PONE-D-25-21928The efficacy and safety of topical wound oxygen therapy for chronic refractory wounds at high altitude: protocol for a randomized controlled clinical trialPLOS ONE

Dear Dr. Ran,

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 submit your revised manuscript by Jul 03 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're 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.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Nan Jiang

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Journal requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match.

When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section.

3. Your ethics statement should only appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please delete it from any other section.

4. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Does the manuscript provide a valid rationale for the proposed study, with clearly identified and justified research questions?

The research question outlined is expected to address a valid academic problem or topic and contribute to the base of knowledge in the field.

Reviewer #1: Yes

Reviewer #2: Partly

Reviewer #3: Yes

Reviewer #4: Yes

**********

2. Is the protocol technically sound and planned in a manner that will lead to a meaningful outcome and allow testing the stated hypotheses?

The manuscript should describe the methods in sufficient detail to prevent undisclosed flexibility in the experimental procedure or analysis pipeline, including sufficient outcome-neutral conditions (e.g. necessary controls, absence of floor or ceiling effects) to test the proposed hypotheses and a statistical power analysis where applicable. As there may be aspects of the methodology and analysis which can only be refined once the work is undertaken, authors should outline potential assumptions and explicitly describe what aspects of the proposed analyses, if any, are exploratory.

Reviewer #1: Yes

Reviewer #2: Partly

Reviewer #3: Yes

Reviewer #4: Partly

**********

3. Is the methodology feasible and described in sufficient detail to allow the work to be replicable?

Descriptions of methods and materials in the protocol should be reported in sufficient detail for another researcher to reproduce all experiments and analyses. The protocol should describe the appropriate controls, sample size calculations, and replication needed to ensure that the data are robust and reproducible.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: No

**********

4. Have the authors described where all data underlying the findings will be made available when the study is complete?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception, at the time of publication. 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

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: No

**********

5. 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

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above and, if applicable, provide comments about issues authors must address before this protocol can be accepted for publication. You may also include additional comments for the author, including concerns about research or publication ethics.

You may also provide optional suggestions and comments to authors that they might find helpful in planning their study.

(Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The efficacy and safety of topical wound oxygen therapy for chronic refractory wounds at high altitude: protocol for a randomized controlled clinical trial

Study Protocol

Generally:

This study is well-structured and scientifically sound, incorporating an appropriate methodology to assess TOT in high-altitude wound care. To strengthen its impact, additional consideration of alternative treatments, environmental confounders, accessibility concerns, and subgroup analyses is recommended.

Abstract:

Strengths:

The title is clear and directly conveys the study’s focus. The abstract concisely summarizes the rationale, methodology, and expected impact, making it accessible to readers.

Comments/Recommendations:

The abstract could briefly highlight potential limitations to set appropriate expectations for the study's findings.

Consider incorporating a sentence acknowledging challenges related to study execution, such as patient adherence or environmental variability.

see attached file

Reviewer #2: This study addresses an important and underexplored clinical problem — the management of chronic refractory wounds in high-altitude populations using topical wound oxygen therapy (TOT). The proposed randomized, double-blinded, placebo-controlled design is methodologically robust, and the outcomes chosen are relevant and clinically significant. However, several key areas require revision to improve the protocol's scientific rigor, reproducibility, and applicability:

1. Wound Type Stratification:

The inclusion of multiple chronic wound types (e.g., diabetic foot ulcers, venous ulcers) without subgroup stratification or analysis is a significant limitation. Different wound etiologies have distinct pathophysiology and may respond differently to oxygen therapy. The authors should consider stratified randomization or include planned subgroup analyses.

2. Standard of Care Consistency:

"Standard wound care" is left to physician discretion without a defined protocol. This introduces variability and may affect the internal validity of the study. A minimum standard wound care protocol should be outlined (e.g., dressing selection, debridement practices, offloading strategies) to ensure consistency across groups.

3. Blinding of Sham Therapy:

The manuscript lacks detail on how sham oxygen therapy (ambient air) will be made indistinguishable from the active TOT, which could compromise blinding. The authors should clarify how the control setup will simulate the sensory experience of TOT (e.g., pressure, temperature, sound).

4. Patient-Centered Outcomes Missing:

Pain and quality of life are briefly mentioned but not evaluated using standardized tools. These outcomes are crucial in chronic wound studies. The authors are encouraged to incorporate validated instruments such as the Visual Analog Scale (VAS) for pain and the EQ-5D or Wound-QoL for quality of life.

5. Literature Review Imbalance:

The background heavily emphasizes positive studies supporting TOT, with little discussion of conflicting or negative findings. A more balanced review of current literature is necessary to frame the rationale objectively.

6. Generalizability and Limitations:

While the setting is appropriate for the study goals, the single-center design limits broader applicability. This should be acknowledged explicitly in the discussion, with a recommendation for future multi-center research.

7. Microbiological Context:

Given the noted differences in microbial profiles in high-altitude wounds, the protocol could be strengthened by including infection tracking or microbial analysis as exploratory endpoints.

8. Figures and Visuals:

Figures 1 and 2 are informative but visually dense. Consider simplifying and clarifying their design for better comprehension.

In conclusion, this is a promising and relevant study. With moderate revisions addressing the points above, it can make a valuable contribution to wound care research in resource-limited, high-altitude environments.

Recommendation: Revision Required Before Acceptance

Reviewer #3: In The efficacy and safety of topical wound oxygen therapy for chronic refractory wounds at high altitude: protocol for a randomized controlled clinical trial, Lin et al. describe a well thought out protocol for a clinical evaluation for TOT therapy. The authors do a good job explaining the rationale and need for the study. The experimental design, inclusion and exclusion criteria, and evaluation plan seem appropriate for the study. Overall, this protocol is appropriate for publication with minor revisions.

Background

- Briefly introduce the basic mechanism of TOT therapy

Methods

- Briefly describe the rationale behind therapy regimen. I.e. is the timing and O2 concentration based upon animal trials? Are there any considerations for implementing in human patients?

Reviewer #4: The primary objective of this randomized, double-blind, placebo-controlled clinical trial involving 250 residents living at high altitudes is to assess the efficacy and safety of topical wound oxygen therapy (TOT) for treating chronic refractory wounds. Participants will be randomly assigned to receive either TOT or sham oxygen therapy over a 12-week period, followed by a one-year follow-up. The primary endpoint is the wound healing rate at the end of the 12-week intervention. Secondary outcomes include reduction in ulcer area, time to complete healing, ulcer recurrence rate, amputation rate, pain levels, and the incidence of adverse events. If proven effective, TOT could serve as a valuable and safe adjunctive treatment for managing chronic wounds in high-altitude settings. The protocol covers all key aspects of study design, randomization, and statistical issues; however, there are several major statistical concerns about the current manuscript.

Statistical critiques:

1. Although the authors discuss power analysis and sample size estimation, they should explicitly clarify the following: (1) the statistical test used to determine the required sample size, (2) whether a one-sided or two-sided test was applied, and (3) that the reported 30% improvement refers to a relative, not absolute, increase in the wound healing rate. Specifically, the authors should state: “Assuming a baseline wound healing rate of 60% in the standard treatment group over a 12-week period, it is hypothesized that the addition of TOT will result in a 30% relative improvement in healing, i.e., 60% vs. 78%.”

2. Although the manuscript discusses randomization, the authors should explicitly specify the method used—for example, simple randomization (not recommended) or stratified permuted block randomization (highly recommended). If stratified permuted block randomization is chosen, the authors should clearly detail the stratification factor(s), block size, and other relevant parameters in the manuscript.

3. The statistical analysis plan covers the major outcomes but lacks essential methodological clarity and precision. While using the chi-square test for comparing wound healing rates is reasonable, the authors should clarify whether assumptions will be assessed and whether Fisher’s exact test will be used for sparse data. The proposed use of logistic regression to adjust for confounders is appropriate, but the model’s covariates, selection criteria, and diagnostics are not specified. Additionally, time-to-healing is better analyzed with survival methods rather than the proposed t-test, and the mention of Kaplan-Meier analysis is vague—for example, there is no indication of what time-to-event outcome is being evaluated or whether log-rank tests or Cox models will be used.

4. In summary, several important elements are missing from the current statistical analysis plan. There is no mention of how missing data will be addressed (a formal multiple imputation approach is recommended), whether analyses will follow the intention-to-treat principle, or how multiple testing across secondary endpoints will be controlled (either Bonferroni adjustment or false discovery rate [FDR] should be applied). The t-tests for ulcer area and healing time assume no severe skewness (skewness < 1), which should be assessed; otherwise, non-parametric methods such as the Wilcoxon rank-sum test should be used. To improve the rigor of the plan, the authors should clarify test assumptions, specify covariates, define outcomes more precisely, and incorporate appropriate strategies for handling missing data and multiplicity.

**********

7. 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

Reviewer #2: Yes: Abdulrahman Abdullah Almalki

Reviewer #3: No

Reviewer #4: 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.]

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 PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Attachments

Attachment

Submitted filename: The efficacy and safety of TOT.pdf

Attachment

Submitted filename: PONE-D-25-21928 Allen review.docx

PONE-D-25-21928R1The efficacy and safety of topical wound oxygen therapy for chronic refractory wounds at high altitude: protocol for a randomized controlled clinical trialPLOS ONE

Dear Dr. Ran,

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 submit your revised manuscript by Jul 31 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're 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.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Nan Jiang

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Does the manuscript provide a valid rationale for the proposed study, with clearly identified and justified research questions?

The research question outlined is expected to address a valid academic problem or topic and contribute to the base of knowledge in the field.

Reviewer #2: Yes

Reviewer #4: Yes

**********

2. Is the protocol technically sound and planned in a manner that will lead to a meaningful outcome and allow testing the stated hypotheses?

The manuscript should describe the methods in sufficient detail to prevent undisclosed flexibility in the experimental procedure or analysis pipeline, including sufficient outcome-neutral conditions (e.g. necessary controls, absence of floor or ceiling effects) to test the proposed hypotheses and a statistical power analysis where applicable. As there may be aspects of the methodology and analysis which can only be refined once the work is undertaken, authors should outline potential assumptions and explicitly describe what aspects of the proposed analyses, if any, are exploratory.

Reviewer #2: Yes

Reviewer #4: Yes

**********

3. Is the methodology feasible and described in sufficient detail to allow the work to be replicable?

Descriptions of methods and materials in the protocol should be reported in sufficient detail for another researcher to reproduce all experiments and analyses. The protocol should describe the appropriate controls, sample size calculations, and replication needed to ensure that the data are robust and reproducible.

Reviewer #2: Yes

Reviewer #4: Yes

**********

4. Have the authors described where all data underlying the findings will be made available when the study is complete?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception, at the time of publication. 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 #2: Yes

Reviewer #4: Yes

**********

5. 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 #2: Yes

Reviewer #4: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above and, if applicable, provide comments about issues authors must address before this protocol can be accepted for publication. You may also include additional comments for the author, including concerns about research or publication ethics.

You may also provide optional suggestions and comments to authors that they might find helpful in planning their study.

(Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: This is a resubmission of a protocol I previously reviewed (PONE-D-25-21928). The authors have addressed the major concerns raised in the initial review, particularly regarding blinding clarification, co-intervention standardization, and ethical transparency. The revised protocol is clearer and more robust overall.

The study protocol presents a novel and clinically significant investigation into the use of topical wound oxygen therapy (TOT) for chronic refractory wounds in high-altitude populations. The rationale is well-articulated, supported by both pathophysiological and epidemiological arguments specific to plateau environments, where wound healing is uniquely compromised due to hypoxia and limited perfusion.

The research design is strong, utilizing a randomized, double-blind, placebo-controlled approach with adequate sample size justification and meaningful primary and secondary endpoints. The use of a sham-controlled group and consistent standard care protocols (including wound off-loading, debridement, and infection control) reflects good clinical trial standards. The methodology is reproducible and clearly described.

Minor suggestions include:

Enhancing standardization in how “standard wound care” will be monitored or audited across patients.

Clarifying if outcome adjudicators for wound healing will be blinded or centralized.

Confirming the data repository platform intended for post-trial public data availability.

This trial, once completed, could significantly contribute to the evidence base for wound care in geographically and physiologically challenging environments. The planned 1-year follow-up is a strength and will support long-term safety and efficacy analysis.

Reviewer #4: The authors have responded well to the statistical issues raised in the previous review. There is no further statistical concern about this revised manuscript.

**********

7. 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 #2: Yes: Abdulrahman Almalki

Reviewer #4: 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.]

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 PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

The efficacy and safety of topical wound oxygen therapy for chronic refractory wounds at high altitude: protocol for a randomized controlled clinical trial

PONE-D-25-21928R2

Dear Dr. Ran,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, 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 help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- 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.

Kind regards,

Nan Jiang

Academic Editor

PLOS ONE

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Does the manuscript provide a valid rationale for the proposed study, with clearly identified and justified research questions?

The research question outlined is expected to address a valid academic problem or topic and contribute to the base of knowledge in the field.

Reviewer #4: Yes

**********

2. Is the protocol technically sound and planned in a manner that will lead to a meaningful outcome and allow testing the stated hypotheses?

The manuscript should describe the methods in sufficient detail to prevent undisclosed flexibility in the experimental procedure or analysis pipeline, including sufficient outcome-neutral conditions (e.g. necessary controls, absence of floor or ceiling effects) to test the proposed hypotheses and a statistical power analysis where applicable. As there may be aspects of the methodology and analysis which can only be refined once the work is undertaken, authors should outline potential assumptions and explicitly describe what aspects of the proposed analyses, if any, are exploratory.

Reviewer #4: Yes

**********

3. Is the methodology feasible and described in sufficient detail to allow the work to be replicable?

Descriptions of methods and materials in the protocol should be reported in sufficient detail for another researcher to reproduce all experiments and analyses. The protocol should describe the appropriate controls, sample size calculations, and replication needed to ensure that the data are robust and reproducible.

Reviewer #4: Yes

**********

4. Have the authors described where all data underlying the findings will be made available when the study is complete?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception, at the time of publication. 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 #4: Yes

**********

5. 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 #4: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above and, if applicable, provide comments about issues authors must address before this protocol can be accepted for publication. You may also include additional comments for the author, including concerns about research or publication ethics.

You may also provide optional suggestions and comments to authors that they might find helpful in planning their study.

(Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #4: The authors have responded well to the statistical issues raised in the previous review. There is no further statistical concern about this revised manuscript.

**********

7. 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 #4: No

**********