Dear Mrs. Cossio,

Thank you very much for submitting your manuscript "Diagnostic performance of a Recombinant Polymerase Amplification Test - Lateral Flow (RPA-LF) for cutaneous leishmaniasis in an endemic setting of 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).

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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,

Philippe Büscher, PhD

Associate Editor

PLOS Neglected Tropical Diseases

Epco Hasker

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: (No Response)

Reviewer #2: This paper describes in details a relatively new technique of combining RPA and latter flow to develop a POC device, in this case for the detection of Leishmania.

The methodology of the study is very well described, and the graphic representation of the study design truly helps to grasp the context of the paper.

Just for the sake of clarity, a few additional details should be added, so that the paper becomes completely clear and will be used a a general refence by others in the future.

1) Line 277: please include primer sequences in the M&M section

2) Line 315 calculation methods (formulas) for sensitivity, specificity and positive / negative predictive should be added, taken that it is not always clear which golden standard is used exactly in the calculation. This reviewer was very pleased by the fact that PPV and NPV were include, something that is very often omitted in experimental diagnostic research papers despite the fact that these are the values that determine if a clinician can 'trust' the test in the field (while sensitivity and specificity are theoretic values that allow very easy masking of serious issues). So, well done !

The reason for this question: see below, result section.

Reviewer #3: lines 148-150 The authors could clarify for which hypothesis and power this sample size is calculated.

line 253 Are 'the samples' the swabs for qPCR?

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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: (No Response)

Reviewer #2: Very clear presentation of results.

Just one question for additional explanation...

Line 369/370: the difference in 'results' obtained in the lab versus the field. Details are outlined in Table 3.

From these results it is clear that the main differences are TP (72 vs 62) and FN (11 vs 21). of course it is obvious that field analysis will never perform as good as good as a lab context, but the question here is how did these 2 difference occur. By providing the formula used to calculate these values, and indicating which factor contributed to the decrease of TP and increase of FN, the work could become a textbook example of how to go about doing these sort of things. Again, the quality of the work, the methodology and paper is outstanding, and by adding this info, 'I' would even be temped to use this paper as a textbook/lecture example in advanced molecular parasitology classes.

Reviewer #3: lines 334-336 If the sample size was estimated to be 118, why did the authors include 128 patients?

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

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: (No Response)

Reviewer #2: The conclusion is very well written, and this reviewer specially appreciated the critical assessment of the limitations of the study. Again, this is something that is very often missing from so many diagnostic papers, and it just adds to the value of this particular study.

One last question however, RPA-LFA is not the cheapest technique, and by adding a 'cheap' readout tool in the future, the 'practicality' of the cost could further be an issue. Would it be possible for the authors just to add a few details on the current cost of the RPA-LFA (something that should be similar to RPA-LFAs for other diseases such as trypanosomosis) versus the cost of the 'current' most used golden standard in the field. the latter will be very different depending on the disease studied. It's not crucial for the quality of the paper, but it just allows the reader to think about the bigger picture and out everything in perspective.

Reviewer #3: line 433 - 443 Could sensitivity have been impacted by first doing swab sampling for qPCR and only then filter disks sampling for RPA-LF?

line 445 - 449 Is the misinterpretation of banding patterns also linked to the <90% specificity of RPA-LF in both field and ref lab?

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

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: See other sections

Reviewer #3: line 59 remove the '('

line 63 duplicated 'in'

line 161 correct 'performed' to 'perform'

line 351 duplicated 'in'

line 397 correct 'Ddiagnotic'

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

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: The authors had previously established a RPA-LF protocol for CL diagnosis (Ref 21). Here, they successfully evaluate its efficiency and usefulness in field conditions by following a rigorous algorithm with multiple comparisons to an exhaustive panel of diagnostic tests (including a challenging composite gold-standard). This is an excellent work, with some real efforts to present the analytic pipelines and their corresponding results in a simple yet precise manner. The manuscript is well-written, the methodology is very rigorous and the discussion is adapted to the results. The results are positive and convincing and they will certainly be of interest for people involved in the control of CL, and possibly in the context of other NTDs for which diagnostic tools require improvement. I only have minor comments and suggestions below.

Minor comments:

• L.33: in the field?

• L.63: in both

• L.225: Leishmania in italic

• L.308-313: The relatively frequent occurrence of contaminations during the RPA protocol, especially in the field, could be explained a bit more in details and discussed in the appropriate section (possible reasons for such contaminations and ways to avoid it in the future).

Reviewer #2: Very clear paper.

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: Yes: Brice Rotureau

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.

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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 Mrs. Cossio,

We are pleased to inform you that your manuscript 'Diagnostic performance of a Recombinant Polymerase Amplification Test - Lateral Flow (RPA-LF) for cutaneous leishmaniasis in an endemic setting of 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.

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,

Philippe Büscher, PhD

Associate Editor

PLOS Neglected Tropical Diseases

Epco Hasker

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: (No Response)

Reviewer #2: (No Response)

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: (No Response)

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: (No Response)

Reviewer #2: (No Response)

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: (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: All suggestions and corrections were taken into account in the revised version.

Reviewer #2: Thank you for addressing the comments made during the first round of reviewing.

Reviewer #3: No further questions.

**********

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: Brice Rotureau

Reviewer #2: No

Reviewer #3: No