Dear Dr Pinlaor,

Thank you very much for submitting your manuscript "Strongyloides stercoralis infection induces gut dysbiosis in chronic kidney disease 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,

Alessandra Morassutti, PhD

Associate Editor

PLOS Neglected Tropical Diseases

Abhay Satoskar

Deputy 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: A clear testable hypothesis "that S. stercoralis infection changes gut microbiome, contributing to progression of chronic kidney disease" is stated. The study design and population are clearly described. This study compared the bacterial composition of the faecal microbiome of infection-driven chronic kidney disease CKD patients who were infected with Strongyloides stercoralis Ss+ with that of those who were uninfected with S. stercoralis Ss-. The groups were pair-matched for sex, age and biochemical factors.

PCR testing was undertaken to ensure that the “uninfected” group was free from S. stercoralis infection.

The microbial composition of the faeces of each patient was characterised by the V3-V4 region of the 16SrRNA. A sequencing library was generated for each sample. These were subjected to data control processes ensuring the quality of the data.

The sample size was limited by the availablity of suitable participants. Nevertheless, given that the Ss+ patients were matched with Ss- patients, meaningful results were obtained.

Suitable statistical analysis was used to support the conclusions.

The manuscript did not indicate whether informed consent had been obtained from the participants.

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

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 matched the analysis plan. The results were clearly presented and the figures of sufficient quality for clarity.

The data showed a relationship between S. stercoralis infection and altered microbial composition in CKD patients.

258 genera from 16 phyla were present in the total group. Overall, alpha diversity was similar in the two groups, but in males, alpha diversity was significantly greater in the Ss- group. Beta diversity was also significantly greater in the Ss- group.

At the phylum level, there was no significant difference in abundance between the Ss+ and Ss- groups, but at the genus level 42 taxa differed in relative abundance. Pathogenic genera Escherichia-Shigella, Steptococcus, Haemophilus, Rothia, Actinomycetes, Aggregatibacter were significantly increased in the Ss+ group. Short chain fatty acids- SCFA-producing bacteria Eubacterium rectale_group, Eubacterium hallii_group, Anaerostipes, Coprococcus and Akkermansia were significantly decreased in the Ss+ group.

The abundance Anaerostipes was significantly lower in those aged over 65 years, in females, in increasingly advanced CKD stage, and in those infected with S. stercoralis. The abundance of Escherichia-Shigella was significantly higher in those aged over 65 years, in females, in increasingly advanced CKD stage and in those infected with S. stercoralis.

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

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 supported by the data presented and the limitations are clearly described.

The authors conclude that the changes in the composition of the microbiome in S. stercoralis infections may result in disruption of the gut barrier that can contribute to toxicity, inflammation and malnutrition and progression of CKD, and its public health importance in areas endemic for S. stercoralis. They also indicate what kind of studies are needed to further elucidate this relationship between S. stercoralis infection and CKD.

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

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: Minor issues

Line 31: In the abstract, “infection-driven” chronic kidney disease is mentioned, but this is the only time “infection-driven” is mentioned in the manuscript. Would you please clarify whether this paper is about infection-driven CKD or CKD of any aetiology. If genuinely “infection-driven” CKD, please clarify in the author summary and the manuscript eg in line 116.

Line 125: renal infection with other intestinal parasites. Should this be “renal infection, infection with other parasites.”?

In Table 1, eosinophilia is elevated in both the Ss+ and Ss- groups, which suggests that there were other helminth infections in the Ss- group. Would you please explain this anomaly?

Line 208: Is the number of species correct here? The number should be more than or equal to the number of genera.

Line 211-212: State in the text which group had the greater beta diversity [the Ss- group, according to Fig 2].

Minor grammatical, spelling or punctuation changes:

Line 72: change “significant” to “significantly”

Line 116: change “age) of” to “age) with”

Line 220: change “significantly” to “significance”

Line 306: change “Anearostipes” to “Anaerostipes”

Line 583 (Caption for Fig 3): change “Oder” to “Order”

Line 618 (Caption for Fig 7): insert “;” after “age”

Recommendation: Minor Revision

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

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: To my knowledge, this study is the first to show in CKD patients that S. stercoralis infection is associated with decreased diversity of the faecal microbiota, increased abundance of pathogenic microbial genera and decreased abundance of SCFA-producing genera. These are very important findings.

Although the study involved a small number of participants, the use of matched pairs ensured that the S. stercoralis-infected group was similar to the uninfected group in characteristics other than S. stercoralis infection status. PCR testing was used to ensure as much as possible that the uninfected group was truly S. stercoralis-free. The authors were careful to ensure that there was sufficient DNA in the extracted samples to carry out gene sequencing.

Although the manuscript states that the study protocol was approved by the human ethical review committee of Khon Kaen University (HE631200), the manuscript does not state whether informed consent was obtained from the participants. Details about informed consent should be included in the manuscript.

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

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: Yes: Jennifer Shield

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 Somchai Pinlaor, We are pleased to inform you that your manuscript 'Strongyloides stercoralis infection induces gut dysbiosis in chronic kidney disease 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,

Alessandra Morassutti, PhD

Academic Editor

PLOS Neglected Tropical Diseases

Abhay Satoskar

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: This is a re-review of the manuscript with minor modifications.

The study meets the requirements listed.

**********

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 and the presentation of results are clearly and completely presented, the figures are appropriate and clear.

**********

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 supported by the data presented, the limitations are clearly described, the authors discuss the relevance of the results to our understanding of CKD, Strongyloides infection and impact on the microbiota. The public health relevance is addressed.

**********

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: Accept

**********

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: To my knowledge, this study is the first to show in CKD patients that S. stercoralis infection is associated with decreased diversity of the faecal microbiota, increased abundance of pathogenic microbial genera and decreased abundance of SCFA-producing genera. These are very important findings.

Although the study involved a small number of participants, the use of matched pairs ensured that the S. stercoralis-infected group was similar to the uninfected group in characteristics other than S. stercoralis infection status. PCR testing was used to ensure as much as possible that the uninfected group was truly S. stercoralis-free. The authors were careful to ensure that there was sufficient DNA in the extracted samples to carry out gene sequencing.

This is an important study, and I look forward to its 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: Yes: Jennifer Shield