Dear Dr. Zhang,

Thank you very much for submitting your manuscript "Unusual dominant genotype NIA1 of Enterocytozoon bieneusi in children in Southern Xinjiang, China" 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.

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,

Abdallah M. Samy, PhD

Guest Editor

PLOS Neglected Tropical Diseases

Todd Reynolds

Deputy Editor

PLOS Neglected Tropical Diseases

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

Editor comments to authors: I invited four reviews for your manuscript; however, we received only three reviews and experienced a delay in receiving the fourth review. My decision is to terminate the review task of this fourth review to avoid any further delay on this manuscript. Please consider all comments addressed by reviewers below before submitting a revised version of your manuscript. Thanks! AMS

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: -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? NO

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested? YES

-Were correct statistical analysis used to support conclusions? YES

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

"Comments to the Author":

Keywords:

- Add: novel genotypes.

Abstract:

- Lines 16-17: “Enterocytozoon bieneusi causes approximately 90% of reported cases of human microsporidiosis”. Add: “mainly pathologies or intestinal disorders”.

Author summary:

- Lines 40-41: “Additionally, we demonstrated the zoonotic potential of E. bieneusi genotype D..”. Change: I prefer to use “we confirmed” or “we contrasted”.

- Comment: The authors must mention the groups 1 and 2 for grouping the nine identified genotypes and highlight the three novel genotypes.

Introduction:

- Line 48: “may cause persistent diarrhea, vomiting and a wasting síndrome”. Add: “dissemination and presence in other organs”.

- Lines 57-58. Comment: The zoonotic role of group 2 and mainly of group 1 should be highlighted. Finally, cite risk factors, sources of infection and animal hosts (reservoirs).

- Line 64: “in China have been reported by several studies”. Add: references [6-14]?.

Materials and Methods:

- Ethics statements: Lines 82-83. “Participants infected with parasites received 83 appropriate treatment according to local policies and national guidelines”. Comment: It is unknown if patients have an active infection or are spores passing through the intestine without causing a real infection in the patient. I prefer to remove “Participants infected” and put “Participants with possible presence of parasites” or “Positive participants in the study with parasites”.

- Sample collection: Lines 86-87. In my opinion it would be very important to indicate if the counties studied are in urban or rural areas. This information would help a greater knowledge of the circulation of different genotypes according to the type of area and the presence or absence of animal hosts as a source of infection.

- Sample collection: Line 91. “No diarrhea was observed during sampling”. Comment: It should be indicated if children have any other signs or symptoms, for example, weight loss, malnutrition, fever, etc. or even some other associated pathology (if this would be possible).

- DNA extraction and PCR amplification. The authors should provide some additional nested PCR information. For example, the pairs of primers used, temperature cycles, independently of the bibliographic references.

Reviewer #2: 1.the sample collection is from 11 counties of Xinjiang ,China, suggest to use a map to fix location.

2."the studies were conducted in diverse geographical locations (Jilin, Heilongjiang, Guangxi, Henan, Shanghai, Hubei, and Chongqing)", need to supply more references, such as PLoS Negl Trop Dis 13(5): e0007356. https://doi.org/10.1371/journal.pntd.0007356.

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: -Does the analysis presented match the analysis plan?.

Yes.

-Are the results clearly and completely presented?

Phylogenetic analysis: Lines 163-164. “(CHN6, CXJH2, and CXJH3) clustered together with the zoonotic genotypes I, J, and BEB6 (Figure 1)”. Add: group 2.

-Are the figures (Tables, Images) of sufficient quality for clarity?

Table 1: Indicate the urban or rural character of the counties studied.

Reviewer #2: 1.the line 141, I think that it make a mistake, "age groupe"? If so, please add it.

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: -Are the conclusions supported by the data presented? Review (comments below)

-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? NO

-Is public health relevance addressed? YES

The authors should modify the conclusions based on the new information provided in the Discussion.

For example:

- Line 201. Remember: In my opinion it would be very important to indicate if the counties studied are in urban or rural areas. This information would help a greater knowledge of the circulation of different genotypes according to the type of area and the presence or absence of animal hosts as a source of infection.

- The authors have recently published the following article that should be discussed in this section and included in the bibliography. “Li D-F, Zhang Y, Jiang Y-X, Xing J-M, Tao D-Y, Zhao AY, Cui Z-H, Jing B, Qi M and Zhang L-X (2019). Genotyping and Zoonotic Potential of Enterocytozoon bieneusi in Pigs in Xinjiang, China. Front. Microbiol. 10:2401. doi: 10.3389/fmicb.2019.02401”.

It is very important because the second genotype (D) and the fourth (EbpA) identified in children are those detected in pigs in the same geographical area of Xinjiang (China).

Therefore, it could reinforce the role or zoonotic character of some of the genotypes found in children and the importance of the geographical area (urban or rural).

Reviewer #2: this study conclusions are proper. the data of analysis is correct. the discuss can help to understanding the topics.

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: (No Response)

Reviewer #2: the objectives of study are very clear, and the sdudy design is appropriate toaddress the objectives.

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: Discussion:

- Line 184: “as in a study which found that 11 of 93 fecal specimens collected from children with”. Add: percentage (%).

- Line 199: “dominant genotype in children in Southern Xinjiang in this study”. Add: percentage (%).

- Line 201. Remember: In my opinion it would be very important to indicate if the counties studied are in urban or rural areas. This information would help a greater knowledge of the circulation of different genotypes according to the type of area and the presence or absence of animal hosts as a source of infection.

- Line 202: “Genotype D was the second most prevalent genotype detected in this study”. Add: percentage (%).

- The authors have recently published the following article that should be discussed in this section and included in the bibliography. “Li D-F, Zhang Y, Jiang Y-X, Xing J-M, Tao D-Y, Zhao AY, Cui Z-H, Jing B, Qi M and Zhang L-X (2019). Genotyping and Zoonotic Potential of Enterocytozoon bieneusi in Pigs in Xinjiang, China. Front. Microbiol. 10:2401. doi: 10.3389/fmicb.2019.02401”.

It is very important because the second genotype (D) and the fourth (EbpA) identified in children are those detected in pigs in the same geographical area of Xinjiang (China).

Therefore, it could reinforce the role or zoonotic character of some of the genotypes found in children and the importance of the geographical area (urban or rural).

References:

- Add: Li D-F, Zhang Y, Jiang Y-X, Xing J-M, Tao D-Y, Zhao A-Y, Cui Z-H, Jing B, Qi M and Zhang LX (2019). Genotyping and Zoonotic Potential of Enterocytozoon bieneusi in Pigs in Xinjiang, China. Front. Microbiol. 10:2401. doi: 10.3389/fmicb.2019.02401.

Reviewer #2: This study reports the zoonotic potential of E. bieneusi genotype D . this study is also the first report of human infection by E. bieneusi genotypes KB-1 and CHN6 in China. This is an important significance .

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.

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Reviewer #1: Yes: Dr. Fernando Izquierdo Arias.

Área de Parasitología e Inmunología.

Facultad de Farmacia.

Universidad CEU-San Pablo.

Urbanización Montepríncipe.

28668 Boadilla del Monte, Madrid (Spain)

Teléfono: +34-91-372-47-00 (ext. 15275)

Fax: 91-351-04-96

e-mail: ferizqui@ceu.es

https://orcid.org/0000-0003-0367-007X

Publons: AAE-3073-2019

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, PLOS recommends that you 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. For instructions see https://journals.plos.org/plosntds/s/submission-guidelines#loc-methods

Attachments

Attachment

Submitted filename: Review comments.docx

Dear Dr. Zhang,

Thank you very much for submitting your manuscript "Unusual dominant genotype NIA1 of Enterocytozoon bieneusi in children in Southern Xinjiang, China" 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 7 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,

Abdallah M. Samy, PhD

Deputy Editor

PLOS Neglected Tropical Diseases

Todd Reynolds

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: -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? YES

-Were correct statistical analysis used to support conclusions? YES

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

Reviewer #3: Yes

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

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: -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

Reviewer #3: 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 #1: -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

Reviewer #3: 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 #1: Comment:

Question 2: - Lines 16-17: “Enterocytozoon bieneusi causes approximately 90% of reported cases of human microsporidiosis”. Add: “mainly pathologies or intestinal disorders”. Response: Thank you very much. We have revised this. Seen in Line 16.

The authors must put all words “mainly pathologies or intestinal disorders”. In the manuscript is only "Enterocytozoon bieneusi is the mainly pathologies".

Reviewer #3: 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: Dear Authors:

I want to thank the authors for the changes done and congratulate them for the work and research presented in the article. And I encourage them to continue in the same line for clearing doubts about the circulation and prevalence of this parasite.

Reviewer #3: The author has already addressed the relevant questions and the authors have made the changes. I have just a few additional comments.

1. “unusual dominant genotype NIA1” is missing in the abstract, please add it.

2. Line 59, ‘of which 96 have been…’ should be revised to ‘of which more than 96 have been…’

3. Line 213, healthcare level also should be included here.

4. Line 251, ‘…isolates identified in ruminants.’. Revised word ‘isolates’ to ‘genotypes’.

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

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: Dr. Fernando Izquierdo Arias.

Reviewer #3: Yes: Jianping Cao

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, PLOS recommends that you 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. For instructions see http://journals.plos.org/plosntds/s/submission-guidelines#loc-materials-and-methods

Attachments

Attachment

Submitted filename: Comments.docx

Dear Dr. Zhang,

We are pleased to inform you that your manuscript 'Unusual dominant genotype NIA1 of Enterocytozoon bieneusi in children in Southern Xinjiang, China' 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,

Abdallah M. Samy, PhD

Deputy Editor

PLOS Neglected Tropical Diseases

Todd Reynolds

Deputy Editor

PLOS Neglected Tropical Diseases