Dear Mr. Fontes,

Thank you very much for submitting your manuscript "Tittle: An integrated analysis of the structural changes and gene expression of spleen in human visceral leishmaniasis with and without HIV coinfection" 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.

The study is an important advancement in understanding the dynamics of HIV/VL co infection in real patient samples. Please respond to reviewers comments and concerns especially the interpretation of the data as well as the parameters studied.

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).

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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,

Hira L Nakhasi, Ph.D.

Section Editor

PLOS Neglected Tropical Diseases

Abhay Satoskar

Section Editor

PLOS Neglected Tropical Diseases

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The study is an important advancement in understanding the dynamics of HIV/VL co infection in real patient samples. Please respond to reviewers comments and concerns especially the interpretation of the data as well as the parameters studied.

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: Methods were well described.

Reviewer #3: The objectives are stated clearly, however, the data are limited and need to be described better to confirm the hypothesis.

The small sample size (3 patients) is adequate for a descriptive clinical study.

Potentially proper statistics are used, they need better description

No concerns about ethics or regulations.

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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: Results are well described.

Reviewer #3: The analysis matches the plan.

The results are extensively presented. However, they are not complete. Gene expression was described as transcripts per gene, yet those numbers were not presented.

The tables are all acceptable. The figures require further explanation.

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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: (No Response)

Reviewer #2: (No Response)

Reviewer #3: The conclusions are largely descriptive consistent with the authors' awareness that the study has few samples. The conclusions drawn often refer to select data to make statements that are not supported by all the data.

The authors focus on stating that the findings in human patients confirm previous results in animals.

The public health benefit is discussed in terms of contribution to the understanding of Leishmania pathogenesis.

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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: (No Response)

Reviewer #2: (No Response)

Reviewer #3: See attached review memo.

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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: (No Response)

Reviewer #2: Strength #

1. This study was performed with rare clinical tissue samples, particularly HIV along with Visceral leishmaniasis.

2. Authors used very well acceptable approach to tease out gene expression at mRNA level.

Weakness # 1. As author mentioned in the discussion section about the low number of samples. Nevertheless, still this study will be a good addition for the Leishmania field as there is not much study report regarding HIV-Leishmania co-infection in endemic region.

Overall, study design to analyze the tissue sample and result analysis was well performed. Please see below my comments:

1. Authors have performed cellular distribution analysis with immuno-histochemical analysis. However, as this technique is very challenging and is varies to one antibody marker to another antibody, the Results depicted in Table 3 needs further validation. Cytokine expression level measured from the serum samples of these patient would be an ideal confirmation of these results.

2. IL-6 signaling is driving force for the IL-17 signaling pathway, which further modulates several inflammatory pathways involve in Visceral Leishmaniasis. Please provide the outcome of gene expression analysis of IL-17 pathway.

3. Please provide an access to the Nano string data analysis for the readers or submit the gene expression analysis in public database upon publication of this manuscript.

Reviewer #3: See attached review memo.

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Reviewer #1: Yes: Parna Bhattacharya

Reviewer #2: Yes: Ranadhir Dey

Reviewer #3: No

Figure Files:

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Reproducibility:

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Attachments

Attachment

Submitted filename: PNTD-D-23-01629.docx

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Submitted filename: Review of PNTD-D-23-01629.docx

Dear Dr. dos-Santos,

We are pleased to inform you that your manuscript 'Tittle: An integrated analysis of the structural changes and gene expression of spleen in human visceral leishmaniasis with and without HIV coinfection' 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,

Abhay R Satoskar

Section Editor

PLOS Neglected Tropical Diseases

Abhay Satoskar

Section Editor

PLOS Neglected Tropical Diseases

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