Dear Prof. Mohebali,

Thank you very much for submitting your manuscript "Contribution of epidermal growth factor (EGF) in the treatment of cutaneous leishmaniasis caused by Leishmania major in BALB/c mice" 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

Academic Editor

PLOS Neglected Tropical Diseases

Abhay Satoskar

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 authors test the effect of EGF in combination with anti-leishmanial therapy on wound healing and parasite load. The sample size used to measure lesion size for the many groups tested is reasonable. However, in the analysis of parasite load only 3 mice per group are included according to the information given in the methods sections. This number is not sufficient.

Moreover, a group only treated with the high dose EFG alone would have been relevant to include since the effect of this growth factor on Leishmania parasite growth is not known and with EFG having effects on both lesion size and potentially parasite load.

The source of EFG should be included in the methods.

No concerns regarding ethics or the statistical method selected.

Reviewer #2: 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? No.

Reviewer #3: - The objective is clear but the methods and the results are not very clear

- Cutaneous leishmaniasis due to L. major is associated with a self-cure rate above 50%–75% at 4–6 months. So it is not clear how this was assessed.

- Any specific reason to euthanize only 3 mice out of 9

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

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 show. Possibly too clear with the same data shown in three different ways.

The text to figure 5 C, D should state which group the samples came from-

Reviewer #2: -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 #3: - Why E1.5 and E4.5 alone groups were not created.

- Why topical paromomyin group was not created which is an established treatment.

- If 3 mice were already euthanized then what was the need to anesthetize the tail (line 213).

-Any specific reason to select only 3 mice per group for analysis

- Why intralesional meglumine antimoniate group was not created

- Why lesions were not observed up to 6 months (24 weeks) when almost half of the lesions in L.major show some self-healing.

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

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 would as stated above benefit from the inclusion of an EFG treated group. It would also be high relevant to address the dissemination of L. major, know to occur in Balb/c mice.

Importantly the effect on parasite load need more data (n=3, as indicated is not sufficient for conclusion) and it would also be relevant to know that the parasites are dead and not incapsulated in a closing wound.

Limitations of the study could be discussed more.

Reviewer #2: -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 #3: - Line 51- A correction is needed. Amphotericin B or its lipid complexes are not the pillar choice for treatment of CL treatment.

- Generally, if the lesion is less than 4 cm, L. major lesions are self-curing in 4-6 months. In this study, none of the lesions showed any signs of self-cure. On the contrary lesions have increased in size in C and N groups.

- It is not explained why lesions in G have increased in size despite of receiving the full course of meglumine antimoniate which is an established and very effective CL treatment.

- Except S+E4.5 and G+E4.5 groups, the other 6 groups have increased in size of lesions week by week, and no explanation is given. It seems the other 6 groups were refractory to the treatment which is difficult to understand.

- Can decrease in size of the lesions in S+E4.5 and G+E4.5 groups be attributed to self-healing? Also had lesions been observed beyond 5 weeks in the remaining 6 groups, lesions might have shown decrease in size?

- It is not clear why lesions increased in size up to the fourth week in G+4.5 and then suddenly decreased in the fifth week. On the contrary in G+E1.5 lesions have continued to increase in size throughout the five weeks' period.

- Difficult to interpret the results from table

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

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: The figures 1-3 show the same thing and should be combined into one figure (1a-c). Also the figure 1 shows the same as table 2 and is repetitive. The table is the more informative.

Reviewer #2: Apart from a few old references that need to be updated and some English terms that need to be improved, the article is well-written and reliably addresses the results discussed.

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: Therapies aiming at reducing the scaring that can be caused by Leishmania are desirable for many and the scope of this study is relevant.

As is stands the scope of the study is very narrow, with one large experiment performed and with the amount of data presented being limited. More extensive analysis of parasite load, dissemination, tissue responses etc would increase the value of the study. Importantly understandning how EFG alone affects parasite load, dissemination and lesion size is highly relevant.

Reviewer #2: Not applicable.

Reviewer #3: Major revisions and explanations needed

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

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

Reviewer #3: Yes: Saurabh Jain

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-23-01171_reviewer.pdf

Dear Dr Mohebali,

Thank you very much for submitting your manuscript "Contribution of epidermal growth factor (EGF) in the treatment of cutaneous leishmaniasis caused by Leishmania major in BALB/c mice" 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,

Abhay R Satoskar

Section Editor

PLOS Neglected Tropical Diseases

Abhay Satoskar

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 #3: There are no issues with the method.

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

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: 1. It is still not clear in the manuscript that why the lesions in the Glucantime treated mice continued to increase in spite of chemotherapy.

2. Generally 50-75% of CL lesions due to L.major heal spontaneously within 4-6 months. How to ascertain that self-healing didnt take place in any of the groups.

3. Although there is a reduction in the lesion size in the two intervention groups (S+E4.5 and G+E4.5) but complete healing of the lesions is not seen in any of the lesions. Authors need to provide clarification for that.

4. As per the Manual for case management of cutaneous leishmaniasis in the WHO Eastern Mediterranean

Region, lesions due to L.major can be treated with Cryo-or-Thermo therapy plus intralesional antimonials or topical paromomycin. However, authors have not mentioned or provide clarification of not using these interventions in any of the groups.

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

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: Major revision is suggested

1. Introduction section- in the first paragraph, there is a need to revise the global situation of CL and number of endemic countries. Authors can refer to WHO's recent publication of Global Leishmaniasis Surveillance Updates 2022 published in WHO weekly epidemiological record.

2. It is still not clear in the manuscript that why the lesions in the Glucantime treated mice continued to increase in spite of chemotherapy.

3. Generally 50-75% of CL lesions due to L.major heal spontaneously within 4-6 months. How to ascertain that self-healing didnt take place in any of the groups.

4. Although there is a reduction in the lesion size in the two intervention groups (S+E4.5 and G+E4.5) but complete healing of the lesions is not seen in any of the lesions. Authors need to provide clarification for that.

5. As per the Manual for case management of cutaneous leishmaniasis in the WHO Eastern Mediterranean

Region, lesions due to L.major can be treated with Cryo-or-Thermo therapy plus intralesional antimonials or topical paromomycin. However, authors have not mentioned or provide clarification of not using these interventions in any of the groups.

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

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: (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 #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 #3: Yes: Saurabh Jain

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 Dr Mohebali,

We are pleased to inform you that your manuscript 'Contribution of epidermal growth factor (EGF) in the treatment of cutaneous leishmaniasis caused by Leishmania major in BALB/c mice' 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,

Abhay R Satoskar

Section Editor

PLOS Neglected Tropical Diseases

Abhay Satoskar

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

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

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: Ok

**********

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: Ok

**********

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: Authors have explained clearly and addressed all previous observations and comments.

**********

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: (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 #3: Authors have explained clearly and addressed all previous observations and comments.

**********

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: Saurabh Jain