Dear Dr. Brown,

Thank you very much for submitting your manuscript "Impact of sampling depth on enteric pathogen detection in pit latrines" 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,

Poppy H L Lamberton

Deputy Editor

PLOS Neglected Tropical Diseases

Poppy Lamberton

Deputy Editor

PLOS Neglected Tropical Diseases

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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: - Sample collection very well described.

- Direct observation used to characterize latrine, good methodology to avoid bias.

- Statistical anaylisis appropriate and well described.

- Only a suggestion, instead of household population and whether the pit latrine was shared with other households, authors could adjusted by the number of members using the latrine (if available).

- Main study limitations identified in discussion.

Reviewer #2: (No Response)

Reviewer #3: - The main objective was to evaluate the spatial distribution of enteric pathogens inside pit latrines with a clear and testable hypothesis.

-The study design is appropriate to address the stated objectives

-The population is clearly described and appropriate for the hypothesis being tested

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

-statistical analysis used to support conclusions are correct using rank order prevalence, regression analysis and the Jaccard similarity coefficient

-There are no concerns regarding ethical or regulatory requirements.Ethical approval and material transfer agreement have been received from the Republicof Malawi National Health Sciences Research Committee

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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: - Table 1, In my opinion it is a bit confusing the total number of latrines sampled, the number of latrines tat were contaminated and the number of parasites detected. I would clarify that.

Reviewer #2: (No Response)

Reviewer #3: The analysis plan is matching perfectly with the final analysis.

The results are clear, complete and tables presented are sufficiet for clarity

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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: - Valuable interpretation of results.

- Study limitations clearly stated.

Reviewer #2: (No Response)

Reviewer #3: Conclusions are in adequation with data presented

Authors described clearly the limitations of their analysis and explain our their founding will be helphul to implement Wastewater based epidemiology (WBE ) in pit latrines.

Public health relevance is adressed.

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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: (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: Relevant question. Problem and knowledge gap very well described and understandable.

Reviewer #2: Line 139 - 145: The SI units should be standardise across the document.

Line 148 - MS2 should be clearly defined

Line 196 - The total in column 3 doesn't seem to add up

Line 206 & 213 - I can't seem to understand the inter and intra latrine section. How are they defined?

Overall the study is ok. Future study should strongly factor eliminating cross-contamination. This study will compliment existing studies on pit latrines as it cut across regions using pit toilet systems.

Reviewer #3: This study provides interesting preliminary information on the collection methodology in small latrines. The methodological approach is good and the results are in line with the initial objective. It would be interesting to delimit the field of application of the results found.

Socio-economic, environmental (oxygen availability,temperature, moisture content, storage time, pH, volatile fatty acid) and microbiological factors were not taken into account in the variables analyzed and could constitute slight biases.

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Reviewer #1: No

Reviewer #2: Yes: Olayinka Osuolale

Reviewer #3: Yes: Abdou Kader Ndiaye

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: PNTD-D-20-02024_review.pdf

Attachment

Submitted filename: PNTD-D-20-02024_KaderNdiaye_Review.pdf

Dear Dr. Brown,

Thank you very much for submitting your manuscript "Impact of sampling depth on enteric pathogen detection in pit latrines" for consideration at PLOS Neglected Tropical Diseases. As editor I am happy with the changes you have made, but would like to see three very minor extra changes to this resubmitted manuscript:

1) add a space on line 82 (fewer than wastewater (25,26).Because fecal sludges can be sampled from shared latrines, pit )

2) I am happy with your response to Reviewer 2 on their point 3 about the median values in the table, and not the means, and hence why they don't add up. However, as other readers may also make a similar misunderstanding, please clarify in the legend that the values are medians within each row.

3) Reviewer 3 point 1, Whilst this is outside the scope of your study, in your discussion please state that whilst this study focuses on the potential of this method for community/area surveillance for specific pathogens, use of these methods could also provide information on risks from these latrines, and then mention the three risks that they have suggested.

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,

Poppy H L Lamberton

Deputy Editor

PLOS Neglected Tropical Diseases

Poppy Lamberton

Deputy Editor

PLOS Neglected Tropical Diseases

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

Three very minor changes to this resubmitted manuscript:

1) add a space on line 82 (fewer than wastewater (25,26).Because fecal sludges can be sampled from shared latrines, pit )

2) I am happy with your response to Reviewer 2 on their point 3 about the median values in the table, and not the means, and hence why they don't add up. However, as other readers may also make a similar misunderstanding, please clarify in the legend that the values are medians within each row.

3) Reviewer 3 point 1, Whilst this is outside the scope of your study, in your discussion please state that whilst this study focuses on the potential of this method for community/area surveillance for specific pathogens, use of these methods could also provide information on risks from these latrines, and then mention the three risks that they have suggested.

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

Dear Dr. Brown,

We are pleased to inform you that your manuscript 'Impact of sampling depth on enteric pathogen detection in pit latrines' 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,

Poppy H L Lamberton

Deputy Editor

PLOS Neglected Tropical Diseases

Poppy Lamberton

Deputy Editor

PLOS Neglected Tropical Diseases

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