The effect of storage conditions on microbial communities in stool

Kristien Nel Van Zyl; Andrew C. Whitelaw; Mae Newton-Foot

doi:10.1371/journal.pone.0227486

Peer Review History

Original SubmissionSeptember 26, 2019
11 Nov 2019 Decision Letter - David N Fredricks, Editor PONE-D-19-27079 The effect of storage conditions on microbial communities in stool PLOS ONE Dear Nel Van Zyl, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please address each concern raised, and explain how this concern has been addressed in the manuscript. We need to see more statistics to support the conclusions. We would appreciate receiving your revised manuscript by Dec 26 2019 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. 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The PLOS ONE style templates can be found at http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. Thank you for including the following information in your ethics statement: "Written consent was obtained as part of the clinical trial". We note that you included minors (age<18) in your study. Please provide additional details regarding minors' consent. In the ethics statement in the Methods and online submission information, please ensure that you have specified whether you obtained consent from parents or guardians. 3. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: No ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The authors presented findings of the stability of targeted bacteria, using qPCR, in stool samples processed and stored using different methods. The authors provide adequate rationale for conducting this study, and its findings could be important toward informing those studies that similarly desire not to use preservative agents for microbial preservation in field studies. However, there are some issues with the authors’ terminology and the statistical analyses presented that need to be addressed prior to publication in PLOS One. Line 55-57: This sentence should be revised: 1) microbial stability should not be limited to ‘specific’ populations, and should theoretically be consistent across populations, given the same conditions/storage/extraction/processing; and 2) the data used by the authors are also ‘limited’ in both sample size (n=10) and population (children); therefore, the rationale in this sentence seems not justified. Line 76: The authors should state if subjects were on antibiotics at the time of collection Line 80-81: It is suggested that authors use a table or diagram to demonstrate the aliquoting of the samples and how many samples were compared within each collection method. They should also include which samples dropped out due to DNA yield/quality Lines 125-132: Statistical analyses were insufficient to appropriately compare the stability of the microbial data between the storage methods. It is suggested that the authors use more quantitative comparisons, rather than basing their findings on the subjective figure interpretations. For example, the authors could compare the adjust mean Ct for each collected method, using linear mixed effects models, and calculate a p-value for each of the collection methods compared to the referent immediately-extracted group. Line 133: The random effect should be stated as the subject and not the sample (since there are multiple measurements on one subject) Line 126, 128, 143: Authors should avoid using terms as ‘cause’ and ‘effect’ Line 165-166: It is unclear what the authors are referring to Figure 2: It is unclear what the 10 boxes represent. It also appears that many of the more abundant taxa were variable (not just the less abundant taxa) Figure 3: This seems redundant, and it is not clear that the subject cluster together. There needs to be a statistical test to quantify variation explained by subject versus storage Line 179: It is unclear why a single p-value is being used to compared multiple taxa abundances across the storage methods, this does not seem like the appropriate statistical test ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0227486.r001
Revision 1
17 Dec 2019 Author Response To the editor and reviewers, Our responses have been detailed fully in the "Response to Reviewers" letter. We request that the responses be read in that document, as the layout and formatting are more pliable. We have summarized those responses below: Response to editor’s comments: With regards to sample homogenization and informed consent: We have expanded on both comments in the edited submission. Response to reviewer’s comments: Line 55-57: Microbiota and what influences them are diverse and unique in different settings; therefore, it is important to study the possible influences of study procedures in each setting. We have revised the sentence to reflect this. Line 76: The authors should state if subjects were on antibiotics at the time of collection • As this paper focuses on the differences related to storage relative to immediate extraction, we do not believe that it impacts the conclusions we have drawn. • Participants were excluded if they had received ≥14 days of isoniazid or a fluroquinolone at enrolment, or if they had been treated for TB in the 12 months before. However, information regarding other antibiotics is not currently available to the authors as the trial is currently ongoing. • Antibiotic usage data have been captured as part of the clinical trial, and have been requested from the trial data management team, but will only be made available for a larger sequencing-based study going forward. • We have noted in the discussion section that differences in subjects, including the influence of antibiotics, will be investigated as part of a larger study going forward. Line 80-81: It is suggested that authors use a table or diagram to demonstrate the aliquoting of the samples and how many samples were compared within each collection method. They should also include which samples dropped out due to DNA yield/quality We have added a workflow (Fig 1) to demonstrate the aliquoting of samples. No samples were excluded from the qPCR analysis due to quality, due to the robustness of PCR. We have included a statement in the results section to this effect. Lines 125-132: Statistical analyses were insufficient to appropriately compare the stability of the microbial data between the storage methods. Additional statistical measures have been described below and in the edited manuscript. Line 133: The random effect should be stated as the subject and not the sample (since there are multiple measurements on one subject) This has been revised in the manuscript. Line 126, 128, 143: Authors should avoid using terms as ‘cause’ and ‘effect’ These terms have been revised in the manuscript where appropriate. Line 165-166: It is unclear what the authors are referring to We have revised this sentence in the edited manuscript to reflect our meaning more clearly. Figure 2: It is unclear what the 10 boxes represent. It also appears that many of the more abundant taxa were variable (not just the less abundant taxa) We have determined that Fig 3 represents our core findings most clearly and have kept it in the main text, but request to keep Fig 2 as a supplemental figure to show the variations for each subject (the ten boxes) across populations. The figure heading has been expanded to refer to the ten boxes as subjects. Figure 3: This seems redundant, and it is not clear that the subject cluster together. There needs to be a statistical test to quantify variation explained by subject versus storage We have added p-values to this figure to demonstrate that statistical tests support our findings. We have described the statistical tests that generated these values below, and in the manuscript in the methods section. Line 179: It is unclear why a single p-value is being used to compared multiple taxa abundances across the storage methods, this does not seem like the appropriate statistical test In consultation with the statisticians, it was felt that the population differences are inherently part of the influence and it was therefore included as a fixed effect in the linear mixed effect model. We used the entire data set, including all populations including the Universal amplification Cts (therefore, non-normalized Ct values) and indicated the p-value for differences related to storage as determined by the lme model. This model also output a p-value for population, and we have added that in the edited manuscript. We agree with the reviewer that different taxa/populations may warrant separate statistical tests. For this reason, we decided to perform statistical testing using the normalized mean/median Ct values for each taxon. Instead of fitting linear mixed effect models for each storage group compared to the immediate as suggested by the reviewer, we performed Kruskal-Wallis multiple group comparisons. The reason for this is that linear mixed-effect models are more useful when more data is available – comparing the mean/median Ct values therefore diminishes the number of data points available. Kind regards, Kristien Nel Van Zyl Division Medical Microbiology, Department of Pathology, Stellenbosch University Attachments Attachment Submitted filename: Response to reviewers_final.doc https://doi.org/10.1371/journal.pone.0227486.r002
20 Dec 2019 Decision Letter - David N Fredricks, Editor The effect of storage conditions on microbial communities in stool PONE-D-19-27079R1 Dear Dr. Nel Van Zyl, We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements. Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication. Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. With kind regards, David N Fredricks, MD Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0227486.r003
Formally Accepted
3 Jan 2020 Acceptance Letter - David N Fredricks, Editor PONE-D-19-27079R1 The effect of storage conditions on microbial communities in stool Dear Dr. Nel Van Zyl: I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. For any other questions or concerns, please email plosone@plos.org. Thank you for submitting your work to PLOS ONE. With kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. David N Fredricks %CORR_ED_EDITOR_ROLE% PLOS ONE https://doi.org/10.1371/journal.pone.0227486.r004

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