Dear Dr. Pandey,

Thank you very much for submitting your manuscript "Metagenomic insights into the dengue patient blood microbiome: Enhanced microbial diversity and metabolic activity in severe patients" 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. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

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

[1] A letter containing a detailed list of your responses to all 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.

Thank you again for your submission to our journal. 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,

Antonio Mas

Academic Editor

PLOS Neglected Tropical Diseases

Andrea Marzi

Section Editor

PLOS Neglected Tropical Diseases

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Dear Dr. Rajesh Kumar Pandey,

We sent your manuscript to two reviewers and both agree in their assessments, minor revisions. The comments are below this email. Please read these comments carefully and respond to them.

Best regards,

Antonio Mas

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: The study design is robust, and the statistical analyses used are appropriate to support the conclusions. There are no concerns regarding ethical requirements. Although the sample size is limited, it is sufficient to substantiate the conclusions.

Reviewer #2: Yes

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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: The analysis presented is well-suited to address the research question, and the results are clearly articulated. The figures are of high quality.

Minor Comments:

• Table 1: Include dispersion values in the data.

• Table 2: Clarify that these are metabolic pathways.

• Page 25: Italicize "S. marcescens."

• Page 26: Remove italics in "although uncommon in humans."

Reviewer #2: Yes

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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: The conclusions are well-supported by the data, and the study's limitations are adequately described. The implications for public health are addressed, and the relevance of the work is clearly articulated.

Minor Comments:

• Page 26: The statement "these species may serve as microbial markers for evaluating the severity of dengue fever" is significant. I suggest elaborating on this idea, emphasizing the potential for early diagnosis. Additionally, consider mentioning as a limitation that it is unclear whether these bacteria are present at the onset of the disease.

• Figure Legend 6: The phrase "active microbial species in the bloodstream exacerbates inflammatory responses and enriched metabolic pathways" implies causation, while the study only shows correlation. The main text does not exhibit this overinterpretation.

Reviewer #2: Yes

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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)

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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: Summary and General Comments

This study is highly compelling, linking the blood microbiome of dengue patients with biometric data on the inflammatory response. The use of RNA-seq to confirm that the bacteria are metabolically active is commendable, and the correlations found between certain species and metabolic pathways with disease severity are significant. The study is well-executed, employing appropriate methodologies and statistical analyses.

I have only a few minor comments, as noted in the Results and Conclusions sections. It was a pleasure to review this manuscript.

Reviewer #2: Yadav et al. report the microbial diversity in dengue patients. Dengue is an emerging problem that is associated with climate change and is becoming an increasing problem in India. The study is straightforward and uses a rigorous data analysis pipeline to report on the microbial diversity in clinically relevant specimens. The study is informative, and I only have minor comments that are appended below.

1. Figure 2A – how many patients had overlapping symptoms? It will be useful to represent the data as overlapping Venn Diagrams to illustrate the overlapping and unique symptoms in the patient population.

2. How does the data compare to healthy individuals? While the comparison of mild, moderate, and severe cases is very relevant and important, including healthy controls will make the data more meaningful and potentially enable the discovery of Dengue-related biomarkers and microbiome disruption, if any.

3. Please submit all raw data, analysis codes and scripts in a public repository, and include a freely accessible link to the code and data in the manuscript. Eg., GitHub, NCBI.

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Reviewer #1: Yes: Mariana Peimbert

Reviewer #2: 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.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

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

References

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.

Dear Dr. Pandey,

We are pleased to inform you that your manuscript 'Metatranscriptomic insights into the dengue patient blood microbiome: Enhanced microbial diversity and metabolic activity in severe patients' 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,

Antonio Mas

Academic Editor

PLOS Neglected Tropical Diseases

Andrea Marzi

Section Editor

PLOS Neglected Tropical Diseases

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