Dear Prof Miró,

Thank you very much for submitting your manuscript "New immunomodulatory treatment protocol for canine leishmaniosis reduces parasitemia and proteinuria" 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,

Camila I. de Oliveira

Academic Editor

PLOS Neglected Tropical Diseases

Hira Nakhasi

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: -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?no

-Is the population clearly described and appropriate for the hypothesis being tested?no

-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 #2: The study meticulously articulates its objectives and presents a well-defined, testable hypothesis, ensuring the study design is suitably tailored to achieve the stated goals. The chosen population aligns perfectly with the hypothesis, the sample size is aptly calculated to provide robust power, appropriate statistical methods bolster the conclusions, and all ethical and regulatory standards are scrupulously upheld.

Reviewer #3: The authors were very careful regarding the methodology. There is only one issue regarding the analysis of parasite load using blood, but the authors duly justified it.

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

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?no

-Are the results clearly and completely presented?no

-Are the figures (Tables, Images) of sufficient quality for clarity?no

Reviewer #2: The analysis aligns seamlessly with the planned approach, and the results are articulated with commendable clarity and completeness. The visual aids, including tables and images, are of high quality, ensuring clear communication of data. However, a detailed discussion is warranted regarding the observed pattern in the IRIS stages within the supplement group, particularly concerning the potential indication of patient relapses over time.

Reviewer #3: Only in the creatinine graph could the authors draw lines indicating the minimum and maximum reference values, making the figure clearer for the reader.

Discussion

Line 273: the authors need to make it clear in this part of the text that the clinical stability of dogs for another year after interrupting the treatment protocol with MGA and Allopurinol occurs in this region of the European continent. This detail is important because in tropical regions the infection pressure is different due to climatic conditions, among other factors.

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

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?no

-Is public health relevance addressed?yes

Reviewer #2: The conclusions are well-grounded in the data provided, with a transparent acknowledgment of the analysis's limitations. The authors effectively discuss the data's contribution to advancing our understanding of the subject, also addressing its public health significance. However, an in-depth exploration of the IRIS stage patterns in the supplement group would enrich the discussion, particularly in relation to the potential for patient relapses over time.

Reviewer #3: The conclusions are in accordance with the results obtained and presented in the manuscript.

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

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: - Line 72: The term "me+2thylglucamine antimoniate" should be corrected to "N-methylglucamine antimoniate."

- The acronym AHCC is not explained in the text; could you please provide its definition?

- Clarification is needed on whether sample analysis was centralized in one lab or conducted separately at the 13 veterinary centers.

- In Figure 4, the meaning of the units U.A. for the total clinical score is unclear. Please clarify this in the axis or figure caption.

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: (No Response)

Reviewer #2: The treatment combining MGA, Allopurinol, and supplements offers a promising alternative for managing canine leishmaniasis. The authors effectively detail how this regimen improves clinical and laboratory outcomes, emphasizing its oral administration convenience for pet owners.

Reviewer #3: The authors recognize the limitations of the study, especially in relation to possible concomitant diseases in animals, which could have some impact on the results. However, any and all field studies in an endemic area are prone to factors of this nature. However, this does not reduce the relevance of the study, which was very well conducted.

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

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

Reviewer #3: Yes: Kelvinson Fernandes Viana

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 Prof Miró,

Thank you very much for submitting your manuscript "New immunomodulatory treatment protocol for canine leishmaniosis reduces parasitemia and proteinuria" 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,

Camila I. de Oliveira

Academic Editor

PLOS Neglected Tropical Diseases

Hira Nakhasi

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

Dear Prof Miró,

Thank you very much for submitting your manuscript "New immunomodulatory treatment protocol for canine leishmaniosis reduces parasitemia and proteinuria" 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,

Camila I. de Oliveira

Academic Editor

PLOS Neglected Tropical Diseases

Hira Nakhasi

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: -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 #4: 1- The objectives of the study clearly articulates with a testable hypothesis

2-The study design is appropriate to address the objectives

3- The population of the study is clearly described

4- The authors had a considerable lost in the number of animals, but in this kind of study is difficult to control this aspect.

5 Statistical analysis needs some clarification in relation to significance of results, since some parameters seem not to be significant and even in the characteristics of the population, there are some significant differences

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

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: 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 #4: 1-Yes

2-Yes

3-needs to be more clear, some of them.

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

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: 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 #4: Yes

Yes

Yes

Yes

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

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: The authors made changes proposed by the reviewers, aiming to improve the presentation of the manuscript.

Reviewer #4: The manuscript is interesting and shows the possibility to improve the treatment of Canine Visceral Leishmaniasis using supplements. Although it is not possible to define what is AHCC, it would be important to mention in since the abstract that is a supplement used in humans to improve the immune response. Another point, it would be discuss the possible mechanism why these compounds contribute to a better results in the outcome of the infection. Is it related to supression of the inflammatory responses? Increase the immune repsonses associated to a Th1 response?

etc

Lines 187-189 Neither were there differences (control vs supplement group,respectively) in LeishVet

clinical stage (2.19±0.40 vs 2.37±0.49; p< 0.01) or total clinical score (9.00±5.58 vs 10.11±4.62; p< 0.01).

There are differences between the two groups concerning clinical stage and total clinical score

Lines 225-226 A significant decrease over time (p= 0.004) in parasite load (blood qPCR) was seen only in the

supplement group (Figure 5). It would be important to perform area under curve, because apparently, the difference is very low.

Lines 232- 233We observe the same issue in the Figure 6 concerning the levels of creatinine, when the authors state a significant difference between the control and supplement groups that seems to occur in the first point before the end of treatment (30 days)

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

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 study has a good experimental design and responds to what was initially proposed, bringing a new adjuvant therapeutic perspective in the treatment of canine visceral leishmaniasis.

Reviewer #4: The manuscript is interesting and shows the possibility to improve the treatment of Canine Visceral Leishmaniasis using supplements. Although it is not possible to define what is AHCC, it would be important to mention in since the abstract that is a supplement used in humans to improve the immune response. Another point, it would be discuss the possible mechanism why these compounds contribute to a better results in the outcome of the infection. Is it related to supression of the inflammatory responses? Increase the immune repsonses associated to a Th1 response?

etc

Lines 187-189 Neither were there differences (control vs supplement group,respectively) in LeishVet

clinical stage (2.19±0.40 vs 2.37±0.49; p< 0.01) or total clinical score (9.00±5.58 vs 10.11±4.62; p< 0.01).

There are differences between the two groups concerning clinical stage and total clinical score

Lines 225-226 A significant decrease over time (p= 0.004) in parasite load (blood qPCR) was seen only in the

supplement group (Figure 5). It would be important to perform area under curve, because apparently, the difference is very low.

Lines 232- 233We observe the same issue in the Figure 6 concerning the levels of creatinine, when the authors state a significant difference between the control and supplement groups that seems to occur in the first point before the end of treatment (30 days)

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

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: No

Reviewer #4: 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 Prof Miró,

We are pleased to inform you that your manuscript 'New immunomodulatory treatment protocol for canine leishmaniosis reduces parasitemia and proteinuria' 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,

Camila I. de Oliveira

Academic Editor

PLOS Neglected Tropical Diseases

Hira Nakhasi

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

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

Dear Dr Miró,

Thank you for submitting the revised version of your manuscript. The points raised by the referees have been addressed and the manuscript shall be of interest to the PLoS NTDs community. Most of all, thank you for your continued patience with the review process and apologies for the delays.