 and 2016 do not show any significant association with the incidence of malaria in Kisumu County (p = 0.153 and p = 0.2014, respectively).\n\nThe total number of patients tested is statistically associated with the incidence of malaria, while adjusting for Kisumu East, Nyakach, Muhoroni, Nyando, Seme, Kisumu Central, years 2015, 2016 and 2017 (p-value ≤ 0.0001)\n\n\nDiscussion\n\nPreliminary and further analysis in this study, investigated the change in the yearly trends of malaria incidence in Kisumu County from 2014 to 2017 to determine if there is any increase or decrease in malaria reported cases. A trend analysis was applied to the weekly malaria reported cases and the appropriate ARIMA model fitted. The yearly cases have exhibited a continuous increasing trend except in 2017, where there was a decrease. The recorded yearly malaria cases for this study were also cyclical, for instance, the year 2016 represent an increase in cases over the years 2014 and 2015, whilst the year 2017 exhibited a decline compared with all other years.\n\nThis increase may be due to improvements in the reporting system by health facilities over time, non-usage of mosquito nets by the residents and low socioeconomic status of the population (Adenomon, 2014). This fluctuating incidence recorded yearly could also be attached to the people not being careful about the illness or inconsistencies by the health care authorities in the management of the illness. The decreasing trend in 2017 could be due to an increase in the use of mosquito nets, general improvement in the malaria awareness by the population of Kisumu owed to the intensive campaigns in malaria education by several organizations. Climatic changes could have also attributed to this(Anokye et al., 2018).\n\nThe overall pattern of the reported malaria cases as reported by this study had seasonal variations. Weekly cases towards midyear recorded the highest number of cases, whereas the first weeks of the year recorded the lowest number of cases. This could be as a result of heavy rainfall experienced in the months of April, May, June and July, which is usually the rainfall season in Kisumu, and the temperature associated with rainfall seasons.\n\nThis confirms the findings of Craig et al. (1999) and Zhou et al. (2004) who revealed that that climate is a major factor in explaining the incidence of malaria. According to Zhou et al. (2004), the incidence of malaria is influenced by rainfall since mosquitoes need stagnant water for a complete life cycle. At the same time, Mabaso et al. (2007) also discovered that rainfall seasonality as well as minimum temperature are related to the number of Plasmodium falciparum-infective bites got by an individual annually or during a season.\n\nThe best fitted time series model developed for predicting the number of weekly reported cases of malaria was ARIMA (2, 0, 1). This suggests that ARIMA (2, 0, 1) can be utilized as a forecasting model to predict the future values of a series. ARIMA works best when data exhibits a constant pattern over time with a minimum amount of outliers (Labys, 2006). Researchers can use this model to forecast malaria reported cases (Mabaso et al., 2001). Nonetheless, it should be updated from time to time with the incorporation of current data.\n\nAccordingly, the study investigated the association between time in years, locality and total number of patients tested for malaria in Kisumu using weekly malaria data from 2014 to 2017.\n\nThe study results revealed that residing in Kisumu East, Seme, Nyando and Kisumu Central sub-counties were statistically associated to the incidence of malaria as compared to residing in Kisumu West sub-county. Residing in Muhoroni and Nyakach sub-counties were not statistically associated with the incidence of malaria as compared to residing in Kisumu West sub-county. The different localities have demonstrated inequalities in malaria incidence mainly due to variations in access to health services, urbanization and wealth distribution. This is similar to findings by Galactionova et al. (2017), who identified regional inequalities in the coverage of malaria interventions because of the inequality of the wealth distribution within and across many countries.\n\nMany studies have also established that there is less access to services in the rural areas compared to the urban areas, leading to less reported malaria cases from the rural sub-counties. This makes this variable a useful determinant of malaria incidence since some localities are more urban than rural and vice versa.\n\nGenerally, there was fluctuation in malaria incidences during the last four years. The year 2017 was statistically associated to the incidence of malaria as compared to the year 2014. Whereas 2015 and 2016 were not statistically associated with the incidence of malaria as compared to the year 2014. Several factors may be responsible for seasonal changes, for example climatic changes, ecologic and environmental factors, social and economic determinants such as change in health care infrastructure. Availability of health facilities and drug resistance also have an impact on incidence of malaria. Although there were different malaria control activities in each year, such as activities to decrease incidence of malaria, insecticide indoor spraying, distribution of ITNs, massive malaria control campaigns and distribution of ITNs the prevalence is still constant. This is similar to findings by Alemu et al. (2012).\n\nThere was a significant association between the total number of patients tested for malaria and the incidence of malaria. Though urban areas have normally been at lower risk of malaria, unpredictable and unplanned population growth has been a key factor in making urban or peri-urban transmission an increasing problem .This could explain the high malaria incidence in 2016.This finding is consistent to a study carried out by (Knudsen & Slooff, 1992).\n\n\nConclusions\n\nThe objective of this research was to determine the factors associated with the incidence of malaria in Kisumu County over time, given the locality where the patients sought health facility intervention, time in years and the total number of patients who were tested for malaria. The study utilized weekly malaria reported cases data from IDSR Disease Surveillance registry, for the period January 2014 to December 2017 in Kisumu County. Reported malaria cases confirmed by laboratory test was used in the analysis and modeling.\n\nA seasonal pattern was observed in the malaria incidences in Kisumu County. ARIMA (2, 0, 1) model was found to be the best fit statistical model to predict malaria incidences in Kisumu County. The results found from this study offer useful information for policy makers to be able to effectively implement timely and effective malaria preventive and control measures.\n\nGoodness of fit model assessment criteria were used in selecting which model will fit the malaria reported cases better by exhausting both Poisson and negative binomial regression models. The negative binomial regression model was found to fit the data better than the Poisson regression model, based on the results. In modeling the incidence of malaria, the quality of the Poisson model was estimated using the AIC. The AIC value produced was 29,318, the deviance was 26,219 and a dispersion parameter of 70.29 showing an over-dispersion in the data leading to a violation of one of its main assumption of the equality of mean and variance parameters. It became a necessary to validate the Poisson regression models using negative binomial because of over-dispersion.\n\nThe results obtained suggested that Kisumu East, Seme, Nyando, Kisumu Central localities, the year 2017 and the total number of patients who underwent a laboratory confirmation test for malaria were significant factors for incidence of malaria in Kisumu County over time. Whereas, Nyakach, Muhoroni localities, the years 2015 and 2016 were not significant factors for malaria incidences. The findings provide better insight of environmental and socio-economic effects on malaria and provide important information for malaria prediction. Nonetheless, there is need for additional studies to consider which other factors influence the incidence of malaria notwithstanding environmental and socio-economic factors. Likewise, health professionals practicing in Kisumu County should be encouraged to regularly review and report cases of malaria in their facilities. This is because, reporting rates, completeness and the consistency of malaria reported cases remain extremely low and importantly was poorer in localities where malaria is endemic.\n\n\nData availability\n\nThe data can be obtained from the Integrated Disease Surveillance and Response (IDSR) registry through the DHIS2 platform, using the standard application programming interface provided https://hiskenya.org/dhis-web-commons/security/login.action. Weekly facility-level data on malaria parasitological testing were extracted from Kenya DHIS2 between January 2014 to December 2017.\n\nAccess to the raw dataset is possible upon placing a formal request to the Ministry of Health, Kenya (emailing pshealthke@gmail.com), since the platform is kept under restricted access.",
"appendix": "Acknowledgments\n\nI thank the Almighty God for his guidance through my study period, without his provisions and guidance, my participation in this program of study would have been futile. Secondly, special thanks to the University of Nairobi administrators and staff at large for regular guidance on the whole research process. I also sincerely thank my friends and family for supporting, advising and encouraging me throughout the time I have worked for my project.\n\n\nReferences\n\nAdenomon MO: Modeling the prevalence of malaria in Niger State: An application of Poisson regression and negative binomial regression models Univariate and Multivariate Time Series Analysis View project Econometrics Analysis in Nigeria View project. 2014. Reference Source\n\nAlemu A, Muluye D, Mihret M, et al.: Ten year trend analysis of malaria prevalence in Kola Diba, North Gondar, Northwest Ethiopia. Parasit Vectors. 2012; 5(1): 173. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAnokye R, Acheampong E, Owusu I, et al.: Time series analysis of malaria in Kumasi: Using ARIMA models to forecast future incidence. Cogent Social Sciences. 2018; 4(1). Publisher Full Text\n\nBarasa E, Nguhiu P, McIntyre D: Measuring progress towards Sustainable Development Goal 3.8 on universal health coverage in Kenya. BMJ Global Health. 2018; 3(3): e000904. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCraig MH, Snow RW, le Sueur D: A climate-based distribution model of malaria transmission in sub-Saharan Africa. Parasitol Today. 1999; 15(3): 105–111. PubMed Abstract | Publisher Full Text\n\nFisher LD: Self-designing clinical trials. Stat Med. 1998; 17(14): 1551–1562. PubMed Abstract | Publisher Full Text\n\nGalactionova K, Smith TA, de Savigny D, et al.: State of inequality in malaria intervention coverage in sub-Saharan African countries. BMC Medicine. 2017; 15(1): 185. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGallup J, Sachs J: The economic burden of malaria. Am J Trop Med Hyg. 2001; 64(1–2): 85–96. PubMed Abstract | Publisher Full Text\n\nKNBS: Kenya Malaria Indicator Survey 2015 - Kenya National Bureau of Statistics. 2015. Retrieved May 2, 2019. Reference Source\n\nKnudsen AB, Slooff R: Vector-borne Disease Problems in Rapid Urbanization: New Approaches to Vector Control. Bull World Health Organ. 1992; 70(1): 1–6. PubMed Abstract | Free Full Text\n\nLabys WC: Modeling and forecasting primary commodity prices. Ashgate. 2006. Publisher Full Text\n\nMabaso ML, Craig M, Ross A, et al.: Environmental Predictors of the Seasonality of Malaria Transmission in Africa: The Challenge. Am J Trop Med Hyg. 2007; 76(1): 33–38. PubMed Abstract | Publisher Full Text\n\nMugenda OM, Mugenda AG: Research Methods: Quantitative and Qualitative Approaches | Text Book Centre. 2013. Reference Source\n\nMusuva A, Ejersa W, Kiptui R, et al.: The malaria testing and treatment landscape in Kenya: results from a nationally representative survey among the public and private sector in 2016. Malar J. 2017; 16(1): 494. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNobre FF, Monteiro AB, Telles PR, et al.: Dynamic linear model and SARIMA: a comparison of their forecasting performance in epidemiology. Stat Med. 2001; 20(20): 3051–3069. PubMed Abstract | Publisher Full Text\n\nPresident’s Malaria Initiative: Kenya Malaria Operational Plan FY 2018. 2018a. Reference Source\n\nPresident’s Malaria Initiative: President’s Malaria Initiative - Kenya Country Profile. 2018b. Reference Source\n\nUS President’s Malaria Initiative: FY 2019 Kenya Malaria Operational Plan. 2019. Reference Source\n\nSDG Indicators. 2018. Reference Source\n\nWorld Health Organization: WORLD MALARIA REPORT 2018. ISBN 978 92 4 156565 3, 2018. Reference Source\n\nZhou G, Minakawa N, Githeko AK, et al.: Association between climate variability and malaria epidemics in the East African highlands. Proc Natl Acad Sci U S A. 2004; 101(8): 2375–2380. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "71238",
"date": "28 Sep 2020",
"name": "Samuel Akech",
"expertise": [
"Reviewer Expertise Paediatrics",
"epidemiology",
"sepsis",
"malaria",
"clinical trials"
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe study reports that is uses a random sample of 384 laboratory-confirmed malaria cases as reported by health facilities in Kisumu County from January 2014 to December 2017. The study concludes that there is a need to encourage health professionals to regularly review and report cases of malaria in their facilities because of incomplete reporting. The study uses time series analysis approaches. The study as written is quite difficult to follow and is not clear to discern a clear message from the paper. The title, the methods, and the conclusions are not connected. It is not clear how data were obtained, the method section is unclear, the analysis section is not clear and generally, the study is not replicable. Even the introduction section is lengthy but not focused and the aim of the study does not come out clearly. Sample size calculation shown is for investigation of prevalence, but the study investigates determinants and time trends. It, therefore, seems that the sample size calculation presented is not appropriate. The sample size is not appropriate to address the results and discussion presented by the authors. The authors state ‘Weekly facility-level data on malaria parasitological testing were extracted from the Integrated Disease Surveillance and Response (IDSR) registry through the DHIS2 platform.’ It is not clear how the authors did this since data in DHIS2 platform has monthly summaries It is not clear how the DHIS2 data were combined with data from the cross-sectional survey\n\nOverall comment: The authors ought to review the data they have and identify a clear message that the data can address and re-write the manuscript to bring out a clear message. This should also involve modifying the title to be appropriate, write methods, and data analysis section clearly.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? No",
"responses": []
},
{
"id": "88444",
"date": "01 Jul 2021",
"name": "João L Ferrão",
"expertise": [
"Reviewer Expertise Modelling and mapping malaria risks. Expert in Information Management Systems (Statistics and Econometrics)."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis topic is very important and contributes to Malaria eradication efforts. The effort in malaria combat is multidisciplinary and mathematical modelling can greatly contribute. I see this study as good attempt. However, major revision should be addressed to fulfill the intention.\nObjective needs a better clarification:\n\nIf the intention is to model, means predicts malaria cases as in the title or if is “to determine the factors associated with the incidence of malaria” as in the abstract or, if is “mapping the corresponding factors contributing to the incidence of malaria”. If the intention is mapping, I would expect to see maps.\nLiterature:\n\nThere is open-source literature in malaria modelling using ARIMA in Africa. I don’t see any in this article.\n\nI don’t see any plausible reason for the use of outdated literature for example: (Gallup & Sachs, 2001), Zhou et al. (2004), (Mabaso et al., 2001) with so many recent literatures.\nMethods:\nData: since they are historic data, the use of five or more years may improve your precision for trends.\n\nI don’t understand why you used a sample instead of all data.\n\n384 data for 208 weeks means less than 2 observations per week. This may not be enough for any trend analysis.\n\nIndependent variables: number of patients tested for malaria is a socio-economic factor? Is location an environmental factor? What factor is time of the year?\n\nWhat was the level of missing data?\nResults:\nFigure 1. Are you presenting incidence or malaria cases - can you clarify?\n\nFigure 3. Are you presenting trend on incidence or malaria cases - can you clarify?\nDiscussion:\nResults are for ARIMA, Negative binomial and, Poisson model. I don’t see any discussion on these models compared to other authors.\nOverall, I don’t see the adding value of this research to science. Adding more data for analysis, defining clearly the objective may result in a better manuscript. Additional file with data used may be useful.\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?\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/9-600
|
https://f1000research.com/articles/9-597/v1
|
12 Jun 20
|
{
"type": "Research Article",
"title": "Flavor perception of wine is unchanged during commercial flight: a comparative field study",
"authors": [
"Anders Gram-Hanssen",
"Cecilie Bøge Paulsen",
"Jacob Rosenberg",
"Cecilie Bøge Paulsen",
"Jacob Rosenberg"
],
"abstract": "Background: It is generally accepted that the human perception of flavor and odor is altered in low-pressure environments such as airplane cabins. This has been demonstrated in several simulation studies, but never in a field study conducted in an authentic environment, and never using wine as the object of study. Methods: We performed a comparative field study composed of two wine tastings. The first tasting was conducted on board an aircraft flying at standard cruising altitude and the second tasting was conducted at ground level. Subjective taste experience and current mood were evaluated through a validated questionnaire. The study was reported according to the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guideline. Results: The study included 22 participants, none of whom had any special training in wine tasting. No statistically significant difference in experienced flavor intensity was found between the high and low altitude tests, with median aromatic intensities of 5 (interquartile range 3.5-6.0) and 5 (interquartile range 4.0-6.5) respectively, measured on a 9-point hedonic scale. Additionally, there was no detectable difference in several other taste parameters. Conclusions: These findings suggest that even though experimental studies have demonstrated that senses of taste and smell may be suppressed on commercial flights, the subjective wine tasting experience of non-professionals in real life testing may not be affected.",
"keywords": [
"Sensation",
"Gustation",
"Aviation",
"Oenology",
"Wine",
"Taste",
"Aircraft",
"Low pressure environment",
"Sense of taste"
],
"content": "Introduction\n\nModern air travel is a multi-billion-dollar industry and more than four billion passengers flew on commercial airlines in 2017, which generated an industry-wide revenue of 755 billion USD1. Labor, fuel, and equipment are the three biggest running expenses of the industry, but airline catering is also a costly venture, estimated to constitute an average of 2–3% of total operating costs2. Despite this, airplane meals generally have a poor reputation among passengers. This may be due to the sheer quality of the food, but could also be a result of altered perception of flavor and odor in an airplane cabin3–5.\n\nFood perception is a complex multisensory experience depending on both taste, smell, and mouthfeel, which collectively form an impression of flavor6. Simulation studies have suggested that our sense of taste and smell is generally reduced in low-pressure environments such as airplane cabins, diminishing the perception of flavor intensity of food products7–10. This is thought to be caused by elevated sensory thresholds for detection of tastants in low-pressure environments5. Moreover, this transitory impairment of the gustatory and olfactory senses may impair the general enjoyment of food as well8. In addition to the low air pressure, several other factors present in a standard commercial airline cabin are also assumed to contribute to this, such as dry recirculating air, cold temperature, and low oxygen levels5,8,9. While the exact physiology behind these phenomena is not definitively determined, some explanatory mechanisms have been suggested: cold and dry air are impeding the interaction between odorants and olfactory receptors, and low oxygen levels are inducing hypoxia which may modulate receptor functioning5,11. Other sensory modalities (e.g. hearing) can also impact flavor perception – a phenomenon known as crossmodal correspondence – and in this regard, the constant background noise in an airplane cabin may be another important sensory influence10,12–15. Psychological stress is also assumed to modulate flavor perception to some degree16,17, and commercial air travel is a source of – perhaps minor – psychological stress18,19. Clearly, flavor perception during flight is a multifactorial process that has not yet been sufficiently characterized, which is ultimately to the disadvantage of the end consumer.\n\nWine has a complex composition of flavors and wine tasting requires a special attention to nuance. Therefore, the perception and appreciation of wine may also be affected by the suboptimal conditions in an airplane cabin. In current literature, the specific mechanisms of this process have not been described in its entirety and not all details are fully understood. In Figure 1 we propose a simplified theoretical framework illustrating how these factors may interact and how environmental and individual factors modulate the process of forming a wine consumption experience. Simulation studies have heavily suggested that wine consumption experience is negatively affected during flights8–10, and reportedly, the bitterness of wine is more prominent when consumed in the air5. However, to the best of our knowledge, the impact of airplane cabin conditions on wine consumption experience has not yet been tested in a field study. In this study, we elected to focus specifically on the concept of flavor intensity and the subjective perception of this.\n\nWe hypothesized that the subjective perception of flavor intensity of wine is reduced while on board a commercial aircraft flying at standard cruising altitude compared with on the ground. Furthermore, we speculated that this would lead to a decreased overall liking and appreciation of wine, likely resulting in a worse consumer experience. To test these pre-specified hypotheses, we performed a comparative field study of high and low altitude wine tastings, and we aimed to verify the discrepancy in taste sensation, as suggested by simulation studies.\n\n\nMethods\n\nThe present study was reported according to the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guideline20 and was performed as a single-blinded, non-randomized field study. The study was carried out in May 2019 and all participants were healthcare professionals (medical doctors, nurses, or medical students) travelling from Denmark to Turkey to attend a scientific conference. Participants were enrolled in February 2019 and all conference attendees were eligible for inclusion in the study. Potentially eligible participants were excluded from the study if they were reluctant to consume alcohol, if they were pregnant or breastfeeding, or if they had a history of dysosmia or dysgeusia (i.e. olfactory or gustatory dysfunction).\n\nDuring the experiment, all participants taste-tested two separate samples of the same wine. The wine samples were distributed to the study participants in two transparent Corning™ Falcon 50 mL Conical Centrifuge Tubes (outside diameter: 30 mm; length: 115 mm). Each tube contained only 25 mL of wine, which corresponds to approximately one sixth of a standard serving size. This conforms to general wine tasting standards and is generally considered sufficient for a simple taste assessment21. The participants were carefully instructed (verbally and in writing) to open the plastic tube and let the wine oxygenate for 60 seconds before drinking the sample directly from the container. Each participant consumed either red or white wine according to personal preference. The red wine of choice was the Majestik Syrah & Kalecik Karasi, 2017 (alc. 13.5%), and the white wine was the Majestik Sauvignon Blanc & Sultaniye, 2017 (alc. 12.5%). Both wines are produced by Sevilen Group, Izmir, Turkey (http://www.sevilengroup.com/en). These wines were chosen as they were the only wines available for purchase on the flight, and because consumption of alcoholic beverages not purchased on the flight was not permitted. Participants were blinded as to the name and production year of the wines, in order to reduce bias22.\n\nAt the first tasting the participants were instructed to fill out a form on basic demographic data and a 12-item questionnaire. The first tasting was conducted on board a Boeing 737–800 (73H) aircraft, which is a short- to medium-range, narrow-body jet airliner that seats 189 passengers and is part of the Boeing 737 Next Generation series. The taste-testing commenced approximately 1h 45m after take-off at an altitude of approximately 37,000 ft (11.278 m) above sea level, which is considered standard cruising altitude. Due to the severe security restrictions on commercial aircrafts, we were not allowed to bring measurement equipment onboard the plane. Therefore, we can only provide a general estimate of the cabin pressure: Aircraft pressurization systems always maintain a cabin pressure above 750 hPa, equivalent to a maximum cabin altitude (i.e. the equivalent altitude with the same air pressure outside the cabin according to the International Standard Atmosphere) of 8,000 ft (2,438 m)23. However, when accounting for the Boeing 737–800’s maximum pressure differential24, the actual cabin pressure would have been between approximately 750 and 790 hPa. This corresponds to a cabin altitude between 6,650 and 8,000 ft (2,027 to 2,438 m).\n\nThe second tasting was performed at ground-level, where the participants were asked to fill out the same questionnaire again. This took place en-route to the hotel on board the airport shuttle bus, an Isuzu Novo Lux, which is a medium-sized coach that seats 25 people. The time of tasting was approximately 1h after landing and the altitude was approximately 305 ft (93m) above sea level. In May of 2019 the average air pressure in Izmir, Turkey was 1013 hPa25.\n\nOur primary outcome was the difference in experienced flavor intensity between high and low altitude testing. The secondary outcomes included differences in the overall liking of the wine and differences in the various other subjective taste parameters.\n\nWe developed and face validated a 2-part questionnaire for this study. The first part was given before taste-testing and concerned basic demographic data and self-assessed wine-tasting expertise on a scale from 1–9 (“poor” to “outstanding”). Part two was given after taste-testing and contained seven items regarding the wine, including overall liking, as well as five items concerning current mood. The questionnaire was developed based on flavor descriptors hand-picked from the literature26. Two additional items on subjective drinkability were included (“chugability” and “inebriating effects”), and four items considered relevant to this study was incorporated from the validated Profile of Mood States for Adolescents (POMS-A) questionnaire27. For the seven items regarding wine, we adopted the widely used 9-point Hedonic Scale28, the de-facto gold standard for sensory testing29.\n\nWe performed a pilot test using two individuals considered representative of the study population. To ensure face validity, these two individuals consumed two sample of wine in a high-and low-altitude setting, respectively. They were subjected to the questionnaire and subsequently interviewed about comprehensibility, relevance, and feasibility of the questionnaire. The pilot study resulted in only minor revisions of the questionnaire to optimize the flow and comprehensibility, specifically regarding Items 5 and 6. Reliability testing was not performed. The questionnaire is provided as Extended data.\n\nData were analyzed using IBM SPSS Statistics for Windows, Version 22.0 (Armonk, NY: IBM Corp). The study characteristics were described with proportions for categorical data (number (%)), and median with interquartile range for non-normally distributed data. Paired data were compared using Wilcoxon signed-rank test, with P < 0.05 considered significant. Bonferroni correction for multiple comparisons did not significantly affect our results (data not presented), and therefore, the presented results are uncorrected. Missing values (n = 2) were assumed to be missing completely at random and were excluded on a test-by-test basis.\n\nDue to the lack of existing comparable data, we were unable to form any reasonable assumptions about magnitude of the hypothesized difference between taste tests. Therefore, participants were selected through convenience sampling and all eligible attendees at the aforementioned scientific conference were enrolled in the study.\n\nAll participants gave verbal consent to participate. According to Danish law, the study did not require approval from the Danish Data Protection Agency nor from the local ethics committee (registration number: H-19013122), because the study did not involve an intervention or collection of biometric data.\n\n\nResults\n\nIn total, 22 participants were enrolled in the study. Of these, 45% were females and the majority were below 40 years of age (77%). 86% chose to drink red wine and the remaining participants drank white wine. The characteristics of the study participants are shown in Table 1.\n\nIQR = Interquartile range. 1 Self-estimated expertise in wine tasting on a scale from 1–9.\n\nThe experienced aromatic intensities at both high- and low-altitude tastings had a median score of 5 on the 9-point hedonic scale (interquartile ranges of 3.5–6.0 and 4.0–6.5 respectively), and Wilcoxon signed-rank test did not demonstrate any statistically significant difference (p = 0.176). The differences between the remaining taste parameters at high and low altitude were also not significant (p-values 0.153 – 0.858). Nor was there any significant difference in the participants' POMS-A scores at high altitude compared with their scores at low altitude (p-values 0.058–0.705). Among these, there was a non-significant tendency towards a higher level of sleepiness at low altitude than at high altitude (p = 0.058). The results are listed in Table 2.\n\nIQR = Interquartile range. POMS-A = Profile of Mood States for Adolescents questionnaire.\n\n1: Ranging from 1 to 9. 2: 1 being “low”, 5 being “neutral”, and 9 being “intense”. 3: 1 being “poor”, 5 being “neutral”, and 9 being “outstanding”. 4: 1 being “dislike extremely”, 5 being “neither like, nor dislike”, and 9 being “like extremely”. 5: Ranging from 0 to 4. With 0 being “not at all”, 2 being “moderately” and 4 being “extremely”.\n\n\nDiscussion\n\nAmong our 22 enrolled participants, the flavor perception and overall liking were unchanged between high and low altitude. Additionally, there was no statistically significant change in participant mood evaluated through the adapted POMS-A questionnaire.\n\nOur findings suggest that lay people do not experience a subjective difference in flavor intensity in a real-life aircraft cabin, when flying at standard cruising altitude compared with at ground level. Furthermore, our study was unable to detect a statistical difference in a variety of taste parameters, including overall liking of wine, fruit notes, and bitterness. This disagrees with multiple simulation studies conducted in controlled environments, simulating the conditions of an aircraft cabin and using test panels of both lay people and gastronomic professionals8–10. Our results suggest that even though the gustatory and olfactory senses may be suppressed, it might not affect the subjective tasting experience of lay people (i.e. the final consumer experience) when tested in a real-life setting. Thus, the poor reputation of aircraft cuisine may not be a reflection of a changed perception of flavor and odor in high altitude environments. The mood of our participants was unaffected by the high and low altitude settings. The only parameter of the POMS-A questionnaire that came close to show a statistical difference was the sleepiness of our participants. This can be explained by the late hour of the low altitude tasting and therefore this result is negligible.\n\nA strength of this study is that participants were neither wine professionals nor sommeliers, but lay people with an average knowledge and experience in wine tasting. Thus, the results of this study have high external validity in the general non-wine-proficient population. However, our study population was fairly limited as only 22 participants were eligible for inclusion, and all participants were Danish healthcare professionals making the study population quite homogenous in that regard. Future studies should include a larger and more heterogenous population in terms of nationality, profession, socio-economic status, etc.\n\nThis study was conducted in a largely authentic setting, which ensures high ecological validity and makes our conclusions generalizable. In this context, “authentic” refers to a setting that approximates normal circumstances for wine consumption during flight. However, such an uncontrolled study environment does allow for a possible influence of unrecognized and potentially confounding external factors. This is the nature of a field study and the influence of undesirable external factors is inherently unavoidable. It could be argued that the two testing environments in this study were fundamentally different, and in the future, these results could be expanded upon by performing two taste tests in the same airplane cabin, one at cruising altitude and another at ground level. This would reduce the influence of possible environmental confounders. Nonetheless, it could also be argued that the two testing environments in this study were comparable in several aspects. For instance, the presence of constant background noise is quite similar in an airplane cabin and a bus cabin, and the possible influence of auditory-gustatory crossmodal correspondence is therefore not expected to be a significant confounder12,13. Since we were not allowed to bring measurement equipment onboard the airplane, the exact levels of background noise were not measured and some degree of auditory influence cannot be ruled out. Psychological stress is also thought to have an impact on flavor perception, but as all forms of public transportation can be considered a minor source of stress, the testing conditions were also in this regard quite similar. Psychological stress is therefore not considered a significant confounder in our study.\n\nSome studies have suggested that the five basic flavors (i.e. sweet, sour, salty, bitter, and umami) may be affected differently by the conditions in an airplane cabin13. Consequently, future studies should include a variety of wines of different characteristics and quality in order to exclude any possible influence of the specific flavor composition of the wines chosen for this study. It may also be interesting to make a similar field study as the present, but including wine experts instead of lay people without special wine tasting abilities or training. It is also recommended that future studies should employ measures to control for confounders related to the individual, such as current satiety, appetite, and thirst. A limitation of the present study is the lack of experimental control. Under optimal circumstances, we would have preferred to implement a randomized crossover design, include a pre-flight taste test, and expand the timeframe in order to reduce the effect of recall bias. Unfortunately, this was not practically or economically feasible given the extensive security restrictions of commercial air travel, which is why we settled on the current experimental design. Another limitation of this study is the use of Falcon tubes as a vessel for the wine, as opposed to normal wine glasses. The experience of drinking a beverage is greatly influenced by the properties of its container30, and these narrow plastic tubes could be an inhibiting factor in the consumption experience, e.g. inhibiting the odor sensation and diminishing the overall aesthetic experience and its emotional impact on the consumer31,32.\n\nIn general, future research into improving the quality of airline catering could be of great benefit to the industry, as we believe that the current relationship between catering expenses and the quality of the final consumer experience may be disproportionate. The theoretical framework presented in Figure 1 was developed for generating hypotheses and is only intended as a preliminary proposal illustrating the mechanisms at work. It is intended as a basis for further discussion of the topic, and actual testing or validation of the framework is beyond the scope of this article. Further research is necessary on the physiology and psychology of flavor perception onboard airplanes, which will inspire new and innovative methods and techniques for designing meals and beverages optimized for consumption on airplanes. This is part of an emerging field of science known as gastrophysics, which will hopefully pave the way for future improvements in airline gastronomy5.\n\nIn conclusion, we were unable to verify the findings of previous simulation studies, as we did not find a difference in either subjective perception of flavor intensity or overall liking of wine between high- and low-altitude tests. This suggests that any possible perceptual change that exists while flying may not affect the subjective taste experience of lay people. Ultimately, current knowledge regarding flavor perception onboard airplanes is inadequate.\n\n\nData availability\n\nOpen Science Framework: Flavour perception of wine is unchanged during commercial flight: a comparative field study, http://doi.org/10.17605/OSF.IO/NMECS33.\n\nThis project contains the following underlying data:\n\n- Participants data.csv (Descriptive participant data)\n\n- Questionnaire data.csv (Raw data from questionnaires). Each column is titled in the following manner: [first/second administration]_[item]_[description]. For example, \"2_03_bitter\" is the second administration of item 3 \"bitterness\".\n\nOpen Science Framework: Flavour perception of wine is unchanged during commercial flight: a comparative field study, http://doi.org/10.17605/OSF.IO/NMECS33.\n\nThis project contains the following extended data:\n\n- Questionnaire.pdf (The adapted questionnaire used in the study)\n\n- STROBE_checklist.docx (Completed STROBE checklist)\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 Mr. Anders Tolstrup and Mr. Joachim Hjalde Halmsted Olsen for their valuable input regarding design and face validation of the questionnaire used in this field study.\n\n\nReferences\n\nInternational Air Transport Association: Industry Statistics Fact Sheet. 2018 [cited 2019 May 31]. Reference Source\n\nAirlines For America: A4A Passenger Airline Cost Index (PACI). 2019 [cited 2019 Jun 28]. Reference Source\n\nGajanan M: Why Airplane Food Is So Bad. Time Magazine. 2017 [cited 2019 May 31]. Reference Source\n\nCalder S: Haute cuisine: Why do airlines persist in serving us in-flight meals? The Independent. 2014 [cited 2019 Dec 13]. Reference Source\n\nSpence C: Tasting in the air: a review. Int J Gastron Food Sci. AZTI-Tecnalia. 2017; 9: 10–15. Publisher Full Text\n\nAuvray M, Spence C: The multisensory perception of flavor. Conscious Cogn. 2008; 17(3): 1016–31. PubMed Abstract | Publisher Full Text\n\nBert P: La pression barométrique. Recherches de physiologie expérimentale. Paris: G. Masson; 1878. Reference Source\n\nBurdack-Freitag A, Bullinger D, Mayer F, et al.: Odor and taste perception at normal and low atmospheric pressure in a simulated aircraft cabin. J für Verbraucherschutz und Leb. 2011; 6(1): 95–109. Publisher Full Text\n\nKuehn M, Welsch H, Zahnert T, et al.: Changes of pressure and humidity affect olfactory function. Eur Arch Otorhinolaryngol. 2008; 265(3): 299–302. PubMed Abstract | Publisher Full Text\n\nRahne T, Köppke R, Nehring M, et al.: Does ambient noise or hypobaric atmosphere influence olfactory and gustatory function? PLoS One. 2018; 13(1): e0190837. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHuppertz T, Freiherr J, Olzowy B, et al.: Reduction of olfactory sensitivity during normobaric hypoxia. Auris Nasus Larynx. 2018; 45(4): 747–52. PubMed Abstract | Publisher Full Text\n\nKnöferle K, Spence C: Crossmodal correspondences between sounds and tastes. Psychon Bull Rev. 2012; 19: 1–15. PubMed Abstract | Publisher Full Text\n\nSpence C, Michel C, Smith B: Airplane noise and the taste of umami. Flavour. 2014; 3(1): 2. Publisher Full Text\n\nSpence C, Velasco C, Knoeferle K: A large sample study on the influence of the multisensory environment on the wine drinking experience. Flavour. 2014; 3(1). Publisher Full Text\n\nWoods AT, Poliakoff E, Lloyd DM, et al.: Effect of background noise on food perception. Food Qual Prefer. 2011; 22(1): 42–7. Publisher Full Text\n\nAl’absi M, Nakajima M, Hooker S, et al.: Exposure to acute stress is associated with attenuated sweet taste. Psychophysiology. 2012; 49(1): 96–103. PubMed Abstract | Publisher Full Text | Free Full Text\n\nIleri-Gurel E, Pehlivanoglu B, Dogan M: Effect of acute stress on taste perception: In relation with baseline anxiety level and body weight. Chem Senses. 2013; 38(1): 27–34. PubMed Abstract | Publisher Full Text\n\nMcIntosh IB, Swanson V, Power KG, et al.: Anxiety and health problems related to air travel. J Travel Med. 1998; 5(4): 198–204. PubMed Abstract | Publisher Full Text\n\nEvans GW, Wener RE: Rail commuting duration and passenger stress. Heal Psychol. 2006; 25(3): 408–12. PubMed Abstract | Publisher Full Text\n\nVandenbroucke JP, von Elm E, Altman DG, et al.: Strengthening the Reporting of Observational Studies in Epidemiology (STROBE): Explanation and Elaboration. Epidemiology. 2007; 18(6): 805–35. PubMed Abstract | Publisher Full Text\n\nJackson RS: Qualitative (general) wine tasting. In: Wine tasting: a professional handbook. 1st ed. Academic Press; 2002; 187–210.\n\nSiegrist M, Cousin ME: Expectations influence sensory experience in a wine tasting. Appetite. 2009; 52(3): 762–5. PubMed Abstract | Publisher Full Text\n\nInternational Air Transport Association: Medical Manual 11th Edition. 2018; [cited 2020 Mar 3]. Reference Source\n\nBrady C: The Boeing 737 technical site; Pressurisation. 2014; [cited 2020 Mar 3]. Reference Source\n\nWorld Weather Online: Izmir monthly climate averages. 2019; [cited 2020 Mar 3]. Reference Source\n\nParr WV, Valentin D, Reedman P, et al.: Expectation or sensorial reality? An empirical investigation of the biodynamic calendar for wine drinkers. Ryabinin AE editor. PLoS One. 2017; 12(1): e0169257. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTerry PC, Lane AM, Lane HJ, et al.: Development and validation of a mood measure for adolescents. J Sports Sci. 1999; 17(11): 861–72. PubMed Abstract | Publisher Full Text\n\nPeryam DR, Pilgrim FJ: Hedonic scale method of measuring food preferences. Food Technol. 1957; 11(Suppl): 9–14. Reference Source\n\nWichchukit S, O’Mahony M: The 9-point hedonic scale and hedonic ranking in food science: some reappraisals and alternatives. J Sci Food Agric. 2015; 95(11): 2167–78. PubMed Abstract | Publisher Full Text\n\nSchifferstein HNJ: The drinking experience: cup or content? Food Qual Prefer. 2009; 20(3): 268–76. Publisher Full Text\n\nMora P, Moscarola J: Representations of the emotions associated with a wine purchasing or consumption experience. Int J Consum Stud. 2010; 34(6): 674–83. Publisher Full Text\n\nCharters S, Pettigrew S: Is wine consumption an aesthetic experience? J Wine Res. Routledge; 2005; 16: 121–36. Publisher Full Text\n\nGram-Hanssen A, Paulsen CB, Rosenberg J: Flavour perception of wine is unchanged during commercial flight: a comparative field study. 2020. http://www.doi.org/10.17605/OSF.IO/NMECS"
}
|
[
{
"id": "64831",
"date": "29 Jun 2020",
"name": "Sanne Boesveldt",
"expertise": [
"Reviewer Expertise (chemo)sensory science",
"eating behavior"
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting topic for investigation, with very little scientific research done so far, but may have large implications.\nHowever, the study has some substantial limitations, mainly the very limited sample size, and the fact that the order of conditions was not randomized. Even though the authors discuss these, I strongly question the validity of the outcomes and conclusions.\nThe authors state that a priori power calculation was not possible, but they could have considered what would be a relevant difference to detect in their outcomes, also based on the available literature on (changes in) smell/taste function under similar circumstances. This small sample size is likely the reason for their null effects, so I am not sure how valid it is to actually publish these findings. At least, phrase this clearly as an exploratory pilot study (e.g. also in the title etc).\nAt what time of day were the assessments done? The authors should then in more depth discuss potential effects of time of day or fatigue (also given the trend towards increased sleepiness during the 2nd assessment). Moreover, specifically for wine tasting, time of day and circumstances/context are important.\nIt does not make sense to me that intensity was assessed on a hedonic scale. Why was this chosen, and not an LMS or VAS? Also, why was intensity chosen as the main outcome (which is better assessed with a trained panel), rather than liking (which is better suited for a consumer panel, such as used here)?\nThe authors missed out on a relevant paper, on liking of foods in airplanes: Holthuysen et al. (20171).\n\nI am not convinced that figure 1 adds any relevance to the paper and study described.\n\nIs the work 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": []
},
{
"id": "66757",
"date": "03 Aug 2020",
"name": "Barry C. Smith",
"expertise": [
"Reviewer Expertise Taste",
"smell and multi sensory flavour perception",
"sensory evaluation of wine."
],
"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\nGeneral:\nThe topic of how flavour perception is affected during commercial flights is a good one. It is currently underexplored and more research would be welcome to help fill out the small set findings in the literature. However, this rather badly designed and poorly conducted study is without proper controls and its findings do not support the declared aim of overturning previous findings in the field. It is not helped by giving different target conclusions at different points in the paper, ranging from the headline grabbing but overclaiming title: ‘Flavor perception of wine is unchanged during commercial flight’ to the more circumspect claim that there is no ‘difference in either subjective perception of flavor intensity or overall liking of wine between high- and low-altitude tests’ to the even narrower claim that ‘on commercial flights, any possible perceptual change that exists while flying may not affect the subjective taste experience of lay people’. However, the findings from the data do not establish any of these conclusions, and as a result not much store can be set by the authors’ assertion that: ‘we were unable to verify the findings of previous simulation studies’. This is not surprising. A more plausible interpretation is that that was not a well enough conducted study either to verify or refute the previous simulation studies, nor to establish that there was no ‘difference in either subjective perception of flavor intensity or overall liking of wine between high- and low-altitude tests’. The current article cannot be recommended for publication. Crucial limitations of the current study are declared by the authors themselves in the discussion section, suggesting the authors may have already faced or anticipate facing these criticisms. The list of limitations almost amounts to a self-review of the problems with the current paper, helping to reinforce the verdict offered here.\nThe authors reveal both a lack of knowledge of the existing literature and at times a failure to understand the literature they do cite. We are told early on that the effects of pressure and altitude on flavour perception has only been conducted in simulated environments and ‘never in a field study conducted in an authentic environment, and never using wine as the object of study’. Later, the authors are more cautious, ‘to the best of our knowledge, the impact of airplane cabin conditions on wine consumption experience has not yet been tested in a field study.’ But in fact it has. Together with British Airways, wine buyer Andy Sparrow carried out a controlled study tasting the same wines in the lounge before the flight, then in flight after two hours, and again after 5 hours. Most of the Bordeaux wines were perceived as less intense and more tannic although a high-altitude produced Catena Angelica Zapata Malbec tasted more intense: https://www.youtube.com/watch?v=0PzeuVCBzQc.\nIn addition, there is The Wine in the Sky Questionnaire (2018 Assouline) where a range of expert and non-expert tasters report on their experiences of wines in-flight.\n\nThe background to the experiment relies on an understanding of multisensory flavour perception and the possible factors affecting it. The authors’ understanding can be questioned given their diagram in Figure 1.\n\nWhy are the authors separating flavour and odour characteristics? Are participants required to attend to each separately? On the far left-hand side should be volatile and non-volatile molecules. Flavour perception is a result of multisensory integration, so does not precede it. Cross-modal effects of one sense on another can happen at the level of sensory inputs that feed into the process of integration. How is the wine consumption experience judged? Is it the same thing as ‘flavour intensity’ or is it more about the intensity of the experience rather than intensity of sensations like bitterness, sweetness, etc. or is is just the hedonic scoring of the wine? It is unclear.\n\nStudy Design:\nIn taking advantage of a commercial flight that colleagues of the authors were travelling on, they were limited to the choice of wine samples sold on the plane. Therefore, there was no opportunity to have the participants taste the same wines on the ground before flying. A limitation the authors acknowledge.\n\nThe authors tell us that ‘Wine has a complex composition of flavors and wine tasting requires a special attention to nuance.’ But it is reasonable to wonder whether the small format bottles available on this flight really fell into this category. There may have been few nuances to attend to and there were no other wines by other producers to allow a comparison in the results. Another limitation of the study acknowledged by the authors.\n\nA claimed strength of the study is its ‘high ecological validity’ and that it is a more authentic study than those carried out in simulated conditions (e.g. pressurised cabin on the ground). Here we have ‘a setting that approximates normal circumstances for wine consumption during flight’. And yet; ‘The wine samples were distributed to the study participants in two transparent CorningTM Falcon 50 mL Conical Centrifuge Tubes (outside diameter: 30 mm; length: 115 mm). Each tube contained only 25 mL of wine’(4). Far from being authentic, naturalistic conditions, this requires participants to sniff and sip from a narrow test tube. The authors tell us: ‘This conforms to general wine tasting standards and is generally considered sufficient for a simple taste assessment’. On the contrary, standardized ISO wine tasting glasses have a much wider aperture than a centrifuge tube, and crucially, the bowl is wider than rim to encourage odour concentration in the headspace. These are far from suitable containers for wine testing purposes. This is yet another possible limitation of the study acknowledged by the authors: ‘these narrow plastic tubes could be an inhibiting factor in the consumption experience, e.g. inhibiting the odor sensation and diminishing the overall aesthetic experience and its emotional impact on the consumer ‘ (60. They most certainly would have been.)\n\nExclusion conditions for participants included those who do not or should not imbibe alcohol, those who ‘had a history of dysosmia or dysgeusia (i.e. olfactory or gustatory dysfunction.’ But normally, for such a test it would include those with cold, hay fever, or sinus problems. Also, were participants asked not to eat or drink anything for more than an hour before the tests, as is standard practice for taste tests. We are not told. They were tested 1hr 45 minutes into the flight.Did they consume any more alcohol or food during the remainder of the flight? Again, we are not told. The authors acknowledge the problem: ‘It is also recommended that future studies should employ measures to control for confounders related to the individual, such as current satiety, appetite, and thirst. A limitation of the present study is the lack of experimental control.’ (6). Some of these could have been controlled for in this study.\n\nTo assess the samples, we are told the authors relied on ‘the concept of flavour intensity’. No attempt is made to establish that participants understand what they are being asked to judge or show reliability in making ‘flavour intensity judgements’. This is also conflated with ‘aromatic intensity’ in places. Is this judged by sniffing or tasting? No attempt is made to assess the participants’ acuity in smelling or tasting. Were they not very discriminating? Could that be the reason ‘the subjective wine tasting experience of [these] non-professionals in real life testing may not be affected’ Were there individual differences?\n\nThe questionnaires draws on terms for evaluation said to be industry standard, though they are taken from Parr et.al. 2016 who is using these terms to test experts. Did the participants, who are largely inexperienced in wine tasting tests know what ‘oak integration’ means and are they reliably able to score it?\n\nHow do participants judge the added dimension of ‘imbibing strength’? Is it from the trigeminal burning sensation of the alcohol, the full-bodied weight of the wine? Are all participants using the same criteria? For those without any experience of wine tasting, it can be hard to make discriminating judgements over a distanced time period – the re-testing is one hour after disembarking from the flight. Thus, the findings could be interpreted as suggesting a lack of ability to discriminate and compare across time rather than making reliably good judgements about the performance of the same wines in two different conditions. Yet a further possible limitation the authors acknowledge.\n\nThe re-testing of the test tubes of the same wine was carried out on the transit bus from the airport to the hotel. Were the samples in the same condition? Freshly poured into the centrifuge tubes or preserved from the first filling? The comparison of response is between tasting test tubes of wine in flight at altitude and then shortly after a 3.5 hour flight on a transit bus (after further food? alcohol?) Many further questions arise from this design.\n\nConducting the experiment:\nThe dependent variable in the hypothesis of the study is the altitude/ air pressure at which the wines were tested and re-tested. Although we are told the altitude of the aircraft was c.37,000 ft, we are not told more importantly what the air pressure was in the cabin. The authors explain that they were not able to take equipment on board to measure the cabin pressure. ‘Therefore, we can only provide a general estimate of the cabin pressure’ However, air crew are usually happy to ask the captain to let a passenger know the cabin pressure. The authors’ estimates look rather high. Mostly at this altitude the cabin pressure corresponds to an altitude of between 5,500-6.500 ft.\n\nSimilarly, “There are standard phone apps for measuring decibel levels in the environment. So special measuring equipment needed to be brought on board. This was a severe limitation since it was not possible to compare the noise levels on the in-flight and on the transit bus where the re-testing of the wines took place.\n\n(The authors admit – yet again – this was a possible limitation of this study. However, they suggest: ‘This reveals a major failure to understand the cross-modal effect at issue and how it provides a confound for this study: a significant failing in the article. If there was an interference effect of noise on the perceived intensity of wine’s aroma or flavour in flight because noise changed the perception of the wine’s flavour, and a similar effect of noise on flavour perception on the transit bus this may explain why the wine was perceived within the same ranges of intensity on the questionnaire in both conditions. This interpretation of the findings, given previous studies confirming the cross-modal effect of hearing white noise on perception of tastes, is more likely than the interpretation that there was no ‘difference in either subjective perception of flavor intensity or overall liking of wine between high- and low-altitude tests’.\n\nThe most the authors can claim is they have not been able to replicate the earlier simulation tests. But given the considerable limitations of the study fully acknowledged by the authors their findings do not, as the authors claim, support their conclusion that ‘Flavor perception of wine is unchanged during commercial flight’.\n\nWithout using a visual analogue scale instead of a 9 point scale subtle shifts can be missed., Besides, the p-values alone are not enough to reject the hypothesis. Baysian statistics would need to be run. A Baysian analysis would tell the authors whether they can reject the hypothesis on the basis of their data or whether it is just insufficiently powered to draw any conclusions of this sort. Why is it likely to be the former rather than the latter? We are not told.\n\nThe crucial weakness of the study – once again acknowledged by the authors – was the inability to test the same wines on the ground first and then in the air and the reasons they were not able to do so were purely logistical. The wines chosen for the study, a Turkish red wine blend (Majestik Syrah & Kalecik Karasi, 2017, alc. 13.5%), and white wine blend (Majestik Sauvignon Blanc & Sultaniye, 2017 alc. 12.5%) ‘were chosen as they were the only wines available for purchase on the flight, and because consumption of alcoholic beverages not purchased on the flight was not permitted.’ No opportunity to test before was possible and only one red and one white were used and this was opportunistic. For such a large conclusion as the authors propose in the title of the article, one would need much more evidence and to consider other, and often more plausible interpretations of the findings.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? No",
"responses": []
}
] | 1
|
https://f1000research.com/articles/9-597
|
https://f1000research.com/articles/9-180/v1
|
11 Mar 20
|
{
"type": "Research Article",
"title": "Computationally going where experiments cannot: a dynamical assessment of dendritic ion channel currents during in vivo-like states",
"authors": [
"Alexandre Guet-McCreight",
"Frances K. Skinner"
],
"abstract": "Background: Despite technological advances, how specific cell types are involved in brain function remains shrouded in mystery. Further, little is known about the contribution of different ion channel currents to cell excitability across different neuronal subtypes and their dendritic compartments in vivo. The picture that we do have is largely based on somatic recordings performed in vitro. Uncovering dendritic ion channel current contributions in neuron subtypes that represent a minority of the neuronal population is not currently a feasible task using purely experimental means. Methods: We employ two morphologically-detailed multi-compartment models of a specific type of inhibitory interneuron, the oriens lacunosum moleculare (OLM) cell. The OLM cell is a well-studied cell type in CA1 hippocampus that is important in gating sensory and contextual information. We create in vivo-like states for these cellular models by including levels of synaptic bombardment that would occur in vivo. Using visualization tools and analyses we assess the ion channel current contribution profile across the different somatic and dendritic compartments of the models. Results: We identify changes in dendritic excitability, ion channel current contributions and co-activation patterns between in vitro and in vivo-like states. Primarily, we find that the relative timing between ion channel currents are mostly invariant between states, but exhibit changes in magnitudes and decreased propagation across dendritic compartments. We also find enhanced dendritic hyperpolarization-activated cyclic nucleotide-gated channel (h-channel) activation during in vivo-like states, which suggests that dendritically located h-channels are functionally important in altering signal propagation in the behaving animal. Conclusions: Overall, we have demonstrated, using computational modelling, the dynamical changes that can occur to ion channel mechanisms governing neuronal spiking in vitro and in vivo. In particular, we have shown that the magnitudes of some ion channel current contributions are differentially altered during in vivo-like states relative to in vitro.",
"keywords": [
"Hippocampus",
"interneuron",
"computational neuroscience",
"ion channels",
"dendrites",
"synapses."
],
"content": "Introduction\n\nSince the days of Hodgkin & Huxley1–4, there have been tremendous advances in techniques to probe cellular activities5–11. However, the “gold standard” of electrophysiological experiments — patch-clamp recordings — is one of the most difficult and laborious types of experiments to perform in live animals10. Although neurons have been recorded intracellularly in vivo12–15, inhibitory interneuron subtypes are more often recorded juxtacellularly16–19. This is because inhibitory interneurons represent a small percentage of the neuronal population, which makes it more difficult to access and patch them relative to pyramidal cells – though there are some studies in barrel cortex where interneuron patch clamp recordings have been obtained20,21. Patch-clamp experiments are vitally beneficial to our understanding since they can provide clear signals of single-cell and single-channel activity at a high temporal resolution. Using this technique in combination with ion channel blockers helps uncover the ion channel mechanisms through which cell excitability is governed. Because in vivo patch-clamp recordings of interneurons are so difficult to perform, not much is known about their ion channel current contribution profiles in vivo or how they might differ from in vitro. Moreover attempting to experimentally assess different ion channel currents in dendritic compartments of interneuron subtypes carries with it a risk for loss of time and resources in return for only a small amount of data. Indeed, most experiments focus on attaining neuronal recordings in somata, since the comparatively thinner dendrites and axons are more difficult to patch.\n\nUsing a combination of biochemical and electrophysiological techniques, it has been shown that neurons, especially inhibitory neurons, can be characterized into many different cellular classes22–27. In the CA1 region of the hippocampus, inhibitory interneurons represent about 10–15% of neurons28, and because they are more diverse than the excitatory cell population, they are also more difficult to isolate and record. As such, a lot less is known about their activities in vivo and the ion channel mechanisms governing their excitability, although we note that there have been several studies using calcium imaging or extracellular recording techniques to uncover interneuron firing or activation patterns during behaviour (e.g. see 16–19,29,30). One such interneuron type whose firing patterns have been characterized experimentally in awake and behaving animals, is the oriens lacunosum moleculare (OLM) cell17,18,31. In CA1, OLM cells have somata and dendrites in the stratum oriens/alveus, and receive inputs from local pyramidal cells32, bistratified cells32, interneuron specific 3 cells33, and long-range projecting GABAergic inputs from medial septum33, among others. They have axons projecting to the stratum lacunosum moleculare where they synapse onto the distal dendrites of pyramidal cells, which allows them to gate the flow of sensory information from entorhinal cortex32,34. Though OLM cell (both putative and confirmed) intrinsic properties have been studied extensively in vitro35–41 and computationally42–47, much remains unknown about the ion channel current profiles across their dendritic trees as well as how ion channel mechanisms affect OLM cell firing in vivo during behaviour.\n\nPrevious experimental studies have shown that neurons exhibit excitability differences in vivo compared to in vitro, due to the effects of synaptic bombardment causing “high-conductance states”12,15,48,49. Though these experiments have mostly focused on recordings from the excitatory cell population, there is some evidence that interneurons show similar differences in vivo versus in vitro50. With morphologically-detailed cellular models containing biophysical ion channel mechanisms it is possible to create in vivo-like states by including a plethora of excitatory and inhibitory synaptic inputs12,51,52. With such models, one can easily probe and record multiple ion channel current types concurrently across different dendritic compartments. In doing so, one can predict how ion channel current contributions may change between in vitro and in vivo-like states as well as across dendritic compartments.\n\nIn this work we use two morphologically-detailed models of OLM cells and bombard them with synaptic inputs so as to create in vivo-like states. We use these models to determine somatic and dendritic ion channel current contributions to excitability that may occur in the behaving animal. In doing so, we highlight changes in dendritic excitability, ion channel current contributions and co-activation patterns relative to in vitro states. Specifically, we find enhanced dendritic hyperpolarization-activated cyclic nucleotide–gated (HCN) channel activation during in vivo-like states, which suggests a specific role for these channels in altering signal propagation in the behaving animal.\n\n\nMethods\n\nWe use two previously developed multi-compartment models of OLM cells (i.e. cell 1 and cell 2)47. Each model was developed using a morphological reconstruction and electrophysiological dataset obtained from the same cell. As such, the passive and active properties of each of the two models are specific to cell 1 or cell 2 and are not identical. For example, we note that cell 2 exhibits electrotonic distances that are up to twice as large as those seen in cell 1 (Figure 1A). These models were developed in NEURON53 and codes for the models are available on https://github.com/FKSkinnerLab/OLMng. Simulations performed for the present paper are run using NEURON 7.5 as a module in Python 2.7.\n\nA: Electrotonic distance (i.e. decay of a 1 mV signal) for voltage flowing into the soma (Vin; top) and voltage flowing out of the soma (Vout; bottom). B: Relationships between holding current and resulting spike rates in cell 1 and 2 models in vitro (red), as compared to the experimental data upon which they were optimized (blue).\n\nIon channel types in our OLM cell models include: hyperpolarization-activated cyclic nucleotide-gated (H), transient sodium (NaT), fast and slow delayed rectifier potassium (Kdr f, Kdrs), A-type potassium (KA), M-type potassium (M), T- and L-type calcium (CaT, CaL), and calcium-dependent potassium (KCa) channels. Equations describing them are given in the Appendix of 43, but specifically for H and NaT channel mechanisms, they are given in 47. Maximal conductances in soma (s), axon (a) or dendrites (d) are represented respectively as GH, GNaT, GKdrf, GKdrs, GKA, GM, GCaT, GCaL, GKCa, as given in Table 1. Note that I{channel} is used to refer to the corresponding ion channel currents of the various ion channel types. Other parameters for activation, inactivation and time constants are given in 43 or 47 as specified above.\n\nFor the synapse model, we use NEURON’s built-in Exp2Syn function, which models synaptic current as a two-state kinetic scheme.\n\n\n\nWhere i is the synaptic current, G is the maximal synaptic conductance, E is the reversal potential, V is the membrane potential, weight is the synaptic weight, factor is a NEURON process that is used to normalize the peak synaptic conductance to the value of weight, t is time, τr is the rise time, and τd is the decay time.\n\nThe input populations to OLM cells that we model include interneuron specific 3 (IS3) cell inputs, GABAergic longrange projecting inputs from medial septum (MS), bistratified (BIS) cell inputs, and local pyramidal (PYR) cell inputs, and, in the absence of specific constraints, distribute them randomly across all dendritic compartments. We focus on these input populations because enough details of EPSCs and IPSCs onto OLM cells have been previously reported in the literature (IS3 cells & MS: see 33; BIS & PYR cells: see 32).\n\nFocusing first on the IS3 cell and MS inputs33, we note that these were recorded under minimal stimulation using voltage-clamp of OLM cells at +10 mV. Note that there is a junction potential correction of +15.4 mV (personal communication from L. Topolnik, Laval University, QC) such that the holding current is actually nearer to -5.4 mV. This is near the reversal potential of excitatory synapses (0 mV) and so would silence current generated from excitatory synapses, leaving only minimally evoked IPSCs from IS3 cell or MS inputs. For BIS and PYR cell inputs to OLM cells32, these were not recorded under minimal stimulation (and so these currents could be generated from multiple synaptic inputs) and only the holding potential for recording excitatory inputs is reported (-60 mV; i.e. holding potential while recording BIS cell inputs is not reported). The target features of the EPSCs and IPSCs reported in the literature are summarized in Table 2.\n\n*20–80% rise time of IPSC\n\n**Peak IPSC amplitude is 67.6 ± 7.8 pA, but this is not with minimal stimulation. If assuming 4 synapses per connection (as per in 51), this means 16.9 pA per synapse.\n\n***Not reported, so the values chosen are midway between the values for IS3 and MS inputs since the BIS amplitude falls about midway between the two.\n\n****Peak EPSC amplitude is -109.3 ± 8.7 pA, but this is not with minimal stimulation. If assuming 9 synapses per connection (as per in 51), this means -12.4 pA per synapse.\n\n*****Not reported, so values used are those that were reported for excitatory inputs from Fimbria Fornix.\n\nAlso note that Erev,exc is assumed to be equal to 0 mV and that Erev,inh should be -87.1 mV, if taking directly from what is reported experimentally with liquid junction potential correction33. For Erev,inh, if assuming that there is voltage decay from the soma to the synapse when measuring reversal potential29, one needs to take a more depolarized value, so we use -80 mV as the inhibitory reversal potential instead. We note that there are other types of inputs to OLM cells that have been reported in the literature (e.g., serotonergic receptors54, metabotropic glutamate receptors55,56, cholinergic receptors57–59, additional complexities in NMDA/AMPA/Kainate receptors60,61, and TRPV1 receptors62), but in the absence of particular constraints, we opted to not include them at this time.\n\nWe set the rise and decay time constants to those reported in the literature (Table 2) and we optimized the synaptic weight parameters. Here, we describe the program that was written to perform this task. Incrementally, for each compartment, we increase the weight until the amplitude of the synaptic current that is generated is approximately equivalent to the target value obtained from the literature. Since past a certain distance from the soma, the electrotonic distance can create an exponential increase in the synaptic weight needed to generate large enough current amplitudes29, we simply stop the optimizations after 100 iterations of increasing the weight. From our previous experience, letting the algorithm optimize to larger weights than this simply generates synaptic conductance predictions outside of the realm of reality when considering single-receptor conductance values and the maximum numbers of receptors per synapse seen experimentally29. During these optimizations we assume that all voltage-gated channels are blocked and we set the leak reversal potential to the voltage-clamped holding potential of the model (0 mV when fitting IPSCs, and -60 mV when fitting to EPSCs). Following the optimizations, the synaptic conductances for each input type have increasing values with distance from soma (GPYR = 0.00020 to 0.00082 µS; GMS = 0.00024 to 0.00132 µS; GIS3 = 0.00018 to 0.00068 µS; GBIS = 0.00021 to 0.00100 µS), most likely due to the effects of electrotonic decay.\n\nIn previous work using IS3 cell multi-compartment models51, we performed high-resolution parameter searches in parallel on the Neuroscience Gateway (NSG) for high-performance computing63 to find input parameter combinations (i.e. numbers of excitatory and inhibitory synapses and incoming spike rates) that could generate in vivo-like (IVL) states. We applied a similar methodology here for creating IVL states for the OLM cell models.\n\nAs done previously51, we range excitatory spike rates from 0 to 30 Hz (resolution: 5 Hz), and inhibitory spike rates from 0 to 100 Hz (resolution: 10 Hz). We estimate maximal ranges for excitatory and inhibitory synapses based on findings from 64, scaled by the lengths and numbers of compartments in the OLM cell models (cell 1: 0 to 4641 excitatory synapses and we use a resolution of 35 synapses, 0 to 1989 inhibitory synapses with a 24-synapse resolution; cell 2: 0 to 6012 excitatory synapses with a 45-synapse resolution, 0 to 3006 inhibitory synapses with a 36-synapse resolution). Also, for every addition of inhibitory synapses, one third are assigned as IS3 cell inputs, another third are assigned as MS inputs, and a final third are assigned as BIS cell inputs (i.e. 8 IS3, 8 MS and 8 BIS synapses for cell 1; 12 IS3, 12 MS and 12 BIS synapses for cell 2). Note that the total numbers of synapses taken from 64 are numbers estimated for calbindin-expressing (CB+) cells. While certain subtypes of OLM cells express CB, this marker is also broadly expressed in several other cell types22. As well, the CB+ morphological reconstructions shown in 64 do not appear to carry resemblances to OLM cell morphologies. Nonetheless, these estimates are still used because they are the only estimates that can reasonably be linked to synaptic densities in OLM cells. We note that when synapses are added we assume common inputs (i.e. each presynaptic spike train is assigned to multiple synapses, that is, akin to cells forming multiple synaptic connections between each other - we use 7 excitatory synapses per connection and 8 inhibitory synapses per connection for cell 1, and 9 excitatory synapses per connection and 12 inhibitory synapses per connection for cell 2).\n\nTo identify IVL states using simulated voltage recordings, we use a previously designed IVL metric51 - see Equation 2. This metric uses threshold values based on experimental values found in the literature to establish whether the average subthreshold membrane potential (V¯m), the standard deviation of the subthreshold membrane potential (σVm), and the interspike interval coefficient of variation (ISICV), are large enough for a given state to be considered IVL12,14,15,48,65,66. It also uses the average spike amplitude (S¯A), to establish whether the model is entering an overly-excited state of depolarization block. Here we use a depolarization threshold value (V¯m; -70.588 mV) tailored to the resting potential of OLM cells and assuming an approximate 3 mV shift in vivo. We used this assumption in previous work51 and it is based on the shift in baseline voltage seen in CA1 place cells in vivo during place field traversals14. We also add a spike rate criterion to ensure that the spike rate is between 3 and 25 Hz since this is known for OLM cells in vivo17,18. The threshold values for membrane potential standard deviation (σVm), interspike interval coefficient of variation (ISICV), and spike amplitude (Samp) are the same as the values used previously for IS3 cell models51.\n\n\n\nGiven this, an IVL metric score of 4 would indicate that the input parameter combination produces an IVL state. Our parameter search yielded a variety of different excitatory/inhibitory input solution sets that could generate IVL states. As well, we observed an input parameter distribution towards low values across all input parameters, and more IVL scenarios were generated whilst in inhibitory-dominant regimes (not shown).\n\nChoosing in vivo-like (IVL) states. We chose representative IVL states from those generated by running ten randomized (i.e., of synaptic locations and presynaptic spike times as done previously51) simulations for each set of input parameters that generated an IVL state, until one was found to be consistently IVL. A state is considered to be consistently IVL if the IVL metric is 4 in at least five out the ten simulations, and the other five simulations have IVL metrics of at least 3. We focused on sampling from a subset of the parameter space with low inputs (i.e., sparse enough) so as to allow cells to have larger input resistances and be more sensitive to additional rhythmically-timed inputs as previously found to be required51.\n\nIn running the full parameter searches and then choosing representative IVL states from the low input subset of the parameter space we found that the amount of input was still far larger than numbers of inputs seen previously in our other models51, corresponding to a more reduced input resistance and therefore a reduced sensitivity to additional inputs. To find even sparser input parameter sets that produce IVL states we sampled at a finer resolution (i.e. at a resolution of 1 synapse for numbers of synapses and 1 additional spike for spike rates) from low input parameter value sets that generate similar excitatory/inhibitory balances to the IVL states found in the full parameter search. This balance is as given by our EI Metric calculation of Equation 3 (i.e. cell 1: -42685 to -8785 synapses × Hz; cell 2: -65700 to -9990 synapses × Hz). Once we identified parameter sets that fell within these EI metric ranges, we sampled the parameter combinations in order from the lowest total input to the highest total input (i.e. see Total input calculation in Equation 3), and simulated each of these possible combinations of input parameters until another consistently IVL state was found.\n\n\n\nwhere NE is number of excitatory synapses, NI is number of inhibitory synapses, fE is excitatory spike frequency, and fI is inhibitory spike frequency. The rationale with this approach is that IVL states should still be generated so long as the balance between excitation and inhibition falls within the right range. Following this, we found IVL states for both models where the total input is reduced (cell 1: 12,938.6 vs. 39,250 inputs; cell 2: 14,510.5 vs 46,350 inputs), which ultimately corresponds to input resistances that would allow enough sensitivity to additional inputs, as based on our previous work51. The resulting input parameter values for cell 1 are 1268 excitatory synapses firing at 1.6 Hz and 1254 inhibitory synapses firing at 8.7 Hz, and for cell 2, they are 1503 excitatory synapses firing at 1.5 Hz and 1532 inhibitory synapses firing at 8 Hz.\n\nTo analyze and compare how the ion channel current contributions differ under in vitro (’isolated slice preparation with synapses blocked’) and in vivo (’behaving animal’) conditions, we use the following approach with the two OLM cell models:\n\n1. Run the models without synapses (i.e., in vitro state) at two different somatic holding currents above rheobase (60 pA and 120 pA). Compute the resulting spike rate and determine the slope (m) and intercept (b) of the line between the two data points of spike rates. Linear extrapolation of these values from just two data points is justified since the F-I curves are fairly linear above rheobase (Figure 1B).\n\n2. Run the models using the input parameter set that produced the chosen IVL state with a given set of random seeds and measure the spike rate (fIVL) of the resulting spike train. Use fIVL together with m and b calculated previously to compute the holding current (Ihold) necessary to elicit a similar spike rate in the in vitro state:\n\n\n\n3. Choose a different set of random seeds and repeat step 2 ten times.\n\nTo consider comparisons in different locations, we choose five different recording sites in each model, which include soma and four increasingly distant dendritic compartments (i.e. soma, D1, D2, D3, and D4; Figure 2).\n\nCurrentscape plots67 are shown above the shape plots in A (cell 1) and B (cell 2). The dots in the shape plots indicate the recording sites (S = Soma; D1–D4 = Dendrite 1–4). All recording sites were chosen such that they are along the same dendritic path. In each currentscape plot, the top trace is the voltage trace (y-axis range = -50 mV to -20 mV), the filled-in black traces above and below the coloured plots are the total outward and inward currents respectively (dotted lines = ±0.5 pA, ±5 pA, ±50 pA), and the coloured plots show the percent contributions of each individual outward (top half of the plot) and inward (bottom half of the plot) current (see colour references in the legend on the right) to the total outward and inward currents. For each recording site location we show one in vitro currentscape plot (top) and one corresponding IVL currentscape plot (bottom), in the last second of simulation time (time axis = 9 s to 10 s). Distance values above each set of in vitro/IVL currentscape plots indicate the recording site distance from soma.\n\nTo analyze the total area under the current traces we use the numpy.trapz(current_trace,time_vector) function in Python. For outward currents, more positive values indicate larger currents, and for inward currents, more negative values indicate larger currents.\n\nTo compute cross-correlations between two time series (i.e. currents and/or voltage traces; a1 and a2), we first normalize the signals in order to generate cross-correlation magnitudes between -1 and 1, as follows:\n\n\n\n\n\nNote that all inward currents are reversed in polarity for these cross-correlations (i.e. from negative to positive), since their “activation” is reversed with respect to the polarity of voltage and outward current activation. This step allows us to better interpret the cross-correlation plots. Note that IL, though mostly an inward current at baseline, is not reversed in polarity since it becomes an outward current during spikes. Analyses of total area under the current traces and cross-correlations across current and/or voltage traces are done using the last 9 seconds of 10 second-long simulations.\n\nTo visualize the contribution of the different ion channel mechanisms to the voltage dynamics, we take advantage of currentscape plots (e.g. Figure 2), a recent visualization technique that plots the percent current contributions to the total inward or outward currents67. Additional relevant code for running simulations and plotting the results shown in this paper is available online at https://github.com/FKSkinnerLab/OLM_IVLCurrents.\n\n\nResults\n\nWhile it is clear that the intense synaptic bombardment present in vivo relative to quiescent in vitro states changes a cell’s response, conferring advantageous computational properties48, how the different ion channel types present in different locations contribute has not been explored. In creating in vivo-like (IVL) states for our computational OLM cell models, as described in the Methods, we are now in a position to compare differences between in vitro and IVL states from the perspective of ion channel currents in somata and dendrites. As well, we can explore how the different ion channel mechanisms might contribute to cell excitability in vivo.\n\nTo ensure that comparable firing rates exist in the in vitro and IVL states of the models, we inject an appropriate amount of current into the soma of the in vitro models (i.e., OLM cell models without any synaptic inputs). The ion channel current contribution changes between in vitro and IVL states for cell 1 and cell 2 are shown in Figure 2 A and B respectively. These changes are shown in somatic compartments as well as across dendritic compartments at locations specified above the plots and as indicated in the reconstructed cell schematics. This is shown through the use of currentscape plots67 where each color represents the percent of the total inward or outward current that each channel contributes across time. In the in vitro cases, across both models there is a shift in the INa/IL balance (pink/brown in Figure 2) with distance from soma, where in more distal dendritic compartments INa contributions become more narrow. However, once moved to the IVL scenario, this shift in INa contributions is no longer apparent. This is likely because somatic current injections decay with distance from soma and thus recruit INa in distal dendrites less, whereas in the IVL state, dis-tal dendritic compartments are bombarded directly with synaptic inputs, and thus engage dendritic INa more directly. A similar observation can be made with the IKdrf contributions.\n\nOne stark difference between cell 1 and cell 2 is that IM (purple) contributions are almost non-existent in cell 2 dendrites during IVL states. When looking at the voltage traces for those compartments where IM is not contributing, we see that spikes fail to form, with large depolarizations occurring instead. Since IM relative contributions appear largest during interspike intervals (see 43), it is not surprising that its contribution is minimized when there is no spiking and interspike intervals are not present. Notably, cell 2 has an almost two-fold larger maximal electrotonic distance than cell 1 (Figure 1A), which may explain why spikes propagate less in the distal dendrites of cell 2.\n\nIn general, it is clear from looking at the voltages and current contributions that dendritic compartments are more de-correlated from each other and the soma when the model is in an IVL state (i.e. relative to the corresponding in vitro state). This could partially be due to the high-conductance effects of synaptic bombardment where it suppresses input sensitivity and can drown out the magnitude of unitary inputs. As a result, these smaller amplitude signals do not propagate as far, and the different morphological compartments may appear more decorrelated from each other.\n\nIn looking at the ion channel current traces (IVL vs. in vitro and soma vs. D4; Figure 3), for both cell 1 and cell 2, it is clear that all ion channel types become sharply activated during spikes, except for IH (cyan) which is suppressed during spikes. IL (brown) is also different but from the perspective that it is primarily an inward current at resting potentials, but sharply transitions into an outward current, specifically during spikes. This is the case across both IVL and in vitro states as well as in soma and in the furthest dendritic location (D4), which makes sense given the biophysical characteristics of these channels.\n\nRecordings show the last second of a ten second simulation from an example pair of IVL (top rows) and in vitro (bottom rows) simulations for cell 1 (A) and cell 2 (B). The colour code for the currents is the same as the legend in Figure 2.\n\nIn looking at changes in the total current (i.e. area under the ion channel current traces; Figure 4) across the two models, four currents changed differently in cell 1 and cell 2 going from the in vitro to the IVL state. These include ICaT (increases dendritically in cell 1, but decreases dendritically in cell 2), IM (increases in cell 1, but decreases dendritically in cell 2), as well as IKCa & IKdrs (decrease dendritically in cell 1, but increase in cell 2). We already observed and discussed this difference in IM contributions changes in the previous section (i.e. the currentscape plots in Figure 2), and it is possible that the other three ion channel currents show this difference for similar reasons (i.e. a larger suppression of dendritic spike propagation in cell 2 during IVL states), since these differences are not readily observable in the somatic compartments Figure 4).\n\nEach line (10 re-randomizations = 10 lines for each recording site) connects in vitro results (left dots) with their corresponding IVL results (right dots). In each plot, from left to right along the x-axis, we plot soma, D1, D2, D3, and D4 (same recording sites shown in Figure 2). Note that outward currents are shown in the top rows (larger contributions = more positive values) and inward currents are shown in the bottom rows (larger contributions = more negative values). As such, the arrows indicate the direction of larger current magnitudes for each row of currents.\n\nAcross both models, many ion channel currents during IVL states relative to in vitro states also showed increased contributions near the soma but decreased contributions in the dendritic compartments, including IKA, IL, IKdrf, INa, ICaL. The only ion channel current that consistently showed increased outputs during IVL states across both models and all compartments was IH.\n\nBelow, we show results from analyzing the timing of ion channel currents using cross-correlations between each current trace and the corresponding location-specific voltage trace (i.e. soma or D4; Figure 5). Across all channel types, with the exception of IH, there were large narrow peaks with timescales in line with the duration of action potentials. Moreover, the lag time of the peaks are consistent across IVL vs in vitro states, as well as in soma vs dendrites, indicating that the relative timing of different ion channel currents is unchanged whilst under synaptic bombardment. Also across all dendritic ion channel currents in the IVL state, the cross-correlation timescale becomes broader, likely due to the general loss of spikes in more distal dendrites and longer timescale plateau-like potentials (i.e. as in the voltage trace plots in Figure 2). Some of the ion channel currents that were tightly linked with spiking had slightly non-zero peak lag times, indicating delayed activation (e.g. IKdrf /IKdrs/IKCa showing delayed rectification properties and ICaT /ICaL becoming more active in the after-spike hyperpolarization period). Others had peaks that were centered almost exactly at zero (IKA, INa, IM, and IL, which could all contribute to balancing action potential amplitude). In particular, IL and voltage cross-correlations were almost entirely symmetrical to the point where they appeared to be auto-correlations, indicating that IL is a good proxy for gauging changes in voltage. Finally, IH was unique in that it is the only ion channel current type that exhibited a negative cross correlation with spiking, which is in line with h-channels being activated during hyperpolarization and not spiking. Moreover, the cross-correlation between IH and voltage is even more negative in dendrites relative to soma, and during IVL states relative to in vitro states. This suggests that IH, which overall is enhanced during IVL states (Figure 4), is comparatively more suppressed during spikes because the relative change in IH during a spike is larger in the IVL state.\n\nEach plot shows the cross-correlations between the current traces and voltage traces using the last 9 seconds of each 10s trace. Note that all inward currents (right columns) are reversed in polarity for these cross-correlations (i.e. from negative to positive) in order to better interpret their activation periods with respect to voltage activation periods. Also note that we did not reverse the polarity for IL since it becomes an outward current during spikes (Figure 3). For both cell 1 and cell 2 we run this analysis across 5 of the 10 re-randomizations of the IVL (red) and in vitro (blue) simulations, as well as across the somatic (darker tone) and D4 (lighter tone) compartments (see legend). The vertical and horizontal dashed lines in each plot are the zeroth axes (x-axis = -20 ms to 20 ms; y-axis = -1 to 1).\n\nBelow, we show cross-correlations between every possible ion channel current combination in order to examine co-activation relationships (Figure 6 and Figure 7). Across all cross-correlations, regardless of state, cell, or recording site, the peak lag time is preserved. While almost all cross-correlations exhibited peaks aligned very near zero, only ICaT consistently exhibited non-zero lag time peaks, indicating a delay in ICaT activation relative to other ion channel current activations. More specifically, most currents activate very close to when spikes are occurring (Figure 5), but ICaT appears to exhibit a considerable delay relative to the timing of the other currents, and is more likely aligned to when the cell is undergoing after-spike hyperpolarization.\n\nCross-correlations are plotted in the same way as in Figure 5 (i.e. same legend but different cross-correlation pairs). The diagonal black dots, highlight channel auto-correlations (e.g. IKA * IKA). Everything above the diagonal is cross-correlated one way and everything below the diagonal is cross-correlated the opposite way (i.e. like a mirror; e.g. IKA * IKdrf vs. IKdrf * IKA). The vertical and horizontal dashed lines in each plot are the zeroth axes (x-axis = -20 ms to 20 ms; y-axis = -1 to 1).\n\nCross-correlations are plotted in the same way as in Figure 5 (i.e. same legend but different cross-correlation pairs). The diagonal black dots, highlight channel auto-correlations (e.g. IKA * IKA). Everything above the diagonal is cross-correlated one way and everything below the diagonal is cross-correlated the opposite way (i.e. like a mirror; e.g. IKA * IKdrf vs. IKdrf * IKA). The vertical and horizontal dashed lines in each plot are the zeroth axes (x-axis = -20 ms to 20 ms; y-axis = -1 to 1).\n\nAlso, of note, is the particular asymmetrical shape of any of the cross-correlations with IH. IH exhibited negative cross-correlation peaks with all of the other ion channel currents (i.e when the other currents increase in magnitude, IH decreases in magnitude). Specifically, this translates to IH showing decreased contributions during spikes, while all of the other ion channel currents show enhanced contributions during spikes (Figure 5). The shape of IH cross-correlations are likely a result of the fact that IH possesses a longer and generally asymmetrical time course relative to the time course of currents that activate most strongly during spikes (Figure 3).\n\nWhen looking at differences between IVL (red) and in vitro (blue) states and across cellular compartments, we see that the amplitude of the peak cross-correlation is dependent on the cell (Figure 6 and Figure 7). For example, with cell 1 (Figure 6), where spiking is more easily propagated across dendritic compartments (Figure 2A), we see that the ion channel currents that are activated during spiking do not show much change in the amplitude of the peak cross-correlations. However, for cell 2 (Figure 7), where spiking is less easily propagated across dendritic compartments (Figure 2B), the peak cross-correlations are decreased and broader in the IVL states. This was particularly the case for dendritic compartments, which exhibit broader cross-correlation peaks. Moreover, the somatic IVL cross-correlations are often closer in shape to the in vitro cross-correlations than they are to the dendritic IVL cross-correlations. One notable exception to this are the cross-correlations of either IM or IL with IH. In all cases, these cross-correlations with IH have more negative peak magnitudes whilst in IVL states (across both soma and dendrites), which parallels the cross-correlations between IH and voltage (Figure 5). This may be related to the IH traces consistently being enhanced across compartments during IVL states (Figure 4). As well, across many cross-correlations with IH (i.e. IKdrf, IKdrs, IKCa, IM, IL, INa, and ICaT), IVL peak cross-correlations appear to be more negative in dendritic compartments than somatic compartments. This is in sharp contrast with other IVL state cross-correlations, which generally all exhibit larger cross-correlation peaks in somatic locations. This observation is seen more clearly with the cell 2 plots (Figure 7). In summary this work predicts that, in vivo, dendritic IH is enhanced and as such, can be suppressed by a larger degree whenever other channels are more active. At the same time, this suggests that other dendritic ion channel currents show weaker co-activation between each other in vivo.\n\nAltogether this suggests that IH activation is more prominent in dendritic compartments than in somatic compartments, as well as during IVL states where dendritic IH is more directly activated through synaptic bombardment. Comparatively, cross-correlations between other dendritic ion channel currents that are more active during spikes will have decreased co-activation during IVL states, potentially because of increased compartmentalization and a resultant decrease in the propagation of spikes across compartments.\n\nHaving observed a decrease in the dendritic propagation of spikes in the IVL context (Figure 2), we can hypothesize that morphological compartments will become more de-correlated with each other during these states. As such, we further analyze channel current cross-correlations between the voltage and current traces in the soma and the corresponding traces in the D4 compartment (in vitro = blue; IVL = red; Figure 8).\n\nEach plot shows cross-correlations (in vitro = blue; IVL = red) of a somatic current type or voltage cross-correlated against the same current type or voltage recorded in the D4 compartment. Cross-correlations are plotted in the same way as in Figure 5.\n\nIt is clear from these plots that ion channel current activity is considerably de-correlated during IVL states since the in vitro amplitudes of the cross-correlation peaks (blue) are considerably larger than the IVL amplitudes of the cross-correlation peaks (red). In the previous currentscape plots (Figure 2) showing the in vitro cases, action potentials recorded in D1-D4 appeared to be the result of back-propagating action potentials generated near the soma. Here we show that this is indeed the case since the blue maximal peaks align at negative lag times (Figure 8).\n\nInterestingly, there is somewhat of a difference between cell 1 and cell 2 for the IVL cases (Figure 8). While they both exhibited very diminished peaks compared to their corresponding in vitro cases, the lag time of the peaks was considerably different. Cell 1 exhibited more pronounced negative lag times, suggesting that spikes were being generated near to the soma and back-propagating to the D4 distal dendrite recording site (though slightly more slowly and with a considerably diminished amplitude compared to the in vitro spike back-propagation). On the other hand, cell 2 exhibited positive and broader peak lag times, suggesting that dendritic postsynaptic potentials are preceding somatic spikes with minimal spike back-propagation. We already know that spikes do not back-propagate easily into cell 2’s distal dendrites due to the combination of increased electrotonic distance (Figure 1A) and synaptic bombardment (Figure 2B). However since there is a positive peak, albeit small, we can presume that distal synaptic input are still coherently integrated such that they can lead to spikes at the soma in cell 2 (Figure 8).\n\nAlthough broad, IVL IH cross-correlations between soma and D4 were larger than the IVL cross-correlations between morphological compartments for other ion channel currents, suggesting that IH is possibly more resistant to the de-correlating effects of synaptic bombardment, possibly due to it being more active during subthreshold periods. This is intuitive given that the dendritic compartments during IVL states are more likely to be in subthreshold regimes due to dendritic plateau/complex spiking effects brought on by synaptic bombardment (Figure 2).\n\n\nDiscussion\n\nIn this work we have computationally explored ion channel current contributions that are seen across different morphological compartments of an interneuronal cell type, the OLM cell, in in vitro and in vivo-like states. In doing so, we assessed the relative timing of ion channel current activation across pairs of ion channel types, across morphological compartments, and relative to voltage — a task that is not possible to perform experimentally in vivo, and that would be almost impossible to do in vitro. We found that the relative timing of ion channel current co-activation is preserved across states and locations. We also found that the magnitudes and relative contributions of the different ion channel currents are altered between states and across locations, and that different morphological compartments become more de-correlated with each other during in vivo-like states. In particular we observe a consistent enhancement in IH across spatial compartments during in vivo-like states relative to in vitro, which could coincide with the de-correlation seen across morphological compartments.\n\nIn this era of big data and channelopathies, it is highly desirable to be able to know and understand how different ion channel types might contribute to normal and pathological states. For example, a link between big potassium channels and epilepsy in Angelman syndrome was recently shown68. In this ’tour de force’ experimental study, genetic, organoid and behavioural platforms were used to suggest this link. Computational modeling approaches as presented here could be harnessed to make such links and hypothesize others by examining particular ion channel types in cellular and networks states that are akin to in vitro and in vivo-like/behavioural states. Interestingly, in light of our results here, recent work has shown that blockage of HCN channels in OLM cells prevented the formation of type 2 theta rhythms (that emerge during immobile, anxiety-laden behavioural states) as controlled by OLM cells in ventral hippocampus69.\n\nWe had noticed an enhancement of dendritic IH during in vivo-like states where subthreshold depolarizations are dominant. This follows since IH is a current that is most active during subthreshold and hyperpolarization regimes where it can be further activated by inhibitory perturbations41,70. In OLM cells, there is experimental evidence and computational support for HCN channels being present in dendrites47. Our previous computational studies showed that dendritic IH in OLM cells can modulate the input frequencies at which they are preferentially recruited to spike46. Since our study here shows that dendritic IH is enhanced during in vivo-like states and operates in the subthreshold regime, it is likely to play a role in subthreshold signal propagation. Previous work has mostly highlighted that IH reduces signal propagation and enhances compartmentalization of dendrites by reducing the input resistance in principal neuron dendrites across hippocampus71,72, cortex73, and basolateral amygdala74. Expression and function of IH is also known to be modulated by long-term synaptic potentiation (LTP) mechanisms, where LTP can upregulate HCN channels, while at the same time suppress channel function75. Altogether, this suggests that enhanced dendritic IH in vivo will contribute towards increasing dendritic compartmentalization. Since we do see a decrease in backpropagating action potentials (Figure 2) and decreased cross-correlations between spatial compartments (Figure 8) in the in vivo-like state, these may be a by-product of enhanced IH. However, it could also be due to a global effect across all of the synaptic and intrinsic currents that are enhanced in the in vivo-like state, which, together, all contribute towards decreasing the input resistance and suppressing the propagation of signals.\n\nIn our in vivo-like states we did not try to directly simulate synaptic inputs linked to any particular behavioral paradigm (e.g. rhythmic or bursting inputs during theta or sharp waves). That is, we generated in vivo-like states that simulate the levels of synaptic bombardment that neurons might receive in vivo12,48. More specifically, we highlighted changes in ion channel current contribution profiles that may exist in vivo, and contextualized how these changes may affect the way in which individual neurons process behaviourally-relevant information.\n\nIn general, using computational simulations alone to investigate biophysical phenomena carries its own set of caveats. Though the models that we use are data driven, no model is ever truly complete76, and degeneracies are to be expected77. Moreover, many assumptions need to be made when constructing a morphologically-detailed multi-compartment model, such as types of ion channels to include and their distribution across the morphology of the model.\n\n\nConclusions\n\nIn summary, this work is a computational investigation into the dendritic ion channel contributions that govern OLM cell excitability in vivo. We highlight that the timing of ion channel currents relative to voltage and each other are invariant across states, though many undergo changes in their current output magnitudes. In particular dendritic IH is enhanced during in vivo-like states, which could indicate altered signal propagation in behaving animals relative to in vitro recordings. Finally we show that during in vivo-like states, voltage and currents across compartments become more de-correlated relative to each, with a shift in the lag time of their maximal cross-correlation peaks. This was indicative of a loss of backpropagating action potentials, which made cross-correlations between subthreshold signals and spikes more apparent. Overall, this work shows a possible way to explore and gain insight into the coordination of ion channel currents that govern neuronal spiking in the “behaving animal”.\n\n\nData availability\n\nCode for running simulations and plotting the results: https://github.com/FKSkinnerLab/OLM_IVLCurrents\n\nArchived code as at time of publication: http://doi.org/10.5281/zenodo.368861978\n\nLicense: GNU General Public License v3.0\n\nModels of cell 1 and 2: https://github.com/FKSkinnerLab/OLMng\n\nArchived models as at time of publication: http://doi.org/10.5281/zenodo.368972479\n\nLicense: GNU General Public License v3.0",
"appendix": "Acknowledgements\n\nWe thank M. Lankarany for reading and providing feedback on this manuscript.\n\n\nReferences\n\nHodgkin AL, Huxley AF: The components of membrane conductance in the giant axon of Loligo. J Physiol. 1952; 116(4): 473–496. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHodgkin AL, Huxley AF: Currents carried by sodium and potassium ions through the membrane of the giant axon of Loligo. J Physiol. 1952; 116(4): 449–472. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHodgkin AL, Huxley AF, Katz B: Measurement of current-voltage relations in the membrane of the giant axon of Loligo. J Physiol. 1952; 116(4): 424–448. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHodgkin AL, Huxley AF: A quantitative description of membrane current and its application to conduction and excitation in nerve. J Physiol. 1952; 117(4): 500–544. 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}
|
[
{
"id": "61212",
"date": "06 Apr 2020",
"name": "Fernando Fernandez",
"expertise": [
"Reviewer Expertise Slice and in vivo intracellular electrophysiology",
"biophysical modeling of neurons."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 manuscript by Guet-McCreight and Skinner, the authors use two variants of an OLM compartmental model to probe the impact of in vivo-like synaptic activity on membrane voltage fluctuation behavior and the differential recruitment of voltage-gated conductances.\nOverall the paper provides interesting insights into how synaptic inputs differentially impact voltage-gated conductances in dendrites and the cell body. Further, the impact of in vivo-like inputs on dendritic membrane voltage dynamics is particularly interesting as it dramatically increases the timescale of fluctuations and creates “plateau-like” depolarizations. In addition, the authors show that differences within the two biologically based OLM models can lead to substantial differences in dendritic voltage behavior and that these differences are enhanced when driven by in vivo-like inputs.\nMajor Issues:\nAlthough many of the results noted above are interesting. It would be helpful to use the models in order to achieve a greater mechanistic understanding of what is observed. In particular, the prevalence of slower voltage depolarization in dendrites and disruption of spiking would benefit from some mechanistic insights that could be generalized to other model neurons. What factors (dendritic geometry, local input resistance etc.) lead to this behavior and does the presence of certain voltage-gated conductances enhance or disrupt these results? At it stands, the manuscript is largely descriptive of modeling data. The enhancement of IH in dendrites under in vivo-like inputs is noted but the mechanism (see above) is not explored. Why does IH conductance recruitment increase in dendrites? Are the results related to the slower time scale of voltage fluctuations under these conditions?\nMinor Issues:\nOverall, the discussion focuses on IH. However, many of the results noted (e.g. slower voltage depolarization, enhanced difference between models under in vivo-like inputs) are very interesting and likely more generalizable than the specific issues of IH in OLM cells. It would be helpful to explore these issues in the context of what others have shown in terms of in vivo recordings and dendritic activity (e.g. SL Smith … M Hausser, 2013).\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "5585",
"date": "16 Jun 2020",
"name": "Alexandre Guet-McCreight",
"role": "Author Response",
"response": "We thank the reviewers for their comments and detailed review of our manuscript. It has led to a better presentation and explanation of our work. We have revised our manuscript accordingly, changing and expanding two of the existing figures to exhibit results more clearly and providing more explanations. We hope that the changes made sufficiently addresses the concerns brought up by the reviewers. Please find below our specific responses (in bold font). Reviewer 1: Fernando Fernandez In the manuscript by Guet-McCreight and Skinner, the authors use two variants of an OLM compartmental model to probe the impact of in vivo-like synaptic activity on membrane voltage fluctuation behavior and the differential recruitment of voltage-gated conductances. Overall the paper provides interesting insights into how synaptic inputs differentially impact voltage-gated conductances in dendrites and the cell body. Further, the impact of in vivo-like inputs on dendritic membrane voltage dynamics is particularly interesting as it dramatically increases the timescale of fluctuations and creates “plateau-like” depolarizations. In addition, the authors show that differences within the two biologically based OLM models can lead to substantial differences in dendritic voltage behavior and that these differences are enhanced when driven by in vivo-like inputs. Major Issues: Although many of the results noted above are interesting. It would be helpful to use the models in order to achieve a greater mechanistic understanding of what is observed. In particular, the prevalence of slower voltage depolarization in dendrites and disruption of spiking would benefit from some mechanistic insights that could be generalized to other model neurons. What factors (dendritic geometry, local input resistance etc.) lead to this behavior and does the presence of certain voltage-gated conductances enhance or disrupt these results? At it stands, the manuscript is largely descriptive of modeling data. The reviewer is correct that passive property factors (geometry, resistance) contribute, and we have noted cell 1 and 2 differences (as shown in Fig 1). That is, spikes propagate less in cell 2 relative to cell 1. We now also report the surface areas, input resistances and time constants for cell 1 and 2 as determined in Sekulic et al (2019) in the Methods. We have also added dendritic diameter values for the locations shown. We also present a new figure that zooms in on a 200 msec window for two dendritic locations (new Fig 3) which more clearly shows the contribution of the different channel types. For example, one can now clearly see that there is an increased relative dominance of Ikdrf and IKCa in dendrites in vivo compared to in vitro and the disruption of firing can be seen with this along with the increased sodium currents as due to not being inactivated. Additional wordings have been included in the paper. The enhancement of IH in dendrites under in vivo-like inputs is noted but the mechanism (see above) is not explored. Why does IH conductance recruitment increase in dendrites? Are the results related to the slower time scale of voltage fluctuations under these conditions? This is brought up by reviewer 2 as well in the context that they do not find this result surprising, and the explanation that she provides is that “IPSCs are extremely effective in activating h-channels, as such, it is not surprising that h currents are more pronounced in the in vivo-like and sub-threshold conditions rather than during depolarizing current injections which would move the membrane outside the activation range for h-channels”. This is certainly a valid assessment and we have added this type of explanation to be more explicit in the results section discussing these findings. This can be seen more clearly in the edited and expanded Figures 3 and 4 where h-current accumulates in magnitude with increased durations of subthreshold voltage periods during the IVL states. Minor Issues: Overall, the discussion focuses on IH. However, many of the results noted (e.g. slower voltage depolarization, enhanced difference between models under in vivo-like inputs) are very interesting and likely more generalizable than the specific issues of IH in OLM cells. It would be helpful to explore these issues in the context of what others have shown in terms of in vivo recordings and dendritic activity (e.g. SL Smith … M Hausser, 2013). We agree that many of these aspects mentioned are generalizable across cell types and we have previously explored the potential impact of sensory-evoked stimuli during in vivo-like states in another hippocampal cell type (Guet-McCreight & Skinner 2019; Luo et al, 2020). Comparison to the results from Smith et al, 2013 in the context of mechanistic insight, is interesting, and our previous work suggests that during synaptic bombardment, sensory-evoked stimuli will largely be dampened (i.e. due to a reduced dendritic input resistance), which suggests the presence of more specialized mechanisms for sensory-evoked recruitment of cells (e.g. a rebalancing of synaptic bombardment, or plasticity mechanisms). We thank the reviewer for this suggestion and have added this reference as a point in our discussion."
}
]
},
{
"id": "62251",
"date": "04 May 2020",
"name": "Vijayalakshmi Santhakumar",
"expertise": [
"Reviewer Expertise Epilepsy",
"inhibition",
"brain injury",
"computational modeling",
"electrophysiology"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nExperimental evaluation of the complex interactions between ion channels in dendritic segments is challenging and inhibitory neuron dendrites are especially difficult to assess using in vivo and in vitro patch-clamp studies. However, understanding channel interactions with synaptic inputs shape integration, AP initiation and propagation is integral to information processing in specific circuits and identifying principles that govern brain function and disease. Guet-McCreight and Skinner approach this issue by adopting detailed multi-compartmental computation models of morphologically reconstructed oriens-lacunosum-moleculare neurons (OLM cells) to examine recruitment of specific ion channels during activity. Specifically, the simulations focus on comparing channel recruitment across somatodendritic compartments under “in vitro-like states” during somatic current injection in the absence of synaptic input and “in vivo-like state” with E-I balanced synaptic inputs. Using morphological simulations of distinct OLM cells, they highlight the specifically salient role for h-currents under in vivo-like conditions of heightened dendritic synaptic activity. The paper is largely descriptive, and findings are intuitive given the model design. The study provides value in demonstrating the potential application of current-scape plots and computational modeling in assessing synaptic/dendritic behavior and could be strengthened with additional consideration/discussion of the role for Ih in synaptic integration.\n\nMajor:\nWhile it is known that several channels show gradients in somatodendritic distribution the models presented in the study assume uniform dendritic distribution of channels. Given the lack of specific data on distribution, the assumption to distribute channels uniformly is understandable. However, the discussion could speculate how differences in channel distribution may alter the overall conclusions. This is particularly relevant to h-channels which are known to have differential dendritic distributions in certain cell types.\n\nThe heavy inhibitory bias of in vivo-like inputs is consistent with experimental data. IPSCs are extremely effective in activating h-channels, as such, it is not surprising that h currents are more pronounced in the in vivo-like and sub-threshold conditions rather than during depolarizing current injections which would move the membrane outside the activation range for h-channels. Additionally, the dendritic synapses appear to be scaled up which could further increase h-channel activation during the IPSCs. These aspects warrant discussion.\n\nGiven the focus on Ih, it may make it easier for the reader if the panels representing Ih data are identified with bold borders or included in a separate figure for discussion. The activation by hyperpolarization and in the subthreshold regimen make Ih different from other channels that activate on depolarization, thus the findings are not surprising. The unique role of Ih in signal integration/ dendritic compartmentalization and how it may contribute to input resistance, the relevance of the relatively slower kinetics of Ih can be better explored.\n\nOverall, it is unclear what Figure 4 adds to the manuscript as it compares currents at very different conditions and would be heavily dependent on membrane conductance that differs greatly between these conditions.\n\nMinor:\nFigure 3: the scales are very different for the in vivo and in vitro conditions even for the same parameter making it difficult to compare the differences. This is the only figure presenting absolute values and appropriate scaling would improve the ability to directly compare amplitudes.\n\nUtilization of NSG “to find input parameter combinations” is a good start but probably does not fully reflect in vivo activity patterns during behavior. The limitations associated with this should be discussed. Were parameters based on any specific behavior (stimulus/inputs)?\n\nApproach and data analysis:\napostrophe backwards in parenthesis (‘isolated…’ and ‘behaving…’)\n\nPg. 7: “dis-tal” àdistal\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "5586",
"date": "16 Jun 2020",
"name": "Alexandre Guet-McCreight",
"role": "Author Response",
"response": "We thank the reviewers for their comments and detailed review of our manuscript. It has led to a better presentation and explanation of our work. We have revised our manuscript accordingly, changing and expanding two of the existing figures to exhibit results more clearly and providing more explanations. We hope that the changes made sufficiently addresses the concerns brought up by the reviewers. Please find below our specific responses (in bold font). Reviewer 2: Vijayalakshmi Santhakumar Experimental evaluation of the complex interactions between ion channels in dendritic segments is challenging and inhibitory neuron dendrites are especially difficult to assess using in vivo and in vitro patch-clamp studies. However, understanding channel interactions with synaptic inputs shape integration, AP initiation and propagation is integral to information processing in specific circuits and identifying principles that govern brain function and disease. Guet-McCreight and Skinner approach this issue by adopting detailed multi-compartmental computation models of morphologically reconstructed oriens-lacunosum-moleculare neurons (OLM cells) to examine recruitment of specific ion channels during activity. Specifically, the simulations focus on comparing channel recruitment across somatodendritic compartments under “in vitro-like states” during somatic current injection in the absence of synaptic input and “in vivo-like state” with E-I balanced synaptic inputs. Using morphological simulations of distinct OLM cells, they highlight the specifically salient role for h-currents under in vivo-like conditions of heightened dendritic synaptic activity. The paper is largely descriptive, and findings are intuitive given the model design. The study provides value in demonstrating the potential application of current-scape plots and computational modeling in assessing synaptic/dendritic behavior and could be strengthened with additional consideration/discussion of the role for Ih in synaptic integration. Major: While it is known that several channels show gradients in somatodendritic distribution the models presented in the study assume uniform dendritic distribution of channels. Given the lack of specific data on distribution, the assumption to distribute channels uniformly is understandable. However, the discussion could speculate how differences in channel distribution may alter the overall conclusions. This is particularly relevant to h-channels which are known to have differential dendritic distributions in certain cell types. We have added a clarification to the methods that the models were developed with IH current recordings from the same cell that predicted the presence of h-channels in the dendrites, and not only in the soma. As discussed in Sekulic et al, non-uniform distributions may be possible in OLM cells, but what that work allowed us to robustly show is that it is very likely that h-channels are present in the dendrites, and any further examinations of non-uniform distributions would require further experimental data to be able to explore. From our various figures that show Ih along the dendritic tree and other works, we now speculate on the situation if h-channels are not uniformly distributed. For example, different spatial Ih distributions (in combination with different spatial synaptic distributions) could contribute towards modulating the input frequency at which cells can be recruited to spike. We have also added some comparative discussion points with respect to the distribution of IH in layer 5 pyramidal cell. The heavy inhibitory bias of in vivo-like inputs is consistent with experimental data. IPSCs are extremely effective in activating h-channels, as such, it is not surprising that h currents are more pronounced in the in vivo-like and sub-threshold conditions rather than during depolarizing current injections which would move the membrane outside the activation range for h-channels. Additionally, the dendritic synapses appear to be scaled up which could further increase h-channel activation during the IPSCs. These aspects warrant discussion. Thank you for making these comments. We agree with these points and have now included them along with expansions to make things more clear with details and explanations, including the point that inhibitory conductances scale up with distance from soma to the discussion and added these points to the results section where increased Ih is first reported. Given the focus on Ih, it may make it easier for the reader if the panels representing Ih data are identified with bold borders or included in a separate figure for discussion. The activation by hyperpolarization and in the subthreshold regimen make Ih different from other channels that activate on depolarization, thus the findings are not surprising. The unique role of Ih in signal integration/ dendritic compartmentalization and how it may contribute to input resistance, the relevance of the relatively slower kinetics of Ih can be better explored. We have added thicker border lines to the IH plots in figure 4 as well as plots of IH traces in expanding Figure 4. We have also added further specific details unique to each cell model (input resistance, diameters, surface areas, etc.) so that model differences are clearer. Overall, it is unclear what Figure 4 adds to the manuscript as it compares currents at very different conditions and would be heavily dependent on membrane conductance that differs greatly between these conditions. We have revised Figure 3 (to show current changes along the dendritic tree and across IVL and in vitro contexts on the same plot). However, as Figure 4 is showing the ‘total current’ in terms of area under the curve, we think that this is helpful in showing that Ih is the main current affected when comparing in vitro and in vivo, and to make the Ih total current more apparent, we show Ih in Figure 4B now – the increase in total current in vivo relative to in vitro can be more clearly seen, as summarized in the datapoints of Figure 4A. We agree that the results are heavily dependent on membrane conductance and this comparison is important for looking at current channel contributions and magnitudes across conditions. We are looking at the effects of in vivo-like states on ion channel current contributions and increases in membrane conductance is one of the primary differences between the in vivo-like state and the in vitro state. We note that to ensure that the comparison is reasonably ‘fair’, we used injected current values in the in vitro state to give rise to similar spike rates (at the soma) in both contexts, as described in the Methods. Minor: Figure 3: the scales are very different for the in vivo and in vitro conditions even for the same parameter making it difficult to compare the differences. This is the only figure presenting absolute values and appropriate scaling would improve the ability to directly compare amplitudes. To facilitate comparisons, we have now moved the in vivo/in vitro and S/D1/D2/D3/D4 traces to the same plots and zoomed in to a 200 ms time range. We have also added h-current plots to figure 4 for the S/D4 compartments to facilitate comparison of these traces across conditions. Utilization of NSG “to find input parameter combinations” is a good start but probably does not fully reflect in vivo activity patterns during behavior. The limitations associated with this should be discussed. Were parameters based on any specific behavior (stimulus/inputs)? We discuss this aspect in the limitations section of our discussion, and we have now extended this point. The decision for the ranges of input parameters is outlined in the section in the methods and are not based on specific sensory-evoked input paradigms. As described in the Methods, the extensive computational exploration to determine IVL states parallels the previously used approach in Guet-McCreight & Skinner (2019) for IS3 cells. Approach and data analysis: apostrophe backwards in parenthesis (‘isolated…’ and ‘behaving…’) Fixed – thank you. Pg. 7: “dis-tal” àdistal Fixed – thank you."
}
]
}
] | 1
|
https://f1000research.com/articles/9-180
|
https://f1000research.com/articles/8-1328/v1
|
01 Aug 19
|
{
"type": "Study Protocol",
"title": "The impact of large-scale deployment of Wolbachia mosquitoes on arboviral disease incidence in Rio de Janeiro and Niterói, Brazil: study protocol for a controlled interrupted time series analysis using routine disease surveillance data",
"authors": [
"Betina Durovni",
"Valeria Saraceni",
"Ana Eppinghaus",
"Thais I.S. Riback",
"Luciano A. Moreira",
"Nicholas P. Jewell",
"Suzanne M. Dufault",
"Scott L. O'Neill",
"Cameron P. Simmons",
"Stephanie K. Tanamas",
"Katherine L. Anders",
"Betina Durovni",
"Valeria Saraceni",
"Ana Eppinghaus",
"Thais I.S. Riback",
"Luciano A. Moreira",
"Nicholas P. Jewell",
"Suzanne M. Dufault",
"Scott L. O'Neill",
"Cameron P. Simmons",
"Stephanie K. Tanamas"
],
"abstract": "Background: Rio de Janeiro and Niterói municipalities in southeastern Brazil experience large dengue epidemics every 2 to 5 years, with >100,000 cases notified in epidemic years. Costs of vector control and direct and indirect costs due to the Aedes-borne diseases dengue, chikungunya and Zika were estimated to total $650 million USD in 2016, but traditional vector control strategies have not been effective in preventing arboviral disease outbreaks. The Wolbachia method is a novel and self-sustaining approach for the biological control of arboviral diseases, in which the transmission potential of Ae. aegypti mosquitoes is reduced by stably transfecting them with the Wolbachia bacterium. This paper describes a study protocol for evaluating the effect of large-scale non-randomised releases of Wolbachia mosquitoes on the incidence of dengue, Zika and chikungunya in the municipalities of Niterói and Rio de Janeiro. This follows a lead-in period since 2014 involving intensive community engagement, regulatory and public approval, entomological surveys, and small-scale pilot releases. Method: The planned releases during 2017-2019 cover a combined area of 121 km2 with a resident population of 1.1 million, across the two cities. Untreated areas with comparable historical dengue profiles and sociodemographic characteristics have been identified a priori as comparative control areas in each municipality. The proposed pragmatic epidemiological approach combines a controlled interrupted time series analysis of routinely notified suspected and laboratory-confirmed arboviral cases, together with monitoring of arbovirus activity utilising outbreak signals routinely used in public health disease surveillance. Discussion: If the current project is successful, this model for control of arboviral disease through Wolbachia releases can be expanded nationally and regionally.",
"keywords": [
"Wolbachia",
"dengue",
"chikungunya",
"Zika",
"vector-borne disease",
"disease surveillance",
"controlled interrupted time series",
"Brazil"
],
"content": "Abbreviations\n\nBG trap: BG-Sentinel trap; IBGE: Instituto Brasileiro de Geografia e Estatística (Brazilian Institute of Geography and Statistics); ITS: interrupted time series; MoH: Ministry of Health; PAHO: Pan American Health Organisation; PCR: polymerase chain reaction; qPCR: quantitative polymerase chain reaction; RCT: randomised controlled trial; SINAN: Sistema De Informação De Agravos De Notificação (Brazilian National Notifiable Diseases Information System); WHO: World Health Organisation\n\n\nBackground\n\nThe global incidence of dengue has increased dramatically in recent decades. Although cases are underreported, it is estimated that 390 million dengue virus infections occur every year, and of these 96 million have clinical manifestations of dengue or severe dengue. Globally, 3.9 billion people in 128 countries are at risk of infection1. The primary vector of dengue is the Aedes aegypti mosquito, which is also capable of transmitting other arboviruses including chikungunya, Zika, yellow fever and Mayaro2.\n\nThe first reported dengue outbreak in Brazil was in 1845, with subsequent outbreaks in 1880–1912 and 1916–19232. As a result of a coordinated effort from the Pan American Health Organization (PAHO) and the World Health Organization (WHO) to eradicate Ae. aegypti, Brazil was considered free of the mosquito in 1955, but the vector was reintroduced into the country two decades later3. In 1986, dengue virus serotype 1 (DENV1) was introduced to Rio de Janeiro and an estimated 1 million people were infected3,4. Since then, dengue has become a major public health problem. From 1986 to 1993, outbreaks occurred approximately every 2–5 years, and from 1993 dengue became endemic with seasonal peaks in cases during the rainy season (December to May), but with ongoing transmission throughout the year2,3. Between 2000 and 2007, more than 3 million dengue cases were reported (caused by DENV serotypes 1, 2 and 3) and in 2010, DENV4 re-emerged after 28 years of absence2.\n\nThe recent introduction of Zika and chikungunya in dengue hyperendemic areas of Brazil has aggravated the situation. The overlapping clinical features, absence of serological assays for the Zika virus that can reliably distinguish between acute disease and past exposure, and the association of pregnancy-associated Zika virus infection with microcephaly and other neurologic complications represents a great challenge for public health that will require new strategies and innovations5,6.\n\nBetween 2016 and 2017, 762 deaths were attributed to severe dengue in Brazil7. Additionally, in 2017, 127 deaths were confirmed to be caused by chikungunya. Yellow fever has spread from the North of Brazil to the Southeast over the last years, affecting humans and non-human primates. From July 2017 to April 2018, 1,266 cases of yellow fever including 415 deaths were confirmed in Brazil, with 223 cases and 73 deaths occurring in Rio de Janeiro State8. No autochthonous cases were reported in Rio de Janeiro city or Niterói, although some residents from those cities acquired yellow fever while traveling to other places in Brazil. A mass vaccination campaign against yellow fever began in 2016.\n\nThe costs of vector control, direct medical costs, and indirect costs related to dengue, Zika and chikungunya in Brazil were estimated to be 2.3 billion Brazilian reais ($650 million USD) in 20169. In the absence of an effective vaccine for these arboviruses, disease prevention depends on vector control. Vector control guidelines in Brazil10 are focused on elimination or larvicide treatment of mosquito breeding sites and the control of adult mosquito populations with insecticides sprayed as ultra-low volume. The limited potential of these traditional vector control strategies to achieve large-scale and sustained reductions in dengue incidence is evidenced by the continuing public health burden of dengue throughout endemic areas where these measures are routinely employed, and the lack of robust efficacy data from well-designed trials to inform their optimal implementation11.\n\nThe Wolbachia method (www.worldmosquito.org) is a novel, natural, and self-sustaining approach to reduce arboviral diseases transmitted by Ae. aegypti mosquitoes. The symbiotic Wolbachia bacterium is found naturally in over 60% of insect species, but not in Ae. aegypti, and is passed from one generation to the next through the insect’s eggs. Stable transinfection of Wolbachia into a local Ae. aegypti colony in the laboratory produces a lineage of Wolbachia-carrying mosquitoes which, upon release over several weeks, can achieve dissemination of Wolbachia into the local Ae. aegypti population through the processes of maternal inheritance and cytoplasmic incompatibility that give Wolbachia-carrying mosquitoes a reproductive advantage. The DENV-transmitting potential of Wolbachia infected mosquitoes is reduced by 66–75%12,13, a phenotype which has been shown to persist in field mosquito populations up to five years after the end of releases. Mathematical modelling of this reduced transmissibility predicts a substantial and sustained reduction in dengue incidence in human populations where Wolbachia is established13. Laboratory data indicate a similar reduction in the competence of Wolbachia-carrying Ae. aegypti for transmitting other viruses including Zika, chikungunya, yellow fever and Mayaro14–17.\n\nWith releases now conducted in eight countries over the past eight years, the World Mosquito Program has demonstrated that Wolbachia can be successfully established and maintained in both small-scale and large-scale urban settings in multiple ecological environments18–22.\n\nA core objective of these releases has been to ensure strong community acceptance and government support for the approach, achieved through embedding community and stakeholder engagement within the project activities in each site. Observational evidence of the impact of Wolbachia releases on arboviral disease in pilot sites has been encouraging, with no evidence of local dengue transmission where Wolbachia has established at high levels. Following city-wide deployment in Townsville, Australia, there has been no confirmed local dengue transmission in Wolbachia-treated areas for four seasons since completion of releases, despite local transmission every year for the prior 13 years and ongoing importation of DENV infection in travelers21. A cluster randomized controlled trial (RCT) to generate a robust and quantitative estimate of the impact of Wolbachia on dengue incidence commenced in Yogyakarta, Indonesia in 2017, with reporting of results expected in 202123.\n\nIn Brazil, planned scale up of Wolbachia deployments from demonstration projects in Rio de Janeiro and Niterói municipalities to large-scale releases was accelerated by the declaration of Zika as a public health emergency by the WHO in early 2016, and the recommendation by WHO’s Vector Control Advisory Group in March 2016 that the Wolbachia method be evaluated in rigorously monitored pilot deployments under operational conditions, to build evidence of epidemiological effectiveness against Aedes-borne viruses24. Given the imperative from stakeholders and funders to scale up deployment within a relative short time frame, and to retain sufficient flexibility to optimize methods for large-scale deployment in the varied micro-environments within Niterói and Rio de Janeiro, an RCT or other carefully controlled deployment was not considered feasible. Instead releases under operational conditions, and with pragmatic evaluation of disease impact using data routinely collected for public health purposes, was favoured.\n\nHere we describe a protocol for evaluating the effect of large-scale non-randomized Wolbachia releases on the incidence of dengue, Zika and chikungunya in the municipalities of Niterói and Rio de Janeiro. The proposed strategy employs a controlled interrupted time series analysis of routinely notified suspected and laboratory-confirmed arboviral cases, together with monitoring of arbovirus activity with outbreak signals routinely used in public health disease surveillance. This methodology allows measurement of the impact of the intervention at the population level over time, accounting for the seasonal trends and inter-annual fluctuations often observed in dengue and other arboviral disease incidence.\n\n\nMethods\n\nThe aim of this epidemiological study is to test the hypothesis that the establishment of Wolbachia in local Ae. aegypti populations in Rio de Janeiro and Niterói leads to a reduction in arboviral disease burden.\n\nThe impact of Wolbachia deployment on arboviral disease incidence will be evaluated using routine notifiable disease surveillance data to describe associations between temporal and spatial trends in arbovirus disease and the deployment of Wolbachia across Niterói and Rio de Janeiro, with two objectives:\n\n1. Estimate the reduction in dengue incidence in the aggregate treated areas of Niterói and Rio de Janeiro compared to an untreated control area, and in each treated zone compared to the untreated control area, each year for five years after Wolbachia establishment.\n\n2. Quantify the occurrence of dengue outbreak signals in Wolbachia treated areas compared to untreated areas in Niterói and Rio de Janeiro, for five years after Wolbachia establishment, using outbreak indicators employed routinely for public health monitoring of dengue activity: i) control diagrams comparing the weekly dengue incidence (five-week moving average) against the five-year historical average in that same area; and ii) outbreak incidence threshold of 300/100,000 population in any month.\n\nRio de Janeiro and Niterói cities are located in the State of Rio de Janeiro, Brazil. Niterói has an area of 134 km2 and a population of 487,562 in 2010. Rio de Janeiro is the second largest city in Brazil with 6,320,446 inhabitants in 2010 and an area of 1,200 km2. The two cities sit on opposite sides of the Guanabara Bay and are linked by a long bridge, which transports a large commuter population between Niterói and Rio de Janeiro.\n\nThe cities are divided into health districts, for the purpose of planning and delivering care - ten in Rio de Janeiro and seven in Niterói. In Rio de Janeiro, Wolbachia deployments will be conducted in one of the administrative areas of the city (Figure 1; produced in ArcMap version 10.5, ESRI, CA), in an area of approximately 90 km2 and with 886,551 inhabitants, 39% of whom live in slums. The total release area in Niterói is approximately 31 km2 covering a population of 268,536 (Table 1). For the purposes of Wolbachia deployment, the Rio de Janeiro and Niterói intervention areas are divided into 4 and 3 release zones, respectively, which are aligned with neighbourhood administrative boundaries.\n\nMap of (a) Rio de Janeiro and (b) Niterói Wolbachia-treated and untreated areas (produced in ArcMap version 10.5, ESRI, CA).\n\nIn Rio de Janeiro, two administrative areas adjacent to the release area have been designated a priori as a comparative control zone (Figure 1), based on comparable sociodemographic characteristics and synchronous historical dengue time series (Figure 2 and Table 1). In Niterói, the remaining untreated area of the city has been designated as the comparative control zone (Figure 1).\n\nDengue incidence in Wolbachia-treated vs untreated areas of (a) Rio de Janeiro (2000–2017) and (b) Niterói (2007–2016) (source: Brazilian National Disease Surveillance System (SINAN)).\n\nStaged Wolbachia-mosquito deployments will be implemented in Niterói and Rio de Janeiro, in order to achieve Wolbachia establishment across the two municipalities. Pilot releases commenced in late 2015, and city-wide deployments are ongoing through to the end of 2019. Wolbachia-containing adult mosquitoes will be released at one location per 50 x 50 meter grid square for a minimum of 16 consecutive weeks in each release zone. In areas where Wolbachia frequency remains low after 16 weeks of releases, or where particularly high wild type Ae. aegypti populations are observed, Wolbachia-containing mosquito eggs will also be released to complement adult releases with the aim of accelerating Wolbachia establishment. Monitoring of Wolbachia frequency will be done using BG-Sentinel mosquito traps (BioGents, Germany), distributed throughout the release area at a density of 16 traps per km2. Traps will be serviced weekly and all collected mosquitoes identified morphologically by microscopy. From eight weeks after the start of releases, a maximum of 10 Ae. aegypti (male and female) per trap will be tested individually for Wolbachia using quantitative polymerase chain reaction (qPCR). Wolbachia screening will be performed biweekly during releases and until establishment, then every 1–3 months thereafter. All surplus Ae. aegypti will be biobanked. Mosquito collection and screening results will be stored in a custom designed web-based data repository. The Wolbachia prevalence in screened Ae. aegypti will be reported aggregated to each release zone, calculated as the total number of Ae. aegypti mosquitoes that tested positive for Wolbachia aggregated across all BG traps in the zone, divided by the total number of Ae. aegypti that were screened in that zone.\n\nThe two proposed strategies for the evaluation of the impact of large-scale Wolbachia deployments on arboviral disease incidence in Niterói and Rio de Janeiro municipalities make use of existing data on dengue, chikungunya and Zika case notifications to the Brazilian national disease surveillance system (SINAN). Dengue surveillance has been in place since its re-emergence in 1986 and data is available from the SINAN system since 2000. Zika and chikungunya became notifiable diseases in 2015.\n\nSuspected cases of dengue and other arboviral diseases are required to be reported to the city health department25, according to a case definition of fever plus two other symptoms including malaise, headache, myalgia, nausea, vomiting, cutaneous rash, and arthralgia. Dengue case notifications include an indication of disease severity (dengue, dengue with alarm signals, severe dengue, fatal dengue). A variable proportion of notified suspected cases are tested by IgM serology or PCR, following the Brazilian guidelines26 and the timing between onset of symptoms and blood collection. The number of cases in a given period of time may limit the availability of tests, and PCR testing is routinely performed only for severe and fatal cases, pregnant women and young children26. In 2016, 4.8% of notified dengue cases in Rio de Janeiro and 11.5% in Niterói were supported by a positive IgM serology result, and only 0.2% of notified cases in Rio and 0.03% in Niterói had a positive PCR result. The ability to confirm dengue cases by serology is impaired since the Zika outbreak due to serological cross-reactivity between the dengue and Zika viruses. As PCR testing is performed only in certain patient populations, the proportion positive is unlikely to be generalisable to all notified cases. Therefore, for the purpose of our analyses we will use all notified dengue cases (suspected and laboratory-confirmed) as the primary endpoint.\n\nIn the absence of a reliable serological test that does not cross-react with dengue, Zika lab diagnosis is done solely on the basis of molecular detection (real-time PCR) up to the first 5 days in serum and 15 days in urine. This has severely limited the ability to confirm Zika virus infection among notified cases (3.5% in 2016, 19.3% in 2017).\n\nChikungunya diagnoses can be confirmed either through PCR or serology, as it does not cross-react with Zika or dengue. The proportion of notified cases with supportive laboratory findings is higher than for dengue: 31.9% and 18.2% in Rio and Niterói, respectively, in 2016.\n\nAnonymized disaggregate (line-listed) data on notified suspected and laboratory-confirmed dengue, chikungunya and Zika cases will be extracted from the SINAN system for the historical pre-intervention period (2000–2016 for dengue, and 2015–2016 for chikungunya and Zika) and the prospective post-intervention period (2017–2023). The dataset will include age, sex, neighbourhood of primary residence, date of illness onset, date of notification, reporting health clinic, disease severity, hospitalisation, death, and where available, geocoordinates of primary residence, type of diagnostic test performed, diagnostic test result, and final diagnostic classification. Population data from the Brazilian census (IBGE) and population by neighbourhood of residence obtained from the municipalities of Rio and Niterói will be used to estimate the population in each release zone. The incidence rate (number of new dengue, Zika or chikungunya cases divided by the population at risk) will be expressed per 100,000 inhabitants.\n\nInterrupted time series (ITS) analysis is a valuable study design for evaluating the effectiveness of a population-level health intervention that is implemented at a clearly defined point in time27. It uses a set of historical observations of an outcome of interest (in this context monthly arboviral case notifications) to establish an underlying trend, which is assumed to be ‘interrupted’ by the introduction of an intervention (in this case Wolbachia releases). Descriptive analyses of seasonal and interannual trends in arboviral disease notifications, by release zone and municipality, will first be made. Comparison of the trend in monthly case notifications in the post-intervention period with the hypothetical scenario of no intervention (the ‘counterfactual’, inferred from the historical time series and untreated control area), provides an estimate of the intervention effect. Segmented regression using an appropriately defined impact model (e.g. negative binomial regression for autocorrelated count data with population offset, assuming a step change post-intervention) will be used to estimate the effect of Wolbachia releases on the arboviral disease endpoints in each release zone, and for the aggregate release areas in each municipality. In Rio and Niterói, the availability of comparative control areas – well-matched to the release area in socioeconomic characteristics and historical dengue incidence (Figure 2) – permits a robust, controlled analysis in which the confounding effects of seasonality and inter-annual variability can be adjusted for. Zone-level ITS analyses will be performed 12 months after completion of releases in each zone, and each 12 months thereafter, with release zones considered ‘treated’ for the purpose of this analysis based on completion of releases, regardless of the long-term Wolbachia monitoring results. Aggregate release-area level analyses will be performed for each municipality 12 months after completion of releases in the last zone and each 12 months thereafter.\n\nPower was estimated for the ITS analysis using 1000 simulated datasets drawn from a negative binomial distribution fitted to a ten-year time series (2007–2016) prior to Wolbachia deployment, of monthly dengue case notifications from release and control zones in Niterói and Rio de Janeiro. The simulated time series of dengue case numbers in the control zones as well as the pre-Wolbachia release dengue case numbers in the treated zones were drawn directly from this model-generated distribution. Post-Wolbachia release dengue case numbers in the treated zones were drawn from the same model-generated distribution, modified by an additional parameter for an intervention effect of Relative Risks = 0.6, 0.5, 0.4, 0.3. For each of these four ‘true’ effect sizes and a null effect (RR = 1), applied to each of the 1000 simulated time series, the ‘observed’ effect size was calculated from a negative binomial regression model of monthly case counts in the treated and untreated zones, as described above. Post-intervention time periods of 1, 2 or 3 years were simulated, with the pre-intervention period fixed at 7 years. The estimated power to detect a given effect size was determined as the proportion of the 1000 simulated scenarios in which a significant intervention effect (p<0.05) was observed. These simulations indicate 80% power to detect a reduction in dengue incidence of 50% or greater after three years of post-intervention observations, and a reduction of 60% or greater after two years.\n\nThe primary endpoint for the ITS analysis will be dengue cases notified to the disease surveillance system. The secondary endpoints will be: a) the count of severe dengue cases reported to the surveillance system, b) the count of fatal dengue cases reported to the surveillance system, and c) Zika and chikungunya cases notified to the disease surveillance system.\n\nAlthough the historical time series for Zika and chikungunya incidence is short, we will nonetheless describe the incidence during and after Wolbachia deployments relative to the a priori defined non-release control areas for both Niterói and Rio de Janeiro.\n\nAs a complementary approach for evaluating the public health impact of large-scale Wolbachia releases, we will also use the following dengue outbreak alert tools routinely used in public health practice. We hypothesise that these dengue outbreak signals will not be triggered in areas where Wolbachia has been established.\n\n1. Control diagram (endemic channel)\n\nThe definition of a dengue outbreak or epidemic has changed over time in Brazil. In Rio de Janeiro and Niterói, a control diagram is currently used to monitor dengue incidence. Briefly, the control diagram is constructed from a five-week centered moving average of weekly notified dengue incidence for the past five years excluding epidemic years. An early signal of a dengue outbreak/epidemic is triggered when the weekly incidence of dengue crosses the upper limit of the control diagram26, with the upper limit defined as [mean+(standard deviation*1.96)]. Incidence that remains above the upper limit of the control diagram for two or more consecutive weeks constitutes a dengue outbreak. For the purpose of monitoring the impact of Wolbachia releases on dengue, we will construct annual control diagrams with weekly dengue incidence, by city and for each release and non-release zone, to monitor the occurrence of dengue outbreaks. The number of dengue outbreak signals triggered per year will be reported.\n\n2. Classical incidence threshold\n\nAnother outbreak definition that has been used by the Ministry of Health (MoH) in previous years20,22,23 is a dengue incidence threshold of ≥300 cases/100,000 population in a given month. Although not included in current MoH guidelines, this provides an alternative endpoint for evaluating dengue activity at a population level in the post-intervention period, compared with pre-intervention, and we will report the number of months in a given year where dengue incidence crosses this threshold.\n\nThis study is ongoing. Wolbachia releases are expected to be completed by the end of 2019, and the collation and analysis of disease surveillance data will continue until 2023.\n\nBased on the results of the power estimation above, the study outcome will be evaluated and reported two years after the completion of releases. The findings will be submitted for peer review and publication in an appropriate open access journal, together with aggregate supporting data.\n\n\nDiscussion\n\nThe municipalities of Rio de Janeiro and Niterói in southeastern Brazil have been affected by dengue for more than 30 years, with epidemics occurring every 2 to 5 years. In recent years, outbreaks of the other Aedes-borne diseases chikungunya and Zika have presented further public health challenges, and since July 2017 at least 221 human cases of yellow fever have been confirmed in Rio de Janeiro State, including in urban areas8. Vector control strategies, based on elimination of mosquito breeding sites and use of insecticides to reduce adult populations, have not been effective in preventing dengue outbreaks28. The Wolbachia method is a novel and self-sustaining approach for the biological control of arboviral diseases. The signature feature of Wolbachia is to reduce the arbovirus-transmission potential of Wolbachia-infected mosquitoes12,14,16,17. The World Mosquito Program29 is deploying Wolbachia-infected Ae. aegypti mosquitoes in Brazil with the purpose of achieving a large-scale and sustained reduction in arboviral disease burden in two cities where these diseases are public health priorities.\n\nIn March 2016, the WHO convened a Vector Control Advisory Group to review new and existing vector control tools for use in the response to the Zika virus outbreak. Based on the available evidence that Wolbachia reduces the Zika, dengue and chikungunya transmission potential of Ae. aegypti mosquitoes and field data showing long-term establishment of Wolbachia in mosquito populations in a range of environmental settings, the WHO recommended carefully monitored pilot implementation of the Wolbachia method in affected countries24.\n\nWhile RCTs are still considered the gold standard, they are not always feasible or agreeable to the community and government. The controlled ITS analysis is a quasi-experimental design that is commonly used to evaluate population-level public health intervention27,30,31 and is a pragmatic alternative design where an RCT is considered infeasible, particularly in the presence of a well-matched untreated control area32,33. Given the public health emergency posed by the Zika epidemic at the time of this study’s inception and the need to scale up Wolbachia deployment in Rio de Janeiro and Niterói within a relative short time frame, an RCT or other carefully controlled deployment was not considered feasible. The controlled ITS is appropriate for the pragmatic evaluation of large-scale Wolbachia deployments given the long and reliable time series of dengue mandatory reporting data from both Rio de Janeiro and Niterói that allows for a longitudinal assessment of dengue trends before and after the Wolbachia intervention. Assessment of an impact of the intervention on chikungunya and Zika may be more difficult given their shorter time series.\n\nNotifiable disease surveillance data can be limited by a lack of specificity in case definitions and inconsistent reporting practices, which may influence our ability to detect a true intervention effect on arboviral disease incidence. A subset of notified dengue cases are supported by laboratory diagnostic results, but these have several limitations: i) laboratory testing occurs infrequently (<15% of notified cases), particularly during outbreaks, ii) the cross-reactivity of IgM serology between dengue and Zika limits the utility of serological data since 2015, and iii) the restricted use of PCR in only certain patient populations limits the generalisability of PCR-positivity rates to all notified cases. We therefore base our analyses on all notified cases (suspected and confirmed). Benefits of using these routinely collected data include the availability of a long time series, reduced costs for data collection and timely acquisition of data.\n\nHuman mobility also presents a challenge to this study as individuals are likely to spend time in both Wolbachia-treated and untreated areas, making it difficult to determine the place of illness acquisition among notified cases of arboviral disease. Travel between areas means that the true Wolbachia exposure status of individuals resident in Wolbachia-treated and untreated areas becomes more similar, thereby diluting the observed intervention effect towards the null.\n\nThe introduction of Zika and chikungunya viruses brought new challenges to health surveillance and a greater willingness for better vector control in affected regions. With the re-emergence of yellow fever in Brazil, there is even more potential public health benefit if the Wolbachia intervention successfully reduces the vector competence of Ae. aegypti mosquitoes in the field and reduces arboviral disease incidence. No specific treatment for dengue, chikungunya or Zika currently exists. Although a vaccine against dengue (Sanofi Dengvaxia®) was licensed in 2015, it was recently found to enhance the severity of subsequent dengue infection in individuals who were seronegative at the time of vaccination. As a result, a serology test prior to the administration of the vaccine is required to confirm previous dengue infection, increasing costs and decreasing feasibility in high-burden areas34.\n\nReleases of Wolbachia are completed or underway in eight countries, with no evidence of local transmission of dengue, Zika or chikungunya in places where Wolbachia is established at high levels21. The implementation of the Wolbachia intervention is complex and has not been done on a large scale in very densely populated urban areas in the Americas before. The implementation of the project was preceded by careful work to engage the community and gain public acceptance for the intervention, even in the most difficult contexts of poverty and urban violence present in both Rio de Janeiro and Niterói. Engagement, entomological monitoring, and public health impact assessment activities were developed in close partnership with local governments. If the current project is successful, this model can be expanded to the rest of the country and the Americas.\n\n\nEthics approval and consent to participate\n\nThe study was approved by the Brazilian Institutional Review Board (Conep) (CAAE: 59175616.2.0000.0008). The study uses pre-existing non-identifiable disease surveillance data, which does not require individuals’ consent.\n\n\nData availability\n\nNo data is associated with this article.",
"appendix": "Grant information\n\nThis study is funded by the Bill and Melinda Gates Foundation (OPP1099252); the Brazilian Ministry of Health (DECIT; TED 09/2016); and the US Centers for Disease Control (CDC).\n\nThe funders had no role in the design of the study or the decision to publish.\n\n\nAcknowledgements\n\nThe authors acknowledge the contributions of Peter Ryan, Simon Kutcher, Jacqui Montgomery, and Frederico Muzzi (World Mosquito Program) to the planning and implementation of Wolbachia deployments in Rio de Janeiro and Niterói.\n\n\nReferences\n\nBhatt S, Gething PW, Brady OJ, et al.: The global distribution and burden of dengue. Nature. 2013; 496(7446): 504–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBrathwaite Dick O, San Martín JL, Montoya RH, et al.: The history of dengue outbreaks in the Americas. Am J Trop Med Hyg. 2012; 87(4): 584–93. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFares RC, Souza KP, Añez G, et al.: Epidemiological Scenario of Dengue in Brazil. Biomed Res Int. 2015; 2015: 321873, Accessed 4 Jul 2018. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSiqueira JB Jr, Martelli CM, Coelho GE, et al.: Dengue and dengue hemorrhagic fever, Brazil, 1981-2002. Emerg Infect Dis. 2005; 11(1): 48–53. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBrasil P, Calvet GA, Siqueira AM, et al.: Zika Virus Outbreak in Rio de Janeiro, Brazil: Clinical Characterization, Epidemiological and Virological Aspects. PLOS Negl Trop Dis. 2016; 10(4): e0004636. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMlakar J, Korva M, Tul N, et al.: Zika Virus Associated with Microcephaly. N Engl J Med. 2016; 374(10): 951–8. PubMed Abstract | Publisher Full Text\n\nBoletins epidemiológicos. Secretaria de Vigilância em Saúde, Ministéio da Saúde. 2018; Accessed 5 Jul 2018. Reference Source\n\nMonitoramento do Período Sazonal da Febre Amarela Brasil - 2017/2018. Ministry of Health. Acessed 28 Jan 2019. Reference Source\n\nTeich V, Arinelli R, Fahhan L: Aedes aegypti e sociedade: o impacto econômico das arboviroses no Brasil. JBES: Brazilian Journal of Health Economics. 2017; 9(3): 267–76. Publisher Full Text\n\nControle de vetores. 2018; Accessed 26 Nov 2018. Reference Source\n\nWilson AL, Boelaert M, Kleinschmidt I, et al.: Evidence-based vector control? Improving the quality of vector control trials. Trends Parasitol. 2015; 31(8): 380–90. PubMed Abstract | Publisher Full Text\n\nYe YH, Carrasco AM, Frentiu FD, et al.: Wolbachia Reduces the Transmission Potential of Dengue-Infected Aedes aegypti. PLoS Negl Trop Dis. 2015; 9(6): e0003894. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFerguson NM, Kien DT, Clapham H, et al.: Modeling the impact on virus transmission of Wolbachia-mediated blocking of dengue virus infection of Aedes aegypti. Sci Transl Med. 2015; 7(279): 279ra37. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDutra HL, Rocha MN, Dias FB, et al.: Wolbachia Blocks Currently Circulating Zika Virus Isolates in Brazilian Aedes aegypti Mosquitoes. Cell Host Microbe. 2016; 19(6): 771–4. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAliota MT, Peinado SA, Velez ID, et al.: The wMel strain of Wolbachia Reduces Transmission of Zika virus by Aedes aegypti. Sci Rep. 2016; 6: 28792. PubMed Abstract | Publisher Full Text | Free Full Text\n\nvan den Hurk AF, Hall-Mendelin S, Pyke AT, et al.: Impact of Wolbachia on infection with chikungunya and yellow fever viruses in the mosquito vector Aedes aegypti. PLoS Negl Trop Dis. 2012; 6(11): e1892. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPereira TN, Rocha MN, Sucupira PHF, et al.: Wolbachia significantly impacts the vector competence of Aedes aegypti for Mayaro virus. Sci Rep. 2018; 8(1): 6889. PubMed Abstract | Publisher Full Text | Free Full Text\n\nO’Neill SL, Ryan PA, Turley AP, et al.: Scaled deployment of Wolbachia to protect the community from dengue and other Aedes transmitted arboviruses [version 2; peer review: 2 approved]. Gates Open Res. 2018; 2: 36. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHoffmann AA, Montgomery BL, Popovici J, et al.: Successful establishment of Wolbachia in Aedes populations to suppress dengue transmission. Nature. 2011; 476(7361): 454–7. PubMed Abstract | Publisher Full Text\n\nHoffmann AA, Iturbe-Ormaetxe I, Callahan AG, et al.: Stability of the wMel Wolbachia Infection following invasion into Aedes aegypti populations. PLoS Negl Trop Dis. 2014; 8(9): e3115. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSchmidt TL, Barton NH, Rašić G, et al.: Local introduction and heterogeneous spatial spread of dengue-suppressing Wolbachia through an urban population of Aedes aegypti. PLoS Biol. 2017; 15(5): e2001894. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGarcia GA, Sylvestre G, Aguiar R, et al.: Matching the genetics of released and local Aedes aegypti populations is critical to assure Wolbachia invasion. PLoS Negl Trop Dis. 2019; 13(1): e0007023. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAnders KL, Indriani C, Ahmad RA, et al.: The AWED trial (Applying Wolbachia to Eliminate Dengue) to assess the efficacy of Wolbachia-infected mosquito deployments to reduce dengue incidence in Yogyakarta, Indonesia: study protocol for a cluster randomised controlled trial. Trials. 2018; 19(1): 302. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWHO: Mosquito (vector) control emergency response and preparedness for Zika virus. Accessed 24 Oct 2018. Reference Source\n\nMinistério da Saúde. 2016; Accessed 25 Oct 2018. Reference Source\n\nGuia de vigilância epidemiológica. Ministério da Saúde, Secretaria de Vigilância em Saúde, Departamento de Vigilância Epidemiológica. - 7. ed. - Brasília: Ministério da Saúde. 2009; Accessed 25 Oct 2018. Reference Source\n\nBernal JL, Cummins S, Gasparrini A: Interrupted time series regression for the evaluation of public health interventions: a tutorial. Int J Epidemiol. 2017; 46(1): 348–55. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGubler DJ: Dengue, Urbanization and Globalization: The Unholy Trinity of the 21st Century. Trop Med Health. 2011; 39(4 Suppl): 3–11. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWorld Mosquito Program. Accessed 25 Oct 2018. Reference Source\n\nGasparrini A, Gorini G, Barchielli A: On the relationship between smoking bans and incidence of acute myocardial infarction. Eur J Epidemiol. 2009; 24(10): 597–602. PubMed Abstract | Publisher Full Text\n\nSteinbach R, Perkins C, Tompson L, et al.: The effect of reduced street lighting on road casualties and crime in England and Wales: controlled interrupted time series analysis. J Epidemiol Community Health. 2015; 69(11): 1118–24. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLinden A: Conducting interrupted time-series analysis for single-and multiple-group comparisons. Stata J. 2015; 15(2): 480–500. Publisher Full Text\n\nLagarde M: How to do (or not to do) … Assessing the impact of a policy change with routine longitudinal data. Health Policy Plan. 2012; 27(1): 76–83. PubMed Abstract | Publisher Full Text\n\nE Clapham H, A Wills B: Implementing a dengue vaccination programme-who, where and how? Trans R Soc Trop Med Hyg. 2018; 112(8): 367–8. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "51960",
"date": "07 Nov 2019",
"name": "Elizabeth L. Turner",
"expertise": [
"Reviewer Expertise Statistical methods in global health research including elimination science and impact evaluation with a focus on malaria and febrile illnesses"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis very well written manuscript describes the protocol for a quasi-experimental study using controlled interrupted time series. The study is currently being conducted in Brazil. The selected design is an innovative use of the controlled interrupted time series which can leverage routinely collected surveillance data to seek to address causal questions of interest. Moreover, the intervention being evaluated, namely the deployment of Wolbachia mosquitoes with the goal to reduce arboviral disease incidence, is innovative and shows great promise.\n\nI have two main comments:\nA desire to see more justification for why randomization was not possible. The difficulties of randomization in this setting are mostly described but, as you’ll see from my more detailed comments below, it’s not absolutely convincing that randomization would not have been possible here, even within the interrupted time series design.\n\nBecause the measurement of incidence of the arbobviruses of interest is mostly through surveillance data and it may be difficult to blind communities to the use of Wolbachia, how can it be determined that in the treatment zones post-Wolbachia release any smaller incidence is not just related to the fact that there is lower reporting as community members expect the incidence of those viruses to go down? I have a big concern about the possibility of differential assessment of outcomes in treated and control zones. Can the authors elaborate on this in relevant places in the text?\nHere I offer some more minor points that may help with clarity for the reader:\n\nAbstract\nIt could help the reader if when “dengue, chikungunya and Zika” are first mentioned in the abstract, they are also named as arboviruses i.e. to help readers not necessarily familiar with them to be certain that all three fall in to this class of viruses.\nBackground\nBased on the title referring to arboviruses, as a reader, I was expecting the first sentence or so to at least mention of arbovirus. Would it be possible to do so? This could be achieved very easily by saying something like “The global incidence of the arbovirus dengue has increased dramatically in recent decades.”\n\nIn the 4th paragraph referring to yellow fever, please clarify if is an arbovirus.\n\nThe last sentence of the 6th paragraph: “Laboratory data indicate a similar reduction in the competence of Wolbachia-carrying Ae. aegypti for transmitting other viruses including Zika, chikungunya, yellow fever and Mayaro”. If all listed are arboviruses, please edit “viruses” to “arboviruses”.\n\nIn the 9th paragraph, I very much appreciate the rationale that there was demand for scale-up of implementation of the Wolbachia releases. I would appreciate a little more clarity on why randomization was not possible since, given that not all areas of the study will receive the treatment during the study, the scale-up argument doesn’t seem sufficient to justify no randomization. I can very well imagine that there are many compelling reasons to not randomize, some of which are alluded to, and so it would help to be even clearer about this e.g. was it because of the existing partnerships and infrastructure making certain areas better for release than others? What else? Some of this is sort of implied in the text and it would be valuable to have just a few more explicit sentences regarding this.\n\nRelated to my previous comments on arboviruses, in the 10th paragraph where it is stated: “Here we describe a protocol for evaluating the effect of large scale non-randomized Wolbachia releases on the incidence of dengue, Zika and chikungunya in the municipalities of Niterói and Rio de Janeiro.”, again, it could really help the reader to edit this sentence to something like “…on the incidence of three arboviruses, namely dengue, Zika and chikungunya, in the……”.\n\nAlso, see a later comment based on the discussion as to why yellow fever is not also included in the study.\nMethods – Study design\nWhy do the two stated objectives only relate to dengue? It will be important to make clear in the manuscript as it is strange that the overall goals are related to three arboviruses but the objectives only related to one of them.\n\nTable 1: Are zones 1-3 in Niteroi and zones 1-3.2 in Rio the release areas? If so, please more clearly label this in the table as it is not immediately obvious to me when I look at the table even though I infer that it is the case. Perhaps add a sub-heading of “release areas” above the rows where the names of the release zones are provided.\nMethods – Study setting and population\n\nOverall, the rationale appears reasonable for the use of controlled interrupted time series but, in reference to my comments above, please look for some opportunities to be even clearer about why randomization could not be used. An interrupted time series design could still be adopted, but one with randomization could offer even stronger evidence.\n\n“In Rio de Janeiro, two administrative areas adjacent to the release area have been designated a priori as a comparative control zone (Figure 1), based on comparable sociodemographic characteristics and synchronous historical dengue time series (Figure 2 and Table 1).” I do not see any information on these SES characteristics except for population whereas I see the “synchronous historical dengue time series” data in Figure 2, which is really helpful. Can more info on SES be added to the appendix or is the data presented the extent of the available data? As a reader, I would want that information when judging the validity of the comparisons between treatment and control areas. Incidentally, is no historical data available on Zika and Chikungunga? If it is available, it would be really valuable to see that compared between treated and control. I see in the last paragraph of pg 6 that some such data is available so why not include it in the current manuscript?\nControlled interrupted time series\nIt is stated: “Zone-level ITS analyses will be performed 12 months after completion of releases in each zone, and each 12 months thereafter, with release zones considered ‘treated’ for the purpose of this analysis based on completion of releases, regardless of the long-term Wolbachia monitoring results.” What does “performed 12 months after completion”? Is it literally about the timing of running the analyses, even though data from each month will be used? The reason I ask for clarification is that on first read I thought that this meant there was a switch to only using data at 12 months after completion of the releases. Also, what is the time frame for control areas where there is no release? Please be more specific.\n\n“Aggregate release area level analyses will be performed for each municipality 12 months after completion of releases in the last zone and each 12 months thereafter.” What are municipalities? Please define this term when it is first introduced as I don’t see this term defined elsewhere. Is it city i.e. Rio and Niteroi? If so, why not just use the same term throughout i.e. municipality or city, and only choose one of those terms?\n\nRegarding analyses, I recognize that there may not be space but it would be beneficial to provide some more details including:\n\nModel diagnostics to be used e.g. what happens if there is even more dispersion than can be accommodated than the proposed negative binomial models?\n\nHow will contrasts be made between the treated and control communities? As far as I can tell, this is not specified, though perhaps I’m missing it? Please specific which parameters will be used to estimate the “treatment” effect.\n\nHow will the fact that the control zones are not randomized be accounted for in modelling i.e. although the set of control zones were selected to be comparable on average to those treated based on historical dengue incidence and SES, what about other factors that may be different between the treated and control zones?\n\nWill there be a “fake” interruption in the series for the control areas? It doesn’t seem necessary and may be problematic but would be valuable to describe explicitly what will be done in the time series modelling for the control zones.\n\nDiscussion\nThe discussion starts with a sentence on dengue, zika and yellow fever. Why is yellow fever not included in the outcome measures for the proposed study?\nOverall, this is a fascinating study and a great use of an interrupted time series.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable",
"responses": [
{
"c_id": "5566",
"date": "11 Jun 2020",
"name": "Katherine ANDERS",
"role": "Author Response",
"response": "Main comments 1. I have a desire to see more justification for why randomisation was not possible. The difficulties of randomisation in this setting are mostly described but, as you’ll see from my more detailed comments below, it’s not absolutely convincing that randomisation would not have been possible here, even within the interrupted time series design. We agree with the reviewer that randomisation could be combined with ITS, and in principle this could be a stronger design. However randomisation of the deployment (either by random allocation of parallel treated and untreated areas, or by randomising the sequence of staged deployments) would have necessitated the study site being divided up into a large number of spatial units for randomisation. Logistically, this would have made the community engagement and deployment activities infeasible within the funding and time constraints of the project, and in the context of the large and complex urban setting. Intensive stakeholder and community engagement, and deployment planning, could only have commenced after the randomisation process had occurred and the intervention areas were determined. Field activities would then have had to be conducted in a spatially fragmented way, complicating the stakeholder and community engagement and the deployment logistics and inevitably lengthening time frames. Smaller non-contiguous release areas interspersed with untreated areas would also have greatly increased the potential for contamination between treatment arms from both mosquito movement and human movement, thereby diluting the observable intervention effect. Please see our responses to comments #7 and #11 below, and Reviewer 2 comment #3. 2. Because the measurement of incidence of the arboviruses of interest is mostly through surveillance data and it may be difficult to blind communities to the use of Wolbachia, how can it be determined that in the treatment zones post-Wolbachia release any smaller incidence is not just related to the fact that there is lower reporting as community members expect the incidence of those viruses to go down? I have a big concern about the possibility of differential assessment of outcomes in treated and control zones. Can the authors elaborate on this in relevant places in the text? It is plausible that community awareness could alter health-care seeking behaviour, or diagnostic / reporting practices, in areas where Wolbachia releases have been conducted. However we find it unlikely that this could lead to a large and sustained difference in dengue case notifications across two cities as large and complex as these, where the total intervention area exceeds 120km2 and 1 million inhabitants. Dengue (and chikungunya and Zika) is a notifiable disease, so there remains a requirement for health care providers to report any suspected cases meeting the standard case definition. We have included a discussion of this as potential limitation, in the fourth paragraph of the discussion. From the beginning of the project's engagement with communities, all messaging has reinforced the continued importance of routine activities related to arbovirus surveillance and care. Minor comments 3. It could help the reader if when ‘dengue, chikungunya and Zika’ are first mentioned in the abstract, they are also named as arboviruses i.e. to help readers not necessarily familiar with them to be certain that all three fall in to this class of viruses. Since the three arboviruses of interest are specifically those vectored by Aedes mosquitoes, for greater clarity we have replaced ‘arboviruses’ with ‘Aedes-borne viruses’ in most instances throughout the abstract and article text, including in the manuscript title. Elsewhere we have replaced ‘arboviral disease’ with the more intuitive term ‘mosquito-borne disease’. In the last sentence of paragraph one, it is made clear that the term ‘arbovirus’ encompasses dengue, chikungunya, Zika, yellow fever and Mayaro viruses, and that these are specifically Aedes-borne arboviruses. 4. Based on the title referring to arboviruses, as a reader I was expecting the first sentence or so to at least mention arbovirus. Would it be possible to do so? This could be achieved very easily by saying something like “The global incidence of the arbovirus dengue has increased dramatically in recent decades”. As above, the manuscript title has been adjusted to replace ‘arboviral disease’ with ‘Aedes-borne disease’ to improve clarity for the reader. The first sentence introduces dengue (the disease) as a global public health challenge, and the last sentence of that paragraph defines dengue, chikungunya and Zika (and yellow fever and Mayaro) as Aedes-borne diseases (and arboviruses). A definition of arbovirus as meaning a mosquito-borne virus has also been added to this sentence. 5. In the fourth paragraph referring to yellow fever, please clarify if it is an arbovirus. Yellow fever has been defined as an Aedes-borne arboviruses in the last sentence of the first paragraph of the introduction. 6. The last sentence of the sixth paragraph: “Laboratory data indicate a similar reduction in the competence of Wolbachia-carrying Ae. aegypti for transmitting other viruses include ZIka, chikungunya, yellow fever and Mayaro”. If all listed are arboviruses, please edit ‘viruses’ to ‘arboviruses’. The text has been corrected as suggested. 7. In the ninth paragraph, I very much appreciate the rationale that there was demand for scale-up of implementation of the Wolbachia releases. I would appreciate a little more clarity on why randomization was not possible since, given that not all areas of the study will receive the treatment during the study, the scale-up argument doesn’t seem sufficient to justify no randomization. I can very well imagine that there are many compelling reasons to not randomize, some of which are alluded to, and so it would help to be even clearer about this e.g. was it because of the existing partnerships and infrastructure making certain areas better for release than others? What else? Some of this is sort of implied in the text and it would be valuable to have just a few more explicit sentences regarding this. Please see our response to Major comment #1 above. We have added text to the end of the ninth paragraph of the introduction, expanding on this justification for not randomising, as suggested. 8. Related to my previous comments on arboviruses, in the tenth paragraph…again it could really help the reader to edit this sentence to something like “…three arboviruses, namely dengue, Zika and chikungunya…” Consistent with our response to comment # above, we have adjusted the wording in the paragraph to refer to dengue, Zika and chikungunya as ‘Aedes-borne diseases’ or ‘mosquito-borne diseases’. 9. Why do the two stated objectives only relate to dengue? It will be important to make clear in the manuscript as it is strange that the overall goals are related to three arboviruses but the objectives only related to one of them. Thank you for pointing out this inconsistency, we have corrected the wording of the aims and first objective to be inclusive of dengue, chikungunya and Zika (as the Aedes-borne diseases of interest). The second objective still relates only to dengue, as the outbreak indicators used routinely for public health purposes in Rio and Niteroi apply only to dengue outbreak monitoring. As explained under ‘Controlled interrupted time series analysis’ (3rd and 4th paragraphs, page 11), the primary endpoint for the ITS analysis will be dengue for which there is the longest historical case time series, with chikungunya and Zika analysed as secondary endpoints in the ITS. 10. Table 1: Are zones 1-3 in Niteroi and zones 1-3.2 in Rio the release areas? If so, please more clearly label this in the table as it is not immediately obvious to me. Perhaps add a sub-heading of “release areas” above the rows where the names of the release zones are provided. Thank you for this suggestion, we have improved the labelling of the areas in Table 1 to make clearer which are the release zones and non-release areas. Please note that we have also updated this table and Figure 2 to reflect an additional release zone in Niteroi (and the consequent reduced size of the control area). 11. Overall the rationale appears reasonable for the use of controlled interrupted time series but, in reference to my comments above, please look for some opportunities to be even clearer about why randomisation could not be used. An interrupted time series design could still be adopted, but one with randomisation could offer even stronger evidence. Please see our responses to comments #1 and #7 above. Additional discussion of the justification for not randomising, and the potential benefits from the pragmatic evaluation of a large-scale contiguous release, has been added to the third paragraph of the discussion section on page 12. 12. “In Rio de Janeiro, two administrative areas adjacent to the release area have been designated a priori as a comparative control zone (Figure 1), based on comparable sociodemographic characteristics and synchronous historical dengue time series (Figure 2 and Table 1).” I do not see any information on these SES characteristics except for population whereas I see the “synchronous historical dengue time series” data in Figure 2, which is really helpful. Can more info on SES be added to the appendix or is the data presented the extent of the available data? As a reader, I would want that information when judging the validity of the comparisons between treatment and control areas. Unfortunately socio-economic data are not available aggregate to the release zones. We have now corrected this text to clarify that the comparability of the untreated control areas is demonstrated by the synchronous historical dengue time series. 13. Incidentally, is no historical data available on Zika and Chikungunga? If it is available, it would be really valuable to see that compared between treated and control. I see in the last paragraph of pg 6 that some such data is available so why not include it in the current manuscript? Historical chikungunya data for the intervention areas and control areas is now included as Figure 3. 14. It is stated: “Zone-level ITS analyses will be performed 12 months after completion of releases in each zone, and each 12 months thereafter, with release zones considered ‘treated’ for the purpose of this analysis based on completion of releases, regardless of the long-term Wolbachia monitoring results.” What does “performed 12 months after completion”? Is it literally about the timing of running the analyses, even though data from each month will be used? The reason I ask for clarification is that on first read I thought that this meant there was a switch to only using data at 12 months after completion of the releases. Also, what is the time frame for control areas where there is no release? Please be more specific. Yes, this is about the timing of running the analyses, ie. the intervention effect will be estimate for each zone when 12 months of post-release observations have accumulated in that zone, and repeated each 12 months thereafter (for five years) on cumulative post-release observations. And the same for the city-level analyses. The text at the end of the first paragraph under the heading ‘Controlled interrupted time series analysis’ has been edited to clarify this. There is no separate analysis for control areas, these are included in the regression model as the comparator for each individual release zone, and for the city-level analysis. This has been clarified in the text. 15. “Aggregate release area level analyses will be performed for each municipality 12 months after completion of releases in the last zone and each 12 months thereafter.” What are municipalities? Please define this term when it is first introduced as I don’t see this term defined elsewhere. Is it city i.e. Rio and Niteroi? If so, why not just use the same term throughout i.e. municipality or city, and only choose one of those terms? We recognise this inconsistency may have been confusing, and have changed throughout to use ‘city/cities’ in place of ‘municipality/municipalities’. 16. Model diagnostics to be used e.g. what happens if there is even more dispersion than can be accommodated than the proposed negative binomial models? Of the common count models, negative binomial models have the most flexible approach for handling overdispersion in count data by assuming that the variance is a quadratic function of the mean. Common causes of overdispersion can range from an excess of zero counts, unexplained heterogeneity, and/or temporal autocorrelation. The models as proposed will be using flexible cubic splines to explain secular and temporal trends, hopefully leaving very little remaining unexplained heterogeneity. To account for any lingering heteroskedasticity or temporal autocorrelation, heteroskedasticity-robust estimation will be used for estimating variance. This has now been made explicit in the description of the Controlled interrupted time series methods. If an excess of zero counts is apparent, the proposed negative binomial model will be nested within a zero-inflated negative binomial model of the same structure. A likelihood ratio test can be performed to test model fit. This is also now described in the methods. 17. How will contrasts be made between the treated and control communities? As far as I can tell, this is not specified, though perhaps I’m missing it? Please specific which parameters will be used to estimate the “treatment” effect The regression model will use monthly dengue case time series data for the treated area (either an individual release zone or aggregate city-level release area, as specified in the text) and the control area (one control area per city). The treatment effect will be estimated from the interaction between a binary ‘area’ variable (treated vs control) and a binary ‘period’ variable (post-Wolbachia vs pre-Wolbachia). This allow explicitly for a level change in the outcome (dengue case incidence) in both intervention and control arms in the post-intervention period, for example in a scenario where other secular events coincident with the Wolbachia deployments may have influenced dengue incidence in both intervention and control areas independent of Wolbachia. The text under ‘Controlled interrupted time series’ has been revised to clarify this. 18. How will the fact that the control zones are not randomized be accounted for in modelling i.e. although the set of control zones were selected to be comparable on average to those treated based on historical dengue incidence and SES, what about other factors that may be different between the treated and control zones? Quasi-experimental designs, such as the proposed ITS analysis, aim to address the validity concerns associated with non-randomization in several ways. By using a region's historic data to inform the counterfactual outcome, the ITS analysis aims to minimize threats to internal validity. Second, by adding control regions and performing a controlled ITS, we better account for seasonal and secular influences during both the pre- and post-intervention periods. While it is true that there may be other factors associated with dengue risk that differ between treated and control zones in a non-randomised design, we make the implicit assumption in the controlled ITS that these differences are consistent throughout both the pre- and post-intervention period. Any divergence in the post-intervention period from the synchrony in dengue time series observed between intervention and control zones in the pre-intervention period is therefore assumed to represent an intervention effect. However we cannot exclude the possibility of a concurrent change in socio-demographic or ecological factors related to dengue risk, or a concurrent change in dengue control practices, between the intervention and control areas independe of the Wolbachia intervention. We have expanded on this limitation in the discussion. 19. Will there be a “fake” interruption in the series for the control areas? It doesn’t seem necessary and may be problematic but would be valuable to describe explicitly what will be done in the time series modelling for the control zones. The inclusion of the control area time series in the regression model and estimation of the intervention effect from an interaction between ‘area’ and ‘period’ effectively does allow for a ‘fake’ interruption in the series for the control area, as it allows for a step change in the control area as well as the treated area, post-intervention. The interaction parameter from which the intervention effect is estimated indicates whether there was any change in dengue incidence following the Wolbachia intervention in areas where Wolbachia releases occurred, over and above any change in dengue incidence that was observed in the untreated control area. 20. The discussion starts with a sentence on dengue, zika and yellow fever. Why is yellow fever not included in the outcome measures for the proposed study? Yellow fever is not included as an endpoint for the study because there is not a history of urban yellow fever in the cities of Rio and Niteroi. The recent outbreak in Rio de Janeiro state that is mentioned early in the discussion included some urban cases, but in other parts of the state. We have removed that sentence from the first paragraph of the discussion, to avoid confusion."
}
]
},
{
"id": "57000",
"date": "09 Dec 2019",
"name": "Adeshina I. Adekunle",
"expertise": [
"Reviewer Expertise Infectious disease modelling and bio-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\nReview: The authors present the study protocol for an ongoing large deployment of Wolbachia-infected Aedes-aegypti mosquitoes in Rio de Janeiro and Niteroi municipalities in southeastern Brazil where Aedes-borne diseases, especially dengue, are common. The study design is a quasi-experimental study (QES) that allows the application of the controlled interrupted time series analysis to evaluate whether Wolbachia strategy is effective in this region. The manuscript is well written and the study, when completed, has great potential for evaluating the promising Wolbachia strategy for controlling Aedes-borne diseases. However, I have some concerns about this study and the ability of the interrupted time series method to infer reduction in disease burden if present:\n\nMain comments:\nAedes-aegypti mosquitoes movement.\nFirst of all, the authors failed to indicate the strain of Wolbachia used in this study. Different strains of Wolbachia have different characteristics that enable them to invade the wild type population and establish themselves. The wMel strain was used in Townsville, Australia1 and was claimed to be successful. However, with climate change and variability in temperature, some Wolbachia-infected mosquitoes with different strains, include wMel, cannot maintain their Wolbachia status2. Hence, even if the intervention works, an introduction of infected patients into such a population will result in an outbreak and all the gains from Wolbachia strategy would have been lost as the majority of the mosquitoes are now uninfected3.\n\nFigure 1 in the manuscript shows the map of the treated and untreated zones. These are adjacent zones, especially for the treated and untreated zones. Mosquitoes movement from untreated zone to treated zone could prevent Wolbachia invasion4 and vice versa. Hence, and since this is a study that takes a few years, mosquitoes migration cannot be ruled out. Thus, the control zones (untreated) is not suitable anymore.\nThe controlled interrupted time-series method.\nThe authors stated that the reason for favoring quasi-experimental design (QED) over randomized controlled trial (RCT) was due to time constraints as stakeholders and funders wanted to scale-up the deployment of Wolbachia to accelerate the control of the emergent Zika virus in this region. However, in this set-up, to carefully randomize zones as treated or not should not take much time. In fact, since the incidence of Aedes-borne virus infections is similar between treated and untreated zones (Figure 2), that should be a motivation for randomizing the zones. I am thinking there are other reasons for ditching RCT. The authors should elaborate on this. For the controlled interrupted analysis, the authors plan to perform first, the descriptive analysis of the seasonal and inter-annual trends in the Aedes-borne infection notifications. It will be great if the authors are more specific about the types of descriptive analysis they intend to carry out. Also, a schematic of the expected trend in the notifications accounting for confounders will greatly help the protocol.\n\nMinor comments:\nTitle\nThe title indicated controlling “arboviral disease incidence”. The authors can be more specific if they change that to either “Aedes-borne disease incidence” or something more specific.\nAbstract\nI think the clause in the fourth line will flow better if “Aedes-borne diseases dengue, chikungunya and Zika” is changed to “Aedes-borne diseases (dengue, chikungunya and Zika). Change “km2” to “km2”.\nMethods\nFigure 2 shows the comparative incidence of dengue for the treated and untreated regions. What about Zika and Chikungunya that the study is trying to control too? I think this is a dengue control study that has the potential to control Zika and Chikungunya in these regions. It will be great if the authors make it appear so. As it is, it seems to be a study deliberately targeting all the three Aedes-borne diseases.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable",
"responses": [
{
"c_id": "5567",
"date": "11 Jun 2020",
"name": "Katherine ANDERS",
"role": "Author Response",
"response": "Major comments 1. The authors failed to indicate the strain of Wolbachia used in this study. Different strains of Wolbachia have different characteristics that enable them to invade the wild type population and establish themselves. The wMel strain was used in Townsville, Australia and was claimed to be successful. However, with climate change and variability in temperature, some Wolbachia-infected mosquitoes with different strains, include wMel, cannot maintain their Wolbachia status. Hence, even if the intervention works, an introduction of infected patients into such a population will result in an outbreak and all the gains from Wolbachia strategy would have been lost as the majority of the mosquitoes are now uninfected. Thank you for drawing our attention to this oversight. We have now indicated the strain used (wMel) in the abstract, the sixth paragraph of the introduction, the ‘Wolbachia release and monitoring’ subheading of the Methods, and the first paragraph of the discussion. While it is true that other Wolbachia strains have been shown to have higher thermostability than wMel in the mosquito larval life stages, the effect of heatwaves on wMel Wolbachia prevalence in Wolbachia-treated areas of northern Australia has been shown to be transient (Ross et al, PLoS NTD 2020). Long-term monitoring of WMP’s release sites in northern Australia and Yogyakarta, Indonesia, has demonstrated sustained high levels of Wolbachia 2 – 8 years post-release. Entomological monitoring of Wolbachia prevalence in local Ae. aegypti populations in the Rio and Niteroi release areas is currently ongoing, and so will provide empirical evidence of the level to which wMel introgression is sustained over time. 2. Figure 1 in the manuscript shows the map of the treated and untreated zones. These are adjacent zones, especially for the treated and untreated zones. Mosquitoes movement from untreated zone to treated zone could prevent Wolbachia invasion4 and vice versa. Hence, and since this is a study that takes a few years, mosquitoes migration cannot be ruled out. Thus, the control zones (untreated) is not suitable anymore. It is true that Wolbachia could theoretically spread from the deployment areas into the untreated control zones, however the rate of spread is known to be relatively slow especially in dengue urban environments like these (100-200m per year; Schmidt et al PLoS Biol 2017). Given the size of the control zone (117km2 in Rio de Janeiro, and 51km2 in Niteroi), it is highly unlikely that Wolbachia establishment will occur in any meaningful way beyond the border area of the control zones within the 5 year timeframe of this study. 3. The authors stated that the reason for favoring quasi-experimental design (QED) over randomized controlled trial (RCT) was due to time constraints as stakeholders and funders wanted to scale-up the deployment of Wolbachia to accelerate the control of the emergent Zika virus in this region. However, in this set-up, to carefully randomize zones as treated or not should not take much time. In fact, since the incidence of Aedes-borne virus infections is similar between treated and untreated zones (Figure 2), that should be a motivation for randomizing the zones. I am thinking there are other reasons for ditching RCT. The authors should elaborate on this. The randomization itself would not necessarily take a lot of time, although there would be additional work required to define an adequate number of spatial units for randomization (ideally based on a power calculation), and define those spatial units with ‘hard’ borders which minimise contamination between treated and untreated areas. The time delays come from the added complexity of conducting the intensive community and stakeholder engagement that is needed prior to releases, and conducting the deployments themselves, across a fragmented set of release areas interspersed with untreated control areas, compared to conducting these engagement and deployment activities in a contiguous release area. Please see our response to Reviewer 1’s comments #1, #7 and #11 for further discussion of the reasons for non-randomisation, and for the references to where we have improved discussion of this point in the manuscript text. 4. For the controlled interrupted analysis, the authors plan to perform first, the descriptive analysis of the seasonal and inter-annual trends in the Aedes-borne infection notifications. It will be great if the authors are more specific about the types of descriptive analysis they intend to carry out. Also, a schematic of the expected trend in the notifications accounting for confounders will greatly help the protocol The descriptive analysis will simply describe the seasonal and interannual trends, as stated. I.e. the calendar months in which dengue incidence has historically been highest and lowest in the two cities, and the range in annual case counts and per capita incidence. We don’t see that this level of detail is of value to the reader here. We don’t understand the reviewer’s suggestion about a schematic of expected trend in notifications. We show in figure 2 the historical trends in dengue incidence in the intervention area and untreated control area for each city; the lines for intervention and control area can be expected to diverge if Wolbachia successfully interrupts dengue transmission. Minor comments 5. The title indicated controlling “arboviral disease incidence”. The authors can be more specific if they change that to either “Aedes-borne disease incidence” or something more specific. We have changed the wording in the title to ‘Aedes-borne disease’ as suggested. 6. I think the clause in the fourth line [of the abstract] will flow better if “Aedes-borne diseases dengue, chikungunya and Zika” is changed to “Aedes-borne diseases (dengue, chikungunya and Zika). We have kept the original wording here, because strictly these three are not the only Aedes¬-borne diseases, but they are the Aedes-borne diseases were are considering in this protocol. 7. Change “km2” to “km2” This change has been made. 8. Figure 2 shows the comparative incidence of dengue for the treated and untreated regions. What about Zika and Chikungunya that the study is trying to control too? I think this is a dengue control study that has the potential to control Zika and Chikungunya in these regions. It will be great if the authors make it appear so. As it is, it seems to be a study deliberately targeting all the three Aedes-borne diseases See response to comment #13, Reviewer 1. Historical chikungunya data for the intervention areas and control areas is now included as Figure 3."
}
]
}
] | 1
|
https://f1000research.com/articles/8-1328
|
https://f1000research.com/articles/8-1725/v1
|
07 Oct 19
|
{
"type": "Research Article",
"title": "Glycemic control monitoring in patients with tuberculosis and diabetes: a descriptive study from programmatic setting in Tamil Nadu, India",
"authors": [
"J. Gurukartick",
"Lakshmi Murali",
"Hemant Deepak Shewade",
"Anil G. Jacob",
"M. M. Samy",
"D. Dheenadayal",
"O. P. Aslesh",
"Ganesh Marimuthu",
"Ramya Ananthakrishnan",
"Nalini Krishnan",
"Lakshmi Murali",
"Anil G. Jacob",
"M. M. Samy",
"D. Dheenadayal",
"O. P. Aslesh",
"Ganesh Marimuthu",
"Ramya Ananthakrishnan",
"Nalini Krishnan"
],
"abstract": "Background: India’s national tuberculosis (TB) programme recommends that among patients with diabetes mellitus and TB, fasting blood glucose (FBG) be recorded at baseline, the end of intensive phase and the end of continuation phase of TB treatment. We conducted this operational research in select districts of Tamil Nadu, India, in 2016 to determine the availability of blood glucose records and glycemic control status during TB treatment. Methods: This was a descriptive study involving secondary programme data. Glycemic control during TB treatment was ‘optimal’ if both baseline and end of intensive phase FBG (during TB treatment) were <130 mg/dl. In the absence of FBG, we used random blood glucose (RBG), with <180 mg/dl as the cut off. Results: Of 438 patients, FBG at baseline, the end of intensive phase and the end of continuation phase were each available in <20%. Glycemic control status was known for 94% (412/438) patients at baseline and for 91% (400/438) during TB treatment. Among those with known glycemic status, glycemic control was not optimal in 77% of patients (316/412) at baseline and in 84% (337/400) during TB treatment. The proportion of patients with unfavourable TB treatment outcomes at the end of intensive phase was 11% (46/438) and at the end of continuation phase was 5% (21/438). We decided against assessing factors associated with glycemic control during TB treatment and association between glycemic control and TB treatment outcomes because glycemic control assessment, if any, was based mostly on RBG values. Conclusion: Among patients with diabetes and tuberculosis, recording of FBG during tuberculosis treatment requires urgent attention.",
"keywords": [
"tuberculosis treatment",
"diabetes mellitus treatment",
"blood glucose",
"treatment outcome",
"glycemic control",
"SORT IT"
],
"content": "Introduction\n\nWorldwide, tuberculosis (TB) remains a major public health problem in low- and middle-income countries. Diabetes mellitus (DM) affected 425 million people in 2017 and is projected to increase to 629 million by 20451. Globally, TB and DM are among the top ten causes of death2. Annually, of the estimated 10 million new people with active TB, one million have DM (TB-DM) and this double burden deserves attention2–4. Considering the bi-directionality of association between TB and DM5, in 2010, a collaborative framework for care and control of TB-DM was developed by World Health Organization (WHO) and the International Union Against Tuberculosis and Lung Disease (The Union)6.\n\nAmong patients with TB, DM increases risk of death, recurrence and ‘treatment failure and death’ combined when compared to not living with DM7. DM may result in early mortality and adverse outcomes among patients undergoing TB treatment8.\n\nIndia has more than 25% of the global TB burden and has the second highest number of people with diabetes after China4. The prevalence of DM among adults with TB was 44% in Kerala state and 25% in Tamil Nadu state9. In 2012, a feasibility study was conducted in India’s revised national TB control programme (RNTCP) settings for bidirectional screening for TB-DM10, based on which in the same year a policy decision was taken to screen all TB patients for DM. The programme also recommends recording of fasting blood glucose (FBG) values at baseline, the end of intensive phase (IP) and the end of continuation phase (CP) of TB treatment11,12.\n\nConsidering that systematic monitoring and recording of glycemic status among patients with TB-DM has been initiated in some districts of Tamil Nadu, this presents a unique opportunity for understanding the same in programme setting. Recording of FBG at baseline and during TB treatment will provide reliable information not only for management of patient’s glycemic status but also to assess the effect of optimal glycemic control on TB treatment outcomes among patients with TB-DM, the evidence for which is limited13.\n\nTherefore, this study was conducted among patients with TB-DM registered for TB treatment in 2016 under the programme in select districts of Tamil Nadu, India. The specific objectives were to determine i) the availability of FBG at various phases of TB treatment as per programme recommendations; and ii) the number (proportion) without optimal glycemic control at baseline and during TB treatment.\n\n\nMethods\n\nThis was a descriptive study involving record review of routinely collected programme data.\n\nGeneral setting. Tamil Nadu is a state in south India along the eastern coast with predominantly plain terrain. (Figure 1) Tamil Nadu has a population of 72 million and is made up of 32 administrative districts. The literacy rate is 80%14.\n\nStudy districts. The study was conducted in programme setting in Tiruvallur, Madurai and Tiruppur districts of Tamil Nadu. Tiruvallur is a suburb of Chennai (the capital of Tamil Nadu). Tiruppur and Madurai districts are located in west and south Tamil Nadu, respectively (Figure 1).\n\nUnder RNTCP in 2016, each district had one district TB centre, sub-district administrative units called as TB units (Tiruvallur-14, Madurai-21, Tiruppur-16) and designated microscopy centers (Tiruvallur-31, Madurai-35, Tiruppur-20). Diagnosed patients were registered for treatment and entered into the treatment register at the TB unit. Patients received domiciliary directly observed treatment (thrice weekly) which was according to the then national treatment guidelines. TB treatment outcomes (end IP and end treatment outcomes) were recorded in line with WHO recommendations and have been summarized in Box 115.\n\n\n\nEnd of TB treatment – minimum 6 month follow up and maximum up to 31 May 2017\n\n- death, treatment failed, loss to follow up, not evaluated (unfavourable treatment outcome)\n\n- treatment completed, cure (favourable treatment outcome)\n\nCured: ’Cured’ defined as “A pulmonary TB patient with bacteriologically-confirmed TB at the beginning of treatment who was smear- or culture-negative in last month of treatment and on at least one previous occasion”;\n\nTreatment completion: ’Treatment completed’ defined as “A TB patient who completed treatment without evidence of failure, but with no record to show that sputum smear or culture results in the last month of treatment and on at least one previous occasion were negative, either because tests were not done or because results are unavailable”;\n\nLoss to follow-up: ’Loss to follow-up’ defined as “A TB patient who did not start treatment or whose treatment was interrupted for two consecutive months or more”;\n\nTreatment failed: ’Treatment failed’ defined as “A TB patient whose sputum smear or culture is positive at month five or later during treatment”;\n\nDied: ’Died’ defined as “A TB patient who dies for any reason before starting or during the course of treatment”;\n\nNot evaluated: ’Not evaluated’ defined as “A TB patient for whom no treatment outcome is assigned. This includes cases “transferred out” to another treatment unit as well as cases for whom the treatment outcome is unknown to the reporting unit”.\n\nEnd of Intensive phase\n\n- Death. Loss to follow up, extension of IP and non-conversion at 3 / 4 months (Unfavourable)\n\n- Microbiological conversion (Favourable)\n\nSputum conversion: those who are sputum negative latest by 3 months for new patients and latest by 4 months for retreatment patients\n\nSputum non-conversion: those who are sputum positive at 3 months for new patients and at 4 months for retreatment patients\n\nTB-DM collaboration. One of the activities under TB-DM collaborative framework consists of screening for DM among all patients with TB (initiated in study districts in 2015) and monitoring glycemic control during TB treatment among all patients with TB-DM (initiated in 2016). Patients with TB are first screened with random blood glucose (RBG) at designated microscopy centers; those with RBG≥140 mg/dl (7.8 mmol/l) are assessed using FBG. Those with FBG≥126 mg/dl (7 mmol/l) are referred to higher centers for clinical confirmation of DM and initiation of treatment. This is followed by treatment continuation at the peripheral health centers11.\n\nPatients with TB-DM undergo FBG at the end of IP and at the end of CP and the same are entered in the TB treatment card and register11. There is no specific modification in either DM or TB treatment because of the co-morbidity. TB and DM management is done free of cost.\n\nThe study population included all patients with TB-DM comorbidity (age ≥15 years) registered under RNTCP between January and September 2016 in Tiruvallur, between January and June 2016 in Madurai and between April and June 2016 in Tiruppur. Patients with multi-drug-resistant TB were excluded from the study.\n\nA review of records was conducted between October 2016 and May 2017. Data were collected from TB treatment registers and patient treatment cards using a paper-based data collection form. Each patient was given a unique identifier derived from district code, tuberculosis unit name and TB registration number.\n\nBaseline socio-demographic characteristics, baseline clinical characteristics and treatment outcomes (end IP and end treatment) were collected. In addition, blood glucose values at baseline, at the end of IP and at the end of CP along with the type of test (FBG or RBG) were collected.\n\nData collected were double-entered, validated and analysed using EpiData (version 3.1 for entry and version 2.2.2.183 for analysis; EpiData Association, Odense, Denmark). Frequency and proportions were used to summarize the key analytic outputs.\n\nThe cut off for optimal glycemic control was <130 mg/dl (7.2 mmol/l) for FBG and <180 mg/dl (10 mmol/l) for RBG. The cut off (to define optimal control) for post-prandial blood glucose was used for RBG16. If both FBG and RBG were known, FBG was used for determining optimal glycemic control. ‘Glycemic control during TB treatment’ was classified as ‘optimal’ (if both baseline and end IP values were optimal), ‘not optimal’ (if baseline and/or end IP were not optimal) or ‘missing’ (if both baseline and end IP values were missing; or if one of the two values were missing and the other was optimal). TB treatment outcomes (both end IP and end treatment) were reclassified as favourable or unfavourable (Box 1).\n\nEthical approval was obtained from the Institutional Ethics Committee of the Resource Group for Education and Advocacy for Community Health, Chennai, Tamil Nadu, India, and the Ethics Advisory Group of the International Union Against Tuberculosis and Lung Disease, Paris, France. As the study involved review of existing patient records, a waiver for informed consent was sought and approved by the respective ethics committees. We conducted the study after receiving approval from the State Tuberculosis Officer.\n\n\nResults\n\nThere were 438 patients with TB-DM: 270 from Tiruvallur, 114 from Madurai and 54 from Tiruppur. The baseline socio-demographic and clinical characteristics have been summarized in Table 1. Extracted values for each participant are available as Underlying data17.\n\nTB-DM, tuberculosis and diabetes mellitus; LFU, loss to follow-up; RNTCP, Revised National Tuberculosis Control Programme.\n\nBlood glucose records (either FBG or RBG) were available for 94% at baseline, 79% at the end of IP and 59% at the end of CP. However, FBG at baseline, at the end of IP and at the end of CP were each available for less than 20% patients. RBG at baseline, at the end of IP and at the end of CP were each available for 76%, 60% and 43%, respectively (Table 2).\n\nRNTCP, Revised National Tuberculosis Control Programme; RBG, random blood glucose; FBG, fasting blood glucose; PPBG, 2-hour post prandial blood glucose. *RBG, FBG, PPBG cut off of 130, 180 and 180 mg/dl respectively; **row percentage; ^FBG preferred over RBG if available; #‘optimal’ (both baseline and IP status optimal), ‘not optimal’ (any one status was not optimal) and missing (both were missing).\n\nGlycemic control status was known for 94% (412/438) patients at baseline and for 91% (400/438) during TB treatment. Among those with known glycemic status, glycemic control was not optimal in 77% (316/412) patients at baseline and in 84% (337/400) during TB treatment.\n\nIn total, 46 patients (10%) had unfavourable end IP outcomes (5% sputum non-conversion, 2% death and 3% not evaluated); 21 patients (5%) had unfavourable end treatment outcomes (1% treatment failed, 3% deaths and 1% not evaluated).\n\nWe decided against assessing factors associated with glycemic control during TB treatment and association between glycemic control and TB treatment outcomes because glycemic control assessment, if any, was based mostly on RBG values, which are unreliable.\n\n\nDiscussion\n\nThis operational research had two key findings. First, though blood glucose values were available during baseline and end IP in four-fifths of patients, most of these values were RBG. FBG was not consistently recorded during baseline, the end of IP and the end of CP (as per programme guidelines). Routinely, patients with TB screened at peripheral health centers using RBG and/or FBG are referred to higher institutions for clinical confirmation. Even if FBG is not measured at peripheral health center at baseline, it ought surely to be measured at the higher center. This FBG at baseline, which is at least expected for all newly diagnosed DM patients at the time of registration for TB treatment, is not reflected in the RNTCP records (only 19% have baseline FBG records). There is one possible reason for this. During data collection, we found that RBG was predominantly measured and recorded as it was the blood glucose test mentioned in the TB treatment card. In a study conducted in 18 randomly selected districts of India, poor documentation of DM status among TB patients from marginalised and vulnerable populations (2016–2017) was observed18.\n\nSecond, a large number of patients did not have optimal glycemic control at baseline and during TB treatment. Though these findings are mostly based on RBG that was available, our figures are comparable with findings from another study by Mahishale et al.19 at a private tertiary care facility in Karnataka, India, where poor glycemic control (HbA1C ≥7%) at baseline was seen in 67% (423/630) patients. Another study in India from programme setting by Nandakumar et al.20 identified 667 patients with TB-DM, 36% cases had known (minimum three blood glucose values available, at least one month apart) diabetic control status during the treatment, of which 43% cases were under glycemic control. However, the cut offs (FBG <100 mg/dl and RBG <140) used were lower than the ones used by us. The cut-off values used by us were as per international recommendations21.\n\nThis study has two key policy implications. First and foremost, systematic recording of FBG should be improved. Though HbA1c has been described as the gold standard for glycemic control, as per programme recommendations FBG should be systematically recorded in the treatment cards (considering the lack of access to HbA1c in programme settings)11,12. One potential solution for this is an incremental administrative change that may easily be incorporated. Newly printed treatment cards replacing RBG with FBG would suffice.\n\nSecond, poor glycemic control indicates that there is an urgent need to improve glycemic management among patients with DM. Tamil Nadu has a well-established primary health care system where DM treatment is provided through the peripheral health centres. Existing DM treatment guidelines, if any, should be reviewed to ensure that management is as per international recommendations16,22–25. Poor glycemic control has been reported among patients with DM in Asian countries26,27. Programmatic interventions to maintain a line list of registered patients with DM (in line with TB and HIV programmes) may be considered. This will ensure proper tracking of DM treatment outcomes28. Integrating care of communicable and non-communicable diseases (such as TB and DM, respectively) has also been recommended in literature29.\n\nTo our knowledge, this is the first study which has systematically reported the recording of blood glucose values among patients with TB-DM at various phases of TB treatment in programmatic setting in India. We found two similar studies; however, they did not review glycemic control measurement as per the recent 2016 programme recommendations19,20. This study was conducted in the programme setting; therefore, findings are reflective of reality on the ground. Double data entry and validation minimized data entry errors.\n\nThere was a major limitation. As RBG was the most frequent type of blood glucose value recorded, glycemic control reported should be interpreted with caution. For the same reason analysis of the association between glycemic control and TB treatment outcome would have been meaningless and hence, was not performed. RBG is a screening test for diabetes and is not recommended for monitoring glycemic control21.\n\n\nConclusions\n\nIn this operational research, involving patients with TB-DM in programmatic setting from South India, we found FBG was not consistently recorded as per programme guidelines (at baseline, the end of intensive phase and the end of continuation phase of TB treatment). The glycemic control status (mostly based on random blood glucose instead of fasting blood glucose or HbA1c) was not reliable to perform a meaningful analysis of its effect on TB treatment outcomes. This calls for an urgent review of the TB-DM collaborative services to improve the recording of glycemic control during TB treatment.\n\n\nData availability\n\nFigshare: Data set for Gurukartick study 2016. https://doi.org/10.6084/m9.figshare.990218017.\n\nFile ‘Dataset_v2’ contains all de-identified variables extracted for this study, alongside a codebook explaining all abbreviations and 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": "Author contributions\n\n\n\nPrincipal Investigator: JG; Mentors: HDS, AGJ, OPA; Senior authors: RA, NK; Conception and design: JG, HDS, AGJ; Data collection: JG, LM, MMS, DD, GM; Development of data capture tool: JG, OPA, HDS; Data analysis and interpretation: JG, HDS, AGJ, OPA, RA, NK; Preparation of first draft; JG, HDS; Critical review and approval of final draft: all authors.\n\n\nAcknowledgements\n\nWe would like to acknowledge Mr. Rajeswaran, Mrs. Sheela Augusteen, Mr.Dilesh and our urban co-ordinators Ms. Rubini Raghunathan, Mr. Srinivasan, Mr. John Paul Jeeva, Mr. Pandiaraj, and Mr. Saravanan (MMCF NGO) who helped us in data collection. We do thank the Senior Treatment Supervisors and District Program Managers for their co-operation and support.\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\nInternational Diabetes Federation: IDF Diabetic Atlas. 8th edition. [cited 2018 Jun 14], 2017; 147. Reference Source\n\nWorld Health Organization (WHO): Global tuberculosis report 2018. Geneva, Switzerland; 2018. Reference Source\n\nKapur A, Harries AD: The double burden of diabetes and tuberculosis - Public health implications. Diabetes Res Clin Pract. 2013; 101(1): 10–19. PubMed Abstract | Publisher Full Text\n\nWorld Health Organization (WHO): Global report on Diabetes. Geneva, Switzerland; 2016. Reference Source\n\nDooley KE, Chaisson RE: Tuberculosis and diabetes mellitus: convergence of two epidemics. Lancet Infect Dis. 2009; 9(12): 737–46. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWorld Helath Organization (WHO) and The International Union Against Tuberculosis and Lung Disease (The Union): Collaborative Framework for Care and Control of Tuberculosis and Diabetes. Geneva, Switzerland; 2011. PubMed Abstract\n\nBaker MA, Harries AD, Jeon CY, et al.: The impact of diabetes on tuberculosis treatment outcomes: A systematic review. BMC Med. 2011; 9: 81. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFaurholt-Jepsen D, Range N, PrayGod G, et al.: Diabetes is a strong predictor of mortality during tuberculosis treatment: a prospective cohort study among tuberculosis patients from Mwanza, Tanzania. Trop Med Int Health. 2013; 18(7): 822–9. PubMed Abstract | Publisher Full Text\n\nWorld Diabetes Federation; International Union Against Tuberculosis and Lung Disease (The Union): The looming co-epidemic of TB-diabetes: a call to action. 2014; 20, [cited 2018 Jun 15]. Reference Source\n\nIndia Tuberculosis-Diabetes Study Group: Screening of patients with tuberculosis for diabetes mellitus in India. Trop Med Int Health. 2013; 18(5): 636–45. PubMed Abstract | Publisher Full Text\n\nRevised National Tuberculosis Control Programme (RNTCP) and National Programme for Prevention and Control of Cancer Diabetes Cardiovascular diseases and Stroke (NPCDCS): National framework for joint TB-Diabetes collaborative activities. New Delhi, India; 2017. Reference Source\n\nRevised National Tuberculosis Control Programme (RNTCP): Technical and operational guidelines for tuberculosis control in India. New Delhi, India; 2016. Reference Source\n\nShewade HD, Jeyashree K, Mahajan P, et al.: Effect of glycemic control and type of diabetes treatment on unsuccessful TB treatment outcomes among people with TB-Diabetes: A systematic review. PLoS One. 2017; 12(10): e0186697. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMinistry of Home Affairs, Government of India: Office of Registrar General and Census Commissioner. Census of India. 2011; [cited 2016 May 27]. Reference Source\n\nWorld Health Organization (WHO): Definitions and Reporting Framework for Tuberculosis- 2013 revision (updated December 2014). Geneva, Switzerland; 2014. Reference Source\n\nAmerican Diabetic Association: 6. Glycemic Targets. Diabetes Care. 2017; 40(Suppl 1): S48–S56. PubMed Abstract | Publisher Full Text\n\nShewade H: Underlying data. figshare. 2019. http://www.doi.org/10.6084/m9.figshare.9902180\n\nShewade HD, Gupta V, Satyanarayana S, et al.: Patient characteristics, health seeking and delays among new sputum smear positive TB patients identified through active case finding when compared to passive case finding in India. PLoS One. 2019; 14(3): e0213345. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMahishale V, Avuthu S, Patil B, et al.: Effect of Poor Glycemic Control in Newly Diagnosed Patients with Smear-Positive Pulmonary Tuberculosis and Type-2 Diabetes Mellitus. Iran J Med Sci. 2017; 42(2): 144–151. PubMed Abstract | Free Full Text\n\nNandakumar KV, Duraisamy K, Balakrishnan S, et al.: Outcome of Tuberculosis Treatment in Patients with Diabetes Mellitus Treated in the Revised National Tuberculosis Control Programme in Malappuram District, Kerala, India. PLoS One. 2013; 8(10): e76275. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAmerican Diabetic Association: Standards of Medical Care in Diabetes. Diabetes Care. 2016; 39(1): S112. Reference Source\n\nAmerican Diabetic Association: Standards of Medical Care in Diabetes-2017 Abridged for Primary Care Providers. Clin Diabetes. 2017; 35(1): 5–26. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAmerican Diabetes Association: Comprehensive Medical Evaluation and Assessment of Comorbidities. Diabetes Care. 2017; 40(Suppl 1): S25–S32. PubMed Abstract | Publisher Full Text\n\nAmerican Diabetes Association: 4. Lifestyle Management. Diabetes Care. 2017; 40(Suppl 1): S33–S43. PubMed Abstract | Publisher Full Text\n\nAmerican Diabetes Association: 8. Pharmacologic Approaches to Glycemic Treatment. Diabetes Care. 2017; 40(Suppl 1): S64–S74. PubMed Abstract | Publisher Full Text\n\nJi LN, Lu JM, Guo XH, et al.: Glycemic control among patients in China with type 2 diabetes mellitus receiving oral drugs or injectables. BMC Public Health. 2013; 13: 602. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChuang LM, Tsai ST, Huang BY, et al.: The status of diabetes control in Asia--a cross-sectional survey of 24 317 patients with diabetes mellitus in 1998. Diabet Med. 2002; 19(12): 978–985. PubMed Abstract | Publisher Full Text\n\nHarries AD, Kumar AM, Karpati A, et al.: Monitoring treatment outcomes in patients with chronic disease: lessons from tuberculosis and HIV/AIDS care and treatment programmes. Trop Med Int Health. 2015; 20(7): 961–964. PubMed Abstract | Publisher Full Text\n\nHarries AD, Kumar AM, Satyanarayana S, et al.: Communicable and non-communicable diseases: connections, synergies and benefits of integrating care. Public Health Action. 2015; 5(3): 156–7. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "57043",
"date": "09 Dec 2019",
"name": "Madhavi Bhargava",
"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\nIt is a well written manuscript. Few suggestions:\n\nFor the 3rd paragraph of the introduction, reference 9: The looming co-epidemic document (https://www.theunion.org/what-we-do/publications/technical/low-resolution/25383_TCB_Report_LR.pdf) mentions figures from TN from a 2012 study (see page 10 of this document). There is one study from Tamil Nadu in which diabetes was documented in 163 (14%) of 1131 TB patients (10.4103/ijcm.IJCM_179_16). Consider using this for more recent, same state figures.\n\nBox 1 seems redundant as the authors decide against analyzing the outcomes based on RBGs. The information available in the results section, fourth paragraph is adequate and self-explanatory.\n\nSince this is a record review, it will be good to know the denominator of all TB patients diagnosed in all these three tuberculosis units of which, the authors found 438 patients with TB-DM. This will add important information for the program, as well as future 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? 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": "5591",
"date": "11 Jun 2020",
"name": "Hemant Deepak Shewade",
"role": "Author Response",
"response": "REVIEWER: Madhavi Bhargava REVIEWER For the 3rd paragraph of the introduction, reference 9: The looming co-epidemic document (https://www.theunion.org/what-we-do/publications/technical/low-resolution/25383_TCB_Report_LR.pdf) mentions figures from TN from a 2012 study (see page 10 of this document). There is one study from Tamil Nadu in which diabetes was documented in 163 (14%) of 1131 TB patients (10.4103/ijcm.IJCM_179_16). Consider using this for more recent, same state figures. AUTHOR RESPONSE Thank you for the comment. We have included this reference in the third paragraph of the introduction. REVIEWER Box 1 seems redundant as the authors decide against analyzing the outcomes based on RBGs. The information available in the results section, fourth paragraph is adequate and self-explanatory. AUTHOR RESPONSE Thank you for the comment. We agree that we have not analysed the outcomes based on glycemic control. However, as we have presented the overall treatment outcomes in the results section, we would request that the box 1 (operational definition of treatment outcomes) be retained. We hope this is fine. REVIEWER Since this is a record review, it will be good to know the denominator of all TB patients diagnosed in all these three tuberculosis units of which, the authors found 438 patients with TB-DM. This will add important information for the program, as well as future studies. AUTHOR RESPONSE Thank you for the comment. We have included this information (438 TB-DM out of 5115 TB patients. Please see first line of results narrative."
}
]
},
{
"id": "62134",
"date": "19 May 2020",
"name": "Aniwada Elias Chikee",
"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 work is good and well-articulated. The authors could not found previous work that hampered testing for association as well as discussion. This is genuine and tenable.\n\nIf possible, the authors to replicate the study using longitudinal or prospective challenge in another study to overcome the limitation of RBG against FBG. This will help in appreciation of difficult associated with TB-DM co-morbidity both for patients and TB control programme\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "5524",
"date": "20 May 2020",
"name": "Hemant Deepak Shewade",
"role": "Author Response",
"response": "Dear Aniwada, Thank you for approval and constructive comments. Your suggestions for a longitudinal study in the future are well taken. With regards, Hemant"
}
]
}
] | 1
|
https://f1000research.com/articles/8-1725
|
https://f1000research.com/articles/9-259/v1
|
14 Apr 20
|
{
"type": "Research Article",
"title": "Length–weight relationships and condition factors of mullets Liza macrolepis and Moolgarda engeli (Pisces: Mugilidae) harvested from Lambada Lhok waters in Aceh Besar, Indonesia",
"authors": [
"Derita Yulianto",
"Indra Indra",
"Agung Setia Batubara",
"Deni Efizon",
"Firman M. Nur",
"Syamsul Rizal",
"Roza Elvyra",
"Zainal A. Muchlisin",
"Derita Yulianto",
"Indra Indra",
"Agung Setia Batubara",
"Deni Efizon",
"Firman M. Nur",
"Syamsul Rizal",
"Roza Elvyra"
],
"abstract": "Background: The mullets fish Liza macrolepis and Moolgarda engeli are predominant in the Lambada Lhok waters in Aceh province. At present, no scientific report on this species in Aceh waters is available. Therefore, the objective of the present study is to examine the growth pattern and condition factor of the species of mullets L. macrolepis and M. engeli harvested from the aforementioned coastal waters. Methods: The sampling was done in three locations in the Lambada Lhok waters from July to November 2018. The fish were captured using gillnets from 6:00 AM to 3:00 PM four times a month for five months. A total of 242 L. macrolepis and 109 M. engeli were used for the analysis. The growth pattern was analyzed using linear allometric model; then, two condition factors, Fulton’s and relative weight, were calculated. Results: The study revealed a b value of 2.49 for the male L. macrolepis and 1.81 for the female. The b value was 3.22 for the male M. engeli and 3.41 for the female. The b value of the fish was higher during the dry season. The Fulton’s condition factor of the male L. macrolepis was 1.19, and that of the female was 1.19. The relative condition factor of this species was 100.11 and 100.01 for males and females, respectively. The Fulton condition factor of male M. engeli was 1.05 and that of the female was 1.06. The relative weight condition factors were 101.08 and 100.61 for the male and female, respectively.\n\nConclusions: The growth pattern of M. engeli tends to be isometric, whereas that of L. macrolepis has a negative allometric growth pattern. The condition factors indicate that the Lambada Lhok waters are still in good condition and support the growth of the mullets, but M. engeli is more adaptable than L. macrolepis.",
"keywords": [
"Growth pattern",
"Relative weight condition factor",
"Fulton’s condition factor",
"Linear allometric model"
],
"content": "Introduction\n\nMullets (Mugilidae) represent a family of euryhaline fish that can tolerate a wide range of salinity1,2. This fish is frequently found in marine environments, brackish and fresh water2–7. To date, a total of 30 genus belonging to 78 species of mullets have been described worldwide8,9. A total of 21 species of mullets have been reported in Indonesian waters, and among them, four species have been recorded in the waters of Aceh province; these are Liza melinoptera, Mugil chepalus, Valamugil cunnesius, and V. speigleri4,10,11. Our previous study recorded three additional mullets from Aceh waters, namely, L. macrolepis, Crenimugil crenilapis, and Moolgarda engeli (Yulianto, thesis in preparation), which accounted for a total of seven species of mullets in the Aceh region. These additional species are commonly found in Lambada Lhok, Aceh Besar district close to Banda Aceh City, the capital of Aceh province, Indonesia. Moreover, our field observation showed that L. macrolepis and M. engeli were the predominant species among other mullets in this area.\n\nThe coastal area of Lambada Lhok has a mangrove forest; however, the forest area has been significantly decreased due to tsunami disaster in 2004, land conversion for settlement and aquaculture ponds. The other potential threats are pollution from domestic waste, fishing port, and tourism activities12,13. Mullets are a species of shoaling and schooling fish commonly found in river mouths for feeding14,15, which then subsequently migrate to deep waters for spawning16. Therefore, these fish are highly susceptible to exposure to pollution from coastal areas; for instance, Chelon subviridis from Donan River estuary, Central Java, have been contaminated by cadmium and copper17. A similar finding has been reported in M. cephalus from the Ligurian Sea in Italy18. In addition, L. macrolepis and M. engeli have been harvested intensively by local fishermen, thereby increasing the pressure on these fish. Thus, research related to bioecology as basic information is crucial in planning an effective conservation strategy. The two important pieces of information are length–weight relationships (LWRs) and growth pattern and condition factors.\n\nThe study of the LWRs and condition factors has become popular and is therefore commonly conducted by fish biologists19. The objectives of LWRs study are to determine the specific weight and length variations of fish individually or the population a whole to determine the age, obesity status, health, productivity, and physiological conditions, including gonadal development20,21. LWRs analysis is also useful to estimate the fish condition or plumpness index, which is an important variable in the evaluation of the health conditions of fish populations or individuals22–24. The condition factor indicates the biological and physical conditions of fish and its fluctuations by interaction among feeding condition, food reserves, and parasite infestation25–27.\n\nSeveral studies on LWRs and condition factor of mullets have been conducted, such as, that on M. dussumieri in Ujung Pangkah, East Jawa, Indonesia14; on LWRs in L. macrolepis from Indian waters28,29, and on L. macrolepis from Taiwanese waters30. In addition, the study of LWRs in several species of mullets in Aceh waters, Indonesia has been reported by Mulfizar et al.31 in M. chepalus from the waters of Kuala Gigieng, Aceh Besar, and by Muttaqin et al.32 in the same species from Madat waters in East Aceh. However, no study has been published on L. macrolepis and M. engeli. Thus, the objectives of the present study are to analyze LWRs and condition factor of these two types of mullet harvested from Lambada Lhok waters.\n\n\nMethods\n\nThe sampling was conducted from July to November 2018 in the Lambada Lhok estuary, Aceh Besar regency in Aceh province, Indonesia (Figure 1). The location is a small river mouth and deforested mangrove areas and fishing port (5°36'57.6\" N, 95° 23'25.6\" E). The sampling was done purposively at these locations as they were easy to access and fish were reported to be present. Sampling was conducted four times a month for five months. The target species in this study were determined based on preliminary survey and observations on the composition of local fishermen catches, where Liza macrolepis and Moolgarda engeli were the dominant species caught.\n\nThe fish was caught using gillnets with a mesh size of 2.0 inch. The gillnets were set up in the waters for 4 h (06.00 AM –03.00 PM) and were monitored in 30 min intervals. The sampled fish was washed, euthanized with cold water 4 °C for 5 min. This euthanize method was chosen as it is easy to apply, non-toxic and inexpensive. Then the fish was preserved temporarily in crushed ice in a styrofoam box, and then transported to a laboratory for further analysis. Other species of fish caught were separated from the mullets, and released back to the waters if still live, but the fish taken for consumption if they died during sampling. All efforts were made to ameliorate harm to the animals by complying to the guidelines of ethics animal use in research of Syiah Kuala University.\n\n\nField observations\n\nDuring this process, the weather condition, tides, and water turbidity are also observed visually during the sampling.\n\nThe length-weight relationships were calculated to predict the growth pattern of the fish. A total of 242 L. macrolepis and 109 M. engeli were measured for total length to the nearest mm using a digital caliper (Mitutoyo CD-6CS, error: 0.05 mm) and for body weight to the nearest mg using a digital balance (ION EPS05, error: 0.1 mg). A linear allometric model (LAM) was used to calculate parameters a and b values based on the work of De-Robertis and Williams33 and Muchlisin et al.22 as follows:\n\nwhere W is total body (g), L is total length (mm), a is regression intercept, b is regression coefficient, σ is residual variation of the LAM, and 0.5 is correction factor. The growth pattern of the fish is divided into three categories; isometric when the b value is equal to 3, negative allometric when the b value lower than 3, and positive allometric when the b value is higher than 3.\n\nThe condition factor indicates the conditions of the fish and water, and their interactions. Two condition factors, namely, relative weight condition factor (Wr) and Fulton’s condition factor (K) were analyzed in this study. The relative weight condition factor of 100 indicates a balance between prey and predator, while if the Wr higher than 100 indicates a surplus of prey, and it vice versa. Based on the work of Rypel and Richter34, the relative weight condition factor was calculated as follows: Wr = (W/Ws) × 100, where Wr is the relative weight condition factor, W is body weight of fish from direct measurement, Ws is the prediction weight of fish, and Ws = aLb.\n\nBased on the work of Okgerman35, Fulton’s condition factor was calculated as follows:\n\nwhere K is the Fulton’s condition factor, W is the body weight of fish from direct measurement (g), and L is the total length of fish from direct measurement (mm). According to Morton and Routledge36, a fish population is in good condition when the K value is higher than 1.\n\nThe raw data of total length and body weight were processed using a Microsoft Excel (Microsoft Office 365). The data were presented as tables and figures, and then the data were analyzed descriptively through comparison with related reports, theories, and field observations.\n\n\nResults\n\nThe field observation of the catch composition showed that Liza macrolepis and Moolgarda engeli were predominant. A total of 242 L. macrolepis and 109 M. engeli were sampled and measured in the study. The length of male L. macrolepis ranged from 141.4 – 202.1 mm (164.8 ± 15.03 mm), and ranged from 129.2–185.4 mm (159.1 ± 12.66 mm) for females. The body weight of male L. macrolepis ranged from 34.7 g to 89.6 g (54.1 ± 13.3 g in average), and 28.8 g to 75.13 g (47.9 ± 9.52 g in average) in females. The length of the male M. engeli ranged from 109.9–188.5 mm (161.9±20.83 mm) and 116.5–182.3 mm (154.1 ± 18.94 mm) for females. The body weight of the males was 13.6 – 108.5 gram (47.6±19.3 gram), and that of the females was 14.2–75.1 gram (41.4 ± 16.43 gram). Raw data are available as underlying data37.\n\nThe results of the LWRs analysis on L. macrolepis showed that the male fish had a b value of 2.49 with a correlation coefficient of 0.93, and the female fish had a b value of 1.81 and a correlation coefficient of 0.82 (Figure 2a and 2b). Therefore, the male and female L. macrolepis displayed negative growth patterns, and a moderate correlation between body weight and total length of the fish. The results of LWRs analysis of male M. engeli revealed that the average b value was 3.22 with a coefficient correlation of 0.89. The female M. engeli had an average b value of 3.41 with a coefficient correlation of 0.93 (Figure 3a and 3b). These data indicate that the male and female M. engeli have a positive allometric growth pattern, and a strong correlation between body weight and total length.\n\nThe length-weight relationship of Liza macrolepis based on linear allometric model (a) male, (b) female; Comparison of observed and predicted growth for male (c), and female (d) of Liza macrolepis. R2 - determination coefficient, r - correlation coefficient, N - number of fish sampled.\n\nThe length-weight relationship of Moolgarda engeli based on linear allometric model (a) male, (b) female; Comparison of observed and predicted growth for male (c), and female (d) of Moolgarda engeli. R2 - determination coefficient, r - correlation coefficient, N - number of fish sampled.\n\nBased on sampling season, the average b value of L. macrolepis (male and female) was 2.78 during the dry season and 2.28 during the rainy season (Figure 4a). The b value of M. engeli was 3.42 during the dry season and 2.48 during the rainy season (Figure 4b). These data indicate that the b value is lower during the rainy season for both species. The scatter plots of predicted standard weight for respective observed length, as calculated from the composite of length–weight regression, are presented in Figure 2c and 2d and Figure 3c and d. The regression models show a difference between the observed and predicted growth patterns in both species.\n\nThe length-weight relationship of Liza macrolepis (a) during dry season (b) during rainy season and Moolgarda engeli (c) during dry season (d) during rainy season. R2 - determination coefficient, r - correlation coefficient, N - number of fish sampled.\n\nCondition factors. The results showed that the male L. macrolepis had a Fulton’s condition (K) factor of 1.19, and relative weight condition factor (Wr) of 100.11; while the females had a Fulton’s condition factor of 1.19, and relative weight condition factor of 100.01. In addition, male M. engeli had a Fulton’s condition factor of 1.05, and relative weight condition factor of 101.08; whereas the females has Fulton’s condition factor of 1.06, and relative weight condition factor of 100.61 (Table 1). Based on sampling season, the Fulton’s condition of L. macrolepis during the dry season was 1.22 and 100.49 for the relative weight; during the rainy season these were 1.19 and 101.74, respectively. In addition, the K and Wr values of M. engeli during the dry season were 1.03 and 102.09, respectively; during the rainy season these were 1.09 and 100.47, respectively (Table 2).\n\n\nDiscussion\n\nThe study revealed that male and female L. macrolepis had negative allometric growth patterns. However, the b value of the females was less than that of the male. The b value of the male M. engeli showed a positive allometric growth pattern. Based on these growth pattern data, the study indicated that M. engeli grows better than L. macrolepis, thereby indicating that M. engeli is more adaptable to the environmental condition of Lambada Lhok waters. Furthermore, the field observation on the catch composition of the fishermen showed that M. engeli was also predominant. The Fulton’s condition factor showed a slight difference in K value between the male and female for both species, where the K value was higher than 1. According to Morton and Routledge36, a fish population is in good condition when the K value is higher than 1. The study showed that the K value of L. macrolepis ranged from 1.16 to 1.22, and 1.03 to 1.09 for M. engeli; therefore, both populations are in good condition, in dry and rainy season, respectively. In addition, the relative weight condition factor of both species is close to 100, indicating a balance between prey and predator20. These results show that these waters provide a sufficient food source for these species. The relative weight condition factor also corresponds to fish health conditions, stock estimates, and management levels23,35,36,38,39. Therefore, the Lambada Lhok waters provide sufficient food sources for mullets.\n\nThe results also showed differences in growth patterns during the dry and rainy seasons, and that the fish grew better during the dry season. The probable reason is that the waters are clear and a maximum rate of sunlight penetrates into the waters, triggering the growth of phytoplankton and algae. Algae is a primary food item for the mullets40–43. By contrast, turbidity and currents were higher during the rainy season44, and thereby presume to inhibit the growth of phytoplankton and algae as important food item for mullets. A similar phenomenon was reported by Chu et al.30, who found a negative growth pattern in L. macrolepis in Taiwan during winter, and an isometric growth pattern during summer and spring. Moreover, Sandhya and Shameem29 observed a negative growth pattern in L. macrolepis in polluted waters, in contrast to an isometric growth pattern in unpolluted waters. According Muchlisin et al.22, besides being affected by the environmental factors, the growth pattern of fish is also influenced by fish behavior; for example, the fish that were active swimmer had a lower b value than those that were passive swimmers45–47.\n\nThe average correlation coefficients of L. macrolepis were 0.93 and 0.89 in females and males, respectively, whereas M. engeli had a correlation coefficient of 0.89 for males and 0.93 for females. In general, the correlation coefficients of L. macrolepis and M. engeli tend to be similar (above 75%), indicating a strong correlation between total length and body weight. The determination coefficients of L. macrolepis were 0.85 and 0.65 for the male and female fish, respectively, which means that approximately 65%–85% of total variants can be explained by the model, while M. engeli had a value of 0.90 and 0.94 for males and females, respectively, indicating that 90%–94% of variants can be explained by the model.\n\n\nConclusion\n\nThe results of this study showed that L. macrolepis had a negative growth pattern, whereas M. engeli had a positive allometric growth pattern. These growth patterns were better during the dry season for both species. Condition factors showed that the environmental condition of Lambada Lhok remains suitable for the growth of the mullets, and the density of prey and predator is balanced.\n\n\nData availability\n\nFigshare: Raw Data of Liza macrolepis.xlsx. https://doi.org/10.6084/m9.figshare.12028062.v137\n\nThis project contains the following underlying data:\n\n- Raw Data of Liza macrolepis.xlsx (Raw data of sampled Liza macrolepis)\n\n- Raw Data of Moolgarda engeli.xlsx (Raw data of Moolgarda engeli)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
"appendix": "Acknowledgments\n\nAll members of Ichthyos Research Group are acknowledged.\n\n\nReferences\n\nChang CW, Iizuka Y: Estuarine use and movement patterns of seven sympatric Mugilidae fishes: the Tatu Creek estuary, central western Taiwan. Estuarine, Coastal and Shelf Science. 2012; 106: 121–126. Publisher Full Text\n\nMinos G, Imsiridou A, Economidis PS: Liza haematocheilus (Pisces, Mugilidae) in Northern Aegean Sea. In: XII European Congress of Ichthyology in Cavtat (Dubrovnik), Croatia. 2007. Reference Source\n\nNzioka RM, Marine K: Fish culture in brackish water. Post Kenya. 1980; 5(2): 22–25. Reference Source\n\nMuchlisin ZA, Siti-Azizah MN: Diversity and distribution of freshwater fishes in Aceh Waters, Northern Sumatera, Indonesia. International Journal of Zoological Research. 2009; 5(2): 62–79. Publisher Full Text\n\nMićković B, Nikčević M, Hegediš A, et al.: Mullet fry (Mugilidae) in coastal waters of Montenegro, their spatial distribution and migration phenology. Archives of Biological Sciences. 2010; 62(1): 107–114. Publisher Full Text\n\nRegner S, Gacic Z, Krpo-Cetkovic J: Mullet fry (Mugilidae) in coastal waters of Montenegro, their spatial distribution and migration phenology. Archives of Biological Sciences. 2010; 62(1): 107–114. Publisher Full Text\n\nTuran C, Gürlek M, Ergüden D, et al.: Systematic status of nine mullet species (mugilidae) in the Mediterranean sea. Turkish Journal of Fisheries and Aquatic Sciences. 2011; 11(2): 315–321. Reference Source\n\nDurand JD, Shen KN, Chen WJ, et al.: Systematics of the grey mullets (Teleostei: Mugiliformes: Mugilidae): molecular phylogenetic evidence challenges two centuries of morphology-based taxonomy. Mol Phylogenet Evol. 2012; 64(1): 73–92. PubMed Abstract | Publisher Full Text\n\nNelson JS: Fishes of the world. USA: John Wliey and Sons, Inc. 2007. Reference Source\n\nBatubara AS, Muchlisin ZA, Thamren MY, et al.: Check list of marine fishes from Simeulue Island waters, Aceh Province, Indonesia. Aceh Journal of Animal Science. 2017; 2(2): 77–84. Publisher Full Text\n\nBatubara AS, Abdan M, Muhammad Z, et al.: Peripheral fishes in the Estuary of Simeulue Island, Indonesia. IOP Conference Series: Earth and Environmental Science. IOP Publishing. 2018; 216: 012011. Publisher Full Text\n\nZainabun, Djuita S, Diba F: Development of region of zoning mangrove rehabilitate land of post tsunami in District of Baitussalam sub-Province of Aceh Besar. Agrista. 2011; 15(1): 10–14.\n\nLiew SC, Kam SP, Chen P, et al.: Mapping tsunami-affected coastal aquaculture areas in Northern sumatra using high resolution satellite imagery. Asian Association on Remote Sensing - 26th Asian Conference on Remote Sensing and 2nd Asian Space Conference. ACRS. 2005; 1: 116–120. Reference Source\n\nSulistiono S, Arwani M, Aziz KA: Growth of mullet, Mugil dussumieri in Ujung Pangkah, East Java. Jurnal Iktiologi Indonesia. 2001; 1(2): 39–47. Reference Source\n\nGuinea J, Fernandez F: Morphological and biometrical study of the gill rakers in four species of mullet. Journal of Fish Biology. 1992; 41(3): 381–397. Publisher Full Text\n\nAl-Ghiffary GA, Rahardjo MF, Zahid A, et al.: Diet composition and niche breadth of mullet Chelon subviridis (Valenciennes, 1836) and Moolgarda engeli (Bleeker, 1858) in Pabean Bay Indramayu Subdistrict, West Java Province. Jurnal Iktiologi Indonesia. 2018; 18(1): 41–56.Publisher Full Text\n\nPrastyo Y, Batu DTL, Sulistiono S: Heavy metal contain Cu and Cd on the mullet in the estuary of Donan River, Cilacap, Central Java. Jurnal Pengolahan Hasil Perikanan Indonesia. 2017; 20(1): 18–27. Publisher Full Text\n\nSquadrone S, Prearo M, Gavinelli S, et al.: Heavy metals in Mugil cephalus (Mugilidae) from the Ligurian Sea (north-west Mediterranean, Italy). Food Addit Contam Part B Surveill. 2013; 6(2): 134–138. PubMed Abstract | Publisher Full Text\n\nFroese R: Cube law condition factor and weight–length relationships: history, meta-analysis and recommendations. Journal of Applied Ichthyology. 2006; 22(4): 241–253. Publisher Full Text\n\nBlackwell BG, Brown ML, Willis DW: Relative weight (Wr) status and current use in fisheries assessment and management. Reviews in Fisheries Science. 2000; 8(1): 1–44. Publisher Full Text\n\nMuchlisin ZA, Batubara AS, Azizah MNS, et al.: Feeding habit and length weight relationship of keureling fish, Tor tambra Valenciennes, 1842 (Cyprinidae) from the western region of Aceh Province, Indonesia. Biodiversitas. 2015; 16(1): 89–94. Publisher Full Text\n\nMuchlisin ZA, Musman M, Siti-Azizah MN: Length-weight relationships and condition factors of two threatened fishes, Rasbora tawarensis and Poropuntius tawarensis, endemic to Lake Laut Tawar, Aceh Province, Indonesia. Journal of Applied Ichthyology. 2010; 26(6): 949–953. Publisher Full Text\n\nMuchlisin ZA, Fransiska V, Muhammadar AA, et al.: Length-weight relationships and condition factors of the three dominant species of marine fishes caught by traditional beach trawl in Ulelhee Bay Banda Aceh City, Indonesia. Croatian Journal of Fisheries. 2017; 75(4): 104–12. Reference Source\n\nBatubara AS, Muchlisin ZA, Efizon D, et al.: Length-Weight Relationships and Condition Factors of the Naleh Fish, Barbonymus gonionotus.(Pisces, Cyprinidae) Harvested from Nagan Raya Waters, Indonesia. Vestnik Zoologii. 2019; 53(1): 75–82. Publisher Full Text\n\nDatta SN, Kaur VI, Dhawan A, et al.: Estimation of length-weight relationship and condition factor of spotted snakehead Channa punctata (Bloch) under different feeding regimes. SpringerPlus. 2013; 2(1): 436. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOscoz J, Campos F, Escala MC: Weight –length relationships of some fish species of the Iberian Peninsula. Journal of Applied Ichthyology. 2005; 21(1): 73–4. Publisher Full Text\n\nPerdana AW, Batubara AS, Nur FM: LWRs, condition factors and isopod parasites infection of the sumbo fish Selar crumenophthalmus (Pisces: Carangidae) in Lampulo fishing port, Banda Aceh, Aceh Province, Indonesia. IOP Conference Series: Earth and Environmental Science. 2019; 348(1): 012017. Publisher Full Text\n\nParida S, Karna SK, Pradhan SK, et al.: Length weight relationship and condition factor of Liza macrolepis (Smith, 1946) in Chilika Lagoon, Odisha, India. Journal of Global Biosciences. 2013; 2(5): 116–120. Reference Source\n\nSandhya V, Shameem U: Length-weight relationship and condition factor of Liza macrolepis (Smith) from polluted and unpolluted waters of Visakhapatnam. Indian Journal of Fisheries. 2003; 50(4): 543–546. Reference Source\n\nChu WS, Hou YY, Ueng YT, et al.: Length-weight relationship of largescale mullet, Liza macrolepis (Smith, 1846), off the southwestern coast of Taiwan. African Journal of Biotechnology. 2012; 11(8): 1948–1952. Reference Source\n\nMulfizar M, Muchlisin ZA, Dewiyanti I: Hubungan panjang berat dan faktor kondisi tiga jenis ikan yang tertangkap di perairan Kuala Gigieng, Aceh Besar, Provinsi Aceh. Depik. 2012; 1: 1–9. Reference Source\n\nMuttaqin Z, Dewiyanti I, Aliza D: Kajian hubungan panjang berat dan faktor kondisi ikan nila (Oreochromis niloticus) dan ikan belanak (Mugil cephalus) yang tertangkap di Sungai Matang Guru, Kecamatan Madat, Kabupaten Aceh Timur. Jurnal Ilmiah Mahasiswa Kelautan dan Perikanan Unsyiah. 2016; 1(1): 397–403. Reference Source\n\nDe-Robertis A, Williams K: Weight-length relationships in fisheries studies: the standard allometric model should be applied with caution. Transactions of the American Fisheries Society. 2008; 137(3): 707–719. Publisher Full Text\n\nRypel AL, Richter TJ: Empirical percentile standard weight equation for the Blacktail Redhorse. North American Journal of Fisheries Management. 2008; 28(6): 1843–1846. Publisher Full Text\n\nOkgerman H: Seasonal variations in the length-weight relationship and condition factor of rudd (Scardinius erythrophthalmus L.) in Sapanca Lake. International Journal of Zoological Research. 2005; 1(1): 6–10. Publisher Full Text\n\nMorton A, Routledge RD: Fulton’s condition factor: Is it a valid measure of sea lice impact on juvenile salmon? North American Journal of Fisheries Management. 2006; 26(1): 56–62. Publisher Full Text\n\nYulianto D, Batubara AS, Efizon D, et al.: Raw Data of Liza macrolepis.xlsx. figshare. Dataset. 2020. https://www.doi.org/10.6084/m9.figshare.12028062.v1\n\nAyoade AA, Ikulala AO: Length weight relationship, condition factor and stomach contents of Hemichromis bimaculatus, Sarotherodon melanotheron and Chromidotilapia guentheri (Perciformes: Cichlidae) in Eleiyele Lake, Southwestern Nigeria. Rev Biol Trop. 2007; 55(3–4): 969–977. PubMed Abstract | Publisher Full Text\n\nOgunola OS, Onada OA: Preliminary investigation of length –weight relationships and condition factors of two commercially important fish species (Mullet, Mugil cephalus (Linnaeus 1758) and Sardine, Sardinella maderensis. (Lowe 1838)) in Okrika creeks (Niger-Delta) of Nigeria. Regional Studies in Marine Science. 2017; 13: 54–58. Publisher Full Text\n\nBlaber SJ: The food and feeding ecology of Mugilidae in the St. Lucia lake system. Biological Journal of the Linnean Society. 1976; 8(3): 267–277. Publisher Full Text\n\nHARI D, Blay J, Yankson K: Food and feeding habits of grey mullets (Pisces: Mugilidae) in two estuaries in Ghana. West African Journal of Applied Ecology. 2005; 8(1): 1–13. Publisher Full Text\n\nFatema K, Omar WM, Isa MM: Identification of food and feeding habits of mullet fish, Liza subviridis (Valenciennes, 1836), Valamugil buchanani (Bleeker, 1853) from Merbok Estuary, Kedah, Malaysia. Journal of Life Sciences and Technologies. 2013; 1(1): 47–50. Reference Source\n\nLuther G: The food habits of Liza Macrolepis (Smith) and Mugil chepalus Linnaeus (Mugilidae). Indian Journal of Fisheries. 1962; 9(2): 604–626. Reference Source\n\nPurnawan S, Setiawan I, Marwatim M: Studi sebaran sedimen berdasarkan ukuran butir di perairan Kuala Gigieng, Kabupaten Aceh Besar, Provinsi Aceh. Depik. 2012; 1(1): 31–36. Publisher Full Text\n\nBulanin U, Masrizal M, Muchlisin ZA: Length-weight relationships and condition factors of the whitespotted grouper Epinephelus coeruleopunctatus. Bloch, 1790 in the coastal waters of Padang City, Indonesia. Aceh Journal of Animal Science. 2017; 2(1): 23–27. Publisher Full Text\n\nMachrizal R, Khaitul K, Nasution J, et al.: Distribution and length-weight relationships of Hilsa shad Tenualosa ilisha. in the Bilah River, Labuhanbatu Regency, North Sumatera Province, Indonesia. Aceh Journal of Animal Science. 2019; 4(1): 42–49. Publisher Full Text\n\nRamses R, Ismarti I, Syamsi F: Length-weight relationships and condition factors of four dominant fish caught by coral bubu trap on the west coast of Batam Island, Indonesia. Aceh Journal of Animal Science. 2020; 5(1): 1–10. Reference Source"
}
|
[
{
"id": "63616",
"date": "01 Jun 2020",
"name": "Esti Handayani Hardi",
"expertise": [
"Reviewer Expertise fish diseases",
"microbiology",
"aquaculture biotechnology"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe method of taking fish has not been clearly described in the Methods.\n\nThe water quality or environment condition has not been mentioned.\n\nMaybe, add the discussion about the relationship between the environment with the fishes' conditions.\n\nMany references were old references, need to add the new articles.\n\nThe conclusion part is not enough.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": [
{
"c_id": "5582",
"date": "16 Jun 2020",
"name": "Zainal Abidin Muchlisin",
"role": "Author Response",
"response": "1. Comment:The method of taking fish has not been clearly described in the Methods.Respond:We have mentioned that “The fish sampling was done purposively at these locations as they were easy to access and fish were reported to be present” (page 4)2. Comment:The water quality or environment condition has not been mentionedRespond:We have not recorded the water quality 3. Comment:Maybe, add the discussion about the relationship between the environment with the fishes' conditions.Respond:The study showed that the fish are growing well during the dry season, this is related to environmental conditions during this season that the turbidity was lower and therefore the sunlight can penetrate into the waters maximumly and trigger the phytoplankton (algae) growing well. The algae are the primary food for mullets. We have mentioned this condition the pages 10-11. 4. Comment:Many references were old references, need to add the new articles.Respond:Very limited reports on mullets were available, but we have added 4 related and up to date references (reference No. 45-48) to support the discussion 5. Comment:The conclusion part is not enough.Respond: We have extended the conclusion (on page 11)"
}
]
},
{
"id": "63617",
"date": "05 Jun 2020",
"name": "Rudy Agung Nugroho",
"expertise": [
"Reviewer Expertise Animal Physiology",
"Fish nutrition",
"Fish Conservation"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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: The title is sufficient and reflects the content of the study.\n\nAbstract: Well-constructed.\n\nIntroduction: Adequate.\n\nMaterials and Methods:\nPlease mention in the Time, site, and sampling or LWRs analyses section that male and female fish were measured/analysis for LWR separately. In the fields observation, it is stated that, the weather condition, tides, and water turbidity are also observed visually during the sampling. Is the observed visual data recorded? If yes, please mention in the result section.\n\nResults:\nPlease add field observation results (if the data was recorded as mentioned in the Materials and methods). In the Condition factors section, it mentions about the dry session and rainy session; which dry or rainy session between July and November? Please add information.\n\nDiscussion:\nDevelop the discussion by mentioning about the relationship between the environment factor and the results of LWRs and CF fishes', ex: what prey and predator of both fish. It is also mentioned in the discussion that the relative weight condition factor also corresponds to fish health conditions, stock estimates, and management levels. This can be an interesting discussion to be developed.\nConclusion: Sufficient, but it is better to add a conclusion regarding the condition factors related to the sex (male/female) of both fish.\n\nReferences: Sufficient.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": [
{
"c_id": "5583",
"date": "16 Jun 2020",
"name": "Zainal Abidin Muchlisin",
"role": "Author Response",
"response": "1. Comment: Please mention in the Time, site, and sampling or LWRs analyses section that male and female fish were measured/analyzed for LWR separately. Respond: We have added this information in the material and method (page 5) 2. Comment: In the field observation, it is stated that the weather condition, tides, and water turbidity are also observed visually during the sampling. Is the observed visual data recorded? If yes, please mention in the result section. Respond: This information was added in the results section (condition factor subsection) on page 8 4. Comments: In the condition factors section, it mentions about the dry session and rainy session; which dry or rainy session between July and November? Please add the information. Respond: This detail description of the dry and wet/rainy seasons have been added at Time, Site and Sampling Section (page 4) 5. Comment: Develop the discussion by mentioning the relationship between the environment factor and the results of LWRs and CF fishes', ex: what prey and predator of both fish. Respond: We have discussed the relationship between environmental factors and LWRs and the condition factor of the fish (page 10-11). Based on some references, and our data (these data will be published separately) that the prey (food) for the mullets are mostly plankton and algae (we have added this information in the discussion section). But, we have not examined the predator for mullets. 6. Comment: It is also mentioned in the discussion that the relative weight condition factor also corresponds to fish health conditions, stock estimates, and management levels. This can be an interesting discussion to be developed. Respond: We have developed the discussion as suggested by the reviewer (page 11 under quotation to Blackwell et al. (2000). 7. Comment: The conclusion is sufficient, but it is better to add a conclusion regarding the condition factors related to the sex (male/female) of both fish. Respond: We have extended the conclusion, but we think that the detailed information male and female fish would be better displayed in the Abstract. In the conclusion section, we mentioned generally."
}
]
}
] | 1
|
https://f1000research.com/articles/9-259
|
https://f1000research.com/articles/9-585/v1
|
10 Jun 20
|
{
"type": "Research Article",
"title": "Word embedding mining for SARS-CoV-2 and COVID-19 drug repurposing",
"authors": [
"Finn Kuusisto",
"David Page",
"Ron Stewart",
"David Page",
"Ron Stewart"
],
"abstract": "Background: The rapid spread of illness and death caused by the severe respiratory syndrome coronavirus 2 (SARS-CoV-2) and its associated coronavirus disease 2019 (COVID-19) demands a rapid response in treatment development. Limitations of de novo drug development, however, suggest that drug repurposing is best suited to meet this demand. Methods: Due to the difficulty of accessing electronic health record data in general and in the midst of a global pandemic, and due to the similarity between SARS-CoV-2 and SARS-CoV, we propose mining the extensive biomedical literature for treatments to SARS that may also then be appropriate for COVID-19. In particular, we propose a method of mining a large biomedical word embedding for FDA approved drugs based on drug-disease treatment analogies. Results: We first validate that our method correctly identifies ground truth treatments for well-known diseases. We then use our method to find several approved drugs that have been suggested or are currently in clinical trials for COVID-19 in our top hits and present the rest as promising leads for further experimental investigation. Conclusions: We find our approach promising and present it, along with suggestions for future work, to the computational drug repurposing community at large as another tool to help fight the pandemic. Code and data for our methods can be found at https://github.com/finnkuusisto/covid19_word_embedding.",
"keywords": [
"Word embedding",
"drug repurposing",
"SARS-CoV-2",
"COVID-19"
],
"content": "Introduction\n\nThe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and associated coronavirus disease 2019 (COVID-19) were first identified in December of 2019 and have since spread to become a global pandemic1. This rapid spread of illness and death demands a rapid response in treatment development. De novo drug development, however, is slow, expensive, and suffers from low probability of success2. In contrast, drug repurposing, identifying new indications for existing drugs, offers the advantages of reduced time and risk to finding treatments. We thus propose that drug repurposing is the most promising approach to treatment development for this pandemic.\n\nThere are several strategies we could employ for drug repurposing. Certainly, getting access to the rapidly growing electronic health record (EHR) histories of those afflicted by COVID-19 could be enlightening. We could, for example, track patient recovery times and look for common prescription histories in those who recover sooner. Gaining access to sufficient EHR data would likely prove challenging though due to privacy concerns and limited data at individual institutions, not to mention the added administrative burden that might entail for an already strained health system. Given the similarity of SARS-CoV-2 to its predecessor SARS-CoV3, we propose leveraging what we have learned about SARS in the intervening years. Specifically, we propose mining a word embedding built on biomedical literature published through early 2019 for candidate FDA approved drugs to treat SARS. Our results show that our proposed approach identifies several promising candidate drugs that have already been suggested or are already in clinical trials for COVID-19. We thus propose other candidate drugs identified by our method as potential leads for further investigation via in vitro and in vivo experimentation.\n\nIn the following sections, we describe our word embedding source, our source and processing method for FDA approved drug names, and our approach to mining the word embedding for drugs to treat SARS. We then present our results and a discussion including manual evaluation of the top candidate drugs proposed by our method, followed by a conclusion and suggestions for future work.\n\n\nMethods\n\nIn order to perform our word embedding mining for COVID-19 drug repurposing, we first need a word embedding. Furthermore, we need drug names to look for within the embedding. Here we briefly describe our sources for both the word embedding and drug names, we describe the data processing we perform on these sources, and we describe our methods for analysis. Code and data used for all of this analysis can be found at https://github.com/finnkuusisto/covid19_word_embedding4.\n\nRather than spend the time building our own word embedding on biomedical text, we instead searched the literature where there are several prebuilt biomedical word embeddings available. For this work, we chose the BioWordVec5 prebuilt embedding, specifically the intrinsic model. We chose BioWordVec because it is the most recent available biomedical word embedding and it has performed well on several benchmark tasks.\n\nIn order to find a vector representation for COVID-19 treatments, we use a simple analogy approach. The original Word2vec publication demonstrated that the structure of a word embedding space could carry semantic meaning by showing that vector(“King”) - vector(“Man”) + vector(“Woman”) resulted in a vector closest to the word vector for Queen6. Effectively, this vector math asks the analogy King is to Man as what is to Woman? We use the same approach here, but instead use common drug-disease pairs as the seed analogy and SARS as the query disease. For example, one analogy we use is: vector(“Metformin”) - vector(“Diabetes”) + vector(“SARS”). Effectively, we get the word vector analogy of Metformin is to Diabetes as what is to SARS? Note that the BioWordVec embedding we are using was published before SARS-CoV-2 was discovered and thus contains no reference to SARS-CoV-2 or COVID-19 in the vocabulary. Given, that SARS-CoV-2 is a strain of SARS-CoV7, we use SARS as an approximation. To get a sense of analogy consistency, we use three separate drug-disease pairs as our seed treatment analogies: metformin and diabetes, benazepril and hypertension, and albuterol and asthma.\n\nGiven the urgency of the situation, we consider drug repurposing the most appropriate approach to finding treatments for COVID-19. We thus chose to tailor our treatment mining toward finding FDA approved drugs, allowing for the potential of off-label prescription in the short term. To get a list of approved drugs for our embedding analysis, we downloaded the FDA’s approved drug database8, extracted the drug names, and processed them for use in the word embedding.\n\nTo extract raw drug names from the FDA database, we first pulled all entries from the DrugName and Active-Ingredient fields of the Products table. We next manually inspected all raw entries that ended with parentheticals (e.g. “prempro (premarin;cycrin)”) to identify entries that contain aliases or combinations versus those that contain tokens related to branding or packaging (e.g. “rogaine (for men)”). From these parentheticals, we manually collected additional drug names and then removed all parentheticals from the drug entries. These manually collected additional names included Ampicillin, Cycrin, Hydrocortisone, Premarin, Sulfabenzamide, Sulfacetamide, Sulfathiazole, Sulfadiazine, Sulfamerazine, and Sulfamethazine. We then split all of the entries by the semicolon character to separate drug names and ingredients entered as lists. Finally, we manually added back in those drugs and ingredients that were manually extracted from the deleted parentheticals. This gave us a list of 8,561 candidate approved drug names.\n\nWe next converted our candidate drug names into word vectors to enable ranking by their similarity with our treatment analogy vector. Here we simply split each candidate drug by white space and averaged the individual token vectors to get a final vector for the drug overall. When a token was not present in the embedding vocabulary, we simply dropped that token from the average and from the initial drug name. We used this approach rather than dropping a drug entirely to allow greater flexibility, for example if the embedding vocabulary is missing an ingredient from a combination drug. Finally, we removed duplicate drug names with the same tokens to account for exact duplicates and those with combinations stated in multiple orders. As a result, we successfully derived 5,833 distinct drug vectors from our initial 8,561 candidate drugs. We then sort these drug vectors by cosine similarity with our treatment analogy vectors and evaluate the closest hits.\n\nAs a preliminary validation that our approach can work to find useful drugs for diseases from treatment analogy vectors, we first considered major diseases and disease families with well-known treatments. Specifically, we used our treatment analogy vector approach to rank drugs for the query diseases Alzheimer’s, allergies, and cancer (see Table 1, Table 2, and Table 3). Note that we still used the same seed drug-disease pairs here (metformin-diabetes, benazeprilhypertension, and albuterol-asthma) but searched for analogous treatments for Alzheimer’s, allergies, and cancer instead of SARS. For example, one analogy we used for initial validation is: vector(“Metformin”) - vector(“Diabetes”) + vector(“Alzheimer’s”). For this preliminary validation, we wanted to find drugs whose main indication is to treat the query disease in the top candidates. We chose these query diseases because they are fairly broad and have minimal treatment overlap with the seed drug-disease pairs that we used for the analogy. After initial validation of our method, we manually reviewed the top 50 drug candidates for SARS using the same method (see Table 4, Table 5, and Table 6).\n\nDrugs with a primary indication for Alzheimer’s are highlighted in gray.\n\nDrugs with a primary indication for allergies are highlighted in gray.\n\nDrugs with a primary indication for cancer are highlighted in gray.\n\nHits containing drugs suggested or under investigation for COVID-19 are highlighted in gray.\n\nHits containing drugs suggested or under investigation for COVID-19 are highlighted in gray.\n\nHits containing drugs suggested or under investigation for COVID-19 are highlighted in gray.\n\n\nResults\n\nHere we present results for validation of our word embedding mining approach along with results from applying our approach for COVID-19 drug repurposing. First, we present validation results for our approach to ranking FDA approved drugs for three diseases or disease families with well-established treatments. Specifically, we use the same three seed drug-disease pairs as analogies to find drugs for Alzheimer’s, allergies, and cancer (see Table 1, Table 2, and Table 3). All drugs with a primary indication for the query disease are highlighted in gray. This is to verify that our complete approach (drug vectors ranked by cosine similarity to treatment analogy vector) can identify effective ground-truth drugs for diseases that are not closely related to the seed disease-drug pair. In nearly every example, a vast majority (if not all) of the top 10 hits have a primary indication for the query disease.\n\nNext, we present the 50 closest FDA approved drugs to the treatment analogy vectors for SARS, thereby filtering to what may be the most promising drugs for repurposing. The top repurposing hits are presented in Table 4, Table 5, and Table 6, and all drugs that have been suggested for or are currently under investigation for treatment of COVID-19 are highlighted in gray. This highlighting serves as a partial evaluation of the repurposing via positive controls, suggesting that other hits may be good candidates for further investigation. We find 22 positive control hits out of 50 for the metformin-diabetes analogy, 12 of 50 for the benazepril-hypertension analogy, and eight of 50 for the albuterol-asthma analogy. We present a Venn diagram of the overlap between the three analogies in Figure 1, and a table containing the drugs shared by all three and by at least two of the analogies in Table 7. Seven drugs are shared by all three analogies in their top 50 hits, and another 10 are shared by at least two of the analogies for a total of 17 higher confidence hits.\n\n\nDiscussion\n\nHere we review the validation results to demonstrate that our approach can find useful drugs for various diseases, followed by manual review of the FDA approved drug repurposing candidates for SARS. First, recall that we have used our drug ranking approach with the same seed analogy vectors for three major diseases with well-established ground-truth treatments. For the validation of our approach on drugs for Alzheimer’s, nearly all of the drugs suggested from each analogy were drugs with primary indications for Alzheimer’s, and several of the seemingly incorrect drugs have a primary indication for Parkinson’s, another neurodegenerative disease. We see a similar result for allergies where only the albuterol-asthma analogy suggests drugs not indicated for allergies in the top 10. Specifically, we see albuterol and levalbuterol show up several times, perhaps as a result of seed drug bias. For the cancer drugs, we see that every drug is indicated for some form of cancer. All of this reassures us that our approach does, in fact, find drugs appropriate for the query disease even if the query disease has no relationship with the seed drug-disease pair.\n\nNext, we manually reviewed every one of our top 50 FDA approved drugs suggested for repurposing with SARS as the query disease, and marked every one that has either been suggested for or is currently under investigation for treatment of SARS-CoV-2 and COVID-19. From the metformin-diabetes analogy, we find 22 of 50 drugs either suggested or under investigation for treatment against SARS-CoV-2 and COVID-19. With the benazepril-hypertension analogy, we find 12 of 50 hits, and from the albuterol-asthma analogy, we find eight of 50. Across the analogies, seven hits are common to all three, and 10 are common to two of the three.\n\nIn the seven hits common to all, four have been suggested for treatment of SARS-CoV-2 and COVID-19. Amantadine and rimantadine are both adamantanes, which have been shown to have antiviral properties in vitro and have demonstrated possible protective effects in a clinical study of patients with neurological diseases11,12. Zanamavir is an antiviral that has been suggested based on in silico molecular docking models of the 3C-like proteinase9, which is a major protease thought essential to viral replication of coronaviruses, including SARS-CoV and SARS-CoV-236,37. Oseltamivir (Tamiflu) is another antiviral that is under investigation via clinical trial17.\n\nIn the 10 hits common to two of the analogies, three have been suggested for treatment of SARS-CoV-2 and COVID-19. Memantine is another adamantane similar to amantadine and rimantadine suggested by all three analogies. Relenza is a trade name for zanamivir, so is essentially a duplicate, though it does perhaps suggest even more confidence in the drug. Virazole is a trade name for ribavirin, an antiviral which has shown antiviral activity against SARS-CoV-2 in vitro29.\n\nWe also note that 13 of all the proposed treatments are in clinical trials: atovaquone, lopinavir and ritonavir, sirolimus (suggested here as the prodrug temsirolimus), oseltamivir, selinexor, ibuprofen, colchicine, bicalutamide, mefloquine, chloroquine, linagliptin, fluvoxamine, and ketamine (suggested here as the enantiomer esketamine). Interestingly, these drugs come from a wide range of primary indications including antiparasitic, antiviral, anti-inflammatory, anticancer, anesthetic, and antidepressant effects. Furthermore, the proposed drugs that are not currently in trials show a similar breadth of primary indication. Overall, we find that our approach shows a great deal of promise as it is able to discover a wide range of drugs that have elsewhere been proposed for COVID-19 from clinical, in silico, in vitro, and in vivo experimentation, all done here with literature published before SARS-CoV-2 was discovered.\n\n\nLimitations\n\nOf course, while our method appears promising, it is not without limitations. First, our method is limited to what has already been published in the scientific literature and cannot propose new drugs or treatments outside of the embedding vocabulary. We also caution readers that, in most cases, these drugs have not been tested for COVID-19 efficacy, and we make no claims other than that some of these drugs deserve further exploration. We can say with confidence that at least a few proposed drugs seem less promising. Peramivir is a neuraminidase inhibitor used to treat influenza. While it is thus an antiviral, coronaviruses do not use neuraminidase, so it would seem less likely to be effective against SARS-CoV-222. On the other hand, zanamivir and oseltamivir, two of our common positive controls9,17, are also neuraminidase inhibitors and should thus be less likely candidates. Given that the potential mechanism of action for zanamivir at least is based on computed binding to the 3C-like proteinase, perhaps some drugs may demonstrate efficacy outside of their traditional mechanism. Nevertheless, the lesson is that we should expect to find false positives in our top hits along with any true positives. Finally, our embedding approach does not take into account the potential of drug-drug interactions to increase or decrease efficacy in any fashion. All of this is to say that further in vitro and in vivo experimentation, and observational EHR or claims data would all be useful additional sources of evidence for or against repurposing candidates listed here.\n\n\nConclusions\n\nIn this work, we present a word embedding mining approach to identifying candidate treatments for SARS-CoV-2 and COVID-19. We first use seed drug-disease pairs to produce treatment analogy vectors for a query disease using a prebuilt biomedical word embedding. We then use a simple word vector averaging approach to get vectors for a list of FDA approved drugs and sort them by their distance to our treatment analogy vectors. We validate that this approach identifies ground truth treatments for well-known diseases. Next, we use the same approach to produce a list of candidate drugs for the query disease SARS, manually evaluate the top candidate drugs, and find several positive controls that have been suggested in the literature or are currently under investigation for SARS-CoV-2 or COVID-19 treatment. While there are certain to be several false positives amongst our top hits as well, we find the presence of positive controls reassuring, and propose the remainder as potential candidates for further investigation. We furthermore propose this word vector embedding approach in general as a useful tool for COVID-19 drug repurposing. These results only scratch the surface of what is possible and we present this work as a suggestion to the community to investigate further. Immediate avenues for future investigation include exploring even more drug-disease analogy vectors, ranking drugs directly by their cosine similarity to proven treatments as they arise, and investigating drug-gene target analogy vectors rather than the disease treatment analogy we demonstrate here.\n\n\nData availability\n\nThe FDA database of approved drugs is available at: https://www.fda.gov/drugs/drug-approvals-and-databases/drugsfda-data-files.\n\nAll code and processed data used to produce these results are available at: https://github.com/finnkuusisto/covid19_word_embedding.\n\nArchived code and data as at time of publication: http://doi.org/10.5281/zenodo.38600574.\n\nLicense: CC0\n\nThe code is provided in Python (v 3.8) as Jupyter Notebooks (v 6.0.3), and additionally requires Gensim (v 3.8.1), Matplotlib (v 3.2.1), and Matplotlib-Venn (v 0.11.5).\n\n\nSoftware availability\n\nThe BioWordVec prebuilt embedding is available via the official GitHub repository: https://github.com/ncbi-nlp/BioWordVec.",
"appendix": "References\n\nWorld Health Organization: WHO director-general’s opening remarks at the media briefing on COVID-19. Geneva, Switzerland. 2020. Reference Source\n\nAshburn TT, Thor KB: Drug repositioning: identifying and developing new uses for existing drugs. Nat Rev Drug Discov. 2004; 3(8): 673–683. PubMed Abstract | Publisher Full Text\n\nWu A, Peng Y, Huang B, et al.: Genome composition and divergence of the novel coronavirus (2019-ncov) originating in china. Cell Host Microbe. 2020; 27(3): 325–328. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKuusisto F: finnkuusisto/covid19_word_embedding: First release for publication. (Versionv1.0). Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3860057\n\nZhang Y, Chen Q, Yang Z, et al.: BioWordVec, improving biomedical word embeddings with subword information and MeSH. Sci Data. 2019; 6(1): 52. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMikolov T, Chen K, Corrado G, et al.: Efficient estimation of word representations in vector space. arXiv preprint arXiv: 1301.3781. 2013. Reference Source\n\nCoronaviridae Study Group of the International Committee on Taxonomy of Viruses: The species severe acute respiratory syndrome-related coronavirus: classifying 2019-nCoV and naming it SARS-CoV-2. Nat Microbiol. 2020; 5(4): 536–544. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFederal Drug Administration: Drugs@FDA Data Files. 2020. Reference Source\n\nHall DC Jr, Ji HF: A search for medications to treat COVID-19 via in silico molecular docking models of the SARS-CoV-2 spike glycoprotein and 3CL protease. Travel Med Infect Dis. 2020; 101646. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHonorHealth Research Institute: Atovaquone and azithromycin combination for confirmed COVID-19 infection. 2020. Reference Source\n\nRejdak K, Grieb P: Adamantanes might be protective from covid-19 in patients with neurological diseases: multiple sclerosis, parkinsonism and cognitive impairment. Mult Scler Relat Disord. 2020; 42: 102163. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCimolai N: Potentially repurposing adamantanes for covid-19. J Med Virol. 2020; 92(6): 531–532. PubMed Abstract | Publisher Full Text\n\nCao B, Wang Y, Wen D, et al.: A trial of lopinavir-ritonavir in adults hospitalized with severe COVID-19. N Engl J Med. 2020; 382(19): 1787–1799. PubMed Abstract | Publisher Full Text | Free Full Text\n\nXu J, Shi PY, Li H, et al.: Broad spectrum antiviral agent niclosamide and its therapeutic potential. ACS Infect Dis. 2020; 6(5): 909–915. PubMed Abstract | Publisher Full Text | Free Full Text\n\nUniversity of Cincinnati: Sirolimus treatment in hospitalized patients with COVID-19 pneumonia (SCOPE). 2020. Reference Source\n\nLiu X, Wang XJ: Potential inhibitors against 2019-ncov coronavirus M protease from clinically approved medicines. J Genet Genomics. 2020; 47(2): 119–121. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTongji Hospital: A prospective/retrospective,randomized controlled clinical study of antiviral therapy in the 2019-nCoV pneumonia. 2020. Reference Source\n\nKaryopharm Therapeutics Inc.: Coronavirus disease 2019 treatment: a review of early and emerging options. 2020. Reference Source\n\nKing’s College London: LIBERATE Trial in COVID-19 (LIBERATE). 2020. Reference Source\n\nMontreal Heart Institute: Colchicine coronavirus SARS-CoV2 trial (COLCORONA). 2020. Reference Source\n\nSidney Kimmel Comprehensive Cancer Center at Johns Hopkins: Trial to promote recovery from COVID-19 with ivermectin or endocrine therapy (RECOVER). 2020. Reference Source\n\nMcCreary EK, Pogue JM: Coronavirus disease 2019 treatment: a review of early and emerging options. In: Open Forum Infectious Diseases. Oxford University Press US, 2020; 7(4): ofaa105. Publisher Full Text\n\nWeston S, Coleman CM, Sisk JM, et al.: Broad anti-coronaviral activity of FDA approved drugs against SARS-CoV-2 in vitro and SARS-CoV in vivo. bioRxiv. 2020. Publisher Full Text\n\nBurnasyan Federal Medical Biophysical Center: An open randomized study of the effectiveness of mefloquine for the treatment of patients with COVID19. 2020. Reference Source\n\nWang M, Cao R, Zhang L, et al.: Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro. Cell Res. 2020; 30(3): 269–271. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFarag A, Wang P, Ahmed M, et al.: Identification of FDA Approved Drugs Targeting COVID-19 Virus by Structure-Based Drug Repositioning. ChemRxiv. 2020; 4. Publisher Full Text\n\nUniversity of Miami: Effects of DPP4 inhibition on COVID-19. 2020. Reference Source\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\nKhalili JS, Zhu H, Mak A, et al.: Novel coronavirus treatment with ribavirin: Groundwork for evaluation concerning covid-19. J Med Virol. 2020. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMourad JJ, Levy BI: Interaction between raas inhibitors and ace2 in the context of covid-19. Nat Rev Cardiol. 2020; 17(5): 313. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLiu C, Zhou Q, Li Y, et al.: Research and development on therapeutic agents and vaccines for covid-19 and related human coronavirus diseases. ACS Cent Sci. 2020; 6(3): 315–331. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWashington University School of Medicine: Doubleblind, placebo-controlled clinical trial of fluvoxamine for symptomatic individuals with COVID-19 infection. 2020. Reference Source\n\nZhang L, Sun Y, Zeng HL: Calcium channel blocker amlodipine besylate is associated with reduced case fatal- ity rate of COVID-19 patients with hypertension. medRxiv. 2020. Publisher Full Text\n\nWilliam Beaumont Hospitals: Study of immunomodulation using naltrexone and ketamine for COVID-19 (SINK COVID-19). 2020. Reference Source\n\nGarg H: Can glycopyrrolate come to the airway rescue in COVID-19 patients? J Clin Anesth. 2020; 64: 109843. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGoetz DH, Choe Y, Hansell E, et al.: Substrate specificity profiling and identification of a new class of inhibitor for the major protease of the SARS coron- avirus. Biochemistry. 2007; 46(30): 8744–8752. PubMed Abstract | Publisher Full Text\n\nZhang L, Lin D, Sun X, et al.: Crystal structure of SARS-CoV-2 main protease provides a basis for design of improved α-ketoamide inhibitors. Science. 2020; 368(6489): 409–412. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "64552",
"date": "30 Jun 2020",
"name": "Quentin Vanhaelen",
"expertise": [
"Reviewer Expertise Computational biology",
"system biology"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors propose a new computational drug repurposing method based on word embedding for FDA approved drugs based on drug-disease treatment analogies. Acknowledging that the onset of the COVID-19 outbreak requires the quick identification of already known drugs which could be repurposed against COVID-19, the authors propose a relatively straightforward method where candidate drug names are converted into word vectors to enable ranking by their similarity with a treatment analogy vector. The paper is well written and organized. The authors have done a good job in describing the rationale behind the development of their method and give an overview of its design process as well as the testing steps. Nevertheless, several points should be addressed regarding the validation of the method and its performance, benchmarking with respect to other similar methods, and applicability of the method for other diseases. The field of computational repurposing methods is very large and in contrast, the introduction of this article is relatively short and does not contain any comprehensive referenced overview of prior works in this field. My recommendation is that the authors should at least shortly cover the existing methods based on word mining and semantic and discuss how their own method positions itself in this context. Discussing those methods could serve as a starting point for a proper assessment of the performance of the proposed algorithm (see comment below).\n\nBelow I summarize my comments and suggestions which I hope could contribute to improving the manuscript.\nThe proposed algorithm is based on drug-disease relationships. It might be interesting to take into account the drug-target relationship as well. Indeed, one drug can often interact with several targets and usually, the drug-disease association is established through one specific target. Including the drug- target and disease-target relationships offer additional possibilities to repurpose the drug against a condition for which the drug-target relationship is relevant.\n\nThe algorithm mainly uses association by keywords. As the authors has noted, this approach has several limitations. It would be difficult to apply the algorithm for finding repurposing candidates for conditions without prior known drugs (This restriction being encountered by many repurposing algorithms). Furthermore, the method might be limited by the vocabulary used. Indeed, it is not uncommon for a condition or a drug to be known by different names in the scientific literature. This issue can be addressed when dealing with formatted databases but it is more difficult to handle with natural language in the literature.\n\nBy its nature, this method does not take into account all the relevant properties at the genomic, transcriptomic, chemical, structural levels which are of paramount importance to correctly establish the compatibility of a drug-target-disease relationship. For instance, the authors have based their study on the assumption that SARS-CoV and SARS-CoV-2 share common features, which is true but it was also pointed out that some structural and non-structural proteins of SARS-CoV-2 have low sequence similarities with the corresponding ones from SARS-CoV and this may have strong implication on the use of SARS-CoV drug to treat SARS-CoV-2 associated disease.\n\nThis method is in my opinion would be interesting as an initial approach to reduce the number of potential repurposing candidates which should be included as input data for a subsequent computational repurposing method capable of taking into account the information about chemical/structural and genomic features of the target- drug-disease relationships. As many methods can be relatively costly from a computational perspective, being able to pre-select the initial set of data to be integrated is of interest.\n\nIt would be interesting to have a more detailed description of how the treatment analogy vectors are built and how the similarities are calculated. The authors have initially a list of 8,561 candidate drugs (after some manual curation) but end up using a reduced list of 5,833 distinct drug vectors. This is a significant reduction in the initial amount of information the algorithm can be applied to. Maybe the authors could suggest alternative methods to be able to keep a higher proportion of the initial list of drugs? Also the table could include the similarity scores of the drugs. The authors decided to list the top 50 drugs, they do not provide a reason why 50 should be chosen as a cut off. Is there any systematic way to define an appropriate cut off for the most relevant results? This should be discussed in more details in the paper.\n\nAs the assembly of the list of drug vectors requires several steps of preparation, it would be interesting for the readers to have those steps summarized in a diagram. This diagram could describe the key steps for the preparation of the drug vectors from a general perspective (so not only for the SARS-CoV-2 case), with an emphasis on what the user should take care of when deciding to include or not a drug. The article already contains many tables summarising the results of the different experiments. Maybe some of them could be moved in the supplementary materials to give space to this diagram.\n\nThe main criteria in this paper to assess the method is to look for predicted drugs that are either already known as being effective against SARS-CoV or already taken into clinical trials for SARS-CoV-2. But those criteria are restrictive and not necessarily generalizable to other type of disease. One can assume that many potential repurposing candidates for a given condition are not undergoing clinical trial for a similar condition. It should be also noted that having a drug entering clinical trial is not a guarantee of success considering the relatively high failure rate in this area. It is true that for assessing the results provided by this type of computational method, a literature review is a first step that needs to be followed by in vitro/in vivo validation of the most promising candidates. Before those more expensive validations could take place, what would be the computational or data-based criteria to assess the accuracy of the predictions? The authors could discuss how this method performs compared to other methods based on word embedding and text mining for instance.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": [
{
"c_id": "5885",
"date": "03 Sep 2020",
"name": "Finn Kuusisto",
"role": "Author Response",
"response": "Dr. Vanhaelen,We greatly appreciate your thoughtful response on this work and apologize for the long delay. This is all really helpful advice. We do not intend for this to be a formal and complete response to your review, but while we wait for a second reviewer, and before resubmitting, we hope to understand if our proposals here might be sufficient to address your concerns.The overarching themes seem to be centered around providing background and context, clarity of methods and evaluation, and overall messaging on the goal of this work. Briefly, rather than being an automated method for validating potential drug candidates, we see this work more as a helpful starting point for researchers to begin literature search. In this way, we hope to accelerate the discovery process by helping researchers prioritize what are the most promising candidates for more in-depth in vitro and in vivo analysis.Regarding a lack of background, that was an oversight. In the resubmission we will add a background section to review methods in computational drug repurposing, including some discussion of drug-drug similarity, matching genetic signatures of phenotypes to drugs, in silico measures of molecular docking, mining electronic health record data for drug side effects, and literature-based discovery. We hope this provides better context for our work.We also have plans for clarifying the methods and intent of the work. For improvement of the evaluation, we intend to include some comparison to the BEST text mining tool and likely Polysearch2 as well. These two are the most similar approaches we could find that would allow for collecting a large number of proposed candidate drugs from the literature quickly.-Specific Points-1. Drug-target relationshipsWe really like this idea and alluded to something similar in the future work section. It may also help alleviate the limitation of only finding drugs that commonly exist in the literature. We are pursuing the idea, but given our initial promising results just using drug names, we decided that angle was out of scope and might even confuse the message of what we have accomplished so far. We will add to our discussion of drug-target relationships as a promising area for future work.2. Keywords/vocabularyYou are absolutely correct that token matching approaches have major limitations in what they can find. They simply cannot directly find something that is not present in the vocabulary. This is the reason for the drug count reducing from 8,561 to 5,833 as explained below. Complicated terms and those with many synonyms are another limitation that would probably be best addressed at the word embedding training phase. Furthermore, some of our other work on more traditional text mining has shown synonyms to be particularly challenging in that they can lead to increases of false positives, and confusion when collisions occur (e.g. drug A and drug B have the same synonym X). It will take more effort to address properly.We tried to limit this work to readily available tools for the sake of expedience and have found that, despite these limitations, our method presents interesting findings. We plan to add your concerns to the limitations/future work discussion.3. Relevant genomic/viral/drug properties not consideredIt is true that our current approach misses several relevant properties related to the drug-target-disease relationship. Some of that may be alleviated by future work investigating word embedding mining at the drug-gene/protein/pathway level, but pursuing those lines of inquiry here would greatly lengthen and complicate this paper. We are aiming here to show simply that analogy vectors are likely useful. We will explain that further analogy-based analysis will be useful for establishing links between drugs and targets and targets to disease which will further filter candidates prior to expensive wet lab validation. We see this method as a potentially helpful tool for quickly prioritizing drug candidates for further investigation. Rather than providing automated in-depth support for candidate drugs, we would expect researchers to use our method to conduct initial literature search, thereby accelerating the process of choosing promising candidates for more in-depth in vitro and in vivo testing. We will attempt to clarify this intent in the resubmission.4. Preselection commentWe wholeheartedly agree that our work is best viewed as an initial preselection step. Our intent is for this method to serve as an early step, providing suggestions for further investigation. As you have pointed out, this approach ignores several relevant properties that define the drug-target-disease relationship. More than anything, we see this work as providing a quick and powerful summarization of leads within the literature and helping to prioritize research in promising directions. We will clarify this message in our resubmission.5. Vector creation and cosine similarity cutoffYou are correct that we do lose a large portion of our initial drug set (8.5k down to 5.8k) when converting them into drug vectors. We will be sure to clarify why this is in our resubmission, but I can briefly explain here. The process of converting from drug names to drug vectors is automated rather than manual and consists of 1) splitting drug names into individual tokens by whitespace, 2) getting word vectors from the embedding for those tokens, and 3) averaging those constituent token word vectors for each drug. When an individual token is not present in the BioWordVec embedding vocabulary, we drop it from the drug name. If none of the tokens from a drug name exist in the BioWordVec vocabulary, we drop the drug entirely. Ultimately, what that means is that there are ~3k drugs in our initial set that simply have no tokens present in the embedding vocabulary and cannot be salvaged. This speaks to some of the limitations previously discussed. We will clarify this in the resubmission.For the cosine-similarity cutoff versus top 50 hits, we are unaware of any particularly principled methods for picking a similarity cutoff. We considered inspecting the cosine-similarity distributions for change points, and they do follow something like an inverted logit, but we found that the change points selected hit counts on the order of hundreds of drugs. Ultimately, we chose to simply pick what seemed a manageable number of drugs to review by hand. We can clarify in the text how this is somewhat an arbitrary decision.6. Visual diagramA generalized visual diagram of the whole pipeline would be helpful. We will add one to the resubmission.7. Evaluation processEvaluation is certainly tough in drug repurposing. As you say, comparing to drugs suggested in other publications and ongoing clinical trials may be quite restrictive and not particularly generalizable to other diseases. At the same time, it may even be too loose of a restriction in this case as the sheer volume of publications on COVID-19 at this point may suggest a vast number of false positive drugs. Again, evaluation in drug repurposing is tough and eventually necessitates experimentation in vitro and in vivo. Thus, we likely still won’t know which hits were true positives for quite some time. That is the main reason we first demonstrated that the treatment analogy vectors could identify gold-standard drugs for well-known diseases in tables one through three. Ultimately though, given our goal of augmenting the initial search process, we simply want to determine if our method sorts drug candidates in a reasonable fashion for this new disease. If so, we can be more confident that other not-yet-considered drugs on the list, and those just a bit farther down the list, may be promising candidates for researchers to consider. We will elaborate on the limitations of this evaluation further.As for other comparable approaches, we plan to include the top drug hits provided by the BEST and Polysearch2 text mining tools. BEST allows perhaps the closest comparison because it allows for limiting the search to literature through 2019, as ours is based on a word embedding built in 2019. Both provide many positive hits according to our manual evaluation process, but we argue that our approach still offers some advantages for two reasons, both related to flexibility. First, our approach here allows for the input of any list of drug names or query entities, whereas BEST and Polysearch2 are limited to prebuilt term lists. Those prebuilt lists can almost certainly be expanded, but our approach is flexible right out of the box. Second, because our method is based on treatment analogy vectors, it can provide a greater number and greater diversity of possible candidates. BEST and Polysearch2 provide a single ranking of drugs to disease based on literature co-occurrence, whereas our approach can be reseeded with a different treatment analogy vector (e.g. metformin/diabetes vs albuterol/asthma) and immediately provide a related but different ranking of drugs. We see this as an interesting advantage to explore in future work.Overall, we mainly hope to demonstrate that our very quick and simple method could be useful in the early stages of research, rather than demonstrating that our method is better than current state of the art methods for deeper evaluation of drug effects. Bear in mind again that these sorted drug suggestions are essentially time-censored to literature from 2019. We were pleasantly surprised to see the quality of suggestions our method provided and how early it may have been used to quickly gather promising candidates that are now under investigation."
}
]
},
{
"id": "75542",
"date": "15 Dec 2020",
"name": "Nansu Zong",
"expertise": [
"Reviewer Expertise Deep Learning",
"computational drug repurposing",
"translational bioinformatics",
"EHR"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 proposed a novel method for drug repurposing to mine a large biomedical word embedding for FDA approved drugs based on drug-disease treatment analogies. The paper is well presented, and the results seem promising. The writing is clear and easy to understand with the help of abundant tables and figures. While the contribution described in the manuscript is worthwhile, there are some limitations needed to be improved:\nThe biggest question is why the three drug pairs are selected? Any metrics used to select those pairs? The authors have other candidate pairs, and what are those results?\n\nEvaluation metrics are missing, e.g., AUCROC, Precision, Recall.\n\nComparison to the state-of-the-art methods is missing\n\nIs the work 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?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/9-585
|
https://f1000research.com/articles/9-584/v1
|
09 Jun 20
|
{
"type": "Research Article",
"title": "Measuring development assistance for health systems strengthening and health security: an analysis using the Creditor Reporting System database",
"authors": [
"Jessica Kraus",
"Gavin Yamey",
"Marco Schäferhoff",
"Hugo Petitjean",
"Jessica Hale",
"Kenan Karakulah",
"Chris Kardish",
"Estuardo Pineda",
"Francesca Sanders",
"Naomi Beyeler",
"Sara Fewer",
"Rachel Nugent",
"Dean T. Jamison",
"Ben Oppenheim",
"Indermit Gill",
"Gavin Yamey",
"Marco Schäferhoff",
"Hugo Petitjean",
"Jessica Hale",
"Kenan Karakulah",
"Chris Kardish",
"Estuardo Pineda",
"Francesca Sanders",
"Naomi Beyeler",
"Sara Fewer",
"Rachel Nugent",
"Dean T. Jamison",
"Ben Oppenheim",
"Indermit Gill"
],
"abstract": "Background: Health systems strengthening (HSS) and health security are two pillars of universal health coverage (UHC). Investments in these areas are essential for meeting the Sustainable Development Goals and are of heightened relevance given the emergence of the 2019 novel coronavirus disease (COVID-19). This study aims to generate information on development assistance for health (DAH) for these areas, including how to track it and how funding levels align with country needs. Methods: We developed a framework to analyze the amount of DAH disbursed in 2015 for the six building blocks of the health system (‘system-wide HSS’) plus health security (emergency preparedness, risk management, and response) at both the global (transnational) and country level. We reviewed 2,427 of 32,801 DAH activities in the Creditor Reporting System (CRS) database (80% of the total value of disbursements in 2015) and additional public information sources. Additional aid activities were identified through a keyword search. Results: In 2015, we estimated that US$3.1 billion (13.4%) of the US$22.9 billion of DAH captured in the CRS database was for system-wide HSS and health security: US$2.5 billion (10.9%) for system-wide HSS, mostly for infrastructure, and US$0.6 billion (2.5%) for system-wide health security. US$567.1 million (2.4%) was invested in supporting these activities at the global level. If responses to individual health emergencies are included, 7.5% of total DAH (US$1.7B) was for health security. We found a correlation between DAH for HSS and maternal mortality rates, and we interpret this as evidence that HSS aid generally flowed to countries with greater need. Conclusions: Achieving UHC by 2030 will require greater investments in system-wide HSS and proactive health emergency preparedness. It may be appropriate for donors to more prominently consider country needs and global functions when investing in health security and HSS.",
"keywords": [
"Development assistance",
"development assistance for health",
"health systems strengthening",
"health security",
"pandemic preparedness",
"universal health coverage",
"DAH",
"UHC"
],
"content": "Introduction\n\nIn September 2015, United Nations member states adopted the Sustainable Development Goals (SDGs), including SDG3 – to “ensure healthy lives and promote well-being for all at all ages”1. Within SDG3 is the ambitious target of achieving universal health coverage (UHC) by 2030 (SDG target 3.8). Defined by the World Health Organization (WHO) as “all individuals and communities receive the health services they need without suffering financial hardship”2.\n\nA core foundation of achieving target 3.8 is health systems strengthening (HSS)3, which Kutzin and Sparkes argue “comprises the means” by which the objectives of UHC can be achieved4. A related foundation is health security – emergency preparedness, risk management, and response capabilities5–7. The incremental investments needed for HSS and health security are substantial. Estimates exist on the resources available for funding certain core components of UHC (such as health systems and pandemic preparedness). Stenberg and colleagues estimate that by 2030 the additional annual funding needed to reach the health-related SDGs across 67 low-income and middle-income countries would be US$274-371 billion, of which three quarters is needed for HSS (and one quarter for program support)8. A more recent estimate from the Disease Control Priorities Project found incremental costs to be about the same (although slightly lower) than did Sternberg and colleagues but that specific program investments account for a much larger fraction of the total (57% rather than 25%) with correspondingly smaller needs for generalized HSS. The DCP estimated the cost for pandemic preparedness to be about US$4 billion per year out of its total estimated cost9. The Commission on a Global Health Risk Framework for the Future estimates that an additional US$3.4 billion annually is needed “to upgrade national pandemic preparedness capabilities”10.\n\nThere are several debates on how development assistance for health (DAH) can best be used to support the health SDGs. Essential to informing these debates is knowing how donor support is currently spent. There has been little research on how much DAH flows to HSS and health security. The Institute for Health Metrics and Evaluation (IHME) publishes annual estimates of global health financing flows, including by year, focus area, donor, and channel, as well as projections on future spending11. IHME found that in 2015, 9.4% of total DAH went towards HSS and 0.8% was directed at pandemic preparedness12. However, given its methodology and scope, IHME’s financing global health estimate cannot be disaggregated to the activity level nor heavily filtered (e.g., to estimate DAH for specific building blocks of the health system, for system-wide health security support, etc.) IHME researchers occasionally provide one-off analyses on specific sub-area(s), e.g., on donor financing for human resources for health13. Little research has also been done on how estimates compare to country need.\n\nThis study provides evidence on donor support for system-wide HSS and system-wide health security, two key UHC pillars. To the best of our knowledge, this study is the first to assess flows of DAH towards these two foundations of UHC in a way that allows the findings to be disaggregated by HSS building block. By providing an in-depth analysis of DAH in 2015, the first year of the SDGs, our study can serve as a baseline for monitoring global progress. In addition, to facilitate evidence-based decision-making, we compared flows against proxies for country needs. By reviewing activities manually through a line-by-line review, financing can be scrutinized and classified in greater detail. Basing the study on a sample of the publicly available, activity level financing data reported to the Organisation for Economic Cooperation and Development’s Creditor Reporting System (CRS) strengthens transparency. Through this approach, we have made available a complementary dataset that we hope can facilitate both donors and recipient countries to better understand which specific activities are contributing to UHC through system-wide HSS and health security.\n\nOur study is designed to inform the global health discourse. There is a continued discussion of the conditions under which DAH for program-specific intervention support (a ‘vertical’ approach), program strengthening (a ‘diagonal’ approach), or system-wide investments (a ‘horizontal’ approach) is more effective14. There is also continued discussion in the global health community on the value of DAH for “global functions” (activities that have transnational benefits, e.g., pandemic preparedness) versus DAH given to a single country for disease control. The Lancet Commission on Investing in Health and the WHO’s Common Goods for Health program suggest that DAH should be increasingly directed to global functions15. These analyses suggest that global functions remain seriously underfunded and that country-specific aid for routine functions can lead countries to relocate domestic resources away from health (‘fungibility’). To inform these discussions, we developed a framework that disaggregates between global versus country-specific functions and distinguishes between system-wide investments and other approaches to health support.\n\n\nMethods\n\nWe developed a new approach for tracking DAH for HSS and health security. We began by developing an analytic framework that classifies DAH aid activity across three health investment areas (Figure 1). The first is country-level program-specific investments (Box 1 of Figure 1), which focus on a single disease/response program, such as DAH given to a country to establish an HIV drug supply system. The second is country-level, system-wide HSS and health security, i.e., investments with a focus across diseases, beyond a single disease response effort (Box 2 of Figure 1). An example is an investment in a comprehensive national health information system. The third is investments in ‘global functions’ – DAH that has transnational benefits (Box 3 of Figure 1), e.g., a platform to foster information exchange across a region16–18.\n\nEach of these three categories was broken down into sub-categories. Program-specific investments were broken down into either program-specific intervention support (Box 1A of Figure 1) or program strengthening (Box 1B of Figure 1). Program-specific intervention support refers to targeted programmatic support (e.g., commodities for a specific disease program). Program-specific investments were further broken down across four sub-categories: three disease areas (reproductive, maternal, newborn, child, and adolescent health [RMNCAH]; infectious diseases; and non-communicable diseases [NCDs]) and a fourth category, programmatic support for one-off health emergencies (e.g., equipment for an Ebola outbreak). Program strengthening investments (Box 1B of Figure 1) aim to improve the quality of a disease-specific program by strengthening just one or a few building blocks of the health system (i.e., diagonal funding). Program strengthening investments were also divided into RMNCAH, infectious diseases, or NCDs.\n\nProgram-specific investments contrast with system-wide HSS (Box 2 of Figure 1), which strengthens the health system in ways that improve health outcomes across multiple diseases and conditions. System-wide HSS investments were broken out across the six building blocks of the health system presented in Stenberg et al.’s conceptual framework8. We also included a component for ‘system-wide emergency preparedness, risk management and response’ for activities that strengthened capacity to deal with epidemic and pandemic preparedness and response, e.g., a mobile emergency alert system.\n\nFinally, global functions (Box 3 of Figure 1) comprised three sub-categories of investments with transnational benefits: HSS (e.g., knowledge exchange on HSS), health security (e.g., research and development of medical countermeasures to control pandemics), and ‘other investments in global functions’ (e.g., in product development for neglected diseases). Appendix, Tables 1–4 (see Extended data)19, provide further detail on the sub-categories.\n\nTo estimate total DAH for UHC through system-wide HSS and health security, we summed the investments for the sub-components highlighted in Figure 1.\n\nWe conducted a detailed analysis of aid activities from the Creditor Reporting System (CRS) database, complemented by a review of additional information sources, to estimate DAH flows to health security and HSS in the year 2015, the latest year for which data were available at the time of the analysis. The CRS is a publicly available database that provides information about DAH resource flows. It covers spending from official bilateral and multilateral organizations – including official development assistance (ODA) from Development Assistance Committee (DAC) members and some non-members – as well as from private philanthropic foundations (at the time of the analysis, limited to the Bill & Melinda Gates Foundation). Unlike other sources, the CRS database is scrutinized by the Organisation for Economic Cooperation and Development (OECD). It avoids double-counting and provides information on individual aid activities (Appendix p. 18, see Extended data)19,20.\n\nWe used our analytic framework to code aid activities in the CRS database. We used the OECD’s June 2017 update of the 2015 CRS database (see Source data), the most recent year at the time of the analysis. We covered ODA, as well as grant financing provided by the Bill & Melinda Gates Foundation. When donors report to the CRS, they assign projects to codes21. DAH was defined using the DAC’s sector codes for health, general (code 121), basic health (122), and population policies/programmes and reproductive health (130)22, as well as social mitigation of HIV (16064). These sectors have previously been used to define health aid23,24. All financing levels were calculated using the ‘USD_Disbursement’ column (provided in US$ 2015 constant prices). At the time of our analysis, donors reported US$22.9 billion in development assistance for these codes in 2015. Each row in this dataset represents one aid activity. The sample of aid activities was selected by ordering the 32,801 \"aid activities\" from highest to lowest in dollar terms using the ‘USD Disbursement’ column, and taking those that represent 80% of all health ODA. This resulted in a sample of 2,427 aid activities (USD$18.3 billion, or 80% of total health ODA). A non-probability sampling method was chosen to capture the majority of DAH. Our approach to extrapolation and reviewing aid activities was based on our earlier effort to track donor financing for health by function12.\n\nA team of analysts assessed what proportion of the aid activity went to each of the investment areas. Measures were put in place to ensure accuracy and consistency across the team of analysts. We developed a codebook with definitions and examples for each area in our analytical framework. Health aid activities were reviewed based on the descriptions in the CRS. Descriptions in a language other than English or French were translated through Google Translate. When descriptive information in the CRS was insufficient to adequately code a project, we obtained additional project information from publicly available sources, e.g., budgets and project logframes from the websites of donors and project implementers. The best source of additional information was typically a donor’s website. When no information was provided on a donor or a recipient’s website, other sources of information were sought, e.g., news articles. Based on the information found, the aid activity amount was then broken down by sub-category.\n\nAnalysts noted the sources and rationale for each aid activity’s allocations. In the best-case scenario, a coder was able to find specific financial information, e.g. budget details of the disbursement or a detailed aid activity document outlining where funding was going. For example, if an aid activity document showed that $300,000 was spent on health information systems of an aid activity worth $1,500,000, then 20% was allocated to health information systems and this would be entered as a formula (“=300,000/1,500,000”). For cases in which no financial data for specific aid activities was identified, the classification was based on non-financial information: calculations were made proportionately to the number of activities presented in each aid activity’s descriptive information, with each objective weighted equally. For example, if no budget information could be identified and five objectives were located, three that relate to emergency preparedness, one that relates to health information systems, and another that relates to global investments in HSS, then 60% was entered in the health security column, 20% to health information systems, and 20% to global investments in HSS.\n\nHowever, given that entries in the CRS database oftentimes are multi-dimensional in nature and do not include enough information for an in-depth analysis, a field was provided for analysts to indicate how certain they were of the aid activity’s classification. A project manager was available to check uncertain classifications against the codebook. In cases of discrepancy, the activity’s classification was discussed and agreed upon as a team.\n\nGiven the contributions that other sectors make to HSS and health security (e.g., support for humanitarian emergencies and water supply improvements), to arrive at a high-level estimate of total donor support for HSS and health security, we expanded our analysis and conducted a light-touch analysis of additional CRS purpose codes)25. We conducted a key term search using Python 3.6 across 14 additional CRS purpose codes: 14030; 14031; 14032; 14050; 15110; 16010; 16050; 16062; 52010; 72010; 72040; 72050; 73010; 7401026. The following keywords were used to identify relevant projects: Accountability System; Antimicrobial Resistan; Care Service; Cholera; Clinic; Clinical Service; Cold Chain; Conditional Cash Transfer; Continuum of Care; Coronavirus; Crimean Congo; Detection; Diagnos-; Diagnostic; Disease; Drug; Early Warning; Ebola; Emergency Management; Epidemic; Essential Intervention; Facil-; Fever; Financial Management System; Financial Risk Protection; Haemorrhagic Fever; Health; Hendra; Hospital; HSS; Hygiene; Illness; Infectio-; Influenza; Information System; Inventory; Laborator-; Lassa; Leptospirosis; Malnutrition; Marburg; Meningitis; MERS; Morbidity; Mortality; Nipah; Outbreak; Pandemic; Plague; Primary Care; Psychosocial; Rift Valley; SARS; Sector-wide approach; Service delivery; Smallpox; Social Determinant; Supply system; Surveillance; SWAP; System strengthen-; System strengthening; System-Wide; Tularemia; UHC; Universal Access; Vaccine; Vector control; Virus.\n\nThe key term search for investments outside of the health sector identified 4,268 aid activities, with a funding volume of US$4.3 billion. We assessed the largest 1,135 aid activities based on the ‘USD Disbursement’ column (US$3.9 billion), equivalent to 90% of the total funding. Most of the aid activities identified through our search included components were not health related. Therefore, a different framework than that used for classifying the health sector was necessary.\n\nThe aid activities identified through the key term search were classified as either health security or system-wide HSS. We used a semi-automated approach to assess these projects. Based on the information in the CRS, the projects were first classified on a numeric system: ‘0’: There is no explicit reference to health or direct benefit to an area relevant to health; ‘1’: Health is only one of many elements explicitly stated in CRS long description. Health accounts for approximately 25% of the project’s resources; ‘2’: Health-relevant components account for approximately half of an activity’s components. Health accounts for approximately 50% of the project’s resources; ‘3’: Nearly all elements explicitly stated in CRS long description are directly relevant to health. Health accounts for approximately 75% of the project’s resources; ‘4’: All elements explicitly stated in CRS long description are directly relevant to health. Such a scaling method was used by Grépin et al. in their assessment of donor funding for health policy and systems approach27. Appendix, Panel 2, Extended data provides examples of our classification method19.\n\nThe decision tree (Figure 2) summarizes how the classification process was operationalized.\n\nDAH, development assistance for health; CRS, Creditor Reporting System; HSS, health systems strengthening; PRNDs, poverty-related and neglected diseases; RMNCAH, reproductive, maternal, newborn child and adolescent health; NCD, non-communicable diseases.\n\nFinally, we analyzed the overall fit between our estimates of DAH flows and country needs through a cross-sectional analysis, fitting ordinary least squares (OLS) regression models that estimate the relationship between flows of DAH to system-wide HSS and health security and measures of country capacity. Analyses were run in Stata 14.2. We used maternal mortality ratio (MMR; modeled estimates of maternal mortality per 100,000 live births) as a proxy for general health system capacity, and joint external evaluation (JEE) core capacity scores as a measure of national preparedness to prevent, detect, and respond to health security threats.\n\nWe controlled for gross domestic product (GDP) and population and estimated three main models to assess the relationship between DAH for health system strengthening and health system capacity, the latter proxied by estimated MMR. Model 1 was a bivariate regression, model 2 included GDP as a covariate, and model 3 included population as a covariate. GDP and population are correlated, and we did not include both variables in a single regression to avoid introducing multicollinearity. MMR is positively and significantly associated with DAH in all specifications. We conducted additional robustness checks, with alternative model specifications including log-transformed independent variables, as well as alternative measurements of maternal mortality (including lagged models and averaging maternal mortality rates over the period 2010–2014), with consistent results.\n\nWe estimated three main models to assess the relationship between DAH health security and health security capacity, measured by JEE core capacity scores. As above, model 1 was a bivariate regression, model 2 included GDP as a covariate, and model 3 included population. We expected that lower JEE scores would be associated with higher DAH health security flows (or conversely, that better-prepared countries would receive lower DAH flows). To check the robustness of our results, we estimated additional models using log-transformed GDP and population variables, dropped extreme outliers, and included a binary variable for three primary countries impacted by the 2014–2015 West Africa Ebola outbreak, which received large inflows of DAH health in 2015, with no substantive changes in results. All models omit two high-leverage outliers (countries with DAH health security exceeding 40,000,000 USD, and with an average (rescaled) JEE measure of 80), but results were robust to their inclusion once control variables were introduced.\n\nData on 2015 country GDP, population, and MMR were extracted from the World Bank World Development Indicators (see Source data). JEE scores were downloaded from Resolve to Save Lives, which abstracted JEE scores from published mission reports; we estimated an aggregate JEE score by taking the mean value of all JEE indicators (results reported here use a rescaled version of the variable, ranging from 0–100). At the time of analysis, 88 countries had completed the JEE process and published mission reports, of which 71 were aid recipients represented in the DAH dataset.\n\n\nResults\n\nIn 2015, of the US$22.9 billion in DAH disbursements in the CRS, we estimated that US$3.1 billion (13.4% of total DAH) was for UHC through system-wide HSS (US$2.5 billion, 10.9% of total DAH) and system-wide health security (US$570.0 million, 2.5% of total DAH) (Figure 3). The remaining US$19.8 billion of total DAH was for program-specific intervention support (56.9% of total DAH), program strengthening (17.7% of total DAH), and other global investments (12.0% of total DAH).\n\nHSS, health systems strengthening.\n\nOf the US$2.5 billion in system-wide HSS support, US$2.2 billion was country-level DAH (9.5% of all DAH; Figure 3), and US$311.4 million (1.4% of all DAH) was ‘global’. Infrastructure, governance, and workforce together accounted for about 72% of all country-level DAH for HSS; the remaining 28% was for the strengthening of countries’ health financing policies, health information systems, and supply chains (Figure 4). In addition, we identified approximately US$417.3 million towards system-wide HSS in purpose codes outside of our definition of DAH, largely for infrastructure and water supply (Figure 5). This brings our total estimate of donor support for HSS to US$2.9 billion.\n\nDAH, development assistance for health.\n\nA total of US$1.7 billion in DAH was disbursed by donors for health security (7.5% of total DAH) (Figure 5). About two-thirds (66.9%) of this funding was program-specific investments in response to individual health emergencies (not counted towards our estimate of funding for UHC through health security), and 18.3% was for more fundamental system-wide support (included in our estimate of UHC through health security). Very little financing was found for health security at the global level (US$255.7 million), equivalent to 14.8% of all DAH for health security, or just 1.1% of total DAH. We identified an estimated US$1.9 billion in additional funding for health security through purpose codes outside of our definition of DAH, largely as humanitarian aid in response to emergencies. This brings the total amount of donor support for all health security to US$3.6 billion.\n\nOf the total DAH invested in 2015, low-income countries (LICs) received 31.2%, lower-middle-income countries (LMICs) received 31.3%, and upper-middle-income countries (UMICs) received 5%. The remainder was regional, unspecified investments, or for global purposes.\n\nSystem-wide HSS. LICs and LMICs accounted for 69.3% of all DAH for system-wide HSS in 2015 (more details are in Appendix, Table 7–8, Extended data)19. In terms of regional distribution, the region that received the largest share of DAH for system-wide HSS was the Africa Region (38.5% of DAH for system-wide HSS at the country or regional level), followed by the Western Pacific Region (13.6%) and the South-East Asia Region (12.4%). The remainder was for the three other WHO regions (Eastern Mediterranean, the Americas, and Europe) and for global support.\n\nHealth security. Of the total disbursements for health security (US$1.7 billion), LICs accounted for 30.8% (US$530.2 million), while LMICs received 22.6% (US$388.6 million) and UMICs 4.5% (US$78.3 million). The remainder was regional, unspecified investments, or for global purposes. Nearly a quarter (23.7%) of the total DAH for health security was allocated to the three countries that were most heavily affected by the 2014-2015 Ebola outbreak in Western Africa – Liberia, Sierra Leone, and Guinea. The countries with the highest ratio of health security support to total DAH were the West Bank and Gaza Strip, Yemen, and Liberia, all on the OECD’s 2015 list of fragile states (Appendix, Table 9–10, Extended data)19.\n\nThe proportion of DAH provided by donors to these areas varied widely by donor. Government donors that prioritized system-wide HSS in their DAH portfolios in 2015 were Australia (43.4% of its DAH portfolio went to system-wide HSS), Korea (42.7%), the European Union (38.1%), Germany (36.3%), and Japan (34.0%). Government donors that prioritized health security (including program-specific investments) in their DAH portfolios in 2015 were France (17.4% of its DAH portfolio went to health security), Japan (13.1%), and Germany (13.0%). The International Development Association (IDA) and WHO contributed over half of all multilateral support for system-wide HSS (41.3% and 21.2%). IDA and the United Nations Relief and Works Agency for Palestine Refugees in the Near East contributed over half of all multilateral support for health security (33.4% and 21.7%, respectively).\n\nWe found a positive, statistically significant association between DAH flows to system-wide HSS and maternal mortality ratios, suggesting that weaker health systems receive higher levels of DAH for HSS. However, we found no statistically significant relationship between JEE scores and DAH for health security (including program-specific support in response to individual health emergencies).\n\nAcross all model specifications, we did not find a correlation between DAH for health security and country capacity to respond to public health risks, as measured by the JEE, a finding that may reflect data limitations - several JEE evaluations are in progress or yet to be completed (Appendix, Panel 3, Extended data)19. This is an important avenue for future research, as LICs and MICs with the weakest health systems and greatest vulnerability to health emergencies are likely to also have greater difficulty mobilizing domestic financing for these functions and a greater need for DAH. Appendix, Panel 3, Extended data provides more detailed findings on the regression analysis findings19.\n\n\nDiscussion\n\nIn 2015, we estimated that donors invested US$2.2 billion in DAH for system-wide health security. In addition, we estimated US$1.9 billion for health security outside of the health sector, including response efforts and US$417 million for system-wide HSS.\n\nOur estimate of overall donor spending for system-wide HSS is similar as a proportion to the estimate by the Institute for Health Metrics and Evaluation (IHME). Although IHME used a very different methodological approach and covers additional financing (Appendix pp. 20–21, Extended data)19, it found that 9.4% of total DAH went towards HSS in 2015, nearly identical to our study’s estimate of 9.5% for system-wide HSS (both estimates exclude investments that are transnational). IHME does not produce detailed project-level data, thus our disaggregated estimates for HSS by cannot be compared by building block. IHME reports that only 0.8% of DAH in 2015 was directed at pandemic preparedness, slightly below our most comparable estimate (1.4% of total DAH for system-wide health security, i.e., excluding programmatic support in response to health crises and global investments)23. We found significantly less financing for the health workforce than Micah et al. found13, which may reflect differences in methodologies.\n\nThe study illuminates three important findings on how DAH is channeled and directed. First, we found that most investments in health system was reactive to emergencies, rather than proactive investments in preparedness (such as pandemic preparedness). Thus, it is likely that donors are massively under-investing in preparedness relative to need11. Second, our study found that 12.5% of all DAH for HSS and 14.8% of DAH for health security in 2015 was delivered at the global level, compared to 14.5% of overall DAH (a total of US$3.3 billion). This suggests that DAH for HSS relative to health as a whole is more focused on benefiting specific, individual countries. A key question going forward is how much donors should be spending on global functions versus country-specific support? Yamey and colleagues argue that donors will see higher returns if they rebalance their portfolio towards the global level28. Thirdly, we found that LICs (the countries with the least ability to fund health domestically) accounted for 31% of all DAH for system-wide HSS in 2015. Going forward, donors should consider targeting LICs when they make their resource allocation decisions as DAH is likely to play an important role in funding HSS in LICs. Collectively, these findings can be used to help guide evidence-based decision-making for global health donors who are looking to understand how much DAH is available for the reduction of cross-border threats and the strengthening of health systems. They also provide important data for monitoring and advocacy related to closing the financing gap for achieving UHC at large.\n\nWe believe our study is particularly timely. Experts believe that the devastating health and economic consequences of the novel coronavirus disease 2019 (COVID-19) serve as a reminder of the vulnerability transnational health threats pose on health systems and the importance of the SDGs, especially UHC, in curbing pandemics and other transnational health threats29. Additional investments in country health systems and the global health system in particular will be needed to more effectively prevent and curb transnational health threats. Going forward, it may be appropriate for national governments to thus lead the response to investment needs in HSS and for donors to lead the response on global functions and health security28. Monitoring support for these areas serves as a way for holding donors accountable for these investments, informing evidence-based policy decisions and measuring progress going forward. Our study creates a framework for monitoring trends that is well-aligned with Stenberg and colleagues’ framework for assessing financing needs. Our framework may thus create a structure and methodology for more closely monitoring the investment gap going forward8.\n\nThere are at least seven key limitations to our study. First, our assessment focused on 2015 investments alone, so we are unable to make any inferences about time trends. Second, our analysis is limited to disbursements by donors who report their investments to the CRS. It includes US$13.2 billion less in DAH financing for 2015 than is captured by IHME. As such, it does not capture corporate donations, DAH from China, and some multilateral funding that is captured by IHME using additional data sources. Our estimates should thus be interpreted as underestimates. Third, our study focuses on disbursements rather than commitments, meaning that aid activities may not reflect the intent of the donors. That said, disbursements are closer to funds available. Our database contains projects without disbursement data at the time of the analysis (approximately US$140 million in additional financing). This reflects that our study relies on a CRS database close to the end of the reporting year. It may also refer to projects for which a donor is reporting a commitment but has not yet disbursed. Future studies could avoid this by predicting disbursements from commitment data. Forth, we included a purpose code outside of the OECD’s definition of aid to health (‘social mitigation of HIV’) in our estimate of DAH. Given that we have not included other related purpose codes in our definition of DAH, this creates a slight bias. Fifth, we based the assessment of aid activities outside of the health sector based only on the limited information available in the CRS. Sixth, our analysis relies on a review by multiple analysts of limited available information against a highly complex framework. Although we put efforts in place to harmonize the classification, our method is vulnerable to imperfect inter-coder reliability. Finally, although the approach of using a non-random sampling technique was taken to capture the greatest amount of financing with available resources, such an approach introduces a sampling bias and means we have not captured the contents of smaller projects.\n\nA future study should be expanded to include domestic financing, to cover multiple years, to more critically assess support outside of the health sector, include commitments in addition to disbursements, and to assess donors that do not report to the CRS. Improvements could also be made to the CRS to better deliver on this method and to measure support for UHC at large – for example, by including more donors, encouraging more descriptive information, or by introducing a ‘policy marker’ for UHC. A policy marker is a way of tagging or marking aid activities that target a particular focus area30. This study reconfirms that tracking techniques need to take into account trade-offs between various factors like timeliness, transparency, precision, and replicability31. It also reinforces the need for the global health community to work towards a common definition of how DAH is defined going forward and to consider how country need is taken into account when making financing decisions, particularly regarding health security investments. We suggest that a future study explores the relationship between country need and financing in more detail (e.g., by examining system-wide health security and other estimates of pandemic preparedness against JEE since the JEE is mainly about managing transboundary risks and not localized humanitarian crises). We hope that our analytical framework can be leveraged for identifying additional health investments needed to achieve UHC and SDG3.\n\n\nData availability\n\nThe original source file for the underlying aid activity data is the OECD DAC’s CRS dataset, available on the OECD’s website: http://stats.oecd.org/Index.aspx?datasetcode=CRS1. The dataset for this analysis is based on the OECD’s June 2017 update of the 2015 CRS database. The file can be found by clicking ‘Export’, ‘Related files’, then by selecting the “CRS 2015 data.zip” file. The file was the most up to date 2015 data file at the start of the analysis.\n\nFor the regression, data on 2015 country GDP, population, and maternal mortality ratios were extracted from the World Bank World Development Indicators (https://data.worldbank.org/indicator/NY.GDP.MKTP.CD; https://data.worldbank.org/indicator/SP.POP.TOTL; https://data.worldbank.org/indicator/SH.STA.MMRT, respectively). The JEE scores were abstracted and structured by Prevent Epidemics, and were extracted in August 2019 (https://preventepidemics.org/wp-content/uploads/excel/all-countries.xlsx).\n\nOpen Science Framework: Development assistance for UHC through health systems strengthening and health security. https://doi.org/10.17605/OSF.IO/B5YAX19.\n\nThis project contains the following underlying data:\n\nDataset.xlsx\n\nOpen Science Framework: Development assistance for UHC through health systems strengthening and health security. https://doi.org/10.17605/OSF.IO/B5YAX19.\n\nThis project contains the following extended data:\n\nAppendix.pdf\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\nCode availability\n\nReproducible code is available at: https://github.com/jessckraus/dahuhc/tree/v1.0.\n\nArchived code at time of publication: https://doi.org/10.5281/zenodo.385807126.\n\nLicense: Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).",
"appendix": "Acknowledgements\n\nWe would like to thank Mathilde Garret for her support developing figures for the original report and Alexander Reitzenstein for his analytical support. We thank Paolo Piva for commenting on the study design, framework, and drafts of the original report.\n\n\nReferences\n\nGoal 3: Sustainable Development Knowledge Platform. Sustainabledevelopment.un.org. [cited 7 August 2018]. Reference Source\n\nUniversal health coverage (UHC). Who.int. [Cited 7 August 2018]. Reference Source\n\nTogether on the road to universal health coverage: a call to action. Geneva: WHO. [Cited 20 Nov. 2019]. Reference Source\n\nKutzin J, Sparkes S: Health Systems Strengthening, Universal Health Coverage, Health Security and Resilience. Bull World Health Organ. 2016; 94(1): 2. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWHO Director-General addresses the Executive Board. World Health Organization. 2018. [cited 7 August 2018]. Reference Source\n\nGhebreyesus T: All Roads Lead to Universal Health Coverage. Lancet Glob Health. 2017; 5(9): e839–e840. PubMed Abstract | Publisher Full Text\n\nSoucat A: FIT for context, FIT for purpose Health Systems. Building health security, Achieving the SDG [cited 29 October 2018]. Unpublished\n\nStenberg K, Hanssen O, Edejer TT-T, et al.: Financing Transformative Health Systems Towards Achievement of the Health Sustainable Development Goals: A Model for Projected Resource Needs in 67 Low-Income and Middle-Income Countries. Lancet Glob Health. 2017; 5(9): e875–e887. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWatkins DA, Qi J, Kawakatsu Y, et al.: Resource requirements for essential universal health coverage: a modelling study based on findings from Disease Control Priorities. The Lancet Global Health. 2020; 8(6): e829–e839. Publisher Full Text\n\nCommission on a Global Health Risk Framework for the Future, National Academy of Medicine: The Neglected Dimension of Global Security: A Framework to Counter Infectious Disease Crises. Washington, D.C: National Academies Press; 2016. PubMed Abstract | Publisher Full Text\n\nDieleman JL, Sadat N, Chang AY, et al.: Trends in Future Health Financing and Coverage: Future Health Spending and Universal Health Coverage in 188 Countries, 2016-40. Lancet. 2018; 391(10132): 1783–1798. PubMed Abstract | Publisher Full Text | Free Full Text\n\nInstitute for Health Metrics and Evaluation (IHME): Financing Global Health Visualization. 2018. [cited 7 August 2018]. Reference Source\n\nMicah AE, Zlavog BS, Chen CS, et al.: Donor Financing of Human Resources for Health, 1990–2016: An Examination of Trends, Sources of Funds, and Recipients. Global health. 2018; 14(1): 98. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMoon S, Oluwatosin O: Development assistance for health: critiques and proposals for change. Health Econ Policy Law. London: Chatham House. Reference Source\n\nYamey G, Jamison D, Hanssen O, et al.: Financing Global Common Goods for Health: When the World Is a Country. Health Syst Reform. 2019; 5(4): 334–349. PubMed Abstract | Publisher Full Text\n\nSchäferhoff M, Fewer S, Kraus J, et al.: How Much Donor Financing for Health Is Channelled to Global Versus Country-Specific Aid Functions? Lancet. 2015; 386(10011): 2436–2441. PubMed Abstract | Publisher Full Text\n\nJamison DT, Summers LH, Alleyne G, et al.: Global Health 2035: A World Converging Within a Generation. Lancet. 2013; 382(9908): 1898–1955. PubMed Abstract | Publisher Full Text\n\nJamison DT, Frenk J, Knaul F: International Collective Action in Health: Objectives, Functions, and Rationale. Lancet. 1998; 351(9101): 514–517. PubMed Abstract | Publisher Full Text\n\nKraus J: Development assistance for UHC through health systems strengthening and health security.2020. http://www.doi.org/10.17605/OSF.IO/B5YAX\n\nThe role of philanthropy in financing for development. Oecd.org. [cited 21 Nov 2019]. Reference Source\n\nPurpose Codes: sector classification - OECD. Oecd.org. [cited 7 August 2018]. Reference Source\n\nAid to Health. OECD: Oecd.org. [cited 7 August 2018]. Reference Source\n\nThe Henry J. Kaiser Family Foundation: The U.S. Government Engagement in Global Health: A Primer. The Henry J. Kaiser Family Foundation. [cited 7 August 2018]. Reference Source\n\nCenter for Global Development: Index of the Quality of Official Development Assistance in Health. [cited 8 May 2020]. Reference Source\n\nUHC2030: Global compact for progress towards universal health coverage. uhc2030.org. [cited 7 August 2018]. Reference Source\n\njessckraus: jessckraus/dahuhc: F100 Manuscript (Version v1.0). Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3858071\n\nGrépin KA, Pinkstaff CB, Shroff ZC, et al.: Donor funding health policy and systems research in low-and middle-income countries: how much, from where and to whom. Health Res Policy Syst. 2017; 15(1): 68. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYamey G, Nonvignon J, Cordelia K: Modernizing Our Public Health Systems to Be Ready for the Next Pandemic. [cited 8 May 2020]. Reference Source\n\nTakian A, Aarabi M, Haghighi H: The role of universal health coverage in overcoming the covid-19 pandemic. [cited 10 May 2020]. Reference Source\n\nThe Policy Marker System: The Policy Marker System. BMZ. [Cited 7 Dec. 2018]. Reference Source\n\nPitt C, Grollman C, Martinez-Alvarez M, et al.: Tracking aid for global health goals: a systematic comparison of four approaches applied to reproductive, maternal, newborn, and child health. Lancet Glob Health. 2018; 6(8): e859–e874. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "83868",
"date": "17 May 2021",
"name": "Leonardo Arregocés",
"expertise": [
"Reviewer Expertise I have previously worked on estiamting development assistance to reproductive",
"manternal",
"newborn",
"and child health using a similar method to the one used by the authors. Also participated in publications that reflec on methods for estimating disbursements of develpoment assistance to health. I know the CRS data quite well."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 from Jessica Kraus, Gavin Yamey, Marco Schäferhoff et al. presents an evolution of methods for estimating development assistance for a specific focus area. It presents an analytic framework that allows to classify the transactions reported to the Creditor Reporting System (CRS) into neatly separated investment areas. The framework was then applied to the 2015 data set from the OECD CRS to produce estimates of total and disaggregated disbursements for Development Assistance for Health (DAH). A latter analysis of targeting of DAH disbursements related to need was assessed through regression analysis.\nThis paper can be considered another step towards having better tools to analyse development assistance. Although is not a completely new approach, it does make an important improvement in defining a neat framework to classify DAH. The text does go through other approaches to estimate DAH and presents how it compares to those, including the benefits of this new approach.\nThe study design shows the knowledge and experience that the authors have on the context and the data sources. The approach decided, which privileges the careful reading of the data in the CRS and further search of missing data in other sources, over including all the records in the dataset that seems appropriate. The process is labour intensive, but the additional effort of searching for additional data was very important. Only one-fourth of the records seem to be based solely on CRS data. The remaining required additional efforts to search for details. That means that not only the effort in classifying was meaningful, but the apportioning of the disbursement into the different categories is quite valuable. Previous similar efforts which included coding all records did not include searching for additional data, relying only on assumptions on the reported information. Although assumptions were adequate, they could not allow for deeper analyses. Technically the work is adequate.\nThe methods have been well described. They are clear despite the complexity of the work done. Process of analysis is well described in the text and in the additional data. The data necessary to replicate the work is available, although quite a bit of the work is analyst dependant. The paper describes all measures taken by the authors to ensure consistency in the coding as much as possible. It could be useful to make some quantitative measure of intercoder consistency. The data file annex to the paper makes much clearer how the coding was made and allow to reproduce the results.\nProbably the weakest part of the method are the searches in purpose codes other that health. It is not described the criteria for creating the list of keywords. The purpose of running searches in other purpose codes is to not leave outside the estimates transactions that can count towards health. Assurance that nothing was left out can be shown by explaining the comprehensiveness of the keywords and the proportion of identified records that were included in the estimates. Authors could reflect on possible ways to identify transactions that can be related to health, but are not included in health purpose codes.\nAssessment of targeting of DAH to needs was done using linear regression. This approach has been used elsewhere. Three models were defined using different covariates. In one GDP was used and in the other population size. Is argued that both could not be used at the same time because of collinearity, but probably using GDP per capita would have been a good alternative to adjust for both. The results for the assessment of targeting to need are according to what is expected. Countries with higher maternal mortality rates receive more support. Coefficients for GDP is also positive suggesting that countries with higher incomes receive more money. This is not discussed in the paper and it seems counter intuitive. This might be suggesting that the relation between DAH and need is not linear, although authors used a log – log specification and found the same results. Probably donors prefer to target the more developed countries. A scatter plot of DAH and GDP labelling countries with different colours according to a categorised level of income.\nData sources are well described, and a processed data set is made available. Besides the paper, producing a dataset that can be further used for other types of analysis is a valuable contribution. Many questions can be answered with the available data. A problem with the dataset for further use is that some columns from the CRS are missing. If the unique ID were generated by the authors, I would suggest making available the same dataset with all columns in the CRS for the selected records so further analyses are feasible.\nDiscussion and conclusion reflect on the main points. All the topics covered in the paper are touched, although they are very broad. Being this a paper that is presenting an improvement on previous methods, which has a big potential for deeper analyses of the effectiveness of DAH in achieving SDGs, I would suggest reflecting more on the precision of the estimates, the effort and plausibility of continuing this work, and probable future improvements. Because this kind of approach is labour intensive, some discussion on the means to keep processing the data could be valuable so this is not a one time only. This can be accompanied by the future uses the authors see of the estimates and further questions that can be answered.\nThis paper covers many topics which makes it quite complicated to stuff all that in a few thousand words. Authors do a great job in that aspect, leaving behind other reflections that are necessary. The way the method is describes and the data that accompanies the paper is good proof of a very hard work and high quality work.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "94393",
"date": "31 May 2022",
"name": "Ashley Fox",
"expertise": [
"Reviewer Expertise Global health policy analysis",
"health politics and policy",
"political economy of development."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article by Kraus et al. offers an important analysis of how much development assistance for health is allocated towards specific activities of health system strengthening (HSS) and health security which are important pillars to achieving universal health coverage. The article provides a great step towards attempting to count development assistance for health (DAH) towards specific health issues and disaggregating the flow by the building blocks of the health system. Furthermore, the study marks an essential step towards expanding our understanding of not only the impact of DAH on HSS and health security but also towards our ability to better comprehend the contribution of each of the building blocks to achieving Sustainable Development Goals including universal health coverage.\nThe study approach coveys the authors' understanding of the data and sources. The study design seems appropriate for tracking DAH flow using Creditor Reporting System (CRS). The authors provide a clear description of data sources and a careful, detailed explanation of the methodology which allows for replication. Although the analysis is pretty complex, the authors presented an excellent and meticulous illustration of their process and provided a useful framework to understand the DAH flow towards HSS and health security.\nThe authors’ classification of DAH flow included country-region-specific health programs, system-wide programs, and funds contributing to global function which provide a good view of DAH health investment. When applying the framework to CRS, the authors attempted to provide an accurate calculation of aid activities. However, their attempt to accurately calculate DAH flow was met with an inability to follow the same process to count the contribution of other sectors to HSS and health security. The authors then had to conduct additional CRS purpose codes using key search terms which was a necessary step to achieve a higher level of accuracy but a complex step because the search terms revealed aid activities that are not related to health. The authors then had to swift through these outputs and further code based on the estimation of health-relevant components of the allocations. Nevertheless, the authors described, in good detail, all decisions that needed to be made and their rationale which shows the high level of transparency accompanying the work.\nThe authors analyze the data using linear regression models to measure the relationship between DAH flow and the country’s health system capacity using maternal mortality rates (MMR) as a proxy controlling for GDP and population and found a strong positive association between DAH flow and MMR. The authors also used a similar model to estimate the relationship between DAH flow and the country’s need using joint external evaluation (JEE) as a measure for health security capacity and found no significant relationship between them which they credited to data limitation from JEE score. I think this might be the weakest point of the study because of the data limitation from JEE (limitation of MMR as a proxy for health care capacity). In addition to the limited ability of linear regression to provide strong associations of country-wide measures without controlling for countries' specific effects beyond GDP and population.\nIn the discussion section, the authors provided an important comparison between their estimate and the IHME estimates of DAH flow and found a close resemblance which provides further confidence in the results.\nThis study is very important and timely due to the expected economic downfalls resulting from the COVID-19 pandemic. The conclusion highlights the important contribution this study provides. The framework allows an opportunity to expand our understanding of the impact of DAH on HSS and health security. The study highlights the importance of creating more investments by allocating funds to strengthen the health care system and improve pandemic preparedness, especially for low- and middle-income countries.\nComments/questions:\nIntroduction\n\nThe third paragraph contains the first reference to the building blocks of the health system and then later in the methodology they reference the six building blocks of the health system presented in Stenberg et al.’s 2017 conceptual framework. Could you please clarify the building blocks framework at the beginning? Stenberg et al.’s 2017 conceptual framework include similar to WHO building blocks, which I believe are introduced in 2010 here. The only difference I noticed is in the terminology of “medical technology: in the WHO and “Supply chain” in Stenberg et al.’s framework.\nMethods\nPage 4: Can you clarify the rationale behind including \"…a component for ‘system-wide emergency preparedness, risk management and response’ for activities that strengthened capacity to deal with epidemic and pandemic preparedness and response, e.g., a mobile emergency alert system\" with the system-wide HSS and not with “Global functions”, given that pandemic and endemic preparedness have as much of a transnational benefit as a local/domestic effect.\n\nPage 5: “…to arrive at a high-level estimate of total donor support for HSS and health security, we expanded our analysis and conducted a light-touch analysis of additional CRS purpose codes). We conducted a key term search using Python 3.6 across 14 additional CRS purpose codes” - Can you clarify what is “light-touch analysis”?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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/9-584
|
https://f1000research.com/articles/8-2140/v1
|
23 Dec 19
|
{
"type": "Research Article",
"title": "Perception of effective access to health services in Territorial Spaces for Training and Reintegration, one year after the peace accords in Colombia: a cross-sectional study",
"authors": [
"Julián Alfredo Fernández-Niño",
"Lud Magdy Chavarro",
"Ana Beatriz Vásquez-Rodríguez",
"Maylen Liseth Rojas-Botero",
"Ginna Esmeralda Hernández-Neuta",
"Ana Maria Peñuela-Poveda",
"David Alejandro Rodríguez",
"Lud Magdy Chavarro",
"Ana Beatriz Vásquez-Rodríguez",
"Maylen Liseth Rojas-Botero",
"Ginna Esmeralda Hernández-Neuta",
"Ana Maria Peñuela-Poveda",
"David Alejandro Rodríguez"
],
"abstract": "Backgrounds: The signing of the peace accords in Colombia created challenges that are inherent to post-conflict transitions. One of those is the process of reintegrating ex-combatants into society, in which ensuring their rights to health is a particularly significant challenge in rural areas affected by armed conflict. These areas, known as Territorial Spaces for Training and Reintegration (ETCR, in Spanish), are geographically dispersed throughout 24 municipalities and 13 departments in Colombia. This study aimed to describe how ex-combatants in ETCR regions perceived access to health services one year after the signing of the peace accords. Methods: A descriptive, cross-sectional study was performed between September and October 2018. It included 591 adults and their families, from 23 ETCRs. The study was designed, culturally validated, and piloted. Interviewers were trained and a structured survey was administered containing five dimensions that characterized the perception of effective access to health services. Results: The majority of interviewees were women, heads of household, young adults, ex-combatants, and residents in an ETCR. In total of 96.4% were enrolled in Colombia’s subsidized health system, and 20.8% indicated that a member of their household required emergency health services. The regional health center provided the majority of the services. Most of those surveyed (96.0%) reported that they did not have to pay for the services, and that they received respectful (91.6%) and good quality (66.6%) care. There were few referrals to disease prevention and health promotion activities, and only 19.0% of households reported having been visited by extramural health care teams, whose activities were highly valued (80%). Lastly, there was little knowledge about community health activities. Conclusions: While residents of ETCR regions have a favorable perception of their access to health services, they need to be made aware of extramural and public health activities.",
"keywords": [
"Health Services Accessibility",
"Armed Conflicts",
"Colombia"
],
"content": "Introduction\n\nAs a human right, health is crucial to human and social development. Societies that reach an optimal level of health are able to ensure that their populations attain higher individual and collective levels of human development1,2. For a society that is going through a period of post-conflict, guaranteeing health as a right demonstrates the State’s interest in taking steps towards renewing the social bonds among the parties that were broken by the conflict. Effective access to health services expresses the recognition of the right to health, making it possible to prevent illness and to address the population’s health needs3.\n\nHistorically, the concept of access to health services has had multiple definitions that correspond to the structural values of each health system. Most of the models and definitions of access are based on the logic of markets and the satisfaction of needs, with very few taking the perspective of health as a right4. United Nations General Observation 14 defines the effective exercise of the right to health as availability, accessibility, acceptability, and quality —elements or dimensions that are essential to enjoying effective access to health services2.\n\nProviding access to health services for the entire population is an enormous challenge for a society that finds itself in the midst of armed conflict or in a period of post-conflict. This is the case of Colombia after the signing of the Final Accord (November 2016) between the federal government and FARC-EP (Fuerzas Armadas Revolucionarias de Colombia—Ejército del Pueblo; Revolutionary Armed Forces of Colombia—People’s Army) revolutionary forces to End the Armed Conflict and Build a Stable and Lasting Peace5. As a result, Colombia finds itself navigating through a critical process that has great social, economic, and political impact. A key factor that ensures the end of the conflict and a stable and lasting peace is preparing for the reintegration of ex-combatants into all areas of civilian life —economic, political, and social. To this end, Territorial Spaces for Training and Reintegration (ETCR, in Spanish) were established to relocate former FARC-EP members and establish the means and inputs needed to fulfill the promises and responsibilities that were set out in the accord, including supplying this population with comprehensive humanitarian aid through national and international organizations6.\n\nOne promise that is aimed at ensuring the reintegration of ex-combatants is promoting and guaranteeing their right to health in rural areas and small towns that lack the institutional and budgetary capacities to provide social and health services, and that historically have not received State investments. Interventions by governments and international agencies are challenged by budgetary limitations due to the economic crisis, as well as by operational limitations in light of the large number of ETCRs and their geographic dispersion throughout Colombia. Added to this is the increased demand for health services, especially by pregnant women and young children, as well as signs of a lack of confidence in the quality of health care and its capacity to respond to needs, all of which could affect the perception of access to health services in the ETCRs.\n\nBased on this approach, perception serves as a differentiating construct. It is defined as “a mental process that enables forming judgments or categories that define reality, of interest to health because it enables identifying the population’s impressions of health status, the resolving of needs, and the interventions performed.”7 Perception more accurately shows “the appreciation of reality by obtaining a set of concepts and attitudes that are associated with the health care provided and received.”8 This is an alternative to representing access to health services based solely on the indicators that are generated by health care providers and authorities, which explore comprehensive access management in a simpler manner. Some examples of this can be found in most of the current surveys that have been developed in Colombia, including the Evaluation of Services by Health Promoter Entities (EPS in Spanish)9, as well as the National Health and Nutrition Survey in Mexico (ENSANUT, in Spanish)10. The objective of the study herein was to describe how ex-combatants living in the ETCRs perceived access to health services one year after the signing of the peace accords. This study captured the specific impressions of effective access to health services by taking into account the health needs of the intervention community, which can be accomplished by measuring two dimensions that make up part of the perception of a community: contact coverage and satisfaction11. From the perspective of the right to health as an analytical framework, these two dimensions come closest to actual access to services, and can best represent that.\n\nTo this end, a survey was developed and administered to the population residing in the ETCRs in order to explore the perception of access to services from the perspective of guaranteeing the right to health.\n\n\nMethods\n\nA cross-sectional, descriptive study was performed. The target population was ex-combatants and their families who were living in 23 Territorial Spaces for Training and Reintegration (ETCR, in Spanish) in September 2018. In each ETCR, a characterization from July 2018 of ex-combatants and their families was used as a sampling framework, which was developed by the IOM (International Organization for Migration) field team. The family was the study unit, although the informant was the head of the household in all cases. According to inclusion criteria, at least one of the family members was a FARC-EP ex-combatant, demobilized after the signing of the Final Accord, residents of an ETCR, who consented to participate. There were not any exclusion criteria.\n\nA proportional sampling was calculated with a finite population, taking each ETCR as a non-proportional stratum, and based on the following parameters: total census population of 3,792 people, grouped into 848 families as of the date of this study; 2) expected prevalence of poor perception of effective access of 40%; 3) 95% confidence interval; 4) 23 strata; and 5) design effect of 2. This resulted in a minimum sample size of 529 people, with 23 participants needed from each ETCR. With this sample, estimators for the total population can be calculated by taking into account the source of variance by cluster of total residents in each ETCR, but it does not permit calculating separate estimates for each ETCR.\n\nThe expected value of 40% for effective access is a conservative estimate given that in the few studies conducted in population without payment capacity in Colombia, the perception of low effective access found is greater than 50%12. However, the sample size needed to be adjusted slightly in order to include parameters having more extreme expected values, such as use of emergency services (20%) and health insurance (80%). The first value derives from the same reports where the emergency services have usually the worst evaluation. On the other hand, given that the commitment of the Peace process was to guarantee universal coverage for all ex-combatants, we considered that a value of 80% for health insurance is conservative considering that it had been a year after the signing of the agreement.\n\nIn this way, the sample would enable obtaining a precise estimation of the majority of the parameters of interest. Given the small number of people living in some of the ETCRs, it was decided that all the families would be included when clusters (ETCR) had less than 25 people, and a minimum of 30 people would be included when clusters had over 25 people. This projection, which was slightly larger than the sample needed, was made in order to compensate for losses in the ETCRs that had less than the required number of families, and to anticipate non-differential losses in information.\n\nThe use of a simple random sampling had been planned for selecting the subjects within each ETCR. However, the ex-combatants considered this to be inconvenient, and therefore, a house-to-house interview (convenience sampling) was conducted in order to identify the sample for this investigation. All the houses located in the ETCR were inhabited by ex-combatants of the FARC-EP and their families. The interviewers applied the survey in all the homes where the head of the household was present and met the inclusion criteria simply following the order of the first houses they found in each ETCR until they completed the required sample size. Results did not present a clear bias, given the high degree of homogeneity in the expected living conditions of the subjects, the high coverage of the sample (which in five cases included the entire population), and the fact that the interviewers did not apply any arbitrary criteria for choosing the interviewees, nor was there any self-selection. This was the best possible approach given the conditions of the population that was required for the sample. The representatives of each ETRC did not accept the possibility of random sampling.\n\nAdditionally, it is important to note that little information exists about the health status of this population, and the construction of a sampling framework was not very feasible because of protection considerations.\n\nA survey was designed in order to identify perception of effective access to health services in the ETCRs.\n\nNo structured instruments exist for measuring effective access to health services for ex-combatants in a context such as Colombia. Therefore, a measuring instrument was designed based on a literature review of perception and effective access to health services from the perspective of the right to health. A review was also conducted of the instruments that are currently available on this topic, particularly those used in rural areas. Based on the findings, perception of effective access to health services was defined as the instrument’s main construct, which was structured according to the domains or dimensions needed for reflecting that construct from the perspective of health as a right, that is: availability, accessibility, acceptability, quality, contact coverage, and satisfaction.\n\nFor the sociodemographic, emergency services, and promotion and prevention modules, some of the items in the instruments related to access to health services for Colombian households were adapted. This included the East Asian Social Survey (EASS) survey that was developed by Arrivillaga, Aristizábal, Pérez & Estrada13 and the instrument that is used to evaluate the services provided by the EPS9. These were adapted in order to ensure traceability with the majority of the indicators that are available for the rest of the Colombian population. The questions that were aimed at determining perception, extramural activities, and community monitoring were designed based on the literature review and the objectives and developments of the Health for Peace project, which served as references.\n\nThe final instrument contained 65 questions that were divided into three modules: 1) sociodemographics of the household (7 questions); 2) perception of effective access to primary health care services (54 questions), which was divided into insurance (2 questions), emergency services (19 questions), health promotion and disease prevention programs (23 questions), and extramural services (10 questions); and 3) community health monitoring (4 questions). All the questions were posed to the head of the family or a member of the household. The head of the family was the person that the family members recognized as most able to provide information about the participants. The study instrument and an English translation are available as extended data14,15.\n\nThe survey was revised and validated using a technique that was applied by professionals from the IOM and MSPS. Face and content validation was performed; the experts evaluated relevance and comprehensiveness of the items in two focal groups reviewing each item of the instrument. Besides, the interviewers used a socio-cultural method to adapt the terms and definitions in the survey for better comprehension on the part of the population of interviewees. The interviewers were nursing professionals located in each municipality and were highly experienced in community work and in collecting information for public health. The team’s researchers trained the interviewers in administering the surveys during a one-day session, which ended with a qualitative evaluation.\n\nA pilot test was conducted, which consisted of administering the survey in six ETCRs, with a total of 43 interviews. Based on this, the first socio-cultural validation of the survey was performed, in which the interviewers had the opportunity to provide feedback based on their experience administering the survey. A second socio-cultural validation was conducted during an in-person training session with the interviewers from each municipality. This involved reviewing each dimension, its acceptability, and the interviewees’ understanding. In this session, role-playing and simulation workshops were conducted and the entire instrument and its application were reviewed.\n\nFor the final administration of the survey, an interviewer was assigned to each one of the 23 ETCRs. These ETRCs were located in the following municipalities: Anorí, Dabeiba, Ituango, Remedios and Vigia del Fuerte (Department of Antioquia); Arauquita (Arauca); La Montañita and San Vicente (Caquetá); Buenos Aires, Miranda, and Caldono (Cauca); “Region between La Paz and Manaure” (Cesar); “Region between Carmen de Darien y Riosucio” (Chocó); Fonseca (La Guajira); San José del Guaviare (department of Guaviare); Vista, Mesetas and La Macarena (Meta), Tumaco (Nariño), Tibú (Norte de Santander), Puerto Asis (Putumayo), Icononzo and Planadas (Tolima).\n\nThe survey was conducted in the homes of ex-combatants, and each interviewer interviewed approximately 25 to 30 families between September 2018 and October 2018, thereby obtaining the sample that had been calculated previously. One of the team’s researchers continually supervised the collection of information in order to ensure the quality of the data.\n\nA basic descriptive analysis was performed, in which the qualitative variables were described with proportions and the quantitative variables were described with measures of central tendency and dispersion. In all cases we worked with the valid percentage. All of the analyses were performed with SPSS © version 23.\n\nThis research was approved by the Ethics Committee of the Health Division of the Universidad del Norte (Committee Minutes # 198).\n\nThe objectives of the study were explained to all the interviewees and oral informed consent was obtained before the survey was administered. The informed consent was oral because we wanted to protect the identity of the ex-combatants and it was preferable not to have any document signed by them with personal information for security reasons as well as to make feel them confident. In order to obtain the oral informed consent, after explaining the objectives of the study, and guaranteeing confidentiality, each person was asked if he/she understood the explanation, if not, the explanation was repeated, and if he/she said yes, it was recorded in the questionnaire whether or not the person agreed to participate in the survey. The answer to this question is recorded in each questionnaire. All these procedures were approved by the Ethics Committee.\n\nWe guarantee that all respondents were informed of the objectives and freely invited to participate. They were also told that no benefit or detriment would result from their decision about whether or not to participate.\n\n\nResults\n\nA total of 591 people were interviewed, with a 100% response rate. They were asked about their sociodemographic characteristics, enrollment in the health system, and health services used by themselves or by a family member. The majority of the interviewees were women, heads of household, young adults, did not identify with a cultural group or ethnicity, and were ex-combatants residing in the 23 Territorial Spaces for Training and Reintegration (ETCR) (Table 1).\n\nThe majority said that they were enrolled in the subsidized General Social Security Health System. Only 3.6% (21 people) said they were not enrolled in the health system, either because they did not apply, or they applied but were not registered by the system.\n\nWith regard to emergency services, 20.8% said that they or a member of their household needed those services for some type of health condition or illness the month prior to the survey. Of those, 16.4% did not seek medical care. The majority of those who did seek care went to the health centers or hospitals in their local area, primarily because they were close to their homes. Obstetric care and illnesses that are prevalent in children were the most reported reasons why people sought medical attention (Table 2).\n\nIPS (in Spanish) - Service provider institutions, EPS (in Spanish) - Health promoter entities.\n\nMore than 50% reported that the waiting time to receive care was short, the majority said they did not have to pay for the services (96.0%), that they had been treated respectfully (91.6%), that the quality of care was good (66.6%), and that they had received all the medications that they needed (75.0%) (Table 2).\n\nIn contrast, 25% said that the waiting time was long or very long (over 2 hours for 13.0% of cases), 21.6% did not receive a diagnosis from the professional who provided the service, and 23.9% could not obtain some or any of the medications that were indicated. The reasons mentioned for not being able to obtain the medications were: they were not available at the IPS (service provider institutions) pharmacy or the pharmacy was not open at night or on the weekend, the place where they are obtained is far from where they received the health services, and the medications were not covered by their benefits plan (Table 2).\n\nThe low number of referrals to prevention and health promotion activities was notable, especially in the case of children under 1 year of age (11.9%) (69) and pregnant women (8.1%) (48) (Figure 1–Figure 3). Nonetheless, this may be due to the time at which the report was requested, which in this case was four weeks before the survey was administered. Of those who indicated that they used promotion and prevention services, the majority (53.4%) said that the appointment was made quickly (the next day) and the majority (68%) perceived the quality of the service to be good, including recommendations, care, and exams that were always appropriate for their age. Most of those surveyed (86.8%) also said that they would definitely return to use the services that these types of programs offered (Figure 4 and Figure 5).\n\nThey were asked about whether they were offered and attended health promotion and disease prevention activities and programs over the four weeks before the survey was taken.\n\nThey were asked about whether they were offered and attended health promotion and disease prevention activities and programs over the four weeks before the survey was taken.\n\nA total of 17.3% of households received visits from extramural health teams. Over 80.0% of participants positively evaluated those activities in terms of the relevance of the health education they received, how they were treated, the opportunity to receive new services, and the quality of the services (Figure 6–Figure 7).\n\nLastly, participation in community health activities and knowledge about them were low. In all, 13.7% knew about the development of a community health monitoring system, and 27.7% considered the development of those types of spaces to be important, where different community organizations get involved (Figure 8).\n\n\nDiscussion\n\nFride (cited by Márquez, 2013 and Molano, 2015) defined post-conflict as “the period in which past hostilities have decreased to the level that is needed for beginning reintegration and rehabilitation activities”16,17. The signing of the accord not only involved the political will of institutions but also a commitment by all sectors in the society to building a stable and lasting peace5,17.\n\nProcesses to disarm, demobilize, and reintegrate (DDR) ex-combatants into civilian life, and their return to legality, are crucial challenges for ensuring a lasting peace in the post-conflict period. Negative repercussions have resulted from inadequately planning these processes or from their absence, such as in the case of Namibia18. These consequences not only affect the quality of life of former combatants, including stigmatization and unemployment, but they also put at risk the strengthening of democratic institutions and the social and economic stability of countries, triggering a reactivation of violence18.\n\nDDR processes are indispensable to building peace. Not only do they contribute to ensuring security after conflict by reducing the number of people who return to armed conflict, but more importantly, these programs can make a significant impact and are socially important in terms of capacity-building, local governance, economic projects, economic development conditions, and the reconstruction and reconciliation needed in countries that have undergone these types of internal conflicts19.\n\nThe Colombian government established eight dimensions for the path of reintegration, one of which is access to the General Social Security Health System (SGSSS, in Spanish). It also established health services as a fundamental right to be guaranteed along with returning these people and their families to legality. The inclusion of these dimensions is notable when compared to other successful national policies aimed at the social and economic reintegration of illegally-armed people and groups. For example, while reintegration polices in Indonesia and the Congo have been successful and innovative, and reconciliation mechanisms have been comprehensive and involved the community, health care was mentioned only as a condition that needed to be met, or it was limited to health days or delivery of medical kits18. Colombia is a good example of the comprehensiveness that these programs need when it comes to ex-combatants who are on the path to reintegration, in the short-term. And long-term, sustainable reintegration into civilian life will be ensured by including crucial elements such as effective health care and ongoing access to health services through enrollment in the health system. This will contribute to individual well-being, continued legality, and a solid ground on which to build stable and lasting peace.\n\nWhile prioritizing the right to health for ex-combatants represents important progress in public policy, Colombia’s health system faces challenges that need to be studied in depth along with academic efforts20,21. This issue is important in two ways. First, because of the dispersion of the locations where these populations reside. This concerns point number 1 in the accord (comprehensive rural reform and the development of the National Rural Health Plan), which for some experts means that the health system needs to expand primary health care and change its tendency towards institutionalization and medicalization, since that tendency has led to the intramural provision of services, with little or no access in rural areas to health services, medications, diagnostic services, and/or treatment22,23. And second, because health is indispensable to the path of reintegration. Ensuring it provides the conditions needed for the reintegration process, and guarantees its success.\n\nProjects were developed with the above challenges in mind and in accordance with the reference framework on which the path was laid out, such as Health for Peace, led by the Ministry of Health and implemented by three United Nations agencies (UNFPA, PAHO/WHO, and IOM). This project was created in order to strengthen institutional and community capacities to address health needs in the rural areas where the ETCRs are located, through already-established regional health centers as well as local primary hospitals. Health services were also complemented by the development of extramural strategies and community monitoring, led by regional health professionals with the support of local public health leaders who were trained by SENA as part of the project.\n\nAfter conducting the literature review on access to health services, we decided to administer a survey in order to estimate the perception of effective access to health services in these rural areas. Obtaining an estimation based on perception provides a clearer picture of how these populations have experienced health care services when seeking those services or being treated, and whether that experience of contact with health services has resulted in actually realizing that they can access the services, be treated, and resolve their health needs. This is what resoundingly represents actual access to health services and the actual quality of those services.\n\nWith regard to the instrument, the survey was designed with this interpretative framework of perception so as to directly evaluate access to health services for ex-combatants within the framework of legality. Given that this type of information is not available from other countries that have implemented post-conflict reintegration processes, the results from the present work provide a new methodology and information that can be used to identify the health status of populations that became invisible because of their situation as active actors in armed conflict. The present work also offers an objective evaluation of progress on one of the dimensions of the reintegration program. This has previously been studied only in terms of percentage of people enrolled in the health system, which does not reflect access to health services for ex-combatants in the rural areas where they are located.\n\nIn terms of the sociodemographic composition and enrollment in the health system, the results very closely reflect the global population that was in the process of reintegration in the ETCRs as of the date the survey was administered. Proportional similarities were found when comparing our data with official 2018 data from the Agency for Reintegration and Normalization (ARN)24, with the same trends continuing until the last report by the agency in March 201925. This suggests that the estimations obtained in the study herein adequately represent the characteristics and perceptions of access to health services in the general population of ex-combatants residing in these spaces. With regard to the composition of the population, the majority (56.43%) in the ARN report were 26 to 40 years of age, and the majority (nearly 80%) of those surveyed by the study herein fell within this same age range. With regard to the highest education level, ARN reported that incomplete or complete elementary school was most frequently (32.29%) reported by the population of ex-combatants, while our results found that the largest percentage of this population reported attending high school, when considering elementary, incomplete high school, and complete high school. For sex, the percentage distributions also differed, which could be explained by who responded to the survey. The unit of observation in the study herein was the household and the majority of the respondents were women, whereas the ARN study surveyed individuals in the process of reintegration, the majority of which were men.\n\nWith regard to enrollment in the health system, ARN reported that 80% of the ex-combatants in the process of reintegration were enrolled in the system in September 2018, the majority of whom belonged to the subsidized system. And the work herein shows that over 90% belonged to this system. It is interesting to note that the small percentage that was not enrolled in the system had not applied, suggesting that health services had high administrative capacities for this population, and that the full orientation and consulting process provided by the path to reintegration was functioning for the ex-combatants in the program. This was also seen in the 2014 survey of perceptions by ARN, in which 82% of those who said they received support to access the health system reported that this strategy helped them to gain access26. These results are similar to the findings from the 2015 National Demographics and Health Survey (ENDS, in Spanish) of the rural population, which reported 94% enrollment, the majority in the subsidized system27.\n\nIt is difficult to compare the results from our survey because of a lack of historical data on the health status of populations belonging to armed groups and their access to health services. This fact also makes these results highly valuable as a baseline for planning the provision of health services for this new population that is joining the system.\n\nFor the purpose of comparing the results from our survey on the health status of the ex-combatant population, we compared our findings with access to health services by the general rural population, which currently may be the best control population given the rural location of the ETCRs. The sociodemographic composition can be compared with the general rural population by using the results from the 2015 ENDS survey28, which contains very precise measurements of the population by location. Similarities with our estimates can be seen, in which the overall characteristics of these populations do not differ and appear to be quite comparable. For example, the age of the majority of the general rural population ranged from 20 to 40 years old, a young population in their productive years, which was similar to our findings. On the other hand, in terms of sex, the composition of the general rural population was mostly male, unlike our results in which the majority was female. This can be explained by the fact that our survey studied the distribution of sex of the informants only and not the composition of sex within each household.\n\nIn terms of types of housing, our results differed from the ENDS survey, where ENDS reported that the majority of the general rural population lived in houses, while the majority of our population reported living in other types of structures. This difference is explained by the fact that the population of ex-combatants were located in the ETCRs, which have characteristics that are different than the rest of the rural population. Lastly, the two surveys also differed with regard to highest educational level, with the highest grade being incomplete elementary school for the general rural area and incomplete high school for our population. These differences may be due to our study having characterized the educational level of the main informant only and not that of the entire family. With regard to emergency services over the previous four weeks, the present study reported low usage of these services, and most of those who sought emergency services went to the local hospital. This is in agreement with the general rural area report by the 2015 ENDS. For both surveys, the populations in these regions were healthy young adults, which is also supported by our findings that obstetric care was the main reason for seeking emergency services. With regard to people with a health problem who did not seek health care services, the percentage was lower in our study than in the ENDS survey, which had a high proportion for the general rural area reportedly due to the health services themselves. While our survey did not directly ask why people did not seek care, it did show that a very small percentage did not seek care, and that the reasons were not related with the services offered in the area but rather with not being enrolled in the health system.\n\nWith regard to paying for the health services received, our findings are similar to the 2010 ENDS survey29, which asked this question in relation to emergency services and found that a high percentage of the rural population reported not having incurred additional health care costs when using those services. This may be because most of the care provided was not complex and the costs were paid by the benefits plan, which is why Colombia has one of the lowest out-of-pocket health care costs in Latin America. Studies of other countries, such as Mexico’s 2016 ESANUT survey, have also reported that those enrolled in state or public insurance systems tend to pay little for emergency and out-patient services10.\n\nNevertheless, the present study found that the EPS did not provide all the medications, which required people to make out-of-pocket purchases. The ENSANUT in Mexico had different results, where nearly 77% of those enrolled received their medications and did not have to pay out-of-pocket10. Therefore, our country continues to experience difficulties with the availability of medications and access to them in rural areas.\n\nWith regard to the perception of emergency health services, it is worth noting that the national and international sources that were consulted did not report specific comparable results as to whether the care received was respectful or culturally appropriate, or whether the same service providers would be used when needing health services again. In terms of access, both of those factors relate to acceptability and satisfaction, and in both cases our study found that high percentages of our population of ex-combatants and their families had favorable perceptions of the emergency care that they received.\n\nIn conclusion, in spite of the limitations in availability and access to medications, the population of ex-combatants who received emergency services generally considered the quality of care given at the local hospitals to be good (60% of the participants in the study). This is similar to that found for emergency services in Mexico, which also reported a good rating in the majority of cases (60.34% of those interviewed)10.\n\nWith regard to perception of access to prevention and promotion services and extramural activities for our study population, only 53% of the total number of households surveyed had some kind of contact with promotion and prevention services, which was because of spontaneous demand more than induced demand. Our study also found reports of little access to these activities by both specific high-risk groups, those under 1 year of age and pregnant women. It is also worth noting that other age groups also reported not having participated in these types of activities.\n\nThe findings were similar for extramural activities offered by the Health for Peace project, with a small percentage of families having received visits (17%), although a large percentage of households knew about the existence of these extramural teams.\n\nBased on these findings, certain situations that are occurring with health services and the community in the territories can explain what has been found. One is related with the logistics and dynamics that are involved in providing health services, namely, induced demand as a strategy to promote not only activities required by law but also those that are voluntarily offered in order to strengthen primary health care (PHC), such as extramural activities. Induced demand is defined as all the actions that are aimed at informing and educating the enrolled population in order to deliver the activities, procedures, and interventions for specific protection and timely detection, as established by the technical guidelines. The Health Promoter Entities, Adapted Entities, and Administrators in the Subsidized System shall develop and implement strategies that ensure that their enrollees have access to procedural activities and to timely specific protection and detection interventions, as well as to health services for diseases that are of interest to public health, in accordance with age, sex, and health conditions27,30. The other situation is that health services are not being widely offered to the population of ex-combatants, thereby affecting both their availability and access to them. These two dimensions can be understood as follows: if they are not offered, then the services will not be known, and thus, they will not be demanded or utilized.\n\nOther studies that have been performed in Colombia have reported similar findings. In Manizales, a household study was conducted of users in the contributory and subsidized systems in order to determine the factors that influenced the use of promotion and prevention programs. In general, it found the frequency of use of those programs to be low, with only 38% of the households surveyed knowing about them. For those who knew about the programs that were available in the subsidized system, vaccination was most well-known and activities for adults were least well-known31. Another study with a similar aim was performed in Medellin, which evaluated the barriers and strategies related to different actors having access to promotion and prevention services. It found that “the users’ lack of knowledge about their rights and responsibilities is a barrier to accessing health services, a lack of information results in users not requesting services about which they do not know they have a right, or in activities involving others that saturate the system,” such as overuse of emergency services32. That supports our findings on promotion and prevention services and extramural activities for the population of ex-combatants, with similar percentages of offerings and access for those under 1 year of age and pregnant women, in which knowledge about these programs promotes soliciting them and actual contact coverage.\n\nAlthough our survey found that access to these services was low, it is important to mention that the people who received health care indicated a high positive perception of availability, accessibility, contact coverage, quality, and satisfaction with both services (prevention and promotion, and extramural services). The evaluations by the ex-combatants mentioned that appointments were given within a maximum of 1 day, they received good treatment in accordance with their culture and beliefs and good care with relevant recommendations, and that they would use the services again. A study performed in Manizales described similar evaluations of the quality of service and relevance of promotion activities, where overall satisfaction was 56% in spite of low use and poor knowledge of the services33, compared to 69% on the part of ex-combatants for promotion and prevention services and 70% for extramural activities. Thus, while these services are technically formulated to meet the health needs of the population, actions are needed in order to potentialize demand, outreach, and knowledge about these services, since that is the most effective way for the population of ex-combatants to exercise actual access to them.\n\nAnother less obvious situation indicated by our findings is that the population did not spontaneously access the services. This should be taken into account, especially since the survey’s data indicates that there was more knowledge about the presence of extramural teams in the territories than there was contact with them. Two reasons can explain this. One is a lack of information that would enable them to know that they could access these services, as explained previously. The other is a very weak or unrecognized capacity for self-agency in health. Similar findings have been reported by other studies of access to primary health services in rural areas, and are supported by the health belief model. Composed of six dimensions related to the person (perception of susceptibility, severity, benefits, barriers, self-efficacy, and cues to action), this model enables identifying the degree of importance or value that a person assigns to caring for their health. It reflects one’s own conception, independently of the health system33. A study performed in Ethiopia used this model to evaluate the determinants involved in rural adolescents using sex and reproductive health services. It found that the characteristics or dimensions that most impacted interest in accessing those services were: the adolescents having at least a ninth grade education, discussing these topics among the family, having a high perception of the severity of the health problems related to this component, and a perception of great benefits and few barriers in terms of accessing these services34. Two other studies determined barriers to health access, one in Uganda with adolescent mothers and another in Pakistan with rural pregnant women. The problem matrix in each of these studies showed that individual barriers were most important, including lack of knowledge about self-care practices and insufficient health education in school. With regard to social barriers, health services were not sought due to social stigmatization related to teenage pregnancy, including at school. Family barriers included the partner not permitting access to health services or family opposition to the health practices performed in hospitals and their preference for traditional health practices34,35. These studies show that even when services are available and offerings are sufficient, something inherent to the person will always enable or prevent someone from accessing health services or seeking advice, which cannot be controlled by the system, but rather, depends on the beliefs and perceptions of the individuals themselves.\n\nWith respect to the finding of a lack of knowledge about the project’s community health monitoring component that was developed for the population of ex-combatants, the reasons for this again pertained to low induced demand, or poor outreach, thereby reaffirming that deficient outreach by the programs or strategies being developed is the main barrier to accessing them. A similar finding was reported by the Medellin study mentioned earlier32. This has a much greater impact in rural areas such as those in the present study, where a dispersed population makes health communication even more difficult. In fact, when asking the population of ex-combatants if community health monitoring strategies were important to accessing health services, the response was resoundingly positive, supporting what some social leaders have expressed in other studies conducted in the country, that “the best way to bring people into health services is to include them in an organized way so that they actively participate in improving the health system”32. Along these same lines about the important role of community health monitoring in the perception of access to services, it is worth mentioning what is known in public health as community-oriented primary care, as presented by Gofin, which highlights the importance of this type of strategy because both health services and the population assume responsibility for the health of the community, which the community also identified as important. By establishing these networks, demographic and health monitoring can be ongoing and both parties can participate in evaluating local action programs, screening the population, and assessing interventions at the individual, family, and community levels. In this way, clinical health care for individuals and families can be integrated into public health36.\n\nParaguay and Spain have demonstrated success with implementing these types of strategies in rural communities, one through surveillance and immediate health care for fever cases and the other through improvements in the quality of health promotion by facilitating the community’s involvement in the process, not only as reporters of the events of interest but also as community health agents. Through this strategy, the community has also been able to play a crucial role in identifying needs and formulating health promotion strategies, as well as detecting people with health risks and referring them to health services. This has improved morbimortality indicators for both case studies, in Asunción (Paraguay) and in Aragón (Spain)37,38, and supports the high interest that stood out in the results from the present study. Informed of these types of initiatives and involved in them, the community of ex-combatants would first consider the health benefits that would be gained from their implementation, and second, actively participate.\n\nThe perception of access to health services on the part of the population of ex-combatants who were surveyed may have been affected by various factors related with the logic by which the demand and supply of health services in the country are organized. One important and indicative factor in the dynamic of these communities may often go unnoticed —the rural areas themselves, where they are located. This is the case not only for ex-combatants in the process of reintegration but also for a large part of the country. When health services planning does not take into account the rural context of the territories and the great importance of guaranteeing effective access, it could be said that the dispersion of the population ends up acting as an intermediary in creating barriers that make it difficult to provide services to the population. This produces problems in the perception of availability and accessibility, and results in a percentage of the rural population’s demand for health services not being effectively addressing, subsequently worsening their health conditions39.\n\nThis type of geographic isolation can spiral into all types of marginalization —economic, political and social— which operate as indirect determinants of the lack of access to basic community services, or the exclusion from the right to enjoy them. Even though physical-natural adverse conditions of place are considered to be the causes of isolation, intrinsic conditions should not result in a failure to take public policy actions to alleviate a situation that is caused by segregated spaces, where social goods such as health are not easily accessible40.\n\nOther studies in Colombia have described similar differences in access to health services among regions, such as one by the Bank of the Republic in 2014 that compared the 2007 and 2012 quality of life surveys41. The main difference found by that study was that the percentage of people with the least access to health care were located in Orinoquia and Amazonia, which are the most remote regions in the country. These disparities are primarily associated with the unequal distribution of hospital services, which are primarily located in the largest cities where the largest number of providers per 100,000 residents are found. Some of the main barriers to services were low perception of the risks caused by health problems, a lack of financial resources for traveling, and a perception of poor service. Paradoxically, enrollment increased during those years, particularly in the subsidized system, while the guarantee of access to services declined. Lastly, regression models to determine the probability of accessing services by region found that the probability of accessing services was lower in the Pacific coast and Orinoquia-Amazonia than in the other regions, primarily because few health providers are located in those regions41. These regional disparities within the same country are of great concern when evaluating perception of access. Rural coverage under these conditions ends up in “no man’s land,” as Borgia said in his study of rural health care in Paraguay, who noted that the government’s recent health reform lacked definitive mechanisms for providing health to the rural population and made the role of rural community clinics invisible42.\n\nIn our country, concerns over differentiation in access to services in these types of regions suggest some urgency to develop and establish a rural health model5, planned in accordance with the perspective of each region so as to not only solve health problems in a timely way and with quality care but also to contribute to improving the living conditions of those located in these areas of the country (as described in point 1 of the peace accords). The Health Project for Peace is an important step in access to services for the population of ex-combatants.\n\nThis study has some limitations. First, it is a measurement of perception, we assume that the responses are loaded with subjectivity and may differ from objective measurements, for example when measuring waiting times. However, we are convinced that this measurement produces a result closer to the experience lived by the subjects when they need and seek medical attention. Second, we could not carry out a simple random sampling. However, and as indicated in the methods, the results are free of bias to the extent that 95.8% of the ETCRs of Colombia were surveyed (23 of 24), in some cases all the households of the ETCR were included, and mainly, due to the high homogeneity in the living conditions of the subjects. Finally, there is the lack of generalization. The results of this study cannot be generalized to the total population of Colombia, nor to all those demobilized at the different moments of reincorporation processes during the internal armed conflict of the country, insofar as it is a population in special conditions, in transition to the legality and in the process of change in multiple dimensions; In this regard, we recognize that the perception of former FARC-EP combatants about the national health system is developed in comparison with the conditions of medical care during their militancy period before the signing of the Final Accord.\n\nAs a practical and experiential experience, it has played a positive role in the majority of the population having positive perceptions, as found by our study. These actions have indirectly strengthened the health dimension component of the path to reintegration, which guarantees this fundamental right and recognizes this population as citizens who are transitioning to civilian life, thereby ensuring the conditions that are needed for a stable and lasting peace. It is important that these types of strategies and actions be sustainable and be extended to the entire rural population through a national plan and state policies aimed at making actual access to health services possible. In practical terms, this is the only mechanism that will subsequently transform the current perception of access to health services.\n\n\nConclusions\n\nThe actions that have been implemented through the Health for Peace framework, which inspired this study, have taken into account WHO recommendations for health equity and universal health coverage for highly vulnerable populations43, such as ex-combatants in the process of reintegration. These two conditions are operationalized by effective access to services through strategies and actions that involve primary care in rural areas. In this way, it is possible to strengthen actual access to services for this population and go beyond the legally-required intra-institutional actions, towards extramural actions and community participation, which as documented herein, have a stronger impact on the highly positive perceptions of the majority of our study population. More of these actions are needed since they reduce the gap between rural areas and cities, and enable ex-combatants to put down roots in civilian life, thereby helping to maintain an extended post-conflict period that can lead to lasting and stable peace.\n\n\nData availability\n\nThe database is stored by the International Organization for Migration’s (IOM) Migration and Health program. Since it contains data about people who are ex-combatants and residents, this database cannot be publicly provided without prior authorization from the administrators, who will evaluate the request. A reviewer or reader interested in acquiring the data should write a request to the Migration and Health Program of the International Organization for Migration in Colombia, specifying: objectives, justification, expected results and dissemination plan of the analysis. Please send your requests to the director or the Program David Rodríguez: darodriguez@iom.int\n\nFigshare: Instrument in Spanish of the Study “Perception of effective access to health services in Territorial Spaces for Training and Reintegration, one year after the peace accords in Colombia: a cross-sectional study”, https://doi.org/10.6084/m9.figshare.1133646814\n\nThis project contains the following extended data:\n\n- InstrumentinSpanishPerception of Effective Access to Health Services in Territorial Spaces for Training and Reintegration.xlsx (Study instrument in Spanish)\n\nFigshare: Instrument in English of the Study “Perception of effective access to health services in Territorial Spaces for Training and Reintegration, one year after the peace accords in Colombia: a cross-sectional study”. https://doi.org/10.6084/m9.figshare.1132834415\n\nThis project contains the following extended data:\n\n- InstrumentinEnglish.xlsx (study instrument in English)\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).",
"appendix": "Acknowledgments\n\nThank you to the ex-combatants and residents in the ETCRs who participated in this study.\n\n\nReferences\n\nFajardo-Dolci G, Gutiérrez JP, García-Saisó S: Acceso efectivo a los servicios de salud: operacionalizando la cobertura universal en salud. Salud Publica Mex. 2015; 57(2): 180–6. Publisher Full Text\n\nONU: Observación General No 14 “El derecho al disfrute del más alto nivel posible de salud” (artículo 12). In: Instrumento Internacionales de Derechos Humanos: Recopilación de las observaciones generales y recomendaciones generales adoptadas en virtud de los derechos humanos. Vol 1. Washington, D.C.: ONU; 2008; 96–117. Reference Source\n\nArrivillaga M, Aristizabal JC, Pérez M, et al.: Encuesta de acceso a servicios de salud para hogares colombianos. Gac Sanit. 2016; 30(6): 415–20. Publisher Full Text\n\nArrivillaga M, Borrero YE: Visión comprensiva y crítica de los modelos conceptuales sobre acceso a servicios de salud, 1970-2013. Cad Saude Publica. 2016; 32(5): e00111415. Publisher Full Text\n\nPresidencia de la República Colombia, FARC-EP: Acuerdo final para la terminación del conflicto y la construcción de una paz estable y duradera. La Habana, Cuba; 2016. Reference Source\n\nRepública de Colombia Presidencia de la República. Decreto 1274 de 2017. Bogotá; 2017; 1–5. Reference Source\n\nHuertas Pedraza C: Percepción de las actividades de educación para la salud en un grupo de personas que asisten a programas de promoción de la salud y prevención de la enfermedad cardiovascular Bogotá, d. c., noviembre a diciembre de 2009. Universidad Nacional de Colombia; 2011. Reference Source\n\nBejarano B: Percepción de los profesionales de enfermería y las Gestantes sobre la asesoría en VIH en una ESE de primer nivel De Bogotá distrito capital. Universidad Nacional de Colombia; 2010. Reference Source\n\nMinisterio de Salud y Protección Social. Encuesta de evaluación de los servicios de las EPS. Bogotá 2012. Reference Source\n\nInstituto Nacional de Salud Pública. Encuesta nacional de salud y nutrición de medio camino 2016. ENSANUT. Instituto Nacional de Salud Pública. México, D.F; 2016. Reference Source\n\nTanahashi T: Health service coverage and its evaluation. Bull World Health Organ. 1978; 56(2): 295–303. PubMed Abstract | Free Full Text\n\nNelson AG, Luís AE, Julio OA: Percepción sobre el Derecho a la Salud y Acceso a Servicios en Usuarios del Régimen Subsidiado en un Municipio Colombiano, 2005. Revista de Salud Pública. 2008; 10(3): 386–394. Publisher Full Text\n\nArrivillaga M, Aristizabal JC, Pérez M, et al.: Encuesta de acceso a servicios de salud para hogares colombianos. AESS. Cali. 2016; [cited 2019 Nov 27]. Publisher Full Text\n\nFernández-Niño JA: Instrument in its original version in Spanish used in the study \"\"Perception of effective access to health services in Territorial Spaces for Training and Reintegration, one year after the peace accords in Colombia: a cross-sectional study\". figshare. 2019. http://www.doi.org/10.6084/m9.figshare.11336468.v1\n\nFernández-Niño JA: Instrument in English of the Study \"Perception of effective access to health services in Territorial Spaces for Training and Reintegration, one year after the peace accords in Colombia: a cross-sectional study\". figshare. 2019. http://www.doi.org/10.6084/m9.figshare.11328344.v1\n\nMárquez I: El papel de la educación en situaciones de posconflicto: Estrategias y recomendaciones.El Impertinente.2014; 11(21): 143–152. Publisher Full Text\n\nMolano Giraldo MF: Del conflicto al posconflicto: temores, retos y reflexiones de la solución negociada. Nov Vetera. 2015; 24: 49–64. Reference Source\n\nCárdenas Díaz JA, Pérez Páez NS, Clavijo Mejía RI: DDR y Comisiones de verdad, límites y puntos de encuentro: evidencia desde los casos de Namibia, El Congo, Indonesia y Colombua. Análisis Político. 2018; 31(93): 20–42. Publisher Full Text\n\nUribe M: Contribución de la reintegración a la reconciliación en Colombia. Universidad de los Andes; 2015. Reference Source\n\nGarcía Peña JJ, Arana Medina CM: Condiciones de base para la reintegración social en el posconflicto.Cienc Soc.2018; 43(4): 69–81. Publisher Full Text\n\nMedina Pérez NA, Yáñez Pérez KJ: Retos para el actual sistema de salud en el macrco de los acuerdos de paz y el post conflicto en Colombia. Universidad Nacional Abierta y a Distancia UNAD; 2018; 52. Reference Source\n\nFranco S: Salud para la paz y paz para la Salud. Rev Gerenc y Polit Salud. 2015; 14(29): 5–8. Reference Source\n\nPuerta Silva CP, Dover RVH: Salud, recursos naturales y el proceso de paz en Colombia. J Lat Am Caribb Anthropol. 2017; 22(1): 183–8. Publisher Full Text\n\nAgencia para la reincorporación y la normalización: Sistema de información para la reintegración: Cuadro 17 Condiciones de salud, régimen. 2018; [cited 2019 May 10]. Reference Source\n\nAgencia para la reincorporación y la normalización: ARN en cifras. Bogotá 2019. Reference Source\n\nAgencia Colombiana para la reintegración: Estudio de percepción y satisfacción dirigido a personas en proceso de reintegración, familias, actores externos y ciudadanos colombianos, frente a los servicios, beneficios y atención ofrecidos por la agencia colombiana para la reintegración de personas. Bogotá (Tercera encuesta), 2014. Reference Source\n\nMinisterio de Salud: Resolución 412 de 2000. \"Por la cual se establecen las actividades, procedimientos e intervenciones de demanda inducida y obligatorio cumplimiento y se adoptan las normas técnicas y guías de atención para el desarrollo de las acciones de protección espec. Ministerio de Salud de Colombia. 2000. Reference Source\n\nMinisterio de Salud, Profamilia: Encuesta nacional de demografía y salud. Bogotá: Profamilia; 2015; 1: 1–430. Reference Source\n\nMinisterio de Salud y Protección Social, Profamilia: Encuesta nacional de demografía y salud 2010. Encuesta Nacional de Demografía y Salud. Bogotá 2010; 108–126. Reference Source\n\nMinisterio de Salud y Protección Social: Resolución 3280 de 2018 Por medio de la cual se adoptan los lineamientos técnicos y operativos de la Ruta Integral de Atención para la Promoción y Mantenimiento de la Salud yla Ruta Integral de Atención en Salud para la Población Materno Perinatal y se es. 2018. Reference Source\n\nRealpe C, Escobar MM, Largo BC, et al.: Utilización del programa de promoción y prevención en las empresas promotoras de salud y administradoras del régimen subsidiado de Manizales, Colombia, 2001-2002. Colomb Med. 2002; 33(3): 102–7. Reference Source\n\nRestrepo-Zea JH, Silva-Maya C, Andrade-Rivas F, et al.: Acceso a servicios de salud: análisis de barreras y estrategias en el caso de Medellín, Colombia. Rev Gerenc y Polit Salud. 2014; 13(27): 236–59. Publisher Full Text\n\nGebreyesus H, Teweldemedhin M, Mamo A: Determinants of reproductive health services utilization among rural female adolescents in Asgede-Tsimbla district Northern Ethiopia: a community based cross-sectional study. Reprod Health. 2019; 16(1): 4. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRiaz A, Zaidi S, Khowaja AR: Perceived barriers to utilizing maternal and neonatal health services in contracted-out versus government-managed health facilities in the rural districts of Pakistan. Int J Health Policy Manag. 2015; 4(5): 279–84. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNabugoomu J, Seruwagi GK, Corbett K, et al.: Needs and Barriers of Teen Mothers in Rural Eastern Uganda: Stakeholders' Perceptions Regarding Maternal/Child Nutrition and Health. Int J Environ Res Public Health. 2018; 15(12): pii: E2776. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGofin J, Gofin R: Atención primaria orientada a la comunidad: un modelo de salud pública en la atención primaria. Rev Panam Salud Publica/Pan Am J Public Heal. 2007; 21(2–3): 177–85. Reference Source\n\nBasogain X, Olabe MA, Espinosa K, et al.: Sistema de vigilancia epidemiológica comunitaria “Bonis”. Estado actual y proyecciones futura.s. Mem Inst Investig Cienc Salud. 2010; 8(2): 44–57. Reference Source\n\nGállego-Diéguez J, Aliaga P, Belén C, et al.: Las redes de experiencias de salud comunitaria como sistema de información en promoción de la salud: la trayectoria en Aragón. Gac Sanit. 2016; 30(S1): 55–62. Publisher Full Text\n\nOPS:: Caracterización de la exclusión en salud en Bolivia. La Paz. 2004. Reference Source\n\nChanampa ME, Tetamanti JM: Accesibilidad a la salud y estrategias de movilidad: caso Aldea Beleiro. ICT-UNPA-9. Santiago de Chile. 2015; 54–77. Publisher Full Text\n\nBanco de la República, Centro de Estudios Económicos Regionales-Cartagena, Ayala Garcia J.: La salud en Colombia: más cobertura pero menos acceso. Cartagena: Banco de la República. (Documentos de trabajo sobre economía regional). 2014. Publisher Full Text\n\nBorgia F, Gularte A, Gabrielzyk I, et al.: De la invisibilidad de la situación de las policlínicas comunitarias-rurales en Uruguay a la priorización de la salud rural como política pública. Saúde Pública, Rio Janeiro. 2012; 36(94): 421–35. Publisher Full Text\n\nOMS, OPS Oficina Regional para las Américas: Cómo tomar decisiones justas en el camino hacia la cobertura universal de salud Informe fi nal del Grupo Consultivo de la OMS sobre. Washington, D.C. 2015; 1–92. Reference Source"
}
|
[
{
"id": "58511",
"date": "25 Mar 2020",
"name": "Cesar Abadía-Barrero",
"expertise": [
"Reviewer Expertise Medical Anthropology",
"Human Rights",
"Colombia"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study addresses one of the most challenging dimensions of the implementation of the peace agreement in Colombia: access to health care services of former combatants. Using a human rights framework, the article aimed to describe how ex-combatants of the Revolutionary Armed Forces of Colombia-People’s Army (FARC-EP), now a political party, perceive access to emergency services, and health promotion and illness prevention programs during their reincorporation process to civil society. All participants inhabited one of the Territorial Spaces for Training and Reintegration (ETCR) which are places established to relocate former fighters. Developing instruments to monitor the socioeconomic dimensions of Disarmament, Demobilization, and Reintegration (DDR) operations, in particular, access to health care services, is not only an important contribution for Colombia, but for all countries that are transitioning from war and conflict to peace. This study also suggests an important contrast between access to healthcare in ETCRs and historical challenges for health provisions in rural areas. While the study is clearly of utmost importance, we identified several areas that warrant significant revisions.\n\nIntroduction:\nThe introduction provides limited context about the reincorporation program, ETCRs, and Health for Peace project. Although Colombia has had multiple experiences of reintegration programs with different armed groups, including the FARC, the process that followed the signing of the 2016 peace agreement is a unique experience that represented a national and international precedent for peace negotiations. The article should mention the main characteristics of the reincorporation program, including the different challenges and developments in the establishment, transformation, and infrastructural conditions of ETCRs in order to understand the living conditions of the participants. There is an important history here that should be mentioned, even in abbreviated form. Regional differences and successful and less successful ETCRs should be briefly mentioned as a way to convey the potential heterogeneity of experiences of ex-combatants. The authors refer to this program as reintegration; however, the distinction between reintegration and collective reincorporation is necessary in the Colombian context since it refers to different programs that determine ex-combatant lives in different ways. Also, the authors do not provide a description of the Health for Peace project. Therefore, it is difficult to know how the intervention of United Nation agencies, the International Committee of the Red Cross, and other medical humanitarian agencies created special conditions in ETCRs. For the context of the present study, it would be important to clarify what were the actions implemented by this project? Did they offer healthcare services or programs? Did they help ex-combatants with the administrative processes of the Colombian healthcare system? Was there a transition or potential overlap between international agencies and the Colombian health care system? Were there services from the Health for Peace project, including ambulances, physicians and nurses, still present at the time of the study? If there was overlap and the agencies were still present in the ETCRs, is the study instrument able to differentiate between the actions implemented by these agencies and the professionals and networks of the Colombian healthcare system? A more extensive description of this historical context of the relationship between the ETCRs, the international humanitarianism that supported the reincorporation process, and the health care networks is very relevant for the comparison with other rural communities and other peace processes. For an international audience, the concepts of “prevention and promotion” and “extramural activities” should be briefly explained.\nMethods:\nThe methods are appropriate to answer the question of the study.\n\nThe high coverage of the sample and the number of ETCRs included are remarkable given the characteristics of these population.\n\nThe study unit seems to be the household not the family.\n\nIt seems that the authors overlooked the name of the survey developed by Arrivillaga, Aristizabal, and Estrada and wrote instead East Asian Social Survey.\n\nThe instrument has an important merit for this kind of study; however, one of its limitations is that it does not include questions about conflict-related health issues, focuses primarily on emergency services and only assesses the four weeks prior to the survey. This should be taken into account in the analysis and discussion.\n\nResults:\nThe core of the results of the study is presented in tables and figures. There are problems in the way the data are reported and we question whether the authors need to present all these data and whether some data deserve further analysis.\n\nFor both tables:\nIt is customary to present results as n (%). Since totals are numbers, it would be preferable to have the tables presented as n (%).\n\nTable 1:\nState that CV is coefficient of variation.\n\nPlace of Residence. It seems that this category was transferred from other studies. However, it is not clear how the category ‘urban’ applies to the context of ETCRs. Furthermore, the differences between rural settlements and dispersed rural areas are not intuitive and deserve an explanation.\n\nNumber of people in household. There seems to be a problem in the reporting of the total column. The total for mean and median doesn’t match the addition of men and women.\n\nCultural identity or physical trait. Please change heading to Cultural identity and/or race/ethnicity.\n\nWe recommend a table that shows the distribution of identity/race/ethnicity among the regions so we can have a better sense of the relationship between these two variables (Region and cultural identity/race/ethnicity).\n\nGroup. The definition of the categories should be explained. For instance, what is the difference between victims and displaced? The authors should also mention that respondents could mark several responses.\n\nHousing. ETCRs, as part of the settlement project of the peace accord, have very similar characteristics. These categories, however, do not seem to reflect the housing conditions of ex-combatants in the collective reincorporation program. We suggest explaining the general characteristics of these types of housing. It seems that some questions were also adapted from other survey studies and it is unclear how they were transferred to the context of the ETCRs. For example, it is hard to think how “rented room” (inquilinato in the original instrument) is even a possibility within the context of ETCR. Equally, it is unclear what “room in another type of structure” refers to.\n\nRole in the family. In this question, if all the interviewees were the heads of the household as it is reported on page 3, it is unclear what relationship categories represent.\n\nTable 2:\nThis table presents the most interesting results of the study. Nonetheless, the variations between regions deserve further analysis and discussion. Importantly, the Pacific and the Amazonian region show important differences from other regions. Depending on the final results and final presentation of the table, relevant discussions about regional variability could take place. In particular, the reader wonders why these two regions rank poorer in people’s perception of effective access. There can be interesting discussions with other Colombian studies around health inequalities and regional variation and this study opens up the possibility to discuss what is specific of the peace accord and what resembles larger problems of the population of these areas. Given the time the data were collected (September and October 2018), we know that some ambulances and health care personnel were still present in some of the ETCRs. Then, it is not entirely clear if the services people received were part of the Health for Peace project that had significant funds from international cooperation or if people were, in fact, using the services of the healthcare system. If ambulances were available, did that facilitate access to services and in a way skewed the data? Further information of the historical moment and specific conditions of the ETCRs at the time of the interview, as mentioned for the introduction, could help with the analysis of these results. Figure 1, 2, and 3 offer interesting information but it is very hard to assess the responses. Are there national and international guidelines that allow us to assess whether those percentages are adequate? Furthermore, Figures 2 and 3 require information about the clinical specificities of the children under 1 or pregnant women to assess whether the services offered were adequate or not, perhaps according to whether their regular or schedule check-ups are in agreement with known guidelines. We are left wondering how important this information is. The fact that the questions ask for the last four weeks further complicates the analysis given that the person could be in a moment in which there is no need to use these services and appointments might have already happened before the four weeks period or are scheduled for after the interview.\n\nFigures 4 and 5 would benefit from an explanation of these programs and the guidelines or parameters established in the country regarding their offering. Are health promotion and illness prevention services replacing regular care? Why would someone have to schedule such an appointment rather than already having an appointment or being contacted to schedule the appointment? How do referrals and enrollments in these programs work? Also, questions arise about the coordination between programs and whether the presented data is adequate or follows any established guidelines. All in all, an explanation of the ways in which prevention and promotion, extramural services, surveillance and monitoring systems, and regular health care work in coordination is needed to make sense of figures 1 through 8. Then, the authors should conduct the analyses of these data based on that description of the multiple pieces of the health care system and the parameters used to assess how adequate or inadequate are. Just to mention one example, what does it mean that 17,3% of the sample said that they had contact with an extramural team over the past 4 weeks? Is this adequate, inadequate, desirable, worrisome? We encourage the authors to rethink the presentation of the results.\n\nDiscussion:\nThe discussion requires major changes. The authors discuss some of the results in relation to existing literature but it is unclear what are the central ideas of the discussion and what results the authors found particularly interesting and relevant.\nPage 13. 2nd paragraph. “While prioritizing the right to health for ex-combatants represents important progress in public policy, Colombia’s health system faces numerous challenges that need to be studied”. What challenges is this sentence referring to?\n\nPage 13. 3rd paragraph. The ARN survey of 2014 describes a different reintegration program. This program includes ex-combatants who demobilized before the peace agreement and the majority of them moved to urban areas. As mentioned, the article should specify from the beginning that the population assessed in the study are part of the collective reincorporation program and describe their main characteristics. Is it possible to discuss some differences between access to health care depending on whether the ex-combatants participated in the reintegration or the reincorporation programs?\n\nPage 13. 1st paragraph. “ (...) the results from the present work provide a new methodology and information that can be used to identify the health status of populations that became invisible because of their situation as active actors in armed conflict.” This phrase is unclear. The present study centers on ex-combatants once they became “visible” and legally recognized after the peace accord. It also has the limitations of asking about the four weeks prior to the interview.\n\nThe contrast with healthcare provision in other rural areas is an interesting and important discussion. However, the main points of this contrast are not clear.\n\nPage 14 3rd paragraph. We suggest avoiding adjectives such as “high”, “low” and “small” without providing specific data.\n\nThe discussion of the results about health promotion and illness prevention services in relation to studies conducted in Manizales and Medellin does not seem appropriate, unless the difference between these services in rural and urban areas is stated.\n\nWhat do the authors mean by good treatment in accordance with their culture and beliefs? Are the notions of culture and beliefs related to their identity as ex-combatant, a regional identity, a rural identity, or an ethnic/racial identity?\n\nThe results do not indicate that people’s behavior to seek health care services can be explained through the health belief model.\n\nWe suggest that the center of the discussion should be around how the current data compare to the challenges described for other rural populations. The authors hint in this direction but could make significant contributions by signaling how ex-combatants’ perception of their access to health care compares to other studies in rural areas. Are they getting better or worse access to health care than other rural communities? What does this say about the reincorporation process and about the health care system? If the analysis is broken down by region, what does it tell us about different regions in Colombia and, perhaps, different “reincorporation processes”?\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": [
{
"c_id": "5504",
"date": "19 May 2020",
"name": "Julián Alfredo Fernández-Niño",
"role": "Author Response",
"response": "Answers Observations, Questions and Requests Introduction:The introduction provides limited context about the reincorporation program, ETCRs, and Health for Peace project. Although Colombia has had multiple experiences of reintegration programs with different armed groups, including the FARC, the process that followed the signing of the 2016 peace agreement is a unique experience that represented a national and international precedent for peace negotiations. The article should mention the main characteristics of the reincorporation program, including the different challenges and developments in the establishment, transformation, and infrastructural conditions of ETCRs in order to understand the living conditions of the participants. There is an important history here that should be mentioned, even in abbreviated form. RTA: In the discussion, we had described the general characteristics of the elements of the reincorporation program that are related with access to the health system, and the relevant components, including a summary of the components of the process, along with some of the challenges for organizing social services, and particularly the right to health and the provision of health services for ex-combatants given the conditions of rurality itself in Colombia. This was aimed at providing the reader with greater context for interpreting the findings. To address the reviewer’s observations, we adapted two paragraphs in the discussion and moved them to the introduction in order to provide the reader with this context at the beginning of the manuscript (new paragraphs 4 and 5 in the introduction):“The Colombian government established eight dimensions for the path of reincorporation, one of which is access to the General Social Security Health System (SGSSS, in Spanish). It also established health services as a fundamental right to be guaranteed along with returning these people and their families to legality. Colombia is a good example of the comprehensiveness that these programs need when it comes to ex-combatants who are on the path to reincorporation, in the short-term. Meanwhile, long-term and sustainable reincorporation into civilian life will be ensured by including crucial elements such as effective health care and ongoing access to health services through enrollment in the health system. This will contribute to individual well-being, continued legality, and a solid ground on which to build stable and lasting peace.Some of the projects were developed with the above challenges in mind and in accordance with the reference framework on which the path was laid out, such as Health for Peace, led by the Ministry of Health and implemented by three United Nations agencies (UNFPA, PAHO/WHO, and IOM). This project was created in order to strengthen institutional and community capacities to address health needs in the rural areas where the ETCRs are located, through already-established regional health centers as well as local primary hospitals. Health services were also complemented by the development of extramural strategies and community surveillance, led by regional health professionals with the support of local public health leaders who were trained by SENA as part of the project”.SENA is the National Learning Service (Servicio Nacional de Aprendizaje), which is the main public institution in Colombia responsible for technical training at the national level.In addition, to addressing the reviewers’ suggestions, a new paragraph was included in the introduction (new paragraph 6 in the introduction). We believe this provides the relevant context from which the survey used in this study arose, more specifically, in the framework for the implementation of the final accord. We briefly mention the Health for Peace project, its coordination with the health system, and the historical change that this represented for the organization of health services in the ETCR:“In this way, the “Health for Peace” contributes to the implementation of Point 1 in the Implementation Framework Plan (IFP) of the Final Accord, which relates to comprehensive rural reform and is associated with the implementation of Territorial Health Plans in the priority municipalities. It also complemented the emergency response by the Colombian government during the first year after the accord, which consisted of a physician, a nursing professional, and an ambulance for each ETCR. These health workers held positions that at first lacked the conditions needed to guarantee the quality of health care, given that the infrastructure and human skills for providing health services in the regions where the ETCRs are located has historically been insufficient, and non-existent in some cases. This emergency measure was gradually phased out as the Health for Peace project was being implemented. In this sense, the main objective of the Project was to strengthen the local capacity for improving access to comprehensive Primary Health Care services —with an emphasis on sexual and reproductive rights, mental health, preventing consumption of psychoactive substances, and children’s health and malnutrition— thereby addressing the needs that had previously been identified by the United Nations Verification Mission in the ETCR. Furthermore, in order to promote access to comprehensive health services, support was provided to implement an extramural strategy as part of Primary Health Care, as well as to develop a strategy for community participation and surveillance. This proposal can be considered to be the specific development of strengthening health services in the rural area through recent technical developments by the Ministry of Health, such as the National Public Health Plan, but adapted to the context of ex-combatants, their families, and the residents of these regions, with the advantage of receiving technical and financial support from agencies in the United Nations system.”With regard to the other points mentioned by the evaluators, we believe that a more detailed description of reincorporation, the “history,” including ongoing challenges, cannot be summarized in only a few paragraphs without risking falling into debatable inaccuracies. This also clearly goes beyond the scope of the aim of our study, which involves a specific objective and moment in time, namely, to describe how ex-combatants perceived effective access one year after the signing of the accord. It is important to consider that, for a merely descriptive quantitative study, the article is already exceedingly long, and the analysis suggested falls outside the authors’ expertise. In addition, an extensive and more detailed discussion of the context is already included in the current version of the article, as well as potential contrasts with other reincorporation processes for ex-combatants in other countries.Given our approach and disciplinary style, the objective of the introduction is to present the specific context that relates with the study. We believe that the most pertinent of what was suggested would be to add more information about the Health for Peace project, how that was implemented in the ETCRs, and where this project was inserted into the reincorporation process. We have added all of this to the introduction (paragraphs 3-5). More detailed information and analyses can be found in the references that are cited, especially in the discussion. Regional differences and successful and less successful ETCRs should be briefly mentioned as a way to convey the potential heterogeneity of experiences of ex-combatants RTA: Agreed. Nevertheless, we believe the sample sizes in the ETCRs do not enable us to make inferences or draw conclusions about differences among regions or among ETCRs. The sampling was proportionally stratified by the size of the population of each ETCR for the purpose of making an overall inference only.We included the regionally stratified analysis in the tables for exploratory purposes, as well as for reflection on the part of the readers and potentially for the formulation of a hypothesis. Nevertheless, we prefer not to speculate about whether the differences found are explained by differences in the program’s success, since they could also be due to randomness, which is why the discussion and conclusion about the results are presented using totals. We would certainly expect that there could be differences in the sociocultural characteristics of the population that could affect the perception of effective access, and that there could be differing degrees of success in implementation across the country. Nevertheless, this is not something that can be evaluated or concluded based on our results. We also do not have valid information from any other source about the status of the implementation of the Health for Peace project in each ETCR or region at that point in time.In order to address the suggestion by the evaluators, we have added a paragraph that describes this aspect, but we prefer that it be presented in the discussion as a limitation, as follows:“The sampling design used by the present study does not permit making inferences at the ETCR or regional level, or comparisons among them. The regional stratification presented in the tables is only exploratory. It is to be expected that differences in the sociocultural characteristics of the population can affect the perception of effective access, as can differing degrees of success with implementation across the country and satisfaction with the services at the territorial level. Nonetheless, this is not something that can be evaluated or concluded based on the results of the present study. Valid information about the status of the implementation of the Health for Peace project in each ETCR or region did not exist at that time, nor does it exist now. In order to identify territorial differences, a different type of analysis needs to be performed using different methods and sources of information. While an overall analysis of the status of the implementation of the Project is undoubtedly research that needs to be conducted, it goes beyond the scope of this study. The authors refer to this program as reintegration; however, the distinction between reintegration and collective reincorporation is necessary in the Colombian context since it refers to different programs that determine ex-combatant lives in different ways. Also, the authors do not provide a description of the Health for Peace project. RTA: These concepts are used in accordance with the terminology in the documents that are cited. The International Organization for Migration (IOM) and the Ministry of Health, as co-authors, have asked us to use the same terminology in order to avoid ambiguity. We understand the debate underlying the distinction mentioned by the evaluators, and we suggest that the reviewers could present this in a public comment about the article, where it could be debated.The Peace Accord is described in the brief introduction to paragraph 3, and a link has been added to the text where it can be consulted in full. The paragraph mentioned above (new paragraph 6 in the introduction) was also added, which describes the Health for Peace project and briefly explains the main components. Therefore, it is difficult to know how the intervention of United Nation agencies, the International Committee of the Red Cross, and other medical humanitarian agencies created special conditions in ETCRs. For the context of the present study, it would be important to clarify what were the actions implemented by this project? Did they offer healthcare services or programs? Did they help ex-combatants with the administrative processes of the Colombian healthcare system? Was there a transition or potential overlap between international agencies and the Colombian health care system? Were there services from the Health for Peace project, including ambulances, physicians and nurses, still present at the time of the study? If there was overlap and the agencies were still present in the ETCRsA more extensive description of this historical context of the relationship between the ETCRs, the international humanitarianism that supported the reincorporation process, and the health care networks is very relevant for the comparison with other rural communities and other peace processes. RTA:An “extensive” description of the historical context of the relationships between the ETCRs and international humanitarian organizations is relevant to this area of study, nonetheless, we believe that it considerably exceeds the scope and objectives of our study. This is not an objective of this report of descriptive information, nor is it of interest to the current research project. A different type of investigation would be warranted in order to respond to this aspect. The current manuscript is over 10,000 words, and we believe that elements that do not enter into dialogue with our specific results would not be relevant. Given our objective, we are unable to identify how it would enter into dialogue with the manuscript in general, or specifically with the results. While the context is undoubtedly relevant, this report has a modest objective, and its intention is not aimed at a historical analyses of the peace process. If the reviewers consider it to be helpful to suggest a reference that discusses this aspect, we can include that.As we mentioned previously, our objective is limited to describing the perception of effective access to health services on the part of ex-combatants in the ETCRs, in the framework of the Health for Peace project. The elements that we consider to be relevant pertain to this context, and are presented in the manuscript, in the introduction and the discussion, and have been expanded. Adding broader elements would warrant a more detailed analysis using other methodologies and sources (sociological, historical, analysis of public policies) that go beyond the objectives of this report, and fall outside our disciplinary background. We believe that this is a task for experts on those types of analyses, who can use our results, among others, as a basis for their studies. For an international audience, the concepts of “prevention and promotion” and “extramural activities” should be briefly explained RTA:We agree. And we would like to add that this article may also be read by people who have a background other than Public Health. Therefore, we want to take this opportunity to suggest incorporating a Glossary of Terms. We suggest including this as a separate linked file, or supplementary material, or as a table in the main article. We include it here in our response and we will defer to the editors with regard to where to place it.This table is referred to in the methodology, specifically in the section “Data collection instruments and procedures” Glossary of termsHealth promotion: This is a global political and social process that encompasses not only actions aimed directly at strengthening the abilities and capacities of individuals, but also at modifying social, environmental, and economic conditions in order to mitigate their impact on public and individual health.Main Strategies: To establish a healthy public policy, create environments that support health, strengthen community action for health, develop personal skills, and redirect health services (WHO,1986)Primary health care: This refers to essential health care that is affordable for the country and the community, and it is conducted using practical, scientifically-founded, and socially-acceptable methods.The primary health care approach includes the following components: equity, community involvement/participation, intersectorality, adequate technology, and affordability.Main activities: Education on individual and community health, including the magnitude and nature of health problems, and indications for how to prevent and control these problems; supply of drinking water and basic sanitation; maternal and child health care, including family planning; immunization, proper treatment of common diseases and injuries; and the supply of essential drugs. (WHO, 1978)Disease prevention: Measures aimed not only at preventing the onset of disease, such as reducing risk factors, but also at stopping its progression and mitigating its consequences once it occurs.Primary prevention is aimed at preventing the initial appearance of a disease or illness.Secondary prevention and tertiary prevention are aimed at stopping or delaying a disease that is already present, as well as its effects, through early detection and appropriate treatment or by reducing recurrence and chronicity, for example through effective rehabilitation. (WHO, 1984)Community action for health: Refers to collective efforts by communities to increase their control over the determinants of health, and consequently, to improve health. (Otawa Letter, 1984)Extramural Activities: This refers to services that are offered to the population in spaces not intended for health, or health spaces in regions that are difficult to access and where health services are not available. These spaces are temporarily setup for the development of specific activities and procedures. (Resolution 1441/2013 Ministry of Health Colombia) Methods: The methods are appropriate to answer the question of the study. The high coverage of the sample and the number of ETCRs included are remarkable given the characteristics of these population. The study unit seems to be the household not the family. It seems that the authors overlooked the name of the survey developed by Arrivillaga, Aristizabal, and Estrada and wrote instead East Asian Social Survey.RTA:Thank you very much for the valuable comments by the reviewers. Indeed the advantage of the study is its ability to encompass, with high representativeness, a population that is difficult to identify with this type of analysis. This was possible because of the availability of a sample of nearly all of the ETCRs that were active in the country at the time the study was conducted, and its availability at a critical time in Colombia’s peace process.The unit of analysis is indeed the household, and not the family, which is why the description of the results refers to households rather than families.In the second paragraph in “Data collection instruments and procedures,” we changed the name of the survey that was used. Thank you very much for the correction. The instrument has an important merit for this kind of study; however, one of its limitations is that it does not include questions about conflict-related health issues, focuses primarily on emergency services and only assesses the four weeks prior to the survey. This should be taken into account in the analysis and discussion. RTA: The main objective of the instrument that we constructed was to determine the perception of effective access to health services in a population of ex-combatants who were located in the ETCRs, and who had been given additional support through the provision of primary health and emergency services. The survey was not intended to establish an individual health diagnosis of ex-combatants in relation to their previous activity as combatants, but rather, to determine whether one of the strategies to guarantee the reincorporation of this population to civil society was met —that is, access to health services from the perspective of a right to health framework, which is a commitment made by Colombia’s government. Since we wanted to identify effective perception and not just access to services, an instrument was constructed to allow us to measure these dimensions in the population of ex-combatants who immediately received services. This was aimed at ensuring that the health services would have been provided primarily through the Health for Peace project. These specific actions were carried out within the framework set forth by Colombia’s health legislation, in order to help ex-combatants and their families access services and have their needs addressed, as well as to promote health. Therefore, by using a window of 4 weeks prior to the interview, we ensured that their answers corresponded to the care they were receiving at that point in time.The study was not aimed at evaluating topics related with the conflict or access to health services by ex-combatants in the past or before the reincorporation process. The health services that were evaluated primarily correspond to the lines of action taken by the Health for Peace project, but also to public health actions taken in rural areas across the entire country. These were not limited to emergencies (which in rural areas tend to be the main point of access to health services), but also involved extramural activities, including Public Health actions and even community surveillance.One month prior to the survey was chosen as the period since the information is cross-sectional and we wanted to use a reduced temporal framework that would be the same for all the study participants, such that we referred to a “snapshot” of what occurred for everyone during virtually the same period. A period of one month is usually standard for studies about health services. If we were to ask about health services over a longer period prior to the interview, the person would be reporting their perception of access at times other than the implementation of the project. A longer window could also result in recall bias.This time frame is always indicated in the description of the results and the figures. And a brief explanation about the reason for using one month was added to the methods. Results:1. The core of the results of the study is presented in tables and figures. There are problems in the way the data are reported and we question whether the authors need to present all these data and whether some data deserve further analysis. RTA:We believe that one of the main strengths of our study is the fact that we present an analysis of all the dimensions of the health services available at that time in the ETCRs, and that not including all of them would be exclusive. The research group’s efforts were directed at measuring all the dimensions of effective access, and the manuscript is aimed at a wide reporting of the results, which is common for a report of a multi-dimensional survey. This way, the readers can draw general conclusions and also extract detailed information on the dimensions that are most relevant to them. This can serve as a key database for more specific analyses by researches and decision-makers.In our opinion, the results are easily readable. Other analyses can of course be performed with this information, for example, exploring associations among dimensions, but our purpose for this report is to provide a simple but exhaustive description that serves as a basis for researchers and decision-makers. There will be few opportunities to publish the complete findings, which is why we chose F1000research, so that the number of words would not be limited and to make this public quickly and openly.Rapid and complete dissemination of the results would take full advantage of the resources used to collect the information, where knowledge management is very relevant.Having said that, if the editor believes it is necessary, we could leave in the two tables, but perhaps dispense with some of the figures, although we think that the way they are structured would be helpful to decision-makers, who may want to extract the information for public presentations that would disseminate the findings. For both tables: It is customary to present results as n (%). Since totals are numbers, it would be preferable to have the tables presented as n (%).RTA:There are a variety of styles. We believe that % should be separate since it is the estimator of interest, as opposed to absolute frequencies, and it should be the first thing that the reader sees. We can change that if the editor believes it is necessary, but we prefer to keep it as is in order to stress that the results are interpreted based on these indicators (percentages), and especially when related to the conclusions. Table 1: State that CV is coefficient of variation.RTA:That’s right. We replaced CV with coefficient of variation. Place of Residence. It seems that this category was transferred from other studies. However, it is not clear how the category ‘urban’ applies to the context of ETCRs. Furthermore, the differences between rural settlements and dispersed rural areas are not intuitive and deserve an explanation. RTA:All of the questions and categories included in the responses to the instrument, and therefore in the report of findings, were specifically designed and adapted for this study. In addition, the contents of the response options were validated in two ways: by experts and by the community. The latter included a review by field nurses who lived in the territory, as well as a pilot in which all the questions were reviewed at the territorial level, in order to study the acceptance, efficiency, and cultural applicability of each response. After completing the review, the option of “urban” was left in because the review found that it was applicable in some cases.We used the definition of “urban” by the National Administrative Department of Statistics (DANE in Spanish), namely: “It is defined as a concentration of a minimum of twenty (20) contiguous, neighboring, or semi-detached dwellings, located in a rural area of a municipality or a Departmental Township.”In accordance with technical criteria, this aspect was studied and verified with the members of the team. Although there are few, the classification is applicable. Number of people in household. There seems to be a problem in the reporting of the total column. The total for mean and median doesn’t match the addition of men and women. RTA: No, the calculation that we present is correct. For the average of persons per household in the total sample, both averages (men and women) cannot be added (summed). It is important to take into account that we are stratifying by the sex of the informant (or head of household). Therefore, each column analyzes different households. That is, they cannot be added together, since what is presented is the average (and median) of persons in the household, for households with women as the head versus those with men as the head of household, as explained in the article. It’s helpful to remember that the unit of analysis is the household, as explained in the article, except that, here, head of household (or informant) data are presented as well as household data.The mean of the total would actually be the result of an average weighted by the sample size of each group, as can be mathematically verified.Cultural identity or physical trait. Please change heading to Cultural identity and/or race/ethnicity.RTA: We changed it to “ethnicity”. We recommend a table that shows the distribution of identity/race/ethnicity among the regions so we can have a better sense of the relationship between these two variables (Region and cultural identity/race/ethnicity) RTA: We created the table that was requested, and we are including it below for the reviewer’s information. As can be seen, the ethnic distribution is as expected for the regions, although the ex-combatants are not necessarily in an ETCR that is in the same region as where they are from, which is an aspect that cannot be determined with the data that we have available. Distribution of ethnicity of the participants by region See the table in this link: https://www.dropbox.com/s/yozcvr3sidutvgg/Distribution%20of%20ethnicity%20of%20the%20participants%20by%20region.docx?dl=0*Presented as absolute frequencies and percentages per columnNevertheless, we do not believe it would be pertinent or congruent to include this table in the article, since it does not correspond with the objectives of our research. As we explained earlier, the sample design does not permit making inferences or comparisons at the regional level, but rather only global. The results in Table 2 are presented by region in an exploratory manner, but are household level data, not individual level. Thus, since the inference in the conclusions is at the household level, and we do not know the ethnicity of the entire family, then we cannot indicate a relationship between ethnicity and the variables of interest in the study that relate with perception of effective access by the household.To summarize, the global description includes the ethnicity of all the informants in the entire sample in an informative manner, but it is not our interest to discuss explanations for regional differences. As we mentioned, the sample design does not permit that analysis, and it is also not one of our objectives. In addition, the sample size does not enable us to explore differences among regions, and ethnicity also cannot be connected with the results since the level of analysis is different (ethnicity at the individual level and effective access at the household level).The table could be consulted in the public reviews, but it seems to us that its inclusion in the article is not warranted. Group. The definition of the categories should be explained. For instance, what is the difference between victims and displaced? The authors should also mention that respondents could mark several responses. RTA:The authors did not use a working definition (theoretical or legal) of the categories in the present article. This work was based on a self-identification and self-affiliation approach, in which each person indicated whether they considered themselves a “victim” or “displaced.” This approach is relevant to these types of quantitative questions, and is even used for complex categories such as ethnicity.Using a working definition defined by the authors would not be useful since the study participants themselves decided how they are classified based on their own criteria, which does not always correspond to theoretical and legal definitions. Thus, for example, “victim” or “displaced” is how one defines oneself. In addition, the methodology does not allow for determining labels in a distinct manner or with “objective” criteria, given that they are based on each individual case and only one simple question. (How it is formulated can be consulted in the questionnaire in Spanish, and its translation into English.)Unfortunately, with regard to the question of group affiliation, at the request of the program, this was defined operationally and in an exclusive manner, though in reality the groups are not mutually exclusive (the same person could be in two or more categories) since people had to choose only one category with which they identified most. These groups were defined because they were the ones considered to be of interest to the program, and we found that the majority of the participants (over 71%) identified as ex-combatants.All of this clearly indicates a limitation with respect to this variable. Therefore, these categories are presented as part of the characterization of the study population, but are not considered to be categories for analysis, nor are they intended to be more deeply explored. We included a comment about the definition of the groups in a footnote in Table 1. Housing. ETCRs, as part of the settlement project of the peace accord, have very similar characteristics. These categories, however, do not seem to reflect the housing conditions of ex-combatants in the collective reincorporation program. We suggest explaining the general characteristics of these types of housing. It seems that some questions were also adapted from other survey studies and it is unclear how they were transferred to the context of the ETCRs. For example, it is hard to think how “rented room” (inquilinato in the original instrument) is even a possibility within the context of ETCR. Equally, it is unclear what “room in another type of structure” refers to RTA:This imprecision clearly stems from our original efforts to create transferability with some of the surveys that have been performed in rural areas in Colombia. We agree that it is not possible to consider whether something like “rented room” (inquilinato) exists in this context. The interviewers were consulted again about this question, and it was decided that all the cases were a type of rented room, while not being able to distinguish which type.Those who indicated “rented room” were probably referring to a room in houses that were not their own and where other families lived, and that is what they called it (very few responded in this way). “Rented room in another structure” refers to a rented room in a house belonging to someone else, but that is not considered to be a rooming house. Therefore, we decided to unify “rented room” into one single category given that it is not possible to distinguish between these. For transparency purposes, we kept the original question as it appears in the instrument.We do not have more information about housing conditions. Role in the family. In this question, if all the interviewees were the heads of the household as it is reported on page 3, it is unclear what relationship categories represent. RTA: The concern about the role of the family was clarified in the methods section (paragraph 1), where the main informant was the head of household or the person who had the most information about the health status of the household members. Therefore, in the results table, we can see that the main informant’s role in the household was not only head of the family but could also be wife. Given the time the data were collected (September and October 2018), we know that some ambulances and health care personnel were still present in some of the ETCRs. Then, it is not entirely clear if the services people received were part of the Health for Peace project that had significant funds from international cooperation or if people were, in fact, using the services of the healthcare system. If ambulances were available, did that facilitate access to services and in a way skewed the data? Further information of the historical moment and specific conditions of the ETCRs at the time of the interview, as mentioned for the introduction, could help with the analysis of these results RTA:We do not have this information and we would not be able to collect it in a valid manner. We do not have records available to us that would enable us to answer this question. Since we only have oral reports from key actors rather than systematic and reliable data, we prefer not to include this. As we mentioned, the objective of this study does not include distinguishing between whether the given services were provided through international cooperation or by the healthcare system. In fact, for us, this difference is not very clear because international cooperation was aimed at strengthening the institutional response in a sustainable manner. Of course this is debatable, but we do not have more information with which to discuss it.Below is an additional response that from the administrator of the IOM’s Migration and Health program, which could be published as part of the response, for readers who would like more discussion: “Yes, there were ambulances in some of the ETCRs and health personnel who strengthened the hospitals of influence in the ETCRs. That was an intervention led and organized by national governmental agencies, as well as hundreds of interventions that were carried out to demobilize the ex-combatants. Comprehensive management of health care also existed by national agencies that played a role in complying with the peace accords. And there were calls for the presidency of the public insurance company to enroll ex-combatants, as well as many other interventions that affect perception. We do not believe there is bias precisely because what was measured was the perception of the multiple and specific social interventions in health that existed at the time. We are not saying that access to health by the rural population in Colombia was measured. We only have a snapshot of a single moment in the history of health in Colombia. It was not of interest to us to evaluate in a distinguishing manner which actions and institutions contributed to this perception.” We do not believe that it is relevant to include more details about coordination between international cooperating agencies and the Health for Peace project. Figure 1, 2, and 3 offer interesting information but it is very hard to assess the responses. Are there national and international guidelines that allow us to assess whether those percentages are adequate? RTA:No standards for these indicators exist, and they are not easily comparable since they depend on demographic and epidemiological profiles, objective morbidity, perceived morbidity, self-perception of health, and the profile of the utilization of services, which are different for different populations and social contexts. The indicators in these figures are primarily a reflection of the offer and use of services, rather than the need, although that does partly determine usage.Nevertheless, their publication does provide a reference for future studies and for making comparisons with similar populations.This comparison is presented in the discussion, as is standard for similar publications. The discussion and analysis portion of the article compares our results with national and international parameters for perception of access to the different dimensions that make up this construct in the survey of emergency services, promotion, prevention, and extramural activities. The discussion describes the literature search that was performed and discusses the results in a detailed manner. The 2015 Colombian Demographic and Health Survey (ENDS in Spanish) was used as one of the main sources for comparing our national-level results with other rural contexts. As we mentioned in the introduction, perception of effective access is not commonly evaluated, and therefore, there are results from other studies with which our parameters cannot be compared, including findings related to the general population. Lastly, the results section of the article was exclusively aimed at presenting and describing the findings of our research.We defer to the editor as to whether it is necessary to include an additional explanatory document (such as a linked file) to clarify how promotion and prevention programs and extramural activities function in Colombia, and how to compare the results in the discussion given the available sources of information. For example: Explanation of the Functioning of Promotion and Prevention Programs in ColombiaIn Colombia, promotion and prevention programs are governed and organized based on Law 1751 of 2015, of the Statutory Law for the fundamental right to health. Article 5 stipulates that the State has the responsibility to respect, protect, and guarantee the effective enjoyment of this fundamental right. Therefore, it must “Formulate and adopt policies to promote and prevent health, and to treat disease through collective and individual actions.” Thus, the Ministry of Health, through all the actors in the Colombian Social Security Health System (SGSSS, in Spanish) (Colombian Health System), must adopt and integrate approaches involving primary health care (PHC) and family and community health, and coordinate individual and collective actions according to a population focus. In order to comply with these levels of care, the Ministry issued Resolution 429 of 2019, which adopted the Comprehensive Health Care Policy (PAIS in Spanish) and the Comprehensive Health Care Model (MIAS in Spanish). One of the components of the MIAS includes comprehensive routes to health care (RIAS in Spanish), which the Colombian Health System is required to use so as to comply with mandatory health care activities.With respect to intramural and extramural promotion and prevention activities, resolution 3280/2018 established a comprehensive route to health promotion and maintenance, a route through specific events, a route for risk groups, and a maternal-perinatal route.These routes involve actions at the individual, collective, population, and public health management levels. They adopt technical and operational guidelines, including service providers’ compliance with procedures and specific consultations by age group, with frequencies stipulated in the technical provisions of the Resolution, for each group served. The Ministry of Health establishes indicators for evaluating and monitoring the health system, nonetheless, their construction is finalized when they are implemented in the territories. Establishing compliance parameters for the indicators depends on the diagnosis of the health situation and the territorial health plan. Given that municipalities have different starting points, comparable standard measurements cannot be established. Therefore, due to the differences that exist among the territories, national standards cannot be established for each program. According to the provisions in the Colombian health system, these routes and programs must be present and operational in at least 80% of each area. (falta cita resolucion 3280/2018).The discussion compared our results with the use of promotion and prevention programs presented in the figures, based on the available literature on population surveys in Colombia and the findings on routes from official sources. Regarding the figures that present the evaluation of our main category of analysis —perception of access— the comparisons in the discussion were based on research studies and the available documented experience in the country and in other countries. Nevertheless, most of the available sources of information on access to health services is based on the perspective of providers rather than the users of the services. Therefore, a comparison with our results would be limited.We do not believe that this text should or could be included in the article. It could be added as an addendum or footnote to the figures (as a linked file). Furthermore, Figures 2 and 3 require information about the clinical specificities of the children under 1 or pregnant women to assess whether the services offered were adequate or not, perhaps according to whether their regular or schedule check-ups are in agreement with known guidelines. We are left wondering how important this information is RTA: This information cannot be recovered from our database. The data is at the household level and the question refers to someone in the household. Unfortunately, we cannot evaluate this indicator because, first, there would be very few pregnant women and children under 1 year of age. And second, we do not have a way to obtain the total children under 1 year or total pregnant women. We do know that there were few, according to the interviewers. Third, this was not the objective. Actually, the information reported does not allow for evaluating adherence to the standards in the guidelines, and that was not the study’s interest. The fact that the questions ask for the last four weeks further complicates the analysis given that the person could be in a moment in which there is no need to use these services and appointments might have already happened before the four weeks period or are scheduled for after the interview. RTA : See the complete response provided previously (comment 2, methods). An explanation about the period of 4 weeks is included. In brief, if we were to use a longer period then the time at which the program was evaluated would not be comparable for all the subjects, and would result in recall bias. Figures 4 and 5 would benefit from an explanation of these programs and the guidelines or parameters established in the country regarding their offering. Are health promotion and illness prevention services replacing regular care? Why would someone have to schedule such an appointment rather than already having an appointment or being contacted to schedule the appointment? How do referrals and enrollments in these programs work? Also, questions arise about the coordination between programs and whether the presented data is adequate or follows any established guidelines. RTA:See the previous response (comment 11), where towards the end we proposed including a short text about the functioning of the programs. There are no standards for the parameters that we are measuring, which we explained in the first part of the response, and it depends on the context. We are not evaluating guidelines or adherence to protocols, but rather, perception of access, subject to indicators of offerings and use of services.We do not believe that primary care or services to prevent illness replace “regular care,” as the evaluator seems to suggest. The strategies are consistent with national public health guidelines for rural areas. We want to stress that we do not conceive of a dichotomy between health services in the context of the project and health services defined by the Public Health Plan, but rather, it is about development and adaptation in the context of the ETCRs. Regarding the last questions, we do not have an answer, we cannot provide one based on the results, and they are not part of the objective of the study. In addition, in order to answer the last three questions, information would have to be collected for that moment in time and for each ETCR, since in practice, this is different in each place and changes rapidly over time. Discussion:1. The discussion requires major changes. The authors discuss some of the results in relation to existing literature but it is unclear what are the central ideas of the discussion and what results the authors found particularly interesting and relevant. RTA: This is a brief report of a multi-dimensional survey. That would likely occur because the objective of this first analysis is to present multi-dimensional findings in a general manner, as a basis and background for more specific analyses. It is complicated to reduce the magnitude of the different results to a few central ideas. Nevertheless, we believe the new pages, 27 and 28, contain central ideas for the discussion. At the request of previous reviewers, the discussion is organized according to the following logic: 1. Overall context of the right to health for ex-combatants and during post-conflict, 2. Context of how this is inserted into the Colombian peace and reincorporation process, 3. Summary of the importance of this study, including strengths and weaknesses of the survey and the instrument, 4. Explanation of the main findings in the context of the ETCRs, 5. Most relevant findings, reference points, and comparison with other studies of ex-combatants, 6. Interpretation of results based on the rural context, and in contrast with other studies on effective access in rural areas, 7. Limitations of the study, and 8. Conclusions. Page 13. 2nd paragraph. “While prioritizing the right to health for ex-combatants represents important progress in public policy, Colombia’s health system faces numerous challenges that need to be studied”. What challenges is this sentence referring to? RTA:The challenges are innumerable. Nevertheless, right after the phrase that we cited, we present the two main challenges for Colombia’s health system in terms of guaranteeing the right to health for ex-combatants. One challenge is rural health, which is explained by the fact that ETCRs are located in rural areas and there is little guarantee of access to health services in those areas in Colombia. The second challenge that we expressed is that by not fulfilling the guarantee of access to health services for ex-combatants, which is one of the cornerstones of compliance with reintegration and reincorporation, the peace process itself could be at risk. Page 13. 3rd paragraph. The ARN survey of 2014 describes a different reintegration program. This program includes ex-combatants who demobilized before the peace agreement and the majority of them moved to urban areas. As mentioned, the article should specify from the beginning that the population assessed in the study are part of the collective reincorporation program and describe their main characteristics. Is it possible to discuss some differences between access to health care depending on whether the ex-combatants participated in the reintegration or the reincorporation programs? RTA:Some of these points have already been addressed in the new version of the introduction, which provides greater context. It is now clear that we refer to reincorporation. Throughout the introduction we now refer to reincorporation and how it is inserted into the Health for Peace project. We corrected where the term “reintegration” was incorrectly used, where it was necessary to replace it with reincorporation. The terminology for discussing reincorporation was verified and is now consistent with the program, IOM, and the institutions.Nevertheless, we are not able to address the last question based on the results that are available to us, since we would not be able to make distinctions based on the information that was collected.Even if differences between access to health services by the different reintegration and reincorporation processes could be compared, it would not fall within the scope of this study since the objective proposed for this work was to determine the perception of effective access to health services according to the activities provided by the project. This is a cross-sectional measurement that is not comparable with other reintegration and reincorporation processes developed in the country, for two fundamental reasons. One reason is because it is organized by official entities that do not have public information about access to health services according to reintegration and reincorporation. Secondly, these programs measure access based only on enrollment in the health system. Furthermore, this research did not differentiate between whether they belonged to one program or the other. The contrast with healthcare provision in other rural areas is an interesting and important discussion. However, the main points of this contrast are not clear. RTA Oficial :In paragraphs 14, 15, and 16 in the discussion, we contrasted the provision of services with other rural areas based on the available literature, which indicated that their availability is very limited. The only source for comparing access to services in rural areas in Colombia is the 2015 National Demographics and Health Survey, which contains some sections that distinguish between type of enrollment and type of services for the rural population. Nevertheless, as can be seen, that survey does not involve perception of access, which is the category analyzed in our study. This greatly limits our ability to compare our results on access in rural areas with those of ENSUT, since the ENSUT results were oriented more towards service provides than users.In general, evaluations of access to services in Colombia are more focused on urban areas, such as the Avillarraga et al survey, and there is still a large gap in knowledge about what occurs in rural areas. Therefore, our results provide an initial basis for this line of study, and contribute to reducing the knowledge gap.Furthermore, in order to discuss our findings in greater depth, we used other international sources, such as the studies obtained from Paraguay and some areas of Africa, where the study of access to services in rural areas is a little more common. But we reiterate that there is a limitation given the type of construct that is used to evaluate access.Therefore, we accept your comments that perhaps our analysis of the subject does not have clear points of contrast. Nonetheless, this is related more to the existing literature and knowledge on the subject, thereby demonstrating the importance of continuing to more deeply explore this subject and generate more knowledge about it. . Page 14 3rd paragraph. We suggest avoiding adjectives such as “high”, “low” and “small” without providing specific data. RTA: These changes were made. The adjectives were replaced with specific data from the surveys. The discussion of the results about health promotion and illness prevention services in relation to studies conducted in Manizales and Medellin does not seem appropriate, unless the difference between these services in rural and urban areas is stated. RTA: This is true, but since the most relevant points of reference do not exist, we prefer to keep this in the text. Although the reviewer is clearly correct and a clarification for the reader is needed.We clarified this in the paragraph before the discussion about the studies. Studies of urban areas were used because we did not find any studies of promotion and prevention services in rural areas in Colombia that presented findings similar to ours, although their correspondence to different socioeconomic and cultural contexts should certainly be taken into account. What do the authors mean by good treatment in accordance with their culture and beliefs? Are the notions of culture and beliefs related to their identity as ex-combatant, a regional identity, a rural identity, or an ethnic/racial identity? RTA:The dimension of treatment according to culture and beliefs was included in the instrument for two reasons: providing culturally appropriate health care for the ex-combatant population is one of the cornerstones of the Health for Peace project, as reflected in the explanation of the components of the project. And, as mentioned in the introduction to the article, one of the dimensions underlying the perception of effective access to health services is that the care provided by health personnel is acceptable to the users. This category does not reflect a specific belief system or culture, but rather, it ensures that when health personnel provide health care to the ex-combatant population and their family, they take into account the user’s opinion, acceptance, and wishes regarding activities and treatments for managing and treating their illnesses and caring for their health. The results do not indicate that people’s behavior to seek health care services can be explained through the health belief model. RTA :This is relevant given how it is presented in the article, and should be kept in the discussion.One of the dimensions of effect access to health services is self-agency, which does not depend on the health system but rather on people’s attitudes and beliefs about seeking health services in a timely fashion. Our results found that 41% of the population knew about the extramural services offered by the project but only 17% used them. It is clear that this was not due to a lack of knowledge about the existence of the service, but rather, to the individual’s own interest in using the services.This belief system is of course a construct that goes beyond the dimension of self-agency in access to health services, and is complemented by bio-psycho-social models that more thoroughly explore the components of a person's belief system, such as the health beliefs model, which can give a more explanatory framework for specific findings related with extramural services. In regard to these services, in addition to knowing about their existence, people need to be motivated to use them, and is determined by the priority that each individual places on health, on each one of its dimensions, as well as by self-agency and self-care. In the discussion, the authors would like to offer possible explanations for our results without establishing a definitive position, but rather, by offering possible explanations that are supported by a rigorous literature search on the subject. . We suggest that the center of the discussion should be around how the current data compare to the challenges described for other rural populations. The authors hint in this direction but could make significant contributions by signaling how ex-combatants’ perception of their access to health care compares to other studies in rural areas. Are they getting better or worse access to health care than other rural communities? What does this say about the reincorporation process and about the health care system? If the analysis is broken down by region, what does it tell us about different regions in Colombia and, perhaps, different “reincorporation processes”? RTA: The questions by the reviewers go well beyond the scope of our results. Our interest is to merely describe a snapshot in time of the process in the ETCRs. We provide context and reference points, but in order to answer the questions that are posed, specifically if it has improved or worsened over time, would require other types of data (probably longitudinal), different sources of primary and secondary information, and different and more complex methodologies, all of which goes beyond the scope of our results.We do not agree with discussing aspects that can not directly enter into dialogue with the results of our study, given that this is a report of a descriptive study. This analysis would warrant another investigation, and we prefer to be cautious since we do not have elements with which to even suggest a response.As we explained, we presented the data by region for informative purposes, and given our sample size and design, regions cannot be compared. Therefore, it would not be possible to explore any comparisons among regions with our results.We limit ourselves to highlighting and comparing only the results from other studies that can enter into dialogue with our results and that pertain to our objective.The main objective of the research presented in this article was to determine the perception of effective access to health services by a population of ex-combatants who were in the process of reincorporation and reintegration in the ETCRs.In this case, the Health for Peace project sought to strengthen the Colombian health system through joint actions, thereby facilitating and enhancing access to health services for this population and their families, in the area of primary health care, since the ETCRs are located in rural environments where access to these types of services presents difficulties, and there are significant gaps for the rural population in general.An instrument was designed to determine effective access to health services in order to identify whether these types of actions were strengthening the services and thereby impacting the health of the ex-combatant population while the project’s actions were underway. The instrument was designed for this population from the perspective of the user. That is, it was based on the construct of perception, which more adequately indicates whether the project strengthened the health system such that ex-combatants and their families had the impression or appreciation that they were effectively accessing the services that they needed —whether faced with an emergency or when seeking health promotion and prevention services through intramural and extramural activities.With regard to the regional comparability of the results, as explained in the discussion, one of the few available sources for making a comparison is a study by the World Bank, which is the only source that was found that evaluates these dimensions by region in Colombia. That is, the regions with the least access are Orinoquia and Amazonia, which is due to the little availability of health centers, whereas central Colombia is characterized by a greater presence of this type of health institution.With regard to comparing different reincorporation processes, the contrast at the global level was included at the beginning of the discussion, in paragraphs 2 to 4. Here, Colombia was described as representing a high standard, as the health component is one of its priorities, whereas other countries, such as Namibia, did not take this component into account or it only involved the supply of health kits for ex-combatants.With regard to the experience with reincorporation processes in other countries, the health dimension has not been prioritized or mentioned in the available sources. Thus, it is important to document Colombia’s experience with the reincorporation route, which we attempted to point out in the beginning of the discussion, in paragraphs 4 and 5.In addition. the data on reincorporation processes and routes that are available from official sources measure access to services based only on enrollment in the Colombian health system and frequency of use of services. Considering the scope of our results, that is not comparable. Based on our findings, we can conclude that the population had a favorable perception of the actions that were taken to strengthen the Colombian health system in rural areas, such as those by the project. We also conclude that compliance with reincorporation processes and their successful completion require actions that ensure that the health dimensions continue to be priorities, but this must be based on the rural context, which is a great challenge for the health system."
}
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}
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https://f1000research.com/articles/8-2140
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https://f1000research.com/articles/9-579/v1
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09 Jun 20
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{
"type": "Research Article",
"title": "Notified dengue deaths in Myanmar (2017-18): profile and diagnosis delays",
"authors": [
"Nwe Ni Linn",
"Khine Wut Yee Kyaw",
"Hemant Deepak Shewade",
"Aye Mon Mon Kyaw",
"Myat Min Tun",
"San Kyawt Khine",
"Nay Yi Yi Linn",
"Aung Thi",
"Zaw Lin",
"Hemant Deepak Shewade",
"Aye Mon Mon Kyaw",
"Myat Min Tun",
"San Kyawt Khine",
"Nay Yi Yi Linn",
"Aung Thi",
"Zaw Lin"
],
"abstract": "Background: Complications in dengue usually occur between day four and day six after fever onset. Hence, early diagnosis and haematological monitoring are vital. Among all hospital reported dengue deaths in Myanmar in 2017-18, we assessed the i) patient profile, ii) proportion of patients who arrived with a dengue diagnosis at admission and iii) delays in diagnosis after fever onset. Methods: This was a descriptive study involving secondary data. For all the notified deaths, death investigation forms were not available in prescribed format and therefore, data were extracted from hospital case records. Results: Of 304 deaths, 184 (60.5%) were female and 233 (76.6%) were less than 10 years old. Township level hospitals or below reported 36 deaths (11.8%) and the remaining deaths were from higher level facilities. Dengue was diagnosed before admission in 26 (8.5%) people and 169 (55.6%) were in shock at admission. Of 208 with date of fever onset recorded, the median diagnosis delay was four (interquartile range-IQR: 3, 5) days. Patient level delay (median three days) was a major contributor to the diagnosis delay. Conclusions: Most of the patients who died did not have a diagnosis of dengue before admission. This calls for an urgent review of health system preparedness in peripheral health facilities to suspect, diagnose, monitor, refer and treat dengue in children and patient level factors for better understanding of the reasons of delay. Timely filling of death investigation forms in a prescribed format and quarterly death reviews based on these is recommended.",
"keywords": [
"Dengue fever",
"Mortality",
"Delay in diagnosis",
"Severe dengue",
"Operational Research",
"SORT IT"
],
"content": "Introduction\n\nDengue is a mosquito-borne viral disease that has rapidly spread in tropical and subtropical regions. Around 3.9 billion people are at risk of dengue in 128 countries where there is good evidence of dengue occurrence1. In recent years, transmission has increased predominantly in urban and semi-urban areas and the incidence of dengue in adults is increasing2,3. In 2016, there were a total of 3.3 million reported dengue patients4.\n\nSevere dengue usually occurs between day four and six after fever onset (called the critical stage, during which fever subsides) and is one of the leading causes of hospitalization and death among children and adults in most Asian and Latin American countries4. There are four distinct serotypes of dengue virus (DEN-1, DEN-2, DEN-3 and DEN-4). People who recover from dengue infection may get lifelong immunity against one particular serotype and cross-immunity for a few months. Subsequent infections by other serotypes increase the risk of developing severe dengue4. Half a million people with severe dengue require hospitalization every year, and around 2.5% of them die4. Mortality is highest in younger age groups and reduces with increasing age5.\n\nDengue mortality can be reduced by early detection and good referral systems especially at the primary health care level, predicting and managing severe dengue with appropriate treatment at the hospital level, reorienting health services to cope with dengue outbreaks, and training health personnel at all levels of the health system6. Once the diagnosis is confirmed or suspected, severe dengue can be detected early by clinical (significant abdominal pain, persistent vomiting, lethargy, restlessness, mucosal bleeding, fluid accumulation) and haematological monitoring7,8.\n\nDelay in diagnosis could be at the level of the patient or health system9,10. Late presentation is associated with severe disease in adult dengue patients10. Dengue death is commonly associated with co-morbidities and clinicians should be aware if dengue patients fulfil the severe case definition on admission11,12.\n\nMyanmar is a high dengue burden country in the Asia Pacific Region. Between 2011 and 2015, of the 89,832 dengue related admissions, 97% were children13. There is limited published literature on diagnosis delays after fever onset among children or adults who died due to dengue. Therefore, we aimed to describe the profile and delays in diagnosis among all dengue deaths in Myanmar.\n\n\nMethods\n\nWe conducted a descriptive study involving secondary data collection. We included all dengue deaths reported to the Vector Borne Disease Control (VBDC) Programme in Myanmar during 2017–18.\n\nMyanmar, a tropical country, is located in the Southeast Asia Region, bordering the Republic of China on the north and northeast, Laos on the east, Thailand on the southeast, Bangladesh on the west, and India on the northwest. Myanmar is still a predominantly rural country, with around 30% of the population living in urban areas14. The country is divided administratively into Nay Pyi Taw union territory and 14 states and regions and consists of 74 districts and 330 townships15.\n\nDoctors are first available at sub-township level station hospitals. Basic health staff at the level of rural health centres (below station hospital) and sub-centres (below rural health centre) provide comprehensive primary health care. Specialists are available at district level hospitals.\n\nSD BIOLINE dengue rapid test kits (includes NS1 Antigen, IgG antibody and IgM antibody) and packed cell volume (PCV) using a centrifuge machine (microhaematocrit or haematocrit) are available at some of the station hospitals. PCV and platelet counts using haematology auto analysers are available at district hospitals and above. The NS1 antigen ELISA test is available only at National Health Laboratory, Yangon, and is used for evaluation of rapid test kits. Diagnosis, treatment and monitoring of dengue patients in health care facilities highly depends on the availability of doctors (at station hospitals), rapid test kits, centrifuge machine and complete haemogram (for platelet count). The laboratory facilities may not be available as assigned or sometimes may malfunction. All the severe or complicated dengue (confirmed or suspected) patients are referred to the nearby district, regional or tertiary hospital.\n\nIn 2011, the Myanmar Paediatric Society published Paediatric Management Guidelines (second edition) and updated them in 201816. The chapters for dengue were adopted from the 2009, 2011 and 2012 WHO guidelines8,12,17. In 2018, the National Guideline for Clinical Management of Dengue for doctors and basic health staff were published18,19. Training has been provided to basic health staff and doctors since 2018 in selected states and regions based on funding availability. It is being expanded to the whole country from 2019.\n\nThe assistant director or team leader is responsible for VBDC Programme planning and implementation at the state and regional levels. All hospitals maintain a dengue register for admitted patients. The VBDC works closely with the department of medical services to collect data on dengue morbidity and mortality by using dengue register. In case of a laboratory confirmed dengue with any grading at hospital, VBDC staff, basic health staff and community volunteers carry out dengue prevention and control activities within a 100 meter radius of the patient’s house.\n\nIf there is any dengue death, hospitals are expected to inform VBDC staff immediately. VBDC staff go to the hospital and fill out a dengue death investigation form and report back to the central VBDC office.\n\nDengue patients that do not get admitted are not reported to the VBDC. The dengue patients managed by the private sector are not routinely reported to the programme, except one private hospital which reported one dengue death during 2017–18.\n\nA line list of all dengue deaths was prepared in March 2019 by using the dengue death reports submitted to central VBDC Programme by State and Regional offices. Though we were planning to extract data from death investigation forms, it was found that they were not filled out in the prescribed format. Therefore, data were extracted from the hospital case records to a structured data collection form. These paper-based hospital case records were sent to principal investigator.\n\nThe data collected included age, sex, state or region, dengue grading at admission, hospital name, date of admission, type of diagnosis (laboratory/clinical), type of first health care provider visited and cause of death. The dates of onset of fever, first health care provider visited after fever, diagnosis, admission and death were also collected. Operational definitions for ecological regions and dengue grading have been summarized in Table 1.\n\nDHF, dengue hemorrhagic fever; DSS, dengue shock syndrome.\n\nThe data were double-entered and validated using EpiData entry software (version 3.1, EpiData association, Odense, Denmark) and analysed using STATA (version 14.2 College Station, Texas). The profile was summarized using frequencies and proportions. Patient level (fever onset to first health care provider visit), health system level (first health care provider visit to diagnosis) and total diagnosis delay (fever onset to diagnosis) were calculated. The hospital stay duration was calculated from date of admission to date of death. The duration and delays in days were summarized using the median and interquartile range (IQR).\n\nAdministrative approval for the study was obtained from the VBDC Programme and ethics approval was received from Ethical Review Committee, Department of Medical Research, Myanmar (Ethics/DMR/2018/149 dated 7 December 2018) and Ethics Advisory Group, The Union, Paris, France (EAG number 45/18 dated 23 August 2018). As this study involved review of routinely collected secondary data, a waiver for informed consent was sought and approved by the ethics committees. This study involved the use of patient medical data in which all data analysed were anonymized.\n\n\nResults\n\nOf 31,288 and 23,273 dengue patients hospitalized in 2017 and 2018, 192 (0.6%) and 112 (0.5%) died, respectively.\n\nThe characteristics of these 304 reported deaths at admission and cause of death are described in Table 2. Of 304 deaths, 184 (60.5%) were among females. The median age was six (IQR: 3, 9) years, 233 (76.6%) deaths were among children (<10 years) and 246 (80.9%) were reported during the wet (June to October) season. Dengue shock syndrome at admission was seen in 169 (55.6%) and at death in 257 (84.5%). Of 304 deaths, 36 (11.8%) were reported from the township level hospital or below and the remaining were from higher level facilities.\n\nDHF, dengue hemorrhagic fever; DSS, dengue shock syndrome; EDS, expanded dengue shock syndrome; BHS, basic health staff.\n\n¥Station hospital = hospital covering villages in a township; township hospital = hospital covering whole township; district hospital = hospital covering the whole district; regional hospital = hospital covering the whole state/region; teaching hospital = hospital that provides medical education and training to health professionals; tertiary hospital = hospital providing tertiary care, which is health care from different specialists.\n\nThe type of first health provider visited after fever onset was not recorded for 142 (46.7%) patients and 57 (18.8%) got directly admitted to hospital where they died. Among patients who had a first health care provider visit (that was recorded) before getting admitted to the hospital where they died (n=105), 32 (30.5%) either went to a chemist or untrained provider or self-medicated (Table 2).\n\nThe date of onset of fever was only available for 208 (68.4%) patients. Of these 208, the patients were admitted after a median of four (IQR: 3, 5) days after fever onset. The median patient level delay was three (IQR: 1, 4) days and median health system level diagnosis delay was zero (IQR: 0, 2) days. Total diagnosis delay was four (IQR: 3, 5) days (Table 3).\n\nIQR, interquartile range.\n\n*Includes those for whom dates were available.\n\nOf 304, 81 (26.6%) patients died within 24 hours of admission. Before admission, dengue fever was diagnosed in only 26 (8.5%) (Figure 1).\n\n\nDiscussion\n\nThis is the first published study from Myanmar that described diagnosis delay from fever onset among all reported dengue deaths in Myanmar.\n\nMost of the patients did not have a diagnosis of dengue fever before admission and more than half of the patients were in shock at admission. This was also seen in a study from Malaysia20. In Brazil, among patients with severe dengue, having a laboratory diagnosis was associated with a lower chance of dying when compared to not having a diagnosis21.\n\nAbsence of or delay in dengue fever diagnosis or lack of suspicion of dengue before admission may not have given time for proper treatment. The patients may have self-referred or were referred to higher level facilities when clinical features worsened, resulting in patients directly presenting with severe dengue at hospitals. Early diagnosis could have ensured detection of patients who were more prone to develop dengue haemorrhagic fever and dengue shock syndrome. Complications or worsening of dengue usually occurs at or after four days of fever onset8,23. This is when the fever also subsides.\n\nPatient level delay (median three days) was a major contributor to the total diagnosis delay. Studies from Myanmar have reported that most people with fever sought care from untrained personnel or self-medicated and the reasons for avoiding care from trained health care providers (basic health staff, village health volunteers) was that minimal action was taken if fever patients were not diagnosed with malaria24,25.\n\nThe training for doctors and basic health staff on updated dengue guidelines was done only in 2018 and all regions were not covered. This could have contributed to the lack of clinical suspicion of dengue in peripheral health facilities.\n\nThe highest numbers of patients were reported in the wet season and were from the less than 10 years old age group. This highly correlates with the epidemiology of dengue disease2,5. The rainy season is also the school start time in Myanmar.\n\nThis study has five implications for policy and practice. First, a review of health system preparedness to suspect, diagnose, monitor, refer and treat dengue is required at peripheral health facilities as most patients were admitted without a diagnosis of dengue fever. This includes review of availability of rapid test kits at station hospitals and above. Considering the burden of dengue is similar to malaria in Myanmar, availability of test kits should be ensured at the level of station hospitals and preferably up to the grass roots level for evaluation of undifferentiated fever17. Training of basic health staff and doctors of all regions regarding the updated 2018 national guidelines is required. Guidelines for the management of undifferentiated fever should be developed26. This will especially be beneficial for health care providers in peripheral health facilities.\n\nSecond, as death investigation forms were not filled out in the prescribed format for all dengue deaths, we recommend quarterly dengue death reviews at regional and national levels. This will indirectly ensure that states and regions fill out the dengue investigation forms in the prescribed format and submit them to the VBDC Programme regularly.\n\nThird, we recommend a similar study among all the reported dengue patients to confirm whether these findings are present (or not). Fourth, to address patient level delay there is a need to raise awareness in the community regarding health seeking for undifferentiated fever. Finally, the following four indicators may be added to the monthly forms in the health management information system in health facilities: i) total suspected with dengue ii) total confirmed dengue and iii) diagnosis within three days of fever among confirmed dengue.\n\nWe collected nationwide data of all reported dengue deaths and this represents the ground reality. Data were robust as they were double entered and validated.\n\nThere were two major limitations in the study. First, death investigation forms were not being filled out. The reason for this is not known. This resulted in many missing variables as these were not available in the hospital case records. This includes details on the first health care provider visited and various dates. Diagnosis delay related findings are limited by the absence of the date of fever onset in one-third of patients. These forms also help the programme at a national level to review the causes of death, identify preventable health system related causes and take timely action.\n\nSecond, the variables recorded in hospital represent the clinical condition of the patients and other important variables for dengue prevention and control such as previous episodes of dengue, comorbidities, housing, income, occupation/school places, travel history, utilization of bed net, use of mosquito repellent were not routinely available.\n\nTo conclude, this study reported a long diagnosis delay after fever onset among dengue deaths reported over two years in Myanmar. This calls for an urgent review of health system preparedness to suspect, diagnose, monitor, refer and treat dengue at peripheral health centres and patient level factors for better understanding of reasons for diagnosis delay. Filling out of dengue death investigation forms should be ensured and corrective action be taken through regular regional and national dengue death reviews.\n\n\nData availability\n\nFigshare: Nwe Ni Linn et al 2020 dataset v2. https://doi.org/10.6084/m9.figshare.12355958.v127\n\nThis project contains the following underlying data:\n\nData_NNL.xlsx (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": "Disclaimer\n\nThe views represented here are those of the authors and do not represent the institutions they are affiliated to.\n\n\nReferences\n\nBrady OJ, Gething PW, Bhatt S, et al.: Refining the global spatial limits of dengue virus transmission by evidence-based consensus. PLoS Negl Trop Dis. 2012; 6(8): e1760. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBhatia R, Dash AP, Sunyoto T: Changing epidemiology of dengue in South-East Asia. WHO South East Asia J Public Health. 2013; 2(1): 23–27. PubMed Abstract | Publisher Full Text\n\nWorld Health Organization (WHO): Comprehensive guidelines for prevention and control of dengue and dengue haemorrhagic fever. World Health Organization. Regional office for South-East Asia. Geneva, Switzerland; 2011. Reference Source\n\nWorld Health Organization (WHO): Dengue and severe dengue fact sheet 2019 [Internet]. 2019. Reference Source\n\nAnders KL, Nguyet NM, Chau NVV, et al.: Epidemiological factors associated with dengue shock syndrome and mortality in hospitalized dengue patients in Ho Chi Minh City, Vietnam. Am J Trop Med Hyg. 2011; 84(1): 127–34. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWorld Health Organization: Global Strategy for Dengue Prevention and Control 2012-2020. World Health Organiszation. Geneva, Switzerland; 2012. Reference Source\n\nWorld Health Organization (WHO): Handbook for clinical management of dengue. Geneva, Switzerland; 2012. Reference Source\n\nWorld Health Organization (WHO): Global strategy for dengue prevention and control 2012-20. Geneva, Switzerland; 2012. Reference Source\n\nSacramento RHM, de Melo Braga DN, Sacramento FF, et al.: Death by dengue fever in a Brazilian child: a case report. BMC Res Notes. 2014; 7: 855. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJain S, Mittal A, Sharma SK, et al.: Predictors of Dengue-Related Mortality and Disease Severity in a Tertiary Care Center in North India. Open forum Infect Dis. 2017; 4(2): ofx056. PubMed Abstract | Free Full Text\n\nThein TL, Leo YS, Fisher DA, et al.: Risk factors for fatality among confirmed adult dengue inpatients in Singapore: a matched case-control study. PLoS One. 2013; 8(11): e81060. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWorld Health Organization (WHO): Dengue: Guidelines for diagnosis, treatment, prevention and control. Geneva, Switzerland; 2009. Reference Source\n\nOo PM, Wai KT, Harries AD, et al.: The Burden of Dengue, Source Reduction Measures, and Serotype Patterns in Myanmar, 2011 to 2015-R2. Trop Med Health. BioMed Central; 2017; 45: 35. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDepartment of Population; Ministry of Labour Immigration and Polpulation; Myanmar. Census Atlas Myanmar: The 2014 Myanmar Population and Housing Census. Nay Pyi Taw; 2014. Reference Source\n\nMinistry of Health and Sports: Health in Myanmar, 2014. Nay Pyi Taw,Myanmar; 2014. Reference Source\n\nMinistry of Health and Sports; Myanmar: Paediatric Management Guidelines. Nay Pyi Taw, Myanmar; 2018.\n\nWorld Health Organization (WHO): Comprehensive guidelines for prevention and control of dengue and dengue haemorrhagic fever. Geneva, Switzerland; 2011. Reference Source\n\nMinistry of Health and Sports; Myanmar: National Guideline for Clinical Management of Dengue. Nay Pyi Taw; 2018. Reference Source\n\nMinistry of Health and Sports; Myanmar: Dengue Prevention and Control for BHS at Township Level. Nay Pyi Taw, Myanmar: Ministry of Health and Sports; 2018.\n\nWoon YL, Hor CP, Hussin N, et al.: A Two-Year Review on Epidemiology and Clinical Characteristics of Dengue Deaths in Malaysia, 2013-2014. PLoS Negl Trop Dis. 2016; 10(5): e0004575. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMoraes GH, de Fátima Duarte E, Duarte EC: Determinants of mortality from severe dengue in Brazil: a population-based case-control study. Am J Trop Med Hyg. 2013; 88(4): 670–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMakroo RN, Raina V, Kumar P, et al.: Role of platelet transfusion in the management of dengue patients in a tertiary care hospital. Asian J Transfus Sci. 2007; 1(1): 4–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMd-Sani SS, Md-Noor J, Han WH, et al.: Prediction of mortality in severe dengue cases. BMC Infect Dis. BMC Infectious Diseases; 2018; 18(1): 232. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNaing PA, Maung TM, Tripathy JP, et al.: Awareness of malaria and treatment-seeking behaviour among persons with acute undifferentiated fever in the endemic regions of Myanmar. Trop Med Health. BioMed Central; 2017; 45: 31. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHein KT, Maung TM, Htet KKK, et al.: Low uptake of malaria testing within 24 h of fever despite appropriate health-seeking among migrants in Myanmar: a mixed-methods study. Malar J. 2018; 17(1): 396. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBhargava A, Ralph R, Chatterjee B, et al.: Assessment and initial management of acute undifferentiated fever in tropical and subtropical regions. BMJ. 2018; 363: k4766. PubMed Abstract | Publisher Full Text\n\nShewade HD: Nwe Ni Linn et al 2020 dataset v2. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12355958.v1"
}
|
[
{
"id": "64469",
"date": "18 Jun 2020",
"name": "Hui Liu",
"expertise": [
"Reviewer Expertise Control of vector borne diseases"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nDengue is now ranking as one of the most critical global mosquito-borne viral diseases and is endemic in over 100 countries. For many countries, dengue is becoming a threat to their public health, and further adversely impacting their health services and economies. Early diagnosis and effective supporting treatment for dengue can reduce transmission and improve patient prognosis. Data on death, delayed treatment are rare. This paper gives reader a profile and diagnosis delays in Myanmar. It is value to get indexed.\nHowever, we suggest the author consider:\nThe part of introduction is not strong enough, not make the objectives clear.\n\n\"Most of the patients who died did not have a diagnosis of dengue before admission\", please make clear in method section, \"how were these patients confirmed as dengue ? just based on records of symptoms or have laboratory test evidence?\n\nMore discussing in detail on reasons of delayed diagnosis and potential solution, this will be significance on dengue control in Myanmar.\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": []
},
{
"id": "65627",
"date": "03 Jul 2020",
"name": "Zhuo Lin Chong",
"expertise": [
"Reviewer Expertise Evaluation of dengue rapid diagnostic test kits",
"dengue surveillance"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nTitle and abstract: Reflective of the content\nIntroduction: The study is important. It would be perfect if the flow of the introduction can be rearranged to make the justification of the study stands out. Beside that, there were many references to adults and children in the text, with emphasis on the latter. Suggest to realign them to better reflect the title and content of the article.\nObjectives: They are clearer in the abstract but in not the main text. Suggest to detail them out in the main text as well.\n\nMethods:\n\nSetting: The second paragraph on the availability of healthcare personnel is important in the interpretation of this study. Suggest making it clearer to readers not familiar with Myanmar healthcare system. E.g. replace the below hospital and center in bracket with an additional sentence right after the mention of sub-township level station hospitals to make clear the organisation.\n\nVariables and data collection: Would be great if more details are given on the data collector/extractor, any quality control in place, how missing values were handled.\n\nResults:\n\nSuggest to state if the number of dengue death extracted for this study matched the number of death reported to VBDC through surveillance. Response rate is important to assess selection bias.\n\nTable 2: Type of first health care provider visited has categories that overlap each other, e.g. directly admitted into hospital and government doctor can be the same thing. Please label the categories more distinctively. Would be perfect if they are also defined in the operational definition.\n\nPatient profile of dengue deaths: Suggest: i) to remove \"Among patients who had a first health care provider visit (that was recorded) before getting admitted to the hospital where they died (n=105)\", ii) use 32 (10.5%), and iii) use \"pharmacist\" instead of chemist. Discrepancies can create confusion and reduce readability.\n\nFigure 1 Timing of dengue diagnosis: If the objective for this comes in second, suggest to present it in the same way. Consider additional subsection corresponding to the objective. Also, number and percentage of patients who died within 24 hours of admission could not be found in this figure.\n\nDiagnosis delay after fever onset: Suggest to convert this table into a figure featuring a horizontal bar, on the left end of which labelled fever onset, the right end - death, and each stage in between. Then put your N, median, and IQR below with tracking lines going upward to different stages on the bar. That way it is understandable at a glance.\nDiscussion:\n2nd paragraph: \"Most of the patients did not have a diagnosis of dengue fever before admission and more than half of the patients were in shock at admission. This was also seen in a study from Malaysia.\" Please make it clear that the reference to Malaysia is pointing to \"shock at admission\" and not \"did not have a diagnosis of dengue fever before admission\". As 73.6% of patients were suspected to have dengue in the Malaysian study.\n\nThe first section of discussion can be strengthened by comparing the results of this study with that of the other studies in depth.\n\nImplication for policy and practice: Strengthening the discussion in the first section would make these recommendations more persuasive. Suggest to add good practices from other countries to make it compelling.\n\nReferences: To reorganize references. Combine identical item into one reference, e.g. WHO Global Strategy 2012 in references 6 & 8.\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": "64470",
"date": "15 Jul 2020",
"name": "Sangay Zangmo",
"expertise": [
"Reviewer Expertise Molecularand virological techniques",
"Molecular epidemiology"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript projects interesting findings and correlations on diagnosis delay with deaths of dengue patients in Myanmar. For a country with high dengue burden, interventions on such findings will greatly enhance dengue diagnosis, referral, timely treatment and patient survival. Methods are clearly elucidated; strengths and weaknesses have been described well. Language and command in general is good, however, may need to be re-looked into in some sentences. I have some suggestions as below:\nIn the abstract: Most of the patients who died did not have a diagnosis of dengue before admission. This Sentence needs rephrasing.\n\nIn the introduction: Dengue death is commonly associated with co-morbidities and clinicians should be aware if dengue patients fulfil the severe case definition on admission. Word “severe” may be in inverted commas\n\nIn the methods: Doctors are first available at sub-township level station hospitals. Remove “first”\nSome general comments:\nDefinition of “delay” is not clear. After how many days would it be considered as delayed? In table 3, would it be more appropriate to use the word “duration” since there is no clear definition as to from which day “delay” would imply?\n\nEcological factors in this manuscript described include rainfall. Rainfall can be one of the factors among other such as temperature, vegetation and urbanization. Information on these variables, if available may be interesting. Some papers have been cited for your reference.\n\nIn the discussion: Most of the patients did not have a diagnosis of dengue fever before admission and more than half of the patients were in shock at admission. Does that mean patient previously visited some kind of health centres prior to admission? For those that were diagnosed, how was the diagnosis made?\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/9-579
|
https://f1000research.com/articles/9-577/v1
|
09 Jun 20
|
{
"type": "Research Article",
"title": "Enclosing a pen to improve response rate to postal questionnaire: an embedded randomised controlled trial",
"authors": [
"Rachel Cunningham-Burley",
"Jenny Roche",
"Caroline Fairhurst",
"Sarah Cockayne",
"Catherine Hewitt",
"Heather Iles-Smith",
"David J. Torgerson",
"SSHeW Study Team",
"Jenny Roche",
"Caroline Fairhurst",
"Sarah Cockayne",
"Catherine Hewitt",
"Heather Iles-Smith",
"David J. Torgerson"
],
"abstract": "Background: Poor response to questionnaires collecting outcome data in randomised controlled trials (RCTs) can affect the validity of trial results. The aim of this study within a trial (SWAT) was to evaluate the effectiveness of including a pen with a follow-up postal questionnaire on response rate. Methods: A two-armed RCT was embedded within SSHeW (Stopping Slips among Healthcare Workers), a trial of slip-resistant footwear to reduce slips in NHS staff. Participants were randomised 1:1 to receive a pen or no pen with their follow-up questionnaire. The primary outcome was the proportion of participants who returned the questionnaire. Secondary outcomes were: time to response, completeness of response, and whether a postal reminder notice was required. Data were analysed using logistic regression, linear regression and Cox proportional hazards regression. Results: Overall, 1466 SSHEW trial participants were randomised into the SWAT. In total, 13 withdrew from the host trial before they were due to be sent their follow-up questionnaire, 728 participants received a pen with their questionnaire, and 725 did not receive a pen. A questionnaire was returned from 67.7% of the pen group and 64.7% of the group who did not receive a pen. There was no significant difference in return rates between the two groups (OR 1.15, 95% CI 0.92 to 1.43, p=0.22), nor level of completeness of the questionnaires (AMD -0.01, 95% CI 0.06 to 0.05, p=0.77). There was weak evidence of a reduction in the proportion of participants requiring a reminder and in time to response in the pen group. Conclusion: Inclusion of a pen with the follow-up postal questionnaire sent to participants in the SSHeW trial did not statistically significantly increase the response rate. These results add to the body of evidence around improving response rates in trials. Trial registration: ISRCTN 33051393 (for SSHEW). Registered on 14/03/2017.",
"keywords": [
"randomised controlled trial",
"embedded trial",
"postal questionnaire",
"response rate",
"pen"
],
"content": "Introduction\n\nRandomised controlled trials (RCTs) are key to evaluating the effectiveness of interventions and often use postal questionnaires to collect outcome data. However, low response rates can limit the validity of the trial findings by reducing the power of the study and introducing bias1.\n\nNumerous strategies to increase response rates have been studied2,3 including sending a pen with the questionnaire. The pen acts both as a facilitator to aid completion of the questionnaire, and an incentive to return it4,5. The effectiveness of this intervention is equivocal with some studies reporting an increase in response rate5–7 whilst others failed to show a positive impact4,8. These studies displayed considerable heterogeneity and only two were embedded in RCTs6,7. A Study within a Trial (SWAT) is a self-contained study embedded within a host trial that can be used to evaluate strategies designed to improve trial efficiency9. This SWAT evaluated the effectiveness of enclosing a pen with a follow-up postal questionnaire on response rates in the SSHeW trial10.\n\n\nMethods\n\nThis two-armed RCT was embedded in the SSHeW trial, a trial evaluating the effectiveness of slip-resistant footwear to reduce slips in NHS staff10. The SSHeW trial was registered (ISRCTN 33051393) and the trial protocol has been published10.\n\nThe SWAT was conducted in seven NHS Trusts in England and included all eligible participants in the SSHeW trial who were due to be sent their 14-week postal questionnaire between 04.07.2018 and 12.02.2019.\n\nThe intervention group were sent a York Trials Unit, University of York branded pen with their questionnaire. The control group did not receive a pen.\n\nThe SWAT outcomes are outlined in Table 1.\n\nAs is usual with an embedded trial, a formal sample size calculation was not undertaken as the sample size was determined by the number of participants due to receive their 14-week questionnaire.\n\nWe anticipated that randomising 2,000 participants into the SWAT would provide 80% power to detect an absolute difference of 6% (two-sided α=0.05) in response rates between the two groups, assuming a control rate of 60%.\n\nParticipants were allocated to either the intervention (pen) or control (no pen) group using simple randomisation in a 1:1 ratio. The allocation sequence was generated by the SSHeW trial statistician, who was not involved in sending out the questionnaires.\n\nParticipants were not aware of their involvement in this SWAT but due to the nature of the intervention participants and study team members could not be blinded to group allocation.\n\nThis SWAT was approved by the Department of Health Sciences Research Ethics Committee at the University of York and the Health Research Authority (HSRGC/2016/187/A).\n\nData were analysed using Stata version 1511 on an intention-to-treat basis, using two-sided tests at the 5% significance level. The models used for each outcome are given in Table 2, the values associated with the pen allocation from each model is presented with its 95% confidence interval and p-value. All models were adjusted for main trial group allocation (slip-resistant footwear or wait-list control) and pen sub-study allocation (pen or no pen).\n\nThe total cost of a standard SSHeW questionnaire pack was £2.42 (envelope and postage: £0.86; questionnaire and cover letter: £0.65; pre-paid envelope and postage: £0.91). The additional cost of including a pen was £0.32. The cost analysis incorporates the changes in number of questionnaires returned and reminders required.\n\n\nResults\n\nA total of 1466 participants were included in the SWAT (pen, n=733; no pen, n=733). In total, 13 participants withdrew from the main SSHeW trial after they had been randomised into the SWAT but before being sent their follow-up questionnaire, leaving 1453 participants (Figure 1). Baseline characteristics are summarised descriptively in Table 3.\n\nResults are presented in Table 4. Overall, 962 (66.2%) questionnaires were returned (pen, 67.7%; no pen, 64.7%) and an average of 4.9/5 items were completed. There was no evidence of a difference in return rate between the groups (OR 1.15, 95% CI 0.92 to 1.43, p=0.22), nor number of items completed (AMD -0.01, 95% CI 0.06 to 0.05, p=0.77).\n\nOD, odds ratio; HR, hazards ratio; AMD, adjusted mean difference\n\nThere was weak evidence of a difference, in favour of the pen group, in both time to return (median time to return 15 vs 18 days; HR 1.12, 95% CI 0.98 to 1.27, p=0.09) (Figure 2), and in the proportion of participants requiring a reminder (OR 0.83, 95% CI 0.68 to 1.02, p=0.08).\n\nA 3% difference in questionnaire response rate and an absolute difference in the percentage of participants who required a reminder of 1.1% were found. Considering these to be true effects, in order to receive one additional questionnaire, 33 participants would have to be sent a pen, at a cost of approximately 33x32p=£10.56. Approximately 91 participants would need to be sent a pen to prevent one reminder mailing and therefore to save £2.42. Hence, roughly one reminder is required per three retained participants, and the cost per retained participant is approximately £10.\n\n\nDiscussion\n\nWhilst the results of all outcomes in this SWAT favoured the pen group, we found that the addition of a pen did not statistically significantly increase the response rate to, or completeness of, a follow-up questionnaire sent at 14 weeks post-randomisation among participants of the SSHeW trial. There was some evidence of a reduction in time to response and the number of reminders required.\n\nIt may be that, in this group of participants, the pen failed to act as a facilitator or was not a sufficient incentive to return the questionnaire, given the fact that participants in the trial already received a free pair of shoes (although offer of shoes was not conditional on returning the questionnaire).\n\nHowever, the trial ultimately only had about 40% power to detect a difference of 3% in response rates (from 64.7 to 67.7%) and is therefore at risk of a type II error. Another potential weakness is that, due to the select population of healthcare workers, the results may not be generalisable to other populations or contexts.\n\nThe strength of this study is that it was a randomised trial.\n\n\nConclusion\n\nThis SWAT suggests that enclosing a pen in a questionnaire mail out may be an effective method to increase response rates but was likely underpowered to detect a statistically significant difference of the 3% observed. Since pens are inexpensive, even a small difference is likely to be cost-effective. The results contribute to the body of evidence regarding this intervention and may be included in future meta-analyses to improve power.\n\n\nData availability\n\nOpen Science Framework: SSHeW Trial Pen SWAT, https://doi.org/10.17605/OSF.IO/YQ76U12.\n\nOpen Science Framework: CONSORT checklist for ‘Enclosing a pen to improve response rate to postal questionnaire: an embedded randomised controlled trial’, https://doi.org/10.17605/OSF.IO/YQ76U12.\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).",
"appendix": "Acknowledgements\n\nThe authors would like to thank the embedded trial participants who returned their trial documentation and the data administration staff at the YTU who worked to support the study.\n\nThe paper was written by the authors on behalf of the SSHeW Trial team, which is made up of the following individuals: Emily Bain (Health and Safety Executive); Sarah Cockayne (University of York); Rachel Cunningham-Burley (University of York); Caroline Fairhurst (University of York); Gillian Frost (Health and Safety Executive); Catherine Hewitt (University of York); Heather Iles-Smith (Leeds Teaching Hospital NHS Trust); Mark Liddle (Health and Safety Executive), Misbah Mogradia (Health and Safety Executive); David Torgerson (University of York), Michael Zand (Health and Safety Executive) and others. With the exception of Emily Bain and Misbah Morgradia, who supported Health Economic Evaluation for the host SSHeW Trial, all members of the SSHeW study team were involved in the designing this SWAT.\n\nWe would also like to acknowledge the support of individuals from the PROMETHEUS programme.\n\n\nReferences\n\nEdwards P, Roberts I, Clarke M, et al.: Increasing response rates to postal questionnaires: systematic review. BMJ. 2002; 324(7347): 1183. 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 JF, Stenning S, et al.: Strategies to improve retention in randomised trials: a Cochrane systematic review and meta-analysis. BMJ Open. 2014; 4(2): e003821. PubMed Abstract | Publisher Full Text | Free Full Text\n\nClark T, Khan K, Gupta J: Provision of pen along with questionnaire does not increase the response rate to a postal survey: a randomised controlled trial. J Epidemiol Community Health. 2001; 55(8): 595–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWhite E, Carney PA, Kolar AS: Increasing response to mailed questionnaires by including a pencil/pen. Am J Epidemiol. 2005; 162(3): 261–6. PubMed Abstract | Publisher Full Text\n\nSharp L, Cochran C, Cotton SC, et al.: Enclosing a pen with a postal questionnaire can significantly increase the response rate. J Clin Epidemiol. 2006; 59(7): 747–54. PubMed Abstract | Publisher Full Text\n\nBell K, Clark L, Fairhurst C, et al.: Enclosing a pen reduced time to response to questionnaire mailings. J Clin Epidemiol. 2016; 74: 144–50. PubMed Abstract | Publisher Full Text\n\nAveyard P, Manaseki S, Griffin C: The cost effectiveness of including pencils and erasers with self-completion epidemiological questionnaires. Public Health. 2001; 115(1): 80–1. PubMed Abstract | Publisher 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\nCockayne S, Fairhurst C, Frost G, et al.: SSHeW study protocol: does slip resistant footwear reduce slips among healthcare workers? A randomised controlled trial.BMJ Open. 2018; 8(11): e026023. PubMed Abstract | Publisher Full Text | Free Full Text\n\nStataCorp: Stata Statistical Software: Release 15. In: College Station, TX:StataCorp LLC; 2017. Reference Source\n\nCunningham-Burley R, Roche J, Fairhurst C, et al.: SSHeW Trial Pen SWAT. 2020. http://www.doi.org/10.17605/OSF.IO/YQ76U"
}
|
[
{
"id": "68921",
"date": "02 Sep 2020",
"name": "Anna Duncan",
"expertise": [
"Reviewer Expertise Hanne Bruhn: Health Services Research and Trial Methodology. Anne Duncan: Trial Management and conduct and Trial Methodology. Magaly Aceves Martins: Nutrition and systematic reviewing."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 study is a SWAT – a Study Within A Trial. A SWAT is a methodological study aiming to add to the evidence base for randomised controlled rial design and conduct. In a SWAT an intervention to improve the conduct of trials is tested in an ongoing ‘host’ trial.\nThe current host trial was Stopping Slips among Healthcare Workers (SSHeW), a trial testing slip-resistant footwear with NHS Staff to reduce slips at work. The SWAT intervention was to include a pen with the follow-up questionnaire to test whether participants who received a pen were more likely to return their questionnaire than those participants who did not receive a pen. The authors also looked at whether inclusion of a pen with the questionnaire affected the time taken to send back the questionnaire; how complete they questionnaire was answered and; whether a reminder had to be sent for return of the questionnaire. The SWAT was run on the 14 week post randomisation follow-up questionnaire. All SSHEW participants were randomised into the SWAT (n=1466). There were no significant difference in return rates between the two groups but weak evidence that including a pen might make participants return the questionnaire sooner and less will need a reminder.\n\nThis study reports findings contributing to the evidence base for how trial teams can increase questionnaire response rates in their trials, using the gold standard randomised comparison. This is a hugely important area as low response rates affect the conclusions that can be drawn from trial findings. Unfortunately, low questionnaire response rates are something which continues to affect a lot of trials.\nWe have a few comments which should be addressed:\nABSTRACT No comments.\nINTRODUCTION It would be interesting to know if the authors expected there to be an issue with the questionnaire return rate in the host trial and what was the basis of this expectation (e.g. low response rate to previous questionnaires)? Please consider adding what the expected response rate for the host trial was. This could be added in the introduction or methods according to the authors preference. Commonly, SWATs are reactive so some background information to the approach in this SWAT would provide a helpful context.\n\nThe authors mention that the pen would act as facilitator and incentive, it would be helpful to have any reference to this use as a behavioural motivator or barrier.\n\nMETHODS It would be interesting to know if the investigators found any issues with posting a pack including a pen, eg additional bulk causing any issues with standard franking machines. Did any changes have to be made to the process of posting out to include the pen?\nRESULTS No comments.\nDISCUSSION Please include some consideration as to whether the timing of the incentive to return questionnaires might have affected the findings. In many trials participants are followed-up for two years or longer and that is the point when it gets really tough to keep the response rate high (reviewers’ personal experiences). It would be really valuable to have the authors thoughts on this for trial teams looking for help.\nParagraph 3 -The authors list a lack of power as a limitation. We’d suggest highlighting that many SWATs will be underpowered because they are generally unable to change the size of the host trial; they are made for meta-analysis really.\n\nCONCLUSION This is a very well conducted and clearly written article on a SWAT that investigated including a pen to improve response rates to a questionnaire, and importantly addressed the cost effectiveness of the intervention as one of the outcomes.\nThe results of the SWAT did not provide strong evidence in itself of the effectiveness of including a pen to improve response rates but the results add to the body of evidence and will be important to include in meta-analysis to examine the effectiveness of a pen as an incentive/facilitator in retention rates.\nWhile the pen itself is inexpensive, when it is multiplied up over the issue of thousands of questionnaires is an additional cost any trials unit would have to justify to funder. The cost for a 3% gain is £10 per retained participant according to statement in Results (Costings) so not an insubstantial amount for weak evidence of an effect. Replication of this SWAT is needed to include other settings, different timepoints etc before concluding that including a pen may be effective and cost-effective way of improving response rates. The authors should expand on this in the concluding paragraph and consider revising sentence 2 of the concluding paragraph.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
},
{
"id": "80788",
"date": "08 Mar 2021",
"name": "Karen Bracken",
"expertise": [
"Reviewer Expertise Trial operations and recruitment"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 result of a SWAT embedded within the SSHeW trial. The SWAT aimed to evaluate whether including a pen with a follow-up postal questionnaire would impact questionnaire response rate. The authors indicate that previous studies have yielded mixed results. The results of this SWAT add to the body of existing evidence. The paper is clear and concise and addresses a common problem faced by trials; low response rates to questionnaires. The paper provides adequate detail for other trialists to replicate the intervention within their own trial. The inclusion of cost analyses is particularly useful. I was interested to see that the cost of including the pen was very low and would have liked an explanation of the components of the cost (cost of pen, additional cost of postage and posting materials). Like many SWATs, this SWAT was unavoidably constrained in sample size by the host trial, making conclusions difficult due to lack of power. It is unclear from the paper whether the authors expected that the sample size of 1466 was sufficient to detect an operationally important difference between groups. If not, then I suggest the conclusion be updated to make this clear, rather than stating that a statistically significant increase was not found. Would the authors suggest that further SWATs are needed in this area based on the Trial Forge guidance? (https://trialsjournal.biomedcentral.com/articles/10.1186/s13063-019-3980-51) Also, I suggest removing the observed power calculation from the discussion (https://lesslikely.com/statistics/observed-power-magic/ provides an explanation as to why observed power calculations should be avoided).\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/9-577
|
https://f1000research.com/articles/9-576/v1
|
09 Jun 20
|
{
"type": "Research Article",
"title": "Potential chimeric peptides to block the SARS-CoV-2 spike receptor-binding domain",
"authors": [
"Debmalya Barh",
"Sandeep Tiwari",
"Bruno Silva Andrade",
"Marta Giovanetti",
"Eduardo Almeida Costa",
"Ranjith Kumavath",
"Preetam Ghosh",
"Aristóteles Góes-Neto",
"Luiz Carlos Junior Alcantara",
"Vasco Azevedo",
"Sandeep Tiwari",
"Bruno Silva Andrade",
"Marta Giovanetti",
"Eduardo Almeida Costa",
"Ranjith Kumavath",
"Preetam Ghosh",
"Aristóteles Góes-Neto",
"Luiz Carlos Junior Alcantara",
"Vasco Azevedo"
],
"abstract": "Background: There are no known medicines or vaccines to control the COVID-19 pandemic caused by SARS-CoV-2 (nCoV). Antiviral peptides are superior to conventional drugs and may also be effective against COVID-19. Hence, we investigated the SARS-CoV-2 Spike receptor-binding domain (nCoV-RBD) that interacts with hACE2 for viral attachment and entry. Methods: Three strategies and bioinformatics approaches were employed to design potential nCoV-RBD - hACE2 interaction-blocking peptides that may restrict viral attachment and entry. Firstly, the key residues interacting with nCoV-RBD - hACE2 are identified and hACE2 sequence-based peptides are designed. Second, peptides from five antibacterial peptide databases that block nCoV-RBD are identified; finally, a chimeric peptide design approach is used to design peptides that can bind to key nCoV-RBD residues. The final peptides are selected based on their physiochemical properties, numbers and positions of key residues binding, binding energy, and antiviral properties. Results: We found that: (i) three amino acid stretches in hACE2 interact with nCoV-RBD; (ii) effective peptides must bind to three key positions of nCoV-RBD (Gly485/Phe486/Asn487, Gln493, and Gln498/Thr500/Asn501); (iii) Phe486, Gln493, and Asn501 are critical residues; (iv) AC20 and AC23 derived from hACE2 may block two key critical positions; (iv) DBP6 identified from databases can block the three sites of the nCoV-RBD and interacts with one critical position, Gln498; (v) seven chimeric peptides were considered promising, among which cnCoVP-3, cnCoVP-4, and cnCoVP-7 are the top three; and (vi) cnCoVP-4 meets all the criteria and is the best peptide. Conclusions: To conclude, using three different bioinformatics approaches, we identified 17 peptides that can potentially bind to the nCoV-RBD that interacts with hACE2. Binding these peptides to nCoV-RBD may potentially inhibit the virus to access hACE2 and thereby may prevent the infection. Out of 17, 10 peptides have promising potential and need further experimental validation.",
"keywords": [
"Antiviral peptides",
"COVID-19",
"SARS-CoV-2",
"nCoV-19",
"peptide design",
"ACE2",
"Spike protein"
],
"content": "Introduction\n\nThe world is currently experiencing the severe coronavirus disease 2019 (COVID-19) pandemic caused by SARS-CoV-2 or novel corona virus (nCoV) that originated from the Wuhan city, China1,2, and spread across the world. So far, two million people have been infected and more than 120,000 deaths are recorded across the globe. The death rate is 3–20% depending on the countries and the most affected countries are the USA, Italy, Spain, the UK and France, which have each recorded more than 10,000 deaths within a couple of weeks (WHO COVID-2019 situation reports). SARS-CoV-2 is highly contagious in humans and so far no medicine or vaccine has been developed to tackle the virus, making it impossible to control its spread across the globe3. Although drugs like hydroxychloroquine, remdesivir, and lopinavir4 are currently being suggested to treat COVID-19 infection, there is no clinical study so far to prove their efficacy in treating these patients. Therefore, currently there is a global search for appropriate drug and vaccine candidates against SARS-CoV-2.\n\nSARS-CoV-2 has shown 80% genome identity with SARS-CoV, which is the causal agent of severe acute respiratory syndrome (SARS) seen in 2002–20035. SARS-CoV binds to the human angiotensin-converting enzyme 2 (hACE2) receptor through its Spike protein (S) to enter into the host cell6, and it is now reported that SARS-CoV-2 also binds to ACE2 to transmit its genetic material to human cells7–9. Therefore, blocking the Spike protein of SARS-CoV-2 could be an attractive and effective way to prevent the SARS-CoV-2 infection.\n\nThe crystal structure of the hACE2 receptor and the receptor binding domain (RBD) of the SARS-CoV-2 Spike protein (nCoV-RBD) (PDB: 6M17>) showed that a total of eight residues, namely, Gln24, Asp30, His34, Tyr41, Gln42 in α1 helix, Met82 in α2 helix, and Lys353 and Arg357 in the β3 and β4 linkers, are important for the binding9. The important interactions between the nCoV-RBD with ACE2 are Lys417 (Spike) --Asp30 (hACE2), Tyr453 (Spike) --His34 (hACE2), Gln474 (Spike) --Gln24 (hACE2), Phe486 (Spike) --Met82 (hACE2), Gln498 (Spike) --Tyr41 (hACE2), Thr500 (Spike) --Gln42 (hACE2), and Asn501 (Spike) --Lys3539.\n\nPeptide-based drugs are a better choice than conventional drugs due to their higher efficiency, lesser molecular weight, and lower toxicity and side effects10. In this regard, antiviral peptides (AVPs), and a subset of antimicrobial peptides (AMPs), are of specific interest due to their higher efficacy in inhibiting viral infection by targeting various stages of the viral life cycle. AVPs can directly invoke innate immune response11 and inhibit viral entry by targeting viral attachment and entry to host cell, and replication, transcription, translation, multiplication, and release inside the host cell12,13. Previously, several AVPs have been reported to inhibit the SARS-CoV Spike protein or viral entry14–16.\n\nIn this report, using bioinformatics strategies, we attempted to design anti-Spike peptides for SARS-CoV-2 towards motivating potential therapeutics against the SARS-CoV-2 infection.\n\n\nMethods\n\nWe adopted three strategies to predict potential AVPs against the SARS-CoV-2 Spike protein.\n\nIn the first strategy, we re-analysed the SARS-CoV-2 Spike RBD with hACE2 to identify the key interacting residues in both the proteins. A recent report suggests that the B chain of SARS-CoV-2 Spike protein interacts with the B or D homodomain of hACE29. Therefore, in this analysis, we used the individual B chain of SARS-CoV-2 Spike RBD (PDB: 6LZG) and the B chain of hACE2 (PDB: 6M18) to dock with each other using the HADDOCK 2.2 server17, providing active residues of both the proteins as described by Yan et al., (2020)9 and using default parameters. Based on the binding interactions and based on previous reports9, we identified the key interacting residues. In the next step, we designed a number of AVPs based on the interacting hACE2 residues to the RBD of the Spike protein. To design the AVPs, we used simple permutation and combination approach of amino acids, keeping the key interacting amino acids and their positions fixed in the peptide. Binding to SARS-CoV-2 Spike RBD with the designed AVPs was determined by the HPEPDOCK protein-peptide docking server18. For the HPEPDOCK analysis, we used the SARS-CoV-2 Spike RBD (PDB: 6LZG) B chain and the sequence of the peptides in FASTA format, specifying eight binding sites (Lys417, Tyr453, Gln474, Phe486, Gln493, Gln498, Thr500, and Asn501) of Spike-RBD9 and 100 peptide binding mode. The final peptides were selected based on their HPEPDOCK docking energy score, number of binding sites, number and position of selected target residue binding, physiochemical properties, and AVPpred prediction13. More negative binding energy and the number and position of residue binding sites were given more importance in selecting the final peptides (described in Results).\n\nIn the second strategy, we screened the available anti-microbial peptides (AMPs) against the SARS-CoV-2 Spike RBD. We used the database of antiviral peptides (AVPdb)19, database of HIV inhibitory peptides (HIPdb)12, Antimicrobial Peptide Database (APD3)20, database of anti-microbial peptides (dbAMP)21, and database of FDA-approved peptide and protein therapeutics (THPdb)22 and screened the peptides against the SARS-CoV-2 Spike RBD. In this process, we collected all the AMP sequences from these databases and then each peptide was docked against the Spike-RBD using the HPEPDOCK protein-peptide docking server18. The process and the parameters of HPEPDOCK docking and selection of peptides were the same as used in first strategy.\n\nIn the third strategy, we adopted a chimeric peptide design approach where the two fragments of two different peptides selected in our previous two approaches are composed in such a way that the resultant peptide can bind to our given target residues in the SARS-CoV-2 Spike RBD. In this approach, we first selected the peptides that bind to any of the three key residues (Phe486, Gln493, and Asn501) of the Spike RBD. Next, we took various lengths of fragments of these peptides (4–15) that interact with the key residues of Spike RBD. Next, we joined these peptides keeping the key residue position fixed to make chimeric peptides of 20–25 amino acids in length. In these peptides, various permutations and combinations of amino acids were made, keeping the key interacting amino acids and their positions fixed. A total of 500 such chimeric peptides were designed and docked with Spike RBD target residues (Lys417, Tyr453, Gln474, Phe486, Gln493, Gln498, Thr500, and Asn501) using the HPEPDOCK server18 as described in the first strategy . The final peptides were selected based on similar criteria as adopted in the first and second strategy.\n\nAntiviral properties of the peptides were predicted using AVPpred13 using its default parameters. The molecular formula, molecular weight, net charge, grand average hydropathy, total hydrophobic ratio, hydrophobicity, and protein-binding potential (Boman index) were calculated using the APD3 antimicrobial peptide calculator and predictor20. The IC50 of the peptides was predicted using the AVP-IC50Pred server, selecting the RSV/INFV/HSV prediction model23 and other default parameters. Hemolytic potency of peptides was determined using the HemoPI server24 with default parameters, where the values tending towards “0” are unlikely to be hemolytic. ToxinPred25, with default parameters, was used to predict the toxicity (toxic or non-toxic) of the peptides. The final peptides were selected based on their: HPEPDOCK docking energy score (cut-off -120 or less), number of H bonds (>2), number of selected target residue bonds (2-10), number of key target residue bonds (>2), physiochemical properties (parameters acceptable for AMPs, see Extended data26), and AVPpred prediction (Yes)13.\n\n\nResults and Discussion\n\nIn SARS-CoV-2 Spike RBD - hACE2 interaction analysis, similar to Yan et al. (2020)9, we found that three stretches of peptides that harbour the active residues of hACE2 interact with Spike RBD. These stretches have amino acid positions: 21-43 (five sites), 78-87 (one site), and 348-361 (two sites). In the Spike RBD, the key interacting amino acid stretches are 480-489 and 490-505. A previous report suggests that among the Spike residues, the most important residues interacting with hACE2 are Phe486, Gln493, and Asn50127. An in-depth analysis revealed that any peptide that potentially blocks the Spike RBD should bind at least three critical positions of the RBD: (i) Gly485 or Phe486 or Asn487, (ii) Gln493, and (iii) Gln498 or Thr500 or Asn501 among which the Phe486, Gln493, and Asn501 are essential.\n\nWe designed three peptides from the first stretch of the hACE2 that shows maximum active binding residues with the Spike RBD. A 26 amino acid peptide (AC26) binds to Thr500 and Asn501 of the Spike RBD (Figure 1A), a 23 amino acid peptide (AC23) binds to Tyr489 and Thr500 of the Spike RBD (Figure 1B), and a third 20 amino acid peptide (AC20) binds to Gln493 and Asn501 of the Spike RBD (Figure 1C). All these peptides show acceptable physiochemical properties to be used as therapeutic peptides (see Extended data26). However, none of these three peptides are able to block all the key three positions of the Spike RBD. Therefore, these peptides may not be suitable for developing very effective anti-SARS-CoV-2 therapeutics targeting its Spike RBD. However, AC20 and AC23 can be further tested.\n\n(A) AC26, (B) AC23, and (C) AC20.\n\nWe identified seven peptides by screening the five different AMP databases. It is known that SARS-CoV directly interacts with hACE2 through their RBD located in the B chain of the Spike protein28,29 and the Spike protein sequence of SARS-CoV-2 is highly similar to SARS-CoV8 and SARS-related coronaviruses. In our peptide database analysis, we also observed that the peptides that have been experimentally proven to be effective against SARS-CoV have the potential of being used against the SARS-CoV-2 Spike protein. All of the seven identified peptides (see Extended data26) are of 20 amino acids in length and are reported to target the Spike protein of SARS-CoV to exhibit their anti-SARS virus activities19. Although the peptides show four to 11 “H” bonds and form between two to four bonds with our eight given target residues, most of these peptides do not bind to all the three positions ((i) Gly485 or Phe486 or Asn487, (ii) Gln493, and (iii) Gln498 or Thr500 or Asn501) to effectively block the Spike RBD.\n\nThe DBP1, DBP2, and DBP3 peptides bind to only the third position (Gln498 and Asn501) of the Spike RBD without binding to the other two sites (Figure 2A–C; Extended data26). DBP4, DBP5, and DBP7 interact with the first (Gly485 or Phe486 or Asn487) and third (Gln498 and Asn501) sites of the Spike RBD without binding to the middle or the second site (Gln493) (Figure 2D, E, G; Extended data26). DBP6 binds to all the three sites within the range of the target residues but does not interact with the key residues of the first and third sites (Figure 2F; Extended data26). DBP6 is also predicted to be an antiviral peptide by AVPpred13. Therefore, DBP6 could be a potential peptide to be tested for SARS-CoV-2 Spike protein-based drug development.\n\n(A) DBP1, (B) DBP2, (C) DBP3, (D) DBP4, (E) DBP5, (F) DBP6, and (G) DBP7.\n\nOut of 500 chimeric peptides generated, only seven were selected for final analysis. All these peptides are non-hemolytic, non-toxic, and meet all the criteria of a therapeutic peptide (see Extended data26). Among these seven peptides, cnCoVP-3, cnCoVP-4, and cnCoVP-7 interact with all the three sites and two key residues of the second (Gln493) and third (Asn501) sites. However, these peptides bind one amino acid apart from the key residue (Phe486) in the first site and potentially block the access of SARS-CoV-2 Spike Phe486 to the hACE2 (Figure 3C, D, G; Extended data26). AVPpred13 also predicted cnCoVP-4 to be an antiviral peptide. Therefore, these three peptides could be selected for further in vitro and in vivo testing.\n\nAlthough the chimeric peptides cnCoVP-2, cnCoVP-5, and cnCoVP-6 interact with all the three sites, they do not interact with the key residue (Phe486) or the immediate to key residue of the first site. Instead they bind a residue that is two to three amino acids apart from the key residue (Phe486) (Figure 3B, E, F; Extended data26). Therefore, these three peptides may not block the first site of the Spike RBD in interacting with hACE2. However, they should also be synthesized and tested for their in vitro effects. The last peptide, cnCoVP-1, was found to interact with all the three sites; however, it only interacts with the key residue (Gln493) of the second site. In the other two sites, it interacts at position (Tyr489) of the first site and (Gln498) of the third site (Figure 3A; Extended data26). Although Gln498 is a key residue of the third site Tyr489, it is not an interacting residue in the original interaction between SARS-CoV-2 Spike RBD and hACE2. Thus, this peptide may partially block the access of SARS-CoV-2 Spike RBD to hACE2 and needs further in vitro and in vivo testing and validation.\n\n(A) cnCoVP-1, (B) cnCoVP-2, (C) cnCoVP-3, (D) cnCoVP-4, (E) cnCoVP-5, (F) cnCoVP-6, and (G) cnCoVP-7.\n\n\nConclusions\n\nIn this article, we screened and designed several peptides that may potentially block the interaction between SARS-CoV-2 Spike RBD and hACE2. Ten peptides (AC20, AC23, DBP6, and cnCoVP-1- cnCoVP-7) have very high potential to achieve this interaction, indicating that these peptides could be attractive therapeutics against SARS-CoV-2. However, peptide synthesis, in vitro, and in vivo experiments are required to evaluate and ensure their potential therapeutic efficacy.\n\n\nData availability\n\nB chain of SARS-CoV-2 Spike RBD from PDB, Accession number 6LZG: https://identifiers.org/rcsb/pdb:6LZG\n\nB chain of hACE2 from PDB, Accession number 6M18: https://identifiers.org/rcsb/pdb:6M18\n\n\nExtended data\n\nHarvard Dataverse: Potential chimeric peptides to block the SARS-CoV-2 Spike RBD. https://doi.org/10.7910/DVN/WSDRTU26\n\nThis project contains the following extended data:\n\n- MS_cnCoVP_Supplementary_Table-S1_F1000.xlsx (detailed physiochemical and docking properties and AVPpred prediction of each peptide)\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\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 | Free Full Text\n\nHuang C, Wang Y, Li X, et al.: Clinical features of patients infected with 2019 novel coronavirus in wuhan, china. Lancet. 2020; 395(10223): 497–506. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLiu NN, Tan JC, Li J, et al.: Covid-19 pandemic: Experiences in china and implications for its prevention and treatment worldwide. Curr Cancer Drug Targets. 2020; 20. PubMed Abstract | Publisher Full Text\n\nSingh AK, Singh A, Shaikh A, et al.: Chloroquine and hydroxychloroquine in the treatment of covid-19 with or without diabetes: A systematic search and a narrative review with a special reference to india and other developing countries. Diabetes Metab Syndr. 2020; 14(3): 241–246. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nYan R, Zhang Y, Li Y, et al.: Structural basis for the recognition of sars-cov-2 by full-length human ace2. Science. 2020; 367(6485): 1444–1448. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCastel G, Chteoui M, Heyd B, et al.: Phage display of combinatorial peptide libraries: Application to antiviral research. Molecules. 2011; 16(5): 3499–3518. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMujtaba MG, Patel CB, Patel RA, et al.: The gamma interferon (ifn-gamma) mimetic peptide ifn-gamma (95-133) prevents encephalomyocarditis virus infection both in tissue culture and in mice. Clin Vaccine Immunol. 2006; 13(8): 944–952. PubMed Abstract | Publisher Full Text | Free Full Text\n\nQureshi A, Thakur N, Kumar M: Hipdb: A database of experimentally validated hiv inhibiting peptides. PLoS One. 2013; 8(1): e54908. PubMed Abstract | Publisher Full Text | Free Full Text\n\nThakur N, Qureshi A, Kumar M: Avppred: Collection and prediction of highly effective antiviral peptides. Nucleic Acids Res. 2012; 40: W199–204. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLiu IJ, Kao CL, Hsieh SC, et al.: Identification of a minimal peptide derived from heptad repeat (hr) 2 of Spike protein of sars-cov and combination of hr1-derived peptides as fusion inhibitors. Antiviral Res. 2009; 81(1): 82–87. PubMed Abstract | Publisher Full Text | Free Full Text\n\nUjike M, Nishikawa H, Otaka A, et al.: Heptad repeat-derived peptides block protease-mediated direct entry from the cell surface of severe acute respiratory syndrome coronavirus but not entry via the endosomal pathway. J Virol. 2008; 82(1): 588–592. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChu LH, Chan SH, Tsai SN, et al.: Fusion core structure of the severe acute respiratory syndrome coronavirus (sars-cov): In search of potent sars-cov entry inhibitors. J Cell Biochem. 2008; 104(6): 2335–2347. PubMed Abstract | Publisher Full Text | Free Full Text\n\nvan Zundert GCP, Rodrigues JPGLM, Trellet M, et al.: The HADDOCK2.2 Web Server: User-Friendly Integrative Modeling of Biomolecular Complexes. J Mol Biol. 2016; 428(4): 720–725. PubMed Abstract | Publisher Full Text\n\nZhou P, Jin B, Li H, et al.: Hpepdock: A web server for blind peptide-protein docking based on a hierarchical algorithm. Nucleic Acids Res. 2018; 46(W1): W443–W450. PubMed Abstract | Publisher Full Text | Free Full Text\n\nQureshi A, Thakur N, Tandon H, et al.: Avpdb: A database of experimentally validated antiviral peptides targeting medically important viruses. Nucleic Acids Res. 2014; 42: D1147–1153. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWang G, Li X, Wang Z: Apd3: The antimicrobial peptide database as a tool for research and education. Nucleic Acids Res. 2016; 44(D1): D1087–1093. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJhong JH, Chi YH, Li WC, et al.: dbAMP: An Integrated Resource for Exploring Antimicrobial Peptides With Functional Activities and Physicochemical Properties on Transcriptome and Proteome Data. Nucleic Acids Res. 2019; 47(D1): D285–D297. PubMed Abstract | Publisher Full Text | Free Full Text\n\nUsmani SS, Bedi G, Samuel JS, et al.: Thpdb: Database of fda-approved peptide and protein therapeutics. PLoS One. 2017; 12(7): e0181748. PubMed Abstract | Publisher Full Text | Free Full Text\n\nQureshi A, Tandon H, Kumar M: AVP-IC50 Pred: Multiple Machine Learning Techniques-Based Prediction of Peptide Antiviral Activity in Terms of Half Maximal Inhibitory Concentration (IC50). Biopolymers. 2015; 104(6): 753–763. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChaudhary K, Kumar R, Singh S, et al.: A Web Server and Mobile App for Computing Hemolytic Potency of Peptides. Sci Rep. 2016; 6: 22843. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGupta S, Kapoor P, Chaudhary K, et al.: In Silico Approach for Predicting Toxicity of Peptides and Proteins. PLoS One. 2013; 8(9): e73957. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBarh D: Potential chimeric peptides to block the SARS-CoV-2 Spike RBD. Harvard Dataverse, V1.2020. http://www.doi.org/10.7910/DVN/WSDRTU\n\nChen Y, Guo Y, Pan Y, et al.: Structure Analysis of the Receptor Binding of 2019-nCoV. Biochem Biophys Res Commun. 2020; 525(1): 135–140. 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\nSong W, Gui M, Wang X, et al.: Cryo-em structure of the sars coronavirus Spike glycoprotein in complex with its host cell receptor ace2. PLoS Pathog. 2018; 14(8): e1007236. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "64504",
"date": "29 Jun 2020",
"name": "Candan Hizel",
"expertise": [
"Reviewer Expertise Pharmacogenomics"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe coronavirus disease 2019 (COVID-19) pandemic has created a worldwide crisis and inspired an urgent search for prevention and treatment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection.\nBarh and his colleagues describe in this article the important role of immuno-informatics by using molecular modeling and docking for chimeric peptide design anti-Spike peptides for SARS-CoV-2 potential therapeutics against the covid-19 infection. The introduction provides a good, generalized background of the topic that quickly gives the reader an appreciation of the applications of the technique. “A large global effort is required to stop viral epidemics and pandemics, and these manuscript insights may lead to identifying novel ways to stop infection.” To this end, this manuscript highlights clearly the potential opportunities several peptides blocking the interaction between SARS-CoV-2 Spike RBD and hACE2 as probable application in clinical practice.\nOverall the topic and the data are very interesting. The manuscript is very well written, well organized, easy to follow, and appropriate in scope and method. Certainly this study will contribute much to the literature.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? 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": "5671",
"date": "01 Jul 2020",
"name": "Debmalya Barh",
"role": "Author Response",
"response": "Thank you for your critical review and approval."
}
]
},
{
"id": "68731",
"date": "06 Aug 2020",
"name": "Shailendra Dwivedi",
"expertise": [
"Reviewer Expertise Molecular Biology",
"Cancer Stem Cell",
"Epigenetics",
"Nutrigenomics",
"Cancer 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 overall article is well written and has properly compiled the report on \"Potential chimeric peptides to block the SARS-CoV-2 spike receptor-binding domain\" which is oriented on the present pandemic of COVID-19. The authors have screened and designed many peptides that can potentially block the interaction between SARS-CoV-2 Spike RBD and hACE2. Thus, the authors concluded that ten peptides (AC20, AC23, DBP6, and cnCoVP-1- cnCoVP-7) have a very high potential of interaction, indicating that these peptides could be attractive therapeutics against SARS-CoV-2.\n\nThe authors have used proper methodologies to conclude the report, so it can be accepted for indexing.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "68734",
"date": "24 Aug 2020",
"name": "Amjad Ali",
"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 study manuscript “Potential chimeric peptides to block the SARS-CoV-2 spike receptor-binding domain” by Barh et al., focuses on chimeric peptides to potentially block the SARS-CoV-2 receptor binding domain; the work is interesting and well designed and written. I have few minor comments and suggestions if they could be considered by the authors for improvement and clarification.\nAs mentioned “SARS-CoV binds to the human angiotensin-converting enzyme 2 (hACE2) receptor through its Spike protein (S) to enter into the host cell6, and it is now reported that SARS-CoV-2 also binds to ACE2 to transmit its genetic material to human cells7–9” In these lines the authors stated that SARS-CoV-2 also binds to ACE2, I think the authors intended to write hACE2 (as stated earlier). The abbreviation is inconsistent in the whole manuscript. Please have a look.\nComment: The authors stated that AVP are a better choice in catering viral infection, but as they are peptides, they are prone to be attacked by proteases. How to overcome this issue?\nComment: Do these AVPs have a role in generating memory response? If the AVPs directly evoke innate immune response there is a possibility the PAMPs present on these can be recognized by PRRs which may lead to degradation.\nComment: A study similar to Strategy 1 has been conducted by Ibrahim et al. (20201) to predict binding residues between RBD and hACE2. Does your study validate those findings? Are there any differences?\nComment: Which tool or database was used to design AVPs in strategy 1?\nComment: As the authors discussed the hemolytic potential of chimeric peptides, were peptides designed using Strategy 1 and Strategy 2 also fulfilling the criteria of therapeutic potential? Also, among the ten peptides mentioned in the conclusion which of them are best? Is it possible to give a brief comparison of these 10 peptides?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/9-576
|
https://f1000research.com/articles/9-574/v1
|
08 Jun 20
|
{
"type": "Software Tool Article",
"title": "flimview : A software framework to handle, visualize and analyze FLIM data",
"authors": [
"Matias Carrasco Kind",
"Mantas Zurauskas",
"Aneesh Alex",
"Marina Marjanovic",
"Prabuddha Mukherjee",
"Minh Doan",
"Darold R. Spillman Jr.",
"Steve Hood",
"Stephen A. Boppart",
"Mantas Zurauskas",
"Aneesh Alex",
"Marina Marjanovic",
"Prabuddha Mukherjee",
"Minh Doan",
"Darold R. Spillman Jr.",
"Steve Hood",
"Stephen A. Boppart"
],
"abstract": "flimview is a bio-imaging Python software package to read, explore, manage and visualize Fluorescence-Lifetime Imaging Microscopy (FLIM) images. It can open the standard FLIM data file conventions (e.g., sdt and ptu) and processes them from the raw format to a more readable and manageable binned and fitted format. It allows customized kernels for binning the data as well as user defined masking operations for pre-processing the images. It also allows customized fluorescence decay fitting functions and preserves all of the metadata generated for provenance and reproducibility. Outcomes from the analysis are lossless compressed and stored in an efficient way providing the necessary open-source tools to access and explore the data. flimview is open source and includes example data, example Jupyter notebooks and tutorial documentation. The package, test data and documentation are available on Github.",
"keywords": [
"FLIM",
"bio-imaging",
"microscopy",
"visualization",
"fluorescence",
"Python"
],
"content": "Introduction\n\nFluorescence lifetime imaging microscopy (FLIM) is an imaging technique where the image contrast is derived from the differences in the exponential decay rate of the fluorescence from a fluorescent sample (Bower et al., 2018).\n\nIn modern two-photon FLIM, the most widely used detection method is known as time-correlated single-photon counting (TCSPC) (Becker et al., 2004). TCPC employs a pulsed excitation source, such as a laser or a light emitting diode and measures the timing of arrival of single photons originating from the fluorescent sample on a detector to reconstruct the fluorescence lifetime decay (McGinty et al., 2016). Typically, different fluorophores can be characterized by their fluorescence lifetimes. Furthermore, fluorescence lifetimes of a fluorophore can be affected by its environment, therefore FLIM have emerged as a valuable tool, providing unique contrast mechanisms for biomedical imaging (Bower et al., 2019; Hirvonen & Suhling, 2016; Ranawat et al., 2019).\n\nTo reconstruct TCSPC based FLIM image, several non-trivial data processing steps are necessary. For extracting the lifetime and amplitude data from initially saved raw files, first the data has to be imported from proprietary files such as Becker and Hickl .sdt, or PicoQuant .ptu. Next, signal preprocessing through denoising or binning has to be applied to ensure robust performance in low photon count scenarios. Finally, after compensating for instrument response function, biexponential decay curves are fitted to extract lifetime and amplitude values for two dominant components at each pixel.\n\nSeveral commercial and open source packages are already available for FLIM analysis (Bergmann, 2003; Fli; Warren et al., 2013). However, in practice, when custom FLIM analysis capabilities are required, this can lead to a need for building complicated signal processing and analysis pipelines (Borhani et al., 2019; Bower et al., 2017; Cao et al., 2020), involving a mix of commercial tools, open-source software and bespoke analysis algorithms. While other packages have been published, (Ballesteros et al., 2019), we expect that flimview, with its focus on simplicity, and versatility, will serve as a useful tool for the FLIM community which can also contribute to its growth.\n\nHere we present a Python package, flimview, which provides a user-friendly toolkit for opening, fitting and visualizing FLIM data. The package can be used as a stand-alone tool for analyzing and visualizing FLIM data, or it can be used as a basis for creating end-to-end streamlined analytical pipelines in Python.\n\n\nMethods\n\nflimview is a library module implemented in Python 3 and can be installed using PyPI (e.g., pip install flimview). Installation instructions using Conda are also included within the repository. A complete list of requirements can be found at the package Github page but include standard Python libraries such as pandas, scipy, matplotlib, h5py, etc. The code is released under the open-source NCSA license which is based on the MIT open source license, allowing full and free use for both commercial and non-commercial purposes. Full usage documentation, Jupyter notebooks (web-browser interface that allows live Python codes), test data and others, including guidelines for contributions and issue reporting, are included in the project repository. This package has been tried in Windows, Mac OS and Linux Operating Systems. In general flimview works better in the latter two for which it is also expected to get supported development.\n\nflimview consists of several utility libraries for storage, visualization and manipulation of FLIM data. After reading and processing the raw data (.ptu or .sdt files), the input data is stored in the main class of the package, a FlimCube, which is an object that represents 3D data (2D spatial dimensions and one temporal dimension) allowing multiple methods to access and manipulate the data cube.\n\nAmong its attributes is the header which includes all the metadata available for the dataset, sizes of the arrays, resolution and whether this cube is binned and/or masked (see Binning and Masking sections below). When the data cube is masked, a process described in the following sections, the pixel-level mask is also included as an attribute in the FlimCube object. Due to its construction, we consider a FlimCube to be self-explanatory as it contains all needed information (including its corresponding metadata) to analyze, visualize and fit the data.\n\nBinning. flimview includes a binning function with several pre-defined kernels, although a user-defined kernel is also possible and can be easily added. The binning procedure is simply a convolution kernel applied to the image to increase the signal or to enhance its features. This function takes a FlimCube as an input and returns a FlimCube as output by copying all of the metadata and properties from the input data. The following snippet shows an example on how to bin an existing FlimCube using a Gaussian kernel of size 9×9 and a sigma of 3 (the size of the kernel is 2 ∗ b + 1, where b is the bin size)\n\n\n\nThe kernel functions included in flimview are Gaussian kernel, Airy disk kernel, linear kernel, and a flat kernel. Customized kernels can be easily incorporated. Figure 1 shows examples of different 9×9 kernels where we can observe how the weight is distributed around the central pixel. In the case of a Gaussian kernel or an Airy disk kernel, the width of the kernel can be customized using the sigma σ parameter. For reference, the Gaussian kernel is given by:\n\nMasking. FlimCube also includes methods for looking at the header information and for masking pixels below a given integrated intensity, or below a given peak threshold in the time series, or even for a custom made geometry mask. The pixels masked this way will not be used during the fitting analysis while the mask is also saved within the same input FlimCube object. The following snippet shows how to apply a mask for a given FlimCube :\n\n\n\nFigure 2 shows different masks applied to the example raw (top) and binned (bottom) data. Masks can be combined and can be transferred between binned and raw data.\n\nMasks can be combined. Masked pixels will not be used in the analysis.\n\nFitting. Once the data is read and binned, and have the low signal and error pixels masked, flimview provides a function to fit a decay function for every pixel in the temporal data using a predefined model (from the module models.py which can be customized and also parallelized. By default a double exponential is used as follows:\n\nBefore fitting the entire cube, a single fit is done to the mean intensity values to obtain initial guess parameters for the fitting procedures as shown in Figure 3, as well as the boundaries to each parameter which can also be provided by hand. The snippet below shows how this is done using flimview :\n\n\n\nNote that the data is normalized, cleaned, and shifted with respect its peak. For example, to generate Figure 3 we can use the following:\n\n\n\nAfter those parameters are defined and a model is selected a function fitCube takes a FlimCube as input and produces another flimview class called FlimFit which contains the fitting information for every pixel, including all parameters from the model, their errors and χ2 values. It is a collection of 2D arrays for the results of the fitting.\n\nTo generate a FlimFit object we can use the following:\n\n\n\nVisualization. The package also includes means to visualize the FlimCube and FlimFit objects using intensity or peak values. Additionally, one can visualize the results from the fitting procedure using a function called plotFit, which takes a FlimCube and a FlimFit as input, along with a pixel coordinate, to produce a figure as the one shown in Figure 4, showing the fit results for a given pixel, the parameters, the residuals, and the residuals distribution which is a useful way to explore the results. These can be combined in an interactive widget showing, for example, the same figure for a given point selected interactively. It is also possible to combine multiple visualizations to generate a sequence of plots into an animation of the datacube. The package repository includes examples on how this can be done to generate a short animation. Other visualization functions are also included and documented in the package.\n\nStorage. FlimCube and FlimFit can be easily stored and easily retrieved by using a hierarchical and compression data store format for array-like data called HDF5 (The HDF Group, 2000–2020). Using this data format, we can store multiple data in different ‘folders’, including parameters, different models, masks, raw, binned and fitted data in one file for easy retrieval, as it only loads what is asked for, saving memory consumption if necessary. In the case of multiple files, we can serve them on-demand using a web server and only load the data as necessary, including over the web, which is an HDF5 feature. The methods saveCube, saveFit, loadCube, loadFit, viewH5 are implemented in the io_utils modules inside flimview. For example, after running fitCube and saving the example data in a file, one can easily see its internal structure with:\n\n\n\nwhich produces the following output, showing the file content of the processed data:\n\n\n\nMore examples on how to save and retrieve FLIM data using this format are included in the example notebooks at the Github repository.\n\n\nUse cases\n\nTo highlight the main features of flimview, we have used two of the most common FLIM data formats, namely, the Becker and Hickl .sdt, and PicoQuant .ptu. We have included the reading routines for both data types but others can be easily extended within the module. All the figures in this paper were made using the two example files included in the package which we describe below. In particular Figure 4 shows both files presented in the same visualization after being processed into a FlimCube. Both example files are included with the package and are briefly described below.\n\nThe example file epidermis.sdt contains 2-photon excited FLIM data from the skin (epidermis) on the upper forearm of a healthy human volunteer captured with commercial optical medical imaging system (MPT-lex CARS, JenLab GmbH, Germany) (Weinigel et al., 2015). The image was captured using the procedure described in (Alex et al., 2018). For generating optical images, the excitation wavelength of the femtosecond laser was set to 760 nm and the incident in situ laser power was set to 30 mW. The light was focused through a 40x 1.35 NA objective. Autofluorescence signals within the spectral range of 405 nm–600 nm were detected. All these parameters are extracted from the file and added to the header inside the FlimCube.\n\nThe example file macrophages.ptu contains the 2-photon excited FLIM data from J774A.1 mouse macrophages grown in Dulbecco’s Modified Eagle Medium + 10% FBA + 1% antibiotic, under 5% CO2. The image was acquired with the laser set to 750 nm and the incident in situ laser power was set to 25 mW.\n\n\nSummary\n\nIn this paper we have presented flimview, a novel Python 3 package to manipulate, visualize, and analyze FLIM images and measurements from specific data formats .sdt and .ptu. We included snippets on how different kernels and different masking strategies can be applied after reading the raw data into a FlimCube object. We also show how the procedure of fitting the whole cube is carried out and how these results can be visualized effectively. Most of the functions provided in this package can be customized for further research or to fit specific needs, including user defined binning kernels, mask functions or exponential decay fitting models. We showed how, using a hierarchical data format, the results from multiple files, multiple formats and multiple views can be stored in a single optimized data format called HDF5 which allows much better data management and data access to the data sets, even in the case of multiple files.\n\n\nData availability\n\nWe have included two data examples along with Jupyter notebooks to explore and get started with the module and its functions. This data examples can be found in the following repository: https://github.com/Biophotonics-COMI/flimview. Individual files can be loaded locally using this snippet. Detailed instructions and description of the data included can be found in the package website:\n\n\n\n\nSoftware availability\n\n1. Package and installation instructions can be found here: https://github.com/Biophotonics-COMI/flimview\n\n2. Source code can be found here: https://github.com/Biophotonics-COMI/flimview\n\n3. Archived source code at time of publication: http://doi.org/10.5281/zenodo.3825694 (Carrasco Kind, 2020).\n\n4. License: NCSA",
"appendix": "References\n\nAlex A, Frey S, Angelene H, et al.: In situ biodistribution and residency of a topical anti-inflammatory using fluorescence lifetime imaging microscopy. Br J Dermatol. 2018; 179(6): 1342–1350. PubMed Abstract | Publisher Full Text\n\nBallesteros GC, Proux R, Bonato C, et al.: readPTU: a python library to analyse time tagged time resolved data. Journal of Instrumentation. 2019; 14(06): T06011–T06011. Publisher Full Text\n\nBecker W, Bergmann A, Hink MA, et al.: Fluorescence lifetime imaging by time-correlated single-photon counting. Microsc Res Tech. 2004; 63(1): 58–66. PubMed Abstract | Publisher Full Text\n\nBergmann A: Spcimage: data analysis software for fluorescence lifetime imaging microscopy. Becker & Hickl GmbH. 2003. Reference Source\n\nBorhani N, Bower AJ, Boppart SA, et al.: Digital staining through the application of deep neural networks to multi-modal multi-photon microscopy. Biomed Opt Express. 2019; 10(3): 1339–1350. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBower AJ, Li J, Chaney EJ, et al.: High-speed imaging of transient metabolic dynamics using two-photon fluorescence lifetime imaging microscopy. Optica. 2018; 5(10): 1290–1296. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBower AJ, Marjanovic M, Zhao Y, et al.: Label-free in vivo cellular-level detection and imaging of apoptosis. J Biophotonics. 2017; 10(1): 143–150. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBower AJ, Sorrells JE, Li J, et al.: Tracking metabolic dynamics of apoptosis with high-speed two-photon fluorescence lifetime imaging microscopy. Biomed Opt Express. 2019; 10(12): 6408–6421. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCarrasco Kind M: Biophotonics-COMI/flimview: flimview 1.2.0 (Version v1.2.0). Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3825694\n\nCao R, Wallrabe H, Siller K, et al.: Optimization of FLIM imaging, fitting and analysis for auto-fluorescent NAD(p)h and FAD in cells and tissues. Methods Appl Fluoresc. 2020; 8(2): 024001. PubMed Abstract | Publisher Full Text\n\nFlimlib: a package for exponential curve fitting of fluorescence lifetime image data. Accessed: 2020-02-26. Reference Source\n\nHirvonen LM, Suhling K: Wide-field TCSPC: methods and applications. Meas Sci Technol. 2016; 28(1): 012003. Publisher Full Text\n\nMcGinty J, Talbot CB, Owen D, et al.: Fluorescence lifetime imaging: Microscopy, endoscopy, and tomography. In: Handbook of Biomedical Optics. CRC Press, 2016; 609–636. Reference Source\n\nRanawat H, Pal S, Mazumder N: Recent trends in two-photon auto-fluorescence lifetime imaging (2p- flim) and its biomedical applications. Biomed Eng Lett. 2019; 1–18. PubMed Abstract | Publisher Full Text | Free Full Text\n\nThe HDF Group: Hierarchical data format version 5, 2000-2020.Reference Source\n\nWarren SC, Margineanu A, Alibhai D, et al.: Rapid global fitting of large fluorescence lifetime imaging microscopy datasets. PLoS One. 2013; 8(8): e70687. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWeinigel M, Georg Breunig H, Uchugonova A, et al.: Multipurpose nonlinear optical imaging system for in vivo and ex vivo multimodal histology. J Med Imaging. 2015; 2(1): 016003. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "65063",
"date": "02 Jul 2020",
"name": "Xavier Michalet",
"expertise": [
"Reviewer Expertise single-molecule FRET",
"wide-field FLIM",
"single-photon detectors"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nReviewer’s Report on the manuscript “flimview…” by Kind et al. This paper describes a python package to load and visualize FLIM datasets. The source code has been deposited on Github, together with two example data files used to illustrate some of the software features.\nThe manuscript is well written and describes the software features with a fair amount of detail. The samples of code I have reviewed appear to be also reasonably well documented. Based on these basic criteria, this work should be valuable as a basis for future developments by researchers interested in developing their own tools for FLIM analysis. I would however encourage the authors to address the following remarks:\nPresentation:\nFor whatever reasons, the figures do not include color scales, which are present in the Jupyter notebooks used to generate them. This can easily be fixed and would be useful.\n\nThe datasets shown correspond to microscopy images of biological samples. It is customary (and very useful) to show such data with a scale bar. Consider making the extra effort to generate one (maybe optionally) as part of the code, to encourage scientific rigor.\n\nSome of the features of the software are incompletely described. For instance, the package opens .sdt and .ptu files containing photon timestamps, but does not dwell on the choice of timestamp binning used internally. This of course has an effect on RAM resources but also potentially on decay artifacts. As a side comment, the package, as is, does not support pre-binned datasets, as far as I can tell (see below). Some .sdt files are pre-binned only and would not be handled by the current software version.\n\nI am not sure what the authors mean by “after compensating for the instrument response function” (p 3)? Clearly the code does fit the decays without IRF convolution (which of course requires the user to provide an IRF file as well).\n\nWhile I understand why binning the original dataset could be interesting to increase SNR and decrease data footprint, I am unclear about the role of convolution with Gaussian or other kernels. What would this correspond to physically? I would understand deconvolution of the image with such kernels (although that would not address the question of how the photon time stamps should be redistributed in the deconvolved image), but the purpose of these kernels, besides image beautification, is mysterious and not an encouraged practice for rigorous analysis.\n\nContent:\nWhile this software is understandably only offering basic features (opening datasets – 2 kinds only supported, pre-processing, displaying an intensity image and fitting), it seems to be missing what I would consider an expected functionality for a software bearing the flimview name, namely visualization of fluorescence lifetime information. A barebone commercial software such as SPCImage from Becker & Hickl, at least does offer a mean lifetime representation, which would not seem to be difficult to implement. Since we are dealing with a software intended for research, I would have naively expected that options to represent the bi-exponential fitted amplitudes and lifetimes as color-coded maps would have been offered.\n\nThe software allows saving the data in the open and multiplatform HDF5 format. While this is commendable, it is not quite clear what principles are governing the structure of the data in this file format, or for that matter, what the file format actually looks like from a bird’s eye view. Our group has made an attempt to offer a similar type of HDF5-based file format for a slightly different purpose (photon-HDF5), which could be taken as an example (if maybe extreme in its level of detail) of what could be provided to potential users1. More importantly, it is not quite clear how this file format will and could evolve, whether it is intended to become a standard, while it has been chosen preferentially to another, etc. A minimum discussion of these considerations within the manuscript, and a more extended presentation online (say on the Github website) would be extremely useful.\n\nIt would be useful to hear from the authors whether and how they intend to encourage contribution and ensure evolution of the software. Of particular importance would be evidence that there is some kind of quality control (test modules – e.g. such as those described in a software cited in the file loading code, FRETBurst2- written using pytest).\n\nAlso related to the lack of bird’s eye view description is the absence of discussion of the principles involved in defining the FlimCube class. How can it be extended to support more than one spectral channel (or any other spectroscopic variable)? An additional spatial dimension? An additional temporal dimension?\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": "68967",
"date": "01 Sep 2020",
"name": "Nirmal Mazumder",
"expertise": [
"Reviewer Expertise Biophotonics",
"Biophysics"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 on flimview is well written and rationale of developing a new software is needed to analysis FLIM data acquired by commercial software or hardware.\n\nThe manuscript can be accepted for indexing after minor revision.\n\nLine 4, in introduction, It should be \"TCSPC\". In this manuscript, definition of \"fluorescence lifetime\" is missing. Author could describe in brief about the instrumentation. Is flimview applicable for single photon or two photon fluorescence measurement?\nA step by step flowchart about the flimview could help to implement.\n\nIn figure 2, only center of the image is used for analysis after masking. However, in some cases cells are distributed in full field of view. In such case, how does this kind of masking work?\n\nAuthor should mention in figure 2, what kind of image it is, what does the color mean, what type of cell is imaged. In fitting, why two lifetimes are used for fitting? If there is only one flurophore then single exponential fitting may work. In case of NADH, it exists in free and bound forms and hence two lifetimes. Author should comment on it.\n\nIn figure 4, it will be more acceptable if author can compare the results of flimview with Becker and Hickl or PicoQuant software analysis. Residues should be close to 1. Is it due to experimental or software error?\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? Partly\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/9-574
|
https://f1000research.com/articles/9-572/v1
|
08 Jun 20
|
{
"type": "Opinion Article",
"title": "A conceptual framework for reopening our society during the Covid-19 pandemic",
"authors": [
"Christopher J. Gill",
"Sandro Galea",
"Sandro Galea"
],
"abstract": "Decisions about how to go about the necessary task of re-opening our society in the midst of the Covid-19 (CV19) have been paralyzed by our extremes. But we can neither afford to insist on a zero-risk response, nor can we pretend that the risk does not exist. What is needed are tools to rationally triage the risk. To this end, we propose a novel ‘risk index’, which is the intersection of two components of risk: 1) the risk of an individual becoming infected due to action ‘X’; and 2) the likely probability of death (or serious harm) if that individual develops CV19. The risk index allows risk to be compared across different scenarios, and may reveal that seemingly very different situations constitute similar degrees of risk. With risk measured in this way, one can then contrast different levels of risk against the social benefits of absorbing that risk, allowing actions to be sorted into those that are tolerable, debatable, or acceptable. While these concepts are presented in abstract based on approximate estimates of risk and influenced by our judgements about social desirability, the concept itself can be refined as more accurate approximations of risk and broadly accepted values of social desirability are derived empirically. In short, this is a tool intended to provide a useful empirical framework for rationale decision making about CV19.",
"keywords": [
"Risk index",
"Covid 19",
"CV19",
"decision making"
],
"content": "Accepting risk is rationale and unavoidable\n\nThe current Covid-19 (CV19) public discussion has taken on absolutist shades, ranging between arguments in favor of ever longer massive national shutdowns to arguments in favor of a return to pre-CV19 interactions. These arguments are ultimately arguments about risk tolerance but are seldom framed that way. We would argue that a reasoned approach to risk and our tolerance of risk can help us sort through the options available and to chart a path forward.\n\nConsider, by way of illustration, a recent example. In the third week of May, a music concert was hosted by the Tupelo Music Hall in Derry New Hampshire1. At first glance, this seemed to be a dangerous proposition. But the details of the event were reassuring: It was to be held outdoors at a rented drive-in movie theater. Guests would be admitted after presenting their email confirmation at the gates, guests would park and watch the show from their cars or, if they preferred, while sitting on their own lawn chairs set up next to their cars. And other than going to the bathroom (ventilated portable toilets), social spacing was enforced. Food and drink could be ordered online in advance in the show but was delivered by concert staff who were wearing face masks.\n\nSo, while it is true that hosting the event incurred more risk than not hosting the event, in this instance, the organizers had been deliberate, thoughtful, and cautious about their risk mitigation strategies, and were aligned with guidelines issued by the State of New Hampshire2. Overall their plan seemed reasonable.\n\nWhat the plan highlights is a tolerance of risk. Certainly, all concert goers would have faced less risk had they stayed home. But they, and the state, judged that risk to be acceptable, and the concert went on. Far from being an outlandish exception, this example illustrates an important general point: we accept risks all the time. That is why it is legal to drive a motorcycle, to skydive, to smoke, to drink soda, to buy sailboats, to kiss a stranger in a night club, or to own a pet tarantula. And we make finely tuned decisions about these risks. While it is legal to drive a motorcycle, even though for every mile driven motorcycle drivers have a 35-fold greater risk than car drivers, it is illegal to drive a motorcycle without a helmet in most states (though ironically not in New Hampshire)3. Everything we do involves parsing risk, deciding what is acceptable to us based on other values (e.g., the pleasure of driving a motorcycle) and what crosses a line of intolerable risk (e.g., driving a motorcycle while intoxicated). CV19 is a particular kind of risk—one that is shared because the disease is contagious, and one where we confer risk on each other. It is not, however, unique in that respect. Kissing in bars is a good example of a shared risk: it may result in cold sores (Herpes simplex infections; common but not life threatening) or bacterial meningitis (Neisseria meningitidis infections; rare but lethal).\n\nThe Tupelo Music Hall concern therefore constituted a pragmatic example of the kind of social experiments that are urgently needed to help us learn how to reopen our society. Such experiments are all variations on the same theme: how do we balance the risk of an action against the value of that action?\n\nBut answering that question is impossible unless we first have a better way of measuring risk.\n\n\nThe proposed risk index\n\nAiming to help organize our thinking on this, we suggest that we can conceptualize risk from CV19 as having two components. These are:\n\n1) the probability of getting infected by doing action ‘X’; and\n\n2) the likelihood of dying if one becomes infected.\n\nThe intersection of these factors yields a conjoint risk index as shown in Figure 1.\n\nUsing best guesses, we could populate the matrix with a set of scenarios illustrating several combinations of risk and vulnerability. For convenience, we color code these combinations from highest to lowest risk index (red>orange>yellow>green>blue). For simplicity, we plot the gradations by primary colors and first order combinations (red and yellow make orange; blue and yellow make green), but obviously the risk index is actually a continuum.\n\nThe risk index is bounded by two extreme scenarios. On the bottom left we envision a child at an outdoor playground. Children are rarely (but not never) harmed by CV19, and the risk of transmission when outdoors is low4. With mitigation, this risk could be lower still. For example, kids could wear masks, sanitize hands after play, and we could limit how many play at a given time to reduce crowding.\n\nBy contrast, the top right presents the opposite extreme and a worst-case scenario. Here, we can imagine an adult who is unknowingly infected with CV19, who now goes to visit their hypertensive and diabetic grandmother at a nursing home. Indoor transmission is very efficient and the odds are high that the grandmother will not survive an encounter with CV19.\n\nIn both cases, the risk index considers the risk solely from the perspective of the individual absorbing the risk (the child or the grandmother). But the framework provides a reminder that we exist in a complicated web of relationships, and that our actions have consequences beyond ourselves. In this way, the framework allows one to separately infer the risk to a hypothetical child or grandmother in isolation, or to model the impact of sequential interactions.\n\n\nAdvantages and implications of the risk index\n\nThis approach has close similarities to the theory underlying Disability Adjusted Life Years (DALYs)5. The DALY is a population level index that measures the impact of any given disease as the combined effect of the total number of years of life lost due to that condition, plus the number of years lived with varying degrees of disability. As with our proposed risk index, it is a way of comparing the net impact of different diseases and can be a valuable tool for public health decision making. Indeed, the DALY is the foundation for the Institute of Health Metrics and Evaluation (IHME) Global Burden of Disease Study6. Other disciplines in public health also index outcomes in different ways.\n\nThe key advantage of using a risk index is that it becomes possible for risk to be compared across different individuals and different scenarios. Within the limits of our approximations, the risk index suggests that scenarios that differ markedly in their details, may actually present similar levels of risk. This makes the concept of risk fungible. For example, by taking some best guesses, this model suggests that sending teens back to high school is roughly the same as an elderly person going to the supermarket while wearing a mask.\n\nA further advantage is that the risk index allows us to hypothesize about the potential effectiveness of different risk mitigation strategies. For simplicity, we have assumed that the impact of risk mitigation strategies is equivalent across each of our scenarios, but in reality, this is surely not the case. Different scenarios present different opportunities for risk mitigation strategies, and these may be variably effective, which would be represented by expanding or reducing the risk of transmission.\n\nFor example, much attention has focused on the value of reopening schools7. Yet the traditional schooling model remains a high transmission proposition. In the base case, schools are characterized by kids clustered in classes, gyms and cafeterias (or just huddled over shared smartphones), and systematically sharing saliva deliberately (by kissing) or inadvertently (via aerosols, droplets, or shared water bottles). But varying degrees of mitigation are certainly possible. Kids can wear masks; schools may alternate in person vs. distance learning week by week; classes taught by elderly/infirm faculty could all go online (to protect the faculty from the students); the semester could be compressed; windows could be open where possible; and so forth. In fact, there are many ways of reducing the risk index while still largely benefitting from the in-person interactions that add value to student education and satisfy the importance of social engagement in the lives of teens.\n\nWhile it is theoretically possible to reduce the level of harm if infected (i.e., moving to the left on the X axis), in practice this may not be realistic within the time scale of an epidemic. For example, we know that hypertension, obesity and diabetes increase the risk of CV19 death8. But we do not know whether a radical diet and exercise regimen would reduce CV19 fatality rates at an individual level, and accomplishing that at scale and in within the time scale of an epidemic would be immensely challenging. Thus, for practical purposes, the risk mitigation strategies focus only on reducing the risk of transmission (i.e., moving down on the Y axis).\n\nThere may be situations for which there are no effective or practical risk mitigation strategies at all. But that too is an acceptable limitation. This is a starting point for risk assessment that is immediately useful when limited to educated guesses, but which becomes more useful and precise when those approximations are replaced by better estimates of actual risk and actual harm. The index is a place to begin discussions and to draw approximate conclusions, not a place to end. It is also an opportunity to identify situations where more precise data from research would be particularly important, making this a helpful tool to guide research.\n\n\nTaking the next step: integrating the risk index with the value of different actions\n\nWhile useful by itself, the risk index is more useful for answering an even more important question: does the value of a given action justify that risk? This is shown in Figure 2, where the Y axis is the risk index as estimated from Figure 1, and the new X axis plots the perceived societal value for each action.\n\nIn this schema, the easiest decision is to open playgrounds. Kids benefit from freedom, exercise and social interaction and parents need their kids to play and to be happy and to have a break from child care. Moreover, playgrounds exist already and have minimal opening costs. Conversely, opening state or national parks are somewhat more expensive and are not immediately accessible in the same way that playgrounds are, making them of slightly lower value to society, particularly during the lock down. Opening playgrounds is a low risk/high value proposition, colloquially known as a ‘no brainer’. It does not, however, mean that those kids can later visit their grandmother at the nursing home.\n\nWhile this is certainly debatable, the social value of opening indoor bars seems to us far lower than playgrounds, even though the economic value of bars may be considerable. While their value to society can be argued, from the perspective of the risk index, spending a Friday night in intimate contact with a crowd of strangers is an ideal strategy for spreading the coronavirus. Moreover, it is hard to envision many strategies for risk mitigation that would be acceptable. Patrons cannot drink beer while wearing masks, and masks are a barrier to social intimacy, and thus undermine a key reason why people go to bars. Outdoor bars would be safer, but that puts the drinking out on the street in most cases, which has its own negative consequences. However, one could legitimately reframe this analysis more discretely in terms of economic value (removing elements of moral judgement), social equity, or many other potential permutations of value.\n\nThrough these risk index/value combinations we can begin to sort actions into those that are acceptable, debatable, or intolerable. In all likelihood, we will spend a lot of time discussing actions that fall into the debatable range, which is precisely the point. This is what rational decision making looks like.\n\n\nConclusions: Opening society means making thousands of individual risk/value assessments\n\nWe fully recognize that this approach leaves many questions unanswered and rests on assumptions about deriving risk index that here are only approximate. However, the goal in proposing this framework is not to categorically rank all different levels of risk or value and thereby answer all potential questions. Rather, the framework is merely a way to start the types of reasoned conversations that must occur if we want to open society in a way that maximizes benefits while minimizing risks.\n\nCurrently, our debate on when and how to reopen society has been paralyzed by our absolutist extremes. While it seems certain that the lock down was essential for buying us valuable time to figure out how we respond to the CV19 strategy, this has had devastating consequences to our economy and cannot be sustained indefinitely. But denying the risk of CV19 is also a mistake, which if pursued will result in death on a scale that the US has never seen before (including the 1918 influenza pandemic) and may also result in economic collapse. Neither extreme is tenable.\n\nRather, we suggest that our way forwards lies somewhere in the rational middle. It means we must accept gradually increasing levels of risk, ideally validated by a rigorous testing strategy to allow us to gauge the impact of these decisions and to shift our strategies if our assumptions later prove to be wrong. To escape the polarizing paralysis, we are convinced that we can - and indeed must - take this kind of approach to reopen our society. Otherwise, we risk consigning millions to economic destitution by trying to live in a zero-risk world that simply does not now, and has never, existed.\n\n\nData availability\n\nNo data are associated with this article.",
"appendix": "References\n\nEdgers G: The era of socially distanced concerts has begun. Here’s what it was like at a New Hampshire venue’s first drive-in show. 2020; Accessed May 21, 2020. Reference Source\n\nHampshire. SoN: Stay at home 2.0 Drive In Movie Theaters. State of New Hampshire; 2020. Reference Source\n\nHow do car accidents compare to motorcycle accidents?2020; Accessed 21 May 2020. Reference Source\n\nTeam CC-R: Severe Outcomes Among Patients with Coronavirus Disease 2019 (COVID-19) - United States, February 12-March 16, 2020. MMWR Morb Mortal Wkly Rep. 2020; 69(12): 343–346. PubMed Abstract | Publisher Full Text\n\nAnand S, Hanson K: Disability-adjusted life years: a critical review. J Health Econ. 1997; 16(6): 685–702. PubMed Abstract | Publisher Full Text\n\nSalomon JA, Vos T, Hogan DR, et al.: Common values in assessing health outcomes from disease and injury: disability weights measurement study for the Global Burden of Disease Study 2010. Lancet. 2012; 380(9859): 2129–2143. PubMed Abstract | Publisher Full Text\n\nEsposito S, Principi N: School Closure During the Coronavirus Disease 2019 (COVID-19) Pandemic: An Effective Intervention at the Global Level? JAMA Pediatr. 2020. PubMed Abstract | Publisher Full Text\n\nRichardson S, Hirsch JS, Narasimhan M, et al.: Presenting Characteristics, Comorbidities, and Outcomes Among 5700 Patients Hospitalized With COVID-19 in the New York City Area. JAMA. 2020; 323(20): 2052–2059. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "64427",
"date": "02 Jul 2020",
"name": "Mauricio T. Caballero",
"expertise": [
"Reviewer Expertise Respiratory viruses disease",
"pediatrics",
"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\nA conceptual framework for reopening our society during the Covid-19 pandemic is a well written insight for a new approach to systematically re-opening our society in the midst of the Covid-19 (CV19). Authors propose tools to rationally triage the risk to be compared across different scenarios. The risk should be builded based on two components: 1) the risk of an individual becoming infected due to action ‘X’; and 2) the likely probability of death (or serious harm) if that individual develops CV19. As this is a theorical approach and I considered correct and interesting.\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": "78046",
"date": "15 Feb 2021",
"name": "Amrita Rao",
"expertise": [
"Reviewer Expertise Epidemiology",
"HIV"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this Opinion Article, the authors argue that much of the public debate around what should and should not be allowed during the COVID-19 pandemic is really an argument about risk tolerance. They note that all decisions made, at the individual and societal level, are a balancing act of how risky an activity might be and how willing we are to absorb that risk. They propose the creation of a risk index that takes into account risk of becoming infected from a certain action, X, and the probability of death or serious harm from participation in that action. Measuring risk in this way, the authors argue, will allow for the rational weighing of risk against the social benefits of absorbing that risk. This article is compelling and clearly written.\nFrom a communication standpoint, I think the use of the two axes and the simplicity of Figure 1 is very useful. It helps us understand the relative risk of different actions in clear terms. It would be helpful, though, to further delineate what the mitigation strategies are and what they might look like.\nWould the mitigation strategies themselves be different in various contexts? For example, mask use at outdoor bar vs. at outdoor national park?\n\nCan we somehow create a guide for the relative importance of different mitigation strategies (e.g. masks vs. sanitizing)?\n\nTo me, I think there are two questions that should play into the risk index: 1) What is the relative risk of different actions? and 2) If I choose to do a certain action, which mitigation strategies should I prioritize?\nThe risk index, as I have read it, answers question 1, but I think these gray areas around mitigation strategies is where additional value could be added in terms of helping people make risk-mitigating decisions. The authors argue that this framework at least allows us to start from the same place and have a rational conversation about what is acceptable/debatable/intolerable, but I wanted to see a bit more here.\nThe discussion of DALYs was crucial. The authors clearly describe the conceptual similarities between the risk index and the use of DALYs and the advantages of their risk index for different individuals and different scenarios.\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": "78042",
"date": "19 Mar 2021",
"name": "Bayarmagnai Weinstein",
"expertise": [
"Reviewer Expertise Epidemiology and Clinical Research"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors recommended a framework to help individuals and policymakers to engage in reopening society amidst the COVID-19 pandemic with a benefit-risk balance. At a minimum, this provides an actionable blueprint that can be modified to each user’s context.\nThe conceptual basis of the risk index in this article corroborates and warrants the acknowledgment of Blumenshine and Diderichsen’s conceptual framework to delineate the potential causes of disparities in death due to respiratory pandemic as following:\nlikelihood of being exposed to the infectious agent\n\nlikelihood of contracting the disease, upon exposure to the infectious agent\n\nlikelihood of receiving timely and effective treatment after disease has developed (Blumenshine et al.,20081; Diderichsen et al.,20012).\nIn Gil and Salea’s framework (Figure 1 & 2) the acceptability of risk and perceived value of actions associated with re-opening of selected businesses appear to be modified using remediation measures. I concur with Amrita Rao to clarify the reliability and feasibility of common remediation strategies for direct actions.\nIn my view, the recommended framework accounts for the extrinsic factors, such as population density (indoor vs outdoor), type of business (school vs cruise ship), household structure, and age (nursing home and family visits). Even though, prior studies show that vulnerability to COVID-19 has been further defined by intrinsic individual characteristics as following:\n\nhealth risk behaviors (i.e., smoking, excessive alcohol consumption)\n\ncomorbidities (i.e., cardiovascular diseases, obesity, diabetes)\n\ndifficulty to social distancing due to inability to afford unemployment resulting in the reduced exertion of protective measures, healthcare access\n\nprogression of the pandemic over time\n\navailability and the efficacy of the COVID-19 vaccine\nSince the reliability of this one-size-for-all framework would be largely modified by the individual’s intrinsic risk factors as specified above, the authors could have provided or proposed future work to develop user-friendly guidance on how to tailor the framework to individual circumstances.\nAlso, for future work, it will be interesting to see if the conceptual model would indicate the data and methods that could bring this to the next level.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Partly\n\nAre all factual statements correct and adequately supported by citations? Partly\n\nAre arguments sufficiently supported by evidence from the published literature? Partly\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/9-572
|
https://f1000research.com/articles/9-570/v1
|
08 Jun 20
|
{
"type": "Software Tool Article",
"title": "Online dashboard and data analysis approach for assessing COVID-19 case and death data",
"authors": [
"Hector Florez",
"Sweta Singh",
"Sweta Singh"
],
"abstract": "The 2019-2020 global pandemic has been caused by a disease called coronavirus disease 2019 (COVID-19). This disease has been caused by the Severe Acute Respiratory Syndrome coronavirus-2 (SARS-CoV-2). By April 30 2020, the World Health Organization reported 3,096,626 cases and 217,896 deaths, which implies an exponential growth for infection and deaths worldwide. Currently, there are various computer-based approaches that present COVID-19 data through different types of charts, which is very useful to recognise its behavior and trends. Nevertheless, such approaches do not allow for observation of any projection regarding confirmed cases and deaths, which would be useful to understand the trends of COVID-19. In this work, we have designed and developed an online dashboard that presents actual information about COVID-19. Furthermore, based on this information, we have designed a mathematical model in order to make projections about the evolution of cases and deaths worldwide and by country.",
"keywords": [
"COVID-19",
"SARS-CoV-2",
"Data analysis",
"Mathematical model"
],
"content": "Introduction\n\nCoronavirus disease 2019 (COVID-19) is caused by Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2), which is a virus strain that causes respiratory illness. This virus was identified in December, 2019 and its first infection case was reported on December 30, 2019 in Wuhan city located in Hubei providence, China1. The World Health Organization (WHO) recognized the disease as a pandemic on March 11 20202. The WHO have reported the following numbers of cases and deaths worldwide: January 31, 9,847 cases and 213 deaths3; February 29, 85,961 cases and 2,941 deaths4; March 31, 754,933 cases and 36,522 deaths5; and April 30, 3,096,626 cases and 217,896 deaths6. These reports show an increment of cases in February of 872.97%, in March of 878.22%, and in April of 410.18%, as well as an increment of deaths in February of 1,387.26%, in March of 1,241.82%, and in April of 596.61%7. Based on these percentiles, we can observe an exponential growth of the disease.\n\nBased on this previous data, it is important to analyze the evolution of the disease in order to make decisions that tackle the growth rate of cases and deaths. Therefore, in this work, we have designed and developed an online dashboard which presents two types of results: (1) actual information about the COVID-19 using different deployment strategies; and (2) based on the actual information, a projection in order to estimate the future behavior of the disease calculated worldwide and by country. To achieve this projection, we have designed a mathematical model based on previous data reported by the WHO.\n\n\nMethods\n\nTo analyze the current state of COVID-19, we have created an online information system called COVID-19 Dashboard, which is accessible from: https://covid19.itiud.org/. For the design of COVID-19 Dashboard, Unified Modeling Language (UML)8 was used allowing the description of the software based on the Object Oriented Paradigm9,10. It has been developed using: a) PHP 7.4.5 (https://www.php.net/) supported by Apache 2.4.43 (https://www.apache.org/); b) Bootstrap 4.4.1 (https://www.getbootstrap.com/) for responsive front-end; c) jQuery 3.3.1 (https://www.jquery.com/) as JavaScript library.\n\nCOVID-19 Dashboard presents full information about COVID-19 focusing on cases and deaths worldwide and by country. In addition, it includes a mathematical model in order to calculate projections for future cases and deaths. The data source used in COVID-19 Dashboard is taken from the WHO (https://covid19.who.int/) and World Population Review (https://www.worldpopulationreview.com/). The first data set is accessed by COVID-19 Dashboard through the URL https://dashboards-dev.sprinklr.com/data/9043/global-covid19-who-gis.json. This information is in JSON format; then, the system includes the algorithms to decode this format before processing the information. The second data set is downloaded in CSV format and included in the persistence layer of the system.This project deploys different graphics to analyze the information from different perspectives11. These graphics are generated based on the JavaScript library Chartkick 2.7.2 (https://www.chartkick.com/), which is supported by Google Charts (https://developers.google.com/chart).\n\nTo perform the projection for estimating future cases and deaths, we developed a mathematical model focused on a quadratic equation12 based on data of present day and 30 previous days. With the quadratic equation, projected cases and deaths for the next days up to 90 days are calculated. The quadratic equation obtained for each projection is deployed supported by the JavaScript library Mathjax (https://www.mathjax.org/).\n\nFigure 1 presents the components of the system, where all mentioned libraries are connected to COVID-19 Dashboard. The conceptual models of COVID-19 Dashboard have been created using the modeling tool UML Designer 9.0.0 (http://www.umldesigner.org/). The selected Integrated Development Environment (IDE) was Eclipse-PHP 2020-03 (https://www.eclipse.org/) and the server used to host the project was Debian Linux Server 10.4 (https://www.debian.org/)13.\n\nFor the mathematical model, let three points P1 = (x1, y1), P2 = (x2, y2), and P3 = (x3, y3) such that y1 corresponds to number of cases or deaths of the date 30 days prior to the present day, x1 = 0, y2 corresponds to number of cases or deaths of the date 15 days prior to the present day, x2 = 15, y3 corresponds to number of cases or deaths of the present day, and x1 = 30. Based on the quadratic equation defined as f (x) = ax2 + bx + c, let the equation system presented in (1), (2), and (3).\n\n\n\n\n\n\n\nSince x1 = 0 from (1):\n\n\n\nSubtracting (2) from (4):\n\n\n\nIsolating the variable b from (5):\n\n\n\nSubtracting (2) from (3):\n\n\n\nReplacing b in (7):\n\n\n\nIsolating the variable a from (8):\n\n\n\nFinally, isolating the variable c from (4):\n\n\n\nTherefore, based on a, b, and c applied to the quadratic equation, the projected cases and deaths are calculated. In addition, calculated quadratic equations will be adjusted when new information is reported by the WHO. Furthermore, COVID-19 Dashboard offers a projection for 90 days; nevertheless, for some countries, the quadratic equation could rise to a maximum point; in these cases, the algorithms will stop calculating and thus the projected days will be lower than 90 days.\n\nConsequently, COVID-19 Dashboard allows analyzing confirmed cases, deaths, and mortality rates as well as projecting cases and death deploying information in: a) geographic charts to understand the country distribution of the disease, b) bar charts to recognize the most impacted countries, c) column charts to present the evolution of the disease over time, and d) line charts to estimate the behavior of the disease.\n\nCOVID-19 Dashboard is an online responsive system; therefore, it is accessible for users via a web browser through desktops, laptops, smart phones, and tablets. Its front-end can automatically adjust to any screen resolution in order to deploy the information in the best possible way.\n\n\nResults\n\nCOVID-19 Dashboard is able to process the data provided by the WHO related to COVID-19 rates and World Population Review related to the current world population. This system deploys up-to-date information as follows:\n\nCases and deaths by country. This information is presented in two ways: geographic charts and bar charts in order to recognize the most impacted countries.\n\nCases and deaths by country per million people. This information is also presented by geographic and bar charts.\n\nMortality rate by country. For this information, the mortality rate is calculated for each country and is presented by geographic and bar charts.\n\nCases and deaths by date worldwide and by selected country. This information is calculated based on the daily information reported by each country. This is presented in two column charts. The first column chart presents the daily cases, while the second column chart presents the cumulative cases.\n\nProjected cases and deaths worldwide and by selected country. This information is calculated using the quadratic equation obtained through the developed mathematical model (as above). It is presented using a line chart with two series. The first presents the actual cases or deaths for the last 31 days. The second presents the projected cases or deaths up to 90 days after the last day reported.\n\nAs mentioned above, the number of cases, deaths, and mortality rates are deployed using geographic charts. Figure 2 presents a geographic chart with the number of deaths by country, where red implies a large number of deaths and light blue implies a low number of deaths. On the date that this chart was obtained from COVID-19 Dashboard, the United States had the largest number of deaths worldwide followed by Spain, France, Italy and the United Kingdom. However, just using the color coding, it is not clear if Spain, France, Italy or the United Kingdom has the second highest number of deaths. Similarly, for the lower number of deaths seen in Belgium, Brazil, Germany, Canada, Turkey, Netherlands, Iran, and China, it is not clear which has the lowest number of deaths using the color coding.\n\nRed = high number of deaths; light blue = low number of deaths. Chart captured on May 29th 2020.\n\nConsequently, it is necessary to sort the countries based on the actual number of deaths in addition to the color coding. Figure 3 presents a fragment of a bar chart with the number of deaths per country sorted by number of deaths. This chart shows that the United States has more than the double of deaths of Italy and the United Kingdom. In addition, Spain and France have almost the same number, but a little less than Italy and United Kingdom, followed by Brazil, while Belgium, Mexico, Germany, Iran, and Canada have similar number of deaths but much less than Spain and France. Finally, Netherlands, India, China, Turkey, Russia, and Sweden have less but a still important number of cases.\n\nChart captured on May 29th 2020.\n\nIt is also important to assess information about COVID-19 based on the population by country. Figure 4 presents a geographic chart with the number of cases by countries per million people. Although United States has the largest number of cases, this chart presents that countries such as Spain, Ireland, and Belgium have more cases per million than United States, while Italy, Switzerland, United Kingdom, Portugal, and Sweden have also a large number of cases per million.\n\nChart captured on May 29th 2020.\n\nAdditionally, the COVID-19 Dashboard shows the mortality rate per country, as shown in Figure 5. Although the United States has the largest number of deaths, it also has a very large number of cases. However, countries such as Italy, United Kingdom, Spain, and France have a higher mortality rate than the United States. Surprisingly, the country with the highest mortality rate by May 29th is Yemen (21.63%), showing the very serious public health issue for this country.\n\nChart captured on May 29th 2020.\n\nNevertheless, it is important to take into account that this country has reported little information. Countries such as France, Belgium, Italy, United Kingdom, and Spain are in the top 10 of this list, which implies that they have a high mortality risk, perhaps due to the high number of active cases.\n\nFurther information to be analyzed is cases and deaths per date. Figure 6 presents two charts. Figure 6(a) shows the number of confirmed cases per day in Colombia, while (b) shows cumulative cases per day in Colombia. Figure 6(a) shows that from April 1 2020, the number of cases was stable with peaks in the last few days, while Figure 6(b) shows stable behavior with a small increment of cumulative cases from April. It implies that COVID-19 keeps impacting health in Colombia and cases will increase until its behavior changes.\n\nChart captured on May 29th 2020.\n\nBased these previous results, it is really important to know the trend of cases and deaths worldwide and by country. Thus, COVID-19 Dashboard includes a very important contribution, i.e. the mathematical model already described, which allows projection for future cases and deaths worldwide and by country. Figure 7 presents four charts with cases and death projections.\n\nChart captured on May 29th 2020.\n\nFigure 7(a) presents projected cases in Colombia. Blue corresponds to the actual cases reported by the WHO, while red corresponds to the projected cases and yellow the increment of cases per day. This projection performed on May 29th 2020 produced the quadratic equation:\n\n\n\nThis projection presents a exponential growth, which implies that COVID-19 in Colombia will continue to impact public health.\n\nFigure 7(b) presents projected cases in Russia. This projection performed on May 29th 2020 produced the quadratic equation:\n\n\n\nThis projection presents a logarithmic growth, which implies that deaths for COVID-19 in Russia are decreasing considerably and might stop in 61 days.\n\nFigure 7(c) presents projected deaths in India. This projection performed on May 29th 2020 produced the quadratic equation:\n\n\n\nThis projection presents a exponential growth, which implies that deaths for COVID-19 in India are still increasing.\n\nFigure 7(d) presents projected deaths worldwide. This projection performed on May 29th 2020 produced the quadratic equation:\n\n\n\nThis projection presents a logarithmic growth, which implies that deaths for COVID-19 worldwide might start decreasing in the first week of July.\n\nQuadratic equations are presented with coefficients with two decimals; however, all calculations are done with the real calculated values for their coefficients.\n\n\nDiscussion\n\nCOVID-19 has required analysis of data regarding cases and deaths reported worldwide. Some projects developed by important organizations such as COVID-19 Dashboard by the Center for Systems Science and Engineering (CSSE) at Johns Hopkins University (JHU) (https://coronavirus.jhu.edu/map.html) and Coronavirus Disease (COVID-19) Dashboard by the World Health Organization (https://covid19.who.int/) have provided very interesting visualizations for reporting the current state of the COVID-19. Nevertheless, these projects lack important information and analysis. With this in mind, the COVID-19 Dashboard presented here has been designed and developed in order to offer additional valuable information. This project uses the data reported by the WHO; therefore, it is able to deploy the same information as the WHO COVID-Dashboard. However, COVID-19 Dashboard is able to deploy more information such as cases and deaths per million people worldwide and mortality rate worldwide, which is also very important to assess the impact of the disease from different perspectives.\n\nMoreover, COVID-19 Dashboard has an additional important contribution not presented in other projects. This is the mathematical model that projects future cases and deaths worldwide and by country. This mathematical model is based on linear algebra and calculates a quadratic equation based on the information reported in the last 31 days. Thus, the mathematical model is able to adjust the quadratic equation once new information is reported. With this model, COVID-19 Dashboard is able to estimate the number of new cases and deaths up to 90 days.\n\n\nConclusions\n\nCOVID-19 Dashboard allows understanding COVID-19 behavior and evolution from different perspectives. Based on those perspectives, it is possible to assess not only the actual number of cases and deaths worldwide and by country, but also the most impacted countries by observing the number of cases and deaths per million people and mortality rate. COVID-19 Dashboard offers a projection of future cases and deaths, which is an important tool to estimate the disease’s evolution. Based on these projections, health organizations around the world might take action in order to minimize the impact, as well as estimate the date when the COVID-19 might stop impacting various countries.\n\n\nData availability\n\nData used for this project has been taken from the following public sources available in JSON and Comma-separated values (csv) format respectively:\n\nCOVID-19 map data by the WHO https://covid19.who.int/.\n\nPopulation by country 2020 data by the WPR https://www.worldpopulationreview.com/countries/.\n\n\nSoftware availability\n\nSoftware available from: https://covid19.itiud.org/.\n\nSource code available from: https://gitlab.com/florezfernandez/COVID-19Dashboard\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.382593914\n\nLicense: GNU General Public License (GPL)",
"appendix": "Acknowledgements\n\nThe authors would like to thank members of the research group ITI https://www.itiud.org/ for its valuable feedback.\n\n\nReferences\n\nXu X, Chen P, Wang J, et al.: Evolution of the novel coronavirus from the ongoing wuhan outbreak and modeling of its spike protein for risk of human transmission. Sci China Life Sci. 2020; 63(3): 457–460. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJiang F, Deng L, Zhang L, et al.: Review of the clinical characteristics of coronavirus disease 2019 (covid-19). J Gen Intern Med. 2020; 35(5): 1545–1549. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWorld Health Organization: Novel coronavirus(2019-ncov) situation report - 11. 2020. Reference Source\n\nWorld Health Organization: Coronavirus disease 2019 (covid-19) situation report - 40. 2020. Reference Source\n\nWorld Health Organization: Coronavirus disease 2019 (covid-19) situation report - 71. 2020. Reference Source\n\nWorld Health Organization: Coronavirus disease 2019 (covid-19) situation report - 101. 2020. Reference Source\n\nSingh S, Florez H: Coronavirus disease 2019 drug discovery through molecular docking [version 1; peer review: awaiting peer review]. F1000Res. 2020; 9: 502. Publisher Full Text\n\nBooch G, Rumbaugh J, Jacobson I: The unified modeling language reference manual, Addison-Wesley Reading, 1999; 2. Reference Source\n\nSalvatierra K, Florez H: Biomedical mutation analysis (bma): A software tool for analyzing mutations associated with antiviral resistance. F1000Res. 2016; 5: 1141. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSalvatierra K, Florez H: Pathogen sequence signature analysis (pssa): A software tool for analyzing sequences to identify microorganism genotypes. F1000Res. 2017; 6: 21. Publisher Full Text\n\nVegega C, Pytel P, Pollo-Cattaneo MF: Evaluation of the bias in the management of patient’s appointments in a pediatric office. ParadigmPlus. 2020; 1(1): 1–21. Reference Source\n\nGregory JA, Delbourgo R: Piecewise rational quadratic interpolation to monotonic data. IMA Journal of Numerical Analysis. 1982; 2(2): 123–130. Publisher Full Text\n\nFlorez H, Salvatierra K: A tool for analyzing mutations in biomedical microorganisms. In Proceedings of the 8th International Multi-Conference on Complexity, Informatics and Cybernetics, 2017; 358–363. Reference Source\n\nFlorez H, Singh S: Covid-19 dashboard (version 1.0). Zenodo. 2020. http://doi.org/10.5281/zenodo.3825939"
}
|
[
{
"id": "64490",
"date": "24 Jun 2020",
"name": "Maria Florencia Pollo-Cattaneo",
"expertise": [
"Reviewer Expertise artificial intelligence - informatic - information systems"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper presents a proposal for a COVID-19 Dashboard to manage significant and relevant information in relation to the pandemic that is currently attacking the whole world.\nFrom a methodological point of view, the solution is developed with solidity and technologies adapted to the technological problem posed.\nThe resulting product is tested with valid data even though it is not developed and analyzed in all countries in detail due to the extension of the work.\nIt would be interesting if they could update and validate if the data obtained by the board match the real data and see if they could take into account second outbreaks of the disease. The solution is validated correctly.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Partly\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes",
"responses": []
},
{
"id": "64489",
"date": "26 Jun 2020",
"name": "Olmer Garcia-Bedoya",
"expertise": [
"Reviewer Expertise Robotics and data science Professor"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 Dashboard's development to analyze the COVID-19 contagious and death base in open data available. The paper is well-written and presents interesting results clearly and concisely. I have enjoyed reading this paper.\nI have the following comments for the authors that should be addressed before indexing:\nThe model presented to predict the time series's tendencies is easy to understand and has a fast implementation. However, I recommend reviewing time series analysis like ARIMA, recurrent neural network, or systems dynamics, which let to include more data in the estimation of the tendencies of the time series.\n\nIt will be interesting to review initiatives like presented in Kaggle wherein collaborative works, have presented alternatives since the data science field could help to combat the COVID-19 disease.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Partly\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly",
"responses": []
},
{
"id": "64488",
"date": "26 Aug 2020",
"name": "Hüseyin Bicen",
"expertise": [
"Reviewer Expertise Educational Technology"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper is significant. The paper result is have an important data analysis approach for assessing COVID-19 case and death data. The Authors analyze the current state of COVID-19, have created an online information system called COVID-19 Dashboard, which is accessible from: https://covid19.itiud.org/. The paper result is relevant to the researchers. For that reason, this paper is necessary to the scientific world.\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/9-570
|
https://f1000research.com/articles/9-302/v1
|
28 Apr 20
|
{
"type": "Method Article",
"title": "A non-fluorescent HaloTag blocker for improved measurement and visualization of protein synthesis in living cells",
"authors": [
"Laurie D. Cohen",
"Ayub Boulos",
"Noam E. Ziv",
"Laurie D. Cohen",
"Ayub Boulos"
],
"abstract": "Background: HaloTag is a modified bacterial enzyme that binds rapidly and irreversibly to an array of synthetic ligands, including chemical dyes. When expressed in live cells in conjunction with a protein of interest, HaloTag can be used to study protein trafficking, synthesis, and degradation. For instance, sequential HaloTag labeling with spectrally separable dyes can be used to separate preexisting protein pools from proteins newly synthesized following experimental manipulations or the passage of time. Unfortunately, incomplete labeling by the first dye, or labeling by residual, trapped dye pools can confound interpretation. Methods: Labeling specificity of newly synthesized proteins could be improved by blocking residual binding sites. To that end, we synthesized a non-fluorescent, cell permeable blocker (1-chloro-6-(2-propoxyethoxy)hexane; CPXH), essentially the HaloTag ligand backbone without the reactive amine used to attach fluorescent groups. Results: High-content imaging was used to quantify the ability of CPXH to block HaloTag ligand binding in live HEK cells expressing a fusion protein of mTurquoise2 and HaloTag. Full saturation was observed at CPXH concentrations of 5-10 µM at 30 min. No overt effects on cell viability were observed at any concentration or treatment duration. The ability of CPXH to improve the reliability of newly synthesized protein detection was then demonstrated in live cortical neurons expressing the mTurquoise2-HaloTag fusion protein, in both single and dual labeling time lapse experiments. Practically no labeling was observed after blocking HaloTag binding sites with CPXH when protein synthesis was suppressed with cycloheximide, confirming the identification of newly synthesized protein copies as such, while providing estimates of protein synthesis suppression in these experiments. Conclusions: CPXH is a reliable (and inexpensive) non-fluorescent ligand for improving assessment of protein-of-interest metabolism in live cells using HaloTag technology.",
"keywords": [
"HaloTag",
"Live Imaging",
"Protein Synthesis"
],
"content": "Introduction\n\nThe 33 kDa HaloTag protein1 is a modified haloalkane dehalogenase enzyme which bonds covalently to a variety of synthetic ligands (reporters), including chemical dyes. HaloTag dyes do not suffer from some of the drawbacks of traditional fluorescent proteins such as slow tag maturation, tendency to oligomerize, and photoswitching artifacts2–4. HaloTag fusion proteins can be followed in living cells or in vivo for long time durations without label dissociation1,5. Thus, the HaloTag system represents an attractive method for studying protein localization, dynamics, trafficking, synthesis and degradation. Besides HaloTag, other labeling systems based on similar principles have been developed (e.g. SNAP tag, CLIP tag;6–9). Compared to SNAP tag, however, the HaloTag system offers superior binding (e.g. 10–12) and brightness13. Moreover, a wide selection of bright, photostable, cell-permeable HaloTag compatible dyes with rapid labeling kinetics and low nonspecific staining (e.g. 14,15) is now available.\n\nBoth the HaloTag1,4,16–20 and the SNAP tag7,21,22 systems have been used with spectrally distinct dyes to visualize newly synthesized proteins, to differentiate between populations of newly formed versus aged proteins, to follow proteins at different subcellular locations and to measure protein half-lifetimes23–31. In such experiments, however, incomplete labeling – that is, binding sites that remain dye-free, as well as fluorescence from residual unbound ligands – represent significant confounds. Notably, ligands can remain in the cells even after multiple washes (due to slow efflux and reduced active clearance capability, especially in unhealthy cells or at low serum levels; Promega “Focus on Imaging” HaloTag protocol; see also 32). Moreover, in some cell types, such as cultured neurons, excessive washes can be detrimental29,33. Saturation of binding sites can be realized by applying fluorescent ligands in large excess (e.g. 34) or Succinimidyl Ester ligands after masking their reactive groups32. This, however, is costly and can introduce other problems, such as the nonspecific labeling mentioned above and even cell toxicity.\n\nIncomplete binding presents a particularly problematic confound when attempting to identify newly synthesized copies of tagged proteins of interest or measure their turnover. Thus for example, 15% unlabeled binding sites, for a protein with a half-life of 5 days, labeled a second time after 24 hours can lead to an erroneous half-life estimate of ~2.5 days, not to mention the misidentification of about half of newly labeled proteins as newly synthesized ones. This confound can be avoided almost entirely by using highly specific, non-fluorescent reagents for blocking residual unbound sites.\n\nAffordable nonfluorescent blockers9,22 are available for the SNAP tag system. Until most recently, however, there has been a paucity of affordable, nonfluorescent HaloTag compatible blockers. Solutions based on commercially available ligands tend to be costly32,35 and may not cross the cell membrane efficiently36.\n\nIn this study we present an inexpensive, non-fluorescent, cell-permeable HaloTag blocker, 1-chloro-6-(2-propoxyethoxy)hexane, which is well-tolerated both in cell lines and in primary neuronal cultures, and demonstrate its application for following newly synthesized protein using single and dual-color HaloTag labeling.\n\nIn the course of this study, four other nonfluorescent compounds were screened as potential HaloTag compatible blockers, of which 7-bromoheptanol was selected as a preferred reagent37. We nevertheless present our findings, in which the characteristics of our alternative blocking reagent and its utility for following protein synthesis in live cells are described, with the hope that it will prove to be useful as well.\n\nAll raw images and quantifications are available as Underlying data38.\n\n\nResults\n\nIn order to quantify blocking efficacy in living cells, a method was needed to estimate the abundance of HaloTag binding sites (total – free and bound) in individual cells. We reasoned that reporter proteins containing one HaloTag binding site and one copy of a fluorescent protein (FP) would be useful in this regard, as this would allow normalization of HaloTag ligand fluorescence to FP fluorescence and thus calculation of fractional HaloTag labeling values, independent of fusion protein expression levels. To that end, we created a fusion protein of HaloTag and the fluorescent protein mTurquoise239 encoded as a single polypeptide (HaloTag-mTurq2; Figure 1A). The DNA coding for the fusion protein was inserted into a third generation lentivirus backbone to allow lentivirus-based expression as well as transfection-based methods (see Methods). As shown in Figure 1B, expression of this construct (in rat cortical neurons in culture, in this case) allowed us to simultaneously measure HaloTag ligand binding (Janelia Fluor JF635HT15; a generous gift from Jonathan Grimm and Luke Lavis, Janelia Research Campus) and FP fluorescence in individual cells. As might be expected, no JF635HT binding was observed in neighboring cells devoid of mTurq2 fluorescence, suggesting that HaloTag ligand binding was highly specific. In our hands, neurons expressing HaloTag-mTurq2 could be imaged for many hours (>15) without noticeable toxicity, damage, or negative effects on cell morphology. This fusion construct was thus used in subsequent experiments to quantify the fractional degree of HaloTag ligand binding in live cells.\n\n(A) A lentivirus-based construct for expressing a HaloTag-mTurquoise2 fusion protein in living cells. UBC = Homo sapiens ubiquitin C; WPRE = woodchuck hepatitis virus posttranscriptional regulatory element. (B) A cortical neuron in primary culture (17 days in culture) expressing the HaloTag-mTurq2 fusion protein, before (left) and after (right) labeling with JF635HT ligand (100nM, 1 hour). Bar, 20µm.\n\nMost HaloTag ligands are based on a chlorinated alkyl chain with a reactive group for attaching fluorescent probes on the opposite side. Here, we synthesized the same ligand without the reactive group, reasoning that its binding properties would be similar but its ability to move across cell membranes improved, and its non-specific reactivity reduced, by the removal of the terminal polar amine group. The resulting molecule {(1-chloro-6-(2-propoxyethoxy)hexane; CID 63684368)} referred to here as CPXH is shown in Figure 2A.\n\n(A) Chemical structure of CPXH {1-chloro-6-(2-propoxyethoxy)hexane}, the non-fluorescent, cell-permeable HaloTag blocker tested in this study. (B) Testing CPXH blocking efficacy. HaloTag-mTurq2 was expressed in HEK293 cells growing in 96 well plates. The cells were treated with 0.01, 0.1 ,1 ,5, or 10 µM CPXH, or carrier solution (0.1% DMSO in cell culture media), for 10, 30, 60, 90, or 120 minutes (all combinations of concentration and duration). The cells were washed with cell culture media and labeled with JF635HT (100nM for 30 minutes). The cells were then washed with HBSS and imaged. Imaging was carried out on an automated high-content imaging system, providing reads from >10,000 cells per concentration and time point per experiment (2 separate experiments). Note that cells at t=0 were treated with CPXH and washed immediately, and were thus exposed to the blocker for anywhere between a few seconds and a minute. (C) Representative images of JF635HT labeling in mTurq2-positive HEK293 cells. Cells were exposed to CPXH (or carrier solution) at indicated concentrations and durations. Bar, 50µm. (D) JF635HT / mTurq2 (background-corrected) fluorescence ratios at the CPXH concentrations and incubation times tested. Only fields of view with at least 90 mTurq2 positive cells were included (8 to 16 fields of view per condition, two separate experiments). Error bars represent SEM of fields of view.\n\nTo test the efficacy of CPXH as a non-fluorescent HaloTag blocker, and characterize its useful concentrations and blocking kinetics, we expressed HaloTag-mTurq2 in HEK293 cells, and treated the cells with 0.01, 0.1, 1, 5, or 10 µM of CPXH, or carrier solution, for 10, 30, 60, 90, or 120 minutes (all combinations of treatment durations and concentrations). The cells were then washed and labeled with JF635HT at a final concentration of 100 nM for 30 minutes (Figure 2B). Imaging was carried out on an automated high-content imaging system, providing reads from >10,000 cells per concentration and time point per experiment (two separate experiments). Background corrected JF635HT/mTurq2 fluorescence ratios were then calculated for all concentrations and durations. As shown in Figure 2C, D, 30-minute exposures to 5 or 10 µM CPXH completely prevented labeling with JF635HT, suggesting saturation of practically all HaloTag binding sites. Nearly full saturation was also attained following exposure to 1 µM CPXH for 120 min.\n\nTo examine the potential cytotoxicity of CPXH, HEK293 cells were treated with CPXH at a concentration of 0.01, 0.1, 1, 5, or 10 µM or carrier solution, for 10, 30, 60, 90, or 120 minutes and cell viability was tested using a Live/Dead assay (see Methods) and high-content screening microscopy. Cell death was found to be negligible at all concentrations and treatment durations tested (0.89±0.44%, average ± standard deviation; maximum ~2% cell death) with no dependence whatsoever on CPXH concentration or exposure duration. Furthermore, in networks of rat cortical neurons grown on multielectrode arrays40, levels of spontaneous network activity - a sensitive measure of neuronal viability - did not decline following chronic exposure to 10 µM CPXH for 18 and 47 hours (two experiments; see Underlying data38). These findings suggest that CPXH is a non-toxic, efficient blocker of HaloTag binding sites.\n\nAn exciting application of HaloTag technology concerns the labeling of newly synthesized copies of proteins of interest and following their fates thereafter. As explained in the Introduction, correct interpretation of such experiments necessitates the complete labeling of all HaloTag binding sites in preexisting protein copies. In preliminary experiments, and in agreement with prior studies32,37 we noted that applying a second fluorescent HaloTag ligand (Oregon Green, Promega, or JF635HT) immediately following apparently saturating labeling with a first ligand (JF635HT or Oregon Green, Promega) still resulted in significant labeling with the second ligand, indicating incomplete saturation of HaloTag binding sites, as exemplified in Figure 3.\n\n(A) mTurq2-positive neurons in cell culture were labeled with Oregon Green HaloTag ligand (Promega 100nM, 1 hour), washed, and labeled immediately with JF635HT ligand. (B) Applying JF635HT resulted in significant labeling, indicating incomplete saturation of HaloTag binding sites. JF635HT labeling occurred also when using a 10-fold higher concentration of Oregon Green (Promega 1 µM, 1hour; not shown). Bar, 20µm.\n\nAs a proof of principle, we examined whether unambiguous identification of newly synthesized proteins would be facilitated by using CPXH to block residual binding sites. To that end, we performed single and dual-labeling time-lapse experiments of neurons expressing HaloTag-mTurq2, using CPXH to block residual binding sites before applying the (second) label. In the first set of experiments (Figure 4 and Figure 5), neurons expressing HaloTag-mTurq2 were first treated with CPXH (10 µM) for 30 minutes. The cells were then washed, JF635HT was added to the cell culture media (without washing it out) and the cells were followed by time-lapse imaging for >12 hours. In a second set (Figure 6), blocking with CPXH was preceded by labeling with Oregon Green ligand (Promega). Changes in JF635HT (and Oregon Green) labeling over time were quantified by measuring, on a cell-by-cell basis, the ratio of JF635HT (or Oregon Green) to mTurq2 fluorescence in mTurq2-positive cells. To correct for nonspecific label accumulation, fluorescence values in neighboring mTurq2-negative cells were obtained at each time point and subtracted from JF635HT / Oregon Green fluorescence in mTurq2-positive cells. As shown in Figure 4C, Figure 5C and Figure 6C, initial JF635HT labeling was negligible; with time, however, JF635HT fluorescence gradually increased, probably reflecting the labeling of newly synthesized HaloTag-mTurq2 copies. This was observed for cell bodies and dendrites (Figure 4 and Figure 6) as well as distal axon ‘beds’ (Figure 5), albeit at slower rates, as might be expected for newly synthesized protein copies delivered from remote somatic protein synthesis facilities. Interestingly, we noted that labeling with Oregon Green ligand was associated with strong, non-specific labeling that washed out relatively slowly. In contrast, very low levels of non-specific JF635HT labeling were observed even after prolonged periods in the presence of this ligand.\n\n(A) Neurons expressing HaloTag-mTurq2 were first treated with CPXH (10 µM) for 30 minutes. The cells were then washed, followed by addition of JF635HT to the cell culture media (100nM, without washing it out), and the cells were followed by time-lapse imaging. (B) Example of mTurq2-positive cortical neuron (17 days in culture) treated as described in A and followed by time lapse imaging. Bar, 20µm. (C) Changes in JF635HT labeling over time were quantified by measuring, on a cell-by-cell basis, the ratio of JF635HT to mTurq2 fluorescence in mTurq2-positive cells. Background corrected JF635HT/mTurq2 fluorescence ratios for three different neurons are shown. Images in B belong to Cell 1. Note the negligible labeling immediately after exposure to JF635HT (compare with Figure 3B).\n\n(A) Imaging of axon ‘beds’ belonging to neurons expressing HaloTag-mTurq2. Neurons were treated as in Figure 4. (B) Example of axon bed belonging to mTurq2-positive cortical neuron(s). Bar, 20 µm. (C) Gradual changes in JF635HT labeling over time in axons enclosed in the yellow rectangle in B. Note the negligible labeling immediately after exposure to JF635HT. Bar, 10 µm. (D) ratio of JF635HT to mTurq2 fluorescence in mTurq2-positive axons (average of 10 regions per field of view). Background corrected JF635HT/mTurq2 fluorescence ratios for three different axon beds are shown. Images in (B) and (C) belong to Region 1. Note the slow increase in JF635HT fluorescence, as might be expected in cellular regions at significant distance from the neurons’ major sites of protein synthesis (i.e., the cell bodies).\n\n(A) Neurons expressing HaloTag-mTurq2 were labeled with Oregon Green ligand (Promega 1 µM, 30 min), washed, and blocked with CPXH (10 µM) for 30 minutes. The cells were then washed and labeled with JF635HT and followed by time-lapse imaging. (B) Example of mTurq2-positive cortical neuron (14 days in culture) treated as described in A and followed by time lapse imaging. Bar, 20µm. (C) Changes in JF635HT, and (D) Oregon Green labeling over time were quantified by measuring, on a cell-by-cell basis, the ratio of JF635HT (or Oregon Green) to mTurq2 fluorescence in mTurq2-positive cells. Background corrected fluorescence ratios for three neurons are shown. Images in B belong to Cell 1.\n\nThese experiments thus suggest that CPXH can greatly facilitate unambiguous identification of newly synthesized proteins in live cells and through continuous HaloTag labeling.\n\nThe accumulation of JF635HT in the experiments of Figure 4–Figure 6 presumably reflected the accumulation of newly synthesized HaloTag-mTurq2. To validate this assumption, we examined JF635HT labeling after cells were blocked with CPXH and then exposed to the potent protein synthesis inhibitor cycloheximide (CHX; 100 µg/ml). Specifically, cortical neurons expressing HaloTag-mTurq2 were treated with CPXH as described above. The cells were then washed and exposed to CHX or carrier solution for 24 hours, followed by JF635HT labeling. As shown in Figure 7, practically no labeling was observed following the 24-hour exposure to CHX; In fact, quantification revealed a nearly 40-fold reduction in labeling intensity (Figure 7C). These findings support the aforementioned presumption, and, at the same time, provide a quantitative assessment of protein synthesis suppression by CHX in these preparations.\n\n(A) Cortical neurons expressing HaloTag-mTurq2 were treated with CPXH. The cells were washed and thereafter exposed to cycloheximide (CHX) or carrier solution (0.1% DMSO in cell culture media) for 24 hours, followed by JF635HT labeling. (B) JF635HT labeling in two such neurons (left: 24-hour CHX; right: 24-hour carrier solution). Scale bar 20µm. (C) 24-hour protein synthesis suppression resulted in a nearly 40-fold (39.2) reduction in JF635HT labeling intensity. There were 81 and 178 neurons, CHX and carrier solutions, respectively, 4 replicates per condition from 2 separate experiments. Average + SEM, t-test assuming unequal variances.\n\n\nDiscussion and conclusions\n\nThe findings described above suggest that CPXH is a potent non-fluorescent, cell-permeable, and non-toxic HaloTag ligand. We first characterized its blocking kinetics and their dependence on CPXH concentration. We then demonstrated that CPXH can improve the fidelity of experiments aimed at following newly synthesized proteins in living cells. Finally, we show how this blocker might be useful for quantifying protein synthesis inhibitor efficacy in living cells.\n\nA recent study37 identified 7-bromoheptanol as an alternative low-toxicity HaloTag-blocking agent, and demonstrated its usefulness for measuring protein turnover at the population and single cell level. The authors suggested that this agent could be potentially used for estimating the synthesis rate of proteins of interest within individual cells. The experiments shown here (Figure 4–Figure 6) confirm this suggestion. Moreover, the use of the HaloTag-mTurq2 fusion protein in our experiments provided confidence that the gradual labeling observed over time in mTurq2-positive cells reflected bona-fide HaloTag labeling, and not, e.g., non-specific accumulation of labels in cells, while providing means for normalizing HaloTag labeling to total numbers of HaloTag binding sites. We note that normalization to mTurq2 levels in such experiments might be somewhat imperfect, as new mTurq2 is synthesized alongside new HaloTag binding sites. Yet at least at initial time points, the contribution of newly synthesized mTurq2 to total mTurq2 fluorescence is probably insignificant, even less so if total HaloTag-mTurq2 levels remain more or less constant.\n\nThe cell entry, binding kinetics and affinity of CPXH were not compared here to those of 7-bromoheptanol and thus their advantages and disadvantages with respect to each other remain unknown. Given that the structure of CPXH is essentially identical to the backbone of most HaloTag ligands, it might be expected to be as affine and effective as the commonly used fluorescent ligands themselves. Hopefully, future studies will provide further information on these and other agents that optimize the utility of HaloTags and their like.\n\n\nMethods\n\nHaloTag blocker 1-chloro-6-(2-propoxyethoxy)hexane (CID 63684368) was synthesized at our request by AKos Consulting & Solutions, GmbH. The dry material was dissolved in DMSO to prepare a 10 mM stock solution, which was stored in small aliquots at -20°C until used.\n\nFU-HaloTag-mTurq2-Wm was generated based on FU-PSD95-mTurq2-Wm41. Briefly, AgeI-HaloTag-XhoI (911 bp; sequence below) was synthesized de novo based on the Halotag sequence from Promega pFN23A HaloTag® CMVd2 Flexi® Vector, 9PIG286 (https://worldwide.promega.com/-/media/files/vector-sequences/flexi/pfn23a.txt). FU-PSD95-mTurq2-Wm was cut with AgeI and XhoI and the PSD-95 segment was removed, replaced by AgeI-HaloTag-XhoI and ligated to obtain FU-HaloTag-Turq2-Wm. Cloning and custom gene synthesis was done by Genscript (Piscataway NJ, US). Positioning the HaloTag at the N-terminus was based on the literature (e.g. 42) and on the structure of the protein of interest.\n\nAgeI-HaloTag-XhoI:\n\nACCGGTCGCCACCATGGCAGAAATCGGTACTGGCTTTCCATTCGACCCCCATTATGTGGAAGTCCTGGGCGAGCGCATGCACTACGTCGATGTTGGTCCGCGCGATGGCACCCCTGTGCTGTTCCTGCACGGTAACCCGACCTCCTCCTACGTGTGGCGCAACATCATCCCGCATGTTGCACCGACCCATCGCTGCATTGCTCCAGACCTGATCGGTATGGGCAAATCCGACAAACCAGACCTGGGTTATTTCTTCGACGACCACGTCCGCTTCATGGATGCCTTCATCGAAGCCCTGGGTCTGGAAGAGGTCGTCCTGGTCATTCACGACTGGGGCTCCGCTCTGGGTTTCCACTGGGCCAAGCGCAATCCAGAGCGCGTCAAAGGTATTGCATTTATGGAGTTCATCCGCCCTATCCCGACCTGGGACGAATGGCCAGAATTTGCCCGCGAGACCTTCCAGGCCTTCCGCACCACCGACGTCGGCCGCAAGCTGATCATCGATCAGAACGTTTTTATCGAGGGTACGCTGCCGATGGGTGTCGTCCGCCCGCTGACTGAAGTCGAGATGGACCATTACCGCGAGCCGTTCCTGAATCCTGTTGACCGCGAGCCACTGTGGCGCTTCCCAAACGAGCTGCCAATCGCCGGTGAGCCAGCGAACATCGTCGCGCTGGTCGAAGAATACATGGACTGGCTGCACCAGTCCCCTGTCCCGAAGCTGCTGTTCTGGGGCACCCCAGGCGTTCTGATCCCACCGGCCGAAGCCGCTCGCCTGGCCAAAAGCCTGCCTAACTGCAAGGCTGTGGACATCGGCCCGGGTCTGAATCTGCTGCAAGAAGACAACCCGGACCTGATCGGCAGCGAGATCGCGCGCTGGCTGTCGACGCTGGAGATTTCCGGCGCTCGAG\n\nJanelia Fluor 635 HaloTag ligand (JF635HT15; 100 nM) was incubated for 30 minutes (or 1 hour; experiment of Figure 3) at 37°C, 5% CO2. Oregon Green cell permeable ligand (100nM for 1 hour / 1 µM for 30 minutes; experiments of Figure 3 and Figure 6, respectively; Promega) was applied at 37°C, 5% CO2, then repeatedly washed to remove unbound ligand.\n\nHEK293 cells were grown in DMEM supplemented with 10% FBS at 37°C, 5% CO2. The cells were transfected with HaloTag-mTurq2 using Calfectin (Signagen) transfection according to the manufacturer’s protocol. In brief, one hour before transfection the media was replaced with complete medium with serum and antibiotics. HaloTag-mTurq2 DNA was diluted in serum-free, high glucose DMEM. Calfectin reagent was added to the tube, and pipetted 3–4 times to mix. The mixture was incubated for 10–15 minutes, and applied dropwise to the cells. The plate was swirled gently to mix and returned to the incubator. At 12–18 hours following transfection the media was replaced with fresh culture media. HEK293 cells were reseeded into 96-well glass-bottom microplates (µClear, Black; Greiner) 24 hours after transfection (20k cells per well). At 24 hours after reseeding, the cells were washed twice in Hanks Balanced Salt Solution (HBSS).\n\nPrimary cultures of rat cortical neurons were prepared as described previously43 using a protocol approved by the Technion committee for the supervision of animal experiments (IL-116-08-71). Briefly, cortices of two 1-2-day-old Wistar rats of either sex (Charles River, UK) were dissected following rapid decapitation, dissociated by trypsin treatment followed by trituration using a siliconized Pasteur pipette, and plated onto 22×22 mm coverslips coated with polyethylenimine (Sigma) inside 8-mm-diameter glass cylinder microwells (Bellco Glass). Cells were initially grown in medium containing Minimum Essential Medium (MEM; Sigma), 25 mg/l insulin (Sigma), 20 mM glucose (Sigma), 2 mM L-glutamine (Sigma), 11 mg/l gentamycin sulfate (Sigma), and 10% NuSerum (Becton Dickinson Labware). The preparation was then transferred to a humidified tissue culture incubator and maintained at 37°C in a 95% air and 5% CO2 mixture. Half the volume of the culture medium was replaced every 7 days with cell culture medium similar to the medium described above but devoid of NuSerum, containing a lower concentration of L-glutamine (Sigma, 0.5 mM), and 2% B-27 supplement (Gibco). Neurons used in these experiments came from approximately 20 separate preparations. HaloTag-mTurq2 DNA was expressed by calcium phosphate transfection, or by addition of HaloTag-mTurq2 lentiviral particles to the neuronal cultures (as detailed below).\n\nCortical neurons were transfected with HaloTag-mTurq2 by calcium phosphate transfection 9–11 days after plating, as described previously44. Neurons were imaged within the cloning cylinders 14–21 days after plating, in the cell culture media.\n\nHaloTag–mTurq2 lentiviral particles were produced in house using a commercially available kit (ViraPower™, ThermoFisher Scientific). Briefly, 80%-confluent HEK293T cells were transfected using Lipofectamine 2000 (Invitrogen) with a mixture of three Virapower kit packaging plasmids (pLP1, pLP2, pLP/VSVG), and the expression vector. Lentiviral stocks were prepared by collecting the supernatant 48 hours after transfection, filtering by a 0.45-μm filter, and storing the liquid in small aliquots at -80°C. To express the DNA in cultures of neuronal cells, 4 days after plating the neurons, 1.5 μl of HaloTag-mTurq2 virus were added to each of the cortical neuronal cultures. Neurons were imaged within the cloning cylinders 14–21 days after plating, in the cell culture media.\n\nExperiments were carried out on neurons grown in culture for 2.5 to 3 weeks. The original cell culture media was set aside and CPXH was applied for 30 minutes (10 µM in cell culture media). The cells were then washed three times in cell culture media and the original media was returned. Cycloheximide (100 µg/ml, Sigma) or carrier solution was then applied and the cells were returned to a 37°C, 5% CO2 incubator. 24 hours later, JF635HT was added to the cells for 30 minutes, and the cells were imaged immediately.\n\nPropidium Iodide and NucBlue Live reagent (Hoechst 33342) staining (ReadyProbes Cell Viability Imaging Kit Blue/Red; Thermo) were applied according to the manufacturer’s instructions to assess cell viability in HEK293 cells. The cells were treated with different blocker concentrations (0.01, 0.1, 1, 5, 10 µM, or carrier solution) for a range of time durations (0, 10, 30, 60, 90, or 120 minutes). After treatment with the blocker, the cells were washed twice in HBSS. The two reagents were applied (two drops per ml of each reagent); NucBlue was applied for 15 minutes, and Propidium Iodide was applied just before imaging (for less than 10 minutes). The cells were washed with HBSS, and imaged. Nine sites (3×3 grid) per well were automatically selected at fixed positions relative to the microplate. Total cell count per field of view (NucBlue positive cells) and dead cell count per field of view (cells stained positive for both NucBlue and Propidium Iodide) were calculated by the imaging system’s integrated software. Cell viability in each well was assayed by calculating the average ratio (from nine fields of view) of dead cell count to total cell count for each concentration and duration. Only fields of view with at least 50 NucBlue-positive cells were included.\n\nNeuronal cultures were imaged on a custom-designed confocal laser scanning microscope43 using a 40X, 1.3 NA Fluar objective. Images were collected by averaging six frames at three focal planes spaced 0.8 µm apart. All data were collected at a resolution of 640×480 pixels, at 12 bits per pixel. Excitation of mTurq2 was performed at 457 nm. Emission was read using either 467-493 nm or 465-485 band pass filters. Excitation of JF635HT was performed at 633 nm. Emission was read using a 635 nm long-pass filter (Semrock). Excitation of Oregon Green was performed at 488nm. Emission was read using a 500–550 nm band pass filter (Chroma).\n\nImage analysis was performed using custom-written software (“OpenView”; available on request; analyses could also be performed using other software packages, such as ImageJ) by placing rectangular regions of interest on cell bodies and dendrites (4 to 7 regions per cell, Figure 4 and Figure 6), axons (10 regions, Figure 5), or cell bodies (Figure 7) of mTurq2-positive neurons using the mTurq2 channel. Fluorescence values of all channels were then collected from these regions and average values were calculated for each neuron or axonal bed. To correct for background fluorescence and non-specific labeling, regions of interest were placed on mTurq2-negative areas (Figure 4–Figure 6) or mTurq2-negative somata (Figure 7) and these values were subtracted from all fluorescence readings for all channels and all time points.\n\nImaging of HEK293 cells was done in a 5% CO2, temperature-controlled 37°C microscope chamber of a high-content imaging system (ImageXpress, Molecular Devices). The cells were washed with HBSS before imaging. A single Z-section (2048×2048 pixels, 16 bits per pixel) was obtained automatically at each site using a 20x objective and the system’s sCMOS camera. For non-fluorescent blocker efficacy measurements, 25 sites (a 5×5 grid, automatically selected) were imaged in each well. mTurq2 images were acquired using the following filter set: 438/29nm (ex.), 458nm (dichroic), 483/32nm (em.); JF635HT images were acquired using the following filter set: 562/40nm (ex.), 593nm (dichroic), 641nmLP (em.). For the live-dead assay, nine sites (3×3 grid) per well were automatically selected at fixed positions relative to the microplate using the following filters sets for NucBlue (377/50 nm, 409 nm, 447/60 nm,) and propidium iodide (562/40 nm, 593 nm, 624/40 nm; ex., dichroic, em., respectively). Data analysis was performed automatically using the imaging system’s integrated software, and data were thereafter exported to Microsoft Excel for further analysis.\n\nImages for figures were processed by uniform contrast enhancement and low-pass filtering using Adobe Photoshop and prepared for presentation using Microsoft PowerPoint.\n\n\nData availability\n\nFigshare: A non-fluorescent HaloTag blocker for improved measurement and visualization of protein synthesis in living cells. https://doi.org/10.6084/m9.figshare.12115509.v138.\n\nThis project contains the following underlying data:\n\nCohen Boulos Ziv Data Fig 1 (ZIP). (Image data underlying Figure 1.)\n\nCohen Boulos Ziv Data Fig 2 (ZIP). (Image data and quantification of blocking efficacy underlying Figure 2.)\n\nCohen Boulos Ziv Data Fig 3 (ZIP). (Image data underlying Figure 3.)\n\nCohen Boulos Ziv Data Fig 4 (ZIP). (Image data underlying Figure 4.)\n\nCohen Boulos Ziv Data Fig 5 (ZIP). (Image data and fluorescence quantification underlying Figure 5).\n\nCohen Boulos Ziv Data Fig 6 (ZIP). (Image data and fluorescence quantification underlying Figure 6.)\n\nCohen Boulos Ziv Data Fig 7 (ZIP). (Image data and fluorescence quantification underlying Figure 7.)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
"appendix": "Acknowledgments\n\nWe are grateful to Jonathan Grimm and Luke Lavis (Janelia Research Campus, HHMI, USA) for the Janelia Fluor 635HT ligand, and to the Ciechanover lab (Technion Faculty of Medicine, Haifa, Israel) for reagents and instrumentation for the HEK293 cells experiments.\n\n\nReferences\n\nLos GV, Encell LP, McDougall MG, et al.: HaloTag: a novel protein labeling technology for cell imaging and protein analysis. ACS Chem Biol. 2008; 3(6): 373–82. PubMed Abstract | Publisher Full Text\n\nChudakov DM, Matz MV, Lukyanov S, et al.: Fluorescent proteins and their applications in imaging living cells and tissues. Physiol Rev. 2010; 90(3): 1103-63. PubMed Abstract | Publisher Full Text\n\nMorisaki T, McNally JG: Photoswitching-free FRAP analysis with a genetically encoded fluorescent tag. PLoS One. 2014; 9(9): e107730. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEngland CG, Luo H, Cai W: HaloTag technology: a versatile platform for biomedical applications. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nCohen L, Boulos A, Ziv NE: A non-fluorescent HaloTag blocker for improved measurement and visualization of protein synthesis in living cells. Dataset. Figshare. http://www.doi.org/10.6084/m9.figshare.12115509.v1\n\nGoedhart J, von Stetten D, Noirclerc-Savoye M, et al.: Structure-guided evolution of cyan fluorescent proteins towards a quantum yield of 93%. Nat Commun. 2012; 3: 751. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMinerbi A, Kahana R, Goldfeld L, et al.: Long-term relationships between synaptic tenacity, synaptic remodeling, and network activity. PLoS Biol. 2009; 7(6): e1000136. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDvorkin R, Ziv NE: Relative contributions of specific activity histories and spontaneous processes to size remodeling of glutamatergic synapses. PLoS Biol. 2016; 14(10): e1002572. 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}
|
[
{
"id": "62839",
"date": "19 May 2020",
"name": "Timothy Ryan",
"expertise": [
"Reviewer Expertise neuronal cell biology & physiology",
"development of quantitative optical 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\nThe authors present a useful and insightful study on an approach that leverages genetically encoded Halo-tags fused to a fluorescent protein to show how new protein synthesis can be readily detected using a novel colorless blocking agent. The experiments are clear, analyzed in quantitative detail and the results appear very robust. I have the following suggestions which I think would improve this manuscript:\nThere is only a side mention of how the data of cell # 2 in Figure 6D should be interpreted. A cell was labeled with OG, chased with CPHX and the pulsed with JF646. Why would the OG fluorescence go down? Does this reflect some reversal of OG binding or purely background washout?\n\nRelated to this point the ability to use this scheme to get estimates of new protein synthesis does depend on the irreversibility of dyes binding to Halo. It seems this point could have been fleshed out a little more by asking how stable fluorescence in a fluorescent label followed by CPHX chase in more than just the anecdote in Fig 6.\n\nIt is probably worth discussing the fact that the measurements depend to a certain extent on the relative maturation times of the mTurq versus Halo proteins. I imagine is it not limiting on long time scales but could become so on shorter ones.\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": "5572",
"date": "08 Jun 2020",
"name": "Noam Ziv",
"role": "Author Response",
"response": "Thank you for the supportive and constructive review. Responses follow, in the same order as the commentary: 1) This decay reflects slow OG ligand washout as determined by measuring OG fluorescence decay in somata and thick dendrites of neighboring, mTurq2 negative cells. The potentially slow efflux of this ligand is also documented by Promega (Technical Manual, HaloTag® Technology: Focus on Imaging, page 35). This is now explained in the Results. 2) We did not test the binding reversibility of any of the ligands, and this is indeed a potential confound in long experiments. We note that a) the literature indicates that HaloTag ligand binding is effectively irreversible and that b) CPXH is essentially the canonical HaloTag ligand lacking the reactive amine used for tagging it with fluorescent groups. Given the magnitude of the slow OG effect (comment #1), this is unlikely to be a major explanation for the observation in Fig. 6. Nevertheless, we mention this potential confound in the Discussion. 3) This is indeed a good point. We now mention it in the Discussion."
}
]
},
{
"id": "62881",
"date": "26 May 2020",
"name": "Susanne tom Dieck",
"expertise": [
"Reviewer Expertise neuronal cell biology",
"protein synthesis"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 Cohen et al. describe a valuable addition to the HaloTag toolbox. They developed and characterized an efficient and inexpensive non-fluorescent blocker (ligand) of HaloTag sites. They make use of a fluorescent protein/HaloTag fusion-based experimental design to evaluate relative labeling of HaloTag sites with fluorescent ligands offering the advantage to normalize and also quantify background labeling. As a result they present a strategy to robustly identify and label newly synthesized HaloTag proteins without several current pitfalls.\nThe need of such a low-cost, efficient blocker is underlined by the simultaneous development of a similar compound in a different lab (as properly cited).\n\nThe manuscript without doubt describes a very useful compound and method in sufficient detail to be used by others as it is. As an improvement, a couple of small points could be discussed in more detail to guide the reader through potential further uses, pitfalls or limitations or could be presented with more of the available information embedded into the figures:\n\nRelated to Figure 2:\nAs important for the method in the text regarding the quantification in D the points of ‘complete’ blocking are discussed. It nonetheless would be helpful to also have a sentence on the data without and with low concentration of CPXH (e.g. high variability between time points in the 0µM).\n\nAdd a line how similar this is for neurons (was the concentration used in the following experiments based on the HEK data or determined independently).\n\nToxicity of new compounds and also influence on physiology is an important issue (especially in neurons). The authors have addressed this by not only live/dead assays in HEK cells but also generated data by long-term CPXH incubation on neuronal network activity. This important dataset should be present in a main figure.\nRelated to Figure 4-6/discussion:\nThe use of a fluorescent protein-HaloTag fusion as a means to normalize the fluorescence from a HaloTag ligand over time helps to follow the time-resolved behavior but also adds a source of influence on the apparent behavior that is not readily visible when only the normalized data are presented. In the discussion the authors mention that this normalization might be imperfect but only discuss one side of the imperfection: the overall influence of newly synthesized mTurq on total mTurq (which is indeed most likely not very high). What is missing from my point of view is the discussion of the influence of mTurq fluorescence decline over time as e.g. visible in Fig. 4B. Is this bleaching? What is the order of magnitude of this effect on the apparent increase in relative ligand fluorescence? At least for one of the figures the non-normalized data of all fluorescent partners could be presented alongside the normalized ones.\n\nThe potential effect of residual CPXH labeling after washout on the onset of ligand labeling of newly synthesized protein should be discussed: Is there any detectable lag phase? Does the shape of the curve in the early time points give any hints that an ongoing labeling after washing is either negligible or should be considered?\n\nRegarding the Oregon Green labeling: it is not clear to me whether the sentence ‘Interestingly, we noted that labeling with Oregon Green ligand was associated with strong non-specific labeling that washed out relatively slowly’ refers to transfected cells or all cells (I guess all). The visible variation of Oregon Green fluorescence in the images in 6B should be discussed. It could also be worth addressing how short labeling with different ligands and CPXH chase might help to determine how ‘reliable’ a fluorescent ligand can be used for time-resolved studies.\nRelated to Figure 7:\nJust out of curiosity and not necessary for description of the method at all: since often protein synthesis inhibitors are used at much shorter timescales – did you check how complete is the protein synthesis block judged by this method and under the conditions given after up to 1 hr?\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": "5573",
"date": "08 Jun 2020",
"name": "Noam Ziv",
"role": "Author Response",
"response": "We thank the reviewer for the supportive and constructive review. Responses below are interleaved with the original comments : Related to Figure 2: As important for the method in the text regarding the quantification in D the points of ‘complete’ blocking are discussed. It nonetheless would be helpful to also have a sentence on the data without and with low concentration of CPXH (e.g. high variability between time points in the 0µM). Indeed. We now address these observations in the legend of Figure 2. Add a line how similar this is for neurons (was the concentration used in the following experiments based on the HEK data or determined independently). The concentrations were based on measurements made in HEK cells. This is now stated in the main text. Toxicity of new compounds and also influence on physiology is an important issue (especially in neurons). The authors have addressed this by not only live/dead assays in HEK cells but also generated data by long-term CPXH incubation on neuronal network activity. This important dataset should be present in a main figure. Data on toxicity, including MEA recordings, were moved to the main text (Figure 2E, F) Related to Figure 4-6/discussion: The use of a fluorescent protein-HaloTag fusion as a means to normalize the fluorescence from a HaloTag ligand over time helps to follow the time-resolved behavior but also adds a source of influence on the apparent behavior that is not readily visible when only the normalized data are presented. In the discussion the authors mention that this normalization might be imperfect but only discuss one side of the imperfection: the overall influence of newly synthesized mTurq on total mTurq (which is indeed most likely not very high). What is missing from my point of view is the discussion of the influence of mTurq fluorescence decline over time as e.g. visible in Fig. 4B. Is this bleaching? What is the order of magnitude of this effect on the apparent increase in relative ligand fluorescence? At least for one of the figures the non-normalized data of all fluorescent partners could be presented alongside the normalized ones. This is an excellent point. These data were analyzed for all neurons and three examples are now shown in Figure 4D. We found that indeed, in some (about half of the neurons), some decay of mTurq2 fluorescence was observed (13% after 10 hours on average, up to 37% in the most extreme case). These observations and their implications are now detailed and discussed in the Results and Discussion. The potential effect of residual CPXH labeling after washout on the onset of ligand labeling of newly synthesized protein should be discussed: Is there any detectable lag phase? Does the shape of the curve in the early time points give any hints that an ongoing labeling after washing is either negligible or should be considered? This too is a good point. At the time scales and imaging frequency of the experiments described here, lag phases on the order of minutes would be undetectable. We found no overt evidence for lag phases on longer time scales, but until rigorous measurements of CPXH efflux rates are carried out, this confound should be brought into account. This is now mentioned explicitly in the Discussion. Regarding the Oregon Green labeling: it is not clear to me whether the sentence ‘Interestingly, we noted that labeling with Oregon Green ligand was associated with strong non-specific labeling that washed out relatively slowly’ refers to transfected cells or all cells (I guess all). The visible variation of Oregon Green fluorescence in the images in 6B should be discussed. It could also be worth addressing how short labeling with different ligands and CPXH chase might help to determine how ‘reliable’ a fluorescent ligand can be used for time-resolved studies. This matter is now explained in much more detail in the main text. Related to Figure 7: Just out of curiosity and not necessary for description of the method at all: since often protein synthesis inhibitors are used at much shorter timescales – did you check how complete is the protein synthesis block judged by this method and under the conditions given after up to 1 hr? No we did not; however, to do so one would need to use a protein that has a much higher turnover rate than mTurq2-HaloTag seems to have (as evidenced from Figures 4 to 6)."
}
]
}
] | 1
|
https://f1000research.com/articles/9-302
|
https://f1000research.com/articles/8-2071/v1
|
10 Dec 19
|
{
"type": "Research Article",
"title": "Functional group and diversity analysis of BIOFACQUIM: A Mexican natural product database",
"authors": [
"Norberto Sánchez-Cruz",
"B. Angélica Pilón-Jiménez",
"José L. Medina-Franco",
"B. Angélica Pilón-Jiménez"
],
"abstract": "Background: Natural product databases are important in drug discovery and other research areas. Their structural contents and functional group analysis are relevant to increase their knowledge in terms of chemical diversity and chemical space coverage. BIOFACQUIM is an emerging database of natural products characterized and isolated in Mexico. Herein, we discuss the results of a first systematic functional group analysis and global diversity of an updated version of BIOFACQUIM. Methods: BIOFACQUIM was augmented through a literature search and data curation. A structural content analysis of the dataset was done. This involved a functional group analysis with a novel algorithm to identify automatically all functional groups in a molecule and an assessment of the global diversity using consensus diversity plots. To this end, BIOFACQUIM was compared to two major and large databases: ChEMBL 25, and a herein assembled collection of natural products with 169,839 unique compounds. Results: The structural content analysis showed that 16.1% of compounds, 11.3% of scaffolds, and 6.3% of functional groups present in the current version of BIOFACQUIM have not been reported in the other large reference datasets. It also gave a diversity increase in terms of scaffolds and molecular fingerprints regarding the previous version of the dataset, as well as a higher similarity to the assembled collection of natural products than to ChEMBL 25, in terms of diversity and frequent functional groups. Conclusions: A total of 148 natural products were added to BIOFACQUIM, which meant a diversity increase in terms of scaffolds and fingerprints. Regardless of its relatively small size, there are a significant number of compounds, scaffolds, and functional groups that are not present in the reference datasets, showing that curated databases of natural products, such as BIOFACQUIM, can serve as a starting point to increase the biologically relevant chemical space.",
"keywords": [
"Consensus Diversity Plot",
"compound databases",
"data mining",
"diversity",
"natural products",
"functional groups",
"in silico"
],
"content": "Introduction\n\nNatural product-based drug discovery continues to be an important part of drug discovery. Recently, the synergy between natural product research with molecular modeling and chemoinformatics is gaining importance, speeding up the drug discovery process1,2. As part of these synergistic efforts, curated databases of natural products have an important role as they are major tools for data mining, hypothesis generation, and starting points of virtual screening. There are several databases of natural products in the public domain as reviewed recently3. Our research group has reported initial efforts to assemble a database of natural products from Mexico called BIOFACQUIM4. As part of that work, it was reported an initial scaffold content, and chemical space diversity, and coverage. However, detailed functional group (FG) content analysis, which has been proven to be valuable to characterize compound databases5, in particular from natural sources6, has not been reported for BIOFACQUIM. One of the main reasons is that most of the currently available software employed for identification of functional groups rely on a predefined set of substructures, even when it has been established that one of the major features that discriminate natural products from synthetic compounds are their unique functional groups.\n\nHerein, we report a functional group content analysis of an updated version of BIOFACQUIM. We employed a validated and novel algorithm that identifies all functional groups in a molecule. As part of the analysis and to compare the results of BIOFACQUIM we also discuss the functional group contents of other large and related databases in the public domain, namely ChEMBL 257 and a herein assembled collection of natural products (NPs) with 169,839 compounds.\n\n\nMethods\n\nAs described elsewhere, the first version of BIOFACQUIM was developed as a proof-of-concept database applying several filters to include compounds4. Briefly, the database was focused on natural products published between 2000 and 2018 by research groups in a major Mexican institution in eight indexed journals. As additional criteria for inclusion of compounds and to increase the quality and reliability of the contents of the database, the articles should have described the procedure for the isolation, purification, and characterization of the natural product. In this work, we decided to expand the contents of the BIOFACQUIM database to further explore the diversity of natural products from Mexico.\n\nThe second version of BIOFACQUIM was assembled using the same methodology described to develop the first version4. To achieve the objective of being representative of Mexico, it was decided to augment the number of Mexican institutions (universities, research laboratories, and research centers) publishing information of novel natural products. For the new version of the database, the same procedure for the curation was performed4, using Molecular Operating Environment (MOE) software. A similar procedure can be done using RDKit. In addition, it was ensured to store in the database, natural products that were collected in Mexico, only. The updated and curated version of BIOFACQUIM contains 531 compounds.\n\nTable 1 summarizes the information of BIOFACQUIM and other major compound databases used in this work as reference: ChEMBL 25 as a representative example of the biologically tested chemical space with 1,667,893 unique compounds; and a collection of known natural products with a total of 169,839 molecules. The reference natural product collection was assembled from three general and publicly available natural products databases: the Universal Natural Products Database (UNPD)8, the Natural Products Atlas9 and Natural Products in PubChem Substance Database10. The data sets were curated using the same procedure. Briefly, compounds consisting of multiple components (e.g. salts) were split and the largest component (defined by the number of heavy atoms) was retained, as long as the number of heavy atoms of the second largest component was less than 70% of the largest component. If this was not the case, the compounds were removed from the dataset, unless the two largest components were identical. Single component compounds as well as the retained component of multiple component compounds were neutralized and canonical simplified molecular-input line-entry system (SMILES) (ignoring stereochemistry information) were generated as molecular representation. Compounds consisting of any element other than H, B, C, N, O, F, Si, P, S, Cl, Se, Br and I, as well as compounds with valence errors, were removed from the dataset. Duplicate structures in the context of each database were also removed. The entire process was performed by using the open source cheminformatics toolkit RDKit.\n\nPCP: physicochemical properties; AUC: area under the cyclic system retrieval curve.\n\nOverlap of BIOFACQUIM with the databases selected as reference was assessed in terms of three different structural levels: compounds, scaffolds, and functional groups. Compound overlap was determined in terms of canonical SMILES. For scaffold comparison we use the definition proposed by Bemis and Murcko11 as implemented in RDKit, while for functional group overlap we selected the recently published definition and implementation suggested by Ertl5. For each structural level, we identified the unique structures belonging to each dataset as well as those belonging to two or three of them.\n\nFor the functional group content analysis we selected the algorithm recently described by Ertl5, which is able to identify all functional groups in a molecule based on an iterative marching through its atoms. In short, the proposed algorithm identifies all heteroatoms in a molecule, all atoms connected by multiple bonds as well as the atoms in oxirane, aziridine, and thiirane rings. Afterwards, all connected atoms are joined together to form a functional group. Single aromatic heteroatoms are retained only if they are connected to an additional aliphatic functionality. Finally, a generalization scheme is applied in which for a defined list of common FGs, information about the parent carbon is retained (e.g. to differentiate between alcohols and phenols) as well as hydrogen atoms (e.g. to differentiate between aldehydes and ketones). The method is fully described in 5. An open source version of this algorithm is available for Python (https://github.com/rdkit/rdkit/tree/master/Contrib/IFG); however, it does not cover the generalization scheme proposed originally. To this end and based on the code available, we implemented with RDKit a fragmentation approach considering the retainment of parent carbons and hydrogens proposed originally, were the remaining carbon atoms are replaced by dummy atoms. This implementation works over a SMILES string and returns a list with the canonical SMILES of the FGs identified in the molecule. The code is freely available at GitHub (https://github.com/DIFACQUIM/IFG_General). After determining the FGs content of the different datasets, we compare the proportion of the most frequent FGs at each library.\n\nIn order to generate a visual representation of the chemical space covered by the analyzed databases, six molecular properties of pharmaceutical interest were computed for each unique compound: averaged molecular weight (AMW) partition coefficient octanol/water (SlogP), number of hydrogen bond donors (HBD), number of hydrogen bond acceptors (HBA), number of rotatable bonds (RB), and topological polar surface area (TPSA). The correlation matrix was computed and a visual representation of the chemical space was generated by using the two properties with the lowest correlation: AMW and SlogP. All calculations were done with RDKit.\n\nThe “global” or total diversity of the datasets was analyzed through the Consensus Diversity (CD) Plot12 that was designed to capture, in a two-dimensional graph, the diversity of chemical libraries considering different and complementary criteria including molecular fingerprints, molecular scaffolds, physicochemical properties, and size. For this work, scaffold diversity was assessed as the area under the cyclic system retrieval curve and the fraction of chemotypes that covers 50% of the dataset (F50). The median of the lower triangle from the pairwise similarity matrix computed as the Tanimoto coefficient of both MACCS keys (166-bits) fingerprint and Morgan fingerprint with radius 2 (Morgan2), were used as molecular fingerprint-based diversity. The mean distance of the lower triangle of the pairwise distance matrix computed as the Euclidean distance of the six molecular properties scaled (mean 0 and unit variance) and the number of compounds in each dataset were selected as measures of the physicochemical properties- and size- based diversity, respectively. The CD Plot was constructed using R.\n\n\nResults and discussion\n\nAs described in the Methods section, the updated BIOFACQUIM database contains the chemical structure of 531 compounds, all collected from Mexico. As with the first version4, each molecule is annotated with information of the chemical structure, the original source of the information (Digital Object Identifier, DOI, to reference paper), kingdom, genus, and species of the plant the natural product was isolated, place of recollection (city and state), and activity value of the reported biological activity. From the original dataset containing 423 compounds, 40 were discarded since they were not collected in Mexico, which means an increase of 148 unique compounds compared to the previous release of the database.\n\nTo assess the chemical space not covered by ChEMBL and NPs but by BIOFACQUIM, we characterized the structural content of the three datasets in terms of unique compounds, scaffolds and functional groups and determined the overlap among them. Figure 1 depicts Venn diagrams showing the overlap among those datasets. It should be noted that despite its small size in comparison with ChEMBL and NPs, 16.1% of compounds present in BIOFACQUIM have not been reported in those other major datasets, as well as 11.3% of its scaffolds and 6.3% of its FGs. Another remarkable observation is the fact that most of the overlap of BIOFACQUIM with the other datasets involves NPs, either alone or in combination with ChEMBL, being 79.7%, 85.5%, and 93.8% for compounds, scaffolds and FGs, respectively.\n\nDatasets content was analyzed in terms of (a) Compounds, (b) Scaffolds and (c) Functional Groups.\n\nA systematic analysis of functional groups was carried out over BIOFACQUIM and the two datasets selected as reference. 80, 4300, and 12,677 functional groups were identified in BIOFACQUIM, NPs and ChEMBL datasets, respectively (the overlap between them is shown in Figure 1). From the total number of functional groups present in each dataset, only 12, 14, and 23 were present in at least 1% of the corresponding library (15.0%, 0.3% and 0.2%, respectively) while 47, 2207, and 5853 (58.7%, 51.3% and 46.17%, respectively) were singletons. This result is consistent with the typical power law observed in other databases6. The most frequent FGs present in BIOFACQUIM are oxygen-containing FGs, being the phenolic hydroxyl group (41.4%), followed by the alcohol hydroxyl group (40.0%), ether (39.8%), alkene (26.6%) and ester (25.8%), which although in a different order, are the most frequent FGs in the herein assembled NPs collection and in other natural product libraries6. This is in contrast to ChEMBL in which only ether is part of the most frequent FGs while the rest of them are nitrogen containing FGs. The complete results of the FGs found of the datasets is included as Extended data (Supplementary File 1). Remarkably, even with the relatively small size of BIOFACQUIM, five compounds with unique FGs were identified, three of them isolated from Mexican plants, two from Salvia ballotiflora (FQNP204 and FQNP211)13 and one from Heterotheca inuloides (FQNP480)14, while the other two correspond to fungal metabolites from Malbranchea aurantiaca (FQNP435)15 isolated from bat guano, and from the endophytic fungus isolated from the Mexican tree Hintonia latiflora, Sporomiella minimoides (FQNP587)16. Figure 2 shows the chemical structure of those compounds.\n\nIdentified functional groups are shown in red.\n\nFor visualization of the chemical space of the datasets compared in this work, we computed six physicochemical properties of pharmaceutical relevance. As described in the Methods section, we computed the correlation matrix between them (Extended data, Table S1 in Supplementary File 2) and since all descriptors were highly correlated (correlation coefficient > 0.60), with the exception of SlogP, we selected this and its lowest correlated property, AMW, as the axes to project the libraries. We also carried out a principal component analysis over the scaled properties. As expected from the correlation matrix, the first principal component was associated with all properties but SlogP and in the opposite way for the second principal component, with explained variances of 69% and 19% respectively. Therefore, the projection of the data over those two components looks quite similar to the selected properties (Slogp and AMW) (Extended data, Figure S1 in Supplementary File 2), with the difference that the principal components do not have the physical interpretation that properties do, e.g. the orally available drugs space as described by Lipinski17. Figure 3 shows a visual representation of the chemical space of the three datasets analyzed in this work. For this plot, in order to better illustrate the unique compounds present in BIOFACQUIM, those compounds belonging to two libraries were assigned to a single one: ChEMBL if they belong to this dataset and NPs if they belong to this dataset but not to ChEMBL. Compounds shared among the three libraries were assigned to a different category. This figure shows that ChEMBL is the most widespread dataset in terms of physicochemical properties and that NPs, BIOFACQUIM, as well as the shared compounds among the three datasets, are mostly contained within this space. It could be noted that a few exceptions from BIOFACQUIM cover a small part of the space currently unexplored by ChEMBL and it is also remarkable that contrary to ChEMBL and NPs, most BIOFACQUIM compounds are part of the orally available drugs space. Different panels showing all different overlaps between datasets can be found in Extended data (Figure S2 in Supplementary File 2).\n\nComparison of BIOFACQUIM with two reference datasets. Axes were truncated to omit the sparsely populated area.\n\nIn order to compare the chemical diversity of the current version of BIOFACQUIM with the previous one and the two datasets selected as reference, we employed a CD plot Figure 4 shows the plot comparing the diversity of all datasets considering four different criteria: scaffolds in the y-axis, molecular fingerprints in the x-axis, physicochemical properties as the filling of the data points in a continuous color scale, and number of compounds as the data points size. This comparison shows the relatively small size of BIOFACQUIM in comparison with the reference datasets. As compared to the previous release of BIOFACQUIM, the current version has increased its diversity in terms of scaffolds and fingerprints but decreased in terms of physicochemical properties. Also, it is shown that its diversity in terms of molecular fingerprints and physicochemical properties, although not the greatest ones of the three datasets, are on the order of those for NPs, contrary to scaffolds, in which is the most diverse.\n\nThe molecular fingerprint diversity of each data set is represented on the x-axis and was defined as the median Tanimoto coefficient of MACCS keys (166-bits) fingerprint. The scaffold diversity of each database is represented on the y-axis and was defined as the area under the corresponding cyclic system retrieval curve. The diversity based on physicochemical properties (PCP) was defined as the mean euclidean distance of six scaled physicochemical properties (SlogP, TPSA, AMW, RB, HBD, and HBA) and is shown as the filling of the data points using a continuous color scale. The number of compounds is represented by the size of the data points.\n\n\nConclusions\n\nThe current version of BIOFACQUIM involved the addition of 148 natural products. This was reflected in a diversity increase based on both scaffolds and molecular fingerprints. It was shown that in terms of diversity and structural content overlap, BIOFACQUIM is more similar to the assembled set of natural products than to the set of biologically tested compounds. The herein reported chemoinformatic study revealed that most of the compounds contained in BIOFACQUIM are focused in the orally active drugs space in terms of physicochemical properties. Interestingly, despite the fact of its relative small size, there were identified a significant number of compounds, scaffolds and functional groups (81, 28 and 5, respectively) that were not present in the two large sets used as reference, showing that curated databases of natural products, such as BIOFACQUIM, can serve as a starting point for the study and increase of the biologically relevant chemical space.\n\n\nData availability\n\nFigshare: BIOFACQUIM_V2. http://doi.org/10.6084/m9.figshare.11312702\n\nThis file contains the chemical structures of 531 compounds in SDF format, alongside ID number, compound name, simplified molecular input line entry system, literature reference, kingdom, genus, species, geographical location and biological activity.\n\nUnderlying data are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).\n\nFigshare: Supporting information for \"Functional group and diversity analysis of BIOFACQUIM: A Mexican natural product database\". http://doi.org/10.6084/m9.figshare.11312735\n\nThis project contains the following extended data:\n\nSupplementary File 1. File with summary results of the functional group analysis.\n\nSupplementary File 2. This file contains the following supporting tables and figures:\n\nTable S1. Correlation matrix for the six PCP computed for BIOFACQUIM and the two reference datasets.\n\nFigure S1. Visual representation of the chemical space covered by the three compound libraries generated by principal component analysis.\n\nFigure S2. Visual representation of the chemical space covered by the possible overlaps between libraries.\n\nExtended data are available under the terms of the Creative Commons Attribution 4.0 International License (CC BY 4.0).",
"appendix": "Acknowledgments\n\nWe thank Roberto Hernández for his valuable contribution toward the update of BIOFACQUIM.\n\n\nReferences\n\nKinghorn AD, Falk H, Gibbons S, et al.: eds. Progress in the Chemistry of Organic Natural Products 110: Cheminformatics in Natural Product Research. Cham: Springer International Publishing. 2019; 110. Publisher Full Text\n\nMedina-Franco JL: New Approaches for the Discovery of Pharmacologically-Active Natural Compounds. Biomolecules. 2019; 9(3): pii: E115. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChen Y, Garcia de Lomana M, Friedrich NO, et al.: Characterization of the Chemical Space of Known and Readily Obtainable Natural Products. J Chem Inf Model. 2018; 58(8): 1518–1532. PubMed Abstract | Publisher Full Text\n\nPilón-Jiménez BA, Saldívar-González FI, Díaz-Eufracio BI, et al.: BIOFACQUIM: A mexican compound database of natural products. Biomolecules. 2019; 9(1): pii: E31. PubMed Abstract | Publisher Full Text | Free Full Text\n\nErtl P: An algorithm to identify functional groups in organic molecules. J Cheminform. 2017; 9(1): 36. PubMed Abstract | Publisher Full Text | Free Full Text\n\nErtl P, Schuhmann T: A Systematic Cheminformatics Analysis of Functional Groups Occurring in Natural Products. J Nat Prod. 2019; 82(5): 1258–1263. PubMed Abstract | Publisher Full Text\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\nGu J, Gui Y, Chen L, et al.: Use of natural products as chemical library for drug discovery and network pharmacology. PLoS One. 2013; 8(4): e62839. PubMed Abstract | Publisher Full Text | Free Full Text\n\nvan Santen JA, Jacob G, Singh AL, et al.: The natural products atlas: an open access knowledge base for microbial natural products discovery. ACS Cent Sci. 2019; 5(11): 1824–1833. Publisher Full Text\n\nMing H, Tiejun C, Yanli W, et al.: Web search and data mining of natural products and their bioactivities in PubChem. Sci China Chem. 2013; 56(10): 1424–1435. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBemis GW, Murcko MA: The properties of known drugs. 1. Molecular frameworks. J Med Chem. 1996; 39(15): 2887–2893. PubMed Abstract | Publisher Full Text\n\nGonzález-Medina M, Prieto-Martínez FD, Owen JR, et al.: Consensus Diversity Plots: a global diversity analysis of chemical libraries. J Cheminform. 2016; 8: 63. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEsquivel B, Bustos-Brito C, Sánchez-Castellanos M, et al.: Structure, Absolute Configuration, and Antiproliferative Activity of Abietane and Icetexane Diterpenoids from Salvia ballotiflora. Molecules. 2017; 22(10): pii: E1690. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDelgado G, del Socorro Olivares M, Chávez MI, et al.: Antiinflammatory constituents from Heterotheca inuloides. J Nat Prod. 2001; 64(7): 861–864. PubMed Abstract | Publisher Full Text\n\nMartínez-Luis S, González MC, Ulloa M, et al.: Phytotoxins from the fungus Malbranchea aurantiaca. Phytochemistry. 2005; 66(9): 1012–1016. PubMed Abstract | Publisher Full Text\n\nLeyte-Lugo M, Figueroa M, González Mdel C, et al.: Metabolites from the endophytic [corrected] fungus Sporormiella minimoides isolated from Hintonia latiflora. Phytochemistry. 2013; 96: 273–278. PubMed Abstract | Publisher Full Text\n\nLipinski CA, Lombardo F, Dominy BW, et al.: Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv Drug Deliv Rev. 2001; 46(1–3): 3–26. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "57653",
"date": "13 Dec 2019",
"name": "Johannes Kirchmair",
"expertise": [
"Reviewer Expertise cheminformatics",
"natural products research"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSánchez-Cruz et al. report on the diversity of an extended version of BIOFACQUIM, a database of natural products extracted from organisms endemic to Mexico.\nThe overall scientific approach is sound. The only technical issue that I believe requires attention is tautomerism, which appears to have been neglected. Consideration of tautomerism will unlikely change the outcomes and conclusions of the statistical analysis but for the functional group analysis it will be important to understand whether the five new functional groups from BIOFACQUIM presented in Figure 2 are indeed unique to that source. Could the authors please use InChI, MolVS or a similar notation/approach for standardizing the molecular (sub-) structures and conduct a tautomer-invariant identification of functional groups?\nLanguage issues are apparent throughout the manuscript and careful proofreading will be required. Some (parts of) statements are unclear, e.g. \"it was reported an initial scaffold content\" \"the articles should have described the procedure\" \"decided to augment the number of Mexican institutions\" \"A similar procedure can be done using RDKit\"\nMinor comment: Could the authors please clarify in the manuscript the sources of the natural products included in BIOFACQUIM (e.g. plants, bacteria, fungi)?\n\nIs the work 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": "5563",
"date": "16 Jun 2020",
"name": "José L. Medina-Franco",
"role": "Author Response",
"response": "The overall scientific approach is sound. The only technical issue that I believe requires attention is tautomerism, which appears to have been neglected. Consideration of tautomerism will unlikely change the outcomes and conclusions of the statistical analysis but for the functional group analysis it will be important to understand whether the five new functional groups from BIOFACQUIM presented in Figure 2 are indeed unique to that source. Could the authors please use InChI, MolVS or a similar notation/approach for standardizing the molecular (sub-) structures and conduct a tautomer-invariant identification of functional groups? RESPONSE: Following the recommendation, we repeated the analysis reported in the manuscript standardizing the structures of all databases using MolVS. The standardization procedure was described in detail in the revised “Methods, Databases and curation” section and the code was made available at GitHub (https://github.com/DIFACQUIM/IFG_General). All the quantities associated with the new and correct number of unique molecules were revised and the main text and figures were updated accordingly (including figure 2). Language issues are apparent throughout the manuscript and careful proofreading will be required. Some (parts of) statements are unclear, e.g. \"it was reported an initial scaffold content\" \"the articles should have described the procedure\" \"decided to augment the number of Mexican institutions\" \"A similar procedure can be done using RDKit\" RESPONSE: The statements pointed out were fixed. We also proofread carefully the second version of the manuscript. Minor comment: Could the authors please clarify in the manuscript the sources of the natural products included in BIOFACQUIM (e.g. plants, bacteria, fungi)? RESPONSE: In the revised “Results and discussion, Update of BIOFACQUIM section” we included the number of compounds from each source (“…406 from plants, 97 from fungus, 15 from propolis and 13 from marine animals….”)."
}
]
},
{
"id": "57650",
"date": "24 Jan 2020",
"name": "W. Patrick Walters",
"expertise": [
"Reviewer Expertise Computational Chemistry",
"Cheminformatics",
"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\nThis paper describes an analysis of the BIOFACQUIM database of natural products derived from Mexico. The paper is well written and provides a useful overview of a resource that will be of use to medicinal and computational chemists, as well as those involved in research on natural products. The authors should be commended for making their database and associated code available as Open Source.\n\nWhile the paper is well written, there are a few areas where changes in the text may make the presentation a bit more clear:\nThe second sentence in the abstract could be changed to read: “An analysis of its structural content, as well as functional group occurrence, provides a useful overview, as well as a means of comparison with related databases.\"\n\nIn the first sentence of the “Methods” section, the authors may want to consider changing “done” to “performed”.\n\nIn the Introduction, the sentence “As part of that work, it was reported an initial scaffold content, and chemical space diversity, and coverage” could be changed to “As part of that work, scaffold content and chemical space diversity were examined”.\n\nIn the first paragraph of the “Results and discussion” section, “place of recollection” would be better stated as “place of collection”.\n\nIn the methods section, the authors detail their procedure for “cleaning” the databases. It would be useful to others if the source code for these procedures was made available as part of the GitHub repository associated with this paper.\n\nThe authors used canonical SMILES to identify exact matches between compounds in different databases. While this is a reasonable approach, canonical SMILES do not standardize tautomers and may miss some duplicates. An alternate approach would be to use InChI keys, which canonicalize tautomeric forms.\n\nThe description of the “Consensus Diversity (CD) Plot” in the “Global diversity” section is a bit vague. An additional paragraph describing this method would be useful.\n\nThe density of points in Figure 3 obscures many of the points in the plot. The authors may want to consider using a plotting technique like hexagon binning which enables the plotting of large datasets without obscuring points. For example, see these links:\n\nhttps://datavizproject.com/data-type/hexagonal-binning/\n\nhttps://rdrr.io/cran/som.nn/man/hexbinpie.html\n\nI’m not convinced that plotting molecular weight vs LogP is the best way to represent the diversity and overlap of datasets. An alternative might be to use a technique like t-SNE to project a set of fingerprints into a lower dimensional space: https://scikit-learn.org/stable/modules/generated/sklearn.manifold.TSNE.html.\nIt might also be interesting to provide another plot which only shows the molecules from the three datasets studied that are in “drug-like” chemical space rather than the very large range shown in Figure 3.\n\nIn summary, this paper provides a valuable resource to researchers working in a number of different areas. Hopefully, these suggestions will provide minor enhancements to the work.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "5564",
"date": "16 Jun 2020",
"name": "José L. Medina-Franco",
"role": "Author Response",
"response": "This paper describes an analysis of the BIOFACQUIM database of natural products derived from Mexico. The paper is well written and provides a useful overview of a resource that will be of use to medicinal and computational chemists, as well as those involved in research on natural products. The authors should be commended for making their database and associated code available as Open Source. While the paper is well written, there are a few areas where changes in the text may make the presentation a bit more clear:RESPONSE: Thank you for the feedback and constructive comments. In the second version of the manuscript we incorporated all your suggestions as described hereunder. The second sentence in the abstract could be changed to read: “An analysis of its structural content, as well as functional group occurrence, provides a useful overview, as well as a means of comparison with related databases.\" In the first sentence of the “Methods” section, the authors may want to consider changing “done” to “performed”. In the Introduction, the sentence “As part of that work, it was reported an initial scaffold content, and chemical space diversity, and coverage” could be changed to “As part of that work, scaffold content and chemical space diversity were examined”. In the first paragraph of the “Results and discussion” section, “place of recollection” would be better stated as “place of collection”. RESPONSE: All four changes in the text were done. In the methods section, the authors detail their procedure for “cleaning” the databases. It would be useful to others if the source code for these procedures was made available as part of the GitHub repository associated with this paper.RESPONSE: In agreement with the suggestion, the code to prepare the databases was made freely available. In the revised version of the manuscript “Methods, Databases and data curation section” we included the sentence: “The code is available at GitHub (https://github.com/DIFACQUIM/IFG_General).” The authors used canonical SMILES to identify exact matches between compounds in different databases. While this is a reasonable approach, canonical SMILES do not standardize tautomers and may miss some duplicates. An alternate approach would be to use InChI keys, which canonicalize tautomeric forms.RESPONSE: To address this point we repeated the analysis standardizing the structures using MolVS that does consider tautomers. The standardization procedure was described in detail in the revised “Methods, Databases and curation” section. All the quantities associated with the new and correct number of unique molecules were revised and the main text and figures were updated accordingly. The description of the “Consensus Diversity (CD) Plot” in the “Global diversity” section is a bit vague. An additional paragraph describing this method would be useful.RESPONSE: We described in more detail the Consensus Diversity (CD) Plots. The density of points in Figure 3 obscures many of the points in the plot. The authors may want to consider using a plotting technique like hexagon binning which enables the plotting of large datasets without obscuring points. For example, see these links:https://datavizproject.com/data-type/hexagonal-binning/https://rdrr.io/cran/som.nn/man/hexbinpie.htmlRESPONSE: To address the reviewer’s point, we used the recently published visualization method TMAP (Tree Manifold Approximation and Projection) (J. Cheminform. 2020, 12, 12.). This approach is suited for visualization of very large data sets (up to millions of data points with high dimensionality). Figure 3 was replaced with a TMAP where the 3 data sets are shown in separate panels and a heatmap in the form of hexbin plot for the distribution of compounds is included (Figure 4 in the revised manuscript). A description of TMAP was added to the Methods section citing the original work and the one generated is discussed accordingly in the Results and Discussion section. I’m not convinced that plotting molecular weight vs LogP is the best way to represent the diversity and overlap of datasets. An alternative might be to use a technique like t-SNE to project a set of fingerprints into a lower dimensional space:https://scikit-learn.org/stable/modules/generated/sklearn.manifold.TSNE.html.RESPONSE: Following the recommendation, we now compare the three data sets using TMAPs. For the description of compounds, we selected Morgan fingerprints with radius 2. It might also be interesting to provide another plot which only shows the molecules from the three datasets studied that are in “drug-like” chemical space rather than the very large range shown in Figure 3.RESPONSE: In agreement with the suggestion, in the newly added TMAPs (Figure 4 in the revised manuscript) we included a visualization of drug-like subsets of the three databases."
}
]
},
{
"id": "58743",
"date": "30 Jan 2020",
"name": "Trong D. Tran",
"expertise": [
"Reviewer Expertise Natural product chemistry",
"Drug Discovery."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 Sánchez-Cruz et al. describes the cheminformatic analysis of the updated BIOFACQUIM database consisting of 531 Mexican natural products. Despite its relatively small size, the results supported the potential of using BIOFACQUIM as a good source for virtual screening in drug discovery. Generally, the study was well designed, and the paper was well-written. However, since stereochemistry plays a significant role in the interactions between small molecules and protein targets, I suggest the authors should add the analysis of the fraction of sp3-hydridized atoms for the new BIOFACQUIM version and compare that value with those of other databases to assess its 3D unique. Can authors detail the “eight indexed journals” which was referred in the “Methods - Databases and data curation”? All in all, I feel this manuscript is suitable for indexing.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "5565",
"date": "16 Jun 2020",
"name": "José L. Medina-Franco",
"role": "Author Response",
"response": "The manuscript by Sánchez-Cruz et al. describes the cheminformatic analysis of the updated BIOFACQUIM database consisting of 531 Mexican natural products. Despite its relatively small size, the results supported the potential of using BIOFACQUIM as a good source for virtual screening in drug discovery. Generally, the study was well designed, and the paper was well-written. However, since stereochemistry plays a significant role in the interactions between small molecules and protein targets, I suggest the authors should add the analysis of the fraction of sp -hydridized atoms for the new BIOFACQUIM version and compare that value with those of other databases to assess its 3D unique. RESPONSE: We thank the reviewer for the suggestion. In the revised “Methods” and “Results and discussions”, we added a new section “Complexity analysis” to compare the distribution of the fraction of carbon atoms that are sp3 hybridized. A new figure (3 in the revised version) was added. Can authors detail the “eight indexed journals” which was referred in the “Methods - Databases and data curation”? RESPONSE: The names of the 8 journals were added: “Journal of Ethnopharmacology, Natural Products Research, Journal of Agricultural and Food Chemistry, Journal of Natural Products, Planta Medica, Phytochemistry, Natural Product Letters, and Molecules.”"
}
]
}
] | 1
|
https://f1000research.com/articles/8-2071
|
https://f1000research.com/articles/9-210/v1
|
25 Mar 20
|
{
"type": "Study Protocol",
"title": "Data extraction methods for systematic review (semi)automation: A living review protocol",
"authors": [
"Lena Schmidt",
"Babatunde K. Olorisade",
"Luke A. McGuinness",
"Julian P. T. Higgins",
"Babatunde K. Olorisade",
"Luke A. McGuinness",
"Julian P. T. Higgins"
],
"abstract": "Background: Researchers in evidence-based medicine cannot keep up with the amounts of both old and newly published primary research articles. Conducting and updating of systematic reviews is time-consuming. In practice, data extraction is one of the most complex tasks in this process. Exponential improvements in computational processing speed and data storage are fostering the development of data extraction models and algorithms. This, in combination with quicker pathways to publication, led to a large landscape of tools and methods for data extraction tasks. Objective: To review published methods and tools for data extraction to (semi)automate the systematic reviewing process. Methods: We propose to conduct a living review. With this methodology we aim to do monthly search updates, as well as bi-annual review updates if new evidence permits it. In a cross-sectional analysis we will extract methodological characteristics and assess the quality of reporting in our included papers. Conclusions: We aim to increase transparency in the reporting and assessment of machine learning technologies to the benefit of data scientists, systematic reviewers and funders of health research. This living review will help to reduce duplicate efforts by data scientists who develop data extraction methods. It will also serve to inform systematic reviewers about possibilities to support their data extraction.",
"keywords": [
"Data Extraction",
"Natural Language Processing",
"Reproducibility",
"Systematic reviews",
"Text mining"
],
"content": "Introduction\n\nResearch on systematic review (semi)automation sits at the interface between evidence-based medicine and data science. The capacity of computers for supporting humans increases, along with the development of processing power and storage space. Data extraction for systematic reviewing is a repetitive task. This opens opportunities for support through intelligent software. Tools and methods in this domain frequently focused on automatic processing of information related to the PICO framework (Population, Intervention, Comparator, Outcome). A 2017 analysis of 195 systematic reviews investigated the workload associated with authoring a review. On average, the analysed reviews took 67 weeks to write and publish. Although review size and the number of authors varied between the analysed reviews, the authors concluded that supporting the reviewing process with technological means is important in order to save thousands of personal working hours of trained and specialised staff1. The potential workload for systematic reviewers is increasing, because the evidence base of clinical studies that can be reviewed is growing rapidly (Figure 1). This entails not only a need to publish new reviews, but also to commit to them and to continually keep the evidence up to date.\n\nLanguage processing toolkits and machine learning libraries are well documented and available to use free of charge. At the same time, freely available training data make it easy to train classic machine-learning classifiers such as support vector machines, or even complex, deep neural networks such as long short-term memory (LSTM) neural networks. These are reasons why health data science, much like the rest of computer science and natural language processing, is a rapidly developing field. There is a need for fast publication, because trends and state-of-the-art methods are changing at a fast pace. Preprint repositories, such as the arXiv, are offering near rapid publication after a short moderation process rather than full peer review. Consequently, publishing research is becoming easier.\n\nAn easily updatable review of available methods and tools is needed to inform systematic reviewers, data scientists or their funders alike on the status quo of (semi)automated data extraction methodology. For data scientists, it contributes towards reducing waste and duplication in research. For reviewers, it contributes towards highlighting the current possibilities for data extraction and empowering them to choose the right tools for their tasks in order to work more efficiently. Systematic reviewers are free to use any published tool that is available to them and need sufficient information to make informed decisions about which tools are to be preferred. Therefore, our proposed continuous analysis of the available tools will not only include the final scores that a model achieves, but it will also assess dimensions such as transparency of methods, reproducibility, and how these items are reported. Reported pitfalls of applying health data science methods to systematic reviewing tasks will be summarised to highlight risks that current, as well as future, systems are facing. Reviewing the available literature on systematic review automation is one of many small steps towards supporting evidence synthesis of all available medical research data. If the evidence arising from a study is never reviewed, and as a result never noticed by policy makers and providers of care, then it counts towards waste in research.\n\nThis review aims to:\n\n1. Review published methods and tools for PICO data extraction to (semi)automate the systematic reviewing process.\n\n2. Review this evidence in the scope of a living review. To keep information up to date and relevant to the challenges faced by systematic reviewers at any time.\n\nWe have identified three publications involving reviews of tools and methods, a document providing overviews and guidelines relevant to our topic, and an ongoing effort to characterise published tools for different parts of the systematic reviewing process with respect to interoperability and workflow integration. In 2014, Tsafnat et al.2 provided a broad overview on automation technologies for different stages of authoring a systematic review.\n\nA systematic review focusing on text-mining approaches was published in 2015. It includes a summary of methods for the evaluation of systems (such as recall, F1 and related scores). The reviewers focused on tasks related to PICO classification and supporting the screening process3.\n\nA further review of the same year also described methods for data extraction, focusing on PICOs and related fields4.\n\nThese reviews present an overview of classical machine learning methods applied to tasks such as data mining in the field of evidence-based medicine. At the time of publication of these documents, methods such as topic modelling (Latent Dirichlet Allocation) and support vector machines constituted the state-of-the art for language models. The age of these documents means that the latest static or contextual embedding-based and neural methods are not included. These modern methods, however, are used in contemporary systematic review automation software5.\n\nBeller et al.6 present a brief overview of tools for systematic review automation. They discuss principles for systematic review automation from a meeting of the International Collaboration for the Automation of Systematic Reviews (ICASR). They highlight that low levels of funding, as well as the complexity of integrating tools for different systematic reviewing tasks have led to many small and isolated pieces of software. A working group formed at the ICASR 2019 Hackathon is compiling an overview of tools published on the Systematic Review Toolbox website7. This ongoing work is focused on assessing maintenance status, accessibility and supported reviewing tasks of 120 tools that can be used in any part of the systematic reviewing process as of November 2019.\n\n\nProtocol\n\nWe registered this protocol via OSF (https://doi.org/10.17605/OSF.IO/ECB3T). PROSPERO was initially considered as platform for registration, but it is limited to reviews with health related outcomes.\n\nThe challenges highlighted in the previous section create several problems. A large variety of approaches and different means of expressing results creates uncertainty in the existing evidence. At the same time, new evidence is likely to emerge. Rapid means of publications necessitate a structured, but at the same time easily updatable review of published methods and tools in the field. We therefore chose a living review approach as the updating strategy for this review.\n\nFor literature searches and updates we follow the living review recommendations published by Elliott et al.8 and Brooker et al.9, as well as F1000Research guidelines for projects that are included in their living evidence collection. We plan to run searches for new studies every second month. This will also include screening abstracts of the newly retrieved reports. The review itself will be updated every six months, providing that a sufficient amount of new records are identified for inclusion. As a threshold for updating, we plan to use 10 new records, but we will consider updating the review earlier if new impactful evidence is published. Figure 2 describes the anticipated reviewing process in more detail.\n\nOur search strategy was developed with the help of an information specialist. Due to the interdisciplinary topic of this review, we plan to search bibliographic databases related to both medicine and computer science. These include Medline via Ovid and Web of Science, as well as the computer science arXiv and the DBLP computer science bibliography. We aim to retrieve publications related to two clusters of search terms. The first cluster includes computational aspects such as data mining, while the second cluster identifies publication related to systematic reviews. The Medline search strategy is provided as Extended data10. We aim to adapt this search strategy for conducting searches in all mentioned databases. Previous reviews of data mining in systematic reviewing contexts identified the earliest text mining application in 20053,4. We therefore plan to search all databases from this year on. In a preliminary test our search strategy was able to identify 4320 Medline records, including all Medline-indexed records included by O’Mara-Eves et al.3. We plan to search the Systematic Review Toolbox website for further information on any published or unpublished tools7.\n\nAll titles and abstracts will be screened independently by two reviewers. Any differences in judgement will be discussed, and resolved with the help of a third reviewer if necessary. The process for assessing full texts will be the same. Data extraction will be carried out by single reviewers, and random 10% samples from each reviewer will be checked independently. If needed, we plan to contact the authors of reports for clarification or further information. In the base review, as well as in every published update, we will present a cross-sectional analysis of the evidence from our searches. This analysis will include the characteristics of each reviewed method or tool, as well as a summary of our findings. Secondly, we will assess the quality of reporting at publication level. This assessment will focus on transparency, reproducibility and both internal and external validity of the described data extraction algorithms. If we at any point deviate from this protocol, we will discuss this in the final publication.\n\nAll search results will be de-duplicated and managed with EndNote. The screening and data extraction process will be managed with the help of Abstrackr11 and customised data extraction forms in Excel. All data, including bi-monthly screening results, will be continuously available on our Open ScienceFramework (OSF) repository, as discussed in the Data availability section.\n\nTsafnat et al.2 categorised sub-tasks in the systematic reviewing process that contained published tools and methods for automation. In our overview, we follow this categorisation and focus on tasks related to data retrieval. More specifically, we will focus on software architectures that receive as input a set of full texts or abstracts of reports. Report types of interest are randomised controlled trials, cohort, or case-control studies. As output, the tools of interest should produce structured data representing features or findings from the study described. A comprehensive list with data fields of interest can be found in the supplementary material for this protocol.\n\nObjective 1: to review published methods for data mining and text classification approaches from the data science perspective. This aims at reducing duplicate efforts and encouraging comparability of published methods.\n\nObjective 2: to highlight contributions of data extraction technologies from the perspective of systematic reviewers who wish to use (semi)automation for data extraction. What is the extent of automation, and is it reliable? Can we identify important caveats discussed in the literature?\n\nIncluded papers\n\nAny full text publication that describes an original natural language processing, machine learning or data mining approach to extract data related to systematic reviewing tasks. Data fields of interest are adapted from the Cochrane Handbook for Systematic Reviews of Interventions12, and defined in the Extended data10.\n\nWe will include papers describing a full cycle of implementation and evaluation of a method.\n\nWe include reports published from 2005 until the present day, similar to O’Mara-Eves et al.3 and Jonnalagadda et al.4. We will translate non-English reports where feasible.\n\nThe data that these methods work with will be reports of randomised controlled trials, cohort or case control studies in the form of abstracts, conference proceedings or full texts.\n\nExcluded papers\n\nMethods and tools related solely to image processing and importing biomedical data from PDF files. This includes data extraction from graphs.\n\nAny research that focuses exclusively on protocol preparation, synthesis of already extracted data, write-up, pre-processing of text and dissemination will be excluded.\n\nMethods or tools that provide no natural language processing approach and offer only organisational interfaces, document management, databases or version control.\n\nAny publications related solely to electronic health reports or data mining genetics data will be excluded.\n\nPrimary:\n\n1. Machine learning approaches used\n\n2. Metrics used for reporting results\n\n3. Type of data\n\n• Scope: Abstract, conference proceeding, or full text\n\n• Target design: Randomised controlled trial, cohort, case-control\n\n• Type of input: The input data format, for example data imported as structured result of literature search (e.g. RIS), API, or in the form of text files.\n\n• Type of output: In which format are data exported after the extraction, for example as text file.\n\nSecondary:\n\n1. Granularity of data extraction: Does the system extract specific entities, sentences, or larger parts of text?\n\n2. Outcomes as defined by paper, for example time saved during screening.\n\nAssessment of the quality of reporting: We will extract information related to the quality of reporting and reproducibility of methods in text mining13. The domains of interest, adapted for our reviewing task, are listed in the following.\n\n1. Reproducibility:\n\n• Are the sources for training/testing data reported?\n\n• If pre-processing techniques were applied to the data, are they described?\n\n2. Transparency of methods:\n\n• Is there a description of the algorithms used?\n\n• Is there a description of the dataset used and of its characteristics?\n\n• Is there a description of the hardware used?\n\n• Is the source code available?\n\n3. Testing:\n\n• Is there a justification/an explanation of the model assessment?\n\n• Are basic metrics reported (true/false positives and negatives)?\n\n• Does the assessment include any information about trade-offs between recall and precision (also known as sensitivity and positive predictive value)?\n\n4. Availability of the final model or tool:\n\n• Can we obtain a runnable version of the software based on the information in the publication?\n\n• Persistence: is the dataset likely to be available for future use?\n\nIs the use of third-party frameworks reported and are they accessible?\n\n5. Internal and external validity of the model:\n\n• Does the dataset or assessment measure provide a possibility to compare to other tools in same domain?\n\n• Are explanations for the influence of both visible and hidden variables in the dataset given?\n\n• Is the process of avoiding over- or underfitting described?\n\n• Is the process of splitting training from validation data described?\n\n• Is the model’s adaptability to different formats and/or environments beyond training and testing data described?\n\n6. Other:\n\n• Does the paper describe caveats for using the method?\n\n• Are sources of funding described?\n\n• Are conflicts of interest reported?\n\nWe plan to publish the finished review, along with future updates, via F1000Research.\n\nAll data will be available via a project on Open Science Framework (OSF): https://osf.io/4sgfz/ (see Data availability).\n\nProtocol published. We did a preliminary Medline search as described in this protocol and the supplementary material. The final search, including all additional databases, will be conducted as part of the full review.\n\n\nData availability\n\nNo underlying data are associated with this article.\n\nOpen Science Framework: Data Extraction Methods for Systematic Review (semi)Automation: A Living Review / Protocol. https://doi.org/10.17605/OSF.IO/ECB3T10\n\nThis project contains the following extended data:\n\nAdditional_Fields.docx (overview of data fields of interest for text mining in clinical trials)\n\nSearch.docx (additional information about the searches, including full search strategies)\n\nOpen Science Framework: Data Extraction Methods for Systematic Review (semi)Automation: A Living Review / Protocol. https://doi.org/10.17605/OSF.IO/ECB3T10\n\nData are available under the terms of the Creative Commons Attribution 4.0 International (CC BY 4.0) data waiver.",
"appendix": "Acknowledgements\n\nWe thank Sarah Dawson for developing and evaluating the search strategy, and providing advice on databases to search for this review. Many thanks also to Alexandra McAleenan and Vincent Cheng for providing valuable feedback for this protocol.\n\n\nReferences\n\nBorah R, Brown AW, Capers PL, et al.: Analysis of the time and workers needed to conduct systematic reviews of medical interventions using data from the PROSPERO registry. BMJ Open. 2017; 7(2): e012545. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTsafnat G, Glasziou P, Choong MK, et al.: Systematic review automation technologies. Syst Rev. 2014; 3(1): 74. PubMed Abstract | Publisher Full Text | Free Full Text\n\nO'Mara-Eves A, Thomas J, McNaught J, et al.: Using text mining for study identification in systematic reviews: a systematic review of current approaches. Syst Rev. 2015; 4: 5. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJonnalagadda SR, Goyal P, Huffman MD: Automating data extraction in systematic reviews: a systematic review. Syst Rev. 2015; 4: 78. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMarshall I, Kuiper J, Wallace B: Robotreviewer on github. 2020; Last accessed 14 Jan 2020. Reference Source\n\nBeller B, Clark J, Tsafnat G, et al.: Making progress with the automation of systematic reviews: principles of the International Collaboration for the Automation of Systematic Reviews (ICASR). Syst Rev. 2018; 7(1): 77. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMarshall C: The systematic review toolbox. 2019; Last accessed 11 Nov 2019. Reference Source\n\nElliott JH, Synnot A, Turner T, et al.: Living systematic review: 1. Introduction-the why, what, when, and how. J Clin Epidemiol. 2017; 91: 23–30. PubMed Abstract | Publisher Full Text\n\nBrooker A, Synnot A, McDonald S, et al.: Guidance for the production and publication of cochrane living systematic reviews: Cochrane reviews in living mode. 2019; Last accessed 06 Mar 2020. Reference Source\n\nSchmidt L, McGuinness LA, Olorisade BK, et al.: Protocol. 2020. https://doi.org/10.17605/OSF.IO/ECB3T\n\nWallace BC, Small K, Brodley CE, et al.: Deploying an interactive machine learning system in an evidence-based practice center: Abstrackr. In Proceedings of the 2nd ACM SIGHIT International Health Informatics Symposium. IHI’12, New York, NY USA, Association for Computing Machinery. 2012; 819–824. Publisher Full Text\n\nHiggins J, Thomas J, Chandler J, et al.: Cochrane Handbook for Systematic Reviews of Interventions. John Wiley & Sons, Chichester (UK), 2nd edition, 2019. Reference Source\n\nOlorisade BK, Brereton P, Andras P: Reproducibility of studies on text mining for citation screening in systematic reviews: Evaluation and checklist. J Biomed Inform. 2017; 73: 1–13. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "61722",
"date": "01 May 2020",
"name": "Emma McFarlane",
"expertise": [
"Reviewer Expertise I am currently conducting a systematic review of automation in systematic reviewing or guideline development. I am also working on a research project on machine learning within the context of guideline recommendations."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis protocol outlines a project to review methods and tools for data extraction to help automate a step in the systematic reviewing process. This will be done in the context of a living systematic review with the aim of providing guidance to reviewers who may want to semi-automate their work.\nThe objectives of the study are described clearly and are set within the current context of increasing publications. However, it would be helpful, as part of the aim and purpose of the study, to note why the focus is around the data extraction stage specifically.\nThe methods of the study are described in enough detail to be replicated. In terms of the methods, can the authors double check the searching approach for accuracy? The abstract notes searches will be conducted monthly whereas the body of the protocol states every two months. The authors indicated they will update their systematic review when evidence expected to impact is identified however, it would be helpful to include some detail to note how impactful evidence will be defined for people doing similar work.\n\nA comment from the authors about their choice of study design for the included papers would be helpful. Identifying RCTs in the context of data science is likely to be challenging so it would be interesting to understand the expectations about the evidence base and how study design could link to the point about impact on the results of the review and need to update.\nThe outcomes listed in the protocol appear to be comprehensive. However, it is not clear if consideration was given to accuracy of the tools identified as that could link to the objective of identifying whether automation is reliable.\n\nOverall, this appears to be an interesting study straddling the fields of systematic reviewing and data science.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable",
"responses": [
{
"c_id": "5557",
"date": "08 Jun 2020",
"name": "Lena Schmidt",
"role": "Author Response",
"response": "Thank you for providing this very helpful peer review. We tried to address the concerns below: “This protocol outlines a project to review methods and tools for data extraction to help automate a step in the systematic reviewing process. This will be done in the context of a living systematic review with the aim of providing guidance to reviewers who may want to semi-automate their work.” “The objectives of the study are described clearly and are set within the current context of increasing publications. However, it would be helpful, as part of the aim and purpose of the study, to note why the focus is around the data extraction stage specifically.” Thank you for pointing this out. In the current revision we added more details about why we chose to focus on data extraction. To summarise this quickly, data extraction is one of the most time-consuming and error-prone tasks in the systematic reviewing process. By reviewing automation of data extraction, we aim to summarise the current knowledge. Furthermore, the area of data extraction has future potential to disrupt the “traditional” systematic review process – if data are extracted and well classified centrally then the searching and screening workflow can change as well. “The methods of the study are described in enough detail to be replicated. In terms of the methods, can the authors double check the searching approach for accuracy? The abstract notes searches will be conducted monthly whereas the body of the protocol states every two months. “ We addressed this, thank you. New articles will be screened every two months. “The authors indicated they will update their systematic review when evidence expected to impact is identified however, it would be helpful to include some detail to note how impactful evidence will be defined for people doing similar work.“ Thank you, this was not clear previously, and we added some further explanation: “We define impactful evidence as, for example, the publication of a tool that is immediately accessible to systematic reviewers and offers substantial automation of the data extraction process, or a tool that aims to change the traditional SR workflow” “A comment from the authors about their choice of study design for the included papers would be helpful. Identifying RCTs in the context of data science is likely to be challenging so it would be interesting to understand the expectations about the evidence base and how study design could link to the point about impact on the results of the review and need to update.” We clarified that we are open to include any paper, as long as the paper reports an automation technology for systematic reviewing that processes texts from clinical studies (not electronic health reports). To clarify further, the texts that the technologies process are likely to be RCT reports, but we are not looking to identify RCTs that compare data extraction tools. “The outcomes listed in the protocol appear to be comprehensive. However, it is not clear if consideration was given to accuracy of the tools identified as that could link to the objective of identifying whether automation is reliable.“ Thank you for this comment. In response we clarified that we will extract the reported performance metrics. We do not plan to rank tools based on these metrics because results can vary throughout different datasets. Instead, we aim to assess quality of reporting (reproducibility, transparency…) in detail. “Overall, this appears to be an interesting study straddling the fields of systematic reviewing and data science.” Thank you very much for your review, it was very helpful in improving the protocol."
}
]
},
{
"id": "62963",
"date": "18 May 2020",
"name": "Matt Carter",
"expertise": [
"Reviewer Expertise Automation of Systematic Reviews"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI believe that the research proposal clearly lays out its objectives and aims. Although there are some minor edits as per the below list:\nThe abstract should specifically mention PICO data extraction rather than data extraction generally. The \"Aims\" section outlines this but seems to contradict with the more general \"Introduction / Objective\" section.\n\nWhile the Tsafnat et al. workflow is considered the first, other papers such as Clark et al. (2019) or Macleod et al. for animal testing are a little more current and also fix some of the gaps with Tsafnat. Since there are a few in this area now a reason should be given for the preference.\n\n\"We plan to run searches for new studies every second month\" - There is currently an effort to get this adjusted to bi-monthly as you suggest but the guidelines specify that this should be monthly. Perhaps just a quick sentence acknowledging this and saying that it is excessive for the subject area?\n\nThe generated search strategy has a specific filter for English / German papers which seems to contradict the more general phrasing of \"We will translate non-English reports where feasible\".\n\nData sets are obviously not provided as this is an ongoing paper.\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? No",
"responses": [
{
"c_id": "5556",
"date": "08 Jun 2020",
"name": "Lena Schmidt",
"role": "Author Response",
"response": "“I believe that the research proposal clearly lays out its objectives and aims. Although there are some minor edits as per the below list: The abstract should specifically mention PICO data extraction rather than data extraction generally. The \"Aims\" section outlines this but seems to contradict with the more general \"Introduction / Objective\" section.” Thank you for pointing this out, we have made some changes to the abstract and to the aims. Firstly, in the abstract we clarified that the data extraction goal is wider (not limited to PICO, it included fields that are generally of interest in systematic reviews as defined in the Cochrane Handbook). We have made changes in the aims section to reflect that, and to make it more consistent throughout. In the method section, we deleted the section “Objectives” and summarised everything under “Aims of this review” in the introduction. “While the Tsafnat et al. workflow is considered the first, other papers such as Clark et al. (2019) or Macleod et al. for animal testing are a little more current and also fix some of the gaps with Tsafnat. Since there are a few in this area now a reason should be given for the preference.” In response to this comment we added a more recent paper review paper to our summary of related research (Marshall and Wallace, 2019). The related research was chosen because it was either in the form of a systematic review or it was an overview that is directly related to our topic of interest. We also added an additional explanation for our preference to focus on the data extraction stage of the systematic review process in response to this, and also a previous peer review comment. To summarise this quickly, data extraction is one of the most time-consuming and error-prone tasks in the systematic reviewing process. By reviewing automation of data extraction, we aim to summarise the current knowledge. Furthermore, the area of data extraction has future potential to disrupt the “traditional” systematic review process – if data are extracted and well classified centrally then the searching and screening workflow can change as well. “\"We plan to run searches for new studies every second month\" - There is currently an effort to get this adjusted to bi-monthly as you suggest but the guidelines specify that this should be monthly. Perhaps just a quick sentence acknowledging this and saying that it is excessive for the subject area?” Thank you, yes. We have added a statement: “The bi-monthly interval for screening was chosen because we expect no sudden rise in relevant publications that could justify daily, weekly or monthly screening”. Furthermore, we added a statement defining impactful research that would lead to a review update even if the threshold of new studies is not met (please see reply to the first peer review for reference) “The generated search strategy has a specific filter for English / German papers which seems to contradict the more general phrasing of \"We will translate non-English reports where feasible\". Thank you, this item was unclear. This initial draft of the search strategy was created for protocol publication and will be minimally altered for the full search and when the remaining databases are searched. Then it will be impossible to use a language filter on most databases. The initial Medline search specifically included German studies because they are feasible to assess, and for any other database search we have no language restrictions. The process of searching all databases will be described in detail in the full review. “Data sets are obviously not provided as this is an ongoing paper.” Thank you for your peer review and for helping us to improve the quality of our paper."
}
]
}
] | 1
|
https://f1000research.com/articles/9-210
|
https://f1000research.com/articles/9-536/v1
|
05 Jun 20
|
{
"type": "Opinion Article",
"title": "Building a successful international research community through data sharing: The case of the Wheat Information System (WheatIS)",
"authors": [
"Taner Z. Sen",
"Mario Caccamo",
"David Edwards",
"Hadi Quesneville",
"Taner Z. Sen",
"Mario Caccamo",
"David Edwards"
],
"abstract": "The International Wheat Information System (WheatIS) Expert Working Group (EWG) was initiated in 2012 under the Wheat Initiative with a broad range of contributing organizations. The mission of the WheatIS EWG was to create an informational infrastructure, establish data standards, and build a single portal that allows search, retrieval, and display of globally distributed wheat data sets that are indexed in standard data formats at servers around the world. The web portal at WheatIS.org was released publicly in 2015, and by 2020, it expanded to 8 geographically-distributed nodes and around 20 organizations under its umbrella.\n\nIn this paper, we present our experience, the challenges we faced, and the answer we brought for establishing an international research community to build an informational infrastructure. Our hope is that our experience with building wheatis.org will guide current and future research communities to facilitate institutional and international challenges to create global tools and resources to help their respective scientific communities.",
"keywords": [
"WheatIS",
"community",
"data sharing",
"bioinformatics",
"wheat"
],
"content": "Introduction\n\nIn 2011, the ministers of agriculture from the G20 nations launched the Wheat Initiative in order to create an international umbrella organization to guide research priorities for developed and developing nations and facilitate communication between international organizations working on wheat (www.wheatinitiative.org/). Under the Wheat Initiative, several “Expert Working Groups“ (EWGs) were formed to fulfill this mission. At the time of writing in 2020, there are 11 EWGs. Realizing the importance of findability and accessibility of wheat data sets distributed around the world, the Wheat Information System (WheatIS) Expert Working Group was established in 2012 to develop data standards for the wheat community and enable data query and access to globally-distributed data sets in standardized formats. The core collaborating groups was chaired by members from The Genome Analysis Centre (TGAC, now Earlham Institute) in the United Kingdom, l’Unité de Recherche Génomique Info (URGI) in France, the United State Department of Agriculture, Agricultural Research Service (USDA-ARS) in the US, and University of Queensland in Australia.\n\nSuch a multi-faceted global data indexing and sharing challenge required close, sustained, and dedicated collaboration among wheat researchers with overlapping expertise. Three years after the inception of the WheatIS EWG, the WheatIS portal was made publicly available in 2015 through wheatis.org. The computational infrastructure, web presence, and data content were created by the EWG committee members, other scientists, programmers, and technicians. Currently, the web portal is maintained at the University of Western Australia, Australia, and the portal WheatIS servers are located at the “Plant Bioinformatics Facility” hosted by URGI France. Only after 7 years, WheatIS “nodes,” i.e., servers that contain indexed and formatted data sets, proliferated and are currently distributed in 3 continents in 5 countries, demonstrating the buy-in from the wheat research communities (Alaux et al., 2018; Blake et al., 2019; Scheben et al., 2019; Wilkinson et al., 2016b; Yuan et al., 2017).\n\nIn this paper, we describe our experience at forming our research community and building wheatis.org in order to provide our answers to the problem of executing such a large-scale project across borders, organizations, and funding mechanisms, so that other research communities can benefit from our experience. We nevertheless wish to mention that there is no single path to create such a global infrastructure and community, and our experience is only an example of how such a productive and successful collaboration can be built.\n\nGoals of WheatIS Expert Working Group. The Wheat Initiative tasked the WheatIS EWG’s to provide the international wheat research community with easy access to wheat genetics, phenotype with environmental information, genomic data and bioinformatics tools, and to support and promote the diverse wheat databases internationally. Specifically, its goals are: 1) provide the wheat research community with a single-entry point of access to genetic, phenotypic, and genomic resources; 2) promote the development of services on top of existing wheat / Triticeae databases; 3) define guidelines for data curation, nomenclature, standards, and integration; and 4) provide a registry of wheat data resources.\n\nBuilding an expert working group. The initial group was formed with a focus on recruiting diverse profiles covering important countries or geographical areas, institutions, interest groups and scientific fields for wheat research. The Wheat Initiative board was instrumental to identify missing profiles. An important success factor has been to include in this group all the key players of international wheat research. This initial group have been complemented by new members along all these years. Being inclusive by nature, the EWG accepted researchers and developers willing to contribute to the project. They meet once a year in a face-to-face meeting and regularly using videoconferences in between. The current EWG members are from the following organizations: l’Unité de Recherche Génomique Info (URGI) at l’Institut national de la recherche agronomique (INRA) (France), University of Western Australia (Australia), National Institute of Agricultural Botany (NIAB) (UK), the U.S. Department of Agriculture, Agricultural Research Service (USDA-ARS) (USA), the International Maize and Wheat Improvement Center CIMMYT (Mexico), the European Bioinformatics Institute (EMBL-EBI) (UK), GARNet (UK), Agriculture and Agri-Food Canada (Canada), Oregon State University (USA), Earlham Institute (UK), University of California, Davis (USA), Howard Hughes Medical Institute (HHMI) (USA), James Hutton Institute (UK), MIPS (Germany), University of Saskatchewan (Canada), Rothamsted Research (UK), International Wheat Yield Partnership (USA), and Royal Botanic Gardens, Kew (UK).\n\nSeeking help from other communities. Achieving data interoperability is a difficult task because of data and tool heterogeneity, but also because of social and scientific challenges. To help, the Wheat Data Interoperability Working Group (WDI-WG) was created as one of the Research Data Alliance working groups, under the umbrella of the WheatIS Expert Working Group. This group was built from scientists taken from diverse fields such as data sciences, web semantics, genomics, phenomics and genetics. Some members belong to the WheatIS EWG, other have a more fundamental or transversal interest. Interestingly, some of them come from communities of other species such as rice. They participate to the work to help defining guidelines for their own community, taking advantage of the diversity of skills brought by the group, but also help us to be more generic in the proposed guidelines. This was a good insurance for the long-term sustainability of our proposals. Moreover, that also demonstrated how our approach can be generalized to other species, so that we found that our experience is valuable outside our community.\n\nThe starting action. Our first common action was to start working on surveys, interrogating the wheat research community on the usage of data standards in the wheat research community through a series of questions sent out to researchers and stakeholders in wheat science. The questions and answers were reported to the community (Subirats et al., 2015). Our successful process leading to the proposed guidelines was described in a community paper (Dzale Yeumo et al., 2017).\n\nThe Wheat Initiative serves as an umbrella organization for eleven EWGs and provides a loose connection between the EWGs to interact, however, it provides very limited funding for the EWGs to meet and organize workshops. For example, in 2018, approximately 9,000 euros were provided by the Wheat Initiative to partially subsidize attendee costs for an annual meeting that took place as a side meeting at the Plant and Animal Genome Conference in San Diego, CA and two workshops in Europe. To this date, no salary is provided to WheatIS EWG members or the members of their research groups to create or contribute to wheatis.org.\n\nThis meager funding from the Wheat Initiative means that many people that are involved in the WheatIS EWG activities, such as curating data, building indexed data sets, configuring and maintaining servers, are doing these tasks on a volunteer basis in addition to their regular daily tasks. Fortunately, both computational and experimental research groups that are part of the WheatIS community recognize the primary importance of data availability, access, and sharing through wheatis.org, and because it is beneficial to the larger scientific community, they consider their service a crucial part of their scientific responsibility. This somewhat guarantees a relative long-term sustainability of the initiative.\n\nThe most significant accomplishment of the WheatIS EWG is the creation of a central hub, called WheatIS that provides a publicly available single-entry point. The WheatIS core server have access to resources at the globally-distributed nodes and enables data query and extraction through the web portal, unifying data discovery for the wheat research community.\n\nSpecifically, the WheatIS portal was created to: 1) provide access to a data file repository storing files with their associated metadata; 2) allow queries to find data available in the WheatIS core and its nodes using keywords through a google-like search engine; 3) Data standards recommendations (Dzale Yeumo et al., 2017); and 4) catalog several dedicated integrative databases that manage data types such as genomic, genetic, phenotypic, and functional genomic.\n\nCurrent WheatIS searchable nodes. The following are the current organizations that manage a WheatIS server node: 1) the International Maize and Wheat Improvement Center CIMMYT (Mexico), 2) the European Bioinformatics Institute (EMBL-EBI) (UK), 3) the GrainGenes database (USA), 4) the Gramene database (USA), 5) the Triticeae Toolbox database (USA), 6) Transplant-IPGPAS (Poland), 7) l’Unité de Recherche Génomique Info (URGI) (France), and 8) wheatgenome.info at the University of Western Australia (Australia). Among them, the URGI node is the main server that queries other servers. Note that the actual contributor list provided previously is larger than the number of nodes, because some groups contribute their data through already existing nodes located at other organizations.\n\nRules of how to become a part of the WheatIS community. The WheatIS community is always expanding, adding new data sets and nodes from groups that never contributed data to wheatis.org. WheatIS contributing members provides know-how and support to those who would like to create and maintain their own WheatIS nodes at their locations or contribute data to WheatIS, a simple request to wheatis-contact@wheatis.org will provide help and support.\n\nGood communication is crucial for the success of such an endeavor. In addition to the website, we set up a Twitter account and use a mailing list to inform on our activities. Regularly, the Wheat Initiative organized meetings of its EWGs. These are useful opportunity to show our progress, to discuss the needs of the wheat research community, and to demonstrate the usefulness of our contribution. We were regularly invited to international conference where we presented our goals and the results. All these events contributed largely to make our initiative known by a number of scientists.\n\nBut, more interestingly, beside these quite obvious actions, an important part of our strategy was to organize training in different circles. At these occasions, we presented our tools to make them adopted by more and more people, but we also got feedback on their usage and the needs that help us to improve our work. In particular we organized joint meeting with other EWG to better answer needs from some scientific communities. Hence the “phenomics” EWG under the Wheat Initiative benefited a lot from such interactions.\n\nOur primary goal for the WheatIS Expert Working Group is to create a single portal that can query indexed data sets distributed worldwide, extract information, and provide access to these data sets to the wheat research community. The task of creating a technical framework for a single portal with access to multiple nodes is now accomplished. The size and types of data sets accessible at WheatIS are growing daily with more nodes being added. In all apparent measures, WheatIS is successful in building up a highly collaborative community of wheat research groups and creating a valuable product that is useful in connecting heterogenous data sets. When other scientific communities learned about our success such as rice (Scheben et al., 2019), grapevine (Adam-Blondon et al., 2016), AgBioData (Harper et al., 2018), we were asked how we accomplished this challenging task, i.e., what our formula was for success. Consequently, some of our approaches have been followed by these groups (Adam-Blondon et al., 2016). In this section, we provide our perspective as a way to guide other current and future research communities.\n\nKeeping data distributed. Keeping data in place of their existing repositories, working on improving their visibility, but also involving people who managed them was a strategy we chose since the beginning. Even if technically more difficult, we thought that it is a key decision that helped us to build a community of data managers. By this we acknowledge the contribution of each contributor to the system, offering them the visibility they need for their own sustainability. Keeping a consistent group of motivated people who can obtain rewards from their efforts when they share their data is essential. In addition, it helps them to obtain funding from their own institutions or countries. This win-win strategy was a key determinant for the long-term success of our group. The social aspect of such a project is needed to be carefully considered and certainly not neglected when facing technological challenges. Here we preferred perhaps to make the challenge technically more difficult by emphasizing and prioritizing the social dynamics and group cohesion.\n\nIdentifying a burning need (a.k.a. “an overarching and shared vision”). The primary starting point to create a community group is to identify a burning need around which the community should be formed. Only such a critical need will encourage researchers to go out of their institutional bubbles to navigate through complicated policies and procedures across national lines and devote their times voluntarily. A group of people can only function as a community as long as a burning need exists. If a need loses its importance and a new burning need is not identified, then the buy-in from scientists weakens and the community falters. If on the other hand, a new need is identified, a community can be transformed, and even evolved with the injection of new people energizing the community, even in some cases replacing some of the “old guard” in the process. In the case of WheatIS, the remarkable need to search, reach, and extract wheat data sets that are generated across the globe was a given among wheat scientists, and still energizes the community as more data sets are being generated with continually cheapening experimental technologies and computational power.\n\nLeadership principles. One of the important features that define a research community is its leadership rational. Cooperation and trust for mutual benefit are of paramount importance. Such an endeavor requires to devote for the needs of the community, so that the community members follow these examples and respond positively. Another important point to emphasize here is that experience and skill sets for managing people and projects are essential. Although it is important for a research community to have leaders who are accomplished scientists, it does not necessarily mean that all accomplished scientists can lead the community to the next level diplomatically and successfully. Natural leadership should take precedence over exceptional publication record; the lack of publications in glamorous journals should not preclude someone to become a leader.\n\nCreating a supra-institutional umbrella group with broad appeal. When establishing a research community, it is also important that the umbrella group should preferably not be led by a single institution, but a wide range of institutions, hopefully international, to create a broad appeal to attract scientists from different institutions. Institutions with big names can provide a great impetus at the beginning, especially with scientists and institutions that are already collaborating with the researchers in those institutions, but then with a single institution, there is a greater chance for the initial momentum to stall with time, and it is a better move to rely on multiple institutions, in a sense to diversify the risk of relying on a single institution. Also, instead of starting with well-known institutions, an alternative is to create an organization above (i.e., “supra-) the partnering organizations, so that partnering organizations feel that they are not being led by a well-known institute, but they are partners on equal terms with each institution under the umbrella group. A feeling of equality will create a greater buy-in from organizations and scientists. In the case of WheatIS EWG, the formation of the Wheat Initiative by G20 ministers of agriculture instantly created such a supra-national umbrella organization. Two crucial aspects presented an opportunity to start such an international organization for wheat: 1) wheat is among the top three crops in the world and 2) it has been produced by a large number of nations in all the continents except Antarctica (Dubcovsky & Dvorak 2007).\n\nNot every supranational organization need to be close-knit and built top-down. For example, the Arabidopsis community went through a stage where the National Science Foundation (NSF) in the U.S. steadily reduced the funding for the centralized Arabidopsis database TAIR (Reiser et al., 2017), forcing the community to seek funding from the funding agencies in different nations to keep the database. In this bottom-up case, the International Arabidopsis Informatics Consortium was formed (International Arabidopsis Informatics Consortium 2010; International Arabidopsis Informatics Consortium, 2019) and provided a venue for scientists and national funding agencies to exchange ideas to support Arabidopsis informatics structure and reached consensus among organizations to maintain and improve on the community’s informatics structure. It is also important to mention that some members of the WheatIS EWG are also members of the maize, Brassica and rice communities and their contributions played a significant role in WheatIS’s success and in turn their experience in the WheatIS initiative are making an impact in their communities.\n\nBroad range of deep, dedicated scientific expertise. The Wheat Information System needed a wide range of expertise to make wheatis.org a reality. It needed technical expertise to build and maintain a strong computational infrastructure and create data formats to make data sets readable; scientific expertise to understand different types of wheat data sets (including genetic, genomic, phenotypic, and metabolic); outreach capability to help build relationships to add new nodes with new data sets; and leaders who not only motivate and manage personnel, but also work with the Wheat Initiative and the broader wheat community to promote and support WheatIS. The need for dedicated and competent personnel with complimentary and overlapping expertise was crucial. For WheatIS, or for any scientific community for that matter, the critical question is the type of the expertise needed and how much time the experts can devote to a fledgling community.\n\n\nConclusions and future work\n\nWhen the WheatIS EWG was formed to create a single portal to make wheat data sets findable, accessible, and shareable (Wilkinson et al., 2016a), the initial focus was primarily on the data sets. However, sharing data sets also lead to strengthening wheat communities as well, which happened for WheatIS working under the Wheat Initiative. Through WheatIS and through sharing data sets, WheatIS has evolved into a fledgling nexus for the other EWGs, a few in the beginning, and more later, to contribute to a single portal where any data points generated would be made accessible. Sharing data does not only require creating data sets in a certain data format and placing them in a certain data directory on a server, but it also requires communication and planning between research groups and between Expert Working Groups. Through this communication network, WheatIS is helping the Wheat Initiative to become a more cohesive group and facilitates future collaborations. These types of collaborations will have a larger impact beyond the Wheat Initiative, first through the wheat research groups that are not part of the Wheat Initiative, and later other plant researchers and researchers working with other species.\n\nThe collaboration across EWGs to develop common data standards is an ongoing effort between and within Expert Working Groups. Following the workshops previously organized in 2017 in Tulln, Austria and Berlin, Germany, and recently in 2019 in the Wheat Initiative Research Committee meeting at the First International Wheat Congress in Saskatoon, Canada, a decision was taken to broaden the participation by including people from other EWGs, and another workshop is in the planning stages to create guidelines for gene naming for genetics and genomics data.\n\nAlthough the current graphical user interface for WheatIS is functional, it needs improvement in several areas. There are ongoing efforts to create a more user-friendly interface to improve user experience. It is not straightforward for users to identify where and how to start their search intuitively, and we plan to provide more information and support links for users. In the new interface, which we colloquially name WheatIS 2.0, we plan not only to work with the cosmetic issues, but also functional issues such as providing a more advanced and easy-to-use search capabilities. Currently some advanced search features are offered to users, but only after a search term is entered and when search results are shown. We plan to incorporate an Advanced Search feature without the need to enter a search term first to show the range of data types. We also need to improve our semantic search capabilities, considering the recent advances in the field. The WheatIS 2.0 will be shaped in these and other specific areas that were identified through personal discussions in the EWG meetings and the feedback we received from actual users.\n\n\nData availability\n\nNo data are associated with this article",
"appendix": "Acknowledgments\n\nWe first thank the “Wheat Information System Expert Working Group” (WheatIS EWG) members for their valuable input to build this infrastructure. We are very grateful to all the personnel including scientists, postdoctoral research associates, technicians, programmers, and others, who made contributions in the past and made wheatis.org a reality. We specifically thank the Plant Bioinformatics Facilities (PlantBioinfoPF) team located at URGI in France. We thank the Wheat Initiative led by Hélène Lucas, Peter Langridge and Frank Ordon that created a remarkable umbrella organization for WheatIS to thrive. We also acknowledge Steve Visscher formerly at the BBSRC as a great facilitator in the early days of the Wheat Initiative.\n\n\nReferences\n\nAdam-Blondon AF, Alaux M, Pommier C, et al.: ‘Towards an Open Grapevine Information System’. Hortic Res. 2016; 3: 16056. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAlaux M, Rogers J, Letellier T, et al.: ‘Linking the International Wheat Genome Sequencing Consortium Bread Wheat Reference Genome Sequence to Wheat Genetic and Phenomic Data’. Genome Biol. 2018; 19(1): 111. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBlake VC, Woodhouse MR, Lazo GR, et al.: ‘GrainGenes: Centralized Small Grain Resources and Digital Platform for Geneticists and Breeders’. Database (Oxford). 2019; 2019: baz065. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDubcovsky J, Dvorak J: ‘Genome Plasticity a Key Factor in the Success of Polyploid Wheat under Domestication’. Science. 2007; 316(5833): 1862–66. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDzale Yeumo E, Alaux M, Arnaud E, et al.: Developing data interoperability using standards: A wheat community use case [version 2; peer review: 2 approved]. F1000Res. 2017; 6: 1843. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHarper L, Campbell J, Cannon EKS, et al.: ‘AgBioData Consortium Recommendations for Sustainable Genomics and Genetics Databases for Agriculture’. Database (Oxford). 2018; 2018: bay088. PubMed Abstract | Publisher Full Text | Free Full Text\n\nInternational Arabidopsis Informatics Consortium: ‘An International Bioinformatics Infrastructure to Underpin the Arabidopsis Community.’ Plant Cell. 2010; 22(8): 2530–36. PubMed Abstract | Publisher Full Text | Free Full Text\n\nInternational Arabidopsis Informatics Consortium: ‘Arabidopsis Bioinformatics Resources: The Current State, Challenges, and Priorities for the Future.’ Plant Direct. 2019; 3(1): e00109. PubMed Abstract | Publisher Full Text | Free Full Text\n\nReiser L, Subramaniam S, Li D, et al.: ‘Using the Arabidopsis Information Resource (TAIR) to Find Information About Arabidopsis Genes’. Curr Protoc Bioinformatics. 2017; 60: 1.11.1–1.11.45. PubMed Abstract | Publisher Full Text\n\nScheben A, Chan CKK, Mansueto L, et al.: ‘Progress in Single-Access Information Systems for Wheat and Rice Crop Improvement’. Brief Bioinform. 2019; 20(2): 565–71. PubMed Abstract | Publisher Full Text\n\nSubirats I, Cooper L, Shrestha R, et al.: ‘Towards a Comprehensive Overview of Ontologies and Vocabularies for Research on Wheat’. Zenodo. 2015. Publisher Full Text\n\nWilkinson MD, Dumontier M, Aalbersberg IJJ, et al.: ‘The FAIR Guiding Principles for Scientific Data Management and Stewardship’. Sci Data. 2016a; 3: 160018. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWilkinson PA, Winfield MO, Barker GLA, et al.: ‘CerealsDB 3.0: Expansion of Resources and Data Integration’. BMC Bioinformatics. 2016b; 17: 256. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYuan Y, Scheben A, Chan CKK, et al.: ‘Databases for Wheat Genomics and Crop Improvement’. Methods Mol Biol. 2017; 1679: 277–91. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "64353",
"date": "26 Jun 2020",
"name": "Runxuan Zhang",
"expertise": [
"Reviewer Expertise high throughput data analysis in plant species",
"transcriptomics and proteomics"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 described the process and successful experiences of forming an international research community, namely the international Wheat Information System (WheatIS) Expert working group. The focus of this article is on the higher level identifying the key factors to build a sustainable and successful community, such as establishing an urgent and shared vision, drawing in broad areas of deep and dedicated expertise, setting up an umbrella group with broad appeal and equal footing for partners, etc. Some technical aspects were also touched on such as keeping the data distributed and setting up a core with access to globally distributed nodes.\n\nAlthough technical issues are not the focus of the paper, here are a few comments regarding to wheatIS.org:\nOn its official webpage, a few tabs on the wheatIS.org, such as \"submit data\" and \"tools\" are still under construction and not available for use.\n\nOne of the aims of the EWG is to allow people to share their data. What is the procedures in place to check the quality of the data? How does the system prevent false info from getting into the system?\n\nCurrently there is very limited funding available for this initiative. Most of the work is done on a volunteer basis. For the long term sustainability, the values created for end-user of the wheatis.org and impact it could make should be considered. A route for funding should be established through charity, governmental funding or industry to fund the initiative directly considering the full economic cost.\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": "5877",
"date": "05 Oct 2020",
"name": "Hadi Quesneville",
"role": "Author Response",
"response": "We thank Runxuan Zhan from the James Hutton Institute (UK) for this review. Below are the replies to its comments. Reviewer comment #1: On its official webpage, a few tabs on the wheatIS.org, such as \"submit data\" and \"tools\" are still under construction and not available for use. Response: We thank the reviewer for his thoroughness. The header links are now fully functional. Reviewer comment #2: One of the aims of the EWG is to allow people to share their data. What are the procedures in place to check the quality of the data? How does the system prevent false info from getting into the system? Response: We agree with the reviewer that data quality is one of the most crucial aspect of data availability and stewardship. At the lowest level of quality check, when a new dataset is indexed at one of the nodes, the WheatIS system automatically checks its formatting, and if the dataset is not formatted correctly, it is not displayed through the web interface, and flagged for further quality check. Although correct formatting and accurate display of the datasets are important, the most important criterion for indexing a dataset at WheatIS is primarily its scientific value for the WheatIS users. The process of selecting and indexing datasets at the WheatIS nodes is crucial; every dataset available through the WheatIS framework needs to be high-quality. Therefore, all indexed datasets at WheatIS are required to be either peer-reviewed, manually curated by professional curators, or both. Most datasets at WheatIS are the products of peer-reviewed scientific research with associated peer-reviewed publications. Some others, such as Wheat Gene Catalogue, are being curated for decades and approved by a committee of wheat genetics experts. Reviewer comment #3: Currently there is very limited funding available for this initiative. Most of the work is done on a volunteer basis. For the long-term sustainability, the values created for end-user of the wheatis.org and impact it could make should be considered. A route for funding should be established through charity, governmental funding or industry to fund the initiative directly considering the full economic cost. Response: We thank the reviewer for bringing this issue to our attention, as well as, the attention of the funding agencies. In addition to our ongoing volunteer efforts, we continuously look for new funding resources for WheatIS."
}
]
},
{
"id": "79260",
"date": "15 Feb 2021",
"name": "Stavros Makrodimitris",
"expertise": [
"Reviewer Expertise bioinformatics",
"plant gene function prediction"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 Sen et al. describes a community-wide effort to create a large information resource about the wheat genome. The authors share their experience to be used as a potential guide for similar endeavors in the future. The manuscript is concise and clearly written. I have a couple of suggestions to the authors from the perspective of bioinformatics:\nDatasets from such large databases often contain large ‘batch effects’, i.e. technical confounders such as differences in experimental protocols, sequencing depth etc. Such batch effects make joint analysis of different datasets more challenging. Are there any efforts to provide batch-corrected data or are only the ‘raw’ data available?\n\nThe authors mention plans to improve the user interface, but as I expect the data in such a big database to continue growing, sooner or later there might be a need for some kind of programmatic access through, for example, an API or an R package. Does such a tool exist? If not, could the authors mention what their plans are in that direction?\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": "75739",
"date": "11 Mar 2021",
"name": "Nevin Gerek Ince",
"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\nSen et.al. described their experience and learning in order to establish an international research community to build a data infrastructure, standards and a single portal for wheat data sets around the world. I think the manuscript is clearly written and explains their endeavours and challenges to build wheatis.org website. Their experience provides a guidance for research communities how to create global tools and resources. I should emphasize that their experience is not only specific to the Wheat Initiative with a broad range of contributing organizations. Their challenges are also valid for other research communities.\nI wonder if they are planning to provide some data analytics or AI type of data prediction in WheatIS in the future.\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/9-536
|
https://f1000research.com/articles/9-517/v1
|
04 Jun 20
|
{
"type": "Research Article",
"title": "Measuring the outcome and impact of research capacity strengthening initiatives: A review of indicators used or described in the published and grey literature",
"authors": [
"Justin Pulford",
"Natasha Price",
"Jessica Amegee Quach",
"Imelda Bates",
"Natasha Price",
"Jessica Amegee Quach",
"Imelda Bates"
],
"abstract": "Background: Development partners and research councils are increasingly investing in research capacity strengthening initiatives in low- and middle-income countries to support sustainable research systems. However, there are few reported evaluations of research capacity strengthening initiatives and no agreed evaluation metrics. Methods: To advance progress towards a standardised set of outcome and impact indicators, this paper presents a structured review of research capacity strengthening indicators described in the published and grey literature. Results: We identified a total of 668 indicators of which 40% measured output, 59.5% outcome and 0.5% impact. Only 1% of outcome and impact indicators met all four quality criteria applied. A majority (63%) of reported outcome indicators clustered in four focal areas, including: research management and support (97/400), the attainment and application of new research skills and knowledge (62/400), research collaboration (53/400), and knowledge transfer (39/400). Conclusions: Whilst this review identified few examples of quality research capacity strengthening indicators, it has identified priority focal areas in which outcome and impact indicators could be developed as well as a small set of ‘candidate’ indicators that could form the basis of development efforts.",
"keywords": [
"Research capacity strengthening",
"Evaluation",
"Indicators",
"Review",
"LMICs"
],
"content": "Introduction\n\nResearch capacity strengthening (RCS) has been defined as the “process of individual and institutional development which leads to higher levels of skills and greater ability to perform useful research”1. National capacity to generate robust, innovative and locally appropriate research is considered essential to population health2,3 and socioeconomic development4,5. However, wide global disparities in research capacity and productivity currently exist: South Asian countries account for 23% of the World’s population yet produced less than 5% of the global output of scientific publications in 20136; and sub-Saharan Africa (accounting for 13% of the global population), contributes 1% of global investment in research and development and holds 0.1% of global patents6. Accordingly, international development partners and research funding bodies are increasingly investing in RCS initiatives in low- and middle-income countries (LMICs). The UK Collaborative on Development Research predicts the United Kingdom’s total aid spend on research will rise to £1.2 billion by 20217, a large proportion of which would be direct or indirect investment in RCS in LMICs. The total global spend on RCS in LMICs, while not yet calculated, would likely be many times this figure.\n\nDespite this substantial investment, few robust evaluations of RCS initiatives in LMIC contexts have been presented in the published or grey literatures with the available evidence base characterised by reflective, largely qualitative individual case studies or commentaries8. RCS evaluation frameworks have been described9–11, but a comprehensive set of standard outcome or impact indicators have not been agreed and common indicators are used inconsistently. For example, publication count has been used as both an output12 and outcome indicator13 sometimes with14 or without10 accounting for publication quality.\n\nThe dearth of robust RCS programme evaluation and, more fundamentally, robust evaluation metrics available for consistent application across RCS programmes, has contributed to a paradoxical situation in which investments designed to strengthen the quantity, quality and impact of locally produced research in LMIC settings are themselves hindered by a lack of supporting evidence. As a substantial proportion of RCS investment is derived from publicly funded development assistance15–17, then ensuring the means to reliably evaluate impact and value for money of research and health system investments assumes even further importance.\n\nThis paper aims to advance progress towards the establishment of a standardised set of outcome and impact indicators for use across RCS initiatives in LMIC contexts. As a first step towards this goal, a systematic review of RCS outcome and impact indicators previously described in the published and grey literatures is presented. The review findings highlight the range, type and quality of RCS indicators currently available and allows inconsistencies, duplications, overlaps and gaps to be identified. These results may then be used to inform planning and decision making regarding the selection and/or development of standard RCS evaluation metrics. In the interim, the resulting list of indicators may also serve as a useful resource for RCS programme funders, managers and evaluators as they design their respective monitoring and evaluation frameworks.\n\n\nMethods\n\nPeer reviewed publications were sought via the following databases: PubMed, Global Health, CINAHL Complete and International Bibliography of the Social Sciences (IBSS). The search was limited to English language publications and was conducted using the keywords: (research capacity) AND (develop* OR build* OR strengthen*) AND (indicator) AND (monitoring OR evaluation). The search was conducted without date limitations up until March 2018. Following removal of duplicates, all retrieved publications were subject to an initial review of the title, abstract and listed keywords. Publications that met, or were suggestive of meeting, the inclusion criteria were then subjected to full text review. Publications subjected to full text review met the inclusion criteria if they: were peer-reviewed; pertained to ‘research capacity’ (as either a primary or secondary focus); and included at least one output, outcome or impact indicator that has been used to measure research capacity or was proposed as a possible measure of research capacity.\n\nThe search was supplemented by a manual review of the references listed in each paper that met the final inclusion criteria and by a citation search using first author names for all papers which met the final inclusion criteria from both the initial electronic and supplementary manual searches. A further 19 papers which met the inclusion criteria were identified in this way and included in the review.\n\nRelevant grey literature was then sought via the following databases: Google Advanced, BASE, Grey Literature and OpenGrey. The same search terms and inclusion criteria as described above were used. This search was supplemented by a request circulated across the authors’ personal networks for relevant research reports pertaining to RCS evaluation which may fit the inclusion criteria. There were seven reports identified this way, resulting in a final sample of 25 publications and seven reports. Figure 1 depicts the overall process and outcomes from this search strategy.\n\nResearch capacity strengthening indicator descriptions and definitions were extracted from each publication/report and recorded verbatim in an Excel spreadsheet (see Underlying data)18. Other information recorded alongside each indicator included: the type of indicator (output, outcome or impact) (Box 1); the level of measurement (individual research capacity; institutional research capacity; or systemic research capacity); source information (author, year and title of publication/report); and a brief summary of the context in which the indicator was applied. Designation of indicator type (output, outcome or impact) and level of measurement (individual, institutional or systemic) were based on those ascribed by the author/s when reported. Where indicator type and measurement level were not reported, we used our own judgement drawing on the reported context from the respective publication/report.\n\nSome publications/reports used the same indicators across different levels (i.e. as both an individual and an institutional measure) and in these cases we reported the indicator at a single level only based on apparent best fit. However, if the same publication reported the same indicator as both an output and an outcome measure, then it was reported twice. Where there was variation between the way that one publication or another classified an indicator (e.g. the same indicator being described as an ‘output’ indicator in one publication and an ‘outcome’ indicator in another), we remained true to the texts and recorded each separately. Indicators that pertained to the evaluation of course materials or content (e.g. how useful were the PowerPoint slides provided?) were excluded from analysis, although indicators that focused on the outcome of course attendance were retained.\n\nOutput indicators - defined as measures of programme or project activities that are directly controllable by the RCS initiative (e.g. number of infectious disease experts from country X training in academic writing).\n\nOutcome indicators - defined as measures of change in behaviour or performance, in the short- to mid-term, that could reasonably be attributed to the RCS initiative in full or large part (e.g. number of manuscripts published by infectious disease experts from country X following an academic writing course).\n\nImpact indicators - defined as measures of longer-term change that may not be directly attributable to the RCS initiative but directly relate to the overarching aims of the RCS initiative (e.g. reduction in infectious disease mortality in country X).\n\nOnce all listed indicators from across the 32 publications and reports had been entered into the Excel spreadsheet, the research team coded all outcome and impact indicators according to their respective focus (i.e. the focus of the indicated measure, such as publication count or grant submissions) and quality. Output indicators were excluded from further analysis. Indicators were coded independently by two researchers, checking consistency and resolving discrepancies through discussion and, if necessary, by bringing in a third reviewer. ‘Focus’ codes were emergent and were based on stated or implied focal area of each indicator. ‘Quality’ was coded against four pre-determined criteria: 1) a measure for the stated indicator was at least implied in the indicator description; 2) the measure was clearly defined; 3) the defined measure was sensitive to change; and 4) the defined measure was time-bound (thus, criteria 2 is only applied if criteria 1 is met and criteria 3 and 4 are only applied if criteria 2 is met).\n\n\nResults\n\nWe identified a total of 668 reported or described indicators of research capacity from across the 32 publications or reports included in the review. Of these, 40% (265/668) were output indicators, 59.5% (400/668) were outcome indicators and 0.5% (3/668) were impact indicators. A total of 34% (225/668) of these indicators were measures of individual research capacity, 38% (265/668) were measures of institutional research capacity and 21% (178/668) were systemic measures of research capacity. Figure 2 illustrates the spread of indicator type across these three categories by level. The full list of 668 indicators, inclusive of source information, is available as Underlying data18.\n\nThe 400 outcome indicators were subsequently coded to nine thematic categories and 36 sub-categories, as described in Box 2. The categories and the total number of indicators in each (across all three levels) were as follows: research management and support (n=97), skills/knowledge (n=62), collaboration activities (n=53), knowledge translation (n=39), bibliometrics (n=31), research funding (n=25), recognition (n=11), infrastructure (n=5) and other (n=77). Figure 3 depicts the number of outcome indicators by category and level.\n\n1. Bibliometrics: Indicators relating to the development, publication and use of written outputs such as peer reviewed journal articles.\n\nSub-categories: peer reviewed publication; publication (any form of publication other than peer review); reference (e.g. records of citations); quality (e.g. rating by impact factor).\n\n2. Collaboration Activities: Indicators relating to networking, collaborating, mentoring type activities.\n\nSub-categories: engagement (evidence of working collaboratively); establishment (creating new networks, collaborations); experience (e.g. perception of equity in a specific partnership).\n\n3. Infrastructure: Indicators relating to research infrastructure including buildings, labs, equipment, libraries and other physical resources.\n\nSub-categories: suitability (the provision of adequate facilities for research); procurement (e.g. purchase of laboratory equipment).\n\n4. Knowledge translation: Indicators relating to the dissemination of research and knowledge, including conferences, media and public education/outreach.\n\nSub-categories: dissemination (examples of research being communicated to different audiences); influence (using research knowledge to influence policy, the commissioning of new research, etc).\n\n5. Recognition:Indicators relating to professional or institutional esteem.\n\nSub-categories: Appointment (e.g. appointed to leadership positions); Awards (i.e. receiving an award); reputation (e.g. invited keynote address).\n\n6. Research funding: Indicators relating to funding for research.\n\nSub-categories: funds received (e.g. competitive grants); allocation (e.g. allocate budget to support local research); expenditure (use of research funds); access (access to research funding/competitive awards).\n\n7. Research Management & Support (RMS): Indicators relating to the administration of university or research institution systems that make research possible (e.g. finance, ITC and project management).\n\nSub-categories: career support (e.g. working conditions, salary and career development); organisation capacity (to manage/support research); research investment; resource access (e.g. to IT, libraries etc); sustainability (of RMS); governance (e.g. formation of ethics review committees); national capacity (to support research); national planning (e.g. developing national research priorities).\n\n8. Skills/training activities: Indicators relating to training and educational activities relating to research or research subject area knowledge.\n\nSub-categories: attainment (of new skills); application (of new skills); transfer (of new skills).\n\n9. Other: Indicators relating to any area other than the eight described above.\n\nSub-categories: research quality (e.g. quality of work undertaken); research production (e.g. increase in research activity); research process (e.g. inclusion of new methods or techniques); research workforce (e.g. growth in number of researchers); career advancement (e.g. promotion); equity (e.g. gender equity); miscellaneous.\n\nTable 1–Table 3 present the number of outcome indicators in each sub-category as well as an example indicator for each, by the three respective research capacity levels (individual, institutional and systemic). The category and sub-category designation assigned to all 400 outcome indicators are available as Underlying data18.\n\nTable 4 presents the percentage of outcome indicators that met each of the four quality measures as well as the percentage that met all four quality indicators by indicator category. As shown, all outcome indicators implied a measurement focus (e.g. received a national grant or time spent on research activities), 21% presented a defined measure (e.g. had at least one publication), 13% presented a defined measure sensitive to change (e.g. number of publications presented in peer reviewed journals) and 5% presented a defined measure, sensitive to change and time bound (e.g. number of competitive grants won per year). Only 1% (6/400) of outcome indicators met all four quality criteria including: 1) Completed research projects written up and submitted to peer reviewed journals within 4 weeks of the course end; 2) Number of competitive grants won per year (independently or as a part of a team); 3) Number and evidence of projects transitioned to and sustained by institutions, organizations or agencies for at least two years; 4) Proportion of females among grantees/contract recipients (over total number and total funding); 5) Proportion of [Tropical Disease Research] grants/contracts awarded to [Disease Endemic Country] (over total number and total funding); and 6) Proportion of [Tropical Disease Research] grants/contracts awarded to low-income countries (over total number and total funding). Indicators pertaining to research funding and bibliometrics scored highest on the quality measures whereas indicators pertaining to research management and support and collaboration activities scored the lowest.\n\nThe three impact indicators were all systemic-level indicators and were all coded to a ‘health and wellbeing’ theme; two to a sub-category of ‘people’, one to a sub-category of ‘disease’. The three impact indicators were: 1) Contribution to health of populations served; 2) Impact of project on patients' quality of life, including social capital and health gain; and 3) Estimated impact on disease control and prevention. All three met the ‘implied measure’ quality criteria. No indicators met any of the remaining three quality criteria.\n\n\nDiscussion\n\nThis paper sought to inform the development of standardised RCS evaluation metrics through a systematic review of RCS indicators previously described in the published and grey literatures. The review found a spread between individual- (34%), institutional- (38%) and systemic-level (21%) indicators, implying both a need and interest in RCS metrics across all levels of the research system. This is consistent with contemporary RCS frameworks10,19, although the high proportion of institutional-level indicators is somewhat surprising given the continued predominance of individual-level RCS initiatives and activities such as scholarship provision, individual skills training and research-centred RCS consortia20.\n\nOutcome indicators were the most common indicator type identified by the review, accounting for 59.5% (400/669) of the total. However, the large number of outcome indicators were subsequently assigned to a relatively small number of post-coded thematic categories (n=9), suggestive of considerable overlap and duplication among the existing indicator stock. Just under two-thirds of the outcome indicators pertained to four thematic domains (research management and support, skills/knowledge attainment or application, collaboration activities and knowledge translation) suggesting an even narrower focus in practice. It is not possible to determine on the basis of this review whether the relatively narrow focus of the reported indicators is reflective of greater interest in these areas or practical issues pertaining to outcome measurement (e.g. these domains may be inherently easier to measure); however, if standardised indicators in these key focal areas are identified and agreed, then they are likely to hold wide appeal.\n\nThe near absence of impact indicators is a finding of significant note, highlighting a lack of long-term evaluation of RCS interventions8 as well as the inherent complexity in attempting to evaluate a multifaceted, long-term, continuous process subject to a diverse range of influences and assumptions. Theoretical models for evaluating complex interventions have been developed33, as have broad guidelines for applied evaluation of complex interventions34; thus, the notion of evaluating ‘impact’ of RCS investment is not beyond the reach of contemporary evaluation science and evaluation frameworks tailored for RCS interventions have been proposed11. Attempting to measure RCS impact by classic, linear evaluation methodologies via precise, quantifiable metrics may not be the best path forward. However, the general dearth of any form of RCS impact indicator (as revealed in this review) or robust evaluative investigation8,20 suggests an urgent need for investment in RCS evaluation frameworks and methodologies irrespective of typology.\n\nThe quality of retrieved indicators, as assessed by four specified criteria (measure for the stated indicator was implied by indicator description; measure clearly defined; defined measure was sensitive to change; and defined measure was time-bound) was uniformly poor. Only 1% (6/400) of outcome indicators and none of the impact indicators met all four criteria. Quality ratings were highest amongst indicators focused on measuring research funding or bibliometrics and lowest amongst research management and support and collaboration activities. This most likely reflects differences in the relative complexity of attempting to measure capacity gain across these different domain types; however, as ‘research management and support’ and ‘collaboration activity’ indicators were two of the most common outcome indicator types, this finding suggests that the quality of measurement is poorest in the RCS domains of most apparent interest. The quality data further suggest that RCS indicators retrieved by the review were most commonly (by design or otherwise) ‘expressions’ of the types of RCS outcomes that would be worthwhile measuring as opposed to well defined RCS metrics. For example, ‘links between research activities and national priorities’19 or ‘ease of access to research undertaken locally’22 are areas in which RCS outcome could be assessed, yet precise metrics to do so remain undescribed.\n\nDespite the quality issues, it is possible to draw potential ‘candidate’ outcome indicators for each focal area, and at each research capacity level, from the amalgamated list (see Underlying data)18. These candidate indicators could then be further developed or refined through remote decision-making processes, such as those applied to develop other indicator sets37, or through a dedicated conference or workshop as often used to determine health research priorities38. The same processes could also be used to identify potential impact indicators and/or additional focal areas and associated indicators for either outcome or impact assessment. Dedicated, inclusive and broad consultation of this type would appear to be an essential next step towards the development of a comprehensive set of standardised, widely applicable RCS outcome and impact indicators given the review findings.\n\nRCS is a broad, multi-disciplinary endeavour without a standardised definition, lexicon or discipline-specific journals8. As such, relevant literature may have gone undetected by the search methodology. Similarly, it is quite likely that numerous RCS outcome or impact indicators exist solely in project specific log frames or other forms of project-specific documentation not accessible in the public domain or not readily accessible by conventional literature search methodologies. Furthermore, RCS outcome or impact indicators presented in a language other than English were excluded from review. The review findings, therefore, are unlikely to represent the complete collection of RCS indicators used by programme implementers and/or potentially accessible in the public domain. The quality measurement criteria were limited in scope, not accounting for factors such as relevance or feasibility, and were biased towards quantitative indicators. Qualitative indicators would have scored poorly by default. Nevertheless, the review findings represent the most comprehensive listing of currently available RCS indicators compiled to date (to the best of the authors’ knowledge) and the indicators retrieved are highly likely to be reflective of the range, type and quality of indicators in current use, even if not identified by the search methodology.\n\n\nConclusion\n\nNumerous RCS outcome indicators are present in the public and grey literature, although across a relatively limited range. This suggests significant overlap and duplication in currently reported outcome indicators as well as common interest in key focal areas. Very few impact indicators were identified by this review and the quality of all indicators, both outcome and impact, was uniformly poor. Thus, on the basis of this review, it is possible to identify priority focal areas in which outcome and impact indicators could be developed, namely: research management and support, the attainment and application of new skills and knowledge, research collaboration and knowledge transfer. However, good examples of indicators in each of these areas now need to be developed. Priority next steps would be to identify and refine standardised outcome indicators in the focal areas of common interest, drawing on the best candidate indicators among those currently in use, and proposing potential impact indicators for subsequent testing and application.\n\n\nData availability\n\nHarvard Dataverse: Measuring the outcome and impact of research capacity strengthening initiatives: A review of indicators used or described in the published and grey literature - Full listing of retrieved RCS indicators. https://doi.org/10.7910/DVN/K6GIGX18.\n\nThis project contains the following underlying data:\n\nList of RCS Impact Indicators\n\nList of RCS Outcome Indicators\n\nList of RCS Output Indicators\n\nList of Source Documents\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\nGolenko X, Pager S, Holden L: A thematic analysis of the role of the organisation in building allied health research capacity: a senior managers’ perspective. BMC Health Serv Res. 2012; 12: 276. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDye C, Boerma T, Evans D, et al.: The World Health Report 2013: research for universal health coverage. Geneva: World Health Organisation; 2013.\n\nPang T, Sadana R, Hanney S, et al.: Knowledge for better health: a conceptual framework and foundation for health research systems. Bull World Health Organ. 2003; 81(11): 815–20. PubMed Abstract | Free Full Text\n\nPastor J, Peraita C, Serrano L, et al.: Higher education institutions, economic growth and GDP per capita in European Union countries. European Planning Studies. 2018; 26(8): 1616–37. Publisher Full Text\n\nDrucker J: Reconsidering the regional economic development impacts of higher education institutions in the United States. Regional Studies. 2016; 50(7): 1185–202. Publisher Full Text\n\nUNESCO: UNESCO Science Report: Towards 2030. Paris, France: UNESCO Publishing; 2016. Reference Source\n\nUK Collaborative on Development Research: UKCDR Strategy: The next 4 years (2018-2022). London: UKCDR; 2018. Reference Source\n\nDean L, Gregorius S, Bates I, et al.: Advancing the science of health research capacity strengthening in low-income and middle-income countries: a scoping review of the published literature, 2000-2016. BMJ Open. 2017; 7(12): e018718. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBates I, Boyd A, Smith H, et al.: A practical and systematic approach to organisational capacity strengthening for research in the health sector in Africa. Health Res Policy Syst. 2014; 12: 11. PubMed Abstract | Publisher Full Text | Free Full Text\n\nESSENCE on Health Research: Planning, Monitoring and Evaluation Framework for Research Capacity Strengthening. Geneva: ESSENCE on Health Research; 2016. Reference Source\n\nKhisa A, Gitau E, Pulford J, et al.: A framework and indicators to improve research capacity strengthening evaluation practice. Liverpool, UK: Liverpool School of Tropical Medicine and the African Population Health Research Centre; 2019. Publisher Full Text\n\nZachariah R, Tayler-Smith K, Van den Bergh R, et al.: Research to Policy and Practice Change: is Capacity Building in Operational Research Delivering the Goods? Trop Med Int Health. 2014; 19(9): 1068–75. PubMed Abstract | Publisher Full Text\n\nKebede D, Zielinski C, Mbondji PE, et al.: Human Resources in Health Research Institutions in Sub-Saharan African Countries: Results of a Questionnaire-Based Survey. J R Soc Med. 2014; 107(1 Suppl): 85–95. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBucur A, Kifor CV, Mărginean SC: Evaluation of the quality and quantity of research results in higher education. Quality and Quantity. 2018; 52(1): 101–18. Publisher Full Text\n\nMorel T, Maher D, Nyirenda T, et al.: Strengthening health research capacity in sub-Saharan Africa: mapping the 2012-2017 landscape of externally funded international postgraduate training at institutions in the region. Global Health. 2018; 14(1): 77. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNational Institutues of Health (NIH): WORLD RePORT: National Institutues of Health (NIH). 2018. Reference Source\n\nUKCDS: Health Research Capacity Strengthening: A UKCDS Mapping. London: United Kingdon Collaborative on Development Sciences (UKCDS); 2015. Reference Source\n\nPulford J, Price N, Amegee J, et al.: List of RCS Outcome Indicators.xlsx. Measuring the outcome and impact of research capacity strengthening initiatives: A review of indicators used or described in the published and grey literature - Full listing of retrieved RCS indicators. V1 ed: Harvard Dataverse; 2020.\n\nCOHRED: Changing Mindsets: Research capacity Strengthening in Low and Middle-Income Countries. Council on Health Research for Development. 2005. Reference Source\n\nFranzen SR, Chandler C, Lang T: Health research capacity development in low and middle income countries: reality or rhetoric? A systematic meta-narrative review of the qualitative literature. BMJ Open. 2017; 7(1): e012332. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCGIAR: Capacity Development Indicators for the second phase of CGIAR Research Programs. [Report]. In press. 2015.\n\nCooke J: A Framework to Evaluate Research Capacity Building in Health Care. BMC Fam Pract. 2005; 6: 44. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCole DC, Boyd A, Aslanyan G, et al.: Indicators for Tracking Programmes to Strengthen Health Research Capacity in Lower- and Middle-Income Countries: a Qualitative Synthesis. Health Res Policy Syst. 2014; 12: 17. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDFID: How to Note: Capacity Building in Research. Department for International Development; 2010. Reference Source\n\nNeilson S, Lusthaus C: IDRC Supported Capacity Building: Developing a Framework for Capturing Capacity Changes. 2007. Reference Source\n\nWilson LL, Rice M, Jones CT, et al.: Enhancing Research Capacity for Global Health: Evaluation of a Distance-Based Program for International Study Coordinators. J Contin Educ Health Prof. 2013; 33(1): 67–75. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMinja H, Nsanzabana C, Maure C, et al.: Impact of Health Research Capacity Strengthening in Low- and Middle-Income Countries: the Case of WHO/TDR Programmes. PLoS Negl Trop Dis. 2011; 5(10): e1351. PubMed Abstract | Publisher Full Text | Free Full Text\n\nUzochukwu B, Mbachu C, Onwujekwe O, et al.: Health Policy and Systems Research and Analysis in Nigeria: Examining Health Policymakers' and Researchers' Capacity Assets, Needs and Perspectives in South-East Nigeria. Health Res Policy Syst. 2016; 14: 13. PubMed Abstract | Publisher Full Text | Free Full Text\n\nErasmus E, Lehmann U, Agyepong IA, et al.: Strengthening Post-Graduate Educational Capacity for Health Policy and Systems Research and Analysis: the Strategy of the Consortium for Health Policy and Systems Analysis in Africa. Health Res Policy Syst. 2016; 14: 29. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHalpaap B: TDR [Tropical Disease Research] Performance Assessment Framework. WHO; 2011. Reference Source\n\nMahmood S, Hort K, Ahmed S, et al.: Strategies for Capacity Building for Health Research in Bangladesh: Role of Core Funding and a Common Monitoring and Evaluation Framework. Health Res Policy Syst. 2011; 9(1): 31. PubMed Abstract | |Publisher Full Text | Free Full Text\n\nMahendradhata Y, Nabieva J, Ahmad RA, et al.: Promoting Good Health Research Practice in Low- and Middle-income Countries. Global Health Action. 2016; 9: 32474. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRogers PJ: Using Programme Theory to Evaluate Complicated and Complex Aspects of Interventions. Evaluation. 2008; 14(1): 29–48. Publisher Full Text\n\nCraig P, Dieppe P, Macintyre S, et al.: Developing and evaluating complex interventions: the new Medical Research Council guidance. Int J Nurs Stud. 2013; 50(5): 587–92. PubMed Abstract | Publisher Full Text\n\nMotari M, Ota MO, Kirigia JM: Readiness of ethics review systems for a changing public health landscape in the WHO African Region. BMC Med Ethics. 2015; 16(1): 82. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChataway J, Ochieng C: Case Studies of the Political Economy of Science Granting Councils in Sub-Saharan Africa. University of Sussex: Science Policy Research Unit (SPRU), University of Sussex, United Kingdom and African Centre for Technology Studies (ACTS), Kenya; 2017. Reference Source\n\nJordans MJD, Chisholm D, Semrau M, et al.: Indicators for routine monitoring of effective mental healthcare coverage in low- and middle-income settings: a Delphi study. Health Policy Plan. 2016; 31(8): 1100–6. PubMed Abstract | Publisher Full Text\n\nMcGregor S, Henderson KJ, Kaldor JM: How are health research priorities set in low and middle income countries? A systematic review of published reports. PLoS One. 2014; 9(9): e108787. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "64400",
"date": "23 Jun 2020",
"name": "Erica Di Ruggiero",
"expertise": [
"Reviewer Expertise Public health research",
"evaluation"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article outlines clear research questions and methods and provides a very useful summary of findings from a structured review of research capacity strengthening indicators for use in low- and middle-income countries (LMICs). Terminology is overall quite clearly defined. Some greater precision relate to the use of the terminology: context for indicator's application; knowledge transfer vs. knowledge translation are used.\n\nThe definition of knowledge translation provided seems limiting and perhaps the authors meant only transfer. It would have been helpful to have some descriptive data on the sources of the publications/reports from which the indicators were derived (i.e. were they all published by academics vs. any from research funders). For example, it's unclear if any indicators developed by funders such as the International Development Research Centre and others that support LMIC research are included.\n\nThe limitations section is clear.\n\nIt would have been helpful to have the authors elaborate a bit more on the dearth of qualitative indicators, appreciating the fact that they would have 'scored poorly by default' because of the methodology used. Could the authors comment in the conclusion on areas for indicator development (like qualitative indicators; equity-related indicators - for e.g. I note that perception of equity in a specific partnership was part of the definition for collaboration and in the 'other' category, but to my knowledge, equity didn't really appear elsewhere)?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the 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": "64403",
"date": "23 Jun 2020",
"name": "Peter Taylor",
"expertise": [
"Reviewer Expertise International development",
"organizational learning and development",
"research capacity strengthening"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article addresses an issue that is receiving renewed interest in recent years - research capacity strengthening (RCS), and the particular challenge of evaluating outputs, outcomes and impacts of RCS initiatives.\nThe study undertook a structured review of RCS indicators in the published and grey literature. Key findings included the identification of rather few examples of quality RCS, with emphasis on four focal areas (research management and support, skill and knowledge development, research collaboration and knowledge transfer. The study concludes that there is significant room for development of indicators, and consequently the potential adoption of these to allow a more systematic approach to RCS approaches and to their subsequent evaluation.\nThe study is clearly presented and has a solid methodology. The validity of the findings rest on the extent to which the systematic review did identify published material that engages in this issue. As the authors note, it is likely that there is a wider body of grey literature in the form of project and program reports that were not located through the search. This suggests that there is need for more published work on this topic (making the paper therefore relevant and useful), and perhaps reinforces a wider view that many RCS efforts are inadequately evaluated (or not evaluated at all). An earlier World Bank Institute report on evaluation of training (Taschereau, 20101), for example, had highlighted challenges in evaluation of the impact of training and institutional development programs. The study refers briefly to RCS interventions, taking training as an example, but this only related to training which makes up a small percentage of the overall efforts towards RCS.\nIt would be very interesting to situate this welcome study in the context of broader discussions and debates on RCS, particularly as a contribution to theory and practice at strengthening research capacity at individual, organizational and system levels. The latter of these is the most complex to conceptualise, to implement, and to measure, and is receiving valuable attention from RCS stakeholders such as the Global Development Network (GDN, 20172) through their Doing Research Program - a growing source of literature for subsequent review.\nAs the authors of the study note, there is a danger in identifying RCS indicators that are seen as having universal application and attractiveness because they are relatively easy to measure. There is an equal, related danger that, due to relative measurability, a majority of RCS interventions become so streamlined in terms of their approach that they begin to follow recipe or blueprint approaches.\nThe study is agnostic on different approaches to RCS. Work undertaken by the Think Tank Initiative (TTI) for example (Weyrauch, 20143) has demonstrated a range of useful RCS approaches, including flexible financial support, accompanied learning supported by trusted advisors/program officers, action learning, training and others. In a final evaluation of the Think Tank Initiative (Christoplos et al., 20194), training was viewed as having had the least value amongst several intervention types in terms of RCS outcomes, whilst flexible financial support and accompanied learning processes were viewed as being significantly more effective. It would be interesting to identify indicators of outcomes or even impacts that might relate to different types of RCS interventions which were not included in the publications reviewed by this study.\nA key indicator of RCS identified by the TTI evaluation, which interestingly does not appear explicitly in the indicator list of this study, was leadership. As the authors indicate, there are likely to be other valuable indicators not surfaced through this review and this requires more work.\nThis study offers a very important contribution to a field currently being reinvigorated and is highly welcome. Rather than being valued because it may potentially offer a future blueprint list of indicators, (not least since, as the authors observe, the indicator list generated in this study is partial in comparison to a much wider potential range), its value lies particularly in its potential for contribution to further debate and dialogue on the theory and practice of RCS interventions and their evaluation; this dialogue can in turn be further informed by access to a more diverse set of grey literature and by engagement with stakeholders who have experience and interest in strengthening this work. Hopefully the authors of this study, and other researchers, will continue this important line of work and promote ongoing discussion and debate.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "64402",
"date": "24 Jun 2020",
"name": "Francesco Obino",
"expertise": [
"Reviewer Expertise Research capacity building methodologies",
"political theory",
"international relations"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 an timely contribution to an urgent question: how do we know if research capacity strengthening is working? The analysis of the problem (a. the lack of a shared reference framework for evaluating research capacity strengthening, which in turn implies that b. the scope for systematic and cumulative learning remains limited) is convincing and valid. The methodology is clearly explained and up to existing standards and expectations for this kind of exercise. The conclusions are straightforward, and the limitations well articulated (the focus on English, and the bias towards quantitative measures being the most important ones.)\nA few overall comments for the authors, keeping in mind the 'agenda' the article is trying to support (i.e. developing good examples of RCS indicators), and its potential uptake:\nRCS lack definition too, not just indicators. The article does not differentiate between research capacity strengthening done at the national level and at the international level, or in different fields (health sciences vs social sciences, etc.). While this is key to the aim of the paper to 'survey' existing indicators, the lack of solid evaluation of RCS can be also understood as the result not so much of 'underdevelopment' of the field, but of its overdevelopment in the absence of a shared definition of what RCS is. In this sense, putting all RCS (indicators) in the 'same box' might in fact reinforce the confusion around what is there to be measured, and how. International donor-funded, project-based RCS efforts differ (in scope, objectives and means) from the RCS effort of a science council or a local research training institution - despite overlaps. Often, the difference in objectives might make indicators hard to include in the same box. In this sense, the paper should acknowledge the lack of a shared definition of RCS, and the limitation it poses to an analysis of indicators. For this specific article, it might be useful to define RCS as international, donor-funded, project-based set of activities. Arguably, the very need of a discussion on RCS evaluation is largely driven by the fact that RCS is part of the evaluation-heavy international donor sector. This might help further defining the relevant timeframe for the search, and situating RCS historically.\n\nRCS is more than the sum of quality outputs. I wonder about the lack of discussion on 'process indicators' given the nature of RCS as a set of activities. These are notoriously difficult (but not impossible) to use in the donor-funded, project-based, time-bound RCS efforts, but might be very relevant to describe change and ultimately impact.\n\nRCS impacts research systems, policy, or development? When it comes to discussion of impacts and impact indicators, the lack of definition of RCS becomes an insurmountable limitation. The study could acknowledge the need for unpacking the link between output, outcome and impact measurement/definition (particularly in light of lack of shared definition of RCS) in internationally funded programs, as a complementary exercise to the surveying of indicators. The fact that the very few impact indicators identified reveal an expectation for RCS to deliver impact on population health outcomes is a good example of the limitations imposed by lack of clear definitions.\n\nHow important is the UK? Given the global audience of the piece, it might be useful to explain why the figures relating to projected RCS funding from the UK are significant to describe larger trends - particularly if figures include both 'direct' and 'indirect' RCS.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "64405",
"date": "29 Jun 2020",
"name": "Meriel Flint-O'Kane",
"expertise": [
"Reviewer Expertise Global health",
"research capacity strengthening",
"higher education capacity"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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: This paper provides a review and analysis of the quality and suitability of M&E metrics for funding that is allocated to strengthen research capacity in LMIC's. Published and grey literature has been reviewed to identify indicators used to measure the outputs, outcomes and impacts of relevant programmes and the findings have been assessed in terms of content and quality. The authors conclude that the outcome indicators identified were of low quality and impact indicators are almost always missing from RCS MEL frameworks and recommend further work to develop appropriate indicators to measure the outcomes and impacts of research capacity strengthening programmes/activities. Through the review of existing outcome indicators the authors have identified four focal areas against which indicators could be developed.\nMethods:\nThe search strategy and study selection is clearly described and links to source data are available. Data extraction and analysis methods are also clearly described.\nMajor:\nNo major points to address. This work is by a leading team in the field of RCS research and makes a useful contribution to the literature in providing a thorough review of indicators used to monitor and evaluate work funded to strengthen research capacity.\nMinor:\nThough the article is not focused on health research, health is specifically referred to in a few places throughout the article e.g. line 4 (and corresponding references) of the introduction, Research Funding example indicators in Table 2, RMS example indicators in Table 3, numbers 5 and 6 of the 6 outcome indicators meeting all four quality criteria refer to TDR, and the impact indicators are all acknowledged as being specific to health and wellbeing. It would be interesting to understand which other research disciplines featured in the literature reviewed, and the spread of results across disciplines in order that analysis and findings could indicate if there is variety between and/or within disciplines in approaches to MEL for RCS and what can be learnt from this.\n\nNo background, references or justification is given for the pre-determined 'quality' criteria.\n\nThe authors note in 'Limitations' that project documents were not available in the public domain and documents not in English were excluded. Further reflection on to what extent Log Frames, ToC's and other MEL docs for programmes that have RCS as a primary or secondary outcome would be in the public domain could be helpful e.g. is it common for delivery partners of RCS programmes to make their MEL docs publicly available? If not, are these indicators representative of those currently/recently being used by actors in the RCS programme delivery space or do they represent a subset that is more likely to have publicly available 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?\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/9-517
|
https://f1000research.com/articles/8-2074/v1
|
10 Dec 19
|
{
"type": "Research Article",
"title": "Different effects of soybean isoflavone genistein on transforming growth factor levels during orthodontic tooth movement among young and old rabbits",
"authors": [
"Verastuti Indriasari",
"Sri Suparwitri",
"Christnawati Christnawati",
"Ananto Ali Alhasyimi",
"Verastuti Indriasari",
"Sri Suparwitri",
"Christnawati Christnawati"
],
"abstract": "Background: Orthodontic treatment to improve aesthetics and for health reasons is performed in children and adults. Elderly individuals have low levels of estrogen, this results in alveolar bone resorption being greater than alveolar bone apposition. Isoflavones present in soybeans may be able to improve the remodeling process through the induction of osteoblastogenesis by increasing transforming growth factor-β1 (TGF-β1) levels. This study aimed to assess the comparative effect of soybean genistein isoflavone to TGF-β1 during orthodontic tooth movement among juvenile and adult rabbits. Methods: In this study, 12 healthy female rabbits were used. Subjects were divided into four groups (n=3); YG group (young rabbits), YGI group (young rabbits + isoflavones genistein), OG group (old rabbits), and OGI group (old rabbits + isoflavones genistein). Two lower incisors of the rabbit were moved distally using an orthodontic force (50 grams force) delivered by an open coil spring, which was inserted between two brackets. During active movements, the genistein isoflavones were given from the initial installation of the device until days 21, at a dose of 1.2 mg/kg BW once a day. Measurement of TGF-β levels were performed on days 1, 7, 14, 21 after appliance installation. TGF-β1 expression was analyzed using enzyme-linked immunosorbent assay (ELISA) and the optical density (OD) of the sample quantifed using a standard curve. The data obtained were analyzed using one-way Anova followed by Tukey HSD test. Results: The TGF-β1 levels were found to highest in the YGI group, and the TGF-β levels were significantly lower in the OG group (p<0.05). ELISA analysis also revealed that TGF-β1 levels of the OGI group were significantly higher when compared with the OG group (p<0.05). Conclusion: The administration of soybean genistein isoflavones could improve TGF-β1 levels in old rabbit’s during active orthodontic tooth movement.",
"keywords": [
"genistein",
"juvenile",
"adult",
"orthodontic orthodontic tooth movement"
],
"content": "Introduction\n\nToday’s society is witnessing an increased interest in cosmetic dentistry, thereby making orthodontics a necessity1. Orthodontic treatment has become one of the most procedures in cosmetic dentistry; it is performed to improve malocclusion, achieve good occlusion and dentofacial harmony2,3. The demand for orthodontic treatment has grown over time, not only in children, but also in adults4. The rising number of adults demanding orthodontic treatment presents a new challenge. A previous study found that adults alveolar bone remodeling is much slower than in juveniles due to decreased cellular activity and vascularity, this suggests that orthodontic treatment duration may be longer in adults due to a delay in orthodontic tooth movement (OTM)5.\n\nThe success of orthodontic treatment depends on the process of alveolar bone tissue remodeling during the treatment which involves bone apposition and resorption by osteoblasts and osteoclasts6. For children and juveniles who are in an early stage of growth and development, bone apposition and resorption are balanced. In contrast, in adults, and following the menopause, the rate of bone apposition is less than that of resorption7. With increasing age, estrogen levels decrease, resulting an increased incidence of osteoporosis with associated complications8. In the context of adult orthodontic patients, it may be beneficial to address this deficiency to improve treatment outcome.\n\nHughes et al.9, demonstrated that estrogen replacement may help to inhibit excessive bone loss by restricting osteoclast life span through promotion of apoptosis, mediated by transforming growth factor beta (TGF-β1). Estrogen effectively modulates TGF-β1 production in osteoblast and other cells. TGF-β1 is one of the most significant factors in bone formation, helping to maintain the balance between the dynamic processes of bone formation and bone resorption10. The development of natural remedies for the promotion of this mechanism, specifically, could be a useful and novel therapeutic approach to enhance bone remodeling by modulating the levels of TGF-β1 in adults during OTM.\n\nNowadays, the use of hormones derived from natural ingredients, namely phytohormones, has gained a lot of popularity. One of these is phytoestrogen, a substrate from plants with estrogen-like activity. The isoflavone genistein is a type of phytoestrogen which is the main polyphenol component of the soybean11. Some derivatives of soybeans have been recognized to have a positive effect on bone remodeling without triggering side effects. The effects of isoflavones on the improvement of osteoblast’s proliferation have previously been observed. Genistein administration has shown potential to increase osteoblast numbers during OTM4. Further study to explore the effect of genistein in enhancing osteoblastogenesis through improving TGF-β1 level is needed to validate its advantage in orthodontic bone remodeling. This study aimed establish the comparative effect of genistein on TGF-β1 during orthodontic tooth movement among juvenile and adult rabbits. The rabbits were selected as a model as it they have previously used to study the effect of medications on OTM12. Rabbit also provide an excellent model system to simulate the response of human tissue and are not aggressive, making them easy to handle and observe13.\n\n\nMethods\n\nAll experimental procedures involving animals were carried out in keeping with the Animal Research: Reporting of in vivo Experiments (ARRIVE) guidelines to ameliorate any suffering of animals. Ethical clearance was obtained from the Research Ethics Committee of the Faculty of Dentistry, Universitas Gadjah Mada, Indonesia, with number 00242/KKEP/FKG-UGM/EC/2019.\n\nWe used 12 female rabbits (Oryctolagus cuniculus) (Integrated Laboratory of Research and Testing, UGM, Indonesia), which were randomly divided into 4 groups (n=3), YG group (young rabbits/controls), YGI (young rabbits + isoflavones genistein), OG (old rabbits/controls), OGI (old rabbits + isoflavones genistein). Young rabbits were 3 months old and weighed approximately 1000 grams, whilst old rabbits were 3 years old and weighed approximately 4000 grams. Sample size (n=3) was determine based on Lemeshow's formula. A sample size of three animals in each group would present more than 85% power to detect significant differences with 0.45 effect size and at a significance level of α= .05. Sample groups were chosen utilizing simple random sampling. Each animal was assigned a tag number, the blind-folded researcher then picks numbered tags from the hat.\n\nAll the rabbits were housed individually in polycarbonate cages (0.90 × 0.60 × 0.60 m) for a week on 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. Animals were fed a standard pellet diet with tap water ad libitum, and were routinely inspected for food consumption and fecal characteristics.\n\nPrior to preforming the experimental procedure, rabbits were anesthetized with ketamine (160095, Kepro™, Netherlands), and xylazine (160096, Xyla™, Netherlands) (ketamine dose 35 mg/kg body weight and xylazine 5 mg/kg body weight), intramuscularly on the gluteus muscle during installation of the orthodontic appliance in their mouth. Two lower incisors of the rabbit were moved distally using a NiTi open coil spring 0.010”x 0.030” (O-951-1200, DynaFlex, the Netherlands) which was inserted between two preadjusted edgewise lower incisor brackets engaged to a 0.016 \"x 0.016\" rectangular stainless-steel wire (126-029977, American Orthodontics®, USA) (Figure 1). An open coil spring was compressed until it produced 50 grams orthodontic force continuously for 21 days (measured by tension gauge, MedKraft Orthodontics, USA). No reactivation of the appliance was done throughout the experiment. During active tooth movements, the genistein (produced by Prof. Mien Karmini from IPB) were given from the initial installation of the device until days 1, 7, 14 and 21, at a dose of 1.2 mg/kg BW dissolved in 5 ml of distilled water. This dose used was determined from evidence from a previous in vivo study14. The solution was given orally using a nasogastric tube once a day. Before gingival crevicular fluid sample collection, all the animals were observed for any general toxicity probability, including edema or deaths, and measured the body weight (using a digital scale, ZB22-P, Zieis®, USA). All these measurements were done by a single blinded observer.\n\nDesign of the orthodontic device: (a) rectangular stainless steel wire, (b) preadjusted edgewise bracket, and (c) open coil spring.\n\nThe GCF samples were collected from the two interproximal areas of the maxillary anterior teeth (mesial and distal sides) at four subsequent time points (1, 4, 7, and 14 days after the installation of the orthodontic appliance) (Figure 2). During GCF collection all animals were sedated. Two #15 sterilized paper points (A-022T, Dentsply, Germany) were used to collect the GCF. The paper points were gently inserted approximately 1 mm into the gingival sulcus and were left in situ for 30 s after removing the supragingival plaque with cotton swabs. Thereafter, the paper points were isolated with cotton rolls and dried. The dipped paper points were then stored in a sterile 1.5 ml tube comprising 350 µl of physiological saline solution (Nova-Tech, Inc., USA). The tube was centrifuged at 2000 rpm for 5 min at 4°C with the help of a microcentrifuge refrigerator (Eppendorf 5424R, USA) to elute the entire GCF element from the paper points. Paper points were removed, and the supernatant solution was kept at a temperature of −80°C in a refrigerator until further analysis. After the collection of the final samples, all rabbits were sacrificed with an overdosed anesthesia (intravenous injection of 100 mg/kg BW Pentobarbital, 1507002, Pubchem, USA) following Guidelines for the Euthanasia of Animals by American Veterinary Medical Association15 to collect bone samples for further histological analysis (Results not reported).\n\nThe TGF-β1 levels were detected and analyzed by enzyme-linked immunosorbent assay (ELISA). The analysis was done using a quantitative sandwich Rabbit TGF-β1 ELISA kit (ERB0119, FineTest, China). The TGF-β1 levels in the sample were found by extrapolating the optical densities of the samples on the standard curve. The optical densities were measured at 450 nm using a microplate reader (Bio-Rad Laboratories Inc., USA), single readings were taken for each animal. TGF-β1 expression levels were calculated as pg/mL. One-way ANOVA tests followed by Tukey’s post-hoc test were used to identify possible differences in TGF-β1 expression between controls and treatment groups. Statistical significance was set at p values < 0.05. Statistical analysis was processed with the SPSS 21.0 software system (SPSS Inc., Chicago, Illinois, USA).\n\n\nResults\n\nIn general, giving genistein isoflavones at the selected dose did not cause any general toxicity, edema or deaths, nor did it affect the body weight of rabbits (see underlying data16). ELISA analysis revealed that in young rabbits with OTM receiving genistein, TGF-β1 levels were significantly higher than other groups on day 1, 7, 14 and 21 (p < 0.05). In older rabbits with OTM receiving genistein, TGF-β1 levels were almost the same as those in young rabbits without genistein isoflavones administration on days 1, 7, 14 and 21 (p > 0.05). In the old rabbits group without genistein administration, TGF-β1 levels were significantly lower than the other groups on days 1, 7, 14 and 21 after bonding (p < 0.05). The YG and OGI group showed no significant difference in TGF-β1 levels at all time points (p > 0.05) (Table 1–Table 4; underlying data17).\n\nValues are presented as mean ± standard deviation or p-value only. *by ANOVA, *Significant differences between groups (p < 0.05).\n\nANOVA: Analysis of variance; YG: young rabbit, YGI: young rabbits+isoflavones genistein, OG: old rabbit, OGI: old rabbits+isoflavones genistein).\n\nValues are presented as mean ± standard deviation or p-value only. *by ANOVA, *Significant differences between groups (p < 0.05)\n\nANOVA: Analysis of variance; YG: young rabbit, YGI: young rabbits+isoflavones genistein, OG: old rabbit, OGI: old rabbits+isoflavones genistein).\n\nValues are presented as mean ± standard deviation or p-value only. *by ANOVA, *Significant differences between groups (p < 0.05).\n\nANOVA: Analysis of variance; YG: young rabbit, YGI: young rabbits+isoflavones genistein, OG: old rabbit, OGI: old rabbits+isoflavones genistein).\n\nValues are presented as mean ± standard deviation or p-value only. *by ANOVA, *Significant differences between groups (p < 0.05).\n\nANOVA: Analysis of variance; YG: young rabbit, YGI: young rabbits+isoflavones genistein, OG: old rabbit, OGI: old rabbits+isoflavones genistein).\n\n\nDiscussion\n\nThis investigation confirms the hypothesis that soybean isoflavone genistein administration could increase the TGF-β1 levels during orthodontic tooth movement, especially in older rabbits. In general, results demonstrated that in the subjects receiving soybean isoflavone genistein, TGF-β1 levels were significantly higher than the other groups. A previous study found that genistein can significantly increase the number of osteoblasts during orthodontic tooth movement4. Soybeans are the most common source of isoflavones, which have physiological effects that mimic native estrogen in maintaining bone formation rates in rats after ovariectomy-induced osteoporosis18. A previous in vitro study reported that genistein increased alkaline phosphatase expression along with protein and DNA content in osteoblastic MC3T3-E1 cells, indicating an anabolic effect19. Osteoblastogenesis is induced by TGF-β1, which is a physiological regulator of osteoblast differentiation and acts as a central component of the coupling of bone formation to resorption during bone remodeling20. TGF-β1 is osteogenic growth factor that has highly osteogenic attributes, enhancing osteoblast activity (by inducing bone marrow mesenchymal stem cells to differentiate into osteoblasts), stimulating osteoclast apoptosis, restraining osteoclastic activity, and resulting in bone formation21. Moreover, TGF-β1 has been found to perform a critical role in tissue regeneration as it potently improves the synthesis of connective tissue elements, such as type I collagen, proteoglycans, osteopontin, fibronectin, and osteonectin, during alveolar bone remodeling. The TGF-β1 superfamily further includes bone morphogenetic proteins (BMPs), which are the most potent inducers of bone formation22.\n\nOur results showed that TGF-β1 levels in old rabbits are lower than young rabbits. Aging can cause a decreasing in the osteoblasts number due to an imbalance in bone remodeling23. Impaired osteoblastogenesis induced by maturation will cause a characteristic of bone loss. It is widely accepted that the loss of bone with aging is a universal phenomenon which is associated with reduced bone strength. In adult individuals, the microarchitecture of trabecular bone becomes thin, while cortical bone becomes thin and porous24. A previous in vivo study demonstrated that in response to mechanical stimuli, the alveolar bone of the young rats was more active and sensitive than that of the adult rats4. As the aging process progresses, the periodontal ligament becomes more fibrotic and may influence how the tissues react to orthodontic forces25.\n\nThe old rabbit group exposed to soybean isoflavone genistein exhibited higher TGF-β1 levels than the old rabbit’s groups without soybean isoflavone genistein and this value was almost similar to the young rabbit’s groups without soybean isoflavone genistein. This is caused by administration of genistein to old rabbit’s groups which can overcome bone formation problems that are caused by a decrease in estrogen. Genistein is a plant compound with potent estrogenic activity that has similarity in structure with the human female hormone 17-β-estradiol. This hormone can bind to both alpha and beta estrogen receptors, and imitate the action of estrogens on target organs, and thereby it can provide many health benefits when used in some hormone-dependent conditions, including old age26. Estrogen prevents loss of bone mineral density through a TGFβ-dependent mechanism, which stimulate TGF-β1 production in the bone marrow with a critical “upstream” mechanism. The main role of TGF-β1 in osteoblastogenesis and bone formation is in recruiting osteoblast progenitors, stimulating their proliferation (increased DNA synthesis) and promoting the early stages of differentiation (bone matrix production) to increases bone formation. In addition, apoptosis of osteoblasts is blocked through maintenance of survival during transdifferentiation into osteocytes by TGF-β1. High levels of TGF-β1 will suppress RANKL expression, which plays a role in osteoclast differentiation27.\n\n\nConclusions\n\nThe administration of the soybean isoflavone genistein could induce TGF-β1 levels during orthodontic tooth movement especially in old rabbits. Further studies are necessary to confirm the efficacy and potency of soybean isoflavone genistein in human.\n\n\nData availability\n\nFigshare: Raw Data TGF beta isoflavone genistein. https://doi.org/10.6084/m9.figshare.1011670417\n\nThis project contains the following underlying data:\n\n- TGF LOCKY ANAVA.xlsx (Recorded TGF beta levels)\n\nFigshare: OUTPUT Statistic Isoflavon TGF.docx. https://doi.org/10.6084/m9.figshare.1011734028\n\nThis project contains the following underlying data:\n\n- OUTPUT Statistic Isoflavon TGF.docx (Output file from statistical analysis)\n\nFigshare: Animal Body Weight of Rabbit. https://doi.org/10.6084/m9.figshare.1031069616\n\nThis project contains the following underlying data:\n\n- Animal Body Weight.xlsx (Animal body weights)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
"appendix": "References\n\nAlhasyimi AA, Pudyani PS, Hafizi I: Effect of mangosteen peel extract as an antioxidant agent on the shear bond strength of orthodontic brackets bonded to bleached teeth. Dental Press J Orthod. 2018; 23(5): 58–64. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNarmada IB, Husodo KRD, Ardani IGAW, et al.: Effect of vitamin D during orthodontic tooth movement on receptor activator of nuclear factor kappa-β ligand expression and osteoclast number in pregnant wistar rat (Rattus novergicus). JKIMSU. 2019; 8(1): 37–42. Reference Source\n\nSofyanti E, Boel T, Soegiharto B, et al.: TMD symptoms and vertical mandibular symmetry in young adult orthodontic patients in North Sumatra, Indonesia: a cross-sectional study [version 2; peer review: 3 approved]. F1000Res. 2018; 7: 697. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSuparwitri S, Pudiyani 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\nLi X, Li M, Lu J, et al.: Age-related effects on osteoclastic activities after orthodontic tooth movement. Bone Joint Res. 2016; 5(10): 492–499. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSuparwitri S, Rosyida NF, Alhasyimi AA: Wheat seeds can delay orthodontic tooth movement by blocking osteoclastogenesis in rats. Clin Cosmet Investig Dent. 2019; 11: 243–249. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBoskey AL, Coleman R: Aging and bone. J Dent Res. 2010; 89(12): 1333–1348. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGambacciani M, Levancini M: Hormone replacement therapy and the prevention of postmenopausal osteoporosis. Prz Menopauzalny. 2014; 13(4): 213–220. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHughes DE, Dai A, Tiffee JC, et al.: Estrogen promotes apoptosis of murine osteoclasts mediated by TGF-beta. Nat Med. 1996; 2(10): 1132–6. PubMed Abstract | Publisher Full Text\n\nDole NS, Mazur CM, Acevedo C, et al.: Osteocyte-Intrinsic TGF-β Signaling Regulates Bone Quality through Perilacunar/Canalicular Remodeling. Cell Rep. 2017; 21(9): 2585–2596. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMing LG, Chen KM, Xian CJ: Functions and action mechanisms of flavonoids genistein and icariin in regulating bone remodeling. J Cell Physiol. 2013; 228(3): 513–521. PubMed Abstract | Publisher Full Text\n\nAlhasyimi AA, Pudyani PS, Asmara W, et al.: Enhancement of post-orthodontic tooth stability by carbonated hydroxyapatite-incorporated advanced platelet-rich fibrin in rabbits. Orthod Craniofac Res. 2018; 21(2): 112–118. PubMed Abstract | Publisher Full Text\n\nAbtahi M, Shafaee H, Saghravania N, et al.: Effect of corticosteroids on orthodontic tooth movement in a rabbit model. J Clin Pediatr Dent. 2014; 38(3): 285–289. PubMed Abstract | Publisher Full Text\n\nDang ZC, Lowik C: Dose-dependent effects of phytoestrogens on bone. Trends Endocrinol Metab. 2005; 16(5): 207–13. PubMed Abstract | Publisher Full Text\n\nLeary S, Underwood W, Anthony R, et al.: AVMA Guidelines for the Euthanasia of Animals, 2013 Edition. American Veterinary Medical Association, Schaumburg, IL. Reference Source\n\nIndriasari V, Suparwitri S, Christnawati C, et al.: Different effects of soybean isoflavone genistein on transforming growth factor levels during orthodontic tooth movement among young and old rabbits. F1000 Research. Dataset. Animal Body Weight of Rabbit. figshare. 2019. http://www.doi.org/10.6084/m9.figshare.10310696.v2\n\nIndriasari V, Suparwitri S, Christnawati C, et al.: Different effects of soybean isoflavone genistein on transforming growth factor levels during orthodontic tooth movement among young and old rabbits. F1000 Research. OUTPUT Statistic Isoflavon TGF.docx. figshare. Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.10116704.v3\n\nBrandi ML: Natural and synthetic isoflavones in the prevention and treatment of chronic diseases. Calcif Tissue Int. 1997; 61 Suppl 1: S5–S8. PubMed Abstract | Publisher Full Text\n\nSugimoto E, Yamaguchi M: Anabolic effect of genistein in osteoblastic MC3T3-E1 cells. Int J Mol Med. 2000; 5(5): 515–520. PubMed Abstract | Publisher Full Text\n\nErlebacher A, Filvaroff EH, Ye JQ, et al.: Osteoblastic responses to TGF-beta during bone remodeling. Mol Biol Cell. 1998; 9(7): 1903–1918. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVan Schepdael A, Vander Sloten J, Geris L: A mechanobiological model of orthodontic tooth movement. Biomech Model Mechanobiol. 2013; 12(2): 249–265. PubMed Abstract | Publisher Full Text\n\nAndriamanalijaona R, Benateau H, Barre PE, et al.: Effect of interleukin-1beta on transforming growth factor-beta and bone morphogenetic protein-2 expression in human periodontal ligament and alveolar bone cells in culture: modulation by avocado and soybean unsaponifiables. J Periodontol. 2006; 77(7): 1156–66. PubMed Abstract | Publisher Full Text\n\nRazi H, Birkhold AI, Weinkamer R, et al.: Aging leads to a dysregulation in mechanically driven bone formation and resorption. J Bone Miner Res. 2015; 30(10): 1864–1873. PubMed Abstract | Publisher Full Text\n\nCompston J: Age-related changes in bone remodelling and structure in men: histomorphometric studies. J Osteoporos. 2011; 2011: 108324. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAlikhani M, Chou MY, Khoo E, et al.: Age-dependent biologic response to orthodontic forces. Am J Orthod Dentofacial Orthop. 2018; 153(5): 632–644. PubMed Abstract | Publisher Full Text\n\nVitale DC, Piazza C, Melilli B, et al.: Isoflavones: estrogenic activity, biological effect and bioavailability. Eur J Drug Metab Pharmacokinet. 2013; 38(1): 15–25. PubMed Abstract | Publisher Full Text\n\nJanssens K, ten Dijke P, Janssens S, et al.: Transforming growth factor-beta1 to the bone. Endocr Rev. 2005; 26(6): 743–774. PubMed Abstract | Publisher Full Text\n\nIndriasari V, Suparwitri S, Christnawati C, et al.: Different effects of soybean isoflavone genistein on transforming growth factor levels during orthodontic tooth movement among young and old rabbits. F1000 Research. Raw Data TGF beta isoflavone genistein. figshare. Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.10117340"
}
|
[
{
"id": "58276",
"date": "20 Jan 2020",
"name": "Ida Bagus Narmada",
"expertise": [
"Reviewer Expertise Orthodontic Research (Orthodontic Tooth Movement)"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nFirst of all, please allow me to congratulate the authors for attempting to undertake this project which I found very interesting and of valuable additional knowledge. The manuscript itself is well-written and well-structured and I have to also commend the authors for this matter.\nHowever, I may require some clarifications on the following issues:\nPaper is well organized and easy to follow.\n\nNovelty is sufficient and high impact.\n\nTo improve the readability, It is recommended that the text is checked by a native English speaking person as many of the sentences might be misunderstood. I suggest a revision of the grammar structures by an expert editor in revising manuscripts.\nAbstract:\nPlease add the p value used for ANOVA and Tukey HSD (p<0.05) add homogenity and normality test p-value used (p>0.05) in the abstract section\n\nPlease add the exact p value obtained after the statistic analysis. (p=.....).\n\nThere is a minor mistake in keyword: orthodontic orthodontic tooth movement. It would be better to choose the keyword according the MeSH.\nIntroduction:\nPlease add your hypothesis\n\nPlease add the statement or sentence about is there any similar studies have been done before or not? or your study is the first study who investigate this topic\n\nThe Introduction section is OK. It would be better if you mentioned why you only examine the TGF-Beta only in this study?\n\nIt would be better add the reason why you choose the genistein instead of others natural active compound?\n\nMaterial and Methods:\nIt would better if you add the detail protocol or cite the protocol reference how to make or obtain the genistein\n\nIt would better if you add the reason why you choose 50 gram force to deliver the orthodontics tooth movement? please state what kind of force 50gf (light force or heavy force).\n\nPlease add the detail about the healthy condition of animal model, how to examine the condition of animal model? did you get a help from veterinarian? please state\n\nPlease explain how to purify the isolated GCF from animal study? how to minimize the GCF contamination and the bias?\n\nThe author mentioned \"During GCF collection all animals were sedated\" please add the detail protocol how you sedated the animal study.\n\nIn this study, the author perfomed the ANOVA and Tukey HSD. It would be better if the author also describe the Levene's and Shapiro Wilk test.\nResults:\nIt would be better to understand and attract the reader if you present the data in the diagrams.\n\nPlease add the result of Levene's and Shapiro Wilk test.\n\nPlease indicate whether the difference is significant or not in your graph/ diagrams as well, using some symbols (asteric).\n\nDiscussion:\nDiscussion of the results is quite comprehensive. In analyzing the results, the authors also show citations from the previous study to support the explanation of these results.\n\nThe answer to the hypothesis of this study have been included at the beginning of the discussion section.\n\nPlease mention the limitation of this study in the discussion section.\nReferences: The supporting references are OK and adequate.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "5559",
"date": "04 Jun 2020",
"name": "Ananto Ali Alhasyimi",
"role": "Author Response",
"response": "Dear Prof Narmada and Dr Nugraha 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. We have added this information in the present manuscript. We thank you again for the valuable insight."
}
]
},
{
"id": "62285",
"date": "29 May 2020",
"name": "Lidia Audrey Rocha Valadas",
"expertise": [
"Reviewer Expertise Pharmacology and Dentistry"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThat´s a well designed study and I have written some considerations below:\nThe introduction is clear and well written;\nOn the methods session I miss details of how you obtained the isoflavone, for me is the most important part to assure the reproducibility of the study.\nThe discussion is small and is necessary to improve it and should add the limitations of the study.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "5560",
"date": "04 Jun 2020",
"name": "Ananto Ali Alhasyimi",
"role": "Author Response",
"response": "Dear Prof Lidia Audrey Rocha ValadasThank 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. That´s a well designed study and I have written some considerations below:The introduction is clear and well written;Thank you so muchOn the methods session I miss details of how you obtained the isoflavone, for me is the most important part to assure the reproducibility of the study.We have edited the methods section and added this information in the present manuscript.The discussion is small and is necessary to improve it and should add the limitations of the study. We have edited the discussion section and added other references and limitation of the study as well. Thank you for the punctiliously review. We have added this information in the present manuscript We thank you again for the valuable insight."
}
]
}
] | 1
|
https://f1000research.com/articles/8-2074
|
https://f1000research.com/articles/9-515/v1
|
04 Jun 20
|
{
"type": "Method Article",
"title": "Identifying the spatial patterning characteristics of HIV positive clients not linked to care using a geographic information system",
"authors": [
"Kevin Rombosia",
"Nelly Rangara",
"Elizabeth Oele",
"Nancy Kirui",
"Douglas Olaka",
"Evans Ondura",
"Bernard Mitto",
"Nelly Rangara",
"Elizabeth Oele",
"Nancy Kirui",
"Douglas Olaka",
"Evans Ondura",
"Bernard Mitto"
],
"abstract": "Background: Linkage to care is a crucial early step in successful HIV treatment. This study sought to identify the spatial patterning characteristics of HIV positive clients that are not linked to care in the Kisumu West HIV program using a geographic information system. Methods: The geocodes of HIV positive, non-linked clients’ residences were exported to ArcGIS software. The spatial patterning characteristics of HIV clients that are testing positive and not linked to care was described using Global Moran’s I statistic, which is a measure of spatial autocorrelation. Results: A total of 14,077 clients were tested for HIV. Of clients testing positive for HIV, 10% (n=34) were not yet linked to care two weeks after the diagnosis of HIV. Of the HIV positive non-linked clients, most (65%; n= 32) had spatially identifiable data about where they resided. Regarding the spatial patterning characteristics of the clients who tested HIV positive but were not linked to care and with spatially identifiable residence information, the Global Moran I statistic for autocorrelation was 0.435 (z score 1.383, p- value 0.167). Conclusion: By using age as an attribute value, the spatial distribution of clients testing HIV positive and not being linked to care is random. Geographical information systems can be used to identify the spatial patterning characteristics of HIV positive clients that are not linked to care. A key requirement to achieving this would require the collection of precise and accurate spatially identifiable locator information but without compromising patient confidentiality.",
"keywords": [
"Geographic Information Systems",
"HIV",
"Linkage to care",
"Spatial patterning"
],
"content": "Introduction\n\nFor many years, the analysis and use of spatial data has guided public health efforts, and Dr. John Snow, who is often credited as the founder of epidemiology, is a classic example of using this type of data1. Geographic Information Systems (GIS) are information management systems for spatial data. Spatial data is data that is associated with specific location features. GIS enables the user to process, analyse and present geographically referenced data2. The use of GIS is increasingly being appreciated in the health sciences. For example, it can be used to map the distribution and accessibility of health facilities and assess the distribution of diseases, hence allowing for planning of their control.\n\nUNAIDS3 has an ambitious 90-90-90 strategy. This strategy stipulates that by the year 2020, 90% of all people with HIV infection will know their HIV status, 90% of HIV positive people will be on antiretroviral therapy (ART) and 90% of all people on treatment will have achieved viral suppression. However, Kenya still has low rates of linking HIV positive persons to HIV care4.\n\nInvestigating the spatial patterning of diseases is an epidemiology tool that provides greater understanding of their possible causes and the prevention steps that can be undertaken. According to Ward et al, using spatial statistics and maps to study diseases can provide better intuition in the study of diseases5.\n\nThere are many barriers to linkage to care following HIV positive results. Previous studies have reported factors such as being male, younger in age, having fears of drug side effects, busy schedules, transport costs, distance to clinics, stigma and fear of disclosure of HIV status, staff shortages at health facilities, and delays in getting services at health facilities as contributing to poor linkage and retention in care4. For these barriers to be effectively addressed and a move made towards the UNAIDS 90-90-90 goal, it requires, firstly, that any non-linked clients are identified and enrolled into care.\n\nThis study aimed to identify the spatial patterning characteristics of clients testing HIV positive who are not linked to care in the Kisumu West HIV program. If spatial clustering of clients is demonstrated for those who test HIV positive but are not linked to care in the Kisumu West HIV program, it would be an entry point for initiating targeted approaches for enrolling and retaining these clients in care. Such approaches include targeted community sensitization sessions on the need for linking into care following HIV positive results through targeted community health education in specific geographic areas that have a high prevalence of non-linkage. Also, it would help in obtaining linkage officers and case managers to target areas that have high densities of non-linked clients that are HIV positive. This targeted approach would be more cost effective, and should be combined with continuous HIV campaigns to address community stigma towards HIV/AIDS4.\n\nSimilarly, if it is demonstrated that the spatial patterning characteristics of clients testing HIV positive and not linked care is non-clustered but geographically randomly distributed, it would reinforce the need to establish rigid client tracking mechanisms post-HIV testing at health facilities.\n\n\nMethods\n\nKisumu County (Figure 1) is one of 47 counties in Kenya6. It lies within longitudes 33° 20’E and 35° 20’E and latitudes 0° 20’South and 0° 50’South. The total land area is approximately 2,086 Km2 and around 567 Km2 is covered by water. The County borders the expansive fresh water lake (Lake Victoria)7 and has 7 sub-counties namely; Kisumu Central, Kisumu East, Kisumu West, Seme, Nyando, Nyakach and Muhoroni. The sub-counties are further divided into administrative wards. The Kisumu West HIV program covers 6 wards namely North-west Kisumu, West Kisumu, Central Seme, East Seme, North Seme and West Seme.\n\nThis HIV care, treatment and prevention program in Seme Sub County and part of Kisumu West Sub County utilizes a “hub and spoke” model. Kombewa County Hospital, being the largest facility, is the “hub” wherein 24 peripheral health facilities (22 facilities in Seme Sub County and 2 facilities in Kisumu West Sub County) pivot for supply of commodities and drugs, training, viral load and HIV PCR sample processing.\n\nA rapid results initiative (RRI) is a management tool that is used to mobilize teams to rapidly achieve tangible results8. During the Kisumu West HIV Program RRI of 1st June 2017 to 31st August 2017, HIV counselling and testing was conducted in the 24 health facilities of the Kisumu West HIV Program. The eligibility criteria of the present study included clients who had never tested for HIV, had tested negative in the past 3 to 12 months or had tested negative in the past 3 months or less but the negative result could not be verified by any available health record. Clients who declined consent to participate or clients whose guardians declined consent were excluded from the study.\n\nPrimary data on clients’ name, age, gender, residence and HIV status was entered into HIV Testing Services (HTS) registers and fed into an information management system, DATIM (Data for Accountability, Transparency and Impact). Data for this study was extracted from DATIM. HIV positivity was calculated as:\n\nHIV positivity = (Number of clients testing HIV positive/Total Number of clients tested) × 100%\n\nThis HIV positivity was calculated for each testing health facility and aggregated at ward level wherein the health facility is located.\n\nThe data mined was entered into Microsoft Excel version 16.35 (RRID:SCR_016137) and then exported to STATA statistical software version 15 (RRID:SCR_012763) for processing and analysis.\n\nThe Kisumu County shapefile and geocodes of HIV positive, non-linked clients’ residences were exported to ArcGIS (ArcGIS for Desktop Basic, RRID:SCR_011081). An alternative open source GIS software for this spatial analysis is QGIS (RRID:SCR_018507). The locations of the clients’ residences, represented by the nearest known landmarks to their homes, were mapped on ArcGIS as points. These landmarks included features such as schools, churches, market centres etc. The geocodes of these landmarks were extracted from Google Maps (Version 2019 by Google Incorporated) by right clicking the “What’s here” Scalable Vector Graphic (SVG) marker and copy – pasting to Microsoft Excel.\n\nThe spatial patterning characteristics of HIV+ clients not linked to care was described using the Global Moran’s I statistic9. Global Moran's I statistic is a measure of spatial autocorrelation developed by Patrick Alfred Pierce Moran10.\n\nIn the Global Moran’s I statistic, spatial autocorrelation is characterized by a correlation of a point among nearby points in a given area and is given by:\n\nwhere n is the total number of observations (points or polygons), i and j represent different locations, Xi and Xj are values of the variable in the ith and jth locations, X¯ is the mean of the variable and Wij is the weight in the spatial weight matrix. The clients’ age was used as the variable of interest because previous studies have demonstrated that younger age is a significant causative factor for non – linkage to care10. The spatial weighted matrix that was used was the inverse distance weighting function in ArcGIS. This is also available in QGIS.\n\nWritten informed consent for publication of clients’ details was obtained from the clients/parents/guardians of the clients as appropriate. The data was de-identified by the use of client numbers (not names). The geocodes of the clients’ residences were represented using their nearest landmarks such as nearby schools, churches and market centres. The exact geocodes of the clients’ residences were not used in this study in order to protect confidentiality.\n\nEthical and institutional approvals for this study were obtained from the Makerere University Research and Ethics Committee and the Kisumu County Department of Health.\n\n\nResults\n\nA total of 14, 077 clients were tested for HIV during the RRI period (men 38%, n=5,304; women 62%, n=8,773). Of the total clients tested for HIV, most (32%, n= 4,552) belonged to the 25–49 year age group, within which the majority were women (62%, n=2,811) (Figure 2).\n\nA total of 345 (2%) clients tested positive for HIV (range = 0–5%; mean = 1.5±1.6). Most clients that were HIV+ were in the 25–49 years age group (5%, n=226), of which more clients were men (52%, n=118; women, 48%, n=108). The <1 year old age group had the lowest number of HIV+ clients (0%, n=0). In the 1 – 4-year age band, West Seme Ward had the HIV highest positivity (89%, (n=8)) (Figure 3). The health facilities with the highest number of HIV+ clients were Kombewa (4.5%, tested n=2,505, HIV+ n= 112), Manyuanda (4.2%, tested n=970, HIV+ n=41), Bodi (3.9%, tested n= 431, HIV+ n = 17) and Oriang Alwala (3.6%, tested n=248, HIV+ n= 9).\n\nOf all clients testing positive for HIV, 10% (n=34) were not yet linked to care two weeks after the diagnosis of HIV. Of these HIV+ non-linked clients, 50% were women (n=17), and most (65%, n= 32) had spatially identifiable data about their residences’ nearest landmarks.\n\nThe geocodes of the clients’ residences’ nearest landmarks were obtained from Google Maps. The geocodes are as shown in Table 1.\n\nThe geocodes were used to map the distribution of HIV positive non-linked clients as shown in Figure 4.\n\nRegarding the spatial patterning characteristics of the clients who tested HIV positive but were not linked to care and with spatially identifiable residence information, the Global Moran I statistic for autocorrelation was 0.435 (z score 1.383, p- value 0.167) (Figure 5).\n\n\nDiscussion\n\nIn this study, ArcGIS, a GIS software, was used to incorporate spatial attributes of clients’ residence to the HIV RRI data. This use of maps helped to better visualize the meaning of the numbers to provide more intuition about the distribution of HIV positivity and the spatial patterning of clients who tested HIV positive but were not linked to care two weeks after the diagnosis in the Kisumu West HIV program.\n\nHaving mapped the HIV positivity by different age groups and by different wards, it was established that most clients who were HIV+ were in the 25-49 year age groups (5%, n=226), which included more men (52%, n=118; women 48%, n=108). This shows that greater preventive efforts among this age group are required, and the establishment of targeted interventions for this cohort, such as viremia clinics, that seek to increase retention to care and viral suppression. Also, it was established that there was high positivity in children below 4 years in the West Seme Ward, which necessitates a need to audit the Prevention of Mother To Child HIV Transmission (PMTCT) programs in the health facilities in West Seme Ward in order to establish why there is a high number of HIV exposed infants seroconverting to active HIV infection. It was noted that while Oriang Alwala is a low volume health facility, it had the fourth highest HIV positivity rate (3.6%, tested=248, HIV positive n= 9); hence a need to redeploy more testers and adherence counsellors to this facility.\n\nRegarding linkage, it was established that 10% of the clients who tested positive during the RRI period for HIV in the Kisumu West HIV program had not been enrolled into care. Through the use of GIS, it was possible to describe the spatial patterning characteristics of these non-linked clients via their residences’ nearest landmarks. The Global Moran’s I statistic for autocorrelation was 0.435 (z score 1.383, p- value 0.167) indicating that the pattern does not appear to be significantly different than random. This means that the distribution of the clients testing HIV positive and not getting linked to care, using age as an attribute value, does not follow the path of spatial clustering. Therefore, the most effective strategy for getting such clients linked to care is not to administer blanket community sensitization sessions on the need for linking into care following HIV positive results through community health education as is routinely done in the Kisumu West HIV program. Instead, through the use of distribution maps, linkage officers can generate a better work plan of navigating the program area as they conduct home visits to these clients.\n\nIn calculating the Global Moran’s Autocorrelation statistic, the clients’ age was used as the y attribute value. It would have been more accurate to use a composite score that quantifies the ‘level of severity of a client’s non linkage’. However, even after an extensive literature review, we could not find such a scale.\n\nThere are some concerns or issues with use of GIS tools for public health efforts. Chief among those is a concern for privacy and confidentiality of individuals11. It would be useful to collect more precise and accurate locator details, e.g. exact residence details, to allow for a more targeted approach towards getting these clients enrolled into care. However, this needs to be balanced with the need to protect clients’ confidentiality. There are certain strategies that can be employed to protect patient confidentiality. For example, data may need to be aggregated to cover larger areas such as a zip code or county, helping to mask individual identities during dissemination of results. Residential geocodes can as well be displaced. Also, maps can be constructed at smaller scales so that less detail is revealed11. In this study, we represented clients’ residences using the landmarks nearest to the residences in order to conceal the pinpoint location of the clients’ residence’s information.\n\n\nConclusion\n\nGeographical information systems can be used identify spatial patterning characteristics of HIV positive clients that are not linked to care. A key requirement to achieving this would require the collection of precise and accurate spatially identifiable locator information. More research needs to be done on how geographical information systems can be used to describe the spatial patterning characteristics of HIV positive clients that are not linked to care without compromising patient confidentiality.\n\n\nData availability\n\nFigshare: HIV testing rapid results initiative 2017, https://doi.org/10.6084/m9.figshare.1224590612.\n\nThis project contains the following underlying data:\n\n- 2017_RRI_ALL_Facilities.csv; used to calculate HIV positivity per facility\n\n- HIV_Positivity.csv; used to calculate HIV positivity for different age bands\n\n- Non_Linkage_To_HIV_Care.csv; used to calculate non-linkage to HIV care for different age bands\n\n- Number_Newly_Enrolled_To_HIV_Care.csv; used to calculate non-linkage to HIV care for different age bands\n\n- Number_Newly_Started_On_ART.csv; used to display number started on ART\n\n- Number_Positive.csv; used to calculate HIV positivity for different age bands\n\n- Number_Tested.csv; used to calculate HIV positivity for different age bands\n\n- Residence_nearest_landmarks.csv; used for spatial 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": "References\n\nBertazzon S: GIS and Public Health. ISPRS Int J Geo-Information. 2014; 3(3): 868–870. Publisher Full Text\n\nFradelos EC, Papathanasiou IV, Mitsi D, et al.: Health based geographic information systems (GIS) and their applications. Acta Inform Medica. 2014; 22(6): 402–5. PubMed Abstract | Publisher Full Text | Free Full Text\n\nUnaids: 90-90-90: An ambitious treatment target to help end the AIDS epidemic. 2020. Reference Source\n\nWachira J, Naanyu V, Genberg B, et al.: Perceived health facility barriers to HIV linkage and retention in western Kenya. BMC Infect Dis. 2014; 14(S2): P30. Publisher Full Text | Free Full Text\n\nWard MP, Carpenter TE: Techniques for analysis of disease clustering in space and in time in veterinary epidemiology. Prev Vet Med. 2000; 45(3–4): 257–84. PubMed Abstract | Publisher Full Text\n\nKisumu County - Wikipedia. [cited 2020 May 18]. Reference Source\n\nKNBS: County Statistical Abstract: Kisumu County 2015. 2015; 1–80. Reference Source\n\nSchaffer R, Ashkenas R: Rapid Results! How 100-Day Projects Build the Capacity for Large-Scale Change. San Francisco: Jossey-Bass; 2005. Reference Source\n\nPfeiffer DU, Robinson TP, Stevenson M, et al.: Spatial Analysis in Epidemiology. Spat Anal Epidemiol. 2008; 1–154.\n\nXu Y, Kennedy E: An Introduction to Spatial Analysis in Social Science Research. 2015; 11. Publisher Full Text\n\nVine MF, Degnan D, Hanchette C: Geographic Information Systems: Their Use in Environmental Epidemiologic Research. Environ Health Perspect. 1997; 105(6): 598–605. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRombosia K: HIV testing rapid results initiative 2017. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12245906.v2"
}
|
[
{
"id": "65484",
"date": "13 Jul 2020",
"name": "Lambrini Kourkouta",
"expertise": [
"Reviewer Expertise Nursing",
"Hiv",
"Public Health. History"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIt is a very interesting and novel manuscript.\nHIV positive patients are often marginalised and a geagraphic information system would enable them to reach such a system. However, the references are not sufficient in number when such a subject is being examined.\nTo my way of thinking, more sources have to be found out in order to support the novelty of a geagraphic information system.\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": "66050",
"date": "07 Aug 2020",
"name": "Brian T. Montague",
"expertise": [
"Reviewer Expertise HIV",
"infectious disease",
"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\nThank you for the opportunity to review this interesting work. The authors describe use of GIS testing to looking at patterns of linkage to care in Western Kenya with the goal of identifying notable areas of less successful linkage that may suggest potential opportunities for program innovation.\n\nThe work is framed as a demonstration of the potential benefits of GIS mapping tools to support linkage to care. There is significant literature on this topic already, some of which I have added in citations here1,2,3,4,5,6. If the authors wish to focus on this as the rationale for the work, more clarity would be needed regarding what their methods add to the existing literature regarding the use of GIS in evaluation of HIV linkage and care in similar settings.\n\nFor their particular results here, the distribution of those not linked was noted to be random which would suggest that the GIS provided relatively less information in this setting. The conclusion drawn from this was a little bit unclear, namely that given the distribution was random blanked distributions of messages was not helpful. One could interpret this in the opposite manner, namely that since there is no clustering, widespread distribution of messages is needed. Targeting interventions to clients identified as out of care wherever they are does not leverage the information from the GIS analysis.\n\nWhile a potentially helpful tool, in this case it appears that the tool did not yield clearly actionable information and some clarification is needed with regard to the focus of the manuscript and what it adds to the existing literature.\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": "5829",
"date": "18 Aug 2020",
"name": "Kevin WW Rombosia",
"role": "Author Response",
"response": "Dear Sir, Thank you for taking your time to review our work. Regarding the conclusions drawn using the GIS analysis, this work influenced a programmatic shift in the Kisumu West HIV program from blanket strategies such as outreaches in areas deemed to have low linkage to care to a more focused approach of messaging (on the need for linking to care) through health education at the health facility level. Also, this may be viewed alongside the CDC revised approach of HIV testing at facility level as opposed to outreach testing (as previously widely practiced) because the former has been shown to have higher yield. This is an area we are continuing to look into and perhaps our next work should evaluate the improvement on linkage to care after adopting this approach. Kind regards, Kevin Rombosia"
}
]
}
] | 1
|
https://f1000research.com/articles/9-515
|
https://f1000research.com/articles/9-513/v1
|
04 Jun 20
|
{
"type": "Case Report",
"title": "Case Report: Can we differentiate mesothelioma from inflammatory pericardial constriction preoperatively?",
"authors": [
"Syed Shahmeer Raza",
"Irfan Ullah",
"Saira Kainat Awan",
"Muhammad Daniyal Nadeem",
"Gulsam Bashir",
"Irfan Ullah",
"Saira Kainat Awan",
"Muhammad Daniyal Nadeem",
"Gulsam Bashir"
],
"abstract": "Primary malignant pericardial mesothelioma (PMPM) is a rare cardiac tumor. The patient usually presents with pericardial constriction, usually misdiagnosed and wrongly managed. We present the case of a 21-year-old woman with a history of pericarditis and cardiac tamponade. The patient was referred from a clinic due to nausea, vomiting and ascites with lower extremity edema, soft and watery diarrhea, and right upper quadrant pain. Surgery (sternotomy and partial pericardiectomy) was proposed after looking at the different relevant investigations; it was not until that the patient was operated on that it was established that this wasn't a mere constriction but a malignancy. The patient shortly died after the operation. Pathology made a diagnosis of PMPM. Along with the classical symptoms those who present with level 1 thoracic adenopathy a decision to operate should be very carefully made, this may lead to a misdiagnosis of PMPM which postoperatively results in patient's death.",
"keywords": [
"Mesothelioma",
"Cardiac Surgery",
"Pericarditis"
],
"content": "Introduction\n\nThe overall outcome of primary malignant pericardial mesothelioma (PMPM) is very poor. Primary pericardial tumors are very rare, 40 times less common than metastatic ones. The patient usually presents with a pericardial constriction and is consequently misdiagnosed and wrongly managed, which can lead to death either on the operating table or right after the surgery1–2. So far there has not been any curative treatment proposed for PMPM. Palliative management is the mainstay of treatment for such tumours, which mostly includes chemotherapy and pericardiectomy. However, even with these measures, the patient doesn't survive for long3.\n\n\nCase report\n\nA 21-year-old woman with a past medical history of pericarditis and cardiac tamponade presented to the Emergency Department of our hospital after a referral from a clinic due to nausea, vomiting and ascites with lower extremity oedema, soft and watery diarrhoea, right upper quadrant pain, scleral icterus, abdominal distention, oedema, rash and joint pain.\n\nAt two months prior to presentation she developed pleuritic chest pain and arthralgias and was diagnosed with cardiac tamponade. Pericardiocentesis was performed, which was negative for any malignant cells, showing only mild nonspecific inflammation. At that time, she had a negative workup for HIV, hepatitis, antinuclear antibody and Anti-Smith antibodies. She was hospitalised for 10 days and discharged without medication.\n\nUpon her arrival to the Emergency Room, she complained of not being able to keep food down and complained of a distended abdomen when taking meals, though she could tolerate some fluid. The distention improved with emesis. She reported a weight loss of 20 lb (approximately 9 kg) in the past month. She was not taking any medication at presentation. The Cardiology Department presented the case to our service for Surgical consultation in the Cardiothoracic Surgery Department of our Hospital.\n\nSystemic review showed the patient had fatigue, shortness of breath, cough, weight change and arthralgias. Upon physical examination, she had a rash in the lower extremities and back. Heart exam showed that the point of maximal impact was not displaced. No murmurs, gallop or bruit or raised jugular venous pressure. Bilaterally edema was positive in both her legs\n\nThe ECG showed low voltage QRS, sinus tachycardia and T wave abnormality. Chest X ray Impression- the cardiomediastinal silhouette Borderline enlarged. Cardiac catheterization-final anatomic diagnosis: constrictive pericarditis (by echo and cardiac magnetic resonance imaging (MRI)). The recommendation was to evaluate for pericardial stripping.\n\nEchocardiography showed that left ventricular systolic function was moderately decreased. Ejection Fraction was 30 (± 5). The right ventricular systolic function was moderate to severely decreased, along with abnormal soft tissue echoes in the anterior mediastinum. Constrictive Pericarditis with markedly thickened Pericardium was appreciated (Figure 1).\n\nMRI was conducted to further examine, as echocardiography alone was difficult to interpret making it hard to arrive at a diagnosis. Cardiac MRI showed diffuse pericardial thickening with only minimal amount of effusion. The chest wall appeared normal (Figure 2). Constrictive pericarditis was the main concern and cardiac magnetic resonance (CMR) was performed for further characterization. The diagnosis was constrictive pericarditis with markedly thickened pericardium (Figure 3).\n\nShort axis (A) and 2 chamber (B) images of steady-state free precession sequence demonstrating diffuse pericardial thickening with loss of normal interphase between the myocardium and pericardium (arrow). Short axis (C) and two-chamber (D) views of inversion recovery images obtained approximately 10 minutes after IV injection of gadolinium, showing diffuse pathological pericardial enhancement (arrows) and to a lesser extent epicardial enhancement and loss of normal myocardial - epicardial interphase (arrowhead).\n\nComputed tomography findings showed there was an underlying Constrictive Pericarditis with a markedly thickened Pericardium. Also observed were anterior mediastinal lymphadenopathy, moderate pleural effusion and associated atelectasis (Figure 4).\n\nThe patient had a full cardiology work-up on the day of presentation, including echocradiogram, cardiac catheterisation and MRI. We agreed that pericardiectomy would be warranted given her degree of constriction. After extensive consideration and discussion with the patient and her family she agreed to undergo sternotomy and pericardiotomy. She was taken to the operating room two days after presenting and after opening the chest, the pericardium was noted to be thick and firm. When we began to dissect the pericardium off the right ventricle, it seemed it was invading the right ventricular mass. We therefore began to suspect a malignant tumor, and after careful stripping of the densely adherent pericardium, a tissue sample was sent to pathology. A frozen section revealed findings consistent with a malignant tumor, possibly a mesothelioma or metastatic breast cancer. Partial pericardiectomy was performed as planned. Shortly after coming off bypass it was clear that the patient was not tolerating cardiac function independently, and high-dose norepinephrine (0.14 µg/kg/min IV infusion), epinephrine (1 mg bolus) and vasopressin (40 Units IV push after second epinephrine dose) was administered. She shortly went into ventricular fibrillation arrest. Multiple shocks and open cardiac massage achieved a viable rhythm with inotropic and intra-aortic balloon pump (IABP) assistance. The chest was left open and she was transferred to the Surgical Intensive Care Unit (SICU) in critical condition. Shortly after arriving the SICU from the operating room she became hypotensive. Multiple efforts including multiple shocks, high dose epinephrine and continuous CPR were made to resuscitate her. After half an hour it was evident that we were unable to resuscitate her. The patient then developed electromechanical dissociation and died shortly after.\n\nFinal pathologic diagnosis of the specimen taken as excisional biopsy revealed biphasic malignant mesothelioma alongside signs of level 1 thoracic adenopathy, which is consistent with cardiac neoplasia PMPM. The morphologic features of the specimen and the immunophenotypic findings support diagnosis of pericardial malignant mesothelioma. Retrospective evaluation of the CMR revealed subtle loss of the normal interface between the epicardial myocardium and the pericardium on steady-state free precession and inversion recovery delayed enhancement images which suggested myocardial invasion by the pericardial process. This should raise concern for neoplastic etiology (Figure 4).\n\n\nDiscussion\n\nAs discussed earlier, this disease is rare and is difficult to diagnose and manage. This is not only tiresome and challenging but can be misleading requiring multiple kinds of imaging techniques and approaches1,2. The overall outcome after all medical or surgical intervention is also relatively poor. The patient is misdiagnosed for pericardial constriction which leads to unnecessary surgical intervention and ultimately results in the death of the patient. Asbestos has always been thought to be the causative agent and is linked with it but not such evidence has yet been documented in the medical literature. A large number of pericardial tumors are of secondary or metastatic nature, with few of primary nature, although the etiology of such tumors is still not known3–5. The disease mimics other diseases of cardiac origin like cardiac tamponade, coronary cardiac diseases and constrictive pericarditis, which further adds to the misery of misdiagnosis. PMPM has a very low incidence—nearly one in 40 million. Notably, a diagnosis of PMPM is mostly made post-mortem and rarely ante-mortem. Palliative management is the mainstay of treatment for such tumors, mostly including chemotherapy and pericardiectomy, but even with this, the patient is unlikely to survive for long.\n\nPMPM is characterized by a sudden onset of symptoms, with patient history mostly misleading and unclear. The tumor may occasionally spread to surrounding mediastinal structures and cause manifestations, but it usually mimics other diseases like myxomas or a tubercular pericarditis. Emboli, conduction blocks and distant metastasis have also been reported6.\n\nTo our knowledge, no information is available about the accuracy of cytologic diagnosis of pericardial mesothelioma; however, in a large study of cytologic samples in 517 cases of pleural mesothelioma, definitive diagnosis was only made in 73% of cases, with a further 13% being suspicious7. Only 25% of reported cases of pericardial mesothelioma were diagnosed antemortem5. Though surgery is often discussed, in general outcomes are poor and not significantly impacted by surgical intervention8.\n\nA key question for surgeons is if there are findings suggestive of mesothelioma that may help avoid unsuccessful procedures. In the presence of very thickened pericardium, CMR might help differentiate the process preoperatively. Ohnishi et al. reported that gadolinium DTPA-enhanced MRI led the authors to suspect a tumor though in their patient there was a discrete soft tissue mass (5). In our case, the mesothelioma was diffuse, not localized, though the loss of the epicardial-myocardial interface was suggestive of tumor invasion. Had mesothelioma been suspected prospectively, the procedure would have been a biopsy, rather than attempted pericardiectomy.\n\nA varied and multidimensional approach needs to be considered by the physician in managing patients who present with such symptoms. The investigations need to be thoroughly evaluated and a decision should then be made. Along with the classical symptoms those who present with level 1 thoracic adenopathy, a decision to operate should be very carefully made in such patients, this may lead to a misdiagnosis of PMPM which may postoperatively result in patient's death. Level 1 Adenopathy presenting with Classical S/S and Pericardial constriction should by far be considered diagnostic for PMPM.\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 parents of the patient.",
"appendix": "References\n\nMaisch B, Seferović PM, Ristić AD, et al.: Guidelines on the Diagnosis and Management of Pericardial Diseases Executive Summary; The Task Force on the Diagnosis and Management of Pericardial Diseases of the European Society of Cardiology. Eur Heart J. 2004; 25(7): 587–610. PubMed Abstract | Publisher Full Text\n\nLanger C, Butz T, Horstkotte D: Multimodality in Imaging Calcific Constrictive Pericarditis. Heart. 2006; 92(9): 1289. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLuk A, Ahn E, Vaideeswar P, et al.: Pericardial Tumors. Semin Diagn Pathol. 2008; 25(1): 47–53. PubMed Abstract | Publisher Full Text\n\nOreopoulos G, Mickleborough L, Daniel L, et al.: Primary Pericardial Mesothelioma Presenting as Constrictive Pericarditis. Can J Cardiol. 1999; 15(12): 1367–72. PubMed Abstract\n\nSuman S, Schofield P, Large S: Primary Pericardial Mesothelioma Presenting as Pericardial Constriction: A Case Report. Heart. 2004; 90(1): e4. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSzczechowski L, Janiec K: [Pericardial Mesothelioma as a Very Rare Cause of Recurrent Cerebral Emboli]. Wiad Lek. 1992; 45(21–22): 857–61. PubMed Abstract\n\nSegal A, Sterrett GF, Frost FA, et al.: A Diagnosis of Malignant Pleural Mesothelioma Can Be Made by Effusion Cytology: Results of a 20 Year Audit. Pathology. 2013; 45(1): 44–8. PubMed Abstract | Publisher Full Text\n\nKardzic R, Kostic-Banovic L, Antovic A, et al.: Primary pericardial mesothelioma presenting as constrictive pericarditis. Arch Oncol. 2005; 13(3–4): 150–2. Publisher Full Text"
}
|
[
{
"id": "64883",
"date": "17 Jun 2020",
"name": "Muhammad Aasim",
"expertise": [
"Reviewer Expertise Cardiac Surgery",
"Adult Cardiac Surgery",
"Congenital Cardiac Surgery",
"Cardiothoracic Surgery",
"Cardiovascular Surgery."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis Case Report is very much pertinent to the practice of cardiothoracic surgeons treating constrictive pericarditis. Primary Malignant Pericardial Mesothelioma is a rare cause of constrictive pericarditis, but associated with poor surgical outcome, hence it shall be understood and diagnosed well. This case is adequately described and presented appropriately to guide the diagnosing cardiologists and treating cardiothoracic surgeons.\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": "99934",
"date": "08 Feb 2022",
"name": "Mehmet Ali Bedirhan",
"expertise": [
"Reviewer Expertise Thoracic & cardiovascular surgery"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nFirstly, I am sorry for the loss of patients. Primary pericardial mesothelioma (PPM) is a rare disease with unfortunate poor prognosis. Most of the PPM patients (75%) receive diagnosis in late stage disease forms (McGehee et al. (20191)). PPM is not a common tumor which makes its diagnosis cumbersome. Clinical dompt and experience is mandatory for its diagnosis; all in all who thinks a 21-year old can have PPM. Nevertheless, my first PPM case was 12-year old male in 1986 who have managed to live only a few months.\nMost pericardial tumors are secondary lesions due to local spread of lung and mediastinal tumors or metastatic lesions originating from lung cancer, breast cancer, lymphoma or melanoma. Primary malignant tumors are rare and 50% are mesotheliomas. Other causes are sarcoma, hemangioma and teratoma.\n\nIn differential diagnosis, radiological examinations such as computed tomography may not help differentiate PPM from inflammatory constrictive pericarditis (ICP). In both diseases, clinician may observe effusion, cardiomegaly, ascite or pedal edema. Cardiac MR is recommended when tumoral invasion of the myocardium exist (Cosyns et al. (20152)). CMR does not provide a histological diagnosis; but provides clues for diagnosis and tissue characterization, with excellent soft tissue separation and wide field of view, and may allow the origin, extent and borders of the tumor to be determined. Most tumors will retain contrast and show delayed contrast (wash-out sign) (Wang et al. (20033) and Hoffmann et al. (19984)) .\n\nAs an invasive diagnostic method of ICP, right sided cardiac catheterization is pretty essential to reach a precise diagnosis. During this method, elevated right atrial pressure, plateau sign or no pressure change during cardiac cycles may be the findings. In addition, ventricular ejection fraction is preserved in ICP whereas it is affected in PPM (Cosyns et al. (20152)).\nLastly, dissection of constrictive layer start from left side of the heart (reverse direction of intracardiac blood flow). Otherwise pulmonary edema develops and acute mortal right sided failure may occur.\n\nPPM remains a rare and poorly understood malignancy of unclear etiology (McGehee et al. (20191)). Despite these diagnostic tools and experience we sometimes still in trouble. I personally thank the authors who share their practice with us.\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/9-513
|
https://f1000research.com/articles/9-505/v1
|
03 Jun 20
|
{
"type": "Case Report",
"title": "Case Report: DSM–5 misses an edge case in tic disorders nosology",
"authors": [
"Kevin J. Black"
],
"abstract": "A boy with multiple phonic tics, one lifetime motor tic, and no impairment or marked distress does not meet criteria for any DSM–5 tic disorder diagnosis. The next version of the Diagnostic and Statistical Manual should adjust the criteria for Tourette's Disorder and/or for \"other specified tic disorder\" and \"unspecified tic disorder.\"",
"keywords": [
"Tourette syndrome",
"tic disorders",
"nosology",
"DSM-5",
"case report"
],
"content": "Introduction\n\nOver time, tic disorder nosology has changed1. The current research criteria changed modestly with the adoption of DSM–52. I describe a case of a child with motor and vocal tics that demonstrates a gap in DSM–5’s diagnostic criteria for tic disorders3.\n\n\nCase report\n\nAt age 9, a right-handed, non-Hispanic white boy and his mother participated in a thorough research assessment as part of the New Tics study4. The study was approved by the Washington University in St. Louis Human Research Protection Office, IRB ID #201109157, and his mother gave informed consent. This visit included questionnaires, K-SADS parent and child interviews, history of illness, neurological exam, 30 minutes of observation of the child alone via video, and YGTSS rating. His mother was a reliable informant, an elementary school teacher well informed about tics, and his father was a physician. His mother dated his tic onset to 8 months ago, at age 8. The child had seasonal allergies but the phonic tics were present when he had no allergy symptoms, and the tics did not respond to cetirizine. He was taking extended-release mixed amphetamine salts (40 mg daily) for ADHD with good response. K-SADS diagnoses were specific phobia, past social anxiety disorder, past nocturnal enuresis, predominantly inattentive ADHD since age 6, and provisional tic disorder.\n\nNeurological exam was normal except for a medium-loud snort occurring once during the exam. He had simple phonic tics (sniff or snort, cough, clear throat), one motor tic (biting lower lip softly, seen during video observation) and no complex tics. He bit his nails sometimes since early childhood, but this was not counted as a tic given the timing and its high prevalence among young children5. He also had one probable simple motor stereotypy (rarely shook his hands up and down near his chest before sports or social events since age 5 or younger; he said “I like doing that” and said it didn’t feel like his tics; seen only once in over an hour of observation). YGTSS scores were: motor tics 5, phonic tics 12, impairment 10.\n\nHe returned at 12 months after tic onset. The stimulant continued at the same dose, now without an antihistamine. The same tics continued within the past week, though not every day, but no tics were observed at the visit. YGTSS scores were: motor tics 4, phonic tics 6, impairment 0. By 24 months after tic onset, he was taking no medications. The lip biting had disappeared but the phonic tics continued. He reported that “they’re kind of annoying and I would like them to go away,” but he did not have marked distress and the tics did not affect self-esteem, family life, friendships or school functioning. No tics were observed during thorough history and a neurological examination, but sniffing, coughing and forceful nasal exhalations were observed by video when he was alone in the room. No motor tics were observed. YGTSS scores were: motor tics 0, phonic tics 9, impairment 0. Diagnostic Confidence Index score was 356.\n\n\nDiscussion\n\nThis boy has a fairly typical history for mild Tourette syndrome, except that he has only one motor tic. (Other clinicians may choose to count the nail biting or hand shaking as tics, but for the present discussion the main point is that some children will have a presentation with vocal tics and one motor tic.) The DSM–5 criteria for Tourette’s Disorder require “multiple” motor tics2. The criteria for Persistent (Chronic) Vocal Tic Disorder exclude patients who have experienced both motor and vocal tics. At the follow-up visits, the duration of ticcing excludes Provisional Tic Disorder, and the history and examination provided no evidence for causation by a substance or non-psychiatric illness. The residual categories, Other Specified Tic Disorder and Unspecified Tic Disorder, require “clinically significant distress or impairment in social, occupational, or other important areas of functioning.” This last criterion is shared with most DSM–5 disorders, but since DSM–IV–TR it has been omitted for Tourette’s Disorder7. Thus this boy does not meet DSM–5 criteria for any tic disorder. Woods and Thomsen addressed the situation in which a patient has vocal tics and exactly one motor tic, and concluded that “the requirement that multiple motor tics exist seems arbitrary and unnecessarily exclusive”8. A DSM–5 work group discussed the nosological issues in detail7. They retained the “multiple” motor tic requirement for Tourette’s Disorder, and did not propose reinstating the impairment or distress criterion in the “not otherwise specified” diagnosis. Roessner and colleagues provided critical feedback on the proposed criteria for tic disorders, but also assumed the impairment or distress criterion would be absent for all tic disorders9. This change appears to have been inadvertent.\n\n\nConclusions\n\nThere is no clinical import for this child, as his symptoms bother him only slightly. But this case demonstrates that the current DSM–5 criteria inadvertently provide no diagnosis in this case, which may occasionally affect research on tic disorders. The exclusion of one motor tic from both Tourette’s Disorder and Persistent Vocal Tic Disorder leaves a gap. The residual diagnostic categories no longer cover this gap since DSM–5 requires the “impairment or distress” criterion for them, though that requirement may have been accidental. I propose that future revisions omit it for all tic disorders. I also agree with Woods and Thomsen’s opinion that one motor tic and multiple phonic tics is best described as Tourette syndrome.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details was obtained from the parent of the patient.",
"appendix": "References\n\nRobertson MM, Eapen V: Tourette’s: syndrome, disorder or spectrum? classificatory challenges and an appraisal of the DSM criteria. Asian J Psychiatr. 2014; 11: 106–13. PubMed Abstract | Publisher Full Text\n\nAmerican Psychiatric Association: Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition: DSM – 5. American Psychiatric Association, Arlington, VA. 2013. Reference Source\n\nRiley DS, Barber MS, Kienle GS, et al.: CARE guidelines for case reports: explanation and elaboration document. J Clin Epidemiol. 2017; 89: 218–235. PubMed Abstract | Publisher Full Text\n\nBlack KJ, Kim S, Schlaggar BL, et al.: The New Tics study: A novel approach to pathophysiology and cause of tic disorders. J Psychiatr Brain Sci. 2020; 5: e200012. Publisher Full Text\n\nWinebrake JP, Grover K, Halteh P, et al.: Pediatric onychophagia: A survey-based study of prevalence, etiologies, and co-morbidities. Am J Clin Dermatol. 2018; 19(6): 887–891. PubMed Abstract | Publisher Full Text\n\nRobertson MM, Banerjee S, Kurlan R, et al.: The Tourette Syndrome Diagnostic Confidence Index: Development and clinical associations. Neurology. 1999; 53(9): 2108–2112. PubMed Abstract | Publisher Full Text\n\nWalkup JT, Ferrão Y, Leckman JF, et al.: Tic disorders: some key issues for DSM–V. Depress Anxiety. 2010; 27(6): 600–10. PubMed Abstract | Publisher Full Text\n\nWoods DW, Thomsen PH: Tourette and tic disorders in ICD-11: standing at the diagnostic crossroads. Braz J Psychiatry. 2014; 36 Suppl 1: 51–8. PubMed Abstract | Publisher Full Text\n\nRoessner V, Hoekstra PJ, Rothenberger A: Tourette’s disorder and other tic disorders in DSM–5: a comment. Eur Child Adolesc Psychiatry. 2011; 20(2): 71–4. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "64373",
"date": "11 Jun 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\nKevin Black’s case report highlights a possible hole in the current classification scheme for tic disorders. The author describes the clinical presentation of a 9-year-old boy who has a fairly typical history for mild Tourette syndrome, except that he has only one motor tic. Since he does not have an impairment or marked distress, he does not meet current diagnostic criteria for any DSM-5 tic disorder diagnosis. This interesting report is useful, as it hints at two possible solutions, not mutually exclusive. Firstly, it would be appropriate to consider relaxing the current criteria for the categories “Other Specified Tic Disorder” and “Unspecified Tic Disorder”, by reviewing the persisting criterion of “clinically significant distress or impairment in social, occupational, or other important areas of functioning”. Secondly, on a more general level, this case report calls into question the rationale for the distinction between motor and vocal/phonic tics. It is hoped that future classification systems will be increasingly more informed by advances in research that would allow them to “carve nature at its joints”.\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": "5605",
"date": "15 Jun 2020",
"name": "Kevin J Black",
"role": "Author Response F1000Research Advisory Board Member",
"response": "I thank Dr. Cavanna for the review. I agree with his comment about the artificial and historical nature of the distinction between motor and vocal tics."
}
]
},
{
"id": "64376",
"date": "17 Jun 2020",
"name": "Carol A. Mathews",
"expertise": [
"Reviewer Expertise Tourette syndrome genetics",
"OCD 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 case report discusses a 9 year old boy who presents with persistent, multiple vocal tics, and one motor tic. Based on history and physical exam, he clearly meets criteria for multiple chronic tics, but has no demonstrated impairment. I would argue that he has mild distress, as evidenced by the fact that he finds them annoying, but from a practical perspective, they do not functionally impact his life. The author argues that this child's tics would not fit into any of the current DSM-5 tic disorder classifications, and most specifically, that he would not fit into either of the two that are closest to his presentation--Tourette's Disorder, because he does not have multiple motor tics, or persistent vocal tic disorder, because he does have one motor tic. This highlights a gap in the current diagnostic criteria (even leaving out the question of whether impairment should be necessary for a diagnosis, which is also addressed). Genetic and emerging clinical data suggest that chronic tics, whether primarily motor,vocal, or a mix of both, emerge from the same underlying genetic causes, and thus are likely to be manifestations of the same disorder. The argument is made, appropriately, for a revisiting of the tic disorder diagnoses in the DSM, to account for this gap, and perhaps also to incorporate the current genetic information when determining the disorder definitions for the next update.\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": "5631",
"date": "26 Jun 2020",
"name": "Kevin J Black",
"role": "Author Response F1000Research Advisory Board Member",
"response": "I appreciate Dr. Mathews's thoughtful input, including the 2 recent references, and agree with her comments about the likely nosological unity of the various DSM–5 chronic tic disorders including TS."
}
]
}
] | 1
|
https://f1000research.com/articles/9-505
|
https://f1000research.com/articles/9-504/v1
|
03 Jun 20
|
{
"type": "Research Article",
"title": "Assessment of adherence level for neonatal hyperbilirubinemia management by various physicians in Iraq: a multi-clinic study",
"authors": [
"Numan Nafie Hameed",
"Hikmat Noori Yousif",
"Hayder Adnan Fawzi",
"Numan Nafie Hameed",
"Hikmat Noori Yousif"
],
"abstract": "Background: Neonatal jaundice is a physiological process that occurs normally for every infant to a varying degree. In some cases, this process becomes pathological and imposes an increased risk of morbidity and mortality for the infant. The aim of this study was to assess the adherence level of various physicians to different guidelines of management of neonatal hyperbilirubinemia in Iraq. Methods: An observational cross-sectional study was conducted in multiple outpatient clinics in various Iraqi provinces, from February 2018 to February 2019. The study involved 130 physicians, who were divided into emergency physicians (EPs), general practitioners (GPs), and pediatricians (PDs), and assessed their compliance to guidelines for management of neonatal hyperbilirubinemia using a questionnaire, which included providing the correct management for a test case scenario. Results: PDs had significantly longer discharge times compared to EPs and GPs. In total, 91.7% of PDs always tested the neonate for bilirubin levels before discharge, while 5.5% of GPs and 0% of EP did so. Regarding follow-up visits, 16.7%, 4.8% and 45.2% of PDs, EPs and GPs, respectively, scheduled a follow-up between 49-72 hours; 47.6% and 38.1% of EPs scheduled a follow-up at ≤24 hours and 25-48 hours, respectively. In addition, 91.7% of PDs gave the correct answer for the management of the test case scenario, followed by 58.9% of GPs, and 38.1% of EPs. About half of PDs extended neonates length of stay beyond 48 hours. Conclusion: GPs and EPs show lower adherence levels for the management of neonatal jaundice than PDs, which indicates that these physicians adhere well to current management guidelines from the WHO, AAP, and NICE.",
"keywords": [
"adherence",
"neonatal hyperbilirubinemia",
"physicians",
"pediatrician"
],
"content": "Introduction\n\nHyperbilirubinemia (HBR) is one of the commonest causes of neonatal readmission to hospital1, it requires urgent medical attention with rapid and prompt management to reduce bilirubin to prevent severe complications, such as kernicterus2.\n\nDespite the development of several guidelines (including the American Academy of Pediatrics (AAP) in 2004) to optimize the management of HBR, still the rate of inappropriate and insufficient therapy is rising3, and consequently, the rate of morbidity will increase4. These facts raise concerns about knowledge translation from and physician awareness of available guidelines in the literature and how much they use these in their daily practice5.\n\nSome surveys indicate that neonatologists are more aggressive than office-based general pediatricians in their treatment of neonatal jaundice in infants6. While others show considerable variation in management depending on the specialty of the physician, with pediatricians being shown to have both exceptionally good awareness and adherence to guidelines. Thus, it is important to enhance knowledge translation to physicians, thereby increasing adherence to these guidelines, especially in physicians other than pediatricians5.\n\nApproximately 66% of pediatricians reported an awareness to neonatal hyperbilirubinemia clinical practice guidelines published in 19947. However, Atkinson et al showed that only 54% of the pediatricians initiated treatment in accordance with recommended parameters8. This study aimed to assess the level of adherence of various physicians to different guidelines of management of neonatal hyperbilirubinemia in a multi-clinic survey in Iraq.\n\n\nMethods\n\nThis is an observational cross-sectional study conducted in multiple outpatient and private clinics in various Iraqi provinces. In this study, the investigator provided a questionnaire-based survey that assessed the adherence of various physicians to neonatal hyperbilirubinemia guidelines.\n\nAll procedures performed in the study were in accordance with the ethical standards of the Institutional Research Committee at the Children Welfare Teaching hospital, Baghdad City Complex, who approved the study protocol (approval date: 10th January 2018; number: 2018/005), and with the 1964 Declaration of Helsinki and its later amendments. Written informed consent was obtained from all participants to be included in the study.\n\nThe study was conducted in various provinces in Iraq (Baghdad, Anbar, Diyala, and Nasiriya provinces) and targeted physicians both in private and public sectors. The period of recruitment was between February 2018 to February 2019. Hospitals at which the physicians were based were selected on whether or not the hospital had a neonatal care clinic. The participants were approached by the investigators at their place of work\n\nIt is estimated that physician awareness about hyperbilirubinemia guideline is 70% according to the Christakis and Rivara study7. Since this is a cross-sectional study, the following equation was used to calculate sample size:\n\nN: sample size, p: prevalence, q = 100 – p, and d is precision (12%) d = (p/100) x p\n\nA sample size of 120 was calculated. To account for a possible drop out of 20%, a sample of 144 physicians was proposed.\n\nInitially, we selected 144 physicians; 14 physicians did not participate in the study, leaving 130 physicians who completed the survey. The selection of physicians was based on whether they participated in the management of neonatal HBR. The physicians were divided according to their specialty: emergency physicians (EPs), general practitioners (GPs), and pediatricians (PDs).\n\nDemographic data was collected from each physician (in an interview), including: age, years of practice, highest level of neonatal intensive care unit (NICU) the physician works in, and number of neonates per month seen by the physician.\n\nA questionnaire9 was used to assess the level of adherence to current guidelines. Questions included: length of hospital stay (discharge time) of neonates, physician policy about pre-discharge bilirubin level test, physician policy about timing of post-discharge follow-up, guideline used by the physician (WHO10, AAP11, and NICE12), nature of guideline access, and problems encountered during management.\n\nThe questionnaire was reviewed and validated by three exports in medical education, neonatology, and biostatistics. After prior pilot study on 12 physicians, the reliability was calculated and found to be 0.89.\n\nCase scenario. Additionally, a case scenario was presented to the physicians, as follows:\n\n“A 30-year-old mother, with blood group O+, gave birth to a 2.8 kg male infant with cephalic hematoma after 37 weeks of gestation. Before discharge, at 36 hours after birth, the infant appeared jaundiced. I would do?”\n\nThe following options were provided to the physicians: discharge and follow-up, laboratory tests for bilirubin and blood group, cancel discharge and start phototherapy, refer to a pediatrician.\n\nDiscrete variables were presented using their number and percentage. Chi-square test was used to analyze discrete variables (or Fisher exact test when chi-square was not valid; due to low sample size <20 and if 2 or more with an expected frequency was less than 5). SPSS 22.0.0 (Chicago, IL) software package was used to conduct statistical analysis. P-value of less than 0.05 was considered significant.\n\n\nResults\n\nIn total, 130 physicians participated in this questionnaire-based study: 21 (16.15%) were EPs, 73 (56.15%) GPs, and 36 (27.67%) PDs.\n\nPDs had a significantly higher age compared to both ERs and GPs. The majority of all physician groups worked at level I NICU (66.7%, 72.6%, 50.0%; respectively); notably more PDs worked at level II and III NICU compared to other groups. Both GPs and PDs had a longer duration of practice compared to an EPs, and additionally, PDs saw the most neonates per month compared to EPs and GPs (Table 1).\n\nED, emergency physician; GP, general practitioner; PD, pediatrician. Levels of NICU 13: Level I: Well newborn nursery, Level II: Special care nursery, Level III: Neonatal intensive-care unit (NICU), Level IV: Regional neonatal intensive-care unit (Regional NICU).\n\nPDs had significantly longer discharge times of neonates compared to EPs and GPs. In addition, 91.7% of PDs always tested the neonate for bilirubin levels before discharge, while only 5.5% of GPs and 0% of EPs did so. Regarding follow-up visits, 16.7%, 4.8% and 45.2% of PDs, EPs and GPs, respectively, scheduled a follow-up between 49–72 hours; 47.6% and 38.1% of EPs scheduled a follow-up at ≤24 hours and 25–48 hours, respectively (Table 2).\n\nED, emergency physician; GP, general practitioner; PD, pediatrician.\n\nTable 3 illustrates the assessment of physician knowledge toward assessment of HBR. It shows that recognizing visual signs and jaundice within 24 hours of birth was performed to a higher extent by PDs compared with EPs and GPs.\n\nED, emergency physician; GP, general practitioner; PD, pediatrician.\n\nIn total, 91.7% of PDs gave the correct answer for the case scenario, followed by GPs (58.9%) and EPs (38.1%) (Table 4).\n\nED, emergency physician; GP, general practitioner; PD, pediatrician. Correct answer: laboratory tests bilirubin and blood group.\n\n\nDiscussion\n\nIn this survey, there was obvious variation in the adherence of various groups of physicians to guidelines for treating neonates with HRB. The results showed that 74% of GPs and 61.9% of EPs did not use any guidelines for the management and identification of neonatal HBR; PDs on the other hand did use guidelines. These findings were higher than reported in other countries. Sampurna et al, in a study conducted in Indonesia that involved 291 midwives, 206 GPs, and 154 PDs, reported that 23% of GPs and 29% of midwives were unaware of the presence of guidelines for identification and management of neonatal HBR14. In a study by Mateo et al, which involved 321 Canadian physicians, they reported that guidelines were used by 41% of family physicians, 75% of PDs, and 69% of midwives, which is partially in agreement with our findings5.\n\nEarly identification is the cornerstone of any guideline created to manage HBR3,10,15. Monitoring of the neonate with HBR is achieved via measurement of bilirubin especially in the first 3 days of life16, with subsequent follow-up every 24-48 hours till oral feeding is assured16. In the present study, before discharge monitoring of bilirubin was being performed by only 5.5% of GPs, 0% of EPs and 91.7% of PDs. In addition, 90.5% of EPs and 94.5% of GPs reported the use of pre-discharge bilirubin testing only if the infant looked jaundiced. This finding is higher than that reported by a previous Iraqi study by Hameed and Abdul Razak17, who reported that 56% of PDs did not consider post-discharge total serum bilirubin monitoring. In the Canadian study by Mateo et al, 42% of family physicians and 22% of midwives compared with 63% of PDs, reported bilirubin measurement before discharge, which is lower than reported by the current study5. In the Indonesian study, 12% of GPs and 8% of PDs reported always performing pre-discharge bilirubin measurement, while 54% of GPs and 65% of PDs reported the use of pre-discharge bilirubin measurement only if jaundice was present, which is partially in agreement with our findings14. In addition, in a study by Petrova et al, which involved 356 PDs in the USA, they reported that 87.4% of all physicians used TSB testing with clinical jaundice before discharge, while 57.7% of them only used TSB testing in clinical jaundice post-discharge, which in agreement with our findings18.\n\nIn the present study, all PDs considered jaundice in less than 24 hours of life as an indicator of severe HBR, compared with 81.0% of EPs and 67.1% of GPs. This is in agreement with the study by Hameed and Abdul Razak that showed that 76% of PDs strongly agreed that neonates presenting with jaundice in the first 24 hours as a predictor of severe HBR17. Petrova et al reported similar findings: 77% of physicians considered that jaundice presenting in the first 24 hours was a marker for severe HBR18. Several studies in the literature indicate the importance of early first-day measurement of bilirubin levels since elevated levels are correlated strongly with later severe complications19–21. There was good knowledge of this by physicians in this study.\n\nIn the present study, PDs relied less on visual assessment of jaundice (5.6%), jaundice in stool and fever (0%), and jaundice at 14 days when the baby is active and growing (0%), compared to EDs and GPs. The Hameed and Abdul Razak study reported few PDs (10%) agreed that jaundice noticed at discharge is a risk factor for severe HBR17, which was in agreement with our findings. Around 61% of primary health care provider assessed the severity of neonatal jaundice by visual cephalocaudal evaluation inspecting the skin or sclera, which is rapid and cost-free, but it is not sufficiently accurate, especially when applied to newborns with dark skin22, which disagrees with our findings.\n\nIn the present study, the majority of PDs gives the correct answer (laboratory tests bilirubin and blood group) to the case scenario (91.7%); however lower correct answer rates were achieved by GPs (58.9%) and EPs (38.1%) (p-value = 0.002). In the Mateo et al study, the response to a case scenario similar to ours showed similar results to our findings, while in the Sampurna et al study, the response to a similar case scenario showed a lower correct answer rate with 54% of PDs, 57% of GPs, and 44% of midwives providing the correct answer14.\n\nHospital affiliation of health care providers is one of the strongest predictors of adherence to practice guidelines6, which is one of the possible explanations of the high rate of good knowledge about the management of neonatal HBR shown in the present study. The variability of the use of guidelines for the management of HBR in this study was similar to others8,23. Barriers to adherence include low awareness, no agreement, and inertia of previous practices24.\n\nThe study included only three types of physicians, other physicians that have role in the management of HBR were not included in the study. A nationwide study is required in the future to assess the overall adherence of physicians to the management of HBR. Another limitation is that we did not examine if the physicians in fact practiced what they had indicated in the survey.\n\n\nConclusions\n\nGPs and EPs in this study, performed in Iraqi hospitals, showed lower adherence levels for the management of neonatal jaundice than PDs, who exhibit excellent adherence to current guidelines (WHO, AAP, and NICE guidelines), especially AAP guidelines. We recommend starting an education program directed toward the enhancement of knowledge for EPs and GPs for the management of neonatal jaundice.\n\n\nData availability\n\nZenodo: Adherence Level for Neonatal Hyperbilirubinemia, https://doi.org/10.5281/zenodo.374561125.\n\nZenodo: Questionnaire about Hyperbilirubinemia, http://doi.org/10.5281/zenodo.38606929\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
"appendix": "References\n\nTan-Dy C, Moore A, Satodia P, et al.: Predicting Kernicterus in Severe Unconjugated Hyperbilirubinemia. Paediatr Child Health. 2004; 9(suppl_a): 17A. Publisher Full Text\n\nJohnson L, Bhutani VK, Karp K, et al.: Clinical report from the pilot USA Kernicterus Registry (1992 to 2004). J Perinatol. 2009; 29(Suppl 1): S25–45. PubMed Abstract | Publisher Full Text\n\nAAP: Management of hyperbilirubinemia in the newborn infant 35 or more weeks of gestation. Pediatrics. 2004; 114(1): 297–316. PubMed Abstract | Publisher Full Text\n\nSgro M, Campbell D, Shah V: Incidence and causes of severe neonatal hyperbilirubinemia in Canada. CMAJ. 2006; 175(6): 587–90. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMateo PC, Lee KS, Barozzino M, et al.: Management of neonatal jaundice varies by practitioner type. Can Fam Physician. 2013; 59(8): e379–86. PubMed Abstract | Free Full Text\n\nGartner LM, Herrarias CT, Sebring RH: Practice patterns in neonatal hyperbilirubinemia. Pediatrics. 1998; 101(1 Pt 1): 25–31. PubMed Abstract | Publisher Full Text\n\nChristakis DA, Rivara FP: Pediatricians' Awareness of and Attitudes About Four Clinical Practice Guidelines. Pediatrics. 1998; 101(5): 825–30. PubMed Abstract | Publisher Full Text\n\nAtkinson LR, Escobar GJ, Takayama JI: Phototherapy use in jaundiced newborns in a large managed care organization: do clinicians adhere to the guideline? Pediatrics. 2003; 111(5 Pt 1): e555–61. PubMed Abstract | Publisher Full Text\n\nFawzi HA: Questionnaire about Hyperbilirubinemia [Data set].Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3860692\n\nWorld Health Organization: Other common neonatal problems: Jaundice; in WHO: Pocketbook of Hospital care for children: guidelines for the management of common childhood illnesses, Second edition. Geneva, Switerland, WHO. 2013; 64–65. PubMed Abstract\n\nAmerican Academy of Pediatrics Subcommittee on Hyperbilirubinemia: Management of Hyperbilirubinemia in the Newborn Infant 35 or More Weeks of Gestation. Pediatrics. 2004; 114(1): 297–316. PubMed Abstract | Publisher Full Text\n\nExcellence NIfC: Jaundice in newborn babies under 28 days. NICE London; 2016. Reference Source\n\nMartin JA, Menacker F: Expanded health data from the new birth certificate, 2004. Natl Vital Stat Rep. 2007; 55(12): 1–22. PubMed Abstract\n\nSampurna MTA, Ratnasari KA, Etika R, et al.: Adherence to hyperbilirubinemia guidelines by midwives, general practitioners, and pediatricians in Indonesia. PLoS One. 2018; 13(4): e0196076. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFawaz R, Baumann U, Ekong U, et al.: Guideline for the Evaluation of Cholestatic Jaundice in Infants: Joint Recommendations of the North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition and the European Society for Pediatric Gastroenterology, Hepatology, and Nutrition. J Pediatr Gastroenterol Nutr. 2017; 64(1): 154–68. PubMed Abstract | Publisher Full Text\n\nBarrington KJ, Sankaran K: Canadian Paediatric Society Fetus and Newborn Committee. Guidelines for detection, management and prevention of hyperbilirubinemia in term and late preterm newborn infants. Paediatr Child Health. 2007; 12(5): 401–407. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHameed NN, Abdul razak OH: Assessment of knowledge and practice of Baghdadi pediatricians regarding the management of neonatal hyperbilirubinemia. J Fac Med Bagdad. 2013; 55(2): 115–20. Reference Source\n\nPetrova A, Mehta R, Birchwood G, et al.: Management of neonatal hyperbilirubinemia: pediatricians' practices and educational needs. BMC Pediatr. 2006; 6: 6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRandev S, Grover N: Predicting neonatal hyperbilirubinemia using first day serum bilirubin levels. Indian J Pediatr. 2010; 77(2): 147–50. Publisher Full Text\n\nAgarwal R, Kaushal M, Aggarwal R, et al.: Early neonatal hyperbilirubinemia using first day serum bilirubin level. Indian Pediatr. 2002; 39(8): 724–30. PubMed Abstract\n\nKhan KA, Alam J, Nahar N, et al.: Predicting Neonatal Hyperbilirubinemia Using First Day Serum Bilirubin Level in Late Preterm and Term Healthy Newborn. Mymensingh Med J. 2017; 26(4): 854–62. PubMed Abstract\n\nMostafa MA, Kamal NM, Eltaher S, et al.: Knowledge of Neonatal Hyperbilirubinemia Among Primary Health Care Physicians: A Single-Center Experience. Clin Med Insights Pediatr. 2019; 13: 1179556518824375. PubMed Abstract | Publisher Full Text | Free Full Text\n\nProfit J, Cambric-Hargrove AJ, Tittle KO, et al.: Delayed pediatric office follow-up of newborns after birth hospitalization. Pediatrics. 2009; 124(2): 548–54. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCabana MD, Rand CS, Powe NR, et al.: Why don't physicians follow clinical practice guidelines? A framework for improvement. Jama. 1999; 282(15): 1458–65. PubMed Abstract | Publisher Full Text\n\nFawzi HA: Adherence Level for Neonatal Hyperbilirubinemia [Data set].Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3745611"
}
|
[
{
"id": "64296",
"date": "10 Jun 2020",
"name": "Robert D. Christensen",
"expertise": [
"Reviewer Expertise Neonatal Hematology"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe submission by Dr. Hameed et al. reviews results of a questionnaire completed by 130 physicians in Iraq, about neonatal bilirubin management. The authors are to be congratulated on this study, analysis, and report. Perhaps they would consider the following somewhat minor issues, all aimed at improving the clarity of the report.\nTitle: Consider deleting the phrase “adherence level for”. It suggests that there are different levels. The title is clearer without that phrase.\n\nAbstract: It is not until the last line of the Abstract Conclusion that we read the guidelines of interest are those of the WHO, AAP, and NICE. Placing this near the beginning of the abstract would be helpful. Also, under Abstract Methods, consider changing …”physicians, who were divided into…” to, “physicians self-identified as either…”. Also, under Abstract Conclusions, delete “adherence levels”, because various levels of adherence was not part of the analysis.\n\nIntroduction: Second line. Instead of, “…to hospital, it requires urgent”, consider, “to hospital, severe cases require…”. Last paragraph before Case Scenario, change “exports” to “experts”.\n\nResults: In Table 3, it is not clear what the “negative” and “positive” indicates. Thus, I can not understand what is being conveyed by this 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": []
},
{
"id": "65857",
"date": "28 Jul 2020",
"name": "Laila M. Sherief",
"expertise": [
"Reviewer Expertise pediatric hematology and oncology and neonatology"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 assesses the adherence of various physicians to guidelines to important and sometimes serious problems in the neonate.\nThe methods: I want to ask the authors, why ER doctors included in the work\nResults: Table 3: need more clarification.\nDiscussion: needs to be deeper with more explanation of the differences between the results\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/9-504
|
https://f1000research.com/articles/9-503/v1
|
03 Jun 20
|
{
"type": "Case Report",
"title": "Case Report: Management of rectal squamous cell carcinoma - a treatment dilemma",
"authors": [
"Nathaniel A. Parker",
"Yasmine Hussein Agha",
"Charles Scott Buess",
"Daniel Lalich",
"Jeremy M. Deutsch",
"Yasmine Hussein Agha",
"Charles Scott Buess",
"Daniel Lalich",
"Jeremy M. Deutsch"
],
"abstract": "Primary rectal squamous cell carcinoma is rare compared to adenocarcinoma, which is the predominant histologic type most commonly discovered at the time of colorectal carcinoma diagnosis. Due to the infrequent nature of this malignancy, data on tumor pathogenesis and risk factors remains sparse. Moreover, no standardized therapeutic regimen exists. This report describes a case of advanced rectal squamous cell carcinoma diagnosed in a 46-year-old female who initially presented with abdominal pain. Her clinical course was uncomplicated and she responded well to the selected therapy. Much work remains to be accomplished for patients with rectal squamous cell carcinoma.",
"keywords": [
"Squamous cell carcinoma",
"Rectum",
"Chemoradiotherapy",
"Radiotherapy"
],
"content": "Introduction\n\nAdenocarcinoma comprises the vast majority of rectal cancers1. As a result, primary rectal squamous cell carcinoma (RSCC) is exceedingly rare, occurring in approximately 0.10–0.25 per 1000 colorectal cancers2,3. The etiology, pathogenesis, and risk factors are poorly defined, and no general consensus exists regarding the optimal treatment regimen due to the rarity of this cancer. Review of the literature encompasses mostly case series and retrospective studies. Nevertheless, evidence-based management is essential for those who are diagnosed. This report describes a rare case of primary RSCC.\n\n\nCase report\n\nA 46-year-old Caucasian female administrative assistant, for whom the only pertinent past medical history was chronic tobacco smoking, presented at the emergency department with the chief complaint of generalized abdominal pain. Symptom onset began two months prior to her initial presentation and had been progressively worsening.\n\nVital signs and measurements were unremarkable. Physical examination was unremarkable. Serum laboratory evaluation was nonrevealing. Computerized tomography (CT) imaging of the abdomen and pelvis showed a sigmoid mass indicating a differential diagnosis of a transmural abscess versus a malignant inflammatory process in the sigmoid colon (Figure 1). There was no evidence of distant metastatic disease. The patient underwent a diagnostic colonoscopy, which showed a rectosigmoid mass that was biopsied between 10 cm and 15 cm from the anal verge. Grossly, the mass was observed to have a flattened and friable mucosa. Histopathology favored a rare, poorly differentiated squamous cell carcinoma of the rectum. To confirm the impression of squamous differentiation, immunohistochemical (IHC) stains were performed on the biopsied specimens. The malignant cells showed strong cytokeratin 5/6 (CD5/6) immunoreactivity (Figure 2). Thus, squamous cell carcinoma of the rectum was diagnosed. Due to the squamous cell origin of her rectal mass, she underwent subsequent gynecologic evaluation. Cervical and endometrial biopsies were negative for malignancy. For tumor staging and evaluate for distant metastatic disease, the patient had a positron emission tomography (PET) scan, which showed a rectosigmoid mass in the colon with a standardized uptake value (SUV) of 16 and multiple PET-avid pelvic lymph nodes with SUVs of 2–3 (Figure 3).\n\nThe rounded thick-walled structure measures approximately 4 cm (arrow). There is some adjacent inflammation in the presacral space as well as prominent lymph nodes. Given the radiological findings the differential diagnosis includes transmural abscess versus inflammatory carcinoma of the sigmoid colon.\n\nAt medium and high power magnification, hematoxylin and eosin (HE) staining reveals sheets of poorly differentiated squamous cells invading the surrounding submucosal tissue (HE x40 and x100). Immunohistochemical staining for the squamous cell marker CK5/6, visualized by a cytoplasmic red-chromogen reaction, is positive (CK5/6 x40 and x100). Together histopathology and immunostaining show a poorly differentiated squamous cell carcinoma originating from rectal tissue.\n\nImaging reveals a large focus of hypermetabolic activity in the rectosigmoid colon with a SUV of 16 and diffuse stranding in the region (arrows). There are multiple slightly prominent perirectal lymph nodes with the maximal SUV of 3.3 (arrowheads). There is presacral fat stranding and retroperitoneal lymphadenopathy, none of which exhibit hypermetabolism. No evidence of malignancy is noted above the diaphragm. Expected physiologic uptake of F-18 fluorodeoxyglucose is observed in the kidneys and brain. Given these findings, the rectal tumor was determined to be stage III rectal squamous cell carcinoma.\n\nSubsequently, she was diagnosed with stage III RSCC. Given the appearance of the tumor on CT scans, as well as the presence of PET-avid external iliac nodes in the perirectal region, neoadjuvant chemoradiation with radiation followed by surgical intervention was recommended. She was started on neoadjuvant chemotherapy with continuous-infusion 5-flurouracil (5-FU) with concomitant radiation. She received radiation therapy (28 treatments; total dose of 180 centiGrays) to her entire pelvis. Follow-up CT scans showed an excellent response and near resolution of the tumor. Subsequent PET scans displayed a low SUV in the primary tumor site with no additional uptake. She proceeded with sigmoid colon resection, with minimal residual carcinoma. Given the patient’s good response to chemotherapy and radiation, she was started on adjuvant chemotherapy with FOLFOX (leucovorin, 5-FU, and oxaliplatin) (Figure 4).\n\nPresented according to CARE guidelines.\n\nFollowing adjuvant FOLFOX chemotherapy for six months, all of the patient’s consecutive surveillance CT scans have showed a complete resolution. This is consistent with a durable and long-lasting response to therapy for a rectal tumor that unusually originated from a poorly differentiated squamous cell carcinoma. The patient remains alive, healthy, and in complete remission following cessation of FOLFOX chemotherapy three years ago (Figure 4).\n\n\nDiscussion\n\nAlthough RSCC has a similar presentation to rectal adenocarcinoma, its pathogenesis remains unclear and response to treatment is highly variable. Some of the most prominent risk factors include tobacco use, inflammatory bowel disease, radiotherapy and infections such as human immunodeficiency virus, human papilloma virus, amebiasis, and schistosomiasis4,5. Compared to adenocarcinoma of the rectum, RSCC occurs more often in younger Caucasian women with an average age of 60 years6,7. Patients clinically present with one or more of the following: gastrointestinal bleeding, changes in stool shape, diarrhea, constipation, tenesmus, weight loss and lower abdominal pain5.\n\nWhen histopathology is suggestive of RSCC, other more common etiologies such as anal squamous cell carcinoma, gynecological malignancy, and bowel fistula should be ruled out prior to establishing a definite diagnosis8. Further evaluation and sampling can be achieved by colonoscopy and colposcopy. IHC plays an important role in differentiating RSCC from other histological subtypes. Although this specific IHC stain was not utilized in this case due to availability, cytokeratin CAM5.2, an epithelial marker, immunoreactivity suggests rectal tissue as the primary tumor site, rather than anal4. Cytokeratin 7 and 20 stain glandular epithelia in the upper and lower gastrointestinal tract, respectively4,9. While these markers identify adenomatous malignancies, both are expected to be negative by IHC in tumors with a squamous cell origin.\n\nHistorically, radical surgery was recommended for RSCC. However, more recent analyses have shown improved outcomes following chemoradiation only in localized disease or preceding salvage surgical resection in advanced disease to reduce tumor burden2–4,6,7,10. One of the main factors contributing to the discrepancy among the results and conclusions drawn is the lack of consistency in staging criteria used among all studies. This raises concern since management is based on tumor staging. Another factor that led to the paradigm shift was the amount of complications that arise following surgical intervention. Resection reduces the risk of death from the cancer itself. Patients often have worse outcomes and reduced overall survival due to the debilitating issues secondary to invasive interventions5. Review of the literature reveals treatment choice can also be influenced by the perceived severity of the illness. As a result, patients with advanced disease and a poorer prognosis were often offered surgical resection rather than conservative management with chemotherapy. However, poor outcomes following surgical resection could have been attributed to complications rather than the extent of the disease itself. The current understanding is based on case series, and results are highly biased. This in turn raises the need for a standardized staging system. Furthermore, randomized controlled trials would help outline an effective management strategy based on disease severity.\n\nIt has been postulated that staging based on size rather depth of invasion is a better predictor of prognosis6. Chemotherapeutic options for RSCC include 5-fluorouracil in combination with capecitabine or cisplatin. A five-year disease-free survival of 86% with chemoradiation only and 93% with chemoradiation plus salvage surgery has helped establish a benchmark for other therapeutic options11. Four other case series involving patients with advanced RSCC have shown improved overall survival with chemoradiation as definitive management, as well as alternative salvage surgery2,10,12,13. However, these retrospective observations are derived from small cohort studies that reported multiple limitations. Thus, it would be difficult to determine if the findings can be generalized.\n\n\nConclusion\n\nThis report presented a unique and rare case of a primary squamous cell carcinoma of the rectum. Most likely due to the extraordinarily low incidence of colorectal tumors having squamous cell origins, the etiology, pathogenesis, and risk factors for RSCC remain poorly understood. As a result, no standardized therapeutic regimen exists. Historically successful regimens for more common colorectal cancers, such as adenocarcinomas, will likely continue to be widely used in practice until additional therapeutic options are elucidated. Recently, overall survival has been shown to be improved for RSCC patients when certain regimens are used. However, this data comes from retrospective small cohort studies. Much work remains to be accomplished for patients with RSCC.\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\nFléjou JF: WHO Classification of Digestive Tumors: The Fourth Edition. Ann Pathol. 2011; 31(5 Suppl): S27–31. PubMed Abstract | Publisher Full Text\n\nYeh J, Hastings J, Rao A, et al.: Squamous Cell Carcinoma of the Rectum: A Single Institution Experience. Tech Coloproctol. 2012; 16(5): 349–54. PubMed Abstract | Publisher Full Text\n\nMusio D, De Felice F, Manfrida S, et al.: Squamous Cell Carcinoma of the Rectum: The Treatment Paradigm. Eur J Surg Oncol. 2015; 41(8): 1054–8. PubMed Abstract | Publisher Full Text\n\nNahas CS, Shia J, Joseph R, et al.: Squamous-cell Carcinoma of the Rectum: A Rare but Curable Tumor. Dis Colon Rectum. 2007; 50(9): 1393–400. PubMed Abstract | Publisher Full Text\n\nGuerra GR, Kong CH, Warrier SK, et al.: Primary Squamous Cell Carcinoma of the Rectum: An Update and Implications for Treatment. World J Gastrointest Surg. 2016; 8(3): 252–65. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGoffredo P, Robinson TJ, Frakes JM, et al.: Comparison of Anal Versus Rectal Staging in the Prognostication of Rectal Squamous Cell Carcinoma: A Population-Based Analysis. Dis Colon Rectum. 2019; 62(3): 302–308. PubMed Abstract | Publisher Full Text\n\nDutta SW, Alonso CE, Waddle MR, et al.: Squamous Cell Carcinoma of the Rectum: Practice Trends and Patient Survival. Cancer Med. 2018; 7(12): 6093–6103. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWilliams GT, Blackshaw AJ, Morson BC: Squamous carcinoma of the colorectum and its genesis. J Pathol. 1979; 129(3): 139–47. PubMed Abstract | Publisher Full Text\n\nSelves J, Long-Mira E, Mathieu MC, et al.: Immunohistochemistry for Diagnosis of Metastatic Carcinomas of Unknown Primary Site. Cancers (Basel). 2018; 10(4): 108. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRasheed S, Yap T, Zia A, et al.: Chemo-radiotherapy: An Alternative to Surgery for Squamous Cell Carcinoma of the Rectum--Report of Six Patients and Literature Review. Colorectal Dis. 2009; 11(2): 191–7. PubMed Abstract | Publisher Full Text\n\nSturgeon JD, Crane CH, Krishnan S, et al.: Definitive Chemoradiation for Squamous Cell Carcinoma of the Rectum. Am J Clin Oncol. 2017; 40(2): 163–166. PubMed Abstract | Publisher Full Text\n\nClark J, Cleator S, Goldin R, et al.: Treatment of Primary Rectal Squamous Cell Carcinoma by Primary Chemoradiotherapy: Should Surgery Still Be Considered a Standard of Care? Eur J Cancer. 2008; 44(16): 2340–3. PubMed Abstract | Publisher Full Text\n\nLoganadane G, Servagi-Vernat S, Schernberg A, et al.: Chemoradiation in rectal squamous cell carcinoma: Bi-institutional case series. Eur J Cancer. 2016; 58: 83–9. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "64793",
"date": "29 Jun 2020",
"name": "Pratiksha Singh",
"expertise": [
"Reviewer Expertise Limited by knowledge of immunohistochemistry."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 described in terms of investigations performed and the treatment modality selected. It helps the practitioners by adding to the limited number of case reports and presentations available. However, the authors fail to describe the patient's symptoms and physical examination in sufficient detail:\nThe chief complaint in an emergency department as abdominal pain requires a further detailed description of presenting symptoms especially pertaining to relevant negative history about bowel habit changes, bleeding per rectum and weight loss.\n\nIt would help to describe smoking as pack years rather than chronic tobacco smoking for better interpretation.\n\nThe authors mention measurements that were unremarkable along with vital signs, it would be helpful to describe what these were (Guerra, 20061).\nDuring the staging process, it would be helpful to know the relevant system used (AJCC 7th or 8th edition staging for rectal carcinoma (Tong et al., 20182).\nDuring discussion, the authors mention \"Compared to adenocarcinoma of the rectum, RSCC occurs more often in younger Caucasian women with an average age of 60 years\". A recent update mentions the age of the patients usually range from 39-93 years with average 63 years with no apparent ethnic predisposition (Guerra, 20061).\nIt would also help if in the discussion the authors could explain how this particular case was unique.\n\nThank you for giving the opportunity to review and learn from your case report.\n\nIs the background of the case’s history and progression described in sufficient detail? No\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly",
"responses": [
{
"c_id": "5750",
"date": "20 Jul 2020",
"name": "Nathaniel Parker",
"role": "Author Response",
"response": "Thank you for the review. The authors responses are listed below peer review commentsThe chief complaint in an emergency department as abdominal pain requires a further detailed description of presenting symptoms especially pertaining to relevant negative history about bowel habit changes, bleeding per rectum and weight loss.- All available details from the case presentation that were pertinent and/or positive have been listed. It would help to describe smoking as pack years rather than chronic tobacco smoking for better interpretation.- Smoking pack years was unknown to the authors. The authors mention measurements that were unremarkable along with vital signs, it would be helpful to describe what these were (Guerra, 20061).- All available and relevant details from the case have been listed. During the staging process, it would be helpful to know the relevant system used (AJCC 7th or 8th edition staging for rectal carcinoma (Tong et al., 20182).- No formal staging process, such as the one mentioned, was not used during this case's work-up and management process. Thus, it would not be factual to present the case in such a manner. During discussion, the authors mention \"Compared to adenocarcinoma of the rectum, RSCC occurs more often in younger Caucasian women with an average age of 60 years\". A recent update mentions the age of the patients usually range from 39-93 years with average 63 years with no apparent ethnic predisposition (Guerra, 20061).- Please see reference numbers 6 and 7, which are more recent.It would also help if in the discussion the authors could explain how this particular case was unique. - Please see the Abstract and Conclusion sections."
}
]
},
{
"id": "65674",
"date": "30 Jun 2020",
"name": "George S. Stoyanov",
"expertise": [
"Reviewer Expertise pathology",
"immunohistochemistry",
"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\nBased on the clinical description, the tumor seems to be in the sigmoid colon, which indeed is a rare localization. The authors however use the terms colon and rectum interchangeably. Rectal SCC carcinoma is common and the ethological factor there is most often HPV infection.\n\nRegarding patient information - it is too much revealing to indicate the profession of the patient. Please replace with the statement that her field of work is not associated with this kind of malignancy.\n\nOn the diagnostic report, please specify TNM of the tumor and G as well. What did the gynecological report show, was there presence of HPV-associated changes in the cervical smear? That information could provide some information on the pathogenesis of the condition.\n\nOn the report of the operative materials, please provide the TRG pf the tumor.\n\nIn the discussion, RSCC does not share many of the hallmarks of AC - no mucus and obstructions are much less common.\n\nGeneral comment on writing style: in some places the text is written very confusingly e.g.: \"Although this specific IHC stain was not utilized in this case due to availability, cytokeratin CAM5.2, an epithelial marker, immunoreactivity suggests rectal tissue as the primary tumor site, rather than anal\". The discussion needs to focus more on the analysis of the patient and the data you have stated in the introduction such as \"no general consensus exists regarding the optimal treatment regimen due to the rarity of this cancer\". Histology is of sub-par quality - in the H&E stained slides I am convinced that that is SCC, but I am not convinced on the location of the tumor, please provide a new slide which is more informative. The quality of the IHC is very bad, there is no counter staining on the cellular nucleus and the pattern on reactivity is more suggestive of tumor necrosis rather than that of any SCC - these need to be addressed. The abstract is way too short and seems a little bit detached from the article main text - please restructure it so be more coherent with the data provided in the manuscript.\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? No\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly",
"responses": [
{
"c_id": "5751",
"date": "20 Jul 2020",
"name": "Nathaniel Parker",
"role": "Author Response",
"response": "Based on the clinical description, the tumor seems to be in the sigmoid colon, which indeed is a rare localization. The authors however use the terms colon and rectum interchangeably. Rectal SCC carcinoma is common and the ethological factor there is most often HPV infection. - This is to reflect the dynamic titles that were used in operative and pathology reports to describe this case (e.g. rectal vs. rectosigmoid). In terms of HPV, these details were not clinically significant to oncologist involved with the case, and thus were not reported. Regarding patient information - it is too much revealing to indicate the profession of the patient. Please replace with the statement that her field of work is not associated with this kind of malignancy. - This is a biased and non-constructive criticism However, if it satisfies the peer reviewer, this vocational information was made available at the request of the Editorial Board. Please ensure you are update on the peer review standards of F1000Research. On the diagnostic report, please specify TNM of the tumor and G as well. What did the gynecological report show, was there presence of HPV-associated changes in the cervical smear? That information could provide some information on the pathogenesis of the condition. - All relevant information has been provided in the case. On the report of the operative materials, please provide the TRG pf the tumor.- No TRG information was available to report. In the discussion, RSCC does not share many of the hallmarks of AC - no mucus and obstructions are much less common.General comment on writing style: in some places the text is written very confusingly e.g.: \"Although this specific IHC stain was not utilized in this case due to availability, cytokeratin CAM5.2, an epithelial marker, immunoreactivity suggests rectal tissue as the primary tumor site, rather than anal\". The discussion needs to focus more on the analysis of the patient and the data you have stated in the introduction such as \"no general consensus exists regarding the optimal treatment regimen due to the rarity of this cancer\". Histology is of sub-par quality - in the H&E stained slides I am convinced that that is SCC, but I am not convinced on the location of the tumor, please provide a new slide which is more informative. The quality of the IHC is very bad, there is no counter staining on the cellular nucleus and the pattern on reactivity is more suggestive of tumor necrosis rather than that of any SCC - these need to be addressed. The abstract is way too short and seems a little bit detached from the article main text - please restructure it so be more coherent with the data provided in the manuscript.- These criticisms are biased and/or non-constructive. - All available and relevant information has been reported in the case."
}
]
},
{
"id": "67425",
"date": "24 Aug 2020",
"name": "Mukesh S. Paudel",
"expertise": [
"Reviewer Expertise Colonoscopy",
"IBS",
"Liver 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 paper describes a rare case of squamous cell carcinoma of the rectum, which was diagnosed by colonoscopy and histopathological examination. The write-up is good and this paper will be a valueable addition to the current existing literature on this topic. The methodology of diagnosis and the treatment protocol have been well explained in the current paper.\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/9-503
|
https://f1000research.com/articles/9-502/v1
|
03 Jun 20
|
{
"type": "Research Article",
"title": "Coronavirus disease 2019 drug discovery through molecular docking",
"authors": [
"Sweta Singh",
"Hector Florez",
"Sweta Singh"
],
"abstract": "Background: The dawn of the year 2020 witnessed the spread of the highly infectious and communicable disease coronavirus disease 2019 (COVID-19) globally since it was first reported in 2019. Severe acute respiratory syndrome coronavirus-2 is the main causative agent. In total, 3,096,626 cases and 217,896 deaths owing to COVID-19 were reported by 30th April, 2020 by the World Health Organization. This means infection and deaths show an exponential growth globally. In order to tackle this pandemic, it is necessary to find possible easily accessible therapeutic agents till an effective vaccine is developed. Methods: In this study, we present the results of molecular docking processes through high throughput virtual screening to analyze drugs recommended for the treatment of COVID-19. Results: Atovaquone, fexofenadine acetate (Allegra), ethamidindole, baicalin, glycyrrhetic acid, justicidin D, euphol, and curine are few of the lead molecules found after docking 129 known antivirals, antimalarial, antiparasitic drugs and 992 natural products. Conclusions: These molecules could act as an effective inhibitory drug against COVID-19.",
"keywords": [
"COVID-19",
"SARS-CoV-2",
"Molecular Docking"
],
"content": "Introduction\n\nCoronavirus Disease 2019 (COVID-19) is caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), that is responsible for respiratory illness and probably many more is yet to be discovered. This novel virus was first identified on 30th December, 2019, with its first infection case infecting a human, which was reported in Wuhan city located in Hubei, China1. Coronaviruses are mainly zoonotic, and are present amongst birds and mammals, causing respiratory, neurological, hepatic and enteric diseases2 as well as comprises of enveloped RNA. The World Health Organization (WHO) declared this disease as a pandemic on 11th March, 2020 and SARS-CoV-2 as the deadliest virus till date on earth claiming 217,896 deaths till 30th April, 20203.\n\nThen, it is necessary for the rapid development and approval of a vaccine, which is not yet available4. Nevertheless, Chang et al.,5 have suggested that some drugs against same type of viruses approved by the US Food and Drug Administration (FDA) might offer promising results. Hydroxychloroquine is one such drug that is used worldwide whereas Remdesivir and Ivermectin have been reported to work against COVID-19 in silico by others.\n\nThe transmission of this coronavirus occurs due to the binding of the CoV spike protein to the angiotensin converting enzyme 2 (ACE2) receptor present on the cell surface of the human host. The ACE2 receptor is present in the respiratory organs, kidneys, gastrointestinal tract (at high levels in the esophagus, colon, and small intestine, but low in the stomach), and testes. Virulence of this novel virus is due to the presence of main protease responsible for virus replication along with many major functions6. Therefore, we have employed the main protease structure 6m03 as the target protein to identify the best inhibitory drugs in silico for our study.\n\nSARS-CoV-2 (negatively stained) when observed under the electron micrograph was found to be spherical in shape with some pleiomorphic characteristic. The epithelial sections of human airway when observed, viruses were found in membrane bound vesicles in cytoplasm along with inclusion bodies. The virions appear similar to solar corona due to 9- to 12-nm distinctive spikes and the virions are 60 to 140 nm in diameter. Thus, it was established due to these morphological characteristics that this virus belongs to the Coronaviridae family along with its genome having more than 85% identity with a bat SARS-like CoV (bat-SL-CoVZC45, MG772933.1) genome as previously assessed via genome sequencing6. SARS-Cov-2 initially infects lower airways, binds to ACE2 receptor on cells activating immune cells, thus, inducing the secretion of inflammatory cytokines and chemokines in human pulmonary system4. Most COVID-19 patients exhibit flu-like symptoms within a span of two weeks from the exposure to the virus whereas there have been a majority rise in the asymptomatic COVID-19 patients.\n\nIn this work, we have performed high throughput virtual screening since it is the fastest approach in finding the probable drug against the target. High-throughput virtual screening (HTVS) of two databases was carried out via PyRx (Python prescription) software, which uses dock, Vina and Autodock as the docking tool. Autodock itself uses MGLTools comprising of computer aided drug discovery (CADD) pipeline for high throughput virtual screening of large databases for probable hits as target drugs. HTVS enables docking of multiple ligands on a single protein. PyRx is a freely available HTVS software. Docking results are based on the identification of pose visually and quantitatively using a scoring algorithm. Docking calculates the free binding energy (∆G) between the ligands and the protein. The free binding energy, thus calculated, is fundamental to the formation of complex systems in biochemistry and molecular biology. Lower free binding energy corresponds to a more favorable ligand binding affinity between a receptor and a ligand7.\n\n\nMethods\n\nMolecular docking is a bioinformatics method that allows predicting the orientation of a molecule, when it is bounded to another molecule8,9. There are two main approaches for molecular docking. The first approach describes the protein and the ligand as complementary surfaces10. The second approach simulates the docking process calculating the ligand protein interaction based on the free binding energy ∆G11.\n\nSelection of database and the COVID-19 main protease structure In this study, we have docked the X-ray crystal structure of main COVID protease protein (PDB ID: 6M03, resolution: 2 Å) with 129 molecules obtained from DrugBank and 992 molecules from the Zinc Natural Product database. The list of 129 molecules are provided along with the link for Zinc natural database in the Extended data12. These 129 molecules chosen were either antimalarial, antiparasitic, antibiotics, or antivirals, since hydroxychloroquine, remdesivir and ivermectin are antimalarial, antiviral and antiparasitic drugs, respectively. The Zinc Natural Product Database was chosen since most of the drugs are natural derivatives used against various diseases at present and it is a freely available database. Similarity search could not be undertaken since there is no known drug to function 100% against this novel disease at time of publication.\n\nDocking requires processing of the macromolecules and the ligands13,14. Water molecules were removed, polar hydrogen bonds were inserted into the crystal structure of 6m03 and it was converted to PDBQT format using AutoDock version 4. The energy of all ligands were minimized and they were converted into PDBQT files using Open Babel version 2.2.3 in PyRX version 0.815.\n\nThe grid box was determined as center the coordinates X:12.2632, Y:12.3998, and Z:5.4737, while as dimension the coordinates X:29.9242, Y:64.1097, and Z:48.1126.\n\nThe docking was done using Vina version 2.0 in PyRX. After the run, the out files stored in the user folder where the path run was specified in the edit preference. These output files were stored in PDBQT files, each having nine poses. The autodock application file was launched which then showed the empty dashboard along with \"File\" on the left hand corner of the page. The out file models were loaded using the \"read molecule\" application from the selected out file folder. Different poses were analyzed in the AutoDock tool. The pose with the lowest binding energy in kcal/mol was selected for further analysis. The docked molecules were then further converted into PDB format in PyMol and their interaction was studied using the software Discovery Studio version 4.1. The interaction can also be studied with PyMol but a better quality picture is obtained via Discovery studio. Three known drugs (hydroxychloroquine, remdesivir and ivermectin) were first docked against the virus main protease to check their binding energy. The interaction of these three drugs with the COVID-19 main protease could later be utilised for getting the hits.\n\n\nResults\n\nThe free binding energy for drugs known to act against COVID-19, which are hydroxychloroquine, remdesivir, and ivermectin, were found to be -5.5 kcal/mol, -6.3 kcal/mol, and -8.7 kcal/mol respectively as indicated in Table 1, which describes: a) PubChem compound ID (CID), which is the compound identifier in the PubChem database from where the 3D mol files of 129 molecules were downloaded; b) common drug name; and c) the free binding energy obtained after docking.\n\nThese drugs are known to improve the condition to some extent and yet their functions against COVID-19 are under study16–19.\n\nTherefore, we used these three molecules as our reference drugs. The interaction of these drugs with the virus main protease can be seen in the Figure 1. We could observe the interaction of these reference molecules as hydroxychloroquine forms a hydrogen bond with Tyr237 residue of the 6M03 main protease with a distance of 2.10 Å. It also interacts with Leu272 and Leu287. Remdisivir forms six hydrogen bonds with Lys137, Thr199, and Tyr239 along with interacting with Leu272, Leu287, Tyr237, and Asn238 residues of the 6M03 main protease. Ivermectin interacts with Leu272, Tyr239, Leu286, Leu287, Gly275, Asn277, and Met276 residues of the 6M03 main protease.\n\nKeeping the free binding energy of our reference molecules in mind, we shortlisted 77 molecules from the database of 129 molecules with a cut of -6 kcal/mol free binding energy. Eprinomectin, artefenomel, doramectin, betulinic acid, atovaquone, and tetrandrine showed the lowest binding energies, at -9 kcal/mol, -8.7 kcal/mol, -8.4 kcal/mol, -8.4 kcal/mol, -8.2 kcal/mol and -8 kcal/mol, respectively. Table 2 presents the details of the best performing 18 of the 77 molecules along with their CID. These molecules have been considered due to the lowest free binding energy between the ligand and protein. Furthermore, their interactions with the virus main protease 6m03 was studied. Artefenomel interacts with Pro108, Val202, Ile249, Pro293, and Phe294 residues of the 6M03 main protease. Eprinomectin forms a hydrogen bond (2.26 Å) with Lys5 residue of the 6M03 main protease and also interacts with Leu286, Leu287, and Asn277. Tetrandrine forms a hydrogen bond with Arg131 and also interacts with Leu272, Leu286 and Leu287. Betulinic acid forms a hydrogen bond with Arg137 with 2.66 Å distance and also interacts with Leu272, Leu286, Leu287, Tyr237, and Tyr239. Doramectin forms three hydrogen bonds with Thr199, Lys5, and Gly138 as well as interacts with Leu286 and Asp289. Atovoquone forms a hydrogen bond with the Thr199 residue of the 6M03 main protease with a distance of 2.48 Å and also interacts with Lys137, Leu272, Leu286, Leu287, and Tyr239. These interactions can be observed in Figure 2. Full results are available in the Extended data12.\n\nA total of 34 molecules of natural origin were chosen from the datasets of 992 molecules with a cut off -8.9 kcal/mol. Table 3 presents the details of the best 20 molecules along with their common name and ZINC ID sorted by ascending order of the free binding energy of the top 20 molecules from natural products database. The interaction of various molecules with the 6M03 main protease was studied. Allegra forms three hydrogen bonds with Thr111, Asn151, and Asp153 as well as interacts with Arg298, Val305, and Phe305 residues of 6M03 main protease. Baicalin forms four hydrogen bonds with Thr111, Thr292, Ile152, and Arg298 along with interacting with Asp153, Asn151, and Val303 residues of the main protease 6M03. Curine forms three hydrogen bonds with Arg131, Thr199, and Leu287 along with having interactions with Asp289, Leu286, and Tyr237 residues of 6m03 main protease. Etamidindole forms two hydrogen bonds with Thr111 and Asp295, it also interacts with Phe8, Phe294, Arg295, Arg298, and Pro252 residues of main protease 6M03. Glycyrrhetic acid forms a hydrogen bond with Lys137(2.24 Å) as well as interacts with Tyr237, Tyr239, Leu272, Leu286, and Leu287 residues of the main protease 6m03. Euphol interacts with Phe8, Val297, Arg298, and Val303 residues of the main protease. The interaction of few of the 20 molecules with the protein can be seen in Figure 3. Full results are available in the Extended data12.\n\n\nDiscussion\n\nHTVS is one of the best methods for identifying molecules acting against drug targets in a very short time period, when compared to traditional drug identification strategies. Remdesivir was detected as COVID-19 inhibitory drug via virtual screening method6. Therefore, keeping the present scenario in our mind, we undertook this study to provide individuals with COVID-19 with drugs already available. Surprisingly, we have found good hits from the databases with medicinal properties.\n\nArtefenomel is known to treat malaria and other parasitic diseases. Betulinic acid is under trial for the treatment of dysplastic nevus syndrome. Atovaquone is an approved drug for the treatment of Pneumocystis carinii pneumonia and malaria. Tetrandrine is in the experimental stage for anticancer, antimalarial, antiparasitic category. Eprinomectin and doramectin are veterinary antiparasitic drugs.\n\nMany of the natural compounds identified have medicinal properties. Taraxerone has allelopathic and antifungal effect20, Morusin has anti-oxidant and anticancer properties21, RA VII compound is an antitumor agent22, and neoruscogenin is used against chronic venous disorders23. Justicidin D exhibits anti-inflammatory properties24, Licoricidin is an antimetastatic molecule25 whereas euphol is used against asthma and cancer along with syphilis, and rheumatism26. Schisandrene has anti-oxidant activity27, curine is reported as a vasodilator27, angoluvarin has antimicrobial activity28, baicalin is used to treat cardiovascular diseases, inflammation and hypertension29. Glycyrrhetic acid shows anti-inflammatory, anti-ulcer, hepatoprotective, anti-allergic, anti-tumor, antioxidant and anti-diabetic activity30. Isomitraphylline has an antioxidant properties31, bikaverin and rutarensin has anti-tumour activities32,33. Jolkinol B has anticancer properties34 and Ethamidindole exhibits antihistamine properties35. Fexofenadine acetate, commonly known as Allegra, is an antihistamine pharmaceutical drug presently used in the treatment of allergy symptoms such as urticaria and hay fever36.\n\nWhen we compared our two datasets, we found that majority of the molecules showed lesser free binding energy as compared to the reference molecules as in Table 1, Table 2, and Table 3. Surprisingly, we obtained good hits from our natural database which is good news since after observing their interaction and biotherapeutic functions, we might have achieved our COVID-19 inhibitory drugs. One such drug from natural database is fexofenadine acetate (Allegra) which is presently in use as anti-allergic medicine. We also have hits from 129 drugs but there is one, atovaquone, which is presently used against pneumonia and malaria and also a very good candidate for COVID-19 treatment.\n\n\nConclusions\n\nThe best therapeutic drugs inferred from our studies are atovaquone, fexofenadine acetate (Allegra), justicidin D, baicalin, glycyrrhetic acid and ethamidindole based on their docking score, interaction studies and their present applications for probable COVID-19 treatment. The rest of the molecules could also be used as COVID-19 inhibitory drugs after further evaluation. When we compared our data with reference molecules score of currently in use drug against COVID-19, we found that atovaquone showed better binding energy than hydroxychloroquine and remdesivir. It is one of the best drug candidate for COVID-19 treatment since it is already in use for treating Pneumocystis carinii pneumonia and malaria. Fexofenadine acetate is another good target drug for COVID-19 treatment since it is naturally derived and presently used for its anti-histamine properties. Ethamidindole could possibly act as COVID-19 inhibitor since it is reported as anti-histamine and this novel virus activates cytokine secretion in human body. Apart from these, anti-inflammatory natural molecules such as justicidin D, baicalin, and glycyrrhetic acid could work against COVID-19 since SARS-CoV-2 virus induces inflammation. The rest of the top 20 molecules could also be considered since they all have some medicinal properties as explained above.\n\n\nData availability\n\nThe COVID-19 main protease structure was downloaded from the Protein Data Bank, ID 6M03: https://www.rcsb.org/structure/6M03.\n\nLigands were obtained from PubChem (https://pubchem.ncbi.nlm.nih.gov/) and the Zinc Natural Products database (http://zinc.docking.org/).\n\nZenodo: Molecular docking COVID-19. https://doi.org/10.5281/zenodo.384062512.\n\nThis project contains the following extended data:\n\nDrugs repurposing list (PDF). (PubChem CID of each ligand along with the minimized energy of each molecule and binding affinity results)\n\nDrugs (subfolder). (Interaction images of the reference molecules as well as the best performing target molecules from the 129 docked drugs.)\n\nNatural compounds (subfolder). (Interaction images of the best performing target molecules from the Zinc Natural Products database.)\n\nResults (PDF). (Complete docking results from the Zinc Natural Products database and the list of 129 drugs.)\n\nExtended data 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 members of the research group ITI for their valuable feedback.\n\n\nReferences\n\nXu X, Chen P, Wang J, et al.: Evolution of the novel coronavirus from the ongoing Wuhan outbreak and modeling of its spike protein for risk of human transmission. Science China Life Sciences. 2020; 63(3): 457–460. 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\n\nWorld Health Organization: Coronavirus disease 2019 (COVID-19) Situation Report - 101. 2020. Reference Source\n\nJiang F, Deng L, Zhang L, et al.: Review of the clinical characteristics of coronavirus disease 2019 (COVID- 19). J Gen Intern Med. 2020; 35(5): 1545–1549. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nLimón MC, Rodríguez-Ortiz R, Avalos J: Bikaverin production and applications. Appl Microbiol Biotechnol. 2010; 87(1): 21–29. PubMed Abstract | Publisher Full Text\n\nYang QY, Tian XY, Fang WS: Bioactive coumarins from boenninghausenia sessilicarpa. J Asian Nat Prod Res. 2007; 9(1): 59–65. PubMed Abstract | Publisher Full Text\n\nLage H, Duarte N, Coburger C: Antitumor activity of terpenoids against classical and atypical multidrug resistant cancer cells. Phytomedicine. 2009; 17(6): 441–8. PubMed Abstract | Publisher Full Text\n\nPatnaik GK, Das PK, Dhawan BN, et al.: Pharmacology of ethamidindole, a new antihistaminic agent. Indian J Pharmacol. 1979; 11(3): 181–187. Reference Source\n\nKumar L, Shahnwaj Alam Md, Lal Meena C, et al.: Fexofenadine hydrochloride. Profiles of drug substances, excipients and related methodology. 2009; 34: 153–192. Elsevier. Reference Source"
}
|
[
{
"id": "64263",
"date": "29 Jun 2020",
"name": "Elio Rodrigo Castillo",
"expertise": [
"Reviewer Expertise Evolutionary Biology",
"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\nI think that all the data is a quite good contribution, but in my opinion, text should be deeply modified to be more attractive to the readers and to point more directly their main objective.\n\nIntroduction\n\nParagraph 2: I suggest the authors to focus in the evidences showed by Chang et al. We know that vaccines are not available for COVID yet but your aim is to identify potential drugs for treatment.\nParagraph 3: Move this paragraph below “The World Health Organization (WHO) declared this disease as a pandemic on 11th March, 2020 and SARS-CoV-2 as the deadliest virus till date on earth claiming 217,896 deaths till 30th April, 2020”.\n\nParagraph 4: Please remove this paragraph because is not discussed in the corresponding section.\nParagraph 5: I recommend the authors to rewrite this paragraph. Authors performed an approach for searching drugs candidates based on databases. Please, focus in the main objective of the work. In my opinion authors could highlight the importance of bioinformatic tools (docking as the first approach) using available databases. I suggest to rewrite this paragraph because most of it content correspond to the Methodology section.\nMethods\nIn my opinion authors should be more clear with the methodology. You could cite a work in the introduction section where docking was the better strategy for drugs candidates search. In this section, you should explain your criteria of protein selection, provide a list and finally, the docking process. I suggest to reorganize this section as: \"Molecule selection and processing (or edition)\" and “Molecular docking”. In my opinion it is not necessary to split this information.\nResults\nFor a better understanding, I strongly recommend to use a general graphical scheme explaining the selection, the macromolecules edition, and the pipe used for the docking analysis. I suggest to focus only in the docking results against different ligands (from databases, natural etc). Some sentences correspond to the discussion section.\nDiscussion\nIt is not clear if you work with information from databases or with clinical samples. Please, rewrite this sentence. The paragraph underline (2nd) is disconnected from this section, please review. In my opinion the third paragraph of this section is not necessary. Authors could write in a sentence all this information and cite. I also suggest to discuss better your results and make a contrast with the available bibliography; authors repeated in this section the results. I understand that your contribution is relevant but from other perspective: bioinformatic tools for a quick detection of potential antivirals or drugs, using as an example the COVID. Several papers cited in the introduction section are not discussed.\nConclusions\nI suggest the integration of this section with the discussion. Authors proposed several treatments, inferred from HTVS, however, you should be careful with your statements. I recommend to discuss and compare the variables or data as indicators for potential treatments.\n\nThe manuscript is written with understandable English, though some English revision is necessary. In my opinion, after several major revisions and reorganization of the text, the study will be acceptable for indexing on F1000Research.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
},
{
"id": "64264",
"date": "06 Jul 2020",
"name": "Fernando Yepes-Calderon",
"expertise": [
"Reviewer Expertise Methods in medicine",
"Medical Imaging",
"Artificial Intelligence"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper is of interest under the current cov2 pandemic. Simulation of the strength of the bonds between molecules can identify inhibitors without human subjects' need to test. This paper did not focus on finding new compounds but in examining those present in existing medicine. This approach expedites the process of finding a cure, and the suggestions done are well-founded. Overall, the materials and methods section is exhaustive and asserts reproducibility.\nThere are some wordy paragraphs with redundancies. Please take a look at the following one and the replacement that I suggest.\nOriginal> The transmission of this coronavirus occurs due to the binding of the CoV spike protein to the angiotensin converting enzyme 2 (ACE2) receptor present on the cell surface of the human host.\nSuggestion>The transmission of this coronavirus occurs due to the binding of the CoV spike protein to the angiotensin-converting enzyme 2 (ACE2) receptor present on the human host's cell surface.\nI might suggest the authors remark the differences between their approach and the one presented by:\nSuyash Pant, Peptide-like and small-molecule inhibitors against Covid-191\nIt could be included in the discussion.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "67037",
"date": "14 Jul 2020",
"name": "Pritha Ghosh",
"expertise": [
"Reviewer Expertise Bioinformatics and 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 manuscript by Singh and Florez presents the discovery of drugs against COVID19 using molecular docking methods.\nMajor:\nThe authors must discuss in further detail as to how this work compares with other published reports of drugs targeted against SARS-CoV-2.\n\nThe authors should cite references for the results mentioned in the Introduction section of the manuscript.\n\nHow was the -6 kcal/mol free binding energy cut-off selected? As I understand this is not the lowest, the highest or the average of the reference molecule binding energies. Then what is the basis for the selection of this cut-off value?\n\nThe authors have discussed the docking results for the reference and the test molecules independently, but have not provided a comprehensive comparison of the observations. In my opinion, such a comparison is necessary for assessing the effectivity of the new drugs as compared to the existing drugs. The authors have also not discussed the importance of the residues Tyr237, Leu272, Leu287 etc. Are these known to be critical residues in the functioning of the main protease?\nMinor:\nThe statistics on the number of cases and deaths need to be updated before the indexing.\n\nThe authors should be more careful about the technicalities mentioned in the paper. For example, \"6m03\" has been mentioned loosely in the Introduction section without pointing to the fact that it is a PDB structure; the HTVS abbreviation has not been introduced in the manuscript before the first instance of its use.\nThe paper requires English editing by a professional and/or a native English speaker.\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? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/9-502
|
https://f1000research.com/articles/9-501/v1
|
03 Jun 20
|
{
"type": "Research Article",
"title": "Analysis of tRNACys processing under salt stress in Bacillus subtilis spore outgrowth using RNA sequencing data",
"authors": [
"Iván Arvizu Hernández",
"José Luis Hernández Flores",
"Juan Caballero Pérez",
"Héctor Gutiérrez Sánchez",
"Miguel Ángel Ramos López",
"Sergio Romero Gómez",
"Andrés Cruz Hernández",
"Carlos Saldaña Gutierrez",
"Erika Álvarez Hidalgo",
"George H. Jones",
"Juan Campos Guillén",
"Iván Arvizu Hernández",
"José Luis Hernández Flores",
"Juan Caballero Pérez",
"Héctor Gutiérrez Sánchez",
"Miguel Ángel Ramos López",
"Sergio Romero Gómez",
"Andrés Cruz Hernández",
"Carlos Saldaña Gutierrez",
"Erika Álvarez Hidalgo",
"George H. Jones"
],
"abstract": "Background: In spore-forming bacteria, the molecular mechanisms of accumulation of transfer RNA (tRNA) during sporulation must be a priority as tRNAs play an essential role in protein synthesis during spore germination and outgrowth. However, tRNA processing has not been extensively studied in these conditions, and knowledge of these mechanisms is important to understand long-term stress survival.\n\nMethods:To gain further insight into tRNA processing during spore germination and outgrowth, the expression of the single copy tRNACys gene was analyzed in the presence and absence of 1.2 M NaCl in Bacillus subtilis using RNA-Seq data obtained from the Gene Expression Omnibus (GEO) database. The CLC Genomics work bench 12.0.2 (CLC Bio, Aarhus, Denmark, https://www.qiagenbioinformatics.com/) was used to analyze reads from the tRNACys gene. Results:The results show that spores store different populations of tRNACys-related molecules. One such population, representing 60% of total tRNACys, was composed of tRNACys fragments. Half of these fragments (3´-tRF) possessed CC, CCA or incorrect additions at the 3´end. tRNACys with correct CCA addition at the 3´end represented 23% of total tRNACys, while with CC addition represented 9% of the total and with incorrect addition represented 7%. While an accumulation of tRNACys precursors was induced by upregulation of the rrnD operon under the control of σA -dependent promoters under both conditions investigated, salt stress produced only a modest effect on tRNACys expression and the accumulation of tRNACys related species. Conclusions:The results demonstrate that tRNACys molecules resident in spores undergo dynamic processing to produce functional molecules that may play an essential role during protein synthesis.",
"keywords": [
"B. subtilis spore germination",
"outgrowth",
"salt stress",
"tRNACys processing",
"RNA-Seq data"
],
"content": "Introduction\n\nWhen unfavorable environmental conditions affect cell growth, spore-forming bacteria activate and coordinate a temporal molecular program to assemble a dormant spore, which sequesters essential biomolecules for subsequent conversion back into growing cells. Bacillus subtilis sporulation has proved to be an extremely valuable model system for the study of the principal biomolecules that are stored and shielded during spore dormancy. Proteome analysis has revealed several categories of proteins that are stored in dormant B. subtilis spores, including proteins involved in ribosome biogenesis, carbon metabolism, RNA processing, and protein synthesis. These protein groups play important roles in spore germination and outgrowth (Swarge et al., 2019). In addition, dormant spores store RNA, which suggests that these RNAs play specific roles during dormancy or germination and outgrowth or are used as ribonucleotide reservoirs for de novo RNA synthesis during spore germination. Some of the RNAs are transcripts involved in the spore formation process, such as those coding for membrane proteins, transporters, RNA processing enzymes, and proteins involved in protein synthesis, modification and degradation (Bassi et al., 2016; Keijser et al., 2007; Nagler et al., 2016; Segev et al., 2012). Among these RNA categories, tRNA has been detected in significant amounts in dormant spores, and represents an essential reservoir (Nagler et al., 2016). One aspect of RNA metabolism that has not been well studied during spore germination and outgrowth is tRNA processing, and knowledge of those mechanisms will be essential to our understanding of long term stress survival by the spores and of the possible role of stored tRNAs in the generation of vegetative cells (Deutscher, 2006; Li & Deutscher, 1996; Li & Deutscher, 2002; Mohanty & Kushner, 2000; Ow & Kushner, 2002; Raynal et al., 1998; Reuven et al., 1997).\n\nIn B. subtilis, tRNACys is encoded by a single copy gene and its study has significant relevance to aspects of processing and to the understanding of the global factors that affect cysteine metabolism. Thus, our previous studies have shown that tRNACys processing during exponential growth in B. subtilis involves the ribonucleases RNase Z, PNPase, RNase R and the 3’ end modifying enzyme, CCAase. The activities of these enzymes converge in ways that have not yet been completely elucidated to facilitate CCA addition and tRNACys repair or degradation (Campos-Guillén et al., 2010; Campos-Guillén et al., 2019). To date, however, similar studies have not been performed on tRNACys metabolism during spore germination and outgrowth in B. subtilis.\n\nA dormant spore is highly organized within a dehydrated spore core, which is enveloped by a dense inner membrane, a germ cell wall, a cortex and a spore coat (Setlow, 2003). Each spore component is formed by molecules that play major roles in protecting the spore from a broad range of detrimental environmental conditions such as radiation, heat, desiccation and chemicals (Setlow, 2006). Under specific conditions, environmental nutrients are recognized by the germinant receptors (GRs) and promote biochemical changes such as the release of monovalent cations and Ca2+-dipicolinate, and the cortex is hydrolyzed and rehydrated. These molecular changes initiate the germination process (Setlow, 2006). After this first step, a phase called outgrowth follows, in which metabolic activity and molecular organization (“ripening”) convert the germinated spore into a growing cell. During the outgrowth phase, at least 30% of B. subtilis genes are activated (Horsburgh et al., 2001; Keijser et al., 2007).\n\nOne interesting aspect of spore germination is the effect of salt stress on the process and thus changes in the gene expression profile of outgrowing B. subtilis spores in the presence of high concentrations of NaCl have been analyzed by RNA sequencing (Nagler & Moeller, 2015; Nagler et al., 2014; Nagler et al., 2015; Nagler et al., 2016). In the last study, the presence of tRNAs in high percentage was detected in dormant spores but the nature of the tRNA population was not analyzed in depth. Taking advantage of new methodologies in high-throughput RNA sequencing, the availability of datasets in the Gene Expression Omnibus (GEO) database and computational analysis, we have investigated the profile of tRNACys in dormant and outgrowing B. subtilis spores in the presence of 1.2 M of NaCl. We were able to identify several categories of tRNACys species in dormant spores and during the first minutes of their conversion into growing cells. This study provides valuable insights into tRNACys processing during this state of transition.\n\n\nMethods\n\nAll experimental conditions to obtain RNA from dormant and outgrowing B. subtilis spores in the presence and absence of NaCl were previously described (Nagler et al., 2016). The raw RNA-Seq data indicated as SRR3488622 to SRR3488635 in Table 1, obtained from the Gene Expression Omnibus (GEO) database under the accession number GSE81238 were analyzed. The RNA-seq raw data correspond to: T0 (dormant spore), outgrowth of spores during 30 (T30), 60 (T60) and 90 (T90) minutes after the initiation of germination in the presence and absence of 1.2 M NaCl. Each sample time was sequenced twice.\n\nTo avoid contributions to our results by tRNA isoacceptors whose genes do not encode CCA at the 3´end in B. subtilis, we examined products of the tRNACys gene, which is present in single copy in the genome of B. subtilis and is located at the distal end of the rrnD operon. The gene does not encode the CCA at the 3´end, which must be added post-transcriptionally. The CLC Genomics work bench 12.0.2 (CLC Bio, Aarhus, Denmark) was used to analyze reads from the tRNACys gene, and small RNA adapters and reads with ambiguous nucleotides were trimmed using default settings. Open source alternatives such as UniPro UGENE, UTAP or Galaxy could also have been used for this purpose. Trimmed reads were then were mapped to the B. subtilis 168 genome downloaded from the NCBI website (accession number NC_000964.3) with short read local alignment mapping using the default setting. We considered alignments with a length fraction of 0.8 and a similarity fraction of 0.8. Two mismatches and three insertions and deletions per read were allowed. The mapped reads results for all experiments ranged from 243 to 1,141.\n\nMapped reads were used in the sequence alignment program MUSCLE in MEGA X v10.1.7 with default settings (Kumar et al., 2018) and high similarity aligned reads to the reference tRNACys sequence were manually counted and categorized as the following subpopulations: I) precursors (containing genomically encoded nucleotides at the 5´or 3´ ends), II) tRNA immature (these species were 71 nucleotides in length and lacked only the CCA-end), III) mature tRNA with 3´-CCA, IV) immature tRNA with 3´-CC. We also observed short, tRNACys-derived RNA fragments (tRFs) in this study. Reads representing such fragments were categorized as V) 3´-tRF fragments detected with CCA 3’ends, VI) 3´-tRF fragments detected with CC 3’ends, VII) fragments detected as 5´-tRF (71 bases – n, where n > 0, from the 3’ end) or internal fragments (between nucleotide positions 2 to 70), VIII) complete tRNACys with incorrect 3´ tails and IX) 3´-tRF fragments with incorrect 3´ tails. The distribution analysis of tRNACys subpopulations was done using the JMP 7 program (SAS Institute Inc., Cary, NC) with the frequency per group algorithm. Trend graphs were constructed in SigmaPlot software version 14.0 (Systat Software, San Jose, CA).\n\nGEO, Gene Expression Omnibus.\n\n\nResults\n\nIn order to provide in-depth insight into tRNACys processing in the dormant spore and during outgrowth in B. subtilis, RNA-Seq methodology and the GEO database (GSE81238) were utilized. The total number of reads for all experiments ranged from 7 to 12 million, but an interesting finding was that there is a distribution of reads mapped for tRNACys in all experimental conditions tested (Table 1).\n\nReads that mapped to the region of the tRNACys gene were used to distinguish between different stages of tRNACys processing. To reduce contributions to the analysis of modification-induced polymerase fall-off during RNA-Seq methodology and due to the high percentage of short tRNACys-derived RNA fragments (5´-tRFs or 3´-tRFs) that were observed in the analysis, for purposes of statistical analysis, all mapped reads were characterized.\n\nThe results show that the dormant spore stores a diverse population. An average of 296 mapped reads were analyzed in dormant spores (T0), and nine categories of tRNACys-related species (see Methods) were characterized and are represented in Figure 1.\n\nReads categorized were: I) precursors, II) immature tRNA, III) mature tRNA with 3’ CCA ends, IV) immature tRNA with 3’ CC ends, V) 3’-tRF fragments detected with 3’ CCA ends, VI) 3’-tRF fragments detected with 3’ CC ends, VII) 5’-tRF or internal fragments, VIII) complete tRNA with incorrect 3´ tails added and IX) 3´-tRF fragments with incorrect 3´ tails added. The numbers after each category indicate the percentage of total tRNACys represented by that species. The table at the right shows the tails associated with the various tRNACys species in green.\n\nFrom these categories, we observed the following percentages of reads stored in dormant spores. A low percentage (1.01%) of reads represented tRNA with genomic encoded nucleotides at the 5´or 3´ ends, which were categorized as unprocessed transcripts (category I). The category with the lowest percentage of reads (0.34%) represented tRNAs with immature 3´ends (category II). The most abundant category of reads (22.85%) represented tRNAs with the correct CCA at the 3´ end (category III). An interesting finding was the relatively high percentage of mapped reads (9.47%) representing tRNAs with CC at the 3´end (category IV), which we suggest are immature tRNAs. We counted a high percentage of reads representing 3´-tRF fragments with correct CCA or CC 3´ends (23.19%) for categories V and VI. Due to the high number of mapped reads of fragments observed at time 0 (dormant spore) and compared with the lower number of mapped reads at the other times analyzed (see results below), we suggest that these reads are most likely fragments generated during spore formation. If these fragments are subsequently degraded, they might support de novo RNA synthesis during outgrowth. However, we cannot rule out the possibility that a percentage of them could be modification-induced polymerase fall-off byproducts resulting from the RNA-Seq methodology.\n\nWe observed a considerable percentage of reads (30.46%) of 5´-tRF or internal fragments, which are represented as category VII. The last categories observed, VIII and IX, were reads with incorrect post-transcriptionally added tails on complete tRNACys or 3´-tRF fragments. The total percentage of these reads was 12.16% and the tails are shown in Figure 1 (table at right side). Both categories of molecules of complete tRNACys or 3´-tRF fragments with CCA at the 3´end were observed to include species that carried the extra nucleotides C or CA. Some of them also had a second CCA terminus, while others possessed poly(A) tails.\n\nOur results show that the dormant spore stores mature tRNACys, which could support the initial phases of translation, while tRNACys processing progress during the outgrowth phase. On the other hand, fragments might be degraded so that the ribonucleotides obtained could be available for de novo RNA synthesis during the outgrowth phase. Therefore, it is reasonable to suggest that during ripening phase, tRNA processing will progress and over time the population frequency of spore-stored tRNACys molecules will change.\n\nTo understand the population dynamics of tRNACys molecules during the outgrowth process, the mapped reads for tRNACys at 30, 60 and 90 minutes after the initiation of germination in the absence and presence of 1.2 M of NaCl were analyzed. From a total of 9,163 mapped reads obtained (Table 1) during 90 minutes of outgrowth in both conditions, the distribution of each subpopulation of tRNACys molecules was obtained, represented as the average at each sample time and visualized in Figure 2.\n\nWe show the last ten nucleotides of tRNACys, with correct or incorrect additions at the 3´end. Fragments (total 5´-tRF and internal fragments), 3´tRF and precursors are indicated at the left side. The average of mapped reads for each subpopulation is indicated by a number and by the bar graph at the right side and its presence at each sampled time by a colored bar in the graph.\n\nWhile some subpopulations of these molecules were observed at lower frequency and detected in at least one RNA sample and at one sampling time, other sub-populations were present at all analyzed time points. In agreement with previous reports, robust transcription was induced during outgrowth. Thus, the sub-population of precursors of tRNACys are accumulated at all analyzed time points and represent a high frequency of tRNA molecules from the reads mapped with an average of 2034.5 mapped reads.\n\nDuring ripening phase, it is reasonable to suppose that a high accumulation of the subpopulation of precursors during outgrowth will change the subpopulation of immature forms of tRNACys (category II). However, the analysis of mapped reads shows a lower frequency, with an average of 34 mapped reads. These results may indicate that tRNACys processing from precursors by endo or exo-ribonuclease activities progresses to some extent during the outgrowth phase to produce the immature forms of tRNACys which are then available for CCA addition. In fact, the mature form of tRNACys with CCA at its 3´end did increase in abundance during the outgrowth phase, with an average of 844 mapped reads, which means that CCA-adding activity is manifested during the ripening phase. Our results indicate that a significant fraction of the tRNACys subpopulations with no CCA-end, or with C- or CC-ends are converted to mature tRNACys during outgrowth. However, some immature species without complete CCA-ends were observed at all outgrowth time points.\n\nAn interesting subpopulation of tRNACys was observed and represented by fragments or 3´-tRFs with different additions at the 3´end; together, these subpopulations represent an average of 1365.5 mapped reads. These results indicate that during ripening phase, degradation by RNases is an important mechanism for obtaining ribonucleotides for RNA synthesis or that a mechanism of RNA quality control is operating in this phase. In this regard, an average of 194 mapped reads were observed for complete tRNACys molecules and an average of 57 mapped reads for 3´-tRF fragments with incorrect additions at 3´end.\n\nEffects on gene expression under conditions of high salinity have been reported for outgrowing spores (Nagler et al., 2016). Therefore, to understand whether tRNACys processing and degradation might be affected by outgrowth in the presence of 1.2 M NaCl, mapped reads for each category mentioned above at the various sample times was contrasted with mapped reads from data obtained in the absence of NaCl. Our analysis results for mapped reads showed remarkably that precursors (category I) for tRNACys strongly increased at 30 minutes after the initiation of germination in both conditions investigated compared with reads from dormant spores (T0), no statistical difference was observed in the presence of NaCl. At 60 and 90 minutes, accumulation of precursors remains relatively stable and both conditions show similar averages of reads mapped (Figure 3).\n\nAverage of reads mapped for each category were plotted. Error lines represent standard errors of means.\n\nThese results mean that during the ripening phase in outgrowing spores, tRNACys gene expression is upregulated. Given this observation, we investigated whether increased tRNACys transcription would differentially affect a particular subpopulation of tRNACys molecules related to the processing mechanisms. Beginning with category II, our results show that for the immature form of tRNACys there was an increase in abundance during outgrowth, but the frequency of reads mapped did not change significantly in the presence or absence of NaCl at any time point (Figure 3). The next step was to investigate whether the subpopulation of tRNACys with CCA at the 3´end (category III) would be affected during salt stress. No statistically significant differences in the abundance of category III species were observed in the presence or absence of NaCl at any time point (Figure 3).\n\nAn intriguing result was obtained in the analysis of immature tRNA with CC at the 3´end (category IV). While that subpopulation of tRNACys molecules decreased dramatically during the first 30 minutes after the initiation of germination in both conditions, in the presence of NaCl the abundance of this species continued to decrease, while in the absence of NaCl at 60 minutes this subpopulation of tRNACys molecules increased somewhat in abundance. These results suggest that CCA repair mechanisms may be operating in the absence of NaCl to produce mature tRNACys during this phase. If we consider the results for tRNACys stored in dormant spores at T0, categories III plus IV represent an average of 95.5±16 mapped reads, compared with an average of 93±1 and 119±12 mapped reads, respectively, for tRNACys with CCA in the presence and absence of NaCl at 30 minutes after the initiation of germination. These observations suggest that CCA-addition mechanisms are operative in the presence of NaCl, albeit less efficiently than in its absence. However, we cannot rule out the possibility that in the presence of NaCl RNase activity might be rerouted as a mechanism to obtain ribonucleotides to support transcription rather than CCA repair.\n\nThis last interpretation correlates with the results for the subpopulations of tRNACys 3´-tRF fragments with CCA or CC addition at the 3´end. The levels for both sub-populations (categories V and VI) decreased significantly, to near zero in the case of category VI, in both conditions during the outgrowth phase (Figure 3). Figure 3 shows further that category VII fragments increased in abundance during outgrowth in the presence and absence of NaCl, but to a lower level in the presence of NaCl than that observed in its absence.\n\nThe abundance of category VIII species increased at T30 then decreased somewhat thereafter in the absence of NaCl, while a continuous increase in this species was observed during outgrowth in the presence of NaCl. At T90, category VIII levels were essentially the same in the presence and absence of NaCl. Category IX levels decreased during outgrowth, initially more dramatically in the absence of NaCl than in its presence.\n\n\nDiscussion\n\nIn B. subtilis, tRNA genes are immersed in operons and tRNAs are therefore synthesized as precursors that undergo post-transcriptional modification to give functional molecules. These modifications include 5´and 3´ processing by ribonucleases, addition of CCA at the 3´end of those tRNAs that do not encode this sequence and post-transcriptional editing of ribonucleosides.\n\nThe tRNACys gene is present in single copy in the genome of B. subtilis and is located at the distal end of the rrnD operon, which codes for three rRNA molecules; 16S, 23S and 5S rRNA, and a cluster of tRNA genes found downstream of the 5S rRNA gene (Vold, 1985; Wawrousek et al., 1984). Regulation of expression of the rrnD operon is mediated by two cognate σA consensus promoter sequences (P1 and P2) upstream of the 16S rRNA gene (Koga et al., 2006). A third σA consensus promotor is localized in the 23S-5S intergenic space, and is associated with mechanisms of the stringent response (Vold, 1985; Wawrousek et al., 1984). A number of reports have revealed that the regulation of expression of P1 and P2 is maintained during spore formation, and that these promoters remain active even during the late stages of sporulation (de Hoon et al., 2010; Koga et al., 2006; Rosenberg et al., 2012). Other reports observed slight differences in the expression of the P1 and P2 promoters under nutritional stress in vegetative cells (Natori et al., 2009; Samarrai et al., 2011).\n\nBased on these reports, it is clear that tRNACys expression under the control of promoters P1 and P2 takes place during sporulation and that diverse sub-populations of tRNACys molecules might thus accumulate in dormant spores. To expand our knowledge of tRNA processing in dormant and germinating B. subtilis spores, RNA-Seq methodology was used to display the diversity of tRNACys species that were present in these conditions and in the spores germinated in the presence of high salt. Our results show for first time the diversity of the sub-populations of tRNACys stored in dormant spores in B. subtilis (Figure 1) and contribute to the knowledge of tRNACys processing during the dormancy and outgrowth phases.\n\nOur results show a progressive generation and degradation of tRNACys fragments (Figure 3), which suggests that RNases are present and active at the various sampling times employed in these experiments. In fact, a recent report by Swarge et al. (2019) demonstrated via proteome analysis that several exo-ribonucleases are present in dormant spores, including PNPase, RNase R, RNase PH, YhaM, RNase Y and others. Transcripts for PNPase, YhaM and RNase Y were only observed at 15 minutes and for RNase R and RNAse PH at 30 minutes after the initiation of germination. Thus, these exo-ribonucleases remaining from sporulation are likely to play essential roles in the pathways of tRNA degradation and/or processing during the sporulation and ripening phases.\n\nIn the particular case of tRNA processing in B. subtilis, in vitro and in vivo studies of the endonucleolytic pathways of RNase P (Evans et al., 2006; Kurz et al., 1998) or RNase Z (Pellegrini et al., 2003) and the exonucleolytic pathways of RNase PH, PNPase, RNase R or YhaM have been performed (Oussenko et al., 2002; Oussenko et al., 2005). From these studies it is clear that tRNA precursors with extensions at the 5´ end are eliminated by RNase P, while RNase Z processes the 3´end of almost all tRNAs lacking an encoded CCA (Pellegrini et al., 2003) and the exonuclease RNase PH processes CCA-containing tRNA precursors (Oussenko et al., 2002; Oussenko et al., 2005). The CCA motif at the 3´end is a key feature of all mature tRNAs and is essential for aminoacylation during protein synthesis (Hou, 2010; Wellner et al., 2018). Our results show that an important subpopulation of mature tRNA with CCA at its 3’ end is stored in the dormant spore and its levels increase during outgrowth. A delay in this increase was observed in the presence of NaCl, due perhaps to an effect of salt on the activities of enzymes related to tRNACys processing. In fact, there are reports demonstrating the effects on CCA addition to tRNACys of RNase Z, PNPase, RNase R and CCAase (Campos-Guillén et al., 2010; Campos Guillén et al., 2019). It remains to be seen how these enzymes are affected under salt stress conditions during spore outgrowth, but Nagler et al. (2016) reported the interesting observation that the cca gene was downregulated during 90 minutes of outgrowth in the presence of NaCl. A relevant feature is that the cca gene is immersed in the operon containing bacillithiol (a REDOX regulator and an α-anomeric glycoside of L-cysteinyl-D-glucosamine with L-malic acid) biosynthetic genes under σA-dependent promoter control, which suggests that cca expression and the integrity of the 3’ terminus of tRNAs may be important during salt stress (Campos Guillen et al., 2017; Gaballa et al., 2010; Gaballa et al., 2013).\n\nAccording to previous reports, incorrect additions at the 3´end of tRNACys were detected in B. subtilis, which suggests that alternative pathways of repair or degradation may exist in that organism (Campos-Guillén et al., 2010; Campos Guillén et al., 2019; Cruz Hernández et al., 2013). Although a high percentage of mature tRNA with the CCA 3´end was observed in dormant spores, the species that would be available to support initial protein synthesis during outgrowth, it remains to be determined whether this stored tRNACys is charged with cysteine. It should be noted in this regard that cysteinyl-tRNA synthetase was detected in dormant spores and its expression was also detected at 60 minutes after the initiation of germination (Nagler et al., 2016; Swarge et al., 2019).\n\nDetrimental responses to salt during spore germination in B. subtilis have been analyzed previously (Nagler & Moeller, 2015; Nagler et al., 2014; Nagler et al., 2015; Nagler et al., 2016). From these studies, 402 genes related to the salt stress response were upregulated, some of them under σA-dependent promoter control (Nagler et al., 2016). Therefore, the observed accumulation of tRNACys precursors during the 90 minutes of outgrowth in our study suggests that upregulation of the rrnD operon, governed by σA-dependent promoters is important for macromolecular synthesis during outgrowth. An unanswered question is which of the three rrnD promoters is responsible for this upregulation.\n\n\nConclusions\n\nThe ability to quantitatively monitor tRNA abundance changes from dormant spore to germination and outgrowth will be critical to understanding these processes and the roles of transcription, post-transcriptional modification and translation in them. The results reported here using RNA-Seq and data analysis provide new insights into the dynamic changes in the sub-populations of RNA species related to tRNACys molecules in dormant spores and during spore germination and outgrowth in the presence and absence of 1.2 M NaCl. An important unanswered question is what role these various subpopulations play in the metabolic economy of the spore and in the processes of germination and outgrowth.\n\n\nData availability\n\nRaw RNA-Seq data on Gene Expression Omnibus, Accession number GSE81238: https://identifiers.org/geo:GSE81238\n\nB. subtilis 168 genome from NCBI Reference Sequence, Accession number NC_000964.3: https://identifiers.org/refseq:NC_000964.3",
"appendix": "Acknowledgments\n\nThe authors would like to acknowledge to Databiology Ltd for supporting the computing to perform this research.\n\n\nReferences\n\nBassi D, Colla F, Gazzola S, et al.: Transcriptome analysis of Bacillus thuringiensis spore life, germination and cell outgrowth in a vegetable-based food model. Food Microbiol. 2016; 55: 73–85. PubMed Abstract | Publisher Full Text\n\nCampos-Guillén J, Arvizu-Gómez JL, Jones GH, et al.: Characterization of tRNA(Cys) processing in a conditional Bacillus subtilis CCase mutant reveals the participation of RNase R in its quality control. Microbiology. 2010; 156(Pt 7): 2102–2111. PubMed Abstract | Publisher Full Text\n\nCampos Guillén J, Arvizu Gómez JL, Jones GH, et al.: Analysis of tRNACys processing in the absence of CCAase in Bacillus subtilis. Braz J Microbiol. 2019; 50(3): 613–618. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCampos Guillen J, Jones GH, Saldana Gutierrez C, et al.: Critical Minireview: The Fate of tRNACys during Oxidative Stress in Bacillus subtilis. Biomolecules. 2017; 7(1): pii: E6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCruz Hernández A, Millan ES, de Jesús Romero Gómez S, et al.: Exposure of Bacillus subtilis to mercury induces accumulation of shorter tRNA Cys species. Metallomics. 2013; 5(4): 398–403. PubMed Abstract | Publisher Full Text\n\nde Hoon MJ, Eichenberger P, Vitkup D: Hierarchical evolution of the bacterial sporulation network. Curr Biol. 2010; 20(17): R735–745. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDeutscher MP: Degradation of RNA in bacteria: comparison of mRNA and stable RNA. Nucleic Acids Res. 2006; 34(2): 659–666. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEvans D, Marquez SM, Pace NR: RNase P: interface of the RNA and protein worlds. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nKeijser BJ, Ter Beek A, Rauwerda H, et al.: Analysis of temporal gene expression during Bacillus subtilis spore germination and outgrowth. J Bacteriol. 2007; 189(9): 3624–3634. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKoga K, Ikegami A, Nakasone K, et al.: Construction of Bacillus subtilis strains carrying the transcriptional bgaB fusion with the promoter region of each rrn operon and their differential transcription during spore development. J Gen Appl Microbiol. 2006; 52(2): 119–124. PubMed Abstract | Publisher Full Text\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\nKurz JC, Niranjanakumari S, Fierke CA: Protein component of Bacillus subtilis RNase P specifically enhances the affinity for precursor-tRNAAsp. Biochemistry. 1998; 37(8): 2393–2400. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nNagler K, Moeller R: Systematic investigation of germination responses of Bacillus subtilis spores in different high-salinity environments. FEMS Microbiol Ecol. 2015; 91(5): pii: fiv023. PubMed Abstract | Publisher Full Text\n\nNagler K, Setlow P, Li YQ, et al.: High salinity alters the germination behavior of Bacillus subtilis spores with nutrient and nonnutrient germinants. Appl Environ Microbiol. 2014; 80(4): 1314–1321. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNagler K, Setlow P, Reineke K, et al.: Involvement of Coat Proteins in Bacillus subtilis Spore Germination in High-Salinity Environments. Appl Environ Microbiol. 2015; 81(19): 6725–6735. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNatori Y, Tagami K, Murakami K, et al.: Transcription activity of individual rrn operons in Bacillus subtilis mutants deficient in (p)ppGpp synthetase genes, relA, yjbM, and ywaC. J Bacteriol. 2009; 191(14): 4555–4561. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOussenko IA, Abe T, Ujiie H, et al.: Participation of 3'-to-5' exoribonucleases in the turnover of Bacillus subtilis mRNA. J Bacteriol. 2005; 187(8): 2758–2767. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOussenko IA, Sanchez R, Bechhofer DH: Bacillus subtilis YhaM, a member of a new family of 3'-to-5' exonucleases in gram-positive bacteria. J Bacteriol. 2002; 184(22): 6250–6259. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOw MC, Kushner SR: Initiation of tRNA maturation by RNase E is essential for cell viability in E. coli. Genes Dev. 2002; 16(9): 1102–1115. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPellegrini O, Nezzar J, Marchfelder A, et al.: Endonucleolytic processing of CCA-less tRNA precursors by RNase Z in Bacillus subtilis. EMBO J. 2003; 22(17): 4534–4543. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRaynal LC, Krisch HM, Carpousis AJ: The Bacillus subtilis nucleotidyltransferase is a tRNA CCA-adding enzyme. J Bacteriol. 1998; 180(23): 6276–6282. PubMed Abstract | Free Full Text\n\nReuven NB, Zhou Z, Deutscher MP: Functional overlap of tRNA nucleotidyltransferase, poly(A) polymerase I, and polynucleotide phosphorylase. J Biol Chem. 1997; 272(52): 33255–33259. PubMed Abstract | Publisher Full Text\n\nRosenberg A, Sinai L, Smith Y: Dynamic expression of the translational machinery during Bacillus subtilis life cycle at a single cell level. PLoS One. 2012; 7(7): e41921. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSamarrai W, Liu DX, White AM, et al.: Differential responses of Bacillus subtilis rRNA promoters to nutritional stress. J Bacteriol. 2011; 193(3): 723–733. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSegev E, Smith Y, Ben-Yehuda S: RNA dynamics in aging bacterial spores. Cell. 2012; 148(1–2): 139–149. PubMed Abstract | Publisher Full Text\n\nSetlow P: Spore germination. Curr Opin Microbiol. 2003; 6(6): 550–556. PubMed Abstract | Publisher Full Text\n\nSetlow P: Spores of Bacillus subtilis: their resistance to and killing by radiation, heat and chemicals. J Appl Microbiol. 2006; 101(3): 514–525. PubMed Abstract | Publisher Full Text\n\nSwarge B, Jonker M, Abhyankar W, et al.: Integrative analysis of proteome and transcriptome dynamics during Bacillus subtilis spore revival. bioRxiv. 2019; 682872. Publisher Full Text\n\nVold BS: Structure and organization of genes for transfer ribonucleic acid in Bacillus subtilis. Microbiol Rev. 1985; 49(1): 71–80. PubMed Abstract | Free Full Text\n\nWawrousek EF, Narasimhan N, Hansen JN: Two large clusters with thirty-seven transfer RNA genes adjacent to ribosomal RNA gene sets in Bacillus subtilis. Sequence and organization of trrnD and trrnE gene clusters. J Biol Chem. 1984; 259(6): 3694–3702. PubMed Abstract\n\nWellner K, Betat H, Mörl M: A tRNA's fate is decided at its 3' end: Collaborative actions of CCA-adding enzyme and RNases involved in tRNA processing and degradation. Biochim Biophys Acta Gene Regul Mech. 2018; 1861(4): 433–441. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "77845",
"date": "05 Feb 2021",
"name": "Anne Moir",
"expertise": [
"Reviewer Expertise Spore germination and outgrowth in Bacillus subtilis. I am not an expert in RNA Seq data or its analysis."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors present their in silico analysis of tRNA-Cys molecules in B. subtilis dormant spores, and during early outgrowth. The data analysed are those deposited by Nagler et al. (2016), in which they used RNA Seq to analyse gene expression during 90 min of outgrowth in an alanine glucose minimal medium. According to their published data, under these conditions the germinated spores do not yet show any increase in size, and this early stage of outgrowth has been referred to as “ripening”. The authors here have examined all the data and have classified, for each relevant sequence read, the form of tRNA-Cys detected, from precursor through mature and degraded forms.\n\nAs the in vivo experiment was carried out in duplicate, with two samples at each time point, it is easy to overinterpret the absolute statistical significance of individual data points in Figure 3. I am not a statistics expert, but it seems that calculating a standard error of the mean of two values does highlight where the levels are particularly variable for an individual time point, but cannot be used in general to claim statistical significance. Such qualitative experiments are extremely useful nonetheless, as data from the succession of time points confirm the general trend.\n\nThe major conclusions are (i) a variety of forms of tRNA-Cys are present in dormant spores; the relative proportions are described (though to a spurious degree of accuracy, as a result of too many significant figures used in the calculated percentages), (ii) that during outgrowth, the total levels of this tRNA species increase approximately three fold, and the relative amounts of the various forms change - most notably, the precursor form increases dramatically, from almost negligible to approximately half of the total, and the mature CCA-tailed form increases approximately 2.5 fold in amount. There is a reduction in the level of several degradation products, except for that most commonly found (Class VII), the amount of which increases approximately three fold. There is no very strong evidence of differences between samples outgrowing in low and high salt in the graphs of Figure 3, although it may be the case for Category VII molecules.\nThis is the first detailed analysis of tRNA-Cys levels in spores, and the implications for its synthesis and processing are fully discussed.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "6369",
"date": "18 Feb 2021",
"name": "JUAN CAMPOS GUILLEN",
"role": "Author Response",
"response": "Thank you for your comments. We agree with you about the interpretation of statistical significance in figure 3. However, we decided to use standard errors of means for the next reasons: 1. When the mapped reads for all categories for tRNACys were analyzed as percentage of total reads for each time, some data can easily be misunderstood. For example, for category III, from time 0 to time 30, a decrease percentage was observed when percentage of total was used. So that, the means for each category in each time was the better option for interpretation. 2. The raw RNA-Seq data were obtained from Nagler et al., 2016, so that only duplicated data were obtained in these experiments. We were curious to analyze other Gene Expression Omnibus (GEO) database for Bacillus subtilis, using different RNA protocol and sequencing methods, however mapped reads for tRNAcys were not obtained. This mean that as the only one copy gene for tRNACys in the genome, probably molar concentration could be less that other tRNAs isoacceptors with two or more copy genes. Additionally, RNA protocol and sequencing method could be affecting the total of mapped read, including in duplicated experiment. 3. In view of the above and for a comprehensive solution to this issue, we use standard error as a mathematical tool used in statistics to measure variability. It is used to estimate the efficiency, accuracy, and consistency of a sample. In other words, it measures how precisely a sampling distribution represents a population. The standard error decrease as the sample size increases, in our case, two values were used, but standard error of means show the distribution of mapped read from these samples at each time for each experiment for a correct interpretation in the presence and absence of 1.2 M NaCl. From these results, no statistical difference was observed in the presence of NaCl."
}
]
},
{
"id": "82204",
"date": "19 Apr 2021",
"name": "Jungwook Kim",
"expertise": [
"Reviewer Expertise tRNA modification"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nBy analyzing RNA-Seq data, the authors demonstrated dynamic population changes of diverse tRNA species from dormant to outgrowth phases of Bacilus subtilis. In the dormant phase, precursor RNA (Category I) is present only at an insignificant level, however, it becomes the most dominant species during the outgrowth phase. Precursor RNA apparently undergoes efficient maturation steps, where a relatively low level of immature species (Category II) was detected at all time points of the ripening stage. Interestingly, 5’-tRFs (designated as Category VII) are the second most abundant species next to precursor RNA during the outgrowth phase, showing a gradual increase in abundance over time. 3’-tRFs (Categories V, VI, VIII, and IX), on the other hand, appear to be formed at a much lower level in general compared to 5’-tRFs, and mature tRNA decrease sharply in the early stage of outgrowth. Authors proposed that these 3’-tRFs are intermediary species on the RNA degradation pathway during spore formation, products of which may serve as building blocks for de novo RNA synthesis in the outgrowth phase. The impact of a high concentration of salts (1.2M NaCl) on the distribution of tRNA subpopulation during the outgrowth phase was also investigated, but no significant effect was observed.\nThe RNA-Seq analysis on dynamics of tRNA metabolism offers interesting biological insights into the regulation of tRNA processing in dormant and outgrowth phases of B. subtilis. Being a non-expert on RNA-Seq, however, interpretation of some data was not clear to me, for example, whether tRNA species with a small number of reads (one or less) are biologically relevant or merely represent sequencing artifacts. If the former, have those species been detected previously in other cases? Secondly, despite Category VII being the second most abundant species in both spore and ripening stages, the discussion on the biological significance of this class in B. subtilis was largely lacking. In particular, the size distribution of 5’-tRFs is not clearly described compared to that of 3’-tRFs. A representative structure of Category VII displayed in Figure 1 implies that the cleavage occurred around the variable loop of mature tRNACys. Interestingly, previously known 5’-tRFs in eukaryotes include species much shorter in length, which are typically generated from the endonuclease activities on D- or stem-loops. Lastly, can tRNA or its fragment with CC instead of CCA at the 3’-terminus (Category IV and VI, respectively) be considered as a degradation intermediate derived from mature tRNA (Category III) as opposed to an intermediate resulting from incomplete addition of CCA from immature tRNA (Category II) as authors claimed?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "6890",
"date": "09 Jul 2021",
"name": "JUAN CAMPOS GUILLEN",
"role": "Author Response",
"response": "Thank for your comments, they are very valuable. Our study is based on the availability of datasets in the Gene Expression Omnibus (GEO) database, remarkably for the data obtained in the research work by Nagler et al., 2016. So that, we only investigated the profile of tRNACys in dormant and outgrowing B. subtilis spores in the presence of 1.2 M of NaCl using this GEO database. As you know, diverse methodologies in high-throughput RNA sequencing exist in the market, however the use of one of them depend principally of the research question. Particularly, for the tRNA sequences is critical the development and use of methodologies for tRNA extraction, tRNA library preparation, sequencing, read processing and mapping, that differ markedly from those used to analyze RNA-seq data when studying microRNAs (miRNAs) or mRNAs. In the case of tRNA, with high secondary structure, it could induce polymerase fall-off during reverse transcription and merely represent sequencing artifacts. Actually, we are development a tRNA methology to resolve this problem. However, in the result we note this: “To reduce contributions to the analysis of modification induced polymerase fall-off during RNA-Seq methodology and due to the high percentage of short tRNACys-derived RNA fragments (5´-tRFs or 3´-tRFs) that were observed in the analysis, for purposes of statistical analysis, all mapped reads were characterized.” Also, we note in conclusion: “The ability to quantitatively monitor tRNA abundance changes from dormant spore to germination and outgrowth will be critical to understanding these processes and the roles of transcription, post-transcriptional modification and translation in them.\" With these criteria, we cannot discuss in dep the significance about the 5’-tRFs or 3’-tRFs and the endonuclease activities on D- or stem-loops. On the other hand, the tRNACys, which lacks an encoded 3’ CCA in Bacillus subtilis, the evidence indicates that CCAase plays an important role in the maturation of the 3´end of tRNACys, but in its absence, other enzymes, possessing C- and A-adding activities in Bacillus subtilis are involved in tRNACys maturation or degradation (Campos Guillen J, Jones GH, Saldana Gutierrez C, et al.: Critical Minireview: The Fate of tRNACys during Oxidative Stress in Bacillus subtilis. Biomolecules. 2017;7(1): pii: E6.). The use of mutants strain in these enzymes and its role in the maturation of the 3´end of tRNA during dormant spores, germination and outgrowth will provide new insights."
}
]
},
{
"id": "83556",
"date": "04 May 2021",
"name": "Fabian M. Commichau",
"expertise": [
"Reviewer Expertise I am not an expert in tRNA modification. My areas of expertise are bacterial physiology & genetics",
"gene regulation",
"second messengers."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn the present manuscript, Hernández and co-workers performed a bioinformatic analysis to uncover the modifications in the populations of tRNACys-related molecules in the Gram-positive model bacterium Bacillus subtilis. The study is based on existing RNASeq data that have been generated with outgrowing spores in the absence and presence of sodium chloride.\n\nThe observation that the spores contain different tRNACys populations is very interesting. To the best of my knowledge, such an analysis has never been done before.\n\nHowever, I have three critical points that I have to make:\n(i) the whole analysis is based on only two data sets. It is therefore not possible to carry out a statistical evaluation. This should be corrected in the text and in the figures.\n(ii) It is not entirely clear what functions the different tRNA populations could have in the cell. The authors could take up this point and speculate.\n(iii) Is it known that the addition of NaCl affects the maturation/modification of tRNAs? If so, what could be the underlying molecular 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? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
},
{
"id": "83460",
"date": "10 May 2021",
"name": "José E. Barboza Corona",
"expertise": [
"Reviewer Expertise Molecular studies with different Bacillus species."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 shows the different populations of tRNAcys-related molecules in dormant spores and during the first time of their conversion into growing cells in the presence or absence of saline stress. Different tRNA-cys species were categorized, and the percent of mature, immature or tRNA-cys fragments were determined.\nIt is common that in nature, dormant Bacillus spores germinate in the presence of nutrients. In this sense, it would be fascinating in future works to determine if similar behavior is found under the presence of saline stress and nutrients. In this regard, I would like to see in the introduction some words explaining the foundation to use saline stress in this work and not the presence of nutrients to pass from dormant to germinated spores. As the primary condition to germinate spores was the presence of NaCl, it would be essential to explain briefly how this experiment was done (beginning of the methods).\nAbout the overview of the subpopulation of tRNA-cys in figure 3, I recommend using the annotation “No NaCl”, “NaCl” showed in the category IX figure, in all figures, or well, show this annotation in the figure 3 legend. Alternatively, one of my primary concerns of this figure 3 is that standard deviations are considerable in more of the times, so it is difficult to consider that statistically significant differences are not found in categories II and III in the presence or absence of sodium chloride as you indicated in the manuscript. You need to clarify this point.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the 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/9-501
|
https://f1000research.com/articles/8-1519/v1
|
28 Aug 19
|
{
"type": "Case Report",
"title": "Case Report: Photo-activated oral disinfection in regenerative endodontics",
"authors": [
"Al-Shaimaa Abdel Hafiz Abdel Rahim",
"Fatma Abdelgawad",
"Ahmed M. Abd Alsamed",
"Dalia Mohamed Moheb",
"Norhan Abdel Wahab El-Dokky",
"Fatma Abdelgawad",
"Ahmed M. Abd Alsamed",
"Dalia Mohamed Moheb",
"Norhan Abdel Wahab El-Dokky"
],
"abstract": "Background: Root canal disinfection is considered critical for achieving successful regenerative endodontic procedures. Photo-activated oral disinfection is a novel disinfection method that can help to achieve the goal of regenerative endodontics. This article reports the clinical and radiographic results after single visit regenerative endodontics using photo-activated oral disinfection. Methods: An 8.5-year-old girl complained of fractured upper right central incisor. Pulp necrosis was diagnosed on the basis of clinical findings. The root canal was irrigated with sodium hypochlorite solution (1.5%) followed by saline. Then, the canal was dried with paper points. A combination of a photosensitizer solution and low power laser light were applied. EDTA solution was used as a final irrigant. Bleeding was induced, followed by placement of collagen resorbable matrix and white mineral trioxide aggregate. Two days later, the tooth was sealed and restored with permanent filling. Results: Clinical findings revealed no pain on percussion or palpation tests. Radiographic examination revealed an increase in root length, an increase of apical root thickness and apical closure at the 12-month follow-up period. Conclusion: Regenerative endodontics using photo-activated oral disinfection achieved successful outcomes in the necrotic immature permanent tooth.",
"keywords": [
"regenerative endodontics",
"necrotic tooth",
"open apex",
"root canal disinfection"
],
"content": "Introduction\n\nThe term regenerative endodontic procedures (REPs) has been widely endorsed. This term describes all procedures that aim to achieve organized repair of the dental pulp1. The clinical considerations for REPs include: disinfecting the root canal system; providing a scaffold with periapical tissue laceration to get a blood clot and introduce stem cell activity within the root canal; and sealing the coronal access properly to prevent reinfection2,3.\n\nAntibiotics appear to be suitable intra-canal medication. Triple antibiotic paste (TAP) consisting of metronidazole, ciprofloxacin and minocycline is considered to be a successful regimen in managing the root canal pathogen of necrotic immature permanent teeth4. Recently, new methods of disinfection have been described to control the limitations of conventional disinfecting methods by neither diminishing the bacterial number to an adequate level or eliminating the toxicity to periapical stem/progenitors5. Other concerns regarding the use of TAP include tooth discoloration and bacterial resistance6.\n\nPhotodynamic therapy (PDT)/photo-activated oral disinfection (PAD) involves a photosensitizer (photoactive dye) that is activated by exposure to light of a specific wavelength in the presence of oxygen. The energy transferred from the activated photosensitizer to available oxygen leads to toxic oxygen species formation, such as singlet oxygen and free radicals. These very reactive chemical species can destroy proteins, nucleic acids, lipids and other cellular components7.\n\nSeveral in vitro studies have shown that PDT is effective in root canal disinfection8–10. A clinical study by Johns et al.11 reported successful outcomes for using PAD in root canal treatment of completely formed root. The case report by Johns et al.12 was the first to document pulp revascularization using PDT and platelet-rich fibrin. Root lengthening, continued thickening of the canal walls and apical closure were demonstrated at 10-month follow-up12.\n\nThis article reports the successful use of PAD in regenerative endodontics as a novel and effective disinfection method, which might present a solution to the problems associated with triple antibiotic paste12.\n\n\nCase report\n\nAn 8.5-year-old Egyptian girl came to the outpatient clinic of Pediatric Dentistry and Dental Public Health Department, Faculty of Dentistry, Cairo University with the chief complaint of fractured upper right central incisor due to trauma one and half months previously (Figure 1A and B). The patient’s medical history was non-contributory. On clinical examination (which included a visual examination for any abnormalities, palpation of labial vestibule, percussion test and sensibility test), the tooth was sensitive to percussion, which was determined by tapping the tooth with the back of the mirror. The surrounding soft tissue had no tenderness to finger palpation and the tooth had no response to the hot test in comparison to the contralateral tooth. Preoperative radiographic examination revealed a wide root canal with an open apex (Figure 2A) using conventional periapical radiograph.\n\nA) front view of preoperative intra-oral photo of traumatized upper right central incisor; B) occlusal preoperative photo; C) three month follow up; and D) 12-month follow-up.\n\nA) preoperative periapical radiograph; B) immediate postoperative radiograph after mineral trioxide aggregate coronal plug application; C) follow up radiograph at six months; and D) follow-up radiograph at 12 months.\n\nThe case was managed by single visit regenerative endodontic procedure using PAD after obtaining written informed consent from the parents (including usage of data and publication) and assent from the child. The procedure was performed according to the American Association of Endodontics (AAE) guidelines13, except PAD instead of TAP was used. A 1.8ml carpule of mepivacaine 3% (Mepivacaine hydrochloride, Alexandria Co. for Pharmaceuticals, Egypt) was administered by infiltration. A rubber dam was applied, followed by access cavity preparation and tooth length detection. Gentle, copious irrigation was done using 20ml NaOCl (1.5% NaOCl 20ml/canal for five minutes). NaOCl of lower concentration was advised and then flushed with saline (20ml/canal for minutes), using irrigating needle located about 1mm from root end then the canal was dried using paper points.\n\nAseptim solution (SciCan Ltd, Denfotex Light Systems Ltd, Inverkeithing, Scotland) was applied to the canal, followed by a low power laser diode red light system (Aseptim system, SciCan Ltd, Denfotex Light Systems Ltd, Inverkeithing, Scotland) with a specific wavelength (635nm) to activate the aseptim solution for 150 seconds. Canals was washed with saline to remove the aseptim solution. Gentle, copious, irrigation with 20ml of 17% EDTA was applied. Dryness with paper points to remove excess EDTA was done. Bleeding was initiated into the canal system by rotating a K-file at 2mm beyond the apical foramen. A resorbable matrix (Collacote dressing Zimmer Biomet, USA) was placed over the blood clot. Then, white mineral trioxide aggregate (MTA) (Angelus, Brazil) was placed over the matrix with placement of a moistened pellet of cotton and glass ionomer (Figure 2B). After two days, the tooth was double sealed using glass ionomer cement (Kromoglass 2, LASCOD- Italy) and composite restoration 3M composite (3M, America Inc).\n\nClinical examination of the patient revealed no adverse signs and symptoms at three, six-, nine- and 12-month follow-up periods (Figure 1C and 1D). Radiographic examination revealed an increase in root length and root thickness at six months (Figure 2C) and complete root closure at 12 months (Figure 2D) using digital radiography (Digora™ Optime UV) and digital software (Soredex, Finland). (Table 1)\n\n\nDiscussion\n\nThis case report documents the successful application of a novel and innovative disinfection technique in regenerative endodontics for management of a necrotic immature permanent tooth in a single visit, although a longer follow up period is recommended.\n\nRoot canal system disinfection is an integral step in the success of REPs. Chemical disinfection of the root canal is dependent on bacteriostatic/bactericidal properties of the agents as well as avoid harming the patient’s stem cells3. Copious and gentle irrigation was done using 20ml NaOCl. The use of NaOCl at lower concentrations was advised (1.5% NaOCl) with the irrigating needle adjusted to be 1mm from the root end to reduce the cytotoxicity to stem cells and to minimize the possibility of irrigant extrusion into the periapical tissues13. Furthermore, 1.5% NaOCl was effective in bacterial reduction of root canals as reported by Trevino et al.14.\n\nThe PAD technique was effective in removing high bacterial concentrations from infected root canals15. Aseptim solution was applied to the canal and agitated in the canal for 60 seconds using an endodontic file to ensure maximum penetration of the dye, since it is essential that the aseptim solution comes in close contact with the bacteria, otherwise the photosensitivity process does not occur16.\n\nBlood clot formation inside the canal is considered a scaffold and a source of growth factors. Inducing bleeding to promote blood clot formation is a commonly used method in many of the reported cases17–20. A resorbable matrix (collagen wound dressing) should be carefully placed on the top of the blood clot to serve as an internal matrix and ease the placement of MTA13. White MTA was applied to act as a coronal plug and achieve effective coronal seal. It was the most frequently chosen material in published regenerative endodontic studies21–24.\n\nNo adverse clinical signs and symptoms were noted during follow up periods, which is considered as a primary goal for regenerative endodontics as reported by AAE13. Apical closure and increasing apical dentin thickness were observed at the 12-month follow-up period. This case report demonstrates that PAD is a promising method for controlling infection in a single visit regenerative endodontic procedure. Further randomized clinical studies are needed to assess this disinfection technique.\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 parents of the patient.",
"appendix": "Grant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nI dedicate this work to the soul of Prof. Mervat Abdelmonem Rashed, Professor of Pediatric Dentistry, Pediatric Dentistry and Dental Public Health Department, Faculty of Dentistry, Cairo University that her death prevents her to see the result of this report.\n\n\nReferences\n\nDiogenes A, Henry MA, Teixeira FB, et al.: An update on clinical regenerative endodontics. Endod Top. 2013; 28(1): 2–23. Publisher Full Text\n\nBezgin T, Sönmez H: Review of current concepts of revascularization/revitalization. Dent Traumatol. 2015; 31(4): 267–73. PubMed Abstract | Publisher Full Text\n\nGaller KM: Clinical procedures for revitalization: current knowledge and considerations. Int Endod J. 2016; 49(10): 926–36. PubMed Abstract | Publisher Full Text\n\nVijayaraghavan R, Mathian VM, Sundaram AM, et al.: Triple antibiotic paste in root canal therapy. J Pharm Bioallied Sci. 2012; 4(Suppl 2): S230–3. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKim SG: Dental pulp regeneration. Curr Ther Endod. 2016; 271–80. Publisher Full Text\n\nHuang GT: A paradigm shift in endodontic management of immature teeth: conservation of stem cells for regeneration. J Dent. 2008; 36(6): 379–86. PubMed Abstract | Publisher Full Text\n\nKonopka K, Goslinski T: Photodynamic therapy in dentistry. J Dent Res. 2007; 86(8): 694–707. PubMed Abstract | Publisher Full Text\n\nBergmans L, Moisiadis P, Huybrechts B, et al.: Effect of photo-activated disinfection on endodontic pathogens ex vivo. Int Endod J. 2008; 41(3): 227–39. PubMed Abstract | Publisher Full Text\n\nGarcez AS, Ribeiro MS, Tegos GP, et al.: Antimicrobial photodynamic therapy combined with conventional endodontic treatment to eliminate root canal biofilm infection. Lasers Surg Med. 2007; 39(1): 59–66. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWilliams JA, Pearson GJ, John CM: Antibacterial action of photoactivated disinfection {PAD} used on endodontic bacteria in planktonic suspension and in artificial and human root canals. J Dent. 2006; 34(6): 363–71. PubMed Abstract | Publisher Full Text\n\nJohns DA, Varughese JM, Thomas K, et al.: Clinical and radiographical evaluation of the healing of large periapical lesions using triple antibiotic paste, photo activated disinfection and calcium hydroxide when used as root canal disinfectant. J Clin Exp Dent. 2014; 6(3): e230–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJohns DA, Shivashankar VY, Krishnamma S, et al.: Use of photoactivated disinfection and platelet-rich fibrin in regenerative Endodontics. J Conserv Dent. 2014; 17(5): 487–90. PubMed Abstract | Publisher Full Text | Free Full Text\n\nConsent Informed, Appointment First: AAE Clinical Considerations for a Regenerative Procedure Revised 4-12-15. 2015: 1–6.\n\nTrevino EG, Patwardhan AN, Henry MA, et al.: Effect of irrigants on the survival of human stem cells of the apical papilla in a platelet-rich plasma scaffold in human root tips. J Endod. 2011; 37(8): 1109–15. PubMed Abstract | Publisher Full Text\n\nPlotino G, Cortese T, Grande NM, et al.: New Technologies to Improve Root Canal Disinfection. Braz Dent J. 2016; 27(1): 3–8. PubMed Abstract | Publisher Full Text\n\nBonsor SJ, Nichol R, Reid TM, et al.: Microbiological evaluation of photo-activated disinfection in endodontics (an in vivo study). Br Dent J. 2006; 200(6): 337–41, discussion 329. PubMed Abstract | Publisher Full Text\n\nReynolds K, Johnson JD, Cohenca N: Pulp revascularization of necrotic bilateral bicuspids using a modified novel technique to eliminate potential coronal discolouration: a case report. Int Endod J. 2009; 42(1): 84–92. PubMed Abstract | Publisher Full Text\n\nShin SY, Albert JS, Mortman RE: One step pulp revascularization treatment of an immature permanent tooth with chronic apical abscess: a case report. Int Endod J. 2009; 42(12): 1118–26. PubMed Abstract | Publisher Full Text\n\nCehreli ZC, Sara S, Aksoy B: Revascularization of immature permanent incisors after severe extrusive luxation injury. J Can Dent Assoc. 2012; 78(1): c4. PubMed Abstract\n\nMao JJ, Kim SG, Zhou J, et al.: Regenerative endodontics: barriers and strategies for clinical translation. Dent Clin North Am. 2012; 56(3): 639–49. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBanchs F, Trope M: Revascularization of immature permanent teeth with apical periodontitis: new treatment protocol? J Endod. 2004; 30(4): 196–200. PubMed Abstract | Publisher Full Text\n\nThibodeau B, Trope M: Pulp revascularization of a necrotic infected immature permanent tooth: case report and review of the literature. Pediatr Dent. 2007; 29(1): 47–50. PubMed Abstract\n\nDing RY, Cheung GS, Chen J, et al.: Pulp revascularization of immature teeth with apical periodontitis: a clinical study. J Endod. 2009; 35(5): 745–9. PubMed Abstract | Publisher Full Text\n\nLenzi R, Trope M: Revitalization procedures in two traumatized incisors with different biological outcomes. J Endod. 2012; 38(3): 411–4. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "58468",
"date": "10 Mar 2020",
"name": "Gianluca Gambarini",
"expertise": [
"Reviewer Expertise Endodontics"
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article presents a case of apicogenesis performed by gold standard methodologies. The novelty is the use of a photoactivated disinfectant instead of a triple antibiotic paste. Since NaOCl and EDTA used are sufficient to remove all the tissues that could avoid a proper regeneration of the apex, the effectiveness of the light activated disinfectant is doubtful.\nThe title of the article is on regenerative endodontics, that is a procedure that aims to regenerate dental pulp, but no evidences of regeneration are presented in the article instead of the production of new dentine, which is obtainable with a simple apicogenesis procedure and also without the use of triple antibiotic paste.\nThe overall novelty of the article is doubtful and the evidences of the regeneration of pulp are not explained at all. Dentine formation and the closure of the apex are evidences that could be obtained with gold standard techniques that do not involve the use of other expensive appliances like diode lasers.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes",
"responses": [
{
"c_id": "5305",
"date": "16 Mar 2020",
"name": "Shaimaa Abdel Hafiz",
"role": "Author Response",
"response": "We appreciate the comments of the reviewer. Herein we address the concerns of the reviewer.1- The article presents a case of apicogenesis performed by gold standard methodologies. The novelty is the use of a photoactivated disinfectant instead of a triple antibiotic paste. Since NaOCl and EDTA used are sufficient to remove all the tissues that could avoid a proper regeneration of the apex, the effectiveness of the light activated disinfectant is doubtful.Response:- Apicogenesis is a root complementation therapy performed in teeth with pulp vitality and consists in the removal of infected coronal pulp, maintenance of vital root pulp and its protection with biocompatible material (1).Pulpal necrosis was diagnosed on the basis of clinical findings in our case report as mentioned in the methods section of abstract. So regenerative endodontic procedure was applied for necrotic tooth with open apex as recommended by American Association of Endodontists (AAE)(2).- Naocl and EDTA may be sufficient to remove all necrotic tissues but in this case report, it was mentioned that the procedure was performed according to the American Association of Endodontics (AAE) guidelines (2), except PAD instead of TAP was used. The AAE guidelines recommend using intracanal medication in adjunctive to the irrigants.- There are several in vitro and in vivo studies proved the effectiveness of PAD system in root canal disinfection. And this article mentioned some of them (4–9).2- The title of the article is on regenerative endodontics, that is a procedure that aims to regenerate dental pulp, but no evidences of regeneration are presented in the article instead of the production of new dentine, which is obtainable with a simple apicogenesisprocedure and also without the use of triple antibiotic paste.Response:- Regenerative Endodontics is defined as a biologically-based procedure designed to predictably replace damaged, diseased or missing structures, including dentin and root structures as well as cells of the pulp-dentin complex, with living viable tissues, preferably of the same origin, that restore the normal physiologic functions of the pulp-dentin complex (10)- The term regenerative endodontic procedures (REPs) has been widely adopted and refers to all procedures that aim to attain organized repair of the dental pulp and include future therapies yet to regenerative endodontic treatment of necrotic immature permanent teeth resulting in continued root development, increased thickness in the dentinal walls and apical closure. evolve in the field of regenerative endodontics (3).3- The overall novelty of the article is doubtful and the evidences of the regeneration of pulpare not explained at all. Dentine formation and the closure of the apex are evidences that could be obtained with gold standard techniques that do not involve the use of other expensive appliances like diode lasers.Response:- Regenerative endodontic treatment of necrotic immature permanent teeth resulting in continued root development, increased thickness in the dentinal walls and apical closure as mentioned by AAE.- AAE Clinical Considerations for a Regenerative Procedure (2) stated that the degree of success of Regenerative Endodontic Procedures is largely measured by the extent to which it is possible to attain primary, secondary, and tertiary goals: Primary goal: The elimination of symptoms and the evidence of bony healing. Secondary goal: Increased root wall thickness and/or increased root length (desirable, but perhaps not essential) Tertiary goal: Positive response to vitality testing (which if achieved, could indicate a more organized vital pulp tissue). - Root canal system disinfection is an integral step in the success of REPs (2). Antimicrobial approaches to disinfect root canals have been proposed that include Photo-activated disinfection (PAD) as it offers a means of destroying bacteria remaining after using conventional irrigants in endodontic therapy (4).References1 Mente Johannes, Leo Meltem, Panagidis Dimos, et al. Treatment outcome of mineral trioxide aggregate in open apex teeth. J Endod 2013. Doi: 10.1016/j.joen.2012.10.007.2 AAE. Clinical Considerations for a Regenerative Procedure (New, Revised 6-18-16). AAE 2016.3 Diogenes Anibal, Henry Michael A., Teixeira Fabricio B., Hargreaves Kenneth M. An update on clinical regenerative endodontics. Endod Top 2013;28(1):2–23. Doi: 10.1111/etp.12040.4 Bonsor S. J., Nichol R., Reid T. M S, Pearson G. J. Microbiological evaluation of photo-activated disinfection in endodontics (An in vivo study). Br Dent J 2006;200(6):337–41. Doi: 10.1038/sj.bdj.4813371.5 Williams Jill A., Pearson Gavin J., John Colles M. Antibacterial action of photoactivated disinfection {PAD} used on endodontic bacteria in planktonic suspension and in artificial and human root canals. J Dent 2006;34(6):363–71. Doi: 10.1016/j.jdent.2005.08.002.6 Garcez Aguinaldo S., Ribeiro Martha S., Tegos George P., Núñez Silvia C., Jorge Antonio O C, Hamblin Michael R. Antimicrobial photodynamic therapy combined with conventional endodontic treatment to eliminate root canal biofilm infection. Lasers Surg Med 2007;39(1):59–66. Doi: 10.1002/lsm.20415.7 Bergmans L., Moisiadis P., Huybrechts B., Van Meerbeek B., Quirynen M., Lambrechts P. Effect of photo-activated disinfection on endodontic pathogens ex vivo. Int Endod J 2008;41(3):227–39. Doi: 10.1111/j.1365-2591.2007.01344.x.8 Johns Dexton Antony, Varughese Jolly Mary, Thomas Kunjamma, Abraham Aby, James Elizabeth Prabha, Maroli Ramesh Kumar. Clinical and radiographical evaluation of the healing of large periapical lesions using triple antibiotic paste, photo activated disinfection and calcium hydroxide when used as root canal disinfectant. J Clin Exp Dent 2014;6(3). Doi: 10.4317/jced.51324.9 Johns DextonAntony, Shivashankar VasundaraYayathi, Krishnamma Shoba, Johns Manu. Use of photoactivated disinfection and platelet-rich fibrin in regenerative Endodontics. J Conserv Dent 2014;17(5):487. Doi: 10.4103/0972-0707.139850.10 Murray Peter E., Garcia-Godoy Franklin, Hargreaves Kenneth M. Regenerative Endodontics: A Review of Current Status and a Call for Action. J Endod 2007;33(4):377–90. Doi: 10.1016/j.joen.2006.09.013."
}
]
},
{
"id": "61455",
"date": "30 Mar 2020",
"name": "Mohamed Mokhtar Nagy",
"expertise": [
"Reviewer Expertise Endodontic regeneration"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 focuses upon two main issues which are single visit regeneration and photo activated disinfection.\nThe title lacks the \"the single visit concept\".\n\nThe title states \"oral\" disinfection, this would be better as \"canal\" disinfection.\n\nThe introduction and discussion did not include the justification of the idea of single visit regeneration.\n\nThe introduction and discussion did not include the possible effects of laser as heat generation and effects on stem cells.\n\nThe manuscript did not state radiographic standardization during follow up.\n\nThe discussion did not justify the supposed mechanism of root canal disinfection by laser (PAD).\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": [
{
"c_id": "5397",
"date": "03 Jun 2020",
"name": "Shaimaa Abdel Hafiz",
"role": "Author Response",
"response": "We are pleased to submit the new version of Case Report: Photo-activated oral disinfection in regenerative endodontics. We appreciate the constructive criticisms of the reviewer that helped us to improve our manuscript. Herein we address their concerns. All changes appear by track changes in the revised version. Hope you find our replies satisfactory.Response to comments of Reviewer:1. The title lacks the \"the single visit concept\".Single visit photo-activated disinfection in regenerative endodontics. The title was modified. 2. The title states \"oral\" disinfection, this would be better as \"canal\" disinfectionI omit the word oral because the most common terminology used in literature is Photo-activated disinfection and according to the remaining title it represents the site of disinfection applied on. So its preferable to state Photoactivated disinfection as mentioned in published literature of endodontics field (1–5). 3. The introduction and discussion did not include the justification of the idea of singlevisit regenerationFor introduction:Photodynamic therapy (PDT)/photo-activated disinfection (PAD) is considered one of the optimized single visit approaches (6). This part was added to introduction section.Several in vitro studies have shown that PDT is effective in root canal disinfection (1,2,7). A clinical study by Johns et al. (3) reported successful outcomes for using PAD in root canal treatment of completely formed root. The case report by Johns et al.(4) was the first to document pulp revascularization using PDT and platelet-rich fibrin. Root lengthening, continued thickening of the canal walls and apical closure were demonstrated at 10-month follow-up (4).All of these mentioned studies applied the PAD system in single visit. As mentioned in introduction section. For Discussion:Regenerative endodontic procedures were completed during a single visit because PAD is considered one of the optimized single visit disinfection approaches (6). A successful single-visit regenerative endodontic therapy of an immature permanent tooth with a chronic apical abscess was published by Shin et al. (8), They stated that a single-visit revascularization procedure has some advantages. It eliminates subsequent appointments to access the root canal environment, thus reducing the possibility of further bacterial contamination of the root canal. It also diminishes the detrimental consequences of poor patient compliance with regular follow-up evaluation (8). Topçuoğlu and Topçuoğlu (9) reported that a single-visit regenerative endodontic procedure may be a favorable treatment option for an asymptomatic immature tooth with a necrotic pulp and no periapical lesion (9). This part was added to discussion section 4. The introduction and discussion did not include the possible effects of laser as heat generation and effects on stem cells.For introduction:Moreover, PAD acts in a selective way. Both photosensitizer and oxygen released during bacterial cell death do not exhibit toxicity to the viable tissues (10). This part was added to introduction section.For Discussion:Photosensitizer and oxygen released during bacterial cell death are not reported to produce any toxicity to normal cells(10).Dickers et al. (11) stated that the average temperature rise was lower than the 7 degrees C safety level for periodontal injury. Moura-Netto et al.(12)reported that studies on tissue engineering using stem cells from human exfoliated deciduous teeth have yielded promising results. Laser phototherapy is able to influence the proliferation and differentiation of these cells. This part was added to discussion section.5. The manuscript did not state radiographic standardization during follow upAn individual acrylic XCP (Extension Cone Paralleling) index was prepared by registering the bite and placed around the XCP plastic tip for radiographic standardization during follow up. This part was added to the case report section.6. The discussion did not justify the supposed mechanism of root canal disinfection by laser (PAD).Response:The photosensitizer stains the bacterial cells in soft and hard tissues and the photo-activated cells release molecular oxygen causing disruption of the bacterial cell wall (10). Moreover, PAD is a newer antimicrobial strategy that involves the combination of a non-toxic PS or dyes and a non-harmful visible light source to disinfect the root canal. Low power laser in itself is not particularly lethal to bacteria, but is useful for photochemical activation of oxygen-releasing dyes (13). This part was added to discussion section. References1 Bergmans L., Moisiadis P., Huybrechts B., Van Meerbeek B., Quirynen M., Lambrechts P. Effect of photo-activated disinfection on endodontic pathogens ex vivo. Int Endod J 2008;41(3):227–39. Doi: 10.1111/j.1365-2591.2007.01344.x.2 Williams Jill A., Pearson Gavin J., John Colles M. Antibacterial action of photoactivated disinfection {PAD} used on endodontic bacteria in planktonic suspension and in artificial and human root canals. J Dent 2006;34(6):363–71. Doi: 10.1016/j.jdent.2005.08.002.3 Johns Dexton Antony, Varughese Jolly Mary, Thomas Kunjamma, Abraham Aby, James Elizabeth Prabha, Maroli Ramesh Kumar. Clinical and radiographical evaluation of the healing of large periapical lesions using triple antibiotic paste, photo activated disinfection and calcium hydroxide when used as root canal disinfectant. J Clin Exp Dent 2014;6(3). Doi: 10.4317/jced.51324.4 Johns DextonAntony, Shivashankar VasundaraYayathi, Krishnamma Shoba, Johns Manu. Use of photoactivated disinfection and platelet-rich fibrin in regenerative Endodontics. J Conserv Dent 2014;17(5):487. Doi: 10.4103/0972-0707.139850.5 Bonsor S. J., Nichol R., Reid T. M S, Pearson G. J. Microbiological evaluation of photo-activated disinfection in endodontics (An in vivo study). Br Dent J 2006;200(6):337–41. Doi: 10.1038/sj.bdj.4813371.6 Siqueira José F., Rôças Isabela N. Optimising single-visit disinfection with supplementary approaches: A quest for predictability. Aust Endod J 2011. Doi: 10.1111/j.1747-4477.2011.00334.x.7 Garcez Aguinaldo S., Ribeiro Martha S., Tegos George P., Núñez Silvia C., Jorge Antonio O C, Hamblin Michael R. Antimicrobial photodynamic therapy combined with conventional endodontic treatment to eliminate root canal biofilm infection. Lasers Surg Med 2007;39(1):59–66. Doi: 10.1002/lsm.20415.8 Shin S. Y., Albert J. S., Mortman R. E. One step pulp revascularization treatment of an immature permanent tooth with chronic apical abscess: A case report. Int Endod J 2009:1118–26. Doi: 10.1111/j.1365-2591.2009.01633.x.9 Topçuoğlu Gamze, Topçuoğlu Hüseyin Sinan. Regenerative Endodontic Therapy in a Single Visit Using Platelet-rich Plasma and Biodentine in Necrotic and Asymptomatic Immature Molar Teeth: A Report of 3 Cases. J Endod 2016;42(9):1344–6. Doi: 10.1016/j.joen.2016.06.005.10 López Fernanda Ullmann, Kopper Patrícia Maria Poli, Bona Alvaro Della, Steier Liviu, de Figueiredo José Antônio Poli, Vier-Pelisser Fabiana Vieira. Effect of different irrigating solutions and photo-activated therapy for in vivo root canal treatment. Braz Dent J 2015. Doi: 10.1590/0103-6440201300154.11 Dickers B., Lamard L., Peremans A., et al. Temperature rise during photo-activated disinfection of root canals. Lasers Med Sci 2009;24(1):81–5. Doi: 10.1007/s10103-007-0526-y.12 MOURA-NETTO Cacio, FERREIRA Leila Soares, MARANDUBA Carlos Magno, MELLO-MOURA Anna Carolina Volpi, MARQUES Marcia Martins. Low-intensity laser phototherapy enhances the proliferation of dental pulp stem cells under nutritional deficiency. Braz Oral Res 2016;30(1). Doi: 10.1590/1807-3107BOR-2016.vol30.0080.13 Mohan Dennis, Maruthingal Sunith, Indira Rajamani, et al. Photoactivated disinfection (PAD) of dental root canal system - An ex-vivo study. Saudi J Biol Sci 2016. Doi: 10.1016/j.sjbs.2015.01.013."
}
]
}
] | 1
|
https://f1000research.com/articles/8-1519
|
https://f1000research.com/articles/9-293/v1
|
24 Apr 20
|
{
"type": "Research Article",
"title": "Effect of different biofloc starters on ammonia, nitrate, and nitrite concentrations in the cultured tilapia Oreochromis niloticus system",
"authors": [
"Iskandar Putra",
"Irwan Effendi",
"Iesje Lukistyowati",
"Usman M. Tang",
"Muhammad Fauzi",
"Indra Suharman",
"Zainal A. Muchlisin",
"Iskandar Putra",
"Iesje Lukistyowati",
"Usman M. Tang",
"Muhammad Fauzi",
"Indra Suharman",
"Zainal A. Muchlisin"
],
"abstract": "Background: High stocking density and intensive feeding in aquaculture systems lead to the accumulation of organic waste, which results in an increase in ammonia, nitrite, and nitrite concentrations in culture media. Biofloc is a potential technology to overcome this problem. The starter is a crucial carbon source for bacteria in the formation of biofloc. The objective of the present study aimed to explore the best starter of biofloc in a red tilapia Oreochromis niloticus culture system. Methods: A completely randomized design with four levels of treatment was used in this study. The tested starter was (A) control treatment, biofloc without starter, (B) biofloc with molasses starter, (C) biofloc with tapioca starter, and (D) biofloc with sucrose starter. The floc was cultured in 100-L tanks with a salinity of 17 ppt. The tanks were stocked with O. niloticus with a size of 3.71±0.11 cm at a stocking density of 30 fish per tank. The fish were fed on a commercial diet two times a day at satiation for 40 days. The ammonia, nitrite, and nitrite concentrations were measured for an interval of 8 days. Results: The study showed that the NH3-N range was 0.02–0.07 mg L−1 (mean, 0.03 ± 0.02 mg L−1), NO2-N range was 0.20–0.43 mg L−1 (mean, 0.25 ± 0.12 mg L−1), and NO3-N range was 0.90–3.20 mg L−1 (mean, 1.42 ± 1.19 mg L−1). Conclusion: Among the starters tested, molasses was found to be the best for biofloc in tilapia culture.",
"keywords": [
"Biofloc",
"Carbon",
"Molasses",
"Water Quality"
],
"content": "Introduction\n\nWater quality is a crucial factor in aquaculture systems. One important water quality parameter is nitrogen. In water, nitrogen can be found in the forms of ammonia, nitrite, and nitrate1–5. Ammonia (NH3-N) is produced from the breakdown of proteins from unconsumed feed, feces, and urine of fish. This compound will turn into nitrite (NO2) when oxygen levels are poor6, which is toxic for fish7. By contrast, ammonia is changed into nitrate when the dissolved oxygen level is sufficient8,9. Fish produces ammonia (inorganic N) through the osmoregulation process; feces and urine contribute about 10%–20% of total nitrogen10. The application of biofloc is one of the alternatives to overcome water quality problems especially in controlling total ammonia nitrogen in the aquaculture system11–13.\n\nBiofloc refers to the use of heterotrophs and autotrophs, which can convert organic waste into floc forms that can be utilized by fish as a food source4,11,14–16. Biofloc technology is cheap, simple, and environmentally friendly13,17. Several organisms, such as bacteria, plankton, fungi, and algae, and suspended particles exist in flocs18,19. These organisms provide nutrition for cultured fish. However, the formation of biofloc needs a starter consisting of probiotics and a carbon source. Molasses, tapioca, and wheat flours are common starters in biofloc culture19–23. Presently, no information is available on the best starter for biofloc in the cultured system of Nile tilapia Oreochromis niloticus. Thus, the present study aimed to explore the best starter for biofloc in a tilapia O. niloticus culture system.\n\n\nMethods\n\nThe research was carried out for 40 days from February 2019 to March 2019 at the Aquaculture Technology Laboratory, Faculty of Fisheries and Marine, Riau University, Indonesia. The experiments were carried out within the ethical guidelines in animal research developed by NC3Rs. In Indonesia, no approval is required to conduct research on fish.\n\nA completely randomized design with four levels of treatment and three replications was performed in this study; the tested treatment was four starters of biofloc, namely control without a starter (treatment A), biofloc with molasses starter concentration 0.48 gL-1 (treatment B), biofloc with tapioca starter concentration 0.35 gL-1 (treatment C), and biofloc with sucrose concentration 0.42 gL-1 (treatment D).\n\nThe amount of carbon added is calculated based on the carbon content (C) in the ingredients and the nitrogen content in the feed given5 using the formula:\n\nC/N = (% C starter x molecule weight of starter + % C feed x feed weight)/ % N feed x feed weight. Based on this formula, to obtain a C/N ratio of 20: 1, a carbon source derived from molasses was 48 g with a C content of 37%24, tapioca flour as much as 35 g with a C content of 50.3%25, and sucrose 42 g with a C content of 42.3%26.\n\nBiofloc was cultured using 0.01 ml/L probiotic bacteria from Boster Multisel (Bacillus sp.)16, and the carbon source was from starters based on the tested treatments with the C/N ratio of 20:1. The starter was applied in cultivation media and probiotic bacteria administration. Probiotics and starter were applied into cultivation cultured media and left for 1 week, and the media were aerated continuously. Floc formation was indicated by green water and foam forming in the media.\n\nThe number of fish used in this study was 360 individuals and they were obtained from a hatchery in the Bengkalis Regency, Riau. The fish were 50 days old, 3.71±0.11 cm TL. As the sex of the fish cannot be identified morphologically, there is no separation of fish sex in this study. The fish was distributed into research tanks as the flocs are formed, 7 days after the starter was cultured in the tanks (total 12 tanks, 100L volume). The density of the fish was 30 fishes/100 L. Stocking of red tilapia larvae in the morning at 8 AM with a water temperature of 25°C The fish were fed on commercial diet containing 38% crude protein, 5% crude lipid, and 6% crude fiber (PF1000, PT Matahari Sakti). Feeding was conducted ad libitum/satiation, 2 times/day (8 AM and 5 PM) for 40 days.\n\nThe ammonia (NH3), nitrite (NO2−), and nitrate (NO3+) contents were measured by spectrophotometry. These parameters were measured every 8 days in each treatment for 40 days. Ammonia (NH3), Nitrite (NO2-) and Nitrate (NO3+) were measured using spectrophotometer (Optima - SP300) at a wavelength of 630 nm27. The measurement was conducted at 8:00 AM with three replications at 8-day intervals over 40 days (six timepoints).\n\nThe data were subjected to one-way analysis of variance followed by the post hoc Newman–Keuls test using SPSS 18.0 software. P<0.05 was considered to indicate a statistically significant difference.\n\n\nResults\n\nOver the six timepoints measured, the mean concentration of ammonia ranged from 0.02 mg L−1 to 0.07 mg L−1 (with an average value of 0.03 ± 0.02 mg L−1), nitrite ranged from 0.20 mg L−1 to 0.43 mg L−1 (with an average value of 0.25 ± 0.12 mg L−1), and nitrate ranged from 0.64 mgL−1 to 3.20 mgL−1 (with an average value of 1.42 ± 1.19 mg L−1). The study revealed that the best concentrations of ammonia, nitrite, and nitrate were recorded in the biofloc starter of molasses. These values did not significantly differ from those obtained with the tapioca and sucrose starters, but they were significantly different from the control treatment without a starter (Table 1). Raw values for each replicate are available as Underlying data28.\n\nData values are the mean and standard deviation. Mean values with different superscripts in the same row were significantly different (p < 0.05).\n\nIn the control treatment, the concentrations of ammonia, nitrite, and nitrate increased gradually with increasing experimental time. However, in the starter treatments, the ammonia concentration dropped during the first week of the experiment (day 8), became stagnant during the second and third weeks (day 16 to day 24), and increased again at day 32 of the experiment. However, the ammonia concentration decreased sharply at day 40 (Figure 1a). The nitrite concentration also decreased during the first week but increased slightly at day 16 and increased gradually until day 40 in treatments C (tapioca) and D (sucrose). By contrast, the nitrite concentration decreased at day 24, increased at day 32, and decreased at day 40 (Figure 1b). The nitrate concentration was relatively stagnant from day 1 to day 8. The nitrate concentration fluctuated during the experiment (Figure 1c). In general, the molasses starter (treatment B) yielded slightly better results than the other starters, but no significant different was found between the treatments except the control. The data of the growth performance of the tilapia fish O. niloticus has been published separately29.\n\n(a) Concentrations of ammonia, (b) nitrate, and (c) nitrate during 40 days of the experiment. A = control treatment, B = molasses starter, C = tapioca starter, and D = sucrose starter.\n\n\nDiscussion\n\nThe study revealed that the concentrations of ammonia, nitrite, and nitrate were significantly lower in the biofloc system using starters compared with those in the system without a starter (control). Biofloc has probiotic bacteria that can change ammonia to nontoxic materials (such as nitrate) that are useful for phytoplankton growth. Therefore, the ammonia and nitrate concentrations are low in the culture media19,21,30. Biofloc does not necessarily only contain bacteria (for example, Bacillus), but is also composed of other useful microorganisms such as microalgae and zooplankton that are trapped by organic particles31. Algae and zooplankton can be used by cultured biota (tilapia) as natural food.\n\nIn general, the starters used in this study were carbohydrate compounds. However, the study showed that the molasses starter yielded slightly better results compared with the other starters. This finding indicated that molasses was the best carbon source for biofloc in the tilapia culture system. Molasses can provide a sufficient carbon level for heterotrophic bacteria that use this carbon as an energy source for growth6,19,21,23,32. Molasses are a liquid byproduct from the sugar industry. This material has a total carbon content of around 37% 24. Therefore, molasses are rapidly soluble in water and can be quickly absorbed by heterotrophic bacteria3. In terms of chemical structure, molasses are classified as a simple carbohydrate containing six C atoms (monosaccharides), while sucrose (treatment D) is a combination of two monosaccharides that contain 12 C atoms (sucrose). Tapioca is classified as a complex carbohydrate (60,000 C atoms) and is more slowly digested by bacteria than molasses25,33.\n\n\nConclusion\n\nCarbon source from molasses is effective in reducing concentrations of ammonia, nitrite, and nitrate in red tilapia culture with biofloc technology.\n\n\nData availability\n\nFigshare: Effect of different biofloc starters on ammonia, nitrate, and nitrite concentrations in the cultured tilapia Oreochromis niloticus system. https://doi.org/10.6084/m9.figshare.1202795128.\n\nThis project contains raw data for the concentration of ammonia, nitrate and nitrite in each tank at each timepoint.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
"appendix": "Acknowledgments\n\nThe authors thank the undergraduate students who assisted the authors during experiments in the laboratory.\n\n\nReferences\n\nEmerenciano MGC, Córdova LRM, Porchas MM, et al.: Biofloc Technology (BFT): A Tool for Water Quality Management in Aquaculture. Water Quality. 2017; 5: 92–109. Publisher Full Text\n\nPutra I: Effectiveness of Nitrogen absorption with different Filter Medium in Tilapia (Oreochromis niloticus) cultured in the recirculation system. Bogor Agriculture University. 2010. Reference Source\n\nSerra FP, Goana CAP, Furtado PS, et al.: Use of different carbon sources for the biofloc system adopted during the nursery and grow-out culture of Litopenaeus vannamei. Aquac Int. 2015; 23(6); 1325–1339. Publisher Full Text\n\nDe Schryver PD, Verstraete W: Nitrogen removal from aquaculture pond water by heterotrophic nitrogen assimilation in lab-scale sequencing batch reactors. Bioresour Technol. 2009; 100: 1162–1167. Publisher Full Text\n\nAvnimelech Y: Carbon Nitrogen Ratio as a Control Element in Aquaculture Systems. Aquaculture. 1999; 176(3–4): 227–235. Publisher Full Text\n\nAzim ME, Little DC: The biofloc technology (BFT) in indoor tanks: water quality, biofloc composition, and growth and welfare of Nile tilapia (Oreochromis niloticus). Aquaculture. 2008; 283(1–4): 29–35. Publisher Full Text\n\nDjokosetiyanto D, Sunarma A, Widanarni: Changes of Ammonia, Nitrite and Nitrate at Recirculation System of Red Tilapia (Oreochromis. sp.) Rearing. Jurnal Akuakultur Indonesia. 2006; 5(1): 13–20. Publisher Full Text\n\nStein LY, Klotz MG: The nitrogen cycle. Curr Biol. 2016; 26(3): R94–98. PubMed Abstract | Publisher Full Text\n\nJiménez-Ojeda YK, Collazoz-Lasso LF, Arias-Castellanos JA: Dynamics and use of nitrogen in Biofloc Technology – BFT. AACL Bioflux. 2018; 11(4): 1107–1129. Reference Source\n\nEbeling JM, Timmons MB, Bisogni JJ: Engineering analysis of the stoichiometry of photoautotrophic, autotrophic and heterotrophic removal of ammonia–nitrogen in aquaculture systems. Aquaculture. 2006; 257(1–4): 346–358. Publisher Full Text\n\nZulfahmi I, Syahimi M, Muliari: Influence of Biofloc Addition With Different Dosages on The Growth of Tiger Shrimp Juvenile (Penaeus monodon. Fabricius 1798). J Biol. 2018; 11(1): 1–8. Publisher Full Text\n\nNurhatijah N, Muchlisin ZA, Sarong MA, et al.: Application of biofloc to maintain the water quality in the culture system of the tiger prawn (Penaeus monodon). AACL Bioflux. 2016; 9(4): 923–928. Reference Source\n\nSupriatna A, Nurhatijah H, Sarong MA, et al.: Effect of biofloc density and crude protein level in the diet on the growth performance, survival rate, and feed conversion ratio of Black Tiger Prawn (Penaeus monodon). IOP Conf. Series: Earth and Environmental Science. 2019; 348: 012131. Publisher Full Text\n\nAvnimelech Y: Control of Microbial Activity in Aquaculture Systems: Aktive Suspension Ponds. Biofloc Technology A Practical Guide Book, 2 edition. United States: The World Aquaculture Society. 2012; 34(4): 19–21. Reference Source\n\nOmbong F, Indra RNS: Application of Biofloc Technology (BFT) in Tilapia (Oreochromis niloticus) Culture. Jurnal Akuakultur Rawa Indonesia. 2016; 4(2): 16–25 [in Indonesian].\n\nPutra I, Rusliadi, Fauzi M, et al.: Growth performance and feed utilization of African catfish Clarias gariepinus fed a commercial diet and reared in the biofloc system enhanced with probiotic [version 1; peer review: 2 approved]. F1000Res. 2017; 6: 1545. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTaw N: Shrimp Farming in Biofloc System, Review and recent developments. FAO project, Blue Archipelago. Presented in World Aquaculture. Adelaide. 2014. Reference Source\n\nRohmana D: Conversion of catfish farming waste, Clarias sp. into heterotrophic bacterial biomass for improving the quality of water and food of giant prawns, Macrobrachium rosenbergii. Bogor Agriculture University. Bogor. 2009; 64. [in Indonesian]. Reference Source\n\nDe Schryver P, Crab R, Defoirdt T, et al.: The Basic of Bioflocs Technology, The Added Value for Aquaculture. 2008; 277(3–4): 125–137. Publisher Full Text\n\nDe Lima PCM, da Silva LOB, Abreu JDL, et al.: Tilapia Cultivated in A Low-Salinity Biofloc System Supplemented with Chlorella vulgaris and Differents Molasess Application Rates. Boletim do Instituto de Pesca. 2019; 45(4): e494. Publisher Full Text\n\nHargreaves JA: Photosynthetic suspended-growth systems in aquaculture. Aquaculture Engineering. 2006; 34(3): 344–363. Publisher Full Text\n\nSuwoyo HS, Abdul M, Gunarto G: Use of Organic Carbon Sources in the culture of Vaname Shrimp (Litopenaeus vannamei) with Biofloc Technology. Indoaqua Proceeding. Aquaculture Technology Innovation Forum.2012. [in Indonesian]. Reference Source\n\nAvnimelech Y: BioflocTechnology, A Practical Guide Book. The World Aquaculture Society. Louisiana, United States 2009; 120. Reference Source\n\nSuastuti NGAMDA: Utilization of By-Products of the Agriculture Industry Molasses and Tofu Liquid Waste as a Source of Carbon and Nitrogen for Biosurfactant Production by Bacillus sp Commercial Strain and Workshop. Bogor Agricultural University. Bogor. 1998; 105. [in Indonesia]. Reference Source\n\nAzhar MH: The Role of External Carbon Resources Different in Biofloc Formation and Its Effect on Water Quality and Production in the Vanamme Shrimp Culture System. Bogor Agricultural Institute, 2013; 39. [in Indonesian]. Reference Source\n\nPurnomo PD: The Effect quaculture Media towards Production of Intensive Tilapia Culture (Oreochromis niloticus). faculty of Fisheries and Marine Science, Universitas Diponegoro. 2012: 1: 161–179. Reference Source\n\nAPHA (American Public Health Assocition): Standard methoda for the examinatation of water and wastewater. 23RD. Edition. 1989. Reference Source\n\nPutra I, Effendi I, Lukistyowati I, et al.: Effect of different biofloc starters on ammonia, nitrate, and nitrite concentrations in the cultured tilapia Oreochromis niloticus system. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12027951.v1\n\nPutra I, Effendi I, Lukistyowati I, et al.: Growth and survival rate of red tilapia (Oreochromis sp.) cultivated in the brackish water tank under biofloc system. Advances in Engineering Research. 2019; 90: 96–99. Publisher Full Text\n\nCrab R, Avnimelech Y, Defoirdt T, et al.: Nitrogen Removal Techniques in Aquaculture for Sustainable Production. Aquaculture. 2007; 270(1–4): 1–14. Publisher Full Text\n\nHastuti S, Subandiyono S: Production Performance of African Catfish (Clarias gariepinus, burch) were Rearing with Biofloc technology. Journal of Fisheries Science and Technology. 2014; 10(1): 37–42. Reference Source\n\nRay AJ, Kevin SD, Jeffrey ML: Water Quality Dynamics and Shrimp (Litopenaeus vannamei) Production in Intensive, Mesohaline Culture System With Two levels of Biofloc Management. Aquaculture Engineering. 2011; 45(3): 127–136. Publisher Full Text\n\nChamberlain G, Avnimelech Y, McIntosh RP, et al.: Advantages of aerated microbial reuse systems with balanced C/N. Nutrient transformation and water quality benefits. Global Aquaculture Alliance Advocate. 2001; 4: 53–56. Reference Source"
}
|
[
{
"id": "62750",
"date": "01 May 2020",
"name": "Christopher Marlowe Caipang",
"expertise": [
"Reviewer Expertise Aquaculture",
"biotechnology",
"aquatic microbiology",
"fish health management"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSummary: This ms aimed to determine the best starter of biofloc in a red tilapia Oreochromis niloticus culture system. Three biofloc starters (molasses, tapioca and sucrose) and control were tested in triplicate over a period of 40 days. Nitrate, nitrite and ammonia were monitored during the study. Based on the findings, molasses is considered to be the best starter for biofloc production among the starters that were tested.\nComments: The work presented the findings in a straightforward manner. However, the ms can be further improved following these suggestions:\nChange the title to: Effect of different biofloc starters on ammonia, nitrate, and nitrite concentrations in the culture of tilapia Oreochromis niloticus.\n\nThe authors mentioned red tilapia in the Abstract but only tilapia in other portions of the text. Please be consistent.\n\nIn the Introduction, the authors wrote: \"Molasses, tapioca, and wheat flours are common starters in biofloc culture\". Please indicate to which species were these starters used.\n\nThe authors wrote: \"Presently, no information is available on the best starter for biofloc in the cultured system of Nile tilapia Oreochromis niloticus\". It is suggested that the authors conduct a thorough literature search, as there have been studies done on biofloc starters in tilapia. Please rectify this statement.\n\nIn the methods, please indicate how the biofloc was maintained in the study. The study only made mentioned the starter. In the 40 days, was there no addition of the starters to maintain the biofloc?\n\nPlease indicate water exchange rates? There was no water change in the control in 40 days, and the fish were quite big during stocking (approximately 3 g). With no water exchange in 40 days in the control and with the addition of feed, there is definitely a deterioration of water quality and lower fish survival. In the actual culture of tilapia, water exchange is done. For biofloc system, water exchange is minimal hence the results are compared in terms of water quality as well as savings in the cost of electricity or volume of water discharge.\n\nWas feeding the same for the biofloc treatments and control? Please explain.\n\nThe authors should explain the water exchange and feeding management between the control and biofloc treatments and how these are controlled/managed relative to the study objectives.\n\nPerform a correlation analysis on the water quality parameter over time. Related the values of the slope and intercept on the effects of water quality for each treatment.\n\nANOVA should also be done on the water quality per sampling point so that the readers will know when the differences became significant.\n\nWhy did the authors conclude that molasses is the best starter? Based on the graph, all three starters had better water quality than the control. In addition, the differences in the water quality values (ammonia, nitrite, nitrate) among the biofloc treatments were not significantly different. It is suggested that authors perform additional statistical test to show that molasses is significantly better than the other 2 biofloc starters.\n\nThe role of the probiotics in the study should be discussed. The authors should provide in the methods the initial counts of the probiotics that were applied initially.\nI hope that these suggestions will be considered by the authors when they revise the manuscript.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": [
{
"c_id": "5481",
"date": "13 May 2020",
"name": "Iskandar Putra",
"role": "Author Response",
"response": "1. Inquiry Change the title to: Effect of different biofloc starters on ammonia, nitrate, and nitrite concentrations in the culture of tilapia Oreochromis niloticus. Response: Thank you for your suggestion. I have already changed it 2. Inquiry The authors mentioned red tilapia in the Abstract but only tilapia in other portions of the text. Please be consistent. Response: Thank you for your suggestion. I have already changed it 3. Inquiry In the Introduction, the authors wrote: \"Molasses, tapioca, and wheat flours are common starters in biofloc culture\". Please indicate to which species were these starters used. Response: We added this information in Introduction 4. Inquiry The authors wrote: \"Presently, no information is available on the best starter for biofloc in the cultured system of Nile tilapia Oreochromis niloticus\". It is suggested that the authors conduct a thorough literature search, as there have been studies done on biofloc starters in tilapia. Please rectify this statement. Response: We have modified this statement 5. Inquiry In the methods, please indicate how the biofloc was maintained in the study. The study only made mentioned the starter. In the 40 days, was there no addition of the starters to maintain the biofloc? Response: The starters were added every week for maintaining the floc 6. Inquiry Please indicate water exchange rates? There was no water change in the control in 40 days, and the fish were quite big during stocking (approximately 3 g). With no water exchange in 40 days in the control and with the addition of feed, there is definitely a deterioration of water quality and lower fish survival. In the actual culture of tilapia, water exchange is done. For biofloc system, water exchange is minimal hence the results are compared in terms of water quality as well as savings in the cost of electricity or volume of water discharge. Response: There is no water exchange during conducting the experiment 7. Inquiry Was feeding the same for the biofloc treatments and control? Please explain. Response: Feeding applied for the biofloc treatments and control were same 8. Inquiry The authors should explain the water exchange and feeding management between the control and biofloc treatments and how these are controlled/managed relative to the study objectives. Response: There was no water exchange during the experiment and feeding was applied the same treatment for the biofloc treatments and control. 9. Inquiry Perform a correlation analysis on the water quality parameter over time. Related the values of the slope and intercept on the effects of water quality for each treatment. Response: We did not perform a correlation analysis on the water quality parameter over time. 10. Inquiry ANOVA should also be done on the water quality per sampling point so that the readers will know when the differences became significant. Response: We calculated ANOVA of the water quality according to data at the end of the experiment 11. Inquiry Why did the authors conclude that molasses is the best starter? Based on the graph, all three starters had better water quality than the control. In addition, the differences in the water quality values (ammonia, nitrite, nitrate) among the biofloc treatments were not significantly different. It is suggested that authors perform additional statistical test to show that molasses is significantly better than the other 2 biofloc starters. Response: Based on the ANOVA, there was no significant difference of water quality among the starter treatments, but the molasses supplementation in the culture media showed the best water quality values compared to other starters 12. Inquiry The role of the probiotics in the study should be discussed. The authors should provide in the methods the initial counts of the probiotics that were applied initially. Response: Thank you for your suggestion. We added this information in methods"
}
]
},
{
"id": "62751",
"date": "11 May 2020",
"name": "Muhammad Rizal Razman",
"expertise": [
"Reviewer Expertise (Agriculture and Biological Sciences",
"Environmental Science",
"Sustainability Science and Governance",
"Environment and Development)"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe work was clearly and accurately presented.\n\nThe work has cited the current literature, (36 %) of (2015-2020).\n\nThe study design was appropriate and the work was technically sound.\n\nThe work presented sufficient details of methods and analysis provided to allow replication by others.\n\nThe statistical analysis and its interpretation was appropriate.\n\nThe source of data underlying the results available to ensure full reproducible.\n\nThe authors should elaborate more for their conclusion. The conclusion should be reflecting the work that has been done. The conclusion given was too brief.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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/9-293
|
https://f1000research.com/articles/9-495/v1
|
02 Jun 20
|
{
"type": "Software Tool Article",
"title": "COPO: a metadata platform for brokering FAIR data in the life sciences",
"authors": [
"Felix Shaw",
"Anthony Etuk",
"Alice Minotto",
"Alejandra Gonzalez-Beltran",
"David Johnson",
"Phillipe Rocca-Serra",
"Marie-Angélique Laporte",
"Elizabeth Arnaud",
"Medha Devare",
"Paul J. Kersey",
"Susanna-Assunta Sansone",
"Robert P. Davey",
"Alice Minotto",
"Alejandra Gonzalez-Beltran",
"David Johnson",
"Phillipe Rocca-Serra",
"Marie-Angélique Laporte",
"Elizabeth Arnaud",
"Medha Devare",
"Paul J. Kersey",
"Susanna-Assunta Sansone"
],
"abstract": "Scientific innovation is increasingly reliant on data and computational resources. Much of today’s life science research involves generating, processing, and reusing heterogeneous datasets that are growing exponentially in size. Demand for technical experts (data scientists and bioinformaticians) to process these data is at an all-time high, but these are not typically trained in good data management practices. That said, we have come a long way in the last decade, with funders, publishers, and researchers themselves making the case for open, interoperable data as a key component of an open science philosophy. In response, recognition of the FAIR Principles (that data should be Findable, Accessible, Interoperable and Reusable) has become commonplace. However, both technical and cultural challenges for the implementation of these principles still exist when storing, managing, analysing and disseminating both legacy and new data.\n\nCOPO is a computational system that attempts to address some of these challenges by enabling scientists to describe their research objects (raw or processed data, publications, samples, images, etc.) using community-sanctioned metadata sets and vocabularies, and then use public or institutional repositories to share them with the wider scientific community. COPO encourages data generators to adhere to appropriate metadata standards when publishing research objects, using semantic terms to add meaning to them and specify relationships between them. This allows data consumers, be they people or machines, to find, aggregate, and analyse data which would otherwise be private or invisible, building upon existing standards to push the state of the art in scientific data dissemination whilst minimising the burden of data publication and sharing.",
"keywords": [
"Open Data",
"Open Science",
"Ontology",
"Ontologies",
"Repository",
"Repositories",
"Open Standards",
"Schemas",
"Annotation",
"Omics",
"Genomics",
"Multi Omics"
],
"content": "Abbreviations\n\nCGIAR: Consultative Group on International Agricultural Research\n\nCOPO: Collaborative Open Plant Omics\n\nFAIR: Findable, Accessible, Interoperable and Reusable\n\nINSDC: International Nucleotide Sequence Database Collaboration\n\nISA: Investigation / Study/ Assay\n\nMIAPPE: Minimum Information About Plant Phenotyping Experiment\n\nJSON: JavaScript Object Notation\n\n\nIntroduction\n\nMetadata, defined as “data about data”, provides contextual information about data and is one of the central concepts required to allow the structuring of datasets in a way that allows automated search, query and retrieval. In the life sciences, this process is called biocuration1.\n\nNaively, people tend to catalogue information within computer systems (and elsewhere) in ways that require subsequent interpretation. For example, naming a file “20190114_final-with-corrections.txt” reminds the author of certain properties of the data inside the file with very little overhead. Documents ending in the suffix “.txt”, are generally written in plain text, but this structure is explicitly given, and must be assumed by the person wanting to reuse this data. Now consider a file called “dogs.csv” in a folder called “behaviour” in a computer file system. The file has the “csv” extension, which stands for “comma separated values” and implies a defined tabular data structure. The file and directory names suggest that the contents relate to some kind of dog behaviour. The contents of these files cannot be fully understood until they are opened and interpreted. The representation of the information conveyed in both these examples has many ambiguities that can only be resolved by a priori knowledge. Without explicit boundaries and rules that describe this kind of data, misinterpretation may be commonplace and if the data is not as expected, it is impossible to know if this is because the data is corrupted or the assumptions of the finder are misplaced. Such descriptive boundaries are called context. Providing structured metadata makes the context for a given piece of data explicit.\n\nConsider a table in a database called “dog_behaviour_observations” which keeps behavioural observations in tabular (row and column) form. Each row of the table represents a single observation, and each column represents a variable about that observation, such as “behaviour_observation_id”, “behaviour_type”, “breed”, “date_observed”, “video_link”, “duration”. Entries within this database now have a structure to the data, and researchers generating observation data have a (relatively) strict format to adhere to when providing data to the resource.\n\nHowever, this methodology still has issues. The names of the variables (column headers in the database) may not be standardised or self-describing, e.g. the variable “duration” might relate to an observation in a number of ways, but without further documentation, it is impossible to know this for sure. “Duration” may also be measured in different ways with different units, and the processes of measuring the duration of an observation may not have been recorded or standardised. Furthermore, without a way of validating the input to this database, errors may find their way in. Whilst databases do have a minimal form of validation in the form of field data types (e.g. “behaviour_observation_id” might be required to be a non-negative whole number, ”video_link” a string of fewer than 255 characters, etc,), these constraints provide almost no semantic insight into the meaning of a field. This type of validation also does not take broader context into account, adding sparse metadata to a dataset. By using well-defined vocabulary terms for field names and values, we can easily provide information such as:\n\n- What was the breed of the dog, based on an agreed set of dog breed names?\n\n- How were the researchers measuring the impact of a given behaviour on other dogs, based on an agreed measurement technique?\n\n- What was the weather like on the day of the test, based on standard meteorological descriptions?\n\n- Are the individuals observed related, based on a common vocabulary of familial relationships?\n\nThe ability to ask and answer questions such as these may well affect the ability of another researcher to find and reuse this dataset. If we wanted to get all behavioural observations collected on a rainy day, between siblings that are all black Labradors, we couldn’t do this with our current database structure. Therefore, effective reuse of data requires that metadata be structured and composed of defined terms according to common agreed standards. Employing such standards enhances the value (and ultimate impact) of the data produced1.\n\nCommon vocabularies, standards, and dictionaries of agreed terms, or “facts”, can be represented as ontologies, i.e. formal descriptions of a domain of knowledge2. Ontologies define axioms, i.e. terms and the relationships between them, that can be used to construct more complex statements of knowledge. For example, we are interested in finding information on crop trials involving Chinese Spring wheat in sub-Saharan Africa. Our first problem is one of vocabulary. There is not a single shared name for this geographical region: we could search for “sub-saharan Africa”, “Africa (sub-saharan)”, or one of many other permutations. The name of the data field containing this information could also be varied e.g. “Location”, “Region”, or “Country”. If trial data is not standardised across experiments, aggregating searches across multiple datasets quickly becomes a time-consuming manual task, if not impossible. However, by labelling with unambiguous ontology terms, we can specify exactly what we mean, thus increasing the usefulness of our data to other researchers.\n\nFigure 1 shows an example of multiple datasets being aggregated in such a way. Three columns from three different spreadsheets are shown, where the field names “Location”, “region” and “LOC” could be resolved by a curator to the same ontology term “LOCATION”, telling us that they are labelling the same entity, i.e. a geographical area. The values “Great Rift Valley” and “GRV” also resolve to the same term, meaning that a common vocabulary is used, removing ambiguity. Similarly, incomplete and erroneous data can also be corrected to a degree. For instance, a curator would likely assume the value “centra;” to be both incomplete and misspelt. However, resolving it to the term “CENTRAL AFRICA” overcomes these inconsistencies. Data is often messy and needs active curation to resolve issues, either through manual editing or through the use of specific software. Tools such as OpenRefine3 are designed to facet human-readable terms and quickly highlight discrepancies.\n\nOntology terms are typically arranged into a directed acyclic graph (DAG) structure based on a model of a domain, where the nodes are the entities and the edges are the properties or relations between the entities. Part of the model may include a hierarchy, which allows us to infer context even when it is not explicitly provided. To continue the example, although none of the data in Figure 1 are labelled with the term “Sub-Saharan Africa”, through the contextual hierarchy of the ontology (the graph defines “Sub-Saharan Africa” as composed of three more specific terms “Central Africa”, “West Africa”, and “East Africa”) a human or a machine is able to easily infer that all these data are part of “Sub-Saharan Africa”. Likewise, countries in East Africa (e.g. Malawi, Uganda, Kenya, Tanzania from the example above) are also inferred to be part of Sub-Saharan Africa, providing a broader context without the need for its explicit specification, and the added effort that would require.\n\nTo summarise, ontologies can be applied to any area of human endeavour where a consensus is required to benefit data access and interoperability. For example, ontologies are becoming recognised as vital elements of the data description and publication process to benefit research into food4 and its security5, and have become widely accepted for description and connection between varying inter-related disciplines such as human health and disease research6, multi-species anatomy7, gene function8 and plants and agriculture9–11.\n\nThe use of ontology terms to mark up data as in the examples above means that information becomes less ambiguous, and less ambiguity makes data more Findable, Accessible, Interoperable, and Reusable (FAIR)12. The FAIR principles provide a framework for data producers to better disseminate and derive value from their data. The consideration of the FAIR principles in policy making in the life sciences was the result of efforts across the wider spectrum of scientific research communities to consolidate good data management practices into a concise set of core values. Whilst these values do not outline the “how’s” of data management, but rather the “why’s”, they provide a Maslow’s Hierarchy of Needs13 for researchers to start thinking more concretely about their data and their responsibility for its reuse by others, often well beyond the timeframes and immediate purposes originally envisaged when generating it. As such, advancing FAIR requires not just technical, but also cultural, considerations.\n\nFor example, as discussed above, the use of defined ontology terms to annotate data and data services helps to render data finadable and interoperable - computers can be used to search, aggregate, and integrate data based on the occurrence of compatible terms (e.g. overlapping concepts or synonyms) between datasets. However, life science datasets tend to be disparate, except for a few large, curated, public data repositories (e.g. INSDC data warehouse resources, FlyBase, WormBase, Ensembl Genomes, etc.) The databases and data hosting services that house them may not mandate ontology terms for data description upon submission. Nor may publishers or funders, even those with mature Open Access policies that govern data as well as other research outputs. Furthermore, funding for data resources often comes at the national or research organisation level and historically only limited funding (e.g. the European Union Horizon 2020 calls for e-Infrastructure) has been available to develop global, or integrate international, standards or tools and systems to enhance and exploit them. However, a consensus is growing within the scientific community to make sure data services describe and expose their data using agreed standards14, in spite of the limited funding that may be available15.\n\nFrom a recent NSF survey, it seems researchers are becoming more mindful of the value of well-described open and FAIR data and their responsibilities to provide it, but they often feel inadequately trained to do so16. The quantity of data produced from scientific experiments is becoming increasingly large and diverse. New approaches, such as BioSchemas17, are attempting to exploit the ubiquity of internet search engines and their metadata harvesting and indexing capacity to expose data through standard search results. Once datasets are discoverable, interoperability then relies on exposure of data through standard application programming interfaces (APIs) of which there are active and notable examples in the life sciences18,19. However, data management tools, especially those used to annotate and describe data assets, tend not to be designed for typical biologists. Rather they are often designed for the small number of professional biocurators, and this may deter other researchers whose participation is essential if curation capacity is to scale to match the growth of data production. Scientific institutions often lack these biocuration experts who could advise researchers20. Moreover, the variety of research data means it is also important to maximise the pool of software engineers developing biocuration systems. This can be best achieved if code is published using an open-source licence.\n\nRecording metadata for structured FAIR data remains difficult for researchers, and represents the “last mile” in scientific data publishing21. We believe the solution is to help users to publish FAIR data through user-friendly systems and workflows explicitly designed for that purpose. Alongside these, training and guidance about the ontologies suitable to annotate their data, and repositories suitable to house it, should be provided.\n\nCOPO is a platform for researchers to publish their research assets, providing metadata annotation and deposition capability. It allows researchers to describe their datasets according to community standards and broker the submission of such data to appropriate repositories whilst tracking the resulting accessions/identifiers. These identifiers can be used as citations for the richer context of a researcher’s output (i.e. Data production, Images, Source Code etc). As such, COPO directly addresses the Accessible and Interoperable tenets of FAIR22. This has been achieved by developing wizards built into a web-based platform to allow users to describe their data without having to resort to complex spreadsheets, multiple redundant data descriptions when handling multiple research object entities, or multiple submissions to different repositories. These wizards facilitate the use of metadata required by individual repositories, compliance with broader community standards, and are presented to users in an incremental linear fashion so as not to overburden them with large numbers of metadata fields at once. Metadata captured in these wizards is then automatically transformed into the format specified by the repository, removing this task from the user. The design is distinct from that of Dendro23, CEDAR24 and *-DCC25, three other platforms with related capabilities. Dendro and *-DCC provides linear web forms, where the latter is specifically designed only for sequencing data submission. CEDAR uses dynamically created templates and, like COPO, allows arbitrary use of ontologies for data description. However, none of these provide rich wizard functionality based on bespoke institutional research data management policies. COPO uses these modular configurable wizard elements based on standard information interchange formats to build complex but easily navigable UIs. Furthermore, COPO is more advanced than *-DCC with respect to data upload management, i.e. broken or corrupted data uploads have to be fixed manually by an administrator within *-DCC, whereas COPO provides chunked uploads (data files are split up into smaller portions and each portion is checked for integrity) and supports upload resuming, making it much easier for users to handle large data files.\n\nCOPO mediates submission to many of the most widely used repositories in the life science community (e.g. the EMBL-EBI European Nucleotide Archive for genomics researchers, see Supported Schemas and Repository Types). As it is open source, COPO can be made specific to a community and/or repository used by that community through implementing new wizards that precisely meet their needs. We are working on supporting a larger set of data repositories as COPO matures and as submission routes to these repositories implement documented APIs. We provide two repository Use Cases below.\n\n\nMethods\n\nCOPO is primarily a Python application. At the time of writing, COPO runs on a Python 3.5.2 interpreter with Django 2.0.2 as the web framework. Python was chosen as it is a well used and documented language with an active development community. This ensures there are mature libraries for common tasks (such as web development, data loading or ingestion, visualisation, tabular data manipulation, statistical analysis, natural language processing, etc.), as well as cutting edge libraries for modern data science (such as Pandas, scikit-learn, TensorFlow and Numpy/SciPy). Python integrates easily into system-level scripting and is therefore suitable for “glue” projects where many components need to interoperate, such as web service platforms.\n\nThe Django web framework was chosen to handle the server side duties of COPO, as it is a tried and tested off-the-shelf solution used by many web services across the international academic, commercial and industrial sectors. A web framework provides developers with many commonly needed functionalities such as URL mapping, page templating, object relational mapping, session handling, user management and security checking. Django is updated regularly, has exceptionally good documentation and a large user base.\n\nCOPO uses an internal JavaScript Object Notation (JSON) data structure, or schema, to store metadata based on modular fragments that can be generated from a range of metadata management processes. JSON is a lightweight, text-based, language-agnostic data interchange format, designed for machines to parse and generate data at scale. JSON is termed a “self-documenting” format, meaning that it is clear enough for a human to understand without reference to any supplementary information. Metadata can be represented in JSON as a collection of name/value pairs. JSON is a common data representation format for many public APIs due to its simplicity and readability. Libraries for manipulating JSON exist for most if not all commonly used programming languages26.\n\nBoth Postgres 9.6 and MongoDB 3.4 databases are used for backend storage: Postgres for structured data such as housekeeping tasks related to Django (user accounts, sessions etc); and MongoDB to store the COPO JSON data objects and metadata. MongoDB is a NoSQL database, i.e. the schema does not need to be predefined, facilitating the support of highly heterogeneous metadata without the need for complex schema design. Data files (e.g. raw sequence data, images, PDFs) uploaded to COPO are held on an Isilon storage array - a robust, scale out network-attached storage (NAS) solution for high-volume storage, backup and archiving of unstructured data27. COPO currently benefits from a shared access to over 3.4 petabytes of storage space on this platform, which provides it with the scope to manage the large volume of data brokered through the system. iRODS 4.1.1128 is a framework for abstracting and federating data storage or repositories in a way that promotes data virtualisation, data discovery, data policy automation and secure collaboration across sites. The use of iRODS is being tested in COPO in order to provide a storage solution that is agnostic of any specific hardware resource architecture.\n\nThe user interface (UI) components of COPO have been developed primarily using the Javascript jQuery 2.1.4 library and the Bootstrap 3 framework. Additional javascript UI libraries used include DataTables 1.10.18 for displaying and paginating lists of data, Semantic UI 2.4.0 for UI controls such as buttons and sliders, Handsontable 7.1.0 for displaying spreadsheet type data, and Fuel UX 3.12.0 for creating the COPO wizards.\n\nCOPO has been dockerized therefore a standard development implementation can be easily built and run. Download the latest version from https://github.com/collaborative-open-plant-omics/COPO/releases and follow the instructions in the README.md file. Minimum requirements vary depending on the work the COPO instance will be running, but for development, we recommend at least 2GB of RAM and 2 CPU cores. The installation method has been tested on Ubuntu 18.04 and OSX 10.14. We recommend using Firefox.\n\nThe primary mechanism COPO uses to collect metadata is the wizard, which represents the simplest form of an expert system. Wizards consist of a series of interactive stages or form pages which are displayed sequentially, showing users paths through a task based on answers they have given to previous questions. Fields in these wizard stages contain labels, help tips and controls, which guide the user to enter the required metadata. Some of these controls look up bespoke lists of values that are not in a formal ontology but are required by a repository, some (such as calendars) always construct data in a valid format, whilst others perform a lookup call on the Ontology Lookup Service29 and complete a valid ontology reference.\n\nNew COPO wizards can easily be configured by defining JSON schemas that comprise the metadata representation for a given repository. Metadata representations between COPO and data repositories are powered by the ISA JSON schema30, allowing information on metadata fields and their rendering to be stored in a central place. For example, fields for a particular type of submission are stored in the same JSON file, with each field having an entry with a unique ID to that field type, specification on type of control to be rendered on a web page to collect the data, whether it’s required, the names it has had in the past, label, help text, etc. (see Box 1). From this JSON representation, UI controls are automatically generated and rendered to collect such metadata during submission (see Figure 2). This simplifies maintenance and rendering of the UI and also means that creating a submission wizard for a new repository/datatype is as simple as copying and pasting an existing JSON fragment and changing the fields as required. This new configuration will then be rendered without having to write any HTML.\n\n\n\n\n\nAs indicated above, COPO uses the ISA JSON representation to keep experimental metadata for further data sharing and data publication. The ISA framework31 comprises an established data model and a set of open source tools supporting users to provide rich descriptions of their experimental data. ISA stands for Investigation (the project context), Study (a unit of research) and Assay (an analytical measurement). The ISA data model is provided in a variety of serialisations: CSV/TSV tabular format, the Resource Description Framework (RDF) for linked data, and as JSON32. There is a large and international community of ISA adopters and developers associated with the ISAcommons31 that ranges from public repositories to institutional repositories, funded research consortia and data journals.\n\nThe ISA model and tools allow the description of experimental details workflows, sample characteristics, technology and measurement types, sample-to-data relationships, and source to sample data processes. The ISA model can also be configured to be compliant with the minimum information requirements for different domains.\n\n‘Omic sequence and feature data. COPO supports the brokering of sequence reads, assemblies, and annotations. We are currently working with repository maintainers to develop wizards for the more esoteric analysis data types, such as optical maps. Data files are uploaded to the EMBL-EBI European Nucleotide Archive servers, and wizards are provided to assist users in fulfilling the requirements of the EBI submission schemas without them needing to create spreadsheets, XML fragments or use the ENA command line tool. The full ENA/SRA metadata fragment is created and the submission happens seamlessly for the user. We describe this in more detail in the Use Case example below.\n\nPhenomics data. Collection and deposition of phenotypic experiments is partially supported. The MIAPPE (Minimal Information About Plant Phenotyping Experiments) schema is implemented to collect the required metadata for such experiments. At the time of writing, there is currently no formal repository into which of these data can be deposited. However, the EMBL-EBI have recently announced their BioImaging repository33, which is able to accept any image-based phenotyping datasets. Submission will be supported in an upcoming COPO version.\n\nAgriculture data. The CG Core metadata schema34 is implemented in COPO as a pilot for agricultural data annotation for the 15 Consultative Group on International Agricultural Research (CGIAR) agricultural research for development centers. A more generalised version of this implementation will soon be more widely available for other entities wanting to describe agricultural data. CG Core is a minimum metadata specification based on the Dublin Core standard to collect descriptive metadata on agronomic, plant breeding, socioeconomic, genomic, and other agricultural information products. Each of the 15 centres store their datasets and publications on centre-specific publicly accessible digital repositories based on one of three repository software implementations, i.e. DataVerse, dSpace or CKAN (see below). The CG Core schema allows consistent description of these data assets for easier discovery and access. We describe this in more detail in the Use Case section.\n\nOther scholarly data types. COPO can be used to annotate other information types such as images, presentations, figures, text documents, and PDFs. The Dublin Core metadata specification35 is also employed for these, and submission is supported to institutional instances of Dataverse36, DSpace37 or CKAN38, all of which are free, open-source, off-the-shelf repository applications. COPO also enables submissions to figshare39. Figshare is a centralised general purpose repository whilst CKAN and Dataverse can be installed on local servers (figshare also supports institutional installations of the software but only through a paid model40). There is a publicly accessible instance of Dataverse available for those who cannot host their own41.\n\nThe COPO infrastructure is currently housed within the CyVerse UK virtual infrastructure42, supported by the Earlham Institute (EI) National Capability in e-Infrastructure (EI NCG NC3)43. COPO uses the Docker44 containerisation software to house the service deployments, i.e. MongoDB, iRODS, the Django web application and the project website. These Docker containers are managed using Docker Swarm technology to ease orchestration, deployment, and load-balancing challenges (see Figure 3). A default swarm configuration is supplied with COPO, but deployment will need certain properties set by a system administrator, i.e. COPO has a central management node that handles the configuration and setup of the swarm, where system administrators need to supply storage volumes, secret keys, and service definitions for swarm worker nodes. These worker nodes comprise the underlying software stack of databases, web servers and indexers. This modern modular approach to deploying web services makes it possible for third parties to quickly and easily initiate their own versions of COPO.\n\nA researcher has undertaken a sequencing experiment, and has a number of files containing the filtered and quality-controlled reads representing the nucleic acids that have been sequenced. Once the researcher is ready to submit this read data, they can log into the COPO system using their Open Researcher and Contributor ID (ORCiD). Once logged in, the user can either upload their read data, or describe their samples. UIs have been developed to streamline this process as much as possible. For example, multiple sample objects can be created at once. If the user has existing sample objects in the system, these can be cloned, saving time and effort if a researcher is working on similar organisms, studies, or protocols. Organism names are auto-completed with reference to the NCBI Taxonomy ontology45. Once the required sample objects have been created, the user uploads their sequence data. COPO will compress (gzip) data files if this has not already been done.\n\nThe user can then choose a number of files to describe simultaneously in a “bundle”, whereby bundled files are labelled with the same metadata. For example, if there are 50 samples and 50 corresponding files in a bundle, the user only has to go through the description wizard once. Users can also work with unbundled datasets which is useful if the metadata is heterogeneous, e.g. if each data file being submitted is from a different organism or sequenced using different sequencing technologies. The metadata fields which are displayed in the wizard are constructed in line with the schema of the target repository for which the submission is intended. In doing so, the burden of managing metadata revisions, as well as the minimum set of requirements, from different repositories is removed from the researcher and offloaded to COPO. Once the metadata description wizard is complete, the user can then view the metadata for all the files and change any values which are not common in a spreadsheet-like tabular user interface. In the wizard the user also defines which corresponding files each sample object relates to. This is a time-consuming task for researchers as care has to be taken to ensure samples are exactly matched to output files, and COPO aims to reduce errors by making simple sanity checks on sample attribution to data files (number of samples per file, duplicated samples, R1 and R2 files for paired end sequencing experiments, etc). Upon completion, the user is taken to the submission page, where they are shown a progress indicator. During the submission process, the required metadata (in this case ENA submission, study, experiment, run and, as required, analysis XMLs) is produced, the datafiles are physically transferred to the ENA servers, the metadata is validated, and the submission completed (see Figure 4). To our knowledge, COPO is one of the first web-based service implementations of the new EMBL-EBI ENA command line validation client46. The validation process is very resource intensive and the public COPO instance uses the CyVerse UK HPC cloud environment42 to undertake validation.\n\nCOPO supports the ENA embargo process whereby researchers can upload data and metadata but not yet make the data public, typically waiting for a publication to be submitted. In any case, ENA accessions for the submission are stored in COPO and are available to the user to publish, and for others to search once public. An example COPO-submitted ENA project can be found under the accession PRJEB30905.\n\nDigital repositories used by CGIAR centres are hosted locally in some cases, and centralised in others, depending on the repository platform and the centre. Metadata for information resources (typically datasets and publications) are entered by the different data curators and information specialists in each centre. The objective of the CG Core metadata schema is to harmonise descriptions of agricultural resources across the CGIAR centres through a minimum metadata set to maximise discoverability and interoperability. COPO provides a unique entry point to the different systems to guarantee that the metadata are captured in a unified way across the different centre repositories. COPO user interfaces are generated based on the CG Core specification, with the advantage of providing data validation checks while the metadata are entered by the end user. COPO captures the full metadata set of the CG Core, although some data repositories only accept a subset of it. By retaining this metadata alongside links to these repositories, COPO improves the provenance of these records despite the full CG Core metadata not being supported by institutional repositories themselves. This is a by-product of repositories effectively being legacy systems, where either the repository wants to remain general to reduce metadata requirement overload, or highly specific but not able to future-proof any and all possible metadata specifications arising from a community. Managing this extra metadata becomes important for specific internal reporting, where metadata needs to be recorded in a harmonised way, i.e. donor information. Thus in future, as COPO is not intended to be a repository in its own right, we will also submit these additional metadata fragments to a public repository, e.g. Zenodo47, to mint DOIs for the metadata and support data citation (where data is published with a unique identifier, a short publication is written to describe that dataset, and a DOI is minted to allow citation of that dataset to demonstrate use). The full metadata information can then be used by GARDIAN48, the CGIAR metadata harvester and aggregator that allows users to discover datasets and publications using rich queries. The integration of COPO and GARDIAN aims to improve user experience to search across the CGIAR information products (see Figure 5), based on the implementation of core ontologies to describe those products4.\n\n\nDiscussion\n\nThe “data deluge” has preoccupied data scientists and knowledge managers for the last decade. It has been the subject of popular news articles49, some of which suggest Big Data will see the end of hypothesis-driven science50. The current investment into data generation far outstrips investment in data curation. Many commentaries about the reproducibility and reuse of code, data, and information within publications make sensible points: researchers need to make their digital research outputs available, with requisite associated metadata for reproducibility, and in formats that are open and computationally accessible50–55. However, many if not all of these studies mention in passing, and omit concrete details and processes for, one crucial step: researchers find it hard and time-consuming to annotate their data with standard vocabularies. Both technical solutions and coordinated community efforts will be required to manage future datasets and the legacy data that resides in individual group workstations and databases which are no longer actively maintained or curated.\n\nThe open source movement in the life sciences is a powerful force, with tools and algorithms developed continuously and released via websites such as Github and Bitbucket. Funders are particularly interested in the impact of algorithms, software tools, and databases to further bioscience. These, including web-based systems, can be submitted as first-class research outputs in many grant reporting platforms, e.g. the United Kingdom Research and Innovation (UKRI) ResearchFish51 system. However, data citation is still a novel concept, and the cost of curation is perceived to be high relative to the cost of producing datasets, particularly raw data generation such as nucleic acid (genome, transcriptome, etc) sequencing. Easing the “FAIRification” of data and enabling the tracking of its impacts can be invaluable for their predestined communities. Further work will enable a rich set of search APIs within COPO to allow users to more accurately track where their data has been used.\n\nThe correct ontologies and terms to use to annotate data might not be immediately obvious to researchers, nor where the data should be deposited. FAIRsharing56 works to fill this gap by providing concrete guidance and tools to enable discovery and use of the appropriate terminology, metadata and data standards. It can also help users determine repositories that implement those standards and which journal and funder policies recommend them. However, individual researchers must still search for and assimilate this information. Finding the relevant standards and repositories is only the first step, and institutions may employ dedicated research data managers to help researchers take the next step of annotating data using community standards. Data management employment positions are rare and very specialised to a given domain of knowledge. Therefore, there remains a professional chasm between researchers who know they need to manage their research data, the tools that are available to allow them to do it, and the people with the epistemological expertise to assist them57.\n\nData brokering systems can help greatly to bridge this gap and help researchers realise the practical aspects of sharing FAIR data. Many years of experience developing the ISA framework and related semantic architectures has shown us that standards-driven annotation and data reformatting for brokering purposes needs to be “made invisible” to users in order to promote uptake. This idea can be brought into line with recent thinking around how to rework the Hierarchy of Needs to something more like a “Hierarchy of Changes”58. COPO is firmly at the “Make it normative” stage, i.e. COPO has been designed with usability in mind, and we have used numerous focus groups to gain feedback on features and the main metadata wizards within the system. We have also presented COPO at events organised by large public data repository managers such as the EMBL-EBI ENA to ensure COPO complements and exploits the submission tools provided by such resources.\n\nNormalising semantic metadata attribution at the researcher and data manager level in COPO means that we now need to consider the “Make it rewarding” and “Make it required” stages. Rewards and incentives for good behaviour help reinforce learning and behavioural change in humans. However, the incentive and reward structures are not yet in place for data (as opposed to journal) publication57. Therefore, to make research data truly FAIR, the scientific community needs to address how sharing and reuse of information outputs is both valued and how it adds value to research in a particular domain. Crop breeding, for example, is an area where standardisation efforts and FAIR data have huge potential to modernise and revolutionise research and applied technological solutions for food security59,60. These changes are fundamental to allow the application of modern data integration and interrogation approaches and tools to help unlock the value of knowledge held in disparate research groups, institutions, and communities.\n\n\nConclusions\n\nCOPO is a semantic metadata annotation and data brokering platform, initially designed to ease data curation and submission to public repositories for plant scientists. However, as it allows for the flexible selection, constraint, and use of any public or user-specified ontology, COPO is applicable to any domain of knowledge, and has already been customised for agricultural data assets through work with the CGIAR System for international agricultural research for development. As well as a free-to-use central production installation running at the Earlham Institute, COPO is open source under the MIT licence, and therefore freely usable by any institution for their own research data management needs. The tool is packaged in Docker containers for both easy testing and deployment in production. COPO aims to address both the “first mile” and “last mile” of data management to make research outputs FAIR: the former, where users are able to address the final stages of preparing data for publication, is an often-overlooked resource-intensive part of the publication cycle; and the latter, where research objects are sufficiently described and made freely available for others through public or institutional repositories.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\n\nSoftware availability\n\nSoftware available from: https://copo-project.org/\n\nSource code available from: https://github.com/collaborative-open-plant-omics/COPO\n\nArchived source code at time of publication: http://doi.org/10.5281/zenodo.383388261\n\nLicense: MIT",
"appendix": "References\n\nInternational Society for Biocuration: Biocuration: Distilling data into knowledge. PLoS Biol. 2018; 16(4): e2002846. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGruber TR: A translation approach to portable ontology specifications. Knowl Acquis. 1993; 5(2): 199–220. Publisher Full Text\n\nopenrefine. Reference Source\n\nDooley DM, Griffiths EJ, Gosal GS, et al.: FoodOn: a harmonized food ontology to increase global food traceability, quality control and data integration. NPJ Sci Food. 2018; 2: 23. PubMed Abstract | Publisher Full Text | Free Full Text\n\nArnaud E, Laporte MA, Kim S, et al.: The Ontologies Community of Practice: An Initiative by the CGIAR Platform for Big Data in Agriculture. Patterns. 2020. Publisher Full Text\n\nKibbe WA, Arze C, Felix V, et al.: Disease Ontology 2015 update: an expanded and updated database of human diseases for linking biomedical knowledge through disease data. Nucleic Acids Res. 2015; 43(Database issue): D1071–D1078. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMungall CJ, Torniai C, Gkoutos GV, et al.: Uberon, an integrative multi-species anatomy ontology. Genome Biol. 2012; 13(1): R5. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHarris MA, Clark J, Ireland A, et al.: The Gene Ontology (GO) database and informatics resource. Nucleic Acids Res. 2004; 32(Database issue): D258–D261. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMatteis L, Chibon PY, Espinosa H, et al.: Crop Ontology: Vocabulary For Crop-related Concepts. 2013. Reference Source\n\nCooper L, Walls RL, Elser J, et al.: The plant ontology as a tool for comparative plant anatomy and genomic analyses. Plant Cell Physiol. 2013; 54(2): e1. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJonquet C, Toulet A, Arnaud E, et al.: AgroPortal: A vocabulary and ontology repository for agronomy. Comput Electron Agric. 2018; 144: 126–143. Publisher Full Text\n\nWilkinson MD, Dumontier M, Jan 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\nMaslow AH: A Theory of Human Motivation. Psychological Review. 1943; 50(4): 370–396. Publisher Full Text\n\nSansone SA, McQuilton P, Rocca-Serra P, et al.: FAIRsharing as a community approach to standards, repositories and policies. Nat Biotechnol. 2019; 37(4): 358–367. PubMed Abstract | Publisher Full Text | Free Full Text\n\nReiser L, Berardini TZ, Li D, et al.: Sustainable funding for biocuration: The Arabidopsis Information Resource (TAIR) as a case study of a subscription-based funding model. Database (Oxford). 2016; 2016: baw018. PubMed Abstract | Publisher Full Text | Free Full Text\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\nMichel F; The Bioschemas Community: Bioschemas & Schema.org: a Lightweight Semantic Layer for Life Sciences Websites. Biodiversity Information Science and Standards. 2018; 2: e25836. Publisher Full Text\n\nSelby P, Abbeloos R, Backlund JE, et al.: BrAPI - an Application Programming Interface for Plant Breeding Applications. Bioinformatics. 2019; 35(20): 4147–4155. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSmartAPI: Building a connected network of FAIR APIs. SmartAPI. Reference Source\n\nGrowing demand for data science leaves Britain vulnerable to skills shortages. Royal Society, 2019. Reference Source\n\nGabridge T: Last Mile: Liaison Roles in Curating Science and Engineering Research Data. RLI 265; 2009. Publisher Full Text\n\nLeonelli S, Davey RP, Arnaud E, et al.: Data management and best practice for plant science. Nat Plants. 2017; 3: 17086. PubMed Abstract | Publisher Full Text\n\nda Silva JR, da Silva JR, Castro JA, et al.: Dendro: Collaborative Research Data Management Built on Linked Open Data. Lecture Notes in Computer Science. 2014; 483–487. Publisher Full Text\n\nGonçalves RS, O’Connor MJ, Martínez-Romero M, et al.: The CEDAR Workbench: An Ontology-Assisted Environment for Authoring Metadata that Describe Scientific Experiments. Lecture Notes in Computer Science. 2017; 103–110. Publisher Full Text\n\nHörtenhuber M, Mukarram AK, Stoiber MH, et al.: *-DCC: A platform to collect, annotate, and explore a large variety of sequencing experiments. Gigascience. 2020; 9(3): giaa024. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJSON. Reference Source\n\nA Closer Look at the Dell EMC Isilon NAS Storage Platform. Virtualization Review. Reference Source\n\niRODS. Reference Source\n\nCote RG, Jones P, Martens L, et al.: The Ontology Lookup Service: more data and better tools for controlled vocabulary queries. Nucleic Acids Res. 2008; 36(Web Server issue): W372–W376. PubMed Abstract | Publisher Full Text | Free Full Text\n\nThe ISA-JSON format. Reference Source\n\nSansone SA, Rocca-Serra P, Field D, et al.: Toward interoperable bioscience data. Nat Genet. 2012; 44(2): 121–126. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGonzález-Beltrán A, Maguire E, Sansone SA, et al.: linkedISA: semantic representation of ISA-Tab experimental metadata. BMC Bioinformatics. 2014; 15(Suppl 14): S4. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBioImage Archive - a new hub for biological images. Reference Source\n\nAgriculturalSemantics: AgriculturalSemantics/cg-core. GitHub. Reference Source\n\nDCMI: Dublin Core. Reference Source\n\nThe Dataverse Project - Dataverse.org. Reference Source\n\nDSpace - A Turnkey Institutional Repository Application. Duraspace.org. Reference Source\n\nckan: ckan. Reference Source\n\nfigshare - credit for all your research. Reference Source\n\nReed RB: figshare for Institutions. J Med Libr Assoc. 2016; 104(4): 376–378. Publisher Full Text | Free Full Text\n\nHarvard Dataverse. Reference Source\n\nCyVerse UK – CyberInfrastructure for life science. Reference Source\n\nNational Capability in e-Infrastructure. Earlham Institute. 2018. Reference Source\n\nWhat is a Container? | Docker. Docker. Reference Source\n\nFederhen S: The NCBI Taxonomy database. Nucleic Acids Res. 2012; 40(Database issue): D136–D143. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEuropean Nucleotide Archive: webin-cli. GitHub. Reference Source\n\nZenodo - Research. Shared. Reference Source\n\nGARDIAN. Reference Source\n\nHannay T: Stop the deluge of science research. The Guardian. 2014. Reference Source\n\nMazzocchi F: Could Big Data Be the End of Theory in Science? A Few Remarks on the Epistemology of Data-Driven Science. EMBO Rep. 2015; 16(10): 1250–5. PubMed Abstract | Publisher Full Text | Free Full Text\n\nResearchfish: Research Impact Assessment Platform. researchfish. Reference Source\n\nSix factors affecting reproducibility in life science research and how to handle them. Reference Source\n\nChen X, Dallmeier-Tiessen S, Dasler R, et al.: Open is not enough. Nat Phys. 2018; 15: 113–119. Publisher Full Text\n\nGrüning B, Chilton J, Köster J, et al.: Practical Computational Reproducibility in the Life Sciences. Cell Syst. 2018; 6(6): 631–635. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFanelli D: Opinion: Is science really facing a reproducibility crisis, and do we need it to? Proc Natl Acad Sci U S A. 2018; 115(11): 2628–2631. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFAIRsharing. Reference Source\n\nLeonelli S: What Difference Does Quantity Make? On the Epistemology of Big Data in Biology. Big Data Soc. 2014; 1(1). PubMed Abstract | Publisher Full Text | Free Full Text\n\nStrategy for Culture Change. Reference Source\n\nRibaut JM, Ragot M: Modernising breeding for orphan crops: tools, methodologies, and beyond. Planta. 2019; 250(3): 971–977. PubMed Abstract | Publisher Full Text\n\nPommier C, Michotey C, Cornut G, et al.: Applying FAIR Principles to Plant Phenotypic Data Management in GnpIS. Plant Phenomics. 2019; 2019: 1671403. Publisher Full Text\n\nShaw F, Etuk A, Minotto A: collaborative-open-plant-omics/COPO: Release for F1000 Paper (Version v1.0.0). Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3833882"
}
|
[
{
"id": "64267",
"date": "11 Jun 2020",
"name": "Eric Lyons",
"expertise": [
"Reviewer Expertise Plant Genome Evolution",
"Comparative Genomics",
"Cyberinfrastructure for Life Science 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\nSummary of work: The overall problem COPO seeks to address are challenges in ensuring that publishing of digital life science research products (data) comply with FAIR data principles. To address this need, COPO enables researchers to more easily describe their digital products using metadata templates that have been developed and approved by various research communities. COPO uses semantic terms to add meaning and relationships to these metadata, allowing researchers and automated systems to discover and use these data products. Comments:\n\nGood:\nGreat reference to “Maslow’s Hierarchy of Needs”.\n\nGreat general overview of the problem space (excellent for those who don’t understand the need for metadata ontologies). Very well written. COPO public website works as advertised Excellent choice of technologies and deployment architecture\n\nSuggestions:\nNot sure why it isn’t made clear at the end of the introduction that COPO is focused on plant metadata (at least, that is what the website for COPO implies). No documentation for an API (searched on project’s ReadTheDocs site). Manuscript does state “We are working on supporting a larger set of data repositories as COPO matures and as submission routes to these repositories implement documented APIs”, but it is clear from the manuscripts introduction that such APIs are essential for automation of data publication and discovery. Documentation: Developer Guide has not been written.\n\nPre-population of data in COPO. It would be great if the authors populate COPO with as much publicly available data as possible. E.g., NCBI’s SRA. While I’m sure not all entries have enough metadata to make this possible, even a significant number of SRA entries available for search and discovery for COPO would be immensely helpful for the research community (as well as be a test for how well COPO works on the discoverability side of FAIR.) However, I could not find any search function in COPO for published datasets. COPO has been in production for several years. Please provide some statistics on usage and adoption of COPO.\n\nMandatory changes:\nIt needs to be made very clear that COPO is helping only with data submission by giving researchers appropriate tools for metadata population and curation. As such, one could argue that COPO does not directly make data FAIR.\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": []
},
{
"id": "72305",
"date": "19 Oct 2020",
"name": "Matthias Hörtenhuber",
"expertise": [
"Reviewer Expertise Bioinformatics",
"data annotation and curation specifically for sequencing data"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nBrief Summary This article describes a web-based platform supporting researchers with step-by-step guidance with so-called wizards to annotate their datasets according to community based standards. The system then allows uploading the data and corresponding annotation to data repositories such as ENA or CGIAR centres. The platform is available as source code, as Docker image and well as on a web server provided by the authors.\n\nMajor issues The documentation for development and deployment is missing. It would be especially helpful to show concrete examples how to introduce a new ontology and how to customize the platform.\n\nMinor issues Text Page 5: “and less ambiguity makes data more Findable, Accessible, Interoperable, and Reusable (FAIR)“: less ambiguity doesn’t result in “more Accessible”.\nPage 6: “(i.e. Data production, Images, Source Code etc). “: Set these to lower case.\nPage 6: “as well as cutting edge libraries for modern data science (such as Pandas, scikit-learn, TensorFlow and Numpy/SciPy).“ Unclear why this aspect of python is needed for a data annotation platform, which is usually hosted on platforms not suited for heavy computation.\nPage 6: “COPO runs on a Python 3.5.2 interpreter with Django 2.0.2 as the web framework.”: According to your docker and requirements file, is the python version 6 and Django is 2.1.4. Related to that, I would recommend upgrading to Django 2.2, which has long term support at least until 2022.\nPage 7 “COPO uses the ISA JSON representation”: Specify version of ISA framework you are using and mention that they are based on json-schema draft 4.\nweb docs Add a video tour through the functionalities of the platform.\nCOPO web demo I can reproduce a 500 Server error when trying to edit the metadata of an upload txt file.\nSource Code\nThe Readme should be expanded. Provide example links to the ORCID dev tools, where you can apply for the needed keys. Give an example of which redirect URIs COPO is expecting from ORCID. Give a link to ENA’s Webin.\n\n`docker-compose.yml` doesn’t contain `$(COPO_KEYS)`, which are mentioned in the readme. Instead still contains the development file paths.\n\nThe building step for coop-web fails with:\n\n``` ERROR: Could not find a version that satisfies the requirement pdfminer3k==1.3.1 (from -r /tmp/requirements/base.txt (line 110)) (from versions: 1.3.2, 1.3.3, 1.3.4) ERROR: No matching distribution found for pdfminer3k==1.3.1 (from -r /tmp/requirements/base.txt (line 110)) ERROR: Service 'copo-web' failed to build : The command '/bin/sh -c pip install -r /tmp/requirements/dev.txt' returned a non-zero code: 1 ``` Changing the required version to `pdfminer3k==1.3.2` fixes this.\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? No\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/9-495
|
https://f1000research.com/articles/9-493/v1
|
02 Jun 20
|
{
"type": "Research Article",
"title": "Phytochemical composition analysis and evaluation of in vitro medicinal properties and cytotoxicity of five wild weeds: A comparative study",
"authors": [
"Pranabesh Ghosh",
"Chandrima Das",
"Swagata Biswas",
"Sudip Kumar Nag",
"Alolika Dutta",
"Maitrayee Biswas",
"Sayantan Sil",
"Labani Hazra",
"Chandreyi Ghosh",
"Shaktijit Das",
"Moumita Saha",
"Nasim Mondal",
"Suprodip Mandal",
"Anirban Ghosh",
"Srabani Karmakar",
"Sirshendu Chatterjee",
"Pranabesh Ghosh",
"Chandrima Das",
"Swagata Biswas",
"Sudip Kumar Nag",
"Alolika Dutta",
"Maitrayee Biswas",
"Sayantan Sil",
"Labani Hazra",
"Chandreyi Ghosh",
"Shaktijit Das",
"Moumita Saha",
"Nasim Mondal",
"Suprodip Mandal",
"Anirban Ghosh"
],
"abstract": "Background: Medicinal plants are a source of phytochemicals and they are used for the treatment of several oxidative stress-related or other diseases for their effectiveness, low toxicity and easy availability. Five traditionally used and less characterized herbaceous weeds of West Bengal, India, namely, Heliotropium indicum, Tridax procumbens, Cleome rutidosperma, Commelina benghalensis and Euphorbia hirta, were investigated for the current research study. Methods: Aqueous and 70% ethanolic extracts of the leaves were analyzed for estimation of essential phytochemicals and to evaluate their in vitro antioxidant status, medicinal properties and cytotoxic effects. To the best of our knowledge, several assays and comparative evaluations using these herbs are reported for the first time. For quantitative study, UV-vis spectrophotometry and high-performance liquid chromatography with diode array detector HPLC-DAD techniques were used. Antibacterial properties were investigated using the Kirby-Bauer disc diffusion method. For in vitro anti-lithiatic study, a titration method was used. The cell viability assay was done using peripheral blood mononuclear cells. Results: The aqueous extract exhibits higher content of polyphenols, flavonoids, tannins and inhibition percentage values for free radical scavenging assays, whereas the 70% ethanolic extract exhibits higher content of alkaloids and cardiac glycosides. HPLC-DAD analysis of 70% ethanolic extracts led us to identify 10 predominant phenolic constituents. Euphorbia hirta extracts showed minimum cytotoxicity (cell death ~2.5% and 4% in water and 70% ethanolic extract, respectively), whereas Cleome rutidosperma and Tridax procumbens’ 70% ethanolic extracts showed higher cell death (~13% and 28%, respectively), compared with the control (cell death ~10-12%). Conclusions: The study concluded that of all the medicinal weeds selected for the current study, Euphorbia hirta possesses the highest amount of bioactive compounds and hence exhibits the highest in vitro antioxidant activity and promising in vitro medicinal properties.",
"keywords": [
"Heliotropium indicum",
"Tridax procumbens",
"Cleome rutidosperma",
"Commelina benghalensis",
"Euphorbia hirta",
"HPLC",
"Antioxidants."
],
"content": "Introduction\n\nAt present, many people are cautious about synthetic drugs usage because of their side effects and high price. On the contrary, herbal medicines have been continuously used in healthcare treatment system due to their cost effectiveness, better feasibility in the human physiological system and with minimal side effects. India is a vast source of medicinal and aromatic plants. Among its diverse and rich collection of medicinal plants, we have chosen five wild ethnomedicinal weeds and those are very less characterized and available with reported various bioactivities1–4.\n\nThese weeds grow in similar habitats, though they belong to different families. Botanically, one species of plant can considered as a valuable crop or medicinal plant, while another species in the same genus can be treated as a dangerous weed. Many plants that are widely accepted as weeds are also grown in gardens and other cultivated areas; these are known as beneficial weeds1–3,5–9. The current study focuses on searching for those medicinal weeds which have enormous future prospects for pharmaceutical industries.\n\nHeliotropium indicum Linn. (Boraginaceae) is an annual herbaceous weed commonly known as the Indian heliotrope and is native to India5. Tridax procumbens Linn. (Asteraceae) is an annual herbaceous weed and commonly known as coat buttons; it is native to tropical America6. Cleome rutidosperma DC. (Cleomaceae) is an annual herb commonly known as Fringed Spider Flower; it is native to tropical Africa7. Commelina benghalensis Linn. (Commelinaceae) is an annual herb native to tropical Asia and Africa, commonly known as Bengal dayflower8. Euphorbia hirta Linn. (Euphorbiaceae) is an annual herb commonly known as asthma plant, and is native to India and Central America9.\n\nLeaf, stem, root or sometime the complete plant possess the property to scavenge reactive oxygen species (ROS) or reactive nitrogen species (RNS), the main causative agents of oxidative stress and cellular damage which results in numerous diseases and disorders10,11. ROS or RNS includes a variety of free radicals such as superoxide anion (O2-), hydroxyl radical (.OH), nitric oxide radical (NO.), and peroxyl radicals (ROO.), and non-free radical species like hydrogen peroxide (H2O2), ozone (O3), hypochlorous acid (HOCl), nitrous acid (HNO2)12. The primary site of ROS production in the cell is the mitochondrial respiratory chain. Natural antioxidants protect us from oxidative stress by reducing the free radicals to nontoxic products. However, healthy cells have their own natural ROS or RNS defense mechanisms which actively eliminate free radicals through enzyme-mediated systems like superoxide dismutases, peroxidases, catalases, and glutathione peroxidases10–12.\n\nLife-threatening microbial infections brought on by various pathogens are a significant cause of morbidity and mortality within immune-compromised individuals. Plant-derived natural antioxidants can play a vital role to protect from those microbial infections13–15. Administration of α-amylase inhibitors are necessary to keep diabetic patient glucose levels under control16. Determination of in vitro anti-diabetic and free radical scavenging activities are vital because it prevents both microvascular and macrovascular complications of diabetes. Medicinal plants are the rich source of α-amylase inhibitors and can be utilized for prevention or therapeutic measures17,18.\n\nPlant derived antioxidants have profound activities in inflammatory disorders. Free radicals are essential mediators that provoke or sustain inflammatory or autoimmune diseases like rheumatoid arthritis, and consequently, their neutralization by antioxidants can give relief from inflammation and swelling19–21.\n\nUrolithiasis is a disorder of the urinary tract, which may happen due to the oxidative stress by free radical generations22–24 and hence natural free radical scavengers are the best answer for that disease as well. Although medicinal plants have many bioactivities, sometimes their use should be prohibited due to their toxic effects; therefore, determination of cytotoxic effects of the medicinal plants’ extracts is a pre-requisite for their ethnomedicinal use against various physiological disorders of human beings25.\n\nThe therapeutic potential of the wild plants can be determined by measuring their phytochemical constituents, in vitro medicinal properties and toxicological effect. Therefore, the need arises to estimate and analyze the phytochemical constituents of these five wild medicinal plants leaves which are basically responsible for curing several physiological disorders and complications. The current research investigation aims to quantify the phytochemical constituents, and to evaluate the in vitro antioxidant activities and other important in vitro medicinal properties of the medicinal weeds under study. Next, cytotoxicity of the leaf extracts is to be determined using a cell viability assay and compared. To the best of our knowledge, the comparative evaluations of these medicinal herbs are reported for the first time.\n\n\nMethods\n\nFresh leaves of the five medicinal plants were collected from Salt Lake City, Kolkata, West Bengal and India and authenticated by Botanical Survey of India, Central National Herbarium, Howrah, West Bengal, and India. The fresh leaves of the herbs were washed with clean water and dried at an ambient temperature for 30 days under shade. Dried leaves were made powdered and extracted using double-distilled water or 70% ethanol (1 g of powdered leaf was extracted with 50 ml solvent). The solution of each extract was then clarified using filter paper and stored at 4°C. The extracts were appropriately diluted for during further studies.\n\nA total of four Gram-positive (Bacillus subtilis, Bacillus cereus, Staphylococcus aureus, and Staphylococcus epidermidis) and four Gram-negative (Escherichia coli, Vibrio cholera, Pseudomonas aeruginosa, and Klebsiella pneumonia) bacterial strains were used to measure the antimicrobial activity. The bacterial strains were obtained from the Department of Microbiology, Calcutta University, West Bengal and India.\n\nAll the chemicals and reagents used in the experiments were of analytical (AR) grade and purchased from Institutional enlisted chemical suppliers. For quantitative assays, Systronics 117 model spectrophotometer was used to determine the specific optical density.\n\nEstimation of total polyphenol content. Quantitative estimation of the total phenolic compounds was done in triplicate by using the Folin-Ciocalteu (Merck, India) method of Singleton et al.26 with slight modifications. In brief, 1500 µl of diluted FC reagent was mixed with 300 µL of leaf extract thoroughly. Next, 1200 µl 7.5% Na2CO3 was added to it, vortexed and the total reaction mixture (3 mL) was incubated at dark for 2 h. The absorbance was measured at 765 nm. Gallic acid (SD Fine-Chem, India) was used as standard. The total phenolics contents were expressed in mg gallic acid equivalent/g dry weight.\n\nEstimation of total flavonoid content. Total flavonoids content was quantified in triplicate by the aluminium chloride (Merck, India) colourimetric assay according to standard protocol of Zhishen et al.27 Quercetin (SRL, India) was used as standard. The absorbance was read at 510 nm. The total flavonoids content was expressed in mg quercetin equivalent/g dry weight.\n\nEstimation of total tannin content. The total tannins content was evaluated by using Broadhurst and Jones28 method in triplicate. Tannic acid (SRL-92101, India) was used as a standard reagent. The absorbance was measured at 500 nm. Total tannins content was expressed in mg tannic acid equivalent/g dry weight.\n\nEstimation of total alkaloid content. Total alkaloids content was determined by using the method of Fazel et al.29 in triplicate. The absorbance was measured at 470 nm. Caffeine (SRL, India) was used as standard. Total alkaloid content was expressed in mg caffeine equivalent/g dry weight.\n\nEstimation of total cardiac glycosides content. Total cardiac glycosides content was estimated according to the method of Solich et al.30 in triplicate. Digoxin (SRL, India) was used as standard. Absorbance was read at 495 nm. The total cardiac glycoside was expressed in mg digoxin equivalent/g dry weight.\n\nEstimation of total saponin content. Total saponin content was quantified using a standard method (20% ethanol) in triplicate31,32. The saponin content was calculated by using the following equation:\n\n\n\nWhere WEP = weight of dried end product, WPS = weight of powdered sample (initial).\n\nHigh-performance liquid chromatography (HPLC) profiling of the 70% ethanolic extracts. The HPLC profiling of ten important bioactive phenolic acids, gallic acid (GA), catechin hydrate (CH), chlorogenic acid (CHA), caffeic acid (CA), syringic acid (SYA), p coumaric acid (pCA), sinapic acid (SIA), coumarin (CM), quercetin (QE) and kaempferol (KMP), was carried out using an Agilent Technologies 1260 Infinity liquid chromatography. Standard reagents were purchased from Sigma-Aldrich (USA). The peak area was calculated by Open Lab CDS version 2.0 software. In the experiments the polyphenolic compounds were separated under the following conditions: Phenomenex-C18 (2)-column (250 mm×4.6 mm i.d.; Luna 5-μm particle diameter 100 Å), the diode array detector (DAD) was set at 280 nm; the mobile phase consisted of 3% acetic acid water and acetonitrile (Himedia, India). Prior to use, the solutions were degassed in an ultrasonic bath and filtered through 0.22-μm membranes. The flow rate was 0.9 ml/min in gradient conditions. The injection volume was 20 μl. All the separations are carried out at ambient temperature. The gradient elution of solvent A (water-acetic acid) and solvent B (acetonitrile) had a significant effect on the resolution of compounds according to the procedure: 0 min, 100% (A); 5 min, 95% (A); 17 min, 85% (A); 40 min, 60% (A); 60 min, 50% (A); 65 min, 50% (A); 70 min, 100% (A)33,34.\n\nABTS free radical scavenging capacity assay. ABTS (2, 2-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid, Tokyo Chemical Industry, Japan) radical cation decolourization assays were carried out in triplicate to determine the stable free radical scavenging property of extracts by the help of the standard method of Re et al.35 with slight modifications. In brief, 5 µL of leaf extract was mixed with 2.995 µl of ABTS reagent thoroughly, vortexed and the total reaction mixture (3 mL) was incubated at dark for 30 min. Next, absorbance was read at 734 nm. Ascorbic acid (Merck, India) was used as a standard. ABTS stable free radical scavenging property was expressed in ascorbic acid equivalent as well as the inhibition percentages were determined using the following equation:35–37\n\n\n\nDPPH free radical scavenging capacity assay. The DPPH (1, 1-diphenyl-2-picrylhydrazyl, SRL, India) stable free radical scavenging property of the extracts was carried out by the help of using the standard method of Shen et al. (2010) with slight modifications. In brief, 0.1 mM DPPH solution was made in methanol and 50 µL of leaf extract was mixed with DPPH solution thoroughly, vortexed and the total reaction mixture (3mL) was incubated at dark for 30 min. Next, absorbance was read at 517 nm. Ascorbic acid (Merck, India) was used as a standard. This experiment was performed in triplicate.\n\nDPPH free radical scavenging property, expressed in ascorbic acid equivalents and inhibition percentages, was determined by the following formula:38,39\n\n\n\nH2O2 free radical scavenging capacity assay. Hydrogen peroxide (H2O2, SD Fine-Chem, Mumbai, India) free radical scavenging ability was measured according to the method of Ruch et al. (1989) in triplicate. The absorbance was taken at 230 nm. Gallic acid was used as standard.\n\nH2O2 free radical scavenging capacity was expressed in gallic acid equivalents and inhibition percentages, which were calculated by the formula:40,41\n\n\n\nThe IC50 values of standards and leaves extracts were also calculated for above three free radical scavenging assays.\n\nβ-carotene bleaching test. The β-carotene bleaching test antioxidant activity (lipid peroxidation) of the aqueous and 70% ethanolic extracts was done by the method of Minh et al. with modifications42. In brief, β-carotene (2 mg) (HiMedia, India) was dissolved in 10 ml of chloroform (Merck, India) and then 1 ml of the chloroform solution was added to 20 μl of linoleic acid (HiMedia-GRM10250, India) and 200 mg of Tween-80 (HiMedia-PCT1513, India). The mixture was evaporated at 50°C, and then 50 ml oxygenated water was added and shaken to form an emulsion. A total of 0.012 ml of leaf extracts were mixed with 0.1 ml of the emulsion. The reactions were left at 50°C, and the absorbance was read at 492 nm. All reactions were read at zero time and every 15 min up to 180 min. This experiment was performed in triplicate. Lipid peroxidation inhibition (LPI) values were expressed using the following formula:\n\n\n\nWhere A0 and A00 is the absorbance measured at the beginning of the incubation for sample and control, respectively. At and Att are the absorbance measured after the incubation of 180 min for sample and control, respectively. Higher LPI value indicates the higher antioxidant activity42,43.\n\nPolyphenol oxidase assay. The polyphenol oxidase (PPO) assay was carried out in triplicate by using the standard protocol of Esterbauer et al.44 A total of 500 mg of fresh leaves was crushed in 2 ml extraction medium containing HCl (Merck, India), sorbitol (Himedia-MB066, India) and NaCl. In the test cuvette, 2.5 ml phosphate buffer was mixed with 0.3 ml of catechol (Loba Chemie, UN281, India) solution. The spectrophotometer was set at 495 nm. Next, 0.2 ml of extract was added, and the change in absorbance was recorded every 30 seconds up to 5 min. PPO activity was expressed as a change in absorbance at 495 nm per minute/g fresh weight44,45.\n\nEvaluation of antimicrobial activity. Antimicrobial activity was determined using the standard Kirby-Bauer disc diffusion method. A total of 40 μl of the extracts was placed into the paper disc with sterile double distilled water (70% ethanol used as a negative control). After diffusion of the extract, the plates were incubated at 37°C for 16-18 h. Then the zone of inhibition was measured14,15,46. Four gram-positive and four gram-negative bacteria (namely, Bacillus subtilis, Bacillus cereus, Staphylococcus aureus, Staphylococcus epidermidis and Escherichia coli, Vibrio cholera, Pseudomonas aeruginosa, Klebsiella pneumonia, respectively) were used for assessing antimicrobial activity against the negative control double distilled water and 70% ethanol. In the antimicrobial activity study, concentration (µg) of total bioactive compounds in 40 µl was also calculated from the extractive values of the particular solvents.\n\nEvaluation of in vitro anti-diabetic activity (α-amylase inhibition assay). The α-amylase activity was measured using a colorimetric method with 3, 5-dinitrosalicylic acid (DNS) reagent. α-amylase (HiMedia, India) inhibition activity was measured using a standard protocol16–18. The absorbance was measured at 540 nm. Acarbose (Sigma-Aldrich, USA) was used as a positive control. The experimental measurements were performed in triplicate.\n\nThe percentage of α-amylase inhibition was determined by using the following formula:\n\n\n\nThe concentration of inhibitors required for inhibiting 50% of the enzyme activity under the assay conditions was presented as the IC50 value.\n\nEvaluation of in vitro anti-arthritic activity (inhibition of protein denaturation). In vitro anti-arthritic activity of extracts was done using a standard protocol, assessing the percentage inhibition of protein denaturation. The experiment was carried out by taking sodium diclofenac (Tokyo Chemical Industry, Japan) as the standard. Absorbance was measured at 416 nm. The experimental measurement was performed in triplicate.\n\nProtein denaturation inhibition percentages were determined by applying the following formula:\n\n\n\nThe IC50 values of standard and extracts were also calculated19–21.\n\nEvaluation of in vitro anti-lithiatic activity (percentage dissolution of calcium oxalates). Evaluation of in vitro anti-lithiatic activity by percentage dissolution of calcium oxalates was done using the standard method22–24 in triplicate with slight modifications. The complete procedure is comprises three different steps, namely (i) Experimental kidney stone (calcium oxalate stones) preparation, (ii) Preparation of semi-permeable membrane from chicken eggs, and (iii) Estimation of calcium oxalate by titration method. All the experimental protocols are given below.\n\nExperimental kidney stone (calcium oxalate stones) preparation\n\nAn equimolar (100 mM) solution of calcium chloride dehydrate (SRL-7065, India), in double-distilled water, and disodium oxalate (Merck, India) in 10 ml of 2 N H2SO4 (Merck, India) was kept to react with distilled water in a beaker. The resulting precipitate was calcium oxalate. The precipitate was freed from traces of H2SO4 using 10% ammonia solution and it was washed with double distilled water and dried at 70°C for 5 h.\n\nPreparation of semi-permeable membrane from chicken eggs\n\nShell was removed chemically by placing the chicken eggs, obtained from a local market, in 10% Glacial acetic acid (Merck, India) for 48 h, which caused complete decalcification. The egg membrane washed thoroughly with distilled water, and put into a 10% ammonia solution and stored in a refrigerator at a pH of 7.4.\n\nEstimation of calcium oxalate by titration method\n\nThe dissolution percentage of calcium oxalate was evaluated by taking precisely 10 mg of calcium oxalate and 10 mg of the lyophilized extracts/standard (1:1 ratio), packed together in the semi-permeable membrane of the egg. This was allowed to suspend in a conical flask containing 100 ml of 0.1 M Tris buffer (SRL-2049170, India). The 1st group was set as blank containing only 10 mg of calcium oxalate. The 2nd group was established as a positive control containing 10 mg of calcium oxalate along with 10 mg of the standard drug cystone (The Himalaya Drugs Company, Bangalore, India). The 3rd and 4th groups, along with 10 mg of calcium oxalate, contained aqueous and 70% ethanol extracts, respectively. The conical flasks of all the groups were placed in an incubator and preheated to 37°C for 3 h. The contents of semi-permeable membranes were removed from each group into separate test tubes, and 2 ml of 1 N H2SO4 was added to each test tube and titrated with 0.9494 N KMnO4 (Avantor, Maharashtra, India), till a light pink colour endpoint was obtained. In the calculation 1 ml of 0.9494 N KMnO4 is counted as equivalent to 0.1898 mg of 4 Calcium. The amount of remaining undissolved calcium oxalate is subtracted from the total quantity used in the experiment, in the beginning, to know the total amount of calcium oxalate dissolved by various solvent extracts22–24.\n\nEvaluation of in vitro cytotoxicity. A healthy male Wistar rat was purchased from Chittaranjan National Cancer Research Institute, Kolkata, West Bengal and maintained subsequently for the experiments were fed with hind liver pellet or equivalent and water ad libitum in 12 h light and dark cycle and at 30°C throughout the experimental period. The experiments were performed under the institutional ethical permission (IAEC/CU/BIOCHEM/SM (1) dated 14.12.2012) and followed the ‘Principles of Laboratory Animal Care’ (NIH publication no. 85-23, revised in 1985). The experiment was performed in triplicate. To conduct the study, a standard rat PBMC isolation technique47 and Cell Viability Assay48 was used. PBMC isolation techniques and Cell Viability Assay are given below in details.\n\nLeaf extract preparation\n\nA total of 100 mg dry weights of each plant leaves were mixed, vortexed in 1 ml of double distilled water and 70% ethanol separately, left overnight, centrifuged and supernatant taken.\n\nRat PBMC isolation\n\nA healthy male Wister rat (160 g) was deeply anaesthetized with sodium pentobarbital (50 mg/kg body weight) followed by thoracotomy. Terminal cardiac puncture was made in ventricle using an appropriate sterile needle and blood drawn slowly and collected in PBS-EDTA solution, diluted by 1X PBS in 1:1 ratio and laid on Histopaque-1077 (HiMedia, India), a density gradient cell separation medium of ficoll and sodium diatrizoate was fixed and adjusted with a density of 1.077 g/ml, an equal volume of media and diluted blood. This Histopaque overlaid with diluted blood was centrifuged at 300g at 25°C for 30 mins. The RBCs were a thick red precipitate at the bottom of centrifuge tube, whereas the blood PBMC were found at the interface of Histopaque and the top layer of serum. PBMCs from the interphase were collected, washed twice with PBS, resuspended in 1 ml of DMEM media and this placed in Neubauer Improved Chamber (Marienfeld, Germany) and observed under Microscope Nikon Eclipse TS 100 (Nikon Corporation, Japan) to measure cell concentration.\n\nCell viability assay\n\nA cell viability assay has been done with Trypan Blue. Cells were then laid in 12 well plates at a concentration of 3.6x107 cells/ml with a volume of 1 ml at each well except media (DMEM) control containing 1% penicillin-streptomycin (P/S, Antibiotic Antimycotic Solution 100X, Himedia, India) along with 10% FBS and incubated in the CO2 incubator for 24 h in 5% CO2 humified environment at 37ºC (CO2 Incubator Galaxy 48S, New Brunswick, Germany). Next, 10, 5 and 2.5 µl of each of the extracts (different dose of aqueous and ethanolic extracts) were added to the wells except controls and continued in culture for another 48 hrs. Culture plates were then taken out and treated with 10 μl of 0.4% freshly prepared trypan blue solution for 5 mins and readily observed under Nikon Eclipse TS 100 Inverted Phase-contrast Microscope and documented by CCD Camera (DS-Fi2-U3) and NIS-BR Software (Nikon Corporation, Japan).\n\nAll the experimental measurements were performed in triplicate (except HPLC-DAD and antimicrobial activity study) and expressed as the average of the three analyses ±standard deviations (SD). The correlation coefficient between variables, means, standard deviations, standard errors, standard curve, IC50 values and one-way ANOVA followed by Bonferroni’s post hoc test was calculated by using MS Excel 2007 Software (Microsoft Corporation, Redmond, WA, USA). Figures are prepared in Origin Pro 8 Software (Northampton, MA, USA). A P-value <0.05 was considered as statistically significant.\n\n\nResults\n\nThe highest concentration of polyphenolics was obtained from EH aqueous and 70% ethanolic extracts (180.59±2.99 and 166.47±3.03 mg GAE/g dry weight, respectively). The lowest concentration was observed in TP aqueous and 70% ethanolic extracts (127.52±1.26 and 80.51±1.21 mg GAE/g dry weight, respectively) (Figure 1). The highest concentration of flavonoids was obtained from EH aqueous and 70% ethanolic extracts (98.05±1.30 and 77.39±1.79 mg QE/g dry weight, respectively). The lowest concentration was observed in TP aqueous and 70% ethanolic extracts (46.78±1.34 and 34.47±0.81 mg QE/g dry weight, respectively) (Figure 2). The highest tannin concentration was obtained from EH aqueous and 70% ethanolic extracts (59.21±0.15 and 42.50±0.37 mg TAE/g dry weight, respectively). The lowest concentration was observed in TP aqueous and 70% ethanolic extracts (14.83±0.37 and 11.61±0.32 mg TAE/g dry weight, respectively) (Figure 3).\n\nIn the figure different lower case letters (a, b, c, d, e, x, y, z, w, v) in the bars indicates significant differences among means (P<0.05). The blue and red colour bars indicate the aqueous and 70% ethanolic extracts, respectively. X-axis denotes the plant name and Y-axis denotes the concentrations of total polyphenols contents (mg Gallic Acid Equivalent/g Dry Weight).\n\nIn the figure different lower case letters (a, b, c, d, e, x, y, z, w, v) in the bars indicates significant differences among means (P<0.05). The blue and red colour bars indicate the aqueous and 70% ethanolic extracts, respectively. X-axis denotes the plant name and Y-axis denotes the concentrations of total flavonoids contents (mg Quercetin Equivalent/g Dry Weight).\n\nIn the figure different lower case letters (a, b, c, d, e, x, y, z, w, v) in the bars indicates significant differences among means (P<0.05). The blue and red colour bars indicate the aqueous and 70% ethanolic extracts, respectively. X-axis denotes the plant name and Y-axis denotes the concentrations of total tannins contents (mg Tannic Acid Equivalent/g Dry Weight).\n\nThe highest concentration of alkaloid content was observed in HI aqueous and 70% ethanolic extracts (0.148±0.006 and 0.171±0.004 mg CE/g dry weight, respectively). The lowest concentration was observed in CR aqueous and 70% ethanolic extracts (0.017±0.006 and 0.024±0.003 mg CE/g dry weight, respectively) (Figure 4). The highest concentration of cardiac glycosides content was observed in EH aqueous and HI 70% ethanolic extracts (2.20±0.20 and 3.86±0.27 mg DE/g dry weight, respectively). The lowest concentration was obtained from TP aqueous and CB 70% ethanolic extracts (0.23±0.04 and 1.70±0.09 mg DE/g dry weight, respectively) (Figure 5). The total saponin content for these medicinal herbs dried powdered leaves 20% ethanolic extracts was estimated. The highest concentration was observed in EH (6.93±0.42%). The lowest concentration was observed in TP (3.50±0.36%) (Figure 6).\n\nIn the figure different lower case letters (a, b, c, d, e, x, y, z, w, v) in the bars indicates significant differences among means (P˂0.05). The blue and red colour bars indicate the aqueous and 70% ethanolic extracts, respectively. X-axis denotes the plant name and Y-axis denotes the concentrations of total alkaloids contents (mg Caffeine Equivalent/g Dry Weight).\n\nIn the figure different lower case letters (a, b, c, d, e, x, y, z, w, v) in the bars indicates significant differences among means (P˂0.05). The blue and red colour bars indicate the aqueous and 70% ethanolic extracts, respectively. X-axis denotes the plant name and Y-axis denotes the concentrations of total cardiac glycosides contents (mg Digoxin Equivalent/g Dry Weight).\n\nIn the figure different lower case letters (a, b, c, d, e, x, y, z, w, v) in the bars indicates significant differences among means (P˂0.05). The gray colour bars indicate the 20% ethanolic extracts. X-axis denotes the plant name and Y-axis denotes the concentrations of total saponin contents (%).\n\nThe analysis of HPLC-DAD profile obtained from the plants leaves 70% ethanolic extracts identified ten bioactive phenolic compounds GA, CH, CHA, CA, SYA, pCA, SIA, CM, QE and KMP (Figure 7 and Extended data, Figures S1, S2, S3, S4)49. Among these three compounds were present in all the extracts (chlorogenic acid, coumaric acid and coumarin). In EH and TP highest ten compounds were identified. In HI lowest six compounds were identified. In EH six compounds were found in the highest amount, and these are GA (83.258 ng/µl), CH (122.149 ng/µl), SYA (9.487 ng/µl, pCA (10.017 ng/µl, SIA (297.315 ng/µl, CM (3.719 ng/µl). In each of TP and CR two phenolic acids were found in the highest amount and these are CHA (6.405 ng/µl), KMP (5.241 ng/µl) and CA (23.006 ng/µl), QE (15.099 ng/µl), respectively. Sinapic acid was the most abundant phenolic acid present in all the leaves extracts as well as it is found in the highest amount in EH extract. Availability of coumarin among these standard phenolics is found minimum in all the extracts. The HPLC-DAD profile showed in chromatogram representation and in Table 1.\n\nPolyphenolic compounds were separated by Phenomenex-C18 (2)-column, the Diode Array Detector was set at 280 nm; the mobile phase consisted of 3% acetic acid water and acetonitrile. The flow rate was 0.9 ml/min in gradient conditions. In the figure X-axis denotes the units of time (minutes) i.e. retention time (tR) and Y axis denotes the intensity of absorbance (milli-Absorbance units) or concentration. Ten compounds were detected in chromatogram.\n\nIn the present study, the maximum inhibition percentage of ABTS free radical scavenging assay was found to be 86.10±0.22% and 79.19±0.36% for EH aqueous and 70% ethanolic extract, respectively. The minimum is 55.33±0.64% and 37.57±0.80% for CR and TP in aqueous and 70% ethanolic extract (Figure 8 and Extended data, Figure S5)49, respectively; the inhibition percentage for standard ascorbic acid was found to be 98.29% at 0.5 mg/ml of concentration.\n\nIn the figure different lower case letters (a, b, c, d, e, x, y, z, w, v) in the bars indicates significant differences among means (P˂0.05). The blue and red colour bars indicate the aqueous and 70% ethanolic extracts, respectively. X-axis denotes the plant name and Y-axis denotes the inhibition percentages by the extracts (%).\n\nThe study reveals that the maximum inhibition percentage of DPPH free radical scavenging assay was 86.75±0.56% and 83.07±0.25% for the EH aqueous and 70% ethanolic extract, respectively. The minimum was 71.02±0.07% and 68.34±0.41% for CB and HI in aqueous and 70% ethanolic extract (Figure 9 and Extended data, Figure S6)49, respectively; this compared with the percentage inhibition of ascorbic acid which was 92.77% at 0.5 mg/ml of concentration in the present study.\n\nIn the figure different lower case letters (a, b, c, d, e, x, y, z, w, v) in the bars indicates significant differences among means (P˂0.05). The blue and red colour bars indicate the aqueous and 70% ethanolic extracts, respectively. X-axis denotes the plant name and Y-axis denotes the inhibition percentages by the extracts (%).\n\nThe maximum inhibition percentage of H2O2 free radical scavenging assay was found to be 85.92±0.31% and 84.83±0.46% for CB aqueous and EH 70% ethanolic extract, respectively. The minimum is 56.05±0.48% and 56.57±0.36% for TP and HI in aqueous and 70% ethanolic extract (Figure 10 and Extended data, Figure S7)49, respectively; the inhibition percentage for standard Gallic acid was 95.10% at 0.5 mg/ml of concentration.\n\nIn the figure different lower case letters (a, b, c, d, e, x, y, z, w, v) in the bars indicates significant differences among means (P˂0.05). The blue and red colour bars indicate the aqueous and 70% ethanolic extracts, respectively. X-axis denotes the plant name and Y-axis denotes the inhibition percentages by the extracts (%).\n\nIn this investigation, the antioxidant activity of lipid peroxidation (LPI) by ß-carotene bleaching test concluded that the highest activity was observed in case of EH aqueous and TP 70% ethanolic extracts, and it is 62.58±6.00% and 63.89±5.87%, respectively. The lowest activity was observed in the case of TP aqueous and HI 70% ethanolic extracts, and it is 25.85±7.51% and 20.44±1.24%, respectively (Figure 11).\n\nIn the figure different lower case letters (a, b, c, d, e, x, y, z, w, v) in the bars indicates significant differences among means (P˂0.05). The red and pink colour bars indicate the aqueous and 70% ethanolic extracts, respectively. X-axis denotes the plant name and Y-axis denotes the inhibition percentages by the extracts (%).\n\nIn the study, the enzyme PPO activity assay concluded that the highest and lowest activity was observed in CB extracts (9.67±6.39) and CR extracts (4.77±0.32), respectively, in terms of change in absorbance at 495 nm per minute/g of fresh weight, which is greater than the control used in the PPO activity assay, i.e. 3.83±3.35 (Figure 12).\n\nIn the figure the bars indicates no significant differences among means (P˃0.05). X-axis denotes the plant name and Y-axis denotes the change in absorbance at 495 nm per minute/g of fresh weight by the extracts (%).\n\nZone of inhibition shown by aqueous and 70% ethanolic leaves extract indicated the presence of high antimicrobial activity. EH aqueous extracts showed the highest zone of inhibition in comparing with all the extracts. TP and CR 70% ethanolic extracts had given better antimicrobial properties than other leaves 70% ethanolic extracts. SE strains have not given any zone of inhibition in case of all the extracts (Table 2 and Extended data, Figures S8, S9, S10, S11)49. The concentration (µg) of bioactive compounds in 40 µl which was the given dose for the assay in antimicrobial activity study was determined from the extractive values of the aqueous and 70% ethanolic leaf extracts (Extended data, Table S1)49.\n\nIn the current study, different concentrations of extract were selected for the in vitro assay between 10 mg/ml and 0.0195 mg/ml. Based on the results, the maximum α-amylase inhibition percentage was found to be 72.19±1.26% and 68.14±0.71% for EH aqueous and 70% ethanolic extract, respectively. The minimum inhibition activity is 52.54±1.94% and 50.48±0.82% for TP aqueous and 70% ethanolic extract (Figure 13), respectively; this compared to an inhibition percentage for standard acarbose of 99.00% at 10 mg/ml of concentration.\n\nIn the figure different lower case letters (a, b, c, d, e, x, y, z, w, v) in the bars indicates significant differences among means (P˂0.05). The blue and red colour bars indicate the aqueous and 70% ethanolic extracts, respectively. X-axis denotes the plant name and Y-axis denotes the α-amylase inhibition percentages by the extracts (%).\n\nThe anti-arthritic activity of aqueous and 70% ethanolic extracts was determined by the in vitro models, i.e. inhibition of protein denaturation. The various concentrations of extracts were selected for the assay between 1 mg/ml and 0.00196 mg/ml. The study observed that the maximum inhibition percentage was 70.36±0.69% and 74.54±0.75% for CR aqueous and 70% ethanolic extracts, respectively. The minimum inhibition activity is 52.28±0.91% and 53.48±0.89% for TP aqueous and 70% ethanol extract (Figure 14), respectively, as compared to inhibition percentage for standard diclofenac sodium was 97.25% at 1 mg/ml of concentration.\n\nIn the figure different lower case letters (a, b, c, d, e, x, y, z, w, v) in the bars indicates significant differences among means (P˂0.05). The blue and red colour bars indicate the aqueous and 70% ethanolic extracts, respectively. X-axis denotes the plant name and Y-axis denotes the inhibition percentages of protein denaturation by the extracts (%).\n\nIn the research investigation, titration was used to assess the in vitro anti-lithiatic property of aqueous and 70% ethanolic extracts using a calcium oxalate dissolution assay. Based on the results obtained in the study, the highest dissolution percentage of calcium oxalate was 79.98±0.95% and 74.41±4.43% for EH aqueous and CB 70% ethanolic extract, respectively. The lowest is 46.37±3.22% and 10.45±1.78% for TP aqueous and 70% ethanolic extract (Figure 15), respectively. The dissolution percentage of calcium oxalate for the standard drug cystone was 86.42±3.57% at 1 mg/ml of concentration.\n\nIn the figure different lower case letters (a, b, c, d, e, x, y, z, w, v) in the bars indicates significant differences among means (P˂0.05). The blue and red colour bars indicate the aqueous and 70% ethanolic extracts, respectively. X-axis denotes the sample name and Y-axis denotes the percentage (%) dissolution of calcium oxalate by extracts.\n\na. Cell control after 48 hrs of seeding; b. Cells after seeding with drugs; c. EH aqueous drug effect after 48 hrs; d. EH 70% ethanolic drug effect after 48 hrs; e. CR aqueous drug effect after 48 hrs; f. CR 70% ethanolic drug effect after 48 hrs; The given dose of extracts is 10 μl.\n\nIn the trypan blue exclusion assay, the absolute count of total cells in each field vs cells with characteristic trypan blue stains were counted for each plant extract-treated group. In our study, 2.5 µl and 5 µl dose of crude drugs did not show any cytotoxic effects towards PBMC. However, 10 µl of the dose showed the effects (Figure 16a–f).\n\nThe dose-dependent (10 µl of crude plant extract) cell viability assay elicited a 10-12% mortality rate of PBMC in 48 hrs of culture in the control; only the aqueous extracts of CR showed higher mortality than control, with other aqueous extracts eliciting lower mortality rates, showing some nontoxic or cytoprotective properties for PBMC or immune cells (Figure 17). However, the EH aqueous extract showed maximum cytoprotectivity showing a mortality of ~2.5%. EH 70% ethanolic extracts showed cytoprotectivity (~4%) nearly comparable to its aqueous soluble counterpart. The 70% ethanolic extracts showed higher mortality in PBMC. Particularly, CR and TP 70% ethanolic extracts are much cytotoxic, where TP found over two-fold cytotoxicity than control. Both extracts of CB showed cytoprotectivity compared to the control.\n\nIn the figure different lower case letters (a, b, c, d, e, x, y, z, w, v) in the bars indicates significant differences among means (P˂0.05). The blue and red colour bars indicate the aqueous and 70% ethanolic extracts, respectively. X-axis denotes the sample name and Y-axis denotes percentage (%) of toxicity of the plants leaves extracts towards PBMC at the dose of 10μl.\n\nFrom the above results, it is observed that except CR aqueous extracts the aqueous medium is overall cytoprotective. Most of the drugs in this form are less cytotoxic than control; hence it is much preferred as a medium in pharmaceutical application purposes. EH aqueous and 70% ethanolic extracts has been found much more cytoprotective to PBMCs. Hence it can be used to treat against immune disorders after necessary processing.\n\nRaw data of each experiment performed are available as Underlying data50.\n\n\nDiscussion\n\nThe oxidation reactions and free radical production lead to some oxidative stress-related disorders such as diabetes, arthritis, urolithiasis and cancer. The activity of phenolic compounds against oxidative stress-related mechanism can have substantial therapeutic applications in the pharmaceutical and nutraceutical industry12,34,51–54.\n\nFlavonoids are the category of plant secondary metabolites with high antioxidant, antimicrobial and chelating activities. Antioxidant activities of flavonoids depend on the structure and substitution pathways of hydroxyl groups. The antioxidative properties of flavonoids classes are due to various reaction mechanisms, such as scavenging of free radicals, chelation of metal ions (e.g. iron and copper), and inhibition of enzymes which are responsible for free radical production. Depending on their specific structure, it can inhibit or regulate all the possible ROS, which can cause cellular damages. Previous studies have concluded the protective ability of flavonoids against various diseases53,55,56.\n\nTannins are found in the stem and barks of various plants rather than in the leaves. The tannins showed excellent antioxidant, antimicrobial and anti-cancer activities. These are astringent, bitter plant polymeric compound which can be capable of tanning leathers, and can sediment proteins, amino acids, alkaloids and nitrogenous substances57. The tannin-protein complex can provide persistent antioxidant and antimicrobial properties53,56,57.\n\nAlkaloids are essential secondary plant metabolites, and have extensive applications in present-day herbal formulations as these compounds have huge potentiality as an antimicrobial agent29,56.\n\nThe amount of cardiac glycoside found in extracts is minor compared to larger quantities that have been reported in various plant leaves. This signifies that the leaves are not toxic as glycosides act as sodium-potassium ATPase inhibitor leading to cell death. Cardiac glycosides are used for the treatment of different oxidative stress-related cardiac diseases such as congestive heart failure and cardiac arrhythmia11,30.\n\nSaponin is an amphipathic (hydrophilic) glycoside moiety with lipophilic triterpene or steroid derivatives58. This is an important bioactive compound which plays a vital role to prevent antimicrobial activity and other biological disorders31,32. In the study, polyphenolic compounds, flavoinoids, tannins, alkaloids and cardiac glycosides contents showed very significant (p-value <0.01) presence in both the extracts as well as they are positively and variably correlated (Tables S2 and S3) with each other and antioxidant properties, too.\n\nIn the current investigation, a simple, accurate, and reproducible online HPLC-DAD method has been used and validated for the identification and quantification of phenolics. Phenolics, especially phenolic acids and flavonoids, are conventional natural products found in plants that have significant antioxidant and anti-inflammatory activity. ROS are produced by various metabolic activities and is a significant contributor to many chronic and oxidative stress-related diseases. Phenolic acids have been demonstrated as potent scavengers of ROS. These are sub-classified as benzoic acid and cinnamic acid backbone structure containing seven (C6-C1) and nine (C6-C3) carbon atoms, respectively. Hydroxybenzoic acid derivatives such as gallic acid, syringic acid and cinnamic acid derivatives like caffeic acid, chlorogenic acid, sinapic acid, p-coumaric acid are widely available in plants33,34,59,60. Masek et al., concluded that caffeic acid is the predominantly and widely available phenolic acid, and it exhibits antioxidant and iron-chelating abilities which are better than p coumaric acid61. Cinnamic acid derivative sinapic acid exhibits potent antioxidant, antimicrobial and anti-arthritic activities62. Katada et al. showed that hydroxycinnamic acid derivative chlorogenic acid act as a dietary supplement which is highly effective in oxidative stress-related diseases such as arthritis and high blood pressure63.\n\nABTS free radical scavenging assay serve as assessing the antioxidant property. The IC50 analysis is used for estimating the activity of an inhibitor. IC50 analysis showed that the standard ascorbic acid is required 0.237 mg/ml, whereas leaf extracts required a higher amount to reach at IC50 (Extended data, Table S4)49. ABTS assay is a free radical cation decolourization assay. In the study, potassium persulfate was used to give a stable form of ABTS radical cation35–37.\n\nIn the DPPH assay, IC50 analysis showed that the standard ascorbic acid is required 0.257 mg/ml, whereas leaves extracts required a higher amount to reach at IC50 (Extended data, Table S5)49. DPPH is a highly used free radical to determine the free radical scavenging activity of natural antioxidants. This free radical scavenging assay is based on the reduction of DPPH radicals in methanol in the presence of hydrogen-donating antioxidant due to the formation of the non-radical shape of DPPH-H. DPPH is unaffected by side reactions, like metal ion chelating and enzyme inhibitions and it is counted as one of the benefits of it. In this method, a change in colour from purple to yellow happens, and this is proportional to the concentration and radical scavenging capacity of the extracts in the form of hydrogen donating capability4,39,46,53,54.\n\nIn the H2O2 radical scavenging assay, IC50 analysis showed that the standard gallic acid is required 0.247 mg/ml, whereas extracts required a higher amount for IC50 (Extended data, Table S6)49. H2O2 becomes toxic to the cell because it is rapidly decomposed into oxygen and water and gives rise to the hydroxyl radical, which can initiate lipid peroxidation mechanisms by obtaining hydrogen atoms from unsaturated fatty acids and cause DNA strands damages. The hydroxyl radical is short-lived severely reactive free radicals, formed in the biological systems, capable of damaging a wide range of biomolecules located in the living cells through diffusion-limited reaction. Its removal is necessary for the protection of food, too4,11,12,40,41.\n\nIt has been well known that carotenoids class of compounds undergo bleaching, i.e. lose their colour, when exposed to free radicals or to oxidative species. This entire mechanism occurs by the addition of the conjugated double bond which is present in the available reaction system either by the help of cleavage or by adding one of the double bonds of molecules. This cleavage occurrence can be identified by analyzing the products that are created, which are primarily carbonyls or epoxides43,44. Membrane lipids are rich in unsaturated fatty acids which are most susceptible to oxidative mechanisms. The free radical chain reaction is widely accepted as a common process of lipid peroxidation, and it is thought that the inhibition of lipid peroxidation is caused by antioxidants due to their free radical scavenging properties13,55. Flavonoids, aglycones and glycosides showed good lipid peroxidation activities by the ß-carotene bleaching test55. Phytomolecules such as saponin, flavonoids and tannins found to protect against lipid peroxidation64.\n\nThe study showed the presence of significant level of antioxidants in the plant extracts (aqueous and 70% ethanol). The study also showed the variable degrees of correlation (Extended data, Table S3)49 between the phytochemicals content and antioxidant activity. The results of the different radical scavenging assays are found to be positively correlated (Extended data, Table S7)49 with each other, signifying the interplay between different antioxidants and their mode of action in combating cellular stress.\n\nFrom previous studies and reports, it was observed that there is a positive correlation exists between antioxidant property and the bioactive compounds. It can be concluded that the bioactive compounds appear to be responsible for the significant (p-value <0.01) antioxidant property of both the solvents extracts12,13. The observation of the investigations agrees with the point that the ethnomedicinal plants are the principal source of therapeutically and nutritionally used natural antioxidants52. The study findings conclude that the aqueous extracts have maximum antioxidant activity in comparison with the 70% ethanolic extracts. EH has huge and intense potentiality towards pharmaceutical and nutraceutical importance due to the presence of high polyphenolic compounds which is comparable with the green and black teas65. Using the above in vitro test, the results suggested that EH exhibited potent antioxidant activity, and it can serve as a new source of the natural antioxidant agent12,66.\n\nPPO is a widely distributed copper-containing enzyme in plants. It is known to be responsible for the enzymatic browning reaction occurring during the handling, storage and processing of the fruits and vegetables. The two molecular oxygen occurs in the two separate reactions when PPO shows the activity. The PPO enzyme catalyzes the oxidation of phenolic compounds into highly reactive quinones. Polymerization of PPO-derived quinones causes the postharvest browning of cut or bruised fruit, but the native physiological functions of PPOs in undamaged, intact plant cells are not well understood44,45,67,68. The p-value <0.05, which showed no significant level of enzyme PPO activity in the experimental extracts. In the present study, variable degrees of correlation (Extended data, Table S8, S9 and S10)49 are found between phytochemicals and free radical scavenging assay with the PPO activity.\n\nThe complete results obtained from the antimicrobial activity study indicated that aqueous extracts had high antimicrobial property than the 70% ethanolic extracts, which supports the previous study of Abubakar, 200969. The presence of bioactive metabolites in the extracts such as flavonoids, tannins, saponins, glycosides, alkaloids, phenolics, steroids and anthraquinone as reported earlier is likely to be responsible for the good antimicrobial property. Polyphenols and other bioactive molecules regulate the synthesis of nucleic acids of both Gram-negative and Gram-positive bacteria; that is why the proper zone of inhibition has been observed in the assay13,56,70. Perumal showed that plants with high caffeic acid levels had shown significant antimicrobial activity. In the study, 70% ethanolic extracts of CR and TP contains the highest amount of caffeic acid, and these two leaves extracts showed better antimicrobial activities which support the previous study as well71. The antimicrobial activity also depends on the ROS formation. Balanced state of the body interrupted by pathological situations due to overproduction of ROS and comparatively low amount of endogenous antioxidants in the body. It results in the reaction between ROS and intra and extracellular species leading to the emergence of oxidative stress-related disorders and pathogenesis. To control oxidative stresses, it is necessary to maintain the balance between ROS and antioxidants by administering exogenous natural antioxidants such as hydroxycinnamic acids46,62.\n\nThe in vitro anti-diabetic property evaluation of the plants leaves extracts was assessed by inhibition of polysaccharides degrading enzyme α-amylase inhibitory method. A large variety of α-amylase inhibitors have been reported from various plants, which protect against oxidative damage resulted in hyperglycemia. Inhibition of enzyme activity may be due to the presence of potentially bioactive compounds like polyphenols, alkaloids, flavonoids, tannins and glycosides. The DNS reagent assay is a widely used procedure to quantify the amount of reducing sugars produced after-treatment of the solution by α-amylase with different types of samples16–18. IC50 analysis showed that the standard acarbose is required 3.55 mg/ml, whereas leaves extracts required a higher amount to reach at IC50 (Extended data, Table S11)49. Bioactive constituents are the natural source of antioxidants and responsible for preventing the destruction of β-cells and diabetes-induced ROS formation. The anti-diabetic and antioxidant properties of the EH leaves might be due to the synergistic effect of biologically active phytochemicals present in the extracts64.\n\nThe in vitro anti-arthritic activity investigation by protein denaturation method indicated that all the 70% ethanolic extract had showed maximum inhibition when compared to the aqueous extracts and IC50 analysis showed that standard sodium diclofenac is required at 0.430 mg/ml, whereas leaf extracts had higher IC50 values (Extended data, Table S12)49. The significant in vitro anti-arthritic activity exhibited by the extracts is caused may be due to the presence of flavonoids, polyphenols and tannins. Denaturation of the protein involves the breakdown of chemical structures of the molecules and finally leads to cell death; it happens due to various stresses like a high level of salt, temperature and acidity. The processes of protein denaturation may be related with the alteration in electrostatic, hydrogen, hydrophobic and disulphide bonding. Denaturation of proteins is leading to oxidative stress-related diseases such as inflammatory situations, rheumatoid arthritis, high blood pressure, diabetes and cancer. The results concluded that leaves extracts of CR have the highest ability to control the oxidative stress-related generation of auto-antigen, which relates to the inhibition of the denaturation of proteins19–21.\n\nThe dissolution of calcium oxalate and the number of calcium ions in the solutions are inversely related. The current in vitro anti-lithiatic investigation concluded that the aqueous extracts showed higher dissolution percentage of calcium oxalate crystals (except CB) than 70% ethanolic extracts. The result of CB aqueous extract contradicts with the previous reports of Celestian et al., who showed that CB aqueous extracts had higher dissolution percentages of calcium oxalates23. According to our knowledge, the present work highlighted the in vitro anti-urolithiasis activity of HI, and CR leaves extracts for the first time. Lithiasis and its co-morbidities proceed to the development of oxidative stresses which is known to be a primary reason for inflammation. Previous research suggests that oxidative stresses and inflammation generated by one disorder, and it may happen in a particular situation which produces the development of the co-morbidities. As observed by the researchers that mildly high calcium or phosphate can promote the deposition of calcium phosphate crystals in the renal interstitium with localized swellings and deposition of collagen resulting to the production of Randall’s plaque or it may be possible that mildly high calcium and phosphate or oxalate and low citrate or magnesium levels in the urine can cause to crystallization in the collecting ducts of kidney that is oxidatively stressed and injured72. The present study reveals that the aqueous and 70% ethanolic extracts of EH can be able to dissolve the calcium oxalate stones which are higher comparing with the other leaf extracts. The result of the study concluded the necessity of EH aqueous extracts for the treatment of renal stones which agrees with the previous investigation as well73, whereas the TP extracts contradict with the previous observations74. The p-value˂0.01, which showed a very significant in vitro bioactivity in case of extracts using each solvent. In this study, the phytochemicals and antioxidant properties are variably and remarkably correlated with oxidative stress-related medicinal properties (Extended data, Tables S13 and S14)49.\n\n\nConclusions\n\nIt is concluded that the medicinal plants are best sources of phytochemicals and remedial agents for several disorders. In the present investigation, the extracts of HI, TP, CR, CB and EH were found to be rich in secondary plant metabolites, which showed significant in vitro antioxidant and medicinal properties as well as minimal level of cytotoxicity75. Though, for all the in vitro experiments, further studies are recommended to know and evaluate the pharmacological efficacy, potentiality and mode of action. The reasonable difference between the presence of phytochemical contents are may be due to leaves maturity, fertility, pest exposure, moisture, relative water content, pH, solubility, solvents polarity, environmental factors like pollution, solar reflectance, rainfalls, precipitation, location and temperature4,37.\n\nIn conclusions, these results suggested that among these five medicinal weeds, EH has showed the highest bioactive component presence as well as the antioxidant properties which agree with the previous studies, too12,52. EH showed better in vitro medicinal properties and prominent cytoprotectivity on dose dependant study in comparison with the other four weeds due to the presence of high amount of vital phytomolecules such as sinapic acid, gallic acid, caffeic acid or chlorogenic acid61–63. In future, Euphorbia hirta Linn. can be used as an important medicinal plant to isolate and identify the active phytocompounds for the therapeutic and natural antioxidant preparation purposes cost effectively.\n\nThus, the study has shown the path that traditionally used easily available weeds can be a low-cost source of important bioactive molecules with potential for herbal drug development.\n\n\nData availability\n\nFigshare: Phytochemical Composition Analysis and Evaluation of In Vitro Medicinal Properties and Cytotoxicity of Five Wild Weeds. https://doi.org/10.6084/m9.figshare.12115827.v250.\n\nFile ‘F1000 Raw Data Final’ contains the raw output data for each repeat of each experiment.\n\nFigshare: f1000 Extended data (Phytochemistry and Phytopharmacology of 5 Medicinal Weeds). https://doi.org/10.6084/m9.figshare.1220563149.\n\nFile ‘F1000 Extended data’ contains the following extended data:\n\nTable S1. Concentration (µg) of bioactive compounds in 40µl in antimicrobial activity study.\n\nTable S2. Correlation between bioactive components.\n\nTable S3. Correlation between antioxidant assays and bioactive components.\n\nTable S4. IC50 of standard reagent and leaves extracts in ABTS free radical scavenging assay.\n\nTable S5. IC50 of standard reagent and leaves extracts in DPPH free radical scavenging assay.\n\nTable S6. IC50 of standard reagent and leaves extracts in H2O2 free radical scavenging assay.\n\nTable S7. Correlation between antioxidant assays.\n\nTable S8. Correlation between PPO and bioactive compounds.\n\nTable S9. Correlation between PPO and Saponin.\n\nTable S10. Correlation between antioxidant assays and PPO.\n\nTable S11. IC50 of standard drug and extracts in α-amylase inhibitory activity.\n\nTable S12. IC50 of standard drug and leaves extracts in inhibition of protein denaturation.\n\nTable S13. Correlation between bioactive compounds and in vitro medicinal properties.\n\nTable S14. Correlation between antioxidant assays and in vitro medicinal properties.\n\nFigure S1. HI HPLC-DAD chromatogram of the phenolic compounds presents in the 70% ethanolic extract.\n\nFigure S2. TP HPLC-DAD chromatogram of the phenolic compounds presents in the 70% ethanolic extract.\n\nFigure S3. CR HPLC-DAD chromatogram of the phenolic compounds presents in the 70% ethanolic extract.\n\nFigure S4. CB HPLC-DAD chromatogram of the phenolic compounds presents in the 70% ethanolic extract.\n\nAntioxidants Concentration in Free Radical Scavenging Assay;\n\nFigure S5. ABTS antioxidants concentration in free radical scavenging assay.\n\nFigure S6. DPPH antioxidants concentration in free radical scavenging assay.\n\nFigure S7. H2O2 antioxidants concentration in free radical scavenging assay.\n\nFigure S8. HI and TP aqueous extracts antimicrobial activity assay.\n\nFigure S9. HI and TP 70% ethanolic extracts antimicrobial activity assay.\n\nFigure S10. CR, CB and EH aqueous extracts antimicrobial activity assay.\n\nFigure S11. CR, CB and EH 70% ethanolic extracts antimicrobial activity assay.\n\nFigure S12. Plant identification voucher.\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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[
{
"id": "64218",
"date": "22 Jun 2020",
"name": "Mariateresa Cristani",
"expertise": [
"Reviewer Expertise Pharmacology",
"Pharmacognosy and Toxicology"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript is provides interesting findings; however, several points need to be addressed:\nAbout plant material: it is important know the time of the year of harvesting.\nLeaf extract preparation: a the beginning you indicate '1 g of powdered leaf was extracted with 50 ml solvent' In Leaf extract preparation ' 100 mg dry weights of each plant leaves were mixed, vortexed in 1 ml of solvent; explain it.\nAbout saponins test it is not clear why you report just one kind of result in figure 6, if you obtain it from the leaf powder and not from the 2 different extracts, I cannot understand why you make correlation with the two different kind of extracts.\nThe same for the ppo test. You did not compare the 2 different extracts.\nAbout the figure 7 HPLC is not clear why you left numbers and names, it is really difficult read it. Please, could you add numbers for each compound in the figure 7 and the same numbers and Rt in the table 1. Moreover, you report 'quantification of different bioactive compounds present in the extracts' . Why have you identified compounds by the HPLC-DAD for ethanolic extract only.\nTable 2: Zone of inhibitions of leaves extracts against different bacterial strains. Please, write 0 or Nil\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
},
{
"id": "64219",
"date": "30 Jun 2020",
"name": "Md Mustafizur Rahman",
"expertise": [
"Reviewer Expertise Medical Pharmacology",
"Natural product pharmacology",
"Biopharmaceutics",
"cancer biology",
"Pharmacogenetics"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 addressed interesting questions and came up with valuable findings. However, I think that the authors need to address a number of points mentioned below:\nOverall:\nIn the introduction, they should mention the rationale of taking the 4 weeds from the same origin.\n\nThey should justify why they used aqueous and 70% ethanolic media for extraction rather than using any non-polar medium. What difference did they expect from aqueous and 70% ethanolic media?\n\nAbstract:\nAlthough the authors described in methods and results they did not mention anything about In vitro antimicrobial activity, anti-diabetic activity, anti-arthritic activity, anti-lithiatic activity findings.\n\nMethods:\nPlease mention the time and duration of the plant collection. In addition, the authors did not mention how they dried the filtrate after clarification by filtration.\n\nCollection of microbial strains - Check the last line West 'Bengal and India' where ánd' to be deleted.\n\nIn PPO test, no comparison shown between two types of extracts.\nResult:\nWhy did the authors carried out HPLC-DAD analysis for only 70% ethanolic extracts, not for aqueous extract.\n\nNumber of Figures in the study is too high. We suggest the authors combine Figures 8-11 under one heading In vitro antioxidant activity.\nConclusions:\nShould be more precise and short. It should contain only their own findings not the findings of others which should be discussed under 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? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
},
{
"id": "64766",
"date": "01 Jul 2020",
"name": "Soumya Sasmal",
"expertise": [
"Reviewer Expertise Biochemical 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\nKindly state why the study conducted using only 70% ethanol. What's the reason behind not using 100% or 50% ethanol?\n\nProvide the best pictures of Petri plates in the manuscript for a better understanding of the zone of Inhibition study.\n\nHow do the authors determine the freshness of the leaves? (Size, Color, age of leaves). Kindly state the same in the \"Methods\" section.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/9-493
|
https://f1000research.com/articles/9-490/v1
|
02 Jun 20
|
{
"type": "Case Report",
"title": "Case Report: Primary melanoma of the gastroesophageal junction",
"authors": [
"Yasmine Hussein Agha",
"Nathaniel A. Parker",
"Joel Alderson",
"Nathaniel A. Parker",
"Joel Alderson"
],
"abstract": "Primary malignant melanoma represents the fifth most common cancer in the United States. It is subdivided into two forms: cutaneous (90%), visceral (8%, including ocular and mucosal) and of unknown primary (2%). The vast majority of gastrointestinal melanomas are secondary lesions until proven otherwise. Primary esophageal melanoma in particular is exceedingly rare, less than 200 cases have been documented in the literature to date. It is highly prevalent in Japan and occurs twice as much in men than women around the 6th decade of life. It has a predilection for the middle and lower esophagus, with only 6 cases occurring at the gastroesophageal junction worldwide. Its etiology and pathogenesis are poorly understood, and no curative treatment has been established given the paucity of cases. We present a case of primary melanoma of the gastroesophageal junction which represents the 2nd incident case in the united states and 7th worldwide.",
"keywords": [
"Esophageal Melanoma",
"Esophageal Neoplasms",
"Melanoma",
"Gastroesophageal junction",
"Rare Diseases"
],
"content": "Introduction\n\nMelanoma is the 5th most common cancer in the United States and accounts for 5.6% of newly diagnosed cancers1. It is characterized by uncontrolled proliferation of melanocytes which are mainly found in the skin’s epidermis and constitute 91.2% of all melanomas2. Non-cutaneous forms of primary melanoma include ocular and mucosal lesions and represent 5.2% and 1.3% of all melanomas, respectively2,3. The majority of mucosal subtypes arise in the head and neck and far less commonly in the gastrointestinal and urogenital tracts2. Primary esophageal melanoma (PEM) in particular, is exceedingly rare and accounts for 0.5% of newly identified primary melanomas4. Compared to primary cutaneous melanoma, the risk factors, etiology and pathogenesis of PEM are not well defined; current understanding is derived from cases reported in the literature. We present an unusual case of primary melanoma of the gastroesophageal junction (GEJ).\n\n\nCase Description\n\nA 90-year-old Caucasian male presented to emergency department in January 2020 with dysphagia to solid food and 30-pound unintentional weight loss over the past three months. Notable past medical history included gastroesophageal reflux disease, iron deficiency anemia, hypothyroidism, coronary artery bypass grafting, cholecystectomy, and prostate cancer treated with radiation therapy ten years ago. A detailed physical exam of the head, neck, and chest were unremarkable. Vital signs, the remainder of the physical examination, and laboratory testing were unremarkable. Notably, the patient was found to be severely anemic below his baseline blood level with a hemoglobin of 8.1 gm/dL (reference range 12 – 16 gm/dL). Stool guaiac testing was positive. Together with clinical history and laboratory testing an upper gastrointestinal (GI) bleed was suspected.\n\nFor further workup, computed tomography (CT) scans of the chest, abdomen, and pelvis with contrast were obtained. Imaging revealed a large esophageal mass. Extending approximately 15 cm, the mass appeared to originate in the mid-esophagus and terminate in the upper gastric tissue (Figure 1). Subsequently, upper GI endoscopy (esophagogastroduodenoscopy; EGD) was performed to determine the underlying cause and exclude malignancy. EGD noted a large, necrotic-appearing ulcerated mass extending 26 cm from the incisors and across the GEJ into the gastric cardia and fundus. Near the GEJ, the mass was obstructive, as it occupied approximately 70% of the esophageal lumen (Figure 2). Multiple biopsies were obtained along the exposed, protruding portions of the tumor to further evaluate the pathology.\n\n[Superior to inferior, alphabetical] The invasive mass originates in the mid-esophagus (top, left) at the level of the carina and extends into the upper portions of the stomach (arrows). At its center along the gastroesophageal junction (GEJ) the mass reaches its greatest dimensions at 6.1 x 9.36 cm. Enlarged gastrohepatic and paraesophageal lymph nodes are concerning for metastatic disease. (Bottom, right) Left renal lesion with intermediate density (arrowhead).\n\nThe large, necrotic-appearing ulcerated mass is observed to be protruding significantly into the esophageal lumen.\n\nMicroscopically, esophageal mass specimens showed sheets of malignant cells. To further classify these tumor cells immunohistochemical (IHC) staining was performed. Tumor cells were positive for MART-1 (melan-A) staining, whereas pankeratin, CD56, synaptophysin, chromogranin, CDX2, p63, and PSA staining was negative (Figure 3). Based on this IHC profile malignant melanoma was confirmed.\n\nAt medium power magnification, histopathology reveals the tumor cells are round with hyperchromatic eccentric nuclei, prominent nucleoli, and mixed eosinophilic and basophilic cytoplasm. Some areas reveal necrosis with neutrophilic exudate. Mitotic activity is frequently seen (HE x100). Pleomorphic melanoma cells containing various amounts of brown melanin pigment (HE x400). At high power magnification, tissue stained with MART-1 (melan-A) by IHC show melanocytes highlighted in brown.\n\nGiven the clinical history, presentation, image findings, endoscopic evaluation, and pathology primary malignant melanoma of the GEJ was diagnosed. The patient was dismissed from the hospital with instructions to immediately establish care with his local oncologist. Out-patient orders to check the tumor’s BRAF and programmed death-ligand 1 status, as well as assess for other tumor markers were placed. Two months after hospital dismissal the patient expired (Figure 4).\n\nPresented according to CARE guidelines.\n\n\nDiscussion\n\nMost primary gastrointestinal melanomas have been identified in the oral mucosa and anorectal region. PEM is exceedingly rare; less than 200 cases have been reported in the literature5,6. It is not only considered a rare subtype of primary melanoma but also accounts for an unusual primary gastrointestinal malignancy as PEM represents 0.1% of all primary esophageal tumors4.\n\nEsophageal melanosis, which is the proliferation of melanocytes and the increase in melanin production within the esophageal mucosa, has been frequently associated with cases describing PEM, however additional risk factors are yet to be defined7,8. To date, no association between PEM and known carcinogens such as alcohol and tobacco have been recognized in the literature. It occurs more frequently in men than women with an average ratio of 2:1 around the 6th decade of life and has a predilection for the Japanese population5. Although melanocytes are mainly skin and choroid bound, Ohashi et al. demonstrated in their study that these highly specialized cells can be found in the lower esophagus of up to 8% of healthy Japanese patients7. This correlation could potentially explain the predominance of PEM in this population.\n\nClinically, patients present with typical symptoms alarming for esophageal malignancy such as dysphagia, sternal chest pain, epigastric pain, hematemesis, melena, decreased appetite and weight loss3. Initial evaluation should include upper endoscopy and biopsy followed by review of the tissue’s pathology and immunohistochemical staining to make a diagnosis9. These tumors are generally located in the middle and lower esophageal lumen, are polypoid and can measure up to 5 cm5,10. 70% of these tumors are non-ulcerated and pigmented, while the rest can be amelanotic and necrotic4,5. S-100 is a highly sensitive marker for melanoma; HBM-45, melan-A, MITF and tyrosinase are highly specific markers for this entity11. Finally, although most PEM are non-metastatic at presentation, re-assessing the patient with a thorough physical exam and full body positron emission tomography / CT is necessary to rule out other primary tumors and to stage the current disease.\n\nGiven the paucity of cases, no general consensus exists regarding the optimal management of PEM. Schizas et al. analyzed in their study the outcomes following treatment of 93 individuals with PEM. Most patients underwent esophagectomy and patients who received adjuvant chemotherapy or immunotherapy had reduced rates of relapse; however overall survival (OS) was unchanged compared to those who had surgery only5,12. The choice of systemic therapy was derived from the guidelines to treat primary malignant melanoma during the time the patient was diagnosed. These drugs included cisplatin, vincristine, and dacarbazine4,13. OS rates for all patients regardless of whether they underwent radical resection only or had adjuvant therapy was 4.5%5. Three additional retrospective studies also showed poor prognosis regardless of management with median OS of 10 and 18 months4,13,14.\n\nTo the best of our knowledge, this is the 7th case of PEM arising at the gastroesophageal junction worldwide and second case documented in the United States15–20. Timely diagnosis and work-up of this patient represent major strengths in managing this case to avoid delay in treatment and worse outcomes. The lack of guidelines for management of patients with PEM however are a major limitation and his unfortunate early death did not allow us to initiate treatment and evaluate his clinical response in an attempt to set ground for management of future patients diagnosed with PEM.\n\n\nConclusion\n\nPEM is an aggressive and uncommon cancer with less than 200 cases worldwide. Its incidence is highest among the Japanese population and could possibly be correlated with the increased prevalence of esophageal melanosis in this group. It occurs around the age of 60 years and has a predilection for men. The etiology, risk factors and tumorigenesis are poorly understood, and no general consensus exists regarding an optimal management for this cancer. Current practices include esophagectomy with adjuvant chemotherapy or immunotherapy and is based on guidelines to treat primary cutaneous melanoma. Prognosis is poor, median survival is less than one year.\n\n\nConsent\n\nWritten informed consent for publication of clinical details and clinical images was obtained from the patient himself prior to his death.\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\nHowlader N, Noone AM, Krapcho M, et al.: SEER Cancer Statistics Review, 1975-2017. National Cancer Institute. Bethesda, MD. 2020. Reference Source\n\nChang AE, Karnell LH, Menck HR: The National Cancer Data Base report on cutaneous and noncutaneous melanoma: a summary of 84,836 cases from the past decade. The American College of Surgeons Commission on Cancer and the American Cancer Society. Cancer. 1998; 83(8): 1664–78. PubMed Abstract | Publisher Full Text\n\nSimons M, Ferreira J, Meunier R, et al.: Primary versus Metastatic Gastrointestinal Melanoma: A Rare Case and Review of Current Literature. Case Rep Gastrointest Med. 2016; 2016: 2306180. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSabanathan S, Eng J, Pradhan GN: Primary malignant melanoma of the esophagus. Am J Gastroenterol. 1989; 84(12): 1475–81. PubMed Abstract\n\nSchizas D, Mylonas KS, Bagias G, et al.: Esophageal melanoma: a systematic review and exploratory recurrence and survival analysis. Dis Esophagus. 2019; 32(12): doz083. PubMed Abstract | Publisher Full Text\n\nSchuchter LM, Green R, Fraker D: Primary and metastatic diseases in malignant melanoma of the gastrointestinal tract. Curr Opin Oncol. 2000; 12(2): 181–5. PubMed Abstract | Publisher Full Text\n\nOhashi K, Kato Y, Kanno J, et al.: Melanocytes and melanosis of the oesophagus in Japanese subjects--analysis of factors effecting their increase. Virchows Arch A Pathol Anat Histopathol. 1990; 417(2): 137–43. PubMed Abstract | Publisher Full Text\n\nYamazaki K, Ohmori T, Kumagai Y, et al.: Ultrastructure of oesophageal melanocytosis. Virchows Arch A Pathol Anat Histopathol. 1991; 418(6): 515–22. PubMed Abstract | Publisher Full Text\n\nWang S, Sun S, Liu X, et al.: Endoscopic diagnosis of gastrointestinal melanoma. Scand J Gastroenterol. 2020; 55(3): 330–7. PubMed Abstract | Publisher Full Text\n\nKaterji H, Childs JM, Bratton LE, et al.: Primary Esophageal Melanoma with Aberrant CD56 Expression: A Potential Diagnostic Pitfall. Case Rep Pathol. 2017; 2017: 9052637. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOhsie SJ, Sarantopoulos GP, Cochran AJ, et al.: Immunohistochemical characteristics of melanoma. J Cutan Pathol. 2008; 35(5): 433–44. PubMed Abstract | Publisher Full Text\n\nUrabe M, Haruta S, Ohkura Y, et al.: Clinicopathological presentations and surgical outcomes of esophageal melanoma. Asian Cardiovasc Thorac Ann. 2019; 27(7): 548–53. PubMed Abstract | Publisher Full Text\n\nGao S, Li J, Feng X, et al.: Characteristics and Surgical Outcomes for Primary Malignant Melanoma of the Esophagus. Sci Rep. 2016; 6: 23804. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWeiner JP, Shao M, Schwartz D, et al.: Patterns of care and survival outcomes in the treatment of esophageal melanoma. Dis Esophagus. 2017; 30(2): 1–6. PubMed Abstract | Publisher Full Text\n\nFukunaga H, Kaneda H, Kumakawa H, et al.: Asymptomatic Primary Malignant Melanoma of the Gastroesophageal Junction. Intern Med. 2016; 55(6): 709–10. PubMed Abstract | Publisher Full Text\n\nImamura K, Adachi K, Enatsu K: Primary Gastrointestinal Malignant Melanoma. Clin Gastroenterol Hepatol. 2016; 14(1): e5–6. PubMed Abstract | Publisher Full Text\n\nKinuya S, Tonami N, Hisada K: Ga-67 SPECT in primary gastroesophageal malignant melanoma. Clin Nucl Med. 1996; 21(4): 325–6. PubMed Abstract | Publisher Full Text\n\nGrilliot MA, Goldblum JR, Liu X: Signet-ring cell melanoma of the gastroesophageal junction: a case report and literature review. Arch Pathol Lab Med. 2012; 136(3): 324–8. PubMed Abstract | Publisher Full Text\n\nIshizaki M, Aibara Y, Furuya K: Primary malignant melanoma of the esophagogastric junction: Report of a case. Int J Surg Case Rep. 2013; 4(8): 700–3. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGao Y, Zhu J, Lin W, et al.: Primary malignant melanoma of esophagogastric junction: a case report. Chin Med J (Engl). 2003; 116(9): 1435–7. PubMed Abstract"
}
|
[
{
"id": "64198",
"date": "05 Jun 2020",
"name": "Mahmoud Al-Rifai",
"expertise": [
"Reviewer Expertise Medicine",
"CVD"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nVery interesting and rare case presentation of gastrointestinal melanoma occurring at the GE junction. A sufficient background and case presentation were presented followed by a well thought of discussion. This case is not only novel but also highly important to describe in the literature given its rarity and lack of proper understanding of risk factors, tumorigenesis, and ultimately treatment. Unfortunately the patient expired which is not surprising given poor prognosis of this disease.\n\nI would suggest the authors include grade and stage of the tumor. It appears that the tumor was not metastatic on presentation but this needs to be clearly established.\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": "65044",
"date": "06 Jul 2020",
"name": "Gregory W. Roloff",
"expertise": [
"Reviewer Expertise Internal Medicine",
"hematology/oncology"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe report by Agha and colleagues is well-researched and well-written, describing an uncommon presentation of a GE junction melanoma. The time course is clearly delineated and the timeline figure allows the reader a full appreciation for the significance of the patient's unfortunate but not unexpected mortality given the debilitation associated with the anatomic location of the lesion. In order to more clearly describe the tumor, it would be helpful if the grade and stage could be included. Namely, was PET-CT conducted to establish distant metastasis? Otherwise, the case is strong and will serve as a helpful addition to the literature.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/9-490
|
https://f1000research.com/articles/9-184/v1
|
12 Mar 20
|
{
"type": "Case Report",
"title": "Case Report: An occurrence of steinstrasse in retrograde intra renal surgery for a large staghorn kidney stone – difficulty managing surgical outcomes",
"authors": [
"Ponco Birowo",
"Nur Rasyid",
"Widi Atmoko",
"Bobby Sutojo",
"Nur Rasyid",
"Widi Atmoko",
"Bobby Sutojo"
],
"abstract": "Immediate removal of staghorn stones is compulsory to prevent life-threatening complications. The advancement of endoscopic technology makes retrograde intrarenal surgery (RIRS) a favorable treatment to remove large stones over the standard percutaneous nephrolithotomy (PCNL). Without careful considerations, it can cause the formation of steinstrasse. Here, we present the case of a 68-year-old man with multiple stones along his right urogenital duct after being treated with RIRS to remove a staghorn stone. After 2 years of multiple interventions, the steinstrasse was completely removed. To prevent this complication, detailed assessment of the stone (size, location) and renal anatomy should be performed prior to the procedure.",
"keywords": [
"retrograde intrarenal surgery",
"staghorn stones",
"steinstrasse",
"complication"
],
"content": "Introduction\n\nThe term “staghorn” describes the configuration of large, branched renal stones that occupy the pelvis and extent to at least two renal calices. Immediate removal of the stones is compulsory to prevent serious kidney injury and life-threatening sepsis1. According to the American Urological Association, percutaneous nephrolithotomy (PCNL) is the standard treatment for staghorn removal2. Recently, urologists started using retrograde intrarenal surgery (RIRS) to treat large stones since it is less invasive and simpler than PCNL3. However, this procedure might cause the formation of steinstrasse as a complication especially in stones of size 2–3 cm, which require more invasive removal procedures. This multi-procedure approach in the treatment of renal stone can impact the patients’ quality of life, especially when the stone is hard (measured with Hounsfield unit of more than 1000)4,5. This study shows and discusses the formation of steinstrasse and the need of follow up procedures, which led to depression in the patient after the use of RIRS for staghorn stone removal.\n\n\nCase presentation\n\nA 68-year-old man came to our hospital in April 2016 with multiple stones along his right urogenital system. He had been experiencing flank pain that was not influenced by body position for 1 month. He denied any treatment relating to the pain that he experienced in this period. He also denied having a family history of this symptom nor ever he had this symptom before. Physical examination only revealed tenderness of the right flank.\n\nComputed tomography (CT) urography at the previous hospital showed a staghorn stone at the right inferior calyx with a size of 45.7 × 59.3 × 27.5 mm (Hounsfield unit not available), a left renal cyst with size of 19.2 × 22.2 × 18.9 mm, and a third-grade right-side hydronephrosis (Figure 1). Post-RIRS photo showed a double J stent with multiple tiny stones from the right pelvio-calyces to vesico-ureteral junction (Figure 2a).\n\nThe first CT Urography of the patient shows right staghorn stone with grade 3 hydronephrosis and left renal cyst.\n\nAn immediate (a) and 1 month (April 2016) (b) Plain abdominal photo following retrograde intrarenal surgery shows multiple tiny stones along the right genitourinary system.\n\nA month later at when he come to our hospital for a second opinion, his plain abdominal X-ray result had not changed (Figure 2b). Right ureteroscopy (URS), right nephrostomy, and right PCNL was performed and post-operative X-ray imaging was conducted (Figure 3a). Another right URS was performed 2 weeks later, resulting in the remaining 8-mm stone at the ureter-pelvic junction (UPJ) (Figure 3b).\n\nAfter right ureterorenoscopy, right nephrostomy, and right percutaneous nephrolithotomy in April 2016 (a), after ureterorenoscopy and percutaneous nephrolithotomy in June 2016 (b), after the second extracorporeal shock wave lithotripsy in June 2016 (c), after the third extracorporeal shock wave lithotripsy in July 2016 (d), after right laser ureterorenoscopy and replacement of right double J stent in July 2016 (e), after extracorporeal shock wave lithotripsy in October 2016 (f), double J stent removal in October 2016 (g), routine control in January 2018 (h & i), retrograde intrarenal surgery which shows no residual stone in June 2019 (j).\n\nExtracorporeal shock wave lithotripsy (ESWL) was performed twice in June 2016, resulting in a decrease in stone size to 6 mm. (Figure 3c). Another ESWL was performed the next month (Figure 3d). In July 2016, he underwent a right laser URS followed by replacement of the DJ stent (Figure 3e). Three months later, another ESWL was performed (Figure 3f). Shortly after, the remaining DJ stent was removed. Plain abdominal X-ray still showed a residual right nephrolithiasis. (Figure 3g). In 2017, the patient was so depressed from the numerous procedures that he decided to end the treatment for his remaining stone. He admitted lacking spirit throughout the day since the failure of the last ESWL procedure and feeling that his stone would not be able to be cured, along with his continuous need for pain medication.\n\nAlmost two years later (January 2018), routine plain abdominal X-ray and CT urography showed no change on his right nephrolithiasis (Figure 3h, i). In June 2019, he was persuaded by his family to re-try stone management and had the last RIRS at another hospital to remove the remaining stone completely (Figure 3j). In November 2019, he visited our hospital for DJ stent removal and plain abdominal X-ray. Neither stone nor DJ stent were observed. The summary of the patient’s history of illness can be seen in Table 1.\n\nRIRS, retrograde intrarenal surgery; URS, Ureteroscopy; PCNL, percutaneous nephrolithotomy; ESWL, extracorporeal shock wave lithotripsy.\n\n\nDiscussion\n\nThe management of nephrolithiasis has been changing dramatically over time, shifting from open surgery to less-invasive procedures, such as PCNL and ESWL6. According to the American Urological Association and European Association of Urology guidelines, the standard treatment for staghorn stone removal is PCNL2,5. PCNL has a high stone-free rate (SFR), similar to that of an open surgery (93%). It also results in lower morbidity, shorter operative time, shorter hospital stays, and earlier back to work compared to open surgery. However, it can cause severe complications, such as renal trauma with severe uncontrollable bleeding7,8.\n\nOn the other hand, the development of flexible ureteroscopes allows for excellent visualization that makes it a favorable procedure for most urologists. The possibility of using holmium lasers alongside a ureteroscope, lowering the cost, has made this procedure even more popular9. Compared to PCNL, RIRS has a slightly lower SFR of 87% and also lower morbidity and complication rate at 2%10. In our case, the first option of RIRS instead of PCNL was because the patient preferred a less invasive method.\n\nIn Indonesia, PCNL is still the first choice for treating large renal calculi according to Ikatan Ahli Urologi Indonesia (the Indonesian Urologist Association). However, the use of PCNL in Indonesia is still limited due to the lack of technology and experts, particularly in remote areas11. The incidence of steinstrasse formation after RIRS is 20% of large renal stone cases followed by hydronephrosis4. The development of SS is also seen in our patient who were initially treated with RIRS. To avoid this complication, a scoring system was developed by Resorlu et al.12 that includes four indicators: a renal stone size >20 mm, lower pole stone with an infundibulum-pelvic angle <45°, a stone number in different calyces >1, and abnormal renal anatomy. A greater score is associated with a lower SFR. This score can be calculated prior to RIRS.\n\nUrolithiasis is a painful chronic disease which significantly impacts the quality of life of the sufferers. In addition to chronic pain, the acute pain of urolithiasis resulting from stone movement often causes fear of recurrence. Recent studies have suggested an association between the disease and anxiety and depression13. In the present study, our patient developed symptoms of depression during the second year of his treatment because he had to undergo multiple surgical procedures in a year. However, after receiving support from his family and reassurance from the clinicians, the patient finally decided to continue treatment for his leftover stones. The patient initially preferred RIRS to PCNL because it was less invasive, but after his negative experiences, he would prefer PCNL to RIRS.\n\nRIRS may be used in cases where open surgery and ESWL are risky or inadequate, such as in patients with obesity, bleeding disorders, musculoskeletal deformities, renoureteral malformations, and infundibular stenosis5.\n\nOne limitation of this study is that we did not know the hardness (Hounsfield units) of the stone prior to the patient presenting to our clinic, meaning we could not specifically determine the cause of his previous failure of treatment other than the size of the stone.\n\n\nConclusions\n\nRIRS can be an alternative choice to remove large staghorn calculi in selected patients even though the efficacy is lower than PCNL. Detailed assessment should be made to prevent any complications. Patients with large staghorn calculi should be informed regarding the possible need for multiple procedures to completely remove the stones, otherwise receiving inadequate information will lead the patient to negative thinking and treatment avoidance.\n\n\nData availability\n\nAll data underlying the result are available as part of the article and no additional source data are required.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and/or clinical images was obtained from the patient/parent/guardian/relative of the patient.",
"appendix": "References\n\nHealy KA, Ogan K: Pathophysiology and management of infectious staghorn calculi. Urol Clin N Am. 2007; 34(3): 363–74. PubMed Abstract | Publisher Full Text\n\nPreminger GM, Assimos DG, Lingeman JE, et al.: Chapter 1: AUA guideline on management of staghorn calculi: diagnosis and treatment recommendations. J Urol. 2005; 173(6): 1991–2000. PubMed Abstract | Publisher Full Text\n\nZengin K, Tanik S, Karakoyunlu N, et al.: Retrograde intrarenal surgery versus percutaneous lithotripsy to treat renal stones 2-3 cm in diameter. Biomed Res Int. 2015; 2015: 914231. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHyams ES, Munver R, Bird VG, et al.: Flexible ureterorenoscopy and holmium laser lithotripsy for the management of renal stone burdens that measure 2 to 3 cm: a multi-institutional experience. J Endourol. 2010; 24(10): 1583–1588. PubMed Abstract | Publisher Full Text\n\nTurk C, Neisius A, Petrik A, et al.: EAU Guidelines on Urolithiasis. 2018. Reference Source\n\nYuri P, Hariwibowo R, Soeroharjo I, et al.: Meta-analysis of Optimal Management of Lower Pole Stone of 10 - 20 mm: Flexible Ureteroscopy (FURS) versus Extracorporeal Shock Wave Lithotripsy (ESWL) versus Percutaneus Nephrolithotomy (PCNL). Acta Med Indones. 2018; 50(1): 18–25. PubMed Abstract\n\nAl-kohlany KM, Shokeir AA, Mosbah T, et al.: Treatment of complete staghorn stones: a prospective randomized comparison of open surgery versus percutaneous nephrolithotomy. [abstract]. J Urol. 2005; 173(2): 469–73. PubMed Abstract | Publisher Full Text\n\nEl-Nahas AR, Shokeir AA, El-Assmy AM, et al.: Post-percutaneous nephrolithotomy extensive hemorrhage: a study of risk factors. J Urol. 2007; 177(2): 576–9. PubMed Abstract | Publisher Full Text\n\nKrambeck AE, Murat FJ, Gettman MT, et al.: The evolution of ureteroscopy: a modern single-institution series. Mayo Clin Proc. 2006; 81(4): 468–73. PubMed Abstract | Publisher Full Text\n\nPrabhakar M: Retrograde ureteroscopic intrarenal surgery for large (1.6-3.5 cm) upper ureteric/renal calculus. Indian J Urol. 2010; 26(1): 46–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRasyid N, Kusuma GW, Atmoko W, editor: Ikatan Ahli Urologi Indonesia: Panduan penatalaksanaan klinis batu saluran kemih Edisi pertama. Jakarta. 2018.\n\nResorlu B, Unsal A, Gulec H, et al.: A new scoring system for predicting stone-free rate after retrograde intrarenal surgery: the “resorlu-unsal stone score”. Urology. 2012; 80(3): 512–8. PubMed Abstract | Publisher Full Text\n\nLien CS, Huang CP, Chung CJ, et al.: Increased risk of anxiety among patients with urolithiasis: A nationwide population-based cohort study. Int J Urol. 2015; 22(10): 937–42. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "61758",
"date": "14 Apr 2020",
"name": "Doddy Moesbadianto Soebadi",
"expertise": [
"Reviewer Expertise General urology",
"voiding dysfunction",
"sexual medicine",
"endourology",
"laser urology."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a good example of how minimally invasive surgeries can take a long journey before they come to a satisfactory result.\n\nThe authors did not mention the drawbacks of these lengthy managements, i.e. the opinion from the patient side and the cost of the serial treatments.\n\nBut this is a very good clinical experience to be one of the considerations before the urologist(s) offer this minimally invasive procedure(s).\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": "61757",
"date": "04 May 2020",
"name": "M. Hammad Ather",
"expertise": [
"Reviewer Expertise Urolithiasis and Bladder cancer"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSteinstrasse is a legacy of SWL (Does ureteral stenting prior to shock wave lithotripsy influence the need for intervention in steinstrasse and related complications? (Ather et al., 20091)) and RIRS for larger stones is no different from performing SWL under endoscopy. The authors should discuss this point.\n\nConclusions are contrary to the case account. The conclusions should be that larger stones (Staghorn) would preferably not be treated by RIRS.\n\nFew minor issues:\n\nIn the abstract I don’t agree with the word “compulsory” - a more preferable alternative would be “desirable\" or “important” etc.\n\nRIRS is not mentioned as a treatment option for large kidney stones.\n\nMention other complications of the use of RIRS besides steinstrasse for large renal stones.\n\nThe phrase “RIRS to remove a staghorn stone” in the abstract is not an appropriate reflection of the abilities of RIRS in that set up.\n\nThere are many grammatical errors that need attention.\n\nThe authors used the phrase “which led to depression in the patient after the use of RIRS for staghorn stone removal”. This needs to be rephrased for clarity as to what the authors are implying. Are they saying prolonged treatment causes psychological issues, like depression? Please cite a reference.\n\nIn the case presentation, the authors write “multiple stones along his right urogenital system” - this needs to be rewritten for clarity. Stones were in the collecting system of the right kidney.\n\nWith the limited access to single section I disagree with the authors' interpretation of the CT. I see a Staghorn stone with a major component in the renal pelvis and branching calculi in lower and middle pole calyces. There is dilatation and obstruction with ballooning of the upper pole calyx due obstruction of the infundibulum of the upper pole calyx. There is no cyst visible in these scans.\n\nIt is grade three, not third grade hydronephrosis, which is incidentally also not clear in the CT cut shown.\n\nThe authors should avoid using term “genitourinary”.\n\nWhat is the stone composition?\n\nIs the background of the case’s history and progression described in sufficient detail? No\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? 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": [
{
"c_id": "5540",
"date": "29 May 2020",
"name": "Ponco Birowo",
"role": "Author Response",
"response": "Comments from Reviewer and Author Response: Steinstrasse is a legacy of SWL (Does ureteral stenting prior to shock wave lithotripsy influence the need for intervention in steinstrasse and related complications? (Ather et al., 20091) and RIRS for larger stones is no different from performing SWL under endoscopy. The authors should discuss this point. Author response:We have revised and added this additional paragraph to our manuscript:The necessity for routine stent insertion before or after RIRS to increase stone clearance remains unclear. The main purpose of stent insertion is to prevent ureteral stricture, accelerate healing, and facilitate stone passing (Cleynenbreugel, et al., 2017). On the other hand, stent insertion increases the possibility of urinary tract infection, dysuria, pollakiuria, hematuria, and repeated cystoscopy may be required in cases of stent migration and to assess the need for extraction (Ozyuvali, et al., 2015). Stent insertion before shockwave lithotripsy (SWL) does not eliminate the need for intervention in the management of steinstrasse (Ather et al., 2009). In the present case, considering the size and the position of the stone, ureteral stent placement prior to RIRS would be difficult and other options should be considered. Conclusions are contrary to the case account. The conclusions should be that larger stones (Staghorn) would preferably not be treated by RIRS. Author response:Thanks for your advice. We have revised our conclusion to become: RIRS is not the preferable option to remove large staghorn calculi due to lower efficacy and other possible complications. It can be used in circumstances where open surgery or PCNL is not possible, but careful assessment is necessary to determine whether the procedure will be beneficial and safe for the patient. In the abstract I don’t agree with the word “compulsory” - a more preferable alternative would be “desirable\" or “important” etc. Author response:Thank you for your advice, we have changed the word according to your advice. RIRS is not mentioned as a treatment option for large kidney stones. Author response:We have revised and added this additional paragraph to our manuscript:Initially, the use of RIRS is limited to patients who cannot undergo PCNL or shockwave lithotripsy (SWL) due to several contraindications. However, with the development of technology, the usage of RIRS for large stone is now possible. Compared to PCNL, RIRS has a slightly lower SFR of 87% and also lower morbidity and complication rate of 2%.10 In our case, the use of RIRS instead of PCNL as the first treatment was due to the patient’s preference for a less invasive method. Mention other complications of the use of RIRS besides steinstrasse for large renal stones. Author response:We have revised and added this additional paragraph to our manuscript:RIRS is a less-invasive procedure compared to PCNL. Complications may arise intra- or post-operatively in some cases but are usually minor and manageable. The common complications of RIRS include hemorrhage, intrapelvic hematoma, mucosal injury, ureteral perforation and avulsion, urinary tract infection, and sepsis. In a study by Niwa et al., the most common complication associated with RIRS in treating staghorn stones was urinary tract infection (Clavien-Dindo II, 28.2%), followed by fever (7.7%), general malaise (2.6%), and malposition of a ureteral stent (2.6%). The phrase “RIRS to remove a staghorn stone” in the abstract is not an appropriate reflection of the abilities of RIRS in that set up. Author response:We have revised our abstract as it is not aligned with the insight of our case presentation that do not recommend the use of RIRS for large stones removal. Here is the revised version:Immediate removal of staghorn kidney stones is important to prevent life-threatening complications. With the advancement of endoscopic technology, retrograde intrarenal surgery (RIRS) is now an alternate treatment to the standard percutaneous nephrolithotomy (PCNL) for stones removal. However, when used to treat large stones (>3cm), RIRS can cause the formation steinstrasse (SS). There are many grammatical errors that need attention. Author response:Thank you for addressing your concern. We have revised the grammatical error that we found. The authors used the phrase “which led to depression in the patient after the use of RIRS for staghorn stone removal”. This needs to be rephrased for clarity as to what the authors are implying. Are they saying prolonged treatment causes psychological issues, like depression? Please cite a reference. Author response:We have added some information to clarify this issue.Urolithiasis is a painful chronic disease that has significant impacts on a patient’s quality of life. In addition to chronic pain, the acute pain of urolithiasis resulting from stone movement often causes fear of recurrence. Recent studies have suggested an association between the disease and anxiety and depression. In the present study, our patient developed symptoms of depression during the second year of his treatment because he had to undergo multiple surgical procedures within a year to remove the SS. In addition, the patient had to endure the pain associated with recovery after each procedure, as well as the pain caused by the remaining stones. In the case presentation, the authors write “multiple stones along his right urogenital system” - this needs to be rewritten for clarity. Stones were in the collecting system of the right kidney. Author response:Thank you for your correction. We have updated the term accordingly. With the limited access to a single section I disagree with the authors' interpretation of the CT. I see a Staghorn stone with a major component in the renal pelvis and branching calculi in lower and middle pole calyces. There is dilatation and obstruction with ballooning of the upper pole calyx due to obstruction of the infundibulum of the upper pole calyx. There is no cyst visible in these scans. Author response:Thank you for your concern. We have decided to change the picture: It is grade three, not third-grade hydronephrosis, which is incidentally also not clear in the CT cut shown. Author response:We have changed “third grade” to “grade three” hydronephrosis. The authors should avoid using term “genitourinary”. Author response:Thank you. We have altered “genitourinary” to urinary system. What is the stone composition? Author response:Unfortunately, in the present case, the stone composition was not analyzed due to financial constraints. This is a good example of how minimally invasive surgeries can take a long journey before they come to a satisfactory result. Author response:Thank you for your appreciation. The authors did not mention the drawbacks of these lengthy managements, i.e. the opinion from the patient side and the cost of the serial treatments. Author response:The patient self-funded the whole treatment but he refused to tell the amount he had to pay. As for his opinion about the lengthy procedure, the patient complained of slight depression due to the continuous pain, either from the remaining stone and from the procedures. This information is mentioned in the case presentation and discussion section. But this is a very good clinical experience to be one of the considerations before the urologist(s) offer this minimally invasive procedure(s). Author response:Thank you. We hope this study provides a handful of insight regarding the disadvantage of RIRS to treat large kidney stone."
}
]
}
] | 1
|
https://f1000research.com/articles/9-184
|
https://f1000research.com/articles/8-1748/v1
|
14 Oct 19
|
{
"type": "Research Article",
"title": "Fertility transition in selected sub-Saharan African countries: the role of family planning programs",
"authors": [
"Vincent Otieno",
"Alfred Agwanda",
"Anne Khasakhala",
"Alfred Agwanda",
"Anne Khasakhala"
],
"abstract": "Background: Change in fertility rate across societies is a complex process that involves changes in the demand for children, the diffusion of new attitudes about family planning and greater accessibility to contraception provided by family planning programs. Among the neo-Malthusian adherents, it is believed that rapid population growth strain countries’ capacity and performance. Fertility has, however, decelerated in most countries in the recent past. Scholars have concentrated on wide range of factors associated with fertility majorly at the national scale. However, considerably less attention has been paid to the fertility preference - a pathway through which various variables act on fertility. The Sub-Saharan African countries’ disparities amid almost similarities in policies is a cause of concern to demographers. Methods: Using Bongaarts reformulation of Easterlin and Crimmins conceptual scheme of 1985 on Demographic and Health Survey Data (DHS) data collected overtime across countries, the understanding of the current transition in general would help to reassess and provide explanations to the observed latest fertility dynamics at play. This study therefore is an attempt to explain the current fertility transition through women’s fertility preference. Results: Results reveal that indeed fertility transition is diverse across countries though generally on a decline course in most of the sub-Saharan countries. The huge disparities in fertility preferences among women of reproductive age and its non-significant change in the implementation indices overtime points at the levels of unmet need to contraception underneath as well as the proportion of demand to family planning commodities satisfied by programs in a bid to allow women implement their fertility desires. Conclusions: It is therefore plausible to conclude that the improvement of the availability and the uptake of quality birth control technologies is one of the most feasible means through which countries can fast track their fertility transitions.",
"keywords": [
"Fertility",
"Preference Implementation index",
"family planning",
"Transition",
"Sub-Saharan Africa"
],
"content": "Introduction\n\nChange in fertility rate across societies is a complex process that involves changes in the demand for children, the diffusion of new attitudes about family planning and greater accessibility to contraception provided by family planning programs1,2. Debates about this transition in Sub-Saharan Africa have almost reached a consensus about its uniqueness since they began in the mid-1990s . The trajectory of African fertility transitions occurred earlier than anticipated if Africa had followed the non-African relationship between fertility and development3. However, the pace of decline in fertility rate at the time of onset of the transition in this rate was slower than the comparable pace at the onsets of non-African transitions. The key features of African fertility regimes indicate that at a given level of development, Africa’s fertility is higher, contraceptive use is lower, and desired family size is higher than in non-African less-developed countries1,2.\n\nThis raises two fundamentally interrelated concepts observed in a number of developing countries, namely: the extent to which changes in fertility levels are due to changes in fertility preference and the extent to which the observed fertility changes result from the ability of women to implement these fertility desires4. In this study we seek to add to our understanding of the fertility transition by examining how countries differ in their patterns of reproductive behavior. We specifically examine trends in the fertility desires and the extent at which the ability to implement fertility desires contributes to the prevailing fertility change.\n\n\nMethods\n\nUsing all the trend data from Measure DHS gathered between 1986 and 2016 across sub-Saharan African countries (listed in Table 1), we apply2 a reformulation of5 a conceptual scheme in which the variable ‘fertility’ is measured by the total fertility rate (F0), a function of the supply of births (natural fertility (Fn)), the demand for births (wanted fertility (Fw)) and the degree of fertility preference implementation index (Ip)6. The latter in turn is dependent on cost of fertility regulation and cost of unwanted childbearing. The degree of Ip is the net result of a decision-making process in which couples weigh the cost of fertility regulation and the cost of unwanted pregnancy. Figure 1 shows the diagrammatic presentation.\n\nAccording to Bongaarts (1993):\n\nC is an index ranging from 0 to 1 measuring the proportional reduction in Fn attributed to deliberate birth control mechanisms. Birth control not only confined to contraception also encompass induced abortion practices though always ignored in studies. F0 data is always available and hence the only additional task required is to compute Fn in (i) with an estimate of C. Bongaarts further provided a procedure for deducing C, with an approach though the limitation was the unavailability of data. Hence:\n\nWhere U is the fraction of women in marriage practicing all forms of birth regulation except during the post-partum in-fecundity period. The error associated with this is negligible hence ignored sometimes. By substituting C in Equation 1 above yields the anticipated approximation of natural total fertility.\n\nFw computation: According to Bongaarts, the favoured approach is dependent on the equation below:\n\nWhere Fw is the proportion of women who want more children, equaling the resulting total fertility after deleting all births to women who want no more children at the time of the survey and Wm (40–49) is the proportion of women in union aged from 40 to 49 who want no more births.\n\nThese two equations helped normalize the biases in order to compute the respective Fn and Fw trends across the regions. With most of the erratic curves expected, a normalization process using the natural logarithms of the equation was applied to give meaning to the various trend curves.\n\nIp is derived from a synthesis of past studies. It begins from the fact that all social and economic factors of fertility operate through a unified pack of proximate factors to exert an impact on fertility5. Easterlin’s economic approach is a model of behavioural and biological factors affecting fertility in developing countries. The model consists of three central concepts: demand for children; the potential supply of children, and the momentary and psychic costs of contraception. According to the model, women whose potential supply of births exceeds demand would consider contraception, taking into consideration the costs involved while choosing suitable family planning methods5.\n\nThe model is simple and attractive; however, it cannot address dynamic issues and has not succeeded in quantifying these factors in acceptable manner4. Emerging from the model is the fact that fertility (measured using F0) is a function of three determinants namely: supply of births (Fn), demand for births (Fw) and the degree of Ip (Figure 1).\n\nSupply of births (Fn) is measured as natural total fertility. Fn infers the rate of birthing likely to prevail minus the premeditated attempts by spouses to limit their number of children. Demand for births (Fw) is the wanted total fertility defined as the rate of prevailing childbearing after eliminating all unwanted births. Under normal circumstances, it is simply calculated as F0 while eliminating the unwanted births from the numerator. Unwanted births are births occurring after an achievement of the ideal family size. Any births that are mistimed though occurring before the achievement of the desired family size are considered wanted births as well.\n\nThe degree of Ip is an index from zero value to unity. Its level of implementation implies the net result of decision-making process. This is the state in which a spouse ponders the cost of fertility regulation as they consider costs of bearing an unwanted child to its end. In general, the index has an inverse variation to the cost of fertility regulation as well as a reverse correlation to the unwanted births. If couples fully implement their fertility preference, the index is equal to unity. This signifies that no unwanted births occur as actual fertility corresponds to w Fw. Conversely, if the index is equal to zero, the observed fertility equals Fn, that is, fertility in the absence of any deliberate fertility control assuming women remain sexually active over their reproductive cycles. The value of the index at play stipulates the position where actual fertility falls as dictated by the range set between wanted and Fn parameter levels.\n\nF0 gives the estimate of the number of children a woman would have by the end of childbearing if she were to pass through her reproductive cycle at the customary age specific birth rates. The model shows that the operation of these variables determines the level of fertility in a community or households. In this variant of the original Easterlin and Crimmins (1985) model, infant and child mortality dynamics affects the desired fertility rather than Fn. Women are deemed to possess precise desired fertility size translated and actualized into numbers through subsequent births after considering past child losses and risks related to future child deaths as well.\n\nAccording to this variant, as development occurs, the trend in prevailing fertility transforms to become a function of the equilibrium between the Fw, Fn and the degree of fertility Ip. Fw is expected to decline over time, as a result of the changes associated with the costs and benefits of child bearing6, as well as reductions in the infant-child mortality. Ip rises as fertility regulation costs decline; with the benefit of fertility regulation focusing on the elimination of any unwanted births5. According to5, the relationship between these variables under discussions and fertility can be expressed in statistical form as follows:\n\nWhere Fu is the unwanted fertility (which can simply be expressed as F0 – Fw).\n\nWhere Ip has a range of 0 to 1. With full Ip, Ip = 1 (which implies that Fu = 0 and F0 = Fw) and Ip = 0 with no prefer Ip (This implies a substantial level of unwanted childbearing and F0 = Fn). Noted here is that as defined by Bongaarts, Fn here is not the same as in total fecundity as in the Bongaarts proximate determinants but taken to mean fertility level achieved in absence of contraception5.\n\nFu is a function of the difference between supply and demand, and the degree of Ip.\n\nSubstitution of Equation 5 in Equation 6 yields:\n\nNoting that Fn is given by:\n\nWhere C implies an index ranging from 0 to 1 measuring the reduction in proportional of Fn attributable to deliberate birth control is estimated as:\n\nWhere U represents the proportion of married women who were practicing contraception at the time of survey. It is measured as the number of married women using contraceptive method to the total number of married women. The values for U and C can be used to estimate Fn\n\nRearranging Equation 6 gives:\n\nEquation 7 can now be used to estimate the degree of Ip once Fn (fertility in absence of contraception), actual fertility and Fw are known. One thing to note is that the estimation of Fw, as previously done overtime contain traits of upward bias as per the recent observation. An alternative estimation of Fw from Bongaarts model detail that the average Fw derived from the wanted status of births as reported by women was 2.8, indicating that this measure of wanted fertility contains an average upward bias of 0.4 birth.\n\nThe analysis consists of two stages. First, we computed the degree of Ip within the variable categories overtime from Fn, Fw and F0. F0 and Fw as a series of indicators are provided by the various country specific DHS reports. This involves compiling all the components of the index within the equation so as to come with the actual figures per the subsequent time intervals. The component variables are Fn, Fw computed to form the degree of Ip. Further correlation analysis between the degree of Ip and the unmet need to contraception, conducted using SPSS v18, was run.\n\nAccording to Bongaarts (1993), the core objective of the demand framework lies in the identification of the causes of fertility decline in a population, with proceeding comparative analysis providing worthwhile insights yet not achieving its sole objective. Turning to the issue at hand, the decomposition of the variations in fertility and to abridge the methodological exposition, trends therefore should inform the basis of focus between two points in time, i.e. T1 and T2 running up to the determinants. The derivation of the decomposition equation also warrants the introduction of the variables listed in Table 2.\n\nSource: Bongaarts (1993)\n\nThe decline in fertility between the two periods is F1-F2, conveyed by substitution as\n\nThe above equation therefore can be written as below\n\nIn Equation 8: ∆F, ∆Fw, ∆Fn and ∆Ip are the change within F, Fw, Fn and Ip respectively.\n\nIn Equation 9: Ѓw, Ѓn and Īp are the mean values of correspondingly, Fw, Fn and Ip. For example, the mean of the degree of implementation index (Īp) is: - [0.5(Ip1 + Ip2)]\n\n\n\nĪp implies the average of the Degree of Fertility Implementation Index (Ip). The influence of change in wanted (∆Fw ) as well as the natural (∆Fn) fertility to prevailing fertility change hinge on the average extent or degree of implementation. Consequently, the outcome of fertility from every shift registered on the degree of fertility implementation index is determined by the corresponding mean change between Fn and Fw (Ѓw-Ѓn). This function requires two successive points in the estimates of the parameter measurers i.e. F0, natural and Fw including the implementation index as well within the population under consideration. It is this function that is used to determine the extent to which implementation of fertility desires contributes to fertility transition.\n\n\nResults\n\nAs Table 1 shows the trend change in fertility parameters measurers, Table 3 further shows the decomposition of fertility changes among countries with two or more surveys. Results reveal there are indeed substantial variations between countries in terms of fertility preference parameter measurers as well as the implementation indices by countries. These results clearly indicate the important role played by the changes in Ip, Fw and Fn. Converse to the expected, eight countries actually increased their F0 over the period 1986–2016. In six out of the eight countries where fertility increased, there was a decline in degree of Ip. Subsequently, in five of the eight countries there was an increase in Fw. The largest decline in fertility rate occurred in Rwanda, Malawi, Kenya and Ethiopia. The four countries subsequently had the greatest contribution of the degree of Ip to fertility decline. On the same note the greatest contribution of Fw decline to fertility change occurred in Malawi, Rwanda and Kenya.\n\nF0, total fertility rate.\n\nIn absolute values, Rwanda, Malawi and Kenya experienced the highest fertility changes as well, while Niger, Mozambique and DRC experienced an increase in fertility rate within the periods 1986–2016. Looking at the contributions made by each of the fertility parameters, the fertility preference (Fw) and the degree of Ip are the reasons for the variations in the changes in fertility. Rwanda registered a 37% decrease in its average wanted fertility desires, with a corresponding degree of Ip of 97% (Table 2). Malawi and Kenya on the same note registered a reduction in Fw of 51% and 54% and corresponding implementation indices of 82% and 84%, respectively.\n\nFigure 2 highlights the graphical correlation between Ip and unmet need for family planning. There is an inverse correlation between Ip and the unmet need for family planning. High unmet need for contraception leads to a low implementation index, since contraception is the sole contributing factor to fertility regulation (also referred to as the extent of Ip). This is because the extent of contraceptive availability and subsequent utilization of contraceptives is what defines Ip level. The absence of these essential birth control commodities leads to non-implementation of family planning, thereby failing to restrain Fn.\n\nFigure 3 echoes the performance of Ip in facilitating the reduction of fertility by each of the countries within the periods under study. Ethiopia, Rwanda and Sierra Leone are the three countries where Ip has most contributed to the fertility decline. However, in some countries, the limited or non-implementation of fertility led to the index not facilitating any declines in F0, thereby allowing the natural increase to take its course. These countries were Mozambique, Chad, Cameroun, Democratic Republic of Congo, Togo, Benin, and Congo Brazzaville.\n\n\nDiscussion and conclusion\n\nBased on the fertility preference and implementation indicators, fertility transition is indeed on course in a number of countries, going by the trend data for each country. The extent at which this occurs varies across countries, with each country exhibiting varied levels of implementation. The Fw and the degree of Ip are therefore key to the prevailing fertility in each country7. Countries where populations desired or Fw are in decline over time are believed to be high in their drive to lower their overall F0. The suppressed desired or Fw correspond to a high index of implementation. Subsequently, exhibit the highest transition changes. The prevailing F0 of a country therefore depends on the interplay between the fertility desires and the degree of Ip which is dependent on the availability of family planning commodities (proportion of demand satisfied). Reduction in fertility hence demands low desired fertility and high index of implementation simultaneously. This implies that those countries with only one high parameter performance (i.e. either suppressed Fw or high implementation index) among the two exhibits only but between moderate to limited reduction in fertility change.\n\nFurther, the generally observed decline in the indices of fertility (i.e. F0, Fn, Fw and Ip) confirms the strength of the family planning program efforts by the various stakeholders in making birth control technologies available (to curb the unmet need thereby satisfying demand), accessible and affordable to their populace as well as improved contraceptive technology. This is due to the fact that only birth control technologies are known to facilitate the implementation of couples’ fertility desires. Looking at the association between Ip and the unmet need for contraception (Figure 2), countries with high unmet need for contraception exhibit low values of Ip3.\n\nThe converse is also true. The unmet need for contraception also reflects the proportion of demand satisfied. High unmet need for contraception is a function of lower total supply of family planning commodities required by all those women in need; implying low proportion of demand satisfied by the birth control commodities under the assumption that women of reproductive age are sexually active. This therefore leads to a surge in births as family planning is reduced owing to lower supplies of commodities than demanded. The unmet need has, however, progressively slowed over the years as births have reduced overtime in the majority of countries within the sub-Saharan Africa; reflected too by the surging implementation index overtime (Table 4). This finding reflects the level of increase in sensitization, advocacy and public education by programs as well as the utilization of birth control technologies. Fw, as a key parameter measure for the fertility change, relies heavily on the proportion of demand for contraception that is satisfied8. Fw is only achievable through the conscious attempt by spouses to deliberately control the number of births they wish to have, assuming all women are reproductive and sexually active at the same time.\n\nSource: Bongaarts (1993). Fn, natural fertility; Fw, wanted fertility; Ip, fertility implementation index.\n\nWith the population well sensitized to trigger conscious decision making with regards to contraceptive use, this sensitization will subsequently influence the couples to demand for specific number of births within their means as opposed to mere natural child bearing with no control. It is therefore plausible to conclude that the improvement of the availability and the uptake of birth control technologies to a sensitized population is one of the most feasible means through which countries can fast track their fertility transitions. The access should not only take into consideration the quantity but also the quality of contraception available. Unconstrained access to contraception is therefore an important marker, with the index level acting as a proxy measure. The association between Ip and the unmet need to contraception suggests that this index can be used as an indicator for program success efforts. Going by the countries’ performances over time, thereby taking into consideration the constants (such as reproductive age and economic situation) and non-constants (such as health system endowment), one can conclude that the current fertility transition witnessed in Sub-Saharan Africa is only modest, but a work in progress at the same time. Further research is recommended on how best Ip can be used as a measure of family planning program efforts.\n\n\nData availability\n\nThe datasets analyzed during the current study are available in 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. The DHS datasets used in this study are shown in Table 1.",
"appendix": "References\n\nBongaarts J: Development: Slow down population growth. Nature. 2016; 530(7591): 409–12. PubMed Abstract | Publisher Full Text\n\nBongaarts J, Casterline J: Fertility Transition: Is sub-Saharan Africa Different? Popul Dev Rev. 2013; 38(Suppl 1): 153–68. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAgwanda A, Amani H: Population growth, structure and momentum in Tanzania. Economic and Social Research Foundation, 2014. Reference Source\n\nIbisomi LD, Odimegwu CO, Otieno AT, et al. editors.: Degree of preference implementation and fertility changes in developing countries. THE XXVTH IUSSP CONFERENCE Tours, France, 2005. Reference Source\n\nBongaarts J: The supply-demand framework for the determinants of fertility: An alternative implementation. Popul Stud. 1993; 47(3): 437–56. Publisher Full Text\n\nEasterlin RA, Crimmins EM: The fertility revolution: A supply-demand analysis. University of Chicago Press; 1985; 211–18. Reference Source\n\nWestoff CF, Ngabo F, Umubyeyi MA, et al.: Rwanda 2010: A dramatic change in reproductive behavior. Calverton, Maryland, USA: ICF International, 2013. Reference Source\n\nAgwanda A, Khasakhala A, Kimani M: Assessment of family planning services in Kenya: Evidence from the 2004 Kenya Service Provision Assessment survey. Calverton, Maryland, USA: Macro International, 2009. Reference Source"
}
|
[
{
"id": "58569",
"date": "15 Jan 2020",
"name": "Julianne Weis",
"expertise": [
"Reviewer Expertise Family planning",
"reproductive health",
"health systems",
"sub-Saharan Africa",
"global health",
"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 is a welcome exploration of a key source of debate in the FP/RH world: what is the more important source of fertility transition: population fertility preferences or contraception service delivery? The analysis was sound, but a qualified statistician needs to review the calculations to ensure accuracy.\nHowever, the conculsions are convincing on the importance of service delivery implementation and its relation to fertility transition.\nI would welcome more exploration of literature exploring this critical question - currently the article does not touch on other studies or the debates and calculations other authors have conducted. This is a huge gap in the study.\nThere is also not enough discussion of implications of findings for policy in FP/RH.\n\nThe article also needs a thorough copy-edit, especially the abstract, where results are written in a confused, unclear manner.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "5527",
"date": "29 May 2020",
"name": "Vincent Otieno",
"role": "Author Response",
"response": "This is a welcome exploration of a key source of debate in the FP/RH world: what is the more important source of fertility transition: population fertility preferences or contraception service delivery? The analysis was sound, but a qualified statistician needs to review the calculations to ensure accuracy. However, the conclusions are convincing on the importance of service delivery implementation and its relation to fertility transition.I would welcome more exploration of literature exploring this critical question - currently the article does not touch on other studies or the debates and calculations other authors have conducted. This is a huge gap in the study. Comment: This is already rectified in the reviewed manuscript and uploadedThere is also not enough discussion of implications of findings for policy in FP/RH. Comment: I have reworked the discussion of findings section to incorporate FP/RH The article also needs a thorough copy-edit, especially the abstract, where results are written in a confused, unclear manner Comment: The abstract has been reworked Is the work clearly and accurately presented and does it cite the current literature?Partly Comment: All citations updated Is the study design appropriate and is the work technically sound?Yes Are sufficient details of methods and analysis provided to allow replication by others?Yes If applicable, is the statistical analysis and its interpretation appropriate?I cannot comment. A qualified statistician is required. Comment: The statistical analysis have been validated by my supervisor Prof Alfred Agwanda of the University of Nairobi - Population Studies and Research Institute (PSRI) Are all the source data underlying the results available to ensure full reproducibility?Yes Are the conclusions drawn adequately supported by the results?Yes Competing InterestsNo competing interests were disclosed.Reviewer ExpertiseFamily planning, reproductive health, health systems, sub-Saharan Africa, global health, policy"
},
{
"c_id": "5529",
"date": "29 May 2020",
"name": "Vincent Otieno",
"role": "Author Response",
"response": "All reviewer coments rectified"
}
]
},
{
"id": "57588",
"date": "24 Jan 2020",
"name": "James Kiarie",
"expertise": [
"Reviewer Expertise Reproductive health and 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\nAbstract Background: “disparities amid almost similarities in policies is a cause of concern to demographers\" – not sure what this is in reference to\n\nAbstract Methods:\n'Using Bongaarts reformulation of Easterlin and Crimmins conceptual scheme of 1985 on Demographic and Health Survey Data (DHS) data collected overtime across countries' – Not clear what the next sentences are justifying. 'The understanding of the current transition in general would help to reassess and provide explanations to the observed latest fertility dynamics at play. This study therefore is an attempt to explain the current fertility transition through women’s fertility preference' – Justification should be moved to background or objectives\nIntroduction:\nThe sentences “African fertility transitions occurred earlier than anticipated if Africa had followed the non-African relationship between fertility and development” and “The key features of African fertility regimes indicate that at a given level of development, Africa’s fertility is higher,” are contradictory.\nThe authors need to discuss the limitation of the model. Such assumption is that F0 = Fw + Fu yet we know there are situations that F0 is less than Fw. How will this explain an increasing observation that due to various factors many women do not achieve their desired fertility.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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/8-1748
|
https://f1000research.com/articles/9-168/v1
|
06 Mar 20
|
{
"type": "Research Article",
"title": "Benefit of cinnamon (Cinnamomum burmannii) in lowering total cholesterol level after consumption of high-fat containing foods in white mice (Mus musculus) models",
"authors": [
"Annisa Pulungan",
"Yunita Sari Pane",
"Annisa Pulungan"
],
"abstract": "Background: Hypercholesterolemia is a condition where cholesterol levels in the body exceed the normal range. In Indonesia, the development of traditional medicine is carried out by examining plants known for their medicinal qualities, including cinnamon (Cinnamomum burmannii). There are many nutritional components contained in cinnamon, such as cinnamaldehyde. This has been suggested as a substance that can reduce cholesterol and triglyceride levels. This study was conducted to determine the efficacy of cinnamon in reducing total cholesterol levels of mice (Mus musculus) given high-fat feed. Methods: This is an experimental study with a pre-post control study design. The groupings were performed by a simple random sampling method. There were five groups (n=6/group): 1) Negative control (aquadest); 2) positive control of high-fat containing food (HFC; quail's yolk); 3) HFC + cinnamon extract (CE; dose 2mg/20g body weight (BW); 4) HFC + CE (dose 4mg/20gBW); 5) HFC + CE (dose 8mg/20gBW). The study was conducted for 28 days. The intervention of CE started on day 15 and ended on day 28. Measurement of total cholesterol and BW of mice was performed on days 0, 14 and 28. Results: A decrease in the total cholesterol of mice (p = 0.001) was found in the groups that consumed CE. However, there was not a significant change between groups in mice BW (p = 0.419). Conclusions: Providing cinnamon extract (Cinnamomum burmannii) for 28 days could decrease total cholesterol levels in mice compared to those not given cinnamon extract when consuming high-fat containing foods.",
"keywords": [
"hypercholesterolemia",
"cinnamon",
"high-fat feed",
"quail's yolk",
"body weight of mice"
],
"content": "Introduction\n\nHypercholesterolemia is a state where the cholesterol level in the body exceeds the normal range. Hypercholesterolemia can increase the risk of atherosclerosis, coronary artery disease, pancreatitis, diabetes mellitus, thyroid disorder, liver disease and renal disease1.\n\nData from the Indonesian Ministry of Health in 2019 reported it’s estimated that coronary artery disease to be the first killer, 26.4% of all deaths in Indonesia2. Out of the 17 million the premature death (under the age of 70) due to non Communicable disease, most cases are in low-and middle income countries, and 37% are caused coronary heart disease and 6.7 million of these were triggered by stroke (WHO, 2015)3.\n\nIn Indonesia, the development of traditional medicine is performed by exploring herbs known to be beneficial for health, and this information is passed from generation to generation. One such herb is such as gambier (Uncaria gambir Roxb.)4, which has antioxidant effects and lowers blood glucose when treating type 2 diabetes mellitus. There is also bangun-bangun leaves (Coleus amboinicus), which are believed to be effective in relieving pain when distilled as ethanol and water extract5. Traditional medicine that comes from plants is used commonly by many people in Indonesia. One of these medicines is cinnamon (Cinnamomum burmannii,) which is often eaten daily with food. Cinnamon is a native plant in Indonesia and can be found abundantly in Central of Java, (Karanganyar), West Sumatra (Padang), Jambi (Kerinci), etc6. Cinnamon has been shown to reduce total cholesterol, low-density lipoprotein and triglyceride levels, as well as increasing high-density lipoprotein levels7. Cinnamon is a flavoring ingredient that has been used in daily routines as a spice. Data from various literature on cinnamon reveal that it mainly contains essential oils and important compounds such as cinnamaldehyde, eugenol, cinnamic acid, and cinnamate6.\n\nThis study was conducted to determine the efficacy of cinnamon in reducing total cholesterol levels of mice (Mus musculus) given high-fat feed. Mice were used in this study because of they have a similar biology to humans, and can therefore be a model for human hyperlipidemia8–10.\n\n\nMethods\n\nThis is an experimental study in an animal model with a pre-post control study design. This study was conducted at the Pharmacology Laboratory of the Faculty of Medicine, Universitas Sumatera Utara, Indonesia.\n\nThis study was approved by the Health Research Ethical Committee of Universitas Sumatera Utara (No:55/TGL/KEPK FK USU-RSUP HAM/2019).\n\nThis study used mice that were given high-fat containing food (HFC) to provide a hyperlipidemic mouse model. The intervention was carried out by giving different CE doses to find the dose that reduces the total cholesterol level of the mice. Cholesterol total levels were obtained from the blood by cutting the mice's tail. To ensure the experimental animals remained in a comfortable condition, the mice were given ketamine anesthesia intraperitoneally (80 mg/kgBW) before cutting of the tail11.\n\nThe experiments took place at the Laboratory of Pharmacology – Universitas Sumatera Utara (USU). The animals were purchased from the Department Biology of Mathematics and Scientific Faculty USU. In total, 30 male white mice (Mus musculus), Swiss Webster strain, 10 to 12 weeks old, and weighing 25 – 40 g were used. Before conducting the study, the mice were adapted to their cages (plastic (30 × 20 × 10 cm) and covered with fine wire mesh; base of the cages was covered with rice husks as thick as 0.5 - 1 cm and replaced every day during the study) for 2 weeks before the experiment started. They had 12 hours of daylight (6:00 A.M. – 6:00 P.M.) and 12 hours of dark (6:00 P.M. – 6:00 A.M.). The mice were fed with standard feed (CP 551) from PT Charoen Pokphand-Indonesia and water was given ad libitum. Room temperature and humidity were kept at normal ranges. They were weighed once a week to avoid stress.\n\nAfter the adaptation period, the mice were randomly divided into five groups, with each group consisting of 6 mice.\n\nThe sample size was calculated according to Federer's formula12:\n\n(t-1) (n-1)>15\n\nt = the number of groups\n\nn=the number of samples\n\nAll 30 mice were given a number (1–30) using a SPIDOL marker pen, then randomized by putting the numbers in an envelope and dividing them into 5 groups according to the numbers are taken from the envelope8.\n\nGroups were as follows: 1) K0, negative control group/placebo, no treatment or high-fat food (only given aquadest 0,2 cc); 2) K1, positive control group, diet of HFC (quail yolk); 3) K2, HFC + cinnamon extract (CE) dose 2mg/20g body weight (BW); 4) K3, HFC + CE dose 4mg/20gBW; 5) K4, HFC + CE dose 8mg/20gBW.\n\nThe study was conducted for 28 days.\n\nCinnamon extract. CE was given to the mice every morning at 8:00 am. Animals were kept during research in the mice laboratory in the Pharmacology laboratory. Before being given CE, it was dissolved with aquadest. The extract was given to the mice orally using feeding tubes. CE interventions began on the 15th day until the 28th day.\n\nCinnamon extract (Cinnamomum burmannii) was obtained as Herbilogy Cinnamon Extract Powder® (PT.Phytochemindo Reksa, Bogor, Indonesia; batch no. 033CT).\n\nHigh-fat containing food. The quail egg yolk was used to induce hypercholesterolemia, because the concentration of the lipid in quail egg yolk is higher than chicken egg yolk13. A dosage of 0.5ml/day was given until day 28. The administration of quail egg yolk was done orally using feeding tubes as per the administration of CE.\n\nDetermination of dose of cinnamon. Based on research by Vanessa et al. in 2013, who saw a decrease in total blood cholesterol levels in white rats (Rattus norvegicus) by administering instant cinnamon powder drink (Cinnamomum burmannii BI.) at a dose of 14.4 mg and 43.2 mg for 14 days. In their research, it was found that a dose of 14.4 mg can reduce cholesterol levels in rats. Therefore we used a dose of 14.4 mg, because it was effective and efficient14. We converted its dose to mice and increased it with variation doses of 2, 4 and 8 mg. The value converted from 200g rat to 20g mice is 0.14.\n\nRatdose=14.4mg\n\nConversiontomice=0.14×14.4mg=2.01mg\n\n~2mg/20gBWmice\n\nMeasurement of total cholesterol and BW of mice was performed on days 0, 14 and 28 (every two weeks). Ketamin 80 mg/kgBW mice intraperitoneally injected for anesthesia before the tail of the mice was cut11.\n\nMice tails were cut 1-2cm to draw blood, which was then assessed for total cholesterol using the digital autocheck® cholesterol measurement tool. Mice were weighed using a digital scale.\n\nData were analyzed using SPSS 24. The number average of sample data presented as mean ± SD. The one-way ANOVA statistical analysis to indicate the effects of treatments for all group. If the result is significant (p < 0.05), then bootstrapping was performed using post-hoc Bonferroni for differentiating between each group.\n\n\nResults\n\nFigure 1 shows that groups K0 (negative controls), K1 (positive controls), K2 (CE 2 mg/20gBW), K3 (CE 4 mg/20gBW), and K4 (CE 8 mg/20gBW), had a decrease in total cholesterol levels. There is a significant difference in total cholesterol among the groups (p=0.001 between groups). A post-hoc Bonferroni test was performed to see the difference in total cholesterol averages within each group.\n\nK0, negative control group, no treatment or high-fat food (aquadest); K1, positive control group, diet of high-fat containing food (HFC; quail's yolk); K2, HFC + cinnamon extract (CE) dose 2mg/20g body weight (BW); K3, HFC + CE dose 4mg/20gBW; K4, HFC + CE dose 8mg/20gBW.\n\nIt was seen that there was a difference in average total cholesterol levels between K0 (107.5 ± 1.87 mg/dl) vs K1 (120.3 ± 5.53 mg/dl), (p = 0.001). This shows that in the K1 group (positive control) given quail yolk succeeded in increasing cholesterol in mice with a significant difference compared to the K0 group (negative control), which was only given aquadest. In addition, differences in total cholesterol levels in the K1 group (120.3 ± 5.53 mg/dl) compared with K2 (107.3 ± 3.61 mg/dl), K3 (106.8 ± 4.57 mg/dl) and K4 (106.7 ± 0.51 mg/dl) groups showed that a significant decrease in total cholesterol levels (p = 0.001). This proves that CE is efficacious in reducing total cholesterol levels. Between groups K2, K3, and K4, which were all given CE, there was not a significant difference between total cholesterol levels (p > 0.05).\n\nFigure 2 shows that there were increases in BW in all five groups. The largest increase of BW was in group K2, which was the positive control group who were given HFC quail egg yolk at 0.5 ml/20gBW. The smallest increase in BW was found in group K5, which was the group provided with HFC quail egg yolk and CE with the dose of 8 mg/20gBW (highest dose in this study). One-way ANOVA showed that there was no significant difference in BW between groups (p=0.419), which could be inferred that the action of giving CE gave no effect in increasing BW in the mice.\n\nK0, negative control group, no treatment or high-fat food (aquadest); K1, positive control group, diet of high-fat containing food (HFC; quail's yolk); K2, HFC + cinnamon extract (CE) dose 2mg/20g body weight (BW); K3, HFC + CE dose 4mg/20gBW; K4, HFC + CE dose 8mg/20gBW. *Mean delta = (average BW at the end of the study) - (average BW at the beginning of the study)\n\n\nDiscussion\n\nThe aim of the study to investigated cholesterol levels was lowered in mice using CE and quail egg yolks for a high-fat diet for 28 days. In this study, it was proven that giving 0.5ml quail egg yolk for 28 days increased the total cholesterol level between the negative and positive control groups (post-hoc Bonferroni K0 vs K1; p=0.001).\n\nThere was a significant difference in the decrease of total cholesterol after treatment among all groups in this research. However, this is not consistent with Vanessa et al. (2013) who stated that there was a decrease, but the difference was not statistically significant. Their study was only conducted for 14 days, whereas our study was conducted for 28 days, leading to differing results14.\n\nCholesterol is formed by the action of HMG-CoA reductase enzyme (3-hydroxy-3-methylglutaryl-CoA)15. If hypercholesterolemia is left without implementing proper diet or treatment, it can cause occlusion in a blood vessel. To treat hypercholesterolemia, some medicines, such as simvastatin, could be given15. The use of statins will competitively block HMG-CoA reductase and efficiently reduce serum LDL cholesterol. But, treatment using simvastatin can cause rhabdomyolysis16. Thus, it needed research on traditional or herbal medicine, e.g. CE, that needs developed and have minimal adverse effects.\n\nCinnamon (Cinnamomum burmannii) has cinnamaldehyde as its biggest compound. Cinnamaldehyde, a phenolic component abundantly found in Cinnamomum6. Bandara et al. (2011) stated that cinnamon had the ability to be an antioxidant, antivirus, antifungal, antimicrobe, antitumor, and can lower cholesterol and blood pressure with low fat compounds. Cinnamon is believed to have a direct role in lipid metabolism and preventing hypercholesterolemia and hypertriglyceridemia, as well as in preventing free fatty acids with its strong lipolytic activity17. In the present study we believe that the increase of cholesterol levels due to quail egg yolk could be decreased with cinnamon by its ability to block HMG-CoA reductase enzyme and suppress lipid peroxidation through increased antioxidant enzyme activity18. In research by Pai et al. (2013), it was stated that cinnamaldehyde could lower cholesterol and triglyceride levels by the action of some enzymes secreted in certain amounts, which might contribute to bile acid synthesis19.\n\nOur study showed that there was no difference in BW of the mice between groups (p = 0.419). This is not consistent with Vafa et al. (2012) who stated that consuming 3 grams of cinnamon for 8 weeks could decrease some biochemical and anthropometric variables compared to previous states significantly, i.e. a decrease of 1.19% body weight, 1.54% body mass index and 1.36% body fat. In addition to lowering BW variables, it could also decrease fasting blood glucose level by about 9.2%, and 6.12% of HbA1c and 15.38% of triglyceride levels20.\n\nLeaf and Antonio (2017) stated that the higher the food intake, the higher the increase in BW21. In the present study, in addition to standard food intake, giving hypercholesterol and interventional food should increase BW, because the mice had increased energy intake. The amount of food intake will affect the amount of energy intake, which will be saved as fat and impact on the mice’s BWs, since energy intake is inversely proportional to physical activity. A high-fat diet group has low sensitivity to leptin, which results in increased appetite and food intake, thus increases the BW21. However, this was not the case in our study.\n\n\nConclusion\n\nIn the present study, a high-fat containing food used in combination with cinnamon extract (Cinnamomum burmannii) for 28 days could decrease total cholesterol levels in mice. Cinnamon extract is believed to have direct effects in lipid metabolism and prevent hypercholesterolemia and hypertriglyceridemia, as well as decreasing free fatty acids by its strong lipolytic activity.\n\n\nData availability\n\nFigshare: data Analysis-cholesterol and bodyweight of mice.docx, https://doi.org/10.6084/m9.figshare.11901174.v222\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\n\nAcknowledgments\n\nThe author gives appreciation and thanks to the Pharmacology Laboratory of the Faculty of Medicine, Universitas Sumatera Utara for allowing the author to use the facility for collecting data in this research. In addition, thanks to all lecturers and examiners who had given advice and suggestions to the author, and all other parties who had contributed to this research.",
"appendix": "References\n\nStapleton PA, Goodwill AG, James ME, et al.: Hypercholesterolemia and microvascular dysfunction: interventional strategies. J Inflamm (Lond). 2010; 7: 54. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKemenkes RI: Hari Jantung Sedunia (World Heart Day): Your Heart is Our Heart Too. 2019. Reference Source\n\nWHO (World Health Organization), 2015. Reference Source\n\nPane YS, Ganie RA, Lindarto D, et al.: The Effect of Gambier Extract on The Levels of Malondialdehyde, Superoxide Dismutase, and Blood Glucose in Type 2 Diabetes Mellitus Patients. Asian J Pharm Clin Res. 2018; 11(10): 121–124. Publisher Full Text\n\nPane YS, Sufitni S, Lumongga, F, et al.: The Effectiveness of Coleus Amboinicus Leaf Extracts as an Analgetic Activity on Mice Exposured to Acetic acid. Asian J Pharm Clin Res. 2018; 11(9): 301–4. Publisher Full Text\n\nPlumeriastuti H, Budiastut, Effendi MH, et al.: Identification of bioactive compound of the essential oils of Cinnamomum burmannii from several areas in Indonesia by gas chromatography–mass spectrometry method for antidiabetic potential. Natl J Physiol Pharm Pharmacol. 2019; 9(4): 279–283. Publisher Full Text\n\nAlsoodeeri FN, Alqabbani HM, Aldossari NM: Effects of Cinnamon (Cinnamomum cassia) Consumption on Serum Lipid Profiles in Albino Rats. J Lipids. 2020; 2020: 8469830. Publisher Full Text\n\nRussell WMS, Burch RL: The Principles of Humane Experimental Technique. London: Methuen& Co.Ltd. (Reissued: 1992, Universities Federation for Animal Welfare, Herts, England.) 1959. Reference Source\n\nFitzpatrick A: Ethics and animal research. J Lab Clin Med. 2003; 141(2): 89–90. PubMed Abstract | Publisher Full Text\n\nFesting MF: Principles: the need for better experimental design. Trends Pharmacol Sci. 2003; 24: 341–5. PubMed Abstract | Publisher Full Text\n\nVertebrate Animal Research. The University of Iowa. Reference Source\n\nFederer WY: Experimental Design, Theory and Application. New York: Mac. Millan. hal. 1963; 544. Reference Source\n\nUkachukwu UG, Ozougwu VEO, Nwankwo NE: A Comparative Study on the Total Cholesterol, Triacylglycerides and Lipid Concentrations of Quail and Chicken Eggs. International Journal of Research in Pharmacy and Biosciences. 2017; 4(10): 11–16, ISSN 2394-5885 (Print) & ISSN 2394-5893 (Online). Reference Source\n\nVanessa R, Purwijantiningsih LME, Aida Y: Pemanfaatan Minuman Serbuk Kayu Manis (Cinnamomum burmanii.BI.) Untuk Menurunkan Kadar Kolesterol Total Darah Pada Tikus Putih. 2014. Reference Source\n\nCherng S, Young J, Ma H: HMG-CoA reductase (3-hydroxy-3-methyl-glutaryl-CoA reductase) (HMGR). The Journal of American Science. 2008; 4(3): ISSN 1545-1003. Reference Source\n\nEzad S, Cheema H, Collins N: Statin-induced rhabdomyolysis: a complication of a commonly overlooked drug interaction. Oxf Med Case Reports. 2018; 2018(3): omx104. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBandara T, Uluwaduge I, Jansz ER: Bioactivity of Cinnamon with Special Emphasis on Diabetes Mellitus: A review. Int J Food Sci Nutr. 2012; 63(3): 380–386. PubMed Abstract | Publisher Full Text\n\nLee JS, Jeon SM, Park EM, et al.: Cinnamate Supplementation Enhances Hepatic Lipid Metabolism and Anti Oxidant Defense Systems In High Cholesterol Fed Rat. J Med Food. 2003; 6(3): 183–191. PubMed Abstract | Publisher Full Text\n\nPai PG, Habeeba PU, Ullal S, et al.: Evaluation of Hypolipidemic Effects of Lycium barbarum (Goji berry) in a Murine Model. Journal of Natural Remedies. 2013. Reference Source\n\nVafa M, Mohammadi F, Shidfar F, et al.: Effects of cinnamon consumption on glycemic status, lipid profile and body composition in type 2 diabetic patients. Int J Prev Med.2012; 3(8): 531–6. PubMed Abstract | Free Full Text\n\nLeaf A, Antonio J: The Effects of Overfeeding on Body Composition: The Role of Macronutrient Composition - A Narrative Review. Int J Exerc Sci. 2017; 10(8): 1275–1296. PubMed Abstract | Free Full Text\n\nPulungan A, Pane YS: data Analysis-cholesterol and bodyweight of mice. docx. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.11901174.v2"
}
|
[
{
"id": "61018",
"date": "02 Apr 2020",
"name": "Tahereh Farkhondeh",
"expertise": [
"Reviewer Expertise metabolic disorder"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 investigated the effect of cinnamon extract on body weight and cholesterol levels of mice-fed HFD. It is a good idea.\nSome revisions should be done:\nAbstract\nIn the method section several point should be cleared:\nType of cinnamon extract. Female or male mice.\n\nIntroduction\nThe aim of study should be better mentioned. it was not clear the question about the effect of plant on the lipid profile. In the past paragraph, the author described the lipid lowing effect of extract. Did this evidence extracted from traditional medicine reports?\nMethod\nDid you used xylazine with ketamine for analgesic effect It should be mentioned. Animal ethics did not accept this anesthesia procedure.\n\nPlease more about cholesterol measurement (technique, instrument, kit).\nConclusion\nIf you tested the quail egg yolks as a high-fat diet that induces hyperchloestromia. It is necessary in the abstract and introduction in the aim section.\n\nIn the first paragraph, you should about your findings without analytic indicator such as p-value.\n\nYou did not measure the changes in TG or FFA therefore, you should not concluded about in the conclusion section.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": [
{
"c_id": "5400",
"date": "07 Apr 2020",
"name": "yunita sari pane",
"role": "Author Response",
"response": "RESPONSE to review's Tahereh FarkhondehCardiovascular Diseases Research Center, Birjand University of Medical Sciences, Birjand, IranThe present study investigated the effect of cinnamon extract on body weight and cholesterol levels of mice-fed HFD. It is a good idea. Some revisions should be done:Abstract1.In the method section several point should be cleared: 1.Type of cinnamon extract.Answer:Type of cinnamon extract used in this study had been explained part of METHODS (page 3 manuscript in F1000research).We used Cinnamomum burmannii in powder extract. 2. Female or male mice.Answer:The sample animal were male mice. This is one of the inclusion criteria of this study. Its mean to homogenized (make smaller of the differences in each samples) for prevent BIAS. As we known, there are differences between male and female will increasing number of lipid and dysfunction metabolic, including of hyperlipidemia. It related to level of hormones. Introduction The aim of study should be better mentioned. Answer:Thank you for your comment, we would confirm our statement on the aim of this study.The aim of the study to determine cinnamomum burmannii effective in reducing total cholesterol levels of mice (Mus musculus) given high fat feed. It was not clear the question about the effect of plant on the lipid profile.Answer:We had explained about the effect of plant on the lipid profile related to few references.Please see in part of DISCUSSION (page 5 manuscript in F1000reseach).Cinnamon is believed to have a direct role in lipid metabolism and preventing hypercholesterolemia and hypertriglyceridemia, as well as in preventing free fatty acidswith its strong lipolytic activity17.In the present study we believe that the increase of cholesterol levels due to quail egg yolk could be decreased with cinnamon by its ability to block HMG-CoA reductase enzyme and suppress lipid peroxidation through increased antioxidant enzyme activity18.In research by Pai et al. (2013), it was stated that cinnamaldehyde could lower cholesterol and triglyceride levels by the action of some enzymes secreted in certain amounts, which might contribute to bile acid synthesis19. (This study needed continuos some research in the future to measured number of cinnamaldehyde in Cinnamon Extract Powder® per 1 mg CE used. Then we’ll do new research to prove how CE mechanism as anti hypercholesterolemia, Is it blocked by HMG-CoA reductase enzyme and suppress lipid peroxidation through increased antioxidant enzyme activity or dysfunction of bile acid synthesis or both mechanism) ? In the past paragraph, the author described the lipid lowing effect of extract. Did this evidence extracted from traditional medicine reports?Answer:Yes, the explain about “this has been suggested as a substance……triglyceride levels” bettermoves to part of DISCUSSION, because we took from one of reference. We will add inform about the quail egg yolks as a high-fat diet that induces hypercholesterolemia in part of ABSTRACT.We can not add inform in the AIM of study. Because, if we added in the Aims of study, it was not consequence to the title of study. If we changed title \"Benefit of cinnamon (Cinnamomum burmannii) in lowering total cholesterol level after consumption of the quail egg yolk as a high-fat diet that induces hypercholesterolemia\". It will make the title very long and not in accordance to the rules of journals F1000Research. Method Did you used xylazine with ketamine for analgesic effect It should be mentioned. Animal ethics did not accept this anesthesia procedure. Answer:Yes, we did. We corrected uncomplete in methods ketamin with xylazine. We choosed of Injectable Anesthetic Agents, we used Ketamine/xylazine* We follow to GUIDELINES - PREPARATION OF KETAMINE/XYLAZINE COCKTAIL FOR MICE. Dose Ketamine 80-100 mg/kg intra peritoneal (IP) and Xylazine 10-12.5 mg/kg IP.(reference 11. Vertebrate Animal Research. The University of Iowa).The administration of effective anaesthetic agents to avoid impact/effects of the experimental procedures when we did cut off mice tail to collect blood sample with anesthesia to make minimize pain and prevent distress caused of pain in mice. Please more about cholesterol measurement (technique, instrument, kit). ANSWER:In Laboratory analysis study showed how was technique to take blood from tail*correction in methods study cut of mice tail.“Mice tails were cut 2 – 5 mm to draw blood, which was then assessed for total cholesterol using the digital autocheck®(merck of digital cholesterol measured tool). We used stick cholesterol to measured total cholesterol of mice. Regarded to follow National Centre for the Replacement, Refinement & Reduction (NC3Rs) of Animals in Research to measured (page 4 manuscript in F1000reseach). Conclusion If you tested the quail egg yolks as a high-fat diet that induces hyperchloestromia. It is necessary in the abstract and introduction in the aim section. Answer:Yes you right. We had tried to add in abstract before, but it was more than 250 words, it was not allow with Journal F1000research rules. But we will try again to put in your comment in abstract. In the first paragraph, you should about your findings without analytic indicator such as p-value. Answer: Im sorry, I can’t catch what you mean in this statement. Would you like to describe more detail? You did not measure the changes in TG or FFA therefore, you should not concluded about in the conclusion section. Answer:Yes, thank you for your comment. We are agree with you. Conclusion:The cinnamon extract (Cinnamomum burmannii) proved it can be decrease of total cholesterol levels of for 14 days in mice consuming high-fat containing compared to group without given CE."
}
]
},
{
"id": "61729",
"date": "14 Apr 2020",
"name": "Mourad Akdad",
"expertise": [
"Reviewer Expertise Ethnopharmacology",
"medicinal plants",
"diabetes",
"hypertension"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 evaluate the effect of cinnamon extract (C. E) on cholesterol levels in mice with hypercholesterolemia. Authors demonstrated that C. E is useful to decrease cholesterol level, so its a promising plant to explore in the treatment of hypercholesterolemia and the related illness (hyperlipidemia, diabetes, atherosclerosis…).\nCinnamon has been shown to reduce total cholesterol, low-density lipoprotein and triglyceride levels, as well as increasing high-density lipoprotein levels. » precise the model of the study.\n\nIn introduction section, you must conclude by the aim of your research, this phrase “Mice were used in this study because of they have a similar biology to humans, and can therefore be a model for human hyperlipidemia8–10. must be include in discussion.\n\nAvoid repetition such as: “This study was conducted at the Pharmacology Laboratory of the Faculty of Medicine, Universitas Sumatera Utara, Indonesia. » « The experiments took place at the Laboratory of Pharmacology Universitas Sumatera Utara (USU). »\n\nWhat is the type of extract (aqueuous, ethanolic…..), and the part of plant by which industry do extraction.\n\nTry to explain why you don’t have a dose-dependent effect in your experiment.\n\n« Figure 1 shows that groups K0 (negative controls), K1 (positive controls), K2 (CE 2 mg/20gBW), K3 (CE 4 mg/20gBW), and K4 (CE 8 mg/20gBW), had a decrease in total cholesterol levels. » why all groups have a decrease of Total Cholesterol, you don’t precise the days of this results you must clarify the idea.\n\nIn the section results, days of measurement must be righted in the description to clarify the idea.\n\nThe smallest increase in BW was found in group K5 », K5 ??, you would like to say K4?\n\nIn the present study we believe that the increase of cholesterol levels due to quail egg yolk could be decreased with cinnamon by its ability to block HMG-CoA reductase enzyme and suppress lipid peroxidation through increased antioxidant enzyme activity18. » the mechanism involved in the antihypercholesterolemic effect must be declared with precaution, ‘may be’.\n\nOur study showed that there was no difference in BW of the mice between groups (p = 0.419). This is not consistent with Vafa et al. (2012)1 who stated that consuming 3 grams of cinnamon for 8 weeks could decrease some biochemical and anthropometric variables compared to previous states significantly, i.e. a decrease of 1.19% body weight, 1.54% body mass index and 1.36% body fat. In addition to lowering BW variables, it could also decrease fasting blood glucose level by about 9.2%, and 6.12% of HbA1c and 15.38% of triglyceride levels20. » model used by the study cited must be declared.\n\nIn the present study, a high-fat containing food used in combination with cinnamon extract (Cinnamomum burmannii) for 28 days could decrease total cholesterol levels in mice. » this conclusion must be reformulated, high-fat containing food was used only to have the model for studying the effect of CE on cholesterol level, so the conclusion must be related only to CE.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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/9-168
|
https://f1000research.com/articles/9-423/v2
|
29 May 20
|
{
"type": "Research Article",
"title": "Glycoalkaloids in commercial potato varieties traded in Nairobi, Kenya",
"authors": [
"Consolata Nolega Musita",
"Michael Wandayi Okoth",
"George Ooko Abong'",
"Duke Gekonge Omayio",
"Consolata Nolega Musita"
],
"abstract": "Background: Glycoalkaloids are naturally occurring toxins in potatoes which, at high levels, pose food safety concerns to consumers. Their concentrations in potatoes are dependent on postharvest handling, variety and stress factors tubers are exposed to. Limited information, however, exists on levels of glycoalkaloids in commercially traded potato tubers in Kenya. The current study sought to determine the glycoalkaloid levels in potatoes traded in Nairobi, Kenya. Methods: Three potato varieties, Shangi, Dutch Robjin and Royal sold in open-air markets and supermarkets were randomly sampled and their glycoalkaloid levels determined by high-performance liquid chromatography. Results: The levels varied significantly (p<0.05). The Shangi variety had the highest glycoalkaloids with a mean of 410.35 mg kg-1 dry weight with samples from supermarkets having the highest levels (550.8 mg kg-1). The same variety from open air markets averaged 382.26 mg kg-1 dry weight compared to the Dutch Robjin (129.2 mg kg-1 dry weight) and Royal variety (98.2 mg kg-1 dry weight) which had the least levels of glycoalkaloids. Conclusions: The levels in sampled tubers did not exceed the recommended levels of 1000 mg/kg on dry weight basis and, therefore, consumption of these potatoes would not raise safety concerns. There is, however, a need to ensure that marketing of tubers is carried out under conditions that minimize occurrence of glycoalkaloids, especially for the Shangi variety, which is the most common in the markets but had relatively high levels of these toxins. There is also a need to educate marketers on the need for proper storage and handling of potatoes during marketing to avoid risk of accumulation of glycoalkaloids.",
"keywords": [
"Glycoalkaloids",
"Commercial potato varieties",
"Nairobi"
],
"content": "Introduction\n\nPotatoes are an important food crop in sub-Saharan Africa (SSA). The estimated annual production of potatoes in SSA in 2016 was 11.6 million tonnes (FAOSTAT, 2018). Potatoes are ranked among the four most important and largely consumed food crops globally, the others being rice, wheat and maize (King & Slavin, 2013). Potatoes provide the consumers with nutrients such as carbohydrates, vitamins and minerals (Furrer et al., 2016) and improve the nutrient density of diets when incorporated into other foods (Gibson & Kurilich, 2013).\n\nGlycoalkaloids, which naturally occur in potato tubers, compromise their safety at higher levels and can render the tubers or the derivative products unsuitable for consumption, with levels beyond 200 mg kg-1 fresh weight being considered unsafe for human consumption (Smith et al., 1996a). The most common glycoalkaloids in potatoes are α-solanine and α-chaconine. α-chaconine occurs in a somewhat higher proportion and is the most potent of the two, being twice as toxic as α-solanine (Friedman, 2006).\n\nGlycoalkaloid levels in potatoes traded in the market may be influenced by transportation and storage conditions (Chuda et al., 2004). Potatoes that are sprouting or subjected to light exposure, inappropriate storage, extreme temperature, wounding, and mechanical injury accumulate glycoalkaloids at a faster rate (Cantwell, 1996). In addition, the level of accumulation of glycoalkaloids in potatoes subjected to these stress factors varies among tuber varieties (Furrer et al., 2016).\n\nWare potatoes are made available to consumers through different marketing systems and handling of these potatoes may influence the formation and accumulation of glycoalkaloids. In Kenya, the potato forms a major part of the diet of many consumers. It is incorporated into local Kenyan dishes or may be eaten boiled, baked or fried. Furthermore, processed potato products such as crisps and French fries (locally known as chips) are in high demand among urban consumers and hence are a great part of menus in restaurants and hotels in major urban centers (Abong’ et al., 2009). Of interest to food safety, however, is that in Kenyan markets, it is not uncommon to find potatoes exposed to stress factors thus exposing consumers to the dangers of glycoalkaloids. However, the exposure of Kenyan consumers to these toxins as a result of consuming potatoes and potato products is not supported by adequate data. Furthermore, not much has been done to quantify the levels of glycoalkaloids in tubers traded in Kenyan markets, meaning that the level of risk to consumers has not been adequately documented. The current study, therefore, sought to establish the glycoalkaloid levels of potatoes traded in various markets and supermarkets in Nairobi, Kenya.\n\n\nMethods\n\nThe study was carried out in Nairobi County, Kenya in February and March 2018. The county hosts the capital city of Kenya and has nine sub-counties: Makadara, Embakasi, Starehe, Langata, Kasarani, Westlands, Kamukunji, Dagorreti and Njiru. The county’s population is estimated to be over 3 million (KNBS, 2009). Most of the people are low income earners hence live in slums. The county has many markets dealing in food stuffs with most of these food markets being open air markets. Five sub-counties were purposively selected for this study: Dagoretti, Westlands, Embakasi, Kamukunji and Starehe. These were purposively selected because most of Nairobi’s population is concentrated in these areas. From these five sub-counties, five major markets were purposively selected from which samples (from both open-air markets and supermarkets) were obtained. The five markets were Kawangware (Dagoretti), Kangemi (Westlands), Wakulima (Starehe), Kona (Embakasi) and Gikomba (Kamukunji).\n\nSample size determination. A total of 48 potato samples were used for the study; this figure was obtained using the formula of Fisher et al. (1991). From the 48 samples, 31 samples were collected from supermarkets and 17 samples were obtained from open-air markets.\n\nSample collection. Three potato varieties were used in the study: Shangi, Dutch Robjin and Royal. These were purchased from open air markets (Shangi only) and supermarkets (all three varieties). Only Shangi variety was obtained from the open-air markets since it was the most common and easily available variety in the markets during the sampling period.\n\nDetermination of moisture content. Moisture content of different potato varieties was determined in triplicate on fresh weight basis as per AOAC (2005) method number 930.15. The oven used was memmert 40500-IP20 (Schutzart, Germany).\n\nDetermination of glycoalkaloids. The levels of glycoalkaloids were determined by high pressure liquid chromatographic (HPLC) methods using Waters HPLC (Waters 2695- Waters Corporation, USA) and detection set at a wavelength of 202 nm using photodiode array detector (Waters 2996, USA).\n\nPotato samples obtained from each market were mixed thoroughly into one batch, from which a single sample was picked randomly for analysis. Randomness was ensured by picking tubers from the top section, middle section and bottom section of the mixed batch. This was done for all the samples from all the five markets thus resulting into five open-air market Shangi samples for analysis (representing the five study sub-counties). The same procedure was followed for the supermarket samples, resulting in five Shangi supermarket samples, two Dutch Robjin samples and two Royal samples for analysis.\n\nTubers were washed under running water, dried using cloth towel, peeled and chopped into 5mm pieces. Next, 20 g of each set were oven dried to a constant weight at 105°C. The dry samples were ground and packaged in sealed plastic bottles and stored for chemical analysis.\n\nExtraction of glycoalkaloids. A 2-g sample was mixed with 20 ml extraction solution comprising of water, acetic acid and sodium hydrogen bisulfite at of 100 ml/5 ml/0.5 g, respectively and shaken for 15 minutes by Burrell vertical shaker (Burrel Corp, Pittsburg, UK). Clarification of the mixture was then done by centrifugation for 30 min at 800 g using Labofuge A (Heraeus, Germany).\n\nCleaning of extract. Acetonitrile (5 ml) followed by 5 ml extraction solution were used in conditioning solid phase extraction (SPE) columns (Strata, Phenomenex). Into these columns, 10 ml of the supernatants were passed through the SPE columns at a controlled pressure after which SPE 4 ml wash solution (15% acetonitrile) was used to wash the glycoalkaloids. This was followed by elution with 4 ml LC mobile phase (60% acetonitrile in 0.01 M phosphate buffer) at a rate of 1–2 drops/s. The final volume collected was adjusted to 5 ml with LC mobile phase filtered through a 0.45-µm filter into vials and frozen ready for injection.\n\nThe HPLC instrument (Waters-2595, USA) was fitted with stainless steel LC column—250 ×4.6 mm, packed with Hypersil ODS (Shandon Southern Products Ltd., Astmoor, UK), 5-µm particle size, C18 phase. The operating conditions included a flow rate of 1.5 ml/min, injection volume of 50 µl, run time of 15 minutes, column temperature of 40°C and wavelength detection set at 202 nm. Glycoalkaloids were calculated based on external calibration curves generated from standards stock solutions and expressed as mg GAS/kg on dry weight basis.\n\nThe data were analyzed using GenStat version 15 software. Descriptive statistics in terms of mean and standard deviation were generated for moisture content and glycoalkaloids levels. Test for significant differences in the means was performed using one-way ANOVA at p<0.05. Means that were statistically different were separated using Fisher’s LSD test (p≤0.05).\n\n\nResults and discussion\n\nThe moisture content between the samples varied significantly (p≤0.05) (Table 1). However, there were no significant differences (p>0.05) in the moisture content for the sampled potato varieties obtained from supermarkets and open-air markets. The Royal variety had the highest moisture content (77.08%), while Shangi had 76.56% and Dutch Robjin had the least with 76.42%. These findings are lower than those reported by other researchers (Elbatawi et al., 2008; Zhang et al., 2018). However, these values are in agreement with other studies where similar findings have been reported (Hafezi et al., 2015; Sablani & Mujumdar, 2006; Xiao et al., 2011). These variations may be attributed to transpiration due to exposure of potatoes to sunlight and higher temperatures during marketing as well as the high relative humidity and long period on shelves before marketing (Chourasia et al., 2005). Moisture content of all potatoes analyzed, alongside glycoalkaloid content, is available as Underlying data (Musita et al., 2020).\n\nValues are given as mean of duplicate samples ± SD (standard deviation), n = 48. Means with different superscript letters are significantly different (Tukey’s test, p ≤ 0.05).\n\nD-Dutch Robjin; R-Royal; CN-Shangi from Kona market; GK-Shangi from Gikomba market; KN-Shangi from Kangemi market; KW-Shangi from Kawangware market; SD-Shangi from supermarkets in Dagoretti; SE-Shangi from supermarkets in Embakasi; SK-Shangi from supermarkets in Kamukunji; SS-Shangi from supermarkets in Starehe; WK-Shangi from Wakulima market\n\nThe total glycoalkaloid levels varied significantly (p<0.05) among the samples (Table 2 and Table 3). The Royal potato variety had the lowest glycoalkaloids levels (104.8 mg kg-1), then the Dutch Robjin (136.1 mg kg-1); the Shangi (483.6 mg kg-1) variety had the highest levels of glycoalkaloids on dry weight basis. The values for Dutch Robjin and Shangi varieties were, however, higher than those reported in a similar study, where the values ranged from 55.6 to 122.3 mg kg-1 (Bejarano et al., 2000). The values in the current study may be indicative of poor postharvest handling of potatoes during marketing as well as the varietal influence on glycoalkaloid levels. Since the Shangi variety had higher levels of glycoalkaloids than the other varieties, it is possible that many Kenyan consumers are exposed to these toxins, given this variety is the most common on the market.\n\nValues are given as mean of duplicate samples ± SD (standard deviation), n = 48. Means with different superscript letters in the same column are significantly different (Tukey’s test, p ≤ 0.05). D, Dutch Robjin; R, Royal; CN, Shangi from Kona market; GK, Shangi from Gikomba market; dw, dry; weight; KN, Shangi from Kangemi market; KW, Shangi from Kawangware market; WK, Shangi from Wakulima\n\nValues are given as means of samples ± SD (standard deviation), n = 48. Means with different lowercase superscript letters along the column are significantly different for the respective glycoalkaloids in the Shangi variety traded in both open-air markets and supermarkets. Means with different capital letter superscripts along the column are significantly different for the respective glycoalkaloid levels obtained from different varieties traded in the sampled supermarkets (Tukey’s test, p ≤ 0.05).\n\nThe levels of glycoalkaloids in potato tubers from the Shangi varieties procured from supermarkets and open air markets did not have significant differences (p>0.05) with the former having the highest levels of glycoalkaloids at 497.2 mgkg-1 compared to levels in Shangi samples obtained from open air markets which averaged 466.1 mgkg-1 (Table 3). The high levels of glycoalkaloids in the supermarket samples may be an indication of long periods of exposure to fluorescent light and longer periods before sales from the supermarket shelves. During these periods, the tubers accumulate chlorophyll and subsequently have increased levels of glycoalkaloids. Light plays a significant role in chlorophyll formation, resulting in “greening” on the surface of the potato. This “greening” has been associated with a rise in the concentration of glycoalkaloids, especially α-solanine (Pavlista, 2001). There is need for the local supermarkets and other retail shops to make use of greening scales that have been developed for discarding greened potatoes from retail displays so as to ensure that the potatoes sold in wholesale and retail shops are safe for consumption (Grunenfelder et al., 2006a; Grunenfelder et al., 2006b). These retail outlets could also substitute the fluorescent lighting in the display shelves with mercury lighting since studies have shown the rate of glycoalkaloids accumulation can be reduced by using mercury lighting instead of fluorescent lighting during display (Percival, 1999). This is attributed to the fact that mercury illumination contains few spectral lines (ultraviolet and infrared), which are less likely to enhance synthesis of glycoalkaloids and chlorophyll, unlike fluorescent light which contains ultraviolet spectra (Nema et al., 2008).\n\nThe concentrations of α-solanine and α-chaconine for the samples varied significantly (p<0.05) and ranged from 47.7 to 206.6 mg kg-1 and 50.5 mgkg-1 to 283.1 mgkg-1, respectively (Table 2). The ratio of α-solanine to α-chaconine ranged from 1:0.7 to 1:2.6. Glycoalkaloid intoxication is relative to this ratio and the ratio should, therefore, be as low as possible since these toxins occur simultaneously in potato tubers (Friedman, 2006). The ratios reported in this study are in agreement with other studies, which have reported variations from 1:0.5 to 1:7 (Kozukue et al., 2008; Lisiĕska et al., 2009; Tajner-Czopek et al., 2008; Valcarcel et al., 2014).\n\nNone of the sampled potato tubers exceeded the recommended safety levels of 1000 mg kg-1 dry weight, assuming a water content of 80% (Valcarcel et al., 2014) which is equivalent to 200 mg kg-1 on fresh weight basis (Smith et al., 1996b). Therefore, consumption of these varieties would result in insignificant glycoalkaloids intoxication to consumers. However, it is important to note that these toxicants have been found to bioaccumulate in the body, especially if daily consumption of foods containing the glycoalkaloids occurs (Omayio et al., 2016).\n\nDisparities in the levels of glycoalkaloids reported could also be a result of varietal effect. Studies have shown that some potato varieties tend to accumulate higher levels of glycoalkaloids compared to others. These findings are, therefore, in agreement with other studies (Aziz et al., 2012; Bejarano et al., 2000; Friedman et al., 2003; Valcarcel et al., 2014). The Shangi variety has also been shown to sprout easily when exposed to stressful conditions which is an indication of elevated levels of glycoalkaloids (Abong' et al., 2015).\n\nThe rate of glycoalkaloids accumulation is also dependent on the storage conditions. Light and temperature have been shown to be stress factors that lead to accumulation of the toxins (Griffiths et al., 1998; Grunenfelder et al., 2006a). The rate of glycoalkaloid accumulation is about 20% higher when potatoes are exposed to light and direct sunlight as compared to storage in dark conditions. Synthesis and accumulation of glycoalkaloids when potatoes are stored at 24°C has also been shown to be about twice the rate at 7°C (Cantwell, 1996; Griffiths et al., 1998; Şengül et al., 2004). It has been reported that generally, there is poor handling of potatoes by traders in Kenya, which continues to increase the risk of consumer exposure to glycoalkaloids (Musita et al., 2019). Differences in storage conditions for the potatoes from both open-air markets and supermarkets could have contributed to the levels of glycoalkaloids reported.\n\n\nConclusions and recommendations\n\nThe glycoalkaloids levels of the marketed potatoes did not exceed the recommended safety levels. Therefore, minimal intoxication would occur to consumers. However, it would be of interest for future studies to be conducted to assess the effect of long-term exposure to low levels of these toxins, a common occurrence among Kenyan consumers. The supermarkets samples had higher levels of glycoalkaloids and there is a need for the traders to be sensitized on appropriate handling of potatoes during marketing.\n\nIt is also essential that consumers and potato handlers select potato cultivars with minimal glycoalkaloid occurrence to ensure that there is minimum glycoalkaloid intoxication. Additionally, consumers are advised to peel potatoes before cooking to reduce the amount of glycoalkaloids since most of the toxins are concentrated in the peel.\n\nMoreover, postharvest practices that will reduce occurrence of glycoalkaloids are paramount. Relevant authorities should, therefore, be involved in raising awareness and implementation of post-harvest handling policies among potato vendors. These include storage of potatoes at lower temperatures, of about 5°C–10°C, keeping potatoes away from direct sunlight and marketing in opaque plastic films and bags that minimize effect of light on tubers. Furthermore, supermarket vendors need to be sensitized to ensure regular rotations of potatoes in retail shops exhibitions and replace fluorescent lighting with mercury lighting for potatoes on display.\n\n\nData availability\n\nFigshare: Sample and data_glycoalkaloids in potatoes_Consolata Musita.xls. https://doi.org/10.6084/m9.figshare.12301424.v1 (Musita et al., 2020).\n\nThis file contains the moisture and glycoalkaloid content of each sample analysed in this study.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
"appendix": "Acknowledgements\n\nThe Food and Nutrition Evaluation Laboratory (FANEL) team at BecA-ILRI Hub is highly acknowledged for providing a platform for HPLC analysis.\n\n\nReferences\n\nAbong GO, Okoth MW, Kabira JN, et al.: Physico-Chemical Changes in Popular Kenyan Processing Potato Varieties as Influenced by Storage Condition. Curr Res Nutr Food Sci. 2015; 3(2): 112–120. Publisher Full Text\n\nAbong GO, Okoth MW, Karuri EG, et al.: Nutrient contents of raw and processed products from Kenyan potato cultivars. J Appl Biosci. 2009; 16: 877–886. Reference Source\n\nAOAC: Official Methods of Analysis of AOAC International. (Arlington,). AOAC International. 2005. Reference Source\n\nAziz A, Randhawa MA, Butt MS, et al.: Glycoalkaloids (α-chaconine and α-solanine) contents of selected Pakistani potato cultivars and their dietary intake assessment. J Food Sci. 2012; 77(3): T58–61. PubMed Abstract | Publisher Full Text\n\nBejarano L, Mignolet E, Devaux A, et al.: Glycoalkaloids in potato tubers: the effect of variety and drought stress on the α-solanine and α-chaconine contents of potatoes. J Sci Food Agr. 2000; 80(14): 2096–2100. Publisher Full Text\n\nCantwell M: A Review of Important Facts about Potato Glycoalkaloids. Perishables Handling Newsletter. 1996; 87(87): 2–27. Reference Source\n\nChourasia MK, Maji P, Baskey A, et al.: Estimation of moisture loss from the cooling data of potatoes. J Food Process Eng. 2005; 28(4): 397–416. Publisher Full Text\n\nChuda Y, Tsuda S, Ohara-takada A, et al.: Quantification of Light-Induced Glycoalkaloids, α-Solanine and α-Chaconine, in Four Potato Cultivars (Solanum tuberosum L.) Distributed in Japan by LC/MS. Food Sci Technol Res. 2004; 10(3): 341–345. Publisher Full Text\n\nElbatawi IE, Ebaid MT, Hemeda BE: Determination of potato water content using nir diffuse reflection method. Misr J Ag Eng. 2008; 25(4): 1279–1292. Reference Source\n\nFAOSTAT: FAOSTAT. FAOSTAT. 2018. Reference Source\n\nFisher AA, Laing JE, Stoeckel JE, et al.: Handbook for family planning operations research (2nd Ed.). Population Council. New York, USA. 1991; 1–77. Publisher Full Text\n\nFriedman M: Potato glycoalkaloids and metabolites: roles in the plant and in the diet. J Agric Food Chem. 2006; 54(23): 8655–8681. PubMed Abstract | Publisher Full Text\n\nFriedman M, Roitman JN, Kozukue N: Glycoalkaloid and calystegine contents of eight potato cultivars. J Agric Food Chem. 2003; 51(10): 2964–2973. PubMed Abstract | Publisher Full Text\n\nFurrer AN, Chegeni M, Ferruzzi MG: Impact of potato processing on nutrients, phytochemicals, and human health. Crit Rev Food Sci Nutr. 2016; 58(1): 146–168. PubMed Abstract | Publisher Full Text\n\nGibson S, Kurilich AC: The nutritional value of potatoes and potato products in the UK diet. Nutr Bull. 2013; 38(4): 389–399. Publisher Full Text\n\nGriffiths DW, Bain H, Dale MF: Effect of Storage Temperature on Potato (Solanum tuberosum L.) Tuber Glycoalkaloid Content and the Subsequent Accumulation of Glycoalkaloids and Chlorophyll in Response to Light Exposure. J Agric Food Chem. 1998; 12(46): 5262–5268. Publisher Full Text\n\nGrunenfelder LA, Knowles LO, Hiller LK, et al.: Glycoalkaloid development during greening of fresh market potatoes (Solanum tuberosum L.). J Agric Food Chem. 2006a; 54(16): 5847–5854. PubMed Abstract | Publisher Full Text\n\nGrunenfelder L, Hiller LK, Knowles NR: Color indices for the assessment of chlorophyll development and greening of fresh market potatoes. Postharvest Biol Tec. 2006b; 40(1): 73–81. Publisher Full Text\n\nHafezi N, Sheikhdavoodi MJ, Sajadiye SM: Evaluation of Quality Characteristics of Potato Slices during Drying by Infrared Radiation Heating Method under Vacuum. International Journal of Agricultural and Food Research. 2015; 4(3): 1–8. Publisher Full Text\n\nKenya National Bureau of Statistics: Population Distribution by Sex, Number of Households, Area and Density by County and District. 2009. Reference Source\n\nKing JC, Slavin JL: White potatoes, human health, and dietary guidance. Adv Nutr. 2013; 4(3): 393S–401S. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKozukue N, Yoon KS, Byun GI, et al.: Distribution of glycoalkaloids in potato tubers of 59 accessions of two wild and five cultivated Solanum species. J Agric Food Chem. 2008; 56(24): 11920–11928. PubMed Abstract | Publisher Full Text\n\nLisiĕska G, Pċksa A, Kita A, et al.: The Quality of Potato for Processing and Consumption.2009. Reference Source\n\nMusita CN, Okoth MW, Abong GO: Postharvest handling practices and perception of potato safety among potato traders in Nairobi, Kenya. Int J Food Sci. 2019; 2019: 2342619. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMusita CN, Omayio D, Abong' G, et al.: Sample Data_glycoalkaloids in potatoes xls (Version 1). figshare. 2020. http://www.doi.org/10.6084/m9.figshare.12301424.v1\n\nNema PK, Ramayya N, Duncan E, et al.: Potato glycoalkaloids: formation and strategies for mitigation. J Sci Food Agric. 2008; 88(11): 1869–1881. Publisher Full Text\n\nOmayio DG, Abong GO, Okoth MW: A review of occurrence of glycoalkaloids in potato and potato products. Curr Res Nutr Food Sci. 2016; 4(3): 195–202. Publisher Full Text\n\nPavlista AD: Green potatoes: The problem and the solution. Agriculture. 2001; 1–4. Reference Source\n\nPercival GC: The influence of light upon glycoalkaloid and chlorophyll accumulation in potato tubers (Solanum tuberosum L.). Plant Sci. 1999; 145(2): 99–107. Publisher Full Text\n\nSablani S, Mujumdar A: Drying of potato, sweet potato, and other roots. In: Mujumdar AS (Ed.), Handbook of Industrial Drying, Third Edition (Issue September 2015). CRC Press. 2006; 647–662. Publisher Full Text\n\nŞengül, M, Keleş F, Keleş MS: The effect of storage conditions (temperature, light, time) and variety on the glycoalkaloid content of potato tubers and sprouts. Food Control. 2004; 15(4): 281–286. Publisher Full Text\n\nSmith DB, Roddick JG, Jones JL: Potato glycoalkaloids: Some unanswered questions. Trends Food Sci Tech. 1996; 7(4): 126–131. Publisher Full Text\n\nSmith DB, Roddick JG, Jones JL: Potato glycoalkaloids: Some unanswered questions. Trends Food Sci Tech. 1996; 7(4): 126–131. Publisher Full Text\n\nTajner-Czopek A, Jarych-Szyszka M, Lisińska G: Changes in glycoalkaloids content of potatoes destined for consumption. Food Chem. 2008; 106(2): 706–711. Publisher Full Text\n\nValcarcel J, Reilly K, Gaffney M, et al.: Effect of Genotype and Environment on the Glycoalkaloid Content of Rare, Heritage, and Commercial Potato Varieties. J Food Sci. 2014; 79(5): T1039–48. PubMed Abstract | Publisher Full Text\n\nXiao J, Zhang H, Niu L, et al.: Evaluation of detoxification methods on toxic and antinutritional composition and nutritional quality of proteins in jatropha curcas meal. J Agric Food Chem. 2011; 59(8): 4040–4044. PubMed Abstract | Publisher Full Text\n\nZhang Z, Guo C, Gao T, et al.: Pilot-scale radiofrequency blanching of potato cuboids: heating uniformity. J Sci Food Agric. 2018; 98(1): 312–320. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "64083",
"date": "13 Jul 2020",
"name": "David K. Weaver",
"expertise": [
"Reviewer Expertise Entomology and plant chemistry"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a useful and significant study on the levels of glycoalkaloids that would be of benefit to consumers. There are two points I think would be helpful to state clearly: 1) Was any sample above recommended health guidelines? Overall, this is of interest to a general reader - especially if none were; 2) In your description of statistical analysis, there was not a statement on how having different numbers of samples for comparisons was handled. It is recommended that this be included for clarity.\nMinor points:\nAbstract, Results \".. lowest levels of glycoalkaloids\"\nMethods, Study area. Perhaps food stuffs should be written as 1 word rather than being separated into 2. Recommend replacing \"purposively\" with \"purposefully\"\nMethods, Determination of moisture content. \"Memmert\" , not \"memmmert\"\nMethods, Sample preparation. Better to say \"all samples \" and \"all markets\". Use \"cloth towels\" instead of \"cloth towel\"\nMethods, Chromatographic analysis. \"....standard stock solutions\".\nAlso author Abong spelling is different for text and listed citations. These should be the same, if possible.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "65456",
"date": "14 Jul 2020",
"name": "Helen Tai",
"expertise": [
"Reviewer Expertise My area of research is plant genetics and genomics. I have experience in statistical analysis of data including the use of ANOVA."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 contributes important information on glycoalkaloids in potato tubers in Kenya. The experimental design was sound and good quality data was collected. The statistical analysis and presentation needs improvement, however.\n\nComments and recommended revisions are below:\n\nAbbreviations used in the manuscript should appear in full name followed by abbreviation in brackets afterwards on first appearance in the text.\nIn the “Sampling” section indicate number of replicate samples used for determination of moisture and glucose. Was there pooling of tubers together for a replicate? Or were there one tuber in each replicate?\nIn the “Sample analysis” section indicate if repeated measures were take for each sample? Recommend that “Sample preparation” section be placed after “Sampling” Recommend that “Chromatographic analysis” section is added to the “Determination of glycoalkaloids” section.\nIn the “Chromatographic analysis” indicate what standard stocks were used and where they were sourced from. What is GAS/kg?\nMore information on the determination of moisture content method summarized in brief would be helpful. Were tuber samples weighed before and after drying?\n\nIn the “Statistical analysis” section, where was the GenStat version 15 software sourced from? Indicate manufacturer and location. Include information on the Tukey’s test that was done.\nTable 1 Why are the samples in column 1 numbered from 1-24? Why are there numbers after the letters representing each variety? Do these represent the replicate number for the variety? It is recommended that one-way ANOVA tables are presented for analysis done on moisture and glycoalkaloid data. For just the Shangi variety there are multiple locations sampled. Here a one-way ANOVA comparing locations for Shangi samples only can be done and presented in separate tables for moisture and glycoalkaloids.\nTable 2 Similar comments as for Table 1. The same one-way ANOVA tables are recommended for the glycoalkaloid analysis as for the moisture analysis in Table 1.\nExample of one-way ANOVA table https://psychohawks.files.wordpress.com/2010/10/anova.png\nIf the F statistic is showing a p-value (Sig.) that is less that 0.05 for between groups (varieties) then a Tukey’s test is done.\n\nThe authors have done a good job in collecting the data. Addition of one-way ANOVA tables showing analysis for each of moisture and glycoalkaloid data will be helpful in interpreting data and enable authors to draw stronger conclusions.\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": []
}
] | 2
|
https://f1000research.com/articles/9-423
|
https://f1000research.com/articles/9-155/v1
|
26 Feb 20
|
{
"type": "Case Report",
"title": "Case Report: Acute kidney failure leading to permanent haemodialysis due to hyperoxaluria following one-anastomosis gastric bypass-related rapid weight loss.",
"authors": [
"Angelo Miranda",
"Andrea Rosato",
"Andrea Costanzi",
"Lucia Pisano",
"Sara Colzani",
"Sara Auricchio",
"Giulio Mari",
"Pietro Achilli",
"Dario Maggioni",
"Angelo Miranda",
"Andrea Rosato",
"Andrea Costanzi",
"Lucia Pisano",
"Sara Colzani",
"Sara Auricchio",
"Pietro Achilli",
"Dario Maggioni"
],
"abstract": "The one-anastomosis laparoscopic gastric bypass (OAGB) has been proven to provide good weight loss, comorbidity improvement, and quality of life with follow-up longer than five years. Although capable of improving many obesity-related diseases, OAGB is associated with post-operative medical complications mainly related to the induced malabsorption. A 52-year-old man affected by nephrotic syndrome due to a focal segmental glomerulosclerosis underwent OAGB uneventfully. At three months post-surgery, the patient had lost 40kg, reaching a BMI of 32. The patient was admitted to the nephrology unit for acute kidney injury with only mild improvement in renal function (SCr 9 mg/dl); proteinuria was still elevated (4g/24h), with microhaematuria. A renal biopsy was performed: oxalate deposits were demonstrated inside tubules, associated with acute and chronic tubular and interstitial damage and glomerulosclerosis (21/33 glomeruli). Urinary oxalate levels were found to be elevated (72mg/24h, range 13-40), providing the diagnosis of acute kidney injury due to hyperoxaluria, potentially associated to OAGB. No recovery in renal function was observed and the patient remained dialysis dependent. Early and rapid excessive weight loss in patients affected by chronic kidney insufficiency could be associated with the worsening of renal function. Increased calcium oxalate levels associated with OAGB-related malabsorption could be a key factor in kidney injury.",
"keywords": [
"OAGB",
"kidney failure",
"Calcium oxalate",
"weight loss"
],
"content": "Introduction\n\nBariatric surgery is known to be the most effective means of inducing durable weight loss in obese populations1–3. In particular, bariatric malabsorptive procedures are effective metabolic therapies capable of improving many obesity-related diseases4,5.\n\nAmong bariatric procedures claiming to be effective in providing a metabolic improvement in obese patients, the one-anastomosis laparoscopic gastric bypass (OAGB) has been proven to provide good weight loss, comorbidity improvement, and quality of life even with follow-up longer than five years6. Despite the positive reported results in terms of surgical outcomes and effectiveness in reducing several obesity-related diseases, OAGB is associated with post-operative medical complications, mainly related to the induced malabsorption7. Focusing on renal function, bariatric surgery is capable of being a protective factor against renal failure8,9; however, the association between induced malabsorption and particular renal syndromes can significantly alter the metabolism of numerous serum metabolites, leading to direct kidney damage10. When reported, such alterations of the metabolic framework tend to appear a relatively long time after surgery and are described as a late complication of weight loss11. However, rapid and early weight loss could aggravate and accelerate such a process.\n\nHere, we report a rare case of acute renal failure occurring three months after laparoscopic OAGB caused by hyperoxaluria associated with rapid and early excessive weight loss.\n\n\nCase presentation\n\nA white Italian 52-year-old man employed at an insurance company underwent OAGB in February 2019 for stage III morbid obesity (135kg, BMI 45).\n\nPast medical history was characterized by pathological obesity, type 2 diabetes, obstructive sleep apnoea syndrome and nephrotic syndrome due to focal segmental glomerulosclerosis. In 2011, at the time of the initial kidney biopsy, his weight was 110kg, his blood pressure and kidney function were normal, and proteinuria was 9g/24h. He was then treated with steroids and after five months cyclosporin was added, leading to complete remission of the nephrotic syndrome. He was also treated with renin-angiotensin-system blockers. In 2015, his weight was 125kg and his serum creatinine (SCr) and proteinuria began to rise, until 2018 when his weight was 134kg, SCr was 1.6mg/dl and proteinuria 4.8g/24h, without nephrotic syndrome. The patient had tried to follow a hypocaloric and hypoproteic diet with no success. The worsening of proteinuria was interpreted as secondary to obesity; in the meantime, steroids and cyclosporin were both contraindicated.\n\nThe patient was then referred to a surgeon for bariatric surgery. In February 2019, the surgical procedure was carried out uneventfully under general anaesthesia using the laparoscopic technique. The gastric sleeve was fashioned with a 60 mm stapler (six charges) and Seamguard. The operating surgeon was highly experienced in laparoscopic bariatric surgery, having performed more than five hundred procedures. The patient was discharged on day three after surgery after the methylene blue test did not reveal any signs of staple line leak. Follow-up was done monthly for the first six months after surgery with outpatient visits. The patient’s SCr was unvaried. In March 2019, the patient’s body weight was 114.5 kg; his sCr was 2.12 mg/dl in March and 2.23 mg/dl in April 2019. In May 2019, his body weight was 95 Kg. His water and food intake were very poor; he had stopped taking vitamin and citrate supplements and his SCr had raised up to 16 mg/dl. The excess weight loss was 64% at two months; at three months after surgery, the patient had lost 40kg, reaching a BMI of 32.\n\nThe patient was admitted to our nephrology unit for acute kidney injury. His blood pressure was 140/80 mmHg, heart rate was 77 bpm and urine output was about 2000 ml/day. On admission, Scr was 16.6 mg/dl, urea was 235 mg/dl, sodium and potassium levels were normal despite metabolic acidosis (HCO3 16 mmol/l), proteinuria was 3 g/day and mild microhaematuria was detected using a urine dipstick. Renal ultrasound was normal. The patient was given fluids (2 litres of polysaline solution intravenously for 10 days and an oral intake of water of 1.5 litres) with only mild improvement in renal function (SCr 8mg/dl). A new renal biopsy was performed; the puncture was ultrasound-guided using 16g × 20 mm Bard Monopty needles and two specimens were obtained for histologic examination. Oxalate deposits were demonstrated inside tubules, associated with acute and chronic tubular and interstitial damage and glomerulosclerosis (21/33 glomeruli). Urinary oxalate level was then found to be elevated (72mg/24h, range 13–40), providing the diagnosis of acute kidney injury due to hyperoxaluria potentially associated to OAGB.\n\nIn July 2019, renal replacement therapy was started. Online hemodiafiltration was administered through a central venous catheter and the treatment was performed three times a week for four hours without ultrafiltration. The patient was also treated with oral sodium bicarbonate (NaHCO3, 2 g/day), potassium and magnesium citrate (2.56 g/day), calcium carbonate (2.5g/day) and sevelamer (2.4 g/day). Renal function was assessed monthly by creatinine and urea clearance tests, but no recovery in renal function was observed and the patient remained dialysis dependent (creatinine clearance 11 ml/min, urea clearance 4 ml/min in January 2020). The patient’s body weight is now steady at 84Kg.\n\n\nDiscussion\n\nKidney failure can complicate the post-operative course after bariatric surgery. Its aetiology can be multifactorial and complex in obese populations. Pre-existing factors such as diabetes, hypertension, and chronic kidney disease (CKD) can play a role in worsening the renal insult during the early post-operative period12,13. In addition to dehydration, which is the main cause of acute kidney failure after bariatric surgery, the alteration of several metabolic assets induced by a rapid and consistent weight loss can become a key factor in triggering kidney failure14. One metabolite potentially involved in the acute onset of kidney failure but rarely associated to it, is calcium oxalate15.\n\nThe reasons for the onset of hyperoxaluria are not yet completely understood. There are various hypotheses: one of the main reasons seems to be the malabsorption of lipids since, in this condition, the calcium found inside the intestinal lumen tends to bind to fatty acids instead of forming insoluble precipitates of calcium oxalate; the oxalate thus remains in a soluble state and can be reabsorbed by the ileal walls16. Another possible explanation is the increase in bile secretion into the colon due to the lack of intestinal reabsorption of bile salts, with consequent increase in the permeability of the colic walls due to a decrease in the function of the epithelial barrier. Finally, another hypothesis is the decrease in colonization of the colon by oxalate-metabolizing bacteria such as Oxalobacter formigenes, able to metabolize oxalate17.\n\nThe association between excessively rapid weight loss and renal damage due to calcium oxalate is not widely documented in the literature and therefore remains a hypothesis based on a valid rational18.\n\nBariatric surgery is able to reduce the most dangerous risk factors for end stage renal disease such as hypertension and diabetes, but post-operative complications in CKD patients are slightly higher than the general population and surgeons must be aware of oxalate nephropathy, because it's a rare but often irreversible cause of acute kidney injury19,20.\n\nPatient with pre-existing chronic kidney disease may be considered to be higher risk for secondary oxalate nephropathy but this has not yet been established. Monitoring the 24-hour urinary oxalate excretion rate might be a useful tool to prevent oxalate nephropathy in high risk patients. For such patients, a careful selection of the right bariatric procedure (malabsorptive vs restrictive) should therefore be performed21.\n\n\nConclusions\n\nThe early and rapid excessive weight loss in patients affected by chronic kidney insufficiency could be associated with the worsening of renal function. Increased calcium oxalate levels associated with OAGB-related malabsorption could be a key factor in kidney injury.\n\n\nConsent\n\nWritten informed consent for the publication of their clinical details and clinical images and video was obtained from the patient.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.\n\nZenodo: Acute kindey failure leading to permanent hemodalysis due to hyperoxaluria following OAGB related rapid weight loss. Case report. https://doi.org/10.5281/zenodo.360935022\n\nThis project contains the following extended data:\n\n- Surgical procedure video (in MP4 file format)\n\n- Creatinine levels pre-surgery (in JPG format)\n\n- Creatinine levels post-surgery (n JPG format)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
"appendix": "References\n\nSchauer PR, Kashyap SR, Wolski K, et al.: Bariatric surgery versus intensive medical therapy in obese patients with diabetes. N Engl J Med. 2012; 366(17): 1567–76. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMingrone G, Panunzi S, De Gaetano A, et al.: Bariatric surgery versus conventional medical therapy for type 2 diabetes. N Engl J Med. 2012; 366(17): 1577–85. PubMed Abstract | Publisher Full Text\n\nDixon JB, O’Brien PE, Playfair J, et al.: Adjustable gastric banding and conventional therapy for type 2 diabetes: a randomized controlled trial. JAMA. 2008; 299(3): 316–23. PubMed Abstract | Publisher Full Text\n\nSugerman HJ, Wolfe LG, Sica DA, et al.: Diabetes and hypertension in severe obesity and effects of gastric bypass-induced weight loss. Ann Surg. 2003; 237(6): 751–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBatsis JA, Romero-Corral A, Collazo-Clavell ML, et al.: Effect of weight loss on predicted cardiovascular risk: change in cardiac risk after bariatric surgery. Obesity (Silver Spring). 2007; 15(3): 772–84. PubMed Abstract | Publisher Full Text\n\nNeuberg M, Blanchet MC, Gignoux B, et al.: Long-Term Outcomes After One-Anastomosis Gastric Bypass (OAGB) in Morbidly Obese Patients. Obes Surg. 2019. PubMed Abstract | Publisher Full Text\n\nBhandari M, Nautiyal HK, Kosta S, et al.: Comparison of one-anastomosis gastric bypass and Roux-en-Y gastric bypass for treatment of obesity: a 5-year study. Surg Obes Relat Dis. 2019; 15(12): 2038–2044. PubMed Abstract | Publisher Full Text\n\nNeff KJ, Baud G, Raverdy V, et al.: Renal Function and Remission of Hypertension After Bariatric Surgery: a 5-Year Prospective Cohort Study. Obes Surg. 2017; 27(3): 613–619. PubMed Abstract | Publisher Full Text\n\nAlkandari A, Ashrafian H, Sathyapalan T, et al.: Bariatric Surgery Modulates Urinary Levels of MicroRNAs Involved in the Regulation of Renal Function. Front Endocrinol (Lausanne). 2019; 10: 319. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGero D, Raptis DA, Vleeschouwers W, et al.: Defining Global Benchmarks in Bariatric Surgery: A Retrospective Multicenter Analysis of Minimally Invasive Roux-en-Y Gastric Bypass and Sleeve Gastrectomy. Ann Surg. 2019; 270(5): 859–867. PubMed Abstract | Publisher Full Text\n\nThakar CV, Kharat V, Blanck S, et al.: Acute kidney injury after gastric bypass surgery. Clin J Am Soc Nephrol. 2007; 2(3): 426–30. PubMed Abstract | Publisher Full Text\n\nNor Hanipah Z, Punchai S, Augustin T, et al.: Impact of Early Postbariatric Surgery Acute Kidney Injury on Long-Term Renal Function. Obes Surg. 2018; 28(11): 3580–3585. PubMed Abstract | Publisher Full Text\n\nCurrie A, Chetwood A, Ahmed AR: Bariatric surgery and renal function. Obes Surg. 2011; 21(4): 528–39. PubMed Abstract | Publisher Full Text\n\nWeingarten TN, Gurrieri C, McCaffrey JM, et al.: Acute kidney injury following bariatric surgery. Obes Surg. 2013; 23(1): 64–70. PubMed Abstract | Publisher Full Text\n\nLiu H, Ye T, Yang X, et al.: H19 promote calcium oxalate nephrocalcinosis-induced renal tubular epithelial cell injury via a ceRNA pathway. EBioMedicine. 2019; 50: 366–378. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNelson WK, Houghton SG, Milliner DS, et al.: Enteric hyperoxaluria, nephrolithiasis, and oxalate nephropathy: potentially serious and unappreciated complications of Roux-en-Y gastric bypass. Surg Obes Relat Dis. 2005; 1(5): 481–5. PubMed Abstract | Publisher Full Text\n\nPatel BN, Passman CM, Fernandez A, et al.: Prevalence of hyperoxaluria after bariatric surgery. J Urol. 2009; 181(1): 161–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNasr SH, D'Agati VD, Said SM, et al.: Oxalate nephropathy complicating Roux-en-Y Gastric Bypass: an underrecognized cause of irreversible renal failure. Clin J Am Soc Nephrol. 2008; 3(6): 1676–83. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChang AR, Grams ME, Navaneethan SD: Bariatric Surgery and Kidney-Related Outcomes. Kidney Int Rep. 2017; 2(2): 261–270. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChang AR, Chen Y, Still C, et al.: Bariatric surgery is associated with improvement in kidney outcomes. Kidney Int. 2016; 90(1): 164–171. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLumlertgul N, Siribamrungwong M, Jaber BL, et al.: Secondary Oxalate Nephropathy: A Systematic Review. Kidney Int Rep. 2018; 3(6): 1363–1372. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAchilli P: Acute kindey failure leading to permanent hemodalysis due to hyperoxaluria following OAGB related rapid weight loss. Case report.2020. http://www.doi.org/10.5281/zenodo.3609350"
}
|
[
{
"id": "60620",
"date": "28 Feb 2020",
"name": "Matteo Uccelli",
"expertise": [
"Reviewer Expertise bariatric surgery"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe case report described here presents a useful suggestion in deciding which bariatric procedure should be chosen in nephropathic patients.\n\nThe patient description is well explained and the discussion is well written as it establishes a sort of guide line that deserves to be taken into consideration.\n\nKidney function in obese patients is a main issue and a main concern. Analyzing which kind of pathological disorder lays underneath kidney insufficiency should be done before choosing the type of bariatric procedure.\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": "63316",
"date": "18 May 2020",
"name": "Mohammad Kermansaravi",
"expertise": [
"Reviewer Expertise Bariatric surgery",
"metabolic surgery",
"dvanced laparoscopic 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\nIt's a well written manuscript, but I have some suggestions:\nIn abstract and introduction, it's better write \" one-anastomosis gastric bypass (OAGB)\" instead of \" one-anastomosis laparoscopic gastric bypass (OAGB)\"\n\nIn case presentation, you mentioned that the patient underwent sleeve gastrectomy! Sleeve or OAGB? Please clarify the surgical technique.\n\nThere is not any strong evidence about rapid weight loss related nephropathy. How do you result in? Please clarify.\n\nHow do you justify the relation between nephropathy and OAGB? Rapid weight loss if imaginary has a relation with rapid weight loss, it could happen in any type of weight loss therapies.\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": "5549",
"date": "29 May 2020",
"name": "Giulio Mari",
"role": "Author Response",
"response": "We corrected the OAGB definition as indicated.We better described the OAGB procedure in the case presentation.As requested by the reviewer we clarified the possible correlation between OAGB, rapid weight loss and kidney failure due to calcium oxalate.The case report that we report describes a kidney failure following OAGB and rapid weight loss. The only change in the renal function across surgery was the calcium oxalate levels increase. We therefore make an hypothesis based on a pathophysiological rationale that will need further analysis to be proven."
}
]
}
] | 1
|
https://f1000research.com/articles/9-155
|
https://f1000research.com/articles/9-199/v1
|
20 Mar 20
|
{
"type": "Method Article",
"title": "Fast analysis of scATAC-seq data using a predefined set of genomic regions",
"authors": [
"Valentina Giansanti",
"Ming Tang",
"Davide Cittaro",
"Valentina Giansanti",
"Ming Tang"
],
"abstract": "Background: Analysis of scATAC-seq data has been recently scaled to thousands of cells. While processing of other types of single cell data was boosted by the implementation of alignment-free techniques, pipelines available to process scATAC-seq data still require large computational resources. We propose here an approach based on pseudoalignment, which reduces the execution times and hardware needs at little cost for precision. Methods: Public data for 10k PBMC were downloaded from 10x Genomics web site. Reads were aligned to various references derived from DNase I Hypersensitive Sites (DHS) using kallisto and quantified with bustools. We compared our results with the ones publicly available derived by cellranger-atac. Results: We found that kallisto does not introduce biases in quantification of known peaks and cells groups are identified in a consistent way. We also found that cell identification is robust when analysis is performed using DHS-derived reference in place of de novo identification of ATAC peaks. Lastly, we found that our approach is suitable for reliable quantification of gene activity based on scATAC-seq signal, thus allows for efficient labelling of cell groups based on marker genes. Conclusions: Analysis of scATAC-seq data by means of kallisto produces results in line with standard pipelines while being considerably faster; using a set of known DHS sites as reference does not affect the ability to characterize the cell populations",
"keywords": [
"single cell",
"scATAC-seq",
"pseudoalignment"
],
"content": "Introduction\n\nRecent technological advances in single-cell technologies resulted in a tremendous increase in the throughput in a relatively short span of time1. The increasing number of cells that could be analyzed prompted a better usage of computational resources; this has been especially true for the post-alignment and quantification phases. As a consequence, it is today feasible to run the analysis of single cell data on commodity hardware with limited resources2, even when the number of observables is in the order of hundreds of thousands. Conversely, the analysis steps from raw sequences to count matrices lagged for some time; alignment to the reference genome or transcriptome is largely dependent on classic aligners, without any specific option to handle single-cell data, with the notable exception of the latest implementation of STARsolo in the STAR aligner3.\n\nMore recently, analysis of NGS data benefit from technologies based on k-mer processing, allowing alignment-free sequence comparison4. Most of these technologies require a catalog of k-mers expected to be in the dataset and, hence, subject of quantification. RNA-seq analysis relies on the quantification of gene/transcript abundances and, while it is possible to perform de novo characterization of unknown species in every experiment, it is common practice5,6 to rely on a well-defined gene model such as GENCODE7 to quantify expressed species. It is then possible to efficiently perform alignment-free analysis on transcripts to quantify gene abundances and, in fact, tools implementing this approach such as kallisto8 or Salmon9 have been quickly adopted on a wide scale. Moreover, a recent implementation of kallisto extended its capabilities to the analysis of single cell RNA-seq data10 by direct handling of cell barcodes and UMIs, allowing the analysis of such data in a streamlined way.\n\nAnalysis of epigenetic features by ATAC-seq requires the identification of enriched peaks along the genome sequence. This is typically achieved using peak callers such as MACS11, opportunely tuned. Since ATAC-seq signal mirrors DNA accessibility as mapped by DNase-seq assays12 and catalogs of DNase I Hypersensitive Sites (DHS) are available13,14 it should be possible to perform reference-based ATAC-seq analysis in a way much similar to what is performed for RNA-seq analysis. In this paper we show it is indeed possible to perform single-cell ATAC-seq analysis using kallisto and bustools, with minor tweaks, using an indexed reference of ~1 million known DHS sites on the human genome.\n\n\nMethods\n\nSingle cell ATAC-seq data were downloaded from the 10x Genomics public datasets (https://support.10xgenomics.com/single-cell-atac/datasets/1.1.0/atac_v1_pbmc_10k) and include sequences for 10k PBMC from a healthy donor. We used the Peak by cell matrix HDF5 (filtered) object as our ground truth.\n\nWe downloaded the DNase I Hypersensitive Sites (DHS) interval list for hg19 genome from the Regulatory Elements DB15, intervals closer than 500bp were clustered using bedtools16.\n\nWe extracted DNA sequences for DHS intervals and indexed corresponding fasta files using kallisto index (v0.46.0) with default parameters, resulting in an index for the full DHS set (iDHSfull) and an index for the merged set (iDHS500). The same procedure was performed for the peak set identified by cellranger-atac and distributed along with the data (iMACS).\n\nkallisto requires the definition of the unique molecular identifiers (UMI) and cellular barcodes (CB) in a specific fastq file. For standard Chromium scRNA-seq data, these are substrings of R1 and RNA is sequenced in R2. Chromium scATAC-seq reads are not structured in the same way, paired end genomic reads are in R1 and R3, R2 includes only the 16bp cellular barcode. In addition, kallisto bus expects only a single read with genomic information. Therefore we simulated appropriate structures in three different ways:\n\n1. by adding 12 random nucleotides and mapping the R1 file (forward read):\n\nkallisto bus -x 10xV2 modified_R1.fastq.gz\n\npbmc_10k_R1.fastq.gz\n\n2. by extracting sequences of different length n (5, 10, 15, 20) from the 5' of R3 (reverse read) and mapping the R1 file:\n\nkallisto bus -x 1,0,16:2,0,n:0,0,0\n\npbmc_10k_R1.fastq.gz\n\npbmc_10k_R2.fastq.gz\n\npbmc_10k_R3.fastq.gz\n\n3. by extracting sequences of different length n (5, 10, 15, 20) from the 5' of R1 and then mapping the R3 file:\n\nkallisto bus -x 1,0,16:2,0,n:0,0,0\n\npbmc_10k_R3.fastq.gz\n\npbmc_10k_R2.fastq.gz\n\npbmc_10k_R1.fastq.gz\n\nWe will refer to the second set of simulation as n-fwd and to the third set as n-rev, where n is the number of nucleotides considered as UMI. We also applied two different summarization strategies for bustools count step. In the first approach, pseudocounts are not summarized, the number of features matches the size of the index; in the second approach, summarized, we let bustools map counts on iDHSfull to the merged intervals (Figure 1A).\n\n(A) Graphical depiction of processing of pseudoalignment over DHS, based on three DHS derived indices. The first (DHS) generated by kallisto on ~2M DNase I sites, the second (DHS500) by merging regions closer than 500bp and the last (DHS500p) by projecting the result of DHS index to DHS500 using bustools capabilities. (B) Heatmaps representing MI scores for the DHS derived matrices. The heatmap on the left reports the pairwise MI values between DHS, DHS500 and DHS500p strategies. The heatmap on the right represents MI values comparing the DHS derived strategies to the cellranger-atac (10x) results or 10-rev strategy. DHS500 strategy achieves the highest scores. (C) MI values comparing DHS (green line) and DHS500 (red line) strategies to cellranger-atac at different thresholds on the number of regions considered in the analysis. When approximately 50,000 regions are included, the MI stabilizes at its maximum.\n\nCounts matrices were analysed using Scanpy (v1.4.2)2 with standard parameters. We filtered out cells that had less than 200 regions and regions that were not at least in 10 cells. The count matrices were normalized and log transformed. The highly variable regions were selected and the subsetted matrices processed to finally clusterized the data with the Leiden algorithm17. Adjusted mutual information (MI) was used to evaluate the concordance between the 10x and our matrices.\n\nThe matrices derived from kallisto and cellranger-atac were also imported into Seurat V318. Gene activity score was calculated using the CreateGeneActivityMatrix function or directly summarized by kallisto. The annotated 10k PMBC scRNA-seq Seurat object was downloaded from the link available in their v3.1 ATAC-seq Integration Vignette (https://satijalab.org/seurat/v3.1/atacseq_integration_vignette.html).\n\nCell labels from the scRNA-seq data were transferred using TransferData function based on the gene activity score. All the analyses were carried out using standard parameters. Jaccard similarities were evaluated using the scclusteval (v0.1.1) package19.\n\n\nResults\n\nAt time of writing, kallisto does not natively support scATAC-seq analysis, though it can be applied to any scRNA-seq technology which supports CB and UMI. According to the kallisto manual, the technology needs to be specified with a tuple of indices indicating the read number, the start position and the end position of the CB, the UMI and the sequence respectively. In this sense, the technology specifier for standard 10x scRNA-seq with v2 chemistry is 0, 0, 16 : 0, 16, 26 : 1, 0, 0 (see kallisto manual for details). Using this logic, a single fastq file contains sequence information and UMI is always required. scATAC-seq from 10x genomics is typically sequenced in paired-end mode and, moreover, there is no definition of UMI as reads can be deduplicated after genome alignment.\n\nkallisto requires an index of predefined sequences, typically transcripts, to perform pseudoalignment and, if applied to scATAC-seq analysis, does not allow for any typical analysis in the epigenomic protocols, including the identification and quantification of enriched regions. Therefore, we computed an index on the genomic sequences for the 80, 234 peaks identified by cellranger-atac and distributed together with fastq files. This ensures that the subsequent analysis were performed on the same regions and allowed us to quantify the bias, if any, introduced by kallisto.\n\nWe tested different strategies to overcome the technical limits and the absence of UMI. We evaluated concordance of different approaches in terms of adjusted mutual information (MI) of cell groups identified with a fixed set of filtering and processing parameters. Analysis based on cellranger-atac results is considered as ground truth. Results are reported in Table 1.\n\nThe table reports adjusted Mutual Information between single cell cluster assignments on cellranger-atac data and kallisto analysis. Different strategies to evaluate pseudoUMI are reported. All simulations raised high MI values, both in the forward and reverse approach, except for the pseudoUMI of length 5. The 10-Reverse configuration reached the highest score.\n\nWe tested two main strategies: in the first the R1 is pseudoaligned and the initial nucleotides of R2, cut at different thresholds, are used as UMI (pseudoUMI hereafter). As UMI is needed for deduplication, we reasoned that a duplicate in scATAC-seq should be identified by the same nucleotides, especially in the first portion of the read, where quality is higher. We observe generally high values of MI, with the notable exception of pseudoUMI 5nt long. Since basecall qualities are generally higher for R1 and kallisto does not use qualities in pseudoalignment, we also tested the strategy in which R2 is used for pseudoalignment and R1 is used to obtain pseudoUMI. Also in this scenario, 5nt pseudoUMI raised the worst results, while MI values were slightly higher than the forward configuration. In particular, we noticed the highest MI values when R2 is used and pseudoUMI is 10nt long (MI = 0.7625). We also tested a configuration using R1 as sequence and 10nt UMI randomly generated. Interestingly, concordance remains in line with previous experiments (MI = 0.7272).\n\nThese data indicate that kallisto is able to properly quantify enrichments in scATAC-seq and does not introduce a considerable bias.\n\nAs one major limitation of a kallisto-based approach to scATAC-seq is the lack of peak calling routines and the need of a index of sequences for pseudoalignments, we reasoned that we could use any collection of regions that putatively would be target of ATAC-seq experiments. Since ATAC-seq is largely overlapping DHS we exploited the regions defined in the ENCODE project20. The DHS data provided by ENCODE includes 2, 888, 417 sites. We generated an additional dataset by merging regions closer than 500bp into 1, 040, 226 sites. We performed pseudoalignment on the full dataset, on the merged dataset and, lastly, on the full dataset summarized to the merged by bustools (see Methods). Pairwise comparison between performances of the three methods reveals lower values of MI (Figure 1B). Comparison with 10x data and the configuration 10-rev previously performed shows high values of MI when considering merged DHS intervals (MI = 0.7164 and 0.743 respectively). When pseudoalignmets are performed on the full DHS set, performance degrades to less than optimal levels. Since the number of DHS intervals is considerably higher than the typical number of regions identifiable by ATAC-seq, we tested the trend of MI at different cutoffs on the number of DHS included in the analysis (Figure 1C). MI reaches a plateau when approximately 50, 000 regions are included into the analysis. This sets a reasonable target for region filtering during preprocessing stages of scATAC-seq data. In all, these findings support the suitability of using kallisto for identification of cell identities in scATAC-seq without any prior knowledge of the epigenetic status of single cells.\n\nA crucial step in the analysis of scATAC-seq data is the identification of marker peaks which can be used to functionally characterize different clusters. We tested the ability of our reference-based approach to identify differential DNase I hypersensitive sites that are overlapping or close to peaks identified with standard analysis. To this end, we first matched cell groups from DHS500 to groups identified after cellranger-atac. We selected the top 1, 000 peaks marking each DHS500 group and evaluated the concordance by mutual distance to the top 1, 000 significant markers in the matched groups (p < 0.05), we could identify significant markers only in five matched clusters. We found that the large majority of peaks (>= 80%) were overlapping between the two strategies or closer than 20kb (Figure 2). These results confirm the substantial equivalence between the standard strategy and the reference-based one.\n\nThe bars represent the proportion of marker peaks that are in common between DHS500 and cellranger-atac-based strategies at different distance thresholds. Only the top 1, 000 significant peaks (p < 0.05) were included in the analysis; the graph reports results for the 5 cell clusters (A–E) that contain the required amount of significant markers. The chart also reports the proportion of peaks without any match (None).\n\nIn addition to the analysis of technical suitability of kallisto for the analysis of scATAC-seq data, we investigated its validity in extracting biological insight. To this end, we performed a more detailed analysis of PBMC data by label transferring using Seurat V318, with the hypothesis that different approaches could lead to mislabeling of cells clusters. Matching is performed with the help of Gene Activity Scores calculated as sum of scATAC-seq counts over gene bodies extended 2kb upstream the TSS, Seurat’s default approach. We applied the same transferring protocol on data derived from cellranger-atac counts and from the DHS500 approach (Figure 3), founding no relevant differences in the UMAP embeddings. A detailed quantification of cluster matches reveals a slight deviance in the characterization of NK subpopulations (Figure 4A). In addition to scores calculated by Seurat, we tested the ability of bustools summarization step to project and sum scATAC-seq values into Gene Activity using the identical mapping to extended gene bodies. In terms of cell labeling, this approach is equivalent to Seurat (Figure 4B), with the additional advantage of reduced run times.\n\nThe UMAP plot on the left represents scRNA-seq data of 10k PBMC as returned by Seurat vignette. The UMAP plots in the middle and on the right represent scATAC-seq analysis on cellranger-atac or kallisto analysis respectively. Cell clusters are consistent in their topology in the three plots, indicating the validity of kallisto for this kind of analysis.\n\n(A) Pairwise Jaccard similarity between cell annotations as a result of label transfer from RNA-seq data using Gene Activity Score evaluated by Seurat. Concordance between results after cellranger-atac (rows) and DHS500 (columns) are largely comparable, with the notable exception of NK subpopulations. (B) Pairwise Jaccard similarity between cell annotations on DHS500 when Gene Activity Score is computed by Seurat (rows) or by bustools summarization step (columns).\n\n\nDiscussion/conclusions\n\nAnalysis of differential chromatin properties, through ATAC-seq and other quantitative approaches, relies on the identification of peaks or enriched regions, this is often achieved with the same statistical framework used in analysis of differential gene expression21,22. Identification of peaks is a key difference between the two approaches, de novo discovery of unannotated transcripts has been shown to be possible in early times of NGS23, but the large majority of analysis is performed on gene models; conversely, analysis of epigenomes involves identification of regions of interest, although a large catalogues of such regions have been provided by several projects, such as the ENCODE project24, the BluePrint project25 or the GeneHancer database26. In single cell analysis, both scRNA-seq and scATAC-seq, identification of novel features may be an issue, especially because of the low coverage at which single cells are profiled. This work is the first, to our knowledge, to test the feasibility of a reference-based approach to ATAC-seq analysis, with a special focus on single cell ATAC-seq. In combination, we tested the suitability of kallisto to this kind of analysis, to maximise the performances of the whole process. Our results suggest that identification of cell groups using a reference-based approach is not different from a standard pipeline. Not only cells could be classified in a nearly identical way, but also differential features are largely matched between the analysis. The most obvious advantage is the gain in speed and efficiency: once reads have been demultiplexed, kallisto analysis requires short execution times, in the order of minutes, with limited hardware resources; this advantage has been known for a while and, in fact, it is has been demonstrated that it can be used on Rock64 hardware27. We also anticipate that adoption of a reference-based strategy comes with additional advantages, in particular functional annotations and gene associations are available for known regulatory regions20 and, more recently, for DNase I Hypersensitive Sites14. Of course, our strategy has some limitations that come from the unavailability of read positioning on the genome: in addition to the impossibility of identifying novel peaks, it is not possible to perform some ATAC-specific analysis, such as nucleosome positioning or footprinting of transcription factors in accessible regions. Indeed, these two can be overcome if standard alignment is used in place of pseudoalignment, at cost of time. As concluding remark we would like to underline that, although we showed that kallisto can be effectively used for analysis of scATAC-seq data, we are aware that it has not been conceived for that purposes and, indeed, its interface needs some tweaks to make it work; for this reason we advocate the development of tools which support scATAC-seq natively and other tools for postprocessing and data visualization.\n\n\nData availability\n\nSingle cell ATAC-seq data were downloaded from the 10x Genomics public datasets (https://support.10xgenomics.com/single-cell-atac/datasets/1.1.0/atac_v1_pbmc_10k) and include sequences for 10k PBMCs from a healthy donor. Access to the data is free but requires registration.\n\nZenodo: vgiansanti/Kallisto-scATAC v1.0. https://doi.org/10.5281/zenodo.370317428.\n\nThis project contains a detailed explanation of the procedures described in this work and the list of DHS sites; this is also available at https://github.com/vgiansanti/Kallisto-scATAC.\n\nExtended data are available under the terms of the Creative Commons Attribution 4.0 International license (CC- BY 4.0).",
"appendix": "Acknowledgements\n\nThe authors want to thank the people and supervisors who supported their work, in particular Giovanni Tonon, Catherine Dulac and Tim Sackton.\n\n\nReferences\n\nSvensson V, Vento-Tormo R, Teichmann SA: Exponential scaling of single-cell RNA-seq in the past decade. Nat Protoc. 2018; 13(4): 599–604. PubMed Abstract | Publisher Full Text\n\nWolf FA, Angerer P, Theis FJ: SCANPY: large-scale single-cell gene expression data analysis. Genome Biol. 2018; 19(1): 15. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDobin A, Davis CA, Schlesinger F, et al.: STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2013; 29(1): 15–21. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZielezinski A, Vinga S, Almeida J, et al.: Alignment-free sequence comparison: benefits, applications, and tools. Genome Biol. 2017; 18(1): 186. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVan den Berge K, Hembach KM, Soneson C, et al.: RNA sequencing data: hitchhiker’s guide to expression analysis. Annu Rev Biomed Data Sci. 2019; 2(1): 139舑173. Publisher Full Text\n\nConesa A, Madrigal P, Tarazona S, et al.: A survey of best practices for RNA-seq data analysis. Genome Biol. 2016; 17(1): 13. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHarrow J, Frankish A, Gonzalez JM, et al.: GENCODE: the reference human genome annotation for The ENCODE Project. Genome Res. 2012; 22(9): 1760–1774. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBray NL, Pimentel H, Melsted P, et al.: Near-optimal probabilistic RNA-seq quantification. Nat Biotechnol. 2016; 34(5): 525–527. PubMed Abstract | Publisher Full Text\n\nPatro R, Duggal G, Love MI, et al.: Salmon provides fast and bias-aware quantification of transcript expression. Nat Methods. 2017; 14(4): 417–419. 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Publisher Full Text\n\nRoadmap Epigenomics Consortium, Kundaje A, Meuleman W, Ernst J, et al.: Integrative analysis of 111 reference human epigenomes. Nature. 2015; 518(7539): 317–330. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAnders S, Huber W: Differential expression analysis for sequence count data. Genome Biol. 2010; 11(10): R106. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYan F, Powell DR, Curtis DJ, et al.: From reads to insight: a hitchhiker's guide to ATAC-seq data analysis. Genome Biol. 2020; 21(1): 22. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRobertson G, Schein J, Chiu R, et al.: De novo assembly and analysis of RNA-seq data. Nat Methods. 2010; 7(11): 909–912. PubMed Abstract | Publisher 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\nAdams D, Altucci L, Antonarakis SE, et al.: BLUEPRINT to decode the epigenetic signature written in blood. Nat Biotechnol. 2012; 30(3): 224–226. PubMed Abstract | Publisher Full Text\n\nFishilevich S, Nudel R, Rappaport N, et al.: GeneHancer: genome-wide integration of enhancers and target genes in GeneCards. Database (Oxford). 2017; 2017. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTan QW, Mutwil M: Inferring biosynthetic and gene regulatory networks from Artemisia annua RNA sequencing data on a credit card-sized ARM computer. Biochim Biophys Acta Gene Regul Mech. 2019; 194429. PubMed Abstract | Publisher Full Text\n\nGiansanti V, Cittaro D: vgiansanti/kallisto-scatac v1.0. Zenodo. 2020. http://www.doi.org/10.5281/zenodo.3703174"
}
|
[
{
"id": "61566",
"date": "30 Mar 2020",
"name": "Iros Barozzi",
"expertise": [
"Reviewer Expertise Genomics",
"Transcriptional Regulation",
"Single-cell Transcriptomics."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 paper “Fast analysis of scATAC-seq data using a predefined set of genomic regions” Giansanti et al. suggest an efficient strategy to analyse scATAC-seq data using kallisto and bustools.\n\nThe paper is clearly written, the proposed strategy is well conceived and tested, and it will be useful for many researchers in the field of regulatory genomics. Clear advantages of this strategy are the reduced requirements in terms of computational resources and shorter execution times, when compared to other pipelines such as cellranger-atac. This comes at a cost, most notably the chance of missing signals at regions that are not present in the reference set. Nevertheless, in my opinion evaluations about this being a limitation has to be made on a case-by-case basis, and the authors clearly pointed this out (among other limitations) in the discussion. The authors also provide access to the full code, datasets and documentation to reproduce the analyses.\n\nA wide range of parameters was tested, both in terms of handling and modifying the input sequences to make them suitable for kallisto, and in terms of pre- vs post- processing the genomic partition considered for indexing. Combinations that return results that are highly concordant with those obtained with cellranger-atac were highlighted. The authors then demonstrated the robustness of the biological inferences made using their strategy by showing a very large overlap with the results achieved by cellranger-atac (in terms of different groups of regions marking distinct clusters and clusters annotation based on label transferring from scRNA-seq data).\n\nI am wondering if a natural application of this strategy would simplify the characterization of chromatin state at highly repetitive regions of mammalian genomes (e.g. indexing a database of transposable elements). This task would otherwise be quite difficult to handle explicitly with pipelines such as cellranger-atac.\n\nI only have two minor comments:\nCan the authors provide more details about the analysis described in the paragraph “Identification of marker regions”? How were the cell groups defined? How were the top 1,000 peaks for each group selected/identified?\n\nFig. 1C: description of the blue curve seems to be missing.\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": "5525",
"date": "28 May 2020",
"name": "Davide Cittaro",
"role": "Author Response",
"response": "Thank you for reviewing our work and for the comments. We have addressed your minor concerns as follows.Can the authors provide more details about the analysis described in the paragraph “Identification of marker regions”? How were the cell groups defined? How were the top 1,000 peaks for each group selected/identified? We apologize for lack of clarity in the manuscript. Cell groups were identified with the Leiden method, while markers were identified with Wilcoxon rank-sum test. The complete list of instructions used in the analysis is part of the repository linked in the main text, nevertheless we modified the text adding these specific details. Fig. 1C: description of the blue curve seems to be missing. Thank you for pointing this out. The blue line represented the fit of the DHS data. We acknowledge colouring scheme was not appropriate and, moreover, the fit DHS500 data was missing. In the revised manuscript we modified the figure accordingly."
}
]
},
{
"id": "62150",
"date": "06 May 2020",
"name": "Qiangfeng Cliff Zhang",
"expertise": [
"Reviewer Expertise Bioinformatics",
"genomics",
"RNA structure",
"Genome structure",
"AI algorithms in biomedicine."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nComments: The work by Giansanti et al. presents a novel and smart idea for scATAC-seq data analysis. It demonstrates the possibility of using a reference-based, pseudo-alignment method to reduce the computational requirement for scATAC-seq data analysis, with only a little sacrifice on precision. The idea is inspired by the using of pesudoalignment for bulk and single-cell RNA-seq quantification. Here they showed that with some tweaking of the input sequencing reads, they could use kallisto to analyze scATAC-seq data on a pre-defined set of DNase hypersensitive sites. They compared their results with the standard protocol (e.g. cellranger-atac) for peak quantification, single cell clustering, marker peaks identification, and gene activity score calculation.\n\nThe results very nicely revealed the consistency on peak quantification between kallisto-based method and cellranger-atac. The cell clusterings were almost identical between the new reference-based method and canonical mapping strategy. And the gene activity scores by two different methods also agreed well with each other. The approach presented in this study thus could be a very efficient way for scATAC-seq data analysis.\n\nThe following are a few comments/questions:\nThe method was only tested with one dataset - PMBC. In fact, single cell ATAC-seq data is usually very sparse. The PMBC dataset used in this study is of relatively high quality. The method remains to be tested on more datasets, especially on those of more sparse, lower-quality.\n\nThe key advantage of the method is presumably the much improved computational efficiency – there may be other advantages brought by the reference-based method. However, there is no results/statistics on the running time and memory usage in the manuscript. From the description, the improvement should be dramatic. I think it would be very nice to include a table or a figure to demonstrate the increase of computational efficiency. This could be a very helpful way to convince potential users.\n\nAs in the above, this whole strategy is so different. It is thus possible for the method to be used for some other scATAC-seq data analysis with advantages not only in computational efficiency. It would be good for the authors to explore.\n\nThe manuscript is well organized with the core ideas clearly described. But the presentation could be improved - there are a lot of very long sentences unnecessarily connected by “and”, “while”, etc.\n\nThe legend for Fig 1A says “The first (DHS) generated by kallisto on ~2M DNase I sites … ”, but according to the figure and the main text, it should be“~3M”?\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": "5526",
"date": "28 May 2020",
"name": "Davide Cittaro",
"role": "Author Response",
"response": "Thank you for reviewing our manuscript and for the helpful comments. We have addressed major and minor points as detailed in the point-by-point response:The method was only tested with one dataset - PMBC. In fact, single cell ATAC-seq data is usually very sparse. The PMBC dataset used in this study is of relatively high quality. The method remains to be tested on more datasets, especially on those of more sparse, lower-quality. We agree that the PBMC dataset is of high quality. It was used as it could be considered a de facto standard in single cell analysis as it includes several populations at different degrees of separations (i.e. B-cells and T-Cells are well separated, while NK and CD8 are less clearly distinguished). We also would like to point out that it is difficult to identify low quality scATAC-seq datasets for two reasons: one is the relative novelty of this technique and the other is the positive bias in publications, which generally lack of negative or low-quality results. Nevertheless, we tried to address this question analyzing data for K562 cell line. Cell lines are supposedly more homogeneous, data were obtained on a low-throughput platform (Fluidigm C1). We believe that it could be considered a good example of “lower quality” dataset, compared to the PBMC, at least considering the information content. We show that our strategy is consistent with standard approaches based on alignment and peak identification, we can identify the same level of residual heterogeneity. The key advantage of the method is presumably the much improved computational efficiency – there may be other advantages brought by the reference-based method. However, there is no results/statistics on the running time and memory usage in the manuscript. From the description, the improvement should be dramatic. I think it would be very nice to include a table or a figure to demonstrate the increase of computational efficiency. This could be a very helpful way to convince potential users. Thank you for this comment. We benchmarked kallisto+bustools and compared it to cellranger-atac, the default application for 10x data. We added a dedicated section in the main text, which shows the large reduction in required resources. Note that the cellranger-atac pipeline includes several steps that are common in downstream analysis (such as Seurat or Scanpy). In order to make it fair, as explained in the text, we did not consider these steps in the comparison. In addition, we added runtime analysis for the approaches used in the analysis of K562 data. As in the above, this whole strategy is so different. It is thus possible for the method to be used for some other scATAC-seq data analysis with advantages not only in computational efficiency. It would be good for the authors to explore. Our work has been mainly motivated by the reduced resources that are needed by a kallisto-based approach, as we predict the number of scATAC-seq experiments will increase as well as the number of cells profiled. We anticipated additional advantages of a reference-based strategy in the first version of our manuscript, e.g. the availability of promoter-enhancer/gene interactions which could be readily applied to scATAC-seq data. During the revision process we had the opportunity to perform the analysis with non-optimal conditions (i.e. peak identification from single end reads instead of paired end), which led to slightly different results. This serendipitous finding suggests that our strategy, not relying on de novo identification, improves the stability of cell characterization and, therefore, the reproducibility of results. We added these observations in the discussion. Of course, the usage of standardized reference could pave the way to a new class of processing steps not currently performed. As an example, one could identify a set of regions known to be generally accessible (or not) to perform standardized QC. Another example could be the identification of regions that could be used to score the cell cycle phases in scATAC-seq data, much like what is normally done with scRNA-seq data. We feel that all these examples require a deeper analysis, which is beyond the scope of this work, and any undemonstrated procedure would be, at best, greatly speculative. Our aim was to show general consistence between diverse approaches, which we believe has been demonstrated. The manuscript is well organized with the core ideas clearly described. But the presentation could be improved - there are a lot of very long sentences unnecessarily connected by “and”, “while”, etc. Thank you for this comment, we modified the text to increase readability. The legend for Fig 1A says “The first (DHS) generated by kallisto on ~2M DNase I sites … ”, but according to the figure and the main text, it should be“~3M”? Thank you for spotting the typo in the figure legend. We corrected accordingly."
}
]
}
] | 1
|
https://f1000research.com/articles/9-199
|
https://f1000research.com/articles/9-249/v1
|
09 Apr 20
|
{
"type": "Research Article",
"title": "Typing and classification of non-tuberculous mycobacteria isolates",
"authors": [
"Thomas Clarke",
"Lauren Brinkac",
"Joanna Manoranjan",
"Alberto García-Basteiro",
"Harleen Grewal",
"Anthony Kiyimba",
"Elisa Lopez",
"Ragini Macaden",
"Durval Respeito",
"Willy Ssengooba",
"Michele Tameris",
"Granger Sutton",
"Thomas Clarke",
"Lauren Brinkac",
"Joanna Manoranjan",
"Alberto García-Basteiro",
"Harleen Grewal",
"Anthony Kiyimba",
"Elisa Lopez",
"Ragini Macaden",
"Durval Respeito",
"Willy Ssengooba",
"Michele Tameris"
],
"abstract": "Background: There are a large and growing number of non-tuberculous mycobacteria (NTM) species that have been isolated, identified, and described in the literature, yet there are many clinical isolates which are not assignable to known species even when the genome has been sequenced. Additionally, a recent manuscript has proposed the reclassification of the Mycobacterium genus into five distinct genera. Methods: We describe using a fast average nucleotide identity (ANI) approximation method, MASH, for classifying NTM genomes by comparison to a resource of type strain genomes and proxy genomes. We evaluate the genus reclassification proposal in light of our ANI, MLST, and pan-genome work. Results: We describe here a sequencing study of hundreds of clinical NTM isolates. To aid in characterizing these isolates we defined a multi-locus sequence typing (MLST) schema for NTMs which can differentiate strains at the species and subspecies level using eight ribosomal protein genes. We determined and deposited the allele profiles for 2,802 NTM and Mycobacterium tuberculosis complex strains in PubMLST. Conclusions: The MLST schema and our pan-genome analysis of Mycobacteria can help inform the design of marker-gene diagnostics. The ANI comparisons likewise can assist in the classification of unknown genomes, even from previously unknown species.",
"keywords": [
"non-tuberculous mycobacteria",
"NTM",
"taxonomic classification",
"MASH ANI",
"MLST"
],
"content": "Introduction\n\nNon-tuberculous mycobacteria (NTM) are a ubiquitous group of diverse environmental mycobacteria related to Mycobacterium tuberculosis, the bacterium that causes tuberculosis (TB)1–7. A significant number of NTMs cause clinical disease. Diseases caused by NTM infections are important sources of morbidity and mortality throughout the world, with a resurgence of infections in the United States, especially in immunocompromised individuals. The host-pathogen interactions of NTMs remain poorly characterized. Importantly, the background prevalence of NTMs has been shown to interfere with important aspects of TB disease control and management, including the efficacy of new TB vaccines8,9, established immuno-diagnostic methods, and the specificity of diagnostic and immune biomarkers10. Data on NTM prevalence in TB-endemic countries, however, is limited and often under-reported. Possible factors contributing to under-reporting include overlapping clinical manifestations of TB and NTM disease, lack of awareness of NTMs, and insufficient laboratory infrastructure to identify and culture NTMs11. The NTM-NET published a survey of NTM patient isolates12: “A total of 20 182 patients had NTM species cultured from pulmonary samples in these centres in 2008; 91.3% (n=18 418) of the isolates were identified to species/complex level; the remaining 1764 isolates (8.7%) were not identified beyond Mycobacterium species other than M. tuberculosis complex. A total of 91 different NTM species were encountered in this survey.”. While this was an important survey of NTM geographical distribution the low number of NTM species identified and the inability to classify some samples emphasizes the need for more classification resources. Gupta et al. proposed the Mycobacterium genus be divided into five distinct genera: Mycobacterium, Mycolicibactergen, Mycolicibacterium, Mycolicibacillus, and Mycobacteroides13 in the Mycobacteriaceae family. There are over 197 Mycobacteriaceae species from these five genera according to the List of Prokaryotic Names with Standing in Nomenclature (LPSN)14,15 and elsewhere.\n\nWe undertook to sequence, analyze and characterize NTM strains representing geographically diverse TB-endemic areas with high NTM prevalence. Ancillary findings from adolescent and neonatal cohort studies conducted by Aeras and their collaborators to determine the incidence and prevalence of TB in Cambodia, India, Mozambique, South Africa, and Uganda included the prevalence and typing of NTM infections. The sites that participated in these studies collected NTM samples which were sequenced by JCVI. Extended data, Supplemental table S116 lists the samples sequenced from each site.\n\nOur experience elucidated some of the issues which confound clinicians. Current commercial methods for typing NTMs are limited to a small number of the better known NTMs and even then are often not sensitive or specific enough2,17. Connecting the published literature for NTM type strains and related characterization of the species to a clinical isolate is often difficult even given the genome of the isolate due to non-specific marker gene sequences being available. This is being addressed to some extent as many more type strains are having their genomes sequenced18–20. We propose a fast average nucleotide identity (ANI) method when whole genome sequencing is practical and a multi locus sequence typing (MLST) approach when whole genome sequencing is not feasible.\n\n\nResults\n\nThere are four major facets of our analysis: clustering of Mycobactericeae family genomes using ANI, a pan-genome of representative genomes from each clade determined by ANI, an evaluation of our MLST scheme, and a comparison of our pan-genome to the work of Gupta in terms of genera assignment and genera specific genes. ANI-based clustering has been shown to be well correlated with known species and subspecies definitions21–30. We show how automated ANI clustering and ANI comparisons to representative genomes can inform classification of a novel genome. Pan-genome analysis can be used to evaluate genome relatedness across two major comparators: ANI as previously mentioned across all genes but also across subsets of genes, and gene content similarity. We discuss aspects of both pan-genome ANI and gene content. MLST can be used in the absence of genome sequencing data if the loci used in the MLST scheme are acquired by other methods such as PCR. We propose a distance metric based on the concatenated alignment of the MLST loci and evaluate how this can be used in the absence of ANI for classification. Gupta recently proposed a reclassification of five genera within the Mycobactericeae family13. We evaluate this proposal compared to our pan-genome analysis. Gupta also proposed genera specific genes to help delineate the genera and we evaluate those as well.\n\nA total of 2,802 genome assemblies in the Mycobactericeae family in NCBI RefSeq were downloaded on 8 August 2019 (to reduce the number of genomes analyzed we only used complete M. tuberculosis genome assemblies). This represents the complete set as at 30 April 2019. We added six Mycobactericeae genome assemblies available in GenBank that had been excluded from RefSeq either for no specified reason or for being untrustworthy type strains, though the latter were not used to assign names to other genomes. An additional 11 genomes from other Corynebacteriales families that were sequenced as part of the project (Extended data, Supplemental table S116) were included in the analysis for a total of 2819 genomes. We generated species- and sub-species-level clusters of the Mycobactericeae genomes using an all versus all ANI comparison with MASH21,31. We generated species and sub-species clusters with average linkage and a cutoff value of 95% ANI for species and 98% for subspecies (Extended data, Supplemental tables S2 and S332) using GGRaSP33. This has been shown to be a reasonable way to computationally compute tentative species/subspecies clusters18,21–30. For the species-level 95% clustering, there were 269 clusters of which 263 are from Mycobactericeae, 126 of the Mycobactericeae clusters contained a type strain, of which 13 contained more than one type strain of different species (Extended data, Supplemental tables S2 and S342). For the subspecies-level 98% clustering, there were 336 clusters of which 329 are from Mycobactericeae, 137 of the Mycobactericeae clusters contained a type strain, of which 11 contained more than one type strain, and 1 which contained multiple subspecies (Extended data, Supplemental table S332). To represent as much of the taxonomic space as succinctly as possible, we selected representative genomes using clades generated from the two cluster levels. By default, we used the 95% ANI clusters, but for the nine 95% clusters that contained two or more 98% clusters containing type strains, we used all of the 98% clusters in place of the 95% clusters. This meant that 26 98% clusters replaced these nine 95% clusters, resulting in 280 Mycobacteriaceae clusters out of 286 total clusters, 131 of which contained a type strain, 12 of which contained more than one type strain of different subspecies (Extended data, Supplemental table S434). For each of the final 280 Mycobacteriaceae clusters, a representative genome was determined using GGRaSP based on the minimum of summed pairwise MASH ANI distances within the cluster which is called the cluster medoid genome. For the clusters with a RefSeq-included type strain, only the RefSeq-included type strains were considered for the representative genomes, whereas in the other clades, all genomes were considered.\n\nWe used the J. Craig Venter Institute (JCVI) pan-genome pipeline to generate a pan-genome using 279 of the 280 representative Mycobacteriaceae genomes plus a Norcadia genome (N. nova SAMN02900753, cluster 190) as an outgroup35,36. We were required to exclude one of the clusters (180B) as the sole cluster member (SAMN04216932) is not available in RefSeq, whose consistent annotation we use for the pan-genome pipeline. Using gene presence in at least 95% of genomes to determine core genes, we found 1,680 core genes (723 at 100% including the outgroup genome another 407 missing in one genome only) out of 118,867 orthologous gene clusters including 71,559 gene clusters present in only a single genome. A previously reported NTM pan-genome19 using only 99 genomes struggled to identify many core genes (243), possibly due in part to using a 100% threshold for genome presence and generated a lot of orthologous gene clusters (150,000), possibly due to the 80% nucleotide identity cutoff used. The pangenome results in multiple similarity measures which aid classification of the genomes. For example, identifying core genes is crucial for developing diagnostics, especially for identifying potential MLST loci with the least diversity to allow for primer design. Additionally, non-core genes which are restricted to only a segment of the phylogeny can be used for classification purposes, such as genus-specific genes for genus classification. Finally, the JCVI pan-genome pipeline produces pairwise genome ANI results based on the orthologous clusters determined from which genome ANI trees can be created.\n\nWe believe the ANI based clusters (Extended data, Supplemental table S434) and their representative genomes can be used for novel genome classification. How to classify clusters which contain more than one differently named type strain or contain no type strain at all is complicated. As Tortoli et al.18 point out, historical species definitions made before genome sequencing may need to be revised when existing species are found to have very similar genomes. Species should not be redefined simply based on ANI clustering since other factors such as significant difference in genome size (gene content), significantly divergent host specificity, disease presentation, or significant presence in the literature must be taken into account. We leave it to those more familiar with Mycobactericeae species to make those determinations. Three other problems were pointed out by Tortoli et al.18 which we also observed: some type strains have been genome sequenced more than once where the genomes are clearly different species from each other38; type strain genomes do not conform to species descriptions and previously sequenced marker gene sequences, and non-type strain genomes have been misnamed based on the first two problems. We identified two type strains: M. phlei (SAMEA4040484) and M. chelonae (SAMEA4040023) whose clustering with other type names suggests that their names have been swapped on submission to NBCI. We have used our corrected names for these samples in our tables and figures. As mentioned above, for clusters with more than one differently named type strain, we chose the medoid of the type strains as the cluster representative but in Extended data, Supplemental table S434 we give all of the type strains in the cluster and in Extended data, Supplemental table S537 we use an asterisk with the proposed species/subspecies classification name to indicate the uncertainty for members of these clusters. For species clusters lacking a type strain, a cluster-specific “genomosp.” (this is NCBI’s preferred designation for proposed species clades without a type strain) such as genomosp. X was used as the species name for all the genomes in the cluster. If a subspecies cluster did not have a type strain, then only the species level name is given since NCBI does not support a genomosp. concept at the subspecies level.\n\nFor all the Mycobacteriaceae genomes, we assigned a genus to them using average-linkage clustering based on PanOCT pan-genome pairwise ANI distances (Figure 1)35. For the four new genera which Gupta proposed, we found the least common ancestor node which contained all the Gupta genus type strains for that genus. Since Gupta supplied only one type strain for the Mycobacterium genus, we also found the root node of the largest subtree that included this type strain and no additional Gupta genus type strains. The genomes descending from these five nodes were then assigned to the respective Gupta genus. Other than the single outgroup genome not from Mycobacteriaceae, there is only one clade which is not included in the five Gupta subtrees. In Figure 1, this clade is indicated by black colored branches. By Gupta’s criteria for NTM genera within the Mycobacteriaceae this novel clade should be designated as a new genus. This clade contains four representative genomes but only one type strain, Mycobacterium haemophilum (SAMN01918491). We labeled these genomes as Unclassified genera in Extended data, Supplemental table S434, pending assignment of an additional genus name. NCBI has accepted the Gupta proposal in naming NTM species except for the misidentified genome, Mycolicibacterium vulneris (SAMN06651660)38,39. For this genome, NCBI has accepted the genus reassignment from the Gupta corrigendum, Mycobacterium vulneris which matches our clade assignment, though they have not renamed the species. Neither Gupta nor NCBI has renamed all NTM genomes in GenBank based on some form of clustering or phylogeny. This means the current NCBI names are not consistent with our clade-based assignment, as seen in the occasional differences in leaf and branch colors in Figure 1. Of the representative type strains, four have NCBI genus names which disagree with our assignment. These are labeled as Mycobacterium (neumannii, syngnathidarumi, dioxanotrophicus and aquaticum), but are located in the Mycolicibacterium subtree. One of them (nuemannii) even shares a cluster with a Gupta Mycolicibacterium type strain (Mycolicibacterium flavescens). There are also 58 non-type representative genomes whose NCBI genus assignment disagrees with ours, and we have highlighted these rows in Extended data, Supplemental table S434. Our pan-genome ANI clustering was done for the representative genomes, so we conferred the same genus as determined for each representative genome to genomes in that representative’s species/subspecies cluster (Extended data, Supplemental table S537). This also identified four non-representative type strains which had a different genus than their NCBI name. All four were previously labeled as Mycobacterium, with two renamed as Mycobacteriodes (chelonae subsp. gwanake and subsp. chelonae), and two as Mycolicibacterium (kyoganese and lehmannii).\n\nThe tree was generated from the pan-genome ANI distance metric using average-linkage clustering. The branches are colored according to the assigned genera of the genomes from least-common-ancestors with the Gupta-defined type strains (shown on the tree as the colored circle on the ancestral nodes). The leaves are colored by the genus given by NCBI for the genome on 09/09/2019. On the outside of the tree, the orange circles denote NCBI-defined type strains, whereas farther out circles indicate the type strains identified in Gupta et al. as belonging to a specified genus. The green star identifies the cluster containing two type strains for which the NCBI type strain genus identification and the Gupta assignment differs, and the orange star identifies the cluster containing a type strain previously suggested to be mis-identified.\n\nThe species and sub-species classification of all the non-representative genomes was done based on cluster membership. For genomes within clusters containing a single named type strain, that species name was used for all genomes. For clusters lacking a type strain, a cluster-specific “genomosp.” was used as the species name for all the genomes in the clade. For clades with multiple named type strains, the species name of the best ANI match to one of those type strains was used. We warn that the above caution about assigning species based solely on ANI holds and that further work might be necessary. The classification results are shown in Extended data, Supplemental table S537.\n\nWe propose classifying a novel genome by running MASH ANI of the novel genome compared to the set of representative genomes. To verify this approach, we classified each genome using MASH pairwise ANI to the set of representative genomes compared against the cluster-based naming. For each genome the closest MASH ANI to the representative genomes were almost exclusively within the same cluster. Only for 26 genomes in clusters 28B as well as single genomes in cluster 47B and in cluster 165B was the closest ANI assigned match not in the same cluster. This only occurred for clusters discriminated at the 98% threshold (subspecies). For the 26 genomes in cluster 28B but with a best ANI match to the cluster 28A’s representative genome, clusters 28B and 28A are two of the four subspecies clusters of M. abscessus. There are only three named M. abscessus subspecies and the fourth cluster (28A) is probably artifactual occurring right at the boundary of the 98% MASH ANI threshold. Clusters 28A and 28B should probably be combined based on the simple observation that all genomes in both clusters had their second-best MASH ANI match to the other’s representative sequence and all these ANI distances were less than 2% which is within the clustering threshold. The same is basically true for clusters 47B/E and 165B/A where only a few second-best MASH ANI distance values are slightly above 2%. Regardless of whether these clusters are merged based on these criteria when a novel genome is being evaluated the second-best MASH ANI distance should also be considered to ascertain the certainty of an assignment. Any novel genome exceeding the 95/98% ANI threshold to this set of genomes can be assigned a species/subspecies name using MASH ANI to representative genomes (again with more careful scrutiny if the second-best MASH ANI distance is very close to the best MASH ANI distance). The set of MASH sketches will need to be updated both as more genomes are sequenced falling outside of the existing clusters and as real species/subspecies names are defined or determined. While recent NTM type strain sequencing projects (BioProjects: PRJNA35424818, PRJDB422720, PRJNA29946719) have greatly increased the number of sequenced NTM type genomes, many have still not been sequenced and could correspond to some of the proposed genomospecies. Genomes sequenced as part of our project (Extended data, Supplemental table S116) have assisted in the expansion, contributing genomes to 88 of the 280 Mycobacteriaceae clades. This includes 58 clades for which they are the only representatives and a third of both the Mycobacterium and the Mycolicibacullus clades.\n\nOne common diagnostic tool used for identifying species or strains is multi-locus sequence typing (MLST), where a small set of single copy conserved gene sequences are used to distinguish genotype. MLST can be done computationally using genome sequences or via primer-based sequencing. In either case it is important that the gene sequences be conserved enough to design primers or find the gene via homology search; and divergent enough to be able to distinguish different species or strains. We found that some marker genes that had been used in the past40 tended to be either fragmented in genome assemblies or too divergent to align easily. When working with an earlier pan-genome we chose the core genes with no variation in gene length and highest conservation to serve as marker genes which all turned out to be ribosomal proteins (S7, S8, S12, S14Z, S19, L16, L19, L35). This MLST schema was submitted to PubMLST Mycobacteria41. These eight marker genes are also core genes in the 280 Mycobacteriaceae genome pan-genome with very low gene length variation and high conservation (Extended data, Supplemental file S642).\n\nWhen genome sequence is not available for a clinical sample but MLST is a possibility, we believe that our or a comparable MLST schema could be used for diagnostic species/subspecies assignment, such as seen in Extended data, Supplemental table S743. Using our MLST schema an ST is never shared across species/subspecies clusters (Extended data, Supplemental table S537). For most of the genomes, the ST distance (the fraction of mismatches across the combined multiple sequence alignment of the MLST loci) of a genome to the representative genome of that genomes ANI cluster is smaller than the ST distance to any other representative genome (Extended data, Supplemental table S537). Only for 142 genomes was the closest ST different than the ANI assigned cluster (Extended data, Supplemental table S537). These genomes include 27 out of the 28 genomes whose best MASH hit to a representative were not in the same cluster as themselves, excluding only SAMN07811434 from Cluster 165B. This difference mostly occurred for an ST when the incorrectly chosen ST was from a different subspecies cluster of the same species, though for 59 genomes they were from different species clusters. For the 59 genomes which had best ST hits to different species the ST distance was above the cutoff for same species assignment explained below. For almost all 142 discrepant genomes the second closest ST was the correct one, with the exception of four cluster 47E genomes where the best ST hit was to 47D, and the second-best ST hit was to 47B. A remaining issue is whether a novel genome is within an existing cluster or a novel cluster. Figure 2 shows statistics for ST distances versus species and subspecies ANI ranges. We can see from this data that ST distance for 95–98% ANI ranges from 0.004–0.02, 98–100% ANI from 0–0.01, and <95% ANI from 0.006–0.26. We devised cutoffs to separate out the three groups using two standard deviations from the mean ST distance for 95–98% ANI (0.007 ± 0.003) which separated the three ANI-based groups (Different Species > 0.01 ≥ Same Species ≥ 0.004 > Same Subspecies) with minimal misassignment (Figure 2). Diagnosticians should use this data to inform the likelihood that a novel ST is from a known or unknown species/subspecies cluster.\n\nThe distribution of ST distances between pairs of Mycobacteriaceae genomes classified by ANI clustering. Three categories of pairwise relationships were examined: genomes of different species (MASH ANI < 95%), genomes of the same species (95% < MASH ANI < 98%), and genomes of the same sub-species (MASH ANI > 98%). ST distance cutoffs to divide the groups were calculated from the mean distances of genomes from the same species plus or minus two standard deviation (solid horizontal lines). These successfully classified nearly all of the data. The percentage of each category of ANI matches which fall into the ST distance bins given by the two thresholds (0.01 and 0.004) are shown directly on the figure.\n\nA recent article published after our analysis had been completed describes what is called an MLST approach to determine NTM species/subspecies classification44. Matsumoto et al. do compare their method against several others including our pubMLST scheme. They do not use our LOCUST software and it is not clear exactly which software on the pubMLST website they do use to evaluate our MLST scheme. For their 29 test genomes, they find that our MLST scheme correctly assigns all genomes at the species level but does not give a subspecies classification as theirs does. This makes sense because the profile we uploaded to pubMLST at the time of their analysis did not include subspecies level information. We have recently updated our pubMLST profile to include subspecies information. The MLST scheme presented by Matsumoto et al. is atypical. First, the set of loci used is very large (184), which is not consistent with performing efficient PCR. Second, only 53 of the loci are core genes; whereas, the remaining 131 are species specific genes. Third, because most of the loci are species specific it is not even clear how one defines a sequence type and what the authors discuss instead is a similarity score. Fourth, the msltverse software which is used (and is not compatible with standard MLST schemes) needs whole-genome read data as input obviating any advantage of MLST over whole-genome sequencing. As we have argued, whole-genome data for ANI comparison is preferable to an MLST approach but we also argue that the ANI data is sufficient for species categorization and that mlstverse is not necessary. We have nothing against looking for the presence or absence of genes in whole-genome sequence data to help identify virulence or antibiotic resistance. We do caution that only WGS data which can be assembled into complete chromosomes and plasmids can reliably determine the absence of a gene. We also note that claiming a particular gene is species/subspecies specific is only possible if a large, diverse set of genomes is available for all species/subspecies. Finally, attempting to do species/subspecies classification based on gene content is problematic due to horizontal transfer, particularly across subspecies boundaries.\n\nTo further aid in the genus classification of novel Mycobacteriaceae genomes, it is also possible to look for the presence of conserved, genus-specific genes. Gupta et al. provide 89 conserved signature proteins which can be used to define the family and genera (Extended data, Supplemental table S845)13. These proteins were selected by Gupta et al. based on BlastP comparisons of the genera type strain genomes and either the non-Mycobacteriaceae protein databases or Mycobacteriaceae genomes outside of a given genus Using our Mycobacteriaceae pan-genome with the genus assignments discussed above, we identified genus specific gene clusters which were present in each representative genome in a genus but not in any of the other genomes. If no cluster met this criterion for a genus, we required the difference between a gene clusters presence in the genus genomes and the out of genus genomes to be greater than 90%. We used the medoids of these selected gene clusters as potential CSPs. This resulted in 648 pan-genome based CSPs (Extended data, Supplemental table S945). Only a small number25 of the Gutpa CSPs were mapped to the pan-genome’s CSP clusters using either GenBank identifier matches or best blast matches probably due to different criteria for selection. Most46 of the remaining 65 of the Gupta et al. CSPs were genus-specific, but only had a partial presence in the genomes associated with the genus.\n\nTo test the genus prediction success rate of BlastX searches of these gene sets, we compared them to the 2,802 RefSeq Mycobacteriaceae genomic sequences. We did not include 11 of the Gupta CSPs which have since been removed from RefSeq (Extended data, Supplemental table S845). Classification of the genomes with the CSPs was based on a simplified assignment algorithm, where a genome was assigned to the genus with the greatest percentage of genus-specific genes that had a blast hit to a given genome with an e-value < 1e-5. The Gupta CSPs gave the same answer as the pan-genome based genus assignment for all the RefSeq Mycobatericeae genomes excluding the Unclassified clade 98.7% of the time, whereas the pan-genome CSPs exceeds this with a 99.9% match rate, even when accounting for the Unclassified genus. The one incorrect assignment was an Unclassified genome being assigned as Mycobacteroides. The assignment could potentially be improved by expanding the search beyond only a single medoid gene for each gene cluster. Overall using the CSPs to assign genus seems to have limited utility compared to MASH ANI or MLST. Researching why certain genes are genus specific and what phenotype(s) they confer might be of more value.\n\nAfter our analysis was complete, some novel high-quality NTM genomes became available from Matsumoto et al.44: “In this work, we expanded the NTM genomic data by 63 species, of which 27 species were newly determined and the remaining 36 species were re-sequenced by Illumina and Oxford Nanopore Technologies (ONT).” We compared these 63 genomes to our cluster representatives using ANI and ST distance (Extended data, Supplemental table S1347). We also used our and Gupta’s CSP proteins to predict genera (Extended data, Supplemental table S1347). Of the genomes, 16 are not in any of the clusters by ANI (95% threshold). Of these genomes, 15 currently have genus names that agree with their closest MASH medoid (four Mycobacterium and eleven Mycolicibacterium). The single genus name disagreement is BioSample SAMD00153170, which is currently labeled Mycobacterium gallinarum, but which is closest to a Mycolicibacterium clade with a MASH distance well within the generally accepted ANI range (94.19%) of being in the same genus. Additionally, seven genomes are in previously untyped clusters, thus enabling the clades to be named with a species name (Extended data, Supplemental table S434). For 47 of the 63 genomes the best ANI match to a cluster representative is greater than 95%. Of these 47, 33 have the same species name as the representative, 7 as noted previously do not have a real species name, and 7 have different species names. Mycobacteroides abscessus subsp. massiliense has its best ANI match to cluster 28A which is one of the four subspecies clusters for Mycobacteroides abscessus but the previously sequenced type strain for Mycobacteroides abscessus subsp. massiliense is in cluster 28B. We had already observed that clusters 28A and 28B should probably be merged based on ANI and ST mapping and this only reinforces that observation. For the 7 with different species names, there are four categories: split the existing cluster into two or more clusters at 98% ANI to represent different species (three genomes with 95.1–96% ANI to cluster representative), split the existing cluster into two or more clusters at 98% ANI to represent different subspecies (two genomes with 97.6–98.3% ANI to cluster representative), consider declaring two type species to be the same species and one of them to be a heterotypic synonym (1 genome with 99.0% ANI to cluster representative), and the genome type strain is already in the cluster but the chosen medoid between the two species is not the same (paraintracellulare and intracellulare – cluster 47D). This leaves 16 genomes with a best match to a cluster representative below 95% ANI. These 16 genomes would found new clusters where each would be a singleton cluster and be the representative for that cluster. Of the 63 genomes 25 had novel ST types which we added to the pubMLST schema. The ST distances were generally well correlated with the ANI values with only 4 genomes with ANI below 96.1% having ST distances above the same species threshold. For the CSPs neither Gupta (five discrepant) nor ours (one discrepant) performed perfectly for genus assignment. Mycolicibacterium aubagnense was classified by both methods as Mycobacterium, and four additional genomes had discrepant Gupta CSP based genus assignments. This confirms our skepticism about this approach.\n\n\nDiscussion\n\nOur analysis shows that the universe of NTM species/subspecies is still far from complete: 142 of our clusters do not even have an associated type strain, many NTM species still do not have a genome sequence, and there are probably many NTM clades which are not represented in our clusters. Our suspicion of the sparseness of the NTM family tree is reinforced with the addition of the Matsumoto genomes, a quarter of which were not assigned to our species-level clusters. The incompleteness of NTM genomic data could easily lead to misdiagnosed or undiagnosed NTM infections. We have proposed an efficient structure based on ANI and species/subspecies cluster representative genomes to classify any patient sample with genomic data. This includes recognizing that a patient sample is from a novel NTM clade. As more NTM genomic data becomes available, our representation is easily extended with new species/subspecies cluster representative genomes. Many NTM species are slow or very slow growing making isolation of DNA for genomic sequencing challenging. When genomic data is not available, we show that our pubMLST scheme is sufficient for the classification of most patient samples. Diagnostic companies may not prefer our choice of MLST loci, so we have included all of our data on the pan-genome clusters (Extended data, Supplemental file S642) from which to choose for PCR or hybridization-based methods. We chose our loci based on being more highly conserved at the nucleotide level for PCR primer design, being present in all genomes, being full length in all genomes, and having low length variability so that gaps would not complicate multiple sequence alignment-based analysis (Extended data, Supplemental file S642). When more extensive analysis is desired, for example for the presence of noncore antibiotic or virulence genes a pan-genome analysis can be helpful. We showed that the pan-genome can be used to determine core and genus-specific genes.\n\n\nMethods\n\nA total of 288 samples of genomic DNA were collected from five different geographical sites (Extended data, Supplemental Table S116). The genomic DNA was isolated from cultures grown from samples of sputum or gastric aspirates that were originally collected from TB presumptive patients with the exception of an adolescent cohort study (E-004-IN-A) from the St. John’s Research Institute (SJRI), where the specimens were collected from healthy children. Samples were initially treated with a decontamination and digestion step using N-acetyl-L-cysteine-sodium hydroxide (NALC-NaOH) and then screened for acid-fast bacilli (AFB) using smear microscopy with Ziehl-Neelsen (ZN) or Auramine O fluorescent staining (South African Tuberculosis Vaccine Initiative (SATVI) and Makerere University (MU). Samples obtained from SJRI, Centro de investigação de Saúde de Manhiça (CISM), and MU were also examined directly by ZN and fluorescent microscopy prior to decontamination. After decontamination and digestion, samples were cultured on Lowenstein Jensen (LJ) media and also inoculated into Mycobacterial growth indicator tubes (MGIT) 960 BD, following manufacturer’s instructions. Samples from SATVI that were MGIT positive for growth were then stained for AFB by ZN and Gram stain. Samples from all sites that were AFB-positive were further identified at the genus and species levels. Samples at Institut Pastuer – Cambodia (IPC) and SATVI were initially analyzed by AccuProbe, Gen-probe, Inc., while samples at MU were first typed by mycobacteria specific 16S rDNA PCR identification. All sites, with the exception of SATVI, used the Hain GenoType Mycobacterium CM/AM test system, according to manufacturer’s instructions, to identify NTMs at the species level. Samples at SATVI that were Accuprobe negative were further identified by methods of Brunello et al.48 and Telenti et al.49 (Briefly, PCR amplification of hsp65 gene, digestion of product by 2–3 restriction enzymes, identification by RFLP – examination of banding patterns on agarose gel electrophoresis); see Hatherill et al.50 for further details.) If any other bacteria were detected during smear microscopy examination for AFB, the cultures were subject to a second round of NACL-NaOH decontamination and reinoculated into LJ and MGIT. The contaminated cultures were also plated onto blood agar to identify contaminants. All decontaminated specimens positive for AFB were saved and stored at either -20°C (CISM) or -80°C (IPC, SJRI, MU, SATVI). Positive growth cultures from the MGIT were also stored at -80°C in the MGIT media of Middlebrook 7H9.\n\nIn an attempt to provide broad coverage of the NTM strains both by species and geographic location, no more than 10 strains of the most frequent species from each site were chosen for regrowth. At the same time, no fewer than five strains from the same species were collected when possible. Additionally, a number of unidentifiable NTM cultures were also collected from each of the sites. Samples within these parameters were chosen at random for regrowth; however, samples that failed to regrow were replaced with new samples. Sites noted that samples frozen at more recent dates were easier to regrow. Regrowth of cultures was done from MGIT Middlebrook 7H9 or decontaminated specimen frozen stocks by inoculating MGIT 7H9 media (CISM, IPC, MU, SJRI), LJ medium (CISM, SATVI/CSU), and 7H11 (SATVI/CSU, CISM). Subculturing on LJ media for single colony isolation was done for samples from CISM, IPC, SATVI/CSU, and SJRI. Regrown samples from MU were observed by ZN staining to re-confirm presence of AFB and also rule out contaminants by culturing on blood agar.\n\nNTM cultures were grown in 7H9 medium up to an OD >0.6 and then centrifuged for five minutes at 10,000 rpm. Supernatant was removed and pellets resuspended in TE buffer before incubating at 95°C for 10 minutes. Several sites grew cultures on solid media instead of 7H9 prior to DNA extraction. This media included 7H11 agar (SATVI) and LJ agar (SJRI, CISM, CISM). For these samples, single colonies were used for DNA extraction in TE buffer. After the incubation step, samples were centrifuged for 10 minutes at 10,000 rpm and supernatant removed and passed through a sterile syringe Filter (0.2 µm). Samples were stored at 4°C.\n\nIllumina NextSeq library preparation and sequencing was used. Genome assembly was performed using SPAdes v 3.1.151, which produced good quality assemblies as detailed in supplementary materials (Extended data, Supplemental Table S116).\n\nThe NTM genomes sequenced for this project were annotated by NCBI’s prokaryotic annotation pipeline, PGAP52.\n\nA total of 2,802 NTM and TB complex strains were downloaded from NCBI RefSeq on 08/08/2019 and their PGAAP annotation used for comparative analysis. 6 Mycobactericeae strains excluded from RefSeq for either being untrustworthy as type or for no given reason and 11 Cornybactereae sequenced by as part of this project were also downloaded. ANI between genomes was calculated using MASH v2.0, a very fast tool for determining approximate pairwise ANI values based minHashing of kmer overlap31. Pairwise comparisons of all the genomes was run with 10,000 21-mers per genome. We used the GGRaSP R package v1.0 which generated a complete linkage tree from the distance matrix calculated by subtracting 100 from the MASH ANI results33. The resulting tree was divided into clusters at 95% (species level) and 98% (sub-species) ANI cutoffs using R commands ggrasp.cluster(threshold =2) and ggrasp.cluster(threshold =5). For 95% ANI clusters which contains at least two daughter 98% clusters with species or sub-species type strains, all the 98% clusters are retained; otherwise, only the 95% clusters are retained. All clusters are named by the GGRaSP 95% numeric order, and the 98% cluster having a sequential additional letter. For each cluster, a representative was selected by picking the type strain that has the lowest combined ANI to all the other genomes in the cluster. For clusters with no type strain, the genome with the lowest combined ANI to all the other genomes was selected. If a cluster contained a single type strain or a Matsumoto genome was later assigned to a cluster with no type strain, then genomes in that cluster were named based on that type strain. For clusters with multiple type strains genomes were also individually named from MASH ANI comparisons with type strains (both those downloaded on 8/8/2019 and those subsequently sequenced by Matsomoto) using the name deriving from the type strain with the highest ANI value. Species names are only used when the ANI value is greater than or equal to 95% and sub-species for values greater than or equal to 98%.\n\nWe used the JVCI pan-genome pipeline (default settings using nucleotide sequences) to generate a pan-genome using 279 of the 280 representative Mycobacteriaceae genomes plus a Norcadia genome (N. nova SAMN02900753, cluster 190) as an outgroup35,36. We were required to exclude one of the clusters (180B) as the sole cluster member (SAMN04216932) is not available in RefSeq whose consistent annotation we use for the pan-genome pipeline. When representative assemblies were not available in RefSeq (two clusters: 141, 188A), but assemblies from other genomes within the cluster were, a secondary representative present in RefSeq was used.\n\nThe genus of the 279 mycobacteriaceae medoiods was assigned using the pan-genome ANI average-linkage clustering generated from the JCVI pan-genome pipeline35,36. For four of the genera, the Least-Common Ancestor for all genus type strains designated by Gupta et al. (found either within the text or in Tables 8–11) was identified13. Since the genus Mycobaceterium only had a single designated genus type strain, the most ancestral node containing the genus type strain and no other type strains was used as the genus ancestral node. All genomes descending from these nodes were assigned to that genus. Any remaining Mycobacteriaceae genomes were in a single cluster and labeled as unclassified. All non-representative genomes were assigned the same genus as the representative strain in their cluster.\n\nCore genes identified by pan-genome analysis35,36 were evaluated to identify candidate marker genes for MLST analysis. Eight highly conserved ribosomal proteins (S7, S8, S12, S14Z, S19, L16, L19, L35) were selected. Distinct allelic profiles and sequence type definitions were generated by searching the consensus sequences of the eight ribosomal proteins against 2,802 NTM and TB complex strains using the novel schema function of LOCUST v3.053. The corresponding allele sequences and full MLST schema was submitted to https://pubmlst.org/mycobacteria/. Species and subspecies assignments were made for 2,802 NTM and TB complex strains using the cluster approximation function of LOCUST version 3.0 (manuscript in preparation) based on the 280 type and proxytype strains discussed previously (Extended data, Supplemental table S434) with the command line typer.pl –biosample_list biosample.list –download_schema “Mycobacteria” –cluster_approximation –cluster_approximation_list type_strain.list -t fasttree\n\nThe programs used in this study are available through GitHub at the JCVI repository at https://github.com/orgs/JCVenterInstitute, including: JCVI pan-genome pipeline (PangenomePipeline), LOCUST, and GGRaSP.\n\nGenus Conserved and Unique Proteins were selected for each of the six Mycobacteriaceae genera by identifying clusters which are present in all the genomes of a genus and absent in all other genomes. When there were no such clusters for a genus, clusters where the percentage difference between presence in the genus and presence outside the genus was greater than 90% were selected. The medoids for each of the selected clusters were used. Protein sequences of the cluster centroids were obtained using transeq in the EMBOSS suite. Protein identifications for all of the Gupta et al. genes were collected from tables 2, 5, and 713 and the sequences were downloaded from NCBI. Comparisons of the presence and absence of the CSPs in the genomes was done using tblastn for the whole genome sequences and the CSPs. Blast matches with e-values < 1e-5 were considered a hit.\n\n\nData availability\n\nNCBI BioProject: Sequence and Analysis of Non-tuberculous Mycobacteria (NTM) strains. Accession number PRJNA305922.\n\nThis BioProject contains all clinical NTM sample sequences; Individual biosample IDs of the genomes can be found in Extended data, Supplemental table 116.\n\nMLST allele sequences and corresponding schema is available at the PubMLST Mycobacteria genome database (https://pubmlst.org/mycobacteria/). All MLST profiles generated as part of these analysis contain the specific genome names in the organism column in Extended data, Supplemental table 743. Other MLST types have NA in the column.\n\nFigshare: Supplemental Table 1. JCVI NTM Genomes. https://doi.org/10.6084/m9.figshare.12016290.v116.\n\nFigshare: Supplemental Tables 2 & 3: NTM Genomes MASH ANI-based Clusters. https://doi.org/10.6084/m9.figshare.12024051.v132.\n\nFigshare: Supplemental Table 4: NTM Clusters. https://doi.org/10.6084/m9.figshare.12024057.v134.\n\nFigshare: Supplemental Table 5. NTM Genomes. https://doi.org/10.6084/m9.figshare.12024117.v137.\n\nFigshare: Supplemental File 6. Pangenome Matchtable. https://doi.org/10.6084/m9.figshare.12024126.v142.\n\nFigshare: Supplemental Table 7. NTM ST Definitions. https://doi.org/10.6084/m9.figshare.12024144.v143.\n\nFigshare: Supplemental Tables 8 and 9. Analysis of Gupta et al. 2018 and Pangenome-Derived Conserved Signature Proteins in NTM Genomes. https://doi.org/10.6084/m9.figshare.12024159.v245.\n\nFigshare: Supplemental Table 10: NTM Pangenome-Based ANI All Versus All Table. https://doi.org/10.6084/m9.figshare.12024024.v254.\n\nFigshare: Supplemental Table 11: NTM Genome MASH ANI Distances. https://doi.org/10.6084/m9.figshare.12024030.v246.\n\nFigshare: Supplemental Table 12. NTM All versus All ST-based Distance Matrix. https://doi.org/10.6084/m9.figshare.12024303.v155.\n\nFigshare: Supplemental Table 13. Analysis of Genomes published in Matsumoto et al. 2019. https://doi.org/10.6084/m9.figshare.12024324.v147.\n\nExtended data 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 Mario Vaz at the Saint John’s Research Institute and Esther Lau at Aeras for their assistance with the collection of the NTM samples, and Karen Beeri, Karrie Goglin, and Indresh Singh at JCVI for their assistance with the sequencing and assembly of the genomes.\n\n\nReferences\n\nJang MA, Koh WJ, Huh HJ, et al.: Distribution of nontuberculous mycobacteria by multigene sequence-based typing and clinical significance of isolated strains. J Clin Microbiol. 2014; 52(4): 1207–12. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTortoli E: Microbiological features and clinical relevance of new species of the genus Mycobacterium. Clin Microbiol Rev. 2014; 27(4): 727–52. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTortoli E: Impact of genotypic studies on mycobacterial taxonomy: the new mycobacteria of the 1990s. Clin Microbiol Rev. 2003; 16(2): 319–54. 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PubMed Abstract | Publisher Full Text\n\nJolley KA, Bray JE, Maiden MCJ: Open-access bacterial population genomics: BIGSdb software, the PubMLST.org website and their applications. Wellcome Open Res. 2018 [cited 2020 Jan 13]; 3: 124. PubMed Abstract | Publisher Full Text | Free Full Text\n\nClarke Thomas: Supplemental File 6. Pangenome Matchtable. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12024126.v1\n\nClarke Thomas: Supplemental Table 7. NTM ST Definitions. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12024144.v1\n\nMatsumoto Y, Kinjo T, Motooka D, et al.: Comprehensive subspecies identification of 175 nontuberculous mycobacteria species based on 7547 genomic profiles. Emerg Microbes Infect. 2019; 8(1): 1043–53. PubMed Abstract | Publisher Full Text | Free Full Text\n\nClarke Thomas: Supplemental Tables 8 and 9. Analysis of Gupta et al. 2018 and Pangenome-Derived Conserved Signature Proteins in NTM Genomes. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12024159.v2\n\nClarke Thomas: Supplemental Table 11: NTM Genome MASH ANI Distances. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12024030.v2\n\nClarke Thomas: Supplemental Table 13. Analysis of Genomes published in Matsumoto et al. 2019. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12024324.v1\n\nBrunello F, Ligozzi M, Cristelli E, et al.: Identification of 54 mycobacterial species by PCR-restriction fragment length polymorphism analysis of the hsp65 gene. J Clin Microbiol. 2001; 39(8): 2799–806. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTelenti A, Marchesi F, Balz M, et al.: Rapid identification of mycobacteria to the species level by polymerase chain reaction and restriction enzyme analysis. J Clin Microbiol. 1993; 31(2): 175–8. PubMed Abstract | Free Full Text\n\nHatherill M, Hawkridge T, Whitelaw A, et al.: Isolation of non-tuberculous mycobacteria in children investigated for pulmonary tuberculosis. PLoS One. 2006; 1(1): e21. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNurk S, Bankevich A, Antipov D, et al.: Assembling single-cell genomes and mini-metagenomes from chimeric MDA products. J Comput Biol. 2013; 20(10): 714–37. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTatusova T, Ciufo S, Fedorov B, et al.: RefSeq microbial genomes database: new representation and annotation strategy. Nucleic Acids Res. 2015; 43(7): 3872. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBrinkac LM, Beck E, Inman J, et al.: LOCUST: a custom sequence locus typer for classifying microbial isolates. Bioinformatics. 2017; 33(11): 1725–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nClarke Thomas: Supplemental Table 10: NTM Pangenome-Based ANI All Versus All Table. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12024024.v2\n\nClarke Thomas: Supplemental Table 12. NTM All versus All ST-based Distance Matrix. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12024303.v1"
}
|
[
{
"id": "62617",
"date": "27 Apr 2020",
"name": "Charles Warden",
"expertise": [
"Reviewer Expertise Genomics and Bioinformatics"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you very much for putting together this publication.\nI think this is a useful paper where you have done a good job overall with being transparent, and I hope it can become a peer-reviewed version soon.\nI think these may be my most important questions (related to clustering in Figure 1):\nIt might already be described somewhere (or split among different locations), but I think some discussion is needed for the large cluster with mixed Mycobacterium and Mycolicibacterium (in the top-right of Figure 1). I have general genomics experience (including working with some pathogen data), but the biological meaning for this mixed portion of the dendrogram was not clear to me (since I don’t have specific experience working with Mycobacteria). I would guess this could be true for other F1000Research readers. Is it possible to provide this in a way that is easy to find (such as a section in the results, or a paragraph in the discussion)?\n\nFor example, am I correctly understanding Figure 1 and Figure 10 in the Gupta et al. 2018 paper show classifications that cluster together (which would be different than the Figure 1 in this paper)? I think this has something to do with whether or not there is a green Mycolicibacterium dot on the outside of the tree, but then the question would be what to think about the Gupta Mycobacterium genomes within that branch (in Figure 1 of this paper)?\n\nLikewise, it looks like the “subgenus” separates more clearly in Figure 1 of the Matsumoto et al. 2019 paper. You mention this paper multiple times, but I don’t believe that I see a sentence that directly compares the results presented in that study to Figure 1 of this study. Unless I have missed something, I think that may be useful to add.\n\nOtherwise, these are my questions related to the interpretation of the results:\nThe separation in Figure 2 looks pretty good, but there are some outliers. Are these well-characterized examples, or could these be misclassifications?\n\nIs there a way to connect Figure 1 and Figure 2? Figure 2 was what I noticed first and felt like I could understand. However, could you do something like separate “Same sub-species” into separate clusters (such as the 5 clusters in Figure 1, if I understand the content)? So, if some clusters have more variation then others, then that would become easier to see. Maybe this should be a Figure 3, for reasons of space.\n\nIf you have performed automated assembly for all of the samples, what sort of checks have you done to justify important differences are due to true biological sequences rather than assembly artifacts (which may or may not be random)? You do mention that “good assemblies” were used from SPAdes and provide some statistics in Supplemental Table 1. However, I think it might help if i) more information about the criteria for “good” can be provided and ii) if any additional factors can be considered.\n\nFor example, there is noticeable variation in the N50 values. Are there any values that tend to be more common in assemblies that don’t have a type strain or previous genome sequence (and/or that appear in the branch with mixed clusters in Figure 1, form small clusters, etc.)?\n\nAre there any features that vary with other expected (or known) genome metrics, such as the proportion or maximum length of repeat sequences?\n\nAdditionally, in Supplemental Table 1, some samples are listed as “No Assembly.” Are there QC measurements that might be able to be used to flag a sample to be removed prior to analysis. This by itself provides a possible flag, but this and the earlier questions relate to whether some samples can generate an assembly but it may be less reliable than others (whether that is due to some limitation in the sample, a limitation in the total number of reads, etc.).\n\nI also have some other minor notes that might help with communicating your results:\nIt may help readability to add numbers for the items listed after “There are four major facets of our analysis” in the Results. While the list is smaller, something similar could be done for “genome relatedness across two major comparators”. This is already in the results section (versus the introduction section, for example), but maybe it would even help to split this into 2 paragraphs and provide more of an explanation? Or, may be a visualization can help with the analysis strategy as a figure? I had a hard time quickly and effectively understanding what was meant to be communicated here.\n\nIt looks like you have done a good job of sharing the raw data and providing a lot of details in supplemental files/tables. I had to start reading the paper to find it, but you also mention sharing code on GitHub. However, am I correctly understanding that these are for the programs that you used (versus code to reproduce the results for this specific study)? If you essentially have 1 wrapper script, perhaps adding that within a .zip file (to see the versions and parameters used) could be sufficient?\n\nThe abstract mentions using the method “MASH”. The methods provide a link (https://github.com/marbl/Mash) that matches the cited reference for a pre-existing method called Mash (Ondov et al. 2016, Genome Biol. 2016 Jun 20;17(1):132). When I re-read the abstract, I can see that this doesn’t mean you *introduced* a method called MASH. I am not sure what other readers think (and this should be obvious to those with experience using that program), but I think one thing that could make this more clear to other reads would be to say “We used Mash to classify NTM genomes” (versus “We describe using a fast average nucleotide identity (ANI) approximation method, MASH, for classifying NTM genomes”). Or, am I overlooking something?\n\nIt seems like a lot of the authors are described as “Investigation, Resources, Writing – Review & Editing”, which kind of sounds like the contributions listed for those in the acknowledgments. Is there some sort of threshold for the amount of work used to identify somebody as an author versus being listed in the acknowledgments? Maybe this even means more authors should be added – I just thought the number of individuals with this exact set of contributions was noticeable.\n\nOn the flip side, I noticed that you said “We leave it to those more familiar with Mycobactericeae species to make those determinations” – while I can understand that it might be difficult to provide all possible checks for all possible samples, is there at least one collaborator that might be able to list some exceptions/limitations to purely using the ANI strategy for assignments (which could be an acknowledgment, if we are talking about a contribution of a few points without revising any analysis)?\n\nI think there should only be one period in a sentence in the introduction (either before or after the quote): different NTM species were encountered in this survey.”.\n\nI think there is a minor formatting issue where only part of the word is italicized in the Introduction: Mycolicibactergen (“gen” is not in italics)\n\nI think there should be a comma in the following sentence in the introduction: “While this was an important survey of NTM geographical distribution the low number of NTM species” à “While this was an important survey of NTM geographical distribution, the low number of NTM species”.\n\nI think there is some unintended capitalization in the results: “of a given genus Using our” à “ of a given genus using our” (this don’t look like the end of a sentence, so I don’t think the “U” should be capitalized)\n\nI think changes are needed (beyond an optional “suggestion”), but I think they can probably be made so that the next revision can be accepted. This is what I was trying to communicate with a decision of “Accept with Reservations”.\n\nThanks Again!\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": [
{
"c_id": "5508",
"date": "28 May 2020",
"name": "Thomas Clarke",
"role": "Author Response",
"response": "We would like to thank Dr.Warden for his thoughtful and critical comments for our manuscript. Below, we have listed the comments (bold font) and our responses (italics font).I think these may be my most important questions (related to clustering in Figure 1): It might already be described somewhere (or split among different locations), but I think some discussion is needed for the large cluster with mixed Mycobacterium and Mycolicibacterium (in the top-right of Figure 1). I have general genomics experience (including working with some pathogen data), but the biological meaning for this mixed portion of the dendrogram was not clear to me (since I don’t have specific experience working with Mycobacteria). I would guess this could be true for other F1000Research readers. Is it possible to provide this in a way that is easy to find (such as a section in the results, or a paragraph in the discussion)? Thank you for bringing this up. It is our understanding that the genera mixture in the top-right clade is indicative solely of the delay in NCBI renaming species according to their Gupta-specified genus, and that the species labeled Mycobacterium are in fact Mycolicibacterium. We have added a sentence to the figure 1 legend clarifying this view (“Some of the NCBI genus names listed do not account for the Gupta et al genus definitions, and thus branch and leaf color disagreements probably only reflects a difference in genus naming conventions”), as well as added a clause to the sentence (shown in bold):“This means the current NCBI names are not consistent with our clade-based assignment, as seen in the occasional differences in leaf and branch colors in Figure 1 and that these differences do not necessarily reveal divergent classification of the species using Gupta based genera.” However as we have not investigated the biological properties of each of the genomes in this tree, we can not conclusively state that they match the Gupta et al physical definitions of the genera as we clarify in an added sentence at the conclusion of the paragraph. For example, am I correctly understanding Figure 1 and Figure 10 in the Gupta et al. 2018 paper show classifications that cluster together (which would be different than the Figure 1 in this paper)? I think this has something to do with whether or not there is a green Mycolicibacterium dot on the outside of the tree, but then the question would be what to think about the Gupta Mycobacterium genomes within that branch (in Figure 1 of this paper)? As specified in the response above, we do not feel that the genome labeled as Mycobacterium are in fact Gupta Mycobacterium but instead old style Mycobacterium where the name has not yet been updated. We found no instances of a genome classified using the Gupta definitions within a clade that was not assigned that genus. We have attempted to clarify the language describing this as noted above, including in the Figure 1 legend. Likewise, it looks like the “subgenus” separates more clearly in Figure 1 of the Matsumoto et al. 2019 paper. You mention this paper multiple times, but I don’t believe that I see a sentence that directly compares the results presented in that study to Figure 1 of this study. Unless I have missed something, I think that may be useful to add. We agree with this. Currently, the data in figure 1c in Matsumoto currently forms part of our analysis as described in the paragraph and in supplemental table 13, so we have added a clarifying clause in the text. We further agree that additional discussion comparing our findings would be useful. We have added three sentences comparing our results with those of Matsumoto when discussing the placement of the Matsumoto et al genomes in Figure 1C, including our disagreement of the M. avium placement and the deeper resolution of our full pagenome tree in Figure 1 in our manuscript.Otherwise, these are my questions related to the interpretation of the results: The separation in Figure 2 looks pretty good, but there are some outliers. Are these well-characterized examples, or could these be misclassifications? The species and subspecies pairs in figure 2 was defined as noted using MASH-based ANI to generate as complete and consistent a dataset as possible for the comparison, since there are no sub-species pairs included in the Pan-Genome analysis, and the similarly named species could have included both pairs at both species and sub-species levels and thus given inconclusive results. As such, though the ANI values might elide or remove biologically defined subtypes, we feel it is the most consistent, though it is possible that genomes listed as sub-species are biologically more realistically defined as species Is there a way to connect Figure 1 and Figure 2? Figure 2 was what I noticed first and felt like I could understand. However, could you do something like separate “Same sub-species” into separate clusters (such as the 5 clusters in Figure 1, if I understand the content)? So, if some clusters have more variation then others, then that would become easier to see. Maybe this should be a Figure 3, for reasons of space. We were able to separate the different sub-species into clusters using the 98% Mash ANI cutoff. The phylogeny of the full genome set including sub-species set was too large to show details of the spread of different sub-species groups, but we can point to Supplemental Table X and Y for more detailed descriptions of the subspecies ANI ranges for the different sub-species, though by definition all of our sub-species clusters are limited to a 2% ANI range If you have performed automated assembly for all of the samples, what sort of checks have you done to justify important differences are due to true biological sequences rather, than assembly artifacts (which may or may not be random)? You do mention that “good assemblies” were used from SPAdes and provide some statistics in Supplemental Table 1. However, I think it might help if i) more information about the criteria for “good” can be provided and ii) if any additional factors can be considered. At the time of assembly and submission the JCVI pipeline ran two assemblies: SPAdes and Velvet. SPAdes tended to give more contiguous assemblies and wasn’t obviously inferior by other metrics. JCVI generated excel spreadsheets of a variety of statistics at the assembly level such as genome size and N50 but also produced data on a per contig basis. One flag of possible assembly issues was when the SPAdes and Velvet statistics such as genome length were not similar. Contig level stats included coverage levels of all contigs and best Blast matches against NCBI genomes. Built into the assembly process contigs with very low coverage versus the median coverage of large contigs were filtered out of the assembly but overall statistics as well as per contig statistics were retained for the filtered contigs. It was not uncommon to see small contigs with coverage depth 2-5x versus primary large contigs with coverage depth 100x or higher where the small contigs would match Ralstonia a common sequencing materials contaminant. Sometimes it was clear that low coverage contigs were a second NTM species but still low enough coverage to be effectively filtered out. Mixed samples where contigs were not at least in a 10 to 1 ratio were deemed inseparable even if 90% of contigs could be easily separated and were not submitted. Besides coverage levels, mixed samples could be seen by outlying genome sizes, varied Blast matches, and more hard to interpret poor assembly (lots of contigs and low N50). We did not submit mixed samples which could not be corrected by filtering out very low coverage contigs. We did not submit assemblies with more than 500 contigs (after filtering out very short and very low coverage contigs) or an N50 < 15kp. While the assembly pipeline was automated as well as the gathering of data to assess the quality of the assembly and the chance of a sample being inseparably mixed the final decision was taken by Granger Sutton who has more than 20 years of experience evaluating assemblies following JCVI guidelines. That being said the NTM samples exhibited a higher than normal rate of mixed samples possibly due to slow growth rate and the isolation of the DNA at the collaborating laboratories. While the authors stand behind the quality of the 201 submitted samples for the BioProject, it is certainly possible that not all contamination/mixing was detected and removed. For example, there is noticeable variation in the N50 values. Are there any values that tend to be more common in assemblies that don’t have a type strain or previous genome sequence (and/or that appear in the branch with mixed clusters in Figure 1, form small clusters, etc.)?This is a very good question which we did not consider for either the JCVI genomes or non JCVI genomes. While genome assembly quality, particularly the non-removal of contamination or mixing of strains, could cause bad ANI values other factors could affect the likelihood that a genome is classified into a cluster with or without a type strain. For instance, genome studies targeting a single outbreak such as the case for many of the M. abscessus genomes which numerically dominate the genomes under study here would bias the results of any simple correlation. We did look at the correlation between N50 and number of contigs to whether a genome was placed into a cluster with a type strain. No significant logistic correlation between MASH mapping with existing type strain and quality of assembly was observed either using the number of contigs or the N50. Are there any features that vary with other expected (or known) genome metrics, such as the proportion or maximum length of repeat sequences?That is another good question that was not the focus of our work. We did not look at repeat content but it has been our experience that there are four major factors influencing N50 size: sequence quality (separate metrics are kept for this and usually flagged before assembly), repeat content, coverage depth (usually once above 30x it is not much of an issue), and the presence of multiple strains of the same species (this is not the same as mixed samples with different species which still tend to assemble well but can be difficult to separate entirely with confidence due to high copy repeats, low copy plasmids, etc). Lower than expected N50 and higher contigs counts in our experience can indicate multiple strains of the same species where variable regions between the conserved core genome regions can chop up the assembly – one call also see an increase in “SNPs” indicating population variation. We try to recognize these mixed strains and not submit them. Studying the actual repeat profile of different NTM species and how that affects assembly while interesting is beyond the scope of this paper. Additionally, in Supplemental Table 1, some samples are listed as “No Assembly.” Are there QC measurements that might be able to be used to flag a sample to be removed prior to analysis. This by itself provides a possible flag, but this and the earlier questions relate to whether some samples can generate an assembly but it may be less reliable than others (whether that is due to some limitation in the sample, a limitation in the total number of reads, etc.). The No Assembly samples are samples that we created BioSamples for based on collaborator input but for which no DNA ever arrived, library prep failed, or assembly was deemed low quality and not submitted. After that initial experience we waited to create BioSamples until a successful assembly existed. In total we received 273 samples of which we submitted assemblies for 201 samples.I also have some other minor notes that might help with communicating your results: It may help readability to add numbers for the items listed after “There are four major facets of our analysis” in the Results. While the list is smaller, something similar could be done for “genome relatedness across two major comparators”. This is already in the results section (versus the introduction section, for example), but maybe it would even help to split this into 2 paragraphs and provide more of an explanation? Or, may be a visualization can help with the analysis strategy as a figure? I had a hard time quickly and effectively understanding what was meant to be communicated here.We agree and have added “first”, “second”, “third”, and “final” and changed the commas to semicolons to the list for clarification, and a comma to the second list to also help accentuate the different type. It looks like you have done a good job of sharing the raw data and providing a lot of details in supplemental files/tables. I had to start reading the paper to find it, but you also mention sharing code on GitHub. However, am I correctly understanding that these are for the programs that you used (versus code to reproduce the results for this specific study)? If you essentially have 1 wrapper script, perhaps adding that within a .zip file (to see the versions and parameters used) could be sufficient?All the programs listed above were run separately and in parallel so we feel the addition of a wrapper script would not add much to assist in their usage beyond what is listed here. The abstract mentions using the method “MASH”. The methods provide a link (https://github.com/marbl/Mash) that matches the cited reference for a pre-existing method called Mash (Ondov et al. 2016, Genome Biol. 2016 Jun 20;17(1):132). When I re-read the abstract, I can see that this doesn’t mean you *introduced* a method called MASH. I am not sure what other readers think (and this should be obvious to those with experience using that program), but I think one thing that could make this more clear to other reads would be to say “We used Mash to classify NTM genomes” (versus “We describe using a fast average nucleotide identity (ANI) approximation method, MASH, for classifying NTM genomes”). Or, am I overlooking something? The reviewer is correct that Mash is a previously released and highly used program. We have added a couple of descriptors to MASH in our abstract to more fully clarify this. It seems like a lot of the authors are described as “Investigation, Resources, Writing – Review & Editing”, which kind of sounds like the contributions listed for those in the acknowledgments. Is there some sort of threshold for the amount of work used to identify somebody as an author versus being listed in the acknowledgments? Maybe this even means more authors should be added – I just thought the number of individuals with this exact set of contributions was noticeable. This manuscript is dependent on and describes novel NTM sequences which were collected from multiple sites around the globe as is seen in supplemental table 1 with JCVI providing the sequencing and assembly. The authors listed were instrumental in both collecting and assisting in the collection of the samples that were used in the paper, and thus they had very similar contributions. We reached out to all the authors and some felt that their contributions were more consistent with an acknowledgement and those we have placed in that section. On the flip side, I noticed that you said “We leave it to those more familiar with Mycobactericeae species to make those determinations” – while I can understand that it might be difficult to provide all possible checks for all possible samples, is there at least one collaborator that might be able to list some exceptions/limitations to purely using the ANI strategy for assignments (which could be an acknowledgment, if we are talking about a contribution of a few points without revising any analysis)?We agree that this is a good idea, but all of the collaborators listed above are involved in health care, sequencing, or bioinformatics and thus not experts in microbiological techniques that we think would be required to analyze the traits of the different strains as described in the manuscript. We feel that this would be necessary to confirm the genera assignment similar to how Gupta et al. clarified their division as stated, and that this is currently outside the scope of this manuscript and the authors. I think there should only be one period in a sentence in the introduction (either before or after the quote): different NTM species were encountered in this survey.”. I think there is a minor formatting issue where only part of the word is italicized in the Introduction: Mycolicibactergen (“gen” is not in italics) I think there should be a comma in the following sentence in the introduction: “While this was an important survey of NTM geographical distribution the low number of NTM species” à “While this was an important survey of NTM geographical distribution, the low number of NTM species”. We agree with all the above suggestions, and have implemented the changes suggested. I think there is some unintended capitalization in the results: “of a given genus Using our” à “ of a given genus using our” (this don’t look like the end of a sentence, so I don’t think the “U” should be capitalized) We agree that this is not correct. It should be the end of a sentence and we have added a period. Thomas Clarke on behalf of all the authors"
}
]
}
] | 1
|
https://f1000research.com/articles/9-249
|
https://f1000research.com/articles/9-452/v1
|
27 May 20
|
{
"type": "Research Article",
"title": "The impact of community containment implementation timing on the spread of COVID-19: A simulation study",
"authors": [
"Attayeb Mohsen",
"Ahmed Alarabi",
"Ahmed Alarabi"
],
"abstract": "Background: Community containment is one of the common methods used to mitigate infectious disease outbreaks. The effectiveness of such a method depends on how strictly it is applied and the timing of its implementation. An early start and being strict is very effective; however, at the same time, it impacts freedom and economic opportunity. Here we created a simulation model to understand the effect of the starting day of community containment on the final outcome, that is, the number of those infected, hospitalized and those that died, as we followed the dynamics of COVID-19 pandemic. Methods: We used a stochastic recursive simulation method to apply disease outbreak dynamics measures of COVID-19 as an example to simulate disease spread. Parameters are allowed to be randomly assigned between higher and lower values obtained from published COVID-19 literature. Results: We simulated the dynamics of COVID-19 spread, calculated the number of active infections, hospitalizations and deaths as the outcome of our simulation and compared these results with real world data. We also represented the details of the spread in a network graph structure, and shared the code for the simulation model to be used for examining other variables. Conclusions: Early implementation of community containment has a big impact on the final outcome of an outbreak.",
"keywords": [
"Simulation",
"Outbreak",
"COVID-19",
"Community containment",
"Social distancing"
],
"content": "Introduction\n\nAs a public health tool, community containment measures are employed to curb/slow the spread of infectious disease outbreaks inside communities, regions, and cities. It is considered a last resort when all other tools such as case-isolation and quarantine fail to yield the required results1. Community containment can be achieved by applying a number of measures, which can vary from social distancing (by closing schools, cancelling big population events, or travel ban) to a complete shutdown/lock-down of entire region or city1. The main goal of community containment measures is to lower the pace and the extent of an outbreak, or “flattening the curve”2, which helps in preventing bottlenecks in the healthcare system. During outbreak acceleration phase, the number of people needing hospitalization and critical care grows exponentially and that can overwhelm healthcare system and lead to sub-optimal treatment, which eventually is translated to higher morbidity and mortality.\n\nThe social distancing policies implemented during pandemic are not without a cost; these measures lead to an economic slowdown. To manage that, authorities might push for relaxation of the containment methods to improve economic opportunity, a step that might be too early and could lead to increase in the spread of infection. Therefore, authorities must maintain a delicate balance between the country economic performance and the health of its citizens3.\n\nTo this end, simulation is a commonly used method to inform our understanding of the impact of several factors/variables on the dynamics of infectious disease. The different types of simulation methods and algorithms are reviewed by Siettos et al.4. Recently, many studies have been published to simulate the dynamic of COVID-19. One particular paper5 examined the effect of case isolation. However, The results of simulating the timing for community containment, to our knowledge, have not been published yet. In this paper, we aim to show this using a recursive simulation model of COVID-196,7 infection spread. Anderson et al.2 published a detailed commentary discussing the dynamics of COVID-19 spread and how its spread can be mitigated, which we have used as the basis of our simulation. Although this simulation can be tuned to examine the effect of multiple factors, we examined/showed only the impact of the timing of community containment implementation. Moreover, we presented the spread in a graph network structure to facilitate further investigation.\n\n\nMethods\n\nThis simulation started by assuming the presence of one patient in equally infection-susceptible community and remained infectious for 6 days. Every day he came to contact with random number of people between 10 to 15, with 3% probability for each one of them to be infected (secondary case). Each secondary case get into incubation period with random assigned length between 3 to 14 days and started to be contagious 1 or 2 days before the end of that period, all other possibilities are randomly modeled to resemble COVID-19 dynamics, the possible outcomes are diagnosed cases, hospitalizations (20%), deaths (6%) or cures2,7.\n\nTo imitate a real life situation, the patient is counted (active case) only after being diagnosed, there is a possibility for a patient to be diagnosed even before developing any symptoms (end of incubation period).\n\nTo simulate community containment measures, we simplify the effect to reduction of number of contacts. Originally the number of contacts are set to be 10–15 per day, We set two levels of containment measures: loose, where the number of contacts is reduced from 10–15 to 5–10 per day (40% reduction); strict, where the number of contacts is reduced to 2–5 per day (~ 72% reduction). We calculated the effect of starting community containment (35, 45, 55, 65, 75, and 85) days after first patient diagnosis. This simulation continued for 100 days. And we recorded the details of infected cases and their origin of infection, so they can be used to build a network graph if other observations are intended like simulating the effect of big transmitters, however, we did not consider any of them in this short article.\n\nWe tested the effect of community containment relaxation by going back to normal number of contacts after one month in the case of starting community containment after 45 days of first patient diagnosis. Moreover, we compared our simulation result with real data from Our World in Data8. We compared the number of cases of India and San Marino from the report of first case to the date we did the analysis on May 10th, 2020.\n\nWe used Python programming language (version 3.7.6), with the following packages: Networkx 2.4 for network analysis, json 2.0.9 and gzip packages for data storage, pandas 1.0.1 and numpy 1.18.4 for data processing and Matplotlib 3.1.3 for visualisation.\n\n\nResults\n\nThe created simulation code is available at www.github.com/attayeb/corona_simulation/9. It is a simple python script that takes input of two items, the details of first patient and the simulation parameters which need to be inserted in form of a python dictionary. This code can be easily modified to fulfil the requirement of wide spectrum of parameters, like the number of contacts, quarantine measures, hospitalization, and death rates.\n\nBecause of the stochastic nature of our simulation, and to get the most reliable result of our work, we did the simulation 100 times, and we calculated the median, we choose median to reduce the effect of outliers.\n\nThe number of active cases (Figure 1), shows how implementing community containment measures earlier after first patient diagnosis has greater effect. For example, starting community containment measures 10 days earlier (65 compared to 75) leads to 60% reduction in number of active cases at day 90. Same behaviour can also be seen with the number of hospitalized cases (Figure 2). The effect on number of dead cases are more prominent at day 45 compared to 65 or later because longer time is required for death to happen in comparison to diagnosis and hospitalization.\n\nThe x-axis shows the day after first patient diagnosis, colored lines correspond to the day when community containment starts.\n\nThe x-axis represents the start day of the community containment measures, y-axis represents the number of cases.\n\nTo show the effect of community containment measures relaxation, we simulated going back to usual number of contacts after one month from starting as an example. Figure 3 showed that, after one month of community containment measures, we go back to the same curve slope compared to the original curve without applying any measures. Which means that relaxing community containment after one month of applying them is delaying the infection spread without curve flattening. Our model can be used for further consideration of the effect of duration of community containment.\n\nS85: starting community containment measures at day 85, S45-R75: starting community containment at day 45 with relaxation at day 75, S45: starting community containment at day 45 without relaxation.\n\nWe also compared our model results to real world data. Figure 4 shows that our loose-45 model is comparable to India spread. In India, the first case was recorded on January 30th, and major lockdown measures were introduced by the government on March 22nd, 50 days after first patient diagnosis. Meanwhile our strict-35 model is similar to the situation in San Marino, which had the first case reported on February 27th and on March 14th a major lockdown started.\n\nleft side panel shows loose measures in India corresponds to loose-45 (applying loose measures 45 days after first patient diagnosis) model, while right side panel shows strict-35 model (applying strict measures 35 days after the diagnosis of first patient) model in comparison with San Marino statistics.\n\nTo show the effect of community containment on the number of COVID-19 infections transmitted by every single patient, we built a spread network graph (Figure 5). Nodes represent infected cases and edges represent infection transmission. Therefore, the calculation of nodes degree over time reflects the transmission rate for each patient. This figure shows that strict measures leads to greater reduction in number of new infected patients per case compared to loose measures and before community containment. This network can be further used to study the effect of other factors such as the incubation period and self-quarantine.\n\nLeft panel shows miniature example of built network graph for strict-55 model. Green colored nodes are the cases infected prior to introduction of community containment. Right panel shows the mean of node degrees, which corresponds to the number of cases got infected per patient. CC, community containment.\n\n\nDiscussion\n\nCOVID-19 is a highly contagious disease with a devastating morbidity and mortality burden10, which led to a debate/uncertainty about the likelihood of its spread in the community11. It is estimated that 20% of COVID-19-infected people suffer severe clinical complications that need urgent and complex treatment, and therefore have the potential to overwhelm health care facilities such as hospitals11. Overburdened hospitals effect might also be compounded by the shortage of trained health care professionals who deliver treatment, and hospital staff who organize the workflow of the day-to-day operation12. Collectively, this can lead to poor quality of care and in some cases a complete exhaustion of the health care system, similar to what happened in the Italy13. With this in mind, community containment methods such as social distancing had been employed to slow down the spread of COVID-19 in affected communities and consequently relieve overburdened healthcare systems and reduce the number of deaths.\n\nFor this purpose, in this simulation study, we have shown that early implementation of social distancing measures (reduction in the number of contacts of people as a general rule) in a whole society has a great impact on the numbers of infections, hospitalizations and deaths as a result of COVID-19 infection. In addition, we demonstrated that early relaxation of social distancing leads to a rebound increase in the number of active cases in the community without curve flattening. We achieved these results by stochastic simulation modelling of the spread with simulation assumptions determined by following the COVID-19 published literature2. We have done the simulation 100 times for each parameter set and calculated the median to reduce the impact of randomization and outliers on the final result.\n\nOne major limitation in our model is the fact that it does not consider the Susceptible, Infected and Removed (SIR) model; we purposefully did that because we are targeting the impact of early application of the measures rather than the extended effect. Moreover, there is no clear conclusion regarding the effectiveness of post-infection COVID-19 immunity14 for SIR model to rely upon. We also considered that applying the measures at a later stage (75 or 85 days) is not practically possible, assuming that the population will follow self-quarantine guidelines if wide disease spread happens; however, we are still showing these results for comparison as extreme case when all protective measures are ignored. We also did not consider the effect of self-quarantine or case isolation strategy if the patient was not hospitalized, because our aim is to show the impact of global society rather than individual preventive measures which other simulation studies have considered5. However, small modifications of our script simulation variables input could be done to simulate such effects.\n\nDuring this manuscript preparation, similar results were reported by Matrajt et al.15. They followed age-stratified compartments similar to the population of Seattle metropolitan area. Our model in comparison did not consider age distribution; however, one important addition of our model is the presentation of the simulation results in graph structure. Our simulation also was limited to the first 100 days of the pandemic without assuming the total community population. Finally, our simulation was compared to an actual COVID-19 containment measures implemented in San Marino and India, which gives more robustness to our model.\n\n\nConclusions\n\nThis simulation shows that the early implementation of social distancing and reducing the number of contacts per day reduces the number of hospitalized cases, deaths and number of infected people. Furthermore, early relaxation of social distancing is associated with rebound increase in the number of active cases in the community. This study can provide some evidence that can help increase awareness among health care professionals, policy makers, and general public about the importance of early implementation of community containment measures such as social distancing during pandemics.\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\nSource code and input data for the simulation available at: https://github.com/attayeb/corona_simulation/\n\nArchived source code at time of publication: https://doi.org/10.5281/zenodo.38359459.\n\nLicense: BSD 3-Clause License.",
"appendix": "Author contributions\n\n\n\nA. M. conceptualized, designed and conducted the simulation, and wrote the original manuscript, A. A. contributed in the experiment design, results discussion and manuscript writing. Both authors approved the manuscript before submission.\n\n\nAcknowledgements\n\nAll computation was done in Laboratory of Bioinformatics, Artificial Intelligence Center for Health and Biomedical Research (ArCHER), National Institutes of Biomedical Innovation, Health and Nutrition (NIBIOHN), Osaka, Japan. We would like to thank Dr. Kenji Mizuguchi and Dr. Ahmad Haredy for their contribution during various stages of paper preparation.\n\n\nReferences\n\nWilder-Smith A, Freedman DO: Isolation, quarantine, social distancing and community containment: pivotal role for old-style public health measures in the novel coronavirus (2019-nCoV) outbreak. J Travel Med. 2020; 27(2): taaa020. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAnderson RM, Heesterbeek H, Klinkenberg D, et al.: How will country-based mitigation measures influence the course of the covid-19 epidemic? Lancet. 2020; 395(10228): 931–934. PubMed Abstract | Publisher Full Text | Free Full Text\n\nReynolds DL, Garay JR, Deamond SL, et al.: Understanding, compliance and psychological impact of the SARS quarantine experience. Epidemiol Infect. 2008; 136(7): 997–1007. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSiettos CI, Russo L: Mathematical modeling of infectious disease dynamics. Virulence. 2013; 4(4): 295–306. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHellewell J, Abbott S, Gimma A, et al.: Feasibility of controlling COVID-19 outbreaks by isolation of cases and contacts. Lancet Global Health. 2020; 8(4):e488–e496. PubMed Abstract | Publisher Full Text | Free 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\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 | Free Full Text\n\nRitchie H: Coronavirus Source Data. 2020. Reference Source\n\nMohsen A, Alarabi A: The impact of community containment implementation timing on the spread of COVID-19: A simulation study. 2020. http://www.doi.org/10.5281/zenodo.3835945\n\nMizumoto K, Kagaya K, Chowell G: Early epidemiological assessment of the transmission potential and virulence of coronavirus disease 2019 (COVID-19) in wuhan city: China, january-february, 2020. medRxiv. 2020. Publisher Full Text\n\nCowling BJ, Aiello AE: Public Health Measures to Slow Community Spread of Coronavirus Disease 2019. J Infect Dis. 2020; 221(11): 1749–1751. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEmanuel EJ , Persad G, Upshur R, et al.: Fair allocation of scarce medical resources in the time of covid-19. N Engl J Med. 2020; 382(21): 2049–2055. PubMed Abstract | Publisher Full Text\n\nSalathé M, Althaus CL, Neher R, et al.: COVID-19 epidemic in Switzerland: on the importance of testing, contact tracing and isolation. Swiss Med Wkly. 2020; 150: w20225. PubMed Abstract | Publisher Full Text\n\n\"Immunity passports\" in the context of COVID-19. 2020. Reference Source\n\nMatrajt L, Leung T: Evaluating the Effectiveness of Social Distancing Interventions to Delay or Flatten the Epidemic Curve of Coronavirus Disease. Emerging Infect Dis. 2020; 26(8). PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "65343",
"date": "07 Jul 2020",
"name": "Salem I.M Alkoshi",
"expertise": [
"Reviewer Expertise Public Health"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nit is a good work with sufficient data analyzed. It was good written, but I have minor comments which should be done for final approval:\nThe result in the abstract should include some numbers and clear results rather than narrative. this will strengthen the abstract.\n\nPlease provide more explanation about 85 days in fig 1 page 4, it seems to be same results in loose and strict, Please clear it in result part or discussion part.\nGreat work and hope all the best to authors.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "67222",
"date": "01 Sep 2020",
"name": "Indumathi Venkatachalam",
"expertise": [
"Reviewer Expertise Infectious disease epidemiology",
"including emerging infectious diseases and multi-drug resistant bacterial infections."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this global Covid-19 pandemic, all societal sectors have been impacted. As the pandemic spread across the globe, countries have responded differently, some instituting community containment measures early while others awaiting in anticipation for development of protective herd immunity. Each strategy is fraught with deep uncertainties and unprecedented far reaching consequences.\n\nMohsen and Alarabi present a simulation study that predicts the impact of community containment measures on the numbers of Covid-19 cases, hospitalizations and deaths. They assess, (1) strict versus loose containment measures and (2) time from first case to implementation of these measures. They then compare their models to real data from India and San Marino, from report of first case to date of analysis on May 10th, 2020.\n\nThough the results have to be interpreted in light of the assumptions such as infectious period of 6 days, 3% probability of being infected once exposed, 20% hospitalization and 6% death rates from Covid-19, the overall finding that early implementation of community containment measures may be more important than the strictness of these measures is interesting and worth exploring further.\n\nResults in Figure 2 can be elaborated further; the results highlight the time to initiation of community containment measures has a greater impact than the type of measures. Looser measures early on have better outcomes, and when interventions are implemented later on, stricter measures don't seem to have much benefit over looser measures.\n\nThe validity of these findings have potential to impact policy and outcomes for subsequent waves of this pandemic.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "70458",
"date": "21 Sep 2020",
"name": "Mahmoud Heshmat",
"expertise": [
"Reviewer Expertise Simulation."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 manuscript, the authors developed a simulation model to study the impact of community containment implementation timing on the spread of COVID-19. The manuscript can be accepted provided that the following issues are solved.\nIn the abstract, the results section should be highlighted in a better way. For example, the first sentence is part of methodology not result.\n\nYou mentioned in the conclusions in the abstract that Early implementation of community containment has a big impact on the final outcome of an outbreak. How much does that impact? How do you measure it?\n\nThe authors used the work in references [3, 6, 7] from the literature, is it a considerable contribution? The authors should clearly illustrate the contribution.\n\nThe literature review is limited, the authors must expand it by mention similar cases of using simulation as a tool in healthcare in general and COVID 19 in particular. Examples: 1,2\n\nThe manuscript should be revised with respect to grammatical and structure issues. I am giving examples below: Abstract: The first sentence is very short, I suggest being merged with second one or moved to the introduction. Introduction: Paragraph 3 Line 7: “However, The results of simulating the timing for community containment, to our knowledge, have not been published yet.” Line 4: the structure should be enhanced. Paragraph 2: Heshmat et al. takes…. Page 4, second paragraph: “….which we studied only the treatment unit..” not clear structure. Page 4, last line in the second paragraph: c in Chemotherapy should be small. Conclusion: This simulation shows that the early implementation of social distancing and reducing the number of contacts per day reduces the number of hospitalized cases, deaths and number of infected people.\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?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/9-452
|
https://f1000research.com/articles/8-1923/v1
|
15 Nov 19
|
{
"type": "Research Article",
"title": "Cajuputs candy impairs Candida albicans and Streptococcus mutans mixed biofilm formation in vitro",
"authors": [
"Siska Septiana",
"Boy M. Bachtiar",
"Nancy Dewi Yuliana",
"Christofora Hanny Wijaya",
"Siska Septiana",
"Boy M. Bachtiar",
"Nancy Dewi Yuliana"
],
"abstract": "Background: Cajuputs candy (CC), an Indonesian functional food, utilizes the bioactivity of Melaleuca cajuputi essential oil (MCEO) to maintain oral cavity health. Synergistic interaction between Candida albicans and Streptococcus mutans is a crucial step in the pathogenesis of early childhood caries. Our recent study revealed several alternative MCEOs as the main flavors in CC. The capacity of CC to interfere with the fungus-bacterium relationship remains unknown. This study aimed to evaluate CC efficacy to impair biofilm formation by these dual cariogenic microbes. Methods: The inhibition capacity of CC against mixed-biofilm comprising C. albicans and S. mutans was assessed by quantitative (crystal violet assay, tetrazolium salt [MTT] assay, colony forming unit/mL counting, biofilm-related gene expression) and qualitative analysis (light microscopy and scanning electron microscopy). Result: Both biofilm-biomass and viable cells were significantly reduced in the presence of CC. Scanning electron microscopy imaging confirmed this inhibition capacity, demonstrating morphology alteration of C. albicans, along with reduced microcolonies of S. mutans in the biofilm mass. This finding was related to the transcription level of selected biofilm-associated genes, expressed either by C. albicans or S. mutans. Based on qPCR results, CC could interfere with the transition of C. albicans yeast form to the hyphal form, while it suppressed insoluble glucan production by S. mutans. G2 derived from Mojokerto MCEO showed the greatest inhibition activity on the relationship between these cross-kingdom oral microorganisms (p < 0.05). Conclusion: In general, all CC formulas showed biofilm inhibition capacity. Candy derived from Mojokerto MCEO showed the greatest capacity to maintain the commensal form of C. albicans and to inhibit extracellular polysaccharide production by S. mutans. Therefore, the development of dual-species biofilms can be impaired effectively by the CC tested.",
"keywords": [
"Cajuputs candy",
"essential oil",
"caries",
"mixed biofilm",
"Candida albicans",
"Streptococcus mutans"
],
"content": "Abbreviations\n\nCC, Cajuputs candy; MCEO, Melaleuca cajuputi essential oil; G0, untreated biofilm, control group, without the addition of CC formula; G1, biofilm group treated with Cajuputs candy made with Melaleuca cajuputi essential oil from Pulau Buru; G2, biofilm group treated with Cajuputs candy made with Melaleuca cajuputi essential oil from Mojokerto; G3, biofilm group treated with Cajuputs candy made with Melaleuca cajuputi essential oil from Ponorogo; G4, biofilm group treated with Cajuputs candy made with Melaleuca cajuputi essential oil from Pasuruan; G5, biofilm group treated with Cajuputs candy made with Melaleuca cajuputi essential oil from Kuningan.\n\n\nIntroduction\n\nCandida albicans is the most prevalent fungus in oral microbiota1. This opportunistic fungus grows as yeast, pseudohyphae, and hyphae based on environmental conditions2. The hyphal form is relevant for its virulence as it allows penetration and invasion of epithelial cells3.\n\nStreptococcus mutans is a strong acidogenic and aciduric bacteria, defined as the major cause of dental caries. The critical virulence factor of S. mutans is its capacity to convert dietary sugars to produce an extracellular polysaccharide (EPS) matrix, mainly through glucosyltransferase enzymes (Gtfs)4. EPS is the main building block of the biofilm. It can provide a binding site for colonization by other microbes and creates an acidic environment5,6.\n\nSeveral studies have reported that C. albicans is frequently found with S. mutans in early childhood caries (ECC)7–9. The presence of both microbes indicates cross-kingdom feeding10. Furthermore, GtfB from S. mutans plays a significant role in mediating this dual-species interaction11. Their co-species interaction enhanced cell accumulation, biofilm formation, and Gtf gene expression8,12. Therefore, targeting the synergism of C. albicans and S. mutans in mixed biofilms has become a promising strategy for oral antimicrobial exploration13–15.\n\nIn accordance with the efficacy of essential oils as natural antimicrobial substances, Cajuputs candy (CC) has been developed using Melaleuca cajuputi essential oil (MCEO) as the main flavor ingredient. Previous work in our lab revealed the efficacy of CC in inhibiting biofilm formation by single oral microbes such as S. mutans (unpublished report) and C. albicans16. As C. albicans and S. mutans have been noted for their synergistic relationship5,7,8,10, we evaluated the capacity of CC to impair their symbiotic interaction in this study. This finding will provide novel evidence for CC in interfering with the traits of cariogenic oral microorganisms.\n\n\nMethods\n\nA C. albicans and S. mutans Xc were used for this study. C. albicans was obtained from the Oral Biology Laboratory stock culture previously isolated from the patients with their consent in the dental hospital of Universitas Indonesia17. S. mutans Xc was kindly provided by Prof. Yoshihisa Yamashita, Department of Preventive Dentistry, Kyushu University, Japan17. They were maintained as glycerol stocks at -80°C in our laboratory. C. albicans was grown in Sabouraud dextrose broth (SDB) (Oxoid, UK) for 24 hours at 37°C. S. mutans was cultured in brain heart infusion (BHI) (Himedia Laboratories, India) for 24 hours under anaerobic conditions (10% CO2, 10% H2, 10% N2). The cell densities of each culture were quantified using total plate count on an agar medium. Five essential oils were obtained. MCEO from Mojokerto, Ponorogo, Pasuruan, and Kuningan were provided by Perhutani Indonesia, whereas MCEO from Pulau Buru was obtained from local villages where they produce the MCEO on a small scale by home distilling. For this, approximately 300kg of sun-dried leaves of Melaleuca cajuputi are placed in the boiler of the distilling apparatus and hydrodistillation is performed for six hours. After passing through the condenser, the extracted oil is collected and separated from the residual water. The essential oil is stored in a dark bottle.\n\nThe candies were prepared by mixing 98 g isomalt (Beneo-Palatinit GmbH, Germany), 0.1 g Acesulfame K (Anhui Jinhe Industries, China) and 0.1 g water18. The mixed ingredients were heated to 150°C with continuous stirring. As the temperature decreased to 135°C, 820 µL MCEO and 180 µL peppermint oil (Brataco Chemika, Indonesia) was added and the dough was molded. Peppermint oil was used as a secondary flavor in addition to MCEO. To identify the most active MCEO, the MCEOs were varied among the candies. Pulau Buru was used as the targeted reference as it has been utilized from the beginning of our research series16,18 and needed to be replaced with other potent MCEOs due to its currently limited amounts. Four MCEOs were selected from our previous work as they had similar sensory characteristics to MCEO Pulau Buru (Siska Septiana, unpublished report). Five kinds of CCs were prepared using MCEO from different origins with Pulau Buru as the reference, and Mojokerto, Ponorogo, Pasuruan, and Kuningan as the alternative MCEOs.\n\nA mixed biofilm was prepared on a 96-well plate by inoculating approximately 2 × 104 colony forming units per milliliter (CFU/mL) of C. albicans suspended in SDB and 2 × 106 CFU/mL of S. mutans in BHI in an equal suspension volume (50 µL). The well was previously coated with fetal bovine serum (Biosera, South America) with one-hour incubation at 37°C. Supernatants were removed after a 90-minute incubation under anaerobic conditions. Then, 140µL of tryptic soy broth (Oxoid, UK) supplemented with 1% sucrose was added to each well followed by 60 µL of CC formula (each CC was dissolved in sterile distilled water (1:2 w/v) prior to the analysis). The biofilm group treated with CC made from Pulau Buru MCEO (as the reference) was represented as G1. Other treated groups G2, G3, G4, and G5 represented biofilms with the addition of CC made from Mojokerto, Ponorogo, Pasuruan, and Kuningan MCEOs, respectively. The untreated control (G0) was mixed biofilm without addition of the test CC formula. A light microscope equipped with a mobile camera (Primo Vert, Zeiss, Germany) was used to observe biofilm formation.\n\nThe plates mentioned previously were incubated for zero, three, and 24 hours at 37°C under anaerobic conditions. The supernatants were aspirated and washed twice with 200 µL PBS. Attached biofilms were stained using 100 µL crystal violet (CV) 0.5% (v/v). Total biomass was extracted using absolute ethanol and absorbance at 600 nm was measured. This CV assay was performed in triplicate from two independent experiments.\n\nSimilar to the CV assay, the mixed biofilms on 96-well plates were washed twice with PBS after zero, three, and 24 hours of incubation at 37°C. Next, 50 µL of 5 mg/mL MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) were added for total cell viability analysis. The plates were incubated for three hours followed by tetrazolium salt extraction using 100 µL acidified isopropanol. After re-incubation for two hours at 37°C under anaerobic conditions, absorbance was measured at 600 nm. Three independent experiments were conducted in triplicate.\n\nThe 24 hour biofilms on 96-well plates were washed twice with PBS. The biofilms at the bottom of the well were manually scraped and diluted with 300 µL PBS. The solutions obtained from each well underwent serial dilution and were grown for 24 hours at 37°C in separate media in triplicates. Sabouraud dextrose agar was used for C. albicans, whereas brain heart infusion agar was used for S. mutans.\n\nA 24-well plate supplemented with 8 mm acrylic resin discs inside was used to grow the mixed biofilms. The biofilms were fixed by immersion in 1 mL of 2.5% glutaraldehyde for 1 hour followed by 20 minutes dehydration with each ethanol series (10, 25, 50, 75, and 90%). They were then immersed in 100% alcohol for one hour. The plates were dried at 37°C for 24 h19. The mixed biofilm on the acrylic resin disc was analyzed using an FEI Quanta 650 Scanning Electron Microscope (SEM) (Thermo Scientific, Chicago).\n\nThe biofilm was harvested after 24 hours incubation. RNA was extracted using Trizol reagent (Sangon Biotech, China). cDNA synthesis was performed using ReverTra Ace qPCR RT Master Mix (Cat. No. FSQ-301; Toyobo, Japan) following the manufacturer’s protocol. cDNA concentration was measured using a Qubit RNA HS Assay Kit (Cat. No. Q32852; Thermo Fisher Scientific, USA). The PCR mixture contained 10 µL SensiFAST SYBR Hi-ROX (Cat. No. BIO-92020; Bioline Reagents, UK), 0.8 µL of the forward and reverse primer, nuclease free water, and 50 ng/mL of template-diluted cDNA to achieve a 20 µL final volume. Table 1 shows the list of primers used for C. albicans and S. mutans specific genes based on the literature13. The PCR program for C. albicans genes was started with five minutes initial denaturation at 95oC, followed by 40 cycles of 15 seconds at 95°C and 60°C for one minute. For S. mutans, the PCR was run at 95°C for two minutes followed by 40 cycles of 95°C for five seconds and 60–61°C for 30 seconds. qRT-PCR was run on a StepOnePlus Real-Time PCR System (Applied Biosystems, USA). The relative gene expression was calculated as 2-ΔΔCt and normalized to 18S rRNA and 16S rRNA for C. albicans and S. mutans genes, respectively.\n\n*Primer sequences were produced based on a previous study13.\n\nData analysis was performed using IBM SPSS Statistics 22 (IBM Corp., New York, USA). A one-way analysis of variance (ANOVA) followed by Duncan’s test (p <0.05) were used to analyze total biomass, cell viability, and CFU/mL. The means of gene expression were evaluated by Student’s t-test. All the graphs were produced using GraphPad Prism 8.0.2 (GraphPad Software, San Diego, California)\n\n\nResults\n\nAll the CC formulae significantly inhibited biofilm development during the early-prematurity phase (0–3 hours) until the maturity phase (24 hours) (Figure 1A). Total viable cell analysis showed comparable results20. CC effectively suppressed both C. albicans and S. mutans viable cells (0–3 hours) (Figure 1B). Cell viability (24 hours) was also reduced in the presence of the CC formula, with G2 exhibiting the strongest capacity, similar to the reference group (G1). Figure 2 showed that both microbes had similar susceptibility to CC exposure. G3, G4, and G5 did not interfere significantly with the number of C. albicans and S. mutans organisms, whereas G2 exhibited the highest inhibition capacity.\n\nCajuputs candy exposure inhibited C. albicans and S. mutans biofilm development: (A) total biomass on C. albicans and S. mutans dual-species biofilm, evaluated by crystal violet (CV) assay; (B) total viable cells on C. albicans and S. mutans dual-species biofilm based on MTT assay. The values were presented as mean and standard deviation of absorbance at 600nm. Different letters represented the significantly different values among the groups according to Duncan’s test (p <0.05). G0: untreated control, biofilm group treated with Cajuputs candy made with Melaleuca cajuputi essential oil from different origins denoted by G1: Pulau Buru, G2: Mojokerto, G3: Ponorogo, G4: Pasuruan, and G5: Kuningan.\n\nTotal plate count (colony forming units/mL) of C. albicans and S. mutans on mixed biofilm (in vitro): (A) mean and standard deviation of the C. albicans colonies; (B) mean and standard deviation of the S. mutans colonies. Different letters represented the significantly different values among the groups according to Duncan’s test (p <0.05). G0: untreated control, biofilm group treated with Cajuputs candy made with Melaleuca cajuputi essential oil from different origins denoted by G1: Pulau Buru, G2: Mojokerto, G3: Ponorogo, G4: Pasuruan, and G5: Kuningan.\n\nBiofilm development started with the germ-tube formation of C. albicans in the 90 minutes before formula treatments (Figure 3A). In the maturity stage (24 hours), the hyphal form of C. albicans dominated the biofilm, surrounded by S. mutans accumulation in the untreated control (G0) (Figure 3B). A corncob-like structure21 was observed in the mixed biofilm (Figure 3C).\n\nLight microscopy analysis of C. albicans and S. mutans interaction in mixed biofilm: (A) germ tube formation in the first stage of biofilm development in 90 minutes (40× magnification); (B) hyphal growth on untreated control of mixed biofilm after 24 hours incubation (20× magnification); (C) dual-species interaction formed a corn cob-like structure for biofilm treated with Cajuputs candy (40× magnification). Grayscale color adjustment has been performed in order to clarify the figures.\n\nSEM analysis confirmed the germ tube formation in the first 90 minutes in which S. mutans was found close to C. albicans (Figure 4A). As shown in Figure 4B, hyphal cells grew progressively in the untreated biofilm (G0), enclosed within the self-produced EPS matrix. This co-species population formed a complex structure within the biofilm. Interestingly, the presence of CC altered the architecture of the mixed biofilm. C. albicans tended to be maintained in yeast form, whereas S. mutans adherence to C. albicans was obviously reduced, especially for G2 (Figure 4C–D). The microcolonies formed were not as many as those in the untreated control. However, exposure to G5 did not significantly affect the interaction and a matrix-rich biofilm was still formed (Figure 4E–F).\n\nIn vitro dual-species biofilm formation of C. albicans and S. mutans by scanning electron microscopy: (A) initial germ-tube formation (3000× magnification); (B) mixed biofilm of untreated control group (G0) (1000× magnification); (C–D) mixed biofilm under G2 exposure (1000× and 5000× magnification, respectively); (E–F) mixed biofilm under G5 exposure (1000× and 5000× magnification, respectively). The presence of Cajuputs candy reduced the hyphal cells of C. albicans and inhibited matrix production after 24 hours biofilm formation. (1. S. mutans cell; 2. C. albicans yeast and hyphal cells; 3. water channel; 4. extracellular polysaccharides matrix; 5. microcolony).\n\nAll of the CC groups demonstrated significant downregulation of ALS3, the adhesion-specific gene of C. albicans. HWP1, which is responsible for hyphal filamentation, was still expressed in G1, G2, and G3. However, the expression of YWP1, the yeast-specific gene, was upregulated dominantly in almost all of the CC groups (Figure 5A). This result confirmed the results of the SEM imaging, that CC exposure tends to maintain the commensal form of C. albicans.\n\nqRT-PCR assay of C. albicans and S. mutans biofilm-related genes: (A) C. albicans-specific genes expression; (B) S. mutans specific genes expression. An untreated control (G0) was defined as ‘1’. The values were shown as mean and SD. *Significantly regulated than the untreated control (G0) according to Student t-test (p<0.05). G0: untreated control, biofilm group treated with Cajuputs candy made with Melaleuca cajuputi essential oil from different origins denoted by G1: Pulau Buru, G2: Mojokerto, G3: Ponorogo, G4: Pasuruan, and G5: Kuningan.\n\nAs for S. mutans gene expression, the greatest downregulation was observed for gtfB in the mixed biofilm exposed to G2, whereas exposure to other formulas still allowed the expression of this insoluble glucan-specific enzyme (Figure 5B). Regarding gtfD expression (the gene for the soluble glucan enzyme), none of the CC groups had a significant effect on gene regulation compared to the untreated control (G0).\n\n\nDiscussion\n\nCC is a lozenge that has been known as an emerging functional food in Indonesia. Further studies have shown its capability in maintaining oral cavity health due to the antimicrobial capacity of MCEO as its flavor against pathogenic oral microbes16,18. In addition to the existing MCEO (PB), we successfully identified four additional MCEOs as potential CC flavors (Siska Septiana, unpublished report). However, the mechanism by which CC interferes with the relationship between the fungus and cariogenic bacteria (S. mutans) remains unknown. CC consists of isomalt and peppermint oil in addition to MCEO as the main flavor. These ingredients were each added at the same concentration in all of the formulas. Hence, their effect can be assumed as background activity. So far, no studies have been performed to evaluate the efficacy of CC derived from several alternative MCEOs in attenuating the mixed biofilm of S. mutans and C. albicans. Our data show that all the CC groups showed a potent capacity in reducing the biofilm mass composed of these oral microflorae, as well as the viability of biofilm cells, until the biofilm reached its maturation stage. We observed that a higher total biomass in the early prematurity phase (three hours) dominantly contributed to matrix production since cell viability was maintained at a low level. The colony number confirmed that viability reduction in the mature biofilm was contributed by the reduction in cell numbers of both microbes, with G2 demonstrating the strongest inhibition capacity, similar to our existing MCEO (G1) used as the reference18.\n\nThe interkingdom interaction might begin in the first 90 minutes of biofilm growth, in which a corn-cob-like structure was observed (shown in Figure 3C). This result is in accordance with that of Zijnge et al.21, who first found that S. mutans cells adhere to the hyphal cells of C. albicans to form this structure. This occurred due to the high affinity of S. mutans cells to the O-mannan group in the C. albicans cell wall7,22. Our study showed that G2 exposure intervenes in the C. albicans and S. mutans interaction, indicated by reduction in total biomass and cell viability (CV and MTT assays, respectively). SEM imaging confirmed these quantitative results. The inhibition effect was related to the morphology alteration of C. albicans into the yeast form, inhibition of S. mutans adherence, and lack of microcolonies compared to the untreated control (G0).\n\nThe molecular mechanism underlying the CC inhibition capacity was explained by the expression patterns of selected biofilm-related genes. The adhesion trait of C. albicans was suppressed by ALS3 downregulation when the CC formulas were present. As observed in this study, HWP1 was still expressed in G1–G3. These two genes contribute to hyphal formation as the critical factors in C. albicans biofilm formation23. However, the gene for the alteration from hyphal to yeast cell (YWP1) was more dominantly expressed under CC exposure, indicating that CCs tend to impair biofilm development by maintaining the yeast form of C. albicans with lack of adhesion and further filamentation. This was confirmed by observation of the hyphal form using SEM imaging (Figure 4).\n\nA parallel investigation of S. mutans genes showed that insoluble glucan production (gtfB) was inhibited as an effect of G2 exposure, which showed greater inhibitory capacity compared to the G1 reference. In contrast, gtfD was still expressed, similar to the untreated control (G0) in all the CC groups. This means that these genes were still expressed in the biofilm. Furthermore, gtfB is one of the key factors for initiating dual-species interaction8,11. It has thus been found to bind C. albicans due to its low dissociation rate, resulting in strong and stable binding such as a covalent bond22. Lower gtfB expression indicated that fewer binding sites of S. mutans were available to form a polymicrobial biofilm with C. albicans, as shown by the CV and MTT assays in this study. This result also clearly explained the lack of a matrix on G2 SEM images (Figure 4C and 4D).\n\nRelated to our finding, farnesol (quorum sensing molecule [QSM] of C. albicans) at low concentrations could induce S. mutans growth besides GtfB10. A lower concentration of farnesol could induce the hyphal form of C. albicans. QSMs are also produced by S. mutans, such as Autoinducer-2 (AI2), which is responsible for suppressing the inhibition capacity of farnesol. Another QSM of S. mutans is competence-stimulating peptide (CSP), which stimulates hyphal-to-yeast alteration24. The result of this study showed that G2 caused a morphology alteration, which might also be correlated with the impairment of these QSMs. This inter-species signaling might induce the yeast form of C. albicans, which inhibits S. mutans cell accumulation. The limitation of this study is that QSMs in the mixed biofilm were not measured quantitatively.\n\nMCEO, as a plant-based antimicrobial used in this experiment, significantly suppressed biofilm formation by reducing the cell number of both the microbes and also inhibited the total biomass production similar to other natural antimicrobials14,15,25. Interestingly, the expression profile of morphology-related genes from C. albicans showed a comparable trend with the synthetic antimicrobial S-8 reported by Fieldman et al.13. G2 also showed an additional activity of inhibiting S. mutans insoluble glucan production. This observation strengthens the potential of this formula to suppress mixed biofilm formation in vitro.\n\nIn general, all of the CC groups indicated potent inhibitory capacity against mixed biofilm formation. Mojokerto performed as the strongest MCEO in CC against the co-species C. albicans and S. mutans biofilm, comparable with the existing MCEO (Pulau Buru). This could be related to their similar metabolite composition as found in our recent work (Siska Septiana, unpublished report). MCEO from Mojokerto is dominated by 1,8-cineole (46.43%), caryophyllene (6.00%), α-terpineol (3.70%), γ-terpinene (3.09), and α-pinene (2.45%). The antibiofilm capacity of this MCEO could be related to these terpenic metabolites, as reported by several studies that essential oils from the Melaleuca genus have various antimicrobial activities26,27. Nazzaro et al.28 summarized their potential mechanisms such as cell wall degradation, affecting the quorum sensing system, and altering adherence capability.\n\n\nConclusions\n\nCC showed the ability to impair mixed C. albicans and S. mutans biofilm formation, with Mojokerto being identified as the most effective MCEO. Inhibition of the total biomass and cell viability were related with the candy’s capacity to maintain the commensal phenotype of C. albicans and to suppress insoluble glucan production by S. mutans.\n\n\nData availability\n\nOpen Science Framework: Cajuputs candy impairs Candida albicans and Streptococcus mutans mixed biofilm formation in vitro. https://doi.org/10.17605/OSF.IO/YT3HQ20.\n\nThis project contains the following underlying data:\n\n- Raw-unedited image files (original JPG files for images in Figure 3 and Figure 4)\n\n- Raw Data of total biomass and cell viability.xlsx\n\n- Raw Data of total plate count of each microbial strains on mixed biofilm.xlsx\n\n- Raw Data of total qPCR assay on specific genes.xlsx\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).",
"appendix": "Acknowledgements\n\nWe would like to thank Prof. Yoshihisa Yamashita, Kyushu University for providing the S. mutans Xc. We also thank Editage for English language editing.\n\n\nReferences\n\nGhannoum MA, Jurevic RJ, Mukherjee PK, et al.: Characterization of the oral fungal microbiome (mycobiome) in healthy individuals. PLoS Pathog. 2010; 6(1): e1000713. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHan TL, Cannon RD, Villas-Bôas SG: The metabolic basis of Candida albicans morphogenesis and quorum sensing. Fungal Genet Biol. 2011; 48(8): 747–63. PubMed Abstract | Publisher Full Text\n\nMoyes DL, Richardson JP, Naglik JR: Candida albicans-epithelial interactions and pathogenicity mechanisms: scratching the surface. Virulence. 2015; 6(4): 338–46. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBowen WH, Koo H: Biology of Streptococcus mutans-derived glucosyltransferases: role in extracellular matrix formation of cariogenic biofilms. Caries Res. 2011; 45(1): 69–86. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKoo H, Bowen WH: Candida albicans and Streptococcus mutans: a potential synergistic alliance to cause virulent tooth decay in children. Future Microbiol. 2014; 9(12): 1295–7. PubMed Abstract | Publisher Full Text\n\nForssten SD, Björklund M, Ouwehand AC: Streptococcus mutans, caries and simulation models. Nutrients. 2010; 2(3): 290–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMetwalli KH, Khan SA, Krom BP, et al.: Streptococcus mutans, Candida albicans, and the human mouth: a sticky situation. PLoS Pathog. 2013; 9(10): e1003616. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFalsetta ML, Klein MI, Colonne PM, et al.: Symbiotic relationship between Streptococcus mutans and Candida albicans synergizes virulence of plaque biofilms in vivo. Infect Immun. 2014; 82(5): 1968–81. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBachtiar EW, Bachtiar BM: Relationship between Candida albicans and Streptococcus mutans in early childhood caries, evaluated by quantitative PCR. [version 2; referees: 2 approved] Referee Status. F1000Res. 2018; 7(1645): 1–15. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKim D, Sengupta A, Niepa TH, et al.: Candida albicans stimulates Streptococcus mutans microcolony development via cross-kingdom biofilm-derived metabolites. Sci Rep. 2017; 7: 41332. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGregoire S, Xiao J, Silva BB, et al.: Role of glucosyltransferase B in interactions of Candida albicans with Streptococcus mutans and with an experimental pellicle on hydroxyapatite surfaces. Appl Environ Microbiol. 2011; 77(18): 6357–67. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSztajer H, Szafranski SP, Tomasch J, et al.: Cross-feeding and interkingdom communication in dual-species biofilms of Streptococcus mutans and Candida albicans. ISME J. 2014; 8(11): 2256–71. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFeldman M, Ginsburg I, Al-Quntar A, et al.: Thiazolidinedione-8 Alters Symbiotic Relationship in C. albicans-S. mutans Dual Species Biofilm. Front Microbiol. 2016; 7: 140. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFarkash Y, Feldman M, Ginsburg I, et al.: Polyphenols Inhibit Candida albicans and Streptococcus mutans Biofilm Formation. Dent J (Basel). 2019; 7(2): pii: E42. PubMed Abstract | Publisher Full Text | Free Full Text\n\nIkono R, Vibriani A, Wibowo I, et al.: Nanochitosan antimicrobial activity against Streptococcus mutans and Candida albicans dual-species biofilms. BMC Res Notes. 2019; 12(1): 383. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWijaya CH, Rachmatillah AF, Bachtiar B: Inhibition of Cajuputs candy toward the viability of Candida albicans by using in vitro assay. J Teknol dan Industri Pangan. 2014; 25(2): 158–67.\n\nBachtiar BM, Srisawat C, Bachtiar EW: RNA aptamers selected against yeast cells inhibit Candida albicans biofilm formation in vitro. Microbiologyopen. 2019; 8(8): e00812. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWijaya CH, Fickie A, Nurramdhan IF, et al.: The composition of Cajuputs Candy which inhibits the growth of dental caries microbes. Indonesia; IDP000040695. 2016.\n\nBarbosa JO, Rossoni RD, Vilela SF, et al.: Streptococcus mutans Can Modulate Biofilm Formation and Attenuate the Virulence of Candida albicans. PLoS One. 2016; 11(3): e0150457. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSeptiana S: Cajuputs candy impairs Candida albicans and Streptococcus mutans mixed biofilm formation in vitro. 2019. http://www.doi.org/10.17605/OSF.IO/YT3HQ\n\nZijnge V, Van Leeuwen MB, Degener JE, et al.: Oral biofilm architecture on natural teeth. PLoS One. 2010; 5(2): e9321. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHwang G, Marsh G, Gao L, et al.: Binding Force Dynamics of Streptococcus mutans-glucosyltransferase B to Candida albicans. J Dent Res. 2015; 94(9): 1310–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLee KH, Park SJ, Choi SJ, et al.: Proteus vulgaris and Proteus mirabilis Decrease Candida albicans Biofilm Formation by Suppressing Morphological Transition to Its Hyphal Form. Yonsei Med J. 2017; 58(6): 1135–43. PubMed Abstract | Publisher Full Text | Free Full Text\n\nXu H, Jenkinson HF, Dongari-Bagtzoglou A: Innocent until proven guilty: mechanisms and roles of Streptococcus-Candida interactions in oral health and disease. Mol Oral Microbiol. 2014; 29(3): 99–116. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJafri H, Khan MSA, Ahmad I: In vitro efficacy of eugenol in inhibiting single and mixed-biofilms of drug-resistant strains of Candida albicans and Streptococcus mutans. Phytomedicine. 2019; 54: 206–13. PubMed Abstract | Publisher Full Text\n\nde Campos Rasteiro VM, da Costa AC, Araújo CF, et al.: Essential oil of Melaleuca alternifolia for the treatment of oral candidiasis induced in an immunosuppressed mouse model. BMC Complement Altern Med. 2014; 14: 489. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhang J, Wu H, Jiang D, et al.: The antifungal activity of essential oil from Melaleuca leucadendra (L.) L. grown in China and its synergistic effects with conventional antibiotics against Candida. Nat Prod Res. 2019; 33(17): 2545–8. PubMed Abstract | Publisher Full Text\n\nNazzaro F, Fratianni F, D’Acierno A, et al.: Essential oils and microbial communication. In: Essential Oils-Oils of Nature. IntechOpen; 2019. Publisher Full Text"
}
|
[
{
"id": "58903",
"date": "05 Feb 2020",
"name": "Mohd Hafiz Arzmi",
"expertise": [
"Reviewer Expertise Oral microbiology and immunology",
"natural products",
"polymicrobial biofilms"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 elucidates the activity of Cajuputs to Candida albicans and Streptococcus mutans in mixed biofilms. The effect was assessed using CV assay, MTT, CFU, genes expression and SEM. The authors claim that Cajuput inhibits biofilm and production of polysaccharide by S. mutans.\nThis study is fascinating; however, major revision is suggested to improve the manuscripts. The following are the comments for authors to consider:\nSection: Introduction\nIt would be best if the authors can include the objective/s and the hypothesis of the study in the introduction section.\n\nSection: Methodology\nMicrobial strains and MCEO sample:\nDid C. albicans was grown in aerobic or anaerobic?\n\nFor MCEO Pulau Buru, what is the temperature in the boiler? If it is 100 degrees Celcius, won't it affect the active compound of the extract? Is this the same method used for the other extracts? If yes, it would be great if you mention that in the paragraph.\n\nCC preparation:\nWon't 150 degrees Celcius affect your active compound? If that is the method, it would be better if you can put a citation on the method. As the expertise in natural product, it seems the temperature is too high for an extract. Unless if the method is commonly used in the preparation by the local.\n\nMixed biofilm formation:\nWhen you prepare the mixed biofilm, you did mention that you grew C. albicans in SDB and S. mutans in BHI in equal volume. Did you mix both media to prepare biofilm? If not, please make it clear in your text.\n\nWhy did you coat the well with fetal bovine serum? Better if you can explain why because that is not the standard method used in polymicrobial biofilms study.\n\nFor you control G0, you mentioned the biofilm was developed without CC. What is the final volume of the well? Did that remain to 200 uL or 140 uL of TSB? If it was 140 uL, how do you compare with other wells which the total volume is 200 uL?\n\nMorphology analysis of dual-species biofilm formation:\nWhy did you choose acrylic as the surface to form a biofilm, and how do you relate this with well that have a different surface?\n\nSection: Results\nEffect of CC on dual-species biofilm development:\n'Susceptibility' is generally referred to as the antimicrobial activity of the extract. In your research, you are emphasizing on biofilm (Figure 2) and not the antimicrobial effect of the extract.\n\nFigure 1. In the legend, the author mentioned that different letters represent significantly different. What does the author comparing too? Significant compared to what? Better to mention.\n\nWhat is a, b and c represent? E.g. 'a' in Figure 1A doesn't show significant at 3 hours. The figure seems confusing. I would suggest improving the figure to ensure understanding of readers. Similar to figure 2.\n\nEffect of CC on the morphology of dual-species biofilm:\nFigure 3A doesn't look corncob to me. Please read this article: The microbial infection of biomaterials: A challenge for clinicians and researchers. A short review. Journal of Applied Biomaterials & Biomechanics 2005; Vol. 3 no. 1: 1-101\n\nWhat do you mean by microcolonies with reference to the figure? Please explain.\n\nHow do you determine significant from SEM image? What was the statistical analysis did you use to claim the significant of your SEM? Did you use image J or any software to measure the biofilm based on your SEM? If yes, better to mention in your methodology. If not, please remove the 'significant'.\n\nEffect of CC on the expression of biofilm-related genes:\nThe author mentioned 'upregulated dominantly'. What do you mean by dominantly and compare to what? G0? G1?\n\nWhat do you mean by 'to maintain the commensal form of C. albicans'?\n\nSection: Discussion\nThe author claims that all CC groups reduce biofilm biomass. What is the author comparing too? Based on my observation, there is no significant difference in total biomass for 24 h and 0 h when all formulation are compared to G0. Please check your statistics.\n\nFigure 4D. This doesn't look cocci shape to me. More like bacilli/rod. I hope you did verify your sample prior to the experiment. Attached is the SEM of commonly seen Streptococcus mutans under SEM. https://www.nature.com/articles/s41598-017-08558-x2\n\nThe author mentioned that lower gtfB indicate the fewer binding site. Glucosyltransferases (Gtfs) are enzymes and not a 'binding site'. This is too speculative. Please consider to rephrase or to remove the statement.\n\nThe author also emphasised a lot on quorum sensing molecule which is not studied in the manuscript. The author also mentioned that QSM is not measured quantitatively. This statement can be misinterpreted none of the section showed the authors have conducted study on QSM either quantitatively or qualitatively. Please consider rephrasing.\n\nOverall, this is a good study; however, more improvement is needed to fit with F1000research, a Q1 journal.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "5532",
"date": "28 May 2020",
"name": "Christofora Hanny Wijaya",
"role": "Author Response",
"response": "Dear reviewers,Please accept our sincere apology for this delayed response. We do appreciate the opportunity to revise our paper entitled, “Cajuputs candy impairs Candida albicans and Streptococcus mutans mixed biofilm formation in vitro”. We also extend our deep gratitude to the reviewers for providing valuable comments and correction on our paper. We have read and understood the comments from the reviewers. Please find below our point-by-point response to the reviewers' comments and concerns. We hope our additional explanations will be able to answer the reviewers' inquiry.We strived to improve the quality of our paper based on the guidance and constructive suggestion provided by the reviewers. We hope that the revisions will be sufficient to make our paper worthy for publication in F1000 Research. We would like to thank you for your hearty support and willingness to find the parts of our paper needing corrections or improvement. Thank you again for your consideration, suggestions, and insightful feedback in order to greatly improve our paper. Sincerely yours,Authors: Siska Septiana, Christofora Hanny Wijaya*, Boy Muchlis Bachtiar and Nancy Dewi Yuliana. Reviewer Comments and Author ResponsesSection: Introduction Comment 1: It would be best if the authors can include the objective/s and the hypothesis of the study in the introduction section.Response to reviewer’s comments 1:Thank you very much, we agree with your suggestion. We have emphasized our sentence in the respective paragraph as follows (third sentence in the last paragraph of the introduction section): “As C. albicans and S. mutans have been noted for their synergistic relationship5,7,8,10, we evaluated the capacity of CC to impair their symbiotic interaction in this study” into “This functional candy may have interfered with their synergistic relationship in dual-species biofilm5,7,8,10. Therefore, this study aimed to evaluate the capacity of CC to impair their symbiotic interaction.”... Section: Methodology Comment 2:Microbial strains and MCEO sample: Did C. albicans was grown in aerobic or anaerobic?Response to reviewer’s comments 2:Thank you for your comments on this issue. We prepare the separate culture of microbial strain based on the previous reports (Ikono et al. 2019). As mention in the literature, the C. albicans as single biofilm was cultured aerobically, after mixed with S. mutans they were cultured anaerobically (CO2 concentration up to 10%). C. albicans commonly have optimal growth in aerobic condition, however, it also can grow in anaerobic conditions (elevated CO2 concentration) (Anand and Prasad 1991; Thein et al. 2007). Comment 3: For MCEO Pulau Buru, what is the temperature in the boiler? If it is 100 degrees Celcius, won't it affect the active compound of the extract? Is this the same method used for the other extracts? If yes, it would be great if you mention that in the paragraph.Response to reviewer’s comments 3:Thank you for the comments and suggestions. MCEO has been widely reported to have antimicrobial activity (Amri et al. 2012; Rini et al. 2012; Wińska et al. 2019). Related to your concern about the MCEO production, hydrodistillation is the most common method for the essential oil extraction, in which the boiler temperature would possible to achieve 100 °C approximately. It is reasonable that this high temperature might affect several bioactive compounds on the leaves and twigs of the plants. However, the extracted essential oil still had antimicrobial activities due to the presence of various terpene compounds as it has been reported in the above-mentioned publications. Based on our recently published article, it was also known that after passing the high-temperature process in candy making, the important terpene compounds in the essential oil were persisting (Wijaya et al. 2020).Regarding a similar method that has been used for the other extracts, we have been added the information in the article as suggested by the reviewer (last sentence in Microbial strains and MCEO samples). “….. the residual water. A similar method was also used for the other extracts. The essential oil is stored in a dark bottle.” Comment 4: CC preparationWon't 150 degrees Celcius affect your active compound? If that is the method, it would be better if you can put a citation on the method. As the expertise in natural product, it seems the temperature is too high for an extract. Unless if the method is commonly used in the preparation by the local. Response to reviewer’s comments 4:Thank you very much for your comment on this CC preparation. We agree with your opinion, the high temperature might affect several compounds in MCEO. However, our study was concern about the capacity of CC as functional food after passed this high-temperature process (150 °C) during the preparation, whether the CC still active or not. Moreover, 1,8-cineole, caryophyllene, and a-terpineol as the highest abundance compounds on MCEO and were predicted as the most responsible compound on the MCEO antimicrobial activity commonly had a higher boiling point than the temperature of CC preparation. It had over 176°C for 1,8-cineole (https://www.chemicalbook.com/ChemicalProductProperty_EN_CB2853653.htm) and over 200°C for caryophyllene, and a-terpineol (https://www.chemicalbook.com/ProductChemicalPropertiesCB6229317_EN.htm). Moreover, our preliminary study revealed that most of them were still present in the candy after the heating process (Wijaya et al. 2020).Regarding the CC preparation method, it has been followed the patented procedure as mention in our paper (Wijaya et al. 2016). Based on our result, this functional food showed the expected antimicrobial capacity against dual-species biofilm. Comment 5: Mixed biofilm formationWhen you prepare the mixed biofilm, you did mention that you grew C. albicans in SDB and S. mutans in BHI in equal volume. Did you mix both media to prepare biofilm? If not, please make it clear in your text.Response to reviewer’s comments 5:Thank you for your interest in this issue. Yes, we did. 50 µL C. albicans in SDB and 50 µL of S. mutans in BHI were inoculated together in the same well to promote initial adhesion. As mention in the article (fourth sentence of mixed biofilm formation section) After the 90 min incubation time, the medium was discarded followed by washing, then, we added Tryptic Soy Broth (TSB)+ 1% sucrose as the medium for the dual-species growth (Ikono et al. 2019). We have also added the reference. Comment 6: Mixed biofilm formationWhy did you coat the well with fetal bovine serum? Better if you can explain why because that is not the standard method used in polymicrobial biofilms study.Response to reviewer’s comments 6:Thank you for your comments regarding our selected method (second sentence in the mixed biofilm formation). Yes, we agree with your opinion. In the dual-species biofilm formation especially for C. albicans and S. mutans, it is common to use saliva coating. In this in vitro study, an ethical clearance to use human saliva was not enclosed. Therefore, we used FBS since either saliva or serum can be used to induce phenotype-associated C. albicans biofilm formation (Barbosa et al. 2016; Krzyściak et al. 2017; Rodrigues et al. 2020). We have embedded this additional information in the third sentence as follows:” Similar with saliva, FBS coating aims to induce phenotype-associated C. albicans biofilm formation (Barbosa et al. 2016; Krzyściak et al. 2017; Rodrigues et al. 2020). Comment 7: Mixed biofilm formationFor you control G0, you mentioned the biofilm was developed without CC. What is the final volume of the well? Did that remain to 200 uL or 140 uL of TSB? If it was 140 uL, how do you compare with other wells which the total volume is 200 uL?Response to reviewer’s comments 7:Thanks for your deep evaluation of this mixed biofilm formation. The final volume for control (G0) was similar to other wells (200 uL). To achieve the equal volume, we added PBS (pH 7) on the control to replace the CC formula. We have added this information as the sixth sentence on this mixed biofilm formation paragraph: ” For the untreated control, the formula was replaced by 60 µL sterile phosphate-buffered saline (PBS)“. We expect that this PBS had no inhibition capacity on the culture. Therefore, as expected, all of the formulae (G1-G5) tested showed a significantly different effect compared to the control. Comment 8: Morphology analysis of dual-species biofilm formationWhy did you choose acrylic as the surface to form a biofilm, and how do you relate this with well that have a different surface?Response to reviewer’s comments 8:Thank you so much for your question. We conducted this analysis based on the previously published paper which used a similar surface (acrylic disc) (Barbosa et al. 2016). The use of acrylic disc (on 24-well plates) was one of the common methods for the morphology analysis of microbial biofilm to facilitate the biofilm sample to be analyzed by using SEM. Moreover, by using SEM, we only focused on the altered morphology between the organisms in control and those treated biofilms (within the group), thus we grew the biofilm on the same surface as mention in the literature. Section: Results Comment 9: Effect of CC on dual-species biofilm development'Susceptibility' is generally referred to as the antimicrobial activity of the extract. In your research, you are emphasizing on biofilm (Figure 2) and not the antimicrobial effect of the extract. Response to reviewer’s comments 9:Thank you so much for your concern. We would like to confirm that in our case, we used the term susceptibility to describe the condition of the cultures by the presence of the CC formula. In another word, this term was focus on the object exposed by the extract whether it is susceptible or resistant. However, we have rephrased this term in the whole text as you suggested. we used “efficacy” to describe the measurement of antimicrobial activity of the CC formula to impair the dual-species biofilm formation. Comment 10: Effect of CC on dual-species biofilm developmentFigure 1. In the legend, the author mentioned that different letters represent significantly different. What does the author comparing too? Significant compared to what? Better to mention.Response to reviewer’s comments 10:Thank you for your review. The statistical analysis on CV assay and MTT assay (Figure 1) was conducted using ANOVA followed by Duncan’s test. In this post hoc analysis, all of the formulae were compared to each other (G0 to G1, G2, G3, G4, G5; G1 to G2, G3, G4, G5; and so on with similar way) within the group in 0, 3, and 24 hours. The significantly different results within the group were then indicated by different letters. We have revised the figure and completed the figure legend with the additional information as suggested.“Figure 1. … absorbance at 600nm. The letters on histogram represented the significantly different values compared to each other formula within the groups in 0, 3, or 24 hours according to Duncan’s test (p <0.05)…” Comment 11: Effect of CC on dual-species biofilm developmentWhat is a, b and c represent? E.g. 'a' in Figure 1A doesn't show significant at 3 hours. The figure seems confusing. I would suggest improving the figure to ensure understanding of readers. Similar to figure 2.Response to reviewer’s comments 11:Similar as the response to the reviewer’s comment no. 10, the use of the different letter (a, b, and c or x, y, and z) was only to describe the significantly different values between the formula (G0-G5) within the group in 0, 3, and 24 hours of incubation time, e.g: in 24 hours the statistic analysis result showed by x, y, and z for the significantly different value among the formulae, whereas “xy” was not significantly different with x and y.Based on Duncan’s test, G1-G5 in 3 hours incubation was showed by ‘a’ and it was significantly different from the negative control which had ‘b’ (Figure 1A). This was our expected result, in which the exposure of CC after 3 hours could inhibit the biofilm formation effectively. However, different superscript symbol/letter between 0, 3, or 24 hours does not correlate each other since the values were analyzed within the group.Thank you for your suggestion to revised our figure. Unfortunately, it seems ineffective to separate each incubation time for CV and MTT assay similar to Figure 2. It will need six additional figures for this revision. Therefore, we have revised the symbol/letter on the histogram to differentiate each incubation time (the revised Figure 1). We hope that this revision would be sufficient to enhance the readers' understanding.(the revised Figure 1 was uploaded separately)“Figure 1. … absorbance at 600nm. The letters on histogram represented the significantly different values compared to each other formula within the groups in 0, 3, or 24 hours according to Duncan’s test (p <0.05)…” Comment 12: Effect of CC on the morphology of dual-species biofilmFigure 3A doesn't look corncob to me. Please read this article: The microbial infection of biomaterials: A challenge for clinicians and researchers. A short review. Journal of Applied Biomaterials & Biomechanics 2005; Vol. 3 no. 1: 1-101Response to reviewer’s comments 12:Thank you, we appreciate your deep review on our figure and your attached literature. Figure 3A describes how the growth of the microbes in the first 90 minutes period, before the exposure to the CC formula. Figure 3B showed the biofilm growth after 24 h, whereas figure 3C indicated an interaction between C. albicans and S. mutans after the exposure of CC. The interaction between those two microbes commonly known as corn-cob like structure (Zijnge et al. 2010). Although Figure 3C still not clearly showed this structure due to the limitation of the light microscopy that we used in the analysis, it indicates that this structure might occur since S. mutans was attached to the C. albicans hyphae as describe in the figure. Comment 13: Effect of CC on the morphology of dual-species biofilmWhat do you mean by microcolonies with reference to the figure? Please explain.Response to reviewer’s comments 13:Regarding the SEM figure, there is part of the figure which indicates the presence of microcolonies (no. 5 in Figure 4B). This microcolony was referred to a microscopic colony of S. mutans cells without considering its interaction with C. albicans. Comment 14: Effect of CC on the morphology of dual-species biofilmHow do you determine significant from SEM image? What was the statistical analysis did you use to claim the significant of your SEM? Did you use image J or any software to measure the biofilm based on your SEM? If yes, better to mention in your methodology. If not, please remove the 'significant'.Response to reviewer’s comments 14:Thank you so much for your correction. We didn’t conduct the quantitative analysis on SEM images. We have removed this term through the whole text as suggested. Comment 15: Effect of CC on the expression of biofilm-related genesThe author mentioned 'upregulated dominantly'. What do you mean by dominantly and compare to what? G0? G1?Response to reviewer’s comments 15:Thank you for your review of this case. The mRNA expression level was presented as the relative result of its comparison to control (G0) which had value as 1 (one). After this relative quantification, YWP1 has upregulated tens of times higher than other C. albicans specific genes (HWP1 and ALS3), this is what we stated as YPW1 'upregulated dominantly' which compares to other genes. As it might be confusing the readers, we have revised this sentence into “…expressed in G1, G2, and G3. However, the expression of YWP1, the yeast-specific gene, was had a higher upregulation in almost all of the CC groups than other specific genes (HWP1 and ALS3) (Figure 5A).” (third sentence of Result section on paragraph Effect of CC on the expression of biofilm-related genes) Comment 16: What do you mean by 'to maintain the commensal form of C. albicans'?Response to reviewer’s comments 16:Thank you for your interest in this part. C. albicans is commonly recognized as the most prominent human commensal fungi which could grow by taking nutrient on the human body without interfering the human body homeostasis. This condition occurs when C. albicans is in the yeast state. Unfortunately, the phenotype switching into hyphae often caused this commensal fungus to become a pathogen (Finkel and Mitchell 2011). This morphological change represented several virulence genes which might be induced as mention in the third sentence of our introduction section “The hyphal form is relevant for its virulence as it allows penetration and invasion of epithelial cells (Moyes et al. 2015)”. Therefore, the exposure of CC on the dual-species biofilm was expected to maintain the yeast state of C. albicans so that its commensal properties remain. In other words, the commensal form describes the yeast state of C. albicans. This yeast form would suppress its virulence and to minimize the interaction with S. mutans. Section: Discussion Comment 17: The author claims that all CC groups reduce biofilm biomass. What is the author comparing too? Based on my observation, there is no significant difference in total biomass for 24 h and 0 h when all formulation are compared to G0. Please check your statistics.Response to reviewer’s comments 17:Thank you for your further review of this data. Our statement was based on Duncan’s test as our statistical approach. As describe in the response no.10-11, this test showed that G1-G5 had different superscript letters with G0 on each groups (0, 3 and 24 h) for the total biofilm which indicated significantly different values have occurred (Figure 1A). It also means that the CC formula significantly affected biofilm formation on 0, 3 and 24 h without comparing the group. Comment 18: Figure 4D. This doesn't look cocci shape to me. More like bacilli/rod. I hope you did verify your sample prior to the experiment. Attached is the SEM of commonly seen Streptococcus mutans under SEM.Response to reviewer’s comments 18:Thank you very much for your deep review of our SEM images. We have verified our sample prior to the analysis. The different shapes with your attached literature might be correlated with the different strains that have been used. We used S. mutans serotype c strain, the most isolated strain from dental plaque, which possible to have a different shape with S. mutans ATCC 25175 in your attached literature (Lim et al. 2017). Our SEM analysis showed that S. mutans serotype c had longer shape than ATCC 25175 strain.Our analysis method was conducted based on literature (Barbosa et al. 2016), which also used S. mutans UA159 (a serotype c strain). In their report, S. mutans also had a long shape (showed in Figure 3) similar to our result. Our image also supported by Feldman et al. (Feldman et al. 2016) which reported that biofilm was dominated by the long shape of S. mutans UA159 (a serotype c strain) which was described in Figure 3 in the literature. Sztajer et al. (Sztajer et al. 2014) had a clear visualization of dual-species biofilm of C. albicans and S. mutans UA159 (Figure 1e-f), in which the long shape S. mutans attached to the hyphae of C. albicans. Therefore, it was reasonable that our S. mutans strain also have a long shape. Comment 19: The author mentioned that lower gtfB indicate the fewer binding site. Glucosyltransferases (Gtfs) are enzymes and not a 'binding site'. This is too speculative. Please consider to rephrase or to remove the statement.Response to reviewer’s comments 19:Thank you for your correction. We understand your opinion about this issue. Based on our result, the exposure of G2 caused the fewer gtfB expression level. This fewer gtfB contributed to the fewer extracellular polysaccharide matrix formation, in which this matrix is needed as a mediator of interaction between C. albicans and S. mutans. Therefore, we have revised our sentence as suggested (fifth sentence in fourth paragraph of Discussion section) into : “…Lower gtfB expression indicated a fewer matrix formation of S. mutans which important to form a polymicrobial biofilm with C. albicans, as shown by the CV and MTT assays in this study.” Comment 20: The author also emphasised a lot on quorum sensing molecule which is not studied in the manuscript. The author also mentioned that QSM is not measured quantitatively. This statement can be misinterpreted none of the section showed the authors have conducted study on QSM either quantitatively or qualitatively. Please consider rephrasing.Response to reviewer’s comments 20:Thank you for your response. The quorum-sensing molecule information was added in the text as the most possible mechanism that supports our result based on the literature. Several QSM has been reported to be involved in the dual-species interaction between C. albicans and S. mutans. We assumed that we should embed this explanation as additional information to enrich the readers' insight related to the dual-species biofilm formation. Although QSM was not measured in our study, these molecules might have a role in their synergism. Therefore, we have revised our last sentence in this QSM paragraph into “…QSMs in the mixed biofilm was not measured quantitatively or qualitatively in our study. However, this assumption needs to be studied further.” References Amri I, Mancini E, de Martino L, Marandino A, Lamia H, Mohsen H, Bassem J, Scognamiglio M, Reverchon E, de Feo V. 2012. Chemical composition and biological activities of the essential oils from three Melaleuca species grown in Tunisia. Int. J. Mol. Sci. 13(12):16580–16591. doi:10.3390/ijms131216580. Anand S, Prasad R. 1991. Growth and Respiration Characteristics of Candida albicans. In Candida Albicans, R. Prasad (Ed. by), Springer, Berlin, Heidelberg, pp. 46–61. Barbosa JO, Rossoni RD, Vilela SFG, De Alvarenga JA, Velloso MDS, De Azevedo Prata MC, Jorge AOC, Junqueira JC. 2016. Streptococcus mutans can modulate biofilm formation and attenuate the virulence of Candida Albicans. PLoS One 11(3):1–16. doi:10.1371/journal.pone.0150457. Feldman M, Ginsburg I, Al-quntar A, Steinberg D. 2016. Thiazolidinedione-8 Alters Symbiotic Relationship in C . albicans - S . mutans Dual Species Biofilm. Front. Microbiol. 7(140):1–12. doi:10.3389/fmicb.2016.00140. Finkel JS, Mitchell AP. 2011. Genetic control of Candida albicans biofilm development. Nat. Rev. Microbiol. 9(2):109–118. doi:10.1109/isdrs.2005.1596138. Ikono R, Vibriani A, Wibowo I, Saputro KE, Muliawan W, Bachtiar BM, Mardliyati E, Bachtiar EW, Rochman NT, Kagami H, et al. 2019. Nanochitosan antimicrobial activity against Streptococcus mutans and Candida albicans dual ‑ species biofilms. BMC Res. Notes 12(383):1–7. doi:10.1186/s13104-019-4422-x. Krzyściak W, Kościelniak D, Papież M, Vyhouskaya P, Zagórska-Świeży K, Kołodziej I, Bystrowska B, Jurczak A. 2017. Effect of a Lactobacillus salivarius probiotic on a double-species streptococcus mutans and Candida albicans caries biofilm. Nutrients 9(11):1–23. doi:10.3390/nu9111242. Lim JH, Song SH, Park HS, Lee JR, Lee SM. 2017. Spontaneous detachment of Streptococcus mutans biofilm by synergistic effect between zwitterion and sugar alcohol. Sci. Rep. 7(1):1–9. doi:10.1038/s41598-017-08558-x. Rini P, Ohtani Y, Ichiura H. 2012. Antioxidant, anti-hyaluronidase and antifungal activities of Melaleuca leucadendron Linn. leaf oils. J. Wood Sci. 58(5):429–436. doi:10.1007/s10086-012-1270-x. Rodrigues ME, Gomes F, Rodrigues CF. 2020. Candida spp./Bacteria Mixed Biofilms. J. Fungi 6(5):1–29. Sztajer H, Szafranski SP, Tomasch J, Reck M, Nimtz M, Rohde M, Wagner-Dobler I. 2014. Cross-feeding and interkingdom communication in dual-species biofilms of Streptococcus mutans and Candida albicans. Int. Soc. Microb. Ecol. 81–16. doi:10.1038/ismej.2014.73. Thein ZM, Samaranayake YH, Samaranayake LP. 2007. In vitro biofilm formation of Candida albicans and non-albicans Candida species under dynamic and anaerobic conditions. Arch. Oral Biol. 52(8):761–767. doi:10.1016/j.archoralbio.2007.01.009. Wijaya CH, Sari BR, Bachtiar BM. 2020. The potency of cajuputs candy in maintaining the competitive capacity of Streptococcus sanguinis upon Streptococcus mutans. J. Funct. Food Nutraceutical 1(2):55–65. doi:10.33555/jffn.v1i2.29. Wijaya CH, Fickie A, Nurramdhan IF, Bachtiar BM, Afandi FA, Indariani S. 2016. The Composition of Cajuputs Candy which inhibits the growth of dental caries microbes. Indonesia; IDP000040695. Wińska K, Mączka W, Łyczko J, Grabarczyk M, Czubaszek A, Szumny A. 2019. Essential Oils as Antimicrobial Agents—Myth or Real Alternative? Molecules 24(11):1–20. doi:10.3390/molecules24112130. Zijnge V, Leeuwen MBM Van, Degener JE, Abbas F, Thurnheer T, Gmu R. 2010. Oral Biofilm Architecture on Natural Teeth Oral Biofilm Architecture on Natural Teeth. PLoS One 5(2):1–9. doi:10.1371/journal.pone.0009321."
}
]
},
{
"id": "58901",
"date": "17 Feb 2020",
"name": "Dikdik Kurnia",
"expertise": [
"Reviewer Expertise Bioactive Natural Products"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe main structure of the research and main data are according and relevant, with all references, and showed the new findings effect on mix biofilm formations.\n\nAs an addition if possible, in table 1, the effect of CC against individual bacterial biofilm can be added as a reference or control.\n\nAs a suggestion, in the discussion, it would be better to explain the bioactive compounds of Melaleuca cajuputi reported in other published papers. In this way, we can predict the active constituents of M. cajuputi that show an effect to biofilm formation.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "5531",
"date": "28 May 2020",
"name": "Christofora Hanny Wijaya",
"role": "Author Response",
"response": "Dear reviewer,Please accept our sincere apology for this delayed response. We do appreciate the opportunity to revise our paper entitled, “Cajuputs candy impairs Candida albicans and Streptococcus mutans mixed biofilm formation in vitro”. We also extend our deep gratitude to the reviewers for providing valuable comments and correction on our paper. We have read and understood the comments from the reviewers. Please find below our point-by-point response to the reviewer’s comments and concerns. We hope our additional explanations will be able to answer the reviewer's inquiry.We strived to improve the quality of our paper based on the guidance and constructive suggestion provided by the reviewers. We hope that the revisions will be sufficient to make our paper worthy for publication in F1000 Research. We would like to thank you for your hearty support and willingness to find the parts of our paper needing corrections or improvement. Thank you again for your consideration, suggestions, and insightful feedback in order to greatly improve our paper. Sincerely yours,Authors: Siska Septiana, Christofora Hanny Wijaya*, Boy Muchlis Bachtiar and Nancy Dewi Yuliana. Reviewer Comments and Author Responses Comment 1: The main structure of the research and main data are according and relevant, with all references, and showed the new findings effect on mix biofilm formations.Response to reviewer’s comments 1:Thank you so much for your positive feedback. We greatly appreciate your reviews in our paper. Comment 2: As an addition if possible, in table 1, the effect of CC against individual bacterial biofilm can be added as a reference or control.Response to reviewer’s comments 2:Thank you for the comments. Your suggestion is reasonable, unfortunately, we couldn’t conduct an additional experiment due to the limited sample. However, as reported in the previous study (unpublished report, Wijaya et al. 2014), CC had been proven to has antibiofilm activity on a single biofilm either on S. mutans or on C. albicans. Our study was then conducted as further evaluation of CC against synergistic interaction between the organisms. Therefore, we focused on the effect of CC against the dual-species biofilm, and the effectivity of CC against individual strain was further evaluated using Total Plate Count, as shown in Figure 2. Comment 3: As a suggestion, in the discussion, it would be better to explain the bioactive compounds of Melaleuca cajuputi reported in other published papers. In this way, we can predict the active constituents of M. cajuputi that show an effect to biofilm formation.Response to reviewer’s comments 3:Thank you for your valuable suggestion, your suggestion would enrich our paper. We have added the additional information regarding the possible bioactive compound. The new version of the paragraph can be found as the sixth sentence in the last paragraph in the discussion section:Based on the previously published article, 1,8-cineole, a-terpineol, caryophyllene, linalool, terpinene-4-ol, and several other terpene compounds on MCEO were commonly reported as the responsible bioactive compounds on the MCEO antifungal and antibacterial activities (Rini et al. 2012; Wińska et al. 2019). Simsek and Duman (2017), further reported the capacity of 1,8-cineole that increases the antimicrobial activity of chlorhexidine gluconate due to its synergistic effect and is expressed as a penetration enhancer. Moreover, Caryophyllene which found most in the MOJ sample (Septiana et al. 2020) also thought to be correlated with the effect of CC in the biofilm formation as it has been widely reported responsible for the antimicrobial activity (Yoo and Jwa 2018).References: Rini P, Ohtani Y, Ichiura H. 2012. Antioxidant, anti-hyaluronidase and antifungal activities of Melaleuca leucadendron Linn. leaf oils. J. Wood Sci. 58(5):429–436. doi:10.1007/s10086-012-1270-x. Septiana S, Yuliana ND, Bachtiar BM, Putri SP, Fukusaki E, Laviña WA, Wijaya CH. 2020. Metabolomics approach for determining potential metabolites correlated with sensory attributes of Melaleuca cajuputi essential oil, a promising flavor ingredient. J. Biosci. Bioeng. 129(5):581–587. doi:10.1016/j.jbiosc.2019.12.005. Simsek M, Duman R. 2017. Investigation of effect of 1,8-cineole on antimicrobial activity of chlorhexidine gluconate. Pharmacognosy Res. 9(3):234–237. doi:10.4103/0974-8490.210329. Wijaya CH, Rachmatillah AF, M. Bachtiar B. 2014. Inhibition of Cajuputs Candy Toward the Viability of Candida albicans by using In Vitro Assay. J. Teknol. Dan Ind. Pangan 25(2):158–167. doi:10.6066/jtip.2014.25.2.158. Wińska K, Mączka W, Łyczko J, Grabarczyk M, Czubaszek A, Szumny A. 2019. Essential Oils as Antimicrobial Agents—Myth or Real Alternative? Molecules 24(11):1–20. doi:10.3390/molecules24112130. Yoo HJ, Jwa SK. 2018. Inhibitory effects of β-caryophyllene on Streptococcus mutans biofilm. Arch. Oral Biol. 8842–46. doi:10.1016/j.archoralbio.2018.01.009."
}
]
}
] | 1
|
https://f1000research.com/articles/8-1923
|
https://f1000research.com/articles/9-445/v1
|
26 May 20
|
{
"type": "Research Article",
"title": "The application of multi-criteria decision analysis to inform in resource allocation",
"authors": [
"Carina Schey",
"Maarten Postma",
"Paul Krabbe",
"Goran Medic",
"Mark Connolly",
"Maarten Postma",
"Paul Krabbe",
"Goran Medic",
"Mark Connolly"
],
"abstract": "Background: There is a perception held by payers that orphan products are expensive. As a result, the current health technology assessment systems might be too restrictive for orphan drugs, therefore potentially denying patients access to life-saving medicines. While price is important, it should be considered in relation to a broader range of disease-related product attributes that are not necessarily considered by many health technology assessment agencies. To overcome these challenges, multi-criteria decision analysis has been proposed as an alternative to evaluate technologies. Methods: A targeted literature review was conducted to identify the most frequently cited attributes in multi-criteria decision analysis (MCDA) in rare diseases. From the leading attributes identified, we developed a multi-criteria decision analysis framework with which to aggregate the orphan drug values. We subsequently reviewed and plotted the relationship between single attributes and the average annual treatment costs for 8 drugs used in the treatment of rare endocrine diseases. The annual treatment costs were based on UK list prices for the average daily dose per patient. Results: The five most frequently mentioned attributes in the literature were as follows: Disease severity, Unmet need (or availability of therapeutic alternatives), Comparative effectiveness or efficacy, Quality of evidence and Safety & tolerability. Results from the multi-criteria decision analysis framework indicate a wide range of average annual per-patients costs for drugs intended for the same diseases, and likewise for diseases with a similar level of Disease severity. Conclusions: Multi-criteria decision analysis may offer a viable alternative to support discussion in reimbursement decisions for orphan drugs. The analyses can be used to inform investigations on the application of MCDAs in rare diseases.",
"keywords": [
"orphan drug",
"multi-criteria decision analysis",
"criteria",
"attributes",
"healthcare",
"lysosomal storage disorders"
],
"content": "Abbreviations\n\nHTA, health technology assessment; MCDA, multi-criteria decision analysis; QALY, quality-adjusted life-year.\n\n\nIntroduction\n\nThe increasing demand on healthcare resources has created the need to minimize costs, resulting in more rigorous pricing and reimbursement pathways in most of Europe, potentially causing delays for patients to receive valuable treatments1. As the pressure on health technology assessment (HTA) bodies increases, so does the need to demonstrate the cost-effectiveness of orphan drugs. There is a perception among payers that orphan drugs are over-priced and therefore not cost-effective2. Patients have been refused access to drugs based on cost-effectiveness perceptions that might possibly be linked to the application of cost-effectiveness thresholds3. The pharmaceutical media go to great lengths to highlight that orphan drugs are expensive interventions with costs being raised by the involvement of “big pharma”4,5. Therefore, orphan drugs are increasingly under scrutiny by health authorities as part of cost-containment initiatives and are frequently only reimbursed when negotiated under a patient access scheme6,7. The HTA processes are aimed at bridging the gap between evidence and healthcare policy and reimbursement decisions8. The current HTA systems adopted in many European countries may facilitate financial decisions that largely disregard disease impact on patients and a host of other disease-related attributes, and potentially ignore some unique features of innovative orphan drugs, thereby delaying or denying patients access to much needed treatments9–11. While the cost and budget impact of orphan drugs are important in relation to affordability, cost should be considered in relation to a broader range of drug- and disease-related attributes that are not necessarily considered in the usual HTA processes for most non-orphan drugs. This is increasingly problematic for markets linking cost-effectiveness analysis to reimbursement decisions as the cost per quality-adjusted life-year (QALY) approach is not necessarily sensitive enough to capture the broader attributes of the therapy such as unmet need, disease severity, patient and societal preferences and other disease-related elements, possibly including disease rarity12.\n\nDisease rarity implies that the level of clinical evidence and the uncertainty surrounding the treatment effectiveness generated in clinical trials is likely to differ from that of conventional diseases due to several key factors13. In particular, small heterogenous populations increase the level of uncertainty of the outcomes14,15. In some rare diseases, there is limited natural history data and often a lack of consensus on the choice of treatment comparator and clinical endpoints16,17. Ethical constraints regarding placebo treatments in the control group when no other treatment options are available may arise15,18. Consequently, these factors might preclude a thorough analysis. Therefore, the likelihood of orphan drugs achieving the expected robust cost per QALY levels is limited19. Thus, the cost per QALY approach might not be optimal to assess the real value and benefits of orphan drugs and should, at least, not be the dominant tool to establish reimbursement.\n\nIn response to criticism regarding the variation in reimbursement decisions and potential inconsistent patient access to orphan drugs20–22, the use of multi-criteria decision analysis (MCDA) has been proposed as a viable method for orphan drug assessments. The MCDA framework provides a consistent, transparent and accountable approach for the decision-making process for orphan drugs. The MCDA approach assumes that the value framework adopts clear criteria and that the criteria are weighted appropriately20,23. The premise is that because the framework is based on multiple criteria, it will provide a result that reflects not only the efficacy and cost of the drug, but also wider aspects such as disease severity, unmet need, patient’s health-related quality of life and target patient age groups. Techniques for MCDA enable expert panels to perform “trade-offs” between multiple aspects and outcomes of a product against the product’s cost as well as a combination of different criteria24. The Evidence and Value: Impact on Decision Making MCDA framework was developed to support the prioritization of a broad range of healthcare interventions, with priority given to the intervention that obtains the highest rank. It has evolved and is now in its 10th edition25, yet it is not designed specifically for assessing the value of orphan drugs. MCDA frameworks could also inform on societal preferences, if designed to capture such information. The International Society for Pharmacoeconomics and Outcomes Research Taskforce in multi-criteria decision analysis published recommendations on MCDA, highlighting that MCDA is a tool to support decision-making by HTA bodies and payers26,27.\n\nBased on our earlier research28, the aim of this study was to further review the literature to establish what criteria are most frequently discussed in the context of assessing orphan drugs with a view to re-testing these criteria on a group of drugs used in the treatment of rare endocrine disorders and to analyze the possible association between the key criteria and the average annual cost of each of the treatments. All the endocrine diseases included in this study are rare (maximum prevalence of 50/100,000 population in Europe29 diseases and the drugs included in the study have orphan drug designation although some have lost marketing exclusivity.\n\n\nMethods\n\nThis study entailed two parts, the first being a targeted literature review of Pubmed, Google Scholar and Google to identify any publications featuring MCDAs specifically in orphan drugs and rare diseases from January 1983 to December 2018. Search terms included “multi-criteria decision analysis”, “MCDA”, “orphan drug”, “health technology assessment”, and “quality-adjusted life-year”.\n\nThis time frame was chosen to capture as wide a range of studies as possible and to reflect the start of the Orphan Drug Act in the USA30. The search strategy was restricted to English-language studies and only those reporting on medicines in human use. Each shortlisted study was reviewed to establish what criteria had been used or mentioned in the studies. Criteria were listed by the frequency in which they were reported. The second part of the study aimed at testing the eight most reported criteria identified in the literature review by developing a MCDA framework in which to compare the average annual costs of the eight endocrine drugs included in the study with their aggregated MCDA-framework score. The drug cost calculations were based on the average dose according to the Summary of Product Characteristics, taking into account the different body weights for adults and pediatric patient cohorts. Prices were obtained from the British National Formulary (March 2019) and published price lists (March 2019) for the UK and converted to Euros at the December 2018 exchange rate31. A further targeted literature review was conducted to source disease- and drug-related data to populate the MCDA framework for each drug and each criterion. In this search, we looked for the data that would be used to populate the different criteria that would be used in developing the MCDA framework. Only studies that met with the Oxford Centre for Evidence-based Medicine32 criteria levels of 1a to 3a were included, i.e. Individual case control studies, case series and expert opinion without explicit critical appraisal were excluded. A hand-search of several health technology assessment bodies (National Institute for Health and Care Excellence, Scottish Medicines Consortium, Zorginstituut Nederland, Haute Autorité de santé) was also performed. The data for each drug and each disease was extracted and categorized in tables by each criterion.\n\nIn developing the MCDA framework, we had planned to use the ten most cited criteria. However, on reflection, we reduced this to six, on the basis that Disease Rarity (prevalence) is a given when developing assessment frameworks for orphan drugs, as is the Size of the Population. The Quality of the Evidence and Level of Research Undertaken were considered to report the same data and were therefore reported as Level of Research Undertaken. Uncertainty of Effectiveness was deemed to reflect efficacy (or lack thereof) and was not used in this framework. A numerical scoring system was developed for each criterion, as shown in Table 1. For the Disease Severity, the higher score denotes a worse impact of disease on the patient. For the treatment-related criteria, the higher the score, the more marginal the benefit of the treatment is deemed to be. Overall, for the average score of each drug, the lower the score, the better the treatment is. Each criterion (and sub-criterion for Disease Severity) was scored by two people working together, and then checked by two further people to ensure that the same approach was used for all the criteria. Disease Severity is comprised of 4 sub-criteria: Life threatening or life-shortening, Severity of disease symptoms, Mental status due to the disease (anxiety, depression) and Impact of the disease on physical ability. Each sub-criterion was scored from 1 to 4 (Table 1) and an average total score was calculated for Disease severity. The data for the sub-criterion were based on studies and publications of the included diseases and their overall impact on patients as well as treatment guidelines to establish how treatment should expect to manage the disease and its symptoms33–63. The Level of Research Undertaken was scored on an adaptation of the Levels of Evidence from the Oxford Centre for Evidence-based Medicine64. Efficacy was scored according to the degree that the primary endpoint was met in the clinical trials (Table 1). The scores for Safety were ranked according to the level of serious adverse events reported in the clinical trials. Unmet Need was defined by the number of other available treatments for each of the endocrine diseases. Similarly, each drug was given a score to represent how many indications the drug was licensed for in total. Criteria scores were compared with the average annual cost of the respective drug, based on list prices published in the British National Formulary for 2019.\n\n\nResults\n\nEven though this was a targeted literature review, we followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses method65. The literature review identified 161 publications (after removal of duplicates), of which 15 were relevant to our study21,23,25,66–77 (Figure 1). A bibliometric analysis described the publication by origin of the primary author (e.g. academic, industry) as shown in Figure 2. First authors were from academia in 53% of the publications23,66–68,70,73,75,77. The first authors in 13% of the publications were each from consultancy backgrounds72,74, government or parastatal origins25,76, and healthcare positions69,71. A final publication (7%) was first-authored from industry21.\n\nA total of 45 criteria were identified (Table 2). Geographic variation in the importance of the different criteria was observed. For example, in publications in The Netherlands and Germany, the most important criteria in MCDA for orphan drugs were Life-threatening nature of disease and Evidence of clinical efficacy and patient outcome, respectively68. As anticipated when focusing on rare diseases, the most frequently cited criterion is Disease severity. This is inevitable, given that many of the rare diseases are life-threatening and result in significant impact on quality of life78. The same applies to Unmet Need. By contrast, and rather surprisingly, Treatment convenience was only reported on by 7% of the studies despite the fact that some drugs may need to be administered regularly by non-oral routes, i.e. injection or intravenously and may impact on overall treatment costs and quality of life. In a landscape where increasingly Innovation (of new treatments) is of significant interest to HTA bodies, it’s a criterion that was only reported on by 20% of the studies.\n\nThe average annual per patient cost in relation to the total drug score is shown in Figure 3 and the comparison of Disease severity score to the average annual cost per patient (Figure 4) highlights the cost differences for diseases with the same severity.\n\nThe Unmet Need is a criterion that featured frequently in the literature as would be anticipated in relation to treatments for rare diseases, yet a plot of Unmet Need and the average annual cost suggests there is no discerning relationship based on this single criterion. In contrast, the three products used to treat acromegaly all address the same unmet need, yet considerable variation in average annual cost is observed.\n\nThe analysis reviewed the relationship between average annual cost and the number of licensed indications that each respective product has. Based on the results of this small sample size of products, the results do not suggest that the average annual cost decreases as a product is licensed for additional indications.\n\n\nDiscussion\n\nThis study sought to assess the key criteria used in MCDA frameworks for orphan drugs and having given each criteria a score based on an ordinal scoring system, to compare the aggregated outcomes for each drug with its average annual per-patient cost without applying different weights (importance) to any criteria. We used the criteria that appeared most in the literature, although because all the diseases studied are rare diseases, the criteria Disease rarity (Prevalence) which was cited by 47% of the studies, and Size of the affected population (40%) were not included in this MCDA framework. We observed that there is room for inclusion of others. Notably, the Treatment convenience was not included in the criteria for orphan drugs. In the context of some complex orphan drug treatments that may be used for the duration of life, such as in the lysosomal storage disorders, the cost and management implications of a drug that has to be administered intravenously on a very regular basis are greater than those of a drug injected subcutaneously once a month (e.g. treatment for acromegaly) or an of a drug taken orally. Therefore, we believe that it is a criterion worth including in MCDA frameworks for orphan drugs.\n\nThe Target age (of the treatment population) is a criterion that featured only in two of the studies we reviewed. Indeed, this could be regarded to impact on the assessment of the drug in so much as it may result in absenteeism from school for children and work for parent(s) caring for ill children. Access to orphan drug treatment could have significant impact on education attainment and productivity.\n\nThe Level of research undertaken prior to licensing of a drug is likely to establish the drug’s efficacy and safety with a view to reducing the level of uncertainty. However, we question the comparability of the level of research between products. To our knowledge, no studies have suggested a methodology to score the duration of studies versus the population size of a study. For example, would a study performed in a small population sample over several years earn a higher score than one conducted on several thousand patients over less than a year? In the scope of drugs used for rare diseases, the population size of a study is frequently very small due to the low prevalence of the disease. In diseases where there is no other treatment option, it may be necessary to reduce the impact or weight of this criterion in order to facilitate access to the medication. Irrespective of the level of research, it was seen to have little correlation with the average annual per-patient cost of each drug.\n\nWhile some other studies have shown an inverse relationship between Disease rarity and price79,80, this study was not designed to analyze this relationship. However, it provided the opportunity for a within therapy comparison of treatments for acromegaly. One might expect treatments for the same disease to have a narrow average annual per-patient cost range. Nevertheless, the average annual prices range from €617 to €20,520 demonstrating that whilst disease severity is the same for two or more drugs for the same indication, their costs may vary widely.\n\nThe criteria can be measured on various scales: binary, nominal, ordinal, cardinal or ratio81. For simplicity and transparency, we chose a numerical scoring system whereby the data for each criterion in the framework are converted into consistent numerical values from which an overall score is calculated. In their pilot study to test a MCDA for orphan drugs, Sussex et al.74 used a numerical rating scale from 1 (worst score) to 7 (best score). Their rationale was that it permitted sufficient discrimination between levels for each criterion. By contrast, Hughes-Wilson et al.21 suggested a simplistic three-level scale. One of the limitations of the model might be the application of the scores to the criteria, and the subjectivity of those scores. However, as each criterion was treated equally and scored by the same team, the differences are likely to be minimal on the overall results.\n\nThe literature review highlights some of the limitations of MCDAs. Defining the criteria at the outset is crucial to ensure that overlap between criteria are avoided. As in this study, we chose to avoid confounding the overall score by excluding Disease rarity and Size of the population. It is essential that the criteria are not selected merely to favor a preferred outcome. Ultimately, disease rarity in itself should not be the reason for paying premium prices. Other factors, such as severity of the disease, may be more important82. Furthermore, it is unlikely that all criteria should carry the same importance (weight)23, and as yet, very little work has been done to elucidate weighting preferences in a way that could best inform in healthcare reimbursement decisions. Weighting the criteria may be complicated, and dependent on the perspective of the assessment21.\n\nGiven that the criteria used might vary from one health economy to another, and similarly the importance of these criteria perceived differently, the MCDA framework will not necessarily lead to the same reimbursement decisions across different countries. Choices of criteria and weighting might be influenced by the health policy focus and the inclusion of the societal perspective83.\n\nSince regulatory organizations, such as the Haute Autorité de Santé84 in France, have indicated that drug innovation is valued in assessing drugs, it is interesting to observe that Level of innovation did not feature more frequently in the literature. It should serve as a powerful criterion in the judgement of drugs, especially in the current climate of priority setting, not only for orphan drugs but equally for all other drugs too.\n\nIn their work on MCDAs, Hughes-Wilson et al.21 highlighted the need for several key criteria that should form part of an MCDA tool for orphan drugs. One such criterion is the Manufacturing complexity. Our study found this criterion to be reported by 27% of the literature review results. In the landscape of plasma-derived medicines (clotting factors, immunoglobulins) that are reputed to be more complex in their manufacturing processes85,86 it is likely that this criterion will feature more prominently if MCDAs are to become useful assessment tools in drug assessments.\n\nThe scoring systems for each of the criteria that were adopted in this study were of the numerical form, with equal importance between grades. Despite potential criticism about the imprecision of a simple numerical scoring system, the rationale for its use is due to its simplicity and that it does not require an expert panel to adjudicate the value of one criterion against another, as would be the case in outranking methods87, satisficing methods20 and value measurement methods20. Furthermore, it does not require the use of special computer software. Similarly, the same rationale applies to lack of using any weighting or preferences for a particular criterion, since the primary aim was to apply the key identified criteria as a tool for within therapy and across therapy comparisons. Since the methodology was applied uniformly across the criteria and drugs in the study, the outcomes provide valuable tools for comparison. However, the aim of the study was to demonstrate the trends and above all, to highlight the need for a different approach to assessing orphan drugs.\n\n\nConclusion\n\nTo date much work has been done in describing MCDAs and the criteria which could be considered core to value despite the evident lack of relationship between Disease severity and the overall drug score derived in the MCDA framework. Whilst no consensus on their design and applicability has yet been reached, MCDAs are frameworks that are worth adopting in the assessment of orphan drugs, specifically because they include disease-related and drug-related criteria that are likely to impact on patients and the healthcare system. They are not intended to replace the current HTA methodologies, but should certainly be used in conjunction to assist in decision making processes and prioritizing allocation of healthcare resources.\n\n\nData availability\n\nAll data underlying the results are available as part of the article and no additional source data are required.",
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PubMed Abstract | Publisher Full Text\n\nFarrugia A, Quinti I: Manufacture of immunoglobulin products for patients with primary antibody deficiencies - the effect of processing conditions on product safety and efficacy. Front Immunol. 2014; 5: 665. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMousseau V, Dias L: Valued outranking relations in ELECTRE providing manageable disaggregation procedures. Eur J Oper Res. 2004; 156(2): 476–482. Publisher Full Text"
}
|
[
{
"id": "63937",
"date": "09 Jun 2020",
"name": "R. Brett McQueen",
"expertise": [
"Reviewer Expertise Health economics"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript, \"The application of multi-criteria decision analysis to inform in resource allocation\" is an exploration of current attributes for MCDA in rare diseases through a targeted literature review and suggested MCDA framework specific to rare disease. The study found the five most frequently mentioned attributes were disease severity, unmet need, comparative effectiveness/efficacy, quality of evidence, and safety & tolerability. The study then plots per patient costs against total drug score and disease severity to demonstrate the wide range in costs.\n\nOverall comments:\n\nThis is a nice contribution and needed for the field to move forward with adopting MCDA. I agree the use of MCDA is an add-on to most HTA processes. However, I believe you can expand on some areas and I have a two main suggestions for consideration:\n1) Your work is mainly focused on the performance metrics of orphan drugs that are based on literature-derived criteria. For example, your discussion comment, \"Defining the criteria at the outset is crucial to ensure that overlap between criteria are avoided.\" This is quite key to informing MCDA scoring/frameworks and flows directly into measurement and weighting of the criteria. On measurement, instead of simply listing this as a limitation can you provide any suggestions to future researchers on how to define the levels within criteria a priori instead of depending on past literature? Is it worth identifying criteria through engaging with patients, providers, etc. on criteria that may not be driven by regulatory approval? Meaning is there value in developing (and eventually weighting) criteria using ex ante preferences? This could aid in evidence generation, societal preference alignment, etc. Maybe that was the point you were making but didn't seem to be clear to me.\n\n2) Related to 1), importance/weighting is quite complicated and should have it's own dedicated paragraph. Not only does an overall MCDA depend on perspective as you note in the Discussion, certain criteria may depend on perspective, i.e., clinical benefits are largely accrued by patients but cost is accrued by health systems. So it may be wise to dig in a bit more here with a separate paragraph noting these challenges. Weighting itself also comes with multiple methods that vary in bias.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "6152",
"date": "04 Dec 2020",
"name": "Carina Schey",
"role": "Author Response",
"response": "Dear Prof. McQueen, Many thanks for your thorough review of the manuscript and valuable insights, which I had addressed in a subsequent publication. However, I shall also address it in this manuscript. Engaging with patients in evidence generation is to meet the increasing awareness of representing the patient needs, a feature of reimbursement decisions that has increased in recent years in several European markets. The issues of weighting, as you aptly point out, are complex and open to \"over-counting\" if criteria overlap in anyway, or are already accounted for in the orphan drug incentives (such as disease rarity, which alone is not a good enough reason to reimburse a drug). Ultimately, the issue is not one of favouring MCDA over cost-effectiveness (or vice versa), but rather an ever-increasing dilemma of affordability and sustainability of healthcare provision especially in light of the horrifically expensive gene therapies coming to market amidst substantial uncertainty of their long-term benefits and other effects. Interesting times ahead! Kind regards Carina"
}
]
},
{
"id": "70233",
"date": "08 Sep 2020",
"name": "Olha Zaliska",
"expertise": [
"Reviewer Expertise Pharmacoeconomic analysis. HTA",
"MCDA",
"HEOR",
"marketing",
"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 manuscript, \"The application of multi-criteria decision analysis to inform in resource allocation\" contens the new results of systematic analysis of MCDA in rare diseases in published literature. The authors found the five most frequently mentioned criterias were disease severity, unmet need, comparative effectiveness/efficacy, quality of evidence, and safety & tolerability. The study then plots per patient costs comparing total drug score and disease severity to demonstrate the wide range in costs.\n\nOverall comments:\n\nThis is a great contribution for MCDA area and needed for the field to move forward with adopting MCDA in rare diseases. I agree the use of MCDA is an add-on to most HTA processes, especially for expensive orphan drugs. Authors can use citation the abstract \"Eliciting Payers Preferences In Central And Eastern Europe: Results Of MCDA Case Study\" ( Piniazhko O., Zalis'ka O., Zah V., 20161). I believe you can expand results for many stakeholders.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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/9-445
|
https://f1000research.com/articles/9-443/v1
|
26 May 20
|
{
"type": "Brief Report",
"title": "Differences in the ratio of soil microbial biomass carbon (MBC) and soil organic carbon (SOC) at various altitudes of Hyperalic Alisol in the Amazon region of Ecuador",
"authors": [
"Benito Mendoza",
"Jaime Béjar",
"Daniel Luna",
"Miguel Osorio",
"Mauro Jimenez",
"Jesus R. Melendez",
"Benito Mendoza",
"Jaime Béjar",
"Daniel Luna",
"Miguel Osorio",
"Mauro Jimenez"
],
"abstract": "Protecting soil fertility represents a fundamental effort of sustainable development. In this study we investigate how different altitudes affect soil microbial biomass carbon (MBC) and soil organic carbon (SOC), and their ratio, MBC/SOC in Hyperalic Alisol. MBC and SOC are well established and widely accepted microbial quotients in soil science. Our work hypothesis was that a decrease in MBC and SOC should be observed at higher altitudes. This initial assumption has been verified by our measurements, being attributed to the increase in MBC and SOC at low altitudes. Our approach should contribute to the better understanding of MBC and SOC distribution in soil and changes in MBC/SOC at various altitudes in the region.",
"keywords": [
"microbial biomass carbon (MBC)",
"soil organic carbon (SOC)",
"MBC/SOC ratio",
"rainforest",
"Hyperalic Alisol",
"and Amazon region Ecuador"
],
"content": "Introduction\n\nProtection of soil quality is a pillar of sustainable development1–3. Recent studies considered effects of minerals4–6 and soil cultivation techniques7,8 on soil quality and soil microbial activity9–12. These studies reported interactions between soil minerals and decomposition of organic matter; positive correlations between soil microbial activity (measured by number of bacteria and fungi, soil respiration, and C and N cycle-related enzyme activity) and cultivation methods, and fertilizer usage. The undertaken efforts in agriculture practice, offer favorable conditions for soil-plant interactions. Additionally, these studies on soil respiration and enzymatic activity report correlation analyses between soil properties and microbial activity, documenting that soil quality is mainly determined by soil microbial activity, as verified by other authors’ work13–15. A highly cited review paper on the topic16 concludes that soil processes and properties are affected by climate change, which causes changes in soil organic matter (SOM) and microbial biomass carbon (MBC). Based on this, one can state that monitoring the soil carbon cycle is of increasing importance, given that microbial processes in soil may serve as an indicative of climate change. Among processes to be monitored, decomposition of organic matter is of primary significance17, considering that this process is strongly influenced by soil microbiological activity. Soil management is another research area of increasing interest18 owing to its significance in soil microbial activity19 that can be expressed by enzymatic activity, soil respiration, and MBC11. Local farmers have demonstrated willingness to start permaculture systems involving tropical orchards, like those previously operated successfully in another plant, Guayusa (Ilex guayusa)20, in the Amazon region. Because of the sensitivity of orchards to nutrient deficiency21–23 we believe that measuring soil microbial activity is of priority when analyzing soil properties, as soil microbial activity is in strong correlation with nutrient uptake in plants24. For this reason, monitoring changes of soil MBC and soil organic carbon (SOC) allow to select favorable locations for orchard plantation in permaculture systems, contributing to sustainable agricultural practices. MBC is the living microbial component of soil organic matter25 and is considered an indicator of microbial activity, owing to its rapid response (less than a year turnover time) to conditions that may alter soil organic matter26. SOC contributes positively to soil fertility and crop production27. Here we report variations with altitude of MBC, SOC, and MBC/SOC in Hyperalic Alisol.\n\n\nMethods\n\nWe were inspired by a prior study conducted in 201828, in which soil properties at various altitudes were measured in the region. We collected samples at similar altitudes as in the prior study28 (395, 1006, and 1554 m.a.s.l, meters above sea level) to gather data on soil properties at different altitudes. We collected 15 soil samples, 5 from each altitude, following the protocol proposed by Singla and co-workers28, where a minimum of 3 sampling points per altitude were recommended: 420, 1000, and 1600 m.a.s.l. in the Ecuadorian Amazonian region (latitude: 4.628247894396525; longitude: -74.95615214109422), on December 10, 2019. Homogeneous soil populations can be used for analysis, when sample size (s) = 15, as determined by Cline (1944)29 (Equation 1):\n\n\n\nWhere,\n\nrn, r1 are the extremes of the range of a parameter,\n\nC is a constant, equal to 3, 4, 5, and 6 for 10, 25, 100, and 500 sampling units, respectively (Cine, 1944)29.\n\nTo estimate extremes of a given range, we used prior literature data24 rom a study performed in the Amazon region in which SOC values vary between 34.69 and 54.62. Cline (1944)29 recommended the use of 20 samples, when the range of parameter is 110. In our case, counting with 19.93 range value obtained from a prior study in the region24 Equation (1) becomes s = 19.93/4 = 4.98 ~ 5 samples. So, the total number of samples for the study is Total number of Samples = 5 samples multiplied with the three altitudes, yielding a number of 15 sampling points.\n\nSamples originated from the upper layer (top 20 cm) of Hyperalic Alisol (Ultisols in U.S. Soil Taxonomy) soil. Sampling points and physical-chemical soil properties were described in a prior study30, parameters being in the following ranges: pH 4.99–5.98; moisture content: 43.9–66.6 %; Allophane: volcanic; and total Fe content (mg/kg): 367–1104.\n\nMBC was measured in quadruplicate (see supporting data) by the fumigation-incubation method, according to Jenkinson and Powlson31. Briefly, four aliquots of oven-dried (105 °C for 24 h) soil samples (25 g each) were placed into glass vials, two aliquots being fumigated with CHCl3 (Fisher Scientific, 67-66-3,64-17-5) at room temperature, while two aliquots were kept untreated at 2 °C for 24 h. All samples were incubated at 25 °C for 10 days, in the dark. Their respiration was measured after incubation with Barcroft-type (differential) respirometer, which enables simultaneous measurements of gas volume in real-time32. Produced raw CO2 values (SIR) were read by Respirometer software RV10 (version 10.03) obtaining data directly from the respirometer’s electronic sensor (model: Respicond VIII, Nordgren Innovations, Sweden). Soil respiration of both fumigated and unfumigated samples were calculated from SIR values according to Anderson and Anderson (1978)32 using the equation (Equation 2):\n\n\n\nMBC was calculated by dividing the difference in soil respiration between fumigated and unfumigated samples with 0.38 according to Jenkinson and Powlson31 (Equation 3):\n\n\n\nWhere,\n\nFumigated is the produced CO2 by fumigated soil samples, expressed in C µg/ml.\n\nUnfumigated is the produced CO2 by not fumigated soil samples, expressed in C µg/ml.\n\nTo calculate SOC we determined humus content according to Székely et al.33. In this procedure, 1.0 g of air-dried soil was placed into a 300-mL Erlenmeyer flask. Then, 10. mL of 5% K2Cr2O7 (Fisher Scientific, 7778-50-9) solution was added and mixed with the soil. Next, 20 mL of concentrated H2SO4 (Fisher Scientific, 7664-93-9) were supplemented, and mixed with 100. mL distilled water. Solution was filtered with Grade 42 Whatman slow filter paper (pore size 2.5 μm) and analyzed with Model 240Z Atomic Absorption Furnace Spectrophotometer (Agilent), from 230 to 700 nm, and absorbances at 600 and 400 nm (E600 and E400), respectively, correspond to A,B, P, and Rp types of humic acids, relevant to humus content, according to Watanabe et al.34. Humus content varied from 2.34 (at 1600 m.a.s.l.), to 4.82, at lower laying areas (420 m.a.s.l.) (see raw data in Supporting material.) From the obtained humus content, we calculated SOC, using a conversion factor, according to Walkley and Black35 (E3):\n\n\n\nMBC/SOC quotient was determined by dividing MBC with SOC.\n\nLinear regression (Z-test) was performed to reveal possible statistical differences (p<0,05) between parameters and altitudes, using SPSS (version 26).\n\n\nResults and discussion\n\nOur results are similar in magnitude with prior studies in the Amazonian region36; the lowest SOC and MBC values are observed at the highest altitude (1600 m.a.s.l.): 1.77% and 267.3 mg/kg, where the average MBC/SOC is 161.17. At 1000 m.a.s.l. both SOC and MBC increased; SOC augmented by 74%, while MBC by 45%. Average MBC/SOC increased to 268.2. At the lowest altitude (420 m.a.s.l.) both SOC and MBC reached their highest values: 3.65% and 2214.4 mg/kg, respectively. The average MBC/SOC also reached its maximum: 667.8. Altitude significantly affects both SOC (R2 = 0.95) with p< 0.0001 and MBC (R2 = 0.89) with p< 0.0001 (Table 137).\n\n* represents significant correlation at 95% probability as accepted in agriculture31. Note: humus % is not included in the table, given that SOC is calculated from humus %, as indicated in the methods section.\n\nSOC - soil organic carbon, MBC - microbial biomass carbon\n\nMBC/SOC increases with decreasing altitude (Figure 137), which means that MBC increases more rapidly with decreasing altitude than SOC. As MBC corresponds to microorganisms’ weight (mainly bacteria and fungi), and is estimated at approx. 5% of the SOC32, the increased MBC value indicates faster microbial activity at lower laying sampling points. This finding yields three main observations:\n\n1. Organic matter decomposition is accelerated by soil microbial activity38,39 in lower-laying areas, because of more favorable conditions40 offered to cellulose decomposing bacteria. Presence of these bacteria is confirmed by MBC/SOC increase at lower altitudes, reported here. Microbial carbon increases more significantly than organic carbon, which suggests increased microbial activity40.\n\n2. Availability of SOC depends on soil properties, and soil depth41, rather than on altitude or coverage42. SOC may also depend on several other soil properties, therefore, enzymatic activities and physical-chemical soil properties should be measured. Total iron increases with decreasing altitude, as well30. With these findings in MBC/SOC value changes, general leaching can be documented.\n\n3. Metabolic efficiency depends on the availability of substrate (organic matter). The greater the MBC, the greater the temporary immobilization of micro and macro-nutrients43–46. To confirm this, immobilization of nutrients should be measured in soil-plant interactions (mainly uptake), as planned for future work in which orchards will be investigated, given the local farmers’ need to plantation in the area and the particular sensitivity of orchards to nutrient deficiency21–23.\n\nSamples numbered correspond to the following altitudes 1–5: 420 m.a.s.l.; 6–10: 1000 m.a.s.l.; 11–15: 1600 m.a.s.l.\n\n\nConclusions\n\nAltitude affects SOC significantly. Decrease in MBC/SOC quotient is observed with increasing altitude. From obtained results we can conclude that lower-laying areas favor increased soil microbial activity. We recommend lower-laying areas for orchard plantations, considering that orchards are particularly sensitive to nutrient deficiency47, while soil microbial activity is in strong correlation with nutrient uptake in plants24.\n\n\nData availability\n\nFigshare: Supporting raw data for MBC and SOC. Dataset. https://doi.org/10.6084/m9.figshare.12264698.v137.\n\nThis project contains the following underlying data:\n\n- Supporting Data_ Melendez-V3.xlsx (raw respiration data for MBC calculation and ABS raw data for humus % and SOC calculation)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
"appendix": "References\n\nVárallyay Gy: A talaj és a víz (Soil and Water). In Stefanovits P. and Michéli E. (Eds.) A talajok jelentősége a 21. században (Importance of soils in the 21st century). MTA Társadalomkutató Központ (Center for Social Research, the Hungarian Academy of Science), Budapest, Hungary. 2005; 61–64.\n\nKocsis T, Biró B, Ulmer Á: Time-lapse effect of ancient plant coal biochar on some soil agrochemical parameters and soil characteristics. Environ Sci Pollut Res Int. 2018; 25(2): 990–999. PubMed Abstract | Publisher Full Text\n\nRumpel C, Amiraslani F, Chenu C, et al.: The 4p1000 initiative: Opportunities, limitations and challenges for implementing soil organic carbon sequestration as a sustainable development strategy. Ambio. 2020; 49(1): 350–360. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSingh M, Sarkar B, Bolan NS, et al.: Decomposition of soil organic matter as affected by clay types, pedogenic oxides and plant residue addition rates. 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Publisher Full Text\n\nAllen DE, Singh BP, Dalal RC: Soil Health Indicators Under Climate Change: A Review of Current Knowledge. In: Singh B, Cowie A, Chan K, (eds) Soil Health and Climate Change. Soil Biology, Springer, Berlin, Heidelberg. 2011; 29. Publisher Full Text\n\nTóth E, Gelybó Gy, Kása I, et al.: A művelés hatása a talaj széndioxidkibocsátására. – A talajvízpotenciál értékelés CO2 emissziója közötti összefüggések vizsgálata laboratóriumi módszertani tesztelés során (Effect of cultivation on carbon dioxide emission of soils - Investigation of the relationship between the estimation of groundwater potential and CO2 emissions over laboratory method testing). Agrokémia és Talajtan (Agrochemistry and Soil Science). 2014; 1: 299–310.\n\nSchlesinger WH, Amundson R: Managing for soil carbon sequestration: Let's get realistic. Glob Chang Biol. 2019; 25(2): 386–389. PubMed Abstract | Publisher Full Text\n\nBenites B: The importance of soil organic matter. Food and Agriculture Organization of the United Nations. Rome. 2005. Reference Source\n\nRocha P, Niella F, Keller H, et al.: Ecological Indigenous (EIK) and Scientific (ESK) Knowledge Integration as a Tool for Sustainable Development in Indigenous Communities. Experience in Misiones, Argentina. In: Montagnini F. (eds) Integrating Landscapes: Agroforestry for Biodiversity Conservation and Food Sovereignty. Advances in Agroforestry, Springer, Cham. 2017; 12: 235–260. Publisher Full Text\n\nMetzidakis IT, Koubouris GC, Kassidonis E, et al.: IMPACT OF SOIL MANAGEMENT PRACTICES ON PHYSICAL AND CHEMICAL PROPERTIES OF SOILS FORMED IN MARLS, CONGLOMERATES OR SCHISTS IN SLOPING OLIVE GROVES. Acta Hortic. 2012; 949: 371–378. Publisher Full Text\n\nSimoes MP, Belo AF, Pinto-Cruz C, et al.: Natural vegetation management to conserve biodiversity and soil water in olive orchards. Span J Agric Res. 2014; 12(3): 633–643. ISSN 2171-9292. Date accessed: 22 mar. 2020. Publisher Full Text\n\nVignozzi N, Agnelli AE, Brandi G, et al.: Soil ecosystem functions in a high-density olive orchard managed by different soil conservation practices. Appl Soil Ecol. 2018; 134: 64–76. Publisher Full Text\n\nGuaca L, Contreras AT, Rodríguez L, et al.: Microbial properties of soil in different coverages in the Colombian Amazon. Floresta e Ambiente. 2019; 26(4): e20171051. Publisher Full Text\n\nDoran JW, Jones AJ, Rice CW, et al.: Role of Microbial Biomass Carbon and Nitrogen in Soil Quality. SSSA Special Publication. 1996. Publisher Full Text\n\nPaul EA: Dynamics of organic matter in soils. Plant Soil. 1984; 76: 275–285. Publisher Full Text\n\nWest TO, Post WM: Soil Organic Carbon Sequestration Rates by Tillage and Crop Rotation. Soil Sci Soc Am J. 2002; 66(6): 1930–1946. Publisher Full Text\n\nSingla A, Bautista G, Mátyás B, et al.: Altitudinal variations in H and Al ions in- terchange along with Fe content in Amazonian rainforest soil. La Granja: Revista de Ciencias de la Vida. 2018; 28(2): 42–50. Publisher Full Text\n\nCline MG: Principles of soil sampling. Soil Sci. 1944; 58(4): 275–288. Reference Source\n\nMendoza B, Guananga N, Melendez JR, et al.: Differences in total iron content at various altitudes of Amazonian Andes soil in Ecuador [version 1; peer review: 2 approved]. F1000Res. 2020; 9: 128. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJenkinson DS, Powlson DS: The effects of biocidal treatments on metabolism in soil—I. Fumigation with chloroform. Soil Biol Biochem. 1976; 8(3): 167–177. ISSN 0038-0717. Publisher Full Text\n\nAnderson JPE, Domsch KH: Mineralization of bacteria and fungi in chloroform-fumigated soils. Soil Biol Biochem. 1978; 10(3): 207–213. Publisher Full Text\n\nSzékely Á, Schlick B-né, Szabó T-né: Szerves kötésű szén fotometrikus és kolorimetrikus meghatározása (Spectrophotometric and colorimetric determination of organically bound carbon). Agrokémia és Talajtan. 1960; 9(1–4): 111–120.\n\nWatanabe A, Rumbanraja J, Tsutsuki K, et al.: Humus composition of soils under forest, coffee and arable cultivation in hilly areas of south Sumatra, Indonesia. Eur J Soil Sci. 2001; 52(4): 599–606. Publisher Full Text\n\nWalkley A, Black IA: An examination of the Degtjareff method for determining soil organic matter,andaproposedmodificationofthechromic acid titration method. Soil Sci. 1934; 34(1): 29–38. Publisher Full Text\n\nGuaca L, Contreras AT, Rodríguez L, et al.: Microbial properties of soil in different coverages in the Colombian Amazon. Floresta e Ambiente. 2019; 26(4): e20171051. Reference Source\n\nMelendez: Supporting data (MBC/SOC). figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12264698.v1\n\nTállai M: (Effect of bentonite and zeolite on the properties and biological activity of acidic sand soils) in original language: ‹Bentonit és zeolit hatása savanyú homok talajok tulajdonságaira és biológiai aktivitásának változására.› Doktori (PhD)értekezés. University of Debrecen. Debrecen. Reference Source\n\nGonzalez-Quiñones V, Stockdale EA, Banning NC, et al.: Soil microbial biomass—Interpretation and consideration for soil monitoring. Soil Res. 2011; 49(4): 287–304. Publisher Full Text\n\nKotroczó Z, Koncz G, Halász LJ, et al.: Litter decomposition intensity and soil organic matter accumulation in síkfőkút dirt site. Acta Microbiol Immunol Hung. 2008; 56: 53–54. Reference Source\n\nSpohn M, Klaus K, Wanek W, et al.: Microbial carbon use efficiency and biomass turnover times depending on soil depth: implications for carbon cycling. Soil Biol Biochem. 2016; 96: 74–81. Publisher Full Text\n\nFekete I, Kotroczó Z, Varga C, et al.: The effects of detritus inputs on soil organic matter content and carbon-dioxide emission in a Central European deciduous forest. Acta Silv Lign Hung. 2011; 7: 87–96. Reference Source\n\nFuentes JP, Bezdicek DF, Flury M, et al.: Microbial activity affected by lime in a long-term no-till soil. Soil Till Res. 2006; 88(1-2): 123–131. Publisher Full Text\n\nMoreira A, Malavolta E: Dynamic of organic matter and microbial biomass in a soil under different management systems in the Western Amazon. Pesq Agropec Bras. 2004; 39(11): 1103–1110. Reference Source\n\nWang WJ, Dalal RC, Moody PW, et al.: Relationships of soil respiration to microbial biomass, substrate availability and clay content. Soil Biol Biochem. 2003; 35(2): 273–284. Publisher Full Text\n\nOulbachir K, Dellal A, Bekki A: Les variations de la biomasse microbienne etudiée sous des environnementales différentes dans quatre sols superposés séparés deux à deux par une croûte calcaire. Eur J Sci Res. 2009; 36(3): 407–417.\n\nSimon L, Szilágyi M: Mikroelemek a táplálékláncban (Microelements in the food chain). Bessenyei György Kiadó, Nyíregyháza. 2003."
}
|
[
{
"id": "63917",
"date": "01 Jun 2020",
"name": "Lenin Ramirez-Cando",
"expertise": [],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors report changes in soil microbiological parameters and their ratio at different altitudinal levels of the Ecuadorian Amazon region. The study is well written and straightforward; anyways, in parts needed to be improved to increase readability, and lead readers to better understand the importance of the research findings. First, in the introduction, authors discuss the role of MBC and, then SOC in assessing soil microbiological activity, but what missing for me is the lack of explaining their ratio MBC/SOC as a microbial indicator. The importance of ratio is mentioned in the paper, but its role is unclear. Up to date and relevant literature is cited. Methods are well written, but missing some information. How many samples were measured for MBC and SOC, 15 (as sampled) in quadruplicated? It is not clear until we open the supporting material. Observation: generally in mobilization studies, apart from the altitudinal variant, sampling depths are also involved in the analysis. It is not clear to me why the authors disregarded different depths in the study that could provide a more complex image of examined changes. Results are presented logically, and succinctly. Conclusions are correct. Overall, it can be said that it is a well-designed study. In any case, I suggest making the following minor changes before article indexing:\n\nExpand introduction, and highlight the role of ratio MBC/SOC.\n\nState number of samples used for biomass analyses.\n\nPlease clarify why depth was not involved in the analysis.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? 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",
"responses": []
},
{
"id": "63948",
"date": "08 Jun 2020",
"name": "Istvan Fekete",
"expertise": [
"Reviewer Expertise Soil ecology",
"soil carbon content"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 very accurate latitude and altitude data are given for the location of the site, which belongs to one specific site; however, there are 3 different altitudes in the text, at which 3-3 sampling points per altitude were mentioned. In the Material and Method, it would have been good to include a site description section, in which the vegetation of the study area could have been described. Also describing what is grown in agricultural areas; how much biomass remains in the area; how long the natural vegetation has been removed (since when the area has been under cultivation). It would be good to know if there was a difference between the slope of each slope because this can also affect SOM and MBC through erosion. Soil moisture and temperature fundamentally affect the MBC of soils, so it would be good to mention the values for specific sites in this manuscript as well. Furthermore, it would be good to know the particle composition of the soil, as the rate of the clay fraction significantly influences the SOC content. The above-mentioned variables and parameters can all influence the final result.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/9-443
|
https://f1000research.com/articles/9-427/v1
|
22 May 20
|
{
"type": "Study Protocol",
"title": "The Benefits and Risks of Iron interventionS in Children (BRISC) trial: Statistical analysis plan",
"authors": [
"Sabine Braat",
"Leila Larson",
"Julie A. Simpson",
"Mohammed Imrul Hasan",
"Jena Derakhshani Hamadani",
"Sheikh Jamal Hossain",
"Shamima Shiraji",
"Mohammad Saiful Alam Bhuiyan",
"Beverley-Ann Biggs",
"Sant-Rayn Pasricha",
"Leila Larson",
"Julie A. Simpson",
"Mohammed Imrul Hasan",
"Jena Derakhshani Hamadani",
"Sheikh Jamal Hossain",
"Shamima Shiraji",
"Mohammad Saiful Alam Bhuiyan",
"Beverley-Ann Biggs",
"Sant-Rayn Pasricha"
],
"abstract": "Background: The Benefits and Risks of Iron interventionS in Children (BRISC) trial will evaluate the impact of universal supplementation with iron supplements or iron-containing multiple micronutrient powders (MNPs) compared with placebo given for 3 months on child development, growth, morbidity, laboratory indices of anaemia, iron deficiency, and inflammation at end of intervention and after a further 9 months post intervention in children aged 8 months living in rural Bangladesh. This paper describes the statistical analysis plan. Methods: BRISC is a multi-site, three-arm, double-dummy blinded, parallel group, randomised control superiority trial in 3300 children. The statistical analysis plan was developed by the trial statistician in consultation with the trial steering committee and trial management committee based on the protocol, data collection forms, and study outcomes available in the blinded study database.\n\nConclusion: This detailed statistical analysis plan published prior to unblinding the allocated treatments will support the statistical analyses and reporting of the BRISC trial to be undertaken after unblinding. It allows for transparency as well as reproducibility of statistical analyses and reporting. Registration: Australian New Zealand Clinical Trials Registry ACTRN12617000660381 (registered on 8 May 2017); World Health Organization Universal Trial Number U1111-1196-1125.",
"keywords": [
"Iron",
"randomised control trial",
"child development",
"cognition",
"Bayley Scales",
"anaemia",
"statistical analysis plan",
"Bangladesh"
],
"content": "Introduction\n\nThe World Health Organization (WHO) recommends daily iron supplementation to all children (universal provision) aged 6–23 months residing in settings where anaemia prevalence is 40% or above, or alternatively, home fortification with iron-containing multiple micronutrient powders where the prevalence of anaemia is 20% or above, with the goal of reducing anaemia and improving child development1,2. However, there is limited evidence for the effects of iron supplementation on early child development3,4; conversely, in high infection burden settings, iron may promote infection, including diarrhoea5.\n\nThe Benefits and Risks of Iron interventionS in Children (BRISC) trial is a placebo-controlled, randomised trial undertaken in rural Bangladesh designed to examine the effect of universal provision of iron syrup or iron-containing MNPs on child development, growth, morbidity from infections, and haematological and iron indices6. The trial recruited the first participant in July 2017 and completed follow-up of the last participant in February 2020. The final results of the trial are expected to be submitted for publication by late 2020.\n\nIn this paper, the planned analyses for the BRISC trial are described. This plan supersedes the plan provided in the registry and published protocol6. Finalisation of the statistical analysis plan prior to study unblinding has been undertaken to ensure transparency in the methods used to analyse and report the data and ultimately create the evidence for the effects of iron supplementation on early child development, growth, haemoglobin, iron status and infection.\n\n\nMethods\n\nThe trial protocol is summarised elsewhere6.\n\nThe primary objective of this study is to determine whether 3 months of iron supplementation or home fortification with MNPs is superior to placebo on cognitive development in 8-month old children at the end of the intervention. The secondary objectives are to evaluate the impact of iron supplementation and home fortification with MNPs, compared with placebo, on developmental indices, prevalence of anaemia and iron deficiency, growth, and infection risks at the end of the intervention and 9 months post-intervention.\n\nBRISC is a three-arm, blinded, double-dummy, parallel group, placebo-controlled, individually-randomised, superiority trial. Starting at 8 months of age, children were randomised to either Arm 1: iron syrup (12.5 mg elemental iron) + placebo MNPs (powder sachet); Arm 2: MNPs (including 12.5 mg elemental iron) + placebo syrup; or Arm 3: placebo syrup and placebo powder/sachet (control), for 3 months (Figure 1). Children were then followed up for an additional 9 months post-intervention. The study received ethics approval from the Ethical Review Committee of the International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b) and the Melbourne Health Human Research Ethics Committee (Melbourne, Australia). It was prospectively registered at the Australian New Zealand Clinical Trials Registry (ACTRN1261700066038) and the World Health Organization (WHO) International Clinical Trials Registry Platform (U1111-1196-1125).\n\nThe trial is conducted in Rupganj, a rural subdistrict/upazila of Narayanganj district about 50 km from Dhaka, in Bangladesh. Three unions (Bhulta, Golakandail, Rupganj) within the subdistrict are included, with each union covered by a dedicated field team.\n\nChildren eligible for enrolment were children who were 8 months of age ±14 days, were not expected to leave the study site for more than 1 week over the next 3 months or for more than one month over the next 12 months and had a legally acceptable representative capable of providing consent. Children were excluded if they had severe anaemia (haemoglobin <80 g/L), if their drinking (ground) water iron content was >1 mg/L, if their mid upper arm circumference <11.5 cm, if they had been previously diagnosed with inherited red cell disorders, or if they had a current infective illness with fever.\n\nChildren were randomly allocated to one of the three arms with 1:1:1 allocation using a computer-generated schedule of randomly permuted blocks of fixed size stratified by union and sex to achieve balance between the arms within each stratum. The randomisation list was computer-generated by an independent statistician. Blinding of the team visiting the site, the caregiver(s) and study participants was achieved through the use of identical packaging of sachets and syrup. Researchers, caregivers, persons involved with data collection (i.e., field team) or analysis will be blinded to the allocation code until the database has been cleaned and is ready for analysis.\n\nAll efficacy and laboratory outcomes were measured at baseline, 3 months post-intervention and after a further 9 months follow-up. Data related to infectious morbidity and hospitalisation were collected weekly during the intervention period and monthly during the follow-up period. Serious adverse events were measured at any time. The primary time-point of interest for all outcome variables is at the end of the intervention.\n\nThe primary outcome of the study is cognitive development, as measured using the cognitive composite score of the Bayley Scales of Infant and Toddler Development, Third Edition (Bayley III)7. Bayley-III is a validated index of child development and the preferred field assessment tool. It is a standard series of measurements primarily to assess cognitive, motor (fine and gross) and language (receptive and expressive) development of infants and toddlers aged 0–3 ½ yrs. The total number of credited items is converted into scaled scores based on child’s age, which are then converted to composite scores of each subscale.\n\nKey secondary outcomes include motor and language composite scores assessed by Bayley III, growth (length-for-age z-score, weight-for-age z-score), and laboratory indices (haemoglobin and ferritin concentrations). Secondary outcomes include other anthropometry measures (weight-for-length z-score, stunting, wasting, underweight, head circumference) and haematological and iron diagnoses (anaemia, iron deficiency, iron deficiency anaemia). Anthropometry outcomes (z-scores) will be derived using the child’s length and weight together with age and sex of the child according to age and sex specific WHO international reference growth standards8. Using the z-scores, stunting will be defined as length-for-age z-score <-2, underweight as weight-for-age z-score <-2, and wasting as weight-for-length z-score <-2. Using the child’s haemoglobin (g/L), ferritin (µg/L), and C-reactive protein (mg/L), anaemia will be defined as haemoglobin <110 g/L, iron deficient as ferritin <12 µg/L or ferritin <30 µg/L if C-reactive protein >5 mg/L, and iron deficient anaemia as iron deficient and anaemia.\n\nExploratory outcomes include child’s behaviour using items from the Wolke’s Behaviour rating scale9, consisting of nine behaviours each scored on a nine-point scale with higher scores indicating more favourable behaviour, and the temperament questionnaire10, consisting of 33 questions each scored on a four-point scale with higher scores indicating a better temperament which will be grouped by summing into seven temperament summary scores.\n\nSafety outcomes include infectious morbidity (includes fever, diarrhoea, bloody stool, vomiting, cough/ difficulty breathing), (serious) adverse events, and C-reactive protein (an inflammatory biomarker) and inflammation defined as C-reactive protein >5 mg/L.\n\nAdditional data collected included household baseline characteristics (union, religion, number of household members, parity, number of children under five years of age living in the household, maternal and paternal education, maternal and paternal occupation, wealth index11, maternal depression12,13, household food insecurity14) and child baseline characteristics (sex, age, currently breastfed, home stimulation as measured using the family care indicator score15). Daily study medication intake was collected weekly during the 3-month intervention period and included reasons for non-adherence to the allocated treatment regimen.\n\nThe sample size for the trial was to recruit 3300 children (1100 per treatment arm) in order to have 80% power to detect a two-point difference in Bayley III composite cognitive score between the iron supplementation and placebo arm and the MNPs and placebo arm (two-sided 2.5% level of significance per comparison), assuming a standard deviation of 15 and a 20% loss to follow up after 3 months of intervention. No interim analyses to stop the trial early were planned, and no interim analysis was conducted.\n\nThe analysis will be conducted by statisticians from the University of Melbourne. After all study data are available and clean, a blinded data review meeting to review protocol violations, overall compliance, and missing data will be held prior to database lock. The final statistical analysis plan will be signed off during this meeting. The analysis of the primary outcome will be checked by an independent statistician. Discrepancies will be discussed and resolved by consensus.\n\nThe intention-to-treat population will be used for the analysis of all primary, key secondary, secondary, and exploratory outcomes and will include all children who were randomised. In case of missing outcome data, we will follow the missing data handling strategy outlined below. The safety population will be used for the analysis of all safety outcomes and consists of all children who received at least one study treatment (including control). Children who have withdrawn informed consent for use of all their data will be excluded from all analyses. Children will be reported and analysed according to their randomised treatment allocation. Time-windows will be applied to all visit-wise data collected at baseline, month 3, and month 12, with assessments outside the predefined visit windows excluded from the analyses. Outcomes will be summarised using frequencies and percentages (based on the non-missing sample size) for categorical variables, mean and standard deviation for continuous variables, or median and quartiles (25th and 75th percentile) for non-symmetrical continuous variables. All analyses models will be adjusted for the stratification variables used during the randomisation (union and sex). All confidence intervals and P-Values will be two-sided.\n\nThe flow of children through the trial will be presented in a Consolidated Standards of Reporting Trials (CONSORT) diagram, reasons for exclusion will be reported (Figure 2).\n\nMNPs denotes multiple micronutrient powders.\n\nDemographic and baseline variables of household and child characteristics will be summarised descriptively and presented by treatment group (Table 1). No formal comparisons between groups will be made.\n\nMNPs, multiple micronutrient powders.\n\nPercentages may not total 100 because of rounding.\n\n*Values are mean (SD).\n\n†Values are median (25th to 75th percentile).\n\nThe two primary comparisons of interest for the primary and key secondary outcomes are iron versus placebo and MNPs versus placebo. We will use a Bonferroni adjustment combined with a gatekeeping procedure to ensure control of the Type I error rate for the primary outcome across all three pairwise comparisons. Using the Bonferroni correction, we will test each of the two primary null hypotheses of no difference between iron and placebo and MNPs and placebo for the primary outcome at the two-sided 2.5% level of significance. If at least one of the two primary null hypotheses can be rejected (multiplicity unadjusted P-Value < 0.025), a comparison of iron versus MNPs will take place at either the two-sided 2.5% (if either iron or MNPs is superior to placebo) or 5% level of significance (if both iron and MNPs are superior to placebo). Estimates and two-sided confidence intervals will be presented with the same level of significance as used for the testing of these null hypotheses, along with multiplicity unadjusted and adjusted P-Values. If neither iron nor MNPs is superior to placebo, we will report the estimate and two-sided 95% confidence interval but not the P-Value for the comparison of iron versus MNPs.\n\nFor the set of key secondary outcomes, we will use the Hochberg procedure for each of the three pairwise comparisons at the level of significance used for the primary outcome only if the corresponding null hypothesis is rejected for the primary outcome. No multiplicity adjustment is planned for secondary and exploratory outcomes, and no P-Values will be presented.\n\nWe will follow the above outlined multiple testing approach for the primary and key secondary outcomes both for the primary time-point at month 3 and the secondary time-point at month 12 separately. The primary objective of the trial will have been met if one of the two primary null hypotheses for the primary outcome at the primary time-point can be rejected at the predetermined significance level.\n\nA constrained longitudinal data analysis method proposed by Liang and Zeger16 will be used to examine the primary outcome (Bayley III cognitive composite score at baseline, 3 months and 12 months post-baseline). The model will incorporate study visit as a categorical variable, treatment and treatment by study visit interaction and adjust for the stratification randomisation factors (union and sex) as main effects. The model will assume a common baseline mean across the three treatment arms and an unstructured variance-covariance among the repeated measurements. In case of non-convergence, we will consider alternative structures (first-order autoregressive, Toeplitz, compound symmetry). The treatment effect will be estimated from this model as the difference between two treatments in mean change from baseline to 3 months post-intervention (Table 2).\n\nMNPs, multiple micronutrient powders; CI, confidence interval\n\n*Values are mean (SD) or no. (%), with the exception of ferritin which is median (25th to 75th percentile).\n\n†Multiplicity-adjusted according to the pre-specified multiple testing strategy.\n\nContinuous key secondary, secondary, and exploratory outcomes will be analysed similarly as the primary outcome. Ferritin (µg/L) will be loge transformed before analysis. Binary outcomes will be analysed using a generalised linear mixed model with a log-link function and binomial distribution, including child as a random intercept. In case of non-convergence, we will use a logit link function instead.\n\nThe total number of times at least one infection (fever, diarrhoea, bloody stool, vomiting, cough/ difficulty breathing) was reported will be summarised per infection type during the intervention period (weekly reports), extended follow-up period (monthly reports), and (total) study period. The incidence rate ratio will be estimated using a Poisson regression model, with a logarithm of the time at risk as offset (Table 3).\n\nMNPs, multiple micronutrient powders; CI, confidence interval\n\nIntervention period – 0–3 months, Extended follow-up period 4–12 months, Study period 0–12 months.\n\n*Values are median (25th to 75th percentile).\n\n†Ratio is incidence rate ratio.\n\nThe number and percentage of children who died, had at least one (overnight) hospitalisation, reported at least one serious adverse event, and had at least one clinic visit (due to any infection, fever, diarrhoea, bloody stool, vomiting, cough/ difficulty breathing, other infection) during the intervention period, extended follow-up period, and study period will be reported and compared between treatments using a log-binomial regression model (Table 4).\n\nMNPs, multiple micronutrient powders; SAE, serious adverse event.\n\nIntervention period – 0–3 months, Extended follow-up period 4–12 months, Study period 0–12 months.\n\n*Hospitalisation is defined as an overnight stay.\n\n†Clinic visit is defined as visit to the clinic not resulting in hospitalisation.\n\n‡Other is defined as stool with mucous, runny nose, skin problem, eye problem, oral problem, ear problem, constipation, check up, other.\n\nC-reactive protein levels will be analysed using similar models as those described for the primary outcome and for inflammation similar models to those described for the binary key secondary and secondary outcomes.\n\nWe will present the multiplicity unadjusted P-Values for the safety outcomes, no multiple testing adjustment is planned.\n\nTo describe the missing data, the frequency and percentage of children with missing data at baseline, month 3 and month 12 will be summarised for the child development, anthropometry, and laboratory outcomes. In addition, baseline and demographic characteristics will be summarized by those with and without missing data for the cognitive composite score (at baseline, month 3, and month 12) to explore the missing data assumption and identify any variables not included in the target analyses that are potentially associated with missingness (known as auxiliary variables).\n\nAs the primary strategy to handle missing data, the analysis will use a likelihood-based approach. This approach relies on the underlying assumption that the probability of missing outcome data is not related to the missing data but to some of the observed measured data in the model (Missing At Random [MAR]).\n\nAs the secondary strategy (sensitivity analysis), missing data on the outcomes will be multiply imputed using chained equations. The imputation model will include union, sex, visit (categorical), the family care indicator total score (continuous), maternal education (continuous), all variables listed as specified for subgroup analyses, and it will be performed separately by treatment group. In addition, auxiliary variables identified during the blinded data review meeting may be included. The variables with missing data will be imputed using a linear regression model if continuous and logistic regression if binary, whereby ferritin will be loge transformed prior to imputation as this outcome will be loge transformed in the analysis of interest. The missing outcome data at baseline, 3-month and 12-month visits will be imputed using the “just another variable” approach (also known as imputing in wide format) which requires a separate imputation model for imputing the variable at each assessment time. The number of imputed data sets will be greater than or equal to the percentage of missing data in the available case analyses. The imputed data sets will be analysed using the models described. The estimates from the analyses of the imputed data sets will be combined to obtain a pooled common estimate and corresponding confidence interval using Rubin’s rules. For the above standard implementation of multiple imputation, we have assumed the outcome data are MAR.\n\nOverall compliance across the 3-month intervention period will be derived as the total number of days the child has reported taking both the syrup and the sachet divided by the child’s study participation duration, with ‘complier’ defined as those with overall compliance ≥70%. If no data on treatment intake is available, compliance will be assumed to be 0%.\n\nIn addition to the analyses specified for the primary, key secondary, and secondary outcomes, the following sensitivity analyses will be applied for these outcomes:\n\n1. Analyses consisting of models adjusted for potential prognostic or predictive variables:\n\na. Adding to the model adjusted for union and sex, the main effect of family care indicator score (continuous) and maternal education (No education; 1–8 years schooling completed; 9–12 years schooling completed; >12 years schooling completed).\n\nb. Adding to the model adjusted for union and sex, the main effect of variables in Table 1 demonstrating unexpected imbalance between the treatment arms after unblinding.\n\nc. Adding to the model adjusted for union and sex, the main effect for rater and the interaction between rater and study visit (only applies to Bayley III, Wolke’s Behaviour rating scale, and temperament questionnaire).\n\nd. Adding to the model adjusted for union, sex, family care indicator, and maternal education, the main effect for rater and the interaction between rater and study visit (only applies to Bayley III, Wolke’s Behaviour rating scale, and temperament questionnaire).\n\n2. Analyses of the secondary strategy to handle missing data.\n\n3. Analyses of the model adjusted for union and sex for the per-protocol population defined as randomised children who were compliant to treatment, and without protocol violations (no informed consent or withdrawn informed consent for use of all data, violation in/ exclusion criteria, or improper unblinding of the child’s allocated treatment).\n\n4. Analyses using the Complier Average Causal Effect method to estimate the average effect of treatment among compliers17.\n\nExploratory subgroup analyses will be performed for cognitive motor and language composite scores assessed by Bayley III at 3 and 12 months. Subgroup (main effect) and the subgroup-by-treatment-by-visit interaction (as well as subgroup-by-treatment and subgroup-by-visit interaction) terms will be added to the constrained longitudinal data analysis model to evaluate whether the treatment effect differs between subgroup categories. The following subgroups will be explored: sex (male/female), baseline anaemia status (yes/no), baseline iron deficient status (yes/no), baseline iron deficient anaemia status (yes/no), baseline stunting (yes/no), baseline home stimulation as measured by the family care indicator questionnaire (below/ above median family care indicator total score), baseline household food insecurity status (yes/no), baseline wealth status (below/ above median wealth index score), and union (Bhulta, Golakandail, Rupganj). No multiplicity adjustments are planned for the subgroup analyses due to their explorative nature, we will present the estimates and two-sided 95% confidence interval along with (multiplicity unadjusted) P-Values. Results of the subgroup analyses will be displayed using Forest plots.\n\nThis paper includes changes to the statistical analysis plan of the BRISC trial in the registry (registered May 2017) and protocol paper (accepted September 2017). These changes are:\n\nThe register states that the regression models will incorporate key confounders and unbalanced baseline factors into the model. Instead, the primary analysis model will include the stratification factors used during the randomisation and additional models (sensitivity analyses) will incorporate key confounders, or key confounders and unbalanced baseline factors.\n\nThe protocol states on the topic of:\n\nMultiple testing: We detailed a multiple testing procedure for the primary and key secondary outcomes, thus the confidence level for the associated two-sided confidence interval will be less than 5% for some treatment effects.\n\nAnalysis of binary outcomes: We changed the analysis model from generalised estimating equations to a mixed model.\n\nThis paper documents version 1 of the statistical analysis plan dated April 20, 2020. Any changes to this version between publishing and breaking of the code will be tracked and still considered as planned analyses. The statistical analysis plan will be approved during the blinded data review before breaking of the allocation code. Any changes after this signed version will be considered post-hoc.\n\n\nDiscussion\n\nIron interventions in early childhood are recommended as an effective intervention to reduce the prevalence of anaemia and improve child development. However, robust evidence for the effects of iron on child development and, importantly, its possible risks is lacking. The BRISC trial will provide definitive evidence for the effects of universal provision of iron, in the form of iron syrup and iron-containing MNPs (the WHO recommendations) on child development, growth, anaemia, and morbidity both immediate and medium term. If effective, it may also establish whether iron supplements or MNPs are better. The results of this rigorous randomised controlled trial will influence global policy guidelines and programmatic practices around universal iron interventions in infants and young children.\n\n\nData availability\n\nNo data are associated with this article.",
"appendix": "Acknowledgements\n\nicddr,b acknowledges with gratitude the commitment of the NHMRC to its research efforts. icddr,b is also grateful to the Governments of Bangladesh, Canada, Sweden and the UK for providing core/unrestricted support. We acknowledge ACME Laboratories Ltd, Bangladesh for manufacturing Iron syrup and the corresponding placebo; and Renata Ltd, Bangladesh for manufacturing the Micronutrient powders and corresponding placebo.\n\n\nReferences\n\nWorld Health Organization: Guideline: daily iron supplementation in infants and children. Geneva: World Health Organization. 2016. Accessed Jan 2018. Reference Source\n\nWorld Health Organization: WHO guideline: Use of multiple micronutrient powders for point-of-use fortication of foods consumed by infants and young children aged 6– 23 months and children aged 2– 12 years. Geneva: World Health Organization, 2016. Reference Source\n\nPasricha SR, Hayes E, Kalumba K, et al.: Effect of daily iron supplementation on health in children aged 4— 23 months: a systematic review and meta-analysis of randomised controlled trials. Lancet Glob Health. 2013; 1(2): e77–e86. PubMed Abstract | Publisher Full Text\n\nLarson LM, Phiri KS, Pasricha SR: Iron and Cognitive Development: What Is the Evidence? Ann Nutr Metab. 2017; 71(Suppl 3): 25–38. PubMed Abstract | Publisher Full Text\n\nSoofi S, Cousens S, Iqbal SP, et al.: Effect of provision of daily zinc and iron with several micronutrients on growth and morbidity among young children in Pakistan: a cluster-randomised trial. Lancet. 2013 ; 382(9886): 29–40. PubMed Abstract | Publisher Full Text\n\nHasan MI, Hossain SJ, Braat S, et al.: Benefits and risks of Iron interventions in children (BRISC): protocol for a three-arm parallel-group randomised controlled field trial in Bangladesh. BMJ open. 2017; 7(11): e018325. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBayley N: Bayley Scales of Infant Development Manual. Third ed. Antonio,TX: The Psychological Corporation. 2006. Reference Source\n\nWorld Health Organization: WHO child growth standards: length/height-for-age, weight-for-age, weight-for-length, weight-for-height and body mass index-for-age: methods and development. 2006. Reference Source\n\nWolke D, Skuse D, Mathisen B: Behavioral style in failure-to-thrive infants: a preliminary communication. J Pediatr Psychol. 1990; 15(2): 237–54. PubMed Abstract | Publisher Full Text\n\nBaker-Henningham H, Hamadani JD, Huda SN, et al.: Undernourished children have different temperaments than better-nourished children in rural Bangladesh. J Nutr. 2009; 139(9): 1765–71. PubMed Abstract | Publisher Full Text\n\nSchellenberg JA, Victora CG, Mushi A, et al.: Inequities among the very poor: health care for children in rural southern Tanzania. Lancet. 2003; 361(9357): 561–66. PubMed Abstract | Publisher Full Text\n\nRadloff LS: The CES-D scale a self-report depression scale for research in the general population. Applied psychological measurement. 1977; 1: 385–401. Publisher Full Text\n\nWolf AW, De Andraca I, Lozoff B: Maternal depression in three Latin American samples. Soc Psychiatry Psychiatr Epidemiol. 2002; 37(4): 169–76. PubMed Abstract | Publisher Full Text\n\nCoates J, Swindale A, Bilinsky P: Household Food Insecurity Access Scale (HFIAS) for measurement of food access: indicator guide: version 3. 2007. Reference Source\n\nHamadani JD, Tofail F, Hilaly A, et al.: Use of family care indicators and their relationship with child development in Bangladesh. J Health Popul Nutr. 2010; 28(1): 23–33. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLiang KY, Zeger SL: Longitudinal data analysis of continuous and discrete responses for pre-post designs. Sankhyā: The Indian Journal of Statistics, Series B. 2000; 62(1): 134–48. Reference Source\n\nWhite IR: Uses and limitations of randomization-based efficacy estimators. Stat Methods Med Res. 2005; 14(4): 327–47. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "65657",
"date": "10 Jul 2020",
"name": "Deborah Ridout",
"expertise": [
"Reviewer Expertise Statistician"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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-designed, large randomised trial investigating the use of iron supplementation or home fortification with multiple micronutrient powders, on cognitive development outcome in 8-month old children. The study is set in Bangaldesh and will be conducted over 3 sites. 3,300 children will be recruited and randomised to one of 3 arms, one of which is a placebo. The study is blinded, so that both family and assessor is unaware of group allocation. The study is designed to assess superiority. Secondary outcomes will assess motor and language ability on the Bayley scale as well as prevalence of anaemia and iron deficiency, growth, and infection risks.\nThe investigators plan to stratify the randomisation by union and sex, which seems sensible and I wonder if they should also consider weight for age as a stratifying factor.\nAn area that may need clarification is around the superiority aspect of the trial, this is mentioned several times, although it is not clear whether the power calculation and planned analyses are considering this design feature.\nThere is a possibility that a comparison will be made between the 2 treatments groups, groups 1 & 2, and the plan for this is described well. It is not so clear how these results will be interpreted, is the same effect size of 2 units on the Cognitive Bayley scale appropriate here?\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable",
"responses": [
{
"c_id": "5881",
"date": "02 Sep 2020",
"name": "Sabine Braat",
"role": "Author Response",
"response": "Thank you for the opportunity to respond to the comments of reviewer #1. We appreciate the review provided and have addressed each point below. This is a well-designed, large randomised trial investigating the use of iron supplementation or home fortification with multiple micronutrient powders, on cognitive development outcome in 8-month old children. The study is set in Bangaldesh and will be conducted over 3 sites. 3,300 children will be recruited and randomised to one of 3 arms, one of which is a placebo. The study is blinded, so that both family and assessor is unaware of group allocation. The study is designed to assess superiority. Secondary outcomes will assess motor and language ability on the Bayley scale as well as prevalence of anaemia and iron deficiency, growth, and infection risks. Response: We thank the reviewer for the feedback. 1. The investigators plan to stratify the randomisation by union and sex, which seems sensible and I wonder if they should also consider weight for age as a stratifying factor. Response: We thank the reviewer for this suggestion. The trial is no longer recruiting and finished in 2020. In the “Sensitivity analyses” section we have pre-specified an analysis model that may adjust for characteristics that are unbalanced between treatment arms. This may include weight if it is unbalanced. 2. An area that may need clarification is around the superiority aspect of the trial, this is mentioned several times, although it is not clear whether the power calculation and planned analyses are considering this design feature. Response: We thank the reviewer for the comment. In the “Aims” section we refer to the study as a superiority study with the primary objective “...to determine whether 3 months of iron supplementation or home fortification with MNPs is superior to placebo on cognitive development ...” Both the sample size and the analyses of cognitive development are based on a superiority hypothesis. 3. There is a possibility that a comparison will be made between the 2 treatments groups, groups 1 & 2, and the plan for this is described well. It is not so clear how these results will be interpreted, is the same effect size of 2 units on the Cognitive Bayley scale appropriate here? Response: We thank the reviewer for this question. The sample size calculation assumed the same effect size for both active treatment groups versus placebo, with 2 units representing a clinically meaningful effect size. The two primary comparisons consist of each active dose vs placebo. When interpreting the results for both active doses, we will take into consideration the findings of the primary outcome (cognitive Bayley scale), secondary outcomes, and safety outcomes."
}
]
},
{
"id": "119673",
"date": "05 Apr 2022",
"name": "Sonia Lewycka",
"expertise": [
"Reviewer Expertise Epidemiology - design and evaluation of public health interventions"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 comprehensive and well thought through statistical analysis plan. The main issue that needs clarification is around the timeline. It seems like data has already been collected and possibly analysed. If so, it needs to be made clear that this statistical analysis plan is being published post hoc. I have some specific comments below:\nP1 – introduction – The concern about iron supplementation and infection risk could be elaborated further, though perhaps this is done in the study protocol. Do you mean that iron supplementation might have adverse side-effects? I would say \"furthermore\" rather than \"conversely\", and be more specific that there may be adverse effects.\n\nP1 – Introduction – I’m not clear on the timeline. In the introduction you say that results are expected to be submitted for publication in late 2020. Have they been submitted yet?\n\nP1 – methods – Although it’s too late to address it at this stage, I did wonder why the study team chose 3-months as the supplementation period. This doesn't seem long enough to have a measurable effect on development and growth outcomes. Why not follow WHO recommendation of 6-23 months? Why instead follow up for 9-months without supplementation? Kids may become anaemic again by this time and have lost out on any earlier benefits.\n\nP4 – Outcome variables – In the section on additional variables, is there any measure of other dietary intake? Particularly foods rich in vitamin C, which aid iron absorption.\n\nP5 – general principles – What do you mean by visit windows? It would also be useful to adjust for baseline measures of outcomes to remove any imbalances due to imperfect randomisation and to increase precision.\n\nP8 – multiple testing adjustment – Why no p-values for secondary and exploratory outcomes? I don't think this is necessary as long as you have stated that these are secondary/exploratory outcomes, not adjusted for multiplicity, and should be interpreted with caution. A p-value of <0.001 would be interpreted differently than one of 0.04 even if it is an exploratory analysis. This information is useful for generating hypotheses for testing in future research, even if it can’t provide robust evidence in this study.\n\nP8 – efficacy – Three points: 1) As above, why not adjust for baseline as a covariate rather than assuming a common mean baseline? Or even calculate the difference from baseline to 3 months for each individual and use this as the outcome measure in the regression model? Can then also still be adjusted for baseline.\n2) I don't understand how child can be a random intercept when outcomes at each time-point are included in separate models, so there is only one measurement for each child in each model. If I have misunderstood, this needs to be made clearer.\n3) It would be better to use the same link function for all models, rather than comparing models with different link functions. Will you use GLM for continuous outcomes? Please state this and what link function will be used, since most of your outcomes are continuous.\n\nP14 – sensitivity analyses 1b – Why are adjusted models considered as sensitivity analyses? If there is imbalance at baseline and the variables are associated with outcomes, then these could potentially confound the associations, and you would want to include them in your main models. On p15 it states that this was how it was in the original protocol, so it’s not clear why it was changed. There is no need to unblind to see if there is imbalance between arms.\n\nP15 – changes from the registry and published study protocol – Has the blinded data review been done yet? Is this version of the statistical analysis plan being published before or after the analyses have been done? If after, then why not publish the final approved version?\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/9-427
|
https://f1000research.com/articles/9-426/v1
|
22 May 20
|
{
"type": "Opinion Article",
"title": "Internet-mediated research in the wake of Covid-19: Dealing with disclosure of serious risk of harm",
"authors": [
"Natalie Edelman"
],
"abstract": "In response to the Covid-19 pandemic many studies are using internet-mediated research (IMR) methods. However, IMR guidance omits the issue of disclosure of serious risk of harm to self or others (DSRHSO). Studies involving face-to-face data collection on sensitive topics or with participants experiencing high adversity, often stipulate that participant confidentiality be broken in the face of DSRHSO. In IMR, participation may have been anonymous, either as part of a research strategy or because participants have chosen a pseudonym. Lack of valid contact details and other identifiers can also make tracing participants—to invoke safeguarding procedures such as social services involvement—more difficult. The need for guidance is also pressing as the pandemic impacts emotional and psychological well-being, so that DSRHSO may be more likely, even when researching non-sensitive topics. Those suffering abuse or severe mental illness may also take the opportunity afforded by research participation to DSRHSO in order to access help, but may still need guidance or prompts to provide contact details. Lack of existing guidance likely reflects that studies on sensitive topics are recommended for face-to-face data collection precisely because researchers have low control over risks in online settings. Prioritising participant safety (by requiring participant identifiers so that confidentiality can be broken if needed) may reduce data validity and participation by the most vulnerable. Nonetheless, the current pandemic presents both an opportunity and a need to establish IMR guidance on handling DSRHSO. In the post-pandemic era, such guidance may also facilitate safe online participation by those who are unable to participate in face-to-face data collection for reasons such as domestic abuse, care and work responsibilities or stigmatisation fears. Planned production of DSRHSO guidance will take an open, transparent and inter-disciplinary approach.",
"keywords": [
"Internet-mediated",
"research",
"risk of harm",
"disclosure",
"confidentiality",
"ethics"
],
"content": "\n\nAmong the many debates sparked by the Covid-19 pandemic is how best to ensure appropriate bio-ethical guidance is in place to inform medical and public health interventions and the research which underpins and guides them. The research community has been galvanised by expedited funding, approval and publication processes, and innovations such as crowd-sourced approaches to international collaboration1 and living evidence reviews2. Alongside these new ventures, many existing and newly-designed studies are moving to internet-mediated research (IMR) – defined by the British Psychological Society as ‘any research involving the remote acquisition of data from or about human participants using the internet and its associated technologies’.\n\nExisting guidance on IMR from bodies such as the British Sociological Association (BSA)3, the British Psychological Society (BPS)4, the Association of Internet Researchers (AoIR)5 and the British Society of Criminology (BSC)6 have been complemented by newer and more broad-reaching guidance such as the crowd-sourced ‘Doing Fieldwork in a Pandemic’7. However, a review of these documents suggests a lack of existing guidance on handling participant disclosure of serious risk of harm to self or others (DSRHSO) during IMR, although AoIR and BSC do acknowledge that researchers may have legal or professional obligations to report such disclosures5. The business of IMR is still often ‘uncharted territory’3 and the timely production of such guidance is vital as the pandemic not only necessitates online methods, but also creates and exacerbates vulnerabilities.\n\nFor studies involving face-to-face data collection, it is often stipulated that if a participant discloses serious risk of harm to themselves or others, the confidentiality usually afforded in the research environment should be broken in order to address those safety concerns. This action is most commonly specified for studies that will predictably touch on very sensitive topics and/or studies involving populations who have experienced significant adversity or trauma. This caveat is written into the participant information sheet along with a brief account of how the researcher will respond to such a disclosure, e.g. who they will notify and when, and who else might be involved in escalating the issue. Though always difficult, the hope is that in the context of a face-to-face interview conducted in an organisational setting any revelation could be handled sensitively.\n\nIn the case of IMR, such participant disclosures may occur in very different contexts. Participation may have been anonymous, either as part of a deliberate research strategy to improve response validity, or because participants have chosen to provide a pseudonym. Even when a bona-fide name is provided, further action may be difficult in the absence of valid contact details and other identifiers such as date of birth. This can mean that tracing participants—in order to invoke safeguarding procedures such as informing police, social services or other agencies—may be much more difficult, particularly as online studies may more easily draw participants from across the world4. Certainly the ‘act’ of participation may inadvertently cause harm, with certain topics awakening distress and other strong emotions, and/or bringing to the fore issues concerning risk of harm to self or others. In particular, disclosures may occur more commonly when gathering qualitative data, particularly interviews, and to a lesser degree, other forms of open-ended responses such as focus groups, diaries or open-ended questionnaire items.\n\nOf particular relevance at the moment is the emotional and psychological cost of the Covid-19 pandemic. Reports of domestic abuse and mental illness have spiralled8,9, such that we might anticipate more frequent DSRHSO even when researching seemingly innocuous topics. Furthermore, victims of abuse or those suffering severe mental illness may take the opportunity afforded by research participation to disclose serious risk of harm in order to access help, but may still need guidance or prompts to provide contact details.\n\nThe lack of existing guidance on this issue is perhaps indicative of the small proportion of studies to which it was previously thought to be relevant; before the Covid-19 pandemic, studies on sensitive topics were recommended for face-to-face data collection precisely because researchers have low control over risks in online settings4. Conducting such studies in online settings presents significant procedural difficulties which may be difficult to surmount in order that ethical and methodological concerns can be adequately balanced. Specifically, the prioritisation of participant safety (by requiring participant identifiers and contact details so that confidentiality can be broken if needed) may reduce the validity of the data they provide, and prevent the inclusion of disenfranchised voices—such that the most vulnerable in society may in fact elect not to participate at all.\n\nNonetheless, the current pandemic presents an opportunity to establish some guidance on handling DSRHSO in the context of IMR—both on sensitive topics, and on topics previously considered less sensitive but conducted with people more likely to be experiencing extreme adversity due to the pandemic. This would be to the benefit of not only participants’ wellbeing but also researchers, who might otherwise experience the distress of being unable to address serious safety concerns following a disclosure. Although the moral ‘context specificity’5 is unique to each study, warranting a situational ethics approach3, guidance on this specific issue is warranted, particularly as it intersects with the core concerns of IMR—i.e. confidentiality and informed consent3. Going forward into the post-pandemic era, such guidance may also facilitate safe online participation by those who are unable to participate in face-to-face data collection for reasons such as domestic abuse, informal care and work responsibilities or fear of stigmatisation.\n\nThe nature of guidance itself, how it is created and adopted and by whom, has itself become the subject of scrutiny during the pandemic. The production of guidance on this issue is therefore planned as an open and transparent process, an inter-disciplinary endeavour, drawing on those with lived experience of participating in research as well as those expert in conducting IMR and in researching disenfranchised populations, stigmatised conditions and sensitive topics.\n\n\nData availability\n\nNo data are associated with this article.",
"appendix": "References\n\nCovid-19; C: Crowdfight Covid-19. 2020. [Website dedicated to bringing together scientific expertise from across the globe to tackle Covid-19 research]. Reference Source\n\nThombs BD, Bonardi O, Rice DB, et al.: Curating evidence on mental health during COVID-19: A living systematic review. J Psychosom Res. 2020; 110113. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDavies H, Halford S, Hine C, et al.: Ethics Guidelines and Collated Resources for Digital Research: Statement of Ethical Practice Annexe. Durham, UK: British Sociological Association. 2017. Reference Source\n\nThe British Psychological Society: Ethics guidelines for internet-mediated research. INF206/04.2017. Leicester, Uk: British Psychological Society; 2017. Reference Source\n\nFranzke AS, Bechmann A, Ess CM, et al.: Internet Research: Ethical Guidelines 3.0. 2020. Reference Source\n\nCriminology; BSo: British Society of Criminology Statement of Ethics. London, UK: British Society of Criminology; 2015. Reference Source\n\nLupton D: Doing fieldwork in a pandemic. 2020. Reference Source\n\nMazza M, Marano G, Lai C, et al.: Danger in danger: Interpersonal violence during COVID-19 quarantine. Psychiatry Res. 2020; 289: 113046. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRajkumar RP: COVID-19 and mental health: A review of the existing literature. Asian J Psychiatr. 2020; 52: 102066. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "144998",
"date": "23 Aug 2022",
"name": "David Rothschild",
"expertise": [
"Reviewer Expertise Most relevantly",
"I have written extensively on online panels and platforms",
"and data quality for behavioral economics and finance studies."
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper focuses on the cost and benefits of a rapid shift from face-to-face to internet-mediated research due to COVID for sensitive topics and at-risk subjects. The specific focus is on the potential for serious risk of harm to self or others, and how to provide the right guidance and support, while ensuring the value of research. The author includes three key milestones in the research process (1) what should be disclosed up-front that gives the subjects option of continuing (2) potential harm of the study itself to the subject (3) potential of what could be learned about subject that may necessitate follow-up.\nThe abstract is like a jigsaw puzzle because the author keeps going back and forth on various aspects, costs and benefits without making it clear what things they are optimizing for and which aspects are good or bad for each of these goals (I am inferring the three milestones from several reads of the abstract and paper).\nI trust the author that various British academic societies have not updated their guidance to explicitly ask for disclosure of serious risk in internet research, but most academics are most concerned with their IRB board, which will certainly require a clear disclosure (at least, I can confirm that for myself and all of my recent colleagues). It is important for the author to tie in the IRB as key guidance for subject research both face-to-face and on the internet.\nIn a world in which a subject is told about potential risks in person or online: are they not in a better position to drop-out and protect themselves in an online environment versus in person? This would be bad for the sample, but better for subjects? The author frames this completely from the face-to-face side: I get the benefits of stating something versus reading it, I get the benefits of reading the subject and saying things with sensitivity, but the author needs to be way more specific of various costs and benefits to these two different modes of research on this outcome.\nConditional on doing the study, the author is concerned not about the impact of the study, but what may be learned requiring follow-up (e.g., determining from results of the study that the subject may be contemplating self-harm). In that case, I agree that if this is a possibility from a study, the ability to securely re-identify could be helpful, if there is an agreed upon intervention, which is hard. This is not as simple is requiring the possibility of re-identification, this is about what is disclosed to the subject and how re-contact could plausibly be done. This should be an open discussion in the field, not something where there is obvious guidance to add.\nFace-to-face studies have less nuance than internet (which can run the gambit of various methods of targeting, onboarding, interfacing, and offboarding subjects. The paper should tap into fact that internet, if necessary could mimic face-to-face in almost every aspect (it could be video call).\nI think this is great topic for discussion, but the writing is confusing, biased towards face-to-face, and too focused on adding guidance versus opening up a debate on what guidance could entail.\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": []
}
] | 1
|
https://f1000research.com/articles/9-426
|
https://f1000research.com/articles/9-424/v1
|
22 May 20
|
{
"type": "Brief Report",
"title": "Sequence variation at 8q24.21 and risk of back pain",
"authors": [
"Frances M.K. Williams",
"Yakov A. Tsepilov",
"Maxim B. Freidin",
"Tatiana I. Shashkova",
"Pradeep Suri",
"Yurii S. Aulchenko",
"Yakov A. Tsepilov",
"Maxim B. Freidin",
"Tatiana I. Shashkova",
"Pradeep Suri"
],
"abstract": "Back pain (BP) is a common condition of major social importance and poorly understood pathogenesis. Intervertebral lumbar disc degeneration in all its guises is one of the major biological risk factors for BP. Previously, we identified the locus at 8q24.21 associated with chronic BP, which has been found elsewhere associated with sciatica after surgery for lumbar disc herniation. In the current study we used co-localisation methods to identify the gene most likely to harbor the causal variant. We show that the same functional variant at the 8q24.21 locus is responsible for both lumbar disc degeneration and BP, and we also studied the effects of this locus on related phenotypes. Our results link the locus to intervertebral disc and bone mineral density, but not to anthropometric measurements, thus corroborating the epidemiological evidence. Moreover, the same functional variant at the locus is more likely to affect the expression of the nearby FAM49B gene, rather than the GSDMC gene, which was previously proposed as a causative one for BP.",
"keywords": [
"lumbar disk degeneration",
"back pain",
"transcriptomics",
"genome wide association study"
],
"content": "Introduction\n\nBack pain (BP) is a common debilitating condition with a lifetime prevalence of 40% and a major socioeconomic impact1. According to the Global Burden of Disease 2016 study, it leads the list of disabling conditions in many parts of the world2. The greatest biological risk for episodes of severe BP is thought to be lumbar disc degeneration (LDD)3. Rather than a discrete disease, LDD is now considered a continuous lifelong aging process that usually starts in the third decade of life but may also be seen in children4. It is highly heritable5, as well as being influenced by smoking6, age7 and body mass index8. The overall burden of lumbar disc degeneration on magnetic resonance imaging is a strong predictor of BP episodes9–11. There is a clear genetic predisposition to both BP and LDD with estimates of heritability in the range of 30%–70%12–15. Also, these traits have been shown to share genetic risk factors16. However, little is known about the genetic architecture of BP and only three associated genetic loci have been identified17,18.\n\nThis work was focused on the locus at chromosome 8 (8q24.21), which was identified in our previous BP studies17,18. The same locus has been reported associated with having lumbar disc herniation (LDH) in Icelanders undergoing surgery for sciatica19. Bjornsdottir et al. additionally reported an association of this region to height, but concluded that the region does not influence LDH through height. They noted that the most highly associated SNP, rs6651255, was linked to expression of the nearby gasdermin (GSDMC) gene in GTEx data20. Further, the immune-related function of gasdermins implied that the inflammatory and immunological consequences of disk herniation may be linked to sciatica symptom severity.\n\nHowever, closely located SNPs associated with different traits do not always reflect pleiotropy21. Furthermore, in the case of eQTL studies, causal hypotheses generated through single-variant look up are highly likely to be false positives21. Consequently, a variety of co-localization methods have been introduced22–25 to distinguish the two common scenarios: pleiotropy (the same functional genetic variant is associated with both traits) and linkage (two different functional genetic variants, each associated with a different trait, that are in linkage disequilibrium).\n\nThe aim of this study was to (1) verify whether the same functional variant at 8q24.21 is responsible for BP and LDH and (2) to study the effects of this variation on related phenotypes. As a result, we offer an alternative explanation to the assertion by Bjornsdottir et al.17 regarding the role of immunity and inflammation in generating symptoms of sciatica and BP due to variation in expression at the locus in between genes CCDC26 and GSDMC.\n\n\nMethods\n\nWe used the BP GWAS performed in all genetically confirmed white British UK Biobank participants (N= 453,862) as target data for future analysis17. We used the summary level-data of LDH GWAS from the original paper of deCODE group19. They do not provide full GWAS scan, only the statistics for the significant SNPs from the 8q24.21 region. We used information about 40 SNPs from this locus for which information about effect size, effective and reference alleles and p-value was available (Supplementary Table 1,3 of the work19).\n\nWe derived summary statistic of 2,419 complex traits provided by the Neale Lab and 34 traits from the Gene ATLAS database. Summary statistics for gene expression levels were obtained from Westra Blood eQTL26 (peripheral blood, http://cnsgenomics.com/software/smr/#eQTLsummarydata, file: westra_eqtl_data_hg19.zip (hg19)) and the GTEx database27 (14 tissues28, https://www.gtexportal.org/home/datasets, file: GTEx_Analysis_V6_all-snp-gene-associations.tar).\n\nSummary-level mendelian randomization and heterogeneity in dependent instruments (SMR-HEIDI)24 approach was used to test for potential pleiotropic effects of the 8q24.21 region on back pain (BP) and LDH and other complex traits including gene expression levels in certain tissues. SMR analysis provides evidence for pleiotropy, but is unable to define whether both traits are affected by the same underlying causal polymorphism. The latter is specified by a HEIDI test that distinguishes pleiotropy from linkage. Zhang F. and colleagues developed the software SMR tool implementing the SMR-HEIDI methods24.\n\nSMR tool is suitable to analyze pleiotropic association using summary-level data from GWAS and eQTL studies. We conducted two groups of analysis: target trait vs eQTL and target trait vs other complex traits. Summary-level data from BP GWAS17 was used as target (--gwas-summary option) in both cases. For eQTL analysis we used Westra Blood eQTL data (converted to suitable BSED format by developers) and GTEx v7, converted using --make-besd option (smr --eqtl-flist Gtex_tissue_1.txt --make-besd --out Gtex_tissue_1, where Gtex_tissue_1.txt file correspond to summary-level data of selected tissue). In order to apply SMR tool for complex traits we selected the region +-250kb around the target SNP from each complex trait GWAS. After that, we add new columns Probe_ID with trait_name to corresponded SNP subset. Finally, we merge these SNP subsets into one file and converted it in BESD format using --make-besd option.\n\nIn both cases we set the minor allele frequency threshold as 0.03 and included in the HEIDI test SNPs inside +-250kb window centered around the target SNP; other parameters used default settings. The LD matrix for the HEIDI test was estimated using PLINK 1.9 software and individual data of white UK Biobank participants, the subset of individuals used to calculate BP GWAS (N=10,000).\n\nNominal P for SMR test was set at 6e-4 (0.05/89, where 89 is the total number of probes for the gene expression analysis) and 2e-5 (0.05/2,453, where 2,453 is the total number of complex traits used in analysis). For HEIDI analysis we used a conservative threshold of P = 0.05 (P < 0.05 corresponds to the rejection of pleiotropy hypothesis).\n\n\nResults and discussion\n\nWe had previously performed the largest-to-date GWAS for chronic BP using the UK Biobank and CHARGE consortium cohorts, with total of 119384 cases and 334478 controls17. Cases were defined as those having BP of duration >3-6 months; controls included those without back pain or with back pain of <3-6 months. We found variants at 8q41.21 locus that showed a genome-wide significant association (OR=1.05, p = 4.4 × 10-13, after genomic control correction)17. The location chr8:130,717,716 is tagged by intergenic variant rs10956487 which is in perfect LD with rs6651255 (r2=1 according to 1000 Genomes EUR samples), the lead SNP in the Icelandic study of LDH by Bjornsdottir et al.19. The strongest association signal for BP is situated near GSDMC gene; yet another protein-coding gene, FAM49B, is located within a 500 kbp window. The summary of the previously reported results and our findings for locus 8q24.21 are shown in Figure 1.\n\nThe BP-related traits and genes analyzed in our work are blue. The locus 8q24.21 is connected to associated traits (p-value < 5*10-8) by green arrows. Possible relations between traits, as inferred from previous studies, are marked by grey arrows. Pairwise pleiotropic effects tested by SMR-HEIDI are denoted by a red cross if the test failed, and by a blue check if the test passed. A new detected pleiotropic effect on expression of FAM49B and BP based on SMR-HEIDI results is denoted by the blue arrow. LDH, lumbar disk herniation; SRDP, self-reported disk problems; HBMD, heel bone mineral density t-score.\n\nWe used the SMR-HEIDI approach24 to test for potential pleiotropic effects of the 8q24.21 region (rs6651255) on BP and other complex traits including gene expression levels in relevant tissues. SMR analysis provides evidence for pleiotropy, but is unable to determine whether both traits are influenced by the same underlying causal polymorphism. The latter question was addressed by the HEIDI test which distinguishes pleiotropy from linkage. For SMR-HEIDI testing we used as the target trait the BP GWAS previously performed in all genetically confirmed white British UK Biobank participants17.\n\nFirst of all, we checked relationships between BP and LDH. We found strong evidence that co-association of these traits to 8q24.21 is due to the pleiotropic action of the same functional variant (PSMR=2.8 × 10-7 and PHEIDI = 0.87; extended data29). We further applied SMR-HEIDI to test for pleiotropic effects of the region in the collection of 2,453 traits available from the UK biobank study and GeneAtlas. We found that the BP risk allele of rs6651255 was negatively associated with heel bone mineral density t-score and positively associated with self-reported prolapsed or slipped disc (both FDRSMR<0.01, underlying data30 and extended data29) and also strongly associated with standing height (underlying data30 and extended data29) and several measures of fat-free mass (all FDRSMR<0.001). However, HEIDI tests showed strong evidence against pleiotropic effects of the same functional variant in this region for BP and these anthropometric traits (all PHEIDI < 0.001), while associations with heel bone mineral density t-score and self-reported prolapsed or slipped disc were likely the result of pleiotropy, controlled by the same functional variant (all PHEIDI > 0.05). This suggests that genetic variants influencing structural spine features – the vertebral body and intervertebral disc – provide the link to BP symptoms, supporting previous work in twins highlighting shared genetic influence between spine degeneration and BP16,31.\n\nThe unanticipated results were obtained in the study of expression data. We have previously17 demonstrated that variants in LD with the lead SNP at 8q24.1 were located in potential regulatory regions in chondrocytes and osteoblasts (Roadmap Epigenomics Consortium32). In the current study, we tested expression of genes in the 8q24.21 region in all available tissues considered potentially relevant to development of pain (underlying data33), including musculoskeletal, brain and nerve tissue (from GTEx,20). Given the suggestion by Bjornsdottir et al. that the locus may act through regulation of immune function19, we also investigated expression in whole blood using the blood eQTL database26 and five immune cell subsets from CEDAR34. Similar to the Bjornsdottir et al. study19, we observed that rs6651255 is co-associated with expression of GSDMC in skeletal muscle (FDRSMR=0.01, PSMR=0.0001, underlying data30), with the BP-associated C allele of rs6651255 being associated with increased expression of the gene. However, HEIDI results show this overlap to be unlikely due to pleiotropic action of the same functional variant (PHEIDI = 3×10-7, underlying data30 and extended data29). Our findings suggest that GSDMC is not the gene through which the functional variant acts to influence BP. The next strongest co-association was with increased expression of FAM49B in brain anterior cingulate cortex (PSMR=0.004, underlying data30 and extended data29) and decreased expression in cytotoxic T (CD8+) cells (PSMR=0.02, underlying data30 and extended data29), although these associations were only nominally significant (p<0.05), yet not statistically significant after multiple testing correction (FDRSMR=0.18 and 0.28, respectively). All corresponding PHEIDI were > 0.05, indicating these co-associations are likely due to pleiotropy. The product of FAM49B was found to be expressed in multiple tissues including bone, lymph node and spleen; according to Proteomics DB35, it has highest expression in lymphocytes, and was implicated as a key negative regulator of actin dynamics and T-cell activation36. The precise mechanism of which gene is involved in the development of BP is difficult to infer due to lack of eQTL data available for relevant tissues, such as intervertebral disc.\n\n\nConclusion\n\nWe hypothesize that the locus 8q24.21 exhibits its influence on BP phenotypes via disk and bone remodeling, and not through influence on anthropometrics or pain processing. Our findings suggest that FAM49B and not GSDMC is the most likely mechanism through which the functional variant leads to BP.\n\n\nData availability\n\nZenodo: Table S1. List of relevant studies of gene expression with available eQTL data. http://doi.org/10.5281/zenodo.371500533\n\nThis project contains the following underlying data:\n\nList of relevant studies of expression data.csv (List of relevant studies of gene expression with available eQTL data)\n\nZenodo: Tables S2-S3. SMR-HEIDI results for 8q24.21 locus between back pain and other phenotypes. [Data set]. http://doi.org/10.5281/zenodo.371502730\n\nThis project contains the following underlying data:\n\nSMR-HEIDI results ST2.csv (SMR-HEIDI results for 8q24.21 locus between back pain and other complex traits)\n\nSMR-HEIDI results ST3.csv (SMR-HEIDI results for 8q24.21 locus between back pain and expression of genes)\n\nFigures S1–S7. SMR-HEIDI analysis results for 8q24.21 locus between BP and selected phenotypes. Zenodo: http://doi.org/10.5281/zenodo.371512029\n\nThis project contains the following extended data:\n\nFigure S1.png (SMR-HEIDI analysis results for rs6651255 between BP and LDH)\n\nFigure S2.png (SMR-HEIDI analysis results for rs6651255 between BP and GSDMC expression in skeletal muscle (GTEx v6))\n\nFigure S3.png (SMR-HEIDI analysis results for rs6651255 between BP and FAM49B expression in Brain anterior cingulate cortex BA24 (GTEx v6))\n\nFigure S4.png (SMR-HEIDI analysis results for rs6651255 between BP and FAM49B expression in CD8 cell line (CEDAR))\n\nFigure S5.png (SMR-HEIDI analysis results for rs6651255 between BP and heel bone mineral) density (UKBB).\n\nFigure S6.png (SMR-HEIDI analysis results for rs6651255 between BP and disc problem phenotype (UKBB))\n\nFigure S7.png (SMR-HEIDI analysis results for rs6651255 between BP and height (UKBB))\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
"appendix": "Acknowledgements\n\nWe thank Denis Gorev, Anna Torgasheva and Eugeny Pakhomov for help with summary level data collection, processing, and analyses. We thank Michel Georges for permission to use the CEDAR results for this work, and for discussion related to CEDAR. We also thank Natalia Aulchenko for help with the manuscript preparation.\n\n\nReferences\n\nHoy D, March L, Brooks P, et al.: The global burden of low back pain: estimates from the Global Burden of Disease 2010 study. Ann Rheum Dis. 2014; 73(6): 968–74. PubMed Abstract | Publisher Full Text\n\nVos T, Abajobir AA, Abate KH, et al.: Global, regional, and national incidence, prevalence, and years lived with disability for 328 diseases and injuries for 195 countries, 1990-2016: a systematic analysis for the Global Burden of Disease Study 2016. Lancet. 2017; 390(10100): 1211–59. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZheng CJ, Chen J: Disc degeneration implies low back pain. Theor Biol Med Model. 2015; 12: 24. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKjaer P, Leboeuf-Yde C, Sorensen JS, et al.: An epidemiologic study of MRI and low back pain in 13-year-old children. Spine (Phila Pa 1976). 2005; 30(7): 798–806. PubMed Abstract | Publisher Full Text\n\nSambrook PN, MacGregor AJ, Spector TD: Genetic influences on cervical and lumbar disc degeneration: a magnetic resonance imaging study in twins. Arthritis Rheum. 1999; 42(2): 366–72. PubMed Abstract | Publisher Full Text\n\nBattié MC, Videman T, Gill K, et al.: 1991 Volvo Award in clinical sciences. Smoking and lumbar intervertebral disc degeneration: an MRI study of identical twins. Spine (Phila Pa 1976). 1991; 16(9): 1015–21. PubMed Abstract | Publisher Full Text\n\nBattié MC, Haynor DR, Fisher LD, et al.: Similarities in degenerative findings on magnetic resonance images of the lumbar spines of identical twins. J Bone Joint Surg Am. 1995; 77(11): 1662–70. PubMed Abstract | Publisher Full Text\n\nWilliams FM, Popham M, Livshits G, et al.: A response to Videman et al., “challenging the cumulative injury model: positive effects of greater body mass on disc degeneration”. Spine J. 2010; 10(1): 571–2; author reply 572. PubMed Abstract | Publisher Full Text\n\nMacGregor AJ, Andrew T, Sambrook PN, et al.: Structural, psychological, and genetic influences on low back and neck pain: a study of adult female twins. Arthritis Rheum. 2004; 51(2): 160–7. PubMed Abstract | Publisher Full Text\n\nHartvigsen J, Christensen K, Frederiksen H, et al.: Genetic and environmental contributions to back pain in old age: a study of 2,108 danish twins aged 70 and older. Spine (Phila Pa 1976). 2004; 29: 897–901; discussion 902. PubMed Abstract | Publisher Full Text\n\nVideman T, Battié MC, Gibbons LE, et al.: Associations between back pain history and lumbar MRI findings. Spine (Phila Pa 1976). 2003; 28(6): 582–8. PubMed Abstract | Publisher Full Text\n\nBattié MC, Videman T, Levalahti E, et al.: Heritability of low back pain and the role of disc degeneration. Pain. 2007; 131(3): 272–80. PubMed Abstract | Publisher Full Text\n\nNyman T, Mulder M, Iliadou A, et al.: High heritability for concurrent low back and neck-shoulder pain: a study of twins. Spine (Phila Pa 1976). 2011; 36(22): E1469–76. PubMed Abstract | Publisher Full Text\n\nMacGregor AJ, Andrew T, Sambrook PN, et al.: Structural, psychological, and genetic influences on low back and neck pain: a study of adult female twins. Arthritis Rheum. 2004; 51(2): 160–7. PubMed Abstract | Publisher Full Text\n\nKalichman L, Hunter DJ: The genetics of intervertebral disc degeneration. Familial predisposition and heritability estimation. Joint Bone Spine. 2008; 75(4): 383–7. PubMed Abstract | Publisher Full Text\n\nLivshits G, Popham M, Malkin I, et al.: Lumbar disc degeneration and genetic factors are the main risk factors for low back pain in women: the UK Twin Spine Study. Ann Rheum Dis. 2011; 70(10): 1740–5. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSuri P, Palmer MR, Tsepilov YA, et al.: Genome-wide meta-analysis of 158,000 individuals of European ancestry identifies three loci associated with chronic back pain. PLoS Genet. 2018; 14(9): e1007601. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFreidin MB, Tsepilov YA, Palmer M, et al.: Insight into the genetic architecture of back pain and its risk factors from a study of 509,000 individuals. Pain. 2019; 160(6): 1361–1373. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBjornsdottir G, Benonisdottir S, Sveinbjornsson G, et al.: Sequence variant at 8q24.21 associates with sciatica caused by lumbar disc herniation. Nat Commun. 2017; 8: 14265. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGTEx Consortium, Laboratory DA & Coordinating C (LDACC)—Analysis WG, Statistical Methods groups—Analysis Working Group, et al.: Genetic effects on gene expression across human tissues. Nature. 2017; 550(7675): 204–213. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLiu B, Gloudemans MJ, Rao AS: Ingelsson E and Montgomery SB.Abundant associations with gene expression complicate GWAS follow-up. Nat Genet. 2019; 51(5): 768–769. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNica AC, Montgomery SB, Dimas AS, et al.: Candidate causal regulatory effects by integration of expression QTLs with complex trait genetic associations. PLoS Genet. 2010; 6(4): e1000895. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHormozdiari F, Van De Bunt M, Segre AV, et al.: Colocalization of GWAS and eQTL signals detects target genes. Am J Hum Genet. 2016; 99(6): 1245–1260. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhu Z, Zhang F, Hu H, et al.: Integration of summary data from GWAS and eQTL studies predicts complex trait gene targets. Nat Genet. 2016; 48(5): 481–7. PubMed Abstract | Publisher Full Text\n\nGiambartolomei C, Vukcevic D, Schadt EE, et al.: Bayesian test for colocalisation between pairs of genetic association studies using summary statistics. PLoS Genet. 2014; 10(5): e1004383. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWestra HJ, Peters MJ, Esko T, et al.: Systematic identification of trans eQTLs as putative drivers of known disease associations. Nat Genet. 2013; 45(10): 1238–43. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLonsdale J, Thomas J, Salvatore M, et al.: The Genotype-Tissue Expression (GTEx) project. Nat Genet. 2013; 45(6): 580–5. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHemani G, Zheng J, Elsworth B, et al.: The MR-Base platform supports systematic causal inference across the human phenome. eLife. 2018; 7: pii: e34408. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWilliams FMK, Tsepilov YA, Freidin M, et al.: Figures S1-S7. SMR-HEIDI analysis results for 8q24.21 locus between BP and selected phenotypes. Zenodo. http://www.doi.org/10.5281/zenodo.3715120\n\nWilliams FMK, Tsepilov YA, Freidin M, et al.: Tables S2-S3. SMR-HEIDI results for 8q24.21 locus between back pain and other phenotypes. [Data set] Zenodo. http://www.doi.org/10.5281/zenodo.3715027\n\nLivshits G, Ermakov S, Popham M, et al.: Evidence that bone mineral density plays a role in degenerative disc disease: the UK Twin Spine study. Ann Rheum Dis. 2010; 69(12): 2102–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKundaje A, Meuleman W, Ernst J, et al.: Integrative analysis of 111 reference human epigenomes. Nature. 2015; 518(7539): 317–30. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWilliams FMK, Tsepilov YA, Freidin M, et al.: Table S1. List of relevant studies of gene expression with available eQTL data. [Data set]. Zenodo. http://www.doi.org/10.5281/zenodo.3715005\n\nMomozawa Y, Dmitrieva J, Théâtre E, et al.: IBD risk loci are enriched in multigenic regulatory modules encompassing putative causative genes. Nat Commun. 2018; 9(1): 2427. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSchmidt T, Samaras P, Frejno M, et al.: ProteomicsDB. Nucleic Acids Res. 2018; 46(D1): D1271–81. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShang W, Jiang Y, Boettcher M, et al.: Genome-wide CRISPR screen identifies FAM49B as a key regulator of actin dynamics and T cell activation. Proc Natl Acad Sci U S A. 2018; 115(17): E4051–60. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "70799",
"date": "23 Sep 2020",
"name": "Lisbet Haglund",
"expertise": [
"Reviewer Expertise Molecular mechanisms of back pain"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe study \"Sequence variation at 8q24.21 and risk of back pain\" is well written and is addressing an important clinical problem. The design and data analysis are well performed.\nIt would be interesting to know more about the function of FAM49B and GDSMC and how a dysregulation could potentially lead to back pain. The fact that it is involved in mitochondrial dysregulation and reactive oxygen species may have implications for IVD degeneration and low back pain.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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/9-424
|
https://f1000research.com/articles/8-2139/v1
|
23 Dec 19
|
{
"type": "Software Tool Article",
"title": "R package “QRISK3”: an unofficial research purposed implementation of ClinRisk’s QRISK3 algorithm into R",
"authors": [
"Yan Li",
"Matthew Sperrin",
"Tjeerd van Staa",
"Yan Li",
"Matthew Sperrin"
],
"abstract": "Cardiovascular disease has been the leading cause of death for decades. Risk prediction models are used to identify high risk patients; the most common model used in the UK is ClinRisk’s QRISK3. In this paper we describe the implementation of the QRISK3 algorithm into an R package. The package was successfully validated by the open sourced QRISK3 algorithm and QRISK3 SAS program. We provide detailed examples of the use of the package, including assigning QRISK3 scores for a large cohort of patients. This R package could help the research community to better understand risk prediction scores and improve future risk prediction models. The package is available from CRAN: https://cran.r-project.org/web/packages/QRISK3/index.html.",
"keywords": [
"CVD",
"risk prediction model",
"R",
"QRISK3"
],
"content": "Introduction\n\nCardiovascular disease (CVD) was responsible for 17.9 million deaths in 2016, which represents 31% of overall global deaths, and over 75% of these deaths happened in low/middle-income countries1. People who are at high risk of CVD need to be identified and treated early1. Risk prediction models that use risk factors to calculate the probability of patients developing diseases are often used to identify high risk patients2. QRISK3 is the most popular risk prediction model for CVD developed in the UK. It calculates risk of patients developing CVD in the next 10 years and has been incorporated into the electronic health records (EHRs) system in the UK in order to detect high risk CVD patients and help clinicians make treatment decisions3,4. NICE guidelines recommend clinicians to consider prescribing statins to patients with a risk over 10% identified from QRISK35. QRISK3 was developed from historical patients’ EHR data using Cox proportional hazard model6 and has been well validated at population level corresponding to discrimination and calibration3,4,7.\n\nThe implementation of QRISK3 into R would not only benefit researchers to improve future risk prediction but also enable them to use QRISK3 scores to identify patients at certain risk levels, e.g. for clinical trial recruitment. There is also scope to improve these risk predictions; it has been found that QRISK3 has uncertainty on individual risk prediction7,8 due to unmeasured heterogeneity between practices, which was not captured. A follow-up study suggests that QRISK3 may need to include additional causal risk factors as this uncertainty on individual risk prediction was not related to data quality and variation of association between disease and outcome9. The current QRISK3 can only be accessed through an online web calculator or specialised commercial software10 and its original algorithm was written by C, which is a low level programming language appealing to software engineering rather than data science11. R is the most popular statistical programming language in the data science field due to its great advantage as free and open-source, with fast computing and a well-supported community12. This paper explains the incorporation of the QRISK3 algorithm into R for ease of research concerning QRISK3 and how the package was developed and validated. The package aims to help researchers to improve risk prediction models and better detect high risk CVD patients.\n\n\nMethods\n\nThe original QRISK3 algorithm was written in C by ClinRisk under a GNU Lesser General Public License11. Their previously published QRISK3 paper was used to understand the original algorithm and the associations between variables used in the original algorithm and risk factors of QRISK33.\n\nThe QRISK3 algorithm was written in both R (3.4.2) and SAS (9.4)13 independently, in order to mimic double programming, with a plan to use the SAS implementation to validate the R package. An additional C program, which could directly call the original QRISK3 algorithm to calculate risk, was written for validation. Two validation datasets (QRISK3_2017_test and QRISK3_2019_test) were then created and included in the R package. Dataset QRISK3_2017_test was created by manually recording the calculated QRISK3 risk score from the original QRISK3 algorithm for a group of simulated patients. The simulated patient groups were generated by changing each risk factor sequentially covering the changes of all QRISK3 risk factors. For example, patient 1 in QRISK3_2017_test does not have any positive CVD risk factors, patient 2 is similar to patient 1 expect he has atrial fibrillation, patient ID 3 is similar to patient 2 except he is on atypical antipsychotic medication rather than atrial fibrillation and so on until all the change of CVD predictors are covered. Therefore, each patient is similar to the previous patient except the change of one CVD predictor. QRISK3_2019_test was the version recorded using the original QRISK3 algorithm with different value changes for each risk factor. Risk scores of the same simulated patient groups (QRISK3_2017_test and QRISK3_2019_test) was compared among different versions of QRISK3, including QRISK3 R package, QRISK3 SAS program and QRISK3 C function for validation. The R package was created using R CMD tool14 with several useful online tutorials15–18.\n\nThe QRISK3 package can be directly installed from CRAN19 using “install(QRISK3)” or GitHub respiratory20 with “install_github(\"YanLiUK/QRISK3\")”. The package contains one function (QRISK3_2017) to calculate the risk of patients developing CVD in the next 10 years using the QRISK3 algorithm11 and the two datasets for testing.\n\nVariables used by the QRISK3 package were summarised and compared to the original algorithm in Table 1. All variables have the same definition as the QRISK3 paper3, most of variables were coded into numeric variables similar to the original algorithm. The coding of ethnicity and smoking was different from the original algorithm (written in C), as the C index starts from 0 but R’s index starts from 1.\n\nThe two datasets QRISK3_2017_test and QRISK3_2019_test were used for validation. Risk scores calculated from this QRISK3 package, the original algorithm and the SAS version on the same group of patients was exactly the same. The external validation of this QRISK3 package in a big CPRD cohorts with 3.6 million patients shows a good and similar discrimination (C statistic: 0.85) and calibration to a previous study7 compared to the original QRISK3 paper3.\n\nA patient cohort with anonymous patient identifiers and CVD risk factors should first be extracted and coded similarly to QRISK3 by the user. Missing values in the dataset should be handled (e.g. multiple imputation) before using this package. Column names of CVD risk factors (e.g. “age”) should then be specified correctly to the QRISK3_2017 function. The function returns calculated risk scores through a dataset with three columns, including patient identifier, calculated QRISK3 score and calculated QRISK3 score with one digit. It also reminds users to double check whether the definition of their variables was the same as the definition of QRISK3. The package also automatically detects whether all variables were coded as numeric and whether age of patients was ranged between 28 and 84, if not an error message returns (explained in Table 2).\n\n\nWorkflow\n\n1. Set path and read data from CSV file\n\n\n\n2. See the data structure and other information\n\n\n\n\n\n3. Call the QRISK3 function to calculate risk score\n\n\n\n\nUse case\n\nUsers first need to create a statistical analysis dataset similar to the provided test dataset (e.g. QRISK3_2019_test) which contains information of patients’ identifier and QRISK3 risk factors and mimics QRISK3’s training cohort3. The structure of this statistical analysis dataset should be set out so that each row (observation) represents one individual patient and each column represents one QRISK3 predictor. The exact definition of all QRISK3 predictors can be found from Box 1 of the original QRISK3 paper3. Variables used by QRISK3 can be extracted from EHR databases, such as CPRD21 or QResearch22. Code lists (Read code) for the outcome variable (CVD) can be obtained from the supplementary materials of the QRISK3 paper3. Code lists for variables included in QRISK2 can be extracted from a previous study23. Code lists for other variables including anxiety, alcohol abuse, atypical anti-psychotic medication, erectile dysfunction, HIV/AIDS, left ventricular hypertrophy, migraine and systemic lupus erythematosus could be found from CPRD24 or clinical codes website25. All CVD risk factors should be coded as numeric, binary variables should be coded as 0 or 1, categorical variables such as smoking status should be coded as the same as this package. Any differences between users’ variables and QRISK3 predictors (e.g. different criteria to define smoking status) should be mentioned in users’ final report. Once the analysis dataset was extracted, it is recommended to compare the distribution of users’ analysis dataset to Qresearch’s cohort using their baseline table3,26. Missing values should be imputed with multiple imputation27. Finally, users should follow the above workflow and carefully match their variable names to pre-defined QRISK3 predictors to calculate risk score. The function will return a dataset with patient identifier, calculated score and calculated score with 1 digit.\n\n\nDiscussion\n\nThis R package successfully implements the QRISK3 algorithm into R, which allows researchers to calculate CVD risk of patients in the next 10 years. The R package was validated by the original algorithm and a SAS version. This is also the first R implementation of the QRISK3 algorithm at the date of writing.\n\nThough QRISK3 was already published and released from the online website, it is time consuming for researchers to calculate QRISK3 risk score, as the online calculator cannot be used as a service to obtain QRISK3 scores for a large cohort, and the original algorithm is written in C rather than a well-established data science language such as R. This package bridges this gap. It allows researchers to obtain QRISK3 scores for large cohorts, which could help to improve model accuracy of QRISK3 and help with any more applied tasks that require knowing CVD risk at a patient level.\n\nAlthough it is easy to use this R function to calculate a risk score, researchers should carefully check whether their variables are coded the same as the original QRISK3 cohort, otherwise the calculated score might not be the correct risk of the patient in the cohort. For example, a patient who is a smoker is coded as “1” in the variable “smoking” would be in conflict with the definition of the QRISK3 algorithm (“smoking” equals 1 in this R package means non-smoker). Since QRISK is updated annually every spring, researchers who are interested in the latest work should refer to their website10.\n\nIn conclusion, we developed this R package to allow researchers to obtain QRISK3 scores for large cohorts. It allows the research community to better understand and apply a currently used risk prediction model for CVD risk.\n\n\nData availability\n\nOriginal QRISK3 algorithm: https://qrisk.org/three/src.php\n\n\nSoftware availability\n\nPackage available from CRAN: https://cran.r-project.org/web/packages/QRISK3/index.html\n\nSource code available from: https://github.com/YanLiUK/QRISK3\n\nArchived source code as at time of publication: https://doi.org/10.5281/zenodo.357068228\n\nLicense: GPL-3\n\nC source code, SAS version and QRISK3_2017_test and QRISK3_2019_test datasets used for validation available from: https://github.com/YanLiUK/QRISK3_valid\n\nArchived C code, SAS version and test datasets as at time of publication: https://doi.org/10.5281/zenodo.357130429\n\nLicense: GPL-3",
"appendix": "References\n\nCardiovascular diseases (CVDs). Accessed November 16, 2019. Reference Source\n\nGrant SW, Collins GS, Nashef SAM: Statistical Primer: developing and validating a risk prediction model. Eur J Cardiothorac Surg. 2018; 54(2): 203–208. PubMed Abstract | Publisher Full Text\n\nHippisley-Cox J, Coupland C, Brindle P: Development and validation of QRISK3 risk prediction algorithms to estimate future risk of cardiovascular disease: prospective cohort study. BMJ. 2017; 357: j2099. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCollins GS, Altman DG: An independent and external validation of QRISK2 cardiovascular disease risk score: a prospective open cohort study. BMJ. 2010; 340: c2442. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCVD risk assessment and management - NICE CKS. Accessed November 16, 2019. Reference Source\n\nCox DR: Regression Models and Life-Tables.1972; 34: Accessed February 21, 2019. Reference Source\n\nLi Y, Sperrin M, Belmonte M, et al.: Do population-level risk prediction models that use routinely collected health data reliably predict individual risks? Sci Rep. 2019; 9(1): 11222. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPate A, Emsley R, Ashcroft D, et al.: The uncertainty with using risk prediction models for individual decision making: an exemplar cohort study examining the prediction of cardiovascular disease in English primary care. BMC Med. 2019; 17(1): 134. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLi Y, Sperrin M, Martin GP, et al.: Examining the impact of data quality and completeness of electronic health records on predictions of patients' risks of cardiovascular disease. Int J Med Inform. 2020; 133: 104033. PubMed Abstract | Publisher Full Text\n\nQRISK3. Accessed November 16, 2019. Reference Source\n\nhttps://qrisk.org/three/src.php. Accessed November 16, 2019.\n\nR: The R Project for Statistical Computing. Accessed April 28, 2019. Reference Source\n\nSAS® 9.4 Statements: Reference, Fifth Edition. Accessed August 20, 2017. Reference Source\n\nR Installation and Administration. Accessed November 17, 2019. Reference Source\n\nSubmitting your first package to CRAN, my experience | R-bloggers. Accessed November 17, 2019. Reference Source\n\nWriting an R package from scratch | Not So Standard Deviations. Accessed November 17, 2019. Reference Source\n\nR package primer. Accessed November 17, 2019. Reference Source\n\nCollins D, Lee J, Bobrovitz N, et al.: whoishRisk – an R package to calculate WHO/ISH cardiovascular risk scores for all epidemiological subregions of the world [version 2; peer review: 3 approved]. F1000Res. 2016; 5: 2522. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCRAN - Package QRISK3. Accessed December 8, 2019. Reference Source\n\nYanLiUK/QRISK3: A QRISK3 R package implements QRISK3 algorithm into R. Accessed December 12, 2019. Reference Source\n\nClinical Practice Research Datalink - CPRD. Accessed August 20, 2017. Reference Source\n\nHome - QResearch. Accessed December 8, 2019. Reference Source\n\nvan Staa TP, Gulliford M, Ng ES, et al.: Prediction of cardiovascular risk using Framingham, ASSIGN and QRISK2: how well do they predict individual rather than population risk? PLoS One. 2014; 9(10): e106455. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCPRD @ Cambridge - Code Lists - Primary Care Unit. Accessed November 18, 2019. Reference Source\n\nClinicalCodes Repository. Accessed November 18, 2019. Reference Source\n\nPate A, Emsley R, Ashcroft DM, et al.: The uncertainty with using risk prediction models for individual decision making: an exemplar cohort study examining the prediction of cardiovascular disease in English primary care. BMC Med. 2019; 17(1): 134. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSterne JA, White IR, Carlin JB, et al.: Multiple imputation for missing data in epidemiological and clinical research: potential and pitfalls. BMJ. 2009; 338: b2393. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYanLiUK: YanLiUK/QRISK3 v1.0.0 (Version v1.0.0). Zenodo. 2019. http://www.doi.org/10.5281/zenodo.3570682\n\nYanLiUK: YanLiUK/QRISK3_valid: QRISK3_valid (Version v1.0.0). Zenodo. 2019. http://www.doi.org/10.5281/zenodo.3571304"
}
|
[
{
"id": "59681",
"date": "12 Feb 2020",
"name": "Mohieddin Jafari",
"expertise": [
"Reviewer Expertise Computational biologist"
],
"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 provided an R package for the QRISK3 algorithm to predict the risk of cardiovascular disease. My main comment is related to the size of this package, which has only one simple function with two simple datasets. I am not sure that this tiny package can be presented as an F1000Research article. There are not any new findings represented in this article compared to the author's previous works. I suggest enriching this package with more visualization and data manipulation functions and datasets. Besides, I do not think it needs to highlight validation of R performance with SAS or C. This is part of your quality control, not an outcome. Also, I have some minor comments:\nWhy didn't you use the R 3.6.2 instead of 3.4.2?\n\nWhy you did not make the categorical variables as ordered factor R object instead of a simple numeric object? (in this case, you do not need to think about the conflict of the meaning of \"1\" and \"0\").\n\nThe range of patient ages is not consistent between text and table.\n\n\"Height\", \"Weight\" and \"Weight/Height\" in table 1 should be lowercase the same as the package.\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": "5229",
"date": "28 Feb 2020",
"name": "Yan Li",
"role": "Author Response",
"response": "We appreciate reviewers’ time and effort in review this package paper, we would like to make following reply to these comments. Reviewer’s comments: The authors provided an R package for the QRISK3 algorithm to predict the risk of cardiovascular disease. My main comment is related to the size of this package, which has only one simple function with two simple datasets. I am not sure that this tiny package can be presented as an F1000Research article. There are not any new findings represented in this article compared to the author's previous works. Reply: This implementation of QRISK3 algorithm was needed due to lack of metadata, explanations of variables and accessible source code. There is a need of this R package as researchers cannot easily estimate risks based on the QRISK3 algorithm1. This is also the first implementation of QRISK3 into R (validated by SAS and C version) and was approved by CRAN. By the date of 12-02-2020 (67 days since the first release), this R package has been downloaded by 1343 times and rank 42 among total 66 packages which were published in the same day (data available from “cranlogs”). We believe the novelty of this package and this paper is: QRISK3 is different from other model such as Framingham model (one other popular American CVD risk prediction model). For Framingham model, the model formula was simple and clearly presented in its original paper, and user could easily implement it into R. While for QRISK3, this is different and difficult. The main challenge implementing QRISK into R is the original algorithm written in low level language C and there is no document to explain the meaning of these variables in original algorithm. Users also need to validate their implemented version is the same as the original version of QRISK3, this requires users to have programming knowledge of both R and C while R is the dominated language in data science. Therefore, researchers don’t have table 1 of this paper if they start to implement QRISK3 from the original algorithm. This creates challenges that it is hard to acquire a similar statistic model formula as presented in Framingham, and it is hard to identify which variable represents what risk factors especially considering they were written in C. For example, how to know variable such as “b_impotence2”, “ethrisk” and “fh_cvd” represents “erectile disfunction”, “ethnicity” and “relative of CVD”. QRISK was used to predict 10 years risk, why there are 11 values in a vector which represents baseline risk from the original algorithm, and which variable represents this baseline risk? There is a long distance between input value of predictors to output a correct predicted risk. This package solved all the challenges mentioned above and bridges the gaps. This R package acts like a bridge which enables researchers to easily access and use QRISK3 model in R like using Framingham model. Without this R package and this paper described how this R package was developed and validated, researchers may spend months to re-implement QRISK into R and they also need to spend a lot of effort on validating whether their implemented version of QRISK3 does the similar thing as the original algorithm. Overall, our package and this paper solved these, and saved time and energy for researchers who only require calculated QRISK3 score as part of their new research. Reviewer’s comments: I suggest enriching this package with more visualization and data manipulation functions and datasets. Besides, I do not think it needs to highlight validation of R performance with SAS or C. This is part of your quality control, not an outcome. Also, I have some minor comments: Reply: We appreciate reviewers’ suggestion, but this package was a simple implementation of QRISK3 algorithm. We decide to make this package as simple as we can as: It would be easier for user to quickly obtain QRISK3 score. Rather than searching a list of redundant functions, user could instantly test and use this package. There are mature R packages which support reviewer suggested functions. Due to the nature of this R package (i.e. only write with base R language without dependent packages), it is well compatible to all the other R packages. For visualization, users could easily use R package like “ggplot2” with this R package. For data manipulation, R package like “dplyr” is well compatible to our package. The main function of this R package is to provide QRISK3 calculated risk score. What visualization and datasets user might require were purely dependent on their research question. In the current version, we have provided a dataset with patient identifier and calculated QRISK score, which could be easily used by researchers to draw their own visualization. Though there are only two datasets present in this package, it contains all the key information of what the datasets would look like and what variables were needed. They contain enough information for user to understand and use this package easily. Overall, simplistic is a part of design of this R package and there are well established R packages which could be used with this simple R package. We do not find any necessary reasons to add more functions in this R package, as the aim of this R package is to help users to obtain a QRISK3 score from large dataset quickly and current version fulfilled this purpose. We are happy to add more functions or tools if there are more specific requirements from users in future. We also believe it is essential to show user how this R package was validated with the original algorithm, as this R package aims to provide a correct QRISK3 predicted score. Users who decide to use our package may wish to replicate our validation process. Reviewer’s comments: Why didn't you use the R 3.6.2 instead of 3.4.2? Reply: R 3.4.2 was used to develop the prototype of this R package in 2017. However, all functions in this package used R base language, so it can be used in any R version including 3.6.2 (tested). Reviewer’s comments: Why you did not make the categorical variables as ordered factor R object instead of a simple numeric object? (in this case, you do not need to think about the conflict of the meaning of \"1\" and \"0\"). Reply: This was due to how QRISK3 score was calculated. It was calculated from a mathematic formula where we need to calculate a linear predictor which requires numeric value of predictors. We coded all variables into numeric rather than some are numeric, and some are factor is for the ease of users (follow the simplistic principle). In this case, users would know all variables need to be numeric rather than to remember which variable should be a factor especially when there are over 20 predictors in the model. We also provided an automate function to help user double check which variables were not coded into numeric. However, we do realize that we may never prevent all unforeseen mistake, so we highlight this in descriptions of both of paper and package. Reviewer’s comments: The range of patient ages is not consistent between text and table. Reply: thanks. Corrected in the next version. Reviewer’s comments: \"Height\", \"Weight\" and \"Weight/Height\" in table 1 should be lowercase the same as the package. Reply: thanks. Updated. Other reviewer’s comments: Is the rationale for developing the new software tool clearly explained? Yes Is the description of the software tool technically sound? Yes Are sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes Is sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes Are the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes References 1. I need SAS, or SPSS or Stata or R code for :Two cardiac risk scores: QRISK and SCORE . May you share? https://www.researchgate.net/post/I_need_SAS_or_SPSS_or_Stata_or_R_code_for_Two_cardiac_risk_scores_QRISK_and_SCORE_May_you_share. Accessed February 12, 2020."
}
]
}
] | 1
|
https://f1000research.com/articles/8-2139
|
https://f1000research.com/articles/9-418/v1
|
21 May 20
|
{
"type": "Research Article",
"title": "Contextualizing COVID-19 spread: a county level analysis, urban versus rural, and implications for preparing for the next wave",
"authors": [
"J. Sunil Rao",
"Hang Zhang",
"Alejandro Mantero",
"Hang Zhang",
"Alejandro Mantero"
],
"abstract": "Background: Contextual determinants of health including social, environmental, healthcare and others, are a so-called deck of cards one is dealt. The ability to modify health outcomes varies then based upon how one’s hand is played. It is thus of great interest to understand how these determinants associate with the emerging pandemic coronavirus disease 2019 (COVID-19). Methods: To this end, we conducted a deep-dive analysis into this problem using a recently curated public dataset on COVID-19 that connects infection spread over time to a rich collection of contextual determinants for all counties of the U.S and Washington, D.C. Results: Using random forest machine learning methodology, we identified a relevant constellation of contextual factors of disease spread which manifest differently for urban and rural counties. Conclusions: The findings also have clear implications for better preparing for the next wave of disease.",
"keywords": [
"COVID-19",
"Pandemic",
"Contextual Factors",
"County Level",
"Growth Curves",
"Random Forests",
"Machine Learning"
],
"content": "Introduction\n\nLike politics, all public health is ultimately local. This tenet is particularly important in trying to understand the dynamics of an emerging outbreak like the pandemic coronavirus disease 2019 (COVID-19). While much attention has focused on modeling infection spread and containment and mitigation strategies to better plan for potential surges, and the impact they may have on hospital capacities, less attention has been paid to understanding the contextual nature of the pandemic.\n\nThis means modeling area level data like at the county level and including information on demographics, race/ethnicity, housing, education, employment and income, climate, transit scores and healthcare metrics1. Many of these are often classified under the umbrella of social determinants of health that have been documented to influence the success or setbacks in steps to a successful public health emergency response2. Being able to do this at a local level, like the county, has significant implications for understanding how where you live influences disease spread, differences between rural and urban settings, and better planning for future anticipated waves of disease. In the absence of comprehensive geocoded patient level data where multilevel interactions can be assessed, the study of the area level influences alone on COVID-19 spread is informative in its own right. In addition, with the realization that racial and ethnic minority groups are suffering disproportionately in the pandemic, social determinants of health (which are important contributors to health disparities) have garnered more attention.\n\nTo that end, recently Killeen et al.1 designed a dataset containing a machine-readable file with demographic, socioeconomic, healthcare and education data for each county in the 50 states and Washington, D.C. organized by FIPS (Federal Information Processing Standard) codes which are unique county code identifiers. This data was cobbled together from 10 governmental and academic sources on the county level. The resulting dataset contains more than 300 contextual county level variables. They then merged this contextual data with county-level time series data from the JHU (Johns Hopkins University) CSSE (Center for Systems Science and Engineering) COVID-19 dashboard3 which gives continuously updated confirmed infection and death counts over time.\n\nOur goal in this paper is to estimate growth rate increases (slopes) of the COVID-19 disease for each county in the U.S. and then relate these estimates to contextual level factors. In addition, we would like to understand differences between urban and rural county (i.e. populations < 50,000) settings in terms of the contextual factors that most relate to growth rates. Since we expect these factors to interact in complex ways, we will employ random forests machine learning methodology4 for model fitting. We expect that our findings will help to inform more nuanced strategies for better preparing for the next wave of disease.\n\n\nMethods\n\nKilleen et al.1 assembled the dataset under focus. COVID-19 infection volume time series came from the Johns Hopkins University CSSE COVID-19 Tracking Project and Dashboard which when the data was pulled ranged from 01/22/2020 until 04/12/2020. Climate data came from NOAA. County level demographic data was extracted from the US Census Bureau and the USDA. Healthcare capacity related data came from the Kaiser Family Foundation. Traffic score information came from the Center for Neighborhood Technology. ICU bed information came from Kaiser Health Network. Additional COVID-19 case information came from USAfacts. Physcian workforce data came from AAMC.\n\nMany of the variables were not usable in their original forms; many involved counts within a county but did not take into account the overall size of the county itself. This would make it impossible to directly compare between counties. Therefore, all count variables were standardized by their totals for example counts of racial and gender groups were divided by the total population of the county, and household counts were divided by the total number of households in the county. These variables are listed in the list of columns section of the GitHub repository created to provide this data1.\n\nThere was also an issue of state level variables that were included in the dataset, either by copying the state level data for every corresponding county, or by extrapolating the state characteristic as a fraction of population. Both types of variables were excluded from the analysis. Our reasoning for this is that the state level information could muddle the mechanism for the individual counties since our goal is to discern the slopes specifically at this level. If the forest were to start grouping counties by data it would defeat the purpose of the analysis. As for the extrapolated values by population, this is a very strong assumption that will most likely overstate the resources available to rural counties. Furthermore, this again may cause the forest to group by state which we want to avoid, our point is to have it group by specifically county level characteristics thus giving us groups of similar counties that experience similar increases in infection rates.\n\nHighly correlated variables were removed because they represented very similar information. The vast majority of these removed variables were upper and lower bound estimates of means included in the dataset. There is one pair of variables for migration that we suspected would be highly correlated, one was absolute count and the other was rate, for these the correlation was not very high and we though both could be particularly important in predicting slopes so both were included.\n\nWe also removed any variables with a missingness rate of over 45%, in order avoid overly high levels of imputation. This resulted in a final set of 186 variables that were analyzed. Full codebook for variable descriptions on Github site accompanying Killeen et al.1. Missing values in these were handled using random forest imputation (discussed in the next subsection).\n\nSome counties had to be omitted from the analysis and this was mostly due to poor fit in the growthcurver procedure (described next). Either the model was not estimable or the estimated midpoint time was much farther out than the data that was available at the time of analysis. We wanted to avoid such far extrapolations when the actual data only existed for far fewer days. This resulted in a loss of 16.8% of the counties. Also, for any counties that continued to have zero infections although the model could not be fit, we assumed the slopes to be zero.\n\nCounty-specific confirmed infections growth curve fitting and growth rate estimation. The infections time series county-level data was obtained from Killeen et al.1 that updated on Apr 13, 2020. The counts of infection in each county were standardized by corresponding county population and then fit into growth curve model. The growth curves for each county was estimated based on logistic equation which is commonly used in population ecology5–7 utilizing R package growthcurver version 0.3.08:\n\n\n\nwhere Ni(t) represents the proportion of infected people at time t for a given county i, ni as the size of initial infection proportion, Ki as the carrying capacity, ri is the growth rate with respect to the infection proportion, and ϵi(t) the model errors assumed to have zero mean and constant variance. Non-linear least squares is used to estimate model parameters.\n\nThe growth curve slopes were further obtained by taking derivative of Ni(t) with respect to t for each county as:\n\n\n\nwhere estimated parameter values are indicated by a hat sign. Then to calculate the maximal slope, we plug in the midpoint of time for t which for this type of function represents when the change in the outcome is greatest. Bootstrap standard errors (SE) were estimated by bootstrapping scaled model residuals. A total of 1000 bootstrap samples were used for each SE estimate.\n\nRandom forest (RF) is a machine learning technique developed by Leo Brieman4 which has been implemented into many subject matters. The technique takes advantage of base learner aggregation to develop a complex learner that has much improved prediction performance and fit on the data. In this case the base learners are CART trees9, but in order to prevent having the same tree appear multiple times in the forest, randomness is introduced to the learning procedure. This takes the form of both bootstrapping the observations for the training of each tree and random feature selection where the optimal split is not taken from all variables available but instead a randomly selected subset. This results in the trees being more independent of each other thus preventing the instability that is present in CART. The issue with CART tree is the balancing between bias and instability via tree depth, if the tree grows too deep, then it is likely have overfit the data (high instability) but will have low bias; while, if it grows too shallow, it will be more stable but have higher bias. Therefore by aggregating very deep and unstable trees random forest is able to obtain overall low bias and average away the instability of each tree thus giving us a very effective overall learner.\n\nWe used the rfsrc version 2.9.3 function in R version 3.6.010 for fitting random forests. Growth curve slope estimates were treated as responses (y) and the 186 contextual variables described above as county level predictors (xj; j = 1, . . . , 186). We chose not to transform the responses because of the robustness of random forests and to facilitate model interpretations11. Random forest procedures default settings were utilized which included mtry of square root of p (i.e. the number of predictors). Additionally, the automatic imputation function that randomForestSRC10 was activated in order to avoid losing county information. The imputation procedure is built into the forest growing procedure and run all at once. We included imputation because if we were to run the model only on the complete data, we would have lost approximately one third of the counties with the biggest loss being in the rural counties. In order to avoid losing so much data, we opted for imputation in our RF training. Separate forests were fit to rural and urban counties. Relative variable importance plots were generated and models validated using the % variance explained defined as,\n\n\n\nwhere MSE is defined as the out-of-bag test error estimated on observations not showing up in bootstrap re-samples. Values near zero (or negative for that fact) are indicative of modeling noise4. Relative variable importance is obtained by dividing all the calculated importances by the maximal one, this then provides a value with a possible range between 0 and 1 which makes it easier to interpret. Values near zero (or negative for that fact) are indicative of modeling noise4. Visualization of RF fits were done using parallel coordinate plots and multi-variable co-plots. These are created based on clusters which were determined with the PAM method using random forest distances as described in 12. For PAM, k was set to 10, the goal here was not to obtain the optimal number of clusters but to separate out a few groups with differing average slopes in order to depict how the contextual variables change as the slopes increase or decrease by group.\n\nRandom forest distances work similarly to random forest proximity but are more sensitive to the tree topology therefore able to provide better clusters. Like random forest proximity, a pair of points are considered highly proximal if they are in the same terminal node, but unlike proximity, instead of simply assigning zero if they are not in the same terminal node, a number between 0 and 1 is used depending on how far down the tree the two points became disjoint.\n\nIn order to attempt to ascertain potential differences in how rates of infection increases relate to the contextual variables between urban and rural settings, rural county data was dropped down the RF trained on urban data and urban county data was dropped down the RF trained on rural data. For this type of reverse prediction, the resulting errors were calculated and 95% confidence intervals determined. This allow us to see if there were regions of non-overlap suggesting the presence of possible heterogeneity between the associations between the rates with their contextual variables depending on rural or urban settings.\n\n\n1 Results\n\nFigure 1 shows sample growth curves fit to two large urban counties (New York, NY (population 1628701) and Miami-Dade, FL (population 271581)) and two rural counties (Randolph county, GA (population 6833) and Barbour county, AL (population 24881)). New York county and Randolph county had large slope estimates and Miami-Dade and Barbour counties had much smaller slopes estimates (note the much smaller values of the y-axis for Babour county). Figure 2 shows the estimates growth curve slopes versus log10(population) based on 2018 census population values or all counties in the U.S. Blue coloring indicates rural counties and red coloring urban. While the majority of estimated slopes are not large, it is clear that there are a mixture of urban and rural counties with large slope estimates. Table 1 gives the slope estimates colored by urban (red)/rural (blue) status. Many of urban counties were widely publicized, but interestingly many of the rural counties that comprise the list also have made the news as localized hot spots or points of concern. Random forests were fit separately to urban and rural counties. For the urban forest the % variance explained was 32.26% and 32.56% for the rural forest. This is very much on par with other studies that focus on area-level variables only (see for example 13,14). This shows good model validation for both forests.\n\nTable 2 and Table 3 display relative variable importance tables (i.e. mean decrease in accuracy using out-of-bag prediction when the variable is permuted) for urban counties and rural counties respectively. These were extracted from the relative variable importance scree plots in Figure 3 by choosing those variables with large relative importance values left of the visible “elbow\" in the plots.\n\nIn order to better visualize the fitted random forests, we next categorized counties by slope estimate groupings as determined by the fitted forests. Parallel coordinate plots are shown in Figure 4 and Figure 5 for urban and rural counties respectively. Each group is represented by lines moving horizontally and each variable by a vertical line (whose range of values is above and below the vertical line). Lines connect mean values of each variable for that group. The mean value of the slope for the group is shown in the first vertical line. The variables are sorted according to relative variable importance.\n\nIt’s clear that there are clusters of variables that clearly separate slope groups and that the variables differ for the urban versus rural random forest. For the urban parallel coordinate plot, such variables included percentage of Hispanic, black or African American male and female (HBA_FEMALE, HBA_MALE), international migration (INTERNATIONAL_MIG_2018), housing density, population density and May 2018 average and maximum temperature values in degrees F. For rural counties, such variables included percentage of non-Hispanic, black or African American male and female (NHBAC_FEMALE, NHBAC_MALE, NHBA_MALE, NHBA_ FEMALE), 2018 and percent poverty as estimated in 2018. Other variables of rural importance also included age - specifically the percent of the population that were elderly.\n\nFigure 6 and Figure 7 show co-plots focusing on these key variables. This allows a closer inspection of how the growth curve slope estimates continuously change as a function of other variables. In particular each co-plot showed co-dependence on 4 other variables. For the urban co-plot, variables plotted include HBA_MALE, population density, and 2018 international migration numbers. Each line is shown with a different color and plotting character. As the legend indicates, they represent quintiles of the 2018 average May temperature. There are some clearly interesting patterns where slopes are seen to rise dramatically. For instance, the top right panel indicates that for counties with higher average 2018 May temperatures, higher percent black male populations and higher degrees of international migration, that COVID-19 spread much more dramatically than in other urban counties.\n\nContrast this with the rural co-plot. There are multiple interesting panels here but focus again on the top right panel. Here each line represents a group of counties classified by rural-urban continuum codes in 2013 with larger numbers indicating smaller population counties either adjacent or not adjacent to a metro area. Smaller, (and also more isolated) counties saw a much more rapid increase in COVID-19 spread when accompanied by having a proportionately more black, older population who also had a higher percentage of their population in 2018 living in poverty.\n\nReverse predictions for urban counties were generated from the rural forest and for rural counties from the urban forest. Absolute error 95% confidence intervals were [4.967 x 10−5, 5.513 x 10−5] and [7.224 x 10−5, 8.535 x 10−5] respectively. The non-overlap further confirms some differences in county level structure being captured by both forests.\n\n\nDiscussion\n\nSocial determinants of health are known to influence the spread of infectious diseases15. These include the impact of poverty, illiteracy, poor sanitation, food insecurity, overcrowding, access to healthcare, workplace conditions and transit options amongst others. Other important factors are immigration patterns, age distributions, weather and environmental conditions. These factors can play a large role in determining how successful the response to a public health emergency is 2. They also are intimately involved in health disparities that arise in more acute fashion during a pandemic. This has already been observed for the COVID-19 pandemic where people of color have borne a disproportionate burden of mortality and morbidity of the disease16.\n\nAbiding by the tenet that public health is ultimately local in nature, we focused on modeling county level data about disease spread and county-level determinants. By linking confirmed infection numbers via growth curve modeling to a large number of area-level factors, we used random forest modeling to show interactions between area-level factors that associate with increased disease spread. These associations were quite different for urban versus rural counties. Urban county spread was more influenced factors like racial distributions (in particular the concentration of African American individuals in a county), population and housing density, international immigration patterns and cyclical weather patterns. Rural county spread was also influenced by similar racial composition, but also by poverty, the degree of elderliness and the degree of rurality.\n\nThe random forest models demonstrated objective goodness-of-fit on par with other studies that focused on area-level predictors alone14. These models can be particularly good at identifying complex interactions and so were well suited for this analysis. As noted, random forest imputation was performed and so we did a sensitivity analysis to confirm that the results were not overly influenced by the imputation scheme17.\n\nThere are some ways in which this analysis could be improved. First, we did not take into account particular county level interventions that were made over time - for example lockdowns. This data does in fact exist as another data file in the same repository at Johns Hopkins University. One strategy to incorporate this data would be to add in lockdown status as a time-varying covariate in the growth curve model and then re-estimate growth curve slopes. One strategy for this is to use group counties by lockdown dates and then use the technology of non-linear mixed models using additional covariates18. Further information that would have been useful to include in the model but was not available at the county level includes number of nursing home facilities and traffic flow or movement data. The nursing home facilities although somewhat presented in the age demographics of the county, are important in their own right since they often served as sites of localized case clusters. Traffic flow or movement data would indicate the degree to which lockdown measures were being adhered to. Lastly, one missing confounding variable is the uneven use of testing across different regions of the country. This would clearly affect the confirmed infection count time series numbers.\n\n\nData availability\n\nAll data are sourced from the GitHub deposit associated with Killeen et al.1: https://github.com/JieYingWu/COVID-19_US_County-level_Summaries\n\nThe specifc files required are:\n\ncounties.csv Contains attributes of the included counties used for predicting rates in random forest\n\ninfections_time.csv Contains the time series data for estimating the growth curves.",
"appendix": "Acknowledgements\n\nThe authors would like to thank Dr. Kevin Patel at the University of Washington for very helpful discussions.\n\n\nReferences\n\nKilleen BD, Wu JY, Shah K: A County-level Dataset for Informing the United States’ Response to COVID-19. 2020. Reference Source\n\nMorano JP, Holt DA: The social determinants of health contextualized for the zika virus. Int J Infect Dis. 2017; 65: 142–143. PubMed Abstract | Publisher Full Text\n\nDong E, Du H, Gardner L: An interactive web-based dashboard to track covid-19 in real time. Lancet Infect Dis. 2020; 20(5): 533–534. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBreiman L: Random forests. Machine Learning. 2001; 45: 5–32. Reference Source\n\nSprouffske K, Wagner A: Growthcurver: an r package for obtaining interpretable metrics from microbialgrowth curves. BMC bioinformatics. 2016; 17(1): 172. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCrow JF, Kimura M, et al.: An introduction to population genetics theory. An introduction to populationgenetics theory. 1970. Publisher Full Text\n\nRockwood LL: Rockwood: Introduction to population ecology. John Wiley & Sons, 2015. Reference Source\n\nKathleen sprouffske: growthcurver: Simple Metrics to Summarize Growth Curves. 2018. R package version 0.3.0. Reference Source\n\nBreiman L, Friedman J, Olshen R, et al.: Classification and Regression Trees. Wadsworth Books, 1984.\n\nIshwaran H, Kogalur UB: Fast Unified Random Forests for Survival, Regression, and Classification (RFSRC). 2020. R package version 2.9.3. Reference Source\n\nShah AD, Bartlett JW, Carpenter J, et al.: Comparison of random forest and parametric imputation models for imputing missing data using mice: a caliber study. Am J Epidemiol. 2014; 179(6): 764–774. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMantero A: Unsupervised Random Forests and Target Outcomes Relationship Exploration. PhD thesis, University of Miami, 2018. Reference Source\n\nRichardson HJ, Hill DJ, Denesiuk DR, et al.: A comparison of geographic datasets and field measurements to model soil carbon using random forests and stepwise regressions (british columbia, canada). GIScience & Remote Sensing. 2017; 54(4): 573–591. Publisher Full Text\n\nOliveira S, Oehler F, San-Miguel-Ayanz J, et al.: Modeling spatial patterns of fire occurrence in mediterranean europe using multiple regression and random forest. Forest Ecology and Management. 2012; 275: 117–129. Publisher Full Text\n\nBishwajit G, Ide S, Ghosh S: Social determinants of infectious diseases in south asia. Int Sch Res Notices. 2014; 2014: 135243. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLaurencin CT, McClinton A: The covid-19 pandemic: a call to action to identify and address racial and ethnic disparities. J Racial Ethn Health Disparities. 2020; 7(3): 398–402. PubMed Abstract | Publisher Full Text | Free Full Text\n\nResseguier N, Giorgi R, Paoletti X: Sensitivity analysis when data are missing not-at-random. Epidemiology. 2011; 22(2): 282. PubMed Abstract | Publisher Full Text\n\nPinheiro J: Model building using covariates in nonlinear mixed models. Journal de la societe francaise de statistque. 2002; 143: 79–101. Reference Source"
}
|
[
{
"id": "63855",
"date": "08 Jun 2020",
"name": "Susmita Datta",
"expertise": [
"Reviewer Expertise Biostatistics",
"Bioinformatics and Computational Biology"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this paper the authors modeled county level data about COVID-19 disease spread and county-level determinants as the disease spread is highly local. They linked confirmed infection numbers via growth curve modeling to a large number of area-level factors. They finally, used random forest modeling to model the estimated rate of infection to show interactions between area-level factors that associate with increased disease spread. They found interesting results such as the rate of change of the proportion of infected individuals in a county depends heavily on whether the counties are in urban or rural areas. These results are important for following the infection rate at a county level and helpful for developing possible mitigation strategy. I find that the paper is useful and interesting However, I have some specific minor comments for clarifications, typos and sentence constructions. I strongly, encourage the authors to read the manuscript carefully for the languages. Specific Comments:\nIntroduction, 2nd paragraph “how where” should be “how and where”. Please break up the sentence for a better understanding “In addition, with the realization that racial and ethnic minority groups are suffering disproportionately in the pandemic, social determinants of health (which are important contributors to health disparities) have garnered more attention.”\n\nPlease use some commas/and in “They then merged this contextual data with county-level time series data from the JHU (Johns Hopkins University) CSSE (Center for Systems Science and Engineering) COVID-19 dashboard3 “\n\nFor Random Forest forest regressions please consider changing “…..many subject matters.” into classification, regression etc. etc.\n\nLine 6 of the same section has “This results…..” should “The resulting….” Or “These results……”\n\nThe authors use “randomForestSRC” procedure for automatic missing value estimation. I wonder whether for a specific random forest iteration, it imputes only once or multiple times and also whether one can find the difference in the final result with respect to the proportion of missing data. Some comments regarding that will be helpful.\n\nPlease write the complete name of PAM for general readers.\n\nTable 2 has some empty values for relative importance, what does that mean?\n\nThere are still some correlated variables considered as the classifying variables, although you mentioned about removing them. Any comment on that?\n\nFigure 6 co-plot could be rescaled to understand better.\n\nSecond paragraph after Figure 7 talks about “rural forest”, what is that?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? 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/9-418
|
https://f1000research.com/articles/9-417/v1
|
21 May 20
|
{
"type": "Research Article",
"title": "Optimised patient information materials and recruitment to a study of behavioural activation in older adults: an embedded study within a trial",
"authors": [
"Peter Knapp",
"Simon Gilbody",
"Janet Holt",
"Ada Keding",
"Natasha Mitchell",
"David K. Raynor",
"Jonathan Silcock",
"David J. Torgerson",
"Simon Gilbody",
"Janet Holt",
"Ada Keding",
"Natasha Mitchell",
"David K. Raynor",
"Jonathan Silcock",
"David J. Torgerson"
],
"abstract": "Background: Printed participant information about randomised controlled trials is often long, technical and difficult to navigate. Improving information materials is possible through optimisation and user-testing, and may impact on participant understanding and rates of recruitment. Methods: A study within a trial (SWAT) was undertaken within the CASPER trial. Potential CASPER participants were randomised to receive either the standard trial information or revised information that had been optimised through information design and user testing. Results: A total of 11,531 patients were randomised in the SWAT. Rates of recruitment to the CASPER trial were 2.0% in the optimised information group and 1.9% in the standard information group (odds ratio 1.027; 95% CI 0.79 to 1.33; p=0.202). Conclusions: Participant information that had been optimised through information design and user testing did not result in any change to rate of recruitment to the host trial. Registration: ISRCTN ID ISRCTN02202951; registered on 3 June 2009.",
"keywords": [
"SWAT",
"trial",
"recruitment",
"patient information",
"user testing",
"behavioral activation"
],
"content": "Introduction\n\nPotential participants in randomised controlled trials are given information that is often long, technical and difficult to navigate1–3. Consequently, they may lack understanding of important details about the trial1,4,5, which limits their ability to make an informed decision about consent.\n\nImproving information materials is possible through optimisation and user-testing. This involves making changes to the design and text based on good practice in information design and people’s ability to find and understand information during testing6. Materials revised after user-testing have been shown to be preferred7,8, although a recent review concluded that optimised information has little or no impact on trial recruitment9. However, the evidence base remains limited10–13, and a recent ‘review of reviews’ reported that information for patients can be a facilitator of research participation14.\n\nThis embedded study within a trial (SWAT) assesses whether optimisation of patient information materials through user testing could increase participant recruitment to the CASPER study15.\n\n\nMethods\n\nThe SWAT was conducted within CASPER, which investigated the effectiveness of behavioural activation in patients aged 65 years or older with sub-threshold levels of depression15. CASPER used a cohort multiple randomised controlled trial design16.\n\nParticipants were registered patients at one of six UK medical practices in Durham, Harrogate, Leeds and York. They were included if they were potentially eligible for CASPER.\n\nAll participants in the SWAT were posted an invitation letter, participant information sheet (PIS), screening questionnaire and consent form for the CASPER trial. The control group received the standard CASPER developed PIS (see Extended data)17 whilst the intervention group were sent an optimised version (see Extended data)18 developed through three rounds of user testing and revision.\n\nPatients returned the questionnaire and a consent form indicating a willingness to participate, after which they were recruited to the CASPER cohort. Following a telephone diagnostic interview, eligible patients were recruited to the CASPER intervention trial.\n\nUser testing involved 30 people reflecting the CASPER target population. In the first round of testing, 10 participants read the standard invitation letter and PIS. They were then asked to locate and demonstrate their understanding of 18 items of information within the PIS (on the study’s nature and purpose; process and meaning of consent; study procedures; nature of the CASPER trial intervention). The PIS was then revised based on participant responses. A second round of testing was completed, in which 10 new participants read the invitation letter and a revised PIS and were asked to find and show understanding of the same 18 information items. The PIS was further revised and tested on 10 new participants through the same 18 information items.\n\nThrough testing, changes to the PIS included adding a title page, a summary of key points and a contents page, highlighting headings using colour and larger font, and simplifying wording. The final optimised PIS was printed as an A4 booklet (Figure 3).\n\nThe primary outcome measure was the proportion of patients in each group who were recruited to the CASPER trial. The secondary outcomes were (i) the proportion of patients recruited to the CASPER cohort, and (ii) the proportion of invited patients returning forms to express interest in participation in CASPER.\n\nIt was predicted that 30% of invited patients would return the consent form and indicate interest in CASPER participation, of whom 20% (600) would be eligible to take part in the CASPER trial. An improvement in response rate of 10% (i.e. from 30% to 33% participants) would be a significant increase in uptake. A sample size of 8,000 potential participants would be sufficient at 80% power to detect a difference of 10% in recruitment rate.\n\nIndividual patients were allocated randomly (1:1) to receive either the standard or optimised PIS by an independent statistician at York Trials Unit.\n\nOdds ratios (ORs) and corresponding 95% confidence intervals (CIs) were calculated to compare the proportion of patients from each group that were recruited to the CASPER trial; recruited to the CASPER cohort; or expressed interest in participation. Analyses were conducted in Stata version 14.2.\n\nCASPER and the SWAT were approved by the NHS Leeds North-East Research Ethics Committee (10/H1306/61).\n\n\nResults\n\nOverall, 11,531 patients were invited to participate19; 5,765 (50.0%) were randomised to the optimised PIS and 5,766 (50.0%) to the standard PIS (Figure 1).\n\nA total of 2,169 patients returned the consent form indicating a willingness to take part: 1,102 (19.1%) in the optimised PIS group and 1,067 (18.5%) in the standard PIS group (odds ratio (OR) 1.04; 95% confidence interval (CI) 0.95 to 1.14; p=0.402).\n\nA total of 229 patients were recruited to the CASPER trial: 116 (2.0% of those invited) in the optimised PIS group and 113 (1.9%) in the standard PIS group (OR 1.027; 95% CI 0.79 to 1.33; p=0.202).\n\nIn total, 1,667 patients expressed interest in participating but were ineligible for the CASPER trial and were recruited to the CASPER cohort: 851 (14.8% of those invited) in the optimised PIS group, and 816 (14.1%) in the standard PIS group (OR 1.05; 95% CI 0.95 to 1.16).\n\n\nDiscussion\n\nOptimisation of the PIS resulted in no statistically significant difference in the rates of recruitment to the CASPER trial or CASPER cohort, or rates of consent form returns. This is consistent with previous research9, including other embedded trials within the MRC START programme, which have observed little or no effect on recruitment11–13,20.\n\nWhilst there was no impact on recruitment, the optimised materials may have improved understanding of the trial thus enabling patients to make a more informed decision. Improved comprehension could also increase retention, due to greater understanding of the trial prior to recruitment. These outcomes were not assessed and further research examining this is warranted.\n\n\nConclusion\n\nOptimised patient information materials did not increase recruitment to the host trial or expressions of interest in participation.\n\n\nData availability\n\nFigshare: CASPER SWAT data.csvCASPER SWAT recruitment data and evaluated information sheets. https://doi.org/10.6084/m9.figshare.1230267220.\n\nThis project contains the underlying data\n\nFigshare: Figure 2 CASPER PIS (original). https://doi.org/10.6084/m9.figshare.1230267517.\n\nThis file is the original CASPER participant information sheet.\n\nFigshare: Figure 3 CASPER PIS (revised). https://doi.org/10.6084/m9.figshare.1230267818.\n\nThis file is the revised CASPER participant information sheet.\n\nFigshare: CONSORT checklist for ‘Optimised patient information materials and recruitment to a study of behavioural activation in older adults: an embedded study within a trial’. https://doi.org/10.6084/m9.figshare.12312206.v121.\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 also like to thank the embedded trial participants and the CASPER study team, particularly Helen Lewis. We thank Luto Research Limited (luto.co.uk) for undertaking the user testing, and Making Sense (makingsense.co.uk) for graphic design input.\n\n\nReferences\n\nLarson E, Foe G, Lally R: Reading level and length of written research consent forms. Clin Transl Sci. 2015; 8(4): 355–356. PubMed Abstract | Publisher Full Text | Free Full Text\n\nManta CJ, Ortiz J, Moulton BW, et al.: From the Patient Perspective, Consent Forms Fall Short of Providing Information to Guide Decision Making. J Patient Saf. 2016. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMontalvo W, Larson E: Participant comprehension of research for which they volunteer: a systematic review. J Nurs Scholarsh. 2014; 46(6): 423–431. PubMed Abstract | Publisher Full Text\n\nGriffin JM, Struve JK, Collins D, et al.: Long term clinical trials: how much information do participants retain from the informed consent process?. Contemp Clin Trials. 2006; 27(5): 441–448. PubMed Abstract | Publisher Full Text\n\nFortun P, West J, Chalkley L, et al.: Recall of informed consent information by healthy volunteers in clinical trials. QJM. 2008; 101(8): 625–629. PubMed Abstract | Publisher Full Text\n\nRaynor DK, Knapp P, Silcock J, et al.: \"User-testing\" as a method for testing the fitness-for-purpose of written medicine information. Patient Educ Couns. 2011; 83(3): 404–10. PubMed Abstract | Publisher Full Text\n\nKnapp P, Raynor DK, Silcock J, et al.: Performance-based readability testing of participant information for a Phase 3 IVF trial. Trials. 2009; 10(1): 79. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKnapp P, Raynor DK, Silcock J, et al.: Can user testing of a clinical trial patient information sheet make it fit-for-purpose?--a randomized controlled trial. BMC Med. 2011; 9: 89. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTreweek S, Pitkethly M, Cook J, et al.: Strategies to improve recruitment to randomised trials. Cochrane Database Syst Rev. 2018; 2: Mr000013. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChen F, Rahimi K, Haynes R, et al.: Investigating strategies to improve attendance at screening visits in a randomized trial. Trials. 2011; 12(1): A111. Publisher Full Text | Free Full Text\n\nMan MS, Rick J, Bower P, et al.: Improving recruitment to a study of telehealth management for long-term conditions in primary care: two embedded, randomised controlled trials of optimised patient information materials. Trials. 2015; 16(1): 309. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCockayne S, Fairhurst C, Adamson J, et al.: An optimised patient information sheet did not significantly increase recruitment or retention in a falls prevention study: an embedded randomised recruitment trial. Trials. 2017; 18(1): 144. PubMed Abstract | Publisher Full Text | Free Full Text\n\nParker A, Knapp P, Treweek S, et al.: The effect of optimised patient information materials on recruitment in a lung cancer screening trial: an embedded randomised recruitment trial. Trials. 2018; 19(1): 503. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSheridan R, Martin-Kerry J, Hudson J, et al.: Why do patients take part in research? An overview of systematic reviews of psychosocial barriers and facilitators. Trials. 2020; 21: 259. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGilbody S, Lewis H, Adamson J, et al.: Effect of Collaborative Care vs Usual Care on Depressive Symptoms in Older Adults With Subthreshold Depression: The CASPER Randomized Clinical Trial. JAMA. 2017; 317(7): 728–737. PubMed Abstract | Publisher Full Text\n\nRelton C, Torgerson D, O’Cathain A, et al.: Rethinking pragmatic randomised controlled trials: introducing the “cohort multiple randomised controlled trial” design. BMJ. 2010; 340: c1066. PubMed Abstract | Publisher Full Text\n\nKnapp P, Gilbody S, Holt J, et al.: Figure 2 CASPER PIS (original). figshare. Figure. 2020. http://www.doi.org/10.6084/m9.figshare.12302675.v1\n\nKnapp P, Gilbody S, Keding A, et al.: Figure 3 CASPER PIS (revised). figshare. Figure. 2020. http://www.doi.org/10.6084/m9.figshare.12302678.v1\n\nKnapp P, Gilbody G, Holt J, et al.: Optimised patient information materials and recruitment to a study of behavioural activation in older adults: an embedded study within a trial (dataset). Publisher Full Text\n\nRick J, Graffy J, Knapp P, et al.: Systematic techniques for assisting recruitment to trials (START): study protocol for embedded, randomized controlled trials. Trials. 2014; 15: 407. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKnapp P, Gilbody S, Holt J, et al.: CONSORT 2010 Checklist Knapp et al. figshare. Journal contribution. 2020. http://www.doi.org/10.6084/m9.figshare.12312206.v1"
}
|
[
{
"id": "67160",
"date": "30 Jul 2020",
"name": "Mike Clarke",
"expertise": [
"Reviewer Expertise Health services research. Randomized trials. Systematic reviews. Methodology research."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an impressive and important piece of methodology research, which has been reported in a clear and succinct manner. I am pleased to have been asked to review it.\nThe reported randomized trial is an example of a very large SWAT, that was embedded in the CASPER trial of behavioural activation therapy for older patients with sub-threshold levels of depression, to test the impact on recruitment of an optimized information materials for people being invited to consider participation in the clinical trial.\nIt might be helpful to readers if the authors could include a little more information about the SWAT concept, perhaps by citing one or both of the relevant Trial Forge papers: (a) Treweek S, Bevan S, Bower P, et al. Trial Forge Guidance 1: what is a Study Within A Trial (SWAT)? Trials 2018; 19: 139; and (b) Treweek S, Bevan S, Bower P, et al. Trial Forge Guidance 2: how to decide if a further Study Within A Trial (SWAT) is needed. Trials 2020; 21: 33.\nAlthough the optimized information materials did not improve the proportion of invitees who were recruited into CASPER, it would be interesting to know if there were other benefits, such as a better understanding of the clinical trial. The paper concludes with some comments about this but the authors' comment that “further research examining this is warranted” seems to suggest that this type of qualitative research might not be done with the CASPER trial patients. However, given that they will be collecting retention data for CASPER, I hope they will revisit this SWAT later in the CASPER trial to answer their own question about the potential for an impact on retention.\nIf they have not already done so, I suggest that the authors register this SWAT in the SWAT repository. It might also help readers if they referred to two similar SWAT that are already registered there (SWAT 101 and SWAT 105).\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "67713",
"date": "03 Aug 2020",
"name": "Shaun P. Treweek",
"expertise": [
"Reviewer Expertise Randomised trial methodology",
"including SWATs."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a well-written article describing a large, well done SWAT. I only have a few comments, which are listed below.\nStudy aims The authors might want to add a sentence (and possibly a reference) that explains in brief what a SWAT is.\nMethods Design– linked to the host trial ‘cohort multiple randomised controlled trial design’. This design is sometimes called a ‘Trials Within Cohort (TwiCs)' design. The authors might want to mention this name too, just in case readers are more familiar with it.\n\nUser testing– the authors mention users were asked to locate and demonstrate understanding of 18 items of information within the participant information leaflet. I wasn’t sure what the 18 items were. Would it be possible to list them? Apologies if I have missed them within the article or extended data.\nRandomisation– could the authors give a little more information on how the randomisation sequence was generated?\nDiscussion The authors mention that their results are consistent with previous research, which is my understanding of the literature too. Do the authors think further evaluation of this SWAT evaluation is required, and if so, could they give some pointers to what sorts of evaluations would add most value to the evidence base?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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/9-417
|
https://f1000research.com/articles/9-416/v1
|
21 May 20
|
{
"type": "Research Article",
"title": "Analysis of the health product pipeline for poverty-related and neglected diseases using the Portfolio-to-Impact (P2I) modeling tool",
"authors": [
"Shashika Bandara",
"Nick Chapman",
"Vipul Chowdhary",
"Anna Doubell",
"Amelia Hynen",
"George Rugarabamu",
"Alexander Gunn",
"Gavin Yamey",
"Shashika Bandara",
"Nick Chapman",
"Vipul Chowdhary",
"Anna Doubell",
"Amelia Hynen",
"George Rugarabamu",
"Alexander Gunn"
],
"abstract": "Background: To estimate how much additional funding is needed for poverty-related and neglected disease (PRND) product development and to target new resources effectively, policymakers need updated information on the development pipeline and estimated costs to fill pipeline gaps. Methods: We previously conducted a pipeline review to identify candidates for 35 neglected diseases as of August 31, 2017 (“2017 pipeline”). We used the Portfolio-to-Impact (P2I) tool to estimate costs to move these candidates through the pipeline, likely launches, and additional costs to develop “missing products.” We repeated this analysis, reviewing the pipeline to August 31, 2019 to get a time trend. We made a direct comparison based on the same 35 diseases (“2019 direct comparison pipeline”), then a comparison based on an expanded list of 45 diseases (“2019 complete pipeline”). Results: In the 2017 pipeline, 538 product candidates met inclusion criteria for input into the model; it would cost $16.3 billion (B) to move these through the pipeline, yielding 128 launches. In the 2019 direct comparison pipeline, we identified 690 candidates, an increase of 152 candidates from 2017; the largest increase was for Ebola. The direct comparison 2019 pipeline yields 196 launches, costing $19.9B. In the 2019 complete pipeline, there were 754 candidates, an increase of 216 candidates from 2017, of which 152 reflected pipeline changes and 64 reflected changes in scope. The complete pipeline 2019 yields 207 launches, costing $21.0B. There would still be 16 “missing products” based on the complete 2019 pipeline; it would cost $5.5B-$14.2B (depending on product complexity) to develop these products. Conclusion: The PRNDs product development pipeline has grown by over a quarter in two years. The number of expected new product launches based on the 2019 pipeline increased by half compared to 2017; the cost of advancing the pipeline increased by a quarter.",
"keywords": [
"neglected diseases",
"product development",
"emerging infections",
"research and development",
"research funding"
],
"content": "Introduction\n\nA growing body of evidence has shown that the ambitious global health goals established by the international health community, including the health-related Sustainable Development Goals (SDGs), are unlikely to be achieved with today’s health technologies alone1–4. New breakthrough technologies will be needed to accelerate the current rate of mortality reduction.\n\nFor example, the 2013 report of the Lancet Commission on Investing in Health (CIH) showed that the development and widespread deployment of new technologies would be needed to achieve a “grand convergence” in global health1. The CIH defined grand convergence as a universal reduction in deaths from infections and maternal and child health conditions down to the mortality levels achieved in 2011 by the best-performing middle-income countries (e.g. China and Chile)1. Unfortunately, progress on mortality from several conditions has been slow since this report was published. If the global trends in maternal mortality and mortality from tuberculosis (TB) achieved in 2010–16 were to continue, the convergence targets would not be achieved until 2067 for maternal mortality and 2074 for TB mortality, respectively4 . This slow progress underscores the need for new medicines, vaccines, diagnostics, and other disease control tools.\n\nThere are several signs suggesting that policymakers at global and national levels do recognize the importance of funding product development for poverty-related and neglected diseases. First, funding levels for health research and development (R&D) were included in the indicators that are linked to the SDG health targets. For example, SDG Indicator 3.b.2 includes “official development assistance (ODA) for medical research and basic health sectors as a % of gross national income (GNI) and as a % of all ODA, by donor country.” SDG Indicator 9.5.1 is “gross domestic R&D expenditure on health (health GERD) as a % of gross domestic product (GDP).” Second, the new Global Action Plan for Healthy Lives and Wellbeing for All, which commits 12 multilateral health and development organizations to improving their collaboration on achieving the SDGs, includes an effort to scale up health R&D. Third, several new initiatives have been launched in recent years to mobilize additional funding for global health product development, such as the Global Health Innovative Technology Fund and the Coalition for Epidemic Preparedness Innovations5. Fourth, a number of new global health R&D coordination mechanisms have also been launched, such as the Global Antimicrobial Resistance R&D Hub and the World Health Organization’s Global Observatory on Health R&D5.\n\nTo estimate how much additional funding is needed for global health product development and to target any new resources effectively, policymakers need up-to-date information on the status of the development pipeline and the estimated costs to fill gaps in the pipeline. In particular, as we have previously argued, they need information on “which candidates are currently in the pipeline and at what development phase; the estimated costs to accelerate this portfolio of candidates to production; the anticipated product launches that would result from such acceleration; and the critical, highly needed products that would still be “missing” under the status quo.”6\n\nIn order to generate such information, in 2018 we published the first ever study to use a new financial modeling tool, called the Portfolio-to-Impact (P2I) tool, that can be used to estimate the costs of moving a portfolio of candidates along the pipeline6. In that 2018 study, we first conducted a comprehensive, rigorous pipeline portfolio review to identify product candidates for 35 neglected diseases as of August 31, 2017. We found 538 candidates that met inclusion criteria for input into the modeling tool. Using this tool, we estimated that it would cost about $16.3 billion (range $13.4B-19.8B) to move these 538 candidates through the pipeline, with three-quarters of the costs incurred in the first 5 years, resulting in about 128 (89-160) expected product launches by 2029.\n\nAmong these launches, our modeling suggested that there would be few launches of complex new chemical entities (NCEs). We also found that, based on the pipeline as of August 31, 2017, launches of highly efficacious HIV, TB, or malaria vaccines would be unlikely. We estimated that the additional costs to launch one of each of 18 priority “missing” products would be $13.6B, assuming lowest product complexity or $21.8B assuming highest complexity ($8.1B-36.6B).\n\nIn this current study, we have conducted a new pipeline portfolio review and we have updated the cost modeling. There are several reasons why this updated study was needed. First, the product development pipeline is never static—it changes frequently—and so we wanted to gain an understanding of how the pipeline has changed. We conducted a new pipeline review up to August 31, 2019, i.e. exactly two years on from our last study, allowing us to show how much change can be expected over such a two-year period. Second, we wanted to assess whether the prospects have improved for launching critically needed products, such as vaccines for HIV, TB, and malaria. Third, our new study was timely given the launch of a new target product profile (TPP) directory developed by the Special Programme for Research and Training in Tropical Diseases (TDR). We hoped that we could potentially compare our new findings on product gaps with the gaps identified in the new TPP directory. Finally, we wanted the new findings to help identify the most promising ways in which the pipeline can be better managed to accelerate the development and deployment of critically needed health technologies for poverty-related and neglected diseases.\n\n\nMethods\n\nIn this section, we begin, in sub-section (i), by summarizing the development of the P2I tool. While the tool development has previously been published in detail6,7, we wanted to summarize the key steps so that readers could understand the underlying principles of—and the assumptions and data sources underlying—the tool. In sub-section (ii), we describe how we conducted the pipeline portfolio review and in (iii) we explain how we classified each candidate into its product type (which we call “archetype”, e.g. simple NCE, complex NCE). In sub-section (iv) we describe how we inputted the pipeline of candidates into the P2I model. In sub-section (v), we explain how we estimated the costs of priority “missing” products. Finally, in sub-section (vi), we describe our sensitivity analysis.\n\nThe P2I model is a deterministic modeling tool that enables users to estimate funding needs to move a portfolio of candidate health products through the pipeline from advanced preclinical to phase III clinical trials, as well as potential product launches over time. The first version of the P2I tool, called P2I version 1 (P2I.v1) is a Microsoft Excel-based tool that is freely available online7. In the first study to apply this tool to the pipeline of candidate products for neglected diseases, we made a number of adaptations to P2I.v1, and called the adapted version P2I version 2 (P2I.v2); this adapted version is also an open access Microsoft Excel-based tool6. Both the P2I.v1 and P2I.v2 models are based on assumptions for costs, attrition rates (probability of success), and cycle times per phase for four phases of research—advanced preclinical to phase III—for different product types (archetypes). Figure 1 shows a conceptual overview of the P2I model, and Figure 2 shows which phases are included.\n\nThis figure is taken with permission from Young R, Bekele T, Gunn A et al. Developing new health technologies for neglected diseases: a pipeline portfolio review and cost model. Gates Open Research 2018, 2:23.\n\nThis figure has been adapted with permission from Health Product Research and Development Fund: A Proposal for Financing and Operation. WHO/TDR, 2016.\n\nThe P2I.v1 model has 11 archetypes: simple or complex vaccines; simple or complex NCEs; simple, innovative, or complex repurposed drugs; simple or complex biologics; and two diagnostic archetypes—assay development or simple technical platform development. We made a number of adaptations in the development of P2I.v2, including the addition of “unprecedented vaccines” (for HIV, TB, and malaria) and vector control products. Table 1 describes the 14 archetypes in the P2I.v2 model, which is the version we have used in this new study.\n\nAdapted from Young et al. (2018)6 under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).\n\nFor each of the 14 different archetypes in P2I.v2, the model has assumptions on costs, attrition rates, and cycle time per phase. As described in detail in Terry et al, assumptions on development costs for each phase of product development for the archetypes included in the P2I.v1 model were initially based on an analysis of clinical trial costs from Parexel’s R&D cost sourcebook7. The assumptions on attrition rates and cycle times for each phase were initially based on the historical attrition rates and cycle times of more than 25,000 development candidates. All of the assumptions were further refined and validated based on (i) academic literature, (ii) industry publications and databases, and (iii) 133 stakeholder interviews with a wide variety of product development partnerships (PDPs), pharmaceutical companies, and major funders of global health R&D7. As described in detail in reference 6, additional sources of assumptions for the new archetypes in P2I.v2, such as vector control products and unprecedented vaccines, were the McKinsey Risk-Adjusted Portfolio Model and clinical trial data shared by the Bill & Melinda Gates Foundation6.\n\nIn order to use the model to analyse the pipeline of product candidates for poverty-related and neglected diseases, each candidate must first be classified into its archetype and phase, and then these candidates can be inputted into the model. There are two main model outputs. The first is “launches”; in this paper, the term launch refers to a candidate making it through phase III and thus being ready for the next steps, e.g. the regulatory and manufacturing steps. The second is the total costs to move all candidates through the pipeline from their current phase from now to 2031 (the model also gives a breakdown of these total costs into annual costs by year, from 2019 to 2031).\n\nThe model only includes candidates in advanced preclinical to phase III research, and thus the cost estimates are an under-estimate of the full costs of product development. In particular, the model excludes all costs related to basic research through lead optimization; chemistry, manufacturing, and controls; good manufacturing practice; manufacturing, build-up, and scale-up costs; regulatory or registration fees (post-phase III); and all post-market commitments (e.g., phase IV pharmacovigilance studies).\n\nWe undertook a global review of candidate products for neglected diseases, Ebola and sexual & reproductive health issues in order to capture a snapshot of the R&D pipeline as of August 31, 2019 (“2019 review”); this followed a previously published review6 of the R&D pipeline as of August 31, 2017 (“2017 review”).\n\nThe scope of candidate inclusion aligned with the G-FINDER survey on global funding for R&D for neglected diseases, emerging infectious diseases and sexual & reproductive health issues, conducted by Policy Cures Research. The G-FINDER scope is based on three key principles: that the disease or health issue disproportionately affects low- and middle-income countries; that there is a need for new products (i.e. there is no existing product, or improved or additional products are needed); and that there is a market failure (i.e. there is insufficient commercial market to attract private R&D investment).\n\nThe scope of the G-FINDER project is reviewed annually in consultation with an expert Advisory Committee. As a result of these consultations, and following an initiative to survey the landscape of sexual and reproductive health R&D funding, the disease scope used for the pipeline review in 2019 was wider than in 2017, with the addition of the following diseases: chlamydia, gonorrhea, hepatitis B, HPV-cervical cancer, human T-cell lymphotropic virus-1, herpes simplex-2, mycetoma, pre-eclampsia, post-partum hemorrhage and syphilis. Snakebite envenoming was added to the G-FINDER scope after the conclusion of the 2019 pipeline review and was therefore not included in the scope of this analysis. “Reproductive health”—which included both contraception and the prevention of sexually transmitted diseases through the use of multipurpose prevention technologies (MPTs) that prevent pregnancy and sexually transmitted infections—was included as a single health issue.\n\nAccordingly, the R&D pipeline review presented here comprises 45 individual disease or health issues and six multiple-disease groups (Underlying data: Data File 1 shows the full list of diseases included in the pipeline reviews for both 2017 and 2019).\n\nThe G-FINDER scope includes drugs, microbicides, biologics, vaccines, diagnostics, vector control products, devices and MPTs. Not all product types are included for every disease or health condition: the product category is excluded if a viable commercial market is thought to exist, and additional restrictions are applied to some disease and product categories if there is potential commercial (high-income country market) overlap. For example, the G-FINDER scope excludes dengue vaccines, and HIV/AIDS drugs are included only if they are label-extensions or reformulations specifically intended for developing country use (e.g., pediatric or slow-release formulations; fixed dose combinations; low-dose formulations for prophylaxis; long-acting injectables for treatment or prophylaxis). Medical devices (except for diagnostics and contraceptives), and general or supportive therapies (e.g., oral rehydration or nutritional supplements) are not included in the scope of G-FINDER and were therefore not included in the pipeline reviews. Due to changes in the G-FINDER scope between 2017 and 2019, as described above, two disease-specific product areas were included for the first time in 2019: vaccines for leprosy, and biologics for HIV.\n\nThe pipeline review included candidates at all stages of development, from discovery through to product registration. Drugs, vaccines, and reproductive health candidate products were classified in the following five development stages: discovery; pre-clinical studies; and clinical development (Phase I, Phase II and Phase III). Diagnostics and vector control products follow different product development and regulatory pathways, so these candidate products were classified under the following stages: concept and research; feasibility and planning; design and development; and clinical validation and launch readiness. Candidates were no longer considered to be part of the R&D pipeline – and therefore were excluded from this analysis – once granted regulatory approval by a stringent regulatory authority, or if their development had been placed on hold indefinitely. Candidates in the discovery phase were also excluded, as only candidates undergoing advanced pre-clinical development up to Phase III are included in the P2I.v2 model.\n\nFurther details on the diseases and product areas within the scope of the pipeline analysis are provided in the G-FINDER R&D matrices and scope documents.\n\nWe reviewed and cross-referenced all major sources of available data on the R&D pipeline within the scope outlined above. Sources included: the G-FINDER R&D funding database; the World Health Organization “rainbow tables”; publications of the WHO Initiative for Vaccine Research (IVR) which included background documents and meeting reports and presentations prepared for the WHO Product Development for Vaccines Advisory Committee (PDVAC) and Strategic Advisory Group of Experts (SAGE) on immunizations; reports of the WHO Vector Control Advisory Group (VCAG) reports; WHO R&D roadmaps; the WHO vaccine pipeline tracker; WHO prequalification lists; multiple Unitaid landscape and technical reports; multiple clinical trial portals including ClinicalTrials.gov, the AIDSinfo clinical trial database, the Chinese Clinical Trial Registry, the Clinical Trials Registry – India and the WHO International Clinical Trials Registry Platform; disease-specific pipeline updates by the HIV Vaccines Trials Network and the Treatment Action Group; publicly available company and product development partnership R&D portfolios; journal publications; conference and meeting proceedings; and university, government, and non-profit organization websites.\n\nCandidates were only included if an authoritative source could confirm they were in active development. The following sources were considered to be authoritative:\n\n▪ the website of the candidate developer, if recently updated\n\n▪ recent reports or other materials from international organizations such as WHO and Unitaid\n\n▪ clinical trial portals\n\n▪ correspondence with product developers\n\n▪ correspondence with experts in the field, including FIND; the Innovative Vector Control Consortium (IVCC); the International AIDS Vaccine Initiative (IAVI); Netherlands Leprosy Relief; Program for Appropriate Technology in Health (PATH); the Sabin Vaccine Institute; and the US National Institute of Allergy and Infectious Diseases (NIAID).\n\nIn the pipeline portfolio review, we identified a total of 1160 product candidates for neglected diseases, Ebola and sexual & reproductive health as of August 31, 2019. We excluded 406 of these from the model because (a) they were already marketed, or in a development phase that is excluded from the P2I.v2 model (n=230), (b) there was insufficient information about the candidate from an authoritative source to be able to confirm its development phase or classify it into an archetype (n=10), or (c) the candidate was no longer in development (n=166). After these exclusions, 754 candidates were included in the model.\n\nWe then classified each of the included candidates into six primary archetypes: repurposed drugs, NCEs, vaccines, biologics, diagnostics, and ‘other products’ (which included only vector control products). Candidates classified as repurposed drugs, NCEs, or biologics were further classified as either simple or complex (with a further distinction for simple NCEs and simple biologics targeting TB); vaccine candidates were classified as either simple, complex, or unprecedented; and diagnostic candidates were classified as either assay development or simple platforms. This resulted in each candidate being assigned to one of the 14 unique archetypes outlined in Table 1.\n\nContraceptives, microbicides and MPTs were classified according to the constituent molecule (NCE, repurposed drug, or biologics). If there was more than one active drug ingredient in the MPT, the candidate was classified according to the most complicated component; we did not consider if the polymer or technology itself was innovative. TB drug candidates beyond Phase IIa were listed as regimens instead of individual compounds because TB treatment is regimen-based rather than monotherapy. Eight diagnostics candidates that were classified in the 2017 review as simple platforms were reclassified as assay development, reflecting the fact that ongoing activity was restricted to disease-specific assay development based on a previously validated proprietary diagnostic test technology.\n\nThe evidence used to support the classification of candidates into archetypes came from a wide range of sources, including academic literature (both original research and review articles); relevant publicly available product databases such as NCATS Inxight: Drugs; information from international clinical trials registries, including ClinicalTrials.gov and the WHO International Clinical Trials Registry Platform; websites of PDPs; patent databases; and relevant reports and news releases from bilateral and multilateral funding agencies, companies, PDPs, other product developers, and non-government organizations. In assigning each candidate product to an archetype, we documented any relevant source material that guided the classification, such as a peer-reviewed research article on the candidate’s mechanism of action. Regimens were classified according to the complexity of the most complicated component.\n\nFollowing the pipeline portfolio review and classification of product candidates into their archetypes, the candidates that met our inclusion criteria were inputted into P2I.v2. For each disease and archetype, we entered the number of candidates that were in each phase of development at the time of our review. For the candidates classified as repurposed drugs, NCEs, biologics, vaccines, and “other products” (vector control products), the phases were preclinical, phase I, phase II, or phase III. For diagnostic candidates, the phases were concept and research, feasibility and planning, design and development, or clinical validation and launch readiness. The analysis was conducted in November 2019, and hence we selected 2019 as the start year for the modeling.\n\nBased on the previously established parameters for cost, attrition rate, and time per phase for each archetype, the model estimates the costs and outcomes of moving product candidates through the pipeline from their current phase. When a candidate is classified into a particular phase in the model, it is assumed that it is at the beginning of the phase, which means that the cost of moving that candidate through to the end of its current phase is included in the total estimated costs for any given product.\n\nWe did not apply a discount rate to our cost estimates. Probability of success, length of phase, cost variables, and archetype were assumed to remain constant throughout the lifecycle of the model. Only candidates that met our inclusion criteria at the time of the pipeline portfolio review were included, meaning that the analysis did not include new candidates entering the pipeline over time.\n\nFor this paper, we have chosen a conservative approach to presenting the launches—we have considered a launch to be a binary event, i.e., we have rounded down (in this case, 2.7 rounds down to 2 launches). However, in Underlying data: Data File 2, we also present the results without any rounding (e.g., in this example, 2.7 launches) and with rounding to the nearest integer (2.7 would round to 3). Both of these other approaches give less conservative estimates of the number of launches. Where rounding was used, it was only applied at the very end of the model. For example, for disease X, if there were 3 simple vaccine candidates at Phase II that led to 1.3 expected launches and 3 simple vaccine candidates at Phase III that led to 1.4 expected launches, we rounded the cumulative total (in this case 2.7).\n\nIn this paper, we call the current pipeline of candidates the “2019 pipeline” (i.e. the pipeline of candidates as of August 31, 2019). We call the previously identified pipeline, published by Young et al, the “2017 pipeline” (i.e. the pipeline as of August 31, 2017)6. As described below, we conducted two distinct analyses. First, we did an analysis to obtain a time trend—that is, we examined changes over time in the number of candidates in the pipeline, the estimated costs to move them through the pipeline, and the likely resulting launches. For this time trend analysis, we used the exact same scope of diseases—an “apples to apples” comparison (we call this the “direct comparison” between the 2017 and 2019 pipelines). We call the 2019 pipeline used for the direct comparison analysis the “2019 direct comparison pipeline.” Second, for the 2019 pipeline, we also used an expanded list of diseases. We call the pipeline used for the complete 2019 analysis the “complete 2019 pipeline.”\n\nThe direct (“apples-to-apples”) comparison analysis between the 2017 and 2019 pipelines. The direct comparison analysis included product candidates in the 2019 pipeline that were identified using the exact same scope and search methodology used in 20176. As described in the Results section, we found a higher number of candidates in the 2019 direct comparison pipeline than in the 2017 pipeline. Since we used the same approach to identify candidates for both the 2017 and 2019 direct comparison pipelines, the higher number of overall candidates identified in 2019 was due to “true” changes in the pipeline since 2017. Despite the overall increase, when disaggregated by disease and by archetype the pipeline showed both increases and decreases in the numbers of candidate products across different diseases/archetypes. The product candidates included in the 2019 direct comparison pipeline belonged to the exact same 35 diseases and 5 multiple disease categories as those included in the 2017 pipeline analysis6.\n\nThe complete 2019 pipeline analysis. The complete 2019 pipeline analysis included all candidates from the pipeline portfolio review that met our inclusion criteria based on an expanded scope of diseases (45 diseases and 6 multiple disease categories). Again, as described in the Results section, we found a higher number of candidates in the complete 2019 pipeline than in the 2017 pipeline, but in this case the increase was partly due to the expansion of the disease scope, as well as true changes in the pipeline. Additionally, eight diagnostic candidate archetype classifications were changed between the 2019 direct comparison pipeline and the complete 2019 pipeline. These were existing candidates captured in the 2017 pipeline, and so their archetype classification was left unchanged in the direct comparison analysis to allow a true apples-to-apples comparison. This classification was updated for the complete pipeline analysis to reflect the fact that ongoing development activity for these candidates was now restricted to disease-specific assay development based on a previously validated proprietary diagnostic test technology.\n\nAs described in the Results section below, the complete 2019 pipeline is unlikely to lead to launches of several critically needed technologies (e.g. a hepatitis C vaccine). To estimate the costs to develop products that are likely to be “missing” but are highly needed, we used the same methodology adopted by Young et al.6 Young et al. used the list of “important” or “game changing” diagnostics, drugs, and vaccines prioritized by the CIH, which was developed through expert consensus1. We examined the overlap between the CIH’s list of needed products and those that our modeling suggested would still be missing by 2031 based on the complete 2019 pipeline. For each missing product, we used the P2I.v2 model in a retrospective manner to estimate the number of additional candidates that would be needed at preclinical phase—over and above the existing candidates—to lead to one expected launch of that product, and the associated additional cost.\n\nFor example, we found 39 malaria vaccine candidates in the pipeline and the modeling suggested that these would result in 0.41 launches. Thus, to estimate the additional costs to reach one launch, we estimated the number of additional candidates needed at preclinical phase and the associated additional costs to achieve an additional 0.59 launches (in this case, an additional 144 candidates would be needed at preclinical phase to achieve 0.59 launches, at an additional cost of $3.3 billion).\n\nFor the sensitivity analysis we adopted a method detailed by Mestre-Ferrandiz et al. at the United Kingdom Office of Health Economics in their study “The R&D Cost of a New Medicine”8. Young et al. also used this method6. Based on this methodology, we altered the model’s parameters for cost per phase and attrition per phase for each archetype by 10 percent higher and 10 percent lower. We also examined the impact of all possible combinations of these changes (e.g., 10% higher probability of success per phase and a 10% higher cost per phase, 10% higher probability of success per phase and a 10% lower cost per phase, etc.). We conducted this sensitivity analysis for moving current candidates through the pipeline both in our direct comparison analysis and our analysis of the complete 2019 pipeline. Additionally, we conducted this sensitivity analysis for the costs of developing priority “missing” products. Since the length of time is independent of the cost variables in P2I model, we did not conduct a sensitivity analysis varying the length of time per phase.\n\n\nResults\n\nFor the direct comparison analysis (the “apples-to-apples” approach), we only included candidates that were identified using the same method and scope used in 2017, as described in sub-section (ii) above. Using these inclusion criteria, we identified 690 candidates in the 2019 direct comparison pipeline that met the inclusion criteria for being entered into the P2I.v2 tool, an increase of 152 candidates from 2017 (see Underlying data: Data File 3).\n\nTable 2 shows the change in the number of candidates from 2017 to 2019 due to changes in the pipeline. The largest increase was for diagnostics, an increase of 67 candidates from 2017 to 2019, followed by vaccines (increase of 48 candidates), biologics (increase of 19 candidates), and repurposed drugs (increase of 13 candidates). For NCEs, there was a reduction in the number of candidates (2 fewer candidates in 2019 than in 2017).\n\nTable 3 compares candidates between the 2017 and 2019 direct comparison pipelines by disease and indicates the change in the number of candidates from 2017 to 2019 due to changes in the pipeline. The largest increase in the number of candidates was for Ebola (an increase of 62 candidates), followed by reproductive health (28), and TB (22). The largest decreases were for HIV/AIDS (a decrease of 10 candidates), schistosomiasis (7) and non-typhoid Salmonella (3).\n\nThe complete 2019 pipeline included a total of 754 product candidates that were identified in the pipeline portfolio review and met the inclusion criteria for inputting into the P2I.v2 model, representing an increase of 216 product candidates compared with the 2017 pipeline (see Underlying data: Data File 3). Table 4 and Table 5 show the product candidates in the 2017 and the complete 2019 pipelines, broken down by archetype (Table 4) or by disease (Table 5). In both tables, the difference in the number of candidates between 2017 and 2019 is further broken down into “difference due to changes in the pipeline” and “difference due to changes in scope.”\n\nOf the 216 additions from 2017 to 2019, 152 candidates were added from a “true” change in the pipeline and 64 product candidates were added due to a scope expansion. Out of these 64 product candidates, 31 products were identified due to either including a new archetype in the scope or due to change in the inclusion criteria of existing archetypes (Table 6). The remaining 33 candidates identified due to the scope expansion belong to new diseases included in the 2019 pipeline. These were 5 candidates for chlamydia, 11 candidates for gonorrhea, 8 candidates for hepatitis B, 7 candidates for herpes simplex-2, and one candidate each for human papilloma virus-cervical cancer and mycetoma. These candidates are included in Table 4 under scope expansion.\n\nTable 7 summarizes the changes in classification for the eight diagnostic product candidates based on the updated information available in 2019.\n\nThe 2019 direct comparison pipeline is expected to lead to 196 launches by the year 2031 at a total cost of $19.9 billion, and the complete 2019 pipeline is expected to lead to 207 launches by the year 2031 at a total cost of $21 billion (Table 8) (see Underlying data: Data File 4 for the 2019 direct comparison pipeline model and Data File 5 and Data File 6 for the complete 2019 pipeline models).\n\nThe direct comparison 2019 pipeline is expected to lead to 105 combined launches of products to control HIV/AIDS, TB, and malaria; in comparison, the 2017 pipeline was expected to lead to 85 combined launches for these three diseases (Figure 3). For all other poverty-related and neglected diseases, the direct comparison 2019 pipeline is expected to lead to 91 launches compared to 43 launches from the 2017 pipeline. Among diseases included in the direct comparison analysis, the largest increase in the number of expected launches based on the 2017 versus direct comparison 2019 pipelines would be for Ebola: the 2017 pipeline was expected to lead to two launches, and the direct comparison 2019 pipeline is expected to lead to 20 launches. This increase reflects the significant growth in the number of Ebola pipeline candidates under active development at the time of the 2019 review compared to the 2017 review.\n\nA key finding of the direct comparison analysis is that the 2019 pipeline is expected to lead to launches of HIV and TB vaccines. In our previous study, based on the 2017 pipeline, launches of HIV and TB vaccines were not expected (i.e. there was a probability of launch of under 1.0 for each)6. Table 9 summarizes the expected number of launches based on the 2017 and the direct comparison 2019 pipeline, broken down by disease. The difference in expected launches between the 2017 and the direct comparison 2019 pipelines are summarized under the fourth column, labeled “Expected launches: difference due to changes in the pipeline,” in Table 9.\n\nAs shown in Table 8, the complete 2019 pipeline is expected to lead to 207 launches by the year 2031 compared with 196 expected launches by the year 2031 from the direct comparison 2019 pipeline. The 11 additional launches from the complete 2019 pipeline analysis compared to the 2019 direct comparison pipeline are due to (a) the scope expansion (which led to 64 newly identified candidates, as summarized in Table 5 and Table 6), and (b) the reclassification of eight diagnostics, as summarized in Table 7. The reclassification of diagnostics increased the number of candidates classified as assay development for some diseases resulting in additional launches compared to the 2019 direct comparison pipeline. The expected launches resulting from the complete 2019 pipeline analysis, which had an expanded scope of diseases, include six additional expected launches for diseases not included in the 2017 pipeline review. These launches are one NCE for hepatitis B; three diagnostics for chlamydia; and two diagnostics for gonorrhea.\n\nThe scope expansion also added an expected launch of one biologic for HIV/AIDS and one repurposed drug for reproductive health. The remaining three additional launches were all diagnostics—for hepatitis C, TB, and malaria—and these additions are related to the changes in classification for diagnostic candidates.\n\nThe expected additional launches based on the complete 2019 pipeline analysis compared to the direct comparison 2019 pipeline are summarized under “Difference in expected launches due to: scope expansion” and “Difference in expected launches due to: classification changes” in Table 10.\n\nIt would cost a total of $19.9 billion to move the candidates in the direct comparison 2019 pipeline through to launch, compared with $16.3 billion to move candidates in the 2017 pipeline to launch, an increase of $3.5 billion from 2017 to 2019. The largest cost increase for any single disease category is for Ebola: it would cost an estimated $1.2 billion to move candidates in the 2017 pipeline to launch, compared with $2.8 billion to move candidates from the 2019 pipeline to launch, an increase of $1.7 billion. Comparing the direct comparison 2019 pipeline against the 2017 pipeline as a baseline, the cost to move candidates through the pipeline to launch increased by $238 million and $133.4 million for TB and malaria, respectively, while the cost to move candidates through the pipeline fell by $66.3 million for HIV/AIDS. Figure 4 compares the costs of moving candidates for HIV/AIDS, TB, Ebola, and malaria through the pipeline to launch based on the 2017 versus the direct comparison 2019 pipelines. Table 11 compares the costs to move candidates through the pipeline based on the 2017 and direct comparison 2019, disaggregated by disease (the cost differences are summarized under the column “Cost difference due to changes in the pipeline”).\n\nCompared to the direct comparison 2019 pipeline, an additional $1.2 billion is required to move candidates in the complete 2019 pipeline through the pipeline to launch (in the narrative part of this section, the individual costs do not always add up to the totals mentioned due to rounding; Table 12 gives the individual costs). This $1.2 billion increase in cost is due to the inclusion of additional product candidates as a result of the scope expansion. The largest increase in cost was for HIV/AIDS: it would cost an estimated $2,241.5 million to move candidates in the direct comparison 2019 pipeline through to launch, compared with $2,661.9 million to move candidates in the complete 2019 pipeline to launch, an increase of $420.4 million. This increase is explained by the addition of 16 biologic candidates for HIV/AIDS control in the complete 2019 pipeline compared with the direct comparison 2019 pipeline. For reproductive health, it would cost an estimated $1,394.1 million to move candidates in the direct comparison 2019 pipeline through to launch, compared with $1,579.9 million to move candidates in the complete 2019 pipeline to launch, an increase of $185.9 million. This increase is explained by the addition of 13 candidates for reproductive health in the complete 2019 pipeline compared with the direct comparison 2019 pipeline.\n\nThe changes in classification for the eight diagnostic candidates (Table 7) from simple platform development in the direct comparison 2019 pipeline to assay development in the complete 2019 pipeline reduced the overall cost by $719 million. This cost reduction is due to the cost per phase being lower for assay development classification than the cost per phase for simple platform development classification for diagnostics in the P2I.v2 model.\n\nBased on the complete 2019 pipeline analysis, Table 12 summarizes the changes in cost by disease based on the changes in the pipeline, scope expansion and classification changes. In the complete 2019 pipeline, of the total cost, 62% ($12.9 billion) would be for phase III candidates, 22% ($4.7 billion) for phase II candidates, 4% ($921 million) for phase I candidates and 12% ($2.5 billion) for pre-clinical candidates. Furthermore, in the complete 2019 pipeline, over 75% of the cost ($15.9 billion) would be incurred during the first five years.\n\nFor the direct comparison 2019 pipeline, the sensitivity analysis estimated that the total cost to move the current candidates through the pipeline ranges from $16.4 billion to $23.8 billion and the expected launches range from 150 to 240 (Table 13). For the complete 2019 pipeline, based on the sensitivity analysis results, the total cost to move the current products to launch ranges from $17.3 billion to $25.2 billion and the expected launches range from 159 to 256 (Table 14).\n\nOur first study using the P2I.v2 tool, based on the 2017 pipeline portfolio review, suggested that there would be 18 “missing products”, which were identified using the list of needed priority products outlined in CIH report6. In the 2017 analysis, the estimated cost for launching these 18 missing products was between $13.6 billion and $21.6 billion, depending on the complexity of the product.\n\nIn the 2019 analysis, the model projects that there are likely to be launches of HIV and TB vaccines and, as a result, there would now be 16 missing products. Since the number of candidates do not change for the 16 missing product categories between the 2019 direct comparison pipeline and the complete 2019 pipeline, the missing products analysis is applicable to both pipelines. To launch one of each of the 16 key missing products, starting from pre-clinical phase, we estimate the costs to be between $5.5 billion (assuming lowest complexity products) and $14.2 billion (assuming highest complexity products). Table 15 shows the summary of these costs by disease and archetype. The ranges are calculated using the sensitivity analysis described in sub-section (vi) of the Methods.\n\n* These diseases are Buruli ulcer, Chagas disease, dengue, hookworm, human African trypanosomiasis (HAT), leishmaniasis, leprosy, lymphatic filariasis, onchocerciasis, schistosomiasis, trachoma and trichuriasis\n\nFor the 16 missing products, about 70% of the costs would be incurred during the first five years of product development. Therefore, the costs for the first five years range between $3.85 billion and $9.94 billion.\n\nOver the next five years, the combined cost to move all current candidates through the pipeline and to launch a product each for 16 “missing products” ranges between $19.7 to $25.8 billion, or $3.9 to $5.2 billion per year.\n\n\nDiscussion\n\nOur study found that the product development R&D pipeline for poverty-related and neglected diseases has grown by more than a quarter in just two years, driven in particular by the global response to 2018–19 outbreaks of Ebola virus disease (EVD), as well as reproductive health, TB and malaria. As a result, the number of expected new product launches expected based on the 2019 pipeline increased by half compared to 2017, while the cost of advancing the pipeline increased by a quarter. Most of the increase in the number of pipeline candidates and the number of launches was in diagnostics, but most of the increased (and overall) costs were associated with vaccine development. The distribution of the R&D pipeline broadly aligns with the landscape of funding going to neglected diseases as assessed in the G-FINDER reports.\n\nAt August 31, 2019, there were 754 product candidates that fell within the study scope and had sufficient publicly available information to allow them to be entered into the P2I.v2 model. Of these 754 candidates, 690 were in disease and product areas that were included in our 2017 review, representing an increase of 152 candidates (or 28%) since 2017.\n\nThe pipeline growth was primarily in just four areas. Ebola, reproductive health, TB and malaria collectively accounted for 130 (86%) of the 152 additional candidates. The increase in pipeline candidates occurred for all archetypes except for NCEs, but the largest increases were in diagnostics (up by 67 candidates, a 66% increase) and vaccines (up by 48 candidates, a 23% increase), which together accounted for more than three quarters (76%) of the overall increase in product candidates.\n\nEbola alone was responsible for a significant proportion (41%) of the total growth of the R&D pipeline between 2017 and 2019. The number of candidates in the pipeline for Ebola increased fourfold in this period, reflecting the robust international response to two outbreaks of EVD in the Democratic Republic of the Congo (DRC) in 2018–19, the second of which – originating in North Kivu province – became the second largest EVD outbreak on record9.\n\nAlthough TB and malaria were among the diseases to see the most significant pipeline growth, the surge in candidates for Ebola and reproductive health helped reduce the degree to which the pipeline is dominated by HIV/AIDS, TB and malaria. While HIV/AIDS, TB and malaria still collectively accounted for close to half of all candidates in the pipeline in 2017, this proportion was down from 57% of candidates in 2017. From 2017 to 2019, the vaccine pipeline for malaria and TB was essentially stagnant, with each disease registering an increase of just a single candidate between 2017 and 2019.\n\nIt is concerning that the size of the R&D pipeline for diarrheal diseases, salmonella infections, helminth infections and kinetoplastid infections either fell or was at best unchanged from 2017 to 2019. Of the 35 diseases included in the 2017 review, 15 still had fewer than four candidates each in the pipeline in 2019. Many of these are neglected tropical diseases, whose funding has remained basically flat over the course of the last decade, while funding for HIV/AIDS, TB, malaria and Ebola has grown significantly. Also concerning is the fact that there was no increase in the number of NCE candidates in the R&D pipeline (in fact there were two fewer in 2019 than in 2017), despite the overall pipeline growing by more than a quarter. There was a roughly even split in the number of new candidates in early-stage development and those in clinical trials or field development.\n\nThe model suggests that the 754 candidates currently in the pipeline will deliver 207 launch-ready health technologies targeting poverty-related and neglected diseases. Looking just at the “apples-to-apples” subset, the 690 candidates in the direct comparison pipeline would be expected to deliver 196 launches, an increase of more than half (53%, an additional 68 launches) compared to the 2017 review.\n\nDiagnostics accounted for the vast majority of the increase in expected launches in 2019 over 2017, mostly for assays rather than platforms. This increase reflected the large number of candidates entering the pipeline (especially in Ebola and TB), as well as effect of underlying assumptions of the P2I.v2 model, which applies a high probability of success for diagnostics development. More than half of the increase was for just three diseases: Ebola (up 18, 23%), TB (up 13, 37%) and malaria (up 11, 41%).\n\nAs with the pipeline distribution, expected launches are less concentrated on HIV/AIDS, TB and malaria than in the 2017 review, but again this is largely due to the surge in Ebola pipeline activity between 2017 and 2019, reflecting the coordinated global response towards unmet R&D needs to contain two back-to-back EVD outbreaks in the DRC in 2018. Based on the pipeline as of August 31, 2019, the model predicts three launch-ready Ebola vaccines. One Ebola vaccine, Merck’s Ervebo, was approved by both the European Commission and the U.S. Food and Drug Administration in late 2019. Another investigational vaccine, Janssen’s MVA-BN-Filo, is currently deployed in East and Central Africa as part of an expanded response to the DRC outbreak, and has been submitted to the European Medicines Agency for regulatory approval.\n\nThe global estimate of launch-ready HIV/AIDS products decreased from 23 in the 2017 review to 19 in the 2019 review, due to a drop in the number of HIV/AIDS NCE candidates in the pipeline. This drop reflected both the successful approval of several late-stage NCE candidates since 2017 and a lack of new candidates entering the pipeline; however, it must be noted that the HIV/AIDS therapeutics category has strict inclusion restrictions in the G-FINDER scope.\n\nOne of the most significant changes in the 2019 review compared to the 2017 review is the estimated launch of vaccines for HIV/AIDS and TB by 2031 (the 2017 pipeline would not have led to these two launches6). This is a positive reflection of progress in the pipeline for these two areas since our last review, but it is worth emphasizing that the P2I model makes no assessment of the appropriateness of the projected launch-ready candidate, nor of any additional research that may be required to support product introduction. So while the model predicts the likely launch of a TB vaccine, the profile of the most advanced candidates in the pipeline suggest that – at the date of the launch projected by the model – such a vaccine may only be approved for use in limited populations. In the case of HIV/AIDS, caution may be warranted given that thus far no candidate has been able to match even the 31% efficacy achieved in the 2009 RV144 Thai Phase III clinical trials, and that during the preparation of this report one of the two most advanced HIV vaccine candidates in the pipeline (a modified version of the RV144 vaccine regimen) was found to be ineffective.\n\nIn May 2019, TDR launched the Health Product Profile Directory (HPPD), described as an “online database describing 8–10 key characteristics (such as target population, measures of efficacy and dosage) of product profiles for medicines, vaccines, diagnostics and other products that are intended to be accessed by populations in low- and middle-income countries.”10\n\nOut of the 50 WHO-authored target product profiles (TPPs) contained in the HPPD, 28 align with the scope of this study, covering 11 diseases. Whilst not all areas with the largest pipelines have WHO-authored TPPs, most of the areas for which WHO-authored TPPs exist have healthy pipelines.\n\nThe continued expansion of the TB diagnostics pipeline (which grew by 50% between 2017 and 2019) is a case in point, but also one that highlights a potential imbalance in the current pipeline. In 2014, the WHO published a report of a consensus meeting highlighting high-priority TPPs for new TB diagnostics, divided into four use cases: (a) a sputum-based test to detect pulmonary TB; (b) a rapid biomarker-based non-sputum-based test; (c) a drug-susceptibility test; and (d) a community-based triage or referral test for identifying people suspected of having TB. In 2019, almost two-thirds (21, 64%) of all diagnostics in late-stage development for TB are rapid drug susceptibility tests; this is compared to just four rapid biomarker-based non-sputum-based test, two triage tests and a single sputum-based test to detect pulmonary TB (the remaining five late-stage candidates are not indicated for a specific TPP).\n\nFurther work to map the pipeline against the TPPs in the HPPD would provide useful insights into the nature of the R&D pipeline.\n\nOverall, the P2I.v2 model suggests that advancing the current pipeline of 754 candidates to deliver 207 launch-ready health technologies would cost an estimated $21 billion by 2031. More than half of the total amount ($12 billion, 59%) of the cost of advancing the candidates is projected to be spent during the first three years (2020–2022), at an average cost of $3.9 billion per year.\n\nHalf of the projected costs ($10 billion, 50%) were for four diseases: Ebola ($2.8 billion, 13%), HIV/AIDS ($2.7 billion, 13%), TB ($2.6 billion, 13%) and malaria ($2.3 billion, 11%). Development of the candidates for the remaining 37 diseases each accounted for less than 10% of the total costs, most of which had less than 1% of the total share of costs.\n\nFrom an archetype perspective, the main driver of the cost of development were vaccines, which accounted for almost two-thirds ($13 billion, 61%) of the total projected cost through to 2031. This was followed by diagnostics ($2.6 billion, 12%), NCEs ($2.0 billion, 9.6%), biologics ($1.9 billion, 9.3%), repurposed drugs ($1.5 billion, 7.3%) and vector control products ($79 million, 0.4%).\n\nLooking just at the “apples-to-apples” subset, the P2I.v2 model suggests that advancing the pipeline of 690 candidates to deliver 196 launch-ready health technologies would cost an estimated $19.9 billion by 2031. This represents an increase of $3.5 billion (up 22%) over the estimate from the 2017 pipeline review.\n\nThe bulk of this $3.5 billion cost increase was due to an additional $3.1 billion in the cost of advancing vaccine candidates, and almost half (47%) was from the more than doubling of projected costs for advancing Ebola candidates (up $1.7 billion, 145%). This makes sense, as Ebola was responsible for a massive increase in the number of vaccine candidates, many of them in pre-clinical development; however the fate of these early-stage candidates is likely to be affected by the approval of Merck’s Ervebo (and pending likely approval of Janssen’s MVA-BN-Filo), as well as the waning of the current DRC outbreak.\n\nProduct development costs also increased dramatically over 2017 for meningitis (up $857 million, from a low base; although this was partly due to better sources, primarily the draft publication of the new 2030 global roadmap for meningitis vaccines) and pneumonia (up $605 million, 94%; reflecting new vaccine candidates advancing to Phase III clinical trials). Although malaria and TB account for a significant proportion of the projected costs, there was no significant increase in projected costs for these diseases between 2017 and 2019, with pipeline growth in both diseases being mainly in diagnostics rather than in other more costly archetypes.\n\nThe additional cost to launch all 16 “missing products” ranges from $5.5 billion to $14.2 billion, depending on the complexity of the products, around 70% of which would be spent in the first 5 years. Combined with the cost of advancing the current pipeline, this means that in the first 5 years the total estimated costs to move all current candidates through the pipeline and develop the 16 missing products would be around $3.9 billion to $5.2 billion per year. This total does not include funding for basic research, discovery, and post-registration studies, all of which are outside the scope of the P2I model cost projections.\n\nHow does this compare to current global funding levels? According to the most recent G-FINDER neglected disease report and two soon to be published analyses of R&D for sexual and reproductive health and emerging infectious diseases, total funding for the poverty-related and neglected diseases included in the scope of this paper totaled $3.6 billion in 2018, once funding for basic research and post-registration studies is excluded. This suggests an annual product development funding gap of at least $0.3 billion to $2.6 billion even at current record levels of neglected disease R&D funding.\n\nTo the best of our knowledge, this is the first study to provide time trends for the R&D pipeline of poverty-related and neglected diseases, as well as for product launches and cost estimates. Information on these trends is valuable for R&D funders, researchers, product development partnerships, and policymakers more broadly. Additional diseases were included in this new analysis including the WHO Product Development for Vaccines Advisory Committee’s priority pathogens, such as HSV-2 and gonorrhoea.\n\nA major strength of this study, as well as our previous study using the P2I model to examine the 2017 pipeline6, is that it goes beyond analysing a single therapeutic portfolio (e.g. the pipeline of drugs for TB control11) to examine the full portfolio of candidates for a very broad range of poverty-related and neglected diseases. This broader approach “aligns with the way in which funders pursue a diversified portfolio of product development projects.”6 Our study helps to identify critical gaps in the global health product development pipeline by showing (a) which diseases and archetypes have few candidates, (b) which product launches are most likely, and (c) which products will probably still be missing based on existing candidates.\n\nWe have previously described the strengths of the P2I.v2 tool in detail6. In brief, we highlight three particular strengths here. First, the tool is publicly available online, as are the full details of the pipeline review, model assumptions, and model inputs and outputs. Thus readers can replicate, improve on, and further adapt our work. Second, the tool is highly flexible — users can make modifications to the underlying assumptions to see how such changes would influence the results. Third, the underlying model assumptions are likely to be realistic. These assumptions are based on a very large number of data points (e.g., assumptions on success rates and cycle times were based on data from of 25,000 development candidates), and were validated through examining peer-reviewed literature, industry reports/databases, and expert interviews7. Our assumptions are roughly in line with reported industry standards (the amount of variation differs by product types)12,13.\n\nThe pipeline data used in this study represents a snapshot of the product development pipeline at a single point in time (up to August 31, 2019). Since the pipeline is constantly evolving, any changes—such as the advancement or termination of pipeline candidates—that occurred after this date are not reflected in the pipeline count or the resulting analysis.\n\nOur research was limited to publicly available sources and relied on confirmation from authoritative sources, including the website of the candidate developer, recent reports from international organizations such as WHO and Unitaid, and clinical trial portals. Where we could not confirm the status of a candidate, we excluded it from the active candidate list. The lack of publicly available information on some products under development means that our count of active candidates may be an underestimate. In particular, there is comparatively less information available on pre-clinical candidates, due to the lack of dedicated public registries mirroring those available for clinical trial candidates, and limited sharing of proprietary data by companies. Our reliance on English language sources also means that the undercounting of early-stage R&D activity may disproportionately affect particular countries or regions. Early-stage candidates from the Middle East, Russia and China are likely to be underrepresented.\n\nOur reliance on publicly available data sources also meant that we were unable to identify when each candidate had entered its current phase of development, meaning that from a model input perspective, a candidate that was close to the conclusion of its phase was treated identically to a candidate that had just entered it.\n\nAs in our 2017 study6, our new study uses the consensus prioritization exercise conducted by the 2013 Lancet Commission on Investing in Health to identify the critical “missing” products that the current pipeline of product candidates would fail to deliver, according to the P2I.v2 model. While we believe this remains a robust global consensus, it is now seven years old. In addition, we note that the P2I. v2 model does not account for whether sufficient research and trial site capacity exists to conduct the additional studies that would be needed to deliver these missing products in the timeframe projected by the model.\n\nWe have previously described the limitations of the P2I.v2 tool in detail6. In brief, we highlight six particular limitations here.\n\nFirst, the P2I tool is a simple, static, and deterministic model that does not consider (a) possible changes in the product development process, such as improved techniques, novel clinical trial designs (such as highly efficient trials14), new regulatory pathways, or (b) product-related decisions, such as the decision to discontinue candidates.\n\nSecond, the tool requires users to classify every candidate based on the categories available in the model, yet it can be challenging to categorize some candidates into the P2I.v2 archetypes. In particular, there are too few archetypes for diagnostics and vector control products—for example, there are no specific archetype fields for vector control products, with both chemical and biological products included as a single archetype.\n\nThird, the assumptions on probabilities of success for unprecedented vaccines in phases II and III are based on a relatively small number of data points—around 10-25 data points per estimated value.\n\nFourth, the model assumptions used for each archetype are the same for almost every disease, with just a few exceptions (“unprecedented vaccines” refers to HIV, TB, and malaria vaccines, and there are two TB-specific archetypes, “simple NCE for TB” and “simple biologic for TB”). Therefore, the model does not take into account the specificities involved for developing a product against a specific disease. So, for example, in the model, the costs, success rates, and cycle times per phase for developing a simple vaccine for schistosomiasis would be the same as those for developing a simple vaccine for hepatitis C.\n\nFifth, the model does not take into account the public health value and clinical utility of a candidate, or the impact of successful product launches on the remaining candidates in the pipeline.\n\nFinally, the model does not include discovery, early pre-clinical development and post-phase III costs such as regulatory review, marketing authorization, Phase IV and pharmacovigilance and other implementation costs. These other costs can be substantial—for example, funders have already committed $49 million to the first phase of implementation studies for the RTS,S malaria vaccine, with further funding required.\n\nDespite these limitations, efforts are now underway, funded by TDR, to address these weaknesses and to develop further iterations of the tool. TDR is funding many different product development partnerships, including MMV, FIND, and DNDI, to use the tool to analyze their own portfolios; these analyses will also help to refine and improve the tool. TDR is also funding research institutions, such as the Graduate Institute of Geneva, to collect data that can help make further tool modifications. While we did reach out to PDPs to ask them to share new data on product development that could potentially be used to develop a version 3 of the tool (P2I.v3), the PDPs did not have large enough numbers of data points to meaningfully make tool refinements.\n\n\nConclusions\n\nThis study has provided additional evidence that the P2I tool can be used effectively to estimate the costs and probable launches associated with moving a portfolio of current candidates for neglected diseases through the pipeline. It can help to identify gaps in the R&D pipeline and to estimate the size of the gap between funding requirements and current levels of R&D investment. Importantly, this study has also shown how the model can be used to estimate the impact of changes in the pipeline over time.\n\n\nData availability\n\nOpen Science Framework: Analysis of the health product pipeline for poverty-related and neglected diseases using the portfolio-to-impact (p2i) modeling tool, https://doi.org/10.17605/OSF.IO/WT6VN15.\n\nThis project contains the following underlying data:\n\n- Data file 1: Full list of diseases included in 2017 and 2019 pipelines\n\n- Data file 2: Expected launches: unrounded, rounded to nearest integer, and rounded down,\n\n- Data file 3: Candidates in the pipeline for neglected diseases, as of August 31, 2019\n\n- Data file 4: P2I.v2 tool showing anticipated launches and costs by disease and archetype for the 2019 direct comparison pipeline\n\n- Data files 5 and 6: P2I.v2 tool showing anticipated launches and costs by disease and archetype for the 2019 complete pipeline\n\n- Data files 4,5 and 6, which include the P2I modeling tool, are provided in Microsoft Excel format as the files cannot be uploaded in any other format. To use these data files please download the file and use Microsoft Excel software to open the files.\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).",
"appendix": "References\n\nJamison DT, Summers LH, Alleyne G, et al.: Global health 2035: A world converging within a generation. Lancet. 2013; 382(9908): 1898–955. PubMed Abstract | Publisher Full Text\n\nVerguet S, Norheim OF, Olson ZD, et al.: Annual rates of decline in child, maternal, HIV, and tuberculosis mortality across 109 countries of low and middle income from 1990 to 2013: an assessment of the feasibility of post-2015 goals. Lancet Glob Health. 2014; 2(12): e698–709. PubMed Abstract | Publisher Full Text\n\nBoyle CF, Levin C, Hatef A, et al.: Achieving a “Grand Convergence” in Global Health: Modeling the Technical Inputs, Costs, and Impacts from 2016 to 2030. PLoS One. 2015; 10(10): e0140092. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWatkins DA, Yamey G, Schäferhoff M, et al.: Alma-Ata at 40 years: reflections from the Lancet Commission on Investing in Health. Lancet. 2018; 392(10156): 1434–1460. PubMed Abstract | Publisher Full Text\n\nBeyeler N, Fewer S, Yotebieng M, et al.: Improving resource mobilisation for global health R&D: a role for coordination platforms? BMJ Glob Health. 2019; 4(1): e001209. PubMed Abstract | Publisher Full Text | Free Full Text\n\nYoung R, Bekele T, Gunn A, et al.: Developing new health technologies for neglected diseases: a pipeline portfolio review and cost model. Gates Open Res. 2018; 2: 23. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTerry RF, Yamey G, Miyazaki-Krause R, et al.: Funding global health product R&D: the Portfolio-To-Impact Model (P2I), a new tool for modelling the impact of different research portfolios. Gates Open Res. 2018; 2: 24. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMestre-Ferrandiz J, Sussex J, Towse A: The R&D Cost of a New Medicine. Office of Health Economics (OHE) Monograph. 2012. Reference Source\n\nAruna A, Mbala P, Minikulu L, et al.: Ebola Virus Disease Outbreak — Democratic Republic of the Congo, August 2018–November 2019. MMWR Morb Mortal Wkly Rep. 2019; 68(50): 1162–1165. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTerry RF, Plasència A, Reeder JC: Analysis of the Health Product Profile Directory - a new tool to inform priority-setting in global public health. Health Res Policy Sys. 2019; 17(1): 97. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGlickman SW, Rasiel EB, Hamilton CD, et al.: Medicine. A portfolio model of drug development for tuberculosis. Science. 2006; 311(5765): 1246–7. PubMed Abstract | Publisher Full Text\n\nSchuhmacher A, Gassmann O, Hinder M: A Review of the Pharmaceutical R&D Efficiency: Costs, Timelines, and Probabilities: The Critical Path to Innovation. In: Value Creation in the Pharmaceutical Industry. Edited by Schuhmacher A, Hinder M, Gassmann O. Wiley-VCH Verlag GmbH & Co. KGaA. 2016. Publisher Full Text\n\nHay M, Thomas DW, Craighead JL, et al.: Clinical development success rates for investigational drugs. Nat Biotechnol. 2014; 32(1): 40–51. PubMed Abstract | Publisher Full Text\n\nThorlund K, Golchi S, Haggstrom J, et al.: Highly Efficient Clinical Trials Simulator (HECT): Software application for planning and simulating platform adaptive trials [version 2; peer review: 2 approved, 2 approved with reservations]. Gates Open Res. 2019; 3: 780. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBandara S, Yamey G, Gunn A, et al.: Analysis of the Health Product Pipeline for Poverty-related and Neglected Diseases Using the Portfolio-to-impact (P2I) Modelling Tool. 2020. http://www.doi.org/10.17605/OSF.IO/WT6VN"
}
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[
{
"id": "63798",
"date": "09 Sep 2020",
"name": "Mikel Berdud",
"expertise": [
"Reviewer Expertise Industrial organisation with specific background on the economics of innovation",
"regulation of pharmaceutical markets",
"incentives for R&D",
"pharmaceutical pricing and competition."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe work by Gavin Yamey and colleagues ‘Analysis of the health product pipeline for poverty-related and neglected diseases using the Portfolio-to-Impact (P2I) modelling tool’ is a relevant and original research on an important topic: the additional funding needed for poverty-related and neglected disease (PRND) product development. Based on a modelling exercise, the study provides evidence to answer two relevant questions:\nHow much funding is needed to progress the PRND related 2019 pipeline to the market based on an analysis of the period 2019-2031.\n\nHow large is the funding gap needed to cover to develop and produce at least one launch in each disease listed for the modelling – based on a list of a total of 45 diseases?\n\nMethods are sound and well-described, and data are transparent and publicly available. The model is also accessible and therefore analyses can be easily replicate and scrutinised by readers. My first comment is that this study is a follow-up analysis of the previous work by Young et al. (2018)1. The current analysis replicates the same estimates using the same methods but with an updated pipeline data and expanded disease list – from 35 diseases to a 45 diseases. It is important to contextualise the present work this way in order to correctly assess its contribution. In that sense, I would like to highlight which in my opinion are the two the contributions that can be drawn from the reviewed article:\n\nThe study demonstrates that PRNDs product R&D pipeline is a vivid organism which only in two years has experienced a huge evolution. The analysis that authors call “2019 direct comparison pipeline” which is based in the same list of 35 diseases of Young et al. (2018)1 shows that the pipeline has increased in 152 new drugs (+28%) with vaccines (+48) and diagnostics (+67) leading this increment and diseases like Ebola (+62), TB (+22) and Malaria (+18) being the most benefited. This can be considered an endogenous change of the PNRDs related pipeline. A change of this magnitude in just two-year period evidences the necessity for policymakers to track the evolution of the pipeline closely and frequently to update the expected launches and required funding to see where and by how much the focus has to be placed to encourage the most needed and impactful innovation. Learnings from continuous analysis of the pipeline may help to shape the Pharmaceutical market regulation and incentives (i.e. push and pull funding programmes).\n\nThe PRNDs is also by itself an evolving set of diseases. The previous work in which this new study is based was only published two years ago. In that study a total number of 35 PNRDs were listed in 2017. Replicating criteria, a mix of clinical and socio-economic variables, the number has been increased to 45 in 2019. This shows that the changing socio-economic environment can affect the pipeline for PNRDs by “exogenously” affecting the list PNRDs. This is also an extremely relevant finding as it suggests that the optimal policy to tackle public health issues related to PNRDs requires a continuous update of what is included in the PNRDs list and which implications these inclusions may in terms of the required funding to progress the pipeline or to guarantee at least one treatment option in a reasonable time horizon.\n\nIn general, results are clear and well explained and easy to follow even for a non-expert reader, discussion is well written and goes through the most relevant findings and methodological details and the conclusion draws form results and discussion. There are many comments that I would like to provide to authors though, most of the requests for clarification and some other points that will require authors' attention and response. In order to be “indexed” i.e. listed in academic research output databases, I think authors should respond to all of them:\nComment 1: In page 4, section (i) authors discuss assumptions on development costs, attrition rates, and cycle times.\nOn development costs, there is a reference to Terry et al. (2018)2. All development costs are well described and showed in the reference. However, I suggest to discuss and compare the cost per phase estimates used in this work with other presented in the literature. How do the used estimates compare with estimates of each development phase costs of DiMasi et al. (2016)3, Prasad and Mailankody (2017)4, and Wouters et al. (2020)5? I have three questions for the assumptions on attrition rates:\nHow are the success/attrition rates applied? How exactly do they affect the cost of R&D? is the cost of failed projects not accounted for in the calculation? This, surely, can be inferred from the P2I.v2 model which is available, but as a reader, I miss an explanation in the paper. More generally, the diagram of figure 1 does not describe in detail how the assumptions affect the cost estimate. I think the box in the middle of the diagram ‘P2I Model’ requires further visual explanation. What about something like Figure 2.4. (p. 15) of Mestre-Ferrandiz et al. (2012)6.\n\nAre attrition rates being applied by development phase? Or are authors basically applying the overall likelihood of approval (LOA) to each archetype without phase specific discrimination? I think this is important to clarify.\n\nHave they been updated from the 2017 work? There is a new paper published in 2019 with new estimates by Wong et al. (2019)7 that may be worth comparing. An interesting sensitivity may be to replicate estimates by using probabilities of success of other recently published works like Hay et al. (2014)9 or Thomas et al. (2016)8 or even those used by DiMasi et al. (2016)3 and DiMasi et al. (2020)10 in their analyses.\n\nComment 2: This is a question for clarification. How are the probabilities of success for unprecedented HIV, TB, and malaria vaccines calculated/estimated/assumed? There is no approved HIV vaccine yet so how can one assume a positive probability alpha and consequently estimate the minimum necessary products in the pipeline required to have at least one launched in the future? Are authors applying the archetype probability of success (PoS) which means that HIV vaccines have the same POS than vaccines for meningitis B or mRNA vaccines? This is what I understand is the approach, otherwise PoS would be equal to zero for these unprecedented vaccines. therefore, If the complex vaccine archetype PoS is taken as reference for unprecedented vaccines, should not we have a HIV vaccine approved already given the huge investment devoted to the disease? But we don’t and the analysis predicts one is expected to be launched based on the current pipeline, is not it tricky to assume? This is discussed by authors in the discussion section (page 22) where evidence that the HIV vaccine projects are highly uncertain to success and being launched is provided. In my opinion these specific cases should be subject to more detailed discussion perhaps acknowledging that it is likely that the estimated HIV vaccine will never progress to launch.\nComment 3: In the methods section, (ii) Pipeline portfolio review, page 7, authors explain the review and cross-reference all major data sources used. This seems to be a huge work producing results by itself. As a reader, I would benefit from an explicit/descriptive summary explanation of the most relevant findings in each. It is also very useful for further work that other researchers would want to start. Also, a summary of what was used from each of the data sources would help understand the data. Which universities, companies, references, governments, and non-profit organizations were checked? Might this information be presented in a table, an annex or as another supplementary material file? More details will provide more credit to methods. Authors should at least consider and assess this possibility.\n\nComment 4: In the next paragraph of the above, authors explain a validation process leading to the inclusion of candidates. They were included only “if an authoritative source could confirm they were in active development”. As a reader I miss more details about both, the process and the authoritative sources. Which clinical trial portals were used and for which products? How was the process of correspondence interaction with product developers? How many products were (in numbers and in percentage) checked by each authoritative source? What was the percentage of testing? Were all products checked for inclusion/exclusion by these means?\nComment 5: In the methods section, (iv) inputting the pipeline of candidates into the adapted P2i model, page 8, authors explain that “when a candidate is classified into a particular phase In the model, it is assumed it is at the beginning of the phase […]”. This approach overestimates the cost of progressing the pipeline as many of the included products can be at mid- or late point of their current development phase at the time of the inclusion hence the full phase cost should not be considered. How large is the size of this overestimation is uncertain and will depend on the distribution of the actual degree of progress within each phase of the products? Why do authors take this approach? Why don’t take the approach of assuming that all products are at the mid-point of each development phase? Some explanation and discussion of the degree of overestimation are missed.\nComment 6: In the following paragraph of the above’s, authors recognise that they do not apply a discount rate to the cost estimates. They neither apply the rate of the cost of capital as the usual practice is in this type of estimation exercises (see DiMasi et al. (2016)3, Prasad and Mailankody (2017)4, Wouters et al. (2020)5, Shuchmacher et al. (2016), Mestre-Ferrandiz et al. (2012)6). Authors then neglect an important and well-established driver of the R&D cost: the cost of capital. An omission that is not properly discussed in the paper. Additionally, they neither simply discount costs although the timelines for cost estimation used is over a full decade and the impact of the discounting significant. This approach implies that the time of development for each phase and archetype is only relevant for the cost stream and time horizon calculation, but it does not have any effect on the total funding required. I suggest to at least discuss this caveat in depth and in relation with what R&D cost estimation literature have been doing to this respect, which as I have said is opposite approach.\nComment 7: In the following paragraph of the above, authors discuss on the conservative rounding approach they use to estimate number of launches. They round down all expected launches number estimates the model provides. Are all launches between 0 and 1 (if any) considered equal to 0? If the estimate is an expected output and cost, I suggest (especially for the cost estimate) to consider results with any rounding to be the base case– explicitly mentioning the integer component of the number as the minimum expected launches – and then present results rounded down for comparison.\nComment 8: On page 9, in the paragraph ‘The complete 2019 pipeline analysis’ authors explain that there are eight diagnostic candidate archetype that were changed between the 2019 direct comparison pipeline and complete 2019 pipeline. My opinion is that relevant assumptions applied to the different diagnostic archetypes vary significantly, this approach may result in misleading. If these changes are rationalised then comparison should be done by assuming them. Alternatively, should not be a better approach to change the archetype in the 2017 pipeline for direct comparison? Or is it equivalent?\nComment 9: in results section, subsection ‘(vii) Costs to move candidates through the pipeline based on the complete 2019 pipeline”, page 18, authors assess the share of total cost / funding that belongs to each phase of development. For example, it is said that 62% of the cost is attributable to phase III. But language is loose as the reader may get confused. Is it 62% of the cost attributable only to projects currently in phase III? or is it the total cost of phase III attributable to all projects, those already in phase III and those that are not yet, but which they will be once they progress until to? I guess it is the second case but the dynamics should be explained, which means, the 62% also includes phase III cost of those projects still in phase I and phase II but which the P2I model predicts they will pass to phase III in the future.\n\nComment 10: In the results section, subsection ‘(viii) Results of sensitivity analysis…’ authors briefly describe the result of the sensitivity analysis and refer the reader to check these results in tables 13 and 14. I consider this discussion on the sensitivity analysis to be insufficient. Sensitivities should be discussed appropriately by parameter and also for all parameters as a whole. Assessment of how sensitive the model and the estimation to the different parameters are and why is necessary so as a reader one can get an idea of what is driving the cost and why. Authors should speculate on the potential implications of these sensitivities and the robustness of their assumptions. In addition, given the dynamic and evolving nature of pipelines and pharmaceutical R&D processes, it should be also discussed the implications of potential changes on the parameters that may likely happen during the time horizon of the estimation exercise. Such discussion will provide valuable information to assess the model's robustness of the estimations and the appropriateness of updating the estimates recurrently.\nComment 11: In tables 13 and 14 )and related to comment 1/second bullet/point 1.), sensitivity analysis of the PoS result in an increase of the cost when PoS increases and vice versa. My concern here is whether the cost of failures of a given phase of development are included or not. For example, if for a given archetype, the pipeline is composed of 10 products and the PoS is 50%, then are the ten considered to calculate the cost of phase II of that archetype, or only the successes are considered? Can authors explain, please? I assume the cost of the ten products is included.\nComment 12: In the discussion section, page 20, authors state that “As a result, the number of expected new product launches expected based on the 2019 pipeline increased by half compared to 2017, while the cost of advancing the pipeline increased by a quarter”. I guess the answer is in the model but the first question that comes to the reader’s mind is, how an increasing return relationship may be possible. How an increase in the input of 25% can produce an increase in the output of 50%? It’s true that we are measuring input in value and output in units, but even if this is taken into account, it is an input-output relationship that requires attention and explanation. Assuming the probabilities of success have not changed, the other possible explanations are (i) the rounding of the expected launches (ii) biased increase of the pipeline for archetypes with higher PoS or lower cost of development. I suggest expanding the discussion on this result to explain why these non-linear relationships happens. This is also important to find out which of the archetypes may be the most efficient to fund (note we are no taking the effectiveness considerations which in a value based assessment should also be included).\nComment 13: I discussion section, in the subsection “new cost estimates”, page 23, authors explain the additional cost to launch “missing products”. These estimates depend on the complexity of the products. It seems like the complexity has an impact of around +158% in terms of the cost increase. However, what 'complexity' means exactly in this context is not discussed, described or explained. As a reader, I miss more details here about what authors mean by complexity in this context, how they model it, and how such complexity translates to higher cost.\n\nComment 14: In the discussion section, in the subsection ‘Strengths of the P2I.v2 tool’, page 23, authors state that their assumptions (i.e. success rates, costs per phase, and per archetype, duration of phase) are aligned with reported industry standards. They cite two references, Hay et al. (2014)9 and Schuhmacher et al. (2016)11. As far as I am concerned there are few newer works that have been published later that would be good to check and compare with the authors’ assumptions (see DiMasi et al. (2020)10, Wong et al. (2019)7, Thomas et al. (2016)8 for probabilities of success and development times and Wouters et al. (2020)5, Prasad and Maylankody (2017)4, for research and development costs). Additionally, methods used for this work and those used by the literature are neither collated nor comparatively discussed.\nComment 15: I'm not the right person to judge the English language as I am not a native English speaker. As a non-native English reader, my assessment is that the manuscript is very well and clearly written and it does not require any work to improve apart from the usual proofreading and QA.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "134990",
"date": "19 May 2022",
"name": "Dzintars Gotham",
"expertise": [
"Reviewer Expertise Public health",
"health economics",
"health 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\nOverall, I do not have many or major comments. The article is very interesting, valuable, and clearly explained. It is essentially an interval update to earlier work. My congratulations to the authors.\nComments: In methods, regarding the search for candidates, the authors state that ‘government websites’ were consulted. An appendix should be provided with a precise list of sources. Was the breadth of sources the same as was used for the 2017 study? If broader, this could be added to limitations as a possible source of overestimating the 2017-2019 change.\nAs the other reviewer has noted, it would be helpful to give some pointers to the reader regarding where this study’s assumed R&D costs and probability of success lie in comparison to other available estimates for per-Phase costs/POS (e.g. versus Sertkaya et al 2014, DiMasi, Prasad & Mailankody, Wouters, and versus DNDi’s reported costs of about 180 EUR million per NCE including failures – if I recall correctly).\nAs the other reviewer has noticed, the authors did not apply discounting or ‘cost of capital’. In my view, this is appropriate, as there is the (implicit or explicit) assumption that the gaps in funding will/should be filled by government or philanthropic funding. In other words, COC estimation is appropriate for a business proposal, but this is not a business proposal. That said, the authors should give their reasoning behind not including COC in calculations.\nOne interesting output – which I did not see in the current draft – would be an estimate of the total cost per approved product (including failures). Based on the sentence “Overall, the P2I.v2 model suggests that advancing the current pipeline of 754 candidates to deliver 207 launch-ready health technologies would cost an estimated $21 billion by 2031.” I get to a back-of-envelope estimate of 21 billion divided by 207 giving about US$101 million per launch-ready technology. Could the authors confirm whether that is correct and/or include the correct per-technology estimate? And additionally give a per-product average estimated development cost in the NCE group, in the diagnostics group, and in the vaccines group.\nMinor points: The authors use the wording ‘move through pipeline’ and variations throughout the article. It was not clear to me whether this means ‘all the way’ through the pipeline i.e. to product approval. For example, in the Methods section (i), they write ‘through the pipeline from advanced preclinical to phase III clinical trials’ … does ‘to phase III clinical trials’ mean not including Ph III trials? Figure 2 does make it clear that what they mean is all the way to approval. Including the phrase ‘to approval’ in relevant places may be helpful.\nIn Table 1, the authors imply that for imatinib the underlying disease pathogenesis was not understood, which is not true; imatinib was developed specifically to inhibit the BCR/ABL tyrosine kinase.\nI found this sentence confusing: “Product development costs also increased dramatically over 2017 for meningitis (up $857 million[)]and pneumonia (up $605 million[)]” - current wording implies the costs have INHERENTLY become higher for developing products for these diseases. Unless I am mistaken, the authors really mean ‘product development EXPENDITURES increased…’\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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/9-416
|
https://f1000research.com/articles/8-1327/v1
|
01 Aug 19
|
{
"type": "Study Protocol",
"title": "The impact of city-wide deployment of Wolbachia-carrying mosquitoes on arboviral disease incidence in Medellín and Bello, Colombia: study protocol for an interrupted time-series analysis and a test-negative design study",
"authors": [
"Ivan D. Velez",
"Eduardo Santacruz",
"Simon C. Kutcher",
"Sandra L. Duque",
"Alexander Uribe",
"Jovany Barajas",
"Sandra Gonzalez",
"Ana Cristina Patino",
"Lina Zuluaga",
"Luis Martínez",
"María Camila Mejia",
"María Patricia Arbelaez",
"Henry Pulido",
"Nicholas P. Jewell",
"Scott L. O'Neill",
"Cameron P. Simmons",
"Katherine L. Anders",
"Stephanie K. Tanamas",
"Ivan D. Velez",
"Eduardo Santacruz",
"Simon C. Kutcher",
"Sandra L. Duque",
"Alexander Uribe",
"Jovany Barajas",
"Sandra Gonzalez",
"Ana Cristina Patino",
"Lina Zuluaga",
"Luis Martínez",
"María Camila Mejia",
"María Patricia Arbelaez",
"Henry Pulido",
"Nicholas P. Jewell",
"Scott L. O'Neill",
"Cameron P. Simmons",
"Stephanie K. Tanamas"
],
"abstract": "Background: Dengue, chikungunya and Zika are viral infections transmitted by Aedes aegypti mosquitoes, and present major public health challenges in tropical regions. Traditional vector control methods have been ineffective at halting disease transmission. The World Mosquito Program has developed a novel approach to arbovirus control using Ae. aegypti stably transfected with the Wolbachia bacterium, which have significantly reduced ability to transmit dengue, Zika and chikungunya in laboratory experiments. Field releases in eight countries have demonstrated Wolbachia establishment in local Ae. aegypti populations. Methods: We describe a pragmatic approach to measuring the epidemiological impact of city-wide Wolbachia deployments in Bello and Medellín, Colombia. First, an interrupted time-series analysis will compare the incidence of dengue, chikungunya and Zika case notifications before and after Wolbachia releases, across the two municipalities. Second, a prospective case-control study using a test-negative design will be conducted in one quadrant of Medellín. Three of the six contiguous release zones in the case-control area were allocated to receive the first Wolbachia deployments in the city and three to be treated last, approximating a parallel two-arm trial for the >12-month period during which Wolbachia exposure remains discordant. Allocation, although non-random, aimed to maximise balance between arms in historical dengue incidence and demographics. Arboviral disease cases and arbovirus-negative controls will be enrolled concurrently from febrile patients presenting to primary care, with case/control status classified retrospectively following laboratory diagnostic testing. Intervention effect is estimated from an aggregate odds ratio comparing Wolbachia-exposure odds among test-positive cases versus test-negative controls. Discussion: The study findings will add to an accumulating body of evidence from global field sites on the efficacy of the Wolbachia method in reducing arboviral disease incidence, and can inform decisions on wider public health implementation of this intervention in the Americas and beyond. Trial registration: ClinicalTrials.gov: NCT03631719. Registered on 15 August 2018.",
"keywords": [
"Wolbachia",
"dengue",
"chikungunya",
"Zika",
"vector-borne disease",
"disease surveillance",
"interrupted time series",
"Colombia"
],
"content": "Abbreviations\n\nBG trap: BioGents Sentinel trap; CI: cytoplasmic incompatibility; ELISA: enzyme-linked immunosorbent assay; IRB: Institutional Review Board; PECET: Programa de Estudio y Control de Enfermedades Tropicales; qPCR: qualitative polymerase chain reaction; SAE: serious adverse event; WEI: Wolbachia exposure index; WHO: World Health Organisation; wMel: Wolbachia pipientis; WMP: World Mosquito Program\n\n\nBackground\n\nDengue is a major public health challenge in tropical regions, with 50 – 100 million symptomatic cases estimated to occur each year1,2. The World Health Organisation (WHO) cites a 30-fold increase in global incidence during the past 50 years1, and among endemic regions the greatest relative increase in dengue disease burden over the past two decades has been seen in Latin America2. The primary vector for dengue, the Aedes aegypti mosquito, also transmits the chikungunya and Zika viruses. Chikungunya emerged in epidemic fashion in several Indian Ocean islands in 2004 before spreading to southern Europe and South and South East Asia, then in 2013 re-emerged in epidemics in the Caribbean and several Latin American countries3. Following Zika virus outbreaks in the Western Pacific in 2013 and in Latin America in 20154, it was declared a public health emergency of international concern by the WHO in 20165 as evidence accumulated that congenital Zika virus infections can result in severe outcomes including foetal death and severe microcephaly. No specific treatment for dengue, chikungunya or Zika currently exists. Although a vaccine against dengue (Sanofi Dengvaxia®) was licensed in 2015, the WHO recommends vaccination only in persons with proven past dengue infection6,7. While efforts to develop a safe and effective vaccine continue, the WHO has emphasised the need for innovations in vector control to achieve reductions in dengue virus transmission and disease burden8. The evidence base for the effectiveness of commonly used vector control interventions is limited, with few having been rigorously evaluated against a clinical disease endpoint9. This highlights a vital need for carefully designed studies to evaluate vector control methods for arboviral and other vector-borne diseases10.\n\nThe World Mosquito Program (WMP; formerly the Eliminate Dengue Program) is an international research collaboration that is delivering a paradigm shift in the control of arboviral diseases transmitted by Ae. aegypti mosquitoes. Our method utilises Wolbachia, obligate intracellular endosymbionts that are common in insect species11–14 but were not present in Ae. aegypti mosquitoes until they were stably transinfected in the laboratory. In insects, Wolbachia is maternally inherited and manipulates insect reproduction to favour its own population dissemination via cytoplasmic incompatibility (CI). Strikingly, the presence of Wolbachia in Ae. aegypti mosquitoes reduces their ability to transmit viruses including dengue, Zika, chikungunya, and yellow fever15–17. Introgression of Wolbachia into wild Ae. aegypti populations is thus expected to severely reduce the vectorial capacity of local mosquito populations to transmit these arboviral infections. WMP’s field teams release male and female Wolbachia-infected Ae. aegypti as eggs or adults over a number of weeks. These mosquitoes then breed with the wild mosquito population and over time, through the actions of CI, the prevalence of Wolbachia in the local mosquito population increases, until such time as the majority of mosquitoes in the area carry Wolbachia. The WMP has demonstrated reduced vector competence in Wolbachia-infected mosquitoes obtained from the field, using human dengue viremic blood and a novel read-out to measure infectious mosquito saliva18. Wolbachia viral interference effects were found for all four DENV serotypes, resulting in estimated reductions of 66–75% in the basic reproduction number R0 for DENV-1-4. Reductions of this magnitude are predicted to result in local elimination of DENV transmission in most epidemiological settings18.\n\nColombia, located in the northwestern region of South America, is home to one-third of the population of Latin America. The Ae. aegypti mosquito is highly prevalent and dengue is endemic. In 2010 Colombia recorded its largest dengue outbreak with more than 150,000 confirmed cases, 217 deaths, and simultaneous circulation of all four dengue serotypes19. The first autochthonous chikungunya case was detected in Colombia in September 201420,21 and the first case of Zika in October 201522. Since then, numerous cases of both have been reported in the country.\n\nThe protocol presented in the current paper describes a pragmatic approach to measuring the efficacy of large-scale Wolbachia deployments in reducing the burden of arboviral diseases, in the municipalities of Medellín and Bello in northwestern Colombia, which have urban populations of 2.2 million in an area of ~100km2 and 476,000 in ~20km2, respectively. Staged deployment at the city-wide scale and within a relatively short time frame was favoured over a randomised controlled trial or other randomised design both by funders and local stakeholders. This was driven by what was, at the time of project conception, an urgent need for novel scalable strategies to combat the threat of Zika, and also a desire for the flexibility to optimise methods for scaled deployment under operational conditions, rather than the more restrictive implementation required for a formal randomised controlled trial. The proposed strategy for evaluating the impact of these staged deployments on the incidence of arboviral disease is a combination of an interrupted time-series analysis of notified arboviral disease incidence in all of Medellín and Bello, together with a more rigorous test-negative design study implemented in a sub-section of the Medellín municipal area. The primary aim of the study is to investigate whether large-scale deployments of Wolbachia-infected Ae. aegypti mosquitoes in Medellín and Bello, Colombia, lead to a measurable reduction in arboviral disease incidence.\n\n\nMethods\n\nTwo complementary approaches will be used to evaluate the disease impact of Wolbachia releases in Medellín and Bello:\n\ni An interrupted time-series analysis utilising routine disease surveillance data collected by the Medellín and Bello Health Secretariats, which aims to compare incidence of dengue, chikungunya and Zika pre- and post-Wolbachia release. This analysis will be applied separately to Medellín and Bello.\n\nii A prospective case control study using a test-negative design, which aims to quantify the reduction in disease incidence among people living within a Wolbachia-treated zone compared with an untreated zone that has a similar dengue risk profile at baseline. This study will be conducted in only one quadrant of Medellín (Figure 1).\n\nWolbachia-containing adult Ae. aegypti mosquitoes and eggs will be deployed sequentially through the study area, starting with the early-release zone of the case-control study (yellow shading in Figure 1; produced in ArcMap version 10.5, ESRI, CA), followed by releases in parts of Medellín and Bello outside the case-control area (grey shading in Figure 1), then lastly in the late-release area of the case-control study (green shading in Figure 1). For the purpose of analysis, release zones will be considered Wolbachia-treated from the date of completion of releases.\n\nAn initial period of Wolbachia deployment was undertaken in the early-release zone of the case-control area and in parts of Bello between April and December 2017, resulting initially in a high prevalence of Wolbachia. However, Wolbachia declined after cessation of releases and a subsequent round of deployments was conducted in these areas between mid-2018 and early 2019. It is this deployment period that will be considered for the purpose of the analyses described in this protocol.\n\nWolbachia prevalence is monitored through a network of BG-Sentinel adult mosquito traps (BioGents) that are evenly spaced throughout the study area at a density of approximately 16 BG traps per km2. BG traps are serviced weekly, with trapped mosquitoes screened for Wolbachia at weekly, fortnightly or monthly intervals throughout the duration of the study, depending on the stage of release and establishment. Trapped mosquitoes will be identified using microscopy. Individual Ae. aegypti mosquitoes (male and female) will be tested for Wolbachia by quantitative polymerase chain reaction (qPCR) assay. The Wolbachia prevalence in screened Ae. aegypti will be reported aggregated to the zone (i.e. early- and late-release zone in case-control area) or comuna (for parts of the city outside of the case-control area) level, calculated as the total number of Ae. aegypti mosquitoes that tested positive for Wolbachia aggregated across all BG traps in the zone/comuna, divided by the total number of Ae. aegypti mosquitoes that were screened in that zone/comuna.\n\nNotifiable disease surveillance data. In Medellín and Bello, routine public health surveillance for dengue is passive. There are more than 400 public and private health institutions that routinely report clinically-suspected and laboratory-confirmed dengue cases to the Secretary of Health as part of the Epidemiological Surveillance System. Approximately 10% of clinically-suspected dengue cases from hyperendemic or epidemic territories in Colombia are laboratory tested, however this proportion varies and was as high as >60% in 2016 in Medellín. Laboratory evidence suggestive of dengue is usually acquired via detection of anti-dengue IgM antibodies. Laboratory testing for chikungunya and Zika is not routinely performed in Colombia and is only done to demonstrate viral circulation in the area rather than for diagnostic purposes.\n\nFor the interrupted time-series analysis, disaggregate (line-listed) data will be requested for notified (clinically-suspected) dengue, chikungunya and Zika cases, and also the subset of dengue cases with IgM ELISA test results, from 2009 to 2025 for both Medellín and Bello. The dataset will include age, sex, address of primary residence, date of illness onset, date of notification, reporting health clinic, disease severity, hospitalisation, death, and, where available, geo-coordinates of the primary residence, type of diagnostic test performed, diagnostic test result, and final diagnostic classification.\n\nPrimary and secondary endpoints. The primary endpoint is the incidence of all dengue case notifications to the Epidemiological Surveillance System.\n\nThe secondary endpoints are: i) the number of cases who were IgM test positive for dengue; ii) the incidence of severe dengue cases reported to the surveillance system; and iii) the incidence of Zika and chikungunya cases reported to the surveillance system.\n\nStatistical analysis. For the primary endpoint and secondary endpoints above, the impact of Wolbachia deployment on disease incidence will be evaluated using an interrupted time series analysis of arbovirus cases reported to the Epidemiological Surveillance System before and after Wolbachia establishment. A generalised linear mixed-effects model will be used to model monthly case counts as the outcome variable, with an offset for population size and controlling for seasonality using flexible cubic splines or polynomial functions, to estimate the level change in incidence following the intervention (i.e. completion of Wolbachia releases). This analysis will be done separately for Medellín and Bello, 12 months after the completion of releases and each 12 months thereafter until five years post-intervention.\n\nStudy design. The prospective clinic-based case control study uses a test-negative design. The impact of Wolbachia deployments on arboviral disease incidence will be assessed by comparing the exposure distribution (probability of living in a Wolbachia-treated (early-release) vs. untreated (late-release) area) among virologically-confirmed arboviral disease cases presenting to a network of primary healthcare clinics, against the exposure distribution among patients with febrile illness of non-arboviral aetiology presenting to the same network of clinics in the same temporal window. Arboviral disease cases and arbovirus-negative controls will be sampled concurrently from within the population of patients who reside in the case-control area and present with febrile illness to the study clinic network, with case or control status classified retrospectively based on the results of laboratory diagnostic testing. The distribution of Wolbachia exposure in the sampled arbovirus negative controls is assumed to reflect the distribution of Wolbachia exposure in the underlying source population that gave rise to cases, as long as a core assumption is met that the relative propensity to seek healthcare for febrile illness at the study clinics in early-versus late-release arms is the same for arboviral disease cases as other febrile illness controls. This should be upheld if cases and controls are clinically indistinguishable until laboratory diagnosis. The concurrent sampling of cases and controls means that the odds of Wolbachia-exposure among sampled arboviral disease cases relative to febrile controls (i.e. odds ratio), is an unbiased estimate of the relative incidence of medically-attended arboviral disease in Wolbachia early-release versus late-release areas (i.e. relative risk or incidence rate ratio), from which protective efficacy can be estimated directly.\n\nStudy setting. The case-control study will be conducted only within a focused study area in northeast Medellín, including six contiguous release zones within four comunas (Figure 1), with a total population of 580,000 and area 15km2. Among these six release zones, three have been allocated non-randomly as the first zones in Medellín to receive Wolbachia deployments (early-release), and three as the last (late-release), such that a parallel two-arm trial is approximated for the period during which Wolbachia exposure remains discordant between arms. There are no buffer areas between treatment arms, but natural borders (roads, rivers, non-residential areas) were used to define study arm boundaries as much as possible, to limit the spatial spread of Wolbachia from treated areas into untreated areas, and of wild-type mosquitoes into Wolbachia treated areas. No attempt will be made to alter the routine dengue prevention and vector control activities conducted by public and private agencies throughout the case-control study area.\n\nAllocation of the intervention. The allocation of the six zones into two arms was done in a way that maximises balance between the arms with respect to measured factors that may be associated with baseline dengue risk, including historical dengue incidence, population characteristics, and geographical area (Table 1).\n\nStudy participants. Participants will be invited to participate, by trained research staff, from within the population of patients presenting with undifferentiated fever to a network of primary health care facilities that serve the population who reside in the study area. Participants (or their guardian if <18 years) must provide written informed consent, and meet the following inclusion criteria to be eligible for the study: fever (either self-reported or objectively measured as ≥38oC), with a date of onset between 1–4 days prior to the day of presentation to the health care facility; aged ≥3 years old; and lived (i.e. slept) in the study area for the 10 days preceding illness onset. Participants will not be eligible for inclusion if localizing features suggestive of a specific diagnosis (e.g. severe diarrhoea, otitis, pneumonia) are identified. An individual presenting to the clinic on repeat occasions for different febrile episodes will be eligible for enrolment during each different episode. However, an individual may only be enrolled once during a single illness episode, which is defined as illness occurring within 4 weeks of a previous febrile episode.\n\nData and sample collection. A unique identifier will be assigned to each participant at enrolment. Basic demographic details, eligibility against the inclusion criteria, illness onset date, and a retrospective travel history will be recorded in a standardised electronic data collection form. Figure 2 summarises the data and sample to be collected from each participant.\n\n*Routine dengue prevention and vector control activities will not be altered in treated or untreated areas. DENV: dengue virus; CHIKV: chikungunya virus; ZIKV: Zika virus.\n\nA brief travel history interview will be conducted at enrolment to determine the main places visited by each participant within the 3–10 days prior to illness onset, i.e. the incubation period for dengue. The duration of time spent at home, work or school, and other visited locations during the hours of 5am to 9pm in the 8-day period will be recorded, and the geographic coordinates of those locations derived by geo-locating them on a digital map, with the assistance of the study participant. These data will be used to determine the proportion of time spent in Wolbachia treated and untreated areas, for the per-protocol analysis.\n\nA single 6 ml venous blood sample will be collected from all consenting participants on the day of enrolment. Blood samples from all participants will be transferred to the project laboratory on the day of collection and batch-tested within one month to determine case or control status (Figure 3).\n\nBlue boxes indicate participant recruitment and enrolment activities undertaken at clinics. Yellow boxes indicate the laboratory diagnostic testing to be performed at the project laboratory, the results of which (white boxes) will be used to classify participants (grey boxes) as virologically confirmed dengue, Zika or chikungunya cases, arbovirus-negative controls, or excluded due to inability to rule out arbovirus infection according to the algorithm shown. DENV: dengue virus; CHIKV: chikungunya virus; ZIKV: Zika virus; PCR: polymerase chain reaction; NS1: non-structural protein 1; ELISA: enzyme-linked immunosorbent assay; IgM/IgG: immunoglobulin M/G.\n\nLaboratory investigations. An internally controlled triplex RT-qPCR assay (Bio-Rad) will be used to detect dengue, chikungunya and Zika viruses in serum samples from all enrolled participants. Dengue NS1 ELISA (Dengue Early ELISA, Panbio) will be performed according to the manufacturer’s instructions, on serum samples which have tested negative by DENV RT-qPCR. Dengue IgM and IgG capture ELISA (IgM/IgG Capture ELISA, Panbio) will be performed on serum samples which have tested negative by DENV RT-qPCR and NS1, and would otherwise be classified as controls, to determine whether they have detectable dengue IgM or IgG antibodies indicating potentially acute secondary dengue or another cross-reactive flavivirus infection in order to prevent misclassification. All research diagnostic investigations will be performed by the Programa de Estudio y Control de Enfermedades Tropicales (PECET), at the University of Antioquia, Colombia.\n\nCase and control classification. Dengue cases are defined as patients with virologically-confirmed DENV infection, meeting the clinical criteria for enrolment and also with a positive result in NS1 ELISA or DENV RT-qPCR. Controls are patients meeting the clinical criteria for enrolment, but with negative test results for DENV RT-qPCR, CHIKV RT-qPCR, ZIKV RT-qPCR, DENV NS1 ELISA, and DENV IgM and IgG ELISA (Figure 3).\n\nFor the secondary endpoints, Zika or chikungunya cases are defined as patients with virologically-confirmed Zika or chikungunya infections, meeting the clinical criteria for enrolment and with a positive result in ZIKV RT-qPCR or CHIKV RT-qPCR, respectively, and controls are defined as above.\n\nExpected study duration. Pilot clinic-based sampling of febrile patients commenced in November 2017, with enrolment into the intention-to-treat dataset to commence after completion of Wolbachia releases in the early-release area. The study will continue to enrol participants until such time as Wolbachia deployment commences in the late-release area, which is expected to be at least 12 months after Wolbachia establishment in the early-release area, i.e. enrolment can continue even when the target sample size is reached. The study timeline is depicted in Figure 4.\n\nITS: interrupted time series.\n\nPower calculations. It is estimated that 88 test-positive cases plus four times as many controls will be sufficient to detect a 50% reduction in dengue incidence with 80% power. Thus, we set the target sample size as 100 test-positive dengue cases and expect that by including in the analysis all participants enrolled after Wolbachia is established in the early zone, there will be in excess of 400 test-negative controls for 100 test-positive cases. Although we expect the true effect of Wolbachia on dengue transmission may be greater than a 50% reduction, the observable reduction in effect is expected to be lower because individuals are likely to spend a substantial proportion of their time outside their release zone of residence. These sample size estimates are based on standard formulae for calculating sample size/power in a case control study. They align with the proposed approach for estimating the intervention effect.\n\nStatistical analysis. The analyses described here will be performed on datasets of cases and controls defined firstly using the primary endpoint of virologically-confirmed dengue cases, and then using the endpoints of virologically-confirmed chikungunya and Zika cases.\n\nThe intention-to-treat analysis will consider Wolbachia exposure as a binary classification based on residence in the early or late-release area. Residence will be defined as the primary place of residence during the 10 days prior to illness onset. The intervention effect will be estimated from an aggregate odds ratio (for data aggregated across all three Wolbachia-release zones within each study arm) comparing the exposure odds (residence in the Wolbachia early-release area) among test-positive cases versus test-negative controls. The null hypothesis is that the odds of residence in a Wolbachia early-release area is the same among test-positive cases as test-negative controls.\n\nThe per-protocol analysis will consider Wolbachia exposure as a quantitative index based on measured Wolbachia prevalence in local Ae. aegypti mosquitoes in the locations visited by the participant during the 10 days prior to illness onset, both within the case control area and in elsewhere in Medellín and Bello. The per-protocol analysis therefore allows for Wolbachia exposure to vary in a location over time, and also accounts for human mobility, in terms of the exposure-time that individuals spend outside their area of residence as reported in the travel history interview at enrolment. A weighted ‘Wolbachia exposure index’ (WEI) will be defined for each participant, as follows. The aggregate Wolbachia prevalence for each release zone will be calculated each month from all Ae. aegypti trapped in that zone. Time spent outside a Wolbachia release area will be treated as not Wolbachia exposed. The WEI for each participant will then be calculated by multiplying the zone-level Wolbachia prevalence (in the month of participant enrolment) at each of the locations visited, by the proportion of time spent at each location, to give a value on a continuous scale from 0 to 1. Cases and controls will be classified by strata of their WEI (e.g. 0-0.2; 0.2-0.4; 0.4-0.6; 0.6-0.8; 0.8-1). This acknowledges that the WEI is not a highly precise measure and serves to reduce error in exposure classification. This analysis can also account for the temporal matching of arboviral disease cases and test-negative controls: risk sets of cases and controls will be defined by frequency matching enrolled confirmed arboviral disease cases to arbovirus-negative controls with illness onset in the same quarter of the year. In the unlikely event that a minimum of four controls cannot be found for a case within the same quarter, the window for matching can be extended until four controls are identified, for that case only. For a time-adjusted analysis, a Cox proportional hazard model will be fitted, which can incorporate the temporal case-control risk sets and participants’ WEI stratum as a categorical variable, using time since completion of Wolbachia releases as the time scale.\n\nData management. Clinical study data will be stored in a custom designed relational database hosted on a secure web-based server. Role-based, tiered access permissions will be used to control access to the clinical database and associated data capture applications. User logs will document the activities of all users. An audit trail will be preserved within the database to capture the history of any changes made to data records after their initial capture.\n\nData collected from participants in the case-control study will be captured through standardised electronic data capture forms and digital mapping interfaces, deployed as web-based applications on mobile tablets. Laboratory diagnostic results will be captured directly from laboratory assay output and uploaded to a web-based application for storage in the same relational database. Validity controls will be applied at the point of data capture into electronic forms, by predefining value ranges, specifying categorical option lists, and minimizing the use of free text fields. The use of carefully designed electronic forms will facilitate the coding of participant responses at the point of data collection. Quality control in the form of logic and consistency checks will be applied at the point of data capture into an electronic form and at the point of upload into the web-based database. All data relating to the case-control study, including field entomology and epidemiological data, will be retained indefinitely, and for a minimum of five years after study completion, in accordance with International Council for Harmonization on Good Clinical Practice requirements.\n\nMonitoring of adverse events. Any severe adverse events (SAE) associated with collection of blood samples from study participants will be reported to the relevant institutional ethics committees within three days of notification. Standard SAE reporting forms will be used.\n\nStudy governance. A steering committee will be assembled to provide operational and strategic advice on planning and operations of the study. A WHO-convened independent evaluation group will review the study within a year of active enrolment as part of a program-wide evaluation of WMP Colombia activities in Medellín and Bello. The independent evaluation group will report its findings to all stakeholders at completion of the review.\n\nEthical considerations. The study protocol (version 3.0) and the informed consent document have been reviewed and approved by the Institutional Review Boards (IRBs) of the IPS Universitaria of Universidad de Antioquia, Colombia (No. 115, 25 Oct 2017 and No. 127, 12 Oct 2018), and Monash University, Melbourne (ID 11534). Any future protocol amendments will be submitted for review and approval by the same IRBs, prior to implementing protocol changes. The trial protocol was registered on ClinicalTrials.gov (NCT03631719) on 15th August 2018.\n\nConfidentiality of participant information will be strictly maintained at all times by the participating investigators, research staff and the sponsoring institution (Universidad de Antioquia) by means of a coded ID number. This confidentiality is extended to cover testing of biological samples in addition to all laboratory specimens, reports, data collection forms and log books, and geolocated records relating to participating subjects. All records that contain names or other personal identifiers, such as informed consent forms, will be stored separately from study records while identified by ID numbers. All local databases will be secured with password-protected access systems. No information concerning the study or the data will be released to any unauthorised third party, without prior written approval of the sponsoring institution. Clinical or personal information will not be released without written permission of the subject, except as necessary for monitoring by an ethical review board or regulatory agencies. Reporting of study results will not be done in any way that permits identification of individual participants, or the location of their homes or other visited locations.\n\nParticipant recruitment into the prospective case-control study commenced in early 2019 and is ongoing. Wolbachia deployments across the two municipalities are ongoing through to 2020, and collation and analysis of disease surveillance data will continue until 2025.\n\nAnalysis and reporting of the results of the prospective case-control study will occur only at completion of participant enrolment, and subject to the prior approval of the steering committee; there will be no interim analysis or dissemination of results. The interrupted time series analysis will be conducted 12 months after the completion of releases and annually thereafter. Findings from both studies will be submitted for peer review and publication in an appropriate open access journal, together with aggregate supporting data.\n\n\nDiscussion\n\nMosquito suppression remains the primary method used to control dengue virus transmission. A recent evaluation found little reliable evidence for the effectiveness of any dengue vector control method, and concluded that standardised studies of higher quality must be prioritised9. Aedes aegypti mosquitoes are the primary vectors of dengue, chikungunya and Zika viruses, therefore evidence-based interventions targeting this species have the potential to reduce multiple arboviral diseases where they co-circulate. The study described here will evaluate the impact of Wolbachia-infected mosquitoes on dengue and other arboviral diseases in Bello and Medellín municipalities, Colombia, using a combination of routinely collected disease surveillance data throughout the municipalities, and a prospective clinic-based test-negative study focused in one area of Medellín.\n\nThe test-negative design, a variant on the case-control design, which has been widely applied in non-randomised influenza vaccine effectiveness studies, uses outcome-based concurrent sampling of dengue cases and non-dengue controls to measure the efficacy endpoint23–26. Effect estimates (odds ratios) from a test-negative design are equivalent to direct estimates of relative risk in the source population, under the assumption that the distribution of test-negative illness is not associated with the intervention, and that test-negative controls are allowed to include participants who may be classified as dengue cases at other times during the study period.\n\nThe interrupted time-series design is commonly used to evaluate the impact of public health interventions introduced at a population level and targeting population-level health outcomes27–29. A series of repeat observations over time is analysed to establish a baseline trend which is assumed to be ‘interrupted’ by the introduction of an intervention. The subsequent post-intervention trend is compared to the counterfactual that would be expected in the absence of the intervention based on the baseline trend. A quantitative estimate of the intervention effect is derived from a segmented linear regression model of the outcome of interest (e.g. dengue case count or incidence) as a function of time, in which the intervention status is captured by a binary variable coded 0 for pre-intervention time points and 1 for post-intervention time points30,31. Seasonality and other secular trends and time-varying confounders can also be controlled for by the inclusion of additional model parameters. A limitation of using notifiable disease surveillance data is the imperfect specificity of the clinical case definition used for notifications, meaning an unknown and time-varying proportion of notified cases are not true dengue infections. Inconsistent reporting practices, outbreaks of non-dengue febrile diseases, or other factors may induce secular trends in the surveillance data that are independent of, but contemporaneous with, the Wolbachia intervention and thus may influence our ability to estimate the true intervention effect from time series data. A subset of the notified dengue cases in Colombia have supportive laboratory diagnostic results, but these have several limitations: i) laboratory testing can be infrequent, particularly during outbreaks, ii) the cross-reactivity of IgM serology between dengue and Zika limits the utility of serological data where Zika co-circulates, and iii) no virological confirmation (PCR or NS1 antigen detection) of cases is performed. We therefore base our primary analysis on all notified dengue cases (suspected and dengue IgM test positive). The benefits of using these routinely collected surveillance data include the availability of a long time series, reduced costs for data collection and timely acquisition of data.\n\nThe pragmatic approach described here to evaluate disease impact arose from the imperative from funders and local stakeholders to achieve rapid scale-up of Wolbachia deployments and to retain flexibility in the release sequence and methods, in the context of the declaration of the Zika public health emergency at the time of project conception and funding8. These imperatives precluded implementation of the proposed test-negative design study across all of Medellín/Bello, given the time required to obtain approvals, establish clinical enrolment processes, train staff, and then maintain an untreated comparison area for the duration of clinical enrolment. Randomised allocation of the early and late Wolbachia release areas in the focused case-control study was also not feasible, given the small number of zones within the case-control area. In general, there is greater potential for selection bias in non-randomised studies than in randomised studies, which, in the context of the current study, may present as a differential distribution of non-arboviral febrile illness (i.e. test-negative controls) by study arm due to chance imbalance in the care-seeking populations between study arms or differential propensity for care-seeking among those in areas where Wolbachia is released compared with untreated areas. As long as a core assumption of the test-negative design is met that the relative propensity to seek healthcare for undifferentiated febrile illness at a study clinic, in early vs late zones, is the same for dengue cases as for other febrile illness controls, then an imbalance in participant enrolment between early and late zones should not in itself bias the results.\n\nA staged approach to city-wide deployments following a ‘stepped-wedge’ design was also considered in planning the disease impact assessment for Medellín, however this carried a requirement to define a deployment sequence that maintained balance between ‘already treated’ and ‘not yet treated’ areas in factors associated with baseline dengue risk, to avoid selection bias. This had resource implications for the field entomology and community engagement teams’ activities that were inconsistent with the necessarily pragmatic approach to deployment in Medellín and Bello needed to achieve large-scale coverage within a short time frame.\n\nThe non-blinded deployment of the intervention means community members may alter their care seeking or vector control behaviour due to the belief that they are protected from dengue by Wolbachia. Blinding was determined to be cost prohibitive in this study as it would have doubled the resources and time required to conduct field releases of mosquitoes. Under the test-negative design, test-positive cases and test-negative controls are drawn from the same population of patients presenting to health clinics with febrile illness, who are clinically indistinguishable at the time of enrolment. Thus, the threat of bias due to non-blinding in the test-negative study is considered minimal as long as any modified behaviour applies equally to test-positive cases and test-negative controls. Routine vector control by health authorities were not altered as part of the study, and it is possible that some public health activities or change in community behaviour occurred which impacts arboviral disease incidence independently of Wolbachia, leading to an under- or over-estimation of the Wolbachia intervention effect in our study.\n\nHuman mobility is a potential confounding factor in the estimation of Wolbachia’s impact on arboviral disease. Individuals in whom the efficacy endpoint is measured may spend a proportion of their time outside their allocated study area (i.e. their area of primary residence) resulting in at least some exposure misclassification. There are no buffer zones separating the early-release and late-release areas of the case control study, nor the other areas of Medellín and Bello where staged deployments will occur, and Wolbachia contamination from one area to another is possible but also measurable. The combined effect of human mobility and Wolbachia contamination is that the exposure status of the populations in the nominally ‘treated’ and ‘untreated’ areas become more similar to each other, thereby diluting any estimated intervention effect towards the null. By powering the case-control study to detect a relatively conservative effect size of 50%, we have allowed for some of this effect dilution while targeting a reduction in dengue incidence of public health significance. The case-control study per-protocol analysis, where recent travel history is documented and a quantitative Wolbachia exposure status is calculated for each participant, will account for both for the time spent in areas away from home and the local measure of Wolbachia prevalence in visited areas.\n\nInter-annual fluctuations in dengue transmission mean that the case-control study might fall in a period of lower incidence just by chance. Nevertheless, the target sample size of 100 dengue test-positive cases is seen as feasible even in the event of a low transmission year. The interrupted time-series analysis, which spans a longer period than the case-control study, is expected to have at least three to five years of post-Wolbachia release data, and thus should be more tolerant to periods of low dengue transmission.\n\nThe generalisability of the current study’s findings to other dengue endemic settings will likely be influenced by setting-specific factors such as the local entomological context, which influences Wolbachia introgression. Generalisability may also depend on the local distribution of circulating DENV serotypes and the intensity of virus transmission, which might influence the observed impact of Wolbachia on arboviral disease incidence. A cost-effectiveness analysis is underway in Indonesia, and the findings are expected to help inform cost optimisation for different deployment scenarios, target settings, and scale-up methods that can be applied to other countries. If the results of the current study do demonstrate a reduction in arboviral disease incidence associated with Wolbachia deployment in Medellín and Bello, a key next step would be to scale this intervention into wider public health implementation in Colombia and elsewhere in the Latin American region, using epidemiological, ecological and cost-effectiveness data to inform an optimal strategy for scaled deployment.\n\nThis study has been approved by the Institutional Review Boards of the IPS Universitaria of Universidad de Antioquia, Colombia, and Monash University, Melbourne. Written informed consent will be obtained from all participants, or their guardian where the participant is a minor (<18 years), prior to enrolment in the clinic-based case-control study. The interrupted time-series analysis uses pre-existing non-identifiable disease surveillance data, which does not require individuals’ consent.\n\n\nData availability\n\nNo data is associated with this article.",
"appendix": "Grant information\n\nThis work was supported by the Wellcome Trust working in partnership with the UK Department for International Development (Grant 102591/Z/13/A), United States Agency for International Development (Grant AID-OAA-AA-16-00081), and Bill & Melinda Gates Foundation (Grant OPP1159497).\n\nThe funders had no role in the design of the study or the decision to publish.\n\n\nAcknowledgements\n\nThe authors acknowledge the contributions of Peter Ryan (World Mosquito Program) for the planning and implementation of Wolbachia deployments in Medellín and Bello, Tomas Santamaria (World Mosquito Program) for administrative support, and Rita Almanza and Carlos Montes (Medellín Health Secretariat) for support and provision of Medellin surveillance data.\n\n\nReferences\n\nWorld Health Organisation: Global strategy for dengue prevention and control 2012–2020. Geneva: WHO; 2012; Reference Source\n\nStanaway JD, Shepard DS, Undurraga EA, et al.: The global burden of dengue: an analysis from the Global Burden of Disease Study 2013. Lancet Infect Dis. 2016; 16(6): 712–23. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRolph MS, Foo SS, Mahalingam S: Emergent chikungunya virus and arthritis in the Americas. Lancet Infect Dis. 2015; 15(9): 1007–8. PubMed Abstract | Publisher Full Text\n\nWeaver SC, Costa F, Garcia-Blanco MA, et al.: Zika virus: History, emergence, biology, and prospects for control. Antiviral Res. 2016; 130: 69–80. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWorld Health Organisation: Outcome of the Emergency Committee regarding clusters of microcephaly and Guillain-Barre syndrome. 2016; Accessed 2 May 2018. Reference Source\n\nWorld Health Organisation: WHO Global Advisory Committee on Vaccine Safety Statement on Dengvaxia (CYD-TDV). 2017; Accessed 2 May 2018. 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}
|
[
{
"id": "51965",
"date": "28 Aug 2019",
"name": "Stephen Waterman",
"expertise": [
"Reviewer Expertise Epidemiology",
"dengue and arbovirology",
"tropical 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 paper describes the protocol for a clinical trial releasing Wolbachia bacterium infected male and female Aedes aegypti mosquitoes in Medellín and Bello, Colombia, with the primary endpoint objective of demonstrating reduction of human dengue infections in the population living in the treated areas. Wolbachia infected female release over time results in replacement of the uninfected Aedes aegypti population with Wolbachia infected mosquitoes that have reduced ability to transmit arboviruses. The study design has two components, an interrupted time series (ITS) approach using passive arboviral disease surveillance data from Bello and a large area of Medellín, and a prospective case-control in a smaller area of northeast Medellin using arbovirus laboratory test negative acute febrile illness controls in 6 early release and late release zones. The studies begin approximately one year after mosquito releases (in 2018 for the early release zone and 2019 for the releases outside the case-control area) to allow for mosquito population replacement.\n\nThis study employs a novel Aedes aegypti vector control approach to address the major and growing tropical public health problem of Aedes transmitted arboviruses including dengue, Zika, and chikungunya viruses. The authors point out that such studies are badly needed as conventional Aedes aegypti vector control approaches have failed or been shown to be non-sustainable, and the only currently licensed dengue vaccine is partially effective and requires a laboratory test prior to immunization.\n\nThis study protocol builds on laboratory and fieldwork in Australia and elsewhere, and is well designed and well written with considerable detail (design, statistical power and analysis, laboratory testing, data management, etc) to address the question of human arboviral disease impact of Wolbachia replacement in a highly endemic tropical urban setting in Latin America.\n\nThe protocol is intentionally “pragmatic” to roll out the intervention at large scale under operational conditions in a timely and cost effective manner acceptable to the study community. The authors thus effectively answer potential criticisms of the study design not being a cluster randomized clinical trial as recommended by the WHO Vector Control Advisory Group. The case control study design also importantly accounts for the extent of replacement with Wolbachia infected mosquitoes in the treated area and human mobility out of the treatment area with a calculated Wolbachia exposure index calculated for each acute febrile illness patient.\n\nThe test negative case control study design obviates the need for expensive clinical cohort follow up to monitor disease incidence and treated and untreated areas. The authors correctly point out that the risk estimate from the test negative case control approach is likely to be unbiased and can be used to calculate efficacy confidence limits. The ITS approach is a less rigorous experimental design in that the vast majority of passively reported cases are not virologically confirmed, and surveillance may not be always be done in a consistent manner, but is another practical approach using available data from the Ministry of Health that allows for monitoring of reductions in arboviral case reports over a long time period (5 years) after the Wolbachia replacement treatment during which arbovirus outbreak cycles would be expected to occur.\n\nThe discussion appropriately comments on the potential generalizability of the findings to other settings and that cost effectiveness analysis of this approach is underway.\n\nThe following are suggestions to clarify aspects of the protocol:\nBackground:\nWhile Medellín is clearly highly endemic for dengue, including the historical annual average incidence of dengue would be of interest, as the case-control study will be just be done over a one year time period. Dengue transmission dropped to unusually low levels throughout most of South America after the Zika epidemic, and is on the increase in the region as of mid-2019.\n\nMethods:\nThe entomologic details regarding the mosquito releases and strain(s) of Wolbachia could be expanded upon. What is being done differently in 2018 and 2019 compared to the 2017 releases that achieved temporary high Wolbachia prevalence?\n\nMore details on the Wolbachia sampling and monitoring strategy would be helpful. What is the extent of the monitoring area? All comuna outside the case-control area? Checking BG traps once a week does not ensure that the mosquito specimens will be identified correctly because of damage from the trap fans to captured mosquitoes. Many samples will be lost because of loss of power, ants, etc. BG traps should be checked daily.\n\nFor the ITS component and the secondary endpoint of severe dengue incidence, how does the Ministry of Health define severe dengue, and has severe dengue been consistently defined over time?\n\nRegarding the case control study participant recruitment, what is the possibility that patients with acute febrile illness in the study areas will seek care in clinics outside the network?\n\nWith regard to the laboratory testing algorithm to exclude potential dengue serologic positives from being control patients, the authors could more explicitly state that PanBio IgG ELISA test is designed with cutoffs to only detect high IgG titers consistent with acute secondary infections, and not past infections. Excluding all patients with IgG antibody due to past dengue infection in this setting would eliminate a large percentage of the potential control patients.\n\nConsider observing changes in the vector-bacteria-virus interaction through time. Coevolution is likely. Whether virus blocking ability changes over time (e.g., lowered bacteria concentration in the mosquito cells) is not clear.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable",
"responses": [
{
"c_id": "5502",
"date": "21 May 2020",
"name": "Katherine ANDERS",
"role": "Author Response",
"response": "Background: 1. To clarify, the case-control study period was planned to be a minimum of 12 months duration but now has funding to continue in order to attain the target sample size, given the low dengue event rate throughout this region of Colombia following a large epidemic in 2016. We have removed the timeline in Figure 4, which is no longer applicable. We have now included the historical annual average incidence of dengue in Medellin and Bello in the background section, as suggested. Methods: 1.Text has been added to the second paragraph under ‘Wolbachia deployment’ to clarify the changes in Wolbachia release material from the 2017 to the 2018/2019 releases, as follows: ‘An initial period of Wolbachia deployment was undertaken in the early-release zone of the case-control area and in parts of Bello between April and December 2017, resulting initially in a high prevalence of Wolbachia. However, Wolbachia levels declined after cessation of releases. We believe this was due to fitness issues with the Wolbachia-infected mosquitoes, specifically that they were less resistant to insecticides than the wild population. Insectary processes were subsequently updated and a new colony of Wolbachia mosquitoes was produced that matched the wildtype insectide resistance profile. Subsequent rounds of deployments were then conducted in these areas between mid-2018 and late-2019. It is this deployment period that will be considered for the purpose of the analyses described in this protocol.’ 2. The text describing the Wolbachia sampling and monitoring strategy has been edited to clarify that ‘Wolbachia prevalence is monitored through a network of BG-Sentinel adult mosquito traps (BioGents) that are evenly spaced throughout all of Bello and Medellin, including the case-control study area, at a density of approximately 16 BG traps per km2’, and that ‘BG traps that do not catch any mosquitoes in three consecutive weeks are moved to another location’. Servicing BG traps more frequently than once a week is not feasible for city-wide projects, and weekly collection is standard across all our project sites. There is certainly the potential for loss of samples due to damage, predators, loss of power etc, but this would result only in a loss of sample size for estimation of Wolbachia prevalence, not a bias in these estimates. Trap failures for various reasons are noted at the time of trap servicing and documented in the entomological database. 3. The case definition for severe dengue has now been added to the text describing the primary and secondary endpoints for the ITS analysis, as follows: ‘Severe dengue is defined as any case of dengue that has one or more of the following manifestations: i) shock or respiratory distress due to severe plasma leakage; ii) severe bleeding according to the evaluation of the treating physician; or iii) severe organ involvement, such as liver damage, impaired consciousness, myocarditis, or other organ involvement.’ 4. We know that a subset of patients with acute febrile illness in the study areas certainly will seek care in clinics outside the network of study clinics, and indeed outside the case-control study area. However the test-negative case-control design is robust to this, based on the assumption that the propensity of patients from the treated and untreated case-control areas to seek care at the network of study clinics is equivalent among test-negative controls (patients with other causes of febrile illness) and test-positive dengue cases. Ie. complete sampling of acute febrile illness patients in the study area is not required, as long as the sampling fraction can be assumed to be equivalent for test-positive cases and test-negative controls. 5. This clarification has been added to the text describing methods for laboratory investigations. 6. Work is ongoing to monitor the co-evolution of Wolbachia and Aegypti over time in our Australian and international field sites. Preliminary unpublished WMP work has established that the wMel Wolbachia genome remains highly stable eight years post-release in northern Australia. Others have shown that pathogen blocking in Wolbachia-infected Ae. aegypti is stable due to an evolutionary trade off between the mosquito host and the bacteria (Ford et al (2019) Nature Microbiology), whereby mutations in Ae. aegypti that reduce Wolbachia-mediated virus blocking carry a significant fitness cost."
}
]
},
{
"id": "57177",
"date": "16 Dec 2019",
"name": "Penelope Hancock",
"expertise": [
"Reviewer Expertise Spatial modelling and statistics",
"population dynamics",
"Wolbachia."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper describes protocols for assessing the efficacy of Wolbachia releases on reducing dengue incidence based on data from release trials conducted in municipalities in Columbia. The Wolbachia has been released into 6 adjacent zones covering a sub-area, where 3 of the zones have early releases and the other 3 have later releases – the zones which receive late releases are regarded as non-intervention arms for the purposes of the trial analysis.\nIt is always difficult to measure the efficacy of vector control interventions using randomized trials because of community level effects such as herd immunity, so that the intervention has impacts on both treatments and controls, and the estimated effect of the intervention is diluted – an effect known as contamination. In the case of Wolbachia releases, the effect is particularly difficult to quantify because the Wolbachia may spread to mosquito populations in the control arms, or Wolbachia may only have an intermediate (and variable) prevalence in the intervention arms. The trial described in this paper has additional complications which the authors describe in the manuscript, including the non-random allocation of intervention clusters and the small size of the study area with intervention arms adjacent to control arms and no buffer between them. The authors explain that these limitations mean that a pragmatic approach to analysis of Wolbachia efficacy is required.\nI think the paper would benefit from a more rigorous quantitative protocol for how intervention efficacy can be quantified in the presence of contamination (see Silkey et al. 2016, Trials1). The power calculation that the authors provide doesn’t explore impacts of contamination, or impacts of incomplete spread of Wolbachia in the intervention arm. In addition the issue of herd immunity should be pointed out in the paper. It seems that given the configuration of the release zones, an effective Wolbachia intervention could greatly interrupt dengue transmission in the non-intervention (later release zones) as well, thus making efficacy very difficult to evaluate.\nI find the Wolbachia Exposure Index proposed for individual participants (to account for their movements outside/into release zones) to be problematic. Aedes aegypti have a very heterogeneous local distribution, and I don’t think variations in exposure can be calculated using travel histories – these estimates are likely to be misleading at best.\nRegarding the interrupted time series approach, I think a more detailed and formal mathematical description of the method is needed, detailing the sampling distribution of the infection data with respect to the location of Wolbachia interventions, and the statistical model for handling spatiotemporal autocorrelation. It does not seem straightforward to distinguish the effect of Wolbachia from non-related fluctuations in disease incidence caused by environmental factors. More explanation of how this will be achieved is required.\nPlease put a scale on the map in Figure 1.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Partly\n\nAre sufficient details of the methods provided to allow replication by others? No\n\nAre the datasets clearly presented in a useable and accessible format? Not applicable",
"responses": [
{
"c_id": "5503",
"date": "21 May 2020",
"name": "Katherine ANDERS",
"role": "Author Response",
"response": "I think the paper would benefit from a more rigorous quantitative protocol for how intervention efficacy can be quantified in the presence of contamination (see Silkey et al. 2016, Trials). Authors’ response: Contamination will be addressed in the analysis by incorporating area-level measured Wolbachia frequencies in the model, rather than presuming an area’s exposure status based on whether Wolbachia releases had occurred in that area. For the interrupted time-series analysis of dengue cases notified to the Medellin and Bello health surveillance system, additional analyses will now explore the effect of Wolbachia on the incidence of notified dengue using Wolbachia frequency either as a continuous covariate or categorised into quintiles of exposure. In the per-protocol analysis of the case-control study, each participant will be assigned a Wolbachia Exposure Index calculated using zone-level Wolbachia frequency and time spent in that release zone. These methods thus additionally take into account potential incomplete spread of Wolbachia in release areas as well as contamination into areas where Wolbachia releases have not yet occurred. The method proposed by Silkey et al to quantify the magnitude of the contamination effect – i.e. comparing disease incidence among naïve (non-intervened) groups living close to an intervention area to those in naïve groups living remote from the intervention - cannot account for the possibility that Wolbachia fails to establish in a communa where releases have occurred and would instead assume these communas to be Wolbachia-treated. The power calculation that the authors provide doesn’t explore impacts of contamination, or impacts of incomplete spread of Wolbachia in the intervention arm. Authors’ response: Contamination and/or incomplete spread of Wolbachia is expected to dilute any intervention effect by making the intervention and untreated areas more similar. Our sample size is powered to detect the conservative effect size of 50%, though we expect the true reduction in dengue incidence to be much greater than 50% based on preliminary findings from other WMP project sites which demonstrate reductions ranging from 75% to 97% at 12 months or more post-intervention. Our existing sample size therefore allows for a dilution of the true intervention effect by contamination, incomplete Wolbachia establishment in intervention areas, and/or human mobility. In addition the issue of herd immunity should be pointed out in the paper. It seems that given the configuration of the release zones, an effective Wolbachia intervention could greatly interrupt dengue transmission in the non-intervention (later release zones) as well, thus making efficacy very difficult to evaluate. Authors’ response: Dengue transmission is known to be quite focal (<500m) in urban areas (Salje et al PNAS 2012; Morrison et al PloS NTD 2010) so it is unlikely, although possible, that interruption of transmission in the early release zone would have spillover effects into the adjacent late release areas, independent of the potential movement of the Wolbachia mosquitoes themselves. A brief discussion has been added to the paper (see Discussion, paragraph 7). I find the Wolbachia Exposure Index proposed for individual participants (to account for their movements outside/into release zones) to be problematic. Aedes aegypti have a very heterogeneous local distribution, and I don’t think variations in exposure can be calculated using travel histories – these estimates are likely to be misleading at best. Authors’ response: The Wolbachia Exposure Index aims to capture not only human mobility in and out of release zones, but also heterogeneity in Wolbachia establishment among the early-release zones. The WEI will be calculated using the zone-specific aggregated Wolbachia prevalence in the month of participant enrolment, which recognises that Wolbachia establishment in intervention areas may be imperfect, that some Wolbachia spread into the control area may occur, and that Wolbachia prevalence varies across time. We acknowledge that there will be heterogeneity in Wolbachia frequency even within each zone (and also in Ae. aegypti distribution as the reviewer notes), however, no accurate interpolation at a geographical scale smaller than communa level is possible. We have added an additional per-protocol analysis in which the WEI is calculated using only the zone-level Wolbachia prevalence in the participant’s zone of residence (in the month of participant enrolment), ignoring the participant’s recent travel history. This recognises that dengue exposure risk may be higher at home versus other locations, rather than assuming an even distribution of exposure risk across daytime hours and locations visited (see paragraph 4 of Methods – Statistical analysis). Regarding the interrupted time series approach, I think a more detailed and formal mathematical description of the method is needed, detailing the sampling distribution of the infection data with respect to the location of Wolbachia interventions, and the statistical model for handling spatiotemporal autocorrelation. It does not seem straightforward to distinguish the effect of Wolbachia from non-related fluctuations in disease incidence caused by environmental factors. More explanation of how this will be achieved is required. Authors’ response: We previously planned on using a mixed-effects negative binomial model to assess the impact of the Wolbachia intervention on dengue incidence, modelling between-communa variability as a random effect by including a random intercept at the communa level and allowing for a random-slope on the intervention. However, given that modelling the random effects themselves are not of particular interest in this analysis, it was decided that a generalised estimating equation approach would be more efficient as it most directly targets the group-level parameter of the intervention effect and requires fewer modelling assumptions than a mixed-effects model. The outcome distribution is assumed to be negative binomial to allow for overdispersion. Monthly case counts will be modelled as the outcome variable, with an offset for population size and controlling for seasonality using flexible cubic splines. Robust standard errors will be used to account for clustering of cases by communa. An autoregressive correlation structure will be specified to account for temporal autocorrelation. A mixed-effects model will then be used to check for potential small sample issues in inference estimation, given the modest number of clusters in Medellin (18 communas) and Bello (11 communas) and the large number of observations per cluster. We acknowledge that there may be other environmental factors contributing to natural fluctuations in dengue incidence which we are unable to explicitly account for in the model. The availability of historical data (range 5-8 years) and the staged nature of the intervention means the statistical model includes between-communa (within-period) comparisons as well as before-and-after (within-communa) comparisons, which is expected to control for at least some of these natural fluctuations that are unrelated to the Wolbachia intervention. Please put a scale on the map in Figure 1. Authors’ response: A scale has been added to the map."
}
]
}
] | 1
|
https://f1000research.com/articles/8-1327
|
https://f1000research.com/articles/9-414/v1
|
20 May 20
|
{
"type": "Research Article",
"title": "COVID-19 countermeasures, Major League Baseball, and the home field advantage: Simulating the 2020 season using logit regression and a neural network",
"authors": [
"Justin Ehrlich",
"Shankar Ghimire",
"Shankar Ghimire"
],
"abstract": "Background: In the wake of COVID-19, almost all major league sports have been either cancelled or postponed. The sports industry suffered a major blow with the uncertainty of sporting events being held in the near future. Various scenarios of how and when sports might recommence have been discussed. This paper examines various scenarios of how Major League Baseball team performance is going to be impacted by the presence of fans, or the lack thereof, in the context of physical distancing and other COVID-19 countermeasures Methods: The paper simulates, using a neural network and a logit regression model, the win-loss probabilities for various scenarios under consideration and also estimates the home effect for each team using data for the 2017-2019 seasons. Results: The model demonstrates that individual team home effect is symmetric between home and away and teams will not necessarily have a win or loss of any additional games in neutral stadiums, as teams with a high home field effect will lose more neutral games that would have been at home but will win more neutral games that would have been away. However, the result of individual games will be different since home effect is asymmetric between teams. Our simulation demonstrates that these individual game differences may lead to a slight difference in Play-Off Berths between a full season, a half season, or a full season without fans. Conclusions: Without fans, any advantage (or disadvantage) from home field advantage is removed. Our models and simulation demonstrate that this will reduce the variance. This stabilizes the outcome based upon true team talent, which we estimate will cause a larger divide between the best and worst teams. This estimation helps decision makers understand how individual team performance will be impacted as they prepare for the 2020 season under the new circumstances.",
"keywords": [
"MLB",
"Baseball",
"COVID-19",
"Neural Network",
"Logit"
],
"content": "Introduction\n\nThe 2019–2020 pandemic from the novel coronavirus (COVID-19) has brought unprecedented countermeasures to every sector of the economy, including individuals, groups, institutions, and industries. The sports industry took one of the biggest hits, with all major leagues in the U.S. cancelling or halting their events. While these actions were necessary to address the public health concern, each segment is now floating various proposals to resume operations and give some relief to the significant portion of the economy that the sports industry comprises.\n\nMajor League Baseball (MLB) is likely to be the first American professional sporting league to resume, probably in May or June (Passan, 2020). Players are willing (although not all players agree to the method), the League is willing, the Arizona government is willing, and health professionals have approved a plan to move forward, known as the Arizona Plan. This plan calls for players, coaches, and staff to be quarantined in hotels around the Phoenix area, and to play in empty ballparks that include the ten Cactus League Spring Training parks, Chase Field, and other Phoenix ballparks. One interesting aspect of this arrangement is that the stadium size will not matter because there will not be any fans. This will be an opportunity for MLB to get back into the spotlight and accumulate massive television viewership that MLB has not seen in decades. The experience will be completely optimized for TV viewing, and so the league will finally be able to experiment with proposed rule changes, including removing mound visits to make the game go faster, adding a Robo Umpire, which has already been successfully tested last season via a partnership with the independent Atlantic League (Bogage, 2019), and an expanded roster giving players more rest due to the extremely hot temperatures of Phoenix. While all of this will alter predictions on who's going to the playoffs, probably the biggest impact that this plan will have on the games is the lack of the home field advantage (HFA): the advantage that the home team has over the visiting team due to the home team having fans, the familiarity of the home team to their own ball park, and the away team having to travel.\n\nBaseball has been shown in previous studies to be less susceptible to the HFA effect than other professional sports (Edwards & Archambault, 1979; Gómez et al., 2011; Pollard et al., 2017). Despite this, there is a measurable home field advantage in baseball, as shown by Jones (2015); Jones (2018)). Building on this, we extend the analysis for the MLB under uncertainty of which scenario the League will be following for the 2020 season. In particular, we simulate the win-loss probabilities for three different scenarios as well as estimate the home advantage for each team using the past three seasons’ data. This estimation helps us understand how individual team performance is going to be impacted as they prepare for the 2020 season in the new circumstances.\n\n\nMethods\n\nWe use the MLB 2017–2019 season data for the 30 teams represented in the league. The data were obtained from the MLB Advanced Media’s Baseball Savant Website using the Python package PyBaseball 1.0.4 (LeDoux, 2017/2020). The data shows that out of the 7,290 home games played during the 2017–2019 seasons, 3,881 (53.237%) resulted in wins and the remaining 3,409 (46.763%) resulted in a loss. Next, we seek to quantify the HFA’s role in this difference.\n\nThere are various techniques to calculate the home advantage depending on the sport, gender, league, and the nature of scoring (Jones, 2015). Pollard et al. (2017) use a general linear model to fit the home advantage. However, because we have a categorical variable of win or lose, we need to follow a non-linear approach. To test the hypothesis that teams have home-field advantage, we apply a logit regression model to predict the probability of winning as a function of home game dummy, team fixed effects, opponent fixed effects, and the win-loss records. We estimate the following regression equation:\n\nA neural network model was also created to act as a robustness check for the logit win prediction model. The software to train the model is hosted on GitHub (Ehrlich, 2020a). We used the R package nnet 7.3–14 (Ripley & Venables, 2020) for the neural network platform, and trained and tuned the model with the R package caret 6.0–86 (Kuhn et al., 2020). We developed a simulator to estimate what might happen if: 1) The full 2020 season continued on in a parallel universe devoid of COVID-19; 2) MLB waits and is able to return and play half a season to packed stadiums around the All Star Break, which is assuming an extremely optimistic timeline of a return to normal life; 3) a full season is played without fans, which is likely the only way they will be able to play this season (i.e., the Arizona Plan). The simulation was executed 100 times, and the logit win prediction model was used as the basis for predicting each win. A random number between 0 and 1 was generated and checked against the win probability provided by the model. If the random number was below the probability, then the team won, otherwise the team lost.\n\n\nResults\n\nThe summary statistics of the training data is contained in Table 1. The logit results from Equation 1, without the fixed effects, are reported in Table 2. Both log odds ratios and the MEs are reported in this table. The results show that the individual regressors included in the model show plausible impacts. Looking at the log-odds ratios, home games and the home team’s previous win-loss percentage (WL%) are more likely to result in a win but the opponent’s WL% is less likely to result a loss for the home team. These results support the presence of the HFA. The right half of the table shows the MEs for each variable. We are mainly interested in the MEe for the Home variable, which is 0.064. This means, the marginal probability of winning a game at home versus away field goes up by 6.4%. This is the average HFA for all of the teams as a whole. The HFA for each team is presented in Figure 1. In our sample, PHI seems to have the highest home advantage and HOU seems to have the lowest (negative, in fact) home advantage.\n\nThe model was trained using the 2017–2019 MLB regular season games. The schedule for the 2020 season was estimated using the schedule from the 2019 season. While the dates will be off slightly, the team pairings will be nearly the same. The wins and losses of the 100 simulations were added to form the result of the 2020 season. The overall results are visualized in Figure 2, while the divisional results shown in Table 3. Table 4 provides statistics calculated during each season and averaged. This includes the correlation between the full season and the half and no-fan seasons using both the overall rankings and the win-loss percent (WL%). The full seasons rank correlations are higher with the no-fans seasons (0.825) than the half seasons (0.735). The correlations using WL% is similar. The standard deviation of the predicted win probabilities is lower for the no-fans seasons (0.073) than the full (0.085) and half seasons (0.085). The home effect was correlated with the win probabilities’ standard deviations and is negative for the no fans seasons (-0.221). In other words, the higher the home effect, the lower the variance.\n\nNote: These statistics are calculated for each season and averaged. WL% is predicted based upon the Win Prediction.\n\nThis neural network was also used as the win predictor in 100 simulations and the results are very similar to the logit win prediction model, which shows robustness in the simulation results. Table 4 shows the statistical results of both models. The correlation and standard deviation differences are approximately the same between the two models.\n\nThe results of the simulations are available as Extended data (Ehrlich, 2020b) and the code necessary for replicating the results, including training the models, are hosted on GitHub (Ehrlich, 2020a).\n\n\nDiscussion\n\nBased on the above results, since the team-home effect is symmetric between home and away, teams will not necessarily win or lose any additional games in neutral stadiums as teams with a high home field effect will lose more neutral games that would have been at home but will win more neutral games that would have been away. The greater the home-team ME, the less variance there will in of the predicted win probabilities. To verify this assumption, we calculated the correlation of HomeEffects and the standard deviation (SD) of win probabilities between a full (0.361) and no-fan season (-0.221). Since the home effect is symmetric for each team (the away field disadvantage = -the home field advantage), decreasing the variance does not affect the overall expected WL% for each team. However, the result of individual games will be different since home effect is asymmetric between teams. For example, if the Cubs (highest home effect in the NL Central) plays the Cardinals (lowest home effect in the NL Central), the Cubs will have a larger advantage playing at home then the Cardinals will have playing at home (besides team fixed effects). These differences are removed with the No-Fan scenario and the outcome will be solely based upon the talent of the teams. However, on average there only a slight change of overall WL% (or playoff berth), just the SD of the results (see Table 3). Without fans, any advantage (or disadvantage) from home field advantage, which cause higher levels of variance, is removed. This stabilizes the outcome based upon true team talent. As fewer games have been played, the half-season will have more upsets, but the SD is close to the same as the full season.\n\n\nConclusion\n\nThis paper analyzes the previous season MLB data to estimate the win-loss probabilities for the 2020 season for each of the 30 teams in the League using logit regressions and a neural network. The Arizona Plan’s neutralization of HFA would not significantly affect the overall outcome of the season. In fact, our model predicts that the Arizona Plan season will produce season results that are based more on the true talent of the teams. Further, our simulation demonstrates that there will be less variance in the win probability between any two teams, which we estimate will cause a larger divide between the best and worst teams. In conclusion, we believe that the results of the Arizona Plan will be similar to a regular season with fans, and that the teams’ standings at the end of the regular season will be more predictable than a normal season.\n\n\nData availability\n\nZenodo: Syracuse-University-Sport-Analytics/MLBCovid19. https://doi.org/10.5281/zenodo.3775959 (Ehrlich, 2020a).\n\nThis project contains the following source data files:\n\ndata/2008_2019Games.csv. (Input data scraped using the scrapingMLB.ipynb.)\n\ndata/divisions.csv. (Input team division data for grouping by division.)\n\ndata/mlbTeamColors.csv. (Input team colors for the visualizations.)\n\nSource data are also available on GitHub: https://github.com/Syracuse-University-Sport-Analytics/MLBCovid19.\n\nHarvard Dataverse: Replication Data for: COVID-19 Countermeasures, Major League Baseball, and the Home Field Advantage. https://doi.org/10.7910/DVN/OOMWSD (Ehrlich, 2020b).\n\nThis project contains the following extended data files:\n\ndivisionRankings. (Results of simulation using the logit model.)\n\ndivisionRankingsNN. (Results of simulation using the neural network model.)\n\nhomeEffectLogit. (Team home effects using the logit model.)\n\nhomeEffectNN. (Team home effects using the neural network model.)\n\nmodelCorrelationsSummaryWithNNResults. (Simulation statistics from both the logit and neural network models.)\n\nZenodo: Syracuse-University-Sport-Analytics/MLBCovid19. https://doi.org/10.5281/zenodo.3775959 (Ehrlich, 2020a).\n\nThis project contains the following source files:\n\npythonStatcastScraper/scrapingMLB.ipynb. (Python Jupiter Notebook code for scraping Statcast.)\n\nhalfSeasonPrediction.Rmd. (R Markdown Notebook code for developing the logit and neural network models. Also contains the code for running the simulations.)\n\nAll the other data is intermediate output from the simulations. The important output files are located in the above Harvard Dataverse repository.\n\nSource code is also available on GitHub: https://github.com/Syracuse-University-Sport-Analytics/MLBCovid19.\n\nMixed data and code hosted on GitHub and Zenodo are available under the terms of the GNU General Public License v3.0.\n\nData hosted on Harvard Dataverse are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).",
"appendix": "References\n\nBogage J: National Baseball Hall of Fame accepts Atlantic League ‘robo ump’. items. Washington Post. 2019. Reference Source\n\nEdwards J, Archambault D: The home field advantage. Sports, games, and play: Social and psychological viewpoints. 1979; 409–438. Reference Source\n\nEhrlich J: Syracuse-University-Sport-Analytics/MLBCovid19: First Release (Version v1.0.0). Zenodo. 2020a. http://www.doi.org/10.5281/zenodo.3775959\n\nEhrlich J: Replication Data for: COVID-19 Countermeasures, Major League Baseball, and the Home Field Advantage. Harvard Dataverse, V1, UNF:6:LiInpTKr15iER0wC31Bb9g== [fileUNF]. 2020b. http://www.doi.org/10.7910/DVN/OOMWSD\n\nGómez MA, Pollard R, Luis-Pascual JC: Comparison of the home advantage in nine different professional team sports in Spain. Percept Mot Skills. 2011; 113(1): 150–156. PubMed Abstract | Publisher Full Text\n\nJones MB: The home advantage in major league baseball. Perceptual and motor skills. 2015; 121(3): 791–804. PubMed Abstract | Publisher Full Text\n\nJones MB: Differences in home advantage between sports. Psychol Sport Exerc. 2018; 34: 61–69. Publisher Full Text\n\nKuhn M, Wing J, Weston S, et al.: caret: Classification and Regression Training (Version 6.0-86) [Computer software]. 2020. Reference Source.\n\nLeDoux J: Jldbc/pybaseball [Python]. 2020; (Original work published 2017). Reference Source\n\nPassan J: Sources: MLB, players eye May return in Arizona. ESPN.Com. 2020. Reference Source\n\nPollard R, Prieto J, Gómez MÁ: Global differences in home advantage by country, sport and sex. Int J Perform Anal Sport. 2017; 17(4): 586–599. Publisher Full Text\n\nRipley B, Venables W: nnet: Feed-Forward Neural Networks and Multinomial Log-Linear Models (Version 7.3-14) [Computer software]. 2020; Reference Source."
}
|
[
{
"id": "140121",
"date": "10 Jun 2022",
"name": "Garry Kuan",
"expertise": [
"Reviewer Expertise Exercise and sports 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\nGENERAL COMMENTS\n\nThe aim of this paper was to examine various scenarios of how Major League Baseball team performance is going to be impacted by the presence of fans, or the lack thereof, in the context of physical distancing and other COVID-19 countermeasures. Although this article addresses an interesting topic, some issues should be addressed before indexing.\n\nSPECIFIC COMMENTS\nINTRODUCTION The introduction needs major revision and clarification. First, the aim of the study is not clear. The manuscript should answer a specific research question, which is lacking in this study. Also, the way the authors build up their introduction does not lead to the research question. The introduction is too shallow, and the context of the manuscript is not well-elaborated. Thus, those who are not living in the USA might have no clue about what the authors are explaining. I suggest that the authors should re-structure their introduction, explaining why their research is important and how is it related to COVID-19.\nIt is recommended that the authors expand this part: What are the environmental factors, besides the presence of the fans, that might affect the players’ performance. More importantly, this should lead to a clear research question.\n\nMETHODS The methods section needs major revision. As it stands, it is not possible to replicate their study. Firstly, COVID-19 started in early 2020 and if data from the 2017-2019 seasons were used it could not represent the COVID-19 situation.\nWhy is only home advantage calculated? Please explain.\nWhat is the sample size, and what is the effect size?\n\nRESULTS The results section seemed okay. Probably, including a research question would help the authors to structure their results. I think the authors put too much information and abbreviation in the tables, but no explanation of the abbreviation, making the entire manuscript hard to follow. Figure 2 needs more clarification.\n\nDISCUSSION The discussion is only a paragraph - the authors should further discuss their findings and the implication of these findings in more depth. Is there any limitation of this study? Suggest revising this section.\n\nThank 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? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
},
{
"id": "164098",
"date": "28 Mar 2023",
"name": "Ismail Dergaa",
"expertise": [
"Reviewer Expertise Sports Medicine and exercice 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 aim of this study is to examine various scenarios of how Major League Baseball team performance is impacted by the presence or absence of fans in the context of physical distancing and COVID-19. While the article is well-structured and falls within the scope of Psychology Research and Behavior Management, it requires major revisions before it can be accepted for indexing.\nThe duplication of keywords with the title needs to be adjusted to increase the visibility of the manuscript. Keywords should not be duplicated with the title. For example, if COVID-19 is in the title and the keywords, it should be replaced with SARS-CoV-2.\n\nThe biggest concern is that the article presents a simulation of the 2020 season using logit regression and a neural network, while it is now 2023. To address this, the authors should add a section with the actual results of the season and compare them to the study's outcome. They should keep the analysis as it is and change the aim of the study. This will provide two aims for the study, the actual one and an assessment of the accuracy of the simulation.\n\nThe manuscript needs to be rewritten in past tense as we are in 2023.\n\nIn addition to the analysis, the authors should compare their results with the actual outcomes of the season.\n\nThe references need to be updated.\n\nThe methodology is not clear, and the authors are requested to add a flowchart explaining the study protocol to make it easier for the reader.\n\nFor the first time in my life, I have seen a discussion section without any references. The authors need to compare their results with similar articles. Additionally, the authors need to mention that sports team performance is a complicated component that depends on several factors, such as players' mentality, team play, motivation, injury of key players, etc. These points need to be supported by credible references to strengthen the arguments presented in the discussion and conclusion section. Special emphasis should be given to the limitation section, which is absent in the study, to provide a more comprehensive analysis of the study's scope and potential impact.\nOverall, the manuscript has potential, but it requires significant revisions before it can be accepted for indexing.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? 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": "164101",
"date": "28 Mar 2023",
"name": "Federico Fioravanti",
"expertise": [
"Reviewer Expertise Mathematics",
"Sports",
"Social Choice"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nGeneral comments The work examines three possible scenarios for the Major League Baseball 2020 season, motivated by the fact that due to Covid-19 countermeasures, there will be no presence of fans. The topic is interesting, but the authors should work more on describing the motivation and the results they find.\n\nSpecific comments Abstract: Line 13 - first sentence is too long.\nIntroduction: It is worth noticing that Covid-19 was a worldwide problem. So it is preferable to start telling that, and then say the study will focus on a particular sport in a particular country. A bit of effort should be done to explain the baseball terminology (or reduce its use), in order to facilitate the understanding for people from countries where baseball is not mainstream. Even how the Baseball environment is, requires an explanation, so it is easier to interpret the results. Use papers such as Schwarz and Basky (1977)1 or Agnew and Carron (1994)2 to expand the explanation of the HFA and the possible factors causing it. Line 10 - it says: “Major League Baseball (MLB) is likely to be the first American professional sporting league to resume” and it should say: “Major League Baseball (MLB) is likely to be the first professional sporting league in the United States” (if it is the first one in the American continent then cite accordingly).\nMethods: This section needs further development. The regression equation needs more explanation and why every variable is introduced in the equation. What does every variable mean? What is the range of every variable?\n\nIt is recommendable to describe a bit more the different simulations that are considered.\nResults: For ease of understanding, there must be a description of the abbreviations that are used in tables and figures (not the name of the teams). Some statistics are missing, such as sample size. Line 6 is difficult to understand. What do we look at? The log-odds ratio? The log-odds ratio, home games and WL%? What is less likely to result in a win?\nDiscussion: Why is the home effect symmetric for each team? If it is a hypothesis, it must be justified. Line 7 - it says: “the less variance there will in of the predicted” and it should say: “the less variance will be in the predicted” or “the less variance of the predicted” Line 18 - it says: “at home then the Cardinals” and it should say: “at home than the Cardinals” Line 22 - it says: “there only” and it should say: “there is only” There is a lack of discussion in this section, commenting on limitations, etc…\nConclusion: The conclusion leads me to think that there is not a real HFA, as the standings will be similar between a regular season and a no-fans season. The message in this section is not so clear.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/9-414
|
https://f1000research.com/articles/9-382/v1
|
20 May 20
|
{
"type": "Research Article",
"title": "Trends and factors associated with long-acting reversible contraception in Kenya",
"authors": [
"Wambui Kungu",
"Anne Khasakhala",
"Alfred Agwanda",
"Anne Khasakhala",
"Alfred Agwanda"
],
"abstract": "Background: Kenya has 12 million female adolescents and youths aged 10-34 years whose reproductive behavior will determine the growth and size of its population for the next decade. The anticipated momentum of births can be slowed by the use of long-acting reversible contraception (LARC) methods as they are more effective, need no user adherence, and hence have no risk of incorrect or inconsistent use. However, in spite of the many health and social benefits, LARC is underutilized because of myths and misconceptions. Kenya is in the ultimate decade towards Vision 2030 and investing in LARC can save costs of health care and accelerate the achievement of the development goal. The objective of this study was to establish factors associated with LARC use, with a view of establishing the potential for increasing demand. Methods: The study was national and used secondary data from the three waves of the Kenya Demographic Health Survey from 2003, 2008/09 and 2014 in a sample of all women of reproductive age who reported currently using modern contraceptive methods at the time of interview. Descriptive and logistic regression analysis was employed to profile and examine LARC users. Results: LARC use was low but picking up rapidly, especially among contraceptive users of higher social economic status in a major shift between 2008/09 and 2014. Consistent factors that influenced its use were age, wealth, and number of living children, while education and residence were of influence some of the time. Conclusions: There is huge unexploited potential for more LARC uptake based on the identified predictors of its use. Scaling up of LARC uptake is critical to deal with issues of poor user adherence, incorrect and inconsistent use, and method failure that characterize short-acting contraception, resulting in increased unintended pregnancies, incidences of unsafe abortions and maternal and infant mortality.",
"keywords": [
"LARC",
"Benefits",
"Highly Effective",
"Predictors",
"Unintended pregnancy"
],
"content": "Introduction\n\nFamily planning is a critical component of Kenya’s development agenda and it is addressed in the Vision 2030 social pillar on provision of reproductive health for the poor and vulnerable population. Kenya is a model of fertility transition, having moved from a very high level of fertility of over eight children in the 1970s to about four children in 2014, with a contraceptive prevalence (CPR) of 58%. A major recent growth in CPR was especially evident in the use of modern methods between 2003 and 2014. CPR increased by 17% between 2003 and 2014, while use of modern methods, which has driven the CPR up, increased by 21% over the decade1,2. The increased demand for contraception was generated by robust campaigns that were initiated with the repositioning of the family planning program. The agenda of family planning is also central in the International Conference on Population and Development after 25 years (ICPD25) Commitments as well as in Sustainable Development Goal (SDG) 3.73.\n\nInvesting in family planning is highly cost-effective and can lower healthcare costs and cascade benefits to Kenya in an unrivalled manner. As of 2015, Kenya had saved US$4.48 directly in healthcare costs for every US$1 that was spent on family planning. If county governments accelerated demand and hence uptake of modern contraceptive methods, the savings would rise to US$5.46 for every US$1 used in family planning and result in a direct total saving of US$80 million by 20204.\n\nLong acting reversible contraception (LARC) refers to contraceptive methods that can be used beyond one year upon insertion and comprises the implant and intrauterine devices (IUDs). Evidence from the 2014 Kenya Demographic and Health Survey (KDHS) shows increased use of LARC in comparison to other modern contraceptive methods, with the implant becoming the second most popular method after injection1. The reproductive behavior of the 12 million female adolescents and youths aged 10–34 years5 will determine the growth and size of the population of Kenya for the next decade.\n\nThe anticipated momentum of births from the huge female population can be slowed by use of LARC as these methods are longer acting, reversible, more effective, need no user adherence and hence have no risk of incorrect or inconsistent use common to young contraceptive users and have lower rates of discontinuation6. They are also credited with higher user acceptability/satisfaction, fewer side effects and user contraindications as well as little partner involvement. They are estrogen free, hence pose fewer risks to a woman’s health, and involve less visits to health facilities, hence less strain on the health system7,8. Despite these great health and social benefits, LARC is underutilized because potential users are not comprehensively counselled about them and additionally, the methods are initially expensive. Evidence on cost has, however, shown they are cost effective in the long-term and can result in savings of $12,000 in five years9. In concurrence, Trussel et al.10 found cost savings can be realized from LARC use within three years whether or not the methods are used to the end of their efficacy period. They arrived at costs of $304 for the IUD and $308 for the implant per woman, per annum in the United States.\n\nEvidence has shown that unintended pregnancies and induced abortions could be considerably reduced by as much as 25% if women used more effective methods and specifically LARC11. Using KDHS 2003 data, the study found a 0.4% failure rate for long acting methods against 2.7% for short acting methods and 15.8% for traditional methods. Total unintended births were 44%, out of which 5% were terminated. The number of unintended births that could be averted was estimated at 11% (69,000) out of the 44%. The expansion of access to LARC is critical in family planning programs that aim to address high rates of unintended pregnancy and curb high unmet need for family planning12.\n\nThe current Kenya Family Planning Costed Implementation Plan (FP CIP) has several targets towards rights-based family planning and improved maternal and child health outcomes for the period 2016 to 2020. The targets are; increasing implant use from 12% to 16%; increasing IUD use from 5% to 7%; avert 2 million unintended pregnancies, 62,500 unsafe abortions, 4700 maternal deaths and reduce unmet need from 15% to 13%. An underlying target is to increase modern contraceptive prevalence (mCPR) from 54% to 58%, accompanied by high quality information and contraceptive services13.\n\nA challenge in the access to contraceptive commodities is that the method mix in Kenya is dominated by the injection, and increasing the share of LARC can reduce this domination as well as expand the method mix. Method mix heavily influences rates of method failure because short acting methods have higher failure rates as they are less effective. Evidence has shown that women are more likely to choose more effective methods if exposed to information and services about them14,15.\n\nIn contraceptive use dynamics, the role of method choice is critical in gauging the quality of services because client satisfaction, acceptance of a method and its continuation depend on it14. The convenience, effectiveness and availability of contraception methods vary, hence the side effects likely to be experienced by different users and resultant discontinuation rates vary by method and also by user characteristics. It is therefore crucial for managers of family planning programs to ensure clients use the most suitable method to enhance continuity.\n\nLARC use has only picked up in the last decade and there is still a paucity of data and a lot of barriers to its uptake. Evidence on predictors of its use is critical in the next decade of promotion of LARC methods to overcome the challenges that hinder women from enjoying the many benefits of LARC.\n\n\nObjective\n\nThe objective of the study was to establish the factors associated with LARC methods with a view to establishing potential for increasing demand for LARC.\n\n\nMethods\n\nSpecific ethical approval is not required for re-analysis of DHS data, but permission to use the data for this study was obtained from ICF. This study uses existing KDHS data and re-analysis was done under the original consent provided by the participants.\n\nThe study was national and used secondary data from the three waves of KDHS 2003, 2008/09 and 2014, mainly from the contraceptive calendar contained in the woman’s questionnaire for all eligible women and also the household file and individual woman file. The sample was all women of reproductive age, 15–49 years, who reported current use of any of the major methods of modern contraception at the time of the interview, whether married or not. Women who did not report current use of the modern contraception were excluded. Full details of sampling procedure, data collection procedure and variables for which data were collected are available in the Kenya DHS Final Reports16–18.\n\nThe dependent variable of interest was the current method of contraception, which was categorized as LARC if the method was IUD or implant or ‘other modern’ if the method was condom, pill, injection or sterilization. The independent variables selected were woman-level characteristics of age, education, marital status, number of living children, desire for children and household level characteristics of place of residence, wealth status, type of contraceptive region. Age was grouped into three 10-year categories (15–24, 25–34, 35+) while education was also grouped into three categories (none, primary, secondary +). Marital status categories were two (married/not married) and those of number of living children were four (none, 1–2, 3–4, 5+). Desire for children was either that the woman wanted more children or did not want more children, while place of residence was either rural or urban. Wealth status reclassified the five DHS categories as follows; lower (lowest/low), middle (middle) and higher (high/highest) while type of contraceptive region was classified into two categories; high contraceptive (Nairobi, Central, Eastern) and low contraceptive (the five remaining regions).\n\nThe first stage of analysis profiled the sampled women using cross-classification analysis by their background characteristics. An analysis to establish the differentials of LARC against other modern methods based on the independent variables was conducted where the chi-square test identified if there existed any statistical significance. The confidence level was set at 95%.\n\nThe dependent variable, contraceptive method, was binary in two categories of LARC or ‘other modern’, hence the binary logistic regression model was employed to determine any influence of the independent variables on modern contraceptive method choice. LARC use was the outcome of interest, therefore ‘other modern’ was used as the reference category in the regression. The independent variables were recoded into fewer categories for ease in the analysis.\n\nThe software used for the data analysis was SPSS version 22.\n\n\nResults\n\nIn an attempt to obtain the profile of the LARC user, cross tabulations were undertaken to show the share of LARC use against other modern contraceptives and examine any significant relationships between the socio demographic factors and use of modern methods categorized under LARC or ‘other modern’ for each year under study. Table 1 presents the results.\n\nNotes: P-value= 0.05.\n\nLARC, long-acting reversible contraception.\n\nTable 1 shows low shares of LARC, ranging from 5% to 15% among modern contraception in 2003, reducing slightly in 2008/9, before a 2-6 fold increase across all socio demographic factors to reach 20–26% in 2014. Other modern contraception had huge shares of 85–95% in 2003 and 2008/09, which declined to 75–80% in 2014. For women with no children, LARC use was very low at 3% across the data sets. The emerging general profile of the LARC user was that of secondary educated, mostly married women of higher wealth, living in urban areas, with at least one child and aged 25 to 34. All the factors apart from region and desire for children exhibited significant relationships with use of modern methods.\n\nTo find out levels and trends in LARC use against other modern methods, descriptive analysis was done based on age and regions. Figure 1 to Figure 9 present the different results. Figure 1 complements Table 1 results on age.\n\nLARC use by age. Figure 1 shows LARC use in 2003 and 2008/09 was highest among women aged over 35 years, but in 2014 was highest among those aged 25–34. There was a decline in LARC use between 2003 and 2008/09, and then major surges between 2008/09 and 2014. Use among 25–34 and 35+ age groups each increased two-fold, while for the 15–24 group, the increase was seven-fold.\n\nFurther analysis (shown in Figure 2 and Figure 3) on age showed that for the IUD, the majority of users were in the 35–49 age group, while for the implant, the majority were in the 25–34 age group. The lowest use for both was among the 15–24 age group.\n\nLARC use by education. Analysis by education (shown in Figure 4 and Figure 5) revealed that in 2014, the majority of users for both IUDs and implants, with a 50–60% share each, had primary level education, while the lowest use for both was among women with higher education.\n\nIUD use by region. Figure 6 shows that the share of IUD use among modern contraceptive use in the Central region was highest in 2003 and 2014 at 13%, while its share in Nairobi was highest in 2008/09 at around 10%. The North Eastern region had 5% of its modern contraceptive users using the IUD in 2014. There was no data for the region in 2003 and there were no IUD users in 2008/09. The Eastern region showed consistent use, with 5% of its modern method users using the IUD, while the Western region showed a gradual rise in IUD use across the three data sets. Results between 2008/09 and 2014 show an increase in IUD use in all regions except for the North Eastern region, with the biggest change being in the Central region, where IUD share among modern methods increased two-fold from 3.2% to 6.3% in prevalence.\n\nImplants use by region. Figure 7 shows the low share of implants among modern contraceptive users in 2003 and 2008/09, before a major rise in the share of implants across all regions in 2014. The Western region in 2014 led, with 27% of its modern method users using the implant, followed by Nyanza, Nairobi and Central, each with around 20% share.\n\nLARC use in 2014. The status of LARC share among modern contraception in 2014 in all regions is shown in Figure 8.\n\nFigure 8 shows the level of LARC use after the surge that occurred between 2008/09 and 2014, wherein the share of LARC among modern contraceptive use rose to comprise 20% to 30% of the share of modern contraceptive use in all regions apart from the Eastern region. The Western region, which earlier had very low shares of IUD and implant use, caught up with Nairobi and Central to reach a 30% share for LARC among modern contraceptive users.\n\nTrends in LARC use by fertility desire. Another analysis done on LARC users was by fertility desire (whether one was spacing or limiting children) in an attempt to establish whether family planning clients were using the appropriate methods for their reproductive goals. The results are presented in Figure 9.\n\nThe results show that majority of LARC users are limiters in all the data sets but there is a sustained total decline of 12% from 71% in 2003 to 59% in 2014. Running counter to this is an equal increase among spacers from 29% in 2003 to 41% in 2014.\n\nFor the regression analysis, the dependent variable was modern contraceptive use with ‘other modern’ as the reference category. Reference categories for the independent variables are indicated for each variable. The results of the regression analysis are presented in Table 2.\n\nNotes: P-value = 0.05 <.05* < .01** <.001 ***.\n\nRef, reference category; LARC, long-acting reversible contraception; B,Beta coefficient; SE, Standard error; EXP B, exponentiation of the B coefficient (odds ratio).\n\nThree consistent predictors of LARC use emerged across the study period. These were age, wealth and number of living children. Education was a predictor in the 2003 data set, while residence was a factor of influence in the 2014 data set.\n\nRegarding age, in 2003, it showed little influence among the 25–34 age group, while for the 35+, its influence was very strong. Women aged 25–34 were almost twice as likely to choose LARC than those aged 15–24, while those aged 35 years and above were almost three times more likely to choose LARC than the 15–24-year-olds. In 2008/09, the influence was very strong in both age groups. Women aged 25–34 were 3.5 times more likely to use LARC than their younger counterparts, while those aged at least 35 years were almost five times more likely to choose LARC than the 15–24 age group. In 2014, the influence of age was minimal and was only exhibited for the women aged 25–34, who were 15% more likely to choose LARC than the younger 15-24 cohort.\n\nEducation showed some influence in 2003, with secondary educated women being 2.8 times more likely to use LARC as compared to those with no education. The variable had no influence in the other data sets. Residence also had very strong influence in one data set, 2014. Rural women were 20% more likely to use LARC than their urban counterparts.\n\nWealth exhibited different strengths ranging from low (p<.05), moderate (p<.01) to very strong (p<.001) in influence towards LARC. In 2003 the influence was moderate for women of lower wealth, who were 55% less likely to choose LARC than their wealthier counterparts. For women of medium wealth, the influence was low and they were 42% less likely to use LARC than the wealthier women. In 2008, wealth showed moderate influence in women of lower wealth, who were 55% less likely to choose LARC than women in the higher wealth category. In 2014, wealth showed very strong predictive ability for women of lower wealth, who were 26% less likely to use LARC than women with higher wealth status. Moderate influence was seen for women of medium wealth, who were 20% less likely to choose LARC when viewed against women of higher wealth.\n\nParity had low or very strong influence towards LARC use. In 2003, the influence was low, showing that women with 1-2 children were 3.6 times more likely to choose LARC than those with no children. In 2008, influence was again low, showing women with 1–2 children as three times more likely to choose LARC in relation to those with no children, while those with 3–4 children were 3.3 times more likely to use LARC than those without children. In 2014, the influence was very strong, showing those with 1–2 children as 11 times more likely to choose LARC than nulliparous women, while those with 3–4 children were 12 times more likely to choose LARC over those without children. Women with five or more children were 13 times more likely to choose LARC than those without children.\n\n\nDiscussion\n\nThe study shows LARC having very low usage initially, before a big shift in use from other modern towards LARC between 2008/09 and 2014. The period of increased use coincided with LARC promotion campaigns that were conducted in Kenya and several African countries, which generated great demand by advancing LARC as the longer lasting, safer and more effective methods. The demand created was met by a steady supply of the methods. The implant has had higher uptake than the IUD, suggesting higher acceptability of the method, which may be due to ease and method of insertion, length of efficacy or preference for hormonal methods7. The increase in LARC use was seen in many sub-Saharan African countries but the uptake of the implant in Kenya was the highest in the world at 18% prevalence as of 201719.\n\nThe network of Tunza clinics in Kenya also had a project to increase LARC uptake through demand generation between 2009 and 2014. The project increased implant uptake by around 12% and IUD uptake by 5% within Tunza clinics20. The Tupange project also scaled up use of implants in five urban centers over a five-year period21. Overall, the relaunch of the Kenyan family planning program and launch of FP2020 in 2011, as well as the development of FP CIP 2012–201622, aided the increase in LARC nationally.\n\nThe uptake of the IUD is low in Kenya in spite of the aggressive LARC campaigns, mostly because of rumors and misconception about their convenience and effectiveness as well as provider attitudes and bias23. Of concern is its suitability for certain populations; for example, young women and women with a tendency towards multiple partners. The situation was different in the 1980s and it commanded a good share of the method mix in Kenya at 31% but thereafter started declining and is currently heavily underutilized23.\n\nInfluence of age on LARC use was seen in all the data sets. LARC use was initially seen to be more common among women of at least 35 years, who are traditionally limiters as they have mostly attained their desired number of children and do not want more children. However, globally LARC was recommended for adolescents and young women24 and those who were spacing, as fertility returned soon after the method was discontinued25. Other studies have also associated age with LARC use26, while others have found it insignificant27.\n\nAs of 2014 in Kenya, a shift had occurred such that the leading users of LARC were now 25–34-year-olds, who are also the majority users of modern contraception, fulfilling the huge demand for spacing births. However, IUD use in Kenya shows dominance by the over 35s, who are limiting births. As expected, low LARC use was seen among the 15–24-year-olds and the reason for this may be their general lower use of modern contraception and their tendency towards short term methods because they are cautious about using methods that may interfere with their future fertility intentions28. The group showed major increases in LARC use from 3% in 2008/09 to 20% in 20141,29.\n\nThis study showed LARC use was more prominent among women of higher socio-economic status, perhaps because of their better exposure and access to contraceptive information and services. Other studies have found LARC to increase with education26 and wealth30,31, while others did not find any effects from education27. LARC methods are generally more available in urban areas and a contributing factor is the lack of trained providers in rural and low-income areas to handle insertions and removal27. For the IUD, the issue of insertion is sensitive and must be done correctly for a user to be satisfied and continue using it for the intended period. This contributes to high discontinuation rates for the IUD32.\n\nAnalysis based on region showed that the Central region and Nairobi had the biggest shares of LARC against other modern methods. However, between 2008/09 and 2014, a shift was seen with regions with previously low contraception rates registering big surges in LARC uptake and some even overtaking the previous high contraception rate regions. The Western region was seen to overtake Central and Nairobi to have the biggest share of implant use among modern method usage. The North Eastern region, the region with the lowest contraceptive prevalence in Kenya, is also seen to be making good strides in terms of its proportion of LARC against other modern methods.\n\nThe reproductive goals of a woman are about the number and timing of the children she desires to have and the method she chooses is supposed to enhance the goal of either spacing or limiting births. This was emphasized by the strong influence seen from the number of living children, with LARC use increasing with the number of children. A possible explanation is that higher parity may expose a woman to a lot of information and experience about contraception during prenatal and postnatal clinics31,33.\n\nLevels of LARC use seen in the study are low when viewed against its many contraceptive and non-contraceptive benefits. One barrier to LARC uptake is myths and fears of side effects. Qualitative studies reveal that among the believed side effects for the IUD is that it causes cancer, while other women think LARC will interfere with their future fertility, hence do not opt for them until they have attained their desired number of children27. This study complements this view as analysis on fertility desire showed most LARC users are limiters.\n\nThe high demand shown for LARC should be accompanied by efforts to address barriers in the supply chain by reducing commodity stock outs.\n\nLARC needs more funding than short term methods and with reduced donor funds, sustainable financing needs to be secured and ringfenced. However, LARC are cost-effective in the long term because resupplies and clinic visits needed are fewer.\n\nLARC are provider dependent methods and the rising demand for them should be accompanied by investment in quality services for insertion and removal procedures. This might improve the acceptability and continuation of the IUD.\n\nHigh demand for implants was evident among 15–24-year-old women. Given that this group is the fastest growing within the reproductive age, it is vital that easy access to LARC information and services is available for them.\n\nThe rise in the use of implants among women aged 15–24 has been accompanied by a huge decline in condom use, which may mean little or no protection against HIV/AIDS and other sexually transmitted infections. Perhaps use of condoms can be recommended alongside LARC for this group depending on the need.\n\nThe slow growth of IUD uptake suggests the challenges of insertion and side effects abound and calls for a change of strategy borrowed from regions that have higher IUD prevalence.\n\nLarge proportions of those aged 25–34 using LARC indicates the success of the policy shift to promoting LARC among spacers, while the increase in uptake among those aged 15–24 also shows acceptability among young, nulliparous women.\n\nThe huge uptake in the Western region and other regions that previously had low LARC use indicates the success of the LARC promotion campaigns and may point to some major changes in strategy in the promotion of LARC, which can guide family planning program managers in the regions where uptake is not as high.\n\nNumber of children has shown an increasingly strong influence on the choice of LARC and the share of spacers using LARC is increasing. Service providers need to package contraceptive counselling information to appeal to both spacers and limiters in future attempts to increase demand for LARC and especially for IUDs.\n\nIn general, results suggest contraceptive users are using methods that are suitable for fulfilling their fertility desires but the large disparities between proportions using LARC against those using other modern methods reveal a huge untapped potential for LARC uptake.\n\n\nConclusions\n\nThe study has compared use of LARC to that of other modern contraception and documented how it has evolved from very low to significant shares in modern contraceptive use and the factors that have influenced its use over different time settings. It has established huge unexploited potential for more LARC uptake based on the identified predictors of its use. The data will be useful to inform strategies to improve LARC use and help Kenya increase mCPR and reduce unwanted fertility, unsafe abortions and unmet need for contraception.\n\nScaling up of LARC uptake is critical to deal with issues of poor user adherence, incorrect and inconsistent use and method failure that characterize short acting contraception, resulting in increased unintended pregnancies, incidences of unsafe abortions and maternal and infant mortality. Strategies are needed to provide accurate information to counteract rumors and misconceptions surrounding the IUD to increase its uptake.\n\n\nData availability\n\nThe Women Individual Recode datasets from the Kenya Demographic and Health Survey 2003, 2008/09 and 2014 were used for this 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\nKNBS: Kenya Demographic and Health Survey 2014. Kenya National Bureau of Statistics and ICF MACRO. 2015. Reference Source\n\nKNBS: Kenya Demographic and Health Survey 2003. Kenya National Bureau of Statistics and ICF MACRO. 2004. Reference Source\n\nNew JR, 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\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), Marie Stopes International (MSI) and Futures Group. Health Policy Project. 2014. Reference Source\n\nKNBS: Kenya Population and Housing Census: Vol III. Distribution of Population by Age and Sex. Kenya National Bureau of Statistics. 2019. Reference Source\n\nShoupe D: LARC methods: entering a new age of contraception and reproductive health. Contracept Reprod Med. 2016; 1: 4. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNgo TD, Nuccio O, Pereira SK, et al.: Evaluating a LARC Expansion Program in 14 Sub-Saharan African Countries: A Service Delivery Model for Meeting FP2020 Goals. Matern Child Health J. 2017; 21(9): 1734–1743. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTsui AO, Brown W, Li Q: Contraceptive Practice in Sub-Saharan Africa. Popul Dev Rev. 2017; 43(Suppl Suppl 1): 166–191. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBlumenthal PD, Voedisch A, Gemzell-Danielsson K: Strategies to prevent unintended pregnancy: increasing use of long-acting reversible contraception. Hum Reprod Update. 2011; 17(1): 121–37. PubMed Abstract | Publisher Full Text\n\nTrussel J, Hassan F, Lowin J, et al.: Achieving cost-neutrality with long-acting reversible contraceptive methods. Contraception. 2015; 91(1): 49–56. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBradley S, Croft T, Rutstein S: The impact of contraceptive failure on unintended births and induced abortions: Estimates and strategies for reduction. DHS analytical studies 22. ICF Macro, Calverton, Maryland. 2011. Reference Source\n\nHiggins JA: Celebration meets caution: LARC's boons, potential busts, and the benefits of a reproductive justice approach. Contraception. 2014; 89(4): 237–241. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMinistry of Health: National Family Planning Costed Implementation Plan 2017-2020. Ministry of Health, Kenya. 2017. Reference Source\n\nHubacher D, Spector H, Monteith C, et al.: Long-acting reversible contraceptive acceptability and unintended pregnancy among women presenting for short-acting methods: a randomized patient preference trial. Am J Obstet Gynecol. 2017; 216(2): 101–109. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSecura GM, Allsworth JE, Madden T, et al.: The Contraceptive CHOICE Project: reducing barriers to long-acting reversible contraception. Am J Obstet Gynecol. 2010; 203(2): 115.e1–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCentral Bureau of Statistics - CBS/Kenya, Ministry of Health - MOH/Kenya, and ORC Macro: Kenya Demographic and Health Survey 2003. Calverton, Maryland, USA: CBS MOH, and ORC Macro. 2004. Reference Source\n\nKenya National Bureau of Statistics - KNBS, National AIDS Control Council/Kenya, National AIDS/STD Control Programme/Kenya, Ministry of Public Health and Sanitation/Kenya, and Kenya Medical Research Institute: Kenya Demographic and Health Survey 2008-09. Calverton, Maryland, USA: KNBS and ICF Macro. 2010. Reference Source\n\nKenya National Bureau of Statistics, Ministry of Health/Kenya, National AIDS Control Council/Kenya, Kenya Medical Research Institute, and National Council for Population and Development/Kenya: Kenya Demographic and Health Survey 2014. Rockville, MD USA. 2015. Reference Source\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\nPSI: Increasing LARC uptake in Kenya through Improved Demand Creation Strategies and Provider Support. Best Practices from the Women’s Health Project 2009-2014. Population Services International. 2015. Reference Source\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\nMinistry of Health: National Family Planning Costed Implementation Plan 2012-2016. Ministry of Health, Kenya. 2012. Reference Source\n\nThe ACQUIRE Project/EngenderHealth: Revitalizing the IUD in Kenya. Acquiring Knowledge. 2006; 2.\n\nChandra-Mouli V, Mccarraher DR, Phillips SJ, et al.: Contraception for adolescents in low and middle income countries: needs, barriers, and access. Reprod Health. 2014; 11(1): 1. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJoshi R, Khadilkar S, Patel M: Global trends in use of long-acting reversible and permanent methods of contraception: Seeking a balance. Int J Gynaecol Obstet. 2015; 131(S1): S60–3. PubMed Abstract | Publisher Full Text\n\nBiza N, Abdu M, Reddy PPS: Long acting reversible contraceptive use and associated factors among contraceptive users in Amhara Region, Ethiopia, 2016. A community Based cross sectional study. Med Res Chron. 2017; 4(5): 469–480.\n\nTibaijuka L, Odongo R, Welikhe E, et al.: Factors influencing use of long-acting versus short-acting contraceptive methods among reproductive-age women in a resource-limited setting. BMC Womens Health. 2017; 17: 25. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPayne JB, Sundstrom B, DeMaria AL: A Qualitative Study of Young Women's Beliefs About Intrauterine Devices: Fear of Infertility. J Midwifery Womens Health. 2016; 61(4): 482–8. PubMed Abstract | Publisher Full Text\n\nKNBS: Kenya Demographic and Health Survey 2008/09. Kenya National Bureau of Statistics and ICF MACRO. 2010. Reference Source\n\nAdedini SA, Omisakin OA, Somefun OD: Trends, patterns and determinants of long-acting reversible methods of contraception among women in sub-Saharan Africa. PLoS One. 2019; 14(6): e0217574. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBhandari R, Pokhrel KN, Gabrielle N, et al.: Long acting reversible contraception use and associated factors among married women of reproductive age in Nepal. PLoS One. 2019; 14(3): e0214590. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWildemeersch D: Intrauterine contraceptives that do not fit well contribute to early discontinuation. Eur J Contracept Reprod Health Care. 2011; 16(2): 135–141. PubMed Abstract | Publisher Full Text\n\nAnguzu R, Sempeera H, Sekandi JN: High parity predicts use of long-acting reversible contraceptives in the extended postpartum period among women in rural Uganda. Contracept Reprod Med. 2018; 3: 6. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "63727",
"date": "15 Jun 2020",
"name": "Jeffrey F. Peipert",
"expertise": [
"Reviewer Expertise Women's Health",
"family planning",
"contraception",
"sexually transmitted infections",
"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\nI enjoyed reading the article, Trends and factors associated with long-acting reversible contraception in Kenya, by Dr. Kungu and colleagues. The article is well written and provides useful information for researchers and clinicians interested in providing comprehensive contraception care globally.\n\nThe study is a secondary analysis of 3 waves of the Kenya Demographic Health Survey. I am not an expert in large survey design and analysis; thus, my review will focus on the usefulness of the information provided. It was encouraging to read that LARC use is “picking up rapidly” in Kenya. The authors group LARC together (IUDs and implants) and separate out implants and IUDs in the text, but they do not cover IUD types (copper and hormonal). This would be useful information to the reader. I believe hormonal IUDs are not as available as copper in Kenya. Is this true? Has it changed over time? There are some sections of the manuscript that lack balance (advantages/disadvantages of LARC). For example, 4th paragraph of the INTRO: the authors highlight the many advantages of LARC, but make no mention of the disadvantages (e.g. irregular bleeding with the implant and heavier bleeding with the copper IUD).\n\nThe Discussion provided some interesting information. I had no idea that the uptake of the implant in Kenya was “the highest in the world.” In the third paragraph of the Discussion, the authors mention the “rumors and misconceptions.” What are the most common misconceptions? Do women believe the IUD is linked to infertility? Looks like this is mentioned in paragraph 5; is this misconception limited to younger women? There is some discussion of these myths in paragraph 9 (e.g. mention of cancer).\n\nIn the discussion of age and LARC, it may be helpful to stratify. In many populations, the implant is more popular in young women, and the IUD is more accepted in older women.\n\nThe authors also comment on the decline in condom use. Has there been an epidemic of STIs as a result? Is there any evidence of a detrimental effect on the population? I do agree with a renewed emphasis on dual-use (barrier plus effective method) for STI and pregnancy prevention.\n\nOverall, I found the Discussion a bit long, wordy, and somewhat disorganized. Avoid one-sentence paragraphs. Group topics (age) together. It would benefit from consolidation and being more concise.\n\nSpecific comments:\n\nABSTRACT: Background: I do not think you can say there is NO risk of incorrect use. Unrecognized expulsion of an IUD is an example. Methods: You might provide the percentage of the population that are “currently using modern contraceptive methods” here. Results: It would be helpful to provide some data, effect estimates, and some confidence intervals. Which factors have the biggest effect estimate on the association with LARC use?\n\nINTRO: 4th paragraph, last sentence: I did not understand “they arrive at costs of _____.” Is this cost savings? I assume the cost of the methods are estimates of the true cost. Please explain more clearly.\n\nMETHODS: Please state the type of regression used (logistic regression) in the text How did you assess confounding? Did you look for interactions?\n\nRESULTS: I would prefer an adjusted analysis that shows the OR and 95% CI. I do not think we need the B, SE in Table 2. A simplified table showing the effect estimates and confidence intervals would be better.\n\nDISCUSSION: Comments above.\n\nThere’s LOTS of information, tables, and Figures. I trust the editor will decide if all of these are needed.\n\nOverall, a well done report.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "5624",
"date": "15 Jun 2020",
"name": "Margaret Kungu",
"role": "Author Response",
"response": "As the corresponding author, I appreciate the expert, thorough, and very useful comments of the reviewer and will address them in the revised article so as to improve it for readers."
}
]
},
{
"id": "138105",
"date": "18 May 2022",
"name": "Kate Cheney",
"expertise": [
"Reviewer Expertise Pregnancy planning",
"contraception",
"preconception",
"postpartum contraception",
"nursing",
"midwifery"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to 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 your manuscript titled: Trends and factors associated with long-acting reversible contraception in Kenya\nThis is interesting and important work for the reader Understanding the barriers to the LARC options are essential (not just in developed countries) to move forward to proving LARC methods to those who wish it.\nAbstract: No comments.\nIntroduction:\nIs this a government or health agenda?\n\nCan you provide a reference of LARC?\n\nNot sure you need the word HUGH before female population?\n\nCan you reference a quick example of heath and social benefits for the reader?\n\nIs there data about postpartum contraception opportunities? This is often a time of engagement with health midwives and doctors.\n\nThe paragraph starting with \"A challenge in the access...\" is a bit unclear for the reader - can this be clarified a little please?\n\nThere are other partners outside family planning programs, like postpartum, so would be good to include all opportunities.\n\nObjective: clear\nMethods:\nMethods - what is ICF?\nResults:\nThere are a great many tables but are they all needed?\n\nParity is very interesting any reason for that? Is contraception well provided in the postpartum period, or is it free then?\nDiscussion:\nWhat are Tunza clinics? Could you explain a little, no all readers will know about Kenya or this.\n\nCould the statement about omen with tendency to multiple partner be better expressed?\n\nDoes type of IUD matter? i.e. copper, mirena, etc.\n\nWhat is it about those regions where LARC use is more common?\n\nWhere is the ref about reproductive goals of women, some may have no goals or plans, or feel they have no choice?\n\nWho inserts LARC, are all workforce being included? i.e. nurses and midwives too.\n\nAre health professionals trained in LARC?\n\nDoes it come down to cost, local support and supplies?\nConclusion: Thank you.\nI am not able to assess the statistical analysis\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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": "8266",
"date": "18 May 2022",
"name": "Margaret Kungu",
"role": "Author Response",
"response": "Thank you Kate for the very insightful comments which will help improve the paper further. Below are immediate responses as I work on an improved revision. Is this a government or health agenda? It is both as the Government has been encouraging uptake of FP to rpomote women health and attain UHC Can you provide a reference of LARC? Yes, I will. Not sure you need the word HUGH before female population? Huge - It was an emphasis on the size. Can you reference a quick example of heath and social benefits for the reader? Yes Is there data about postpartum contraception opportunities? This is often a time of engagement with health midwives and doctors. The data is not available in KDHS in this context. The paragraph starting with \"A challenge in the access...\" is a bit unclear for the reader - can this be clarified a little please? It is about expanding the method mix so that injection is not the dominant option. I will revise in the final version. There are other partners outside family planning programs, like postpartum, so would be good to include all opportunities. Yes in line with the focus of the study (trends and factors associated with use of LARC) and available data. Methods - what is ICF? ICF Macro are the administrators of the DHS worldwide program. There are a great many tables but are they all needed? Yes . The tables are only 2 not many but the graphs are quite a number to show LARC use by different key socio-economic variables hence they are necessary. Parity is very interesting any reason for that? Is contraception well provided in the postpartum period, or is it free then? It may be because LARCS were previously encouraged among those limiting children but not spacers. Contraception is freely available in Kenya especially for postpartum clients Discussion: What are Tunza clinics? Could you explain a little, no all readers will know about Kenya or this.Tunza clinics were an Non- Governmental initiative for scaling up family planning. I will clarify in the final version. Could the statement about women with tendency to multiple partner be better expressed? Yes in the final version. Does type of IUD matter? i.e. copper, mirena, etc. Yes but KDHS does not have the data on types. What is it about those regions where LARC use is more common? This is where the FP campaigns to market LARC were very successful. Where is the ref about reproductive goals of women, some may have no goals or plans, or feel they have no choice? It will be incorporated if available. Who inserts LARC, are all workforce being included? i.e. nurses and midwives too. LARCs are inserted by nurses. Are health professionals trained in LARC? Yes very well. Does it come down to cost, local support and supplies? Cost is not a major issue but maybe acceptance and supplies in some far flung areas."
}
]
},
{
"id": "138102",
"date": "30 May 2022",
"name": "John Cleland",
"expertise": [
"Reviewer Expertise Social demography with focus on 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\nThe increasing uptake of LARCs in Kenya, and in many other African countries, is an important development and a paper on factors that are associated with use of these methods is welcome. The sharp focus on method choice (LARCs versus other modern methods) is appropriate. The overall message of the paper is clear but several problems need to be addressed.\nYour handling of region should be reconsidered. In Tables 1 and 2, you show results for high versus low use regions but differences are minor and not statistically significant. However in figures 6, 7 and 8 you show results for Kenya’s 8 regions. I cannot understand why results for these 8 regions are not included in tables 1 and 2. My suggestion is that you do include them and omit the low/high use characterisation of region.\n\nA total of nine figures seems to me excessive. All the results in these 9 figures could be compressed into a single table. It would look like table 1 but instead of % LARC and % other modern, it would show % implant and % IUD. This would have the advantage of showing results across the three surveys for variables such as wealth and urban-rural residence which are excluded from the figures. Please consider this option.\n\nEven if you insist on retention of figures, some of them should be re-designed. Figures 2-4 and 9 shows profiles of users. It would be more informative to re-design as in figure 1. For instance in figure 4, it is impossible to tell whether the very small % of uneducated women using implants is due to an aversion to implants among these women or to the fact that there are very few uneducated modern method users. A line graph as in figure 1 with denominators of all modern method users of specified educational status would be better. Whether you follow the suggestion in (2) or re-design figures, the text would need to be amended.\nMinor points\nHeadings of tables and figures should be more precise. For instance the title for table 1 should be “Percentage of all modern method users who use long-acting and other methods by background characteristics, 2003-2014.\n\nIn table 2 I see no need to show B values and standard errors. It is also more customary to label EXP B as Odds ratios. In the footnote, the reference category is Other methods NOT LARCs.\n\nThe Discussion would benefit from consideration of the relevant Kenyan evidence about method-specific beliefs and method choice e.g., Odwe et al., (2021)1; Obare et al., (2020)2; Machiyama et al., (2018)3.\n\nSome brief discussion concerning the introduction to Kenya of the levonorgestrel intrauterine system should be added.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "8319",
"date": "31 May 2022",
"name": "Margaret Kungu",
"role": "Author Response",
"response": "Dear Prof. Cleland, Thank you for the review and very useful comments. We will endeavor to address the issues you have raised in a revised version which I am sure will be much more improved. Dr. Wambui Kungu"
}
]
}
] | 1
|
https://f1000research.com/articles/9-382
|
https://f1000research.com/articles/8-2120/v1
|
18 Dec 19
|
{
"type": "Software Tool Article",
"title": "Generalized EmbedSOM on quadtree-structured self-organizing maps",
"authors": [
"Miroslav Kratochvíl",
"Abhishek Koladiya",
"Jiří Vondrášek",
"Abhishek Koladiya",
"Jiří Vondrášek"
],
"abstract": "EmbedSOM is a simple and fast dimensionality reduction algorithm, originally developed for its applications in single-cell cytometry data analysis. We present an updated version of EmbedSOM, viewed as an algorithm for landmark-based embedding enrichment, and demonstrate that it works well even with manifold-learning techniques other than the self-organizing maps. Using this generalization, we introduce an inwards-growing variant of self-organizing maps that is designed to mitigate some earlier identified deficiencies of EmbedSOM output. Finally, we measure the performance of the generalized EmbedSOM, compare several variants of the algorithm that utilize different landmark-generating functions, and showcase the functionality on single-cell cytometry datasets from recent studies.",
"keywords": [
"dimensionality reduction",
"self-organizing maps",
"single-cell cytometry"
],
"content": "Introduction\n\nEmbedSOM is a dimensionality reduction (DR) algorithm for single-cell cytometry data, designed for high scalability, computational efficiency and performance1. The design is based off FlowSOM2, which utilizes unsupervised manifold learning by self-organizing maps (SOMs) to find structure in the high-dimensional data, and process the result into a meaningful and easily interpretable clustering of the dataset. So far, FlowSOM and SOMs in general seem to be the manifold learning and clustering method of choice for all kinds of cytometry based on protein-targeting antibodies, surpassing other clustering methods in precision, speed and scalability3. EmbedSOM utilizes the same manifold learning method to extract information about the topology of an approximate manifold that describes the high-dimensional cell expression space, and uses it to quickly compute low-dimensional image of the cells that is suitable for visualization.\n\nIn this work, we focus on fixing inconsistencies and problems of the first version of EmbedSOM: First, we describe an updated version of EmbedSOM that improves the approximation to achieve perfect smoothness of the projection. The brief description of EmbedSOM provided in the original paper is supplemented here by fully commented pseudocode, in order to aid scrutinization and interpretation of the method. Second, we review EmbedSOM as a generalized function for enriching a projection of selected landmarks to a projection of entire spaces. We demonstrate this by replacing the original SOMs with less-demanding t-SNE on random landmarks. Additionally, we describe GQTSOM, a novel variant of growing self-organizing maps (GSOMs, described e.g. by Rauber et al.4) that was designed to alleviate precision and overcrowding problems of the original EmbedSOM. GQTSOMs utilize quad-tree space-partitioning structure to grow inwards, thus allowing the training algorithm to increase the resolution of manifold approximation on demand, and to benefit from the performance gain in early stages of training that is common to all GSOMs.\n\nThe functionality of the new algorithm is showcased on datasets that were recently used for studying other DR techniques. We show the differences between individual variants of landmark-generating functions, and provide visualizations comparable to those produced by current state-of-art algorithms. Finally, we demonstrate how the dynamic resolution of GQTSOMs aids detection of various small cell populations and rare cell types.\n\n\nMethods\n\nEmbedSOM projection can be viewed as an embedding enrichment method: From a set of landmarks in the high-dimensional space and a set of corresponding landmarks in the low-dimensional space, it produces a smooth function that maps all points from the higher-dimensional space to the low-dimensional space and preserves the relative neighborhoods of the landmarks. EmbedSOM was originally designed to work with simple SOM-originating landmarks, as shown in Figure 1.\n\nEmbedding process starts by reducing the input dataset (data flow is visualized as orange arrows) to landmarks (black arrows and dots) in high-dimensional (top) and low-dimensional space (bottom). EmbedSOM quickly places the relatively large amount of individual input points into matching neighborhoods of the low-dimensional landmarks. The landmark-generating methods from left: A simple SOM-based grid, a random selection of input points with 2-D topology reconstructed by t-SNE, and a GQTSOM-based grid. GQTSOM-based landmarks are labeled by their level in the quadtree.\n\nWe will refer to the high- and low-dimensional landmarks as L ϵ ℝn×D and l ϵ ℝn×2. EmbedSOM embedding of a single high-dimensional point is achieved by reducing it to a collection of coordinates of its projections into subspaces that are generated by affine combinations of landmark pairs from L, and reconstructing it in low-dimensional space by reversing the process with corresponding landmark pairs from l.\n\nThe procedure is detailed as Algorithm 1. First, the algorithm chooses k landmarks closest to X, which are expected to give sufficient approximation. In lines 2–6 it computes scores for the k landmarks. The affine projection of X to a space defined by a pair of landmarks from L is computed at line 12 as d, its value is used to create a linear equation which has solutions at positions that would project to the same position d in the affine space generated from corresponding landmarks in l. After adding all parts of the approximation together, the linear system stored in M is very unlikely to remain singular. The position of embedded point is then obtained by simply solving the linear equation of 2 variables. Alternatively, one can view the algorithm as a minimization of the total squared error in all projected d:\n\n\n\n1: procedure EmbedSOM(X ϵ ℝn, L ϵ ℝn×D, l ϵ ℝn×2, k ϵ {4 . . . n}, m > 0, a > 0)\n\n2: c ← a sequence of ci = 〈〈X − Li〉〉 for i ϵ {1 . . . n}\n\n3: o ← indexes of k smallest elements of c in order\n\n4: μ ← ∑i≤kco(i)i⋅k ▷ estimate the distribution of landmark distances\n\n5: σ ← ∑i≤k(co(i)–μi)2\n\n6: S ← a sequence of Si=expb⋅(μ–co(i))σ⋅(1–expco(i)–co(k)m⋅co(k)) for i ϵ {1 . . . k} ▷ compute scores\n\n7: M←(0000|00) ▷ accumulator for the linear equation system\n\n8: for i′ ϵ {1 . . . k − 2} do ▷ iterate through pairs of k − 1 closest landmarks\n\n9: for j′ ϵ {i + 1 . . . k − 1} do\n\n10: i ← o(i′) ▷ obtain non-permuted landmark indexes\n\n11: j ← o(j′)\n\n12: d←〈X–Li,Lj–Li〉〈〈Lj–Li〉〉 ▷ projection position in the affine space\n\n13: h ← lj − li ▷ helper values\n\n14: y←d+〈h,li〉〈〈h〉〉\n\n15: s←(1+〈〈h〉〉)–a⋅e(d–12)2⋅Si′⋅Sj′, ▷ score for this pair of landmarks\n\n16: M←M+s⋅(h⋅hT〈〈h〉〉|y⋅h1y⋅h2) ▷ add this approximation to the linear system\n\n17: end for\n\n18: end for\n\n19: return solution (x1, x2 ) of the linear system in M\n\n20: end procedure\n\nSince the squared term is linear in x, the inner function is a quadratic form that can be minimized algebraically by finding zero of its derivation. This procedure gives the formulas used in the algorithm.\n\nThe algorithm can be easily expanded to embedding into general P-dimensional spaces by taking the low-dimensional landmarks l from ℝn×P, increasing the size of the matrix M for a linear equation of P variables, and solving a larger linear system at the end.\n\nNotably, the initial reduction of the input data to one-dimensional projections to affine spaces (d in the algorithm) prevents various complications from fitting the high-dimensional distances into low-dimensional space, avoiding many problems that arise from dimensionality overhead in other DR algorithms. A similar approach has been taken e.g. by TriMap5, where the transferred information is reduced to mere binary relations between point distances.\n\nEmbedding parameters The embedding procedure admits several tunable parameters: k is the number of nearest landmarks used for the approximation, m > 0 is an arbitrary parameter that selects the steepness of score decay for distance order approaching k, b > 0 chooses the steepness of score decay for landmarks far from X, and a lowers the score of approximations to pairs of relatively far low-dimensional landmarks.\n\nParameter m is specifically designed to lower the score of landmarks with distances that approach k-closest landmark. As a result, small changes in the input point X can not cause sharp changes in the scores assigned to individual parts of the approximation. Consequently, EmbedSOM function is smooth in X.\n\nValues of parameters k, m, m and a must be chosen to avoid singularities and near-singularities when computing the final approximation, which may happen if the set of si,j contains insufficient number of higher-than-negligible scores. That may be caused mainly by setting too low values of k or m, or too high value of b. Argument setting of k≃|L|, m = 10, b = e−1 and a = 1 worked well in a majority of tested use cases and can be considered a good default.\n\nEmbedding complexity To compute a P-dimensional projection of a single point from a D-dimensional space, EmbedSOM projection conducts the following operations: |L| measurements of distances in high-dimensional space, sorting the k smallest elements of the distance vector of size |L|, and conversion of k distances to scores. On the landmark pairs, it conducts at most k2 computations of scores s, the same number of computations of di,j from 2 dot-products in high-dimensional space, and computation of a partial P-by-(P + 1) matrix for solving the linear system in ℝP. Finally, the linear system is solved using Cramer’s rule. The total of computation times is thus, in respective order,\n\n\n\nAssuming the default parameter setting and P ϵ {2, 3}, this complexity sums to 𝒪(D · |L|). The procedure can be trivially repeated for any number of input points.\n\nDifferent distance measures We have assumed that the metric used in both high-dimensional and low-dimensional spaces is Euclidean. Generally, EmbedSOM behaves well even if the distance measure used for the scoring function is swapped for any function that acts as a metric on vector spaces, including the popular L1 and L∞ metrics.\n\nNevertheless, the computation of ‘projections’ using dot-products may then be viewed as a rather questionable reinterpretation of the point coordinates in an inner product space. Fortunately, the minimal-distance projection to a fixed subspace is a linear operator under both L1 and L∞6, which is sufficient for EmbedSOM computation even without requiring the inner product property.\n\nWhile the SOMs are a great method to generate landmarks L and l that carry various beneficial properties that simplify human interpretation of the result (notably the regularity of l), other methods are admissible as well, as long as they can cover the input space sufficiently by L and generate the corresponding landmarks l in the low-dimensional output space so that the topology is similar to L.\n\nFor example, the embedding process can be simplified to a great extent by completely removing SOMs: Instead of constructing L in a complicated way so that it reflects the input space topology, we can take only a small random sample of input points as the landmarks, and use a general DR method to find its topology and arrange landmarks l in a matching way, as shown in Figure 1 on an example with t-SNE. While this is often sufficient, for the purposes of embedding it is more beneficial to find a smaller set of landmarks that provide better description of the various features in the input space than the random sampling.\n\nMany variants of the SOM algorithm have been created to optimize this metric: For example, the Growing SOMs (GSOMs) by Dittenbach et al.7, start with a simple 2×2 SOM grid, and dynamically add new SOM grid vertices at the SOM perimeter only if it is necessary to keep the total quantization error low. A hierarchical variant of GSOM called GHSOM introduced by Rauber et al.4 aims to improve the description of small details in the input data space that were not described sufficiently by GSOMs. Depending on the heuristic, the vertices of GHSOM grid are converted to small independent versions of GHSOMs, which map the corresponding local parts of the input space; this continues recursively to create a layered structure of SOMs that describe increasingly fine and subtle details in the data.\n\nGQTSOMs Although the GHSOMs improve the classification of small-scale features in the datasets, the hypertree structure complicates their use as landmarks for planar visualization with EmbedSOM. We propose the Growing QuadTree-structured SOMs (GQTSOMs) to alleviate this problem: The GQTSOMs grow by recursively splitting the nodes to form a hypertree, but unlike GHSOMs the hypertree shape is restricted to a quadtree, which possesses straightforward interpretation as a 2-dimensional structure8.\n\nThe nodes in GQTSOMs are identified by their position and depth in the quadtree, represented as an integer triple (L, x, y). The corresponding 2-dimensional coordinates are obtained as (2x + 1, 2y + 1) ⋅ 2−L. Initial nodes in training occupy positions (1, 0, 0), (1, 0, 1), (1, 1, 1) and (1, 1, 0). Upon growing, a node (L, x, y) is split into 4 nodes identified as (L +1, 2x, 2y), (L +1, 2x +1, 2y), (L +1, 2x +1, 2y +1), and (L +1, 2x, 2y +1). Figure 1 shows an example of 3-level GQTSOM in a 2-dimensional space, where the initial 2×2 SOM growed three times to produce 13 landmarks.\n\nGQTSOM training proceeds by batches as in the usual batch SOM training. After each epoch, several nodes with greatest position change in the input data space are split, so that the total number of nodes grows linearly during the whole training. Initial positions for the new nodes are interpolated from the topological SOM neighborhood, using the same neighborhood function as for training the SOM (e.g. a Gaussian). To avoid overcrowding of the map by small nodes and promote their specialization to fine details, the nodes are penalized by a factor of L−1 in the growing heuristic, and by a factor of 4L applied to their neighborhood volume in both input space and SOM space.\n\nThe current version of EmbedSOM is available as R package EmbedSOM from http://github.com/exaexa/EmbedSOM, together with the customized versions of SOM and GQTSOM algorithms. The implementations are conducted in C++; the code is independent from the actual package or R language, and can be reused in other environments. The integration into R serves mostly as a bridge to the large number of cytometry-oriented packages in the ecosystem.\n\nLow-level implementation has provided several additional ways to improve the performance of the algorithms when compared to the original implementation: For example, cache-efficient version of the SOM training has improved the performance by up to 15× on large SOMs; SIMD-based acceleration of the vector operations by up to 4×, and parallelization of the batch SOM training and embedding by a factor roughly equivalent to the number of used CPUs.\n\nOverall, the computation time required for typical datasets was reduced by a factor greater than 10× on commonly available hardware, and more than 30× in case of processing complicated datasets using large SOMs.\n\nEmbedSOM is now used primarily from R environment; the package can be downloaded from GitHub using R command devtools::install_github('exaexa/EmbedSOM'). The package installation will automatically compile the code that uses the SIMD capabilities if they are enabled on the target platform.\n\nGenerally, the SOM and embedding process can be executed on any real matrix with individual data points in rows, and parameters in columns. This expectation is consistent with many other DR or clustering packages, including FlowSOM, Rtsne and umap. For example, a user may obtain an embedding of the Iris dataset as such:\n\n\n\nIn the code, the landmarks are first created using a SOM and saved in the map, which is then passed to the EmbedSOM function that produces the final 2-column matrix e with embedded coordinates. These can be plotted e.g. using the standard plot function.\n\nOn data larger than Iris dataset, GQTSOMs may be used to generate the landmarks and a map usable with EmbedSOM function in a similar way:\n\n\n\nHere, target_codes chooses the desired final number of the landmarks in the fully grown SOM, and parameter parallel=T allows the computation to use multiple available CPUs. Functions SOM and EmbedSOM support parallelization as well, using the same parameter.\n\nOther DR methods may create the landmarks. In case of t-SNE, the following code generates a map object with 500 landmarks suitable for embedding:\n\n\n\nThe parameters of the SOM, GQTSOM and EmbedSOM functions are extensively documented in the supplied R manual pages.\n\n\nUse cases\n\nThe output of new EmbedSOM variants is demonstrated on two use-cases: First, using the described variants of landmark-generating functions, we reproduced the visualizations by Becht et al.9 of a dataset that maps specific trafficking and cytokine signatures of human T cells across tissues, created by Wong et al.10. Second, we visualized a human gastrointestinal disorders dataset by van Unen et al.11 using GQTSOMs, showing that EmbedSOM provides a viable alternative to the semi-interactive analysis of rare cell types using the HSNE algorithm12.\n\nThe primary purpose of EmbedSOM is to provide quickly available and highly comprehensible data visualization in situations where processing speed is critical. The embedding time of the demonstration datasets was measured on an AMD Ryzen 7 2700U CPU with 16GB of RAM running Debian Linux (Bullseye), R version 3.6.1 compiled with gcc version 9.2.1; the timing is reported in the corresponding figures as t, together with number of cells (n) and landmarks (|L|). As the main result, the measurements show that a high-quality visualisation of a data file from a common experiment (around 300 thousand cells) can be obtained in less than 10 seconds using common office hardware.\n\nTo visualize the Wong dataset, we have embedded it using EmbedSOM with SOM landmarks (i.e. the original EmbedSOM), EmbedSOM with t-SNE generated landmarks, and EmbedSOM with GQTSOM-generated landmarks. As seen in Figure 2, the original EmbedSOM implementation has managed to separate and visualize both the different cell types and their layout according to source organ. However, the result may seem unsatisfactory due to overcrowding and loss of both detail and global layout, especially when compared to UMAP visualizations of the same dataset [9, Figure 1a,b]. Despite the overcrowding, it is still possible to identify separate clusters of CD69+CD103+ Trms (resident-memory T cells) in all organs except cord blood, and naive (CD69+CD45RA+), central memory (CCR7+CD62L+) and effector memory T cells (CD45RA– CD45RO+CCR7–CD62L–) within both CD4 and CD8 T cell types; this is in agreement with findings of van Unen et al. [11, Figure 3a,b]. Plots of all marker expressions are available as Extended data.\n\nTop row: cells embedded using 3 different landmark-generating methods, colored by the tissue of sample origin. Middle row: The same embedding colored by major cell types. The colors used for annotation are purposefully reproduced from the article of Becht et al.9 to simplify comparison. Bottom row: visualizations of the low-dimensional landmark images, colored by their corresponding marker expressions.\n\nWe have observed that the improved methods of landmark positioning have successfully alleviated both overcrowding and layout problems. In particular, the layout of MAIT (mucosal-associated invariant T) and γδ T cells in the embedding with t-SNE-generated landmarks reflects the expected properties of cell populations, and the individual population clusters are clearly separated by low-density areas with intermediate cell states and noise. Additionally, the smoothness of EmbedSOM projection has displayed even features and similarities that were not captured by t-SNE. That can be seen on the cluster of γδ T cells, where EmbedSOM shows a clear connection of the gut-originating part of the cluster to both the very similar gut-originating CD8+ T cells and the other γδ T cells. In comparison, this connection is preserved by all tested types of SOMs, but neglected by both plain t-SNE and UMAP, which show the population separated to 3 resp. 2 separate clusters [9, Figure 1a]. The embedding based on GQTSOM landmarks has provided similar global layout of the output as the one with t-SNE landmaks, additionally capturing the continuity of γδ T cell cluster and its similarity to MAIT and NK cells, and providing separation of individual clusters differentiated by tissue of origin comparable to that of UMAP. Regardless of that, the main advantage of using GQTSOMs comes with the ability to generate smaller amount of more precise landmarks than plain SOMs. Compared to the SOM used with the original EmbedSOM approach, this resulted in significant computation speed increase (around 50%) and better description of the small and rare cell populations by landmarks (and, therefore, also by subsequent FlowSOM-style clustering). In particular, the small subpopulations of γδ T cells were assigned roughly twice the number of landmarks by GQTSOM than by the standard SOM, which resuted in spatially correct separation of the cell subtypes in the embedding.\n\nWe showcase the ability of GQTSOM landmark generation method to capture and display various rare cell types using a dataset by van Unen et al.11. The dataset was created as such: A total of 5.2 million single cells were collected from duodenum biopsies, rectum biopsies, perianal fistulas, and PBMC from patients undergoing various gastrointestinal disorders and healthy individuals (as controls). The gastrointestinal disorders included celiac disease (CeD), refractory celiac disease type-II (RCDII), enteropathy associated T-cell lymphoma type II (EATLII), and Crohn’s disease. Cells were stained using 32 metal conjugated monoclonal antibodies to identify cells within the innate and adaptive immune system. This dataset was later reanalyzed by van Unen et al.12 using a hierarchical version of t-SNE algorithm called HSNE, showing that the hierarchical dissection of the data was able to identify several rare cell types within the innate lymphoid cell (ILC) compartment.\n\nFor the purpose of demonstration, we preprocessed the same dataset by removing debris, doublets and dead cells based on simple thresholds on the DNA, Event length and Viability parameters. The 32 antibody markers of the 4.14 million cleaned cells were then transformed by hyperbolic arcsine and used to train the GQTSOM and produce an embedding. The result in Figure 3 allows easy observation of both the ILC compartment and the CD4+ T cell subset, corresponding to the observations produced by second-level HSNE [12, Figures 3 and 5]. Additionally, the embedding shows presence of many clusters from lower levels of the hierarchical dissection: In the figure, it is possible to identify clusters of CD4M+CD28–CCR7–CD56– and CD4+CD28–CCR7–CD56+ rare cell types within the CD4+ compartment, and of the CD127–CD45RA–CD56partial cluster within the ILC (CD7+CD3–) compartment. These clusters were identified by HSNE at 4th resp. 3rd levels of dissection [12, Figures 5b and 3c]. Recently, Belkina et al.13 showed that the opt-SNE algorithm can additionally identify CD4+CD28–CCR7+ CD56– rare cell type, which is also clearly separated by the GQTSOM-based embedding, using much less computational resources than optSNE.\n\nTop left: Overview of the cleaned and embedded Unen dataset, colored by expression of main cell lineage markers. The contour based on Gaussian difference is added for easier identification of changes in cell density. Labels mark the rare cell types identified by van Unen et al.12, Belkina et al.13: (a) CD4+CD28–CCR7+, (b) CD4+CD28–CCR7–CD56–, (c) CD4+CD28–CCR7–CD56+, and (d) CD7+CD3–CD127–CD45RA– CD56partial. Top right: Expressions of separate markers used for the identification. Bottom: Cells color-coded by sample origin (left) and separated by disease status of the patient (right).\n\nThe plot of cells separated by disease status in Figure 3 confirms the observation that the rare CD4+CD28–CD56+ phenotype is enriched in the samples from patients with Crohn’s disease. Moreover, the plot gives a useful overview for identifying cell types specific for the other diseases, showing two specific and one enriched cluster for RCDII, a single specific cluster of CD8+CD56+CD127+c-KIT+ cells for EATLII, and one specific and some enriched cell types in patients with CeD.\n\n\nSummary\n\nWe have presented an improved and generalized version of EmbedSOM, supported by the new model of quadtree-structured growing self-organizing maps. The functionality of the new algorithm was demonstrated on data and analyses from recent studies, showing that the new combination provides superior embedding speed and good rendering of various cell types, including tissue-specific and rare phenotypes.\n\n\nSoftware availability\n\nSource code available from: https://github.com/exaexa/EmbedSOM\n\nArchived source code available from: https://doi.org/10.5281/zenodo.356898014\n\nLicense: GNU GPLv3\n\n\nData availability\n\nThe used datasets are freely available from FlowRepository.org under accession IDs:\n\nFR-FCM-ZZTM (Wong dataset; the data was preprocessed exactly as described by Becht et al.9)\n\nFR-FCM-ZYRM (Unen dataset)\n\nFigshare: Wong and Unen datasets, EmbedSOM embedding based on SOMs, t-SNE and GQTSOMs, http://doi.org/10.6084/m9.figshare.1132803515.\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 all authors of the original EmbedSOM article for supplying the interesting problems and use-cases that motivated the development of the current version of EmbedSOM.\n\n\nReferences\n\nKratochvíl M, Koladiya A, Balounova J, et al.: SOM-based embedding improves efficiency of high-dimensional cytometry data analysis. bioRxiv. 2019. Publisher Full Text\n\nVan Gassen S, Callebaut B, Van Helden MJ, et al.: FlowSOM: Using self-organizing maps for visualization and interpretation of cytometry data. Cytometry A. 2015; 87(7): 636–645. PubMed Abstract | Publisher Full Text\n\nWeber LM, Robinson MD: Comparison of clustering methods for high-dimensional single-cell flow and mass cytometry data. Cytometry Part A. 2016; 89(12): 1084–1096. PubMed Abstract | Publisher Full Text\n\nRauber A, Merkl D, Dittenbach M: The growing hierarchical self-organizing map: exploratory analysis of high-dimensional data. IEEE Trans Neural Netw. 2002; 13(6): 1331–1341. PubMed Abstract | Publisher Full Text\n\nAmid E, Warmuth MK: TriMap: Large-scale dimensionality reduction using triplets. 2019. Reference Source\n\nBorodin PA: Linearity of metric projections on Chebyshev subspaces in L1 and C. Mathematical Notes. 1998; 63(6): 717–723. Publisher Full Text\n\nDittenbach M, Merkl D, Rauber A: The growing hierarchical self-organizing map. In Proceedings of the IEEE-INNS-ENNS International Joint Conference on Neural Networks. IJCNN 2000. Neural Computing: NewChallenges and Perspectives for the New Millennium, IEEE, 2000; 6: 15–19. Publisher Full Text\n\nSamet H: The quadtree and related hierarchical data structures. ACM Computing Surveys (CSUR). 1984; 16(2): 187–260. Publisher Full Text\n\nBecht E, McInnes L, Healy J, et al.: Dimensionality reduction for visualizing single-cell data using UMAP. Nat biotechnol. 2019; 37(1): 38. PubMed Abstract | Publisher Full Text\n\nWong MT, Ong DE, Lim FS, et al.: A High-Dimensional Atlas of Human T Cell Diversity Reveals Tissue-Specific Trafficking and Cytokine Signatures. Immunity. 2016; 45(2): 442–456. PubMed Abstract | Publisher Full Text\n\nvan Unen V, Li N, Molendijk I, et al.: Mass Cytometry of the Human Mucosal Immune System Identifies Tissue- and Disease-Associated Immune Subsets. Immunity. 2016; 44(5): 1227–1239. PubMed Abstract | Publisher Full Text\n\nvan Unen V, Höllt T, Pezzotti N, et al.: Visual analysis of mass cytometry data by hierarchical stochastic neighbour embedding reveals rare cell types. Nat commun. 2017; 8(1): 1740. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBelkina AC , Ciccolella CO, Anno R, et al.: Automated optimized parameters for T-distributed stochastic neighbor embedding improve visualization and analysis of large datasets. Nat commun. 2019; 10(1): 5415. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKratochvil M: EmbedSOM 2.0 (Version v2.0). Zenodo. 2019. http://www.doi.org/10.5281/zenodo.3568980\n\nKratochvíl M, Koladiya A, Vondrášek J: Wong and Unen datasets, EmbedSOM embedding based on SOMs, t-SNE and GQTSOMs. figshare. Figure. 2019. http://www.doi.org/10.6084/m9.figshare.11328035.v1"
}
|
[
{
"id": "57989",
"date": "11 Feb 2020",
"name": "Evan Newell",
"expertise": [
"Reviewer Expertise Cellular immunology"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors generalize their EmbedSOM approach to examine two additional ways of selecting the respective sets of landmarks in the high- and low-dimensional spaces, beyond the standard SOM, to address. Example data analyses are appreciated.\nThat selection is the first stage in the embedSOM approach. The second stage is the actual embedding enrichment process.\n\nThe authors explain: \"EmbedSOM projection can be viewed as an embedding enrichment method: From a set of landmarks in the high-dimensional space and a set of corresponding landmarks in the low-dimensional space, it produces a smooth function that maps all points from the higher-dimensional space to the low-dimensional space and preserves the relative neighborhoods of the landmarks.\"\nTesting:\nWe followed the paper’s guidance on some in-house fcs files and had success with embedSOM and the GQTSOM function.\n\nSome naming confusion:\nBy \"generalized EmbedSOM” the authors refer to using different ways of generating landmarks, other than the original (self-organizing map) SOM approach.\n\nIt seems preferable to drop the “SOM” rather than refer to these variants as \"generalized EmbedSOM” methods. The authors might use the more general notion of landmarks, rather than SOM. As they note, the random-sampling, followed by tSNE, version of “generalized EmbedSOM” doesn’t use SOMs at all.\n\nRe “compacting noise\"\nThe first reference of the manuscript includes some background on differences between the “generalized EmbedSOM” approach and what the authors call “plain tSNE and UMAP,” and attributes these differences to the respective designs of the algorithms.\n\nIn that background paper, the authors explain: “neither UMAP nor tSNE aim to preserve local linearity of the transformation, which allows them to take apart the clusters with noisy data and attach the residual noise to nearest clusters.”\n\nThey concluded in that paper: “Compacting the residual or unexplained noise is desirable for providing a clean display of the data for publication. On the contrary, almost-immediate availability of all information about very large datasets, including the (often informative) noise, is more important for producing comprehensive graphics for high-throughput analysis.\"\n\nThis paper marks an attempt to explore those differences, and the apparent trade-offs, in more detail, so it would benefit from discussing these tradeoffs in the context of the algorithm designs.\n\nThe authors noted in the first reference, \"While the observed cluster separation may be desirable if the embedding is expected to approximate the population boundaries, it may be inappropriate if the population environment is relevant for analysis.\"\n\nGQTSOMs:\nThe manuscript introduces a new landmark-generating algorithm that simplifies a hierarchical variant of an adaptive SOM approach, namely, growing quad tree SOMS (GQTSOMs) as a simplified growing hierarchical SOM (GHSOM), which is in turn a variant of growing SOMs (GSOMs).\n\nThe aim is to identify and incorporate features in the input space more efficiently than random sampling, by using a \"layered structure of SOMs\". This is a natural thing to do to improve on SOM.\n\nOn page 8 the authors report that GQTSOM leads to using a “smaller amount of more precise landmarks” and thus faster computation, and appears to be a nice contribution.\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": "5252",
"date": "27 Feb 2020",
"name": "Miroslav Kratochvíl",
"role": "Author Response",
"response": "Thank you for the review and comments. We will wait for the additional reviews and address some of your suggestions in the second version of the manuscript.Regarding the name of EmbedSOM, we are aware of the issue with textual \"specialization\" to SOMs which ignores the modifiable parts of the workflow, but since the package is already published over a year and we had not been able to invent a strictly better name so far, we expect that the name will stay. We will gratefully accept suggestions (also from readers) for a reasonably short name that sufficiently characterizes the projection procedure."
}
]
},
{
"id": "59220",
"date": "25 Feb 2020",
"name": "Nikolay Oskolkov",
"expertise": [
"Reviewer Expertise Computational Biology",
"Bioinformatics",
"Mathematical Statistics and 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 authors present an improved version of EmbedSOM that was optimized for speed to address extra large single cell data sets. In addition, a novel way of growing SOM based on quad-tree was proposed.\nDimension reduction of large single cell RNAseq and flow cytometry data sets is very challenging due to the crowding problem and algorithm scalability issues. Therefore, developing methods alternative to the current golden standards such as PCA, tSNE and UMAP is of high importance. More specifically, tSNE and UMAP are capable of preserving only local structure while PCA keeps the global structure information. However, no method is currently available that can preserve both local and global structure.\nSelf-Organizing Maps (SOMs) and the modified EmbedSOM that are discussed in the manuscript represent an interesting and promising algorithm in this respect. However, I would like to raise a few questions and concerns to be addressed by the authors.\nFirst, based on the cost function mentioned on the page 3, the algorithm seems to resemble MDS / PCA type of dimension reduction. Therefore, I would like to see a comparison of EmbedSOM with MDS / PCA. If a connection between the gamma-delta T cells and CD8 T cells was not captured by tSNE and UMAP as it is mentioned on the page 8, probably due to the lack of global structure preservation by tSNE and UMAP, was this connection captured by MDS / PCA?\nSecond, what would be the benefit of using EmbedSOM compared to PCA / MDS, tSNE and UMAP? Do we discover any new biology using EmbedSOM that is not captured by PCA / tSNE / UMAP? Do we benefit from the computational speed of EmbedSOM compared to PCA / tSNE / UMAP? If so, is it really faster (and how much faster) than PCA? I would like to see a clear formulation of the role of the EmbedSOM among other dimension reduction methods.\nThird, I was really impressed by the Figure 1 and how well GQTSOM-based embedding was able to reconstruct the original 3D S-shaped non-linear manifold. To my experience, tSNE / UMAP and especially PCA / MDS would have difficulty reconstructing the 3D S-shaped manifold as 2D embeddings. I have not found any links to the codes for reproducing this embedding and would be very curious to see whether GQTMSOM / EmbedSOM is really capable of capturing the internal 2D structure of the 3D S-shaped non-linear manifold.\n\nIs the rationale for developing the new software tool clearly explained? Partly\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Partly\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes",
"responses": [
{
"c_id": "5430",
"date": "27 Apr 2020",
"name": "Miroslav Kratochvíl",
"role": "Author Response",
"response": "Thank you for the review and for pointing out the deficiencies. We believe that the raised points should be addressed -- especially regarding the comparison of EmbedSOM with other dimensionality reduction methods (adding some helpful hints for the readers to decide whether they should use EmbedSOM) and the code that produces the Figure 1 and processes the other dataset (thus improving availability and reproducibility). We plan to submit the extended version of the article in several days."
}
]
}
] | 1
|
https://f1000research.com/articles/8-2120
|
https://f1000research.com/articles/9-378/v1
|
19 May 20
|
{
"type": "Research Article",
"title": "Analgesic efficacy of nefopam for cancer pain: a randomized controlled study",
"authors": [
"Koravee Pasutharnchat",
"Wichita Wichachai",
"Rungrawan Buachai",
"Wichita Wichachai",
"Rungrawan Buachai"
],
"abstract": "Background: Nefopam is a non-opioid, non-steroidal, central acting drug used effectively for postoperative pain. The efficacy of nefopam for cancer pain remains unclear. We aimed to evaluate the analgesic efficacy of nefopam for cancer pain in a randomized controlled trial. Methods: Patients with moderate to severe cancer pain (n=40) were randomly divided into two groups. The nefopam group (n=20) received three 20 mg doses of nefopam every 8 hours. The placebo group (n=20) received normal saline. Intravenous patient-controlled analgesia with morphine was given for breakthrough pain for 48 hours. The primary outcome was significant pain reduction. Secondary outcomes were morphine consumption over 48 hours and incidence of side effects. Results: The nefopam group showed pain reduction at 12 hours (65% of patients), 24 hours (80%), 36 hours (85%), and 48 hours (65%). The placebo group showed pain reduction at 12 hours (70%), 24 hours (75%), 36 hours (80%), and 48 hours (60%). However, there were no statistically significant differences between the groups (p>0.05). The median dosage of morphine consumption in 48 hours was lower in the nefopam group (25.5 mg) compared with the placebo group (37 mg), but this was not statistically significant (p=0.499). There were no statistically significant differences in blood pressure and heart rate between the groups. Side effects in both groups were comparable. Conclusions: At dosage of 60 mg in 24 hours, nefopam did not provide significant pain reduction in moderate to severe cancer pain patients. However, there was a trend of reduced opioid consumption. Further studies with larger sample sizes, longer duration, or higher doses of nefopam are warranted. Registration: Thai Clinical Trail Registry (TCTR) ID TCTR20181016001; registered on 12 October 2018.",
"keywords": [
"cancer",
"pain",
"nefopam",
"analgesia",
"efficacy",
"patient controlled analgesia",
"morphine consumption",
"side effect",
"analgesia",
"pain reduction"
],
"content": "Introduction\n\nPain is one of the most common and distressing cancer symptoms. Despite development of new drugs and modalities for treating cancer pain during the past decade, around half of patients with cancer suffer from moderate to severe pain, which impacts their quality of life1 and may result in various psychosocial responses. The prevalence of pain among patients with cancer remains high worldwide. A systematic review and meta-analysis showed pooled pain prevalence rates of 39.3% after curative treatment, 55% during cancer treatment, 66.4% in advanced or terminal stages, and 50.7% in all cancer stages2.\n\nWorld Health Organization guidelines recommend starting medications such as non-steroidal anti-inflammatory drugs (NSAIDs), paracetamol, or opioids in all adults with cancer-related pain when pain management is initiated3. However, these drugs may be contraindicated for some patients. Additionally, these medications may result in various intolerable adverse effects. For example: opioids can cause nausea, vomiting, constipation, drowsiness, and dry mouth4; paracetamol may induce hepatotoxicity5; and NSAIDs should be used with caution in high-risk patients who are aging or with pre-existing renal or gastrointestinal disease, hypovolemia, prior peptic ulcer disease, and renal impairment6.\n\nNefopam is a centrally acting non-opioid, non-steroidal analgesic agent that inhibits the reuptake of serotonin, norepinephrine, and dopamine7. It also inhibits calcium influx and blocks voltage-sensitive sodium channels, which leads to decreased activation of post-synaptic glutamatergic receptors such as N-methyl-D-aspartate (NMDA) receptors8. Nefopam does not bind to the opioid receptors that may suppress respiration and has no anti-inflammatory or antiplatelet effects.\n\nThe role of nefopam in treating pain has been investigated in several animal models and clinical human studies. Nefopam was found to be an effective analgesic adjuvant, with studies showing some benefits in reducing postoperative opioid consumption following abdominal and orthopedic surgeries9,10. However, few studies have evaluated the efficacy of nefopam for cancer pain. It remains unclear whether nefopam has value as an adjuvant or alternative to analgesia in management of cancer pain. This study aimed to evaluate the analgesic efficacy of nefopam for moderate to severe cancer pain. We also evaluated concurrent morphine consumption and adverse effects during nefopam administration.\n\n\nMethods\n\nThis double-blinded randomized controlled trial was approved by the Ramathibodi Hospital Ethical Committee (approval number, MURA2018/368) and was registered to the Thai Clinical Trials Registry (TCTR) on 12 October 2018 (TCTR20181016001). Written informed consent was obtained from patients to participate in the study. This study was conducted among patients with cancer who were admitted to Ramathibodi Hospital between October 2018 and March 2019.\n\nInclusion criteria were patients with cancer who: 1) were over 18 years of age, 2) had moderate to severe cancer pain defined by a numeric rating scale (NRS) score of 4/10 or over, and 3) required a strong opioid. Exclusion criteria were patients who were: 1) unable to report on the NRS, 2) unable to use the intravenous patient-controlled analgesia (IV-PCA) machine, 3) currently receiving radiotherapy or chemotherapy (within the past 3 weeks), 4) within 1 week postoperative, 5) receiving a monoamine oxidase inhibitor (within the past 30 days), 6) with a history of convulsive disorder, ischemic heart disease, or allergy to morphine, 7) pregnant, and 8) breastfeeding.\n\nAfter eligible patients signed the informed consent form, they are blindly randomized into two groups (a nefopam group and placebo group) by stratified permuted block randomization in a 1:1 ratio base on two stratification factors: sex (male vs. female) and current opioid use (naïve opioid user vs. non-naive opioid user). The randomization was performed by a research assistance who was not involved in the study. Allocation of the patients to each study group was concealed in an opaque envelope. All participants and outcome assessors were blinded to the group allocation.\n\nThe nefopam group received 20 mg of nefopam in 100 ml normal saline (NSS) intravenously via an infusion pump over 30 minutes every 8 hours for a 24-hour period. The placebo group received 100 ml NSS intravenous infusion with a similar protocol. The agents, prepared by a nurse who was not involved in the study, were in identical appearance bottles; 100 ml of transparent, colorless solution, containing either nefopam (Acupan® BIOCODEX) or placebo. Other current medications were continued throughout the study period. IV-PCA with morphine was given for 48 hours using a similar protocol for all patients (PCA only with morphine 1 mg/ml, PCA dose 1 mg, lock-out interval of 5 minutes).\n\nAll patients were educated on rating their pain from 0–10 using the NRS and operating the IV-PCA machine. Morphine consumption, NRS, blood pressure, and heart rate were measured every 4 hours over a 48-hour period and reported side effects were also collected. Significant pain reduction was defined as a decrease on the NRS of at least 30% from the initial score. New onset of tachycardia was defined as a heart rate equal to or higher than 120 beats per minute (bpm) that had increased by 20% from baseline measurements.\n\nThe patients were withdrawn and labels were opened if they exhibited a heart rate over 150 bpm, arrhythmia, development of extreme unexpected events (e.g. pulmonary embolism, acute ischemic heart disease), or if the patient could not continue to use the PCA machine during the study period.\n\nWith a desired power of 0.8, alpha level of 0.05, and a 30% dropout rate, the calculated sample size was 20 patients per group11. Student’s t-tests or Mann-Whitney tests were used to test differences between groups for continuous variables such as body weight and cumulative morphine consumption. Fisher’s exact or chi-square tests were used for categorical variables such as gender and side effects. Repeated measures analysis of variance was used to compare blood pressure and heart rate between the groups over time. SPSS version 20.0 (IBM Corp. Released 2011, IBM SPSS Statistics for Windows, Armonk, NY) was used for all analyses. A p-value less than 0.05 was considered to indicate statistical significance.\n\n\nResults\n\nIn total, 40 patients were assessed for eligibility and randomized to a nefopam group or a placebo group (20 patients in each group). Figure 1 and Figure 2 show the study flow chart and patient enrollment, respectively.\n\nThe most common cancer type was gastrointestinal cancer (32.5%), and the majority of patients (67.5%) had metastases; consequently, the majority (67.5%) already had prescriptions for opioids for pain management at the time of enrollment. Some patients had also been prescribed analgesic adjuvants, such as tricyclic antidepressants and gabapentinoids. Demographic data for both groups are presented in Table 1. There was homogeneity across all parameters considered.\n\nIn the nefopam group, 65% of patients had significant pain reduction at 12 hours, 80% at 24 hours, 85% at 36 hours, and 65% at 48 hours. In the placebo group, there was significant pain reduction for 70% of patients at 12 hours, 75% at 24 hours, 80% at 36 hours, and 60% at 48 hours. There were no statistically significant differences between the groups at any measurement point (p>0.05; Table 2).\n\nData presented as mean ± standard deviation or frequency. NRS, numeric rating scale.\n\n** Significant pain reduction defined as at least a 30% decrease on the numeric rating scale from the initial value. Chi-square or Fisher’s exact test.\n\nThe median cumulative dose of morphine consumption in 48 hours was lower in the nefopam group (25.5 mg) compared with the placebo group (37 mg), but there were no statistically significant differences at any measurement point (Figure 3).\n\nFigure 4 shows the median cumulative dose of morphine in the opioid naive (Figure 4a) and non-opioid naive subgroups (Figure 4b). In the opioid naive subgroup, the median cumulative dose of morphine was lower in patients in the nefopam group at all measurement points, but this was not statistically significant.\n\n(A) Opioid naïve subgroup; (B) non-opioid naive subgroup.\n\nFigure 5 shows the comparison of the mean systolic and diastolic blood pressure between the two groups. Figure 6 compares the mean heart rate between the two groups. There were no statistically significant differences in mean systolic blood pressure, mean diastolic blood pressure, or mean heart rate at all measurements (p=0.066, p=0.213, and p=0.84, respectively). However, we found new onset of tachycardia in three patients (15%) in the nefopam group and four patients (20%) in the placebo group (p=0.68).\n\nMean systolic/diastolic blood pressure and standard error of the mean for the two groups of patients.\n\nMean heart rate and standard error of the mean for the two groups of patients.\n\nSide effects such as nausea, vomiting, sweating, dry mouth, dizziness, and drowsiness were found in both groups (Table 3). However, there were no statistically significant differences between the groups for any side effects.\n\nChi-square or Fisher’s exact tests.\n\n\nDiscussion\n\nIn published guidelines, most recommendations concerning nefopam have focused on acute and postoperative pain control8,12–15. A quantitative systematic review drew three main conclusions for nefopam in postoperative pain prevention: 1) nefopam had a morphine-sparing effect, 2) it decreased pain intensity at 24 hours postoperatively, and 3) it increased the risk for tachycardia and sweating16. Although the effectiveness of nefopam in chronic cancer- and non-cancer-related pain remains unclear, recent studies have investigated the analgesic protocols for nefopam, its effect on hyperalgesia, and its role in neuropathic pain8,17,18.\n\nEvidence for the use of nefopam for cancer pain remains limited. Minotti and coworkers19 evaluated the analgesic efficacy of oral diclofenac, nefopam, and aspirin with codeine for chronic cancer pain, and found statistically significant pain relief for all treatments; however, that study had a high dropout rate (73.7%). In our study, the rescue of any breakthrough pain by IV-PCA was thought to prevent dropout because of insufficient pain control. In addition, the intravenous infusion protocol might have been able to prevent dropout because it decreased the risk for side effects.\n\nThe pathophysiology of cancer-related pain is complex and may include several mechanisms such as local and systemic inflammatory responses, direct tumor-related pain, metastatic cancer-induced bone pain, and neuropathic pain20–23. Central sensitization can also make cancer pain management more complicated. Its “wind up” phenomenon is also activated via NMDA receptors24. Nefopam is a non-opioid analgesia that is considered to act centrally. Its main mechanisms of action involve the inhibition of serotonin, norepinephrine, and dopamine reuptake and modulation of calcium and sodium channels, which leads to decreased activation of postsynaptic glutamatergic receptors (e.g., NMDA receptors) that are involved in development of hyperalgesia8. Consequently, we hypothesized that nefopam could be used as an adjuvant or alternative analgesia for cancer pain based on its various mechanisms of analgesic action.\n\nTo the best of our knowledge, this study is the first randomized controlled study to evaluate efficacy of intravenous nefopam in moderate to severe cancer pain. However, nefopam did not show statistically significant pain reduction compared with placebo. This may be explained by various reasons. First, the drug potency might have been too low to control moderate to severe cancer pain. Some studies have suggested that in surgical settings, nefopam 20 mg should be equipotent to morphine 6–12 mg25 or meperidine 50 mg26. Its plasma half-life is 3–5 hours and plasma peak concentration is reached after 30 minutes of continuous intravenous infusion16. Previous studies10,27 that evaluated higher doses of nefopam (maximum dose of 120 mg per day) in hepatic resection and orthopedic surgery found nefopam had a superior analgesic effect. We investigated the efficacy of nefopam at 60 mg per day, which had been found to be the effective dose 80 for moderate post-operative pain28. However, this dose might have been too low for moderate to severe cancer pain. Second, the treatment period might have been too short to allow discrimination of any differences. Third, the complex mechanisms of chronic cancer pain would be considerably hindrances of effective pain reduction.\n\nAlthough many previous studies9,10,28–32 on postoperative pain have shown that nefopam reduced morphine consumption at 24–48 hours for 30–50% of patients, our study did not confirm this finding. However, the median cumulative dose of morphine showed a lower trend in the nefopam group at all measurement points, especially in opioid naive patients receiving nefopam. Further studies focusing on nefopam in opioid naive patients with cancer that include a larger sample size, have a longer duration, or use higher doses of nefopam may be beneficial.\n\nWe found that side effects such as nausea, vomiting, sweating, dry mouth, dizziness, tachycardia, and drowsiness were comparably low in both groups. In one quantitative review, the results showed an increased risk for tachycardia (21.3%) and sweating (8.8%) in postoperative patients receiving nefopam16. However, the rate of side effects may be dissimilar in different patients and clinical settings. The low rate of adverse effects in this study might have been due to the mode of drug administration. Continuous intravenous administration avoids the peaks associated with periodic administration, as used in earlier study protocols.\n\nThere were some limitations in this study. We only investigated one dosage of intravenous nefopam (60 mg per day). This may be considered the lowest effective dose and, combined with the short duration of administration, was considered to reduce the risk for unfavorable adverse effects28. Therefore, the low dose of nefopam and the short study period may explain why our study could not show differences in pain reduction between the two groups. Additionally, the baseline opioid dose in the non-opioid naive subgroup was not controlled, which might have affected the results. In addition, we did not evaluate pain characteristics, psychological parameters, and quality of life, which may have provided helpful information. Moreover, the incidence of analgesic-related side effects might have been underestimated because evaluation of side effects was only performed after 48 hours. Therefore, the incidence of temporary side effects that developed might have been omitted.\n\n\nConclusion\n\nAt a dosage of 60 mg per day, nefopam did not provide significant pain reduction in patients with moderate to severe cancer pain. However, this study showed a trend towards reduction of opioid consumption in the nefopam group. Further studies with larger sample sizes, longer duration, or higher doses of nefopam are warranted to further investigate the efficacy of nefopam for cancer pain.\n\n\nData availability\n\nFigshare: Data of nefopam in cancer pain, https://doi.org/10.6084/m9.figshare.1217742333.\n\nFigshare: CONSORT 2010 Checklist for \"Analgesic efficacy of nefopam for cancer pain: A randomized controlled study\", https://doi.org/10.6084/m9.figshare.1224956934.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
"appendix": "Acknowledgements\n\nThe authors would like to thank all patients who willingly participated in this study. The study was not possible without them. We thank the staff of Ramathibodi Hospital’s Pain Clinic, particularly, Ms. Rojnarin Komonhirun, M.Ed., for coordination and administrative support.\n\n\nReferences\n\nWiffen PJ, Wee B, Derry S, et al.: Opioids for cancer pain - an overview of Cochrane reviews. Cochrane Database Syst Rev. 2017; 7: CD012592. PubMed Abstract | Publisher Full Text | Free Full Text\n\nvan den Beuken-van Everdingen MH, Hochstenbach LM, Joosten EA, et al.: Update on prevalence of pain in patients with cancer: systematic review and meta-analysis. J Pain Symptom Manage. 2016; 51(6): 1070–90.e9. PubMed Abstract | Publisher Full Text\n\nWorld Health Organization: WHO guidelines for the pharmacological and radiotherapeutic management of cancer pain in adults and adolescents. 2018. Reference Source\n\nOosten AW, Oldenmenger WH, Mathijssen RH, et al.: A systematic review of prospective studies reporting adverse events of commonly used opioids for cancer-related pain: a call for the use of standardized outcome measures. J Pain. 2015; 16(10): 935–46. PubMed Abstract | Publisher Full Text\n\nYoon E, Babar A, Choudhary M, et al.: Acetaminophen-induced hepatotoxicity: a comprehensive update. J Clin Transl Hepatol. 2016; 4(2): 131–42. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMercadante S: The use of anti-inflammatory drugs in cancer pain. Cancer Treat Rev. 2001; 27(1): 51–61. PubMed Abstract | Publisher Full Text\n\niNova Pharmaceuticals NZ Ltd: Acupan NZ data sheet. 2018. Reference Source\n\nGirard P, Chauvin M, Verleye M: Nefopam analgesia and its role in multimodal analgesia: a review of preclinical and clinical studies. Clin Exp Pharmacol Physiol. 2016; 43(1): 3–12. PubMed Abstract | Publisher Full Text\n\nMcLintock TT, Kenny GN, Howie JC, et al.: Assessment of the analgesic efficacy of nefopam hydrochloride after upper abdominal surgery: a study using patient controlled analgesia. Br J Surg. 1988; 75(8): 779–81. PubMed Abstract | Publisher Full Text\n\nDu Manoir B, Aubrun F, Langlois M, et al.: Randomized prospective study of the analgesic effect of nefopam after orthopaedic surgery. Br J Anaesth. 2003; 91(6): 836–41. PubMed Abstract | Publisher Full Text\n\nNa HS, Oh AY, Koo BW, et al.: Preventive analgesic efficacy of nefopam in acute and chronic pain after breast cancer surgery: a prospective, double-blind, and randomized trial. Medicine (Baltimore). 2016; 95(20): e3705. PubMed Abstract | Publisher Full Text | Free Full Text\n\nUK Medicines Information pharmacists for NHS healthcare professionals: What is the evidence to support the use of nefopam for the treatment of persistent/chronic pain?. 2018; Accessed October 21, 2019. Reference Source\n\nVan Elstraete AC, Sitbon P: Median effective dose (ED50) of paracetamol and nefopam for postoperative pain: isobolographic analysis of their antinociceptive interaction. Minerva Anestesiol. 2013; 79(3): 232–9. PubMed Abstract\n\nSon JS, Doo A, Kwon YJ, et al.: A comparison between ketorolac and nefopam as adjuvant analgesics for postoperative patient-controlled analgesia: a randomized, double-blind, prospective study. Korean J Anesthesiol. 2017; 70(6): 612–618. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChompoobai P, Chaiboon P, Taecholarn P: Analgesia after administration of one or two dose of nefopam for appendectomy: a randomized controlled trial. Thai J Anesthesiol. 2019; 45(1): 27–33. Reference Source\n\nEvans MS, Lysakowski C, Tramèr MR: Nefopam for the prevention of postoperative pain: quantitative systematic review. Br J Anaesth. 2008; 101(5): 610–7. PubMed Abstract | Publisher Full Text\n\nKim KH, Abdi S: Rediscovery of nefopam for the treatment of neuropathic pain. Korean J Pain. 2014; 27(2): 103–11. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJoo YC, Ko ES, Cho JG, et al.: Intravenous nefopam reduces postherpetic neuralgia during the titration of oral medications. Korean J Pain. 2014; 27(1): 54–62. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMinotti V, Patoia L, Roila F, et al.: Double-blind evaluation of analgesic efficacy of orally administered diclofenac, nefopam, and acetylsalicylic acid (ASA) plus codeine in chronic cancer pain. Pain. 1989; 36(2): 177–83. PubMed Abstract | Publisher Full Text\n\nMantyh PW, Clohisy DR, Koltzenburg M, et al.: Molecular mechanisms of cancer pain. Nat Rev Cancer. 2002; 2(3): 201–9. PubMed Abstract | Publisher Full Text\n\nDelaney A, Fleetwood-Walker SM, Colvin LA, et al.: Translational Medicine: Cancer Pain Mechanisms and Management. Br J Anaesth. 2008; 101(1): 87–94. PubMed Abstract | Publisher Full Text\n\nColvin L, Fallon M: Challenges in Cancer Pain Management--Bone Pain. Eur J Cancer. 2008; 44(8): 1083–90. PubMed Abstract | Publisher Full Text\n\nFalk S, Bannister K, Dickenson AH: Cancer Pain Physiology. Br J Pain. 2014; 8(4): 154–62. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRegan JM, Peng P: Neurophysiology of Cancer Pain. Cancer Control. 2000; 7(2): 111–9. PubMed Abstract | Publisher Full Text\n\nSunshine A, Laska E: Nefopam and Morphine in Man. Clin Pharmacol Ther. 1975; 18(5 Pt 1): 530–4. PubMed Abstract | Publisher Full Text\n\nTigerstedt I, Sipponen J, Tammisto T, et al.: Comparison of Nefopam and Pethidine in Postoperative Pain. Br J Anaesth. 1977; 49(11): 1133–8. PubMed Abstract | Publisher Full Text\n\nMimoz O, Incagnoli P, Josse C, et al.: Analgesic Efficacy and Safety of Nefopam vs. Propacetamol Following Hepatic Resection. Anaesthesia. 2001; 56(6): 520–5. PubMed Abstract | Publisher Full Text\n\nBeloeil H, Eurin M, Thévenin A, et al.: Effective Dose of Nefopam in 80% of Patients (ED80): A Study Using the Continual Reassessment Method. Br J Clin Pharmacol. 2007; 64(5): 686–93. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTramoni G, Viale JP, Cazals C, et al.: Morphine-sparing Effect of Nefopam by Continuous Intravenous Injection After Abdominal Surgery by Laparotomy. Eur J Anaesthesiol. 2003; 20(12): 990–2. PubMed Abstract | Publisher Full Text\n\nKapfer B, Alfonsi P, Guignard B, et al.: Nefopam and Ketamine Comparably Enhance Postoperative Analgesia. Anesth Analg. 2005; 100(1): 169–74. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTirault M, Derrode N, Clevenot D, et al.: The effect of nefopam on morphine overconsumption induced by large-dose remifentanil during propofol anesthesia for major abdominal surgery. Anesth Analg. 2006; 102(1): 110–7. PubMed Abstract | Publisher Full Text\n\nAveline C, Gautier JF, Vautier P, et al.: Postoperative analgesia and early rehabilitation after total knee replacement: a comparison of continuous low-dose intravenous ketamine versus nefopam. Eur J Pain. 2009; 13(6): 613–9. PubMed Abstract | Publisher Full Text\n\nPasutharnchat K: Data of nefopam in cancer pain. figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12177423\n\nPasutharnchat K: CONSORT 2010 Checklist for \"Analgesic efficacy of nefopam for cancer pain: A randomized controlled study\". figshare. Dataset. 2020. http://www.doi.org/10.6084/m9.figshare.12249569"
}
|
[
{
"id": "63652",
"date": "01 Jun 2020",
"name": "Kok Yuen Ho",
"expertise": [
"Reviewer Expertise Pain Management"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a placebo-controlled double-blind RCT comparing nefopam and placebo in moderate to severe cancer pain. The methodology and analysis is properly done and the results are clear. Unfortunately, the sample size is small with only 20 patients per group.\n\nAs the patients in both groups were given an IV-PCA pump to use, I am not surprised that there was no difference in the NRS pain scores for 48 hours. A better indication would be a difference in the morphine consumption - which could have been the primary outcome of this study.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "63651",
"date": "04 Jun 2020",
"name": "Abdul Hadi Mohamed",
"expertise": [
"Reviewer Expertise Pain and palliative"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 relevant to our today practice and important for the patients’ well-being and continuous health-care after suffering from cancer pain.\n\nOverall coverage and discussion are appropriate. The main limitation for this study is the number of sample and monitoring of the results which may hinder the purpose of the study.\nAdequate and covering the area of research but some area may need sources of the references. The methodology and flow of the study was clearly organized inclusive the randomization performed. This study was well constructed which included intravenous administration and back up with the patient controlled administration of morphine to hinder potential pain during the study. The table and the analysis of the result are adequate.\nThe coverage of the discussion and suggestion are adequate. I would agree that the result might be different if the samples more that 20 patients for each arm.\nHowever, if the monitoring interval be shorter for examples 4 hourly or 6 hourly then we might see the significant different results of the study with the similar sample.\nEven-though Nefopam have multiple mode of action but how much they covered nociceptive and neuropathic pathway were not covered in this study and discussion.\nCancer pain can be prominent either nociceptive or neuropathic pain and sometimes both which causing dilemma for palliative to manages.\nIt is good if in this study or discussion to highlight more on the aspect of nefopam coverage either on nociceptive or neuropathic or both to certain degree.\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/9-378
|
https://f1000research.com/articles/9-207/v1
|
24 Mar 20
|
{
"type": "Research Article",
"title": "Visualizing highly cited scientific output of Indian physiotherapists: A bibliometric study",
"authors": [
"Arun Vijay Subbarayalu",
"Manuelraj Peter",
"Mohamed Idhris",
"Sivasankar Prabaharan",
"Muhil Sakthivel",
"Vinoth Raman",
"Palanivel R.M.",
"Ola Ibrahim Ramzi",
"Manuelraj Peter",
"Mohamed Idhris",
"Sivasankar Prabaharan",
"Muhil Sakthivel",
"Vinoth Raman",
"Palanivel R.M.",
"Ola Ibrahim Ramzi"
],
"abstract": "Background: Physiotherapy research supports the advancement of evidence-based practice and development of a highly skilled workforce. This study aims to visualize highly cited scientific output of Indian physiotherapists from 1999 to 2018. Methods: A descriptive study design was adopted to visualize highly cited scientific output of Indian physiotherapists using the Web of Science (WoS) database from 1999 to 2018. A search was carried out using the following term \"((TS=(physiotherapy) OR TS=(\"physical rehabilitation\") OR TS=(\"physical therapy\")) AND AD=(India))Indexes=SCI-EXPANDED, SSCI, A&HCI, CPCI-S, CPCI-SSH, ESCI, CCR-EXPANDED, IC Timespan=1999-2018”. Data collected were analyzed using Incites from WoS and VOSviewer software. Results: A total of 489 articles were published between 1999 and 2018, with a peak of 103 in 2016 with 2420 citations. A decline in publication count was observed after 2016. The journal International Journal of Physiotherapy published the highest number of articles (n=35). Manipal University (n=26) was found to be the most active institution for physiotherapy research in India, as determined by producing the most articles. Indian physiotherapists published the highest number of research articles in collaboration with US authors (n=24). Conclusion: There is an increasing trend in the scientific output of Indian physiotherapists over the past two decades; however, a decline is observed after 2016. It is recommended that research collaborations across the globe are increased and scientific output should be improved, leading to a higher number of citations. Future research should explore factors influencing scientific production of Indian physiotherapists and devise appropriate strategies to attain further improvement.",
"keywords": [
"Bibliometric study",
"India",
"Physiotherapy",
"Scientific output"
],
"content": "Introduction\n\nScientific output of a profession is recognized by the frequency of publications, which are published in peer-reviewed journals and indexed in bibliographic databases1–3. In physiotherapy, this scientific output is utilized to enhance existing knowledge and develop guidelines for highly effective clinical practice4. Accordingly, the analysis of scientific output allows definition of baseline indicators in knowledge and clinical practice in physiotherapy5,6. Various studies investigated the scientific output of physiotherapists across the globe6–14. Among these studies, several utilized electronic searches6,7,9–11,13,14, whereas others were limited to document reviews8,12. Concerning the Indian context, only two studies have been performed to reveal the research productivity of Indian physiotherapists from 2000 to 2014, which were limited to the Medline database10,11. Moreover, Li et al. (2018) recently stated that Clarivate Analytics’s Web of Science (WoS) is the World’s foremost scientific citation search and analytical platform, which can be used as both a research tool and dataset15. Hence, there is a need for further research that should involve the WoS database to detect high-quality research publications by Indian physiotherapists up until 2018 (this study took place in 2019). Therefore, this study intended to conduct a bibliometric study on the scientific output of Indian physiotherapists using WoS during the last two decades (from 1999 to 2018).\n\n\nMethods\n\nA descriptive study design was adopted to reveal the scientific output of Indian physiotherapists using an electronic literature search in the WoS database during the period from 1999 to 2018.\n\nThe search was conducted on 14th October 2019. The term 'Indian physiotherapists' denotes physiotherapy professionals employed in any of academic or clinical establishments in India in the study period. The search was carried out in WoS using the following term \"((TS=(physiotherapy) OR TS=(\"physical rehabilitation\") OR TS=(\"physical therapy\")) ANDAD=(India))Indexes=SCI-EXPANDED, SSCI, A&HCI, CPCI-S, CPCI-SSH, ESCI, CCR-EXPANDED, IC Timespan=1999–2018”. The search started from 1999 since this study aimed to retrieve data from the past two decades.\n\nThe search methodology is described in Figure 1. Based on the inclusion criteria, 489 publications were included and proceeded for further analysis.\n\nRetrieved articles were analysed using Incites in WoS and VOSviewer 1.6.11. Incites was used to gather information publication year, authorship ranking, source journal productivity, collaborating institutions, country-wise research collaboration, and citations. In addition, the information related to h-index was obtained from the Incites in WoS. The data, which is exported from the WoS database as an ISI common export (.ciw) format, were imported into VOSviewer to explore the co-occurrences of keywords used by the authors in their articles. The flowchart describing the procedures for carrying out both Incites and VOSviewer analysis is depicted in Figure 2.\n\nSince Incites in WoS used in this study are proprietary software, the researchers could alternatively use a tab-delimited file downloaded from WoS and use it in VOSviewer.\n\n\nResults\n\nA total of 489 articles were included in the study; 381 research articles, 53 reviews, 34 proceedings, 9 meeting abstracts, 8 letters, 3 editorial material, and one correction.\n\nBetween 1999 and 2008, scientific output of Indian physiotherapists is minimal, reaching a peak of 9 in 2008. Subsequently, the publication count starts to rise gradually from 2009 to 2016, which has a peak of 103 articles. Publications decreased after 2016 and rose slightly in 2018, but not to levels seen in 2016 (Table 1 and Figure 3). Regarding citation count, there was a gradual rise over the research period, with a total of 2420 citations between 1999 and 2018, more than 100 of which have been documented since 2012. The highest average citation (citations/article) of ≥10 was observed only in 2006 (mean, 11.00) and 2014 (mean, 10.20).\n\nData obtained from Web of Science. Includes all articles types. N articles = 489.\n\nData obtained from Web of Science. Includes all articles types.\n\nA total of 264 journals had published the 489 retrieved articles. The top 20 journals in which Indian physiotherapists published over the study period are displayed in Table 2. The top 20 journals published 174 articles, 35.58% of total publications (N=489) in the research period. Out of the top 20 ranked journals, 11 were journals based in India, 26.79% of the total publications.\n\nData obtained from Web of Science. Includes all articles types. N articles = 489.\n\nThe International Journal of Physiotherapy published 35 articles, with six citations for these 35 articles, an average citation of 0.17. It was the most active journal found in this study and contributed to 7.16% of total publications. In contrast, the journal Haemophilia published seven articles with 68 citations for these articles, an average citation of 9.71. Similarly, Annals of Indian Academy of Neurology published four articles, with citations of 43, giving it the highest average citation of 10.75 (Table 2).\n\nTable 3 shows the top 20 authors who worked with Indian physiotherapists to publish physiotherapy articles. These authors contributed 22.09% of total publications (N=489) in collaboration with Indian physiotherapists. Kumar S, Mahadevappa M, and Samuel AJ collectively have accounted for 5.73% of total publications (N=489). An Indian author named Kumar R (ICMR-National Institute of Occupational Health) is the Indian physiotherapist with the highest h-index (18) and had published five articles, which were cited 1372 times.\n\nData obtained from Web of Science. Includes all articles types. N articles = 489.\n\nThe top 20 institutions that collaborated and published articles with Indian physiotherapists for physiotherapy research are displayed in Table 4. Among these institutions, Manipal University (India) has the highest number of publications, with 7.36% of total publications, followed by Christian Medical College Hospital (India; 3.89%), the Indian Institute of Technology (India; 3.68%) and King Saud University (KSU; Saudi Arabia; 3.68%). In total, 90% of collaborating institutions were based in India. Internationally, KSU and the University of London (UK; 1.23%) had the most active cooperation with Indian physiotherapists over this time period.\n\nData obtained from Web of Science. Includes all articles types. N articles = 489.\n\nOut of the total publications (N=489), articles published by Indian physiotherapists in collaboration with authors belonging to international countries was as follows: United States (4.91%), Saudi Arabia (4.50%), UK (3.68%), Canada (1.84%), and Sweden (1.02%). Australia, Brazil, Italy, Malaysia, Mexico, and Pakistan contributed 0.82% each to total publications (Table 5). Out of the top 20 countries, Indian physiotherapists collaborated the most with the US (after India), publishing 24 articles, which secured 370 citations (average citation 15.42). Notably, articles published by Indian physiotherapists in collaboration with German authors had the highest number of average citation (41.00), though only three articles were published.\n\nData obtained from Web of Science. Includes all articles types. N articles = 489.\n\nUsing VOSviewer, the authors created a keyword table and graphic representation of the co-occurrence of keywords across articles. The top 20 keywords used in articles (from a total of 2477 keywords) are shown in Figure 4. An article's keyword may represent its primary material, and to some degree, the frequency of occurrence. Likewise, co-occurrence can indicate centered themes of research in a field. Among the top 20 keywords, those with a considerable rate of occurrence are “Physiotherapy” (n=72; 2.91%), “Rehabilitation” (n=48; 1.94%), “Management” (n=46; 1.86%), and “Exercise and Therapy” (n=25; 1.01%). The keyword “Randomized Controlled-Trial” had the highest average citations (16.50%) among the top 20 keywords.\n\nGraphic created using VOSviewer.\n\n\nDiscussion\n\nPreviously, using data from Scopus, it had been observed that Italian physiotherapists published 1083 articles with 13,373 citations. The number of publications by these authors had gradually increased over the years from before 1995 (10 articles) to 2016 (143 articles). More than 50% of the total publications were produced between the years 2012 and 201613. In India, Hariohm et al. observed a considerable increase in the research output of Indian physiotherapists, using the MEDLINE database, between 2000 and 2014, with a total of 182 articles11. This study observed that Indian physiotherapists had published 489 articles in WoS from 1999 to 2018, with a peak of 103 articles in 2016. However, there was a drastic drop in publication count from 103 in 2016 to 74 in 2018. Remarkably, the number of articles from 2016 to 2018 accounted for 50% (n=252) of total publications (N=489). From these results, it is inferred that Indian physiotherapists are increasingly aware of publishing more articles in high-quality journals in recent years and have enhanced their research competencies gradually to raise their scientific output. Nevertheless, a drastic drop in their publication count after 2016 indicates that there is a need for further research to reveal individual and institutional factors causing this decline and create appropriate strategies to improve the scientific output of Indian physiotherapists.\n\nA total of 489 articles published by Indian physiotherapists from 1999 to 2018 secured a total of 2420 citations. Citations in each year gradually increased and reached 507 by 2018, and this rise was observed with a substantial count from 2012 to 2018. Littman et al. analyzed the research output of 45 physical therapy faculty in southeastern US from 2000 to 2016 using their curriculum vitae. The range of publications and the citations of these faculty was observed as 0 to 43, and 0 to 943, respectively12. Another study by Sturmer et al. found that 222 articles were published by Brazilian physical therapy researchers in WoS in 2010, which had a total of 1805 citations6. Contrary to these findings, this study reported that Indian physiotherapists published only 65 articles with 171 citations up to the year 2010 in WoS. Even though the articles published by Indian physiotherapists were suitable enough for several researchers to cite them often, there is a need to improve the citation count of their publications in the future.\n\nOut of 264 journals, the top 20 journals that Indian physiotherapists published in contributed to 35.58% of total publications (N=489) in the WoS database. Notably, it is observed that Indian-based journals published 26.79% of the total publications; no publications were observed in US-based journals. Further, the highest count of publications was observed in an Indian-based journal International Journal of Physiotherapy. This affinity of Indian physiotherapy researchers towards Indian-based journals might be due to the nature of their research articles, or interest in country-based journals. However, those researchers should expand their contribution to other high-quality international journals. Exploring the reasons behind Indian physiotherapists’ choice to publish in these Indian journals is beyond the scope of this study, and further research is warranted to address this critical issue. Besides, a high number of articles published by Indian physiotherapists in International Journal of Physiotherapy had only a small number of citations, giving a low average citation. Nevertheless, other journals, such as Haemophilia and Annals of Indian Academy of Neurology, showed considerable citations and a high average citation for only a few articles published in these journals. This implies that these articles must possess information that is cited often by other researchers.\n\nA previous study by Man et al. found that four Hong Kong physiotherapy professors had a median h-index of 30.5 and their average total number of citations was 2930.314. Moreover, Brazilian physical therapy researchers had a median h-index of 3, according to WoS6. Recently, Vercelli et al. reported that the mean h-index of 363 Italian physiotherapists was 2.2, which ranged from 0 to 16; mean citations per author were observed as 5813. This study reported that the top 20 authors who worked with Indian physiotherapists contributed to 22.09% of total publications (N=489). Kumar S (India) was the leading author who collaborated with other Indian physiotherapists in physiotherapy research with 12 publications. Another Indian author, Kumar R, had the highest h-index of 18.\n\nAmong the top 20 collaborating institutions, Manipal University in India was the leading institution that had the most collaborations with Indian physiotherapists, contributing to 7.36% of total publications (N=489). In line with our findings, Hariohm et al. revealed that Manipal University is an active research institution with 59 articles in the MEDLINE database from 2000 to 201411. Of the top 20 institutions, 90% were based in India, whereas only two institutions were based in the UK and Saudi Arabia. This implies that Indian physiotherapists had more collaborations with institutions in their own country. However, there is a need for Indian physiotherapists to collaborate with international institutions to improve their scientific output.\n\nThe international country with the highest number of articles published with Indian physiotherapists (contributing to 4.91% of total publications in this research period) was the US, followed by Saudi Arabia (4.50%). The total percentage of publications with other countries is minimal. Articles published in collaboration with the US had a high citation count, and Germany had the highest average citation. Hence, this study recommends that Indian physiotherapists should enhance their research collaboration with other countries since collaborative research allows the development of networks with early-career researchers in other countries16–18, and improves the quality of their scientific output19. Furthermore, earlier studies have also stressed the importance of international research collaboration in health care, and it is frequently regarded as an indicator of quality to develop and disseminate scientific knowledge to newly developing countries20,21.\n\nLastly, this study revealed the top 20 keywords that occurred in various articles using VOSviewer software. The term “Physiotherapy” had the highest occurrence rate (2.91%). \"Randomized controlled trial\" was associated with the highest average citation (16.50).\n\n\nConclusion\n\nThis study observed that the scientific output of Indian physiotherapists shows an uptrend in performance since 1999, excluding 2017/18 where a drastic decline was noticed. Indian physiotherapists are mostly publishing and collaborating with Indian-based journals and institutions, respectively. Even though there are high-quality publications, there is a need to enhance both the quality and quantity of scientific papers, to increase the high number of citations and average citation. This study also recommends that Indian physiotherapists should expand their research collaboration internationally to improve their scientific output. Future studies can focus on analyzing individual and institutional factors influencing research productivity of Indian physiotherapists and develop suitable strategies to enhance their scientific production.\n\n\nData availability\n\nOpen Science Framework: Visualization pattern of the highly cited scientific output of Indian Physiotherapists: A bibliometric study, https://doi.org/10.17605/OSF.IO/YC6VK22\n\nThis project contains the following underlying data:\n\n- Article level and citation data for all 489 articles retrieved.\n\n- Journal, author, institution and country data for all 489 articles retrieved.\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\nIlott I, Bury T: Research capacity. Physiotherapy. 2002; 88(4): 194–200. Publisher Full Text\n\nIlott I: Challenges and strategic solutions for a research emergent profession. Am J Occup Ther. 2004; 58(3): 347–352. PubMed Abstract | Publisher Full Text\n\nCoury HJCG, Vilella I: Profile of the Brazilian physical therapy researcher. Rev Bras Fisioter. 2009; 13(4): 356–363. Publisher Full Text\n\nRichter RR, Schlomer SL, Krieger MM, et al.: Journal publication productivity in academic physical therapy programs in the United States and Puerto Rico from 1988 to 2002. Phys Ther. 2008; 88(3): 376–386. PubMed Abstract | Publisher Full Text\n\nMugnaini R, Packer AL, Meneghini R: Comparison of scientists of the Brazilian Academy of Sciences and of the National Academy of Sciences of the USA on the basis of the h-index. Braz J Med Biol Res. 2008; 41(4): 258–262. PubMed Abstract | Publisher Full Text\n\nSturmer G, Viero CC, Silveira MN, et al.: Profile and scientific output analysis of physical therapy researchers with research productivity fellowship from the Brazilian National Council for Scientific and Technological Development. Braz J Phys Ther. 2013; 17(1): 41–48. PubMed Abstract | Publisher Full Text\n\nRobertson VJ: Research and the cumulation of knowledge in Physical Therapy. Phys Ther. 1995; 75(3): 223–332. PubMed Abstract | Publisher Full Text\n\nFrantz JM, Rhoda A, Struthers P, et al.: Research productivity of academics in a physiotherapy department: a case study. Afr J Health Prof Educ. 2010; 2(2): 17–20. Reference Source\n\nCoronado RA, Riddle DL, Wurtzel WA, et al.: Bibliometric analysis of articles published from 1980 to 2009 in Physical Therapy, journal of the American Physical Therapy Association. Phys Ther. 2011; 91(5): 642–655. PubMed Abstract | Publisher Full Text\n\nHariohm K, Prakash V, Saravankumar J: Quantity and quality of randomized controlled trials published by Indian physiotherapists. Perspect Clin Res. 2015; 6(2): 91–97. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHariohm K, Prakash V, Saravankumar J: Research productivity of Indian physiotherapists: A review of MEDLINE. Curr Sci. 2016; 110(12): 2226–2230. Publisher Full Text\n\nLittman MA, Sonne JW, Smith GV: Research productivity of doctor of physical therapy faculty promoted in the southeastern United States. Med Educ Online. 2017; 22(1): 1368849. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVercelli S, Ravizzotti E, Paci M: Are they publishing? A descriptive cross-sectional profile and bibliometric analysis of the journal publication productivity of Italian physiotherapists. Arch Physiother. 2018; 8: 1. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMan DWK, Tsang WSF, Lu EY, et al.: Bibliometric study of research productivity in occupational therapy and physical therapy/physiotherapy in four Western countries and five Asian countries/regions. Aust Occup Ther J. 2019; 66(6): 690–699. PubMed Abstract | Publisher Full Text\n\nLi K, Rollins J, Yan E: Web of Science use in published research and review papers 1997–2017: A selective, dynamic, cross-domain, content-based analysis. Scientometrics. 2018; 115: 1–20. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCamargo AA, Simpson AJ: Collaborative research networks work. J Clin Invest. 2003; 112(4): 468–471. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCarroll JK, Albada A, Farahani M, et al.: Enhancing International Collaboration Among Early Career Researchers. Patient Educ Couns. 2010; 80(3): 417–420. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZutshi A, McDonald G, Kalejs L: Challenges in collaborative writing: Addressing authorship attribution. Eur Bus Rev. 2012; 24(1): 28–46. Publisher Full Text\n\nDakik HA, Kaidbey H, Sabra R: Research productivity of the medical faculty at the American University of Beirut. Postgrad Med J. 2006; 82(969): 462–464. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKim KW: Measuring international research collaboration of peripheral countries: taking the context into consideration. Scientometrics. 2006; 66(2006): 231–240. Publisher Full Text\n\nFreshwater D, Sherwood G, Drury V: International research collaboration: Issues, benefits and challenges of the global network. J Res Nurs. 2007; 11(4): 295–303. Publisher Full Text\n\nSubbarayalu AV, Peter M, Idhris M, et al.: Visualization pattern of the highly cited scientific output of Indian Physiotherapists: A bibliometric study. 2020. http://www.doi.org/10.17605/OSF.IO/YC6VK"
}
|
[
{
"id": "61694",
"date": "06 Apr 2020",
"name": "Gopal Nambi",
"expertise": [
"Reviewer Expertise Rehabilitation",
"Sports injuries",
"Physical Therapy"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for the chance to review this paper titled “Visualizing highly cited scientific output of Indian physiotherapists: A bibliometric study”. The major strength of this paper is that they have attempted to undertake a study on this title. The title is interesting. The design and outcome measures are appropriate to answer the question and I’m sure a lot of hard work went into conducting this study.\nAbstract:\nThe search is limited with one database (WOS), what about the other databases?\n\nIntroduction:\nThe introduction part is superficial.\n\nWhy is the search duration limited to 1999 – 2018?\n\nMethods:\nHow many reviews were collected in the articles and what are their kappa score?\n\nIndian physiotherapist means, who works only in India or Indian physiotherapists works throughout the world?\n\nSelection criteria should be more specific.\n\nDiscussion:\nThe strength and the limitations of this study are not mentioned.\n\nConclusion:\n\nRephrase the conclusion and make it more precise and clear.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "5494",
"date": "19 May 2020",
"name": "Arun Vijay Subbarayalu",
"role": "Author Response",
"response": "REVIEWER 1 Abstract: The search is limited with one database (WOS), what about the other databases? As this study only focused on revealing the scientific output of Indian physiotherapists in the WoS database, the authors did not include other databases. However, this statement has been written as a limitation, and a recommendation is suggested. The description is provided under the heading 'Limitations and Recommendations' as follows. Limitations and Recommendations The findings of this study are only limited to the WoS database. Future research can focus on studying the research output of the Indian physiotherapists in other databases to ascertain their research productivity. Future studies can focus on analyzing individual and institutional factors influencing the research productivity of Indian physiotherapists and develop suitable strategies to enhance their scientific production. Introduction: The introduction part is superficial. Since it is a medium article, the introduction part is briefly written to cover the scientific output of physiotherapists and physiotherapy research in the Indian context. Why is the search duration limited to 1999 – 2018? In the Indian context, Hariohm et al. have studied the research productivity of the Indian physiotherapists in the Medline database from 2000 to 2014. Hence, this study aimed to reveal the scientific output of Indian physiotherapists in the WoS database for the past two decades, i.e., 1999 to 2018. This study was conducted in the year 2019. Methods: How many reviews were collected in the articles, and what are their kappa score? This study found 53 reviews by Indian physiotherapists during the period between 1999 and 2018. As this study only focused on revealing the scientific output of Indian physiotherapists in the WoS database, the authors did not explain the Kappa score for analysis and discussion. Indian Physiotherapist means, who works only in India or Indian physiotherapists works throughout the world? We mean those Indian physiotherapists who work in any of the academic or clinical establishments located in India (refer to article selection paragraph in the manuscript). It is explained Under \"Article Selection\" of the Methods section The search was conducted on Oct 14, 2019. The term 'Indian physiotherapists' denotes physiotherapy professionals employed in any of the academic or clinical establishments in India in the study period. The search was carried out in WoS using the following keywords \"((TS=(physiotherapy) OR TS=(\"physical rehabilitation\") OR TS=(\"physical therapy\")) AND AD=(India))Indexes=SCI-EXPANDED, SSCI, A&HCI, CPCI-S, CPCI-SSH, ESCI, CCR-EXPANDED, IC Timespan=1999–2018\". The search started from 1999 since this study aimed to retrieve data from the past two decades. Selection criteria should be more specific. We have included four selection criteria (i) Only those publications made by Indian Physiotherapist who works in India (subject understudy); (ii) Publications made between the year 1999 to 2018 (Timespan) (iii) database explored and the exact time where this search was carried out and (iv) key words used to retrieve the articles. (refer to article selection paragraph & figure 1) Discussion: The strength and limitations of this study are not mentioned. The strength of this study is observed as the uptrend in the scientific output of Indian physiotherapists since 1999. It is mentioned in the conclusion section. Further, the limitations and recommendations of this study are mentioned under the heading \"Limitations and Recommendations\". The following description is added Under 'Conclusion section.' This study observed that the scientific output of Indian physiotherapists shows an uptrend in performance since 1999, excluding 2017 and 2018, where a considerable decline was noticed. The results showed that Indian physiotherapists had mostly published in Indian-based journals, and collaborated with Indian institutions. Even though there are high-quality publications, there is a need to enhance both the quality and quantity of scientific papers to increase the high number of citations and average citations. This study also recommends that Indian physiotherapists should expand their research collaboration internationally to improve their scientific output. The following description is added Under 'Limitations and Recommendations' section The findings of this study are only limited to the WoS database. Future research can focus on studying the research output of the Indian physiotherapists in other databases to ascertain their research productivity. Future studies can focus on analyzing individual and institutional factors influencing the research productivity of Indian physiotherapists and develop suitable strategies to enhance their scientific production. Conclusion: Rephrase the Conclusion and make it more precise and clear. The Conclusion is revised to improve the clarity of the readers as follows: Under Conclusion This study observed that the scientific output of Indian physiotherapists shows an uptrend in performance since 1999, excluding 2017 and 2018, where a considerable decline was noticed. The results showed that Indian physiotherapists had mostly published in Indian-based journals, and collaborated with Indian institutions. Even though there are high-quality publications, there is a need to enhance both the quality and quantity of scientific papers to increase the high number of citations and average citations. This study also recommends that Indian physiotherapists should expand their research collaboration internationally to improve their scientific output."
}
]
},
{
"id": "61693",
"date": "07 Apr 2020",
"name": "Aamir Raoof Memon",
"expertise": [
"Reviewer Expertise Physical activity",
"rehabilitation",
"exercise",
"scientometrics",
"bibliometric analyses"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI enjoyed reading the bibliometric analysis of scientific output of physiotherapists from India. The manuscript is interesting but requires improvement in its content. My suggestions are given below:\nAbstract:\nAuthor 1: “….determined by producing the most articles” should be revised to “determined by publishing the most articles”\n\nIntroduction:\nAuthor 2: “…….Indian physiotherapists up until 2018 (this study took place in 2019)” please delete “(this study took place in 2019)”\n\nAuthor 3: “…..this study intended to conduct a bibliometric….” should be revised to “this study was planned to conduct a bibliometric”\n\nMethods:\nAuthor 4: “…..the following term” should be revised to “……the following keywords” or “……the following query”.\n\nAuthor 5: Instead of “physical rehabilitation”, authors could have used “rehabilitation”. Also, authors could use the term “exercise therapy” or “manual therapy” or “manipulative therapy” because not all papers use the terms physical therapy or physiotherapy. Please add space between “AND” and “AD”.\n\nAuthor 6: Please explain the Web of Science indexes (i.e. SCI-EXPANDED, SSCI, A&HCI, CPCI-S, CPCI-SSH, ESCI, CCR-EXPANDED, IC) in full-form.\n\nAuthor 7: I think there is a typo here “……to gather information….” and should be revised to “…..to gather information on…”.\n\nAuthor 8: Inclusion criterion 3 (Fig 1) should be “publications with at least one physiotherapist with institutional/author affiliation from India listed as a co-author”. In addition (related to criterion 1), did the authors include peer-reviewed journal publications or meeting abstracts and other document types?\n\nAuthor 9: What is the significance of adding “Correction”? The common practice is that this category of papers is EXCLUDED from the analysis.\n\nAuthor 10: “……the information related to h-index was obtained from the Incites in WoS”: please explain h-index and its use here.\n\nAuthor 11: Please explain the VOS parameters used and cite their original source.\n\nAuthor 12: “…..used in this study are proprietary…” should be “…..used in this study is a proprietary….”\n\nAuthor 13: Statistical analysis should present the bibliometric parameters in detail.\n\nResults:\nAuthor 14: “Between 1999 and 2008….” this paragraph should be in the past tense, please revise.\n\nAuthor 15: “….published 174 articles, 35.58%” should be revised to “published 174 articles i.e. 35.58%”.\n\nAuthor 16: “…11 were journals based” should be revised to “…..11 journals were based”.\n\nAuthor 17: “average citation of 0.17” should be revised to “average citation per paper as…”.\n\nAuthor 18: Fig 2: Please check the heading for Flowchart for VOSviewer analysis – I suspect some errors there.\n\nAuthor 19: Table 1 (please cross-check with the textual description): I see publications depicting a fluctuating pattern - the only rise is seen after 2010 until 2016. Why don't the authors compare the growth according to the strata of 5 years?\n\nAuthor 20: Fig 3: It would be good to present the citation trend along with the number of publications - please see these papers. For example, see these papers: https://www.ncbi.nlm.nih.gov/pubmed/31766944; https://doi.org/10.3390/clockssleep2020010.1,2\n\nAuthor 21: Table 2-5: I think it would be appropriate to list the top 10 instead of 20 – again, this is a common practice in bibliometric studies. I would also want to see top-10 highly cited papers.\n\nAuthor 22: Table 2: It would be interesting to discuss the impact factor and SJR ranking (https://www.scimagojr.com/journalrank.php) of these journals. This will reflect the quality and visibility of these journals. I see that most of these journals are indexed in the ESCI - the lowest level of WoS indexes.\n\nAuthor 23: “International Journal of Scientific Study”: I could not find this journal in the Master List search (https://mjl.clarivate.com/search-results). I am not really sure about the authenticity of this journal – it looks rather like a questionable journal to me, so I am not sure how its content came up in the results. Please double-check.\n\nAuthor 24: “The top 20 institutions ……. displayed in Table 4” – this does not look grammatically correct and needs revision. These could be organized in two tables as national (within India) and international (outside India) collaborations.\n\nAuthor 25: “An article’s keyword may represent its primary…..” needs citation.\n\nAuthor 26: It would be better to exclude “Physiotherapy” and “Rehabilitation” from the map because they will certainly be the most used. The readers of this paper would rather be interested to see what other terms had frequent occurrence. The authors should also discuss the map (Fig 4) to reflect upon which keywords tend to occur together. For instance, keywords in green (i.e. management, trial, children, diagnosis, surgery, India) tend to belong to the same cluster and are likely to co-occur together. What was the minimum value set for the co-occurrence of a keyword? For example, see this paper: https://www.ncbi.nlm.nih.gov/pubmed/31766944.1\n\nAuthor 27: Table 3: the column for h-index should come to the right of total citations. Iqbal ZA does not have Indian affiliation, so these names don’t correspond well with the caption of the table. I believe these could be organized in two tables as national (within India) and international (outside India) collaborations. If this is not possible then the authors are advised to revise the caption of the table to bring more clarity in it.\n\nAuthor 28: Table 4: Please revisit the caption for clarity. Authors should add a column “collaboration” and categorize it as national and international.\n\nAuthor 29: Table 5: Six institutions with 4 papers are presented but I don't understand how they are ranked (according to the number of citations or citations/paper). As of now, it appears as if they are listed alphabetically. The common method in bibliometric studies is to rank them based on the number of citations.\n\nDiscussion:\nAuthor 30: Overall, this section is just the repetition of the results and fails to integrate what is known about the topic and what was found.\n\nAuthor 31: Concerning these two points: “Exploring the reasons…..critical issue” and “This implies that……other researchers”. These are vague arguments – I believe that this would be due to several factors, including the quality of the journal (indexing, metrics), ease of publication (acceptance rate), focus (audience/readership), APCs and reputation to the scientific community. I think authors have failed to give a balanced view here. Most of these journals appear to be of low-quality. Just a point for your understanding is that the IJPT was once involved in deceptive practices (please see this paper: https://www.ncbi.nlm.nih.gov/pubmed/27924967)3, but it has probably improved its practices and has gotten into ESCI.\n\nAuthor 32: Collaborating authors: It would be appropriate to discuss the average h-index (and its range) and average citations to the papers by Indian authors.\n\nAuthor 33: I still have confusion regarding the organization and presentation of findings of this paper in the results and discussion sections. Collaborating authors, institutions, and countries should have been leading authors, institutions and countries. Collaborations between countries, institutions, and authors should better be presented through VOSviewer as done in these papers: https://www.ncbi.nlm.nih.gov/pubmed/31766944; https://doi.org/10.3390/clockssleep2020010.1,2\n\nAuthor 34: What is the clinical implication of these keywords? Please discuss.\n\nAuthor 35: Please add a section about the limitations of this study.\n\nConclusions:\nAuthor 36: The decrease in papers during 2017 and 2018 was not “drastic”, please rephrase this.\n\nAuthor 37: “…….though there are high-quality publications…” this stands as a mere claim and won’t be meaningful without presenting the top-10 highly cited publications.\n\nLanguage & grammar:\n\nAuthor 38: I noticed grammatical errors at several places, please revise accordingly.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": [
{
"c_id": "5495",
"date": "19 May 2020",
"name": "Arun Vijay Subbarayalu",
"role": "Author Response",
"response": "REVIEWER 2 Abstract: Author 1: \"….determined by producing the most articles\" should be revised to \"determined by publishing the most articles\" The correction has been incorporated in the manuscript (Refer Results section of the Abstract).Under the Results section of the Abstract, it is written as follows: Manipal University (n=26) was found to be the most active institution for physiotherapy research in India, as determined by publishing the most articles. Introduction: Author 2: \"…….Indian physiotherapists up until 2018 (this study took place in 2019)\" please delete \"(this study took place in 2019)\" The term \"this study took place in 2019\" is deleted in the Introduction of the manuscript.Under Introduction, it is written as follows: Hence, there is a need for further research that should involve the WoS database to detect high-quality research publications by Indian physiotherapists until 2018. Author 3: \"…..this study intended to conduct a bibliometric….\" should be revised to \"this study was planned to conduct a bibliometric\" The correction has been incorporated in the text of the manuscript. Under Introduction, it is updated as follows: Therefore, this study was planned to conduct a bibliometric study on the scientific output of Indian physiotherapists using WoS during the last two decades (from 1999 to 2018). Methods: Author 4: \"…..the following term\" should be revised to \"……the following keywords\" or \"……the following query\". The correction has been incorporated in the Abstract and Methods sections of the manuscript.Under Methods section of the Abstract, following updates has been carried out: A search was carried out using the following keywords \"((TS=(physiotherapy) OR TS=(\"physical rehabilitation\") OR TS=(\"physical therapy\")) AND AD=(India))Indexes=SCI-EXPANDED, SSCI, A&HCI, CPCI-S, CPCI-SSH, ESCI, CCR-EXPANDED, IC Timespan=1999-2018\". Under \"Article Selection\" of the Methods section The search was conducted on Oct 14 2019. The term 'Indian physiotherapists' denotes physiotherapy professionals employed in any of the academic or clinical establishments in India in the study period. The search was carried out in WoS using the following keywords \"((TS=(physiotherapy) OR TS=(\"physical rehabilitation\") OR TS=(\"physical therapy\")) AND AD=(India))Indexes=SCI-EXPANDED, SSCI, A&HCI, CPCI-S, CPCI-SSH, ESCI, CCR-EXPANDED, IC Timespan=1999–2018\". The search started from 1999 since this study aimed to retrieve data from the past two decades. Author 5: Instead of \"physical rehabilitation\", authors could have used \"rehabilitation\". Also, authors could use the term \"exercise therapy\" or \"manual therapy\" or \"manipulative therapy\" because not all papers use the terms physical therapy or physiotherapy. Please add space between \"AND\" and \"AD\". The search conducted based on TS=\" physiotherapy\" TS means Topic We have initially tried our search using all these alternative keywords, i.e., 'rehabilitation', which brought lots of irrelevant content that was not related to physiotherapy rather related to other medical rehabilitation, after that we have excluded the term \"rehabilitation\" from our search term. Likewise, while using the keyword \"manual therapy\" or \"manipulative therapy,\" as of today, we got 510 articles with very meager differences, and few of them irrelevant to physio records. Even we noticed a few extra articles were also added in WoS using our same previous search. Next, the space between \"AND\" and \"AD\" is added in the Abstract and Methods sections of the manuscript. Under Methods section of the Abstract, we made the following update: A search was carried out using the following keywords \"((TS=(physiotherapy) OR TS=(\"physical rehabilitation\") OR TS=(\"physical therapy\")) AND AD=(India))Indexes=SCI-EXPANDED, SSCI, A&HCI, CPCI-S, CPCI-SSH, ESCI, CCR-EXPANDED, IC Timespan=1999-2018\". Under \"Article Selection\" of the Methods section The search was conducted on Oct 14 2019. The term 'Indian physiotherapists' denotes physiotherapy professionals employed in any of the academic or clinical establishments in India in the study period. The search was carried out in WoS using the following keywords \"((TS=(physiotherapy) OR TS=(\"physical rehabilitation\") OR TS=(\"physical therapy\")) AND AD=(India))Indexes=SCI-EXPANDED, SSCI, A&HCI, CPCI-S, CPCI-SSH, ESCI, CCR-EXPANDED, IC Timespan=1999–2018\". The search started from 1999 since this study aimed to retrieve data from the past two decades. Author 6: Please explain the Web of Science indexes (i.e., SCI-EXPANDED, SSCI, A&HCI, CPCI-S, CPCI-SSH, ESCI, CCR-EXPANDED, IC) in full-form. (SCI-EXPANDED)-Science Citation Index Expanded(SSCI)-Social Sciences Citation Index(A&HCI)-Arts & Humanities Citation Index(CPCI-S)-Conference Proceedings Citation Index - Science(CPCI-SSH)-Conference Proceedings Citation Index - Social Sciences & Humanities(ESCI)-Emerging Sources Citation Index(CCR Expanded)-Current Chemical Reactions(IC)-Index ChemicusAs suggested, we have added these full forms in figure 1, instead of Article Selection paragraph under the Methods section (This is done as it is a medium-sized article where we need to fit into the word count provided by the Journal). Author 7: I think there is a typo here \"……to gather information….\" and should be revised to \"…..to gather information on…\". The correction has been incorporated in the text of the manuscript.Under Data Analysis of the Methods section Incites was used to gather information on publication year, authorship ranking, source journal productivity, collaborating institutions, country-wise research collaboration, citations, and collaboration pattern of articles. Also, the information related to h-index was obtained from the Incites in WoS. Author 8: Inclusion criterion 3 (Fig 1) should be \"publications with at least one physiotherapist with institutional/author affiliation from India listed as a co-author\". In addition (related to criterion 1), did the authors include peer-reviewed journal publications or meeting abstracts and other document types? The authors have updated the correction in Figure 1. With regard to criterion 1, we have included all types of documents, not restricted to peer-reviewed journal publications. Author 9: What is the significance of adding \"Correction\"? The common practice is that this category of papers is EXCLUDED from the analysis. As suggested, one item excluded as it is a correction article, and all the tables & graphs updated accordingly.Author 10: \"……the information related to h-index was obtained from the Incites in WoS\": please explain h-index and its use here. We have added the following information concerning h-index in \"Data Analysis\" of the methods section: Under Data Analysis of the Methods section, we have updated it as follows: In addition, the information related to h-index was obtained from the Incites in WoS. Here, the h-index reflects the productivity of authors based on their publication and citation records. It is useful because it discounts the disproportionate weight of highly cited papers or papers that have not yet been cited. Author 11: Please explain the VOS parameters used and cite their original source. We have added the following literature source to cite VOC parameter within the manuscript: Under Data Analysis of the Methods section, we have updated it as follows: Retrieved articles were analyzed using Incites in WoS and Visualization of Similarities (VOS) viewer 1.6.11. VOS is a new method used for visualizing similarities between objects 16,17. Under References, both the articles reference are added as follows: 16. Van Eck NJ, Waltman L, Van den Berg J, Kaymak, U: Visualizing the computational intelligence field. IEEE Comput. Intell. M. 2006;1(4):6-10.17. Van Eck NJ, Waltman L: VOS: A new method for visualizing similarities between objects. In Lenz H-J, Decker R, editors. Advances in data analysis: Studies in Classification, Data Analysis, and Knowledge Organization. Springer: Berlin, Heidelberg; 2007. p. 299-306. https://doi.org/10.1007/978-3-540-70981-7_34 Author 12: \"…..used in this study are proprietary…\" should be \"…..used in this study is a proprietary….\" The required correction carried out. The word 'is a' has been added in front of the word 'proprietary software'. Under Data Analysis of the Methods section, we have updated it as follows: Since Incites in WoS used in this study is a proprietary software, the researchers could alternatively use a tab-delimited file downloaded from WoS and use it in VOSviewer. Author 13: Statistical analysis should present the bibliometric parameters in detail. We have included bibliometric parameters related to publication year, authorship ranking, source journal productivity, collaborating institutions, country-wise research collaboration, citations, and h-index. As suggested by the reviewer, we have added a year-wise collaboration pattern of the articles as an additional parameter (Table 4). Refer to the following table caption in the text of the manuscript.Table 4: Collaboration Patterns of Articles between 1999 and 2018.Data obtained from Web of Science. Includes all articles types. N articles = 488. Results: Author 14: \"Between 1999 and 2008….\" this paragraph should be in the past tense, please revise. The respective paragraph has been revised and presented in the past tense. The revised paragraph is provided in the Results section as follows: Under the Results section The chosen period of study was divided into four strata with five years each. The strata were 1999-2003; 2004-2008; 2009-2013; and 2014-2018. In the first two strata, the publication count was observed in a single digit (≤9). In the third strata, this count has reached two digits with a maximum of 26 in 2013. However, in the fourth strata, an abrupt rise in the publication count was observed with a peak of 103 in 2016. Notably, the publication count after 2016 decreased and rose slightly in 2018, but not to those levels seen in 2016 (Table 1 and Figure 3). Regarding citation count, there was a gradual rise over the research period, with a total of 2419 citations between 1999 and 2018, more than 100 of which have been documented since 2012. The highest average citation (citations/article) of ≥10 was observed only in 2006 (mean, 11.00) and 2014 (mean, 10.20). Author 15: \"….published 174 articles, 35.58%\" should be revised to \"published 174 articles i.e. 35.58%\". The correction has been incorporated in the text of the manuscript.Under the Results section as follows: A total of 264 journals had published the 488 retrieved articles. The top 20 journals in which Indian physiotherapists published over the study period are displayed in Table 2. The top 20 journals published 174 articles, i.e., 35.66% of total publications (N=488) in the research period. Out of the top 20 ranked journals, 11 journals were based in India, 26.84% of the total publications. Author 16: \"…11 were journals based\" should be revised to \"…..11 journals were based\". The correction has been incorporated in the text of the manuscript.Under the Results section as follows: A total of 264 journals had published the 488 retrieved articles. The top 20 journals in which Indian physiotherapists published over the study period are displayed in Table 2. The top 20 journals published 174 articles i.e., 35.66% of total publications (N=488) in the research period. Out of the top 20 ranked journals, 11 journals were based in India, 26.84% of the total publications. Author 17: \"average citation of 0.17\" should be revised to \"average citation per paper as…\". The correction has been incorporated in the text of the manuscript.Under the Results section, we have updated it as follows: The International Journal of Physiotherapy published 35 articles, with six citations for these 35 articles, an average citation per article as 0.17. It was the most active Journal found in this study and contributed to 7.17% of total publications. In contrast, the journal Haemophilia published seven articles with 68 citations for these articles, an average citation of 9.71. Similarly, Annals of Indian Academy of Neurology published four articles, with citations of 43, giving it the highest average citation of 10.75 ( Table 2). Author 18: Fig 2: Please check the heading for Flowchart for VOSviewer analysis – I suspect some errors there. In the flow chart, we have mentioned the subject, and, in the graph, we have indicated the keyword. To address this issue, we have updated the Figure 2 by providing the following corrections on it. In the heading box “Flowchart….VOSviewer software”, we changed the term \"co-occurrence of subjects\" to \"co-occurrence of keywords.\" The heading in the second box of figure 2, we changed the term \"co-occurrence of subjects\" to \"co-occurrence of keywords.\" In addition, in Step-5 box of Figure 2, the number 489 is replaced with 488. Author 19: Table 1 (please cross-check with the textual description): I see publications depicting a fluctuating pattern - the only rise is seen after 2010 until 2016. Why don't the authors compare the growth according to the strata of 5 years? As advised, we have added the following description in the results section: Under the Results section The chosen period of study was divided into four strata with five years each. These strata were 1999-2003; 2004-2008; 2009-2013; and 2014-2018. In the first two strata, the publication count was observed in a single digit (≤9). In the third strata, this count has reached two digits with a maximum of 26 in 2013. However, in the fourth strata, an abrupt rise in the publication count was observed with a peak of 103 in 2016. Notably, the publication count after 2016 decreased and rose slightly in 2018, but not to those levels seen in 2016 (Table 1 and Figure 3). Author 20: Fig 3: It would be good to present the citation trend along with the number of publications - please see these papers. For example, see these papers: https://www.ncbi.nlm.nih.gov/pubmed/31766944; https://doi.org/10.3390/clockssleep202001 0.1,2 The corresponding figure 3 is modified as the same provided in the example study. Also, the figure caption is revised as follows: Figure 3. The growth trend for publications and citations by Indian physiotherapists between 1999 and 2018. Author 21: Table 2-5: I think it would be appropriate to list the top 10 instead of 20 – again, this is a common practice in bibliometric studies. I would also want to see top-10 highly cited papers. The focus of our study is to review the results based on the top 20 journals, authors, institutions, and countries collaborating with Indian physiotherapists. However, as per the reviewer's feedback, we have provided information about the top 10 highly cited papers in Table 7 of the manuscript. The description of the top 10 highly cited articles is provided in the Results and Discussion section. Under the Results section, we have updated it as follows: Besides, the top 10 highly cited papers during the study period were provided in Table 7. Among those papers, an article published by Singh et al. (2008) in the Digest journal of Nanomaterials and Biostructures received 236 citations until 2018 with the average citation of 18.15 per year. (Refer Table 7) Under Discussion section, we have updated it as follows: CitationsLittman et al. analyzed the research output of 45 physical therapy faculty in the southeastern US from 2000 to 2016 using their curriculum vitae. The range of publications and the citations of these faculty was observed as 0 to 43, and 0 to 943, respectively 12 . Further, Italian physiotherapists published 1083 articles with 13,373 citations in the Scopus database from before 1995 to 2016.13 Compared to these findings, this study revealed that 488 articles published by Indian physiotherapists in WoS from 1999 to 2018 secured only 2419 citations. Specifically, an article by Singh M published in 2008 had a high citation count of 236 till 2018. Besides, Sturmer et al. found that 222 articles were published by Brazilian physical therapy researchers in WoS in 2010, which had a total of 1805 citations 6 . In contrast, this study reported that Indian physiotherapists published only 65 articles with 171 citations up to the year 2010 in WoS. Even though the articles published by Indian physiotherapists were suitable enough for several researchers to cite them often, there is a need to improve the citation count of their publications in the future. Author 22: Table 2: It would be interesting to discuss the impact factor and SJR ranking (https://www.scimagojr.com/journalrank.php) of these journals. This will reflect the quality and visibility of these journals. I see that most of these journals are indexed in the ESCI - the lowest level of WoS indexes. This study is focused on revealing the scientific output of Indian physiotherapists in the WoS database during the period from 1999 to 2018. The criteria have been fixed that this study is limited to the WoS database, and the Web of Science indexes covered was mentioned in the search items (i.e., SCI-EXPANDED, SSCI, A&HCI, CPCI-S, CPCI-SSH, ESCI, CCR-EXPANDED, IC). As advised by the reviewer, the h-index, JCR impact factor and SJR ranking of the observed journals are additionally provided in Table 2 (Refer Table 2.) Author 23: \"International Journal of Scientific Study\": I could not find this Journal in the Master List search (https://mjl.clarivate.com/search-results). I am not really sure about the authenticity of this Journal – it looks rather like a questionable journal to me, so I am not sure how its content came up in the results. Please double-check. This Journal is indexed in Web of Science till 2017. Kindly refer to the following screenshot. Author 24: \"The top 20 institutions ……. displayed in Table 4\" – this does not look grammatically correct and needs revision. These could be organized in two tables as national (within India) and international (outside India) collaborations. The corresponding sentence is revised for grammatical correction and provided in the Results section. Under Results, we have updated it as follows: The top 20 institutions collaborating with Indian physiotherapists for physiotherapy research are displayed in Table 5. In addition, the authors have added a column titled \"Collaboration\" in Table 5 to mention the collaborating institutions as \"National\" or \"International\" (Refer Table 5). Author 25: \"An article's keyword may represent its primary…..\" needs citation. As advised, the reference for the following sentence \"An article's keyword may represent its primary…..\" is provided in the Results section. Under the Results section An article's keyword may represent its primary material, and to some degree, the frequency of occurrence 18. Author 26: It would be better to exclude \"Physiotherapy\" and \"Rehabilitation\" from the map because they will certainly be the most used. The readers of this paper would rather be interested to see what other terms had frequent occurrence. The authors should also discuss the map (Fig 4) to reflect upon which keywords tend to occur together. For instance, keywords in green (i.e., management, trial, children, diagnosis, surgery, India) tend to belong to the same cluster and are likely to co-occur together. What was the minimum value set for the co-occurrence of a keyword? For example, see this paper: https://www.ncbi.nlm.nih.gov/pubmed/31766944.1 The authors have discussed the map (Figure 4) to reflect upon which keywords tend to occur together. They have also described the minimum value set for the co-occurrence of a keyword. The description is provided in the Results section (Also Refer to Figure 4 and Table 8). Under the Results section, it is updated as follows: Using VOSviewer, the top 20 keywords used in articles (from a total of 2477 keywords) are shown in Figure 4. An article's keyword may represent its primary material, and to some degree, the frequency of occurrence 18. Likewise, co-occurrence can indicate centered themes of research in a field. Through VOSviewer, the authors observed the top 20 keywords, and it is shown in Table 8. Among the top 20 keywords, the minimum number of occurrences of each keyword was set to 11 and excluded the keyword \"Physiotherapy,\" \"Rehabilitation,\" and \"Physical Therapy\" from the formation of the cluster. There were three co-citation clusters formed using this criterion. The results showed that the keyword \"Management (cluster 1 red color)\" had the highest linkages (N=50) with all the 3 clusters, followed by keywords \"Exercise (cluster 2 green color)\" and \"Reliability (cluster 3 blue color)\" had 40 and 39 linkages respectively with all 3 clusters. Author 27: Table 3: the column for h-index should come to the right of total citations. Iqbal ZA does not have Indian affiliation, so these names don't correspond well with the caption of the table. I believe these could be organized in two tables as national (within India) and international (outside India) collaborations. If this is not possible then the authors are advised to revise the caption of the table to bring more clarity in it. As advised, in Table 3, we have relocated the h-index column to the right side of the total citations column. The new column \"citations per article\" is also added. In addition, Table 3 caption has already been edited as follows: Table 3. Top 20 Authors collaborated between 1999 and 2018. Author 28: Table 4: Please revisit the caption for clarity. Authors should add a column \"collaboration\" and categorize it as national and international. Table 4 is changed into Table 5. We have changed the caption of Table 5 as \"Top 20 institutions collaborating with Indian physiotherapists between 1999 and 2018. A new column, \"collaboration,\" has been added to categorize the institutions as national or international collaboration (Refer to Table 5). Author 29: Table 5: Six institutions with 4 papers are presented, but I don't understand how they are ranked (according to the number of citations or citations/paper). As of now, it appears as if they are listed alphabetically. The common method in bibliometric studies is to rank them based on the number of citations. Table 5 is renamed as Table 6. The order of the collaborating countries is listed on the basis of the number of articles. Further, the countries with similar number of articles are ranked based on the number of citations and it is presented in Table 6. Discussion: Author 30: Overall, this section is just the repetition of the results and fails to integrate what is known about the Topic and what was found. As advised, the discussion is revised to avoid the repetition of the results and integrate what is know about the Topic and what was found. Author 31: Concerning these two points: \"Exploring the reasons…..critical issue\" and \"This implies that……other researchers\". These are vague arguments – I believe that this would be due to several factors, including the quality of the journal (indexing, metrics), ease of publication (acceptance rate), focus (audience/readership), APCs and reputation to the scientific community. I think authors have failed to give a balanced view here. Most of these journals appear to be of low-quality. Just a point for your understanding is that the IJPT was once involved in deceptive practices (please see this paper: https://www.ncbi.nlm.nih.gov/pubmed/27924967)3, but it has probably improved its practices and has gotten into ESCI. As suggested, we have modified the sentence as follows: Under the heading \"Journals\" in the Discussion section, we have updated it as follows:Exploring the reasons behind Indian physiotherapists' choice to publish in these Indian journals is beyond the scope of this study, and further research is warranted to address this critical issue. In general, the choice of researchers to publish in a journal depending on the prestige, impact factor, quality of peer reviews, acceptance rate, readership, article publishing charges, and reputation to the scientific community 19,20. Under the heading \"Journals\" in the Discussion section, we have updated it as follows:As the journals such as Haemophilia and Annals of Indian Academy of Neurology with a considerable citation and a high average citation for only a few articles, the authors mentioned the following sentence:This implies that these articles might be more useful for the researchers to cite them often 21. Author 32: Collaborating authors: It would be appropriate to discuss the average h-index (and its range) and average citations to the papers by Indian authors. As the average h-index and average citations to the papers by Indian authors were not observed, this study described the range of h-index and total citations of the top 20 authors. A description is provided under the heading ‘Collaborating authors’ in the discussion section as follows: Collaborating authors A previous study by Man et al. found that four Hong Kong physiotherapy professors had a median h-index of 30.5 and their average total number of citations was 2930.3 14 . Moreover, Brazilian physical therapy researchers had a median h-index of 3, according to WoS 6 . Recently, Vercelli et al. reported that the mean h-index of 363 Italian physiotherapists was 2.2, which ranged from 0 to 16; mean citations per author were observed as 58 13 . On the other hand, this study observed the top 20 authors who worked with Indian physiotherapists with the range of total citations from 0 to 1372 and h-index from 0 to 18. Particularly, Kumar R (India), had the highest h-index of 18, total citations of 1372, and citations per article of 274.40. Author 33: I still have confusion regarding the organization and presentation of findings of this paper in the results and discussion sections. Collaborating authors, institutions, and countries should have been leading authors, institutions and countries. Collaborations between countries, institutions, and authors should better be presented through VOSviewer as done in these papers: https://www.ncbi.nlm.nih.gov/pubmed/31766944; https://doi.org/10.3390/clockssleep202001 0.1,2 As recommended by the reviewer, the authors have provided information about the top 10 author collaborations and the top 10 collaborating countries in figures (Refer Figure 5 and Figure 6) and cited within the text (Refer Results section). Under Results section Besides, the collaboration observed among the top 10 authors and top 10 countries were presented in figures 5 and 6 respectively. Concerning the top 10 authors collaboration, co-authorship network analysis produced a map for authors with at least four papers and formed six clusters. The most profile authors in terms of citation were observed as Kumar R and Mahadevappa M. These authors showed more collaboration. Furthermore, network visualization of countries with a minimum of four papers showed the top 10 countries in three clusters. The following pairs of countries showed a strong collaboration between them: India-USA (link strength =22), India-Saudi Arabia (link strength=22), and India-England (link strength=17). Author 34: What is the clinical implication of these keywords? Please discuss. The clinical implications of the keywords were discussed with the appropriated literature under the heading \"Keywords by co-occurrence\" in the Discussion section. Refer to the heading \"Keywords by co-occurrence\" in the Discussion section Dash et al. stated that the keywords are one of the three pillars of a biomedical research article. Using the right keywords would augment the article being found by other researchers as these are used by abstracting and indexing services 28. Hence, this study revealed the top 20 keywords that occurred in various articles using VOSviewer software. It is observed that the keyword \"Management\" had the highest of 50 linkages with all the three co-citation clusters. Author 35: Please add a section about the limitations of this study. As advised, a new heading \"Limitations and Recommendations\" is added in the manuscript. Limitations and Recommendations The findings of this study are only limited to the WoS database. Future research can focus on studying the research output of the Indian physiotherapists in other databases to ascertain their research productivity. Future studies can focus on analyzing individual and institutional factors influencing the research productivity of Indian physiotherapists and develop suitable strategies to enhance their scientific production. Conclusions: Author 36: The decrease in papers during 2017 and 2018 was not \"drastic\", please rephrase this. The term \"drastic\" is revised and described as follows in the Conclusion section. Under Conclusion Conclusion This study observed that the scientific output of Indian physiotherapists shows an uptrend in performance since 1999, excluding 2017 and 2018, where a considerable decline was noticed. The results showed that Indian physiotherapists had mostly published in Indian-based journals, and collaborated with Indian institutions. Even though there are high-quality publications, there is a need to enhance both the quality and quantity of scientific papers to increase the high number of citations and average citations. This study also recommends that Indian physiotherapists should expand their research collaboration internationally to improve their scientific output. Author 37: \"…….though there are high-quality publications…\" this stands as a mere claim and won't be meaningful without presenting the top-10 highly cited publications. As advised, the top 10 highly cited papers are provided in Table 7 of the manuscript. A description of the top 10 highly cited papers is provided in the Results and Discussion section. Refer to the Results section to see the changes made as follows: Besides, the top 10 highly cited papers during the study period were provided in Table 7. Among those papers, an article published by Singh et al. (2008) in the Digest journal of Nanomaterials and Biostructures received 236 citations until 2018 with the average citation of 18.15 per year. Refer to the Discussion section to see the changes made as followsCitationsLittman et al. analyzed the research output of 45 physical therapy faculty in the southeastern US from 2000 to 2016 using their curriculum vitae. The range of publications and the citations of these faculty was observed as 0 to 43, and 0 to 943, respectively 12 . Further, Italian physiotherapists published 1083 articles with 13,373 citations in the Scopus database from before 1995 to 2016.13 Compared to these findings, this study revealed that 488 articles published by Indian physiotherapists in WoS from 1999 to 2018 secured only 2419 citations. Specifically, an article by Singh M published in 2008 had a high citation count of 236 till 2018. Besides, Sturmer et al. found that 222 articles were published by Brazilian physical therapy researchers in WoS in 2010, which had a total of 1805 citations 6 . In contrast, this study reported that Indian physiotherapists published only 65 articles with 171 citations up to the year 2010 in WoS. Even though the articles published by Indian physiotherapists were suitable enough for several researchers to cite them often, there is a need to improve the citation count of their publications in the future. References Seven new references are added in the revised manuscript. The newly added references are as follows: 16. Van Eck NJ, Waltman L, Van den Berg J, Kaymak, U: Visualizing the computational intelligence field. IEEE Comput. Intell. M. 2006;1(4):6-10.17. Van Eck NJ, Waltman L: VOS: A new method for visualizing similarities between objects. In Lenz H-J, Decker R, editors. Advances in data analysis: Studies in Classification, Data Analysis, and Knowledge Organization. Springer: Berlin, Heidelberg; 2007. p. 299-306. https://doi.org/10.1007/978-3-540-70981-7_3418. Memon AR., Vandelanotte C, Olds T, Duncan MJ, et al.: Research combining physical activity and sleep: A bibliometric analysis. Percept. Mot. Skills, 2020;127(1):154–181. https://doi.org/10.1177/003151251988978019. Pepermans G, Rousseau S: The decision to submit to a journal: Another example of a valence‐consistent shift? J Assoc Inf Sci Tech. 2016;67(6):1372–1383.20. Mukherjee D: Choosing the Right Journal-A Comprehensive Guide for Early-career Researchers. 2018 Aug 03. https://blog.typeset.io/choose-right-journal-early-stage-researchers-guide-ea2cf236dde4 [Accessed April 20, 2020].21. Aksnes DW, Langfeldt L, Wouters P: Citations, citation indicators, and research quality: An Overview of basic concepts and theories. SAGE Open. 2019 Feb 07. https://doi.org/10.1177/215824401982957528. Dash M: Three pillars of a biomedical research article: The title, Abstract and keywords. J Health Spec. 2016;4:186-189."
}
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}
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https://f1000research.com/articles/9-207
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https://f1000research.com/articles/9-374/v1
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18 May 20
|
{
"type": "Research Article",
"title": "Current status of global research on novel coronavirus disease (COVID-19): a bibliometric analysis and knowledge mapping",
"authors": [
"Md. Mahbub Hossain"
],
"abstract": "Background: Novel coronavirus disease (COVID-19) is a major global health concern due to its pathogenicity and widespread distribution around the world. Despite a growing interest, little is known about the current state of research on COVID-19. This bibliometric study evaluated the contemporary scientific literature to assess the evolution of knowledge on COVID-19, identify the leading research stakeholders, and analyze the conceptual areas of knowledge development in this domain. Methods: Bibliometric data on COVID-19 related studies published until April 1, 2020, were retrieved from Web of Science core collection. Further, a quantitative evaluation and visualizations of knowledge areas in COVID-19 research were created by statistical and text-mining approaches using bibliometric tools and R software. Results: A total of 422 citations were retained in this study, including journal articles, reviews, letters, and other publications. The mean number of authors and citations per document was 3.91 and 2.47, respectively. Also, the top ten articles, authors, and journals were identified based on the frequencies of citations and publications. Networks of contributing authors, institutions, and countries were visualized in maps, which highlight discrete developments in research collaborations. Major areas identified through evaluating keywords and text data included genetic, epidemiological, zoonotic, and other biological topics associated with COVID-19. Conclusions: Current status of COVID-19 research shows early development in different areas of knowledge. More research should be conducted in less-explored areas, including socioeconomic determinants and impacts of COVID-19. Also, global research collaboration should be encouraged for strengthening evidence-based decision-making preventing and addressing the COVID-19 pandemic and aftermath.",
"keywords": [
"COVID-19",
"Coronavirus",
"Communicable diseases",
"Bibliometric study"
],
"content": "Introduction\n\nCoronaviruses are RNA viruses widely found among many mammal species, including human beings1. Although these viruses generally have low virulence, two epidemics by severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) are considered as major public health events in the past two decades. The case fatality rates were 10% and 37% for SARS-CoV2 and MERS-CoV3, respectively. In December 2019, a novel coronavirus emerged in Wuhan City, Hubei Province of China4. This outbreak was unique in terms of high pathogenicity and mortality compared to the earlier epidemics by coronaviruses5. Soon cases affected by novel coronavirus were found outside Wuhan and eventually around the world. On January 30, 2020, the World Health Organization (WHO) declared the outbreak a public health emergency of international concern6. Later, the WHO named the disease a novel coronavirus as “COVID-19”, which is a short form of “coronavirus disease 2019” on February 11, 20207. With a growing number of new cases and increased mortality attributable to COVID-19 pandemic8, global health discourses among the scientific community, policymakers, and the general population are emphasizing on what is known about this virus. Although it is known that COVID-19 is uniquely different to SARS-CoV and MERS-CoV, the scientific knowledge on COVID-19 remains limited within the scope of recently published articles. It is essential to understand the evolution of emerging scientific knowledge on COVID-19 to inform further research as well as evidence-based policymaking.\n\nBibliometric analysis quantitatively examines the research progress of any topic and offers a comprehensive assessment of scientific research trends, which is widely used for mapping knowledge in different scientific disciplines9–11. As a research method, bibliometric study was used back in 1917 by Cole and Eales, who studied the growth of scientific production of articles published in the field of comparative anatomy12. This approach was subsequently termed as “Bibliometrics” by eminent British scientist Allen Richard13. Over the years, bibliometric studies have been used for analyzing a topic or field emerging in the global knowledge landscape and evaluating the evolution of research over time14–16. More importantly, it provides critical insights on most prolific authors, institutions, countries of affiliation, thematic change within the domain itself, co-citations, co-authorships, and holistic development of the field(s) of interest9,13,16.\n\nWith a growing interest in COVID-19 related research across the globe, a bibliometric study may inform the current status of global research and provide meaningful insights on future research. An earlier bibliometric analysis evaluated the scientific literature on different coronaviruses17. However, there is no bibliometric analysis available to date that specifically focuses on contemporary scientific development on COVID-19. This study aimed to address this knowledge gap and conducted a bibliometric analysis to evaluate the characteristics of the current body of literature on COVID-19, identify the prolific authors, institutions, and countries involved in COVID-19 research, and examine the evolution of key knowledge areas within COVID-19 related studies.\n\n\nMethods\n\nFor this study, bibliometric data were collected from the Science Citation Index Expanded, Social Sciences Citation Index, and Emerging Sources Citation Index databases within Web of Science (WoS) core collection. These databases within WoS are maintained by Clarivate Analytics, which offer the world’s leading scientific citation search and analytical information platform18. Collectively WoS collection provides enriched bibliometric data useful for citations analytics and mapping the knowledge in a given domain by examining leading authors, institutions, and collaborating nations working in a given domain of scientific research.\n\nThe following query was administered to retrieve COVID-19 related bibliometric data: “Novel coronavirus” OR “Novel coronavirus 2019” OR “2019 Novel coronavirus” OR “2019 nCoV” OR “COVID-19” OR “Wuhan coronavirus” OR “Wuhan pneumonia” OR “SARS nCoV” OR “SARS-CoV-2”. Considering the timing of the outbreak in late 2019, the search strategy was limited to 2019–2020 to retrieve data that may contain publications on COVID-19 rather than earlier coronaviruses. Also, all search fields were selected including topics, titles, and abstracts to retrieve the bibliometric data ensuring the sensitivity of the search strategy. This search was conducted in February 22, 2019 and updated in April 1, 2020, for the last time. Moreover, no restrictions on languages or publication types were applied due to the low number of publications on this recent topic. The inclusion criteria for this bibliometric study was as followings: a) journal articles published on COVID-19 topic, b) language of the publication was English, c) articles irrespective of their methodology were included, d) studies published between January 1, 2019, to April 1, 2020, were included. Furthermore, articles were excluded if they had conflicts with any of the above-mentioned inclusion criteria. The references of the retrieved articles were not evaluated, therefore, articles retrieved through citations search are the only source of data in this bibliometric study.\n\nAfter extracting bibliometric data from WoS, the citations were uploaded to RefWorks (freely available alternative: Mendeley), which is a cloud-based software for citation management. Further, they were screened as per the criteria described earlier, and then finally recruited citations were uploaded to R (Version 3.6.1). Using this software, descriptive analyses were conducted to evaluate the characteristics and types of documents. Also, the WoS metrics were used to assess the top ten impactful articles in the literature in terms of citations, the top 10 authors and journals based on the number of published documents on COVID-19. In addition, co-authorship among all the authors in the bibliography was assessed, and an evaluation of how many of them were connected within documents authored or co-authored by individuals was conducted.\n\nFurther, the affiliating institutions and countries of the respective authors were mapped using a network analysis approach. This set of analyses allowed to evaluate the nature and magnitude of collaboration at the individual, institutional, and international levels and how such collaborated impacted the knowledge base on COVID-19. Also, keywords and texts in titles and abstracts within scientific documents were identified and evaluated using text-mining approaches using shiny package in R (Version 3.6.1). At this stage, network analyses were conducted to assess the connectedness among those documents and related keywords. Furthermore, the cooccurrence of multiple authors, keywords, institutions, and countries, different thresholds were used to create visualizations of frequency distributions for each variable, whereas all entries within each variable were assessed for the same threshold to ensure equitable comparisons within respective fields of analyses. Furthermore, the relational mapping among the authors, institutions, countries, and common keywords were created using VOSviewer software, which is a bibliometric tool for visualization of citations data. In this mapping process, networks were developed at different thresholds for authors (n = 3 documents per author), institutions (n = 7 documents per institutions), countries (n = 1 document per country), and keywords (n = 3 cooccurring keywords). In addition, a multi-dimensional scaling approach was used to conduct a factorial analysis of 50 most-occurring research terms in the bibliometric data in R package as stated earlier. This allowed constructing a conceptual structure map depicting hierarchical relationships among knowledge areas within the research landscape of COVID-19.\n\n\nResults\n\nA total of 422 bibliometric records were recruited in this study, which were authored by 1652 authors with 3.91 authors per document (Table 1). Most documents (n = 1581) had multiple authors, and the mean citations received per document was 2.47. A major proportion of the publications were articles (33.41%) and editorials (32.23%).\n\nIn addition, top ten articles based on the number of citations in WoS were identified (Table 2), which included genetic, epidemiological, and clinical studies on COVID-19. Among the authors, Mahase E. has the highest number of publications (n = 13) followed by Akhmetzhanov AR., Linton NM., Nishiura H., and Zhang W. with seven publications per author. Also, top ten journals were identified that published the highest number of documents, which include British Medical Journal (n = 47) followed by The Lancet (n = 37), Eurosurveillance (n = 22), Journal of Medical Virology (n = 22), and Intensive Care Medicine (n = 13).\n\nA network map of co-authors who contributed to COVID-19 research was created at the threshold of 3 documents per author, which found 52 collaborating authors, as illustrated in Figure 1. Scattered zones show groups of collaborating authors, whereas connections between individuals and groups are plotted accordingly. Figure 2 shows the network of collaborating institutions that were affiliated with at least seven documents on COVID-19 research. This threshold identified 16 collaborating institutions, including Capital Medical University (number of documents, nd = 13; number of citations, nc = 237), Huazhong University of Science and Technology (nd = 18, nc = 167), and Wuhan University (nd = 15, nc = 154).\n\nFurther, the bibliometric records were analyzed for the contributing countries and a network was developed using co-authorship among scholars from those nations. Any collaborating country with at least one publication was included in this map (Figure 3). Among global nations, China has the highest number of documents (n = 185), followed by the US (n = 68), UK (n = 36), Italy (n = 23) and Canada (n = 23).\n\nIn this study, a visualization guided by quantitative evaluation of the cooccurrence of keywords was prepared, as depicted in Figure 4. A threshold of at least three cooccurrence of a keyword was set to identify the most frequent research terms indexed in the literature, which revealed a total of 69 keywords. The top ten cooccurring words were “coronavirus” (n = 69), “sars” (n = 47), “2019-ncov” (n = 43), “covid-19” (n = 44), “sars-cov-2” (n = 26), “pneumonia” (n = 25), “wuhan” (n = 18), and “outbreak” (n = 18).\n\nA factorial analysis was conducted among the leading 50 key terms in the bibliometric data using a multi-dimensional scaling approach (Figure 5). This analysis resulted in a dendrogram of repeatedly co-appearing keywords in hierarchical clustering, which highlights conceptual structures in the research field. The first cluster in this dendrogram (in blue) included research terms including diversity, multiple sequence alignment, and sars-like coronaviruses. Another cluster (in red) comprised of research terms related to pathogenicity of coronavirus outbreak, earlier outbreaks with other typologies, epidemiology, and diagnostic approaches. Both structures shared several common thematic areas, including zoonotic connections in COVID-19 epidemiology and genetic and molecular properties of interest in COVID-19 research.\n\n\nDiscussion\n\nThis bibliometric study and knowledge mapping identified contemporary scientific documents on COVID-19 from scholarly sources. The findings of this study reflect the recent scholarly growth of the global body of knowledge on COVID-19. Most documents had multiple authors from different collaborating institutions and nations, which highlight the productivity of scientific activities. Moreover, research keywords presented in the bibliometric data reflect the complexity and inclusion of multiple disciplines like virology, microbiology, infectious diseases, clinical medicine, public health, allied health sciences, social sciences, and other branches of knowledge. Such scholarly growth of the knowledge base may help in understanding the ontology and phenomenology of a new global health challenge imposed by COVID-19.\n\nNotably, the affiliating institutions and nations collaborated in COVID-19 research may inform the utility of global research collaboration, particularly during complex public health problems where multiple stakeholders from different institutions and contexts may offer diverse resources and competencies in addressing knowledge gaps in a more efficient manner compared to individualistic approaches. This can be profoundly challenging for low- and middle-income countries, like nations in Africa and South Asia, who have suboptimal research capacities and poor evidence-base to make informed decisions on highly prevalent health problems19.\n\nAnother challenge is a critical lack of technological infrastructure in such contexts, which reinforces the need to strengthen global collaborations for research and evidence synthesis. For example, resource-constrained contexts have lesser availability and accessibility to advanced technologies, which may limit their abilities to conduct research requiring tools like deep learning or other computational approaches20,21. Also, this may restrict opportunities for substituting time-intensive lab-based research through simulation or increase the speed and quality of research processes. This may be a reason for the lack of representation of studies from countries in South Asia, South America, and Africa. Future efforts should focus on strengthening research capacities in those contexts is essential to improve regional and global knowledge on persisting and emerging diseases affecting global populations.\n\nAlso, it is essential to acknowledge the need for global collaborations as the magnitude of the problem necessitates a series of large-scale analyses, exchange of perspectives, knowledge synthesis, and translating the same to inform evidence-based policies and practices22,23. More importantly, increased collaborations in research are likely to facilitate trust and cooperation in developing scalable solutions globally, minimizing the cost and maximizing human benefits beyond borders24,25. Lessons learned from research collaboration can foster hope in existing global health disparities, particularly in developing vaccines and other preventive solutions26,27. These aspects are critical for the overall development of COVID-19 related research and practice as the current evidence on collaboration shows scattered growth of research groups, which may affect the true potential that collaborative efforts may offer in this scenario.\n\nThis study identified top keywords that appeared in scientific literature and demonstrated how they co-appeared across studies taking intellectual roots from earlier studies. Moreover, an evolution of conceptual structure using those keywords inform the current scenario of uncontrolled observations retrieved from global studies. Keywords are useful not only to retrieve studies from databases or topics within studies, but they also tell the scientometric themes underlying the information presented in a document28,29. In addition, these conceptual constructs may inform future scientific measures to define and distinguish how sub-domains within the knowledge base on COVID-19. Furthermore, similar keywords appeared in multiple documents in this study, among which a one-third were original articles, which informs the early stage of research. This early finding may offer critical directions of scientific development in this knowledge domain. Also, this study found more frequent appearances of epidemiological, genetic, and molecular biological keywords in COVID-19 studies, whereas some keywords indicated an emergence of zoonotic topics, including other animals related to the human food chain or ecology.\n\nIt is notable that the cooccurrence of keywords analysis or conceptual structure mapping did not find significant presence of social, economic, political, or cultural determinants of COVID-19 in the global landscape. It is increasingly being recognized that neither disease nor health can happen in isolation from the complex web of those determinants of human lives30–33. The findings of this study highlight this gap, which necessitates further multi-sectoral research on how different determinants can be associated with higher or lower risks of COVID-19 among individuals or populations. In addition, programs and policies for addressing epidemic outbreaks may influence physical and psychosocial health outcomes in diverse population groups34,35, which remains another potential area for future research. Moreover, there is a lack of research that may inform the preventive measures like vaccinations, pharmacological interventions, clinical prognosis, and outcomes of COVID-19. Maybe such studies yet to be available in the future, which will enrich future scientometric analyses and evidence mapping processes.\n\nAnother issue is the existing literature mentions little about the psychosocial and economic consequences of COVID-19. A major public health crisis like COVID-19 can affect those aspects of lives and create lasting problems among the affected populations36,37. Perhaps it is too early to estimate such impacts or get them published in indexed sources, which would need more research and timely communication across journals and other media. In addition to epidemiological and genetic studies, psychological, econometric, and social sciences research assessing those concurrent and future challenges should be prioritized to improve the knowledge base in those areas.\n\nThis study has several limitations that must be acknowledged to apprehend the findings and address those limitations through future research. First, this study used three databases from WoS core collection, which may have included most studies in a given domain, whereas it may exclude studies that are exclusively indexed in other databases. This may affect the generalizability of the findings. Second, newly published studies may take some time to get indexed in WoS, which could be sourced from searching individual journals that published articles related to COVID-19. A similar gap exists in terms of preprints that are available in respective servers, and insights from those articles cannot be reflected in this study. Also, research studies may take time to get published as journal articles and to be indexed in associated databases, which may also limit the scope of current literature to reflect contemporary knowledge. Third, a bibliometric analysis provides an overview of the evolution of a knowledge domain that is methodologically different than approaches used in clinical reviews. Such reviews may have different objectives and methods of synthesis, which were beyond the scope of this study. However, this study evaluated the knowledge evolution on COVID-19, which may have long-term impacts on the field of COVID-19 studies and future discourses on public health emergencies. The above-mentioned issues should be considered to use the findings of this study and conduct future research and evidence synthesis on COVID-19 addressing those challenges.\n\n\nConclusions\n\nA public health emergency, like the COVID-19 pandemic, may affect different frontiers of human lives globally. To solve such problems, it is necessary to fully understand the problem and solutions that may address this. This need for knowledge is a fundamental force that keeps science alive and allows scientists to thrive in their research domains bringing the best possible methods and materials to answer real-life questions. Solving a complex public health problem like COVID-19 needs robust knowledge generated through rigorous methods specific to each problem related to different dimensions of COVID-19 as well as the lives of millions of people around the world. This study provided a global bibliometric evaluation of COVID-19 related studies, which may facilitate ongoing and future research. Such academic and professional efforts in understanding COVID-19 and addressing the same will be informed by the knowledge base we have today, which will continue to evolve over time, enriching science and societies globally.\n\n\nData availability\n\nOpen Science Framework: Bibliometric Analysis of COVID-19 Research, https://doi.org/10.17605/OSF.IO/SJB8638. Registered on 11th May 2020, https://osf.io/65v2z",
"appendix": "Acknowledgements\n\nThe author is thankful to Dr. Noor Al Quddus, Post-doctoral Researcher at the Mary Kay O'Connor Process Safety Center, Texas A&M University, for his valuable support and encouragement in conducting this study.\n\nA previous version of this article is available on the preprint server SSRN: http://dx.doi.org/10.2139/ssrn.3547824.\n\n\nReferences\n\nRichman DD, Whitley RJ, Hayden FG: Clinical virology. John Wiley & Sons. 2016. Reference Source\n\nCenters for Disease Control and Prevention (CDC): Update: Severe acute respiratory syndrome -- Worldwide and United States, 2003. MMWR Morb Mortal Wkly Rep. 2003; 52(28): 664–5. PubMed Abstract\n\nWorld Health Organization (WHO): Middle East respiratory syndrome coronavirus (MERS-CoV). (accessed 2 March 2020). Reference Source\n\nWorld Health Organization (WHO): Coronavirus disease (COVID-19) outbreak. (accessed 2 March 2020). 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PubMed Abstract | Publisher Full Text\n\nCohen S, Gottlieb B, Underwood L: Social Relationships and Health. 2000. Publisher Full Text\n\nMazumder H, Hossain MM, Das A: Geriatric Care during Public Health Emergencies: Lessons Learned from Novel Corona Virus Disease (COVID-19) Pandemic. J Gerontol Soc Work. 2020; 1–2. PubMed Abstract | Publisher Full Text\n\nHossain MM, Sultana A, Purohit N: Mental Health Outcomes of Quarantine and Isolation for Infection Prevention: A Systematic Umbrella Review of the Global Evidence. SSRN Electron J. 2020. Publisher Full Text\n\nVirus will have huge socioeconomic impact in China. Oxford Analytica, 2020. Reference Source\n\nLee SI: Costly Lessons from the 2015 middle east respiratory syndrome coronavirus outbreak in Korea. J Prev Med Public Health. 2015; 48(6): 274–276. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHossain MM: Bibliometric Analysis of COVID-19 Research. 2020. http://www.doi.org/10.17605/OSF.IO/SJB86"
}
|
[
{
"id": "65722",
"date": "17 Jul 2020",
"name": "Roger Chun-Man Ho",
"expertise": [
"Reviewer Expertise COVID-19 research"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI have the following comments and need to review them again.\nCan the authors discuss a recent COVID-19 review on this topic? What is the value-added by this new review by contrasting with an existing review?\nTran BX, Ha GH, Nguyen LH, et al. Studies of Novel Coronavirus Disease 19 (COVID-19) Pandemic: A Global Analysis of Literature. Int J Environ Res Public Health. 2020;17(11):4095. Published 2020 Jun 8. doi:10.3390/ijerph171140951.\nIn Figure 4, can the authors explain why mental health or negative mental health consequences associated with COVID-19 was not found to be there in the Figure?\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": "67701",
"date": "23 Jul 2020",
"name": "Siddharth Sridhar",
"expertise": [
"Reviewer Expertise Emerging 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 perform bibliometric analysis of the COVID-19 literature at a relatively early stage of the pandemic. There are some interesting observations, but I have the following reservations:\n\nBasically, most research is expected to originate from areas where the case load is highest. Most of the bibliometric analysis was performed at a relatively early stage of the pandemic at which time the bulk of COVID-19 case load was in China. Africa, for example, had negligible case loads in February/ March 2020. In fact, COVID-19 spread to high-income countries in Europe first. This would explain why the majority of research was conducted in China and Europe rather than other regions. The author's point about less research from low-income countries is valid in general, but I would like to point out that this disparity is probably not well illustrated by this study. Such a conclusion can only be reached from bibliometric analysis of several year's data, which is not possible at this stage.\n\nA large volume of early work on COVID-19 was published in Chinese. Not including works in other languages has probably skewed the data. Also many many pre-prints, which are still not published due to heavy backlog of journals.\n\nNot sure what the value of including individual researcher's names is.\n\nThe other author has pointed out another publication on a similar topic. Please address additional value provided by this study.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/9-374
|
https://f1000research.com/articles/9-373/v1
|
18 May 20
|
{
"type": "Brief Report",
"title": "A snapshot of the ongoing clinical research on COVID-19",
"authors": [
"Daniele Piovani",
"Claudia Pansieri",
"Laurent Peyrin-Biroulet",
"Silvio Danese",
"Stefanos Bonovas",
"Daniele Piovani",
"Claudia Pansieri",
"Laurent Peyrin-Biroulet",
"Silvio Danese"
],
"abstract": "The pandemic of coronavirus disease 2019 (COVID-19) presents an unprecedented challenge to rapidly develop new diagnostic, preventive and therapeutic strategies. Currently, thousands of new COVID-19 patients are quickly enrolled in clinical studies. We aimed to investigate the characteristics of the COVID-19 studies registered in ClinicalTrials.gov and report the extent to which they have incorporated features that are desirable for generating high-quality evidence. On April 28, 2020, a total of 945 studies on COVID-19 have been registered in ClinicalTrials.gov; 586 studies are interventional (62.0%), the most frequent allocation scheme is the parallel group assignment (437; 74.6%), they are open-label and the most common primary purpose is the research on treatment. Too many of the ongoing interventional studies have a small expected sample size and may not generate credible evidence at completion. This might lead to a delayed recognition of effective therapies that are urgently needed, and a waste of time and resources. In the COVID-19 pandemic era, it is crucial that the adoption of new diagnostic, preventive and therapeutic strategies is based upon evidence coming from well-designed, adequately powered and carefully conducted clinical trials.",
"keywords": [
"SARS-CoV-2",
"2019-nCoV",
"2019 novel coronavirus",
"severe acute respiratory syndrome coronavirus 2",
"Covid-19"
],
"content": "Introduction\n\nThe pandemic of coronavirus disease 2019 (COVID-19) caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) presents an unprecedented challenge to rapidly develop new diagnostic, preventive and therapeutic strategies1. Currently, thousands of new COVID-19 patients present for care every day, and many are quickly enrolled in clinical studies. We aimed to investigate the characteristics of the COVID-19 studies registered in ClinicalTrials.gov2, and report the extent to which they have incorporated features that are desirable for generating high-quality evidence.\n\n\nMethods\n\nWe investigated the ClinicalTrials.gov website on April 28, 2020, using the search term: SARS-CoV-2 OR 2019-nCoV OR 2019 novel coronavirus OR severe acute respiratory syndrome coronavirus 2 OR Covid-19. No restrictions were applied. No screening of trials was performed; all results were included regardless of their content.\n\nStata 15.0 (Stata Corp., College Station, TX, USA) was used for the analysis of study characteristics.\n\n\nResults\n\nA total of 945 studies on COVID-19 have been registered in ClinicalTrials.gov up to April 29, 2020; 586 studies are interventional (62.0%), and 435 of them (74.2%) are randomized. Among interventional studies, the most frequent allocation scheme is the parallel group assignment (437; 74.6%), followed by single group (111; 18.9%], sequential (18; 3.1%), factorial (9; 1.5%), and cross-over assignment (11; 1.9%). The majority of the clinical trials are open-label (no masking, 338 [57.7%]); however, 57 (9.7%) trials are double-blinded, 41 (7.0%) triple-blinded, 90 (15.4%) quadruple-blinded, and 60 (10.2%) single-blinded. Among observational studies, cohort (222; 64.3%) is the most common study design (Table 1).\n\nMost studies target adult or elderly participants, while 178 (18.8%) enroll children, with only five (0.5%) recruiting exclusively children. Median expected study size is 200 (interquartile range, 66–504), although sample sizes vary from ≤100 (344; 37.0%) to >1,000 individuals (148; 15.9%). Overall, only 27 of 945 studies (2.9%) have completed recruitment, 453 (47.9%) are actively recruiting subjects, while a large number of studies (414; 43.8%) are not yet actively recruiting participants. Most of the studies are conducted in Europe (n=327), North America (n=217, of which 186 in the US), East Asia (n=102), Africa (n=27), and in South America (n=26). No study has reported results yet.\n\nAmong the interventional studies, the most common primary purpose is the research on treatment (441; 75.3%), followed by prevention (79; 13.5%), supportive care studies (22; 3.8%), and diagnostic investigations (17; 2.9%). Regarding the drugs under scrutiny, hydroxychloroquine (110; 28.6%), azithromycin (38; 9.9%), lopinavir/ritonavir (24; 6.2%), interferon-α and -β (24; 6.2%), glucocorticoids (22; 5.7%), chloroquine (14; 3.6%), favipiravir (10; 2.6%), remdesivir (8; 2.1%), tocilizumab (21; 5.5%), anti-SARS-CoV-2 immunoglobulins (15; 3.9%) and sarilumab (9; 2.3%) account for the majority of interventional studies. Additional details are featured in Table 2.\n\n\nDiscussion\n\nOur survey presents the current COVID-19 clinical research landscape. Several hundreds of clinical studies have been initiated all over the globe, and the number is growing. Most interventional studies incorporate randomisation, which is considered the hallmark of high-quality clinical trials3, while more than 40% are blinded.\n\nStudies are being conducted especially in the most affected areas: Europe and US. The number of COVID-19 cases in low to middle-income countries is still relatively low, also reflecting scarce testing, but is expected to rise in the next period. These countries will need more research on organizational measures, and trials on interventions that are affordable and applicable to those settings4.\n\nMost studies focus on adults and elderlies, while only few target children, possibly reflecting the observed burden of the disease. Additional effort is needed to ensure that minors are included in COVID-19 clinical research, so that therapeutic decisions are based upon high-quality evidence.\n\nNo drug with proven clinical efficacy currently exists for SARS-CoV-2 infection. Despite the absence of solid evidence, several treatments are being currently used in clinical practice in several countries, with sometimes disastrous consequences5. Too many of the ongoing interventional studies have a small expected sample size, and may not generate credible evidence at completion4. This might lead to a delayed recognition of effective therapies that are urgently needed, and a waste of time and resources. In the COVID-19 pandemic era, it is crucial that the adoption of new diagnostic, preventive and therapeutic strategies is based upon evidence coming from well-designed, adequately powered and carefully conducted clinical trials.\n\n\nData availability\n\nThe Clinical Trials website can be accessed here: https://clinicaltrials.gov/",
"appendix": "References\n\nSanders JM, Monogue ML, Jodlowski TZ, et al.: Pharmacologic Treatments for Coronavirus Disease 2019 (COVID-19): A Review. JAMA. 2020. PubMed Abstract | Publisher Full Text\n\nClinicalTrials.gov. 2020; Accessed at 21 April 2020. Reference Source\n\nMoher D: CONSORT: an evolving tool to help improve the quality of reports of randomized controlled trials. Consolidated Standards of Reporting Trials. JAMA. 1998; 279(18): 1489–1491. PubMed Abstract | Publisher Full Text\n\nCOVID-19 Clinical Research Coalition: Global coalition to accelerate COVID-19 clinical research in resource-limited settings. Lancet. 2020; 395(10233): 1322–1325. PubMed Abstract | Publisher Full Text\n\nOwens B: Excitement around hydroxychloroquine for treating COVID-19 causes challenges for rheumatology. Lancet Rheumatol. 2020; 2(5): e257. PubMed Abstract | Publisher Full Text | Free Full Text"
}
|
[
{
"id": "63548",
"date": "26 May 2020",
"name": "Demetris Lamniosos",
"expertise": [
"Reviewer Expertise Epidemiology",
"Statistics",
"Research Methods",
"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 brief report is a survey of the current COVID-19 clinical research landscape. The number of clinical studies on COVID-19 is rapidly growing and it is important the investigation of whether these studies are incorporating features that are desirable for generating high-quality evidence. This survey performed this investigation and found that too many of the ongoing interventional studies have a small expected sample size. This might lead to delayed recognition of effective therapies and a waste of time and resources. This important evidence should guide the design of any future clinical study on COVID-19 and the decision of any funding body as well as the approval of any bioethics committee. For this reason, I consider this brief report important for the scientific community. There are a few minor suggestions for the authors:\nTable 1: The presentation of the studies' characteristics will be improved if they are presented separately for interventional and observational studies (particularly for the intervention type and expected study size). In this way, the table will provide more information about the studies' characteristics for each study type (interventional and observational).\n\nTable 1: It will be useful to report the type of randomization (such as blocking and block 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? 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": "63547",
"date": "28 May 2020",
"name": "Ioannis Mamais",
"expertise": [
"Reviewer Expertise Molecular 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\nI have read it through, and it seems to be an interesting brief report.\nSome comments below:\n\n74.2% of those studies are mention as \"Randomized\" and 40% as blinded, from researchers. Authors of this brief report can \"discuss\" those numbers and prepare readers that most of those may have a high risk of bias (maybe with no control group, or bias arising from the randomisation process, etc).\n\n\"Additional effort is needed to ensure that minors are included in COVID-19 clinical research, so that therapeutic decisions are based upon high-quality evidence\". I am not very sure if it is necessary to discuss this in the brief report\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Not applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
}
] | 1
|
https://f1000research.com/articles/9-373
|
https://f1000research.com/articles/9-367/v1
|
15 May 20
|
{
"type": "Case Report",
"title": "Case Report: Hurler syndrome (Mucopolysaccharidosis Type 1) in a young female patient",
"authors": [
"Sadaf Saleem Sheikh",
"Dipak Kumar Yadav",
"Ayesha Saeed",
"Sadaf Saleem Sheikh",
"Ayesha Saeed"
],
"abstract": "Hurler syndrome is a rare autosomal recessive disorder of mucopolysaccharide metabolism. Here, we present the case of a young female patient who presented with features of respiratory distress. In addition, the patient had gingival hypertrophy, spaced dentition, misaligned eruptive permanent dentition, microdontia, coarse facial features, low set ears, depressed nasal bridge, distended abdomen, pectus carinatum, umbilical hernia and J-shaped Sella Turcica on an X-ray of the skull. A diagnosis of Hurler syndrome (Mucopolysaccharidosis Type I) was made. The patient was kept on ventilator support from the third day; however, she died on the fifth day of admission. Enzyme replacement modality of treatment can increase a patient's survival rate if an early diagnosis can be made. To the best of our knowledge, only a few cases of Hurler syndrome have been reported in Pakistan.",
"keywords": [
"Hurler syndrome",
"mucopolysaccharide",
"enzyme",
"genetic"
],
"content": "Introduction\n\nMucopolysaccharidosis (MPS) represent a set of metabolic disorders, which are autosomal inherited disorders. MPS are lysosomal storage disorders that occur as a result of the deficiency of one group of enzymes, which degrade three classes of mucopolysaccharides: heparan sulphate, dermatan sulfate, and keratan sulfate1. MPS occurs due to mutations in the gene encoding human α-L-iduronidase2. Glycosaminoglycans (GAGs) accumulate chronically and progressively in the lysosomes of the cells throughout the body. Such accumulation of GAGs leads to multiorgan dysfunction and significant morbidity. Patients with Hurler’s syndrome (MPS Type 1) experience progressive debilitation of the musculoskeletal, cardiorespiratory, and central nervous systems, leading to death before 10 years of age if they remain untreated. There are very few cases of Hurler syndrome reported in Pakistan especially in last few years. Here, we present the case of Hurler’s syndrome in a young female patient.\n\n\nCase report\n\nAn 8-year-old female patient was referred to the Children’s Hospital, Faisalabad, Pakistan with complaints of respiratory distress, massive abdominal distension, and generalized body swelling. According to her parents, developmental milestones slowed down at the age of 1.5 years with gradual mental decline expressed in terms of lost physical skills. The parents mentioned multiple joint stiffnesses and that the patient was having difficulty in walking for last few years and was unable to walk in the last year of her life. Though they were unaware of any hearing difficulties in the patient, they reported that she was not able to speak. The patient had noisy breathing noticeable since the third day of birth. In the past few years, the patient had multiple chest infections and had been constipated for around the last 3–4 months before hospital admission. Family history revealed that the parents had a consanguineous marriage (first cousins), and the patient had one older brother who had no obvious mental and physical abnormalities. This was the patient’s first tertiary hospital visit.\n\nGeneral physical examination revealed coarse facial features, hirsutism, low set ears, depressed nasal bridge, rotated legs, talipes varus, short neck, distended abdomen, pectus carinatum, thick short claw-like hands, umbilical hernia, and kyphosis (Figure 1). Abdominal examination revealed massive hepatosplenomegaly. On cardiac auscultation, a murmur was not heard but loud P2 was audible. Moreover, on chest auscultation, bilateral crepitations were present. On oral examination, gingival hypertrophy, spaced dentition, misaligned eruptive permanent dentition, microdontia, delayed eruption of the permanent tooth and delayed shedding of the deciduous tooth were observed (Figure 2).\n\nChest X-ray showed cardiomegaly and oar shaped ribs (Figure 3). X-ray of the patient’s skull showed J-shaped Sella Turcica (Figure 4), while X-ray of her hand showed proximal pointing of metacarpals (Figure 5), and X-ray of the lumbosacral spine showed inferior beaking of vertebrae (Figure 6). The patient’s echocardiogram showed normal left ventricular function, and hematological investigations were all in the normal range.\n\nA differential diagnosis of Hunter syndrome (MPS Type II; MPS-II) and Sly syndrome (MPS-VII) was made. However, after clinical analysis and imaging findings, a provisional diagnosis of Hurler syndrome was made. The patient’s MPS urine was 1673 mL MPS/g creatinine (normal levels, 116.4–324.4 mL MPS/g creatinine). The patient’s α-L-iduronidase activity in the leukocytes was non-detectable (normal levels, 0.105–0.327µmol phenol per 18 hours per mg protein). A final diagnosis of Hurler syndrome (MPS-I) was confirmed after the result of urinary excretion of MPS and an enzyme assay.\n\nOn arrival the patient was having difficulty breathing. She was supplied with oxygen through nasal prongs and was managed conservatively. On the third day, the patient was intubated and kept on ventilatory support. On diagnosis of Hurler syndrome (MPS-I), the patient’s parents were advised for further management; however, they denied further management because of socio-economic reasons. The patient died on her fifth day of hospital stay.\n\n\nDiscussion\n\nHurler syndrome manifests as an autosomal recessive disorder of mucopolysaccharide metabolism. There is an excess accumulation of lipids in the central nervous system as well as other viscera. This condition manifests in early infancy. Patients have developmental retardation, an expressionless face, and increase in the size of the head with deformed shape. They also have various skeletal abnormalities, contracture on flexion, hernias, and an increase in the size of the liver and spleen. Corneal clouding can be found in some patients. Hurler syndrome is a metabolic disorder of mucopolysaccharide metabolism to defect in lysosomal degradation pathways. It is seen in approximately 1:100,000 live births3.\n\nHurler syndrome is diagnosed based on the reduced level of α-L-iduronidase activity in leukocytes or cultured fibroblasts4. Prenatal diagnosis is confirmed by the presence of unusual glycosaminoglycan components in the amniotic fluid, or abnormal metabolic activity in cultures amniotic fluid cells, and a deficiency of the lysosomal enzyme α-L-iduronidase in these cell homogenates5. Along with symptomatic treatment, enzyme replacement therapy with α-L-iduronidase, as well as bone marrow transplantation, increases the probability of life expectancy6. Couples who have a positive family history must be provided with genetic counseling and testing.\n\n\nConclusion\n\nHurler syndrome is a rare genetic disorder of mucopolysaccharide metabolism and is caused by a defect in lysosomal degradation pathways. For patients with MPS-I, enzyme replacement modality of treatment can increase the patient's survival rate if an early diagnosis can be made.\n\n\nConsent\n\nWritten informed consent was obtained from the father of the patient for the publication of this case report and 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\nScott HS, Litjens T, Hopwood JJ, et al.: A common mutation for mucopolysaccharidosis type I associated with a severe Hurler syndrome phenotype. Hum Mutat. 1992; 1(2): 103–108. PubMed Abstract | Publisher Full Text\n\nBach G, Moskowitz SM, Tieu PT, et al.: Molecular analysis of Hurler syndrome in Druze and Muslim Arab patients in Israel: multiple allelic mutations of the IDUA gene in a small geographic area. Am J Hum Genet. 1993; 53(2): 330–8. PubMed Abstract | Free Full Text\n\nLowry RB, Renwick DH: Relative frequency of the Hurler and Hunter syndromes. N Engl J Med. 1971; 284(4): 221–222. PubMed Abstract | Publisher Full Text\n\nManley G, Hawksworth J: Diagnosis of Hurler's syndrome in the hospital laboratory and the determination of its genetic type. Arch Dis Child. 1966; 41(215): 91–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHenderson HE, Nelson MM: Antenatal diagnosis of Hurler's syndrome. S Afr Med J. 1977; 51(8): 241–243. PubMed Abstract\n\nKirkpatrick K, Ellwood J, Walker RW: Mucopolysaccharidosis type I (Hurler syndrome) and anesthesia: the impact of bone marrow transplantation, enzyme replacement therapy, and fiberoptic intubation on airway management. Paediatr Anaesth. 2012; 22(8): 745–751. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "69761",
"date": "07 Sep 2020",
"name": "Sanghamitra Satpathi",
"expertise": [
"Reviewer Expertise Pathology"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nCase report is well written besides few lacuna.\nThe authors have diagnosed the case with urinary test and demonstration of enzyme deficiency, but could not do genetic testing. This can be mentioned.\n\nIn the discussion a general description about Hurler syndrome is given, but nothing has been mentioned about the present case like whether the case has classical presentation or any other deviation.\n\nThe authors have mentioned about bone marrow transplantation, but actually nowadays stem cell transplantation is the treatment of choice which should be done early preferably before 2.5 years.\n\nThe case report can be accepted with corrections.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly",
"responses": []
},
{
"id": "70716",
"date": "21 Sep 2020",
"name": "Alla N. Semyachkina",
"expertise": [
"Reviewer Expertise hereditary diseases",
"storage 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 article is devoted to one of the most common forms of storage diseases - mucopolysaccharidosis type I - Hurler syndrome. The authors note that for the Pakistani population, Hurler syndrome is a rare disease, and on this basis, the publication of this case is undoubtedly relevant.\nIn the introduction, the authors give literary information about this form of mucopolysaccharidosis.\nThe clinical results describe in great detail the patient's phenotype and the data of the clinical, laboratory and X-ray examinations. The results obtained are perfectly illustrated by the attached figures, which undoubtedly increases the practical significance of the article. However, there are a number of remarks to the scientific fragment of the article: the authors note that the diagnosis was confirmed on the basis of a study of the GAG fractionation indices and the activity of the lysosomal enzyme alpha-L-iduronidase, but the results of urinary glycosaminoglycan excretion were not presented in the article.\nIt is regretful that molecular genetic studies have not been carried out, namely the search for mutations in the IDUA gene. Previous articles by Pakistani authors indicate a high frequency of occurrence of the variant p. L490P among the Pakistani population, and it has even been suggested that this option be included in Pakistan's newborn screening program. If possible, the authors of the article should be advised to conduct a molecular genetic examination of the parents and siblings of the dead girl. These data will undoubtedly increase the scientific value of the article.\nIn the Discussion section, which provides data on the prenatal diagnosis of Hurler syndrome, the authors cite outdated methods of antenatal diagnosis. Currently, all over the world, methods of DNA diagnostics of chorionic biopsy performed at 10-11 weeks of gestation are used. The advantage of this method is in the early diagnosis of pathology, when the pregnancy can still be terminated (at the request of the parents) by means of medical abortion, and not premature birth.\nThus, the peer-reviewed article is especially relevant for practitioners, and in the current version can be published in the section titled \"Notes and observations from practice.\"\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes",
"responses": []
},
{
"id": "70715",
"date": "28 Sep 2020",
"name": "Fortunato Lonardo",
"expertise": [
"Reviewer Expertise Clinical genetics",
"Molecular genetics",
"Prenatal diagnosis",
"Genetic counseling"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors describe a case of Hurler syndrome in an 8-year-old girl. The clinical description is good and well supported by the images. It may be useful to index this article, but it is necessary to make some corrections.\nIn the Introduction the authors describe mucopolysaccharidosis in an incomplete way, neglecting the recessive X-linked transmission form (MPS II), and stating that they are due to mutations in the gene encoding for alpha-L-hyduronidase, which only concerns MPS I. Furthermore, no mention is made of the subdivision within MPS I into a severe form (Hurler syndrome) and an attenuated form (Hurler-Scheie syndrome and Scheie syndrome). The distinction is important because it reflects on the therapeutic choices.\nFor the definitive diagnosis of MPS I it is sufficient to demonstrate the enzymatic deficit, but it would be appropriate to add also the molecular examination of the IDUA gene, as the results would also be useful in the family for the prevention of the risk of recurrence. Since the test was not performed on the patient, it would be appropriate to perform it on the parents, and then extend it to family members, starting from those of first degree.\nIn the Discussion the authors cite outdated methods of prenatal diagnosis. Currently Prenatal testing is possible for pregnancies at increased risk for MPS I by measuring α-L-iduronidase enzyme activity in cultured cells obtained by amniocentesis (usually performed at ~15-18 weeks' gestation) or CVS (at ~10-12 weeks' gestation). Difficulty with prenatal diagnosis for MPS I may result from the low α-L-iduronidase enzyme activity in normal chorionic villi; however, difficulties in interpreting borderline low α-L-iduronidase enzyme activity can be overcome by assaying enzyme activity in cultured rather than uncultured CVS cells, provided analysis for possible maternal contamination is performed.\nWith regard to treatment options, the authors correctly cite enzyme replacement therapy, while haematopoietic stem cell transplantation is preferred to bone marrow transplantation cited by the authors.\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": []
}
] | 1
|
https://f1000research.com/articles/9-367
|
https://f1000research.com/articles/7-1405/v1
|
04 Sep 18
|
{
"type": "Research Article",
"title": "The signs of adaptive mutations identified in the chloroplast genome of the algae endosymbiont of Baikal sponge.",
"authors": [
"Sergey Feranchuk",
"Natalia Belkova",
"Lubov Chernogor",
"Ulyana Potapova",
"Sergei Belikov",
"Natalia Belkova",
"Lubov Chernogor",
"Ulyana Potapova",
"Sergei Belikov"
],
"abstract": "Background: The study of ecosystems of the great lakes is important as observations can be extended to ecosystems of larger scale. The ecological crisis of Lake Baikal needs investigations to discover the molecular mechanisms involved in the crisis. The disease of Baikal sponges is one of the processes resulting in the degradation of the littoral zone of the lake. Methods: The chloroplast genome fragment for the algae endosymbiont of Baikal sponge was assembled from metagenomic sequencing data. The distributions of polymorphic sites were obtained for the genome fragment, separately for samples from healthy sponge, diseased sponge and dead sponge tissues. Results: The comparative analysis of chloroplast genome sequences suggests that the symbiotic algae from Baikal sponge is close to Choricystis genus of unicellular algae. Also, the distributions of polymorphic sites allowed detection of the signs of extensive mutations in the chloroplasts isolated from the diseased sponge tissues. Conclusions: The study demonstrate the particular case of evolution at the molecular level due to the conditions of a severe crisis of a whole ecosystem in Lake Baikal. The detection of adaptive mutations in the chloroplast genome is an important feature which could represent the behavior of an ecosystem in the event of a severe crisis.",
"keywords": [
"Chlorophyta",
"Lake Baikal",
"Chloroplas Genome",
"Genetic Polymorphism",
"Mutation Rate"
],
"content": "Introduction\n\nLake Baikal, located in Southeastern Siberia, is the largest by volume and the oldest great lake on the planet, and several signs of ecological crisis in Lake Baikal have been observed since 2010–2011 [Bormotov, 2012; Khanaev et al., 2018; Kravtsova et al., 2014; Timoshkin et al., 2016]. One of these signs is severe disease and death of sponges which is now observed in almost all parts of the lake. The symptoms of the disease begin with the appearance of pink and brown spots on the surface of the sponge and terminate with complete destruction of the sponge tissues. The cause of the disease is still unclear.\n\nEndemic freshwater Baikal sponges (Demospongiae, Lubomirskiidae) dominate their biomass among the benthic organisms of the littoral at depths from 3 to 25 m covering 47% of the available surfaces [Pile et al., 1997]. In healthy condition sponges have a green color, mainly explained by the presence of a photosynthetic symbiont, an intracellular coccoid green algae. This algae belongs to the Cholorophyta division, and is close in taxonomy to the Choricystis genus [Chernogor et al., 2013]. It is natural to assume, that the photosynthetic symbiont is the source of feeding for sponge cells. And, the change of color of sponge tissue could indicate that the chloroplasts of the symbiont are damaged in the early stages of the disease. So, precise study of this algae symbiont could be of critical importance in investigating the cause and consequences of sponge disease.\n\nThe sequencing and comparative analysis of chloroplast DNA is a conventional method for detailed study of planctonic algae [Lemieux et al., 2014; Lemieux et al., 2015]. Normally, chloroplast DNA sequences are determined using cultivated algae and de novo assembly of genomic DNA reads [Twyford & Ness, 2017]. But for uncultured species, the chloroplast genome can be obtained using metagenome sequencing [Worden et al., 2012]. For symbiotic algae from Baikal sponges, this strategy is probably more efficient, and the comparative analysis of samples of healthy and diseased sponges can provide a deeper look into the features of chloroplast genome affected by sponge disease. The presence and properties of polymorphic sites on the chloroplast genome could be an effective way to investigate the variations of genome sequence depending on the disease state of the sponge. The study of the distribution of bacterial strains depending on geographic location [Truong et al., 2017] can be mentioned as a precedent, where gene-batteries typical for gut microbiome were compared using the distribution of polymorphic sites in genome sequences, using metagenome sequencing.\n\n\nMethods\n\nThree samples of freshwater sponge Lubomirskia baicalensis were collected from Lake Baikal in the Bol'shiye Koty area (51° 90´ 69 N ´´, 105° 07´ 05 E´´) at a depth of 10 m by scuba divers in June 2016. One sample was obtained from the sponge that was healthy in appearance (exhibiting a green colour), one sample was taken from diseased sponge and one from dead rotten sponge tissues. The collected samples were immediately placed in containers with Baikal water and ice and transported to the lab, maintaining a constant water temperature. For all three samples Illumina pair-end reads were obtained by DNA metagenome sequencing in Novogene Inc. (Illumina PE 150). The extraction and sequencing of RNA in the samples was also performed at Novogene Inc., to represent their metatranscriptome content. This was possible only for healthy and diseased sponge tissues; not enough RNA was extracted from the rotten tissues. The DNA metagenomic reads were processed by a conventional bioinformatics pipeline implemented at Novogene Inc, including the filtering of sequencing errors and the assembly of contigs using SoapDeNovo assembler [Luo et al., 2012]. RNA metatranscriptomic reads were filtered and trimmed using Trimmomatic 0.35 software [Bolger et al., 2014].\n\nThe sequence of chloroplast genome Choricystis parasitica (NC_025539) was used as a template for assembly and for comparative analysis because it is the chloroplast genome closest to the available genomes. For the assembly of the targeted genome, the following steps were performed:\n\n1. Scaffolds were obtained by de novo assembly of each DNA metagenomic sample by conventional utilities with the use of the SoapDeNovo assembler\n\n2. Scaffolds were aligned to the reference sequence of chloroplast using Blastn (evalue threshold 1e-40)\n\n3. The paired-end sequence reads from 3 DNA metagenomics libraries and 2 RNA metatransriptomic libraries were aligned to the selected scaffolds using Bowtie2 (v. 2.2.6)\n\n4. The aligned reads from all samples were collected and assembled de novo using the Inchworm assembler from the Trinityrnaseq 2.6.5 package [Grabherr et al., 2011] with the lowest possible tolerance to sequencing errors and the highest possible value of k-mer size (K=31)\n\n5. The contigs obtained after the de novo assembly by Inchworm were compared with the reference genome; this allowed the selection of a single contig of length 55638 with high homology to the reference genome. It was the only contig which could be reliably identified as a fragment of the chloroplast genome.\n\nOpen reading frames were identified in the obtained contig, and most of the identified proteins are annotated following the annotations of the reference genome. TrnaSCAN 1.4 software [Lowe & Eddy, 1997] was used for identification of the transport RNA in the putative chloroplast sequence, and the locations of 18S rRNA and 23S rRNA were identified by a direct alignment with reference rRNAs using the Mummer 3.23 package [Kurtz et al., 2004].\n\nIn order to separate it from traces of sequencing errors, the selection of the polymorphic sites in the genome was implemented following the approach described in [Truong et al., 2017]. Each of the RNA and DNA samples represented as pair-end reads was separately aligned to the assembled fragment of the chloroplast genome using Bowtie2; the alignments were then processed using the Samtools 1.7 software pipeline with conventional settings, and the approach proposed in [Truong, 2017] was used to identify the polymorphic sites.\n\nDescribing the algorithm, for each position s on the alignment of the reads against the Ns is defined as the total number of reads covering it, and Ts is defined as the number of reads supporting the most abundant allele. Given the sequencing error rate E, the non-polymorphic null hypothesis was rejected if the probability that the number Ns − Ts of reads coming from the non-dominant allele is <α = 0.05. This is estimated using the probability mass function of a binomial distribution with Ns trials and the successful rate 1 − E. The error rate was set to 0.01 for Illumina sequencing. The bases with quality below 30 were removed and the reads with an average identity to the reference below 99% were ignored before applying the statistical test. Failing to reject the null hypothesis reflects the absence of alternative alleles or inability of distinguishing between low-coverage potential alternative alleles and sequencing noise.\n\nThus, the number of polymorphic sites could be counted for each gene. Another property of each gene is the number of polymorphic sites where the count of alternative alleles is higher than the count of dominant allele (Ts < Ns - Ts). This property could detect mutations in the sample genotype and phenotype, for each gene.\n\nThe chloroplast genome sequences of Picocystis salinarum (NC_024828), Myrmecia israelensis (KM462861), Botryococcus braunii (KM462884), Coccomyxa subellipsoidea (NC_015084), Hydrodictyon reticulatum (NC_034655), Mychonastes jurisii (NC_028579) and Chlorella vulgaris (NC_001865) were used for a reconstruction of the phylogenetic trees for the 16S ribosomal RNA (rrs gene) and the ATP synthase subunit beta (atpB gene). The nucleotide sequences of the selected genes were aligned using Mafft 7.27 software [Katoh & Standley, 2013]. The trees were constructed using the FastMe 2.1.5.1 software [Lefort et al., 2015], with the distance-based neighbor-joining method to select tree topology and Jukes-Cantor measure to calculate the distances between genes.\n\nAnalysis was performed using custom scripts in Python 2.7 (see Data and software availability section).\n\n\nResults\n\nThe chloroplast genome of the Choricystis parasitica algae is a circular DNA 94206 base pairs in length. The comparison of open reading frames of the candidate genome fragment from the metagenomic samples with annotated genes of C. parasitica support the statement that this genome fragment of length 55638 is a large part of the chloroplast genome of algae close to the C. parasitica species. Figure 1 illustrates the order of genes in these two related chloroplasts. The comparison of gene sequences shows them to be up to 98% identical in these two species.\n\nUpper track: C.parasitica chloroplast; Lower track: Chloroplast of sponge symbiont. The start position of the C.parasitica chloroplast sequence was changed to fit the location of the fragment shown at the bottom. The rRNA and tRNA locations are shown in brown color for both tracks. Text labels show the locations of several selected genes in both genomes.\n\nThe phylogenetic trees for the two selected genes, 16S ribosomal RNA and ATP synthase beta (Figure 2) in general confirm the conventional relations between Cholorophyta algae [Lemieux et al., 2014; Lemieux et al., 2015]. Figure 2 suggests that the symbiotic algae of L. baikalensis sponge is close in taxonomy to the Choricistys genus.\n\nBars located at the node for the studied chloroplast genome represent the relative number of polymorphic positions, in all 5 studied samples, at the 1:1 scale.\n\nThe bars on Figure 2 which show the proportion of polymorphic positions in the genes of symbiotic algae in metagenomic samples is comparable in scale with the distances between genera. This observation needs discussion, because a timescale which separates the origins of the close genera in Figure 2 implies a much larger timescale than that which could characterize the separation of the chloroplast strains detected in the metagenome. Partially this can be explained by the RNA editing and similar modifications which lead to the accumulation of polymorphic positions.\n\nA different view of the unexpectedly high proportion of polymorphic sites in the metagenomic samples is illustrated in Figure 3. Here, the proportion of polymorphic sites, and the proportion of polymorphic sites with a low abundance of dominant allele (“mutations”) is shown separately for each DNA and RNA sample. The results of Figure 3 are presented separately for each gene frame, and for a whole set of genes.\n\nThe results for each of the annotated genes are show in the bottom part. The upper line presents the integrated results for each sample. The proportions of polymorphic sites and the sites with high levels of alternative alleles (“mutations”) are shown as pie charts, relatively to a total number of polymorphic sites in all samples. The proportion of sites which are polymorphic in some other samples, but not in the given sample, are shown in light blue. The legend on the right shows a color scheme used to represent three types of sites. The circle radius represents a total number of sequencing reads aligned to the gene segment and used to identify polymorphic sites. The scale of the circle radii is transformed for better appearance, to compensate for the high variations in the numbers of aligned reads.\n\nThe proportion of polymorphic sites in the DNA and RNA metagenomes for the sample of healthy sponge tissues reflects the natural situation, where the quantity of matrix RNA in the chloroplast organelle is in general higher than the quantity of DNA. Here the polymorphic sites in the DNA sequences may arise due to natural heterogeneity of chloroplast genomes, but the dominant strain is clearly identified. The number of polymorphic sites in the RNA sequences is slightly higher than in DNA sequences due to RNA editing and other modifications.\n\nIn contrast, in the DNA and RNA samples of the diseased tissue, the quantity of chloroplast DNA is decreased, and the quantity of RNA is decreased even more, reflecting the fact of disease and the low level of chloroplast activity. Importantly, the number of polymorphic sites is sharply higher in the remaining DNA sequences, and the alternative alleles are presented in high proportion. And, for RNA sequences, the dominant allele is present much less than alternative alleles. For the case of dead tissue, where RNA couldn't be extracted, the discussion about the observed number of DNA molecules and the proportion of polymorphic sites is beyond the scope of this study.\n\nThe natural assumption about the diseased but alive tissue is that living cells are desperately trying to survive. Adaptation to a changed environment is the one of the best ways to survive. The accumulation of mutations is a straightforward form of adaptation, and this could be confirmed by the results of Figure 3 for the sample of diseased sponge tissue. The rapid increase of mutations in the genome can be observed in the chart for chloroplast DNA, and as it may be suggested from the chart for chloroplast RNA, that the mutations which help survival are fixed in the cells which still continue to develop.\n\nThe observed signs of extensive mutations in response to severe stress are somewhat controversial when compared to a the widely accepted concept of molecular clocks and a theory of neutral evolution [Kimura, 1968; Margoliash, 1963; Zuckerkandl & Pauling, 1962] where mutations are appear randomly and are independent from the environment. But in several studies the presence of adaptive mutations in response to stress has been detected in certain species, as reviewed in [Rosenberg, 2001; Wright, 2004]. So the present result cannot be treated as completely inadequate, in conditions of severe and unusual crisis of the whole ecosystem.\n\n\nDiscussion\n\nThe signs of the large-scale ecological crisis in Lake Baikal are confirmed from many sources, and ecological crises on such a large scale are rare in the documented history of water ecosystems. Sequencing technologies have appeared only in a recent years, and, to the authors best knowledge, no cases of large and sharp changes in ecosystems have been documented using the tools of molecular biology.\n\nThe importance of Lake Baikal itself as an ecosystem with an unusual diversity of endemic species, and as a glorious source of pure drinking water, is a subject high above the economic and pragmatic reasons which are usually considered in molecular biology studies. However the present results suggest that the conventional approaches of molecular biology may be insufficient to adequately describe situations of ecological crisis. In particular, the observations of rapid accumulation of mutations in the chloroplast genomes in the diseased tissues could indicate that the concept of molecular clocks is inappropriate in rapidly changing ecosystems.\n\nWhat’s more, using the tools of molecular biology to study the Baikal ecosystem has another importance; it is a unique chance to accumulate observations about a rapidly changing environment. Great lakes are themselves simplified cases of large-scale marine ecosystems. The presented results, as a part of all Baikal ecosystem studies, could find application, not only in the challenge of minimizing the consequences of the crisis in Baikal, but also in the possible future global challenges caused by sudden changes in ecosystems of any scale.\n\nIn particular, the reconstructed genome of the symbiotic algae may improve knowledge about a cause of sponge disease, and indirectly narrow the possible strategies to prevent the spread of destruction in the Baikal ecosystem. The presented description of the genome may be helpful in the evolutionary studies of marine and freshwater Cholorophyta algae.\n\n\nData availability\n\nThe nucleotide sequence of the chloroplast genome fragment is deposited to Genbank under the accession number: MH591948\n\nNucleotide sequences have also been deposited with the European Nucleotide Archive (ENA) of the European Molecular Biology Laboratory-European Bioinformatics Institute (EMBL-EBI) under study number: ERP110335.\n\n\nSoftware availability\n\nThe sequencing reads and source codes of scripts sufficient to reproduce the presented results are available from GitHub: https://github.com/sferanchuk/bsponge_chloroplast\n\nArchived source code at time of publication available at: https://doi.org/10.5281/zenodo.1326765 [Feranchuk, 2018].\n\n(License: CC BY 4.0).\n\nCustom scripts on Python (v 2.7) were used to run the pipeline and present the results. Python libraries pysam (0.14.1), biopython (1.66) and matplotlib (2.2.2) are required to run the scripts.",
"appendix": "Grant information\n\nThis study was supported by the Ministry of Education and Science of the Russian Federation by Government contract project no. 0345-2015-0002, “Molecular Ecology and Evolution of Living Systems of Central Asia in Terms of Fishes, Sponges, and the Microbial Flora Associated with Them” [VI.50.1.4], and the Russian Foundation for Basic Research [16-04-00065, 16-54-150007, 18-04-00224].\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe thank Dr. Colin Brown for valuable help in the work presented in the manuscript; and we thank Dr. Dmitry Kuzmin and Vadim Sharov from the Siberian Federal University for assistance in the data processing.\n\n\nReferences\n\nBolger AM, Lohse M, Usadel B: Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014; 30(15): 2114–20. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBormotov AE: What has happened to Baikal sponges? SCIENCE First Hand. 2012; 2: 32–35. Reference Source\n\nChernogor L, Denikina N, Kondratov I, et al.: Isolation and identification of the microalgal symbiont from primmorphs of the endemic freshwater sponge Lubomirskia baicalensis. (Lubomirskiidae, Porifera). Eur J Phycol. 2013; 48(4): 497–508. Publisher Full Text\n\nFeranchuk S: Supplement scripts and data files for the manuscript entitled \"The signs of adaptive mutations identified in the chloroplast genome of the algae endosymbiont of Baikal sponge.\" (Version august 2018). Zenodo. 2018. http://www.doi.org/10.5281/zenodo.1326765\n\nGrabherr MG, Haas BJ, Yassour M, et al.: Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol. 2011; 29(7): 644–52. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKatoh K, Standley DM: MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol. 2013; 30(4): 772–80. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKhanaev IV, Kravtsova LS, Maikova OO, et al.: Current state of the sponge fauna (Porifera: Lubomirskiidae) of Lake Baikal: Sponge disease and the problem of conservation of diversity. J Great Lakes Res. 2018; 44(1): 77–85. Publisher Full Text\n\nKimura M: Evolutionary rate at the molecular level. Nature. 1968; 217(5129): 624–626. PubMed Abstract | Publisher Full Text\n\nKravtsova L, Izhboldina LA, Khanaev IV, et al.: Nearshore benthic blooms of filamentous green algae in Lake Baikal. J Great Lakes Res. 2014; 40(2): 441–448. Publisher Full Text\n\nKurtz S, Phillippy A, Delcher AL, et al.: Versatile and open software for comparing large genomes. Genome Biol. 2004; 5(2): R12. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLefort V, Desper R, Gascuel O: FastME 2.0: A Comprehensive, Accurate, and Fast Distance-Based Phylogeny Inference Program. Mol Biol Evol. 2015; 32(10): 2798–800. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLemieux C, Otis C, Turmel M: Chloroplast phylogenomic analysis resolves deep-level relationships within the green algal class Trebouxiophyceae. BMC Evol Biol. 2014; 14: 211. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLemieux C, Vincent AT, Labarre A, et al.: Chloroplast phylogenomic analysis of chlorophyte green algae identifies a novel lineage sister to the Sphaeropleales (Chlorophyceae). BMC Evol Biol. 2015; 15: 264. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLowe TM, Eddy SR: tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res. 1997; 25(5): 955–64. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLuo R, Liu B, Xie Y, et al.: SOAPdenovo2: an empirically improved memory-efficient short-read de novo assembler. GigaScience. 2012; 1(1): 18. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMARGOLIASH E: Primary Structure and Evolution of Cytochrome C. Proc Natl Acad Sci U S A. 1963; 50: 672–679. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPile AJ, Patterson MR, Savarese M, et al.: Trophic effects of sponge feeding within Lake Baikal's littoral zone. 2. Sponge abundance, diet, feeding efficiency, and carbon flux. Limnol Oceanogr. 1997; 42(1): 178–184. Publisher Full Text\n\nRosenberg SM: Evolving responsively: adaptive mutation. Nat Rev Genet. 2001; 2(7): 504–515. PubMed Abstract | Publisher Full Text\n\nTimoshkin OA, Samsonov DP, Yamamuro M, et al.: Rapid ecological change in the coastal zone of Lake Baikal (East Siberia): Is the site of the world’s greatest freshwater biodiversity in danger? J Great Lakes Res. 2016; 42(3): 487–497. Publisher Full Text\n\nTruong DT, Tett A, Pasolli E, et al.: Microbial strain-level population structure and genetic diversity from metagenomes. Genome Res. 2017; 27(4): 626–638. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTwyford AD, Ness RW: Strategies for complete plastid genome sequencing. Mol Ecol Resour. 2017; 17(5): 858–868. PubMed Abstract | Publisher Full Text\n\nWorden AZ, Janouskovec J, McRose D, et al.: Global distribution of a wild alga revealed by targeted metagenomics. Curr Biol. 2012; 22(17): R675–7. PubMed Abstract | Publisher Full Text\n\nWright BE: Stress-directed adaptive mutations and evolution. Mol Microbiol. 2004; 52(3): 643–50. PubMed Abstract | Publisher Full Text\n\nZuckerkandl E, Pauling LB: Molecular disease, evolution, and genic heterogeneity. In Kasha M. and Pullman B (eds). Horizons in Biochemistry. New York: Academic Press, 1962; 189–225. Reference Source"
}
|
[
{
"id": "42738",
"date": "08 Jan 2019",
"name": "Michael G. Sadovsky",
"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\nWell, on one hand I should say ``Yes’’ to this submission. At least, it completely meets all the up-to-date customs and observances in genomics and molecular biology. On the other hand, the authors rely on a number of (quite complex and apparent) software packages, workbenches and pipelines, and this is the matter of my general scepticism. Actually, we all become the hostages of those software tools; one has to trust software designers and believe the output of the programs is correct, free of mistakes, stably working, etc. Somebody may say ``You, physicists and mathematicians, do use Wolfram’s Mathematica and there is no collapse in math, nor in physics’’. Reciprocally, I would like to draw attention to an Elsevier journal devoted to Microsoft Excel errors solely (McCullough and Heiser, 20071).\nI am far from the idea to say that the study is wrongly arranged or badly accomplished; I just want to stress the point that there should be some special efforts done to ensure the results are at least stable. For example, what happens with assembled contigs, if we randomly remove a small part of reads? If a series of runs of an assembler yields (almost) the same contigs set, then the results could be used for further analysis. The problem arises, if one gets a number of sets of contigs with a sounding difference between them. The paper has no answer on that point; a comparative study (like that one presented by the authors) should have some proofs of the absence of artefacts affecting the comparison of fine differences between biological objects involved in the study. Meanwhile, I pretty well understand that such output testing falls beyond the customs and habits of NGS sequenced data treatment and I am in the smallest minority. So, from that point of view the paper completely meets all the custom data treatment procedures and in such capacity should be recommended for indexing.\nAnother important issue of the paper is that it presents an attempt to tie together ecological (environmental) processes, and some genetic background that may stand behind. Here the word `crisis’ used by the authors makes a point: regularly, ecological crisis is stipulated as a rather fast running process in a community resulting in serious (and inevitable) loss of the greater part of species from the community. Maybe, this word is too strong here: what if the observed infection intrusion is just a regular (while long ranged) periodic event in the community? Nonetheless, the scientific merit of the paper is obvious, the results and conclusions are sounding and up-to-date, and paper should be indexed.\n\nThe paper needs major revisions in its English. The paper is written in a version I dare say is Runglish. There are too many lines in the manuscript that look like a literal translation from Russian of (quite boring) scientific Russian-style. I myself can decipher what the authors mean, since my mother language is also Russian. I am absolutely sure that the current version of the paper will fall out of comprehension for the greatest majority of readers who have no active Russian. To begin with, the title must be changed. No signs, at all. The correct version should be like ``Evidences of the adaptive mutations in chloroplast genomes of some algae endosymbionts of Baikal sponge’’.\nSame in the Abstract (Background paragraph): instead of “The study of ecosystems of the great lakes is important as observations can be extended to ecosystems of larger scale. The ecological crisis of Lake Baikal needs investigations to discover the molecular mechanisms involved in the crisis. The disease of Baikal sponges is one of the processes resulting in the degradation of the littoral zone of the lake” there should be something like “Monitoring and investigation of the great lakes ecosystem provides a sounding background to forecast the greater scale ecosystem dynamics. Changes in the Baikal lake biota observed nowadays demand deeper investigations of the molecular mechanisms standing behind these former. The endemic Baikal sponge disease may cause a degradation of littoral ecosystem of the lake”. I am far from the idea that my version is the best, but the original one must be rewritten.\nUnfortunately, there are many more similar problem lines in the manuscript, so very strong revisions in the English are absolutely necessary.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "5393",
"date": "14 May 2020",
"name": "Sergey Feranchuk",
"role": "Author Response",
"response": "I'm grateful to Prof. Sadovsky for his decision to review this manuscript. I did carefully consider his remarks and prepared a revised version with a respect to his position.First of all, he was right that crises like the crisis on Baikal could anyway happen in the past. The need to survive in the times of severe crises can be encoded in genome. This idea was introduced to the revised version, as an additional support to the hypothesis about adaptive mutations.To answer the remark about \"closeness\" and insufficient robustness of the software, I did several other runs of the assembly. I agree with Prof. Sadovsky about \"closeness\" and over-complication of some software, and this is why I did choose Inchworm assembler in the initial version of the pipeline, as the most lightweight and straightforward of the available assemblers. In additional runs I tried another assemblers. The correctness of the assembled chloroplast sequence was anyway confirmed, and the fact of verification was pointed out in the second revision.To answer the remark about \"Russian\" style of language. This question is in part beyond the scope of the discussion. It is unlikely that me who is Russian will speak the same English as a man from England. But Prof. Sadosvky was right that the meaning of the text in the first edition was unclear in many parts. And in the revised version I put much more attention to a choice of words and grammatic constructions, to use only those words, for which I am certain in their meaning. The text can anyway look unusual to one who know in perfect the context of all words in English, but at least I do my best to make the meaning of the text the most clear."
}
]
},
{
"id": "42769",
"date": "23 Jan 2019",
"name": "Roman Kondratov",
"expertise": [
"Reviewer Expertise Gene expression",
"biological rhythms",
"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 massive presence of sponges is known as playing a leading role in the process of biofiltration of Baikal's water. Recently the cases of sponge disease have been expanded rapidly. The significance of the current study is coming from the attempt to investigate molecular aspects of sponge disease using a systems biology approach. In the present submission the authors provide data on the genome and transcriptome of chloroplasts from symbiotic algae. Metagenomic and metatranscriptomic sequencing data was used for a template-based assembly of the chloroplast genome of algae, and a mapping of sequencing reads to the obtained genome sequence was a subject of interpretation, based on a detection of polymorphic sites. The advantage of the study is the exploration of natural samples, improvement of methods of bioinformatical analysis and provocative ideas. I think that the manuscript deserves to be indexed, but there are concerns which the authors need to address:\n\nWhat was the number of analyzed biological replicas or independent samples? Do the authors expect the same spectra if another sample(s) of sponge tissues would be processed with the same pipeline? What would be the distribution of variations in polymorphic sites in another series of experiments?\n\nWere the “healthy”, “diseased” and “dead” samples collected from the same spot? How can the authors be sure that they are dealing with the same species? Could it be that what the authors proposed as “adaptive mutations” is in fact a difference between different subspecies?\nI will recommend to tone down the conclusions and to discuss possible alternative interpretations. The statement on a presence of adaptive mutations in the chloroplast genome is too strong. It requires confirmation with more independently obtained samples. There is also no evidence that even if these mutations will be confirmed, that they are “true adaptive” mutations and provide the organism with any physiological advantage. Therefore the title is misleading in my opinion.\nI suggest also to provide more details of the overall design of the study and on novelty in bioinformatics approaches.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "5394",
"date": "14 May 2020",
"name": "Sergey Feranchuk",
"role": "Author Response",
"response": "Many thanks to Prof. Kondratov that he had found a possibility to read carefully the manuscript, so that he had found deep and precise issues in which where the proposed results can be doubted. I did consider his remarks in a most full extent.I respect the experience of Prof. Kondratov who did suggest a presence of several subspecies of algae in a sponge host. In the revised version I explicitly told about possible presence of subspecies in the samples and add an extra chart which shows the distribution of algae subspecies around Baikal. And I did explicitly specify some of most important details of the study, and did remove some extra material from the second edition, trying to keep the format and to put more accents on the main of the declared results.I did change the declared statement of the manuscript, to propose a proof of a possibility rather than a proof of an observation. In the revised version I tried to provide a more explicit \"proof of possibility\", that the adaptation can be just one of the many contributions to the observed distribution of polymorphic sites. And I add an additional argumentation why just a possibility of the adaptation is worth to be presented as a scientific result. In short, the situation around Baikal is complicated, and just an idea how this complication can be resolved is worth to be said."
}
]
}
] | 1
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https://f1000research.com/articles/7-1405
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https://f1000research.com/articles/9-36/v1
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23 Jan 20
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{
"type": "Research Article",
"title": "Determinants of prenatal depression among women attending the antenatal clinic at a referral facility in Mombasa County, Kenya: a case control study",
"authors": [
"Harriet K. Mirieri",
"Marshal M. Mweu",
"Joyce M. Olenja",
"Marshal M. Mweu",
"Joyce M. Olenja"
],
"abstract": "Background: Despite prenatal depression being a public health burden and the major predictor of postnatal depression, it has not received as much attention as postnatal depression in research and policy globally. There is limited evidence on the factors associated with prenatal depression and therefore understanding these factors will inform the design of specific interventions and formulation of guidelines for the effective prevention and control of prenatal depression particularly in high-risk regions. Methods: A hospital-based case control study design was used to identify the determinants of prenatal depression among 170 women attending an antenatal clinic. Prenatal depression was assessed using the Edinburgh Postnatal Depression Scale (EPDS). A semi-structured questionnaire was administered to collect data on the socio-demographic, social network and family, lifestyle and obstetric characteristics of the participants. All eligible cases were enrolled into the study while a simple random sample of depression-free women attending the antenatal clinic were enrolled as controls. The relationship between the predictors and prenatal depression was evaluated by logistic regression. Results: In the multivariable analysis, only marital status (adjusted odds ratio (aOR)=17.1; 95% confidence interval (CI):4.0-73.0), occupation (aOR=2.4; 95% CI:1.4-4.2), domestic violence (aOR=18.3; 95% CI: 5.7-58.7) and social support (aOR=0.2; 95% CI:0.05-0.8) were identified as significant determinants of prenatal depression. Conclusion: Marital status, occupation, domestic violence and lack of social support were the major predictors of prenatal depression in this setting. There is therefore need to implement screening for prenatal depression among pregnant women in health facilities as part of the routine antenatal care package, establish social support networks and spaces to provide an avenue for the prenatally depressed women to meet, share challenges and coping mechanisms and revise the government policy on sexual and gender based violence (SGBV) so as to strengthen efforts towards elimination of all forms of SGBV.",
"keywords": [
"Prenatal depression",
"Edinburgh Postnatal Depression Scale",
"Determinants",
"Case control"
],
"content": "Introduction\n\nThe prevalence of depression in women is about 20% with pregnancy increasing the susceptibility to depression1. Depression related to child bearing can develop either during pregnancy (prenatal depression), after birth (postnatal depression) or both (perinatal depression)2. Prenatal depression refers to a form of clinical depression which occurs during pregnancy and is characterized by chronic anxiety, insomnia, guilt, fatigue, irritability, forgetfulness, headaches and isolation3. Despite prenatal depression being a significant health problem, regrettably, it has received less attention than postpartum depression4–6. This is partly attributable to misconceptions about existing socio-cultural structures that could shield one from mental disturbance during this period4. Besides, more attention is paid to the physical health of the mother and fetus than on maternal mental health during pregnancy, with a propensity to dismiss emotional episodes as the result of hormonal imbalances. Thus, depression may persist silently during this period7.\n\nBesides prenatal depression being a major determinant of post-natal depression, it is also a risk factor for adverse maternal and fetal outcomes such as premature births, preterm labor, low birth weight and poor infant feeding patterns8–10. Moreover, prenatal depression is associated with impaired neurocognitive and socio-developmental disorders in the offspring such as poor motor and regulation skills, anti-social behavior and increased risk of depression and attention problems11 Additionally, higher health expenses, poor immunization rates and frequent hospitalization have also been reported in children who are born to depressed mothers12.\n\nPredictors of prenatal depression can be categorized into three domains: social, psychological and biological risk factors. Social risk factors comprise low socio-economic status, lack of social support and stressors such as economic deprivation and unplanned pregnancy13. Women from a low socio-economic class are likely to have fewer financial resources which may be insufficient to meet the increasing financial demands of pregnancy and this may result to prenatal depression12. Likewise, women who lack social support are likely to have little emotional support from their spouses, family and friends and this can lead to social instability which subsequently leads to prenatal depression14. Gestational age, maternal age, genetic and hormonal susceptibility and obstetric complications are some of the biological risk factors for prenatal depression15. Women who are in the first trimester of pregnancy are likely to have higher depression rates than those in the third trimester that are partly ascribable to the first trimester pregnancy symptoms like fatigue, nausea, food aversions and heartburn, which most women find difficult to cope with16. A history of stillbirth or miscarriage can be traumatic and may result in anxiety or depression14,17,18. The main psychological and psychiatric predictors of prenatal depression are history of mental or anxiety disorder19.\n\nGiven the paucity of research on prenatal depression in Kenya there is need to understand the predictors of prenatal depression with a view to informing the design of specific interventions and formulation of guidelines for the effective prevention, control and surveillance of prenatal depression particularly in high-risk regions in Kenya. Consequently, the objective of this study was to investigate the sociodemographic, lifestyle, obstetric and social network and family determinants of prenatal depression among women attending the antenatal clinic (ANC) at a referral facility in Mombasa County, Kenya.\n\n\nMethods\n\nA hospital-based case control study design was used to identify the determinants of prenatal depression. The choice of study design owes to its suitability in the investigation of rare outcomes that may be missed through random sampling. Although a population-based study would have been more optimal, a hospital-based design was selected due to the ease of recruitment of pregnant mothers (cases and controls) presenting to the antenatal clinic for care.\n\nThe study was conducted at the Coast Provincial General Hospital (CPGH) which is a level five public health facility located in Mombasa County, Kenya. The facility’s total catchment population is roughly four million people and includes the neighboring Coastal counties20. The ANC clinic visits are scheduled monthly with the average number of clinic visits per pregnant woman being four. The number of first-time antenatal visits per month is on average 140. Notably, prenatal depression is not screened for during ANC visits.\n\nThe study population comprised pregnant women ≥15 years attending routine ANC at CPGH during the data collection period between April and June 2019. All pregnant women who consented to participate were included in the study. Women who had already been diagnosed with depression prior to pregnancy or had concurrent chronic illnesses were excluded from the study.\n\nA case was a pregnant woman aged ≥15 years residing in the hospital’s catchment area and had been attending the ANC clinic at CPGH during the two-month study period and registered an Edinburgh Postnatal Depression Scale (EPDS) score of ≥1321. All cases meeting this definition were prospectively recruited into the study. Considering prenatal depression was not routinely screened for during ANC visits, the EPDS tool was completed in the triage room just before administration of routine ANC services.\n\nControls were pregnant women similarly defined as cases but with EPDS scores of <1322 presenting to the same ANC clinic for care. Owing to the comparably large number of controls, these were simple randomly sampled and frequency-matched to cases by day of presentation.\n\nThe appropriate sample size was estimated as per Kelsey et al.23 for case control studies:\n\n\n\n\n\n\n\n\n\nWhereby:\n\nn1 = number of cases and n2 = number of controls. p1 = proportion of cases with previous history of intimate partner violence (IPV) or domestic violence (primary exposure) and p2 = proportion of controls with previous history of IPV (set at 0.40)24. Of note, Zα/2 (1.96) is the value for two-tailed confidence level of 95% and Z1–β (-0.84) is the value for a statistical power of 80%. The odds ratio for the IPV-prenatal depression association was hypothesized to be 3.4124. To enhance statistical power, a ratio of 1:4 (cases to controls) was employed. Based on these figures, a total sample size of 34 cases and 136 was derived.\n\nThe predictor variables were collected using a semi-structured questionnaire (see Extended data)25 and included demographic characteristics, lifestyle, social network and family risk factors and obstetrical factors. The questionnaire has not been validated. The demographic predictors included age, level of education, occupation and marital status. Social network and family related predictors consisted of social support and domestic violence. Lifestyle factors comprised smoking, use of alcohol and substance abuse. Obstetric factors were unplanned pregnancy, gestational age, history of still birth, history of miscarriage/pregnancy loss and parity. Table 1 shows the method of assessment of the study variables. Figure 1 displays the relationship between the predictor variables and outcome.\n\nApproval to conduct the study was obtained from the Kenyatta National Hospital-University of Nairobi (KNH-UON) Ethics and Research Committee (P787/11/2018). Written informed consent was secured from the participants prior to engaging in the study.\n\nFollowing data collection, the questionnaires were manually checked for completeness and accuracy. Data were then double entered by two data entry clerks into an Excel Spreadsheet, after which the resulting datasets were compared and revisions made accordingly. Interviewer bias was minimized by training the research assistants on the standard operating procedures (SOPs) to ensure consistency in elicitation of information from the respondents. In a bid to minimize recall bias information such as the gestational age, the obstetric history and number of antenatal clinic visits was abstracted from the mother and child health booklet.\n\nThe Excel dataset was exported to Stata version 13.0 (Stata Corporation, College Station, Texas, USA) for analysis.\n\nDescriptive statistics (medians, means, standard deviations and inter-quartile ranges) were used to summarize continuous variables. Proportions and percentages were generated for categorical variables. In the univariable analysis, the effect of each predictor on the odds of prenatal depression was assessed using logistic regression at a liberal P-value (P≤0.20)30. Since inclusion of age as a continuous variable was insignificant in the univariable analysis, it was categorized into three groups: 18–25 years, 26–29 years and 30–34 years and reassessed for significance as a categorical variable.\n\nVariables that were found to be significant in the univariable analysis were offered to a multivariable model, where a backward step-wise approach was used to eliminate variables from the model at P>0.05. Notably, the non-significant variables were eliminated from the model if their exclusion from the model did not result in a greater than 30% change in the effects of the remaining variables30. Two-way interactions were fitted between the remaining variables in the final model and their significance assessed. A Hosmer-Lemeshow test was used to assess the goodness of fit of the logistic model, with a P-value of > 0.05 being suggestive of a good fit.\n\n\nResults\n\nA total of 170 pregnant women (34 cases, 136 controls) were enrolled into the study.\n\nA study flow chart illustrating the enrollment process is shown in Figure 2. Table 2 shows the descriptive statistics of the respondents.\n\nThe mean age of the respondents was 27.8 years (range: 18–44 years) with the mean age of cases being 27.0 years (range: 19–36 years) and that of controls being 28.0 years (range: 18–44 years). On the level of education, 44.1% (n=75) of the respondents had attained a tertiary level of education; this comprised 50.0% (n=17) of the cases and 42.7% (n=58) of the controls. Only 45.3% (n=77) of the respondents were employed, of which 26.5% (n=9) were cases and 50% (n=68) were controls.\n\nRespondents who reported to have consumed alcohol, tobacco or abused substances during the pregnancy period constituted 14.7% (n=25) of the population. Amongst these 29.4% (n=10) were cases and 11% (n=15) were controls.\n\nA fifth (20%, n=34) of the respondents reported that the current pregnancy was unplanned. Of these, 38.2% (n=13) were cases while 15.4% (n=21) were controls. Approximately 19% (18.8%, n=32) of the participants reported to have experienced obstetric complications in previous pregnancies. Of these, 26.5% (n=9) were cases and 16.9% (n=23) were controls.\n\nMajority of the participants had received social support (88.2%, n=150). In particular, 61.8% (n=21) of the cases reported to have had social support compared to 94.9% (n=129) of the controls. The proportion of women who experienced domestic violence was 27.6% (n=47), with this proportion being higher among cases at 64.7% (n=22) than in controls at 18.4% (n=25).\n\nOf the factors assessed, only age, marital status, occupation, alcohol and substance abuse, unplanned pregnancy, gestational age, social support and domestic violence were associated with prenatal depression at P≤0.2. (Table 3). These variables were subsequently included in the multivariable model. In the multivariable analysis, only marital status, occupation, social support and domestic violence were shown to be significant predictors of prenatal depression at 5% significance level (Table 4). Exclusion of the non-significant variables from the model did not result in ≥30% change in the effects of the remaining variables.\n\n*Variables eligible for inclusion in the multivariable model (P≤0.20). CI, confidence interval.\n\naOR, adjusted odds ratio; CI, confidence interval.\n\nCompared to participants who were married, those who were single had 17.1 times the odds (adjusted odds ratio (aOR)=17.1; 95% confidence interval (CI): 4.0-73.0) of prenatal depression controlling for their occupation, domestic violence and social support status. Unemployed respondents had 2.4 times the odds of prenatal depression (aOR=2.4; 95% CI: 1.4-4.2) as employed participants holding their marital status, domestic violence experience and social support constant. Participants who experienced domestic violence had 18.3 times the odds of prenatal depression (aOR=18.3; 95% CI: 5.7-58.7) compared to those who did not experience domestic violence regardless of their marital status, occupation and social support level. Respondents who had social support had one-fifth the odds of prenatal depression (aOR=0.2; 95% CI: 0.05-0.8) in comparison to those who did not have social support controlling for their marital status, occupation and domestic violence experience.\n\nThe model had a good fit (P = 0.403).\n\n\nDiscussion\n\nMarital status was shown to be a significant predictor of prenatal depression among women in the study with single women having higher odds of prenatal depression compared to those who were married. This finding is corroborated by other studies17,31. Being single as a result of a break up or abandonment by a partner can result in emotional problems and lack of social support from the male partners and this could lead to depression. Moreover, single parenting is stigmatized in the African culture and this may predispose one to antenatal depression32 .\n\nThis study found that unemployed women had higher odds of prenatal depression compared to their employed counterparts. This finding is similar to that reported by a study in Italy33 which found that participants who were unemployed had 2.17 times the odds of prenatal depression compared to those who were employed. Another study conducted among Japanese women revealed that employment is protective against prenatal depression34. Pregnant women who are unemployed have fewer financial resources which may be insufficient to meet the increasing demands of pregnancy and this may predispose to prenatal depression.\n\nDomestic violence was strongly associated with prenatal depression in this study. These findings are consistent with those from other studies, which described gender-based violence as an important predictor of prenatal depression with women who experience psychological, physical and sexual violence being prone to antenatal depression35–37. Domestic violence may cause physical injury with attendant emotional and psychological trauma that can lead to depression38.\n\nSocial support was protective against prenatal depression in this study. These findings support the results of other studies14,32,39–41. Social support from a spouse, friends or relatives provides psychosocial resources during pregnancy and these act as a cushion against difficulties that may be experienced during pregnancy hence can protect one from antenatal depression. In contrast, women who lack social support are likely to have little emotional support from their spouses, family and friends bringing about social instability which can heighten the risk of prenatal depression42,43.\n\nAge did not significantly influence the likelihood of prenatal depression in this study. However, other studies have revealed that age is a significant predictor of prenatal depression owing to the fact that young pregnant women are likely to be financially unstable and may not be socially and psychologically prepared to cope with pregnancy demands and this may predispose them to depression44. Contrarily, some studies have demonstrated that older women are at a higher risk of developing prenatal depression as ageing increases the possibility of experiencing difficulties in conceiving and anxiety of experiencing obstetric complications. Besides, there is a high likelihood of experiencing stigma when you conceive later in life45,46 .\n\nUse of alcohol and abuse of drugs did not significantly predict a participant’s probability of developing prenatal depression taking into account the effect of other variables. The findings of this study concur with the results of a study conducted among African American women47 On the contrary, other studies have reported a significant relationship between alcohol and drug abuse and prenatal depression31,48,49. Alcohol being a depressant may inhibit neurotransmitters that regulate mood such as serotonin and norepinephrine, and this can lead to depression50.\n\nAfter accounting for other variables, unplanned pregnancy was not found to be significantly related to developing prenatal depression in this study. This is partly ascribable to the fact that although an unplanned pregnancy might be unwanted at first, as the pregnancy progresses the shock associated with the undesired occurrence decreases and it becomes increasingly accepted, hence reducing the symptoms of depression51. Other studies have reported a significant association between unplanned pregnancy and prenatal depression which is related to the fact that unplanned pregnancy is associated with lack of preparedness to deal with the financial and psychological demands of pregnancy35,52,53.\n\nGestational age was not found to be associated with prenatal depression after controlling for other variables. This finding is similar to another study that was conducted in KwaZulu-Natal54. Nonetheless, other studies have demonstrated that women who are in the second or third trimester are less likely to be depressed antenatally compared to women in the first trimester owing to the fact that during the first trimester some women find it difficult to cope with pregnancy symptoms like nausea and food aversions and this can lead to depression7,31,55.\n\nA couple of limitations were present in this study. There was likely to be differential recall of past exposures between the cases and controls with cases having better recall than controls. Moreover, cases were more likely to over-report their exposures and this could bias the effect estimates away from unity. The case definition of prenatal depression only relied on EPDS, which is a screening tool; this could have been supplemented by a clinical examination of the participants to improve on detection of prenatal depression. The results from this study are generalizable to similar settings in other low- and middle-income countries.\n\n\nConclusions\n\nThe present study showed that marital status, occupation, domestic violence and lack of social support were the major predictors of prenatal depression in this setting. There is therefore need to: (1) implement screening for prenatal depression among pregnant women in health facilities as part of the routine antenatal care package, (2) establish social support networks and spaces to provide an avenue for the prenatally depressed women to meet, share challenges and coping mechanisms and (3) review the government policy on sexual and gender based violence (SGBV) so as to strengthen efforts towards elimination of all forms of SGBV and improve the quality of life of the victims.\n\n\nData availability\n\nHarvard Dataverse: prenatal depression CPGH. https://doi.org/10.7910/DVN/QIFMOT.\n\nThis project contains the following underlying data:\n\nPrenatal depression_data.xlsx (containing responses to each question of the questionnaire from all participants).\n\nHarvard Dataverse: prenatal depression CPGH. https://doi.org/10.7910/DVN/QIFMOT.\n\nThis project contains the following extended data:\n\nPrenatal depression_questionnaire.pdf (questionnaire used in this study).\n\nprenatal_depression_code.do (STATA commands file for determinants of prenatal depression evaluation).\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 are grateful to the study participants and staff members of the Coast Provincial General Hospital antenatal clinic for their support throughout the data collection process and their contribution to the success of this study. We also wish to express our appreciation to the Coast Provincial General Hospital administration for authorizing use of the facility for the study.\n\n\nReferences\n\nRich-Edwards JW, Kleinman K, Abrams A, et al.: Sociodemographic predictors of antenatal and postpartum depressive symptoms among women in a medical group practice. J Epidemiol Community Health. 2006; 60(3): 221–7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOgbo FA, Eastwood J, Hendry A, et al.: Determinants of antenatal depression and postnatal depression in Australia. BMC Psychiatry. 2018; 18(1): 49. 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PubMed Abstract | Publisher Full Text\n\nNeter E, Collins NL, Lobel M, et al.: Psychosocial predictors of postpartum depressed mood in socioeconomically disadvantaged women. Womens Health. 1995; 1(1): 51–75. PubMed Abstract\n\nAgnafors S, Bladh M, Svedin CG, et al.: Mental health in young mothers, single mothers and their children. BMC Psychiatry. 2019; 19(1): 112. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMuraca GM, Joseph KS: The Association Between Maternal Age and Depression. J Obstet Gynaecol Can. 2014; 36(9): 803–10. PubMed Abstract | Publisher Full Text\n\nWeobong B, Akpalu B, Doku V, et al.: The comparative validity of screening scales for postnatal common mental disorder in Kintampo, Ghana. J Affect Disord. 2009; 113(1–2): 109–17. PubMed Abstract | Publisher Full Text\n\nLuke S, Salihu HM, Alio AP, et al.: Risk Factors for Major Antenatal Depression among Low-Income African American Women. J Womens Health (Larchmt). 2009; 18(11): 1841–6. PubMed Abstract | Publisher Full Text\n\nAbuidhail J, Abujilban S: Characteristics of Jordanian depressed pregnant women: a comparison study. J Psychiatr Ment Health Nurs. 2014; 21(7): 573–9. PubMed Abstract | Publisher Full Text\n\nZhu SH, Valbo A: Depression and smoking during pregnancy. Addict Behav. 2002; 27(4): 649–58. PubMed Abstract | Publisher Full Text\n\nBanerjee N: Neurotransmitters in alcoholism: A review of neurobiological and genetic studies. Indian J Hum Genet. 2014; 20(1): 20–31. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLee AM, Lam SK, Sze Mun Lau SM, et al.: Prevalence, course, and risk factors for antenatal anxiety and depression. Obstet Gynecol. 2007; 110(5): 1102–12. PubMed Abstract | Publisher Full Text\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\nLeigh B, Milgrom J: Risk factors for antenatal depression, postnatal depression and parenting stress. BMC Psychiatry. 2008; 8: 24. PubMed Abstract | Publisher Full Text | Free Full Text\n\nManikkam L, Burns JK: Antenatal depression and its risk factors: an urban prevalence study in KwaZulu-Natal. S Afr Med J. 2012; 102(12): 940–4. PubMed Abstract | Publisher Full Text\n\nGavin NI, Meltzer-Brody S, Glover V, et al.: Is population-based identification of perinatal depression and anxiety desirable? A public health perspective on the perinatal depression care continuum. Identifying perinatal depression and anxiety: Evidence-based practice in screening, psychosocial assessment, and management: Wiley-Blackwell. 2015; 11–31. Publisher Full Text"
}
|
[
{
"id": "60464",
"date": "13 Mar 2020",
"name": "Linnet Ongeri",
"expertise": [
"Reviewer Expertise I am a psychiatrist and mental health researcher. I have published papers on perinatal depression"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a well written manuscript. However, I have a few comments that may help strengthen the manuscript.\nIn the introduction, the authors state that Depression related to child bearing can develop either during pregnancy (prenatal depression), after birth (postnatal depression) or both (perinatal depression)\". The word both is hanging, consider both periods.\n\nStill in the introduction, the authors state: The main psychological and psychiatric predictors of prenatal depression are history of mental or anxiety disorder\". This is confusing, since anxiety disorder is a mental disorder. Consider correcting to read: The main psychological predictor of prenatal depression is a history of mental disorders.\n\nIn the methods, the study lacks a description of study procedures, specifically: Were the questions administered through interviews, or self reporting? Were the tools translated, what language was used, who administered the tools, where was this done in the hospital, a private room perhaps?\n\nIn the methods, kindly clarify if the social provisions scale and the composite abuse scale were translated and validated for the setting. Have they been used in this setting?\n\nUnder statistical analysis, the authors describe the categorization of ages \"Since inclusion of age as a continuous variable was insignificant in the univariable analysis, it was categorized into three groups: 18–25 years, 26–29 years and 30–34 years and reassessed for significance as a categorical variable.\" What informed this categorization and where were the women older than 34 years included?\n\nIn the discussion: the authors state \"Age did not significantly influence the likelihood of prenatal depression in this study. However, other studies have revealed that age is a significant predictor of prenatal depression owing to the fact that young pregnant women are likely to be financially unstable and may not be socially and psychologically prepared to cope with pregnancy demands and this may predispose them to depression\". The authors may need to elaborate or postulate why this study found no association with age, seeing that other studies have found some link. What is a possible explanation. Perhaps the age range of your sample was not wide enough? Similarly to alcohol and substance use, kindly give an explanation for the negative findings.\n\nUnder limitations the authors state \"The results from this study are generalizable to similar settings in other low- and middle-income countries.\" I believe the authors meant not generalizable. Please correct.\n\nAlso include limitation that this study was a facility based study as opposed to population based. We know majority of depressed ladies may not go for antenatal care, hence some bias in sample selection.\n\nThe conclusion can be considered an over reach. It appears as if the authors did an intervention study that identified these specific interventions to work while instead they have simply identified risk factors in a small non generalized population. I believe the conclusion should call for more research around specific risk factors identified (violence and social support and how it relates to prenatal depression). This study goal was not to screen nor examine the interventions postulated in the conclusion but to identify predictors/risk factors. Also results were not generalizable. Consider including future studies on the same for a larger more generalizable studies with these predictors included or more studies that replicate similar findings to establish these predictors. Perhaps also test interventions that can address this predictors.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the 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? No",
"responses": [
{
"c_id": "5479",
"date": "14 May 2020",
"name": "Harriet Mirieri",
"role": "Author Response",
"response": "1. In the introduction, the authors state that Depression related to child bearing can develop either during pregnancy (prenatal depression), after birth (postnatal depression) or both (perinatal depression)\". The word both is hanging, consider both periods The word 'periods' has been added to the statement on types of depression during childbirth as recommended 2. Still in the introduction, the authors state: The main psychological and psychiatric predictors of prenatal depression are history of mental or anxiety disorder\". This is confusing, since anxiety disorder is a mental disorder. Consider correcting to read: The main psychological predictor of prenatal depression is a history of mental disorders The statement 'The main psychological predictor of prenatal depression is a history of mental disorders ' has been added to the manuscript 3. In the methods, the study lacks a description of study procedures, specifically: Were the questions administered through interviews, or self-reporting? Were the tools translated, what language was used, who administered the tools, where was this done in the hospital, a private room perhaps? In addition to the modification included in the manuscript, recruitment and interview of participants was carried out by two hospital-based research assistants (registered nurses). Upon obtaining informed consent from the participants, the Edinburgh Postnatal Depression Scale (EPDS) (either the English or Kiswahili version depending on the individual’s preference) was administered via a face-to-face interview in a private room within the ANC clinic. Information on predictor variables was collected from the case and control participants using a semi-structured questionnaire (administered in a similar manner as the EPDS tool) 4. In the methods, kindly clarify if the social provisions scale and the composite abuse scale were translated and validated for the setting. Have they been used in this setting? As modified in the manuscript, the English version of the social provisions scale and composite abuse scale were used and both have been validated to be used in this setting. 5. Under statistical analysis, the authors describe the categorization of ages \"Since inclusion of age as a continuous variable was insignificant in the univariable analysis, it was categorized into three groups: 18–25 years, 26–29 years and 30–34 years and reassessed for significance as a categorical variable.\" What informed this categorization and where were the women older than 34 years included? Inclusion of age as a continuous variable assumes that age is linearly associated with the log odds of prenatal depression. If this assessment is insignificant, categorization of age allows the exploration of a potential non-linear association. 6. In the discussion: the authors state \"Age did not significantly influence the likelihood of prenatal depression in this study. However, other studies have revealed that age is a significant predictor of prenatal depression owing to the fact that young pregnant women are likely to be financially unstable and may not be socially and psychologically prepared to cope with pregnancy demands and this may predispose them to depression\". The authors may need to elaborate or postulate why this study found no association with age, seeing that other studies have found some link. What is a possible explanation? Perhaps the age range of your sample was not wide enough? Similarly, to alcohol and substance use, kindly give an explanation for the negative findings. We believe that the lack of association between ‘age’ and ‘alcohol and substance abuse’ and prenatal depression can be explained statistically. The effects of these two variables are clearly distorted (mixed-up) with the effects of the more stronger predictors (marital status, occupation, domestic violence and social support) so that upon the removal of the effects (control of confounding) of these latter predictors in the multivariable analysis, age’ and ‘alcohol and substance abuse’ cease to important predictors. Thus, for instance, revealing that a woman’s marital status, employment status, whether or not she’s violated or has social support is more telling of her depression status than simply her age. 7. Under limitations the authors state \"The results from this study are generalizable to similar settings in other low- and middle-income countries.\" I believe the authors meant not generalizable. Please correct. We inadvertently included the generalizability statement under “study limitations” section thus creating some confusion. We have however relocated this statement. We believe these findings would be readily extendable to populations of pregnant women presenting for ANC services in similar low- and middle-income settings. 8. Also include limitation that this study was a facility-based study as opposed to population based. We know majority of depressed ladies may not go for antenatal care, hence some bias in sample selection. Since this was a facility-based study, we have included the possibility of selection bias as a potential study limitation in the manuscript. 9. The conclusion can be considered an over reach. It appears as if the authors did an intervention study that identified these specific interventions to work while instead they have simply identified risk factors in a small non generalized population. I believe the conclusion should call for more research around specific risk factors identified (violence and social support and how it relates to prenatal depression). This study goal was not to screen nor examine the interventions postulated in the conclusion but to identify predictors/risk factors. Also results were not generalizable. Consider including future studies on the same for a larger more generalizable studies with these predictors included or more studies that replicate similar findings to establish these predictors. Perhaps also test interventions that can address this predictor. In the conclusion section as recommended, we have indicated opportunities for future research to build on the present study."
}
]
},
{
"id": "59454",
"date": "01 Apr 2020",
"name": "Abiodun Olugbenga Adewuya",
"expertise": [
"Reviewer Expertise Public Mental Health"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study addresses an important topic as depression during the perinatal period is every common and have adverse impact on the women, the child and the entire family especially in LMICs. However, I have many reservations about the justification, the study design, the data collection and analysis\nIntroduction\n\nThere is need to show that a review of the literature from SSA has been carried out and the gap in the knowledge had been identified which this present work is trying to fill. See below a list of published works I could find:\n\nGreen EP, Tuli H, Kwobah E, Menya D, Chesire I, Schmidt C. Developing and validating a perinatal depression screening tool in Kenya blending Western criteria with local idioms: A mixed methods study. Journal of affective disorders. 2018 Mar 1;228:49-591.\n\nOngeri L, Otieno P, Mbui J, Juma E, Mathai M. Antepartum risk factors for postpartum depression: a follow up study among urban women living in Nairobi, Kenya. J Preg Child Health. 2016;3(288):22.\n\nOsok J, Kigamwa P, Vander Stoep A, Huang KY, Kumar M. Depression and its psychosocial risk factors in pregnant Kenyan adolescents: a cross-sectional study in a community health Centre of Nairobi. BMC psychiatry. 2018 Dec;18(1):1363.\n\nOsok J, Kigamwa P, Huang KY, Grote N, Kumar M. Adversities and mental health needs of pregnant adolescents in Kenya: identifying interpersonal, practical, and cultural barriers to care. BMC women's health. 2018 Dec 1;18(1):964.\n\nVelloza J, Njoroge J, Ngure K, Thuo N, Kiptinness C, Momanyi R, Ayub S, Gakuo S, Mugo N, Simoni J, Heffron R. Cognitive testing of the PHQ-9 for depression screening among pregnant and postpartum women in Kenya. BMC psychiatry. 2020 Dec 1;20(1):315.\nMethods\n\nThe authors described the Coast Provincial General Hospital (CPGH) as a “level five” public health facility located in Mombasa County, Kenya. They need to describe what this means to the reading public. The authors also need to explain why the choice of a referral hospital where only the complicated cases (needing doctors attention) are referred. It is expected that majority of the deliveries in Kenya will be via the primary health facilities with attending midwives. The choice of a level 5 facility will definitely bias the outcome\n\nThe procedure did not state how the EPDS was administered. Was it self administered or interviewer administered? What of non-literate women, how were they catered for? Also how valid is the EPDS amongst Kenyan women population? It should be noted that a rate of 20% positive (34/170) was obtained in this study.\n\nSample size: A detailed sample size formulae was provided but not how the sample size of 34 cases in the index arm was obtained. This number I think is definitely too low and do not have enough power to make any prediction for a data that is focused on determinants and significantly associated factors in a population\n\nStudy design: It was not clear how the participants were selected into cases and control. Given that the authors said the EPDS was completed in the triage room before the routine antenatal services, were the individual scores collated there and then before asking them to complete the rest of the questionnaire or did they all completed the questionnaire no matter what they score? There is a concern that the design of this study may not follow the case-control design\n\nResults\nWhy was P≤0.2 chosen as the level of significance during the univariate analysis?\n\nThe very wide Confidence interval in Table 4 again gives cause for concern regarding the sample size and the power of the 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? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "5480",
"date": "14 May 2020",
"name": "Harriet Mirieri",
"role": "Author Response",
"response": "Introduction 1. There is need to show that a review of the literature from SSA has been carried out and the gap in the knowledge had been identified which this present work is trying to fill. As indicated in the manuscript, review of literature revealed that there is paucity of research on prenatal depression in Kenya and this study aimed at contributing towards filling this gap by understanding the predictors of prenatal depression and informing the formulation of guidelines for effective prevention and control of prenatal depression. Methods 2. The authors described the Coast Provincial General Hospital (CPGH) as a “level five” public health facility located in Mombasa County, Kenya. They need to describe what this means to the reading public. The authors also need to explain why the choice of a referral hospital where only the complicated cases (needing doctors attention) are referred. It is expected that majority of the deliveries in Kenya will be via the primary health facilities with attending midwives. The choice of a level 5 facility will definitely bias the outcome Although the CPGH is a referral facility, the ANC clinic also attends to mothers seeking routine antenatal care and is actually the main county facility offering ANC services. 3. The procedure did not state how the EPDS was administered. Was it self administered or interviewer administered? What of non-literate women, how were they catered for? Also how valid is the EPDS amongst Kenyan women population? It should be noted that a rate of 20% positive (34/170) was obtained in this study. As modified in the manuscript, the data collection tools were administered by the research assistants in a face to face interview. Besides, the EPDS has been validated to be used in the Kenyan setting. Given that this was a case control study, we did not give a disease frequency measure since we do not have all the cases present for the study. 4. Sample size: A detailed sample size formulae was provided but not how the sample size of 34 cases in the index arm was obtained. This number I think is definitely too low and do not have enough power to make any prediction for a data that is focused on determinants and significantly associated factors in a population Given a 95% confidence level, 80% statistical power, odds ratio of 3 for the main exposure-outcome association (domestic violence – prenatal depression) and 40% proportion of controls (non-depressed women) that are exposed (have experience domestic violence) and a ratio of 4:1 for controls to cases ( in order to optimize on statistical power) we get a sample size of 170. This is anticipated in case control studies considering that they target outcomes with low frequency in the population. 5. Study design: It was not clear how the participants were selected into cases and control. Given that the authors said the EPDS was completed in the triage room before the routine antenatal services, were the individual scores collated there and then before asking them to complete the rest of the questionnaire or did they all completed the questionnaire no matter what they score? There is a concern that the design of this study may not follow the case-control design In addition to the modification included in the manuscript taken together with the study flow chart, we further provide a clarification here. The EPDS was initially applied (by face-to-face interview) in a private room to identify cases and controls based on cut-off point of 13 (considering that prenatal depression is not routinely screened for). Afterwards, the cases and controls were interviewed to elicit information on the study predictors. Results 6. Why was P≤0.2 chosen as the level of significance during the univariate analysis? A liberal p-value of ≤ 0.20 is commonly used to allow variables that are potentially negatively confounded during the univariable analysis stage (effect has been suppressed by other study variables) to “express” themselves in the multivariable analysis once the effect of the suppressing variables has been removed – See Dohoo et al. 2012. 7. The very wide Confidence interval in Table 4 again gives cause for concern regarding the sample size and the power of the study We believe that the wide confidence intervals for some of the variables (marital status and domestic violence) is more of a reflection of collinearity than sample size. For instance, with marital status and domestic violence being highly correlated, the standard errors of these variables is likely to be elevated and thus their confidence intervals."
}
]
}
] | 1
|
https://f1000research.com/articles/9-36
|
https://f1000research.com/articles/9-217/v1
|
30 Mar 20
|
{
"type": "Research Article",
"title": "Host deficiency in ephrin-A1 inhibits breast cancer metastasis",
"authors": [
"Eileen Shiuan",
"Ashwin Inala",
"Shan Wang",
"Wenqiang Song",
"Victoria Youngblood",
"Jin Chen",
"Dana M. Brantley-Sieders",
"Eileen Shiuan",
"Ashwin Inala",
"Shan Wang",
"Wenqiang Song",
"Victoria Youngblood",
"Jin Chen"
],
"abstract": "Background: The conventional dogma of treating cancer by focusing on the elimination of tumor cells has been recently refined to include consideration of the tumor microenvironment, which includes host stromal cells. Ephrin-A1, a cell surface protein involved in adhesion and migration, has been shown to be tumor suppressive in the context of the cancer cell. However, its role in the host has not been fully investigated. Here, we examine how ephrin-A1 host deficiency affects cancer growth and metastasis in a murine model of breast cancer. Methods: 4T1 cells were orthotopically implanted into the mammary fat pads or injected into the tail veins of ephrin-A1 wild-type (Efna1+/+), heterozygous (Efna1+/-), or knockout (Efna1-/-) mice. Tumor growth, lung metastasis, and tumor recurrence after surgical resection were measured. Flow cytometry and immunohistochemistry (IHC) were used to analyze various cell populations in primary tumors and tumor-bearing lungs. Results: While primary tumor growth did not differ between Efna1+/+, Efna1+/-, and Efna1-/- mice, lung metastasis and primary tumor recurrence were significantly decreased in knockout mice. Efna1-/- mice had reduced lung colonization of 4T1 cells compared to Efna1+/+ littermate controls as early as 24 hours after tail vein injection. Furthermore, established lung lesions in Efna1-/- mice had reduced proliferation compared to those in Efna1+/+ controls. Conclusions: Our studies demonstrate that host deficiency of ephrin-A1 does not impact primary tumor growth but does affect metastasis by providing a less favorable metastatic niche for cancer cell colonization and growth. Elucidating the mechanisms by which host ephrin-A1 impacts cancer relapse and metastasis may shed new light on novel therapeutic strategies.",
"keywords": [
"Ephrin-A1",
"host-tumor interactions",
"breast cancer",
"metastasis",
"metastatic niche"
],
"content": "Introduction\n\nOver the past several decades, the conventional dogma of treating cancer by focusing on the elimination of rapidly dividing tumor cells has been gradually refined to include consideration of the environment in which the tumor thrives – the tumor microenvironment. The tumor microenvironment consists of both cancer cells and host stromal cells, such as endothelial cells, immune populations, and fibroblasts. Prominent discoveries regarding tumor-associated endothelium and immune cells have notably led to breakthrough therapeutic strategies with anti-angiogenic agents and immunotherapies, respectively1–4. Thus, understanding the host-tumor interactions involved in tumor growth and metastasis is critical for the development and application of new anti-cancer therapies.\n\nAs a result of new advancements in targeted and immunotherapies, the majority of patients with early stage disease have a very favorable prognosis. However, patients who later develop distant metastasis or who are diagnosed with disseminated disease at the onset are typically very difficult to treat effectively5,6. This is largely because our knowledge of how cancer cells spread is still limited. Cancer metastasis is a dynamic and complex process that requires tumor cells to undergo many steps, including adopting invasive properties, intravasating into proximal vasculature, surviving in circulation, evading immunosurveillance, extravasating from distant vasculature, and finally adapting to selective pressures of a new environment7,8. Each of these steps involves multiple interactions between cancer cells and different types of host stromal cells. As an example, breast cancer most commonly metastasizes to the lung, bone, liver, and brain, but how and why these cells travel and colonize these particular organs is still unknown5,6. A better understanding of how breast cancer metastasizes to these distant sites is greatly needed in order to develop more effective therapies and prevent spread of malignant disease.\n\nEphrin-A1 is a cell surface protein that regulates cell adhesion and migration9–28, and its role in cancer has recently been investigated in several different solid tumors29–35. It belongs to the group of ephrin ligands that interact with the largest family of receptor tyrosine kinases (RTKs), the Eph receptors, and regulates various developmental processes, such as embryonic cardiovascular development and angiogenic remodeling36–38. It is expressed in various cell types, including epithelial, endothelial, and immune cells and is the primary ligand for EphA2 RTK, which has been implicated in cancer growth and metastasis in various solid tumors36–41. While ephrin-A1 expression in cancer cells has been shown to be tumor suppressive25,26,42,43, its role in the host, has not been fully investigated. Here, we use ephrin-A1 knockout mice to examine how ephrin-A1 host deficiency affects cancer growth and metastasis in a murine model of breast cancer.\n\nTo test the impact of ephrin-A1 host deficiency on cancer progression, we utilized an orthotopic 4T1 mammary tumor model, as well as two different models of metastasis. While primary tumor growth did not significantly differ between ephrin-A1 wild-type (Efna1+/+), heterozygous (Efna1+/-), and knockout (Efna1-/-) mice, metastasis and primary tumor recurrence were significantly decreased in Efna1-/- mice. Results of analysis on tumor-infiltrating immune cell populations and vascularity in the primary tumor did not evidently explain the differences in metastasis between Efna1+/+ and Efna1-/- mice. However, tumor cell lung colonization was reduced in Efna1-/- mice, and lung metastases in Efna1-/- mice were less proliferative than in their wild-type counterparts, suggesting that the metastatic niche in Efna1-/- mice is less hospitable for invading tumor cells. Together, our studies suggest that host deficiency of ephrin-A1 does not impact initial tumor growth but does affect metastasis through inhibiting cancer cell extravasation and proliferation at the metastatic niche.\n\n\nMethods\n\nAnimals were housed in a non-barrier animal facility under pathogen-free conditions, 12-hour light/dark cycle, and access to standard rodent diet and water ad libitum. Experiments were performed in accordance with AAALAC guidelines and with Vanderbilt University Medical Center Institutional Animal Care and Use Committee approval. All mice used in this study were immunocompetent BALB/c mice. Ephrin-A1 knockout (Efna1-/-) mice were previously characterized by our lab44. To generate littermate controls, wild-type BALB/c mice were purchased from Jackson Laboratory and mated with Efna1-/- mice to generate heterozygote mating pairs. Efna1+/+, Efna1+/- and Efna1-/- animals were identified by PCR analysis of genomic DNA using the following primers: Forward primer (5’-TGGTTATATCCCCCCACCTCACAC-3’) and two allele-specific reverse primers (WT 5’-AAGGACTCCCATATCTCAGCGACG-3’) and (KO 5’-AGACTGCCTTGGGAAAAGCG-3’). Mice were co-housed with one to four littermates for at least two weeks prior to and during all experiments and compared with littermate controls whenever possible. All mice used for tumor experiments were six to ten weeks old at the onset on the experiment. Experimental cohorts were limited to litters that were born within two consecutive weeks and that also had at least one Efna1-/- and Efna1+/+ gender-matched littermate pair and, when applicable, at least one Efna1+/- gender-matched littermate. Sample sizes are as shown in the figures and range from three to twelve mice per group. At experimental endpoints, mice were euthanized by cervical dislocation.\n\n4T1 murine mammary adenocarcinoma cells were purchased from ATCC and maintained in DMEM (Corning #MT10013CV) supplemented with penicillin/streptomycin (Gibco #15140163) and 10% FBS (Gibco #A3160502). 4T1-GFP-luciferase clones were generated by serial dilutions of 4T1 cells with lentiviral overexpression of GFP and luciferase genes.\n\nTo reflect human breast cancer, only female mice were used for tumor experiments. For orthotopic mammary tumor implantations, 1×105 4T1 cells suspended in a 1:1 mixture of PBS and Growth Factor-Reduced Matrigel (Corning #354230) were injected through the nipple into the fourth mammary fat pads of six to eight-week-old female mice. Tumor dimensions were measured by digital caliper at given time points every other day, and volume was calculated using the following formula: volume = length × width2 × 0.52. To observe spontaneous lung metastases and primary tumor recurrence, mammary tumors were resected at day 14 post-implantation, along with draining inguinal lymph nodes and surrounding fat pads, and mice were ultimately sacrificed at day 32. At the time of surgical resection of primary tumors on day 14, tumors were weighed and cut in half to provide tissue for both flow cytometry analysis and cryosection staining. At the experimental endpoint on day 32, tumors were weighed, and lung metastases were counted in a blinded manner. For lung colonization experiments, 4T1-GFP-luciferase cells suspended in PBS were injected via tail vein, and mice underwent in vivo bioluminescence imaging with a PerkinElmer IVIS Spectrum several hours post-injection to verify successful and equal delivery of 4T1 cells. To observe gradual formation of GFP+ metastases, 1×105 4T1-GFP-luciferase cells were injected via tail vein, and mice were sacrificed at day 17. GFP+ lung metastases were counted in a blinded manner. The left lung lobe of each mouse was fixed in 10% formalin for subsequent formalin-fixed paraffin-embedded (FFPE) processing, sectioning, and H&E staining, while the other lung lobes were processed for flow cytometry analysis. To observe early colonization and proliferation of 4T1 cells, 5×105 4T1-GFP-luciferase cells labeled with CellTrace Violet dye (Invitrogen #C34571) were injected via tail vein. At 24 hours, mice were sacrificed, and lungs were perfused with PBS and processed for flow cytometry analysis.\n\nFFPE lung sections were prepared and stained for PCNA (1:100, BD Biosciences #555567 raised in mouse, RRID: AB_395947) as described previously45. Slides were blinded, and the number of metastatic foci per section of lobe was quantified. Nuclear PCNA staining was analyzed using ImageJ v1.52o with the IHC Profiler plugin46 and percentage of PCNA+ tumor cell nuclei were quantified. Each data point is an average of two sections of the left lung from an individual mouse. To prepare cryosections, mammary tumors were frozen in OCT Compound (Thermo Fisher Scientific #23-730-571) on dry ice and stored at -80°C. Sections (8 µm) were cut on a Leica Cryostat CM1950, fixed in 4% PFA, washed with PBS, permeabilized with 0.3% Triton X-100 (Sigma-Aldrich #X100), and blocked using M.O.M. Mouse Ig Blocking Reagent and Protein Concentrate (Vector Laboratories #PK-2200) per manufacturer recommendations and with 2.5% goat serum (Sigma-Aldrich #G9023) in PBS. Slides were then incubated over two nights at 4°C with primary antibodies against CD31 (1:150, Biolegend #102501 raised in rat, RRID: AB_312908) and αSMA (1:150, Dako #M085129-2 raised in mouse, RRID: AB_2811108) in blocking buffer. After washing with PBS, slides were incubated for one hour at room temperature in secondary antibodies goat anti-rat Ax594 (1:500, Invitrogen #A11007, RRID: AB_10561522) and anti-mouse Ax488 (1:500, Invitrogen #A11001, RRID: AB_2534069), washed with PBS, and mounted with ProLong Gold Antifade Mountant with DAPI (Invitrogen #P36931). Slides were blinded, and images were taken by an Olympus DP72 camera through a BX60 inverted fluorescence microscope and processed using CellSens Dimension software. A total of 12-40 20x fields of view were analyzed from each section using ImageJ. For αSMA analysis, images were evaluated for colocalization with CD31 staining, and data was displayed as a percentage of αSMA+ out of CD31+ area or integrated intensity. Each data point is an average of all fields of view of two to three tumor sections from an individual mouse.\n\nTumors and lungs were minced and dissociated in RPMI-1640 media (Corning #MT10040CV) containing 2.5% FBS, 1 mg/ml collagenase IA (Sigma-Aldrich #C9891), and 0.25 mg/ml DNase I (Sigma-Aldrich #DN25) for 45 minutes at 37°C. Digested tissue was then filtered through a 70-µm strainer, and red blood cells were lysed using ACK Lysis Buffer (KD Medical #RGF-3015). Samples were washed with PBS and stained with Ghost Dye Violet V510 (Tonbo Biosciences #13-0870) to exclude dead cells. After washing with buffer (0.5% BSA, 2mM EDTA in PBS), samples were blocked in αCD16/32 mouse Fc block (Tonbo Biosciences #70-0161) and stained for extracellular proteins using an antibody master mix made in buffer. After washing with buffer, cells were fixed with 2% PFA. For FoxP3 intracellular staining, cells were permeabilized using the FoxP3 Transcription Factor Staining Kit (Tonbo Biosciences #TNB-0607-KIT) per manufacturer protocol. Flow cytometry data was obtained on a BD 4-laser Fortessa using BD FACS Diva software v8.0.1 and analyzed using FlowJo software v10.6.1. Fluorescence minus one (FMO) samples were used as gating controls when needed. Antibodies used in flow panels are detailed in Table 1, and gating strategies used in analysis are detailed in Table 2. Each data point is generated after analyzing at least 5×105 viable cells from a specimen from an individual mouse.\n\nAll graphs and statistical analysis were completed using GraphPad Prism software v6.07. For comparisons between two groups, an unpaired Mann-Whitney U-test was performed. For comparisons between three groups, a Kruskal-Wallis H-test was performed, followed by post-hoc Mann-Whitney U-tests evaluating differences between the knockout and either the wild-type or heterozygote animals. A P-value less than 0.05 was considered statistically significant.\n\n\nResults\n\nWe initially investigated the impact of ephrin-A1 host deficiency on primary tumor growth by implanting 4T1 cells orthotopically into the mammary fat pads of syngeneic BALB/c female Efna1+/+ and Efna1-/- mice. No difference in primary tumor growth or weight at 21 days post-implantation was observed (Figure 1A). To test the impact of ephrin-A1 host deficiency on spontaneous metastasis, 4T1 cells were implanted orthotopically as described above and surgically resected on day 14 post-implantation to allow for gradual development of endogenous metastases by day 32 (Figure 1B). As expected, primary tumors resected from Efna1+/+ and Efna1-/- mice were not different in size (Figure 1C), and this was additionally verified with Efna1+/+, Efna1+/-, and Efna1-/- littermates (Figure 1D). However, at the experimental endpoint, the number of visible lung metastases and lung weights were significantly decreased in knockout mice (Figure 1E). Many of these mice not only harbored lung metastases but also tumors that had regrown at the original site of the resected primary tumor. Similar to our findings in lung metastases, the size of recurrent primary tumors was significantly reduced in knockout mice (Figure 1F). Together, these results demonstrate that while host deficiency in ephrin-A1 may not affect initial tumor growth, it can impact metastatic spread and recurrence. Underlying data are available47,48.\n\n(A) 4T1 primary tumor growth curves in age-matched female Efna1+/+ (WT) and Efna1-/- (KO) mice and resulting tumor weights at day 21 post-implantation. (B) Schematic diagram showing experimental procedure for evaluating spontaneous metastases. (C) 4T1 primary tumor growth curves in WT and KO mice and resultant tumor weights at time of surgical resection on day 14. (D) 4T1 primary tumor growth curves in WT, heterozygous (+/-) and KO littermates and resultant tumor weights at time of surgical resection on day 14. (E) Blinded quantification of visible lung metastases and lung weights from WT and KO mice at experimental endpoint on day 32. (F) Weights of recurrent 4T1 tumor at primary site 18 days after surgical resection. Data shown are averages ± SD (n=9-12 mice per group). *p<0.05, **p<0.01 (unpaired Mann-Whitney U-test).\n\nTo complement our findings in our model of spontaneous metastasis, we evaluated the impact of ephrin-A1 host deficiency on experimental metastasis. 4T1 cells engineered to express GFP and luciferase (4T1-GFP-luciferase) were injected into the tail veins of Efna1+/+, Efna1+/-, and Efna1-/- littermates. In vivo bioluminescence imaging several hours after injection illustrated comparable signal across all mice (Figure 2A, B), indicating ephrin-A1 host deficiency did not impact tumor cell trafficking and lodging within the lung, at least in this short time frame. After harvesting the lungs 17 days later, we observed decreased GFP+ metastases in Efna1-/- mice, compared to both Efna1+/+ and Efna1+/- littermates, which was additionally confirmed by flow cytometry (Figure 2C, D). Similarly, histological analysis revealed fewer metastatic foci in lungs from Efna1-/- mice (Figure 2E). These data align with our previous observations on endogenous metastasis and suggest that host deficiency in ephrin-A1 inhibits circulating cancer cells from colonizing the lung. Underlying data are available49,50.\n\n(A) Representative image of bioluminescence signal in WT and KO littermates several hours after tail vein injection of 1×105 4T1-GFP-luciferase cells. (B) Quantification of bioluminescence signal in WT, +/-, and KO littermates. (C) Representative images of GFP+ surface lung metastases in WT and KO littermates 17 days after tail vein injection. (D) Blinded quantification of GFP+ lung metastases in WT, +/-, and KO littermates and percentages of GFP+ 4T1 cells in the lung from flow cytometry analysis. (E) Representative H&E staining of left lung lobes from WT and KO littermates and blinded quantification of metastatic foci per lung section. Scale bar: 200 µm. Data shown are averages ± SD (n=4-9 mice per group). *p<0.05, **p<0.01 (unpaired Mann-Whitney U-test for comparisons between two groups, Kruskal-Wallis H-test with post-hoc unpaired Mann-Whitney U-test for comparisons between three groups).\n\nEphrin-A1 is expressed in several types of host cells, including immune cells and endothelial cells39–41. Thus, we sought to determine how the ephrin-A1-deficient host immune system and endothelium may mitigate metastasis. Among immune cells, Ephrin-A1 can be expressed in B and T cells, monocytes, and macrophages39. The role of ephrin-A1 in B cells is largely unknown51. However, in T cells, monocytes, and macrophages, ephrin-A1 has been shown to regulate cell adhesion and migration11,12,15,27,52–55. These immune cell populations play a critical role in overall anti-tumor immunity and immunosurveillance. Dendritic cells and T cells, particularly CD8 cytotoxic T cells, are the primary drivers of the adaptive anti-tumor response in solid tumors and increased infiltration of these cell types is correlated with better prognosis and enhanced response to immunotherapies56,57. Conversely, T regulatory cells (Tregs) suppress effector functions of T cells and typically inhibit the anti-tumor response58,59. Between the two ends of this spectrum, myeloid populations, such as monocytes, macrophages, and granulocytes, can either promote or suppress an anti-tumor response, depending on their polarization and functionality60,61.\n\nBecause of ephrin-A1’s known role in adhesion and chemotaxis of immune cells, we performed flow cytometry analysis on 4T1 primary tumors harvested from Efna1+/+, Efna1+/-, and Efna-/- littermates. To our surprise, we found no significant differences in total infiltrating immune cells, CD4 or CD8 T cells, dendritic cells, Tregs, or myeloid populations in Efna1+/+, Efna1+/-, and Efna-/- littermates (Figure 3A, B). While there were no apparent differences in the immune microenvironment of the mammary tumors, the immune microenvironment of the lung is distinct from that of the mammary gland and may impact the metastatic niche. To investigate this, we performed flow cytometry analysis on 4T1 tumor-bearing lungs generated from our model of experimental metastasis. Similar to the results we obtained from the 4T1 primary tumors, we did not see significant differences in immune populations in tumor-bearing lungs harvested from Efna1+/+, Efna1+/-, and Efna1-/- littermates, except for a modest increase in macrophages in knockout mice (Figure 3C, D). Underlying data are available62,63.\n\n(A) Flow cytometric analysis of total immune cells, T cells, and T regulatory (Treg) cells, as well as (B) monocytes, macrophages, granulocytes, and dendritic cells, in 4T1 primary tumors resected from WT, +/-, and KO littermates at day 14 post-implantation. (C, D) Similar analyses of immune populations in tumor-bearing lungs harvested from WT, +/-, and KO littermates 17 days after tail vein injection of 4T1-GFP-luciferase cells. Data shown are averages ± SD (n=3-7 mice per group). *p<0.05 (Kruskal-Wallis H-test with post-hoc unpaired Mann-Whitney U-test).\n\nAlthough the percentage of tumor infiltrating T cells in Efna1+/+ and Efna1-/- mice is comparable, their activation status and effector function may still be different. Tumor-infiltrating T cells with upregulated expression of activation markers, such as CD44, CD69, and CD25, and downregulated expression of antigen-naïve markers like CD62L and exhaustion markers like PD-1 and CTLA-4 indicate a higher T cell functional status that mediates a stronger and more enduring anti-tumor response58,59. We assessed these markers on T cells in 4T1 primary tumors and tumor-bearing lungs from Efna1+/+ and Efna1-/- littermates using flow cytometry. However, we did not observe consistent increases in activation or decreases in naïve or exhaustion markers in knockout-derived T cells (data not shown, included in Underlying data)62. In summary, host deficiency in ephrin-A1 does not significantly affect tumor-infiltrating immune cells in both primary tumors and tumor-bearing lungs. Thus, the reduction of lung metastases in Efna1-/- hosts in vivo is unlikely due to host immunity.\n\nIn addition to the anti-tumor immune response, another host factor that can impact metastasis is the tumor vasculature. Angiogenesis is the formation of new blood vessels from a pre-existing network and is required for solid tumor growth and progression. Blood vessels can supply nutrients that support tumor growth and provide an entry for hematological dissemination and invasion1,64. These new blood vessels are typically hastily constructed in response to the high release of growth factors, such as vascular endothelial growth factor (VEGF), from tumor cells65,66. Thus, tumor vessels tend to be disorganized, leaky, and poorly covered by pericytes, which normally support the integrity of the endothelium. Ephrin-A1 is expressed in the vascular endothelium and has been shown to promote angiogenesis in vitro and in several in vivo models67–72. Therefore, we hypothesized that tumors in Efna1-/- mice may have reduced tumor vasculature and increased endothelial pericyte coverage compared to Efna1+/+ controls.\n\nTo evaluate tumor vascularity and vessel function, we co-stained cryosections of 4T1 primary tumors from Efna1+/+ and Efna1-/- littermates with CD31 and αSMA, markers for endothelial cells and pericytes, respectively. Colocalization of αSMA with CD31 acts as an indicator for functional endothelium within tumors. Surprisingly, we did not observe a change in CD31+ area or intensity in 4T1 tumors from Efna1+/+ and Efna1-/- littermates (Figure 4A, B). Furthermore, pericyte coverage on tumor vessels remained the same in tumors from Efna1+/+ and Efna1-/- littermates (Figure 4A, C). Together, these data suggest that loss of ephrin-A1 in the host does not affect tumor vessel formation and function in the primary tumor. Underlying data are available73.\n\n(A) Representative images of CD31 (red), αSMA (green), and DAPI (blue) staining on cryosections of 4T1 primary tumors harvested from WT and KO littermates at day 14 post-implantation. Scale bar: 100 µm. (B) Quantification of CD31+ area and integrated intensity in arbitrary units (au) per high power field (hpf) of view. (C) Quantification of αSMA colocalization with CD31 as a percentage of αSMA+ over CD31+ stained area and integrated density. Data shown are averages ± SD (n=3-7 mice per group).\n\nOur results from analysis of the immune infiltrate and vasculature of primary tumors, coupled with the significant difference in experimental lung metastasis between Efna1+/+ and Efna1-/- mice, suggest that host factors critical to this metastatic phenotype are more likely to lie downstream of the primary tumor site. These steps include tumor cell trafficking to the lung vasculature, extravasation, and adaptation to new selective pressures of the lung microenvironment. 4T1 cell trafficking to the lung was not significantly different between Efna1+/+ and Efna1-/- littermates after tail vein injections (Figure 2A, B). Thus, we aimed to evaluate extravasation and adaptation to the lung metastatic niche in Efna1+/+ and Efna1-/- hosts.\n\nTo test extravasation of 4T1 cells in vivo, we injected 4T1-GFP-luciferase cells labeled with CellTrace Violet dye into the tail veins of Efna1+/+ and Efna1-/- littermates. This dye is retained only in the labeled tumor cells and diminished after subsequent cell divisions, enabling quantification of short-term cell proliferation. At 24 hours after injection, we perfused the lungs with PBS to flush out remaining cells in the pulmonary vasculature and processed the lungs for flow cytometry. Decreased GFP+ 4T1 cells were found in ephrin-A1-deficient lungs compared to wild-type controls (Figure 5A), suggesting that fewer cancer cells had extravasated into the lung parenchyma at this timepoint. This result was not due to decreased vascularity of ephrin-A1-deficient lungs at baseline, as CD31+ endothelial cells were not significantly different in knockout and wild-type lungs (Figure 5B). Moreover, this was not due to decreased proliferation of the 4T1 cells within the 24-hour timeframe, as the amount of retained CellTrace Violet dye was not increased in 4T1 cells that had extravasated in knockout lungs compared to wild-type lungs (Figure 5C). Together, these data suggest that extravasation of 4T1 cells is inhibited in knockout mice, compared to wild-type controls, and ephrin-A1 deficiency in the host lung may play a role in this process.\n\n(A) Quantification of GFP+ 4T1 cells and (B) CD31+ endothelial cells by flow cytometry in perfused lungs harvested from WT and KO littermates 24 hours post-tail vein injection. (C) Percentage of GFP+ 4T1 cells in perfused WT and KO lungs that still contained CellTrace Violet dye, indicating reduced proliferation. (D) Representative images of PCNA staining on FFPE sections of tumor-bearing lungs from WT and KO littermates 17 days after tail vein injection of 4T1-GFP-luciferase cells. Higher magnification images and blinded quantification of PCNA+ tumor cell nuclei shown. Scale bar: 50 µm. Data shown are averages ± SD (n=3-6 mice per group). *p<0.05 (unpaired Mann-Whitney U-test).\n\nWhile decreased extravasation of tumor cells may explain in part the decreased lung metastases in Efna1-/- mice, another possibility is that once tumor cells have extravasated and established in the lung, they have reduced fitness of survival in ephrin-A1-deficient lungs, compared to wild-type lungs. There are many stressors in the lung metastatic niche that could impact the adaptability of the tumor cell. We used tumor proliferation index as a marker to evaluate how well tumor cells have adapted to a metastatic niche. Since no differences were observed in proliferation of 4T1 cells that had newly extravasated into the lung parenchyma of Efna1+/+ and Efna1-/- littermates within the short 24-hour timeframe (Figure 5C), we next assessed proliferation of tumor cells in lung micrometastases that had established over 17 days after tail vein injection (Figure 2). There was a significant decrease in cell proliferation in metastatic foci established in Efna1-/- mice, compared to Efna1+/+ controls, as indicated by PCNA staining (Figure 5D). These findings suggest that reduced tumor cell lung colonization in Efna1-/- hosts is due to both decreased extravasation of cancer cells and decreased proliferation in the metastatic niche. Underlying data are available74,75.\n\n\nDiscussion\n\nIn conclusion, host deficiency in ephrin-A1 inhibits metastasis by providing a less hospitable metastatic niche for cancer cell extravasation and colonization of the lung. Our data from 4T1 primary tumor specimens demonstrated no differences in primary tumor growth, infiltrating immune cell populations, and vascularity. This led us to investigate the metastatic process downstream from the primary tumors. We then found that lung colonization in knockout mice was decreased compared to wild-type mice as early as 24 hours post-tail vein injection of 4T1 cells, in part due to decreased extravasation. Moreover, the metastases that established in Efna1-/- lungs were not only reduced in number but also less proliferative compared to those in wild-type lungs. These studies offer insight on how host expression of ephrin-A1 may impact tumor growth and dissemination, but they also lead to additional questions.\n\nOur ephrin-A1 knockout model is not tissue-specific nor inducible, which creates challenges in identifying specific mechanisms that contribute to our observed phenotype. For example, ephrin-A1 is highly expressed in embryonic stages of development and plays a known role in neuronal and mammary development67,76–80. The transcriptional and epigenetic changes that occur in utero and during early physiological development as a result of ephrin-A1 deficiency in various tissues may all contribute to the observed phenotype; however, dissecting which changes are directly downstream of ephrin-A1 and critical to metastasis may be quite difficult. This challenge is further augmented when we consider the many cell types that can express ephrin-A1, especially immune, endothelial, and epithelial cells.\n\nMany studies have demonstrated ephrin-A1’s role in immune cell adhesion and migration. Although we did not observe significant differences in tumor immune infiltrate, this does not preclude a role for ephrin-A1 in these cell populations. Immune cells engage in a complex network of crosstalk, and it is possible that loss of ephrin-A1 in one cell type may mask the effects it has in another. One intriguing difference we observed was an increase in macrophages in Efna1-/- tumor-bearing lungs. However, we have not determined if this difference occurs in the specific context of a stressor, such as tumor metastasis, or if knockout mice have increased macrophages at baseline. Furthermore, it remains to be seen if these macrophages are polarized towards an anti-tumor or a pro-tumor response. Nevertheless, this offers evidence of a novel role of ephrin-A1 in macrophage differentiation or survival, which requires further investigation.\n\nIn addition, ephrin-A1 has been shown to regulate expression of adhesion molecules on endothelial cells and promote angiogenesis. Modulation of surface expression of adhesion proteins, such as ICAM-1 and VCAM-1, on endothelial cells impact binding to immune cells and cancer cells8,40. Thus, it is possible that ephrin-A1 on endothelial cells may mediate cancer cell transendothelial migration through modulation of these adhesion proteins. While this result may be consistent with published literature, in contrast to ephrin-A1’s known role in angiogenesis, we did not observe differences in angiogenesis between tumors from Efna1+/+ and Efna1-/- hosts. This discrepancy may be due to a couple reasons. First, most studies reporting on ephrin-A1’s impact on angiogenesis has shown its effect through EphA receptor signaling on the endothelial cell, not necessarily through ephrin-A1 directly in the endothelium67–70. Loss of ephrin-A1 in the endothelium and other host tissues is unlikely to completely abrogate EphA receptor signaling in the endothelium, as other ephrin ligands are able to promiscuously bind to the same EphA receptors and may even compensate for the loss of ephrin-A137. Second, some of these studies use soluble ephrin-A1, instead of membrane-bound or cell-surface ephrin-A1. In our Efna1-/- model, both cell-surface, membrane-bound ephrin-A1 and soluble, secreted ephrin-A1 are lost in vivo, and these two forms of ephrin-A1 have been shown to have competing effects13.\n\nThe different forms of ephrin-A1, as well as the range of interactions with various Eph receptors, show how potentially complex the molecular mechanisms can be when considering host deficiency of ephrin-A1. A clue into this complicated investigation can be found in our data obtained with ephrin-A1 heterozygote controls. When comparing tumor metastasis and immune infiltrate, results from Efna1+/- mice were much more comparable to wild-type than knockout littermate controls. This suggests that ephrin-A1 has a genetically dominant effect – one wild-type allele may be sufficient to induce the wild-type phenotype.\n\nAlthough we focused our inquiries on primary mammary tumors and lung metastases, there is much more to be explored. 4T1 cells, like human breast cancer, metastasize to other organ sites, such as the bone, liver, and brain. The lungs in Efna1-/- hosts may or may not be the only organ that provides a less favorable environment for colonizing tumor cells than those in Efna1+/+ hosts. We observed differences in recurrent primary tumor, in addition to lung metastases, which may indicate that tumor cell apoptosis or senescence is altered in knockout hosts. Further elucidating the mechanisms by which ephrin-A1 host deficiency limits cancer relapse and metastasis may enhance our understanding of the metastatic process and ultimately shed new light on novel therapeutic strategies.\n\n\nData availability\n\nHarvard Dataverse: Host deficiency in ephrin-A1 inhibits breast cancer metastasis; https://dataverse.harvard.edu/dataverse/hostEfna1metastasis.\n\nThis project contains the following underlying data:\n\nHarvard Dataverse: 4T1 primary tumor dimensions and weights. https://doi.org/10.7910/DVN/AGKDWV47. (4T1 primary tumor dimensions from digital caliper measurements, volume calculations, and weights (related to Figure 1A, C, D.)\n\nHarvard Dataverse: 4T1 recurrent primary and spontaneous lung metastases. https://doi.org/10.7910/DVN/FU8JEY48. (Spontaneous 4T1 lung metastases quantification and recurrent primary tumor weights (related to Figure 1E, F.)\n\nHarvard Dataverse: Images and quantification of 4T1-GFP-luciferase spontaneous lung metastases. https://doi.org/10.7910/DVN/2ANDYX49. (Experimental 4T1-GFP-luciferase lung metastases quantification and images (related to Figure 2C-E.))\n\nHarvard Dataverse: 4T1-GFP-luciferase bioluminescence images and quantification post-tail vein injection. https://doi.org/10.7910/DVN/39D0YR50. (4T1-GFP-luciferase bioluminescence quantification and images (related to Figure 2A, B).)\n\nHarvard Dataverse: 4T1 primary tumor flow cytometry. https://doi.org/10.7910/DVN/ZRX2RG62. (Flow cytometry files (fcs), gating and analysis (wsp), and panels (xlsx) containing immune profiling of 4T1 primary mammary tumors from Efna1+/+, Efna1+/-, and Efna1-/- littermate mice (related to Figure 3A, B))\n\nHarvard Dataverse: 4T1-GFP-luciferase tumor-bearing lung flow cytometry. https://doi.org/10.7910/DVN/S06NQ163. (Flow cytometry files (fcs), gating and analysis (wsp), and panels (xlsx) containing immune profiling of 4T1-GFP-luciferase tumor-bearing lungs from Efna1+/+, Efna1+/-, and Efna1-/- littermate mice (related to Figure 2D, 3C, D).)\n\nHarvard Dataverse: 4T1-GFP-luciferase 24-hr lung colonization flow cytometry. https://doi.org/10.7910/DVN/G7TAAE74. (Flow cytometry files (fcs), gating and analysis (wsp), and panels (xlsx) containing profiling of 24-hr 4T1-GFP-luciferase tail vein injected lungs from Efna1+/+ and Efna1-/- littermate mice (related to Figure 5A-C).)\n\nHarvard Dataverse: CD31 and aSMA images and quantification of 4T1 primary tumors. https://doi.org/10.7910/DVN/MOYPE773. (4T1 primary tumor CD31 and αSMA staining quantification and images (related to Figure 4A-C).)\n\nHarvard Dataverse: PCNA images and quantification of 4T1-GFP-luciferase lung metastases. https://doi.org/10.7910/DVN/8AJKFM75. (Lung metastasis PCNA staining quantification and images (related to Figure 5D).)\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\nSample preparation was supported by the Vanderbilt Translational Pathology Shared Resource and Brain Institute. We also thank the Vanderbilt Center for Small Animal Imaging and Flow Cytometry Shared Resource for their assistance.\n\n\nReferences\n\nFolkman J: Angiogenesis: an organizing principle for drug discovery? Nat Rev Drug Discov. 2007; 6(4): 273–286. PubMed Abstract | Publisher Full Text\n\nFerrara N, Hillan KJ, Gerber HP, et al.: Discovery and development of bevacizumab, an anti-VEGF antibody for treating cancer. Nat Rev Drug Discov. 2004; 3(5): 391–400. PubMed Abstract | Publisher Full Text\n\nZou W, Chen L: Inhibitory B7-family molecules in the tumour microenvironment. Nat Rev Immunol. 2008; 8(6): 467–477. PubMed Abstract | Publisher Full Text\n\nPardoll DM: The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer. 2012; 12(4): 252–264. 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PubMed Abstract | Publisher Full Text | Free Full Text\n\nJellinghaus S, Poitz DM , Ende G, et al.: Ephrin-A1/EphA4-mediated adhesion of monocytes to endothelial cells. Biochim Biophys Acta - Mol Cell Res. 2013; 1833(10): 2201–2211. PubMed Abstract | Publisher Full Text\n\nIeguchi K, Tomita T, Omori T, et al.: ADAM12-cleaved ephrin-A1 Contributes to Lung Metastasis. Oncogene. 2014; 33(17): 2179–2190. PubMed Abstract | Publisher Full Text\n\nBrantley-Sieders DM, Jiang A, Sarma K, et al.: Eph/ephrin profiling in human breast cancer reveals significant associations between expression level and clinical outcome. PLoS One. 2011; 6(9): e24426. PubMed Abstract | Publisher Full Text | Free Full Text\n\nIeguchi K, Omori T, Komatsu A, et al.: Ephrin-A1 expression induced by S100A8 is mediated by the toll-like receptor 4. Biochem Biophys Res Commun. 2013; 440(4): 623–629. PubMed Abstract | Publisher Full Text\n\nYoungblood VM, Kim LC, Edwards DN, et al.: The Ephrin-A1/EPHA2 Signaling Axis Regulates Glutamine Metabolism in HER2-Positive Breast Cancer. Cancer Res. 2016; 76(7): 1825–1836. PubMed Abstract | Publisher Full Text | Free Full Text\n\nEfazat G, Novak M, Kaminskyy VO, et al.: Ephrin B3 interacts with multiple EphA receptors and drives migration and invasion in non-small cell lung cancer. Oncotarget. 2016; 7(37): 60332–60347. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChu M, Zhang C: Inhibition of angiogenesis by leflunomide via targeting the soluble ephrin-A1/EphA2 system in bladder cancer. Sci Rep. 2018; 8(1): 1539. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhuo W, Liu Y, Li S, et al.: Long Noncoding RNA GMAN, Up-regulated in Gastric Cancer Tissues, Is Associated With Metastasis in Patients and Promotes Translation of Ephrin A1 by Competitively Binding GMAN-AS. Gastroenterology. 2019; 156(3): 676–691.e11. PubMed Abstract | Publisher Full Text\n\nLee PC, Chen ST, Kuo TC, et al.: C1GALT1 is associated with poor survival and promotes soluble Ephrin A1-mediated cell migration through activation of EPHA2 in gastric cancer. Oncogene. 2020; 1–17. PubMed Abstract | Publisher Full Text\n\nLisabeth EM, Falivelli G, Pasquale EB: Eph receptor signaling and ephrins. Cold Spring Harb Perspect Biol. 2013; 5(9): pii: a009159. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKania A, Klein R: Mechanisms of ephrin-Eph signalling in development, physiology and disease. Nat Rev Mol Cell Biol. 2016; 17(4): 240–256. PubMed Abstract | Publisher Full Text\n\nBarquilla A, Pasquale EB: Eph receptors and ephrins: therapeutic opportunities. Annu Rev Pharmacol Toxicol. 2015; 55: 465–487. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDarling TK, Lamb TJ: Emerging Roles for Eph Receptors and Ephrin Ligands in Immunity. Front Immunol. 2019; 10: 1473. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShiuan E, Chen J: Eph Receptor Tyrosine Kinases in Tumor Immunity. Cancer Res. 2016; 76(22): 6452–6457. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCoulthard MG, Morgan M, Woodruff TM, et al.: Eph/ephrin signaling in injury and inflammation. Am J Pathol. 2012; 181(5): 1493–1503. PubMed Abstract | Publisher Full Text\n\nLiu DP, Wang Y, Koeffler HP, et al.: Ephrin-A1 is a negative regulator in glioma through down-reguation of EphA2 and FAK. Int J Oncol. 2007; 30(4): 865–871. PubMed Abstract | Publisher Full Text\n\nZhang W, Zeng X, Briggs KJ, et al.: A potential tumor suppressor role for Hic1 in breast cancer through transcriptional repression of ephrin-A1. Oncogene. 2010; 29: 2467–2476. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFrieden LA, Townsend TA, Vaught DB, et al.: Regulation of heart valve morphogenesis by Eph receptor ligand, ephrin-A1. Dev Dyn. 2010; 239(12): 3226–3234. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBrantley-Sieders DM, Zhuang G, Vaught D, et al.: Host deficiency in Vav2/3 guanine nucleotide exchange factors impairs tumor growth, survival, and angiogenesis in vivo. Mol Cancer Res. 2009; 7(5): 615–623. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVarghese F, Bukhari AB, Malhotra R, et al.: IHC Profiler: an open source plugin for the quantitative evaluation and automated scoring of immunohistochemistry images of human tissue samples. PLoS One. 2014; 9(5): e96801. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShiuan E: \"4T1 primary tumor dimensions and weights\". Harvard Dataverse, V1. 2020. http://www.doi.org/10.7910/DVN/AGKDWV\n\nShiuan E: \"4T1 recurrent primary and spontaneous lung metastases\". Harvard Dataverse, V1. 2020. http://www.doi.org/10.7910/DVN/FU8JEY\n\nShiuan E: \"Images and quantification of 4T1-GFP-luciferase spontaneous lung metastases\". Harvard Dataverse, V1. 2020. http://www.doi.org/10.7910/DVN/2ANDYX\n\nShiuan E: \"4T1-GFP-luciferase bioluminescence images and quantification post-tail vein injection\". Harvard Dataverse, V1. 2020. http://www.doi.org/10.7910/DVN/39D0YR\n\nAlonso-C LM, Trinidad Eva MA, de Garcillan B, et al.: Expression profile of Eph receptors and ephrin ligands in healthy human B lymphocytes and chronic lymphocytic leukemia B-cells. Leuk Res. 2009; 33(3): 395–406. PubMed Abstract | Publisher Full Text\n\nWohlfahrt JG, Karagiannidis C, Kunzmann S, et al.: Ephrin-A1 suppresses Th2 cell activation and provides a regulatory link to lung epithelial cells. J Immunol. 2004; 172(2): 843–850. PubMed Abstract | Publisher Full Text\n\nSharfe N, Nikolic M, Cimpeon L, et al.: EphA and ephrin-A proteins regulate integrin-mediated T lymphocyte interactions. Mol Immunol. 2008; 45(5): 1208–1220. PubMed Abstract | Publisher Full Text\n\nAasheim HC, Delabie J, Finne EF: Ephrin-A1 binding to CD4+ T lymphocytes stimulates migration and induces tyrosine phosphorylation of PYK2. Blood. 2005; 105(7): 2869–76. PubMed Abstract | Publisher Full Text\n\nSharfe N, Freywald A, Toro A, et al.: Ephrin stimulation modulates T cell chemotaxis. Eur J Immunol. 2002; 32(12): 3745–3755. PubMed Abstract | Publisher Full Text\n\nChen DS, Mellman I: Oncology meets immunology: the cancer-immunity cycle. Immunity. 2013; 39(1): 1–10. PubMed Abstract | Publisher Full Text\n\nBinnewies M, Roberts EW, Kersten K: Understanding the tumor immune microenvironment (TIME) for effective therapy. Nat Med. 2018; 24(5): 541–550. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJiang Y, Li Y, Zhu B: T-cell exhaustion in the tumor microenvironment. Cell Death Dis. 2015; 6: e1792–e1792. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPauken KE, Wherry EJ: Overcoming T cell exhaustion in infection and cancer. Trends Immunol. 2015; 36(4): 265–276. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGabrilovich DI, Nagaraj S: Myeloid-derived suppressor cells as regulators of the immune system. Nat Rev Immunol. 2009; 9(3): 162–174. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAwad RM, De Vlaeminck Y, Maebe J, et al.: Turn Back the TIMe: Targeting Tumor Infiltrating Myeloid Cells to Revert Cancer Progression. Front Immunol. 2018; 9: 1977. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShiuan E: \"4T1 primary tumor flow cytometry\". Harvard Dataverse, V1. 2020. http://www.doi.org/10.7910/DVN/ZRX2RG\n\nShiuan E: \"4T1-GFP-luciferase tumor-bearing lung flow cytometry\". Harvard Dataverse, V1. 2020. http://www.doi.org/10.7910/DVN/S06NQ1\n\nPotente M, Gerhardt H, Carmeliet P: Basic and therapeutic aspects of angiogenesis. Cell. 2011; 146(6): 873–887. PubMed Abstract | Publisher Full Text\n\nBaluk P, Hashizume H, McDonald DM: Cellular abnormalities of blood vessels as targets in cancer. Curr Opin Genet Dev. 2005; 15(1): 102–111. PubMed Abstract | Publisher Full Text\n\nNagy JA, Chang SH, Shih SC, et al.: Heterogeneity of the tumor vasculature. Semin Thromb Hemost. 2010; 36(3): 321–331. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOgawa K, Pasqualini R, Lindberg RA, et al.: The ephrin-A1 ligand and its receptor, EphA2, are expressed during tumor neovascularization. Oncogene. 2000; 19(52): 6043–6052. PubMed Abstract | Publisher Full Text\n\nBrantley DM, Cheng N, Thompson EJ, et al.: Soluble Eph A receptors inhibit tumor angiogenesis and progression in vivo. Oncogene. 2002; 21(46): 7011–7026. PubMed Abstract | Publisher Full Text\n\nBrantley-Sieders DM, Fang WB, Hicks DJ, et al.: Impaired tumor microenvironment in EphA2-deficient mice inhibits tumor angiogenesis and metastatic progression. FASEB J. 2005; 19(13): 1884–1886. PubMed Abstract | Publisher Full Text\n\nChen J, Hicks D, Brantley-Sieders D, et al.: Inhibition of retinal neovascularization by soluble EphA2 receptor. Exp Eye Res. 2006; 82(4): 664–673. PubMed Abstract | Publisher Full Text\n\nBrantley-Sieders DM, Fang WB, Hwang Y, et al.: Ephrin-A1 facilitates mammary tumor metastasis through an angiogenesis-dependent mechanism mediated by EphA receptor and vascular endothelial growth factor in mice. Cancer Res. 2006; 66(21): 10315–10324. PubMed Abstract | Publisher Full Text\n\nDunaway CM, Hwang Y, Lindsley CW, et al.: Cooperative signaling between Slit2 and Ephrin-A1 regulates a balance between angiogenesis and angiostasis. Mol Cell Biol. 2011; 31(3): 404–16. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShiuan E: \"CD31 and aSMA images and quantification of 4T1 primary tumors\". Harvard Dataverse, V1. 2020. http://www.doi.org/10.7910/DVN/MOYPE7\n\nShiuan E: \"4T1-GFP-luciferase 24-hr lung colonization flow cytometry\". Harvard Dataverse, V1. 2020. http://www.doi.org/10.7910/DVN/G7TAAE\n\nShiuan E: \"PCNA images and quantification of 4T1-GFP-luciferase lung metastases\". Harvard Dataverse, V1. 2020. http://www.doi.org/10.7910/DVN/8AJKFM\n\nVaught D, Chen J, Brantley-Sieders DM: Regulation of mammary gland branching morphogenesis by EphA2 receptor tyrosine kinase. Mol Biol Cell. 2009; 20(10): 2572–2581. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKang M, Jeong W, Bae H, et al.: Bifunctional role of ephrin A1-Eph system in stimulating cell proliferation and protecting cells from cell death through the attenuation of ER stress and inflammatory responses in bovine mammary epithelial cells. J Cell Physiol. 2018; 233(3): 2560–2571. PubMed Abstract | Publisher Full Text\n\nAveraimo S, Assali A, Ros O, et al.: A plasma membrane microdomain compartmentalizes ephrin-generated cAMP signals to prune developing retinal axon arbors. Nat Commun. 2016; 7: 12896. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHarboe M, Torvund-Jensen J, Kjaer-Sorensen K, et al.: Ephrin-A1-EphA4 signaling negatively regulates myelination in the central nervous system. Glia. 2018; 66(5): 934–950. PubMed Abstract | Publisher Full Text\n\nAoki M, Yamashita T, Tohyama M: EphA receptors direct the differentiation of mammalian neural precursor cells through a mitogen-activated protein kinase-dependent pathway. J Biol Chem. 2004; 279(31): 32643–32650. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "61805",
"date": "07 Apr 2020",
"name": "Jinming Yang",
"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 role of the ephrin-A1/EphA2 signaling axis in tumor cells has been studied extensively. However, many cell types in the tumor microenvironment also express ephrin/Eph molecules, the functions of which are not completely understood. This report investigates the EphA2/ephrin-A1 axis-mediated host-tumor interaction. By implanting tumors in wild-type or ephrin-A1 knockout hosts, the authors showed that loss of ephrin-A1 in tumor microenvironment significantly reduced tumor metastasis, both in the spontaneous allograft model and in experimental metastasis assays via tail vein injection.\nOverall, the studies are well designed and the manuscript is well written. Using both spontaneous metastasis and tail vein injection models increases the rigor and reproducibility of the studies. Experiments are also well controlled by using littermates and scoring blindly. The authors showed negative findings in tumor-infiltrating immune populations and tumor vascularity between ephrin-A1 knockout and wild-type control littermates. Nevertheless, the exact molecular mechanism of ephrin-A1 in tumor metastasis has not been determined. For example, decreased lung metastatic lesions could be due to reduced survival of tumor cells in circulation, extravasation into lung, or growth in the lung microenvironment, as ephrin-A1 is expressed in the lung epithelium, vascular endothelial cells, and some of immune cells. Because this is a global knockout, dissecting specific roles of ephrin-A1 in each cell population in the tumor microenvironment are challenging. After all, the fact that ephrin-A1 plays a critical role in metastasis should be of interest to the general audience in the field of cancer research.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "5488",
"date": "14 May 2020",
"name": "Eileen Eileen",
"role": "Author Response",
"response": "Thank you for reviewing this manuscript!"
}
]
},
{
"id": "61809",
"date": "15 Apr 2020",
"name": "Elena B. Pasquale",
"expertise": [
"Reviewer Expertise Signal transduction",
"with focus on Eph receptors and ephrins in cancer and the nervous system."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 novel and interesting study on the role of ephrin-A1 expressed in the host in breast cancer lung metastasis. The role of ephrin-A1 in the tumor microenvironment and the metastatic niche is poorly understood. This manuscript addresses this gap in knowledge by systematically investigating how ephrin-A1 expressed in tumor immune cells, the tumor vasculature, the lung vasculature and the lung microenvironment affects lung metastases formed by 4T1 mouse breast cancer cells. These studies uncover a new role of host ephrin-A1 in promoting the formation of lung metastases by breast cancer cells and in tumor recurrence.\n\nOverall, the manuscript is of high quality and most conclusions are well supported by the data presented. I only have several suggestions that the authors might want to consider.\n\nThe authors conclude that host ephrin-A1 does not affect the growth of the primary tumor. In contrast, they find that ephrin-A1 present in the microenvironment promotes primary tumor recurrence as well as the growth of lung metastases. However, the primary tumors were seeded by injecting tumor cells mixed with Matrigel. I wonder whether the use of Matrigel might create an artificial microenvironment that obfuscates possible effects of host ephrin-A1 on the primary tumor, particularly at early stages of tumor development. The authors should indicate in the Methods the volume of Matrigel used and mention also in the Results that the tumor cells were injected mixed with Matrigel. They could also briefly mention in the text the possible effects of Matrigel on primary tumor development (or discuss why they believe using Matrigel is not a significant issue).\n\nIn some instances (such as Fig. 3A, dendritic cells panel, and possibly Fig. 5B) the authors find that there is no statistically significant difference between mice with different genotypes. However, n (presumably indicating the number of mice) is small in some groups and the variability of the data is high, so it could be argued that analysis of more mice could yield a more definitive result. Perhaps the conclusions could be stated more cautiously in these cases.\n\nOn page 3, left column, third paragraph, ref. 43 does not report a tumor suppressive role of ephrin-A1 but rather the opposite.\n\nVery minor points:\nAnimal models section in the Methods: In a couple of sentences, it would seem better to say “female littermates” rather than “gender-matched littermates” since only females were used.\n\nSpecify in the figure legends for the histograms whether the dots represent the number of mice.\n\nPage 10, right column, 3 lines from the bottom. “Higher” would be better than “increased”.\n\nPage 11, right column, second paragraph, line 13. It should be “have” not “has”.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "5490",
"date": "14 May 2020",
"name": "Eileen Eileen",
"role": "Author Response",
"response": "Thank you for reviewing this manuscript! We have addressed your suggestions and comments with the following revisions:1) Added details regarding use of Matrigel in the Methods and Results and discussed potential confounding effects of Matrigel on primary tumor volume in the Discussion.2) For data with small sample sizes showing a nonsignificant trend, revised interpretation of data to include possibility of significant differences if more mice were used.3) Indicated in all figure legends that each data point corresponds to an individual mouse.4) Minor text changes."
}
]
},
{
"id": "61807",
"date": "22 Apr 2020",
"name": "Nikki Cheng",
"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\nEphrinA1 is a cell surface bound protein that has been shown to be tumor suppressive when expressed in cancer cells. In this study, the authors characterize the effects of the microenvironment on mammary tumor progression using variations of the 4T1 mammary tumor model- spontaneous, and experimental metastasis. By injecting 4T1 cells in ephrinA1 knockout mice, they show that ephrinA1 knockout does not affect primary tumor growth and inhibits metastasis and recurrence. The study is generally well written, and the phenotypes are interesting. Comments are described below:\nThe discussion could be better expanded.\n\nHow do expression of ephrinA1 and corresponding receptors in the stroma of primary tumor vs. lung tissue compare in normal and tumor bearing mice? How do results fit into the bigger picture of what is known about this?\n\nRecommend expanding on the discussion of the mechanism regarding ephrinA1 mediated lung metastasis. In particular:\n\na) How might ephrinA1 signaling might affect macrophage function in the lung.\n\nb) Please expand on what is known about macrophage polarization, effects on T cells in the lung. How would ephrinA1 signaling affect macrophage function in the formation of pre-metastatic vs. metastatic niche?\n\nc) How would ephrinA1 ko affect Eph receptorA1 vs. A2 signaling in the lung stroma?\n\nd) Further discussion the clinical implications of the study would be helpful, especially since the tumor suppressive/oncogenic effects of ephrinA1 appear to be cell type dependent.\n\nMore minor comments:\nFigure 1A, C, D, Figure 3, Figure 4, Figure 5B-C: Statistics are not consistently shown. Even if data show no statistical significance, it would be helpful to include p- value or indicate n.s to indicate the analysis was done.\n\nFigure 4: CO-IF staining is a bit difficult to see. It would be helpful to show magnified insets for clearer details.\n\nFigure 5: recommend revising figure legend. From the way the figure legend is stated “Ephrin-A1-deficient lung microenvironment provides a less favorable metastatic niche,” I would expect data correlating decreased metastasis with changes in the lung microenvironment of ephinA1 KO mice. However, this is not the case - no changes in endothelial cells were observed. Alternatively, for Figures 3 and 5 - recommend reorganizing some of the data so that the messages are clearer. Figure 3 figure legend indicates that “Tumor-infiltrating immune populations are not significantly different in ephrin-A1-deficient hosts” However, Figure 3D show increased in macrophage recruitment to lung tissues in ephrinA1 KO mice. This data would be more appropriate in Figure 5, which shows decreased lung metastasis, along with characterization of endothelial cells in the lung.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "5491",
"date": "14 May 2020",
"name": "Eileen Eileen",
"role": "Author Response",
"response": "Thank you for reviewing this manuscript! We have addressed your suggestions and comments with the following revisions:1) Expanded discussion on potential impact of ephrin-A1 deficiency on other ephrin-A1 ligands and EphA receptors in stromal cells and their signaling.2) Expanded discussion on what is known about the role of macrophages in the lung metastatic niche and how ephrin-A1 deficiency may impact recruitment and function of macrophages.3) Expanded discussion of clinical implications of the study.4) Indicated \"ns\" on graphs where data from WT and KO showed a trending difference that was not statistically significant.5) Minor text changes."
}
]
}
] | 1
|
https://f1000research.com/articles/9-217
|
https://f1000research.com/articles/8-1741/v1
|
11 Oct 19
|
{
"type": "Research Article",
"title": "The Noetic Experience and Belief Scale: A validation and reliability study",
"authors": [
"Helané Wahbeh",
"Garret Yount",
"Cassandra Vieten",
"Dean Radin",
"Arnaud Delorme",
"Garret Yount",
"Cassandra Vieten",
"Dean Radin",
"Arnaud Delorme"
],
"abstract": "Background: Belief in the paranormal is widespread worldwide. Recent surveys suggest that subjective experiences of the paranormal are common. A concise instrument that adequately evaluates beliefs as distinct from experiences does not currently exist. To address this gap, we created the Noetic Experiences and Beliefs Scale (NEBS) which evaluates belief and experience as separate constructs. Methods: The NEBS is a 20-item survey with 10 belief and 10 experience items rated on a visual analog scale from 0-100. In an observational study, the survey was administered to 361 general population adults in the United States and a subsample of 96 one month later. Validity, reliability and internal consistency were evaluated. A confirmatory factor analysis was conducted to confirm the latent variables of belief and experience. The survey was then administered to a sample of 646 IONS Discovery Lab participants to evaluate divergent validity and confirm belief and experience as latent variables of the model in a different population. Results: The NEBS demonstrated convergent validity, reliability and internal consistency (Cronbach’s alpha Belief 0.90; Experience 0.93) and test-retest reliability (Belief: r = 0.83; Experience: r = 0.77). A confirmatory factor analysis model with belief and experience as latent variables demonstrated a good fit. The factor model was confirmed as having a good fit and divergent validity was established in the sample of 646 IONS Discovery Lab participants. Conclusions: The NEBS is a short, valid, and reliable instrument for evaluating paranormal belief and experience.",
"keywords": [
"paranormal",
"noetic",
"paranormal belief",
"paranormal experience",
"exceptional human experiences",
"anomalous information reception"
],
"content": "Introduction\n\n“Paranormal beliefs pertain to phenomena that have not been empirically attested to the satisfaction of the scientific establishment”1. Paranormal beliefs encompass a broad range of concepts, such as ghosts or spirits, extrasensory perception (ESP), extraterrestrial beings, and mind-to-mind communication, or telepathy. Belief in the paranormal is widespread around the world1–13. For example, in a Gallup poll of 1,002 United States adults conducted in 2005, 55% respondents believed in psychic or spiritual healing or the power of the human mind to heal the body, 41% believed in extrasensory perception, and 31% believed in telepathy or mind-to-mind communication14.\n\nHowever, having a belief in the paranormal does not necessarily mean having experienced the paranormal. A paranormal experience refers to an individual’s memory of an experience that one judges to be genuine. The memory of a paranormal experience relies on a different mental substrate than a belief based on environment, education and reasoning and the neural structures underlying memory of an experience and belief are likely different15,16. Paranormal belief and experience are often correlated when measured simultaneously, although this is rarely done17,18. For example, one study found a positive correlation (r = 0.61) between paranormal experience and belief scores12. Another interesting study found that exposure to television programs that regularly depict paranormal phenomena was positively correlated with belief, but only for respondents who had personal experiences19.\n\nPrevalence of reported paranormal experiences evaluated over the last 40 years in a variety of populations has ranged from a low of 10% in Scottish citizens20 to a high as 97% in enthusiasts in the United States12. Two very large prevalence studies have been conducted. One surveyed adults in 13 European countries and the United States (N=18,607). European respondents reported experiencing telepathy (34%), clairvoyance (21%), and contact with the dead (25%). Percentages for the U.S. adults were considerably higher: 54%, 25%, and 30% respectively21. Another large study of British adults (n=4,096) found that 37% of respondents reported at least one paranormal experience defined as precognitions, extra-sensory perception, mystical experiences, telepathy, and after-death communication22. Other smaller prevalence studies have been conducted around the world. Haraldsson et al. conducted two surveys of prevalence in Iceland, one in 1974 with 902 participants5 and one in 2006 with 991 participants3. He found that psychic phenomena actually increased from 59% of men and 71% of women in 1974 to 70% of men and 81% of women in 2006. In Scotland, 10–16% of the general population sample (n – 241) had experienced second sight, with the exception of the Grampian area where prevalence was more than doubled at 33%20. Chinese, Japanese, African-American and Caucasian-American college students (n - 1922) were surveyed and 31–47% reported having at least one experience23. Of 502 adults in Winnipeg, Canada 65%24 and 38% of 622 Charlottesville, Virginia students and townspeople25 reported having at least one experience. In the United States, 67% of the 1460 participants reported having had an ESP experience, 31% a clairvoyant experience, and 42% contact with the dead26. More recently in the United States, 89.3% of the general population, 89.5% of scientists and engineers, and 97.8% of paranormal enthusiasts reported at least one paranormal experience (n – 899)12.\n\nSpecificity of the work in this field is limited by the lack of questionnaires that adequately separate paranormal belief from experience and do so concisely1,27. Using ambiguous measures can lead to confounding the two constructs of belief and experience, and blur results1,6. Instruments that do separate these constructs are long and not conducive to the time constraints of many studies - see Exceptional Experiences Questionnaire28 and Anomalous Experience Inventory29. To address these limitations and as part of a larger research program on extended human capacities, we created the Noetic Experience and Belief Scale (NEBS), a 20-item survey that evaluates paranormal beliefs and experiences separately. The present studies investigate the psychometric properties of the Noetic Experience and Belief Scale in two populations. By studying these phenomena, we aim to gain a deeper understanding of the nature of consciousness and the reach of human potential.\n\nInitial development of the NEBS The NEBS was developed through consensus by the authors and two expert consultants who actively work in the field. This group was informed by our own previous studies and by reviewing other studies and previously used instruments that evaluated paranormal beliefs and/or experiences. One previous study30 evaluated the prevalence of 27 paranormal experiences listed here in decreasing order of prevalence: Claircognizance, Clairempathy, Precognition, Lucid Dreaming, Emotional Healing, Clairvoyance, Clairsentience, Animal Communication, Telepathy, Aura Reading, Astral Projection, Clairaudience, Clairalience, Mediumship, Channeling, Physical Healing, Geomancy, Retrocognition, Psychometry, Remote Viewing, Automatic Writing, Clairgustance, Psychokinesis, Pyrokinesis, Levitation, and Psychic Surgery (please see extended data for definitions of each of these terms31). Based on feedback from participants and a review of these items, we removed emotional healing (very similar to physical healing), psychic surgery (very rare), and clairsentience (very similar to claircognizance), renamed channel to psychophony and mediumship to contact with the dead, and added the item Information from Dreams. We then conducted another prevalence study in a different population. Notably, we did not use the jargon term for each paranormal belief/experience, but instead used as neutral language as possible to describe the experience itself. For example, rather than asking if the participant had ever experienced “pyrokinesis - the ability to create and/or manipulate fire”, the item asked “Have you ever created fire using only your concentration or will?” These neutral language items were then administered to 899 participants consisting of three samples: a general population sample, scientists and engineers, and paranormal enthusiasts12.\n\nIn both studies, we found that some items were highly correlated and represented overlapping constructs. They could also be viewed as specific nuanced experiences within a larger extended human capacities category. For example, psychic physical healing or the purported ability to feel other people’s physical symptoms in your own body and heal, transform, or transmute them would fall under the umbrella category of psychokinesis or the purported ability to influence a physical system without any physical interaction or with mental effort alone. Thus, in an effort to reduce participant burden and allow for quick assessment of experiences and beliefs we collapsed any overlapping constructs into individual items for each of the following categories: 1. Non-local consciousness (e.g. Astral Projection, Lucid Dreaming); 2. Extraterrestrials; 3. Precognition/Retrocausation; 4. Survival of Consciousness (after bodily death); 5. Contact with the dead (Mediumship); 6. Clairvoyance (Claircognizance, Clairempathy, Clairvoyance, Clairsentience, Aura Reading, Clairalience, Clairaudience, Geomancy, Clairgustance, Remote Viewing, Psychometry, Animal Communication); 7. Psychokinesis (Physical Healing, Psychokinesis, Psychic Surgery, Pyrokinesis, Levitation); 8. Telepathy; 9. Automatism (Channeling, Automatic Writing)\n\nWe also reviewed a number of existing questionnaires that measured paranormal experience and/or belief8,26,28,29,32–45. Each scale was evaluated for number of items, belief and experience as separate constructs, and subscales (please see extended data31). From this review, an additional item on intuition, representing perhaps the most common paranormal experience, was added to the new scale for a total of 10-items.\n\nThe instrument was called the Noetic Experience and Belief Scale using noetic from the Greek noēsis/noētikos, meaning inner wisdom, direct knowing, or subjective understanding; and unlike a vague impression, a noetic experience carries a deep sense of authority and certainty. We included “noetic” in the title rather than “paranormal” in part because of the stigma associated with the term paranormal, which could introduce bias that might be mitigated by using an alternate term. Similarly, the paranormal categories were not stated in the scale but only descriptions of the constructs included (please see extended data31.\n\nThe objectives of the following two observational studies were to evaluate the validity and reliability of the NEBS and to confirm the two latent variables of belief and experience in a confirmatory factor analysis. In study 1, the survey was administered to 350 participants for the validity and confirmatory factor analyses and again to a subsample of 96 of these participants for a test-retest analysis. In study 2, the survey was administered to a different population where divergent validity was evaluated and the factor model reevaluated. We hypothesized that NEBS would be valid, reliable, and demonstrate good fit for a model with belief and experience as latent variables in both populations.\n\n\nSTUDY 1: General population sample\n\nProcedures The first study administered the NEBS to a randomly selected general population group in the United States to establish validity, test-retest reliability, and confirm the two latent variables of belief and experience. We contracted with Lucid, LLC (New Orleans, Louisiana) to obtain completed surveys from an unbiased census-distributed sample of 350 participants representative of the general population in the United States. The sample was unbiased in that it was not associated with the Institute of Noetic Sciences or any other paranormal or noetic-related group. Lucid, LLC is a marketplace that connects hundreds of sample suppliers with individual primary research studies to facilitate online surveys. Lucid uses screening questions to qualify respondents for a particular study then through programmatic technology aligns the best suppliers for that individual audience. Once a respondent qualifies through the screener, the appropriate suppliers are notified through an API and an email is triggered from the supplier directly to the survey taker. Each of the suppliers on the marketplace has approximately 200 pre-profiled mapped qualifications. These include age, gender, household income, job role, hobbies, etc. Lucid uses these qualifications as well as the screening questions to ensure efficiency and high quality when matching survey takers with individual projects. All potential volunteers are screened, checked for validity, and emailed a link to the survey. Participants were English-speaking adults in the United States. Inclusion criteria were: Adults aged 18 to 89, who could read and understand English, and were willing to complete questionnaires. Exclusion criteria were: Children (<18 years old) or Elders >89 years old. Elders 90 years old and older were excluded because the survey was designed to be anonymous and recording ages greater than 90 is considered private health information46. Targets for distribution were based on United States census values and were as follows: Gender - 50% males and females, Age - 18-24 - 13%, 25-44 - 41%, 45-64 - 30%, 65+ - 16%; Ethnicity - Hispanic - 11%, Black - 12%, White (non-Hispanic) - 59%, Other - 18%. The study was approved by the Institute of Noetic Sciences Institutional Review Board # WAHH_2018_06. Participants were given a link to a Health Insurance Portability and Accountability Act compliant survey on SurveyMonkey. The first page of the survey was a consent form (please see extended data31). Participants were asked to read the form and check a box acknowledging that they had been informed of the procedures, and risks and benefits of participating in the study. They then completed the survey, which took approximately 15-20 minutes. Data were collected from November 9, 2018 through December 14, 2018. All data were collected anonymously, with no identifiers or IP addresses. Participants were compensated $3 for completing the survey once and $7 ($3 + $4) if they also participated in the retest administration.\n\nMeasures In addition to demographic information (e.g. age, gender, marital status, socioeconomic status), the main instrument in the survey was the Noetic Experience and Belief Scale (NEBS). The scale contains ten statements about beliefs in intuition, non-local consciousness, extraterrestrials, precognition, survival of consciousness, contact with the dead, clairvoyance, psychokinesis, telepathy, and automatism that all begin with the stem “I believe…” and then a description of the concept. The participant rates each belief statement on a slider anchored by Disagree Strongly (0) and Agree Strongly (100). For each of the ten items, participants also answered “I have personally had this experience.” on a slider scale anchored by Never (0) and Always (100). Two experience items were worded differently to accommodate the nature of the concept. The life after death experience item was worded “I have personally had an experience that I interpreted as a proof that consciousness survives the physical body.” and the contact with the dead item was worded “I have personally had the experience of contact with the dead.” Six of the 10 items were from the Australian Sheep-Goat Scale, three of which were exactly the same (#’s 9, 10, 11), and three were modified (#’s 4, 5, 14)45. The scale results in overall scores for paranormal belief and experience by averaging the ten items for each subscale. Item scores can also be used individually for scores on each specific category. Internal consistency of the NEBS scale was calculated with a Cronbach α coefficient, as described subsequent sections (Full scale is available in extended data31).\n\nConvergent construct validity was measured by administering pre-existing survey instruments that evaluate similar concepts: Australian Sheep-Goat Scale45,47, Revised Paranormal Belief Scale43, and Anomalous Experiences Inventory (AEI)29. The Australian Sheep-Goat Scale is an 18-item questionnaire on various beliefs and experiences. Respondents endorse True (2 points), Uncertain (1 point), or False (0 points) for each item. Values are then summed to form a score ranging from 0-36. The Revised Paranormal Belief Scale is a 26-item scale that measures the degree of belief in the paranormal in each of seven dimensions: Traditional Religious Belief, Psi, Witchcraft, Superstition, Spiritualism, Extraordinary Life Forms, and Precognition. Respondents endorse how strongly they believe in each item on a 7-item Likert scale. Subscales and a total score are obtained by calculating means of specific items. The AEI is a 70-item questionnaire that evaluates multiple subscales: anomalous/paranormal experience, anomalous/paranormal beliefs, anomalous/paranormal ability, fear of the anomalous/paranormal, and drug use. Respondents answer True (1) or False (0) for each item and values are summed for each scale. The scales selected have already been assessed as valid and reliable and used in numerous peer-reviewed publications. Correlation matrices of the scores were evaluated for expected patterns of associations between measures of the same construct.\n\nTest-Retest: Some participants repeated the survey approximately one month later so that test-retest reliability could be assessed with a Pearson correlation coefficient.\n\nSample size: Some sources suggest at least 10 people per item for psychometric validation although a recent review suggested that sample size is rarely justified a priori48. We aimed for a sample size of 350 for the 20-item scale. For confirmatory factor analysis, there is also no agreement on the number of participants needed although sources49 recommend approximately 10 participants for each estimated parameter (10 × 20 parameters = 200). We had 361 participants resulting in a ratio of 18.05 participants to each parameter estimated.\n\nConfirmatory factor analysis: A confirmatory factor analysis was used (rather than an exploratory factor analysis) because a theoretical framework was already established for evaluating belief and experience as separate constructs, albeit highly correlated1,12. The latent variables for the model were Belief and Experience. Observed variables were the 20 NEBS items. Univariate variables were tested for normality with the Shapiro-Wilk Test and any outliers assessed with scatter and box plots. Normality of residuals were evaluated with kernal density estimates and standardized normal probability plots. Outliers were evaluated with residuals, leverages, influence and Cook’s distance. Multicollinearity was evaluated with the variance inflation factor (VIF), which is the quotient of the variance in a model with multiple terms by the variance of a model with one term alone and quantifies the severity of multicollinearity. An unstructured covariance matrix was used so as to not impose any constraints on the variance and covariance values. All 20 items were highly correlated and thus, covariances between unique factors for all items were included in the model and then removed if they did not reach significance. All statistical analyses were conducted with Stata 15.0 (StataCorp, LLC, College Station, TX).\n\n\nResults\n\nConstruct validity In total, 444 began the survey; 26 did not agree to the consent form and 57 agreed to the consent form but did not complete the survey. The remaining 361 participants completed the survey (underlying data50). Surveys were collected between November 9, 2018 and December 13, 2018. Participants were on average 44 years old ± 16.8 and had 14.5 ± 5.3 years of education. Of these, 52% were female and 56.8% were in-relationship. Participants were mostly Caucasian (67% Caucasian, 13% Black or African American, 8% Hispanic or Latino, 6% Asian or Pacific Islander, 5% American Indian or Alaskan Native, and 2% preferred not to answer). In terms of salary, 67% of participants had earned between 0 and $75,000 (30% Under $30K, 37% $30K to under $75K, 11% $75K to under $100K, 11% $100K or under $150K, 7% $150K to under $250K, 3% $250K or greater, 2% Decline to answer) with an average household size of 2.6 ± 1.4.\n\nThe means and standard deviations for the paranormal belief and experience questionnaires are shown in Table 1. All correlation pairs were positive and significant at p = 0.05 level or less (all but three being more than p < 0.00005).\n\nNotes: p ≤ 0.0001 except for one shaded item where p = 0.02. With a Bonferroni multiple comparison adjustment, a significant p-value would be <0.0044 (0.05/16*16/2-16). Bold font represents Pearson r values that are moderately correlated (0.50-0.69), bold and underlined fonts represent values that are highly correlated (0.70-0.89), bold and double underlined fonts represent values that are very highly correlated (0.90-1.0). NEBS = Noetic Experience and Belief Scale, AEI = Anomalous Experiences Inventory.\n\nInternal consistency Cronbach’s alpha was calculated for the NEBS Belief subscale items and Experience subscale items to measure the extent to which the items within the subscales correlated with each other and measured a similar construct51. The ten belief items had a Cronbach’s alpha of 0.90 and average inter-item covariance of 429.9. The ten experience items had a Cronbach’s alpha of 0.93 and average inter-item covariance of 610.7.\n\nBelief: On average, intuition, survival of consciousness, and non-local consciousness were the highest rated Beliefs (see means and standard deviations for each item in Table 2). All Belief construct pair correlations were significant (p < 0.00005). Telepathy Belief and clairvoyance Belief were highly correlated (Table 2; Very high: r = 0.90 to 1.00; High: r = 0.70 to 0.89; Moderate: r = 0.50 to 0.69; Low: r = 0.30 to 0.49; Negligible: r = 0 to 0.30 (Mukaka, 2012)). Many of the Belief pairs were moderately correlated.\n\nNote: All values significant at p < 0.00005 except for belief in intuition and the experience of extraterrestrials (p = 0.0004), contact with the dead (p = 0.0001), psychokinesis (p = 0.002), telepathy (p = 0.002), and automatism (p = 0.0016). Bold font represents Pearson r values that are moderately correlated, bold and underlined fonts represent values that are highly correlated.\n\nExperience: On average, intuition and non-local consciousness were the most common Experiences. All Experience pairs were significantly correlated (p < 0.00005). Seven Experience pairs were highly correlated (r = 0.70 - 0.89). Many Experiences were moderately correlated (r = 0.50-0.69).\n\nBelief and Experience: Most Belief and Experience pairs were significantly correlated at the p<0.000005 level except for belief in intuition and the experience of extraterrestrials (p = 0.0004), contact with the dead (p = 0.0001), psychokinesis (p = 0.002), telepathy (p = 0.002), and automatism (p = 0.0016). Belief in telepathy was highly correlated with the Experience of telepathy (r = 0.79). Belief and Experience pairs of the same construct were all moderately correlated except for Survival of Consciousness which had a significant but low correlation. Many beliefs were moderately correlated with Experiences.\n\nTest-retest reliability: Of the original 361 participants who completed the survey, 96 completed the same survey again approximately one month after the first administration (mean 35.3 days ± 3.7) between December 14, 2018 and January 2, 2019. Participants who completed the retest had similar demographics as the original sample (age 44 years old ± 16.9, education 14.3 ± 2.1, 54% male, 64% Caucasian, 53% in relationship, 74% with income under $75,000, and average household size of 2.4 ± 1.4). The NEBS had high test-retest reliability for both the Belief (r = 0.83, p <0.00005) and Experience (r = 0.77, p <0.00005) subscales. The Wilcoxon sign-rank test was used to evaluate individual item and subscale differences because variables were not normally distributed. All items and subscale scores were not significantly different between the two time-points except for the telepathy Experience item which decreased Experiences for the second administration (Table 3). Individual’s responses to the subscales remained relatively consistent across the repeated administration and above standardly accepted values for reliability of r = 0.7052.\n\nB - Belief; E - Experience; NEBS - Noetic Experience and Belief Scale.\n\nConfirmatory factor analysis was performed based on data from 361 respondents; there were no missing data. The retest data of the 96 participants were not included in the confirmatory factor analysis modeling. A correlation table of observed values with means and standard deviations is shown in Table 2. The a priori theoretical model of Belief and Experience items as described in the statistics section is presented in Figure 1.\n\nSignificant covariances are denoted with curved arrows and error terms are displayed.\n\nWe hypothesized a two-factor model to be confirmed in the measurement portion of the model where Belief and Experience were the latent variables. We evaluated the assumptions of univariate and multivariate normality and linearity. Univariate variables were not normally distributed individually. The ADF estimation method was used because it makes no assumption of joint normality or even symmetry for observed or latent variables (StataCorp, 2013). Residuals were normally distributed. There were no observations with a Cook’s distance greater than 1. No variable had a VIF less than 0.1 or greater than 10 (average VIF for all variables 3.29) indicating acceptable multicollinearity. The model chi-square (159) was 283.1, the root mean square error of approximation (RMSEA) was 0.060 (90% confidence interval 0.051-0.069), the comparative fit index (CFI) was 0.94, the standardized root mean squared residual (SRMR) was 0.13, and the Tucker-Lewis fit index (TLI) was 0.90. These values represent a good fit of the model to the dataset as indicated by commonly reported fit statistics (RMSEA < 0.08, CFI ≥ 0.90, SRMR < 0.08, TLI ≥ 0.95)53.\n\n\nSTUDY 2 – IONS Discovery Lab sample\n\nThe NEBS was then administered to participants attending workshops at the IONS Discovery Lab and also online. Participants were recruited through workshop leaders hosting events at the IONS EarthRise Retreat Center in Petaluma, California and through workshop leaders hosting events at their own sites who contacted IONS to participate in the study. This study was approved by the IONS Institutional Review Board. Participants had to be adults (aged 18 years and above) with the ability to understand the consent form, were willing and able to complete the measures, and did not have an acute or chronic illness that precluded completion of the survey. Participants enrolled in the IONS Discovery Lab completed a number of surveys including the NEBS prior to their workshops. First, participants read and agreed to the consent form. Then data were collected anonymously through SurveyMonkey. Relevant measures used to establish NEBS divergent validity, which tests whether concepts or measurements that are not supposed to be related are actually unrelated were: Arizona Integrative Outcomes Scale54, Positive and Negative Affective Well-being Scale55, single-item general health56, acute sleep quality scale57, the Numeric Pain Rating Scale58, and Big Five Inventory-10 scale59, and the compassion subscale of the Dispositional Positive Emotion Scale (1. It’s important to take care of people who are vulnerable; 2. When I see someone hurt or in need, I feel a powerful urge to take care of them; 3. Taking care of others gives me a warm feeling inside; 4. I often notice people who need help; 5. I am a very compassionate person.)60.\n\nArizona Integrative Outcomes Scale (AIOS) is a one-item, visual analogue self-rating scale (VAS) with two alternate forms (one for daily ratings, AIOS-24h; and one for monthly ratings, AIOS-1m). The daily rating version was used for this study. The instructions are: \"Please reflect on your sense of wellbeing, taking into account your physical, mental, emotional, social, and spiritual condition over the past 24 hours. Mark the line below with an X at the point that summarizes your overall sense of well-being for the past 24 hours.\" The horizontally-displayed VAS is 100 mm in length, with the low anchor being, \"Worst you have ever been\" and the high anchor being, \"Best you have ever been.\" The AIOS has demonstrated the ability to discriminate between healthy and unhealthy populations and has adequate convergent and divergent validity54.\n\nPositive and negative affective well-being is measured with a variety of dichotomous indicators asking subjects whether they had experienced an emotional state for much of the day yesterday. For positive affect, the emotional states are happiness, enjoyment and smiling/laughter, which, aggregated together, have a reliability of α = 0.72. For negative affect, the emotional states are stress, worry and sadness, with a reliability of α = 0.6555.\n\nOverall health is a single item question “In general, how would you rate your overall health?” which is answered by choosing one of five options: Poor; Fair; Good; Very good; Excellent56.\n\nAcute sleep scale is a single item scale asking participants to rate their quality of sleep over the past 24 hours on an 11-point numeric rating scale ranging from 0 denoting \"best possible sleep\" to 10 denoting \"worst possible sleep\"57.\n\nThe Numeric Pain Rating Scale (NPRS) is a segmented numeric version of the visual analog scale in which a respondent selects a whole number (0–10 integers) that best reflects the intensity of his/her pain. The NPRS is anchored by terms describing pain severity extremes. Participants are asked to report pain intensity “in the last 24 hours” or an average pain intensity with 0 = “No pain” to 10 = “Worst possible pain”58.\n\nBig Five Inventory-10 (BFI) scale is a 10-item measure of the Big Five (or Five-Factor Model) dimensions: Neuroticism, Extraversion, Openness to Experience, Agreeableness, Conscientiousness. The BFI-10 was developed to provide a personality inventory for research settings with time constraints. It allows assessing the Big Five with only two items per dimension. Previous research has shown that the BFI-10 possesses psychometric properties that are comparable in size and structure to longer five factor inventories such as the NEO-PI-R which has 240 items. The score for each dimension is obtained by summing standard items and reverse scored items for each scale59.\n\nCompassion scale is 5 items from the Dispositional Positive Emotion Scale compassion subscale. It measures dispositional tendencies to feel positive emotions toward others in their daily lives. Items are rated from strongly disagree to strongly agree and scored from 1 to 7. Items are averaged for a total score and higher scores indicate greater levels of positive emotion60.\n\nStatistical Analysis: Demographic information was qualitatively described for categorical variables. Means and standard deviations calculated for all continuous variables. Pearson correlations were conducted for relationships between measures. Cronbach’s Alpha was calculated for the Belief and Experience subscales. All analyses were conducted with Stata 15.0 (StataCorp, LLC, College Station, TX). The confirmatory factor analysis was conducted in the same was study 1.\n\n\nResults\n\nIn total, 646 participants completed the surveys from March 17, 2018 through July 22, 2019 (underlying data50). There were no missing data. Participants were on average 55.0 years old ± 13.3 with 17.4 ± 3.0 years of education, 75% female, 81% Caucasian, and 66% in relationship. In terms of income, 28% earned under $75,000, 16% between $75,000 and $100,000, 19% between $100,000 to $150,000, 15% between $150,000 to $250,000, and 12% above $250,000. The average household size was 2.3 ± 1.3.\n\nThe NEBS Belief items had a Cronbach’s alpha of 0.93 and an average inter-item covariance of 304.4. The NEBS Experience items had a Cronbach’s alpha of 0.91 and average inter-item covariance of 476.4. The experience scale was moderately correlated with the belief scale in this sample (Table 4).\n\nNotes: a = p < 0.00005; b = p < 0.001; c = p < 0.05; NEBS - Noetic Experience and Belief Scale. Bold font represents Pearson r values that are moderately correlated (r = 0.50-0.69).\n\nTable 4 shows the correlations of NEBS Belief and Experience scale with the other measures administered. NEBS Belief had negligible correlations (r = 0 - 0.30) with all items according to Mukaka et al. criteria except for NEBS Experience61. NEBS Experience had negligible correlations (0 - 0.30) with all other scales (except NEBS Belief).\n\nThe same confirmatory factor analysis model developed through the general population data was applied to the IONS Discovery Lab dataset to evaluate its fit. The model chi-square (123) was 318.34, the RMSEA was 0.060 (90% confidence interval 0.052-0.068), the CFI was 0.85, the SRMR was 0.24, and the TLI was 0.77. These values demonstrate a good fit of the model to this new dataset and similar to the original dataset where RMSEA = 0.060, CFI = 0.94, SRMR = 0.13, and TLI = 0.90.\n\n\nDiscussion\n\nThe overall results of the two studies provide psychometric support for the validity and reliability of the NEBS as a brief assessment of self-reported paranormal beliefs and experiences. The participant demographics of study 1 reflected the general population of the United States as designated by the recruitment criteria. Construct validity of the NEBS Belief subscale was strong, as it was strongly correlated with multiple other scales measuring paranormal belief including the Australian Sheep Goat scale, the Psi, Spiritual and Precognition subscales of the Paranormal Belief Scale, and AEI Paranormal Belief subscale. Construct validity of the NEBS Experience subscale was also strong, demonstrating higher correlations to experience items such as AEI-Paranormal Experience and AEI-Paranormal Ability than other items such as Traditional Religious Beliefs. The NEBS did not measure paranormal fear or drug use as reflected in the low correlations on those AEI subscales. For divergent validity, there were only negligible correlations (r’s between 0 and 0.30) to all other measures, providing more evidence that the NEBS is not measuring other constructs. Interestingly, other studies evaluating personality traits and paranormal beliefs have been mixed62,63, with some studies observing positive correlations with neuroticism64,65 (unlike our study which found a negligible negative correlation) and others not finding any correlations65–67. The NEBS reliability and internal consistency was also demonstrated through high Cronbach’s alphas for both subscales in two different samples. Our confirmatory factor analysis for two latent constructs of Belief and Experience in the general population dataset revealed a model good fit (RMSEA = 0.06), controlling for covariances between specific individual items, that was then confirmed with the IONS Discovery Lab sample (RMSEA = 0.06). RMSEA calculates the size of the standardized residual correlations and theoretically ranges from 0 (perfect fit) to 1 (poor fit). A model is considered satisfactory when RMSEA < 0.0868,69. Our conceptual model of Belief and Experience as separate constructs and as evaluated through the NEBS was confirmed.\n\nWhen measured separately, Belief and Experience are highly correlated. We found this in both of our samples (study 1: r =0.77; study 2: r = 0.64). Interestingly, the correlation was stronger in our general population sample that in our IONS Discovery Lab sample. The mean NEBS belief scores for the IONS Discovery Lab group were 21.3 points higher than the general population group (59.7 ± 21.9 general population vs. 81.0 ± 18.1 IONS Discovery Lab). The mean NEBS Experience scores were also greater in the IONS Discovery Lab group but only by 15.1 (44.3 ± 25.6 general population vs. 59.4 ± 22.9 IONS Discovery Lab). The AEI - Paranormal Belief and Paranormal Experience subscales were highly correlated in our study 1 sample as well (0.77). Interestingly, the original study of this scale found a much lower (r =0.57) although significant correlation between the two subscales29. We also found belief and experience to be highly correlated (r = 0.61) for another mixed population of scientists and engineers, the general population, and paranormal enthusiasts12. Other studies that have evaluated belief and experience in general have also found positive correlations17,18. A study examining the correlation between specific religious and classic paranormal beliefs, such as belief in heaven and hell or psychic healing, in relation to the paranormal experiences of illness cured by prayer and the use of the mind to heal the body, found mixed results. For example, belief in the devil and belief in illness cured by prayer had a low significant correlation (r = 0.38), but the relationship between illness cured by prayer and the belief in psychic healing (r = -0.04) was not40.\n\nParanormal belief and experience are highly correlated in most studies that assess them, and yet they are distinctly different constructs that should be evaluated separately. What we do not yet understand is the causal or temporal nature of the relationship between belief and experience. Does paranormal belief precede experience or vice versa? Does someone’s belief in the paranormal prime them to experiencing it or does a subjective experience of the paranormal instill belief in the phenomena? Future longitudinal studies evaluating a baseline level of people’s beliefs and collecting data on how those beliefs change over time in relation to any experiences they have would be helpful in answering this question.\n\nThere are a number of limitations that should be kept in mind when reviewing the results of this study. The individual constructs included in the NEBS are highly correlated. Conceptually, the individual concepts are unique but could also be viewed as overlapping. For example, the items on non-local consciousness (B2. I believe that my consciousness is not limited by my physical brain or body. E2. I have personally had this experience.) and survival of consciousness (B5. I believe in life after death. E5. I have personally had an experience that I interpreted as a proof that consciousness survives the physical body.) could be considered as the same construct worded in a different way. The experience items are administered directly after the belief item of the same construct. The instrument was purposefully designed in this way to keep it concise. However, asking the belief question directly before the experience question could bias responses to the experience question in some way. We also acknowledge that the limited objective format of the survey (answered with a slider from 0-100) with constrained definitions is limiting. A more in-depth phenomenological approach would surely provide greater nuance and depth of understanding of belief and experience. However, the nature of such an instrument in terms of administration and scoring would not solve the problem of needing a simple and concise instrument. Any NEBS results should be interpreted with these limitations in mind.\n\nIn summary, the NEBS is a 20-item survey rated on a sliding scale from 0-100, with 10 Belief and 10 Experience items. Both subscales demonstrated convergent validity, internal consistency, and test-retest reliability. A confirmatory factor analysis model demonstrated a good fit for Belief and Experience as separate latent variables. This model was confirmed in another sample where divergent validity was also established. The NEBS is a concise, valid, and reliable instrument for evaluating individual differences in paranormal beliefs and experiences. This measure provides a new tool for rigorously investigating these beliefs and experiences, and their relationship (as predictors, outcomes, or covariates) with other variables of interest such as psychological well-being, physical health, effects of interventions, coping with death and dying, grief and trauma resilience, and extended human capacities, just to name a few.\n\n\nData availability\n\nFigshare: NEBS Validation Dataset. https://doi.org/10.6084/m9.figshare.9209510.v250\n\nThis project contains the following underlying data:\n\nWahbeh_NEBS Validation Dataset.xlsx (Excel workbook containing three datasets used in the NEBS validation study)\n\nFigshare: NEBS Extended Data. https://doi.org/10.6084/m9.figshare.9770750.v231\n\nThis project contains the following supportive material:\n\nAppendix 1 Noetic Experience and Belief Scale.docx (Noetic Experience and Belief Scale)\n\nSupplemental Data.docx (Table of previous studies and definitions of terms)\n\nConsent Form.pdf (consent form provided for participants)\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 the participants, and the members of the Institute of Noetic Sciences for their support of this project. The authors would also like to thank Jessica Utts for her statistical review of our analyses and Loren Carpenter and Julia Mossbridge for their support in the early phases of this project.\n\n\nReferences\n\nIrwin HJ: The Psychology of Paranormal Belief: A Researcher’s Handbook. Great Britain: University of Hertfordshire Press; 2009. Reference Source\n\nGallup GH, Newport F: Belief in paranormal phenomena among adult Americans. Skept Inq. 1991; 15(2): 137–46. Reference Source\n\nHaraldsson E: Psychic experiences a third of a century apart: Two representative surveys in Iceland with an international comparison. J Soc Psych Res. 2011; 75(903): 76–90. Reference Source\n\nHaraldsson E: West-and east-Europeans and their belief in reincarnation and life after death. Romania. 2005; 28: 68.\n\nHaraldsson E: Representative national surveys of psychic phenomena: Iceland, Great Britain, Sweden, USA and Gallup’s multinational survey. J Soc Psych Res. 1985; 53(801): 145–158. Reference Source\n\nIrwin HJ: Belief in the paranormal: A review of the empirical literature. J Am Soc Psych Res. 1993; 87(1): 1–39. Reference Source\n\nMacDonald WL: The effects of religiosity and structural strain on reported paranormal experiences. J Sci Study Relig. 1995; 366–76. Publisher Full Text\n\nOtis LP, Alcock JE: Factors Affecting Extraordinary Belief. J Soc Psychol. 1982; 118(1): 77–85. Publisher Full Text\n\nRoe CA: Belief in the paranormal and attendance at psychic readings. J Am Soc Psych Res. 1998; 92(1): 25–51. Reference Source\n\nSheils D, Berg P: A research note on sociological variables related to belief in psychic phenomena. Wis Sociol. 1977; 14(1): 24–31. Reference Source\n\nSjödin U: Paranormal beliefs among Swedish youth. Young. 1995; 3(2): 46–57. Publisher Full Text\n\nWahbeh H, Radin D, Mossbridge J, et al.: Exceptional experiences reported by scientists and engineers. Explore (NY). 2018; 14(5): 329–41. PubMed Abstract | Publisher Full Text\n\nWiseman R, Watt C: Belief in psychic ability and the misattribution hypothesis: a qualitative review. Br J Psychol. 2006; 97(Pt 3): 323–38. PubMed Abstract | Publisher Full Text\n\nGallup, Inc: Three in Four Americans Believe in Paranormal. Three if Four Americans Belief in Paranormal: Little change from similar results in 2001. 2005. Reference Source\n\nMoscovitch M, Cabeza R, Winocur G, et al.: Episodic Memory and Beyond: The Hippocampus and Neocortex in Transformation. Annu Rev Psychol. 2016; 67(1): 105–34. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSeitz RJ, Paloutzian RF, Angel HF: Processes of believing: Where do they come from? What are they good for? [version 2; peer review: 2 approved, 1 approved with reservations]. F1000Res. 2016; 5: 2573. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGlicksohn J: Belief in the paranormal and subjective paranormal experience. Personal Individ Differ. 1990; 11(7): 675–83. Publisher Full Text\n\nSpinelli SN, Reid HM, Norvilitis JM: Belief in and experience with the paranormal: Relations between personality boundaries, executive functioning, gender role, and academic variables. Imagin Cogn Personal. 2002; 21(4): 333–46. Publisher Full Text\n\nSparks G, Miller W: Investigating the relationship between exposure to television programs that depict paranormal phenomena and beliefs in the paranormal. Commun Monogr. 2001; 68(1): 98–113. Publisher Full Text\n\nCohn SA: A survey on Scottish second sight. J Soc Psych Res. 1994; 59(835): 385–400.\n\nHaraldsson E, Houtkooper JM: Psychic experiences in the multinational human values study: Who reports them. J Am Soc Psych Res. 1991; 85(2): 145–65. Reference Source\n\nCastro M, Burrows R, Wooffitt R: The paranormal is (still) normal: The sociological implications of a survey of paranormal experiences in Great Britain. Sociol Res Online. 2014; 19(3): 1–15. Publisher Full Text\n\nMcClenon J: Surveys of anomalous experience in Chinese, Japanese, and American samples. Sociol Relig. 1993; 54(3): 295–302. Publisher Full Text\n\nRoss CA, Joshi S: Paranormal experiences in the general population. J Nerv Ment Dis. 1992; 180(6): 357–61; discussion 362–8. PubMed Abstract | Publisher Full Text\n\nPalmer J: A community mail survey of psychic experiences. J Am Soc Psych Res. 1979; 73(3): 221–251. Reference Source\n\nGreeley AM: The sociology of the paranormal: A reconnaissance. Sage Publications; 1975. Reference Source\n\nIrwin HJ, Dagnall N, Drinkwater K: Parapsychological experience as anomalous experience plus paranormal attribution: A questionnaire based on a new approach to measurement. J Parapsychol. 2013; 77(1): 39–53. Reference Source\n\nKohls N, Walach H: Exceptional experiences and spiritual practice: a new measurement approach. Spiritual Health Int. 2006; 7(3): 125–50. Publisher Full Text\n\nGallagher C, Kumar VK, Pekala RJ: The anomalous experiences inventory: Reliability and validity. J Parapsychol. 1994; 58(4): 402–428. Reference Source\n\nWahbeh H, McDermott K, Sagher A: Dissociative Symptoms and Anomalous Information Reception. Act Nerv Super (Praha). 2018; 60(3–4): 75–85. Publisher Full Text\n\nWahbeh H, Delorme A, Radin D, et al.: NEBS Extended Data. figshare. Journal contribution. 2019. http://www.doi.org/10.6084/m9.figshare.9770750.v2\n\nBanham KM: Psi Questionnaire. Duke University Archives, David M. Rubenstein Rare Book & Manuscript Library: Duke University; (Katharine M. Banham). 1996.\n\nDagnall N, Drinkwater K, Parker A: Alien Visitation, Extra-Terrestrial Life, and Paranormal Beliefs. J Sci Explor. 2011; 25(4): 669–720. Reference Source\n\nDrinkwater KG: Belief in the Paranormal: Measurement Development and Evaluation. 2017; 387. Reference Source\n\nEckblad M, Chapman LJ: Magical ideation as an indicator of schizotypy. J Consult Clin Psychol. 1983; 51(2): 215–25. PubMed Abstract | Publisher Full Text\n\nJinks AL: Paranormal and alternative health beliefs as quasi-beliefs: Implications for item content in paranormal belief questionnaires. Aust J Parapsychol. 2012; 12(2): 127–158. Reference Source\n\nLuke D, Kittenis M: A preliminary survey of paranormal experiences with psychoactive drugs. J Parapsychol. 2005; 69(2): 25. Reference Source\n\nNeppe VM: The BEASTS: Brief Experiences of Anomalous Subjective Type Screen. The NEASTS: Neppe Experiences of Anomalous Subjective Type Screen. The SEASTS: Screening Experiences of Anomalous Subjective Type Screen. Seattle, Washington: Pacific Neuropsychiatric Institute; 1999.\n\nRandall TM: Paranormal Short Inventory. Percept Mot Skills. 1997; 84: 1265–6.\n\nRice TW: Believe It Or Not: Religious and Other Paranormal Beliefs in the United States. J Sci Study Relig. 2003; 42(1): 95–106. Publisher Full Text\n\nSchofield M, Baker IS, Staples PA, et al.: Creation and Validation of the Belief in the Supernatural Scale. J Parapsychol. 2018; 82(1): 41–64. Publisher Full Text\n\nStorm L, Drinkwater K, Jinks AL: A Question of Belief: An Analysis of Item Content in Paranormal Belief Questionnaires. J Sci Explor. 2017; 31(2): 187–230. Reference Source\n\nTabacyk JJ: A Revised Paranormal Belief Scale. Int J Transpers Stud. 2004; 23(23): 94–8. Publisher Full Text\n\nThalbourne MA: The Australian Sheep-goat Scale: Development and Empirical Findings. Aust J Parapsychol. 2010; 10(1): 5. Reference Source\n\nThalbourne MA, Delin PS: A new instrument for measuring the sheep-goat variable: Its psychometric properties and factor structure. J Soc Psych Res. 1993; 59(832): 172–86. Reference Source\n\nU.S Department of Health & Human Services: Office for Civil Rights. Methods for De-identification of PHI. HHS.gov Health Information Privacy. 2015. Reference Source\n\nThalbourne MA: Measures of the sheep-goat variable, transliminality, and their correlates. Psychol Rep. 2001; 88(2): 339–50. PubMed Abstract | Publisher Full Text\n\nAnthoine E, Moret L, Regnault A, et al.: Sample size used to validate a scale: a review of publications on newly-developed patient reported outcomes measures. Health Qual Life Outcomes. 2014; 12: 176. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBentler PM: EQS 6 Structural Equations Program Manual. Encino, CA: Multivariate Software Inc. 1995. Reference Source\n\nWahbeh H, Delorme A, Yount G, et al.: NEBS Validation Dataset. figshare. Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.9209510.v2\n\nTsang S, Royse CF, Terkawi AS: Guidelines for developing, translating, and validating a questionnaire in perioperative and pain medicine. Saudi J Anaesth. 2017; 11(Suppl 1): S80–S89. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTerwee CB, Bot SDM, de Boer MR, et al.: Quality criteria were proposed for measurement properties of health status questionnaires. J Clin Epidemiol. 2007; 60(1): 34–42. PubMed Abstract | Publisher Full Text\n\nPerry S: Fit Statistics commonly reported for CFA and SEM. Cornell University: Cornell Statistical Consulting Unit. 2019. Reference Source\n\nBell IR, Cunningham V, Caspi O, et al.: Development and validation of a new global well-being outcomes rating scale for integrative medicine research. BMC Complement Altern Med. 2004; 4: 1. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJebb AT, Tay L, Diener E, et al.: Happiness, income satiation and turning points around the world. Nat Hum Behav. 2018; 2(1): 33–8. PubMed Abstract | Publisher Full Text\n\nDeSalvo KB, Bloser N, Reynolds K, et al.: Mortality prediction with a single general self-rated health question. A meta-analysis. J Gen Intern Med. 2006; 21(3): 267–75. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCappelleri JC, Bushmakin AG, McDermott AM, et al.: Psychometric properties of a single-item scale to assess sleep quality among individuals with fibromyalgia. Health Qual Life Outcomes. 2009; 7(1): 54. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFarrar JT, Young JP, LaMoreaux L, et al.: Clinical importance of changes in chronic pain intensity measured on an 11-point numerical pain rating scale. Pain. 2001; 94(2): 149–58. PubMed Abstract | Publisher Full Text\n\nRammstedt B: The 10-item Big Five Inventory: Norm values and investigation of sociodemographic effects based on a German population representative sample. Eur J Psychol Assess. 2007; 23(3): 193–201. Publisher Full Text\n\nShiota MN, Keltner D, John OP: Positive emotion dispositions differentially associated with Big Five personality and attachment style. J Posit Psychol. 2006; 1(2): 61–71. Publisher Full Text\n\nMukaka M: Statistics corner: A guide to appropriate use of correlation coefficient in medical research. Malawi Med J. 2012; 24(3): 69–71. PubMed Abstract | Free Full Text\n\nCardeña E, Marcusson-Clavertz D: States, Traits, Cognitive Variables and Psi. In: Parapsychology: A handbook for the 21st century. Jefferson, North Carolina: McFarland; 2015; 110–24. Reference Source\n\nMiklousic I, Mlacic B, Milas G: Paranormal beliefs and personality traits in Croatia. Drustvena Istraz. 2012; 21(1): 181. Publisher Full Text\n\nThalbourne MA: Further studies of the measurement and correlates of belief in the paranormal. J Am Soc Psych Res. 1995; 89(3): 233–47. Reference Source\n\nWilliams E, Francis LJ, Robbins M: Personality and Paranormal Belief: A Study Among Adolescents. Pastor Psychol. 2007; 56(1): 9–14. Publisher Full Text\n\nPeltzer K: Paranormal beliefs and personality among black South African students. Soc Behav Personal. 2002; 30(4): 391. Publisher Full Text\n\nRattet SL, Bursik K: Investigating the personality correlates of paranormal belief and precognitive experience. Personal Individ Differ. 2001; 31(3): 433–44. Publisher Full Text\n\nHooper D, Coughlan J, Mullen M: Structural Equation Modelling: Guidelines for Determining Model Fit. 2008; 11. Reference Source\n\nKline RB: Principles and Practice of Structural Equation Modeling, Fourth Edition. Guilford Publications. 2015; 553. Reference Source"
}
|
[
{
"id": "56127",
"date": "17 Jan 2020",
"name": "Malcolm Schofield",
"expertise": [
"Reviewer Expertise Paranormal belief"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 acknowledges the importance of having a scale that distinguishes paranormal belief from paranormal experience and develops a concise measure of twenty items. However, to bring it up to an acceptable standard, the following points should be addressed:\nThe introduction is severely lacking any review of previous scales and how different factors have been conceptualised in the past.\n\nThe development of the NEBS should be in the methods section and not the introduction.\n\nThe methods sections would benefit from traditional subheadings.\n\nThe results section contains demographic data which should be in the participant's section in the methods.\n\nAn exploratory factor analysis would have been useful on the first sample.\n\nFor study one, the CFA should come first followed by test retests.\n\nDouble-check the fit indices (particularly the CFI, the cut of is listed as .90 when in fact it should be .95) and was the chi-square significant?\n\nStudy two – why are there wellbeing measures? They are only mentioned briefly via a table in the results.\n\nThe items relating to experience are all the same.\n\nThe participant is asked if they have experienced something directly after they have been asked if they believe it; this could present a confound.\n\nOverall, while this paper offers an interesting premise, there are several flaws. The scale itself does not have dedicated experience items that refer to specific phenomena, which is problematic. The methodology itself is also flawed. I suspect an Exploratory Factor Analysis (EFA) would reveal that the factors would be around the phenomena rather than the dichotomy of belief and experience. I.e., a person, who believes in particular phenomena is more likely to experience them; therefore, the factors will demonstrate this. I would recommend that this be more of an exploratory study and that an EFA be run on the first sample at the very least. However, this would probably not give the desired outcome. The items that relate to experience should ideally be able to be answered in isolation and not be dependent on the belief items. This could act as a prime, with people who state they believe in something when asked if they have experienced it directly after being more likely to agree.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": [
{
"c_id": "5225",
"date": "21 Feb 2020",
"name": "Helané Wahbeh",
"role": "Author Response",
"response": "We thank the reviewer for their thoughtful comments. We have made revisions to the paper as a result that we believe have strengthened it. Below you will find each reviewers comments and our changes to the manuscript or responses. The introduction is severely lacking any review of previous scales and how different factors have been conceptualised in the past. - The original manuscript included Supplementary Data that summarized a review of 18 previous scales that informed the design of our scale. We have revised the text in the Introduction to clarify and draw attention to this. The revised text reads: \"We also reviewed a number of existing questionnaires that measured paranormal experience and/or belief (8, 26, 28, 29, 32– 45 ). A summary of this review is presented as Supplemental data A. (please see extended data (31)). Each questionnaire was evaluated for the number of items, whether it assesses belief, experience or both, whether it evaluates belief and experience as separate constructs, and subscales if applicable.\" The development of the NEBS should be in the methods section and not the introduction. -The development of the NEBS has now been moved to the methods section. The methods sections would benefit from traditional subheadings. -The methods sections now has subheadings. The results section contains demographic data which should be in the participant's section in the methods. -The participant demographic information was moved to the participant’s section in the methods. An exploratory factor analysis would have been useful on the first sample. -Please see last item for full response of this comment. For study one, the CFA should come first followed by test retests. -The test-restest results have been moved so that the CFA results come first followed by the test-retest results. Double-check the fit indices (particularly the CFI, the cut of is listed as .90 when in fact it should be .95) and was the chi-square significant? - These fit indices were taken from Cornell University Cornell Statistical Consulting Unit. Reference - Perry S: Fit Statistics commonly reported for CFA and SEM. Cornell University: Cornell Statistical Consulting Unit.2019. -Yes, the chi-square was significant and has been added to the manuscript. Study two – why are there wellbeing measures? They are only mentioned briefly via a table in the results. -The well-being measures were included to test divergent validity. This is mentioned in the methods section. “Relevant measures used to establish NEBS divergent validity, which tests whether concepts or measurements that are not supposed to be related are actually unrelated were: Arizona Integrative Outcomes Scale 54 , Positive and Negative Affective Well-being Scale 55 , single-item general health 56 , acute sleep quality scale 57 , the Numeric Pain Rating Scale 58 , and Big Five Inventory-10 scale 59 , and the compassion subscale of the Dispositional Positive Emotion Scale.” The manuscript then goes on to describe each of those measures in more detail. The items relating to experience are all the same. -The wording of the experience items are the same but reflect the belief construct expressed in the item directly before. The participant is asked if they have experienced something directly after they have been asked if they believe it; this could present a confound. -Please see response below about EFA and dependencies between belief and experience items. Overall, while this paper offers an interesting premise, there are several flaws. The scale itself does not have dedicated experience items that refer to specific phenomena, which is problematic. The methodology itself is also flawed. I suspect an Exploratory Factor Analysis (EFA) would reveal that the factors would be around the phenomena rather than the dichotomy of belief and experience. I.e., a person, who believes in particular phenomena is more likely to experience them; therefore, the factors will demonstrate this. I would recommend that this be more of an exploratory study and that an EFA be run on the first sample at the very least. However, this would probably not give the desired outcome. The items that relate to experience should ideally be able to be answered in isolation and not be dependent on the belief items. This could act as a prime, with people who state they believe in something when asked if they have experienced it directly after being more likely to agree. -Exploratory factor analysis (EFA) is a statistical technique used to find the underlying structure of a set of observed variables (Gorsuch, 2015), whereas confirmatory factor analysis (CFA) is used when researchers have formulated a hypothesis regarding the relationship between observed variables and the underlying latent factors (Gorsuch, 2015). There has been a debate regarding the circumstances in which these two analyses should be used in research (Hurley et al., 1997) and whether/when they should be used in tandem (Gerbing and Hamilton, 1996). EFA can be used prior to cross-validation with CFA for the purpose of model specification (Gerbing and Hamilton, 1996). EFA can also be used after CFA to explore poor fits in CFA models, explore factor structures when the original hypotheses are weak, and confirm factor structures when the original hypotheses were strong, but certain assumptions are not reasonable (Schmitt, 2011). The reviewer recommends an EFA on the first sample. It is suggested, but not made clear, that an EFA on the first sample would serve the purpose of exploration regarding other possible factor structures. It is also suggested that EFA could possibly identify elevated correlation in the already correlated belief and experience factors that is due to survey structure alone.In this study, hypotheses are based on theory and practice, and are therefore strong. It is unclear whether EFA is suggested to confirm assumptions that may not hold in this study. If so, what are these specific assumptions and why are they unreasonable or not upheld? If EFA is suggested as a pre-cursor to CFA, then EFA should be conducted followed by cross-validation with CFA on an independent data set, as suggested by Gerbing and Hamilton (1996). If this procedure is followed, either (1) the EFA will support the researchers’ hypotheses or (2) the EFA will not support the researchers’ hypotheses. If (2) occurs, it is unclear what impact this should have on the current study. The reviewer notes that if (2) occurs, “this would probably not give the desired outcome”, and it is highly pertinent to point out that the researchers did not undertake this study to achieve a “desired outcome” but rather to test hypotheses. If (2) occurs, it is not clear whether this invalidates NEBS as a functioning survey tool. CFA does not always confirm a factor structure obtained via EFA (van Prooijen and van der Kloot, 2001; Borkenau and Ostendorf, 1990). If a CFA fits well and satisfies all assumptions but an EFA indicates that the underlying structure of the data can be represented by different factors, is this sufficient evidence to discard the original CFA? If so, can the reviewer provide support for this claim? In Gorsuch’s classic text on factor analysis, he states “Confirmatory factor analysis tests hypotheses that a specified subset of variables legitimately define a prespecified factor” (Gorsuch, 2015). If the researchers have found that a subset of variables legitimately defined their prespecified factors and this was the central goal of their paper, what then is the purpose and role of added exploratory analysis in the context of this paper?If the reviewer’s hypothesis that an EFA “would reveal that the factors would be around the phenomena rather than the dichotomy of belief and experience” is supported by an EFA on the first sample, then it is not clear whether presenting items in isolation would remedy this given the high inherent correlation between belief and experience, and therefore between the items themselves. If this statement is more than opinion or a hypothesis and there is scientific support for it, how should this isolation be achieved and how much isolation is enough isolation to guarantee that this effect is removed from the analysis? If the diagnosis of this specific issue is the only reason for the suggested EFA, then if the researchers were to reorganize and re-administer the surveys according to the reviewer’s specifications, would an EFA still be necessary? It is also worth noting that the question of whether or not the survey structure introduced added dependence between items can be easily tested experimentally by providing the original and modified surveys (with some reasonable span of time in between) to a group of (new) participants and quantifying the difference in responses. If the difference is not statistically significant, then any added dependence should be negligible. Would this kind of adjustment in the methodology remedy the need for an EFA, according to the reviewer? In summary, it is unclear whether the reviewer calls for an EFA to 1) examine other factor structures in the data or 2) test additional dependencies that may have been introduced by the survey structure. If (1), it is unclear what role this EFA would play in the current paper. If (2), it may be more straightforward to address this experimentally.Literature CitedBorkenau, Peter and Ostendorf, Fritz (1990). Comparing exploratory and confirmatory factor analysis: A study on the 5-factor model of personality. Personality and Individual Differences, 11(5), 515 – 524.Gerbing, David W. and Hamilton, Janet G. (1996). Viability of Exploratory Factor Analysis as a Precursor to Confirmatory Factor Analysis. Structural Equation Modeling, 3(1), 62 – 72.Gorsuch, Richard L. (2015) Factor Analysis, Classic Edition. Routledge: New York and London.Hurley, Amy E., Scandura, Terri A., Schriesheim, Chester A., Brannick, Michael T., Seers, Anson, Vandenbery, Robert J., and Williams, Larry J. (1997). Exploratory and confirmatory factor analysis: guidelines, issues, and alternatives. Journal of Organizational Behavior, 18, 667 – 683.Schmitt, Thomas A. (2011). Current Methodological Considerations in Exploratory and Confirmatory Factor Analysis. Journal of Psychoeducational Assessment, 29(4), 304 – 321.Van Prooijen, Jan-Willem and van der Kloot, Willem A. (2001). Confirmatory analysis of exploratively obtained factor structures. Educational and Psychological Measurement, 61(5):777 – 792."
}
]
}
] | 1
|
https://f1000research.com/articles/8-1741
|
https://f1000research.com/articles/8-1938/v1
|
20 Nov 19
|
{
"type": "Research Article",
"title": "Optimization of glutaminase-free L-asparaginase production using mangrove endophytic Lysinibacillus fusiformis B27",
"authors": [
"Asep Awaludin Prihanto",
"Indah Yanti",
"Mohammad Achsanil Murtazam",
"Yoga Dwi Jatmiko",
"Indah Yanti",
"Mohammad Achsanil Murtazam",
"Yoga Dwi Jatmiko"
],
"abstract": "Background: The mangrove, Rhizophora mucronata, an essential source of endophytic bacteria, was investigated for its ability to produce glutaminase-free L-asparaginase. The study aimed to obtain glutaminase-free L-asparaginase-producing endophytic bacteria from the mangrove and to optimize enzyme production. Methods: The screening of L-asparaginase-producing bacteria used modified M9 medium. The potential producer was further analyzed with respect to its species using 16S rRNA gene sequencing. Taguchi experimental design was applied to optimize the enzyme production. Four factors (L-asparagine concentration, pH, temperature, and inoculum concentration) were selected at four levels. Results: The results indicated that the endophytic bacteria Lysinibacillus fusiformis B27 isolated from R. mucronata was a potential producer of glutaminase-free L-asparaginase. The experiment indicated that pH 6, temperature at 35°C, and inoculum concentration of 1.5% enabled the best production and were essential factors. L-asparagine (2%) was less critical for optimum production. Conclusions: L. fusiformis B27, isolated from Rhizophora mucronata, can be optimized for L-ASNase enzyme production using optimization factors (L-ASNase, pH, temperature, and inoculum), which can increase L-ASNase enzyme production by approximately three-fold.",
"keywords": [
"L-asparaginase",
"mutation",
"statistical",
"taguchi",
"Lysinibacillus fusiformis."
],
"content": "Introduction\n\nL-asparaginase (L-ASNase, EC.3.5.1.1, L-asparagine amidohydrolase) hydrolyzes L-asparagine (L-ASN) to L-aspartic acid and ammonia1. This enzyme has an essential role in food safety owing to its acrylamide-mitigating potential2,3. Acrylamide, a compound formed from the reaction of asparagine with reducing sugars, can be converted into L-asparagine (ASN) in raw materials by adding L-ASNase. The food industry mainly uses two commercial products, Acrylaway and Preventase from Aspergillus oryzae and Aspergillus niger, respectively4,5.\n\nL-ASNase can also inhibit the growth of cancer cells, especially leukemia4, and it is potentially suitable for leukemia treatment due to its ability to deplete L-ASN in blood plasma5. Cells in healthy tissue can synthesize L-ASN in sufficient amounts for protein synthesis, but some types of lymphoid malignancies remove it from plasma. Limiting the amount of L-ASN will eventually inhibit the growth of cancer cells. Applying L-ASNase to the treatment of acute lymphoblastic leukemia, acute myelocytic leukemia, Hodgkin’s disease, non- Hodgkin’s disease, and melanosarcoma has been investigated for its effectiveness in animals and humans6.\n\nSome characteristics of this enzyme hinder its application in foods and medicines. For example, it is unsuitable for application in foods due to its sensitivity to high temperatures6. In medical applications, it has a low half-life and can cause allergic reactions in patients6. Some of these operational and technical constraints cause manufacturers to identify new sources of L-ASNase. Therefore, new L-ASNase producers need to be explored.\n\nL-ASNase is commonly found in animal tissues, bacteria, and plants, but is lacking in humans. L-ASNase is produced in large quantities by several microorganisms such as Enterobacter aerogenes, Escherichia coli, Erwinia carotovora, Enterobacter aerogenes, Candida sp., and Corynebacterium glutamicum7,8. Production of L-ASNase sourced from animals or plants encounter several impediments, including those relating to large-scale enzyme production. Therefore, the production of L-ASNase derived from microorganisms is preferred because these multiply rapidly and are easily managed9.\n\nOptimizing the growth parameters improves enzyme production. Enzyme production can be optimized via a statistical approach using Taguchi analysis. The Taguchi method facilitates product or process design10. This approach offers time efficiency and accurate results, using relatively few treatment parameters11.\n\nTo fulfill the demand for L-ASNase in food and medical fields, this study was conducted to isolate and identify potential endophytic bacteria producing the L-ASNase enzyme from the mangrove Rhizophora mucronata. Optimization of L-ASNase enzyme production by selected bacteria was performed by optimizing factors influencing production (L-ASN, pH, temperature, inoculum concentration, and incubation time) using a statistical Taguchi approach.\n\n\nMethods\n\nIsolation and screening of L-ASNase-producing endophytic bacteria was performed as described by Prihanto et al.12 and Mahajan et al.13. Mangrove (Rhizophora mucronata) was obtained from Aeng Sareh Beach, Madura Island, Indonesia with the location of 7°13'5.57\"S and 113°19'8.89\"E. Mangrove stem was aseptically cut to approximately 1 cm diameter. The sample was placed in a plastic bag and immediately transported to the laboratory on ice (4°C). It was crushed and weighed (1 g) and then serially diluted with physiological buffer. Three independent treatments were applied. An appropriately diluted sample (10–3–10–5) was plated onto Luria-Bertani agar (Sigma-Aldrich, USA) using the pour plate method and incubated for 72 h at 35°C. Colonies were purified in Luria-Bertani agar (Sigma-Aldrich, USA) using three quadrants streaking, and stored at −20°C.\n\nA modified M9 medium was used for screening isolates that were produced glutaminase-free L-ASNase. All materials and reagents were purchased from Merck, USA. The composition of the medium was 6 g/l of Na2HPO4, 2 g/l of KH2PO4, 0.5 g/l of NaCl, 20 g/l of L-ASN or L-glutamine, 2 g/l of glucose, 0.2 g/l of MgSO4, 0.005 g/l of CaCl2, agar 2%, and Bromotymol blue (BTB) 0.007%. The pH of the medium was set to 5.5 using a pH meter with 2 N HCl. BTB served as a color indicator. Two media (M9 medium with L-ASN, and M9 medium with L-glutamine) were applied to the isolates. Glutaminase-free L-ASNase isolates were detected by the ability to hydrolyze only L-ASN.For isolate which able to hydrolyze both L-ASN, and L-glutamine was not chosen for further analysis. Initial identification of the selected isolates was performed by Gram staining.\n\nMolecular identification of the selected bacterial isolates from mangrove was performed based on the methods of Prihanto et al.12. Genomic DNA from bacteria was amplified using 27F primers (5’-AGAGTTTGATCATGGCTCAG-3') and 1492R (5'-TACGGCTACCTTGTTACGA-3'). Primers were purchased from 1st BASE (Singapore). Genomic DNA of bacteria were extracted, and amplified by following the company manual procedures (WizardTM DNA purification Kit, Cat. No. A1120, and Gotaq® DNA polymerase, Cat No. M3005, Promega, USA ). Obtained DNA sample (1 μL) was mixed with 18.5 μL double distilled H2O (ddH2O), 2.5 μL Buffer B with Mg2 + 10X, 1 μL dNTPs, 1 μL forward primer, 1 μL reverse primer, and 0.2 μL Taq polymerase. The reaction was performed in a thermocycler (T100 Thermal Cycler, Bio-Rad, USA) (denaturation: 94°C, 45 s; annealing: 61°C, 45 s; elongation: 72°C, 2 min). The amplification process was performed over 32 cycles. The Polymerase Chain Reaction results were checked by gel electrophoresis (Mupid EXu submarine, Takara, Japan) under 80 volt and 40 mA with 1.5% agarose. The gel was visualized using Benchtop UV Transilluminator (UVP, Canada).\n\nThe amplicon was further sequenced with ABI PRISM 3130×1 DNA sequencer (Applied Biosystems, USA). The sequence homology was investigated at the National Center for Biotechnology Information (NCBI) GenBank database using BLAST nucleotide programs, on the nucleotides collection database by excluding human and mouse genomic. The program was optimized for highly similar sequences (megablast). A phylogenetic tree was constructed using the UPGMA method with the MAFFT online ver 7. phylogenetic analysis service14.\n\nAll materials and reagents were purchased from Merck, USA. Enzyme production was performed in a medium containing trisodium citrate 0.375 g, (NH4)2HPO4 0.1 g, K2HPO4 0.00625 g, MgSO4·7H2O 0.01 g, FeSO4·7H2O 0.001 g, yeast extract 0.075 g, and L-ASN 0.25–1 g15. Different volume of inoculum (108 CFU/ml) was inoculated on 50 ml fresh treated-production mediums. The cultures were incubated in a shaker incubator (120 rpm) at different temperatures. Four factors at four levels, L-ASN (0.5–2%), pH (6–9), temperature (30–45°C), and inoculum volume (0.5–2%), were investigated (Table 1). The L16 orthogonal array was selected for experimental design (Table 2). To determine the time for harvest, before the optimization, the length of culture production (24 h, and 48 h) was investigated.\n\nA slightly modified method16 was used for the enzyme activity assay. The samples (150 μl) were transferred to dialysis tubes to which 100 μl liquid L-ASN, 200 μl aquades, and 50 μl phosphate buffer were added before homogenization. The samples were then incubated in a water bath at 30°C for 30 min to react. Subsequently, 1.5 μl of Trichloro Acetic Acid 1.5 M was added to stop the reaction followed by centrifugation at 2,000 rpm for 12 min; the supernatant (450 μl) was then transferred into the microtube. Nessler’s reagent (125 μl) was then added and reacted for 15 min until the color changed; the absorbance was read with a UV–vis spectrophotometer at a wavelength of 480 nm. NH4Cl served as standard ammonia. The enzyme activity was expressed as micromoles ammonia released per minute.\n\nAll L16 data were analyzed using Qualitek-4 software (Nutek Inc., MI). The analysis was used to obtain the predicted optimum conditions for achieving the highest L-ASNase production. Analysis of variance (ANOVA) was used to identify factors that influenced L-ASNase production. The F-ratio was calculated with a 95% confidence interval. Interactions among factors and levels of experiment was analyzed by determining the severity index (SI)\n\nExperimental validation was conducted to confirm the accuracy of the obtained results. This aimed to verify the optimum factors and levels obtained from the experiment. After determining the optimum treatment, the next step was to conduct a confirmatory experiment by creating the optimum conditions based on the predetermined factors and levels.\n\n\nResults and discussion\n\nThe screening results showed that 31 endophytic mangrove isolates could produce the L-ASNase enzyme. Judging from the blue color of the medium, isolate B27 was the best producer (Figure 1A). Isolate B27 had a more extensive and darker blue tone compared to other isolates. Furthermore, only isolate B27 produced glutaminase-L-asparaginase. Hence, further molecular identification was applied only to the B27 isolate. Further, Gram stain analysis revealed that the bacteria was Gram positive (Figure 1B).\n\n(A) Blue color indicating the production of glutaminase-free L-asparaginase. (B). Gram stain of B27 isolate.\n\nThe 16S rDNA molecular identification results showed that B27 isolates had a similarity of 98% with the Lysinibacillus fusiformis species. The phylogenetic analysis results showed that L. fusiformis was most closely related to L. fusiformis strain NBRC15717 (Figure 2). The production of L-ASNase from this bacteria was further optimized using the Taguchi method.\n\nL-ASNase production was controlled to determine enzyme production without optimization. The enzyme production during incubation periods of 24 and 48 h was 3.18 and 1.99 U/ml, respectively. The higher production was observed after 24 h. During the exponential phase, bacteria experience fast growth and produce metabolites for growth and self-defense17. The result for optimization experiment is shown in Table 2.\n\nTo determine the effects of factors that could increase the L-ASNase production, L-ASN concentration (0.5, 1, 1.5, and 2%), pH (6, 7, 8, and 9), temperature (30, 35, 40, and 45°C), and inoculum concentration (0.5, 1, 1.5, and 2%) were evaluated. L-ASN concentration was used to determine the effect of inducers on increasing enzyme production. pH, temperature, and inoculum concentration were subsequently investigated. The effects of these factors are shown in Figure 3.\n\nL-ASN concentration influenced L-ASNase production, with the best concentration being 2% L-ASN with enzyme production of 9.466 U/ml. Therefore, to some extent, L-ASN could increase enzyme activity. However, the results showed that L-ASN concentration 1% could reduce enzyme production. Consequently, the higher the substrate concentration added to the enzyme solution, the lower the enzyme activity until the enzyme reached the optimum substrate concentration18. This data suggested that the enzyme production exhibited an optimum concentration of more than 2% L-ASN in the medium.\n\nL. fusiformis B27 at pH 6 showed an enzyme production of 9.795 U/ml. When the pH was too high or low, enzyme production was reduced. Therefore, the optimum pH for enzyme production must always be considered. L. fusiformis B27 grown at 35°C showed the highest enzyme production of 17.037 U/ml. Temperature plays a vital role in enzymatic reactions. High temperatures increase the reaction rate of the enzyme. Hence, the optimum temperature for enzymes needs to be determined.\n\nThe optimum inoculum concentration for L-ASNase production was 1.5% with an enzyme production of 9.638 U/ml. A very high inoculum concentration can cause competition between microbes in obtaining nutrition. This competition can cause some microbes to lack the nutrients required for growth19,20.\n\nAnalysis of variance (ANOVA) was used to identify factors that influenced L-ASNase production (Table 3). The F-ratio was calculated with a 95% confidence interval. The results showed that all factors significantly influenced enzyme production. The most influential factor affecting L-ASNase enzyme production was temperature, while the inoculum concentration, although it was known to affect the enzyme production, showed the least effect.\n\nInteractions among factors and levels of experiment can be analyzed by determining the severity index (SI), as shown in Table 4. The SI can be used to predict the effect of combined factors on enzyme production.\n\nThe SI measures the interaction between two factors. The analysis showed that the interaction between pH and inoculum was the highest with a SI of 69.72%, while the lowest SI of 4.5% was found for the interaction between pH and temperature. The interaction between pH and inoculum significantly influenced L-ASNase production. Conversely, the ANOVA results showed that the most influential factor for L-ASNase production was temperature. Different SI values could result in different values of individual factors21.\n\nL-ASN at a concentration of 2% (level 4), pH 6 (level 1), temperature 35°C (level 2), and an inoculum concentration of 1.5% (level 3) for 24 h of incubation were deduced as the best conditions for L-ASNase production with an expected enzyme production of 19.2065 U/ml (Table 5).\n\nA validation experiment was required to confirm the expected result. Based on the confirmation test results, the L-ASNase activity obtained from L. fusiformis B27 at 24 h was 8.51 U/ml. The L-ASNase enzyme activity from L. fusiformis B27 at 24 h before and after optimization is shown in Table 6.\n\nThe data showed that before optimization (control) L-ASNase had a low activity of 3.18 U/ml. This was because the bacterial environment for producing L-ASNase was suboptimal. The L-ASNase activity increased to 8.51 U/ml after optimization. The optimized factors for enzyme production could be predicted using the Taguchi method22–24. Confirmation experiment results are usually closer to the predicted values. However, these are sometimes lower than the predicted results. In this study, the optimized factors enhanced the enzyme production from 3.18 to 8.51 U/ml. This represented an almost three-fold increase. This increase was higher than that published in previous research21 but lower than that in another study25.\n\n\nConclusions\n\nLysinibacillus fusiformis B27, which was isolated from mangrove, Rhizophora mucronata, can be optimized for L-ASNase enzyme production using optimization factors (L-ASNase, pH, temperature, and inoculum). Optimization using the Taguchi approach can increase L-ASNase enzyme production by approximately three-fold.\n\n\nData availability\n\nFigshare: Raw data for production improvement of L-Asparaginase from Lysinibacillus fusiformis. https://doi.org/10.6084/m9.figshare.10265396.v226\n\nThis project contains the following underlying data:\n\n- L-ASNase activity for Taguchi analysis for 16 optimization experiments.\n\n- Results for confirmatory experiment\n\n- Images of all 31 isolates\n\n- Uncropped, unedited blots from genome (B27)\n\n- Sequence of B27\n\n- Uncropped, unedited image of Gram stain of B27, as shown in Figure 1B\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 Ministry of Research, Technology, and Higher Education, Republic of Indonesia, for the funding.\n\n\nReferences\n\nChan WK, Lorenzi PL, Anishkin A, et al.: The glutaminase activity of L-asparaginase is not required for anticancer activity against ASNS-negative cells. Blood. 2014; 123(23): 3596–3606. PubMed Abstract | Publisher Full Text | Free Full Text\n\nOnishi Y, Prihanto AA, Yano S, et al.: Effective treatment for suppression of acrylamide formation in fried potato chips using L-asparaginase from Bacillus subtilis. 3 Biotech. 2015; 5(5): 783–789. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSanghvi G, Bhimani K, Vaishnav D, et al.: Mitigation of acrylamide by l-asparaginase from Bacillus subtilis KDPS1 and analysis of degradation products by HPLC and HPTLC. Springerplus. 2016; 5: 533. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCachumba JJ, Antunes FA, Peres GF, et al.: Current applications and different approaches for microbial l-asparaginase production. Braz J Microbiol. 2016; 47 Suppl 1(1): 77–85. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZuo S, Zhang T, Jiang B, et al.: Reduction of acrylamide level through blanching with treatment by an extremely thermostable L-asparaginase during French fries processing. Extremophiles. 2015; 19(4): 841–51. PubMed Abstract | Publisher Full Text\n\nVimal A, Kumar A: Biotechnological production and practical application of L-asparaginase enzyme. Biotechnol Genet Eng Rev. 2017; 33(1): 40–61. PubMed Abstract | Publisher Full Text\n\nPrihanto AA, Wakayama M: Marine Microorganism: An Underexplored Source of l-Asparaginase. Adv Food Nutr Res. 2016; 79: 1–25. PubMed Abstract | Publisher Full Text\n\nEl-Bessoumy AA, Sarhan M, Mansour J: Production, isolation, and purification of L-asparaginase from Pseudomonas aeruginosa 50071 using solid-state fermentation. J Biochem Mol Biol. 2004; 37(4): 387–393. PubMed Abstract | Publisher Full Text\n\nNigam PS: Microbial enzymes with special characteristics for biotechnological applications. Biomolecules. 2013; 3(3): 597–611. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMakowski K, Katarzyna M, Sebastian B, et al.: Optimization of a culture medium using the taguchi approach for the production of microorganisms active in odorous compound removal. Appl Sci. 2017; 7(8): 756. Publisher Full Text\n\nChiranjeevi T, Rani GB, Chandel AK, et al.: Optimization of holocellulolytic enzymes production by Cladosporium cladosporioides using taguchi-l’16 orthogonal array. Journal of Biobased Materials and Bioenergy. 2012; 6: 1–10. Publisher Full Text\n\nPrihanto AA, Jaziri AA, Perwira IY: Purification and characterization of neutral protease from Bacillus substilis UBT7 isolated from terasi, Indonesian fermented fish. Biosci Biotech Res Asia. 2016; 13(3): 1409–1413. Publisher Full Text\n\nMahajan R, Garg S, Sharma PB: Global food safety: determinants are codex standards and WTO’s SPS food safety regulations. J Adv Manage Res. 2014; 11(2): 176–191. Publisher Full Text\n\nKatoh K, Rozewicki J, Yamada KD: MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization. Brief Bioinform. 2019; 20(4): 1160–1166. PubMed Abstract | Publisher Full Text | Free Full Text\n\nErva RR, Goswami AN, Suman P, et al.: Optimization of L-asparaginase production from novel Enterobacter sp., by submerged fermentation using response surface methodology. Prep Biochem Biotechnol. 2017; 47(3): 219–228. PubMed Abstract | Publisher Full Text\n\nChohan SM, Rashid N, Sajed M: Pcal_0970: an extremely thermostable L-asparaginase from Pyrobaculum calidifontis with no detectable glutaminase activity. Folia Microbiol (Praha). 2019; 64(3): 313–320. PubMed Abstract | Publisher Full Text\n\nJaishankar J, Srivastava P: Molecular Basis of Stationary Phase Survival and Applications. Front Microbiol. 2017; 8: 2000. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRobinson PK: Enzymes: principles and biotechnological applications. Essays Biochem. 2015; 59: 1–41. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHibbing ME, Fuqua C, Parsek MR, et al.: Bacterial competition: surviving and thriving in the microbial jungle. Nat Rev Microbiol. 2010; 8(1): 15–25. PubMed Abstract | Publisher Full Text | Free Full Text\n\nStubbendieck RM, Straight PD: Multifaceted Interfaces of Bacterial Competition. J Bacteriol. 2016; 198(6): 2145–2155. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPrihanto AA, Wakayama M: Combination of environmental stress and localization of L-asparaginase in Arthrospira platensis for production improvement. 3 Biotech. 2014; 4(6): 647–653. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKamble R, Gupte A: Cyclodextrin glycosyltransferase production by alkaliphilic bacillus sp. Isolated from rice cultivated soil and media optimization using taguchi method. Int J Pharm Sci Res. 2014; 5(7): 2754–62. Publisher Full Text\n\nAzin M, Moravej R, Zareh D: Production of xylanase by Trichoderma longibrachiatum on a mixture of wheat bran and wheat straw: Optimization of culture condition by Taguchi method. Enzyme Microb Tech. 2007; 40(4): 801–5. Publisher Full Text\n\nKumar M, Singh S, Beniwal V, et al.: Improved production of tannase by Klebsiella pneumoniae using Indian gooseberry leaves under submerged fermentation using Taguchi approach. AMB Express. 2016; 6(1): 46. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChenthamarakshan A, Parambayil N, Miziriya N, et al.: Optimization of laccase production from Marasmiellus palmivorus LA1 by Taguchi method of Design of experiments. BMC Biotechnol. 2017; 17(1): 12. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPrihanto AA, Yanti I, Murtazam MA, et al.: Raw Data (L-ASNase activity for Taguchi analysis).xlsx. figshare. Dataset. 2019. http://www.doi.org/10.6084/m9.figshare.10265396.v2"
}
|
[
{
"id": "56818",
"date": "18 Dec 2019",
"name": "Evans Manyara Nyakeri",
"expertise": [
"Reviewer Expertise Microbiology",
"biotechnology",
"food security and applied insect 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 study topic is important as it clearly illustrates the need for optimising the production of the enzyme L-ASNASE.\n\nI however have a few suggestions that can contribute towards improving the work:\nThe authors should consider revision of the topic to capture the gist of the study which isolation endophytic bacteria that produce the enzyme glutaminase free L-ASNASE mangrove forest. Suggestion: Isolation of L-ASNASE producing endophytic bacteria and optimization of production of the enzyme. The topic as it is suggest that the study was targeting the isolation of the Lysinibacillus fusiformis B27 yet this was the finding of the study rather than the intentional target as from the start.\n\nThe study is not clearly bringing out the problem of the study: For example: the study rightly points out the limitations of L-ASNAS in paragagh 2 and 3. However, it does not point out the source of L-ASNAS with these limitations and the reason for advocating for alternative sources such as the endophytic bacteria from the mangrooves. Furthermore, the reason given for advocating the sourcing from microbes is not consistent in solving the prior identified limitations of the enzyme: the rapid multiplication and easy management of microbes vis avis the sensitivity to high temperatures (ie. need for extremophilic enzyme).\n\nThe study has not clearly stated the relationship between substrate glutamine and the enzyme L-ASNase and the need for microbes able to produce asparaginase and not glutaminase nor both. This needs to be established in the introduction. Is it because the two enzymes are antagonistic? The idea was to screen for bacteria isolates capable of producing only the enzyme asparaginase and not glutaminase or both gluataminase and asparaginase., Why discriminate on glutaminase? What is the basis for this? This has not been explained. Is is because the two enzymes are antagonistic?\n\nAgain, how did you identify isolates that were producing the enzymes? Formation of clearance zones, testing of media for presence of substrates and substrate level reduction, change of media colour especially due to bromothymol blue indicator? Explain to make this clearer.\n\nThe methodology on screening of enzyme producing bacterial isolates is not conclusively described. Suggestion: state that the plating of the bacteria on the two media types was done by replica plating.\n\nThe molecular study is adequate in methodology, results and interpretation.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the 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": "5129",
"date": "14 May 2020",
"name": "Asep Awaludin Prihanto",
"role": "Author Response",
"response": "I, however, have a few suggestions that can contribute towards improving the work:1. The authors should consider revision of the topic to capture the gist of the study which isolation endophytic bacteria that produce the enzyme glutaminase free L-ASNASE mangrove forest. Suggestion: Isolation of L-ASNASE producing endophytic bacteria and optimization of production of the enzyme. The topic as it is suggest that the study was targeting the isolation of the Lysinibacillus fusiformis B27 yet this was the finding of the study rather than the intentional target as from the start. -->As suggested by the reviewer, we revised the title to “Isolation of glutaminase-free L-asparaginase producing endophytic bacteria and optimization of production of the enzyme” 2. The study is not clearly bringing out the problem of the study: For example: the study rightly points out the limitations of L-ASNAS in paragraphs 2 and 3. However, it does not point out the source of L-ASNAS with these limitations and the reason for advocating for alternative sources such as the endophytic bacteria from the mangroves. Furthermore, the reason given for advocating the sourcing from microbes is not consistent in solving the prior identified limitations of the enzyme: the rapid multiplication and easy management of microbes vis avis the sensitivity to high temperatures (ie. need for extremophilic enzyme). -->We added one sentence before the end of paragraph 3 with “…….The drawbacks of the enzyme applications might be overcome by isolating the enzyme from a unique habitats, such as an endophytic bacteria from mangrove. Therefore, new L-ASNase producers need to be explored. 3. The study has not clearly stated the relationship between substrate glutamine and the enzyme L-ASNase and the need for microbes able to produce asparaginase and not glutaminase nor both. This needs to be established in the introduction. Is it because the two enzymes are antagonistic? The idea was to screen for bacterial isolates capable of producing only the enzyme asparaginase and not glutaminase or both gluataminase and asparaginase., Why discriminate on glutaminase? What is the basis for this? This has not been explained. It is because the two enzymes are antagonistic? -->We have revised the third sentence on paragraph 3 with”….In medical applications, it has a low half-life, allergic reactions, and unnecessary glutamine hydrolysis on L-asparaginase due to the lack of asparagine synthetase in cancer cells. 4. Again, how did you identify isolates that were producing the enzymes? Formation of clearance zones, testing of media for the presence of substrates and substrate-level reduction, change of media colour especially due to bromothymol blue indicator? Explain to make this clearer.-->We have revised the paragraph on Section of Result and Discussion, subsection Screening and Identification with “The screening results showed that 31 endophytic mangrove isolates could produce the L-ASNase enzyme shown by blue color-formed surrounding colony (Figure 1A). The change of the color due to the increase of pH resulting from enzyme activity. The best enzyme producer was isolate B27. This isolate had a more extensive and darker blue tone compared to other isolates. Furthermore, only isolate B27 produced glutaminase free-L-asparaginase. Hence, further molecular identification was applied only to the B27 isolate. Further, Gram stain analysis revealed that the bacterium was Gram-positive (Figure 1B).5. The methodology on screening of enzyme-producing bacterial isolates is not conclusively described. Suggestion: state that the plating of the bacteria on the two media types was done by replica plating.-->We have revised on Section Methods, subsection Isolation and screening of L-ASNase producers in paragraph two, sentence six with “All isolates were streaked onto two different media (M9 medium with L-ASN, and M9 medium with L-glutamine) in duplicates. The plates were incubated at 37°C overnight. 6. The molecular study is adequate in methodology, results and interpretation. -->Thank you for your comment"
}
]
},
{
"id": "62909",
"date": "07 May 2020",
"name": "Sathyanarayana N. Gummadi",
"expertise": [
"Reviewer Expertise Industrial Biotechnology",
"Bioprocess Engineering",
"Fermentation",
"Enzymology"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address 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 investigated the ability of mangrove Rhizophora mucronata to produce glutaminase-free L-asparaginase and then production enhancement by optimizing parameters L-asparagine concentration, pH, temperature, and inoculum concentration by Taguchi method.\n\nNumerous reports are available on glutaminase-free asparaginase producing microbial sources and considering this, results presented in this study are too preliminary and authors should think the novelty of this study.\n\nThe following are the comments to improve the quality of the manuscript.\n\nMajor comments:\n\nThe Introduction part is looking very general like in many other published papers. There is no strong rationale to screen another strain that produced very low levels of L-asparaginase.\n\nIt was mentioned in ASNase assay that 1.5 μl of Trichloro Acetic Acid 1.5 M was added to stop the reaction. Eager to know how this lower volume 1.5 μl TCA will stop the reaction and can be accurate? It is better to include the equation to calculate the activity.\n\nThe results of this study in terms of activity measured is very low (8.5 U/ml to 15 U/ml) compared to other commercial L-asparaginases (E. coli) and other novel reported sources. Moreover, the study is titled the enzyme is ‘glutaminase free L-asparaginase’; however, no evident experiments or data reported in this study for claiming this.\n\nDid the authors also check for urease activity?\n\nTables are numbered 1-5, showing only coding and interpretation results obtained from Taguchi statistical analysis. Many can be compiled as a single table and if necessary can be filed as supplementary data. For figure no.3, more description needed and clarify the 2 curves shown in each plot.\n\nWhat is the rationale in choosing only four variables for optimization? Why did the authors screen the variables initially?\n\nDid the authors try to fit the experimental results to any quadratic equation?\n\nHow did the Taguchi experimental design optimize the four variables? How did authors arrive at the optimal values and verify?\n\nAt optimal settings, the expected result is ~ 19 U/ml but the authors obtained only 8.51 U/Ml. The huge variation between experimental and predicted values suggests that the design is not able to perform to good optimization.\n\nThere is no growth data provided in the manuscript. The experimental values represented in Table 1 corresponds to what time of growth?\n\nThe study showed mangrove Rhizophora mucronata as producers of L-asparaginase and optimization of its production parameters. More experiments must be included to reveal its biochemical properties, glutaminase free activity, molecular weight, and purity to explore it into application level as mentioned in the introduction part.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? 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? Partly",
"responses": [
{
"c_id": "5500",
"date": "14 May 2020",
"name": "Asep Awaludin Prihanto",
"role": "Author Response",
"response": "1. The Introduction part is looking very general like in many other published papers. There is no strong rationale to screen another strain that produced very low levels of L-asparaginase. The reason for the need for exploring the new source is added to the first sentenced in the last paragraph. 2. It was mentioned in ASNase assay that 1.5 μl of Trichloro Acetic Acid 1.5 M was added to stop the reaction. Eager to know how this lower volume 1.5 μl TCA will stop the reaction and can be accurate? It is better to include the equation to calculate the activity. We have miswritten the amount of the TCA, The TCA volume was revised.Formula for calculating the enzyme activity was added to the manuscript 3. The results of this study in terms of activity measured is very low (8.5 U/ml to 15 U/ml) compared to other commercial L-asparaginases (E. coli) and other novel reported sources. Moreover, the study is titled the enzyme is ‘glutaminase free L-asparaginase’; however, no evident experiments or data reported in this study for claiming this.Several studies for endophytic bacteria showed lower production than these bacteria. For example the report from Hatamzadeh et al. (2020), PeerJ, DOI10.7717/peerj.8309.The glutaminase free-L-asparaginase activity was confirmed in our preliminary experiment when investigated the activity onto M9 modified agar supplemented L-Asparagine and L-Glutamine. In the L-Glutamine –supplemented M9 media, the bacteria don’t show the activity (the color of media remains yellow). Meanwhile, Asparagine –supplemented M9 media turn blue. We also confirmed the nil activity of the cell-free supernatant. 4. Did the authors also check for urease activity?No, we did not check the Urease activity 5. Tables are numbered 1-5, showing only coding and interpretation results obtained from Taguchi statistical analysis. Many can be compiled as a single table and if necessary can be filed as supplementary data. For figure no.3, more description needed and clarify the 2 curves shown in each plot. We afraid if we compiled the Tables, then the clarity and the flowing of the manuscript will also difficult to be followed. Hence, we prefer to show the detail on the tables as in the manuscript. The additional description of the manuscript was added.6. What is the rationale in choosing only four variables for optimization? Why did the authors screen the variables initially?We have performed the initial investigation toward five individual factors namely, temperature, L-ASN, pH, inoculum size, and shaker speed. In our preliminary investigation, shaker speed failed to give a significant effect on the production of L-ASNase. Hence, we only applied four variables (L-ASN, temp, pH, and inoculum size). 7. Did the authors try to fit the experimental results to any quadratic equation?No, we did not, in the Taguchi method we automatically are recommended the best equation /formula in order to obtain the best production. 8. How did the Taguchi experimental design optimize the four variables? How did authors arrive at the optimal values and verify?By using Qualitek-4 software for Taguchi experimental, we directly we are automatically directed with the formulation for the production of the most optimum enzymes accompanied by an estimate of its production (Table 5.)9. At optimal settings, the expected result is ~ 19 U/ml but the authors obtained only 8.51 U/Ml. The huge variation between experimental and predicted values suggests that the design is not able to perform to good optimization. It is true that the result of the estimated production is higher than that of the exact production. However many researchers also experienced a similar result, where the exact value is lower than the predicted one. As examples is reported by Chenthamarakshan, A., Parambayil, N., Miziriya, N. et al. Optimization of laccase production from Marasmiellus palmivorus LA1 by Taguchi method of Design of experiments. BMC Biotechnol 17, 12 (2017). https://doi.org/10.1186/s12896-017-0333-x; and Shehata AN, Abd El Aty AA. 2014. Optimization of Process Parameters by Statistical Experimental Designs for the Production of Naringinase Enzyme by Marine Fungi. International Journal of Chemical Engineering. ID 273523. https://doi.org/10.1155/2014/273523. The main focus is on the increase in production by comparing before and after optimization.In the optimized formulation, we reached the increase of the production by three folds. Hence, we believe that the optimization worked.10. There is no growth data provided in the manuscript. The experimental values represented in Table 1 corresponds to what time of growth?The culture time is 24 h. we have revised the manuscript to clarify the time of harvesting 11. The study showed mangrove Rhizophora mucronata as producers of L-asparaginase and optimization of its production parameters. More experiments must be included to reveal its biochemical properties, glutaminase free activity, molecular weight, and purity to explore it into application level as mentioned in the introduction part.We have confirmed the glutaminase free activity by investigating using L-glutamine as a substrate. The further characteristic has been under investigation. Next paper will discuss the purification and characteristic of the enzyme from Lysinibacillus fusiformis B27"
}
]
}
] | 1
|
https://f1000research.com/articles/8-1938
|
https://f1000research.com/articles/9-365/v1
|
14 May 20
|
{
"type": "Brief Report",
"title": "Prediction of the effectiveness of COVID-19 vaccine candidates",
"authors": [
"Veljko Veljkovic",
"Vladimir Perovic",
"Slobodan Paessler",
"Vladimir Perovic",
"Slobodan Paessler"
],
"abstract": "A safe and effective vaccine is urgently needed to bring the current SARS-CoV-2 pandemic under control. The spike protein (SP) of SARS-CoV-2 represents the principal target for most vaccines currently under development. This protein is highly conserved indicating that vaccine based on this antigen will be efficient against all currently circulating SARS-CoV-2 strains. The present analysis of SP suggests that mutation D614G could significantly decrease the effectiveness of the COVID-19 vaccine through modulation of the interaction between SARS-CoV-2 and its principal receptor ACE2.",
"keywords": [
"COVID-19",
"SARS-CoV-2",
"mutations",
"vaccine effectiveness"
],
"content": "Introduction\n\nSARS-CoV-2 is the novel highly infectious human coronavirus which by May 2020 has infected 3 million and killed more than 200,000 people. Until immunity is induced in large populations throughout the world this coronavirus is likely to become endemic seasonally causing the coronavirus disease 2019 (COVID-19) in millions worldwide. Scientists and drug companies around the world are working hard to develop a vaccine against the disease with at least five candidate vaccines in clinical evaluation and another 71 in preclinical evaluation. The spike protein (SP) of SARS-CoV-2 is the principal target of most these vaccine candidates. Analysis of 5,700 isolates collected between December 2019 and April 2020 revealed only one mutation which was found in more than 1% of currently circulating viruses1. This finding suggests that a single vaccine based on the consensus sequence of highly conserved SP antigen should be efficacious against current global strains.\n\nHuman coronaviruses SARS-CoV and SARS-CoV-2 recognize the angiotensin converting enzyme 2 (ACE2)2 as the natural receptor but present a distinct binding interface to ACE2 and a different network of residue-residue contacts. SARS-CoV and SARS-CoV-2 have comparable binding affinity but the SARS-CoV-2–ACE2 complex contains a higher number of contacts, a larger interface area, and decreased interface residue fluctuations relative to SARS-CoV3. These suggest that the receptor binding site (RBD)–ACE2 interface of SARS-CoV-2 resembles some properties of antibody-antigen interactions, which allow the accelerated evolution of spike protein (SP) binding to the ACE2 receptor, similar to the rapid evolution along the antibody-antigen affinity maturation process3. This opens the question about the effectiveness of the vaccine against SARS-CoV with mutations, which modulate its interaction with the receptor.\n\nPreviously, a novel bioinformatics approach for assessment of the effectiveness of the seasonal influenza vaccine was proposed4. This approach, which is based on electronic biology, was successfully used for the prediction of the influenza vaccine effectiveness for two successive flu seasons5,6. Here, this approach was used for the assessment of the effectiveness of the COVID-19 vaccine. This analysis showed that mutation D614G, which spreads globally, and which is present in >50% of all circulating viruses, could significantly decrease the effectiveness of the COVID-19 vaccine.\n\n\nMethods\n\nWe analyzed the subunit 1 of S protein (SP1) from SARS-CoV-2 with following mutations7,8:\n\nV367F, G219K, M153T, Q409E, R408I, A435S, N354D, D364Y, H655I, V615I, Q239K, Y28N, T29I, H49Y, L54F, N74K, D111N, F157L, G181V, S221W, S247R, A348T, G476S, V483A, H519Q, A520S, D614G.\n\nThe SARS-CoV-2 S protein reference sequence (GenPept accession YP_009724390) is used in the analysis as the wild type (WT).\n\nThe informational spectrum method (ISM), a virtual spectroscopy method, is developed for a fast and simple structure analysis of proteins and their functionally important domains. Physical and mathematical basis of ISM is described in detail elsewhere9 and here the method will be presented only briefly.\n\nA sequence of N amino acid residues is represented as a linear array of N terms, with each term given a weight. The weight assigned to a residue is the electron-ion interaction potential (EIIP)10,11, determining the electronic properties of amino acids, which are responsible for their intermolecular interactions12. In this way, the alphabetic code is transformed into a sequence of numbers. The signal obtained is then decomposed in periodical function by Fourier transformation. Thus, the initial information defined by the sequence of amino acids can now be presented in the form of an informational spectrum (IS), representing the series of frequencies and corresponding amplitudes. The IS frequencies correspond to the distribution of structural motifs with defined physicochemical characteristics determining long-range interaction properties of the protein.\n\nPhylogenetic analysis of SARS-CoV-2 SP1 was performed with the ISM-based phylogenetic algorithm ISTREE, which was previously described in detail13. This phylogenetic approach that allows the assessment of the biological effect of mutations was previously applied in the analysis of influenza viruses13–16, Ebola virus17 and Zika virus18,19. Figure 1 gives the schematic presentation of this algorithm. Here, we used an ISM distance measure d defined on the specific frequency F = 0.257 which characterizes the interaction between SP1 and the ACE2 receptor20.\n\nFor the development of the conventional phylogenetic tree, based on multiple sequence alignment (MSA), we used the MEGA521 software package. For the MSA calculation of sequences, the MUSCLE algorithm of MEGA5 software was applied.\n\n\nResults and discussion\n\nIn Figure 2a, we show the ISM-based phylogenetic tree for mutant SP1 from SARS-CoV-2. These SP1 are grouped in two separate clusters, A and B, that, according to the informational spectrum (IS) concept, have different interacting and immunological profiles13–17. There is no difference between the analyzed SP1 in the homology-based phylogenic tree (Figure 2b). Presented results suggest that most of the analyzed mutants (cluster A) will interact with ACE2 in a similar way as the WT virus. Only five mutations V367F, R408I, G476S, D111N, and D614G (cluster B) could significantly affect the interaction of SP1 with ACE2. Three of these mutations (V367F, R408I, G476S) are located in the RBD of SP.\n\n(a) The phylogenetic tree constructed using the ISM-based method. (b) The phylogenetic tree constructed by the homology-based method.\n\nThe previous ISM-based phylogenetic analysis of hemagglutinins from seasonal influenza viruses showed that viruses which are in the same cluster with the vaccine virus are responsive to the seasonal flu vaccine and those viruses which are grouped in the separate clusters are resistant to the vaccine. This finding served as a base for the accurate prediction of the efficacy of the flu vaccine several months before the start of the flu season5,6. The same approach was also used for the design of antigens for the vaccine against ZIKA virus18,19. By analogy, it could be expected that SARS-CoV-2 with mutations V367F, R407I, G476S, D111N and D614G in SP are resistant to the COVID-19 vaccines that are based on the WT SARS-CoV-2.\n\nThe D614G mutation frequency is much higher than the others. This mutation was found in 55% of sequences sampled globally as of April 10, 20201. The next most frequent mutation is found in only 0.87% of these sequences. A virus carrying the D614G mutation, which had not then been observed among sequences from China, was transmitted in January 2020 to Germany and became dominant in Europe and then globally within two months (found in 55% of sequences)1.\n\nThe D614G mutation is located in the middle of one of three epitopes of SP122. This change of large acidic residue D into small hydrophobic residue G in the middle of the epitope would compromise the binding affinity to antibodies elicited by vaccines with WT SP.\n\n\nConclusions\n\nThe present results suggest that the highly prevalent D614G mutation in SP, although located outside of the receptor binding site, could decrease the efficacy of the vaccine by modulation of the interaction of SARS-CoV with the ACE2 receptor. In addition, this mutation may cause antigenic drift, resulting in vaccine mismatches which additionally could affect the efficacy of the vaccine. These possible obstacles should be considered for the future development of COVID-19 vaccines. The further ISM-based monitoring of the evolution of SARS-CoV-2 is important for identification of other mutations, which could affect the effectiveness of vaccines against this virus.\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\nDearlove B, Lewitus E, Bai H, et al.: A SARS-CoV-2 vaccine candidate would likely match all currently circulating strains. bioRxiv preprint. 2020. Publisher Full Text\n\nShang J, Ye G, Shi K, et al.: Structural basis of receptor recognition by SARS-CoV-2. Nature. 2020. PubMed Abstract | Publisher Full Text\n\nBrielle ES, Schneidman-Duhovny D, Linial M: The SARS-CoV-2 exerts a distinctive strategy for interacting with the ACE2 human receptor. bioRxiv preprint. 2020. Publisher Full Text\n\nVeljkovic V, Paessler S, Glisic S, et al.: Evolution of 2014/15 H3N2 influenza viruses circulating in US: consequences for vaccine effectiveness and possible new pandemic. Front Microbiol. 2015; 6: 1456. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPaessler S, Veljkovic V: Prediction of influenza vaccine effectiveness for the influenza season 2017/18 in the US [version 1; peer review: 2 approved]. F1000Res. 2017; 6: 2067. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPaessler S, Veljkovic V: Using electronic biology based platform to predict flu vaccine efficacy for 2018/2019 [version 2; peer review: 2 approved, 1 approved with reservations]. F1000Res. 2018; 7: 298. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMutation sites in the Spike (S) protein: China National Center for Bioinformation, 2019 Novel Coronavirus Resource (2019nCoVR). Reference Source\n\nLokman SM, Rasheduzzaman Md, Salauddin A, et al.: Exploring the genomic and proteomic variations of SARS-CoV-2 spike glycoprotein: a computational biology approach. bioRxiv preprint. 2020. Publisher Full Text\n\nVeljkovic V, Cosic I, Dimitrijevic B, et al.: Is it possible to analyze DNA and protein sequences by the methods of digital signal processing? IEEE Trans Biomed Eng. 1985; 32(5): 337–41. PubMed Abstract | Publisher Full Text\n\nVeljkovic V, Slavic I: Simple general-model pseudopotential. Phys Rev Let. 1972; 29: 105. Publisher Full Text\n\nVeljkovic V: The dependence of the Fermi energy on the atomic number. Physics Letters A. 1973; 45(1): 41–42. Publisher Full Text\n\nVeljkovic V: A theoretical approach to preselection of carcinogens and chemical carcinogenesis. Gordon & Breach, New York, 1980. Reference Source\n\nPerovic VR, Muller CP, Niman HL, et al.: Novel phylogenetic algorithm to monitor human tropism in Egyptian H5N1-HPAIV reveals evolution toward efficient human-to-human transmission. PLoS One. 2013; 8(4): e61572. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVeljkovic V, Veljkovic N, Muller CP, et al.: Characterization of conserved properties of hemagglutinin of H5N1 and human influenza viruses: possible consequences for therapy and infection control. BMC Struct Biol. 2009; 9: 21. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVeljkovic V, Niman HL, Glisic S, et al.: Identification of hemagglutinin structural domain and polymorphisms which may modulate swine H1N1 interactions with human receptor. BMC Struct Biol. 2009; 9: 62. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVeljkovic V, Veljkovic N, Paessler S, et al.: Predicted enhanced human propensity of current avian-Like H1N1 swine influenza virus from China. PLoS One. 2016; 11(11): e0165451. PubMed Abstract | Publisher Full Text | Free Full Text\n\nVeljkovic V, Glisic S, Muller CP, et al.: In silico analysis suggests interaction between Ebola virus and the extracellular matrix. Front Microbiol. 2015; 6: 135. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPaessler S, Veljkovic V: Vaccine with reduced enhancement of viral infection. United States Patent 10322171. 2019. Reference Source\n\nJones RF, Balint J, Rice A, et al.: Compositions and methods for flavivirus vaccination. Patent WO 2018/014006 Al. 2018. Reference Source\n\nVeljkovic V, Vergara-Alert J, Segales J, et al.: Use of the informational spectrum methodology for rapid biological analysis of the novel coronavirus 2019-nCoV: prediction of potential receptor, natural reservoir, tropism and therapeutic/vaccine target. F1000Res. 2020; 9: 52. Publisher Full Text\n\nTamura K, Peterson D, Peterson N, et al.: MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol. 2011; 28(10): 2731–2739. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKoyama T, Weeraratne D, Snowdon JL, et al.: Emergence of drift variants that may affect COVID-19 vaccine development and antibody treatment. Preprints (www.preprints.org); Publisher Full Text"
}
|
[
{
"id": "64327",
"date": "15 Jun 2020",
"name": "Sándor Pongor",
"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\nThis work highlights an interesting aspect of the COVID spike protein that may be useful for vaccine developers.\n\nSequences of spike protein mutants are compared with the information spectrum method developed by the authors and it is found that the clustering of spike protein mutants allows one to draw conclusions on the expected effectiveness of the vaccines.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? 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": "68166",
"date": "13 Aug 2020",
"name": "Takahiko Koyama",
"expertise": [
"Reviewer Expertise genomics",
"oncology",
"immunology",
"stem cell",
"virology",
"bioinformatics"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors are trying to find out important residues of spike protein interacting with ACE2 using the ISM method.\nThe application of the methods have not been validated with wide range of proteins comparing with experimental data. Therefore, it is not easy to evaluate the effectiveness of the method. Experimental data is available in the following paper:\nChan, K.K., et al., Engineering human ACE2 to optimize binding to the spike protein of SARS coronavirus 2. Science, 2020: p. eabc0870.1\nI would like to see if how ISM in agreement with the data.\nSecondly, the ISM method is attempting to identify the residue impacting interactions with ACE2. However, for vaccines, neutralizing antibodies attached to spike protein to inhibit interaction or other process. Therefore, direct interaction with ACE2 does not necessarily mean key residue for vaccine. Rather, antibodies from B-cell must bind to the residues and the residues identified by the authors are not necessarily in the B-cell epitopes. Therefore, those residues are irrelevant to vaccines.\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? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": [
{
"c_id": "5816",
"date": "18 Aug 2020",
"name": "Veljko Veljkovic",
"role": "Author Response",
"response": "The Reviewer’s remark: “The application of the methods have not been validated with wide range of proteins comparing with experimental data. Therefore, it is not easy to evaluate the effectiveness of the method.” Response Reviewer can find several articles in PubMed where ISM prediction is experimentally demonstrated (e.g. doi: 10.1038/s41598-018-20185-8. doi: 10.1038/srep11434). Based on previous work we believe the ISM does not need any additional proof and can be used to estimate efficacy as it is. Moreover, the Reviewer overlooked that the ISM analysis concerns the long-range interactions between molecules (interactions at distances between 5 and 1000 A) and not their chemical binding (the interactions at distances <5A). The ACE2 example suggested by the Reviewer is not suitable for the ISM analysis because it concerns the improvement of the chemical binding of SP to this receptor. The Reviewer’s remark: “Secondly, the ISM method is attempting to identify the residue impacting interactions with ACE2. However, for vaccines, neutralizing antibodies attached to spike protein to inhibit interaction or other process. Therefore, direct interaction with ACE2 does not necessarily mean key residue for vaccine. Rather, antibodies from B-cell must bind to the residues and the residues identified by the authors are not necessarily in the B-cell epitopes. Therefore, those residues are irrelevant to vaccines” Response Presented ISM results indicate that the mutation D614G modulate the recognition and targeting between antibodies and SP antigen and not their “direct interaction” (chemical binding). If the mutation prevents the efficient recognition between antigen and an antibody, the neutralization will not be efficient even if the antibody would efficiently bind to the antigen. In other words, the antibody will not efficiently “find” its target in vivo."
}
]
}
] | 1
|
https://f1000research.com/articles/9-365
|
https://f1000research.com/articles/9-363/v1
|
14 May 20
|
{
"type": "Research Article",
"title": "Role of pharmacogenetics and clinical parameters on nevirapine plasma concertation among HIV-1 patients receiving Antiretroviral Therapy in Kenya",
"authors": [
"Mungiria Juster",
"Lucy Gitonga",
"Moses Muraya",
"John Mwaniki",
"Musa Ngayo",
"Mungiria Juster",
"Lucy Gitonga",
"Moses Muraya",
"John Mwaniki"
],
"abstract": "Background: Patients’ clinical outcomes and pharmacogenetic factors are important predictors of nevirapine (NVP) plasma concentration. This study evaluated the association of socio-demographic factors and Cytochrome P450 2B6 (CYP2B6) polymorphisms with NVP plasma concentrations among patients receiving antiretroviral therapy (ART) treatment in western and coastal Kenya. Methods: Blood samples were collected from 377 consenting HIV adult patients receiving an NVP-based first-line ART regimen. A detailed sociodemographic questionnaire was administered. NVP plasma concentration was measured by liquid chromatography - tandem mass spectrometry (LC-MS/MS). CYP2B6 c.516 G>T rs3745274 and c.983T>C genotypes were evaluated using real-time polymerase chain reaction. HIV drug resistance mutations were detected using an in-house genotypic assay. Results: The patients’ mean age was 41.6 (SD ± 11.5) years and the majority (59.2%) were female. The mean duration of ART was 5.1 (SD ± 4.8) years. Overall NVP plasma levels ranged from 4-44207 ng/mL (median 6213 ng/mL, IQR 3097–8606.5 ng/mL). There were 105 (25.5%) participants with NVP levels of <3100 ng/mL, associated with poor viral suppression. Multivariate linear regression analysis showed CYP2B6 516 G>T polymorphism (β 0.71, 95% CI 0.4–0.98; p<0.0001), male gender (β 0.45, 95% CI 0.01–0.9; p=0.047) and presence of HIV drug-resistant virus (β 1.98, 95% CI 1.24–2.72; p<0.001) were the independent factors influencing NVP plasma concentration. Conclusions: The majority of patients receiving an NVP-based ART regimen had plasma concentrations within the therapeutic range. CYP2B6 516 G>T polymorphism, gender and presence of a HIV drug-resistant mutation significantly influences NVP plasma concentration. Routine pharmacogenetic testing and measurement of NVP plasma concentrations, considering gender and presence of HIV drug-resistant mutations are key to ensuring optimal ART treatment outcomes in Kenya.",
"keywords": [
"Nevirapine plasma concentration",
"pharmacogenetics and clinical parameters",
"HIV-1 patients in Kenya"
],
"content": "Background\n\nThe use of antiretroviral therapy (ART) is an integral component in reducing the burden of HIV globally1. In 2018, it was estimated that 68% (826,000) of adults and 73% (71,500) of children in Kenya needing ART were actually receiving it1. A remarkable scale up of ART has put Kenya on track to reach the target of AIDS-related deaths1. Kenya is among the countries that have adopted the 2015 World Health Organization (WHO) recommendations, which require immediate initiation of ART to people diagnosed with HIV, aimed at increasing ART access further2. At the time of the study, the first-line ART guidelines for children, youth and adults in Kenya typically contained a backbone of two nucleoside reverse transcriptase inhibitors (NRTIs; zidovudine [AZT], stavudine [d4T], tenofovir [TDF] or lamivudine [3TC]), plus one non-nucleoside reverse transcriptase inhibitor (NNRTI): either nevirapine (NVP) or efavirenz (EFV)2.\n\nNVP is a particularly extensively prescribed antiretroviral drug in developing countries mainly because of its efficacy, availability, low cost and use in prevention of vertical HIV transmission3. However, a higher incidence of rashes (which can be severe and life-threatening, such as Stevens-Johnson syndrome) associated with NVP4, the association with a rare but potentially life-threatening risk of hepatotoxicity5 and a low genetic barrier to resistance are the fundamental factors that restrict the use of NVP6. A study in Kenya reported that 35% of patients on NVP experienced treatment failure7. Factors such as unavailability of ART stock, poor prescribing practice and prompt antiretroviral refill are known to significantly affect treatment outcomes8.\n\nGenerally, the most important aspect of ART management is ideal drug exposure9; extensive use of, or suboptimal exposure to, ART, especially NNRTIs (NVP and EFZ), bestows countless risks to the success of treatment10. Studies in the USA, Europe and Asia have shown that low NNRTI concentrations are predictive of virologic failure11,12 and highly ART drug-resistant HIV quickly develops when administered in suboptimal regimens13. In Africa, studies have reported expansive person to person variation in plasma antiretroviral levels among patients with management failure, with a large percentage falling out of the therapeutic window14,15. Though countable reports have shown plasma NVP concentrations affect treatment outcome16, therapeutic drug level quantification is currently not part of HIV management in many countries, including Kenya15.\n\nNVP is majorly metabolized by cytochrome P450 2B6 (CYP2B6) and minorly by CYP3A isoforms17. The occurrence of single-nucleotide polymorphisms (SNPs) especially on 516GT and 983TC, are correlated with significant changes in hepatic enzyme metabolic activities17. Other factors such as body weight, age, clinical outcome, gender, CD4 and HIV viral load nadir are also important determinants of NVP plasma levels8,18.\n\nTo build up data aimed at personalizing ART treatment in Kenya, this study assessed the relationship between CYP2B6 516G>T and 983T>C polymorphism, HIV drug resistant mutation, and other clinical parameters with NVP concentrations among HIV-1 patients receiving ART treatment in western and coastal Kenya.\n\n\nMethods\n\nThis study was approved by the Kenya Medical Research Institutes’ (KEMRI) Scientific Review Unit (SERU) (KEMRI/SERU/CVR/002/3214) and NACOSTI (NACOSTI/P/19/11747/28173). Before recruitment in this study, all patients filled in written informed consent for study participation.\n\nThis was a cross-sectional study conducted between August 2018 to January 2020 and was part of an ongoing study designed to establish a cost-effective laboratory method to monitor antiretroviral adherence in HIV-1 infected individuals on treatment in Kenya. Patients were recruited in this study if they were: (i) HIV-1 infected adults (aged above 18 years), attending the two HIV treatment programs, (ii) willing to voluntarily give written informed consent, (iii) able to read either English or Kiswahili, (iii) be on ARV treatment for 12 months, (iv) be on NVP based 1st line ARV treatment regimen and (v) HIV patients with viral load results at month 12 of treatment. The patients were categorized as either failing (HIV viral load >1000 copies/mL) or responding (HIV viral load <1000 copies/mL) to treatment.\n\nUsing the case control sample size formula described by Lemeshow et al.19, and using the previous study of Ahoua et al.20, which showed that among HIV patients with virological failure at month 12, 12.8% of them developed sub-optimal drug level/drug resistance. In this study, we wished to have a 90% chance of detecting an odds ratio significantly different from 1 at the 5 % level. Considering an odds ratio of 2 as an important difference between the two groups, a total of 376 (188 cases and 188 controls) were to be recruited from each of the two sites. A two-stage sampling method was then used to select patients meeting the inclusion criteria from the two sites. First, the overall number of patients meeting the inclusion criteria was generated based on the laboratory records. A total of 272 patients from Kisumu (western Kenya) and 105 patients from Malindi (costal Kenya) were then selected based on probability proportionate to size. Second, a consecutive sampling technique was used to obtain consent from and recruit every patient meeting the inclusion criteria until the required sample size was achieved.\n\nAn exhaustive structured interview (including demographic data, clinical history, adherence, HIV stigma and medical history) was used to collect patient-related information (see Extended data)21. These interviews were conducted by medical clinician or doctor employed by the two organizations and who regularly attends to these patients. Each interview was conducted in a separated private room and lasted for about 45 minutes.\n\nAfter the interviews, the patients were sent to the phlebotomy room where blood specimens were drawn by a trained phlebotomist into ethylenediaminetetraacetic acid (EDTA)-containing Vacutainers® (BD, US) for determination of NVP plasma concentration and CYP2B6 516G>T and 983T>C genetic analysis. From each of the patients, approximately 5ml of intravenous blood was collected in EDTA tubes. Blood samples were centrifuged at 20,000g to collect plasma, which was stored at -20°C at each research site until it was shipped in dry ice to the KEMRI Nairobi laboratory for storage and laboratory testing.\n\nRNA was extracted manually from 1ml of all HIV-1 samples using QIAmp viral RNA mini kit (Cat. No. 52906, Qiagen Inc., USA) according to the manufacturer instructions. Purified RNA was eluted in 60μL of molecular grade water. A volume of 10μL of RNA extract was used for quantification with the Generic HIV Viral Load assay (Biocentric, Bandol-France). The cycling conditions comprised of 50°C for 10 minutes and 95°C for 5 minutes, followed by 50 cycles of 95°C for 15 seconds and 60°C for 1 minute. Amplification and data acquisition were carried out using the ABI Prism 7300 Sequence Detection System (Applied Biosystems) and the detection cut-off value was 60 HIV-1 RNA copies/ml.\n\nThe NVP plasma concentrations were measured using a Xevo TQ-S tandem quadrupole mass spectrometer (Waters Corporation, U.S.A) designed for ultra-high performance as described by Reddy et al.22. First, the HIV virus was inactivated as follows. Into a 1.5ml Eppendorf tube, 50μl of plasma and 5μl internal standard (2μg/ml nevirapine, purity: 100 %, from Vivan Life Sciences, Mumbai, India prepared in methanol) was added. This was heated at 65°C for 10 minutes, followed by 10 minutes cooling at room temperature. 100μl cold methanol (-20oC) was then added to each sample and kept at -20°C for 10 minutes. This was followed by eight minutes of centrifugation at 20,000g, 20°C to collect the supernatant in a clean 1.5ml tube. Then, 850μl ammonium acetate buffer (pH = 3.00) was added to the supernatant and briefly centrifuged. The sample was considered safe to be handled in a non P3 laboratory.\n\nSolid phase extraction was carried out using Bond Elut C18 cartridges. The cartridges were prepared and placed onto the Visiprep Vacuum Manifold with standard lid (Merck, Germany). The Bond Elute C18 150×4.6mm, 5μm column was conditioned by first passing through 1ml methanol, followed by 1ml ultrapure water. Each column was then charged with 150μl samples containing 850μl ammonium acetate buffer (pH = 3.00), followed by twice cleaning using 1ml ultrapure water. The first cleaning was collected into clean separate tube while the second water cleaning collected in the waste tubes. The columns were vacuum dried (5–10 kPa). NVP elution was carried out at a flow rate of 1ml/min twice using 500μl methanol with vacuum drying between the two elutions. Elutes were then completely evaporated using Thermo Scientific™ Reacti-Vap™ Evaporators (Thermo Fisher Scientific Inc, USA) at 37°C for 30 minutes. This was then reconstituted using 100μl of equal parts 1:1 acetonitrile and water, vortexed briefly and transferred into 50ml capped vials and placed into the Xevo TQ-S for quantification. Approximately 1μl of the samples was injected automatically into the LC/MS/MS instrument and quantified within five minutes. NVP plasma concentration was categorized as <3100 ng/mL (below therapeutic range), 3100–4300 ng/mL (therapeutic range) and >4300 ng/mL (above therapeutic range) as previously defined23.\n\nDNA was extracted from patients’ blood samples using the QIAamp DNA Blood Mini Kit (Cat. No. 51106, Qiagen GmbH, Hilden, Germany) according to the manufacturer’s instructions. Briefly, 20μl QIAGEN Protease and 200μl blood sample were added into a 1.5ml microcentrifuge tube and 200μl of Buffer AL added and mixed by pulse-vortexing for 15 seconds. This was incubated at 56°C for 10 minutes. This was followed by a series of washing and eventually the DNA was eluted using 200μl Buffer AE at 6000 × g (8000 rpm) for one minute. The quality of DNA was measured using a ND-1000 UV spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). Genotyping was carried out on an ABI 7500 Fast Sequence Detection System (Applied Biosystems, Foster City, CA, USA). SNPs were analyzed using the validated Taqman Genotyping Assays for CYP2B6 516G>T (rs3745274; assay ID C_7817765_60) and CYP2B6 983T>C (rs28399499; assay ID C_60732328_20), according to the manufacturer’s instructions. Briefly, in a final volume for each reaction of 25μl, consisting of 2x TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA), 20x drug metabolizing genotype assay mix and 10ng genomic DNA. The PCR profile consisted of an initial step at 50°C for 2 minutes and 50 cycles at 95°C for 10 minutes and 92°C for 15 seconds. The plates were read using the allelic discrimination settings. The SNP assay was set up using SDS, version 1.3.0 as an absolute quantification assay. Post-assay analysis was done using SDS software. The results for CYP2B6 516G>T and 983T>C genotypes were defined as follows: homozygous wild type as 516GG or 983TT, heterozygous as 516GT or 983TC, and homozygous mutated as 516TT or 983CC.\n\nThe presence of HIV drug-resistant mutation was tested using an in-house genotypic method previously described Lehman et al.24. This involved the following steps:\n\nRNA extraction. The viral RNA was extracted from plasma using QIAamp Viral RNA Extraction Kit (Cat. No. 52906, Qiagen Inc., USA) according to manufacturer’s instructions.\n\nNested PCR amplification and visualization. A nested PCR was then performed using AmpliTaq Gold (Roche Molecular Systems, Branchburg, NJ). Briefly, in the first round; HIV- 1 pol gene was amplified using primers (RT18: 5′ GGAAACCAAAAATGATAGGGGGAATTGGAGG 3′ and RT21: 5′ CTGTATTTCTGCTATTAAGTCTTTTGATGGG 3′) achieved as follows: one cycle of 45°C for one minute and 94°C for two minutes, followed by 35 cycles of 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for one minute, with a final extension of 72°C for two minutes. The second-round amplification used primers (RT1: 5′ CCAAAAGTTAAACAATGGCCATTGACAGA 3′ and RT4: 5′ AGTTCATAACCCATCCAAAG 3′) was achieved as follows: one cycle of 94°C for two minutes and 30 cycles of 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for one minute, with a final extension of 72°C for 10 minutes. The PCR amplification was confirmed by visualization with ethidium bromide staining of agarose gel. The nested reverse transcriptase-polymerase chain reaction (RT-PCR) amplified a positive sample of 645 base pairs.\n\nBigDye sequencing reactions. The PCR positive samples were first cleaned to remove excess primers and nucleotides in a single step up using ExoSAP-IT™ PCR technology (Cat. No. 78200, Applied Biosystems, Foster City, CA, USA). Briefly, for each positive PCR product visualized on the gel, 5ul of the PCR product was mixed with 2ul ExoSAP-IT reagent and held at 37°C for 15 minutes, followed by 80°C for 15 minutes in a thermocycler.\n\nThe cleaned DNA was then amplified using a BigDye Terminator Kit (Cat. No. 4337457, Applied Biosystems, Foster City, CA, USA) and an ABI Prism 3300 Genetic Analyzer (Applied Biosystems, Foster City, US). Briefly, a 10µl reaction comprising 5.5µl DNA grade water, 2µl 5x BigDye buffer, 1ul BigDye 0.5µl primers (RT 1: 5’ CCAAAAGTTAAACAATGGCCATTGACAGA 3’ and RT4: 5’ AGTTCATAACCCATCCAAAG 3’), and 1ul PCR product template was amplified as follows: one cycle of 94°C for 10 seconds and 25 cycles of 94°C for 10 seconds, 50°C for 5 seconds, and 60°C for 4 minutes24.\n\nThis BigDye PCR amplification product was cleaned up using spin columns impregnated with Sephadex® G-50 (Sigma, US) and denatured using 10 ul Hi-Di™ Formamide and heated at 95°C for two minutes using a thermocycler. The 20ul of the denature BigDye product were sequenced using BigDye technology on an ABI 310 Genetic Analyzer (Applied Biosystems, Foster City, CA).\n\nDrug resistant interpretation. The ART drug resistance mutations were identified using the Stanford University and International AIDS Society-USA website. Genotypic resistance was defined as the presence of resistance mutations associated with impaired drug susceptibility using the Stanford Genotypic Resistance Interpretation Algorithm.\n\nFrequencies and percentages were used to present the sociodemographic data. The relationship between NVP plasma concentrations and CYP2B6 516G>T and 983T>C and the presence of HIV drug-resistant mutations were determined using Kruskal-Wallis test and Dunn’s test. Univariate and multivariate linear regression analyses were performed to determine the relationship between NVP plasma concentration and genetic polymorphisms, presence of HIV drug-resistant mutations and other clinical characteristics at the significance level of p<0.05. All statistical analyses were performed using STATA v 13 (StataCorp LP, Texas, USA).\n\n\nResults\n\nThe results from the 377 patients were assessed, of whom 272 (72.2%) were from Kisumu county (western Kenya), 223 (59.2%) were female and 114 (30.2%) had a HIV viral load of >1000 copies/mL25. The median age of the patients was 41 years (IQR = 34–49 years), with a median duration of living with HIV infection of five years (IQR = 1–11years) and a median duration since ART initiation of three years (IQR = 1–8 years). There were 306 (81.2%) patients currently taking lamivudine, nevirapine, tenofovir regimen, while 97 (25%) had missed an ART scheduled visit due to HIV-related illness and 205 (54.4%) reporting missing taking current ART at least once (Table 1).\n\nARV, antiretroviral; ART, antiretroviral therapy.\n\nThe steady-state NVP plasma concentrations varied widely among patients, ranging from 4 ng/mL to 44,207 ng/mL (median 5179 ng/mL, IQR 2557–7453 ng/mL). Out of the total 377 patients, 96 (25.5%) had an NVP concentration <3100 ng/mL and 26 (6.9%) had an NVP concentration of 3100–4300 ng/mL, with the majority, 255 (67.6%), of the patients having an NVP concentration >4300 ng/mL (Table 2). No significant differences were found with regards to region of origin between patients with NVP levels <3100 ng/mL and those with NVP levels of 3100–4300 ng/mL or >4300 ng/mL (p=0.829). Further, no differences were observed with regards to gender, HIV RNA viral load, age and duration infected with HIV in patients with NVP levels >4300 ng/mL when compared to patients with lower NVP levels. Similarly, no significant correlations were observed with regards to age of sexual debut (p = 0.785), duration since ART initiation (p=0.888), initial ART regimen type (p=0.883), current ART regimen type (p=0.972), missing scheduled HIV care visit (p=0.644), or non-adherence to current ART regimen (p=0.769) in patients with NVP levels >4300 ng/mL when compared to patients with lower NVP levels (Table 1).\n\nThe number of patients with GG, GT and TT genotypes for the CYP2B6 516G>T SNP were 142 (37.7%), 187 (49.6%) and 48 (12.7%), respectively. In the case of the CYP2B6 983T>C SNP, the majority of patients (n = 326, 86.5%) had the homozygous wild type TT genotype. There were 48 patients (12.7%) who had the heterozygous mutant TC genotype, while three (0.7%) patients had the homozygous mutant CC genotype.\n\nIn this study, CYP2B6 516G>T and 983T>C SNPs were correlated with increased mean NVP plasma concentrations (Table 2). For CYP2B6 516G>T, patients who had the homozygous mutation (CYP2B6 516TT) had higher median NVP plasma levels (6753.5 ng/mL, IQR 4595.5–11046 ng/mL), as did those who were heterozygous for the mutation (CYP2B6 516GT; 5579 ng/mL, IQR 2960–8323 ng/mL), compared to those with the wild-type (CYP2B6 516GG; 3920.5 ng/mL, IQR 1416–6278 ng/mL) (p<0.0001). In the case of CYP2B6 983T>C, although the median NVP plasma concentrations were higher among patients who had the heterozygous genotype (CYP2B6 983TC; 5987 ng/mL, IQR 3101.5–9143 ng\\mL) than those who had homozygous wild-type (CYP2B6 983TT; 5132 ng/mL, IQR 2394–7384 ng\\mL), the distribution was not significant (p=0.33).\n\nWith regard to the presence of a HIV drug-resistant mutation, although there were only 31 (8.2%) patients with an NVP-based resistant mutation as opposed to 346 (91.8%) without, the median plasma NVP levels for those with resistant mutations was almost half (2283 ng/mL, IQR 18–5283 ng/mL) that of those without (5498 ng/mL, IQR 2960–7690 ng/mL) (p=0.001) (Table 2). The associations between log10-transformed plasma NVP concentrations and CYP2B6 genotypes and the presence of HIV drug-resistant mutations are shown in Figure 1.\n\nA) Log10-transformed nevirapine plasma levels for each CYP2B6 516G>T genotype: GG, GT, and TT. B) Log10-transformed nevirapine plasma levels for each CYP2B6 983T>C genotype: TC, TT and CC. C) Log10-transformed plasma NVP levels for the presence (yes) and absence (no) of HIV drug-resistant mutations.\n\nIn the multivariate linear regression model, factors that remained significantly associated with a higher NVP plasma levels included: the T allele of CYP2B6 G516T genotype (adjusted β 0.71, 95% CI 0.4–0.98; p<0.0001), male gender (adjusted β 0.45, 95% CI 0.01–0.9; p=0.047) and presence of drug-resistant virus (adjusted β 1.98, 95% CI 1.24–2.72; p<0.001) (Table 3).\n\nART, antiretroviral therapy; SNP, single nucleotide polymorphism.\n\n\nDiscussion\n\nIn this study, in the two regions of Kenya reporting highest prevalence of HIV infection, we found wide variation in person-to-person NVP concentrations, ranging from 4ng/mL to 44,207 ng/mL (median 5179 ng/mL, IQR 2557–7453 ng/mL). This range is wider compared to other studies reported in Kenya26, in South Africa27 and in India23.\n\nThe therapeutic range of NVP plasma concentration is indicated as 3000 to 8000 ng/mL. In our study, the number of participants with plasma NVP levels within this therapeutic range was 255 (67.6%). The NVP plasma concentration required to achieve virologic control is indicated as >3000 ng/mL28. Reports associate NVP plasma concentrations lower than this threshold with poor virologic control and a concomitant increase in the number of NNRTI–resistant mutations29. The current study reports that 25.5% of patients had NVP plasma levels of <3100 ng/ml, associated with poor viral suppression. This prevalence was higher compared to those reported earlier in Kenya26 and in Italy30. This finding may be anticipated among patients with a stable and longer NVP treatment period, with 25% of them reporting non-adherence to medication.\n\nThe prevalence of CYP2B6 516T (37.7% GG, 49.6% GT and 12.7% TT) and CYP2B6 983C (86.5% TT, 12.7% TC and 0.7% CC) genotypes among these patients was comparable to those described among other African ethnic groups26,31. Studies have already established the importance of CYP2B6 gene polymorphisms as a key determinant influencing NVP plasma concentrations32,33. In the current study, patients who had both CYP2B6 516G>T and CYP2B6 983T>T genotypes had higher plasma NVP concentrations. This effect was more pronounced among patients who were homozygous for the mutant (CYP2B6 516 TT). Even though this study was cross-sectional, several longitudinal studies have reported a genotype dose-dependent increase in NVP plasma concentration among carriers of the CYP2B6 c.516 T allele26,30,34. For CYP2B6 983T>C, even though there were only 48 and three participants with the heterozygous mutant genotype (CYP2B6 983TC) and the homozygous mutant (CYP2B6 983CC), respectively, these genotypes were marked with raised NVP plasma concentrations. In agreement with previous work in Kenya26, which reported that the heterozygous TC genotype was associated with 55% higher NVP levels compared to individuals who had the wild type TT genotype. The SNP in CYP2B6 983T>C results in the variant protein CYP2B6*18 with I328T as the only amino acid change35. In vitro experiments have shown no measurable protein or activity with the expression of CYP2B6 983TC and is thus termed as a null allele36. Therefore, in other studies, the null status of CYP2B6 983TC has been associated with a greater impact compared to the CYP2B6 516GT26,34.\n\nIn this study, although there were only 8.2% patients with a HIV drug-resistant mutation to both NNRTIs and NRTIs, compared to 91.8% with susceptible viruses, median plasma NVP levels were almost half that of those who did not have drug-resistant HIV. In agreement with other reports, the presence of a HIV drug-resistant mutation is associated with higher NVP plasma concentrations37. Sustained optimal NVP plasma concentration is vital, given that only a single point mutation at specific position on the HIV-1 pol gene is associated with increased NVP resistance38. Development of HIV drug-resistant strains are shown to occur at the trough plasma NVP concentration of ≤3μg/ml, which has been correlated with increased risk of treatment failure18,39.\n\nBeyond genetic background and viral dynamics, other patient pharmacoecological factors influencing the day-to-day concentration of drugs were evaluated. Although not significant, male patients had slightly higher NVP plasma concentrations compared to females, contrary to a report from South Africa40, which showed that females had higher median NVP levels than males. This difference in NVP plasma levels between genders has been attributed to the differences in body size and drug clearance rates between males and females. Older patients had higher median NVP plasma levels than younger ones, although this was not significant. This corresponds to studies that indicated more rapid NVP metabolism among younger children aged ≤8 years, who may require higher doses of NVP to achieve the therapeutic concentration23,41.\n\nThe importance of ART regimen type, adherence, and duration on ART for the success of HIV treatment and care, although not significant in the current study, has been widely studied9,42. Generally, unstable steady-state levels of NVP have been attributed to poor adherence9,42.\n\nOur study had some limitations worth pointing out. First, due to limited resources available, the study could not recruit a larger number of patients. Second, the use of an NVP-based ART regimen in Kenya and other countries, especially developed countries, has been considerably reduced in the recent past, meaning that this study could be relevant to a restricted number of patients. Third, this study assessed well-known SNPs (CYP2B6 516T and CYP2B6 983C), whose relationship with NVP metabolism has been established. Other SNPs or other regulatory genes could also be important in NVP metabolism. Fourth, the outcomes from one population may not be generalized to other populations due to differences in drug effects between those of different ethnicities and body weights. Fifth, this was a cross-sectional study, which only permitted the description of the relationship between NVP plasma concentrations, patient genetics and a few pharmacoecologic factors and not a causal conclusion. Such outcomes can be confirmed in a longitudinal study.\n\nThese limitations notwithstanding, our study, conducted in two regions of Kenya marked by high prevalence of HIV infection, shows the significant role of CYP2B6 polymorphisms in NVP plasma concentrations. NVP plasma concentration is linked to the emergence of NVP-related resistant mutations. Moreover, patient pharmacoecologic factors, such as gender, age and ART adherence, are key in influencing NVP plasma concentration. With the consistent advancement of technical know-how, even in resource-limited settings like Kenya, individualization of ART is today reachable with the use of pharmacogenomics.\n\n\nData availability\n\nFigshare: Role of pharmacogenetics and clinical parameters on nevirapine plasma concentration among HIV-1 patients receiving antiretroviral therapy in Kenya. https://doi.org/10.6084/m9.figshare.11977680.v125\n\nFigshare: Role of pharmacogenetics and clinical parameters on nevirapine plasma concentration among HIV-1 patients receiving antiretroviral therapy in Kenya. https://doi.org/10.6084/m9.figshare.1203369921\n\nThis project contains the following extended data:\n\n- Copy of participant consent form (DOCX)\n\n- Copy of interview guide (PDF)\n\nData are available under the terms of the Creative Commons Zero \"No rights reserved\" data waiver (CC0 1.0 Public domain dedication).",
"appendix": "Acknowledgements\n\nWe wish to acknowledge the study patients, all the staff of the respective comprehensive care clinics in the two regions of Kenya. The authors thank the late Brian Khasimwa and Erickson Angira for HIV drug resistant genotyping and drug plasma measurement.\n\n\nReferences\n\nUNAID: Global HIV & AIDS statistics — 2019 fact sheet. 2018. Reference Source\n\nNASCOP: Guidelines on Use of Antiretroviral Drugs for Treating and Preventing HIV Infection in Kenya. In: Ministry of Health GoK, editor. 2016. Reference Source\n\nWorld Health Organization: Consolidated guidelines on the use of antiretroviral drugs for treating and preventing HIV infection: recommendations for a public health approach. 2nd ed. Geneva: 2016. 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PubMed Abstract | Publisher Full Text\n\nWyen C, Hendra H, Vogel M, et al.: Impact of CYP2B6 983T_C polymorphism on non-nucleoside reverse transcriptase inhibitor plasma concentrations in HIV-infected patients. J Antimicrob Chemother. 2008; 61(4): 914–918. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSchipani A, Wyen C, Mahungu T, et al.: Integration of population pharmacokinetics and pharmacogenetics: an aid to optimal nevirapine dose selection in HIV-infected individuals. J Antimicrob Chemother. 2011; 66(6): 1332–1339. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZanger UM, Klein K: Pharmacogenetics of cytochrome P450 2B6 (CYP2B6): advances on polymorphisms, mechanisms, and clinical relevance. Front Genet. 2013; 4: 24. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHonda M, Muroi Y, Tamaki Y, et al.: Functional characterization of CYP2B6 allelic variants in demethylation of antimalarial artemether. Drug Metab Dispos. 2011; 39(10): 1860–5. PubMed Abstract | Publisher Full Text\n\nVardhanabhuti S, Acosta EP, Ribaudo HJ, et al.: Clinical and genetic determinants of plasma nevirapine exposure following an intrapartum dose to prevent mother-to-child HIV transmission. J Infect Dis. 2013; 208(4): 662–71PubMed Abstract | Publisher Full Text | Free Full Text\n\nWang Y, Xing H, Liao L, et al.: The development of drug resistance mutations K103N Y181C and G190A in long term Nevirapine-containing antiviral therapy. AIDS Res Ther. 2014; 11: 36. PubMed Abstract | Publisher Full Text | Free Full Text\n\nde Requena DG, Bonora S, Garazzino S, et al.: Nevirapine plasma exposure affects both durability of viral suppression and selection of nevirapine primary resistance mutations in a clinical setting. Antimicrob Agents Chemother. 2005; 49(9): 3966–3969. PubMed Abstract | Publisher Full Text | Free Full Text\n\nShiau S, Kuhn L, Strehlau R, et al.: “Sex differences in responses to antiretroviral treatment in South African HIV-infected children on ritonavir-boosted lopinavir- and nevirapine-based treatment”. BMC Pediatr. 2014; 14: 39. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSwaminathan S, Ramachandran G, Agibothu Kupparam HK, et al.: Factors influencing plasma nevirapine levels: a study in HIV-infected children on generic antiretroviral treatment in India. J Antimicrob Chemother. 2011; 66(6): 1354–9. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTalam NC, Gatongi P, Rotich J, et al.: Factors affecting antiretroviral drug adherence among HIV/ AIDS adult patients attending HIV/AIDS clinic at Moi Teaching and Referral Hospital, Eldoret, Kenya. East Afr J Public Health. 2008; 5(2): 74–8. PubMed Abstract"
}
|
[
{
"id": "63456",
"date": "26 May 2020",
"name": "Andrea Giacomelli",
"expertise": [
"Reviewer Expertise HIV",
"Infectious Diseases",
"SARS-CoV-2."
],
"suggestion": "Not Approved",
"report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI have assessed the manuscript by Juster and co-workers assessing the potential role of pharmacogenetics and clinical parameters on nevirapine plasma concentrations in people living with HIV. The study design was cross-sectional and the authors aimed to correlate CYP2B6 c.516 G>T rs3745274 and c.983T>C genotypes with nevirapine plasma concentration in people living with HIV under their first line nevirapine-based antiretroviral regimens. Although the findings of an association between CYP2B6 c.516 G>T genotype and nevirapine plasma concentration is in line with previous literature reports, the use of nevirapine is becoming less frequent and consequently the generalizability of the results may be limited. Moreover, there are several methodological points that must be fixed.\nMajor comments:\nThe statement regarding the sample size is not clear. Could the authors better explain how they reached their sample size? In fact, if they had a pre-planned sample size, why did they state in the limitation section of the manuscript that the study size is limited?\n\nHow were patients refereed to perform a genotypic test to detect nevirapine drug resistance?\n\nIt’s not stated how the variables have been handled in the linear regression model. Which are the units entered in the model? In the present format the model is not interpretable.\n\nThe authors entered in the linear regression model several variables that are unlikely to be confounders or factors that may influence nevirapine concentration. For example, the use of drug resistance in the model does not make sense because drug resistance does not impact on nevirapine concentration. Moreover, important variables seem to have been missed such as hepatic viruses’ coinfection or body weight. Overall, the model presented is not reliable and should be rebuilt by entering variables which have been demonstrated to potentially impact on nevirapine concentration according to the available literature.\n\nMinor comments:\nPlease refer to HIV patients by using “people living with HIV”.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
},
{
"id": "73322",
"date": "03 Nov 2020",
"name": "Emmanuel Ndashimye",
"expertise": [
"Reviewer Expertise HIV",
"SARS-CoV-2"
],
"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\nMungiria Juster et al., present a manuscript on effect of CYP2B6 polymorphisms as well as social-demographic factors on NVP concentrations among Kenyans on ART. Dolutegravir in combination with NNRTIs is now preferred first-line regimen in Kenya and other low- and middle-income countries. I therefore find the topic not relevant in view of current HIV treatment programs but in addition to current literature. My comments and queries are written below.\nMajor comments:\nIn the multivariate regression analysis, I find some key confounders applicable to study design and patients missing, for instance, the type of combined ART, and drug dosage.\n\nThe sample size determination for the study is not adequately articulated.\n\nMinor comments and queries:\nThe background should include the latest 2020 UNAIDS data on HIV in Kenya and a summary of current NVP use if any in era of tenofivir/lamivudine/dolutegravir.\n\nHow adherence to ART was reported by study patients?\n\nWere factors which could have affected recruitment of the right patients into the study, for instance, rifampicin exposure, body mass index etc. considered?\n\nIt is important to provide a summary of NVP drug resistance mutations observed.\n\nThe web link to Stanford and IAS databases or references should be included in the test.\n\nThough not very critical, direct sequencing on a subset of samples is advisable to compare with QPCR results.\n\nThe sequences data from HIV resistance assays should be submitted to Genbank.\n\nThe last sentence of conclusions for the abstract should be paraphrased considering the current first-line ART recommendations in Kenya and other countries.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/9-363
|
https://f1000research.com/articles/9-362/v1
|
14 May 20
|
{
"type": "Review",
"title": "COVID-19 in pregnancy: What do we really know?",
"authors": [
"Jerome Bouaziz",
"Marc Even",
"Frederique Isnard-Bogillot",
"Eli Vesale",
"Mariam Nikpayam",
"Andrei Mihalache",
"David Krief",
"Rene Frydman",
"Jean-March Ayoubi",
"Marc Even",
"Frederique Isnard-Bogillot",
"Eli Vesale",
"Mariam Nikpayam",
"Andrei Mihalache",
"David Krief",
"Rene Frydman",
"Jean-March Ayoubi"
],
"abstract": "Coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), continues to escalate worldwide and has become a pressing global health concern. This article comprehensively reviews the current knowledge on the impact of COVID-19 over pregnant women and neonates, as well as current recommendations for their management. We also analyse previous evidences from viral respiratory diseases such as SARS, Middle East respiratory syndrome, and influenza that may help to guide clinical practice during the current pandemic. We collected 23 case reports, case series, and case-control studies (18 from China) comprising 174 pregnant women with COVID-19. The majority of mothers showed a clinical presentation of the disease similar to that of non-infected adults. Preliminary evidences point towards a potentially increased risk of pregnancy adverse outcomes in women with COVID-19, with preterm delivery the most frequently observed (16.7%) followed by fetal distress (9.77%). The most commonly reported adverse neonatal outcomes included respiratory symptoms (7.95%) and low birth weight (6.81%). A few studies reported other maternal comorbidities that can influence these outcomes. Mothers with other comorbidities may be at higher risk of infection. Mother-to-child transmission of SARS-CoV-2 appears unlikely, with no study observing intrauterine transmission, and a few cases of neonatal infection reported a few hours after birth. Although the WHO and other health authorities have published interim recommendations for care and management of pregnant women and infants during COVID-19 pandemic, many questions remain open. Pregnant women should be considered in prevention and control efforts, including the development of drugs and vaccines against SARS-CoV-2. Further research is needed to confirm the exact impact of COVID-19 infection during pregnancy. To fully quantify this impact, we urgently need to integrate the current knowledge about viral characteristics, epidemiology, disease immunopathology, and potential therapeutic strategies with data from the clinical practice.",
"keywords": [
"coronavirus disease",
"covid",
"infection",
"pregnancy",
"neonate",
"outcomes",
"vertical transmission"
],
"content": "Introduction\n\nCoronavirus disease 2019 (COVID-19) has emerged as the most pressing current global health issue. Since the identification of the first cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causing COVID-2019 in Wuhan, China, in December 20191, the virus has spread almost to every country in the world. Local transmission of SARS-CoV-2 is occurring in most countries2. The World Health Organization (WHO) declared COVID-19 a 'public health emergency of international concern' on 30th January 2020, and characterized the situation as pandemic on 11th March 2020. According to WHO situation report from 7th April 2020, there were around 1,280,000 COVID-19 confirmed cases and over 72,600 deaths worldwide, with Europe and North America being the current epicentres of the pandemic2. The numbers continue to grow at staggering speed.\n\nSARS-CoV-2 is the seventh known coronavirus that infects humans, and several studies support its probable zoonotic origin3,4. SARS-CoV-2 belongs to the betacoronaviridae and, similarly to other members of this genus such as severe acute respiratory syndrome coronavirus (SARS-CoV), binds to angiotensin-converting enzyme 2 (ACE2) as the host receptor for entering the cell to cause infection5,6. Although current data suggest that the majority of infections with SARS-CoV-2 cause a mild presentation of COVID-19, older adults and people with comorbidities, including cardiovascular and respiratory diseases and diabetes, are at increased risk of severity and death, with men potentially at higher risk than women7,8.\n\nPregnant women are particularly vulnerable to infectious diseases that can cause both maternal and fetal adverse outcomes, compared to their non-pregnant counterparts. Infections caused by influenza virus9,10, hepatitis E virus11, herpes simplex virus12, cytomegalovirus12, human papilloma virus13, and malaria parasites14, among others, more severely affect women during gestation. During pregnancy, women experience a series of immunological transformations that allow the immune system to tollerate and support the growing fetus while still maintaining antimicrobial defence and tissue repair15. These transformations, together with mechanical or anatomical changes, may explain the increased severity or susceptibility to some infectious diseases observed in pregnant women16.\n\nIn particular, the physiological and immunological changes associated with gestation make pregnant women at increased risk for serious viral respiratory illness17. In view of the urgent threat that COVID-19 pandemic is posing worldwide, obstetricians and future mothers are wondering whether SARS-CoV-2 infection could be associated with adverse fetal and maternal outcomes and/or be transmitted to the fetus. Although current evidences are scarce, several case reports, case series, and case-control studies have already been published reporting clinical manifestations and evaluating the possibility of vertical transmission, as well as maternal and fetal outcomes in pregnant women with COVID-19.\n\nThis review collects up-to-date information from the existing literature about the presentation and outcomes of COVID-19 in pregnant women; and discusses current recommendations for their care and management. Moreover, since current knowledge about COVID-19 in pregnancy is scarce, we summarize previous evidences from the impact of other viral respiratory infections, including SARS-CoV, Middle East respiratory syndrome coronavirus (MERS-CoV), and influenza virus on maternal and fetal outcomes to get insights that may help to better understand COVID-19 and guide the clinical practice. We also discuss challenges that pregnant women may face in the context of the pandemic, including the access to safe therapeutic strategies and mental health issues, and the particularly difficult situation of those in low-resource settings.\n\nThe scientific community is intensively working to elucidate the impact of this global health concern over pregnant mothers and their newborns, but still many questions remain unanswered. Since new data are being collected everyday, it is essential to provide healthcare workers with the most updated information about COVID-19 in pregnant women. This information will help to answer the numerous questions that future mothers may have in the current situation and to prevent obstetrical complications.\n\n\nMethods\n\nAfter defining the research questions and objectives, we conducted a literature review using PubMed and Google Scholar. A first search was conducted on 27th March 2020, and a second search was conducted on 8th April 2020 to include the most recently published articles. We searched for articles including the terms 'COVID-19', 'SARS-CoV-2' or 'ncov-2019', and 'pregnancy', 'pregnant', 'gestation', 'newborn', 'neonatal', 'neonate' or 'perinatal' without limits. On 8th April 2020, this search produced 104 results. All abstracts were screened and relevant articles were further inspected. The bibliographic reference lists of the included articles were screened for additional publications. In total, 23 studies comprising case reports, case series, and case-control studies (reporting a total of 174 pregnant women with COVID-19) were included. Relevant information was extracted from the full-text articles, with the exception of one case series and one case-control study that were originally published in Mandarin Chinese (relevant data were extracted from the published English version of the abstract). Publications in Chinese without an English version of the abstract available were discarded. We also reviewed letters, perspectives, and reviews to obtain additional information. We searched for information about COVID-19, SARS-CoV-2, and other viral diseases in pregnancy in PubMed database and WHO webpage.\n\n\nWhat do we know about COVID-19 in pregnancy?\n\nThis section reviews the reported cases of pregnant women with COVID-19 since the beginning of the pandemic until 8th April 2020 (summarized in Table 1 and Table 2).\n\nAbbreviations: LBW, low birth weight; PROM, premature rupture of membranes; ARDS, acute respiratory distress syndrome; SGA, small for gestational age; MODS, multiple organ dysfunction syndrome; ARDS, acute respiratory distress syndrome; PCIS, Pediatric Critical Illness Score; CK-MB, creatine kinase myocardial band; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; IgG, immunoglobulin G; IgM, immunoglobulin M.\n\na Not directly related with COVID-19.\n\nAbbreviation: LBW, low birth weight.\n\na Significantly higher in comparison with control pregnant women.\n\nA previous review including 108 infected pregnant women in the third trimester described fever (68%) and coughing (34%) as the most frequent symptoms of COVID-19, and elevated C-reactive protein (70%) and lymphopenia (59%) as the most commonly altered maternal parameters41.\n\nSeveral authors suggested that pregnancy did not aggravate the symptoms or computed tomography (CT) features of COVID-19 pneumonia18,42. The most common early finding on chest CT was ground-glass opacity, which progressed to 'crazy paving' pattern and consolidations18. The most frequent symptoms at onset of COVID-19 were fever (13/15 patients) and cough (9/15), while the most common abnormal laboratory finding was lymphopenia (12/15). Pneumonia was not aggravated after delivery in these women. The results of a point-of-care lung-ultrasound examination were found to correlate with CT findings in one pregnant women; and therefore, this technique has been proposed as a tool to facilitate the triage of mothers with suspected COVID-1935,43.\n\nLiu H. et al. evaluated clinical and CT imaging on 41 COVID-19 infected pregnant women (16 laboratory-confirmed and 25 clinically-diagnosed), in comparison with 14 infected non-pregnant adults44. Both laboratory-confirmed and clinically-diagnosed COVID-19 pregnant groups more frequently presented leukocytosis, elevated neutrophil ratio, and lymphopenia than non-pregnant adults. Mixed and complete consolidation were more commonly observed in the pregnant groups than in non-pregnant adults. Ground-glass opacity with reticulation was less common in the two pregnant groups than in non-pregnant adults. Moreover, the clinically-diagnosed cases showed more pulmonary involvement than the laboratory-confirmed cases. In view of these results, researchers warned that the atypical clinical findings observed in pregnant women with COVID-19 could complicate the initial identification of the disease44.\n\nIn a case series including three pregnant women infected by SARS-CoV-2, all placental histopathological findings included various degrees of fibrin deposition within and around the villi with increased local syncytial nodules, but without villitis or chorioamnionitis19. One placenta also showed chorionic hemangioma, and another presented massive placental infarction. However, Chen S. et al. found no placental complications, including placenta infarction or chorionic amniotic inflammation, in five infected pregnant women from Hubei province, in China20. Further histolopathological studies of placenta samples are urgently needed.\n\nA vast majority of available publications refers to Chinese pregnant women. In a case series including three pregnant women infected by SARS-CoV-2, one of the infants was born preterm with low birth weight (LBW)19. Other publication presented the case of a 30-week pregnant woman with COVID-19 that gave birth to a LBW but healthy baby21.\n\nLiu et al. presented 13 pregnant women infected with SARS-CoV-2, two with less than 28 weeks of gestation and 11 in the third trimester22. Three patients improved after hospitalization, were discharged from the hospital, and had an uncomplicated pregnancy. The other ten patients underwent caesarean section, of which five were emergency sections because of fetal distress (n=3), premature rupture of membranes (PROM) (n=1), and stillbirth (n=1). Six patients (46%) had preterm labour from 32 to 36 weeks of gestation. The condition of one of the mothers deteriorated during hospitalization, requiring ICU admission with intubation and mechanical ventilation. This woman presented multiple organ dysfunction syndrome (MODS) that comprised of acute respiratory distress syndrome (ARDS), acute hepatic failure, acute renal failure, and septic shock, and was under extracorporeal membrane oxygenation at the time of the study22.\n\nChen H. et al. reported two cases of fetal distress, four cases of preterm delivery (one mother with preeclampsia, one case of PROM and two with previous history of caesarean sections or stillbirth), and two of LBW (the mother with preeclampsia and the case of PROM) among nine pregnant women infected with COVID-1923. Chen R. et al. included 17 infected pregnant women, three of the neonates were prematurely born24. One case of newborn death due to endouterine asphyxia was reported in a critically ill pregnant woman with SARS-CoV-2 (who developed severe acute respiratory distress syndrome, multiple organ dysfunction syndrome, and septic shock)25. The mother successfully recovered after treatment with convalescent plasma.\n\nZhu H. et al. found several adverse pregnancy outcomes among nine infected women (clinical symptoms beginning before delivery in four cases, on the day of delivery in two mothers, and after delivery in other three)26. Of ten newborns (eight singletons and two twins), six were born premature and two were small for gestational age (SGA) infants. Six of the newborns had a Pediatric Critical Illness Score (PCIS) lower than 90, and showed clinical symptoms including shortness of breath (n=6), fever (n=2), thrombopenia accompanied by abnormal liver function (n=2), rapid heart rate (n=1), and vomiting (n=1). Four neonates presented gastrointestinal symptoms including bloating, refusing milk, feeding intolerance, and gastric bleeding, and seven showed radiography abnormalities (infections, n=4; neonatal respiratory distress syndrome, n=2; and pneumothorax, n=1). One infant presented multiple organ failure, refractory shock, and disseminated intravascular coagulation at day 8 after delivery, received a transfusion, and died on day 926.\n\nFan C. et al. reported two cases of COVID-19 during the third trimester of pregnancy showing good outcomes in general27. One infant developed low grade fever and abdominal distension with lymphopenia (16.9%) on day 3, and diffuse haziness in both lung fields without patchy consolidation was observed in a chest radiograph on day 4. The fever and lung infection responded to antibiotics, and was discharged on day 9. The second newborn developed mild neonatal pneumonia and lymphopenia (10.5%), and was treated with antibiotics with good response in two days27. Chen S. et al. and Yu N. et al. observed good maternal, fetal, and neonatal outcomes in 12 infected pregnant women20,28.\n\nOnly a few cases of COVID-19 in pregnancy have been reported outside from China. An infected pregnant women in Spain delivered through an urgent caesarean section due to severe preeclampsia34. The newborn presented LBW, required resuscitation and was transferred to the neonatal unit due to immediate respiratory distress. The neonate received respiratory support for 2 h post birth, showing normal findings on the physical examination at 9 h post birth. The neonate tested negative for SARS-CoV-2 at day 6 post birth, but positive at day 8, showing respiratory symptoms until day 13, while the mother required ICU care with mechanical ventilation34. Kalafat et al. reported the case of a pregnant women in Turkey with lung ultrasound findings consistent with COVID-19, but an initial negative real time PCR (RT-PCR) result that subsequently became positive; the woman delivered a healthy newborn35. One Korean mother with COVID-19 delivered a healthy infant through emergency caesarean section due to obstructed labour36. In Sweden, one SARS-CoV-2-positive woman with severe preeclampsia and gestational diabetes delivered twins, one was LBW and the other showed respiratory problems and vomiting after birth37. In Honduras, a woman with gestational hypertension and hypothyroidism prematurely delivered a LBW infant38. Although general media have reported the birth of healthy non-infected infants from SARS-CoV-2 infected mothers in Italy, there is no published scientific literature on the topic yet.\n\nUntil now, two case-control studies have been conducted in China (Table 2). Zhang L. et al. compared the pregnancy outcomes of 16 women with COVID-19 (one severe case) and 45 non-infected women, all delivering by caesarean section39. Researchers did not observe significant differences in the intraoperative blood loss, meconium-stained amniotic fluid, fetal distress, preterm birth, neonatal asphyxia, or birth weight between infected and non-infected mothers. A second case-control study by Li N. et al. compared 16 pregnant women with confirmed COVID-19 pneumonia and 18 suspected cases, all with mild symptoms who were admitted to labour in the third trimester, with two cohorts of 121 age-matched control pregnant women each (one from 2019 and one from 2020)40. Around 70% of the two cases groups presented other maternal complications (mostly gestational diabetes mellitus, gestational hypertension, and hypothyroidism), a percentage significantly higher than in the control groups (33%). Three confirmed cases (18.8%, two caused by PROM and one by placental bleeding) and three suspected cases (16.7%, one due to gestational hypertension/preeclampsia and one due to placenta previa) delivered prematurely, compared with a 5.8% of the controls. LBW also occurred more often in infants of the two cases groups (17.6% for confirmed and 10.5% for suspected cases) than in those of controls (2.5%). Two newborns from COVID-19 confirmed mothers and one from a suspected case had intrauterine fetal distress, but this percentage was not significantly different from the control groups40.\n\nIn the great majority of COVID-19 cases reported, mothers delivered by caesarean section (Table 1 and Table 2). Of them, several emergency caesarean sections were performed because the condition of the mother deteriorated, or due to fetal risk (including fetal distress or PROM). Only 11 out of 174 mothers had a vaginal delivery (6.32%).\n\nChen R. et al. evaluated 17 infected patients undergoing caesarean delivery, and found that a significant majority of women with COVID-19 receiving continuous epidural anesthesia presented significant intraoperative hypotension24. The team reported an increased use of carbetocin or carboprost tromethamine for the treatment of uterine contraction fatigue in caesarean sections in pregnant women with COVID-19, compared with a non-COVID-19 group39.\n\nNone of the studies reported vertical transmission of SARS-CoV-2 when fetal samples, amniotic fluid, cord blood, or breast milk where tested by RT-PCR18–23,26,27,29,30,34,35,36,39. A neonate born to a pregnant woman with SARS-CoV-2 pneumonia tested positive for the infection in pharyngeal swabs 36 h after birth, but maternal breast milk was negative31. Yu N. et al. found that one out of three neonates tested in throat swabs was positive for SARS-CoV-2 infection 36 h after birth28. Zeng L. et al. reported three neonates born from SARS-CoV-2-positive mothers (out of 33 neonates from infected women investigated) that tested positive for viral infection in nasopharyngeal and anal swabs on days 2 and 4 after birth32. In this study, the babies were all born by caesarean section under strict infection control conditions and were immediately separated from their mothers. The outcomes were favourable, and infected neonates only showed a mild presentation with shortness of breath and non-specific radiographic findings (with the exception of a seriously ill probably due to prematurity, asphyxia, and sepsis, rather than SARS-CoV-2 infection). One case report presented a 2 h newborn with elevated immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies to SARS-CoV-2 born to a mother with COVID-1933. Infant’s nasopharyngeal swabs, maternal vaginal secretions at delivery, and breast milk were negative for SARS-CoV-2. Laboratory analysis suggested inflammation and liver injury in the newborn. Since IgM antibodies cannot cross the placenta, further research is needed to confirm this observation, and whether intrauterine or perinatal SARS-CoV-2 infection occurred in these studies.\n\nZheng Q.L. et al. found that ACE2 expression is minimal in almost all cell types of the early maternal-fetal interface in humans, with the exception of decidual perivascular cells cluster45. This observation may suggest that the susceptibility of cells in the maternal-fetal interface to COVID-19 could be low, limiting the probabilities of transplacental vertical transmission of SARS-CoV-2. On the contrary, Levy et al. found that ACE2 expression and activity are increased during pregnancy in rat models, in particular in the placenta but also in the uterus46. Reduced expression of ACE2 has been linked to fetal growth restriction, both in humans and rats47,48. Further experimental studies are needed to discern the link between ACE2 expression, SARS-CoV-2 susceptibility, and pregnancy outcomes.\n\nA WHO joint mission recently investigated 147 pregnant women (64 confirmed, 82 suspected and 1 asymptomatic; 8% with severe disease and 1% critical) and suggested that pregnancy does not appear as a risk factor for severe COVID-19 disease, as opposed to pandemic influenza A (H1N1)pdm0949. The report concluded that having COVID-19 during pregnancy may have an impact on fetal outcomes.\n\nA previous review about COVID-19 in pregnancy included published studies until the 25th February 2020, and was used to inform the UK Royal College of Obstetricians and Gynaecologists guidance50. This review found that preterm delivery affected 47% of women (n = 32) hospitalized with COVID-19. There were no maternal deaths and serious morbidity requiring intensive care unit (ICU) admission occurred in two out of 32 (6.25%) reviewed cases. A second review including 37 mothers reported 6 cases (16.2%) of preterm labour, 6 (16.2%) of PROM, 2 (5.40%) with abnormal amniotic fluid, and 2 (5.40%) with abnormal umbilical cord51. A review published on 17th March 2020 reported preterm birth in 39% of cases, intrauterine growth restriction in 10%, and miscarriage in 2%52. A review published on 7th April including 108 women noted three cases of maternal ICU admissions, one neonatal death and one intrauterine death, with 91% of deliveries by caesarean section41.\n\nSince the publication of these reviews, several new cases of infected pregnant women have been reported, a great majority of them including Chinese pregnant women and only a few case reports from other settings. Among the 174 cases included in this review, a majority of the mothers did not show severe complications related with COVID-19 infection, in particular from mid-trimester onwards. The predominant presentation of COVID-19 in pregnancy included fever, cough, dyspnea, and lymphopenia, as previously reported52. Data on first-trimester COVID-19 infection and subsequent follow-up are lacking. It is unknown whether pregnant women have an increased susceptibility to SARS-CoV-2 infection, and there is no evidence from SARS or MERS about an increased susceptibility during pregnancy. However, several publications reported adverse pregnancy and neonatal outcomes in pregnant women infected by SARS-CoV-2.\n\nRegarding pregnancy outcomes in the 174 women with COVID-19, there were 29 cases of preterm delivery (16.7%), 17 cases of fetal distress (9.77%), three cases of PROM (1.72%), two cases of SGA (1.15%), one stillbirth (0.57%), and one fetal death (0.57%). Regarding neonatal outcomes (n=176 since there were two births of twins), there were 14 newborns with respiratory problems (including shortness of breath and pneumonia, 7.95%), 12 with LBW (6.81%), five with fever (2.84%), three with gastrointestinal problems (1.70%), three with lymphopenia (1.70%), three with thrombopenia (1.70%), two with tachycardia (1.14%), and one infant death (0.57%) (Table 1 and Table 2). These percentages should be interpreted cautiously, since at this point we cannot exclude that some women have been included in more than one publication. Similarly, we cannot exclude that other maternal conditions may potentially confound the results, or even interact with COVID-19. A few cases of preeclampsia and gestational diabetes, among others, were reported in the pregnant women included in this calculations. Interestingly, a case-control study in China found that other maternal complications, such as gestational diabetes mellitus, gestational hypertension, and hypothyroidism, were more frequent in women with COVID-19 than in control women. Further research is needed to understand whether pregnant women with other comorbidities are more susceptible to SARS-CoV-2 infection.\n\nUntil more evidences are collected in larger studies and in different settings, the current observations suggest that COVID-19 may be potentially associated with adverse pregnancy outcomes, including preterm delivery and fetal distress, and adverse neonatal outcomes including respiratory problems and LBW. Therefore, special attention should be paid to infected women along the gestational period and at the time or delivery, or to those pregnant women that may be eventually exposed to SARS-CoV-2 virus.\n\n\nCan we learn something from other viral respiratory infections in pregnancy?\n\nAcute lower respiratory tract illness caused by viral infection, mostly human rhinovirus, respiratory syncytial virus, and influenza virus, is common during pregnancy and associated with significant morbidity17. In particular, infection is a relevant cause of morbidity and mortality among future mothers53. Pneumonia during pregnancy is related with a higher risk of maternal death and pregnancy-related complications; including preterm delivery, caesarean section, preeclampsia/eclampsia, and LBW and SGA newborns53. The consequences of influenza on pregnant women and fetuses have been widely studied, but less is known about the impact of other viral acute respiratory illnesses in this population. In this section we summarize previous studies about infections with SARS-CoV, MERS-CoV, other coronaviruses, and influenza virus during pregnancy (Table 3).\n\nSymbols indicate: frequently reported (++), reported in some cases (+), not reported (-), presumable in view of the currently limited evidences (?).\n\nAbbreviations: SARS, severe acute respiratory syndrome; CoV, coronavirus; MERS, Middle East respiratory syndrome.\n\na Limited evidence available.\n\nb A majority of studies analysed pandemic influenza, not seasonal influenza. Maternal severe infections, but not mild, seem to have a stronger impact on pregnancy.\n\nSARS-CoV is a zoonotic virus from the betacoronaviridae genus first identified in 200354. SARS-CoV and SARS-CoV-2 are closely related (around 79% of genome sequence identity) and originated in bats, which have been proposed as a reservoir host for the two viruses3. Between 2002 and 2003, SARS-CoV caused an epidemics that affected more than 8000 people and resulted in around 1000 deaths in 29 countries, mostly in China and Hong Kong, as reported by the WHO. The illness caused by SARS-CoV frequently initiated with fever and mild respiratory symptoms, but progressed to pneumonia within a few days, and eventually led to respiratory distress requiring intensive care. The WHO estimated an overall case fatality rate of 14–15%55.\n\nSeveral case reports and small clinical studies documented relatively few cases of SARS occurring during pregnancy, with estimations forecasting around 100 pregnant women infected during the 2003 epidemics55. Although vertical transmission was not observed, SARS-CoV infection was associated with severe maternal illness or even maternal death, spontaneous abortion, severe intrauterine growth retardation, preterm delivery, and life-threatening gastrointestinal complications in the newborn18,56,57. A case-control study investigating the effects of SARS on pregnancy found a higher mortality rate (30% maternal mortality vs. no deaths in non-pregnant), more frequent renal failure and disseminated intravascular coagulopathy, more frequent admission to the ICU, and higher intubation rate in pregnant SARS patients compared with a non-pregnant infected cohort58.\n\nNg W.F. et al. evaluated the placental pathology of SARS-CoV infection in seven women59. The placentas from women with SARS infection detected along the first trimester of pregnancy were normal. On the contrary, the placentas from women convalescent from SARS during the third trimester showed increased intervillous or subchorionic fibrin, probably associated with disturbances in placental blood flow due to the hypoxic respiratory disease. The delivery outcomes of women convalescent during the third trimester included intrauterine growth retardation and SGA newborns. The placentas from women with acute SARS infection were also highly abnormal, showing extensive fetal thrombotic vasculopathy, probably caused by placental hypoxia, with sharply demarcated zones of avascular fibrotic villi at delivery59. Clinical guidelines have been published for the management of obstetric patients and neonates born to mothers with suspected or probable SARS60. The WHO declared SARS epidemics as contained in July 2003, and the latest outbreak in China was contained in May 2004.\n\nMERS-CoV. MERS-CoV is a betacoronavirus causing respiratory disease, which was first identified in Saudi Arabia in 201261. From 2012 until the end of January 2020, the total number of laboratory-confirmed MERS-CoV cases reported globally to the WHO were 2519, with 866 associated deaths (around 35% estimated mortality). Since 2012, 27 countries have reported cases of MERS, although 80% of them occurred in Saudi Arabia, according to the WHO. Humans can be infected with MERS-CoV from direct or indirect contact with dromedaries, although the virus can also be transmitted between humans in close contact. The clinical presentation of MERS-CoV infection widely ranges from absence of symptoms or mild respiratory symptoms to severe acute respiratory disease, and even death. Pneumonia is frequent, and gastrointestinal symptoms have also been described. Severe illness can lead to respiratory failure that requires ICU support with mechanical ventilation. The severity of the disease increases with age, in immunosuppressed people, and in those with chronic conditions such as renal or respiratory diseases, cancer, and diabetes.\n\nInformation about the pathophysiology of MERS-CoV infection during pregnancy is scarce, but pregnant women are thought to develop severe disease and adverse maternal and fetal outcomes. A retrospective study conducted in Saudi Arabia between 2012 and 2016 included five pregnant women infected with MERS-CoV during the second or third trimester of pregnancy, all requiring ICU care and showing adverse pregnancy outcomes62. Among them, two women died, and two pregnancies resulted in perinatal death. A case report presented a stillbirth, while a second one reported the delivery of a healthy infant, with both women requiring ICU care63,64. One mother died in Abu-Dhabi after delivering a healthy baby by caesarean section65. One asymptomatic woman delivered a healthy infant66. In South Korea, a MERS-CoV-infected mother had abrupt vaginal bleeding with membranes rupture, and delivered a healthy baby by caesarean section67. About 10% placental abruption was found in this patient. Considering all these cases, the estimated maternal case fatality rate in pregnant women is not significantly different than the overall case fatality rate by MERS-CoV infection, while the disease has resulted in 27% of fetal/infant deaths66.\n\nIn view of the available information, maternal outcomes in COVID-19 appear less concerning compared to those of SARS and MERS. Pooled data found a case fatality rate of 0% for COVID-19, 18% for SARS, and 25% for MERS, being severe sepsis and respiratory failure the most frequent causes of maternal death in the last two infections52.\n\nOther coronaviruses. At least four other types of coronaviruses (alphacoronaviruses HCoV-229E and HCoV-NL63 and betacoronaviruses HCoV-OC43 and HCoV-HKU1) are known to infect humans causing mild, self-limiting respiratory infections. These viruses cause around 15–30% of respiratory tract infections each year, with more severe presentation in neonates, the elderly, or individuals with underlying conditions68. A prospective pilot study evaluated the potential maternal-fetal transmission of these coronaviruses69. The results suggested the possibility of vertical transmission of human coronaviruses, in particular HCoV-229E, which was detected in three out of four newborn gastric samples whose mothers tested positive in vaginal samples collected at delivery. Two of these mothers were also positive in respiratory samples, and none of the infants showed clinical symptoms. Further studies are needed to understand the potential intrauterine transmission, as well as the effect over pregnancy outcomes, of these coronavirus infections.\n\nA detailed discussion of the epidemiological and clinical impact of SARS, MERS, and other coronavirus infections on pregnant women and newborns can be found in a recently published perspective article70.\n\nInfluenza virus. Influenza viruses are enveloped viruses with segmented negative-sense single-strand RNA genome, members of the family Orthomyxoviridae. This family comprises several genera, including influenza A viruses infecting humans and several animals with pandemic potential; influenza B viruses that circulate among humans and cause seasonal epidemics; influenza C viruses that can infect humans with mild presentation, as well as pigs; and influenza D viruses that primarily affect cattle (see WHO influenza page). Influenza A virus infections can cause human diseases ranging from mild upper respiratory tract infection with fever and cough to severe pneumonia, sepsis with shock, acute respiratory distress syndrome, and even death. Seasonal influenza caused by influenza A or B virus subtypes is characterized by a fast onset of fever, cough, headache, severe malaise, muscle and joint pain, sore throat, and rhinorrhea, which can cause severe illness or death especially in high-risk populations (children under five, the elderly, or individuals with chronic diseases or immunosuppressive conditions).\n\nInfluenza infection in pregnant women has been associated with increased severity, adverse maternal and fetal outcomes, and a higher risk of death. Moreover, women also have an increased risk of death from pandemic influenza during the four weeks post-partum71. Substantial evidences were obtained from the influenza pandemic of 1918 to 1919, 1957 to 1958, and 2009 to 2010, and many studies have been published on this topic. For example, the mortality rate of the 1918 influenza pandemic in Iceland reached 37% among pregnant women, while it was around 2.6% in the general population72. In 2009, pregnant women were 1% of patients in the US infected with influenza A H1N1 virus, accounting for 5% of deaths73. The increased severity of influenza during pregnancy and postpartum is thought to be related to the normal physiologic changes occurring during pregnancy, including the hormone-regulated shift in immune responses, decreased lung capacity, and augmented heart rate and oxygen consumption74,75. The WHO recommends the inactivated influenza vaccine for all pregnant women independently of the trimester of pregnancy76.\n\nA few systematic reviews and meta-analyses have been conducted to summarize the results of the abundant literature about influenza infection in pregnancy. A meta-analysis published in 2017 found that influenza during pregnancy resulted in a 2.5-fold higher risk of hospital admission compared with non-pregnant adults, but the risk of mortality was similar in both groups10. Similar results were found in a meta-analysis published in 2019 including 36,498 women, although the risk of hospital admission was almost seven-fold higher in pregnant women than in non-pregnant individuals, and they observed an association between pregnancy and a reduced risk of ICU admission77.\n\nSeveral clinical studies reported complications related to pandemic influenza infection in fetuses and neonates, particularly those born from mothers with severe infection, including miscarriage, preterm birth, stillbirth, neonatal death, and LBW78–80. A systematic review of comparative studies from 2017 confirmed the increased risk of preterm birth following severe pandemic H1N1 influenza illness, but not in mild-to-moderate pandemic H1N1 or seasonal influenza infections81. This study found that evidences for the increased risk of SGA birth and fetal death were limited. Littauer E.Q. et al. suggested that H1N1 virus infection during pregnancy results in dysregulated inflammatory responses leading to preterm labour, impairment of fetal growth, and increased fetal mortality and maternal morbidity in animal models82.\n\nA meta-analysis including 22 studies showed that first trimester maternal influenza exposure is associated with an augmented risk of several non-chromosomal congenital anomalies, including neural tube defects, hydrocephaly, congenital heart defects, among others, although exposure to influenza-related medication was not assessed83. Previous work suggested a link between influenza infection during pregnancy and psychotic disorders in the offspring, particularly in mothers with psychiatric disorders84,85, but the evidences are currently insufficient to draw a conclusion86. Transplacental transmission of influenza virus has been rarely reported in the literature, but evidence exists that suggests that the virus can cross the placenta87.\n\nA WHO technical consultation identified several quality issues (potential confound biases, methodological limitations, inconsistent definitions, etc.) and evidence gaps in the current knowledge about the risks of severe influenza outcomes among pregnant women, the risks to the fetus, and the effects of maternal immunization88. In particular, evidence is lacking for seasonal influenza, for the impact of other contextual factors such as malnutrition or co-infections, as well as evidence from low-resource settings and long-term safety data regarding vaccination in pregnant women88. As this report suggests, even in the case of influenza infection in pregnant women where numerous studies have been conducted, many questions remain unanswered.\n\nCare and management of pregnant women and newborns during COVID-19 pandemic\n\nInterim recommendations\n\nIn view of the scarce knowledge about COVID-19 in pregnancy and the similar pathogenic potential as SARS-CoV and MERS-CoV, several authors have highlighted in letters and perspectives the need for a systematic screening and careful follow-up of any suspected SARS-CoV-2 infection during pregnancy89,90. Several organizations have issued interim guidance specific to pregnant women, and several individual expert opinions have been published giving recommendations based on their institutional experiences.\n\nThe WHO published on 13th March 2020 an interim guidance for the clinical management of severe acute respiratory infection when COVID-19 is suspected, which includes recommendations for pregnant women91. In this document, the WHO specifically states that pregnant women with suspected or confirmed COVID-19 should be treated with supportive and management therapies, considering the immunologic and physiologic adaptations during and after the gestational period91. It also emphasizes the need to prioritize COVID-19 testing of symptomatic pregnant women to enable access to specialized care. WHO recommends caesarean section to be only undertaken when medically justified, and highlights that the mode of delivery should be decided based on obstetric indications and the woman’s preferences. Infants born to mothers with suspected or confirmed COVID-19 infection should be fed according to standard infant feeding guidelines, and thus breastfeeding is encouraged following strict hygienic practices. Moreover, this interim guidance mentions that mothers and infants should remain together and practice skin-to-skin contact, in particular immediately after birth during establishment of breastfeeding, also when they or their infants have suspected or confirmed SARS-CoV-2 infection. If the mother is severely or critically ill, separation could be needed, with attempts to express breast milk to maintain milk production91. Similarly, the Centers for Disease Control and Prevention (CDC) from the US have prepared some 'interim considerations for infection prevention and control of COVID-19 in inpatient obstetric healthcare settings'. The International Society of Ultrasound in Obstetrics & Gynecology (ISUOG), the European Centre for Disease Prevention and Control (ECDC), and other national health authorities have also published interim recommendations for management of infection during pregnancy and puerperium92–94. In Italy, one of the countries most affected by the pandemic, the health authorities state that caesarean section does not seem appropriate unless there are specific clinical indications. Moreover, they recommend health professionals and mothers to carefully consider together any temporary separation of the infant from the mother during hospitalization, evaluating the risks and benefits of this choice, and advises breastfeeding under strict measures of infection control95. The International Federation of Gynecology and Obstetrics (FIGO) published on 4th April the most up-to-date interim guidance on COVID-19 during pregnancy and puerperium96.\n\nIn the studies including Chinese pregnant women with COVID-19, the majority of patients delivered through caesarean section, the neonates were isolated immediately after birth for at least 14 days, and breastfeeding was not recommended. The National Health Commission of China, after expert consensus, issued recommendations on 8th February 2020 emphasizing that neonates of pregnant women with suspected or confirmed COVID-19 infection should be isolated in a designated unit for at least 14 days after birth, and not be directly breastfed97,98. These recommendations generated some controversy99,100 and have been updated in some Chinese settings to promote breastfeeding and neonatal isolation only of newborns with confirmed SARS-CoV-2 infection101.\n\nSpecial attention should be paid to pregnant healthcare workers, and some countries have already recommended to those after 28 weeks of pregnancy to avoid direct contact with SARS-CoV-2-infected patients102.\n\nThe existence of disseminated intravascular coagulation (DIC) is common in deaths of COVID-19 patients, and abnormal coagulation parameters have been associated with poor prognosis103. During pregnancy, women are at increased risk for hemorrhagic events and obstetrical syndromes that may develop into DIC, usually associated with high mortality and morbidity rates104. Additionally, women with SARS-CoV infection showed higher risk of DIC58. Although alterations in coagulation processes have not been reported in pregnant women infected with SARS-CoV-2, obstetric teams should evaluate this risk and consequently adapt the anticoagulation therapeutic plan during and after the hospitalisation of pregnant women.\n\nDiagnosis of SARS-CoV-2 in pregnant women. A CT scan of the chest is a primary tool to confirm or exclude SARS-CoV-2 pneumonia, complementing RT-PCR diagnosis105. The majority of reported pregnant women with COVID-19 infection showed radiological signs of viral pneumonia23. Chest CT scan has been recommended in suspected cases of COVID-19 in pregnant women, since the risk of radiation exposure to the fetus is small92. However, shared decision-making should be encouraged. In any case, informed consent is needed, and a radiation shield should be applied over the uterus92.\n\nRecommendations for the obstetrics units. In the present emergency situation, the obstetrics units need to be reorganized without delay, and several recommendations have been made in this sense106. These recommendations include the preparation of a closed circuit for all COVID-19 positive patients separated from non-infected patients, whenever possible. The ISUOG has prepared a position statement to guide the safe performance of obstetric and gynecological scans and equipment cleaning in the context of COVID-19 pandemic107. The document refers to ultrasound in particular, which places the patient and the caregiver at high risk because of the impossibility of maintaining the safety distance. Ultrasound is an essential tool of obstetric and gynecological care, and therefore what constitutes an elective or an emergency examination has to be decided locally107. The expert-opinion-based guidance from the ISUOG focuses on two aspects: first, the preparation and cleaning of the room and the ultrasound equipment; and second, the protection of the patient and ultrasound operators (physicians, sonographers and other health professionals).\n\nDrugs and vaccines against COVID-19: what happens with pregnant women? Currently, there are no specific treatments for COVID-19. More than 80 clinical trials are ongoing, some of them on repurposed drugs and, therefore, in more advanced clinical stages. WHO has recently launched the SOLIDARITY trial (ISRCTN83971151) of treatments for COVID-19 infection in hospitalized patients. The study treatments under evaluation are the experimental antiviral remdesivir, the antimalarials chloroquine or hydroxychloroquine, the combination of human immunodeficiency virus (HIV) drugs lopinavir/ritonavir, and interferon-beta. These drugs were selected based on the likelihood of being effective, previous safety data, and expected wide availability. Simultaneously, the DISCOVERY trial (NCT04315948) of treatments for COVID-19 in hospitalized adults will be conducted in several European countries to evaluate the safety and efficacy of the same drugs, with the exception of chloroquine. Similarly to all decisions regarding treatment during pregnancy, careful assessment of the risk-benefit ratio both for the mother and the fetus is indispensable. It seems reasonable not to exclude seriously ill pregnant women from trials of these experimental drugs for COVID-1950.\n\nRemdesivir and lopinavir/ritonavir appear to be safe in pregnancy52. Chloroquine phosphate has shown apparent efficacy and an acceptable safety profile against COVID-19-associated pneumonia in multicentre clinical trials conducted in China108. However, special attention should be put to the use of the antimalarial chloroquine in pregnant women. Previous pharmacokinetic studies of chloroquine and its active metabolite observed differences in the elimination half-lives and in the volumes of distribution and clearance when compared with age-matched non-pregnant women109. This translates in a reduced plasma concentration of chloroquine in pregnant women that could compromise the antiviral effect, while higher doses may increase the risk of adverse hemodynamic effects. Hydroxychloroquine (with a maximum tolerable dose of 1200 mg), also used in women with rheumatic diseases, could be a therapeutic alternative to chloroquine (with a maximum tolerable dose of 500 mg) in pregnant women because of its safety profile, with the potential to be administered at a higher dosage110.\n\nIf the deleterious effect of COVID-19 in pregnancy is confirmed, maternal immunization must constitute a priority and pregnant women should be considered in any prevention effort. As some researchers have highlighted, pregnant women should be included in the design, evaluation, and implementation of vaccine candidates for SARS-CoV-2111.\n\nPhysical and mental health of pregnant women during COVID-19 pandemic. The mental challenges that the pandemic may pose in pregnant women should be carefully considered, and health authorities should implement programs to provide counselling and psychological support to future mothers. Stress and anxiety during pregnancy seem to be related with side effects such as depression, preeclampsia, preterm labour, LBW, and others112,113. Ng J. et al. revealed the pivotal role of midwives in providing care, health education, and counselling to pregnant women during and after the crisis posed by the 2003 SARS epidemics in Hong Kong114.\n\nIn addition to an adequate management of stress and anxiety, a lifestyle including appropriate nutrition, moderate physical activity, and plenty of sleep could help pregnant women to stay healthy during COVID-19 pandemic. Emphasis should be put on the practice of regular exercise at home or away from other people if no contraindications exist. Moderate physical exercise during pregnancy has previously demonstrated benefits both for the mother and the fetus, including the reduction of back pain, the promotion of healthy weight gain, and the reduction of the risk of gestational diabetes, preeclampsia, and caesarean delivery (for example, recommendations from The American College of Obstetricians and Gynecologists)115.\n\nPregnant women in low-resource settings. Maternal mortality remains unacceptably high (around 295000 deaths in 2017), with 94% of deaths occurring in low- and middle-income countries116. Infections remain one of the main causes of maternal death and adverse pregnancy outcomes in these settings. With SARS-CoV-2 present in every country of the world and the number of cases escalating, it is unclear how fragile health systems will be able to confront the pandemic. In this context, pregnant women and neonates in low-resource areas, as well as refugees and those in conflict zones, emerge as one of the most vulnerable populations. Moreover, we have no clues on the potential impact of comorbidities, such as tuberculosis or HIV infections, obstetric complications or malnutrition, on pregnant women with COVID-19. Experience from previous disease outbreaks revealed the disruptive effect on health service delivery and the consequences for diseases such as malaria, which mostly affects children and pregnant women. The WHO has already urged countries to continue to guarantee the delivery of malaria services in the context of the pandemic, including chemopreventive treatment in pregnant women and infants. Similarly, other current efforts regarding women's, infant's, and children's health and healthcare should be guaranteed. Effective coordination, collaboration, and communication are more necessary than ever to protect pregnant women and their infants globally.\n\n\nConclusions\n\nAlthough pregnant women do not seem to present an increased susceptibility to COVID-19 or more severe complications than non-pregnant adults, the available studies suggest that they may be at risk of adverse pregnancy outcomes, mostly preterm delivery, fetal distress, respiratory symptoms and LBW in the newborns. Analysis of the impact of COVID-19 on pregnancy regarding the ongoing trimester is needed in detail. Further research is urgently needed to understand the real effect of COVID-19 on pregnant women and neonates, and to guide the most appropriate recommendations for obstetricians. Only an integrated multiangle assessment of the current knowledge about viral characteristics, COVID-19 epidemiology, disease immunopathology, and potential preventive and therapeutic strategies, together with clinical observations, will help to understand the exact impact of COVID-19 infection during pregnancy.\n\n\nData availability\n\nNo data are associated with this article.",
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PubMed Abstract | Publisher Full Text | Free Full Text\n\nWorld Health Organization: Report of the WHO-China Joint Mission on Coronavirus Disease 2019 (COVID-19). 2020. Reference Source\n\nMullins E, Evans D, Viner RM, et al.: Coronavirus in pregnancy and delivery: rapid review. Ultrasound Obstet Gynecol. 2020; uog.22014. Publisher Full Text\n\nPanahi L, Amiri M, Pouy S: Risks of Novel Coronavirus Disease (COVID-19) in Pregnancy; a Narrative Review. Arch Acad Emerg Med. 2020; 8(1): e34. PubMed Abstract | Free Full Text\n\nDashraath P, Wong JLJ, Lim MXK, et al.: Coronavirus disease 2019 (COVID-19) pandemic and pregnancy. Am J Obstet Gynecol. 2020; pii: S0002-9378(20)30343-4. PubMed Abstract | Publisher Full Text\n\nChen YH, Keller J, Wang IT, et al.: Pneumonia and pregnancy outcomes: a nationwide population-based study. Am J Obstet Gynecol. 2012; 207(4): 288.e1–288.e7. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhong NS, Zheng BJ, Li YM, et al.: Epidemiology and cause of severe acute respiratory syndrome (SARS) in Guangdong, People's Republic of China, in February, 2003. Lancet. 2003; 362(9393): 1353–8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWorld Health Organization (Department of Communicable Disease Surveillance and Response): Consensus document on the epidemiology of severe acute respiratory syndrome (SARS). 2003. Reference Source\n\nShek CC, Ng PC, Fung GP, et al.: Infants born to mothers with severe acute respiratory syndrome. Pediatrics. 2003; 112(4): e254–e254. PubMed Abstract | Publisher Full Text\n\nStockman LJ, Lowther SA, Coy K, et al.: SARS during pregnancy, United States. Emerg Infect Dis. 2004; 10(9): 1689–90. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLam CM, Wong SF, Leung TN, et al.: A case-controlled study comparing clinical course and outcomes of pregnant and non-pregnant women with severe acute respiratory syndrome. BJOG. 2004; 111(8): 771–774. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNg WF, Wong SF, Lam A, et al.: The placentas of patients with severe acute respiratory syndrome: a pathophysiological evaluation. Pathology. 2006; 38(3): 210–218. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMaxwell C, Mcgeer A, Fan K, et al.: SOGC CLINICAL PRACTICE GUIDELINE No. 225-Management Guidelines for Obstetric Patients and Neonates Born to Mothers With Suspected or Probable Severe Acute Respiratory Syndrome (SARS). J Obs Gynaecol Can. 2017; 39(8): e130–e137. Publisher Full Text\n\nZaki AM, van Boheemen S, Bestebroer TM, et al.: Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia. N Engl J Med. 2012; 367(19): 1814–1820. PubMed Abstract | Publisher Full Text\n\nAssiri A, Abedi GR, Al Masri M, et al.: Middle East Respiratory Syndrome Coronavirus Infection During Pregnancy: A Report of 5 Cases From Saudi Arabia. Clin Infect Dis. 2016; 63(7): 951–953. PubMed Abstract | Publisher Full Text | Free Full Text\n\nPayne DC, Iblan I, Alqasrawi S, et al.: Stillbirth during infection with Middle East respiratory syndrome coronavirus. J Infect Dis. 2014; 209(12): 1870–1872. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAlserehi H, Wali G, Alshukairi A, et al.: Impact of Middle East Respiratory Syndrome coronavirus (MERS‐CoV) on pregnancy and perinatal outcome. BMC Infect Dis. 2016; 16: 105. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMalik A, El Masry KM, Ravi M, et al.: Middle East Respiratory Syndrome Coronavirus during Pregnancy, Abu Dhabi, United Arab Emirates, 2013. Emerg Infect Dis. 2016; 22(3): 515–517. PubMed Abstract | Publisher Full Text | Free Full Text\n\nAlfaraj SH, Al-Tawfiq JA, Memish ZA: Middle East Respiratory Syndrome Coronavirus (MERS-CoV) infection during pregnancy: Report of two cases and review of the literature. J Microbiol Immunol Infect. 2019; 52(3): 501–503. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJeong SY, Sung SI, Sung JH, et al.: MERS-CoV Infection in a Pregnant Woman in Korea. J Korean Med Sci. 2017; 32(10): 1717–1720. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFehr AR, Perlman S: Coronaviruses: an overview of their replication and pathogenesis. Methods Mol Biol. 2015; 1282: 1–23. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGagneur A, Dirson E, Audebert S, et al.: Materno-fetal transmission of human coronaviruses: a prospective pilot study. Eur J Clin Microbiol Infect Dis. 2008; 27(9): 863–866. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSchwartz DA, Graham AL: Potential Maternal and Infant Outcomes from Coronavirus 2019-nCoV (SARS-CoV-2) Infecting Pregnant Women: Lessons from SARS, MERS, and Other Human Coronavirus Infections. Viruses. 2020; 12(2): 194. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMertz D, Kim TH, Johnstone J, et al.: Populations at risk for severe or complicated influenza illness: systematic review and meta-analysis. BMJ. 2013; 347: f5061–f5061. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGottfredsson M: The Spanish flu in Iceland 1918. Lessons in medicine and history. Laeknabladid. 2008; 94(11): 737–45. Reference Source\n\nSiston AM, Rasmussen SA, Honein MA, et al.: Pandemic 2009 influenza A(H1N1) virus illness among pregnant women in the United States. JAMA. 2010; 303(15): 1517. PubMed Abstract | Publisher Full Text | Free Full Text\n\nJamieson D, Theiler R, Rasmussen S: Emerging infections and pregnancy. Emerg Infect Dis. 2006; 12(11): 1638–1643. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLittauer EQ, Skountzou I: Hormonal Regulation of Physiology, Innate Immunity and Antibody Response to H1N1 Influenza Virus Infection During Pregnancy. Front Immunol. 2018; 9: 2455. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWorld Health Organization: Vaccines against Influenza WHO Position Paper – November 2012. 2012. Reference Source\n\nMertz D, Lo CKF, Lytvyn L, et al.: FLURISK-INVESTIGATORS. Pregnancy as a risk factor for severe influenza infection: an individual participant data meta-analysis. BMC Infect Dis. 2019; 19(1): 683. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLaake I, Tunheim G, Robertson AH, et al.: Risk of pregnancy complications and adverse birth outcomes after maternal A(H1N1)pdm09 influenza: a Norwegian population-based cohort study. BMC Infect Dis. 2018; 18(1): 525. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRibeiro AF, Pellini ACG, Kitagawa BY, et al.: Severe influenza A(H1N1)pdm09 in pregnant women and neonatal outcomes, State of Sao Paulo, Brazil, 2009. PLoS One. 2018; 13(3): e0194392. PubMed Abstract | Publisher Full Text | Free Full Text\n\nNewsome K, Alverson CJ, Williams J, et al.: Outcomes of infants born to women with influenza A(H1N1)pdm09. Birth Defects Res. 2019; 111(2): 88–95. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFell D, Savitz D, Kramer M, et al.: Maternal influenza and birth outcomes: systematic review of comparative studies. BJOG. 2017; 124(1): 48–59. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLittauer EQ, Esser ES, Antao OQ, et al.: H1N1 influenza virus infection results in adverse pregnancy outcomes by disrupting tissue-specific hormonal regulation. PLoS Pathog. 2017; 13(11): e1006757. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLuteijn JM, Brown MJ, Dolk H: Influenza and congenital anomalies: a systematic review and meta-analysis. Hum Reprod. 2014; 29(4): 809–823. PubMed Abstract | Publisher Full Text\n\nBlomström Å, Karlsson H, Gardner R, et al.: Associations Between Maternal Infection During Pregnancy, Childhood Infections and the Risk of Subsequent Psychotic Disorder—A Swedish Cohort Study of Nearly 2 Million Individuals. Schizophr Bull. 2015; 42(1): sbv112. PubMed Abstract | Publisher Full Text | Free Full Text\n\nCai L, Wan CL, He L, et al.: Gestational Influenza Increases the Risk of Psychosis in Adults. Med Chem. 2015; 11(7): 676–682. PubMed Abstract | Publisher Full Text\n\nSelten JP, Termorshuizen F: The serological evidence for maternal influenza as risk factor for psychosis in offspring is insufficient: critical review and meta-analysis. Schizophr Res. 2017; 183: 2–9. PubMed Abstract | Publisher Full Text\n\nCreanga A: Transplacental transmission of influenza virus: What do we know? Indian J Med Sci. 2010; 64(12): 529. PubMed Abstract | Publisher Full Text\n\nFell DB, Bhutta ZA, Hutcheon JA, et al.: Report of the WHO technical consultation on the effect of maternal influenza and influenza vaccination on the developing fetus: Montreal, Canada, September 30–October 1, 2015. Vaccine. 2017; 35(18): 2279–2287. PubMed Abstract | Publisher Full Text\n\nYang H, Wang C, Poon LC: Novel coronavirus infection and pregnancy. Ultrasound Obstet Gynecol. 2020; 55(4): 435–437. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFavre G, Pomar L, Musso D, et al.: 2019-nCoV epidemic: what about pregnancies? Lancet. 2020; 395(10224): e40. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWorld Health Organization: Clinical Management of Severe Acute Respiratory Infection When COVID-19 Is Suspected. Interim Guidance. 2020. Reference Source\n\nPoon LC, Yang H, Lee JC, et al.: ISUOG Interim Guidance on 2019 novel coronavirus infection during pregnancy and puerperium: information for healthcare professionals. Ultrasound Obstet Gynecol. 2020; 55(5): 700–708. PubMed Abstract | Publisher Full Text\n\nPeyronnet V, Sibiude J, Deruelle P, et al.: SARS-CoV-2 infection during pregnancy. Information and proposal of management care. CNGOF. Gynecol Obstet Fertil Senol. 2020; 48(5): 436–443. PubMed Abstract | Publisher Full Text | Free Full Text\n\nChawla D, Chirla D, Dalwai S: Perinatal-Neonatal Management of COVID-19 Infection - Guidelines of the Federation of Obstetric and Gynecological Societies of India (FOGSI), National Neonatology Forum of India (NNF), and Indian Academy of Pediatrics (IAP). Indian Pediatr. 2020; pii: S097475591600154. PubMed Abstract\n\nDavanzo R, Moro G, Sandri F, et al.: Breastfeeding and coronavirus disease-2019: Ad interim indications of the Italian Society of Neonatology endorsed by the Union of European Neonatal & Perinatal Societies. Matern Child Nutr. 2020; e13010. PubMed Abstract | Publisher Full Text\n\nPoon LC, Yang H, Kapur A: Global interim guidance on coronavirus disease 2019 (COVID-19) during pregnancy and puerperium from FIGO and allied partners: Information for healthcare professionals. Int J Gynecol Obstet. 2020. PubMed Abstract | Publisher Full Text\n\nChen D, Yang H, Cao Y: Expert consensus for managing pregnant women and neonates born to mothers with suspected or confirmed novel coronavirus (COVID-19) infection. Int J Gynecol Obstet. 2020; 149(2): 130–136. PubMed Abstract | Publisher Full Text\n\nQiao J: What are the risks of COVID-19 infection in pregnant women? Lancet. 2020; 395(10226): 760–762. PubMed Abstract | Publisher Full Text | Free Full Text\n\nFavre G, Pomar L, Qi X, et al.: Guidelines for pregnant women with suspected SARS-CoV-2 infection. Lancet Infect Dis. 2020; pii: S1473-3099(20)30157-2. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSchmid MB, Fontijn J, Ochsenbein-Kölble N, et al.: COVID-19 in pregnant women. Lancet Infect Dis. 2020; pii: S1473-3099(20)30175-4. PubMed Abstract | Publisher Full Text\n\nWang S, Zhou X, Lin X, et al.: Experience of Clinical Management for Pregnant Women and Newborns with Novel Coronavirus Pneumonia in Tongji Hospital, China. Curr Med Sci. 2020; 40(2): 285–289. PubMed Abstract | Publisher Full Text\n\nRimmer A: Covid-19: doctors in final trimester of pregnancy should avoid direct patient contact. BMJ. 2020; 368: m1173. PubMed Abstract | Publisher Full Text\n\nTang N, Li D, Wang X, et al.: Abnormal coagulation parameters are associated with poor prognosis in patients with novel coronavirus pneumonia. J Thromb Haemost. 2020; 18(4): 844–847. PubMed Abstract | Publisher Full Text | Free Full Text\n\nErez O, Mastrolia SA, Thachil J: Disseminated intravascular coagulation in pregnancy: insights in pathophysiology, diagnosis and management. Am J Obstet Gynecol. 2015; 213(4): 452–463. PubMed Abstract | Publisher Full Text\n\nAi T, Yang Z, Hou H, et al.: Correlation of Chest CT and RT-PCR Testing in Coronavirus Disease 2019 (COVID-19) in China: A Report of 1014 Cases. Radiology. 2020; 200642. PubMed Abstract | Publisher Full Text\n\nCapanna F, Haydar A, McCarey C, et al.: Preparing an obstetric unit in the heart of the epidemic strike of COVID-19: quick reorganization tips. J Matern Fetal Neonatal Med. 2020; 1–7. PubMed Abstract | Publisher Full Text\n\nPoon LC, Abramowicz JS, Asta AD, et al.: ISUOG Safety Committee Position Statement: safe performance of obstetric and gynecological scans and equipment cleaning in the context of COVID-19. Ultrasound Obstet Gynecol. 2020; 55(5): 709–712. PubMed Abstract | Publisher Full Text\n\nGao J, Tian Z, Yang X: Breakthrough: Chloroquine phosphate has shown apparent efficacy in treatment of COVID-19 associated pneumonia in clinical studies. Biosci Trends. 2020; 14(1): 72–73. PubMed Abstract | Publisher Full Text\n\nKarunajeewa HA, Salman S, Mueller I, et al.: Pharmacokinetics of Chloroquine and Monodesethylchloroquine in Pregnancy. Antimicrob Agents Chemother. 2010; 54(3): 1186–1192. PubMed Abstract | Publisher Full Text | Free Full Text\n\nZhou D, Dai SM, Tong Q: COVID-19: a recommendation to examine the effect of hydroxychloroquine in preventing infection and progression. J Antimicrob Chemother. 2020; pii: dkaa114. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSchwartz DA, Graham AL: Potential Maternal and Infant Outcomes from Coronavirus 2019-nCoV (SARS-CoV-2) Infecting Pregnant Women: Lessons from SARS, MERS, and Other Human Coronavirus Infections. Viruses. 2020; 12(2): pii: E194. 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}
|
[
{
"id": "68159",
"date": "10 Aug 2020",
"name": "Alfonso J. Rodriguez-Morales",
"expertise": [
"Reviewer Expertise I am a Senior Researcher in Tropical and Emerging Infectious Diseases",
"currently Chair of the Latin American Network of Coronavirus Disease 2019 (COVID-19) Research (LANCOVID)",
"http://www.lancovid.org/."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis a very interesting review, addressing a relevant topic in the context of the coronavirus disease 2019 (COVID-19), its impact on pregnancy. Although I congratulate the authors for the efforts, before can be fully acceptable it deserves some improvements, including an update of the review. This review is based on two searches in the literature in two databases. The last review on April 8, 2020. Then, this means, more than four months have passed. For COVID-19 this is a lot. Multiple case reports, studies, and even reviews, and systematic reviews have came out. Then, this needs to be updated, including other additional databases, such as Scopus, and Web of Sciences. The section of Mother-to-Child transmission should be reviewed carefully, as new evidence suggested, that although sporadically, some cases ended in vertical transmission.\nFinally, I would suggest to extend more the Conclusions, specially to better discuss the current and future implications of the COVID-19 on pregnancy.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Partly\n\nAre all factual statements correct and adequately supported by citations? Partly\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Partly",
"responses": []
},
{
"id": "79428",
"date": "22 Feb 2021",
"name": "Caterina Montagnoli",
"expertise": [
"Reviewer Expertise Midwifery",
"Obstetrics",
"education",
"simulation"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI congratulate the authors for the extensive work on the review on COVID-19 effects upon pregnancy and maternal and neonatal outcomes.\nI have few but essential comments for improving the text.\nIn the abstract: \"a clinical presentation of the disease\" is used. Please rephrase this sentence to make it more accessible.\nWhy describing the impact of other viral respiratory infections? Please add a sentence to justify your choice beside the lack of COVID-19 knowledge also in this part of the text.\nThis insight on other coronaviruses and pregnancy is very interesting. Nevertheless, it adds little information about the management of the current COVID-19 disease.\nWere letters, perspectives, and reviews included in your data extraction/shortlisted papers?\nYou write: \"The results of a point-of-care lung-ultrasound examination were found to correlate with CT findings in one pregnant women; and therefore, this technique has been proposed as a tool to facilitate the triage of mothers with suspected COVID-19\" but were the articles case reports or studies? Were there statistical analysis?\nIn the conclusions you could discuss more recent literature and evidence regarding the administration of vaccines to pregnant women.\nI also advice an update with more recent case studies and other databases for the search.\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? Partly\n\nAre the conclusions drawn appropriate in the context of the current research literature? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/9-362
|
https://f1000research.com/articles/9-361/v1
|
13 May 20
|
{
"type": "Brief Report",
"title": "The IMPDH inhibitor merimepodib provided in combination with the adenosine analogue remdesivir reduces SARS-CoV-2 replication to undetectable levels in vitro",
"authors": [
"Natalya Bukreyeva",
"Rachel A. Sattler",
"Emily K. Mantlo",
"Timothy Wanninger",
"John T. Manning",
"Cheng Huang",
"Slobodan Paessler",
"Jerome B. Zeldis",
"Natalya Bukreyeva",
"Rachel A. Sattler",
"Emily K. Mantlo",
"Timothy Wanninger",
"John T. Manning",
"Cheng Huang",
"Slobodan Paessler"
],
"abstract": "Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the novel coronavirus responsible for the ongoing COVID-19 pandemic, which has resulted in over 2.5 million confirmed cases and 170,000 deaths worldwide as of late April 2020. The pandemic currently presents major public health and economic burdens worldwide. No vaccines or therapeutics have been approved for use to treat COVID-19 cases in the United States despite the growing disease burden, thus creating an urgent need for effective treatments. The adenosine analogue remdesivir (REM) has recently been investigated as a potential treatment option, and has shown some activity in limiting SARS-CoV-2 replication. We previously reported that the IMPDH inhibitor merimepodib (MMPD) provides a dose-dependent suppression of SARS-CoV-2 replication in vitro. Here, we report that a 4-hour pre-treatment of Vero cells with 2.5µM MMPD reduces the infectious titer of SARS-CoV-2 more effectively than REM at the same concentration. Additionally, pre-treatment of Vero cells with both REM and MMPD in combination reduces the infectious titer of SARS-CoV-2 to values below the detectable limit of our TCID50 assay. This result was achieved with concentrations as small as 1.25 µM MMPD and 2.5 µM REM. At concentrations of each agent as low as 0.31 µM, significant reduction of viral production occurred. This study provides evidence that REM and MMPD administered in combination might be an effective treatment for COVID-19 cases.",
"keywords": [
"merimepodib",
"remdesivir",
"SARS-CoV-2",
"COVID-19"
],
"content": "Introduction\n\nThe novel coronavirus SARS-CoV-2 is responsible for the current coronavirus disease 2019 (COVID-19) pandemic that originated in Wuhan, China in December of 2019. Since its emergence, the virus has infected 2.5 million people and is responsible for 170,000 deaths at the time of writing. In addition to the significant health burden, the pandemic is causing a worldwide economic crisis as countries attempt to limit the spread of SARS-CoV-2. There is a dire need for efficacious treatments, as the United States has yet to approve a vaccine or therapeutic to combat COVID-19. The most efficient means of approval involves drugs with a history of clinical testing. Two of these drugs, remdesivir (REM) and merimepodib (MMPD), have already been tested in patients. REM is currently in clinical trials as a COVID-19 therapeutic, while MMPD has previously been tested against chronic hepatitis C and psoriasis with an excellent safety profile1,2.\n\nREM is an adenosine analogue that displays broad-spectrum antiviral activity against RNA viruses, and has been developed for the treatment of Ebola virus disease3. The drug causes a delayed chain termination of RNA synthesis roughly five nucleotides downstream of the incorporated nucleotide analogue4. REM has demonstrated antiviral effects against both Ebola and Nipah viruses in nonhuman primates (NHPs)5,6, and has recently shown efficacy as a COVID-19 therapeutic in NHPs7. However, the drug must be administered intravenously limiting the ability for use outside of a hospital setting.\n\nMMPD is a selective and potent inhibitor of inosine-5’-monophosphate dehydrogenase (IMPDH), an enzyme involved in de novo guanine nucleotide synthesis8, which manipulates cellular nucleoside triphosphate levels to control the rapid replication of viruses. The drug has been tested for in vitro inhibition of many RNA viruses including Ebola, Junin, Lassa, Zika, and Chikungunya viruses9. We recently determined that MMPD suppresses the replication of SARS-CoV-2 by 3-log when Vero cells were pre-treated 4 hours prior to infection10.\n\nIn this study, we aimed to compare the antiviral effect of REM to MMPD in a tissue culture model in which cells were pre-treated with either agent prior to SARS-CoV-2 infection, and to determine whether pre-treatment with both REM and MMPD could further reduce the replication of SARS-CoV-2 in vitro.\n\n\nMethods\n\nVero cells (ATCC Cat# CCL-81, RRID:CVCL_0059) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (HyClone) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (HyClone). SARS-CoV-2 USA-WA1/2020 was obtained from the World Reference Center for Emerging Viruses and Arboviruses (WRCEVA), which obtained the original isolate from the Centers for Disease Control and Prevention (CDC). Upon obtaining the isolate, virus titration was performed. A subsequent low multiplicity of infection (MOI) passage was performed in Vero cells (DMEM supplemented with 5% fetal bovine serum and 1% penicillin/streptomycin), and the resulting stock was titrated prior to use. All experiments were conducted within approved biosafety level three (BSL-3) laboratories at the University of Texas Medical Branch (UTMB). All personnel undergo routine medical surveillance.\n\nMMPD clinical lot #2 (VX-497 Bulk Drug Substance # 300445), which is supplied as a white solid, was obtained from the PCI (Newburyport, MA). 10 mM stock of MMPD was prepared in DMSO and stored at -80°C. REM (Synonym GS-5734), Cat. # HY-104077, which is supplied as 10 mM solution in DMSO, was obtained from MedChemExpress (Monmouth Junction, NJ) and stored at -80°C. Both MMPD and REM then were diluted in compound dilution media to reach the assay target concentrations and the final DMSO concentration was equal or below 0.1%. Vero cells were pre-treated 4 hours prior to infection with either REM or MMPD diluted to the indicated concentration in DMEM, or a combination of the two drugs at the indicated concentrations diluted into DMEM (12-well plates, 1 mL per well). The media was removed at the time of infection, and the cells were inoculated with SARS-CoV-2 (MOI = 0.05) in 0.1 mL of inoculum, which was pre-mixed with the appropriate drug compound(s). The cells were incubated for 1 hour at 37°C to allow for infection. The cells were subsequently rinsed three times with 1 mL of DMEM, and then 1 mL of DMEM containing the appropriate drug compound(s) at the indicated concentration was added to each appropriate well. The cells were incubated for 0, 16, or 24 hours at 37°C before collecting a portion of each supernatant for titration. Upon collection, an equal amount of DMEM containing the compound was added back to each well. The infectious titer of SARS-CoV-2 for each sample was determined by TCID50 assay in Vero cells. The collected samples were serially diluted ten-fold a total of six times in DMEM, with dilutions performed in quadruplicate. The resulting dilutions were used to inoculate Vero cells in 96-well plates, and the plates were allowed to incubate for 4 days at 37°C. Cells were then fixed by addition of 10% formalin to each well for 30 minutes, and subsequently stained for 5 minutes with crystal violet solution. TCID50 values were obtained by counting the lowest dilutions in which infection was visible after crystal violet stain for two of four replicates. Each experiment was performed in biological triplicate, and the results were analyzed using a one-tailed t-test conducted in the GraphPad Prism 7 built-in statistical analysis software.\n\n\nResults\n\nWe have previously observed a peak in SARS-CoV-2 titer at 24 hours post-infection, which plateaus through 48 hours post-infection11. During the exponential growth phase, SARS-CoV-2 replication can be inhibited by 3-log with an overnight pre-treatment of MMPD at a concentration of 10µM, or 1.5-log with 4 hour pre-treatment at 5µM10.\n\nBoth REM and MMPD exhibit a potent antiviral effect on SARS-CoV-2 at concentrations of 2.5µM and 5µM after a 4 hour pre-treatment12. REM pre-treatment at a 5µM concentration provides a 2.1-log decrease in titer at 16 hours-post infection (p=0.001) and a 3.9-log decrease in titer at 24 hours post-infection (p<0.001). MMPD causes a similar antiviral effect as REM at the 5µM concentration, exhibiting a 2.1-log decrease in titer at 16 hours-post infection (p=0.001) and a 2.7-log decrease in titer at 24 hours post-infection (p<0.001). The effect of REM was dose-dependent, with the 2.5µM concentration of REM decreasing the titer by 1-log at 16 hours post-infection (p=0.103) and 1.5-log at 24 hours post-infection (p=0.002). However, MMPD antiviral effects appeared to be less dose-dependent, maintaining a 1.9-log decrease in titer at 16 hours post-infection (p=0.003) and a 2.5-log decrease in titer at 24 hours post-infection (p=0.001) at a concentration of 2.5µM (Figure 1). At sixteen hours post-infection 2.5µM REM does not have a statistically significant (p<0.05) difference between REM and untreated samples, whereas MMPD significantly reduced the infectious titer at this time point. When compared directly, MMPD causes a significantly greater reduction in SARS-CoV-2 infectious particles at both the 16-hour (p=0.022) and 24-hour (p=0.014) than REM. Therefore, MMPD provides a stronger inhibitory effect than REM at the 2.5µM concentration.\n\nVero cells were pre-treated with either 2.5µM or 5µM concentrations of either REM or MMPD 4 hours prior to infection with SARS-CoV-2. The media was removed, and the cells were infected with SARS-CoV-2 (MOI = 0.05) pre-mixed with each respective compound. The cells were washed three times with DMEM, and 1mL of media containing the respective compound was added. Cells were incubated at 37°C, and samples were collected at 0, 16, and 24 hours post-infection for TCID50 titration. The experiments were performed in triplicate, and the averages of the three TCID50 experiments are shown. The whiskers represent the standard deviation between the three results for each data point.\n\nSince REM and MMPD inhibit virus replication via separate mechanisms, we tested the inhibitory effect of both drugs in combination. We tested combinations ranging from 5µM + 5µM to 0.31µM + 0.31µM (Figure 2), performing the same 4 hour drug pre-treatment. Inhibition of SARS-CoV-2 was dose-dependent, and the virus titer was reduced to undetectable levels at all time points (p<0.001) for concentrations of 1.25µM +2.5µM, 2.5µM + 1.25µM, 2.5µM +2.5µM, 2.5µM + 5µM, 5µM + 2.5 µM, and 5µM + 5µM concentrations of REM and MMPD, respectively (Figure 2A, 2B). The lowest concentrations tested were 0.31µM REM + 0.31µM MMPD, which reduced the titer by 0.2-log at 16 hours post-infection (p=0.371) and 1.3-log at 24 hours post-infection (p=0.015). All combinations of concentrations between 0.31µM + 0.31µM and 1.25µM + 1.25µM produced a dose-dependent reduction in titer between 0.2-log and 2.5-log at 16 hours post-infection and a dose-dependent reduction between 1.3-log and 3.7-log at 24 hours post-infection (Figure 2C).\n\nVero cells were pre-treated with combinations of REM and MMPD ranging from 0.31µM to 5µM 4 hours prior to SARS-CoV-2 infection. The media was removed, and the cells were infected with SARS-CoV-2 (MOI=0.05) pre-mixed with each respective compound combination. The cells were washed three times with DMEM, and 1mL of media containing the appropriate compound mixture was added. Cells were incubated at 37°C, and samples were collected at 0, 16, and 24 hours post-infection for TCID50 titration. A) Co-treatment with concentrations between 5µM and 1.25µM. B) Co-treatment with equimolar concentrations of REM and MMPD between 5µM and 0.31µM. C) Co-treatment with concentrations between 1.25µM and 0.31µM. The 24-hour post-infection time point is further visualized using a bar graph. The experiments were performed in triplicate, and the averages of the three TCID50 experiments are shown. The whiskers represent the standard deviation between the three results for each data point.\n\nWe noticed a more robust inhibitory effect when the MMPD was provided at a lower concentration as opposed to REM being provided at a lower concentration (p<0.001, p=0.01), as illustrated 24 hours post-infection with the 0.31µM REM + 0.62µM MMPD and 0.62µM REM + 1.25µM MMPD to the 0.62µM REM + 0.31µM MMPD and 1.25µM REM + 0.62µM MMPD combinations (Figure 2C). This further supports that MMPD provides more potent SARS-CoV-2 inhibition than REM within the tested concentration ranges. More importantly, the data provides evidence that a treatment of REM + MMPD at a concentration as low as 2.5µM + 1.25µM reduces the production of infectious SARS-CoV-2 progeny to undetectable levels in cell culture.\n\n\nDiscussion\n\nOur results provide evidence that MMPD potently inhibits SARS-CoV-2 replication in vitro at concentrations as low as 2.5µM, which was the lowest concentration tested. Further work will determine the EC50 and EC90 for MMPD. Previous work established that cellular cytotoxicity for MMPD is greater than 10 μM so the activity against the virus cannot be attributed to the drug being cytotoxic.\n\nThe inhibitory effect was similar for both MMPD and REM at 2.5µM at 24 hours post-infection. However, MMPD was the only drug to significantly inhibit SARS-CoV-2 at the 16 hour time point using a 2.5µM concentration. REM also appears to have a slightly higher range of dose-dependent efficacy in the 1–10µM range than MMPD. Here, we show a 2.4-log difference in antiviral activity for REM between 2.5µM and 5µM concentrations at 24 hours post-infection and only a difference of 0.2-log for MMPD using the same conditions. Previously, we determined that only a 1.5-log difference exists in MMPD inhibitory effect between 10µM and 3.3µM concentrations10.\n\nThe concentration of MMPD used in this study is well below the clinically-achievable concentration based on human PK trials. Oral administration of 50mg MMPD achieves a plasma concentration of around 2500ng/mL (5.5 μM) which makes it highly attractive for antiviral therapy2. Intravenous administration of a 10mg/kg dose of REM results in blood concentrations of 10µM in NHPs13. REM and MMPD affect the replication of RNA viruses through different mechanisms. REM is an adenosine analogue that causes a delayed chain termination of RNA synthesis upon being incorporated into viral RNA4; whereas MMPD is a selective IMPDH inhibitor that manipulates the nucleoside triphosphate levels within cells, acting indirectly as a host-directed therapy. Therefore, we also tested whether the combination treatment using both, direct antiviral REM and host targeted drug MMPD could possibly have synergistic anti-COVID-19 activity. Our data demonstrate that this combination can completely abolish production of infectious virus in our cell culture model. Neither agent by itself completely abrogates viral production. While we are now determining the dose response of MMPD in this model system, REM data demonstrates that a combination of both drugs still gives significant reduction of viral productions with low concentrations of both agent.\n\nOur study indicates that MMPD alone, and/or in combination with REM, could be a viable treatment option for COVID-19 based on the potent inhibitory effect in cells culture at an easily achievable dose in the clinical setting. The drug could be quickly tested in a clinical setting based on its clinical trial history, and might be a useful prophylactic treatment to prevent spread of SARS-CoV-2 to the lower respiratory system10.\n\n\nData availability\n\nHarvard Dataverse: Remdesivir and Merimepodib co-treatment for SARS-CoV-2. https://doi.org/10.7910/DVN/HE6ZYJ12.\n\nThis project contains the following underlying data:\n\nRaw Data REM + MMPD co-treatment for SARS-CoV-2.xlsx (SARS-CoV-2 inhibition data for remdesivir and merimepodib co-treatment in Vero cells)\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\nMarcellin P, Horsmans Y, Nevens F, et al.: Phase 2 Study of the Combination of Merimepodib With peginterferon-alpha2b, and Ribavirin in Nonresponders to Previous Therapy for Chronic Hepatitis C. J Hepatol. 2007; 47(4): 476–83. PubMed Abstract | Publisher Full Text\n\nRustgi VK, Lee WM, Lawitz E, et al.: Merimepodib, Pegylated Interferon, and Ribavirin in Genotype 1 Chronic Hepatitis C Pegylated Interferon and Ribavirin Nonresponders. Hepatology. 2009; 50(6): 1719–26. PubMed Abstract | Publisher Full Text\n\nSiegel D, Hui HC, Doerffler E, et al.: Discovery and Synthesis of a Phosphoramidate Prodrug of a Pyrrolo[2,1-f][triazin-4-amino] Adenine C-Nucleoside (GS-5734) for the Treatment of Ebola and Emerging Viruses. J Med Chem. 2017; 60(5): 1648–1661. PubMed Abstract | Publisher Full Text\n\nTchesnokov EP, Feng JY, Porter DP, et al.: Mechanism of Inhibition of Ebola Virus RNA-Dependent RNA Polymerase by Remdesivir. Viruses. 2019; 11(4): 326. PubMed Abstract | Publisher Full Text | Free Full Text\n\nLo MK, Feldmann F, Gary JM, et al.: Remdesivir (GS-5734) Protects African Green Monkeys From Nipah Virus Challenge. Sci Transl Med. 2019; 11(494): eaau9242. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWarren TK, Jordan R, Lo MK, et al.: Therapeutic efficacy of the small molecule GS-5734 against Ebola virus in rhesus monkeys. Nature. 2016; 531(7594): 381–5. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWilliamson BN, Feldmann F, Schwarz B, et al.: Clinical benefit of remdesivir in rhesus macaques infected with SARS-CoV-2. bioRxiv. 2020. Publisher Full Text\n\nMaurya SK, Gollapalli DR, Kirubakaran S, et al.: Triazole inhibitors of Cryptosporidium parvum inosine 5'-monophosphate dehydrogenase. J Med Chem. 2009; 52(15): 4623–30. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTong X, Smith J, Bukreyeva N, et al.: Merimepodib, an IMPDH inhibitor, suppresses replication of Zika virus and other emerging viral pathogens. Antiviral Res. 2018; 149: 34–40. PubMed Abstract | Publisher Full Text\n\nBukreyeva N, Mantlo EK, Sattler RA, et al.: The IMPDH inhibitor merimepodib suppresses SARS-CoV-2 replication in vitro. bioRxiv. 2020. Publisher Full Text\n\nMantlo E, Bukreyeva N, Maruyama J, et al.: Potent Antiviral Activities of Type I Interferons to SARS-CoV-2 Infection. bioRxiv. 2020. Publisher Full Text\n\nManning John: \"Remdesivir and Merimepodib co-treatment for SARS-CoV-2\". Harvard Dataverse, V1, 2020. http://www.doi.org/10.7910/DVN/HE6ZYJ\n\nWilson AJ, Martin DS, Maddox V, et al.: Thromboelastography in the Management of Coagulopathy Associated With Ebola Virus Disease. Clin Infect Dis. 2016; 62(5): 610–612. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "63764",
"date": "26 May 2020",
"name": "Luka Čičin-Šain",
"expertise": [
"Reviewer Expertise Virology and immunology."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article \"The IMPDH inhibitor merimepodib provided in combination with the adenosine analogue remdesivir reduces SARS-CoV-2 replication to undetectable levels in vitro\" is a timely study on the effects of remdesivir and merimepomid against SARS CoV-2. The anti SARS CoV-2 effects of Remdesivir is not new, and neither is the antiviral a ctivity of mereimepomid, but the effect of merimepomid against SARS CoV-2 has not been shown in other studies and is a new observation, along with the observation on additive effects of remdesivir and merimepomid in combination. The authors demonstrate robust antiviral activity of the two substances in the low micromolar concentration and a robust antiviral activity when both substances are administered in combination at a concentration of 1,25 µM. The major shortcoming of the study is that all results were generated on VeroE6, a green monkey cell line that is not necessarily representative of the natural sites of infection. This issue is particularly important because merimepodib acts by inhibiting inosine-5’-monophosphate dehydrogenase (IMPDH), a cellular enzyme, and potential polymorphisms between this gene in non-human primates and humans may result in a different efficacy of the drug in clinical settings. Therefore, the study would be strengthened by expanding the assay to human epithelial cells, ideally of lung origin. Another smaller shortcoming of the study is that all statistical analysis was performed by t-tests, but the data are for the most part much more complex and require ANOVA analysis, and due to the low n in the experimental groups should be performed by Kruskal-Wallis. This in itself is not a major point of contention in my eyes. Reproducibility in experimental settings trumps statistical analysis by definition, the results in this study are clear already by eyeballing them, and the core results have been reproduced in several experiments shown in the two figures. Nevertheless, if statistical significance is used as an argument to validate the results, then one should use statistical methods that fit the experimental design and the group sizes. In sum, despite these shortcomings, this is an important set of data that brings us closer to the development of antiviral strategies against the COVID19 pandemics and a substantial contribution to the field.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Partly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes",
"responses": []
},
{
"id": "67602",
"date": "04 Aug 2020",
"name": "Mahmoud Kandeel",
"expertise": [
"Reviewer Expertise Pharmacology and drug discovery"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nDespite the presence of several reports about the antiviral activity of remdesivir and merimepodib, the manuscript can provide useful information in the fight against SARS CoV2.\n\nDespite the provided piece of experiments and results of this manuscript, we still unable to precisely know the potency of these two drugs. The description of results was using the two concentrations of the two drugs without further important details. The potency of the drugs must be presented in terms of IC50. In this regard, several compounds were proved to be effective on SARS CoV-2 replication in the low nanomolar range and could be at least several folds more potent than REM and MMPD. In this case, the potency of REM and MMPD can be related to other anti-SARS CoV-2 drugs.\n\nThe relation of the two drugs combination must be discussed in terms of additive effect, synergism or antagonism. For this purpose software as CompuSyn or similar can be used to calculate the combination index, which will scientifically assess the nature of these two drugs combination effect.\n\n“Oral administration of 50mg MMPD achieves a plasma concentration of around 2500ng/mL (5.5 μM) which makes it highly attractive for antiviral therapy” recheck this sentence\n\nFor assay reproducibility, another cell type is required e,g, Calu-3 cells\n\nThe raw data of crystal violet stain pictures and plaques count could be provided as supplementary file.\n\nThe two drugs seems to act on late stages of virus replication, at least not in the attachment and fusion steps, the 4-hr pretreatment needs clarification. The time of addition experiment must be provided during different hours before and after infection.\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? No\n\nAre the conclusions drawn adequately supported by the results? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/9-361
|
https://f1000research.com/articles/9-188/v1
|
13 Mar 20
|
{
"type": "Brief Report",
"title": "Improved reperfusion following alternative surgical approach for experimental stroke in mice",
"authors": [
"Melissa Trotman-Lucas",
"Raymond Wong",
"Stuart M. Allan",
"Claire L. Gibson",
"Melissa Trotman-Lucas",
"Raymond Wong",
"Stuart M. Allan"
],
"abstract": "Background: Following ischemic stroke, recanalisation and restoration of blood flow to the affected area of the brain is critical and directly correlates with patient recovery. In vivo models of ischemic stroke show high variability in outcomes, which may be due to variability in reperfusion. We previously reported that a surgical refinement in the middle cerebral artery occlusion (MCAO) model of stroke, via repair of the common carotid artery (CCA), removes the reliance on the Circle of Willis for reperfusion and reduced infarct variability. Here we further assess this refined surgical approach on reperfusion characteristics following transient MCAO in mice. Methods: Mice underwent 60 min of MCAO, followed by either CCA repair or ligation at reperfusion. All mice underwent laser speckle contrast imaging at baseline, 24 h and 48 h post-MCAO. Results: CCA ligation reduced cerebral perfusion in the ipsilateral hemisphere compared to baseline (102.3 ± 4.57%) at 24 h (85.13 ± 16.09%; P < 0.01) and 48 h (75.04 ± 12.954%; P < 0.001) post-MCAO. Repair of the CCA returned perfusion to baseline (94.152 ± 2.44%) levels and perfusion was significantly improved compared to CCA ligation at both 24 h (102.83 ± 8.41%; P < 0.05) and 48 h (102.13 ± 9.34%; P < 0.001) post-MCAO. Conclusions: Our findings show CCA repair, an alternative surgical approach for MCAO, results in improved ischemic hemisphere perfusion during the acute phase.",
"keywords": [
"Ischemia",
"Cerebral stroke",
"Reperfusion",
"Cerebral blood flow"
],
"content": "Introduction\n\nCerebral ischemic stroke is one of the leading causes of death worldwide, carrying a significant disease burden as the second-leading cause of disability-adjusted life years1,2. Current approved treatment options are limited for acute ischemic stroke patients, involving either thrombolytic clot breakdown or physical clot removal. Recombinant tissue plasminogen activator (rtPA) is the only pharmacological treatment available with proven efficacy3,4, eligibility for this treatment is low and, due to a narrow therapeutic time window (<4.5 h), only ~15% of stroke patients are able to receive iv-rtPA treatment, with recanalisation success at less than 50%5,6. In addition to this, endovascular thrombectomy is increasingly being used in the treatment of large vessel occlusions, where the clot is removed from within the vessel allowing recanalisation, especially in patients who respond poorly to rtPA treatment5. Prompt restoration of blood flow to the ischemic zone is the primary clinical goal, potentially salvaging tissue and preventing the spread of ischemic damage7. Recanalization of occluded vessels is positively correlated with improved survival rates and recovery outcomes for ischemic stroke patients8.\n\nTo allow development of new clinical therapeutics for stroke, the pathophysiology and mechanisms of disease/recovery need to be elucidated. Preclinical stroke models that closely mimic mechanisms of injury (and recovery) allow investigation of potential clinically viable treatments. Although experimental stroke models have shown the positive benefits of various potential therapeutics, these have repeatedly failed during clinical trials, preventing their progression into the clinic. This continued lack of translation requires us to further refine preclinical studies and to allow the evolution of more representative models9,10.\n\nThe most commonly used experimental model of ischemic stroke is the intraluminal filament model of middle cerebral artery occlusion (MCAO). First developed by Koizumi et. al.11 and later modified by Longa et. al.12, with numerous minor variations to the model since then. These two models are widely used in preclinical experimental stroke research largely due to their minimally invasive technique and ability to induce post-occlusion reperfusion13. However, although surgical procedures follow a defined protocol, lesion volumes have large standard deviations14–16. With the Koizumi method, permanent ligation of the common carotid artery (CCA) is required, preventing return of flow through the internal carotid artery (ICA), thus reperfusion is dependent on collateral flow via the Circle of Willis (CoW). However, the CoW in C57BL/6J mice, commonly used in preclinical stroke models, is anatomically variable which affects collateral flow ability17,18. In addition to this, there are adverse consequences to animal wellbeing when impacting the external carotid artery (ECA), either for vessel entry as in the Longa modified model or for surgical ease as seen in the original Koizumi model, such as lick impairment affecting ability to drink alongside increased weight loss19.\n\nStroke in the territory of the middle cerebral artery is the most common presentation seen in patients20, thus targeting this vessel in preclinical models is valid. However, clot lysis induced by rtPA treatment results in gradual reperfusion to the ischemic territory, whereas in the MCAO model, filament removal is sudden giving an immediate blood flow surge to the ischemic area21. In the clinic, endovascular thrombectomy allows physical removal of large vessel clots21,22, permitting rapid reflow through the previously occluded vessel, which is the primary clinical goal and parallels that which occurs in transient MCAO upon filament withdrawal. However, MCAO models where intraluminal access is obtained via the CCA, such as the Koizumi method23, rely on collateral perfusion after filament removal due to the CCA being permanently tied off to prevent blood loss. Previously, we reported a modification to the Koizumi model of intraluminal filament MCAO, which allows bilateral CCA reperfusion, negating the reliance on the CoW10. In commonly used mice strains such as the C57BL/6J, the variability seen in lesion volume may be largely due to anatomical variations in the CoW 17. Removing reperfusion reliance on the CoW reduces the variability seen in lesion volume but the impact on reperfusion is not known. Our aim here therefore is to determine if the adapted MCAO model can improve reperfusion following removal of the filament.\n\n\nMethods\n\nThis study was conducted in accordance with the UK Animals (Scientific Procedures) Act, 1986, under Project license P28AA2253 following ethical approval from the University of Manchester. Animals were group housed (n=4) on arrival in temperature- and humidity-controlled individually ventilated mouse cages (IVCs) with a 12-h light/dark cycle, situated within a specific pathogen free (SPF) facility. Cages contained: woodchip bedding, paper sizzle nesting and cardboard tube. Animals were given adlib access to dry pellet food and water. Experimental sample size was determined using a power calculation from preliminary data (one tailed, α=0.001, β=0.9), taking into account a mortality rate of 10% established from historical MCAO data within our group. A total of 18 adult male C57BL/6J mice (10 weeks old on arrival; Charles River, UK), weighing 22–33 g at the time of MCAO, were used during this study: two animals were excluded due to surgical complications, two due to reaching humane endpoints and one due to repeated wound complications. All remaining animals (n=13) were humanely killed by cervical dislocation under anaesthesia following the 48-h post-MCAO scanning procedure. All experiments are reported in accordance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines 201824.\n\nIndividual animals were randomly assigned using an automated list randomiser (random.org) to one of two experimental groups: CCA ligation or CCA repair. The CCA ligation group underwent 60 minutes transient MCAO with permanent CCA ligation following reperfusion25. The CCA repair group underwent 60 minutes transient MCAO followed by repair of the CCA using a tissue pad and sealant combination, resulting in a patent CCA10. Both groups underwent laser speckle contrast imaging (LSCI). The order of coded LSCI raw data files was randomised prior to analysis using an automated list randomiser. The experimenter was blind to groups during LSCI scanning and image analysis for raw data, data was decoded for statistical analysis only.\n\nAll animals underwent LSCI. Imaging was undertaken 12 days prior to MCAO (baseline imaging), then at 24 h and 48 h post-MCAO (Figure 1A). Anaesthesia was induced and maintained with isoflurane (induction 5% in 100% O2; maintenance 1.5% in N2O/O2, 70/30%), following which mice were placed in a stereotaxic frame on a heat mat set to 37°C. The animals were positioned underneath a moorFLPI2 Full-Field Perfusion Imager (Moor Instruments, UK). Prior to incision Bupivacaine hydrochloride (2 mg/kg, Marcain 0.25%, AstraZeneca, UK) local anaesthesia was injected subcutaneous around the intended incision site. A midline incision was made and skin retracted to expose the intact skull. Ultrasound gel was applied to the skull, followed by a 10-mm2 glass coverslip pressed down slightly to allow complete coverage of the intended scan area. LSCI was conducted for 3 min at 19 frames (20 ms per frame, 10-s intervals). The incision was cleaned free of ultrasound gel using sterile swabs and flushed with sterile saline, the incision was sutured using subcutaneous 6-0 dissolvable sutures followed by tissue adhesive application (Vetbond, US). The animal was returned to a pre-warmed clean cage for recovery. Following baseline LSCI only, animals were given access to paracetamol (200 mg/kg/24 h; Calpol®, Johnson & Johnson Ltd UK) in jelly for 24 h for self-administration analgesia. LSCI images were analysed using moorFLPI2 Full-Field Laser Perfusion Imager Review v5.0 software. For each animal, ipsilateral and contralateral hemisphere ROIs were drawn, ROI flux data was obtained from each hemisphere for each of the 19 images per scan, the mean of these values was used in later analyses. Ipsilateral hemisphere cerebral blood flow (CBF) is expressed as a percentage of the contralateral hemisphere CBF, within each imaging session. All analysis was conducted blinded to experimental conditions.\n\nCommon carotid artery (CCA) vessel repair promotes cerebral blood flow (CBF) recovery following transient middle cerebral artery occlusion (MCAO). Regions of interest (ROIs) surrounding the ipsilateral and contralateral hemisphere were used to quantify CBF. (A) Representative LSCI images at baseline, 24 h and 48 h post-MCAO in CCA Ligated and CCA repair mice. Contra, contralateral hemisphere; Ipsi, ipsilateral hemisphere. (B) CBF obtained by LSCI, measured separately at baseline, 2 4h and 48 h post-MCAO, expressed as ipsilateral flux as a percentage of contralateral flux. Statistical analysis performed using two-way ANOVA followed by Sidak post-hoc (ns P ≥ 0.05, */X P ≤ 0.05, **/XX P ≤ 0.01, ***/XXX P ≤ 0.001). *Represents significance within experimental group versus Baseline. XRepresents significance between experimental groups. All data presented as mean ± SD (CCA Ligated n=7, CCA Repair n=6).\n\nAll mice underwent transient MCAO as previously detailed, and surgical methods used were identical to those published previously by the author10,26, modified only to exclude Laser Doppler Flowmetry recording at MCAO reperfusion.\n\nAll data are expressed as mean ± standard deviation of the mean (SD). All statistical analysis was performed using Prism v8 for windows (GraphPad Software, CA). The criterion for statistical significance was P ≤ 0.05. Distribution of data was assessed using Shapiro-Wilk normality test, prior to statistical analysis. Repeated measure two-way analysis of variance (ANOVA) test was used to determine effect of time and intervention, repair and ligation. Post-hoc Sidak corrected multiple comparison testing was then used to compare within groups against baseline/pre-MCAO, and within time point between groups.\n\n\nResults\n\nThere was a significant interaction between the two factors, time and CCA incision closure method/intervention, suggesting the effect of CCA intervention on CBF differs between the timepoints (F(2,22) = 17, P < 0.001; Figure 1B)). CCA intervention post-MCAO, either ligation or repair, significantly affected ipsilateral CBF perfusion overall (F(1,11) = 7.4, P = 0.02). Mice that underwent CCA ligation showed significantly reduced ipsilateral CBF compared to baseline (102.3 ± 4.57%) at 24 h (85.13 ± 16.09%; P = 0.001) and 48 h (75.04 ± 12.954%; P < 0.001) post-MCAO. Contrastingly, mice that underwent CCA repair showed no difference versus baseline (94.152 ± 2.44%) at 24 h (102.83 ± 8.41%; P = 0.191) or 48 h (102.13 ± 9.34%; P = 0.251) post-MCAO. There was no difference in ipsilateral CBF perfusion between groups at baseline (P = 0.417). At both 24 h and 48 h post-MCAO ipsilateral CBF was significantly increased in CCA repair mice compared to CCA ligated mice (P = 0.0122, P = 0.0001 respectively).\n\n\nDiscussion\n\nHere we report CCA vessel repair following MCAO in mice results in improved reperfusion of the ischemic hemisphere, assessed using LSCI. Ligation of the CCA following MCAO filament removal significantly reduced perfusion to the ischemic hemisphere for a prolonged period post-surgery and the CBF did not return to baseline up to 48 h post-MCAO. Repair of the CCA incision, using homologous tissue pad and TISSEEL sealant as previously reported10, returned hemispheric perfusion to pre-MCAO baseline levels within 24 h post-MCAO. The use of CCA repair in mice has been shown to improve immediate MCA territory blood flow within 5 minutes following repair and reduce lesion volume variability at 48 h post-MCAO10. Data here show the initial post-MCAO return to baseline CBF, as shown previously, is sustained during the acute phase (i.e. 48 h) with no evidence of hypo or hyperperfusion in the ipsilateral hemisphere at 24 and 48 h post-MCAO.\n\nThe data indicate CCA repair improves ischemic area perfusion post-MCAO, we assume this is due to the return to patency of the CCA removing the reliance on collateral flow through the CoW. The CoW has been shown in C57BL/6J mice to be anatomically variable across individual animals17, adding variability to collateral supply to the ischæmic area. Similarly, Smith et al. reported, using MCA territory laser doppler flowmetry to measure perfusion, significantly increased post-MCAO perfusion using the Longa ECA entry method with perfusion returning to comparable baseline levels, whereas use of the Koizumi CCA entry method showed only a return to 50% of baseline levels23. The key difference between the two methods is that the Longa method allows reperfusion through the bilateral CCAs whereas the Koizumi method relies on collateral supply to perfuse the ischemia area due to only the CCA being patent. These results mimic those reported here with CCA repair resulting in bilaterally patent CCAs leading to a return to baseline in perfusion post-MCAO. The use of a muscle pad combined with a fibrin sealant was previously shown in rat MCAO to allow forward blood flow through the CCA with complete occlusion of the vessel not present following the procedure27. Additionally, the CCA repair method ensures that the ECA remains in situ and patent, reducing the impact on animal welfare and recovery19.\n\nAllowing reperfusion to occur through all available vessels transforms the MCAO model to be more representative of clinical endovascular thrombectomy. Endovascular thrombectomy is employed in an increasing number of clinical cases of large vessel occlusion (LVO), allowing mechanical, within vessel, removal of the occluding clot leading to a surge reperfusion of the ischemic zone, such as is seen following MCAO filament removal21. There is discussion in the literature as to the relevance of MCAO for modelling clinical stroke, due to the slow gradual clot dispersion that occurs following rtPA treatment, a mechanism unlike the sudden increase in flow seen at MCAO filament removal28, with suggestion that this slower clinical rtPA treatment reperfusion profile is closer to other embolic stroke models21,29. However, due to the positive effects of using endovascular thrombectomy for ischemic stroke treatment, on blood flow and patient outcome, the intervention is likely to become the primary treatment for LVO21. With this in mind, there is renewed interest in the intraluminal filament model of MCAO as an established model of endovascular thrombectomy.\n\nIn conclusion, this brief report shows that CCA repair following MCAO, in the mouse, using homologous tissue pad combined with sealant improves blood flow to the ischemic hemisphere. Validating the use of CCA repair in MCAO studies, removing the reliance on the CoW and improving localised perfusion. This may be of particular interest for use in endovascular thrombectomy research, mimicking the clinical situation of clot removal without ligation of arteries.\n\n\nData availability\n\nZenodo: Dataset corresponding to scientific paper “Improved reperfusion following alternative surgical approach for experimental stroke in mice”. https://doi.org/10.5281/zenodo.370123130.\n\nThis project contains the following underlying data:\n\nBASE_Repair (baseline raw LCSI export files for animals that underwent repair of the CCA; the three-symbol code at the start of each file represents a different mouse).\n\n24h_Repair (24-hour raw LCSI export files for animals that underwent repair of the CCA; the three-symbol code at the start of each file represents a different mouse).\n\n48h_Repair (48-hour raw LCSI export files for animals that underwent repair of the CCA; the three-symbol code at the start of each file represents a different mouse).\n\nBASE_Ligated (baseline raw LCSI export files for animals that underwent ligation of the CCA; the three-symbol code at the start of each file represents a different mouse).\n\n24h_Ligated (24-hour raw LCSI export files for animals that underwent ligation of the CCA; the three-symbol code at the start of each file represents a different mouse).\n\n48h_Ligated (48-hour raw LCSI export files for animals that underwent ligation of the CCA; the three-symbol code at the start of each file represents a different mouse).\n\n20200206_F1000_LS_Manuscript Raw Data (LCSI Flux values obtained by drawing ROIs in the LSCI software and exporting the values).\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0)",
"appendix": "References\n\nWorld Health OrganisationGlobal Health Estimates 2016: Disease burden by Cause, Age, Sex, by Country and by Region, 2000-2016. 2018.\n\nWorld Health Organisation. Global Health Estimates 2016: Deaths by Cause, Age, Sex, by Country and by Region, 2000-2016. 2018.\n\nNational Institute for Health and Care Excellence. Stroke and transient ischaemic attack in over 16s: diagnosis and initial management. National Institute for Health and Care Excellence Guideline NG128. 2019; Reference Source\n\nO'Collins VE, Macleod MR, Donnan GA, et al.: 1,026 experimental treatments in acute stroke. Ann Neurol. 2006; 59(3): 467–477. PubMed Abstract | Publisher Full Text\n\nBhaskar S, Stanwell P, Cordato D, et al.: Reperfusion therapy in acute ischemic stroke: dawn of a new era? BMC Neurol. 2018; 18(1): 8. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGrech R, Mizzi A, Pullicino R, et al.: Functional outcomes and recanalization rates of stent retrievers in acute ischaemic stroke: A systematic review and meta-analysis. Neuroradiol J. 2015; 28(2): 152–171. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBalami JS, Hadley G, Sutherland BA, et al.: The exact science of stroke thrombolysis and the quiet art of patient selection. Brain. 2013; 136(Pt 12): 3528–3553. PubMed Abstract | Publisher Full Text\n\nRha JH, Saver JL: The impact of recanalization on ischemic stroke outcome: a meta-analysis. Stroke. 2007; 38(3): 967–973. PubMed Abstract | Publisher Full Text\n\nPercie du Sert N, Alfieri A, Allan SM, et al.: The IMPROVE Guidelines (Ischaemia Models: Procedural Refinements Of in Vivo Experiments). J Cereb Blood Flow Metab. 2017; 37(11): 3488–3517. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTrotman-Lucas M, Kelly ME, Janus J, et al.: An alternative surgical approach reduces variability following filament induction of experimental stroke in mice. Dis Model Mech. 2017; 10(7): 931–938. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKoizumi J, Yoshida Y, Nakazawa T, et al.: Experimental studies of ischemic brain edema. I: A new experimental model of cerebral embolism in rats in which recirculation can be introduced in the ischemic area. Japanese Journal of Stroke. 1986; 8(1): 1–8. Publisher Full Text\n\nLonga EZ, Weinstein PR, Carlson S, et al.: Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke. 1989; 20(1): 84–91. PubMed Abstract | Publisher Full Text\n\nMacrae IM: Preclinical stroke research--advantages and disadvantages of the most common rodent models of focal ischaemia. Br J Pharmacol. 2011; 164(4): 1062–1078. PubMed Abstract | Publisher Full Text | Free Full Text\n\nIngberg E, Dock H, Theodorsson E, et al.: Method parameters' impact on mortality and variability in mouse stroke experiments: a meta-analysis. Sci Rep. 2016; 6: 21086. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDirnagl U: Bench to bedside: the quest for quality in experimental stroke research. J Cereb Blood Flow Metab. 2006; 26(12): 1465–1478. PubMed Abstract | Publisher Full Text\n\nCarmichael ST: Rodent models of focal stroke: size, mechanism, and purpose. NeuroRx. 2005; 2(3): 396–409. PubMed Abstract | Publisher Full Text | Free Full Text\n\nMcColl BW, Carswell HV, McCulloch J, et al.: Extension of cerebral hypoperfusion and ischaemic pathology beyond MCA territory after intraluminal filament occlusion in C57Bl/6J mice. Brain Res. 2004; 997(1): 15–23. PubMed Abstract | Publisher Full Text\n\nFoddis M, Winek K, Bentele K, et al.: An exploratory investigation of brain collateral circulation plasticity after cerebral ischemia in two experimental C57BL/6 mouse models. J Cereb Blood Flow Metab. 2020; 40(2): 276–287. PubMed Abstract | Publisher Full Text\n\nTrueman RC, Harrison DJ, Dwyer DM, et al.: A Critical Re-Examination of the Intraluminal Filament MCAO Model: Impact of External Carotid Artery Transection. Transl Stroke Res. 2011; 2(4): 651–661. PubMed Abstract | Publisher Full Text\n\nNg YS, Stein J, Ning M, et al.: Comparison of clinical characteristics and functional outcomes of ischemic stroke in different vascular territories. Stroke. 2007; 38(8): 2309–2314. PubMed Abstract | Publisher Full Text\n\nSutherland BA, Neuhaus AA, Couch Y, et al.: The transient intraluminal filament middle cerebral artery occlusion model as a model of endovascular thrombectomy in stroke. J Cereb Blood Flow Metab. 2016; 36(2): 363–369. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGoyal M, Menon BK, van Zwam WH, et al.: Endovascular thrombectomy after large-vessel ischaemic stroke: a meta-analysis of individual patient data from five randomised trials. Lancet. 2016; 387(10029): 1723–1731. PubMed Abstract | Publisher Full Text\n\nSmith HK, Russell JM, Granger DN, et al.: Critical differences between two classical surgical approaches for middle cerebral artery occlusion-induced stroke in mice. J Neurosci Methods. 2015; 249: 99–105. PubMed Abstract | Publisher Full Text\n\nKilkenny C, Browne WJ, Cuthill IC, et al.: Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research. PLoS Biol. 2010; 8(6): e1000412. PubMed Abstract | Publisher Full Text | Free Full Text\n\nGibson CL, Murphy SP: Progesterone enhances functional recovery after middle cerebral artery occlusion in male mice. J Cereb Blood Flow Metab. 2004; 24(7): 805–813. PubMed Abstract | Publisher Full Text\n\nTrotman-Lucas M, Kelly ME, Janus J, et al.: Middle Cerebral Artery Occlusion Allowing Reperfusion via Common Carotid Artery Repair in Mice. J Vis Exp. 2019; (143): e58191. PubMed Abstract | Publisher Full Text\n\nFehm NP, Vatankhah B, Dittmar MS, et al.: Closing microvascular lesions with fibrin sealant-attached muscle pads. Microsurgery. 2005; 25(7): 570–574. PubMed Abstract | Publisher Full Text\n\nKilic E, Hermann DM, Hossmann KA: Recombinant tissue plasminogen activator reduces infarct size after reversible thread occlusion of middle cerebral artery in mice. Neuroreport. 1999; 10(1): 107–111. PubMed Abstract | Publisher Full Text\n\nSena ES, Briscoe CL, Howells DW, et al.: Factors affecting the apparent efficacy and safety of tissue plasminogen activator in thrombotic occlusion models of stroke: systematic review and meta-analysis. J Cereb Blood Flow Metab. 2010; 30(12): 1905–1913. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTrotman-Lucas M, Wong R, Allan SM, et al.: Dataset corresponding to scientific paper “Improved reperfusion following alternative surgical approach for experimental stroke in mice”. [Dataset v1] . Zenodo.2020; http://www.doi.org/10.5281/zenodo.3685595"
}
|
[
{
"id": "61343",
"date": "30 Mar 2020",
"name": "Arshad Majid",
"expertise": [
"Reviewer Expertise Preclinical models of stroke"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a well written concise study on refinement of the MCAO model in mice. The investigators show that repair of the CCA achieves better reperfusion of the hemisphere than ligation of the CCA This article is a useful addition to the literature.\n\nIt would be useful to know how stable the underlying infarction is using this model. These data are not shown.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "5365",
"date": "31 Mar 2020",
"name": "claire gibson",
"role": "Author Response",
"response": "Thank you for this review. In response to the specific point about lesion volume - we did not investigate that within this study as we have previously (Trotman-Lucas M, Kelly ME, Janus J, et al.: An alternative surgical approach reduces variability following filament induction of experimental stroke in mice. Dis Model Mech. 2017; 10(7): 931–938) demonstrated the impact of this refined surgical approach on lesion volume."
},
{
"c_id": "5483",
"date": "06 May 2020",
"name": "Arshad Majid",
"role": "Reviewer Response",
"response": "Thank you for your response."
}
]
},
{
"id": "61345",
"date": "07 Apr 2020",
"name": "Brad Sutherland",
"expertise": [
"Reviewer Expertise Stroke and cerebral blood flow"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript by Trotman-Lucas is a brief report outlining a technical refinement of the intraluminal filament middle cerebral artery occlusion model that leads to improved reperfusion. The authors have previously shown that CCA repair can increase acute CBF during reperfusion and consequently have reduced infarct volume size and variability. This manuscript provides a nice addition with Laser Speckle Contrast Imaging showing reperfusion changes lasting 48 hours between the traditional Koizumi method and the CCA repair method. This simple repair method could improve our ability to conduct stroke modelling. I have some comments below.\nSpecific comments:\nThe Laser Speckle Contrast Imaging method has great spatial and temporal resolution but does not provide any depth assessment of CBF changes in the brain. It only provides a measurement of CBF on the surface of the brain. Therefore, all that can be stated from this study is that pial CBF can be improved post-MCA during CCA repair compared to the CCA ligated method, as we do not know from this imaging technique whether the underlying brain is fully reperfused. Some discussion around this would be useful.\n\nThe Laser Speckle Contrast Imaging was conducted at baseline (prior to MCAO), 24h post-MCAO and 48h post-MCAO. However, there was no apparent assessment during MCAO to show that a stroke had actually occurred. How can you confirm that these animals actually had a stroke with no CBF assessment during MCAO?\n\nThe sustained (48h) changes shown in Figure 1 are interesting. It would have been excellent to have shown how these CBF values are associated with changes in neurological deficit (could be done by a neuroscore) or infarct volume at 48 hours. Were the brains assessed for this?\n\nThe CCA ligated results show sustained hypoperfusion on the surface of the brain for 48 hours. Some extended discussion on how this could impact the development of the lesion and chronic injury would be useful, particularly since chronic occlusion of the CCA is used in vascular dementia studies with chronic neurodegeneration and impaired cognition present.\n\nThere are many studies showing that CBF in the contralateral hemisphere can be altered during MCAO and during reperfusion (e.g. Lavy et al 1975 Stroke 6:160-31; Taheri et al 2019 Transl Neurosci Res Rev 2:27-372; Premilovac et al 2020 JCBFM 271678X209054933 and others). Given that the quantification of the ipsilateral CBF is a % of the contralateral, any changes in CBF in the contralateral hemisphere in response to MCAO or reperfusion could alter the reference point for this analysis. Some discussion should be added about this as a limitation.\n\nThe second last sentence of the discussion “Validating the use of CCA repair…” is an incomplete sentence and will need rewritten.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "5468",
"date": "05 May 2020",
"name": "claire gibson",
"role": "Author Response",
"response": "The Laser Speckle Contrast Imaging method has great spatial and temporal resolution but does not provide any depth assessment of CBF changes in the brain. It only provides a measurement of CBF on the surface of the brain. Therefore, all that can be stated from this study is that pial CBF can be improved post-MCA during CCA repair compared to the CCA ligated method, as we do not know from this imaging technique whether the underlying brain is fully reperfused. Some discussion around this would be useful. Response: Please see added section into discussion. The Laser Speckle Contrast Imaging was conducted at baseline (prior to MCAO), 24h post-MCAO and 48h post-MCAO. However, there was no apparent assessment during MCAO to show that a stroke had actually occurred. How can you confirm that these animals actually had a stroke with no CBF assessment during MCAO? Response: As now stated in the methods section of the paper the drop in CBF during MCAO was confirmed using laser doppler flowmetry. The sustained (48h) changes shown in Figure 1 are interesting. It would have been excellent to have shown how these CBF values are associated with changes in neurological deficit (could be done by a neuroscore) or infarct volume at 48 hours. Were the brains assessed for this? Response: We would agree that any further analysis under these experimental conditions would be useful. However, that was beyond the remit of this current study which was powered to answer the specific research question. We have previously reported the effect of this modified surgical approach on lesion volume (see Trotman et al., 2017, Disease Models & Mechanisms). The CCA ligated results show sustained hypoperfusion on the surface of the brain for 48 hours. Some extended discussion on how this could impact the development of the lesion and chronic injury would be useful, particularly since chronic occlusion of the CCA is used in vascular dementia studies with chronic neurodegeneration and impaired cognition present. Response: Please see amended section in discussion. There are many studies showing that CBF in the contralateral hemisphere can be altered during MCAO and during reperfusion (e.g. Lavy et al 1975 Stroke 6:160-31; Taheri et al 2019 Transl Neurosci Res Rev 2:27-372; Premilovac et al 2020 JCBFM 271678X209054933 and others). Given that the quantification of the ipsilateral CBF is a % of the contralateral, any changes in CBF in the contralateral hemisphere in response to MCAO or reperfusion could alter the reference point for this analysis. Some discussion should be added about this as a limitation. Response: Please see amended section in discussion. The second last sentence of the discussion “Validating the use of CCA repair…” is an incomplete sentence and will need rewritten. Response: This sentence has been amended."
}
]
}
] | 1
|
https://f1000research.com/articles/9-188
|
https://f1000research.com/articles/9-352/v1
|
12 May 20
|
{
"type": "Research Article",
"title": "School closure in response to epidemic outbreaks: Systems-based logic model of downstream impacts",
"authors": [
"Dylan Kneale",
"Alison O'Mara-Eves",
"Rebecca Rees",
"James Thomas",
"Dylan Kneale",
"Alison O'Mara-Eves",
"Rebecca Rees"
],
"abstract": "Background: School closures have been a recommended non-pharmaceutical intervention in pandemic response owing to the potential to reduce transmission of infection between children, school staff and those that they contact. However, given the many roles that schools play in society, closure for any extended period is likely to have additional impacts. Literature reviews of research exploring school closure to date have focused upon epidemiological effects; there is an unmet need for research that considers the multiplicity of potential impacts of school closures. Methods: We used systematic searching, coding and synthesis techniques to develop a systems-based logic model. We included literature related to school closure planned in response to epidemics large and small, spanning the 1918-19 ‘flu pandemic through to the emerging literature on the 2019 novel coronavirus. We used over 170 research studies and a number of policy documents to inform our model. Results: The model organises the concepts used by authors into seven higher level domains: children’s health and wellbeing, children’s education, impacts on teachers and other school staff, the school organisation, considerations for parents and families, public health considerations, and broader economic impacts. The model also collates ideas about potential moderating factors and ethical considerations. While dependent upon the nature of epidemics experienced to date, we aim for the model to provide a starting point for theorising about school closures in general, and as part of a wider system that is influenced by contextual and population factors. Conclusions: The model highlights that the impacts of school closures are much broader than those related solely to health, and demonstrates that there is a need for further concerted work in this area. The publication of this logic model should help to frame future research in this area and aid decision-makers when considering future school closure policy and possible mitigation strategies.",
"keywords": [
"School closure",
"pandemic",
"logic model",
"conceptual framework",
"COVID-19",
"evidence synthesis",
"novel coronavirus"
],
"content": "Introduction\n\nClosing schools in order to limit the transmission of infection during epidemics has long been a recommended course of non-pharmaceutical action under certain circumstances1. The logic underpinning this policy is clear: school children mix at close proximity for extended periods of time, can have poor hygiene, and can then infect parents, carers and other contacts outside of the school setting. School staff are also at higher risk of infection while in the school environment. Previous evidence has shown that closing schools can have the intended impact of reducing infection rates, although factors such as the timing and length of the closures are likely to be important.\n\nIf reductions in rates of infection were the only outcome resulting from closing schools, then their closure would appear to be a powerful weapon to be deployed in epi/pandemic situations. However, the decision to close schools is not straightforward. Firstly, it is not necessarily a binary decision; there are situations where partial closure of schools may be possible/essential or, likewise, the closure of, for example, secondary but not primary schools. Secondly, closing schools can have many downstream impacts which may result in unintended and undesirable outcomes, making the decision as to whether (or when, or how) to close schools, and how long for, much more complicated than it might first appear.\n\nParticularly undesirable impacts can include a reduction in healthcare staff availability, just when their presence may be most important, if parents then need to stay at home to look after children. Other members of a family, such as grandparents, may be called upon to take care of children while parents are at work and, given that higher age groups may be more at risk (as is the case with COVID-192, the disease caused by the 2019 novel coronavirus) school closures may result in increased infection rates in the ‘wrong’ population groups. While some might welcome the opportunity not to attend school, negative consequences for children might include poorer nutrition, due to missed school meals, and increased exposure to dangerous home environments. School closures are likely to have an impact on children’s educational progress, disrupting the delivery of teaching and assessment, and having potentially far-reaching consequences in the case of long-term closures. Of course, there may be some unintended benefits too, such as improved parent-child relations, reduction in anxiety for some students, or the opportunity to focus learning where needed, but these will be idiosyncratic and very much dependent on the home situation.\n\nDetermining the appropriate policy when considering school closures thus requires consideration of a complex range of possible outcomes, which may differ between epi/pandemic scenarios. The 2019 novel coronavirus pandemic has highlighted that, despite many nations having pandemic preparedness plans, the downstream consequences and side effects of school closures are much greater than anticipated. As a result, there has been a collective scramble to address those impacts that are being identified in a reactive – rather than proactive – way. To be better prepared in the future, we need to have a more comprehensive picture of the different factors and impacts of school closures.\n\nLogic models (sometimes called ‘conceptual frameworks’) are a useful tool for decision-makers when considering broader impacts from this type of policy decision, and they can aid planning regarding other measures that might need to be put in place in order to mitigate unwanted impacts3. Logic models are tools used to depict assumptions graphically about the chains of processes, activities or events expected to occur during the implementation of an intervention (in this case, school closures), and the way in which these lead to changes in outcomes. Logic models provide an initial set of assumptions of how different intervention components or intervention models are expected to change outcomes, and can be used to develop further sub-research questions to investigate the validity of these assumptions. In addition to being tools for theorising impacts, logic models are also tools for communicating findings, and in this case can provide a visual account of the possible impacts of school closures across different domains.\n\nOur aim here is to develop a systems-based logic model as an aid in theorising the complexity of school closures and the broader context and impacts, and how these interact4. System-based logic models may be particularly useful in accounting for the myriad ways in which different interconnecting components of health, educational, economic, and social systems are impacted by school closures. The model will provide a starting point for theorising whether there are aspects of the context (setting and existing health/social/education infrastructure) or population(s) that are likely to facilitate or hinder the implementation and effectiveness of school closures, and whether there are particular groups likely to be disproportionately adversely impacted. It is also hoped that the concepts identified in the literature will be supplemented by input from a range of stakeholders, leading to the creation of a more complete model of potential impacts of school closures.\n\n\nMethods\n\nThe development of this logic model followed a systematic plan in order to ensure that: a) it represents the state of current knowledge and thinking on the subject; and b) that it is rooted in empirical research and not solely the prior opinions of the authors. Although we have adopted a systematic plan and attempted to document all decisions to enhance the transparency of our process, this is not a formal systematic review.\n\nThe aim of finding research to inform the development of a logic model is to enable the team to develop a comprehensive picture of the concepts – and the relationships between them. We do not need to find every paper on the subject, but simply a sufficient range of documents to enable us to create a complete model. Thus, the aims of searching differ here from a typical systematic review, in which we would aim to find as many relevant papers as possible to increase precision and reduce (statistical) uncertainty around a given outcome. Given the aim of searching for logic model development, we used ‘snowballing’ methods to locate relevant papers, beginning with a set of known records and moving out from them in a systematic way. This enabled us to refine the logic model iteratively as new papers were discovered and to maximise the efficiency of the search.\n\nAs the context of this research is the 2019 novel coronavirus outbreak, we began with a set of nine records for published papers included in a review of school closure and management practices during coronavirus outbreaks1. We used the Microsoft Academic dataset (updated date: 30 January 2020) to locate and retrieve, in addition to the set of nine: all the records in the bibliographies of these papers, all records that cite those papers, and all records that Microsoft Academic ‘recommends’ as being ‘related’ to those records. We then filtered this list to identify the records that mentioned “school” and “closure” in their titles or abstracts.\n\nWe conducted supplementary searches to broaden the representation of disciplines and service sectors in our mapping. These included: title- and abstract-focused searches of the educational, psychological and social services literature via the ERIC, PsycINFO and ASSIA databases (see Extended data5); an online hand search (27/03/20) of records containing the word “school” within the Cambridge University Press journal ‘Disaster Medicine and Public Health Preparedness’; searches using Google Scholar for studies that themselves cite selected included studies (studies with implementation, policy and/or attitude as a term in the paper’s title). Several policy documents were also included6,7.\n\nScreening and coding the studies occurred in parallel. Studies were excluded based on their titles and abstracts if they were:\n\nnot focussed on school closures,\n\nnot focussed on planned school closures, or\n\nfocussed on unplanned school closures not related to pandemics.\n\nRecords were flagged as key papers if they were identified (from title and abstract) as having good breadth and depth of the study and a potential for helping us to identify new concepts.\n\nAt the same time, we conducted an initial examination of how each paper contributed relevant concepts to the model by coding terms contained in the title and abstract. The codes were developed as we progressed through the list of records; they were not developed a priori as we wanted the evidence to drive the model.\n\nCoding reflected (i) the type of intervention (i.e., form of school closure and whether there were concurrent interventions); (ii) impacts (short and long-term, societal and individual outcomes); (iii) context, including the reason for closure (e.g., type of pandemic) and location (country/countries); and (iv) potential moderators. The broad study design was also coded at this stage.\n\nImportantly, we decided to not code extensive information about the preconditions of the school closures. This is because we want to draw more attention to the huge range of possible outcomes and impacts that could result from school closures. Where the preconditions (e.g., features of the virus itself) were presented as potentially moderating or mediating the effect of the intervention on the impacts, we recorded that information as a factor. This decision has inevitably constrained the scope of our model to make the closure of the schools the starting point of the model, although clearly much has to happen beforehand to determine the introduction – and form – of the school closure policy.\n\nWe then retrieved the full-text documents of the key papers to check for information not already captured at the title and abstract stage. A number of studies were deprioritised as reporting studies where the concepts had already been identified (i.e., we had reached conceptual saturation), mainly reflecting simulation studies that modelled the hypothesised impact of school closures on viral transmission rates, or peripheral studies where the impact of school closures was not a focus of the study. Full texts were not obtained for these deprioritised studies.\n\nWorking with the extracted codes, and Miro software, we sorted the impacts identified in the literature into logical chains, drawing as much as possible from the information contained within the studies themselves to theorise the sequence of outcomes. This stage was largely conducted in collaborative virtual meetings between the authors, in which we discussed the positioning of the codes that we had identified and added any unrepresented factors (see below).\n\nThe codes were arranged according to broad domains (e.g., impact on children’s education), and chains of outcomes were hypothesised in some cases to help identify more distal outcomes3. Additional factors that could moderate the impacts of school closures including broader context and setting factors only appear as a list in the model due to the complexity of relationships. We also considered whether more complex relationships between outcomes needed to be depicted (particularly vicious or virtuous cycles but also instances of conjunctural causation). Working collaboratively, the team met at several points to discuss the evolving logic model.\n\nA number of factors were not explicitly represented in the literature, the most prominent gap being impacts on children’s educational achievement and development. We supplemented the model with additional hypothesised factors, shaded in a different colour (green), to indicate that these were not explicitly mentioned in the included literature. These are largely drawn from the media, policy documents, or anecdotal evidence (e.g., the experience of the researchers). These were added in acknowledgement that not all academic papers have considered all possible impacts (indeed, this concern was one of the motivating factors for undertaking this work).\n\nAlthough the school closure intervention is the starting point for our model, we decided that it was easier to view the model with the intervention in the centre and the proposed impacts emanating out from there. It is important to bear this in mind when viewing the model: the ordering in the diagram does not reflect temporal or causal ordering, nor does it indicate order of importance—we did not collect data on these aspects. The positioning of the impacts emanating from the centre is largely determined by aesthetics and readability, such as by placing linked concepts near each other to minimise arrows stretching across the model.\n\nIn order to develop our model around potential impacts of school closures, and in line with recommended practice in constructing logic models8, we are publishing an early version of the model for input across a range of stakeholders and peer reviewers. On the basis of this input, a second version of the logic model will be published, following an iterative model, and we hope to investigate the evidence underlying the outcome chains further. We expect that stakeholder involvement will help us to consider further impacts, drawing on the principles of ‘dark’ logic models through incorporating the expertise and insight of individuals or groups from different contexts within the UK (and beyond)9.\n\n\nResults\n\nOur initial search of Microsoft Academic resulted in a set of 205 unique records to examine. We then added additional records from supplementary searches of databases, reference lists (including systematic reviews), and policy documents. When the additional records are included, we had 288 unique papers to utilise to identify the concepts present in our map (Figure 1). A total of 177 papers were used in some way (though some contained concepts we had previously identified and were not examined in detail) and 111 were not relevant. (See Underlying data5 for a list of the 177 references.)\n\nThe resulting logic model (see Figure 2) contains more than 100 concepts that are organised into seven higher level domains: children’s health and wellbeing, children’s education, impacts on teachers and other school staff, the school organisation, considerations for parents and families, public health considerations, and broader economic impacts. As mentioned above, the positioning of the higher-level domains in the model is not designed to indicate their importance or temporal ordering. In addition, a large number of effect moderators were identified, organised under five headings: pandemic related factors, closure related factors, child related factors, social/political factors, and environmental factors. The final component in the logic model is the presence of the ethical principles that apply throughout school closure processes and which are embedded in decision-making.\n\nThe graphical depiction of the logic model is shown in Figure 2. As this is a large image, we have shared a link to the Miro board where an interactive version can be found, here: https://miro.com/app/board/o9J_kuvvCJo=/. Concepts and relationships that were identified in the research literature are coloured in bold with purple connecting lines. Concepts identified through team discussion and their engagement with wider literature (e.g., stories in the media) are depicted in italics with green connectors.\n\nStudy characteristics. The logic model was developed from concepts identified in 177 included studies. These considered the impacts of school closures, either in isolation or along with other pandemic mitigation strategies. The majority of papers considered the impacts of school closures related to the pandemic H1N1/09 virus in 2009 (also known as swine flu) (n = 74 papers)10,11. This strain disproportionately affected children and younger adults10, and school closures were part of pandemic mitigation strategies across a number of different countries. However, the decision to close schools during this pandemic was discretionary in several settings and dependent on the number of cases in the school or the immediate locale (for example12). as well as other factors, and often not part of nationwide pandemic control measures. Furthermore, the closure of schools tended to be for a much shorter period than in the current 2019 novel coronavirus pandemic, for example for a week13, although there may have been recurring closures in some settings (for example14).\n\nOther papers considered the impact of pandemic flu more generally (n = 60 papers) including the 1919 flu pandemic (n = 3 papers), or considered other forms of infectious disease or pandemics (n = 24, e.g., Hand, Foot and Mouth disease), or the response of schools to localised outbreaks of cases within schools (n = 4). A smaller number of papers considered recent school closures in the context of recent outbreaks including severe acute respiratory syndrome in 2002-2004 and Middle East respiratory syndrome, another form of coronavirus outbreak identified in 2012. As with H1N1, closure decisions were localised and reactive or discretionary in some settings (for example15), although they were more widespread and part of public health policy in others (for example16,17); however, closures tended to be shorter than those being implemented in the 2019 novel coronavirus pandemic (for example17). Finally, six studies were identified as being focussed on the2019 novel coronavirus pandemic from different settings including China (n = 3), Hong Kong (n = 1) and the United States (n = 2). As these are studies considering closures currently in progress, they may not fully capture the potential short and long-term impacts of sustained periods of school closures.\n\nOverall, the composition of the included studies means that the concepts in the logic model are generally drawn from studies on short-term school closures that may not be implemented as nationwide policy. Such scenarios are vastly different to the 2019 novel coronavirus pandemic, which has seen the introduction of widespread and long-term non-pharmaceutical interventions, of which school closures is just one. This makes obtaining input from a wide range of stakeholders to supplement the concepts represented in the logic model all the more important.\n\nWe will now briefly outline the main concepts in the logic model, beginning with the intervention, then the seven higher level domains, then the moderators, and finally the ethical frameworks.\n\nThe intervention: school closure. The form of the school closure—that is, the way that the policy intervention was implemented—was seen to vary substantially between epidemics and by country/region/jurisdiction (Figure 3). In most cases, the closure of schools was one of a number of pharmaceutical interventions (PI, such as vaccine) and or non-pharmaceutical interventions (NPI, such as public transport restrictions and social distancing of confirmed cases). This is very important to bear in mind, as the presence of pharmaceutical interventions such as vaccines and treatments may greatly reduce the need for NPIs, while the converse is also likely. Further, in a complex system of NPIs over a long period of time, the relative costs and benefits of all measures need to be considered. For example, some of the impacts covered in the domains below are likely to be exacerbated in a stringent social distancing scenario (“lockdown”), but might be less problematic if other social distancing measures are relaxed.\n\nThe geographical coverage of the closure was also quite variable; in some studies, only schools with confirmed cases closed (referred to as reactive closure), whereas others involved a school district or wider region (e.g., a state or country) and could be considered proactive closures (closing even if the school has not had a confirmed case within its student population).\n\nThere are various models of school closure. Whole school closures are the most intensive, but other partial options indicated include just some types of school (e.g., just nurseries), targeted class or grade closures within a school, or alternating class attendance on different days of the week or alternating weeks.\n\nThe duration of the closure is likely to be one of the biggest factors in determining the impacts, and some impacts may not be relevant for very short closures. Very short-term closures, for example, are unlikely to have much impact on educational achievement or future earning potential, although this is hypothesised rather than detail we extracted from the papers. Linked to duration, some papers mentioned reopening the school in waves—i.e., subsequent reclosing of a school after a period of reopening, usually in response to new confirmed cases or additional peaks in the pandemic.\n\nIn summary, the policy intervention, school closures, took many forms. Although it was not within the scope of this work, we suspect that the form of the school closure is likely to change the logic model quite substantially, with some impacts only being observed in longer term closures or when broader social distancing measures are in place.\n\nHigher-level domains\n\nImpacts on children’s health and wellbeing\n\nAlthough parents may be concerned about the impact of school closures on their children’s health and wellbeing, there were relatively few concepts identified from studies that provided much indication of the nature of these impacts (Figure 4). Studies had considered that closure of schools also entailed losing access to school meals, which we hypothesised would exacerbate health inequalities if access to subsidised meals was removed, as well as broader forms of inequality. The literature had considered that closing schools would disrupt and reduce children’s social interactions, although some studies also suggested that some children would continue to socialise. Such impacts are likely to be affected by the presence or absence of additional social distancing measures, and how stringently they are enforced or adhered to.\n\nClosing schools could impact on children’s health, through increasing levels of anxiety, as well as impacting on access to school-based health care, which in turn was hypothesised to interrupt public health strategies such as vaccination programmes for other diseases (e.g., MMR) and to interrupt care provided to children with special health needs (e.g., asthma). Some studies considered increasing levels of self-care or inadequate care arising from the closure of schools, which in turn was hypothesised by the team to increase the risk of neglect or accidents occurring, the latter in turn being linked with a risk of injury. Given that most of the included studies examined short-term closures, the risk of these factors occurring may be different in the context of the 2019 novel coronavirus pandemic where school closures may last several months, and these potential impacts should be considered in light of the list of moderators (discussed below).\n\nThe logic model also contains a number of impacts that were not considered within the research literature, but instead were proposed by our team, and included potential serious negative impacts (e.g., increased risk of abuse and violence) as well as some positive impacts (improved parent-child relationships and improved mental health for some children). These additional impacts are represented in italics with green arrows in the diagram. Finally, although not based on academic literature, school closures may increase the risk of child marriage and child labour, a concern flagged by the United Nations7. Clearly school closure is going to be experienced differentially across existing lines of inequality, and although some of the hypothesised pathways are represented here, the publishing of this model for public input is expected to enhance our understanding of the impacts of school closure on children’s health and wellbeing.\n\nImpacts on children’s education\n\nRelatively few concepts were identified in the literature that indicated the impact of school closures on children’s education, a likely artefact of the short duration of closures examined in extant studies (Figure 5). Studies considered children missing classes and lessons, which may impact on their future earning potential, as well as disrupting attendance patterns once schools reopen. If the children’s mode of education shifts to online once schools close, this might exacerbate inequalities where children are unable to access suitable equipment at home and may mean that some children are particularly vulnerable to missing classes and lessons. A hypothesised implication of shifting education to online modes is that children are expected to become independent learners earlier than expected, and earlier than is developmentally suitable.\n\nHypothesised consequences of children missing lessons are that the development of their basic skills (e.g., literacy, numeracy) may be compromised, and children may not be taught subjects that are more challenging to deliver through distance learning (e.g., Music or Design & Technology). Although there has been widespread concern in the media about the cancellation of exams18 due to the 2019 novel coronavirus, this has generally not been explored in literature on pandemic-related school closures. Similarly, we can only hypothesise that school closures will impact on transitions between different settings (nursery/kindergarten to primary school; primary to secondary; secondary to college or university). We also hypothesise that children with special educational needs may lose additional support ordinarily received within educational settings, exacerbating educational inequalities. In contrast to other domains, the impacts of school closure on children’s education and potential ways of mitigating these impacts are mainly hypothesised, and the concepts represented tended not to be drawn from the literature.\n\nImpacts on teachers and other school staff\n\nWe found this area of impacts also to be a relatively unexplored one in the research literature. We found research papers that discussed impacts on teachers in terms of increased workloads during school closure and stress in the run up to closure. Concepts not found in the included research or policy literature but evident elsewhere (see italics, Figure 6) include concerns over job losses for school staff with less secure contracts and interruptions to teacher training.\n\nImpacts on the school as an organisation\n\nWe found reference in research papers to changes in teaching provision, in terms of moves to teaching at a distance (Figure 7). Studies also referred to interactions with bodies external to the school such as those with responsibility for public health or social care, as well as communications with parents. The latter of these explored the challenges of handling large numbers of queries, and challenges in reaching all parents with communications. There was reference to resources being diverted to deal with additional communication work.\n\nAspects considered where schools are only partially closed were found only in the policy literature (and so are identified in italics in the logic model). These included the identification of children who should be encouraged still to attend (e.g., those of key workers, or children judged vulnerable by social care authorities); the implementation of procedures to promote social distancing and hand-washing; the reorganisation of the curriculum in order to prioritise safeguarding; the reorganisation of teaching staff rotas (e.g., to cover holiday periods, absent staff, and alternative teaching settings); the managing of furloughing (e.g., for temporary staff); and aspects of student transport.\n\nParent and family level considerations\n\nThe research literature explored the potential for impacts on parents and families in multiple areas, with those associated with obtaining alternative childcare at the fore (Figure 8). This conceptual area, as with impacts of school organisation and school staff, tended to be determined by researchers in their setting of questionnaire items but were also sometimes contributed directly by study participants who had been asked open-ended questions.\n\nHere, parents may find themselves needing to juggle a need to work from home with looking after their children – and possibly taking a role in their education at the same time. In situations where the parent cannot work from home, they may need to take time off which, in extended periods of school closure, may be unpaid, and they may be at risk of losing their job if they cannot work. Sometimes, parents will arrange alternative childcare which might be expensive, increasing household expenditure at a time of economic uncertainty.\n\nThere is also a risk that alternative childcare provision might be unsuitable (for example, there can be increased risk of injury where an older, but nevertheless young, sibling is called upon to look after their younger sibling(s)). Pre-existing employment insecurity may be magnified in these situations, and households with limited disposable income will be less able to pay for childcare; thus, disadvantaged groups are more at risk than others, and gaps and gradients in inequalities might be increased in some situations. Of relevance here are potentially unequal gender impacts, with women more likely to take on increased childcare responsibilities, and single-parent households likely to find the situation more challenging. Uncertainty in the duration of the school closure can make planning difficult, which might also have economic and mental health consequences for those attempting to make alternative working and childcare arrangement without knowing whether they are planning for several weeks, or months. Dealing with the above can have a detrimental psychosocial impact on parents and carers.\n\nBroader economic impacts\n\nResearchers have examined possible broader economic impacts resulting from school closures (Figure 9). Given that the main aim of closing schools is to reduce infection rates in epidemic/pandemic situations, the cost effectiveness of this is considered in a range of ways. Some papers carry out traditional health economic analyses, examining impacts in terms of quality-adjusted life years, disability-adjusted life years and years lost due to disability. Others examine the impact of parental and carer absence on economic productivity and how this might impact negatively on GDP. Other analyses consider how the downstream impact of school closures might result in a reduction in population level healthcare costs.\n\nPublic health considerations\n\nMost of the papers that we identified focused on the public health considerations of school closures (Figure 10). The observed or predicted reduction in the transmission rate was by far the most common issue considered. Some of the papers assessed the likely reduction in transmission rate under certain conditions, such as the infectivity rate, combination with other interventions (e.g., availability of a vaccine or other social distancing measures), or duration of the closure. Some simulation (modelling) studies questioned the cost-effectiveness of school closures in certain scenarios. The consideration of moderators is likely to be very important in understanding the impact of school closures on virus transmission rates.\n\nOther public health considerations were mentioned in the literature. For example, mortality rates were often considered in terms of the relevant sector of the population that might see an improved mortality rate. Some studies noted that school closures may affect mortality rates in age groups beyond the school age via the reduction in transmission rates outside of the school setting.\n\nChanges to the spread of the virus were also considered. In particular, limiting the spread of the pandemic to other geographical areas beyond the initial observed site/s and interruptions to the duration or the nature of the epidemic (including the timing of peaks of viral infection) were mentioned. Unsurprisingly, the impact on the health care service was considered in some studies. This included discussion of hospital and intensive care unit admissions, the purchasing of virus-relevant medications, and visitations to GPs.\n\nThe removal of children from a school setting was proposed to have a potential adverse impact because schools are then unable to reinforce health messaging. There may also be changes in the hygienic behaviours of children, such as hand washing, as they would have in the school, although this could be compensated by changes to hygienic behaviours in the home.\n\nOne public health impact is linked in our model to other impacts discussed above under parent and family considerations and broader economic impacts. This is seen as stemming from a reduction in the healthcare labour force when frontline healthcare workers (doctors, nurses, etc.) need to care for children at home and are unable to work or can only work reduced hours. There is a hypothesised consequent increase in the overall mortality rate (not just due to the pandemic), as health service demand exceeds capacity.\n\nMediators and moderators. Every study that we looked at either directly or in passing considered potential moderators or mediators of the impacts of school closures. These are factors that might change the impact that school closures have on a given outcome. The relation between the school closures intervention and key outcomes is likely to be very influenced by a range of factors working together in a complex relationship. Because of the complexity of these relations, we have not generally tried to include the factors in the pathways of the logic model; instead we are listing them as factors to consider when viewing the relation between school closure and proposed outcomes. We have grouped the factors into similar types of concepts.\n\nPandemic-related factors, which are those that pertain to the virus itself. These include the type of virus (e.g., specific influenza strains); viral infectivity (or virulence, the ability of the virus to establish an infection); transmission rate (the speed at which new transmissions occur, although this sometimes appears to be used interchangeably in some papers with reproductive ratio); availability of antivirals or vaccines; whether the virus exhibits asymptomatic transmission; availability of testing (virus or antibodies). There are likely to be others not observed in our sample of studies, including the infectious period (how long an infected person is able to transmit the infection) and mode of transmission (airborne, faecal-oral, vector-borne, etc).\n\nClosure-related factors, which are those that relate to the way in which the closure is implemented. This overlaps with the features of the intervention itself, which are listed in the central box of the model. Closure-related factors may include the timing of the closure in relation to pandemic (e.g., before or during the peak of infections); length of closure; geographical coverage (one or small number of schools, a whole school district, city, region, nation); partial or full closure within the schools (e.g., only certain grades, or certain students); whether closures are reactive (closure after a confirmed case) or proactive (closure before confirmed case); closure/reopening decision mechanism (e.g., is it discretionary or mandatory, and who decides).\n\nChild-related factors, which are those factors relating to the students whose schools have closed. These include the age of the affected children and whether they continue to meet or socialise outside of the school setting.\n\nSocial/political factors, which are those that relate to the social or political context in which the school closures take place. These include child or household characteristics (e.g., socioeconomic status); scepticism of intervention efficacy (i.e., doubt that the school closure will have the desired effect or the benefits outweigh the costs); level of support; differences in adherence to social distancing; the knowledge/attitudes of people in charge of schools; the location of household/school (e.g., rural versus metropolitan, areas of social deprivation); and type of school (e.g., public/ private).\n\nEnvironmental factors, which are those that relate to the environmental context of the pandemic. These include the weather, season, and geographical scale of the infection’s reach\n\nEthical and local policy implementation factors. School closures raise a number of questions for decision-makers around the way in which they should be implemented and how they can be implemented ethically and equitably. Closure decisions made within small areas (e.g., local or regional districts) raise particular considerations with regards to implementation, and may require considering developing policies across different policy domains. One study drew on an existing framework for decision-making during pandemic preparation19 that includes processes to be implemented, and values to be considered, in ensuring an ethical response to pandemic preparedness. The study20 adapted the original framework to examine the implementation of decisions around school closures as a result of H1N1 pandemic, and considered processes including (i) accountability, openness and transparency (decisions are communicated and open to scrutiny); (ii) inclusivity (decisions are made with the views of stakeholders in mind); (iii) reasonableness (decisions are based on evidence, principles and values); and (iv) responsiveness (decisions are revisited on the basis of new information). In addition, these were supported by values including (i) a duty to provide care; (ii) protection of the public from harm; (iii) stewardship; (iv) solidarity; (v) privacy; (vi) individual liberty; (vii) equity; (viii) proportionality; (ix) reciprocity; and (x) trust.\n\n\nDiscussion\n\nThe logic model presented here presents a summary of the concepts considered by studies on pandemic-related school closures, grouped into domains of impacts. Many of the public health impacts considered in the literature involve examining reduction in viral transmission rates, lowering mortality rates, and limiting the spread of the pandemic, suggesting that the literature has tended to explore the positive epidemiological impacts of closures. Other domains, such as the impact on parents and families, children’s health and wellbeing, and children’s education, are largely composed of concepts relating to the negative impacts of school closures. Similarly, many of the broader economic impacts considered by the literature involve negative impacts on productivity and GDP, as well as consideration of cost-effectiveness of closures. Concepts around the impact on the school as an organisation and on teachers reflected some of the impacts of (sudden) pandemic-related closures and the resulting disruption on school functioning and teacher workload. Some of the impacts considered involved longer-term impacts of closures, such as the impact of the loss of education on children’s future earning potential. Across several of the domains, many of the concepts identified from the literature are hypothesised to exacerbate inequalities, including health and educational inequalities, if left unchecked.\n\nDue to the nature of the extant evidence base, which mainly reflected evidence on shorter-term, often reactive or discretionary, school closures, few of the concepts reflected interventions or steps to mitigate the impact of school closures. Some of the concepts did reflect potential adaptations to the closure of schools, notably the movement of learning to online means, a concept that emerged in studies on the 2009 H1N1 pandemic. However, in turn, there was also consideration of how movement of teaching online could exclude those children without computers or reliable internet access, thereby exacerbating inequalities in the impacts of the pandemic among children from poorer socioeconomic backgrounds.\n\nSome of the studies examined the implementation of school closures. Although these were of peripheral interest given our primary concern around mapping and hypothesising the impacts of school closure, these did nevertheless illuminate the challenges of making school closures decisions. In addition, some that were more focussed on implementation also provided frameworks around how the impacts of school closures could be understood from an ethical perspective20, providing potential insights into the types of process and value considerations that might be useful to incorporate in the design of policies and interventions to mitigate the potential negative impact of school closures.\n\nThis logic model represents a map of concepts from studies identified through a rapid systematic search. The concepts were mainly drawn from those mentioned in the title and abstract of studies, and only those studies that appeared to have greater breadth and depth were selected for full text review to help to identify new concepts. While this is a pragmatic strategy, suitable to our purpose of identifying concepts but not synthesising findings, it does mean that some more marginal concepts may have been missed from the logic model. Similarly, although the model is based on a systematic search with supplementary searches of policy documents, many of the steps that could have been conducted in duplicate in a systematic review (e.g., screening and coding of studies) were instead conducted by a single researcher because of the rapid nature of the study. In addition, although a concept may have been considered within a study, this does not give an indication as to the weight of evidence supporting that concept.\n\nAlthough the model considers some elements in the implementation of school closures, the model does not explicitly represent the rationale or triggers for closure (or non-closure). This includes those factors that influence the decision to close (e.g., legal or jurisdictional factors), levels of public support, the type of closure policy set in place (e.g., all or some schools, or parts of schools, closing), or the adherence to closure. These factors were beyond the direct scope of the model but are likely to further moderate the impact of closures. They may also be important elements for decision-makers to consider in developing policy around the re-opening of schools.\n\nThe map is reflective of the global literature, although there are likely to be heightened challenges in closing schools in a number of different contexts and scenarios. While these factors, which may moderate the severity of impact of closure are represented in the model, few studies were explicitly focussed on any of these groups or settings. For example, few studies explored the impact of closures in the case of special schools, or focussed on children with particular educational or social needs, or focussed on disadvantaged groups. Similarly, although the logic model is representative of the available global literature on pandemic-related school closures, studies exploring the impact of school closures on children in the Global South were largely absent.\n\nAs outlined earlier, most of the studies examined the impacts of short-term school closures. Although most of the concepts outlined are applicable to the current context, the implementation of longer-term school closures with uncertain end dates may introduce a number of new impacts that are not fully represented in the model. Further input is welcome to supplement the concepts represented, and we intend to engage with both reviewers as well as a broad range of stakeholders openly and transparently during the peer review process in creating a revised and expanded model. This iterative approach represents a strength of the study, and obtaining the input of stakeholders in this way is a key recommended process in the development of a logic model to strengthen the salience of the model and its value in subsequent research activities21. Different stakeholders (e.g., evaluators, policy-makers, community leaders, parents, teachers, children) tend to hold different views and understandings, which are useful to incorporate when dealing with the uncertainty and complexity in pandemic emergencies. We hope that this involvement can help us in supplementing the range of concepts identified, provide a useful challenge to the assumptions we have made, ensure that a diversity of perspectives are represented, help us in identifying how contextual factors may amplify or dampen the impacts of school closures, and enhance the usefulness of the model21. The revised logic model is intended to be used as a framework to guide further research, as well as a tool for policy-makers to consider the range of different impacts that follow from school closures.\n\nWhile the model represents some of the downstream impacts resulting from school closures, we know little about the long-term impacts, and the model is underrepresented in a number of domains. Similarly, there were few studies employing quasi-experimental methodologies to understand the causal impacts of pandemic-related closures on outcomes other than health. In the absence of evidence based on a range of methods and considering a breadth of impacts, both long-term and short-term, it is also questionable whether pandemic preparation plans that are being deployed in the 2019 novel coronavirus pandemic also consider a sufficient range of outcomes, and by extension, have developed strategies on how these could be mitigated.\n\nThe model is designed to cover as many impacts as we could identify, but it needs to be emphasised that not all impacts will be observed in all scenarios, or to the same extent. For instance, short term closures or those without concurrent additional social distancing measures are likely to mean some impacts are irrelevant and could be dropped from the model for that scenario. In any scenario, the moderators and mediators might reduce some impacts to negligible levels. And even within the same scenario, different individuals will experience the impacts in different ways. We have not made any attempt to quantify the strength of the relationships partly because of the extreme complexity and the idiosyncratic nature of the pandemics, the closure implementations, the moderating factors, and the individual responses to and experiences of pandemic policy interventions. In other words, we present this model as an aid to thinking about what might be important, rather than suggesting that school closures will necessarily lead to any or all of the proposed impacts; such assessments will need to be done with the full contextual information of a given pandemic scenario.\n\n\nConclusions and future directions\n\nMany papers aim to model the impact of school closure in isolation, rather than as part of a package of social distancing measures. They often conclude that it is difficult to isolate effects on transmission rates, and where these are estimated, they tend to be small. However, identifying the impacts of school closures in isolation is not particularly useful in terms of informing decision-making, since school closures are usually only part of a much broader range of measures. Our examination of the range of potential impacts of school closure shows how widespread and wide-ranging they might be. Modelling transmission within its wider context – and including some of the mediators and moderators we have identified – may result in more actionable recommendations for decision-makers.\n\nPart of understanding the wider impacts of school closures entails ensuring a broad range of perspectives are represented within the research literature. Notable within the body of research encountered in developing the logic model was the absence of studies published in psychological, educational and social care journals, among others. Clearly the impacts of school closures are much broader than those related solely to health, and the logic model and its representation of impacts that are considered in the literature highlights that there is a need for further concerted work in this area. This includes setting a multidisciplinary research agenda that represents the perspectives of educational and social researchers, as well as health and epidemiological researchers, in order to broaden our understanding of the impacts of school closures.\n\nTwo further revisions to the model will be considered after further stakeholder input. Firstly, we may seek input from researchers who have published on the impact of school closures, to discuss our model and the validity of the underlying assumptions. Secondly, we may consider whether different models are needed for different scenarios of interest to better clarify the concepts and outcomes. For example, we will consider whether different models are required based on the closure of different types of school (e.g., primary vs secondary school) or different patterns or models of closure and potential reopening of schools.\n\n\nData availability\n\nZenodo: School closure in response to epidemic outbreaks: Systems-based logic model of downstream impacts. http://www.doi.org/10.5281/zenodo.37803485.\n\nFile ‘Underlying data - Studies used to develop logic model’ contains the list of references used to develop the model described herein.\n\nZenodo: School closure in response to epidemic outbreaks: Systems-based logic model of downstream impacts. http://www.doi.org/10.5281/zenodo.37803485.\n\nFile ‘Extended data - search strings’ contains the search strings used in the literature search performed in model generation.\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).",
"appendix": "References\n\nViner RM, Russell SJ, Croker H, et al.: School closure and management practices during coronavirus outbreaks including COVID-19: a rapid systematic review. Lancet Child Adolesc Health. 2020; 4(5): 397–404. PubMed Abstract | Publisher Full Text\n\nWu Z, McGoogan JM: Characteristics of and Important Lessons From the Coronavirus Disease 2019 (COVID-19) Outbreak in China: Summary of a Report of 72 314 Cases From the Chinese Center for Disease Control and Prevention. JAMA. 2020. PubMed Abstract | Publisher Full Text\n\nKneale D, Thomas J, Harris K: Developing and Optimising the Use of Logic Models in Systematic Reviews: Exploring Practice and Good Practice in the Use of Programme Theory in Reviews. PLoS One. 2015; 10(11): e0142187. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRehfuess EA, Booth A, Brereton L, et al.: Towards a taxonomy of logic models in systematic reviews and health technology assessments: A priori, staged, and iterative approaches. Res Synth Methods. 2018; 9(1): 13–24. PubMed Abstract | Publisher Full Text\n\nKneale D, O'Mara-Eves A, Rees R, et al.:School closure in response to epidemic outbreaks: Systems-based logic model of downstream impacts. 2020. http://www.doi.org/10.5281/zenodo.3780348\n\nThe Alliance for Child Protection in Humanitarian Action: Technical Note: Protection of Children during the Coronavirus Pandemic. Version 1, 2020. [cited 2020 Apr 8]. Reference Source\n\nUnited Nations: Shared responsibility, global solidarity: Responding to the socio-economic impacts of COVID-19. [cited 2020 Apr 8].Reference Source\n\nKneale D, Thomas J, Bangpan M, et al.: Conceptualising causal pathways in systematic reviews of international development interventions through adopting a causal chain analysis approach. J Dev Eff. 2018; 10(4): 422–437. Publisher Full Text\n\nBonell C, Jamal F, Melendez-Torres GJ, et al.: ‘Dark logic’: theorising the harmful consequences of public health interventions. J Epidemiol Community Health. 2015; 69(1): 95–8. PubMed Abstract | Publisher Full Text\n\nCDC: 2009 H1N1Pandemic (H1N1pdm09 Virus). [cited 2020 Apr 8]. Reference Source\n\nKhandaker G, Dierig A, Rashid H, et al.: Systematic review of clinical and epidemiological features of the pandemic influenza A (H1N1) 2009. Influenza Other Respir Viruses. 2011; 5(3): 148–56. PubMed Abstract | Publisher Full Text | Free Full Text\n\nDavis BM, Markel H, Navarro A, et al.: The effect of reactive school closure on community influenza-like illness counts in the State of Michigan during the 2009 H1N1 pandemic. Clin Infect Dis. 2015; 60(12): e90–7. PubMed Abstract | Publisher Full Text\n\nEffler PV, Carcione D, Giele C, et al.: Household responses to pandemic (H1N1) 2009-related school closures, Perth, Western Australia. Emerg Infect Dis. 2010; 16(2): 205–11. PubMed Abstract | Publisher Full Text | Free Full Text\n\nUchida M, Tsukahara T, Kaneko M, et al.: Effect of short-term school closures on the H1N1 pandemic in Japan: a comparative case study. Infection. 2012; 40(5): 549–56. PubMed Abstract | Publisher Full Text\n\nLee IS, Yoon JH, Hong EJ, et al.: Schools’ Response to MERS(MERS-CoV) Outbreak: Schools’ Discretionary Response in Absence of Control Tower. J Korean Soc Sch Heal. 2015; 28: 188–199. Publisher Full Text\n\nCowling BJ, Ho LM, Leung GM: Effectiveness of control measures during the SARS epidemic in Beijing: a comparison of the Rt curve and the epidemic curve. Epidemiol Infect. 2008; 136(4): 562–6. PubMed Abstract | Publisher Full Text | Free Full Text\n\nRao N: Sars, preschool routines and children’s behaviour: Observations from preschools in Hong Kong. Int J Early Child. 2006; 38(2): 11–22. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWeale S, Batty D: Fears that cancelling exams will hit BAME and poor pupils worst. The Guardian. 2020 [cited 2020 Mar 19]. Reference Source\n\nThompson AK, Faith K, Gibson JL, et al.: Pandemic influenza preparedness: an ethical framework to guide decision-making. BMC Medical Ethics. 2006; 7: E12. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBraunack-Mayer A, Tooher R, Collins JE, et al.: Understanding the school community’s response to school closures during the H1N1 2009 influenza pandemic. BMC Public Health. 2013; 13(1): 344. PubMed Abstract | Publisher Full Text | Free Full Text\n\nKneale D, Bangpan M, Waddington H, et al.: Using logic models in research and evaluation of health emergency and disaster risk management interventions. In: WHO guidance on research methods for health emergency and disaster risk management."
}
|
[
{
"id": "69702",
"date": "18 Aug 2020",
"name": "Lisa Pfadenhauer",
"expertise": [
"Reviewer Expertise Public Health",
"Framework synthesis",
"school closures during Corona",
"Qualitative research",
"logic model development"
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you very much for providing me with the opportunity to review this very interesting article. This is a very much needed analytical framework which allows researchers and policy-makers alike to structure their thinking around the downstream effects of school closures as widely debated non-pharmaceutical intervention. Please find below a couple of minor comments and thoughts:\nIntroduction: Overall, I think the introduction would benefit from more literature, e.g. on the effect of school closures on the course of a pandemic and potential side effects thereof. A few detailed comments:\n1st paragraph: previous evidence - please cite reviews on topic (e.g. Viner et al. 2020).1\n\nReduction in rates of infection AND delay of epidemic/shift of course of epidemic.\n\nThere is also a lot of literature on previous undesirable impacts of school closures, e.g. Cauchemez 20092 and Aledort 20073 which should be cited\n\nMethods: This seems like a very reasonable and pragmatic approach to creating a logic model. Two specific comments:\nI am only wondering what types of studies you focused on (and whether that was an inclusion/exclusion criterion).\n\nI am interested in how you derived from a factor, which was integrated into a (quantitative) study as a variable, at a code that would then lead to a category represented in the logic model? Would it have to be a statistically significant association in order to be represented in the model?\n\nWhat qualified as a moderator? How did you determine whether a factor was considered a moderator rather than, e.g., a precondition or something else?\n\nThere is some literature out there on how to derive at frameworks (e.g. determinant frameworks) which you could use to back up your approach\n\nResults:\nFigure: Maybe consider creating a \"smaller\" graphical representation of the framework for easier access; I like the Miro Canvas, but the PPT figure is not readable; maybe reduce the level of granularity and only present the higher level domains.\n\nThe intervention: what about hybrid models as implemented in some countries in order to reduce the number of students in a class?\n\nImpacts on teachers and other school staff: there is some literature on this topic now, e.g. in the French and Israeli context; I would maybe consider thinking about additional need of staff (e.g. more staff is needed for facilitating hybrid forms of teaching as implemented in Germany; some teachers will not be able to teach in person in hybrid models).\n\nAge of students: it might be worthwhile to point out the differing presentations of disease in different age groups already in the result section (as you do in the discussion section).\n\nSocial/political factors as moderators: maybe this should be referred to as context since it not only refers to social/political aspects but also to geographic, socio-economic and other factors.\n\nI am also wondering if the infrastructure in schools (e.g. the setup of classrooms, the possibility for outdoor teaching; cafeterias; rooms for teachers; etc.) plays a role when deciding whether or not to close/reopen schools.\n\nFuture development:\nIt would be interesting to continue working on this model while evidence evolves (e.g. by integrating causality as it arises) which the authors point out in the discussion.\n\nIt would moreover be interesting to also discuss the question (and maybe pointing towards the literature on it) around re-opening; I am aware that the authors touch base on this, but I think more thought about the utility of the framework for thinking about under what circumstances schools can be reopened; what criteria have to be considered when making the decision; which stakeholders should be integrated in making decisions around school closure/opening; what plans have to be in place in order to facilitate reopening? I know that there is a lot we do not know yet, but maybe some food for thought?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre 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": "69701",
"date": "24 Aug 2020",
"name": "Marla Petal",
"expertise": [
"Reviewer Expertise Hazard and disaster impacts on education",
"children's impacts on household safety",
"evidence-based practice & practice-based evidence for child-centred risk reduction and school safety",
"school-safety self-assessment."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nVery timely and valuable contribution. Well presented. Miro presentation is very handy to cover the complex topic. Professional info-graphic design might really enhance differentiating the content for better understanding.\nA couple of things that could be clarified:\n\"We also considered whether more complex relationships between outcomes needed to be depicted (particularly vicious or virtuous cycles but also instances of conjunctural causation).\" insider-language... please unpack.\n\n\"trampolining\"? \"continue to socialise\" - is that in reference to having some normal interaction (eg bubble group?)\n\n+ theoretical: some children doing better at home due to avoidance of bullying and other violence against children...\n\nWhere does this go next:\n#1. A key statement buried in the main text that can be brought out in the discussion: \"Although it was not within the scope of this work, we suspect that the form of the school closure is likely to change the logic model quite substantially, with some impacts only being observed in longer term closures or when broader social distancing measures are in place.\"\n#2. Given that you recognize that a) previous research was on much shorter-term closures b) previous pandemics didn't have the enormous complication of asymptomatic transmission (and you might also mention cost/invasiveness/slow results of testing... ) I think that it becomes important to open the door on\n\na) the very critical 'implementation and effectiveness of public health measures'... which will need tracking and unpacking (for different age groups) b) the variation in interventions emerging which are designed to mitigate many of the impacts. (eg. see NYC and others continuing to distribute school lunches even though \"closed\" etc.)\nI really hope that you can take this to next level (next article, perhaps) to do the same for intervention models, because we don't have the classification/language to discuss the huge range of interventions (radio, tv, teacher phone contact, teacher individual and small group mtgs with caregivers or students, peer learning groups, bubble groups, etc..\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\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/9-352
|
https://f1000research.com/articles/9-350/v1
|
12 May 20
|
{
"type": "Case Report",
"title": "Case Report: A unique case of a retroperitoneal abscess during pregnancy complicated with an ileo-sigmoid-vesical fistula in a patient with active Crohn’s disease",
"authors": [
"Ghizlane Bendriss",
"Zafer Esmandar",
"Zafer Esmandar"
],
"abstract": "The enterovesical fistula is an uncommon condition resulting from complications of inflammatory diseases such as Crohn’s disease. While erosion of an abscess can result in the formation of enterovesical fistula, early diagnosis of abscesses is crucial in order to avoid complications. This case is a retrospective analysis of the unusual combination of events that contributed to the misdiagnosis, further complications in a pregnant 28-year-old female diagnosed with Crohn’s disease. The patient presented a retroperitoneal abscess, which went undiagnosed and complicated with an ileo-sigmoid-vesical fistula during pregnancy. She complained of right sciatica, cruralgia, and a complete inability to lift her right leg as well as recurrent urinary infections, which were treated by multiple courses of antibiotics. The chronic situation led to the restricted growth of the fetus, which made the fetus not tolerate the labor. Emergency C-section was done due to fetal distress, but baby did well and did not need NICU. None of the gynecological examinations, ultrasound, X-ray, CT-scan, cystography, and colonoscopy performed before, during and after pregnancy were able to explain her symptoms. Fecaluria is the symptom that ultimately directed toward the presence of an eventual fistula five months after delivery. The MRI diagnosed the retroperitoneal abscess and an ileo-sigmoid-vesical fistula, which were treated by total parenteral nutrition followed by ileo-colo resection. An ileostomy was maintained for four months before anastomosis. Sciatic pain and psoasitis in a context of Crohn’s disease should alert physicians to the possible presence of a retroperitoneal abscess, which should be monitored by MRI. In addition, recurrent urinary infections during pregnancy in a context of Crohn’s disease can be sign of a formed enterovesical fistula. The multiple antibiotic courses, while allowing the continuity of the pregnancy, also contributed to further delay of diagnosis by decreasing inflammation.",
"keywords": [
"case report",
"sciatic pain",
"recurrent urinary infection",
"abscess",
"appendicitis",
"fistula."
],
"content": "Introduction\n\nEnterovesical fistulae are uncommon conditions that are most commonly diagnosed based on clinical evidence. While only about 2% of patients with Crohn’s disease develop enterovesical fistulae, ileovesical fistulae are the most common type1,2 and the ileo-sigmoid-vesical fistula remains an exceptional case. One of the mechanisms of the formation of an enterovesical fistula the erosion of an abscess3–5. The importance of early diagnosis of such abscess, in order to avoid complications, is illustrated in this case. A retrospective analysis of the case allows us to propose important symptoms that shall alert on the presence of an abscess and/or an enterovesical fistula. In addition, we will comment on the benefit and inconvenient of using prophylactic antibiotic courses in this case.\n\n\nCase report\n\nThe patient was a 28-year-old female, with no past surgical intervention and no known family history of inflammatory bowel disease. She was diagnosed in France with an inflammation of the last 10 cm of the ileum leading to the diagnosis of a Crohn’s disease and was treated with an initial dose of 50 mg/day of prednisolone and 50 mg/day of azathioprine for 8 weeks. As her symptoms started to fade, prednisolone dose was progressively decreased to 5 mg/day.\n\nAt first visit, the primary concern of the patient was the continuous sciatic pain and an inability of her to lift her leg, with no fever. She reported having had an episode a high fever and kidney pain while on holiday in Tunisia two weeks earlier. Recurrent urinary infections and abdominal stiffness were retrieved at her monthly examinations during her pregnancy follow-up and with elevated C-reactive protein levels of 225 mg/l (normal range <5 mg/l). Because of the pregnancy, limited imaging options were available, and ultrasounds could not give a clear diagnosis. Instead, appendicitis and kidney stones were proposed several times. After pneumaturia and fecaluria appeared, MRI was performed and finally revealed the presence of a retroperitoneal abscess and an ileo-sigmoid-vesical fistula.\n\nThe time elapsed between the first complaint and the resolution of the underlying cause of the symptoms was 18 months. This patient was diagnosed by several specialties and in different countries as the case happened during the relocation of the patient to a new country. For these reasons, this timeline is based on reported facts from the patient and from the images, lab analyses and reports she could provide. The case happened in 2012, and this patient has been followed up for eight years.\n\nFebruary 2011: Patient complains from high fever, abdominal pain and is diagnosed by ultrasound with a right kidney dilation. She is given paracetamol 500 mg every 6 hours and 500 mg oral ciprofloxacin every 12 hours for 7 days.\n\nMarch 2011: Patient complains for the first time of sciatic pain, crualgia and inability to lift right leg, while having no fever. She is taking 5 mg/day prednisolone and 50 mg/day azathioprine for Crohn’s disease management. CT-scan for L3–L4 is normal, ultrasound is normal, gynecological examination is normal, hip X-ray is normal. Abdominal CT-scan shows dilation of right ureter and stenosis against the terminal ileum. She is prescribed 500 mg paracetamol every 6 hours.\n\nApril 2011: Patient moves to Qatar.\n\nMay 2011: Patient continues complaining from sciatic pain and flexing incapacity, when pregnancy is discovered. The patient decided by herself to discontinue azathioprine at this point, but was followed up with a family medical doctor to maintain doses of prednisolone at 5 mg/day. At first emergency hospital admission, patient presents with severe abdominal pain, with right lower abdominal stiffness and a C-reactive protein levels showing an inflammation of the ileum region leading to the diagnosis of appendicitis. Appendectomy is proposed but the patient refuses surgery.\n\nJuly 2011: Patient is admitted to hospital with severe kidney pain, general fatigue and paleness. She was taking 500 mg paracetamol every 6 hours. Urine had an abnormal brown and cloudy urine color, which lead to the diagnosis of a urinary infection and beginning of pyelonephritis, confirmed by ultrasound. Augmentin (amoxicillin 250 mg and clavulanic acid 125 mg) is prescribed with a dose of 1g every 12 hours for 7 days.\n\nNovember 2011: The patient is admitted for pre-term labor. Patient is under the same antibiotic and dose as in July 2011 for the recurrent urinary infections and does not present symptoms. Tocolytic medication is given to stop contractions.\n\nJanuary 2012: The patient is admitted for normal labor; however, fetal distress required emergency C-section. The baby weighed 2400 g, but with a good Apgar score and did not need NICU.\n\nFebruary 2012: The patient complains of severe urinary infections and pneumaturia; cystoscopy is normal. She is on auto medication with 500 mg paracetamol every 6 hours. She is again treated with Augmentin (amoxicillin 250mg and clavulanic acid 125mg) at a dose of 1g every 12 hours for 7 days.\n\nMay 2012: Colonoscopy is performed but it was not possible to reach the terminal ileum. A few days after the colonoscopy, the patient is admitted to emergency with vomiting, high fever, severe abdominal pain. Ultrasound and CT-scan show inflammation of the right iliac fossa, leading to diagnosis of appendicitis. The patient is convinced of having a fistula. She refuses surgery and is treated Flagyl (metronidazole hydrochloride) 250 mg twice daily for 7 days.\n\nJune 2012: The patient reports fecaluria and is admitted for an MRI examination that reveals the presence of a retroperitoneal abscess and an ileo-vesical fistula. She travels to her home country (France), where a new MRI reveals that the fistula is actually an ileo-sigmoid-vesical fistula. She is treated with total parenteral nutrition for 30 days, and with a combination of 500 mg metronidazole IV and 400 mg ciprofloxacin IV every 12 hours for 4 weeks, after which ileo-colo resection was performed. An ileostomy was maintained for four months before anastomosis.\n\n\nFollow up\n\nThe patient has been followed up for eight years. After resection, an anti-TNFα treatment was proposed but the patient refused and went onto a restrictive diet (the Specific Carbohydrate Diet) a grain-free diet planned to help people with Crohn's disease6,7. The patient has been followed up for eight years, during which she stayed in remission, with normal levels of C-reactive protein, and two pregnancies with babies of normal birth weight. Significant number of adherences resulted from the surgeries happened in 2012 (c-section , ileo-colo resection, and anastomosis surgery), leading to the formation of a peritoneal cyst that reached 13 cm in 2015, and which reduced by itself during the second pregnancy. Adherences led to the complication of the third C-section by a bladder injury of two cm, which healed after one week of urine catheterization. The patient reports having been more sensitive to fungal infections as important candidiasis happened after each of her three pregnancies, requiring the use of a single dose of fluconazole 150 mg and daktarin cream (miconazole 2%).\n\n\nDiscussion\n\nOur retrospective analysis of the events is that a retroperitoneal abscess was formed in February 2011, which resulted in psoasitis, which explains both sciatica and crualgia. The patient was under prednisolone, which might have increased the risk of infection. Several diagnostic challenges further complicated the case. First, the relocation of the patient led to difficulties to navigate through the healthcare system, as patient visited different specialists (gynecology, urology, internal medicine, gastroenterology, orthopedics). Second, X-ray and CT-scan were not possible during pregnancy, and urinary infection and sciatic pain were attributed to pregnancy. Third, antibiotic courses allowed the patient to enter into a cycle of flare-recovery that delayed understanding of the disease but allowed pregnancy to pursue to term with no fetal damage. As pregnancy progressed, the gastrointestinal tract was moving in relation to the uterus and bladder, which might have allowed the formation and closure of the fistula at various locations along the urinary tract leading to further confusion.\n\nThe pharmacological approach was used to reduce symptoms and allow pregnancy to pursue to term and allowed the patient to continue to work and have a normal active life, principally using paracetamol and antibiotics. However, this contributed to the delay of the diagnosis of the fistula. Nevertheless, as growing evidence on the importance of the gut microbiome during pregnancy are being exposed, it is important to question whether a preferential therapeutic intervention to protect both mother and child is available. Total parenteral nutrition has been shown to successfully resolve enteric fistula in Crohn’s disease and a safe option during pregnancy8,9.\n\nTo conclude, this case highlights the importance of close monitoring of any symptoms occurring during pregnancy in patients with active Crohn’s disease. Sciatic pain and psoasitis in a context of Crohn’s disease shall alert on the possible perforation of the ileum and a retroperitoneal abscess, which need to be monitored by MRI10. Recurrent urinary infections during pregnancy in a context of Crohn’s disease can be sign of the presence of an enterovesical fistula. Finally, courses of antibiotics provide a temporary recovery from symptoms, but also a decrease of the inflammation thus masking the lesions observed on imagery at the time of investigation, leading to further delay of correct diagnosis. Investigations shall be performed far from antibiotic courses.\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 was obtained from the patient.",
"appendix": "References\n\nRandall D, Tittle V, Wright G, et al.: Crohn’s disease and enterovesical fistulae: common things are common. magonlinelibrary.com. PubMed Abstract | Publisher Full Text\n\nGruner JS, Sehon JK, Johnson LW: Diagnosis and management of enterovesical fistulas in patients with Crohn’s disease. Am Surg. 2002; 68(8): 714–9. PubMed Abstract | Publisher Full Text\n\nDaniels IR, Bekdash B, Scott HJ, et al.: Diagnostic lessons learnt from a series of enterovesical fistulae. Colorectal Dis. 2002; 4(6): 459–62. PubMed Abstract | Publisher Full Text\n\nNajjar SF, Jamal MK, Savas JF, et al.: The spectrum of colovesical fistula and diagnostic paradigm. Am J Surg. 2004; 188(5): 617–21. PubMed Abstract | Publisher Full Text\n\nBalsara K, Dubash C: Complicated sigmoid diverticulosis. Indian J Gastroenterol. 1998; 17(2): 46–7. PubMed Abstract\n\nKakodkar S, Mutlu EA: Diet as a Therapeutic Option for Adult Inflammatory Bowel Disease. Gastroenterol Clin North Am. 2017; 46(4): 745–67. PubMed Abstract | Publisher Full Text | Free Full Text\n\nSpecific Carbohydrate Diet: What to Eat and What Not. Reference Source\n\nTriantafillidis JK, Papalois AE: The Role of Total Parenteral Nutrition in Inflammatory Bowel Disease: Current Aspects. Scand J Gastroenterol. 2014; 49(1): 3–14. PubMed Abstract | Publisher Full Text\n\nTheilla M, Ławiński M, Cohen J, et al.: Safety of home parenteral nutrition during pregnancy. Clin Nutr. 2017; 36(1): 288–92. PubMed Abstract | Publisher Full Text\n\nTufo A, Desai GJ, Cox WJ: Psoas syndrome: A frequently missed diagnosis. J Am Osteopath Assoc. 2012; 112(8): 522–8. PubMed Abstract | Publisher Full Text"
}
|
[
{
"id": "67598",
"date": "05 Aug 2020",
"name": "Siv Fonnes",
"expertise": [
"Reviewer Expertise Appendicitis",
"peritonitis",
"hernia",
"microbiome",
"abdominal surgery."
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting case report, which underlines that the patient's medical history (in this case Crohn’s disease) is of great importance when diagnosing and treating “ordinary” symptoms and complaints (e.g. recurrent urinary infection during pregnancy).\nGeneral comments: Throughout your manuscript, it is not sufficiently clear which of the complaints and findings that covers the patient’s complaints and which consist of findings of the clinical examination. Please make this clear throughout the manuscript.\n\nIntroduction:\nThe first sentence in the introduction is unclear and has no reference to either of the statements: “Enterovesical fistulae are uncommon conditions that are most commonly diagnosed based on clinical evidence.” Please revise it.\n\nYour reference 1 concerning the incidence and rate of fistulas for Crohn’s patients is a case report, it does not seem plausible that this is the most accurate reference – please consider revising the reference.\n\nPlease add a verb in the sentence: “One of the mechanisms of the formation of an enterovesical fistula the erosion of an abscess”.\n\nYou write: “One of the mechanisms of the formation of an enterovesical fistula the erosion of an abscess3–5.” Reference 5 is a small case series of 15 patients, consider changing or omitting this reference.\n\nCase report: patient information and clinical findings\nWhat year was she diagnosed with Crohn’s disease?\n\nThe time course is a bit hard to follow - when was “the first visit”, please specify.\n\nWas abdominal stiffness a symptom reported by the patient or a finding of repeated clinical examinations?\n\nThe sentences “After pneumaturia and fecaluria appeared, MRI was performed and finally revealed the presence of a retroperitoneal abscess and an ileo-sigmoid-vesical fistula” is not accurate. I agree that pneumaturia is a key symptom and should be reported here. But pneumaturia was reported in February 2012, but she was not diagnosed until June 2012. Please revise the sentence.\n\nCase report: Timeline of diagnostic and therapeutic interventions\nThe timeline is a bit hard to follow. Have you considered making a figure along with the description?\n\nAll the CT scans were they with or without contrast if with contrast was it given i.v., p.o. or rectally? Did any of the CT scans show intravesical air?\n\nThere are no descriptions of the urine, urinalysis, or analysis of the urine until July 2011. In February and March, there were clinical findings in relation to her right kidney – was there really no information on her urine at these appointments?\n\nMay 2011: “At first emergency hospital admission, the patient presents with severe abdominal pain, with right lower abdominal stiffness and a C-reactive protein levels showing inflammation of the ileum region leading to the diagnosis of appendicitis.” The sentence should be revised – there are no descriptions of scans that show inflammation of the ileum.\n\nIn January 2012 the patient had an emergency C-section, was there any information regarding her abdominal cavity in the surgical record?\n\nDid the patient only report pneumaturia in February 2012 and not during any of the other visits?\n\nHow many times did the patient experience recurrent urinary infections, the information is not clear from the timeline?\n\nMay 2012, “The patient is convinced of having a fistula”, could you please elaborate on how the patient suspected that – and none of the doctors previously?\n\nDiscussion:\nYou write: “Second, X-ray and CT-scan were not possible during pregnancy,” – please make it clear how a fistula should be diagnosed e.g. CT with oral or rectal contrast, as the timeline and your discussion do not clearly state that.\n\nPlease add the importance of the symptoms recurrent urinary infection, pneumaturia, and fecaluria in suspecting the diagnose fistulas.\n\nReferences:\nReference 1 is not cited correctly.\n\nYour reference 7 is missing details.\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes",
"responses": []
},
{
"id": "67589",
"date": "17 Aug 2020",
"name": "Rahul Hegde",
"expertise": [
"Reviewer Expertise Radiology."
],
"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\nAbstract:\n\nAlthough comprehensive, it would be better if the authors would make the abstract concise.\n\nIntroduction:\n\nIt would be better to word that 'ileo-sigmoid-vesical fistula is uncommon rather than calling it 'exceptional' which implies that it is very rare, which its is not, especially in Crohn's disease where severe disease can have complex fistulas.\n\nCase report section:\n'diagnosed by ultrasound with a right kidney dilation'- I am presuming the authors mean right renal hydronephrosis? - Please correct.\n\nMarch 2011- What CT was performed? Lumbar spine of abdominopelvic CT? Without or with contrast?\n\nMay 2011- How does CRP lead to the conclusion of inflammation of ileum region? Was imaging performed at this instance?\n\nMay 20012 and June 2012- Unfortunately, this case has no images shown. It is difficult to believe that CT would not pick up a large intra-abdominal abscess.\n\nOverall, the case is not very novel as complex fistulizing disease is known to happen with Crohn's disease. Multiple shortcomings mentioned above. Lack of figures/imaging is a significant shortcoming.\n\nI would refer authors to read the following review of fistulizing disease in Crohn's: Booya, F., Akram, S., Fletcher, J.G. et al. CT enterography and fistulizing Crohn’s disease: clinical benefit and radiographic findings. Abdom Imaging 34, 467–475 (2009). https://doi.org/10.1007/s00261-008-9419-11\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? No",
"responses": []
}
] | 1
|
https://f1000research.com/articles/9-350
|
https://f1000research.com/articles/9-348/v1
|
11 May 20
|
{
"type": "Case Report",
"title": "Case Report: Suspected COVID-19 death in the community - histological lung findings and the challenges faced by the pathologist",
"authors": [
"Megan Jenkins",
"Oliver Johnson",
"Tim Helliwell",
"Christopher Paul Johnson",
"Oliver Johnson",
"Tim Helliwell",
"Christopher Paul Johnson"
],
"abstract": "Coronavirus disease 2019 (COVID‐ 19) has now been declared a global pandemic. The literature on the histopathological changes associated with COVID-19 infection is currently limited. Early data consistently describe diffuse alveolar damage on lung histology from patients with COVID-19 pneumonia. We present the case of a 65-year-old female who died whilst self-isolating in the community following a short history of symptoms consistent with COVID-19. An invasive autopsy and subsequent lung histology demonstrated diffuse alveolar damage, in keeping with the previously reported cases of COVID-19 pneumonia. We hope to contribute to the growing body of literature available on the pathological findings in such cases. The challenges of ascertaining post mortem virological confirmation of infection are discussed.",
"keywords": [
"Coronavirus",
"COVID-19",
"Severe acute respiratory syndrome coronavirus 2",
"Autopsy",
"Diffuse alveolar damage",
"Acute respiratory distress syndrome"
],
"content": "Introduction\n\nCoronavirus disease 2019 (COVID- 19) is a novel coronavirus that has spread rapidly across the globe and on the 12th March 2020, the World Health Organisation declared the outbreak a global pandemic1. The current data suggests the mortality rate from COVID-19 is 2–5%2. The spectrum of disease severity varies, with 14% suffering severe disease and 5% experiencing critical disease, defined as respiratory failure with shock or multi-organ dysfunction3. Respiratory failure is often the result of acute respiratory distress syndrome (ARDS)4. The current literature on the histopathological changes associated with COVID-19 infection is limited to ten cases and diffuse alveolar damage (DAD) is consistently reported as the primary abnormality5–9.\n\nWe present the case of a 65-year-old female who died in the community amid the COVID-19 pandemic. The aim of this report is contribute to the limited data available on the pathological findings in COVID-19 cases and to highlight the challenge of ascertaining virological PCR confirmation in those patients who have died.\n\n\nCase report\n\nA 65-year-old Caucasian female cleaner with a background of chronic kidney disease and morbid obesity contacted her GP regarding a short history of dry cough and a sore throat. Her GP prescribed antibiotics and advised the patient to self-isolate in line with the NHS COVID-19 guidance. The following day she was discovered deceased at her home address.\n\nThe Coroner requested a post mortem examination. External examination of the body confirmed morbid obesity (BMI 49.6kg/m2). There was no evidence of recent injury. On internal examination, the most striking abnormality was the appearance of the lungs, which were heavy (combined weight 1658g) and diffusely firm (Figure 1). No pus was expressed from the cut surface. The air passages contained frothy oedema and there was mild reddening of the mucosae. Additionally, there was cardiomegaly (heart weight: 524g) and both ventricles appeared dilated. Scattered, coarse scars were present across the renal surfaces. Internal examination showed no other significant abnormality.\n\n(a) Both lungs were firm and heavy; the left lung weighed 778g and the right lung weighed 880g. (b) The cut surface of the lung appeared reddened. There was no consolidation or pus.\n\nHistology of the lungs showed diffuse hyaline membrane formation, typical of DAD. In areas, narrow hyaline membranes lined the alveolar sacs; elsewhere, these changes were more severe (Figure 2 and Figure 3). There was prominent pulmonary oedema and variable congestion of the alveolar septal capillaries (Figure 4). Focally there was an increase in interstitial chronic inflammatory cells and there was type II pneumocyte hyperplasia (Figure 5 and Figure 6). In areas, the bronchiolar epithelium appeared attenuated with possible ulceration, indicating larger airway involvement (Figure 7). No viral inclusion bodies or multinucleated giant cells were seen.\n\n(a) The histologic features showed relatively marked variation; in some areas, the diffuse alveolar damage appears milder. Early hyaline membranes line the alveolar airspaces and ducts. (b). Elsewhere, hyaline membranes are broader and more extensive. (×20)\n\n(a & b) The hyaline membranes are often regarded as the defining feature of the ‘exudative phase’ of diffuse alveolar damage. In the airspaces contained necrotic cellular debris and haemorrhagic oedema rich in fibrin due to the damaged alveolar walls. These materials condense to form the hyaline membranes. (x10, ×20).\n\n(x10).\n\nVery occasional neutrophils are present within the bronchiole walls, but no acute inflammatory cells are seen within the airspaces. Scattered, large cells with multilobed nuclei are present in the alveoli, which likely represent megakaryocytes [not visible at this magnification]. (x10).\n\n(a & b) There is hyperplasia of type II pneumocytes and expansion of the alveolar walls due to fibroblast proliferation. (x10, x20).\n\n(a) There is widespread loss of the respiratory epithelium, which is likely artefactual and is often seen in post mortem lung histology; however, very occasionally this feature appears genuine. Here, the bronchiole lining appears attenuated and the lumen contained a fibrin plug. (b) There is attenuation of the epithelium with focal ulceration (12 to 3 o’clock position). The bronchiole wall and surrounding parenchyma are infiltrated by chronic inflammatory cells. (x10).\n\nHistology of the heart, brain, spleen, thyroid and tonsils were normal. Sections taken from the kidneys revealed mild glomerulosclerosis. The liver showed mild fatty change. Unfortunately, in certain parts of the UK, laboratories are not processing virology samples in patients who have already died and therefore virological confirmation was not available. The cause of death was offered as COVID-19 based on the history and histopathological findings.\n\n\nDiscussion\n\nThis case report describes the autopsy findings in an individual who died in the community whilst isolating with symptoms of COVID-19. The current literature on post mortem findings in cases of COVID-19 infection is limited, but the available data consistently highlights DAD as the predominant pathological process. This case report contributes to the growing body of evidence that describes features of DAD in the setting of COVID-19 associated acute respiratory symptoms. There is also emerging evidence to suggest some individuals may develop cardiovascular complications due to COVID-19 infection10–12. In this case, the heart was enlarged but histology was normal and these changes most likely represent obesity-related cardiomegaly.\n\nThe absence of post mortem virology testing is a limitation in this case. Considering the consistent symptomology and amidst the current pandemic, the histopathological changes were considered diagnostic for COVID-19. Availability and sensitivity of testing seem varied across the UK. In some regions, autopsy examinations are delayed whilst awaiting results from post mortem nasal swabs; an invasive examination is only carried out if these results are negative. Interestingly, in the recent case published by Barton LM et al.,9 nasal swabs were positive for COVID-19 but pulmonary swabs were negative and post mortem examination revealed bronchopneumonia as the cause of death, suggesting post mortem nasopharyngeal swabs may not be an accurate screening method after death. Based on the data available, it appears that lung histology provides the most reliable means to attribute death to COVID-19 infection, providing the clinical history is appropriate. It is worth noting that currently in the UK, suspected COVID cases in the community are not able to access confirmatory swabbing and so the onus will often fall to the pathologists to establish an appropriate cause of death. From anecdotal hospital experience, patients who develop COVID-19 associated ARDS will often succumb rapidly following a dramatic increase in oxygen requirements over a period of several hours (Johnson O, personal communication, 14 Apr 2020). For this reason, the authors are aware that the number of community deaths will inevitably continue to increase given that some patients may not be able to get to secondary care in a timely fashion.\n\nAt our centre, invasive autopsies in cases of suspected COVID-19 infection are now only performed when the history indicates a probable alternative to the cause of death, or where there are concerns that the death may be suspicious. When it seems that death has resulted from respiratory failure, post mortems are limited to an external examination for the purposes of ruling out unnatural causes. Providing there are no unexpected or suspicious findings, a cause of death is offered based on a history consistent with COVID-19 infection. Whilst there are many benefits to an invasive autopsy, such as facilitating accurate diagnosis, excluding internal trauma and improving understanding of the disease, factors such as accessibility of COVID-19 testing, availability of personal protective equipment, infection risk to staff and body storage capacity must be considered. Where COVID-19 is suspected, the appropriateness of performing an invasive procedure must be decided on a case-by-case basis.\n\n\nConsent\n\nWritten informed consent for publication of their clinical details and clinical images was obtained from the next of kin 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": "Acknowledgements\n\nThe authors would like to thank HM Coroner for the Liverpool and Wirral area for his permission to publish this case. The use of this case is in accordance with the requirements of the Human Tissue Act 2004 (UK).\n\n\nReferences\n\nWHO: WHO announces COVID-19 outbreak a pandemic. The World Health Organization, Geneva, Switzerland, 2020. Reference Source\n\nWu YC, Chen CS, Chan YJ: The outbreak of COVID-19: An overview. J Chin Med Assoc. 2020; 83(3): 217–220. PubMed Abstract | Publisher Full Text | Free Full Text\n\nWang D, Hu B, Hu C, et al.: Clinical Characteristics of 138 Hospitalized Patients With 2019 Novel Coronavirus-Infected Pneumonia in Wuhan, China. JAMA. 2020; 323(11): 1061–1069. PubMed Abstract | Publisher Full Text | Free Full Text\n\nHasleton P, Flieder DB, editors: Spencer’s Pathology of the Lung. 6th ed. New York: Cambridge University Press; 2013; 342–365. Reference Source\n\nTian S, Hu W, Niu L, et al.: Pulmonary Pathology of Early-Phase 2019 Novel Coronavirus (COVID-19) Pneumonia in Two Patients With Lung Cancer. J Thorac Oncol. 2020; 15(5): 700–704. PubMed Abstract | Publisher Full Text | Free Full Text\n\nXu Z, Shi L, Wang Y, et al.: Pathological findings of COVID-19 associated with acute respiratory distress syndrome. Lancet Respir Med. 2020; 8(4): 420–22. PubMed Abstract | Publisher Full Text | Free Full Text\n\nTian S, Xiong Y, Liu H, et al.: Pathological Study of the 2019 Novel Coronavirus Disease (COVID-19) through Post-Mortem Core Biopsies. Preprints. 2020. Publisher Full Text\n\nZhang H, Zhou P, Wei Y, et al.: Histopathologic Changes and SARS-CoV-2 Immunostaining in the Lung of a Patient With COVID-19. Ann Intern Med. 2020. PubMed Abstract | Publisher Full Text | Free Full Text\n\nBarton LM, Duval EJ, Stroberg E, et al.: COVID-19 Autopsies, Oklahoma, USA. Am J Clin Pathol. 2020; pii: aqaa062. PubMed Abstract | Publisher Full Text | Free Full Text\n\nInciardi RM, Lupi L, Zaccone G, et al.: Cardiac Involvement in a Patient With Coronavirus Disease 2019 (COVID-19). JAMA Cardiol. 2020. PubMed Abstract | Publisher Full Text\n\nMa KL, Liu ZH, Cao CF, et al.: COVID-19 Myocarditis and Severity Factors: An Adult Cohort Study. Preprints. 2020. Publisher Full Text\n\nZeng JH, Lui YX, Yuan J, et al.: First Case of COVID-19 Infection with Fulminant Myocarditis Complication: Case Report and Insights. Preprints. 2020. Publisher Full Text"
}
|
[
{
"id": "63295",
"date": "22 May 2020",
"name": "Sebastian Lucas",
"expertise": [
"Reviewer Expertise Morbid anatomy and infectious diseases."
],
"suggestion": "Approved",
"report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe case report describes an autopsy of a person who almost certainly died of COVID-19 lung disease, on the basis of the circumstances of death and the lung histology (all other organs were normal for age).\nThe problems of getting virology tests done on cadavers is addressed - though it should be noted that currently this situation has improved and most mortuaries are now able to access viral tests for COVID-19.\nThe lung histology is typical for COVID-19 infection and the illustrations are good.\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": "96474",
"date": "25 Oct 2021",
"name": "Jan H von der Thüsen",
"expertise": [
"Reviewer Expertise Pulmonary pathology"
],
"suggestion": "Approved With Reservations",
"report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn the current report, the authors describe their findings regarding the autopsy of a patient suspected for death due to COVID-19. The most important findings are those of DAD in the lungs, which are described in some detail and with appropriate illustrations. This is a not uncommon finding in fatal COVID-19, and they therefore conclude that this is evidence of COVID-19 infection. Unfortunately, DAD is frequently seen in deceased patients, and can have many causes and associations, and the conclusion may be based on over-interpretation. Viral testing may not have been possible at the time, but IHC for SARS-CoV-2 is currently widely available, and could have been applied to this case.\n\nAlso, presently the literature is certainly not limited to 10 cases anymore, and this statement in the introduction should therefore be deleted or altered.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly",
"responses": []
}
] | 1
|
https://f1000research.com/articles/9-348
|
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