Dear Mr Tilahun,

Thank you very much for submitting your manuscript "Effectiveness of carbon dioxide cryotherapy for the treatment of cutaneous leishmaniasis: systematic review and meta-analysis" 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,

Claudia Ida Brodskyn

Section Editor

PLOS Neglected Tropical Diseases

Claudia Brodskyn

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 #1: The study design is appropriate to address the stated objectives

Reviewer #2: This manuscript presents an intriguing summary of the evidence regarding the effectiveness of carbon dioxide cryotherapy in treating localized cutaneous leishmaniasis, aiming to support its continued clinical use. The authors included 16 studies, of which only 7 underwent meta-analysis, covering a total of 1,357 cases

Overall, the study adhered to the standard methodologies for this type of research and manuscript preparation. Nonetheless, there are several issues within the manuscript that require attention, as well as some that were omitted and could have varying implications for data analysis and conclusion drawing.

Rows 99 and 100 mention the following about carbon dioxide base cryotherapy: It is relatively easy to apply (17), and effective (less than 10 % of failure 100 rate) (15,18), minimal adverse events (16,19) and it takes shorter healing time (15,19). All references cited in this paragraph however are not specifically related to CL, and the reader might be misled to believe that these numbers refer to CL

Rows 109-112 mention issues related to patients' barriers to CL treatment and that AHRI will conduct a study on this topic at a community level. I do not see how it fits in the manuscript (at least in the introduction section)

Row 122-124: mention the use in Ethiopia of laser and the lack of well-organized evidence for the effectiveness of this laser therapy on CL caused by L. aethiopica. Personally, the mention of laser in this paragraph creates confusion and more important is not the topic of this manuscript.

Rows 155-161: I found it quite difficult to understand the whole paragraph: It mentions that The outcome of interest for this review was cure rate (effectiveness) of cutaneous leishmaniasis after application of CO2 laser or cryotherapy. But it was mentioned before that “This study aims to summarize the evidence on the effectiveness of carbon dioxide cryotherapy for the treatment of localized cutaneous”.

How the treatment was applied and patients follow-up after the application across the different studies is not clear: Rows 158-161 mention the following schemes:

• once per two weeks till the lesion gets away (cure)

• or not healed up to three months.

• The lesions were treated 4-6 times every two weeks.

Regarding follow-up, it only mentions the following:

When the lesion gets cured within the 4x or less, the patients were had follow up only to see the presence of any satellite lesion around the intervention sites.

The number of applications, time intervals between applications, and the exact follow-up period to assess cure after the onset of treatment are crucial pieces of information to better assess the study results and all of them are not provided.

There is a lack of information regarding the criteria or parameters used to define ‘cure’ or ‘no cure.’ Additionally, there is no data on the devices used to deliver CO2, which, based on other studies such as those on acne, is an important variable in terms of efficacy

It is well-acknowledged in Clinical Leishmaniasis that treatment outcomes are heavily dependent on the species of Leishmania causing the lesions. No single reference or analysis has been presented here that considers this.

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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 presented match the analysis plan.The analysis are presented match the analysis plan.The author needs to display the location, number, and comparison images of the skin lesions before and after treatment in the results.

Reviewer #2: There are several inconsistencies in the Results section.

Rows 191-193 indicate that 16 studies reporting treatment outcomes of carbon dioxide based cryotherapy for the treatment of CL were included in the systematic review, of which seven studies were used for further meta-analysis.

However in the following section, none of the numbers mentioned add up to 16. See some examples below:

The majority of the primary studies 13(87%) were published between 2004 to 2019, one (6.7%) since 1991 (24), and one (6.7%) study included in the review were not published yet. (Total 15)

Moreover, 5 (3.33%) of the research were conducted in research centers, 4 (26.7%) in dermatology clinic, 3 (20%) private clinic and 2 (13.33%) research area was unknown. (Total 14)

In 9 (53.33%) of the cases, SSS exams were used to diagnose the patients, 5 (33.33%) of the

cases combined SSS and histopathologic exams, and 2 (15%) research used PCR; however, 4

(30%) of the studies' diagnostic procedures were not precisely defined. (Total 20)

In other instances, only % are reported: Overall, the majority (73.3%) of the studies were conducted in old world (25) where as 26.7% of the primary 200 studies were conducted in new world countries (25, 26, 27).

Furthermore, more than one-third of the included publications (30, 31, 37) had lesion durations of more than 6 months, while the other two-thirds (24, 29, 32, 37) had durations of less than 6 months. One-third of 16 is ~5 and only 3 papers are cited, which by the way is the same number of manuscripts described in Table 3. Perhaps its easier to say 3 out of 16 publications reported lesions of >6m

Rows 221–216 mention the number of treatments applied: 2 times: 10; 5 times: 1; 1 time: 3 (Total 14)

Etc, Etc.

217-219: At what time (after onset of treatment) was cure defined?

Rows 237-238: Similarly, when the treatment is applied a second time, the cure rate increases dramatically from 82.10% (95% CI: 50.19 - 114.00) to 93.01 (95% CI: 62.72 - 123.29). I wonder if the statement of “increases dramatically” is based on additional statistics done by the authors, I so, please add it. Looking at the 95% CI of both figures, I believe there are no statistically significant differences to make such statement.

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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 supported by the data presented.

Reviewer #2: Discussion / Conclusions

I believe the study had several limitations, (some of which were only briefly mentioned in Rows 293-299) that drawing such categorical conclusions, as presented in the manuscript, could potentially mislead the readers.” I believe it needs to be rephrased based on the limitations found and other considerations mentioned above.

Rows 254-256: I am not sure if in the following statement “However, it is often used in the new world but infrequently in old world including Ethiopia” the authors are referring to the infrequent use of carbon dioxide-based cryotherapy for CL in particular or for dermatological use in general. For CL, there are clearly more studies in the OW than in the NW.

Rows 267-268 briefly mention the inclusion of control groups in certain studies. It is important to elaborate on this point and compare the cure rates of the control groups with those treated with carbon dioxide-based cryotherapy.

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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 recommend “Major Revision”.

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: This is an interesting article, but some changes need to be made before publication. Firstly, the author needs to display the location, number, and comparison images of the skin lesions before and after treatment in the results. Secondly, discuss the application of this method in skin diseases in the conclusion and compare it with other treatment methods. The author needs to improve the writing and grammar of the article, for example, there is a grammar error in line 254 of the discussion. Should the syntax of "we synthesizes" be singular? Therefore, it is necessary to polish the article and provide a certificate.

Reviewer #2: (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.

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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: Wenzhong Xiang

Reviewer #2: No

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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, 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 Mr Tilahun,

Thank you very much for submitting your manuscript "Effectiveness of carbon dioxide cryotherapy for the treatment of cutaneous leishmaniasis: systematic review and meta-analysis" 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,

Claudia Ida Brodskyn

Section Editor

PLOS Neglected Tropical Diseases

Claudia Brodskyn

Section Editor

PLOS Neglected Tropical Diseases

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

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

PNTD-D-24-00369R2Effectiveness of carbon dioxide cryotherapy for the treatment of cutaneous leishmaniasis: systematic review and meta-analysisPLOS Neglected Tropical Diseases Dear Dr. Tilahun, Thank you for submitting your manuscript to PLOS Neglected Tropical Diseases. After careful consideration, we feel that it has merit but does not fully meet PLOS Neglected Tropical Diseases's publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript within 30 days Dec 12 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosntds@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pntd/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. Please include the following items when submitting your revised manuscript:* A rebuttal letter that responds to each point raised by the editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. This file does not need to include responses to any formatting updates and technical items listed in the 'Journal Requirements' section below.* A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.* An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, competing interests statement, or data availability statement, please make these updates within the submission form at the time of resubmission. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. We look forward to receiving your revised manuscript. Kind regards, Claudia Ida BrodskynSection EditorPLOS Neglected Tropical Diseases Claudia BrodskynSection EditorPLOS 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

 Journal Requirements: Additional Editor Comments (if provided):   [Note: HTML markup is below. Please do not edit.] Reviewers' comments: 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 study design is appropriate to address the stated objectives.

Reviewer #2: Thank you for the opportunity to re-review this interesting manuscript. The authors have kindly addressed previous comments and incorporated some changes, overall improving its quality.

However, there are some additional edits needed to further enhance the manuscript’s readability and clarity.

1. Ethiopian and WHO CL treatment guidelines: These are mentioned multiple times (Rows 86, 95, and 112). I suggest mentioning them once at the beginning of the introduction to avoid repetition.

2. Row 100-102: The statement about Carbon dioxide-based cryotherapy being easy to apply and effective (<10% failure rate) with minimal adverse effects is unclear that it is regarding its application to cervical cancer treatment. This sentence needs to be rephrased for clarity.

3. Row 118: Please provide a reference for the cure rate mentioned.

4. Outcome Measurement and Quality Assessment section (Rows 145 onward): Most of the information here seems more appropriate for the Results section. In this section, it might be sufficient to mention that the assessment considered various variables, including the number of Carbon Dioxide applications, time between applications, follow-up period, etc.

5. Rows 164-167: This sentence introduces confusion to the readers as it seems more like a conclusion. I suggest moving it to the discussion section

**********

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 presented match the analysis plan.

Reviewer #2: 6. Row 200: For the “Characteristics of included primary studies,” it would be simpler and easier to read if all these data were presented in a single table (example below)

Date of Publication

<2004 2(x%)

2004-2019 13

Unpublished 1

Region

NW 1

OW 15

Study site

Private clinic

Research center

Dermatologic clinic

Unknown

Diagnostic tests

# of lesions

etc

7. Row 275: There seems to be a confusion here. Based on the data presented above, only one study was done in NW, hence it is more frequently used in OW (not the opposite as mentioned in the text)

**********

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 by the data presented.

Reviewer #2: Conclusions: The authors are missing the opportunity to send a clear message. It needs to be rephrased, i.e.

• A small number of studies,

• the majority conducted in OW,

• problems of heterogenicity,

• duration and size of the lesion as well as the number of applications impact the effectiveness of carbon dioxide therapy

• Patients with up to 2 lesions receiving at least two applications are the ones benefiting the most (efficacy ~93%) with this intervention.

**********

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: Accept.

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: (No Response)

Reviewer #2: (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: Wenzhong Xiang

Reviewer #2: No

 [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] Figure resubmission: 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. Registration is free. 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 PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. If there are other versions of figure files still present in your submission file inventory at resubmission, please replace them with the PACE-processed versions. Reproducibility: To enhance the reproducibility of your results, we recommend that authors of applicable studies 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. 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 Mr Tilahun,

We are pleased to inform you that your manuscript 'Effectiveness of carbon dioxide cryotherapy for the treatment of cutaneous leishmaniasis: systematic review and meta-analysis' 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,

Claudia Ida Brodskyn

Section Editor

PLOS Neglected Tropical Diseases

Claudia Brodskyn

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

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