Dear Dr. Dreyfus,

Thank you very much for submitting your manuscript "Leptospira spp. seroprevalence in the Wiwa indigenous population from Colombia and the comparison of the Serion IgM ELISA and Microscopic Agglutination Test for diagnosis of Leptospira spp. infections in human sera from different geographical origins" 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.

As some reviewers pointed out, the objectives, methods and analysis needs to be consistent and aligned to answer the research question. This manuscript's objective is to evaluate the performance of the Serion ELISA, therefore, sample size and methods need to focused on that objective. As diagnostic tests need to be evaluated for their specific use, clearly separate the evaluation for i) clinical diagnosis and for ii) prior exposure detection.

For each one these sub-objectives, method and results need to reflect the standard practices for reporting diagnostic test evaluation:

https://journals.plos.org/plosone/s/best-practices-in-research-reporting

https://www.equator-network.org/reporting-guidelines/stard/

Additionally, considering the small sample size of the evaluation for the clinical diagnosis, more details regarding its contribution and comparison with several others papers evaluating the same kit are needed under the rationale for the study.

The sub-objective evaluating the accuracy to detect prior exposure needs a better description of the design and analytical approach as MAT is a reference test but not a perfect "gold" standard and results correspond to relative SE and SP. Authors can consider analytical methods for evaluation of diagnostic tests with no gold standard and two populations. The effective sample size are the number of samples with both ELISA and MAT results.

The goal of estimating sero-prevalence in the Colombian study population is a secondary outcome of the accuracy study, and stated, most of the report should be focused on the diagnostic test evaluation study following recommended guidelines.

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,

Claudia Munoz-Zanzi

Associate Editor

PLOS Neglected Tropical Diseases

Elsio Wunder Jr

Deputy Editor

PLOS Neglected Tropical Diseases

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As some reviewers pointed out, the objectives, methods and analysis needs to be consistent and aligned to answer the research question. This manuscript's objective is to evaluate the performance of the Serion ELISA, therefore, sample size and methods need to focused on that objective. As diagnostic tests need to be evaluated for their specific use, clearly separate the evaluation for i) clinical diagnosis and for ii) prior exposure detection.

For each one these sub-objectives, method and results need to reflect the standard practices for reporting diagnostic test evaluation:

https://journals.plos.org/plosone/s/best-practices-in-research-reporting

https://www.equator-network.org/reporting-guidelines/stard/

Additionally, considering the small sample size of the evaluation for the clinical diagnosis, more details regarding its contribution and comparison with several others papers evaluating the same kit are needed under the rationale for the study.

The sub-objective evaluating the accuracy to detect prior exposure needs a better description of the design and analytical approach as MAT is a reference test but not a perfect "gold" standard and results correspond to relative SE and SP. Authors can consider analytical methods for evaluation of diagnostic tests with no gold standard and two populations. The effective sample size are the number of samples with both ELISA and MAT results.

The goal of estimating sero-prevalence in the Colombian study population is a secondary outcome of the accuracy study, and stated, most of the report should be focused on the diagnostic test evaluation study following recommended guidelines.

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 authors described the study objectives clearly. The study design, including sample size, population sources, and statistical analysis, were explained well. However, the approvals of the Human Research Ethics committee on using serum samples from Group A and B are not described. And I need a rationale on why the study using Bhutanese specimens was granted by the Human Research Ethics committee of the University of Newcastle, Australia, only.

Reviewer #2: Introduction

1.- Objectives can be clearly summarized and stated. Lines 148-162 may be better included in the materials and methods

Material and methods

2.- Line 181, indicate in the first time what “TPH” means

3.- Line 295. What is an ambiguous result? Why an ambiguous result is considered negative? There were duplicates that could be made to disambiguate

Reviewer #3: inclusion of all groups is not clear according with objetive of study: compare two diagnoses test and the seroprevalence of Wiwa population.

Two different topics. Sample size should be different: test for a test or population survey.

Ethical OK

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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 authors demonstrate results and the data analysis pretty clearly and entirely as the study objectives. The Figures and Tables are tidy and easy to read. In Table 4, the "N" for Group E should be 156 instead of 321. To fulfil more on the study objectives, the accuracy of the "Serion ELISA classic Leptospira IgM" test inferring seroprevalence in sera Group D and E--the endemic areas. Additional positive predictive value (PPV) and negative predictive value (NPV) of the ELISA test in Table 4 (MAT used the reference) will directly point out the failure of using ELISA to investigate the seroprevalence in the endemic areas.

Reviewer #2: results

4.- In the table 4, groups A + B should have a single value of "N" (34) to maintain congruence with the other data in the table.

5.- In group C, why can't the 95% CI for specificity be calculated?

6.- In group E, the reference test was carried out in 156 people and not in 321.

7.-Line 359. In this section, authors should not incorporate sensitivity in the interpretation, because in group C only specificity is evaluated.

8.-Figure 1. Were some of the random samples considered MAT (+)? If any were, it would not be correct to include all the "ELISA negative" (476 or 163) in the sero-negative group, because in theory the ELISA-negative have 2X or 5X with respect to the random sample and if they have some MAT + in the random sample group, there would also be some MAT + in the "ELISA negative" group. With this, the sero-prevalence could be re-calculated.

9.-The paragraph on lines 441-451 corresponds to the limitations of the study. They must be sent to the corresponding section.

Reviewer #3: Table 4 should include total, positives by MAT and ELISA.

the use of the term seroprevalence of the study is not clear because are different populations, different countries and sample size, not methodological correct.

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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 authors conclude the study reasonably, including all limitations of the analysis and study design.

Reviewer #2: 9.- The first paragraph (lines 416-424) is part of the objectives and methodology that has already been explained. It is recommended to start the discussion with the main results obtained.

10.- Lines 458-479 support the over and underestimation assumption. Therefore, they must be linked to the sentence that mentions it.

11.-Line 494. Has the hypothesis of low specificity been evidenced in other studies of leptospira or other spirochetes? If so, include those studies as references.

Reviewer #3: It is inconsistent with the introduction the profile of risk of Wiwa population and the conclusion presented.

The statement "ELISA is technically working well..." should include the concrete arguments for that.

The discrepancy between MAT and ELISA should be analice from the theory more than make comparisons with results from other species.

conclusions about seroprevalence in Wiwa population should be discuss and evaluate in a future with comorbilities from the region as other febrile syndromes from infectious diseases.

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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: I suggest a Minor Revision for this manuscript. My significant comments are as follows:

1. show IRB approval for study Group A & B and IRB multi-centre or Bhutanese IRB approval for study Group D

2. correct the "N" number in Table 4 for Group E

3. add PPV (95%CI) and NPV (95%PI) in Table 4 for Group D and E.

Reviewer #2: Be congruent with the number of decimal places to include in the percentages.

In Abstract: Maintain the same units at the conclusion of the% seroprevalence (line 59)

Reviewer #3: Maybe a figure (a different figure than a diagram) to show some results will be more easy to understand (388-399).

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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: Some significant results were published in Pathogens; the whole story is still beneficial for Leptospira diagnosis or seroprevalence study. It emphasizes the essential test validation before use in disease-endemic areas.

Reviewer #2: This study has the strength of having investigated the performance of a serological test for leptospirosis in a non-endemic setting (after onset of disease) and in an endemic setting where further research is still necessary.

Reviewer #3: I think that more importance should be done to wiwa results. And as secondary results should be comparisons with other populations. That implies more emphases at those results and to begin with Wiwa populations context, methodology and results.

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Reviewer #1: No

Reviewer #2: Yes: Antonio Flores

Reviewer #3: Yes: Janeth Perez-Garcia

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

Dear Dr. Dreyfus,

Thank you very much for submitting your manuscript "Comparison of the Serion IgM ELISA and Microscopic Agglutination Test for diagnosis of Leptospira spp. infections in sera from different geographical origins and estimation of Leptospira seroprevalence in the Wiwa indigenous population from Colombia" 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,

Elsio Wunder Jr, D.V.M., Ph.D.

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: Authors describe the objectives of each experiment clearly. The primary aims are evaluating the analysis sensitivity and specificity of the Serion ELISA classic Leptospira IgM using a) 14 confirmed leptospirosis cases originated from Netherland, b) 20 healthy blood donors, c) 41 confirmed other infections originated from Switzerland, d) 321/489 sera from volunteers (healthy or ill) originated from Colombia.

The objectives of the group d investigation (line 191-193) are arguable. In basic immunology, IgG antibody screenings are usually performed for investigating seroprevalence. The IgM ELISA was used for seroprevalence of leptospirosis in this study. Authors should describe reasons for use this unexpected study design. Previous studies on validating IgM and IgG for Leptospira seroprevalence in comparison are recommended in the Introduction section. Emphasizing using MAT on single sera for the seroprevalence (not diagnosis) as a reference method should be done to remind readers.

The study design of the seroprevalence in group d is questionable. There is a cross-sectional study design that perform a reference test first and then performing case-control sampling (based on the reference test results) to further perform index test (the ELISA test in this study). Authors should describe the type of the cross-sectional designs selected in this study and the reason of selections.

PPV and NPV are not needed for group a, b or c.

In my personal opinion, all a,b, c and d should be done for only one primary aim: to investigating the accuracy of the ELISA test in survey sero-prevalence (both past and current infection) compared to MAT using single sera collected during leptospiremia, leptospirurea, and no Leptospira in bodies.

Reviewer #2: objectives.

Please to include the text suggested for the author:

"“The objectives of our study were to evaluate the sensitivity and specificity of the commercially

available Serion ELISA classic Leptospira IgM using the MAT as imperfect reference test with patient

sera from confirmed leptospirosis cases, blood donor sera, with sera from patients with other

infections than leptospirosis (all European) and sera from a cross-sectional study in the indigenous

population called “Wiwas” of the Sierra Nevada de Santa Marta, North-east of Colombia..."

Reviewer #3: (No Response)

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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: Figure 1--It is not correct to pool ELISA and MAT results (called sero-positive and sero-negative).

Table 4 --the 124/32 (instead of NA) should be add in the ELISA (pos/neg) column in row D ii

Estimating sero-prevalence based on the ELISA in Wiwa population were less accuracy as the significant low specificity compared to those estimated by MAT. Combination results from samples having ELISA results only might be another kind of bias.

Reviewer #2: Table 4.

specificity values of Group B are incorrectly added within the group A row. (Group A are cases, there are no "negative samples", so it should not have any specificity value)

In group C, please indicate what the value of 100% specificity corresponds to. At 3 MAT-/ELISA- ? if so, clarify it in the legend below

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: Authors conclude that the IgM ELISA test was less accuracy when use for Leptospira seroprevalence compared to MAT. Authors argue on the less sensitivity of MAT (single sera) when use as a diagnosis tool (line 479-483). However, I did not see the relevant to the group d experiment.

Even the results clearly show low accuracy of the IgM ELISA test, authors insist that ".. IgM detection test can be useful in screening for Leptospira exposure in the recent past.." (510-517) based on a citation of book reference. Serious discussion with previous reports on this issue are lacking.

Reviewer #2: Discussion: About your hypothesis in lines 422-424, It is Ok after your feedback.

Reviewer #3: (No Response)

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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: none

Reviewer #3: 5 In the title could be more accurate to use leptospirosis seroprevalence

51 Sensitivity value at cross sectional study in Colombia is missing in the abstract

184 “most likely seronegative for Leptospira spp..” could means doubt about leptospirosis diagnoses and cannot be objective to evaluate cross reaction.

254 Should be interesting pointed the reason to use OIE standards for human test, and not WHO.

278 If you referred to “endemic places” to Colombia, should be better to describe it explicitly, because its methodology of the study.

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

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 concerns on the objectives and study designs used in this study. It might be better that authors present a major and accuracy key message to readers with evidence based style. The manuscripts can be confusing as the ELISA was evaluated as a diagnosis tool and a sero-prevalence in the same article. As a reader of PLoS NTD, I eager to know if IgM ELISA (using non-pathogen Leptospira antigen) is useful for seroprevalence in the low income countries with leptospirosis endemic areas or not.

I am confused by Abstract. And please check the information accuracy in the abstract as well.

Reviewer #2: none

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: Yes: Antonio Flores

Reviewer #3: Yes: Janeth Perez-Garcia

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. Dreyfus,

We are pleased to inform you that your manuscript 'Comparison of the Serion IgM ELISA and Microscopic Agglutination Test for diagnosis of Leptospira spp. infections in sera from different geographical origins and estimation of Leptospira seroprevalence in the Wiwa indigenous population from Colombia' 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.

There are one minor suggestion for change in the text. On page 80, the authors mention that the Leptospira genus has 64 species. A recent publication (PMID: 34914572) has identified 4 new species for the genus, bringing the total to 68.

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,

Elsio Wunder Jr, D.V.M., Ph.D.

Deputy Editor

PLOS Neglected Tropical Diseases

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