Dear Dr Fahim,

Thank you very much for submitting your manuscript "Infection with Blastocystis hominis and associated outcomes in relation to enteric infections and environmental enteric dysfunction among slum-dwelling malnourished adults in Bangladesh" 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.

All 3 reviewers have indicated that the current study is of interest to the scientific community. The authors should take note of the concerns and suggestions of the reviewers, with particular attention to the comments of Reviewer #2, including the need for some form of validation for the high % of Blastocystis infection among the cohort examined. The reviewers have also indicated more details on the methods, such as the collection procedure (e.g. how many samples collected) should be provided. Please note that the term Blastocystis hominis is no longer used and the accepted terminology is Blastocystis spp. The lack of subtype information should be discussed as a limitation of the study.

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,

Kevin SW Tan

Associate Editor

PLOS Neglected Tropical Diseases

Shan Lv

Deputy Editor

PLOS Neglected Tropical Diseases

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

All 3 reviewers have indicated that the current study is of interest to the scientific community. The authors should take note of the concerns and suggestions of the reviewers, with particular attention to the comments of Reviewer #2, including the need for some form of validation for the high % of Blastocystis infection among the cohort examined. The reviewers have also indicated more details on the methods, such as the collection procedure (e.g. how many samples collected) should be provided. Please note that the term Blastocystis hominis is no longer used and the accepted terminology is Blastocystis spp. The lack of subtype information should be discussed as a limitation of the study.

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 acceptable for publication.

Study design is appropriate for the aim.

Authors mentioned limitations of this study. Lacking of the healthy control group and subtype analysis of Balstocystis are the main limitations for this study.

Some suggestions were written for correction in the main text and supp2.

Yes the sample size is sufficient to ensure adequate power to address the hypothesis being tested

Yes, correct statistical analysis was used to support conclusions

Yes, there concerns about ethical or regulatory requirements are being met

Reviewer #2: - It is a routine practice in parasitological examinations to examine three faecal samples for each patient (such samples being taken at appropriate intervals). It is not specified how many samples per one patient were analyzed and how many times each trial was repeated. This information should be provided.

- The percentage of infected patients is very high. Therefore, the results for positive samples should be confirmed/verified using another method recommended by CDC (e.g. fecal smears, PCR).

Reviewer #3: Minor Revision.

Partly related to STROBE Items 6,9, and 13. While the inclusion and exclusion criteria is specified, the paper refers to the BEED study protocol in BMJ Open for details. The reference is a protocol that describes the recruitment process, but does not describe the outcome of recruitment. The paper can refer to a publication with the outcome of recruitment, or if this is the first from that protocol, perhaps provide more details in the Results section. Undernutrition in Bangladesh between females and males show a higher prevalence of malnutrition among females (23% vs 20%; https://globalnutritionreport.org/resources/nutrition-profiles/asia/southern-asia/bangladesh/). However, the study has 73% females. What steps in the recruitment process resulted in this ratio?

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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: Yes, the analysis was presented match the analysis plan.

Yes, the results are clearly and completely presented

Yes the figures (Tables, Images) of sufficient are quality for clarity

Reviewer #2: - Molecular method used in these study -Quantitative PCR assay using the TaqMan Array Cards- allows obtaining quantitative data. Such data should be presented and correlated with the indications of the studied biomarkers. The ms needs to be supplemented with this.

- Results with regard to association of Blastocystis hominis with fecal biomarkers should be summarized in a table.

Reviewer #3: Minor Revision. Related to the comments in Methods. It might be useful to describe the flow of recruitment to consent and reasons for refusal to better understand the ratio of females to males in this study.

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

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: Yes, the conclusions are supported by the data presented

Yes, the limitations of analysis are clearly described

Yes, the authors discuss how these data can be helpful to advance our understanding of the topic under study.

Reviewer #2: - The most common parasitic infections in Bangladesh are Ascariasis, Hookworm, Trichuriasis, and Cryptosporidium. However, in the ms it is not explained why only co-invasion with T. trichiura was taken into account.

- The high results of Blastocystis hominis vs. study participants and associated factors should be discussed.

Reviewer #3: Minor revisions. Related to Strobe Item 19 and points above. Should the female : male ratio in this study be considered a limitation? How would this affect internal and external validity .

The title uses the phrase "associated outcomes" which tend to suggest to me, that the outcomes are a result of B. hominis infection. The study design is cross-sectional and given that is the case, any association of B. hominis infection and fecal biomarkers and other pathogens is just that - an association. I think the paper is best served by stating this as assocations clearly.

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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: The only modifications needed are minor. So my decision is minor revision. Authors should be made all of them.

Reviewer #2: .

Reviewer #3: Consider the best use of graphs and tables as some of the figures can easily be represented by a table.

For instance, figure 1 shows the prevalence of B. hominis according to different sub-groups, but do not show the confidence intervals that would have demonstrated that the differences are not significant and reinforced the text. That information could be contained in a table or a graph of a point estimate bounded by 95% confidence intervals.

Figure 2 shows the prevalence of infection of each pathogen defined by the TAC assay. This could also have been visualized as a table or heatmap which could also show the extent of co-infection. This information would also reinforce the text.

I am particularly interested in the distribution of positive results of all enteropathogens and enteric biomarkers as a categorical or quantitative variable sorted by BMI. By choosing to show aggregate data, you lose the value of the powerful methods you have used. Hence the suggestion of a heatmap above. I think this might in fact be very useful to explore the how infection / inflammation (whether due to B hominis or not) is distributed across the range of malnourished adults, if only to generate new hypotheses.

Lines 204-207 and 227-230 may be repeating information on REG1B. If it is, perhaps, just place that information in one place.

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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: I think the publication of this study, which is aimed at explaining the pathogenesis of the blastocytsis protist parasite and includes a large number of samples, will make a great contribution to the researcher.

It will benefit both in terms of creating new hypotheses and in terms of evaluating the researchers ' own work with the data of this study.

However, as the authors noted, the absence of a particularly healthy control group and the absence of subtype analysis of Blastocystis, as well as the absence of microscopic examination of stool samples, are an important limitations. It will be useful for the TaqMan array method used to indicate the pathogens it is investigating and present these identified pathogens in a table. A standard investigation of all enteric pathogens and the study of a large number of patients and a detailed survey are the strengths of the study.

Reviewer #2: This ms presents the data on Blastocystis prevalence in malnourished adult patients in Bangladesh. The authors investigated the relationship of B. hominis with the occurrence of intestinal biomarkers. The data are interesting and can be used to develop new diagnostic methods. However, I have some doubts about the parasitological research.

Analysis of results is clear but the parasitological analysis is insufficient. They should be completed.

Reviewer #3: This is a relevant paper and addresses the possible associations of B. hominis infection with other pathogens and host factors.

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Reviewer #1: No

Reviewer #2: No

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.

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

Attachments

Attachment

Submitted filename: PNTD-D-21-00194_reviewer-02.03.2021.pdf

Attachment

Submitted filename: S2 Supporting Figures (1).doc

Dear Dr Fahim,

Thank you very much for submitting your manuscript "Infection with Blastocystis spp. and its association with enteric infections and environmental enteric dysfunction among slum-dwelling malnourished adults in Bangladesh" 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.

The reviewers have completed their assessment of the revised manuscript. Reviewers #1 and #3 are supportive of accepting the current version. However, Reviewer #2 has indicated 2 minor edits to the manuscript before acceptance. These are to provide details and correlations of the TaqMan qPCR data with the assessed biomarkers, and to provide details on lab identification of Ascariasis, Hookworm infestation, Entamoeba histolytica, and Cryptosporidium species, which is currently missing in the ms. Both these aspects can be included in the supplementary section of the manuscript.

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,

Kevin SW Tan

Associate Editor

PLOS Neglected Tropical Diseases

Shan Lv

Deputy Editor

PLOS Neglected Tropical Diseases

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

The reviewers have completed their assessment of the revised manuscript. Reviewers #1 and #3 are supportive of accepting the current version. However, Reviewer #2 has indicated 2 minor edits to the manuscript before acceptance. These are to provide details and correlations of the TaqMan qPCR data with the assessed biomarkers, and to provide details on lab identification of Ascariasis, Hookworm infestation, Entamoeba histolytica, and Cryptosporidium species, which is currently missing in the ms. Both these aspects can be included in the supplementary section of the manuscript.

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: Yes

Reviewer #2: It is a routine practice in parasitological examinations to examine three faecal samples for each patient (such samples being taken at appropriate intervals). The present research has only been done from one trial. The authors suggest that the method used in this study has a high detection rate. However, they do not take into account the fact that dispersive forms of parasites can be excreted periodically and taking only one sample for analysis significantly reduces the detection of, for example, parasite eggs.

Molecular method used in the study -Quantitative PCR assay using the TaqMan Array Cards - allows obtaining quantitative data. Such data should be presented and correlated with the indications of the studied biomarkers. The ms needs to be supplemented with this.

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: Yes

Reviewer #2: (No Response)

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: Yes

Reviewer #2: The discussion in lines 311-314 ["However, the prevalence of Ascariasis, Hookworm infestation, Entamoeba histolytica, and Cryptosporidium species were inconsiderable (4%, 0%, 1%, and 2%, respectively) among the study participants which may explain the insignificant results of co-invasion between Blastocystis and these parasites."] refers to results that are not described in the relevant sections of the paper, i.e.: materials and methods and the results. It is not known how these species of parasites were detected and why these studies were not described in Methods and Results.

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: Revised manuscript Line 311 "Ascariasis, Hookworm infestation" should be changed as "ascariasis, hookworm infection".

After his correction was made there is no need reviewing again by me.

Reviewer #2: (No Response)

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: Dear Editor

Thank you very much for reviewing invitation again for this manuscript.

I checked all my suggestion and other reviewew's comments. Authors tried to make all of corrections according to the reviewer's comments.

In this revised form, the manuscirpt has become better understood and scientifically enriched.

My decision is "accept" of this manuscript for publication.

Kind regards

Reviewer #2: This ms presents the data on Blastocystis prevalence in malnourished adult patients in Bangladesh. The authors investigated the relationship of B. hominis with the occurrence of intestinal biomarkers. The data are interesting and can be used to develop new diagnostic methods. However, I have some doubts about the parasitological research.

The analysis of the results is clear but the parasitological analysis is still insufficient.

Reviewer #3: (No Response)

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

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: No

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.

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 Fahim,

We are pleased to inform you that your manuscript 'Infection with Blastocystis spp. and its association with enteric infections and environmental enteric dysfunction among slum-dwelling malnourished adults in Bangladesh' 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,

Kevin SW Tan

Associate Editor

PLOS Neglected Tropical Diseases

Shan Lv

Deputy Editor

PLOS Neglected Tropical Diseases

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