Response to ReviewersRevised Manuscript with Track ChangesManuscript

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

orcid.org/0000-0003-4304-636XX

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

orcid.org/0000-0003-1765-0002

Additional Editor Comments (if provided):

Thank you for submitting your manuscript to PLOS Neglected Tropical Diseases (PNTD-D-24-01412 - "Schistosoma haematobium Infection Promotes Oncogenic Gene Expression in Cervical Mucosa, with Enhanced Effects Following Treatment").

Expert reviewers in the fields of schistosomiasis and infectious diseases have evaluated your manuscript. They found that your study provides valuable insights into the impact of S. haematobium infection on oncogenic gene expression in cervical mucosa. However, they also identified areas for revision before the manuscript can be fully accepted by PNTD.

For example, reviewers suggested that you clearly address the limitations of the study, particularly the small number of participants enrolled and the high number of RNA-seq samples that failed to pass quality control. Additionally, they were concerned that the lack of significant pathways when comparing women with and without S. haematobium infection might be due to the data analysis pipeline used. They recommend reanalyzing the dataset with a modified pipeline to confirm the findings.

Finally, please ensure that all data used in this manuscript (including all phenotype data) are publicly available, with a functional link to the repository included in the manuscript.

The reviewers' comments are enclosed for your consideration, and I hope their feedback will be helpful for strengthening your analysis and manuscript overall. Please address all reviewers' comments before resubmission.

Again, thank you for your interest in PLOS Neglected Tropical Diseases.

Sincerely,

Winka Le Clec'h

Journal Requirements:Reviewers' Comments:

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: It is unclear if the sample size was adequate, have mentioned this in the uploaded review. It is an exploratory paper and has value as such. Otherwise affirmative.

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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: (No Response)

Reviewer #2: The information is there but needs more succinct portrayal as written in the uploaded review.

**********

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: Conclusions are supported by the data. Limitations have been described but need to be concentrated as a section in the discussion. The authors discuss how these data can be helpful to advance our understanding of the topic and public health relevance is addressed but needs to be laid out properly. The discussion is the weakest section.

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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: Very minor typo:

Lines 91-92 “In S. haematobium-associated bladder cancer, S. haematobium eggs are classified as Group 1 carcinogen” should be “a Group 1 carcinogen”

Reviewer #2: (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: This is a very interesting manuscript but I do have methodological concerns:

24 samples failed to pass QC for RNA-Seq and only 39 passed. Likewise, many women (7) were not eligible for continued follow up after praziquantel treatment. The authors should better emphasize these important limitations

The lack of significant pathways (using IPA) comparing women with and without Sh is concerning. The authors should consider analyzing their data by modifying their pipeline (i.e., GSEA).

The genes involved in oncogenesis in Table 4 – what is the direction of differential gene expression for women who cleared infection post-praziquantel vs. women with baseline Sh?

Reviewer #2: The paper is interesting but it's a small sample size, making it more of a hypothesis generating paper. However, I do think it should be published and PNTD is a good forum, likely read by many who could make use of this information

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Reviewer #1: No

Reviewer #2: No

Figure resubmission:Reproducibility:--> -->-->To enhance the reproducibility of your results, we recommend that authors of applicable studies deposit 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-->?>

Attachments

Attachment

Submitted filename: DNA Tz FGS.pdf

Schistosoma haematobium Infection is Associated with Oncogenic Gene Expression in Cervical Mucosa, with Enhanced Effects Following Treatment

PLOS Neglected Tropical Diseases

Dear Dr. Mertelsmann,

Thank you for submitting your manuscript to PLOS Neglected Tropical Diseases. After careful consideration, we feel that it has merit but does not fully meet PLOS Neglected Tropical Diseases'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 within 60 days. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosntds@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pntd/ 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 editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. This file does not need to include responses to any formatting updates and technical items listed in the 'Journal Requirements' section below.

* 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, competing interests statement, or data availability statement, please make these updates within the submission form at the time of resubmission. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

We look forward to receiving your revised manuscript.

Kind regards,

Winka Le Clec’h, PhD

Academic Editor

PLOS Neglected Tropical Diseases

Krystyna Cwiklinski

Section Editor

PLOS Neglected Tropical Diseases

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

orcid.org/0000-0003-4304-636XX

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

orcid.org/0000-0003-1765-0002

Additional Editor Comments (if provided):

Dear Dr. Mertelsmann,

Thank you for submitting your manuscript to PLOS Neglected Tropical Diseases (PNTD-D-24-01412, "Schistosoma haematobium Infection Promotes Oncogenic Gene Expression in Cervical Mucosa, with Enhanced Effects Following Treatment").

Expert reviewers in the fields of schistosomiasis and infectious diseases have evaluated your revised manuscript. They appreciated the revisions you have made. However, one reviewer has suggested additional areas for improvement before the manuscript can be fully accepted by PNTD.

In particular, validation of the nine genes identified as most differentially expressed in your RNA-seq analysis using RT-qPCR is essential to confirm the reliability of the observed expression patterns.

It is also important to discuss whether cervical cancer in this context may be related to somatic mutations in genes induced by the presence of S. haematobium , and/or whether it is associated with inflammation, fibrosis, and alterations to the epithelial cell microenvironment caused by the presence of S. haematobium eggs.

These are valid scientific questions that need to be addressed.

Finally, please ensure that all data used in this manuscript are publicly available, with a functional link to the data repository included in the manuscript. Sequence data should be submitted to the SRA repository.

The reviewers’ comments are enclosed for your consideration. I hope their feedback will be helpful in further strengthening your analysis and manuscript. Please address all reviewers' comments before resubmitting.

Again, thank you for your interest in PLOS Neglected Tropical Diseases.

Sincerely,

Winka Le Clec'h

Associate Editor

Reviewers' Comments:

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 #3: Objectives are clear, the study design is apropriate, the population appropriate, the sample size is small, statistical analysis is correct, ethical and regulatory requirements seems to be well done.

Reviewer #4: (No Response)

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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 #3: No problems here, also.

Reviewer #4: (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 #3: Conclusions are not supported by data, and they need to be clear. Authors discussion is not helpful to advance our understanding of this topic.

Reviewer #4: (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 #3: (No Response)

Reviewer #4: (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 #3: Manuscript PNTD-D-24-01412R1 entitled “Schistosoma haematobium Infection is Associated with Oncogenic Gene Expression in Cervical Mucosa, with Enhanced Effects Following Treatment” authored by Anna M. Mertelsmann et al.

Global comments: Authors have choice an interesting, relevant and pertinent scientific topic. This is a strong work developed by scientists with domain of conceptual and instrumental tools, medical and scientific. They need to be stimulated to go on working on this scientific topic: urogenital schistosomiasis. However, results described in the manuscript does not sustain authors’ initial proposal. Small amount of patients? Associated infections? Controversial strategy? Authors studding cytobrush samples state “altered cervical mucosal gene expression with oncogenic potential in patients infected with S. haematobium”. What this means? Statistic relevance derived from a p value <0.05 for selected genes is not enough. The nine genes, selected, and expressed differentially should be validated with another test, for instance, Real Time PCR. Because selected patients are too complex with associated infections. “Women with S. haematobium infections compared to uninfected women, had gene expression consistent with enhanced oncogenesis in genital mucosa”. This is not clear to me and need to be proved, I am afraid. And it is not clear also the reason to select LINC02084 gene to discuss. Maybe because is thought to be related with other cancers discussions. Schistosoma haematobium is assumed as a carcinogenic pathogen. However, the mechanisms implicated in carcinogenesis are not yet clear. Cervical cancer related with S. haematobium infection is so important to be studied. The epistemology of this form of cancer is related with somatic mutations in genes or, on contrary, is related with inflammation, fibrosis and epithelial cells microenvironment alterations? In my point of view, somatic mutations are epiphenomena.

Reviewer #4: (No Response)

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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 #3: Yes:  José M. Correia da Costa

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.]

Figure resubmission:

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. If there are other versions of figure files still present in your submission file inventory at resubmission, please replace them with the PACE-processed versions.

Reproducibility:

?>

Dear Dr. Mertelsmann,

We are pleased to inform you that your manuscript 'Schistosoma haematobium Infection is Associated with Oncogenic Gene Expression in Cervical Mucosa, with Enhanced Effects Following Treatment: A pilot 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,

Winka Le Clec’h, PhD

Academic Editor

PLOS Neglected Tropical Diseases

Krystyna Cwiklinski

Section Editor

PLOS Neglected Tropical Diseases

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

orcid.org/0000-0003-4304-636XX

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

orcid.org/0000-0003-1765-0002

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

p.p1 {margin: 0.0px 0.0px 0.0px 0.0px; line-height: 16.0px; font: 14.0px Arial; color: #323333; -webkit-text-stroke: #323333}span.s1 {font-kerning: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 #3: -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? yes

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested? No

-Were correct statistical analysis used to support conclusions? yes

-Are there concerns about ethical or regulatory requirements being met? No

**********

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 #3: -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

**********

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 #3: -Are the conclusions supported by the data presented? yes

-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

-Is public health relevance addressed? yes

**********

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 #3: No

**********

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 #3: I accept the publication of this manuscript according to its reviewed form; a project pilot with more work to be done. The novelty is cervical cancer.

**********

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 #3: Yes:  José Manuel Correia da Costa