Dear Dra. Alves,

Thank you very much for submitting your manuscript "Local amphotericin B therapy for cutaneous leishmaniasis: a systematic review" 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.

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,

Ana Laura Grossi de Oliveira, Ph.D.

Guest Editor

PLOS Neglected Tropical Diseases

Ricardo Fujiwara

Section 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: Major revision: I have recommended the GRADE to evaluate the certainty of evidence.

Reviewer #2: The introduction of the study and its goal is clear. The methods are also clearly defined and in line with reporting guidelines for systematic reviews. There are, however, some concerns about the decision to conduct a meta-analysis and estimate pooled cure rates with very heterogeneous studies. Authors also described having to "arbitrarily assumed as the initial cure rate that assessed between D42 and D90."

Why did the authors decided for this approach instead of a qualitative synthesis of the available literature?

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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: Major revision: -The figures are not of sufficient quality for clarity, and need to be improved.

Reviewer #2: The results presented are clear and in line, again, with reporting guidelines. Authors have presented their findings in a very clear and succinct form. However, in line with the comment on the methods, the pooled estimates of cure rates with AmB treatment are difficult to interpret. The clinical studies included were conducted in populations infected with different Leishmania species, particularly OWCL and American species, which are known to differ in the rates of self healing and response to treatments.

Regarding the presentation of adverse events, did any other of the studies (besides Lopez, 2018) assessed systemic effects (e.g., electrolytes, creatinine, transaminases, etc.)? If so, I would recommend stating in the manuscript whether systemic effects were assessed in these clinical studies and if none of them observed, which is different from lack of collection of systemic effects (e.g., no systematic measurement of blood chemistry and other markers).

In addition, it would be informative to describe the duration of these adverse events, particularly those related to intralesional administration. If not available, at least would be interesting to know if some of these are permanent. For example, table 4 describes prolonged pain (n=17/93) and fibrosis (n=12/93) at the injection site (Goyonlo,2014) in about 13-18% of treated cases, which would be important parameters to consider the feasibility of conducting further evaluations of intralesional AmB, given their potential impact in patient preference.

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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 must be consider the GRADE analysis for conclusion.

Reviewer #2: Abstract: The conclusion stated in the abstract ('Amp.B is a promising strategy") is not well supported. Gicen that the abstract is a standalone section, I would suggest to present more details in the results of the abstract (e.g., safety or other clinical outcome) that supports the 'promising' nature of the strategy (or use other qualifications to describe the intervention).

Main text: The discussion section is clear and many of the limitations of the available literature are presented. Less is discussed about the decisions made by the author team and the implications on result interpretation, for example, the decision to ‘assume’ that evaluations were at D90 in the studies, and mostly, the decision of continuing with a meta-analysis given the high heterogeneity of the studies.

In the pre-clinical studies, were there any PK or toxicity studies retrieved with the search strategy? All the included ones present outcomes related to cure, so, it would be good to know if the lack of PK/toxicity data was an artifact of the strategy selected to conduct the search.

In terms of conclusions, little could be inferred beyond the lack of standards and limited evidence for the use of AmB. This is a finding on its own and very relevant. However, it would be difficult to conclude, as authors have made for example, that continuing with intralesional administration of commercially available AmB is the best option. As mentioned in the results, more details on the safety would also help inform such a conclusion, but this is only briefly mentioned in the manuscript.

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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: The figures are barely legible. This may have been an effect during the creation of the pdf for review, but fig 2 and 2 are almost impossible to read (very small font size and blurred text).

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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 comments were made in the attached file.

Reviewer #2: The study seeks to gather evidence in a very important topic, for which the number and quality of available published articles is limited. This is reflected in the results and discussion of the current systematic review. The main concern is the decision of authors to push for a meta-analysis in this context, which makes the results very difficult to interpret and potentially misleading. For example, the pooled cure rate for topical administration of AmB, was 45.6% [CI: 27.5–64.8%; I2 = 79.6), but the larger studies contributing to this number have very low cure rates (27%) while the smallest study, mostly in L. major (n=22), reported 81% cure. This is mentioned in the discussion, but one may argue that a qualitative synthesis of the available literature would allow to preserve these nuances when describing in the evidence. Given the objective of the study, a more detailed structure in the presentation of safety data (AEs), to see whether e.g., systemic AEs did not occur vs were not collected in the study, would be benefitial.

Other comments to the manuscript are detailed in the respective sections.

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Reviewer #1: Yes: IZABEL GALHARDO DEMARCHI

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

Attachments

Attachment

Submitted filename: PNTD-D-23-00751_reviewer (first review).pdf

Dear Dra. Alves,

Thank you very much for submitting your manuscript "Local amphotericin B therapy for cutaneous leishmaniasis: a systematic review" 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,

Ana Laura Grossi de Oliveira, Ph.D.

Guest Editor

PLOS Neglected Tropical Diseases

Ricardo Fujiwara

Section 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 #2: The study objectives are clear and methodology adheres to the reporting standards for these studies. Authors have addressed most of the comments from the previous round.

Reviewer #3: The objectives of thi study articulated with a clear objective. The population clearly described and appropriate for the hypothesis being tested. The sample size is not large but sufficient to ensure adequate power to address the hypothesis being tested. Correct statistical analysis used to support conclusions. The paper is a review based on the results of clinical treatment, there are no concerns about ethical or regulatory requirements.

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

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 #2: The results are clear and in line with the analysis plan.

Authors have now presented the findings by region and added comment on the GRADE low certainty of the evidence, which are welcome edits and provided important nuance to the interpretation of the results.

Regarding adverse events (table 4 and related texts), it is still not clear whether systemic adverse events were not inquired (i.e., no blood test or questions were performed as part of the study) in some of the studies instead of not reported. This distinction is very important to understand the systemic effects of the intralesional or topical administration of amphotericin B.

Reviewer #3: The analyses presented matched the analysis plan and the results clearly presented. The figures (Tables) are of sufficient quality.

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

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 #2: The discussion has improved and the limitations of the data analyzed are better presented. Some additional comments include:

Lines 340 - 341: please add the confidence intervals when describing the pooled estimate of the clinical studies.

Lines 349 - 351: please add references to this statement

Lines 372 - 373: Please verify the grammar of this sentence, it is not clear what is the meaning of: "It is and the comparison of studies"

Authors indicate that there is "lack of association" between the concentration and frequency of administration with the outcome. However, most of the studies are small and it is difficult to discern whether the lack of observed effect is is real or due to the small sample size. In this case one can argue that there is not evidence of an association (due to the size of the studies), rather than a lack of association.

Reviewer #3: The conclusions are supported by the data presented. The limitations of analysis clearly described.

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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 #2: Figures are slightly better, however, figures 2-4 are still blurred in the pdf available for review and the font size is too small. Please revise the quality of the figures (not just that they meet a file format suggested by the journal, but that the font size and layout are legible).

Some of the tables have missing horizontal lines (e.g., Table 2 and 4)

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 #2: (No Response)

Reviewer #3: Leishmaniasis is still a disease epidemic in many countries, and effective therapeutic and preventive tools are still missing but urgently needed. In this manuscript the authors systematically reviewed the efficacy of treatment approaches with two AmB-based regimes on CL. The data were gathered mainly from studies carried in Iran, Colombia and India in the last 12 years. The cure rates significantly varied with very low certainty, which were likely determined by various factors including parasite species, subject physiological conditions and others. The review is comprehensive, but the significance of the study may be limited due to the lack of novelty of the treatment regimes.

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

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 #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.

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 Dra. Alves,

We are pleased to inform you that your manuscript 'Local amphotericin B therapy for cutaneous leishmaniasis: a systematic review' 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,

Ana Laura Grossi de Oliveira, Ph.D.

Guest Editor

PLOS Neglected Tropical Diseases

Paul Brindley

Editor-in Chief

PLOS Neglected Tropical Diseases

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