LeishmaniaResponse to ReviewersRevised Manuscript with Track ChangesManuscript

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

orcid.org/0000-0003-4304-636XX

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

orcid.org/0000-0003-1765-0002

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Reviewers' comments:

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? yes

Reviewer #2: The study objectives, study design, statistical analysis and conclusion are fine. The sample size for clinical evaluation of the tests is not justified.

Reviewer #3: All methods used are suitable for answering the hypothesis.

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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: -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 #2: The analysis and presentation of the results are clear and tables and figures as well.

Reviewer #3: All results presented are in line with the analysis plan and are clearly presented.

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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: -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 #2: The conclusion is supported by the obtained results. Limitation of the study is that it needs further improvement to comply with WHO ASSURED criteria. They, however, need to indicate approximate cost of the current version of the tests and how they plan to bring it down so that it can be implemented in the LMIC if eventual phase III diagnostic trial confirmed tests' diagnostic accuracy more than 95%.

Reviewer #3: All conclusions are corroborated by the data presented and have an impact on public health. The authors discuss the limitations of the analysis.

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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: NA

Reviewer #2: The current version of the manuscript is too long. Repeats can be avoided. Technical details can be summarized where applicable.

Reviewer #3: The manuscript presents robust results on the use of LAMP combined with CRISPR-Cas12a for the diagnosis of cutaneous leishmaniasis. The two assays evaluated, particularly the one using the kDNA target, showed good analytical and clinical performance. My suggestion is that the article be accepted after minor modifications.

Comments:

1- Include in the Methods section the criterion (qPCR kDNA) used to define cases and non-cases of cutaneous leishmaniasis;

2- Do the authors have information on the diseases of the patients in the non-case group? If so, it would be helpful to include this information in the supplementary table;

3- Were parasitological tests (imprint, scarification, or culture) performed on the patients? If so, it would be important to compare the performance of these techniques with the results obtained with the LAMP-CRISPR-Cas12a assays;

4- Although the authors suggest that the CRISPR-Cas12a system increases the specificity of the reaction, it would be interesting to demonstrate the results of the 18S LAMP and kDNA assays without the use of the CRISPR system. This comparative analysis would demonstrate the real need to use CRISPR to increase specificity;

5- Include in the discussion other articles that evaluated Lamp for cutaneous leishmaniasis, without the use of the CRISPR-Cas12a system.

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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: The manuscript presents the development and validation of CRISPR–Cas12a-based molecular assays coupled with LAMP amplification for the detection of Leishmania spp. The study is well designed, clearly written, and makes a significant contribution to the field of molecular diagnostics for tegumentary leishmaniasis, particularly in the context of low-resource endemic settings. The work is timely and relevant, and the results are convincingly presented. I have only minor comments and suggestions that may help strengthen the manuscript:

Contextualization of field implementation

The authors may wish to expand slightly on potential challenges or considerations for the implementation of these assays in real-world field settings (e.g., sample preparation, training needs, device portability, or cold chain requirements). This would better highlight the practical applicability of the tool.

Clarification of assay comparison

The comparison with reference qPCR methods is clearly described. It could be helpful to add a brief statement on the Ct range or parasite loads of the discordant samples to further illustrate assay performance in low-parasite contexts.

Discussion on 18S assay performance

The lower sensitivity of the 18S assay compared to kDNA is briefly noted. A short discussion of potential biological or technical reasons for this difference (e.g., target copy number, sequence diversity) would be valuable for readers considering assay selection.

Minor editorial points

Ensure consistency in the use of species names (e.g., Leishmania (Viannia) vs. L. (Viannia)).

A few typographical issues were noted (e.g., “crossreactivity” should be “cross-reactivity”). A final language check is recommended.

Overall, this is an excellent manuscript that presents innovative diagnostic tools with clear public health relevance. With minor clarifications, it will make a strong contribution to the literature on Leishmania diagnostics.

Reviewer #2: The study is encouraging to pave the way to take molecular test in a lateral-flow test. However, the proposed DNA amplification tools are challenging in the field which limits its eventual implementation in the LMIC. The sensitivity is

Reviewer #3: Comments above

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Figure resubmission:

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Dear Mrs. Adaui,

We are pleased to inform you that your manuscript 'LAMP-coupled CRISPR-Cas12a assays: a promising new tool for molecular diagnosis of leishmaniasis' 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.

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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

Sarman Singh

Section Editor

PLOS Neglected Tropical Diseases

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

orcid.org/0000-0003-4304-636XX

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

orcid.org/0000-0003-1765-0002

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

Your revised manuscript was reviewed by Reviewer 3 and the Editors and found acceptable for publication in PNTD.

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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 #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 #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 #3: Yes

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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 #3: Accept

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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 #3: The authors made all the requested changes.

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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.

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 #3: Yes: Daniel Moreira de Avelar