Dear Mrs Barbosa,

Thank you very much for submitting your manuscript "Morphological and functional cardiac alterations in children with congenital Zika syndrome and severe neurological deficits Cardiac alterations in children with congenital Zika syndrome" 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.

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,

Andrés F. Henao-Martínez, M.D.

Academic Editor

PLOS Neglected Tropical Diseases

Andrea Marzi

Section Editor

PLOS Neglected Tropical Diseases

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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: For Methods of this study, my primary concerns are: 1) the determination/consideration of the study's sample size or power seems have not been delineated, and 2) the current manuscript only used bivariate analysis to access the epidemiological associations, and extra multivariate analyses adjusting for confounders are needed.

Reviewer #2: -Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

Yes

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

Yes

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

The population description for the CZS group is well described, however there are very few details of the healthy group, I would suggest that they do a better description of the population and specify if they applied any selection criteria.

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

I believe that the 2:1 ratio of cases and controls should be reviewed. If there is any reason why they made the decision of this relationship, it would be interesting if they describe it in the discussion, since for the results achieved I do not believe that the sample size is adequate. I understand the characteristics of the study population but it would be nice to increase the controls. If there is any support for that decision, ignore this recommendation.

-Were correct statistical analysis used to support conclusions?

Yes

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

No

Reviewer #3: The methods section needs to be rewritten as there are several repetitions. Also, Line 127 to 129 is an irrelevant information from the perspective of the manuscript.

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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: For Results of this study, things that can be improved are: 1) the footnotes in Tables can be strengthened, 2) when prevalence estimate of a health outcome is presented, the author can consider providing its 95% confidence interval to indicate uncertainty, and 3) some prevalence estimates mentioned in point 2) may be summarized in new tables to improve readability.

Reviewer #2: -Does the analysis presented match the analysis plan?

Yes

-Are the results clearly and completely presented?

Yes

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

Yes

Reviewer #3: The study Morphological and functional cardiac alterations in children with congenital Zika

syndrome and severe neurological deficits is an interesting one and describes the first to evaluate cardiac alterations, including an evaluation of the ventricular myocardial deformation index, in young children with CZS. In the opinion of this reviewer, the work is clinically relevant and definitely shades light on the pathologies associated with CZS. However, this is a purely clinical work that was done in a single centre. While the clinical data is unequivocal and thoroughly done, the authors do not offer any mechanistic explanations to the same. Such investigations will be important to better understand the disease and also to identify therapeutic approaches. Could the authors attempt to provide such explanations?

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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: For Conclusions, the public health implications for the population at large seems have not been mentioned in the manuscript. Rather, the current conclusion paragraph seems focus solely on the next steps for the study population.

As mentioned in my comments for Methods, the conclusions need to be further corroborated by extra multivariate analysis.

Reviewer #2: -Are the conclusions supported by the data presented?

Yes, but They could improve the recommendation of the line 424 - 426

-Are the limitations of analysis clearly described?

Yes

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

Yes. I have a recommendation in this regard, in line 279 - 283, it mentions one of the strengths of this study that presents original and novel results, however it would be important to mention that in vitro studies have been carried out, you can review the following article, it may help you as an important precedent for its results.

Rossi F, Josey B, Sayitoglu EC, Potens R, Sultu T, Duru AD, Beljanski V. Characterization of zika virus infection of human fetal cardiac mesenchymal stromal cells. PLoS One. 2020 Sep 17;15(9):e0239238. doi: 10.1371/journal.pone.0239238. Erratum in: PLoS One. 2021 Jan 22;16(1):e0246112. PMID: 32941515; PMCID: PMC7498051.

-Is public health relevance addressed?

Yes

Reviewer #3: (No Response)

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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: All editorial comments along with their corresponding highlighted areas in the manuscript are embedding in the attached document.

Reviewer #2: Minor revision

Line 121. The sentence of line 121 I think is unnecessary. The specification that they were born to mothers who were infected during pregnancy could be added to lines 116 and 117.

Reviewer #3: (No Response)

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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: Prior to being accepted for publication, this manuscript can still be significantly improved by considering the comments provided in my former responses and the attachment.

Regarding data availability, although the author mentioned that the data are available in the submitted materials, it seems no supplementary materials containing the individual-level data were available. The authors may upload these data in revision if have not done so.

Reviewer #2: This study is of great impact due to the scarcity of similar studies in vivo, it has an adequate design and the results are interesting in this study population, which has had an impact in several Latin American countries, with the greatest impact in Brazil. This type of study will allow the provision of adequate medical care at an early age to improve the health and quality of life of the population studied.

Reviewer #3: The study reports that the heart size was reduced in the CZS children but there was no difference in the LV ejection volume or myocardial deformation between this group and healthy controls. Also, none of the patients in the study had moderate or severe heart disease dysfunction. These observations raises the question of the implication of the study. Apart from advocating for the need of regular monitoring of cardiac function by echocardiography, the study does not really offer much in terms of progress of the field. Even the cardiac function biomarkers do not show any significant changes.

The other part of the manuscript deals with the neurological abnormalities. Here also, the authors do not offer anything more in-depth than "severe neurological impairment was also observed in these patients.", which has already been reported by previous studies. In sum, the work suffers from the lack of novelty and also lack of mechanistic implications. Further long-term follow-up of the patients with the cardiac anomalies might have provided important clues to the disease pathogenesis.

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Reviewer #1: Yes: Dehao Chen

Reviewer #2: Yes: Evelin Martinez

Reviewer #3: 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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Reproducibility:

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

Dear Mrs Barbosa,

We are pleased to inform you that your manuscript 'Morphological and functional cardiac alterations in children with congenital Zika syndrome and severe neurological deficits Cardiac alterations in children with congenital Zika syndrome' 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,

Andrés F. Henao-Martínez, M.D.

Academic Editor

PLOS Neglected Tropical Diseases

Andrea Marzi

Section Editor

PLOS Neglected Tropical Diseases

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

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)

Reviewer #2: Accept

Reviewer #3: (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)

Reviewer #2: Accept

Reviewer #3: (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)

Reviewer #2: Accept

Reviewer #3: (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: Thanks for considering my suggestions in revision. The following are the final suggestions moving to the publication phase:

1) It seems a "Reference" heading is missed, so please ensure it is added in the manuscript proof.

2) In the uploaded "EKG" datasets, I could not find a codebook/directory explaining the meaning of each variable. Please ensure it is added for the benefit of future readers.

Reviewer #2: Accept

Reviewer #3: (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: (No Response)

Reviewer #2: Accept

Reviewer #3: The authors have adequately addressed the issues and have provided satisfactory explanations to the questions or doubts raised. The Methodology has been re-written to avoid repetitions. In my opinion, the manuscript can now be accepted for publication.

**********

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: Evelin Martinez

Reviewer #3: Yes: Gayatri Mukherjee