Dear PhD De los Santos,

Thank you very much for submitting your manuscript "Sampling is decisive to determination of Leishmania species​" 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.

The Reviewers the Editors acknowledge the relevance of your report to the diagnosis and speciation of Leishmania and the importance/impact of the sampling methods used. The novelty of your report may be limited as a number of earlier studies have compared the various sampling methods for leishmania diagnosis, your study and the major findings are well documented and useful to the scientific community, especially to scientists interested in field diagnosis of leishmaniasis. Further, this report is well within the scope of PNTD. However, please address all the comments and questions of the reviewers in your revised manuscript. Specifically, please address in detail the comment related to false negativity mentioned by reviewer 1.

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,

Alain Debrabant

Academic Editor

PLOS Neglected Tropical Diseases

Walderez Dutra

Section Editor

PLOS Neglected Tropical Diseases

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The Reviewers the Academic Editor acknowledge the relevance of your report to the diagnosis and speciation of Leishmania and the importance/impact of the sampling methods used. The novelty of your report may be limited as a number of earlier studies have compared the various sampling methods for leishmania diagnosis, your study and the major findings are well documented and useful to the scientific community, especially to scientists interested in field diagnosis of leishmaniasis. Further, this report is well within the scope of PNTD. However, please address all the comments and questions of the reviewers in your revised manuscript. Specifically, please address in detail the comment related to false negativity mentioned by reviewer 1.

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: There is no hypothesis clearly stated, however the aims of the study are clearly articulated.

The design, population studied, and sample size are clearly described and appropriate; the statistical methods seem appropriate and there are no ethical concerns as consent was signed by the patients.

It is unclear though whether the study was blinded at any stage. This should be indicated, and the limitations of a non-blind study discussed.

Reviewer #2: See general comments

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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 analysis and results are well described and supported.

The graph in figure 3B is difficult to read. Please make sure to increase the font and submit a high-quality image.

I also suggest creating a graphical abstract figure or a figure summarizing the various methods used and the main results yielded.

Reviewer #2: (No Response)

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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 conclusions are supported and some limitations are mentioned.

The public relevance of the study is addressed.

Reviewer #2: (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: Minor revisions:

1) It is unclear though whether the study was blinded at any stage. This should be indicated, and the limitations of a non-blind study discussed. (See methods part)

2) The graph in figure 3B is difficult to read. Please make sure to increase the font and submit a high-quality image.(See results part)

3) I also suggest creating a graphical abstract figure or a figure summarizing the various methods used and the main results yielded. (See results part)

4) What is the prevalence in the territory of the various species of Leishmania identified in this article? This needs to be discussed in the introduction or discussion sections.

5) The authors talk about false negatives in the discussion. Is this also addressed in the data? This concept should be made more clear.

6) The authors should discuss why it is so important to determine the exact species of Leishmania affecting a certain individual (because of treatment choice, epidemiological research...etc?)

Reviewer #2: (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: This is a very interesting manuscript investigating different methods of Leishmania detection in order to improve species identification in the field. The results found will have a noticeable public health impact in the territory and could be extrapolated to other endemic areas as well. Overall, there are no major issues and I recommend publication with minor revisions. See my comments above.

Reviewer #2: In the paper described by de los santos et al, entitled “sampling is decisive to determination of Leishmania species, they compared the sensitivity of molecular identification of Leishmania species from Peru, using 3 types of sampling: punch biopsy, filter paper and lancet scraping. They showed that different sampling methods can affect the discrimination capacity of molecular tests for the identification of Leishmania species. They demonstrated that lancet scraping is more efficient than filter paper imprints or biopsies to identify more leishmanisais cases. They suggested that scraping with lancets could be used to identify parasite as an alternative to invasive methods such biopsy. It is very interesting study that deals with a topical problem, diagnosis of leishmaniasis, in fact the identification of the parasite are necessary for the efficiency of treatment. However, the authors need to improve the manuscript.

Major points

1. The authors have to add controls: negative control samples from healthy individuals. They have to check the integrity of extracted DNA by using PCR targeting host gene like beta globin. It is very important to check the true negativity. It is also important to compare the recovery of DNA from each sampling methods, especially in the sensitive area (Eyes, lips…).

2. It would be interesting to consider the amplification and sequencing of another molecular marker for the identification of Leishmania species especially for those that were not identified.

3. In the section 2 of results (line 285-line 296), 85 patients were identified infected by Leishmania, but in the section 3 (line 302-line313) and table 2 all the percentages were calculated based on the total 105 patients. In addition they have to add p value to show significant difference between sampling methods (line 307- line 308).

4. Table 2 the parasite load is not clear, in my opinion they have to illustrate the parasite load separately in another figure.

5. For the weak band intensity, it could be interesting to add another molecular method for the identification of parasite.

Minor point

1. In my opinion the title is general, the authors should think about another title reflecting the message of the study

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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: No

Reviewer #2: 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

Dear PhD De los Santos,

Thank you very much for submitting your manuscript "Sampling is decisive to determination of Leishmania species​" 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 address the remaining two comments of Reviewer 2 in your revised 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,

Alain Debrabant

Academic Editor

PLOS Neglected Tropical Diseases

Walderez Dutra

Section Editor

PLOS Neglected Tropical Diseases

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

Please address the remaining two comments of Reviewer 2 in your revised 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. The authors have responded to my comments and addressed my concerns.

Reviewer #2: (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: The results are clearly presented, more controls were added, and the figure quality has increased.

Reviewer #2: (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: The conclusion section is satisfactory.

Reviewer #2: (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: /

Reviewer #2: (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: /

Reviewer #2: The authors answered to all queries and included all the modifications requested. They improved the quality of the manuscript. Two points still need to be revised.

Minor revision

1. The title is still general, not limited to what was carried out. Indeed the authors used just Leishmania viannia species causing CL in Peru. I suggest ex “sampling is decisive to Leishmania viannia species identification…

2. The authors answered that they used samples from healthy individuals as a negative controls. Please mention that in the text in MM (study population).

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

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

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 PhD De los Santos,

We are pleased to inform you that your manuscript 'Sampling is decisive to determination of Leishmania (Viannia) species​' 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,

Alain Debrabant

Academic Editor

PLOS Neglected Tropical Diseases

Igor C. Almeida

Section Editor

PLOS Neglected Tropical Diseases

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