Dear PhD Pérez-Molina,

Thank you very much for submitting your manuscript "Chagas disease is related to structural changes of the gut microbiota in adults with chronic infection (TRIPOBIOME Study)" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

I am really sorry for the delay.

Unfortunately, your manuscript was not handled in an expedient manner. The Academic Editor did not reply to the staff e-mails, and I wasn't cc on this correspondence.

In the mean time, I am sending you the comments we received from the lone reviewer. Upon receipt of your revised manuscript I will handle it personally, and send it out for review.

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Charles L. Jaffe, Ph.D.

Section Editor

PLOS Neglected Tropical Diseases

Charles Jaffe

Section Editor

PLOS Neglected Tropical Diseases

***********************

I am really sorry for the delay.

Unfortunately, your manuscript was not handled in an expedient manner. The Academic Editor did not reply to the staff e-mails, and I wasn't cc on this correspondence.

In the mean time, I am sending you the comments we received from the lone reviewer. Upon receipt of your revised manuscript I will handle it personally, and send it out for review.

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: see below

--------------------

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: see below

--------------------

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: see below

--------------------

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: see below

--------------------

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: In this manuscript, Pérez-Molina et al performed fecal microbiome characterization of Chagas disease patients and controls. Overall, this is a well-designed study that uses appropriate methods and controls. A particular strength of this study is the dietary questionnaire, which enables the exclusion of dietary confounders. However, authors over-interpret minor differences, and make statements that are not supported by the data. Specifically, I have the following major concerns:

1. PLS-DA is prone to over-fitting. Authors should provide metrics to confirm that this is not the case here (explained variation (R2) and predictive ability (Q2)).

2. Heat tree analyses are an atypical way to present the data. Authors should provide additional text on figure interpretations, and on the methods used to generate this figure.

3. Authors should also clarify the discrepancies between the differential bacteria displayed on the volcano plot and on the heat tree.

4. Lines 275-277: this statement is not supported by the data. Indeed, the differences in Enterococcus hirae, Lactobacillus buchneri, and Megamonas between CD and controls appear to be primarily outlier-derived, with group medians and quartile overlapping fully. The differences in Parabacteroides appear more robust.

5. Authors should test whether these outliers come from the six patients with more-severe disease symptoms

6. Authors use the heatmap in figure 6 to justify a lack of impact of dietary, treatment or sex-based confounders. However, given that the samples also do not cluster by Chagas status, the study may simply be under-powered to address confounders.

7. Authors under-used their metagenomics data by merely performing species-level analyses. Important analyses should also investigate whether there are specific bacterial genes or pathways that differ between groups.

8. Authors fail to cite recent work already published on this topic using 16S sequencing (de Souza-Basqueira et al, doi: 10.3389/fcimb.2020.00402). A comparison of findings from the current study vs this prior publication should be performed. This also leads to the incorrect statement of novelty by the authors at lines 387-389.

9. Lines 304-305: “we found a small impact of this parasitosis on bacterial richness”. Richness differences did not reach statistical significance and thus should not be stated as a finding.

10. Authors should specify whether beta-diversity differences were observed between the different CD forms

Minor concerns:

1. Line 223-225: this statement is not supported by Table S2, since significant differences in PUFA and Omega 6 fatty acids were observed.

2. Line 223 (“data not shown”). Data should be provided in SI.

3. There are two color legends in Figure 4b.

4. Line 113-118: the details on REDcap are unnecessary and irrelevant to the study’s findings

5. Given that E. hirae is rarely associated with humans, and yet is found at relatively high abundance in both CD and controls in this study, authors should verify their taxonomic assignments, before making this large interpretation.

6. Lines 380-385: italicize Prevotella.

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Reviewer #1: No

Figure 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. 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.

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Dear PhD Pérez-Molina,

We are pleased to inform you that your manuscript 'Chagas disease is related to structural changes of the gut microbiota in adults with chronic infection (TRIPOBIOME Study)' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Matthew Brian Rogers, Ph.D.

Academic Editor

PLOS Neglected Tropical Diseases

Charles Jaffe

Section Editor

PLOS Neglected Tropical Diseases

***********************************************************

Thank-you for your the re-submission of you manuscript, after consideration of modifications made to your manuscript, your manuscript has been found suitable for publication. Please ensure that your deposited sequence data is released at the time of publication.

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: (No Response)

**********

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: (No Response)

**********

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: (No Response)

**********

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: (No Response)

**********

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: All my comments have been satisfactorily addressed.

**********

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