Dear Prof Shita

Thank you very much for submitting your manuscript "Prevalence of Leishmania RNA virus in Leishmania parasites in patients with cutaneous leishmaniasis: a 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.

The manuscript was revised by three different reviewers. I strongly suggest taking into account the comments of the second and third reviewers. Despite little evidence in the literature, the data presented are of interest, but I suggest that there is a better explanation regarding the presence of LRV and the development of clinical forms of tegumentary leishmaniasis. The review cannot be based only on cutaneous leishmaniasis, since there are data associating mucosal leishmaniasis and the presence of LRV and, therefore, the word cutaneous should be replaced by tegumentary in the title of the manuscript.

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,

José Angelo Lauletta Lindoso

Guest Editor

PLOS Neglected Tropical Diseases

Fabiano Oliveira

Deputy Editor

PLOS Neglected Tropical Diseases

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

The manuscript was revised by three different reviewers. I strongly suggest taking into account the comments of the second and third reviewers. Despite little evidence in the literature, the data presented are of interest, but I suggest that there is a better explanation regarding the presence of LRV and the development of clinical forms of tegumentary leishmaniasis. The review cannot be based only on cutaneous leishmaniasis, since there are data associating mucosal leishmaniasis and the presence of LRV and, therefore, the word cutaneous should be replaced by tegumentary in the title of the manuscript.

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: see general comments below

Reviewer #2: -Are the objectives of the study clearly articulated with a clear testable hypothesis stated? Answer: YES

-Is the study design appropriate to address the stated objectives?Answer: Yes, because the authors used the PRISMA guidelines

-Is the population clearly described and appropriate for the hypothesis being tested?Answer: No , because there are few published studies avialable to be analyzed

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?Answer: NO , because there are few published studies avialable to be analyzed

-Were correct statistical analysis used to support conclusions?Answer: YES

-Are there concerns about ethical or regulatory requirements being met?Answer: Yes

Reviewer #3: The aim of the study is clearly presented and the study design described thoroughly.

It is a review and meta-analysis MS and the selection criteria for the articles included for analysis are clearly presented.

In the introduction, some informations are not based in appropriate references such as case reports and visceral leishmaniasis paper (referring to asymptomatic cases). We notice that the authors are not aware that New World CL rarely evolves to self-cure; this information needs correction.

Among the articles included some were based in small number of parasite isolates and it is not clear wether in these articles the isolates were randomly included or they had any bias for the inclusion in the study.

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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: see general comments below

Reviewer #2: -Does the analysis presented match the analysis plan? Answer: YES

-Are the results clearly and completely presented?Answer:NO, not because the data extraction form is missing

-Are the figures (Tables, Images) of sufficient quality for clarity?Answer: No not because the prevalence could be separated by species

Reviewer #3: To estimate the prevalence of LRV, from the data presented in the Table 2, it seems that the presence of the virus does not relate to particular clinical presentation and the development of severe forms.

There is no other data that show correlation of the presence of the virus and worse disease development. It would be desirable the analysis of the data from articles showing high number of isolates where it would be possible to compare the development of the disease with parasites with and without the virus avoiding the confounding factors such as geographical area and Leishmania species diversity.

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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: see general comments below

Reviewer #2: -Are the conclusions supported by the data presented? Answer: no , Because some conclusions are not a consensus in the area

-Are the limitations of analysis clearly described?Answer:No , because it was not discussed that there is little bibliographical information available

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?Answer:YES but could better discuss

-Is public health relevance addressed?Answer:YES but could better discuss

Reviewer #3: Conclusions are not fully supported by the presented data. Limitations of the analysis are not clearly described.

The relevance of the study for the public health is not clearly addressed.

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

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: could do with a native English speaker improving English in places

Reviewer #2: Introduction line 5 not necessarily leishmaniasis is prevalent in the poorest communities . paragraph 2 line 5

there is no consensus that LV can define the clinical form.paragraph 4 line 8 the authors could clarify whether the isolates were obtained from patients.

methods:

2.1 it was not clear the search strategy, it was not clear whether the keywords were used separately, together or concatenated.

2.2 why? all in vitro and animal model studies were eliminated, as these studies can be done with patient isolates.paragraph 2 lines 6 and 7 authors could provide the exclusion criteria made at this stage of the analysis

2.3 line 1 authors could provide the data extraction form and citeria.line 9 authors could make it clear if the "checklist-JBI" were used as exclusion criteria.

3.1line 4 the authors could indicate that these results are described in figure 1. line 5 the authors can describe how the 83 studies from the last 107 were excluded and indicate that the exclusion criteria is described in figure 1.

3.3 in line 4 what does "p=0.00." means? .

Discussion lines 9-10 there is no consensus that LV defines the clinical form of cutaneous leishmaniasis.lines 13-14 there is no consensus that LV causes therapeutic failure.line 15 separate leishmania from parasite.

Figure 4 was not shown in the results.

page 18-19 line 2 is not a consensus

Reviewer #3: Language revison and edition would be desirable.

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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: Prevalence of Leishmania RNA virus in Leishmania parasites in patients with cutaneous leishmaniasis: a systematic review and meta-analysis.

This a useful and well performed study, and highlights areas relevant knowledge gaps and areas for future research.

My comments mostly relate to areas of text which could be clearer.

Principal findings abstract: ‘total of 1159 samples from 24 studies were tested’ makes it sound as if you tested them.

Author summary: ‘It needs further studies to well understand’ would read better as ‘It needs further studies to better understand…’

Your opening sentence is too vague ‘Cutaneous leishmaniasis (CL) is among neglected tropical diseases affecting millions of the world population since long years back [1, 2].’

I would suggest something like ‘Cutaneous leishmaniasis (CL) is a neglected tropical diseases affecting millions of people worldwide. Leishmania species probably co-evolved with mammals (Steveding et al 2017) and have been identified in humans from at least 4 millennia before present (Zink et al).’

multiplenon-ulcerative nodular lesionswith. should be : multiple non-ulcerative nodular lesions with lar

methods- can you be clearer about your search strategy. You write ‘combinations of search terms: “cutaneous leishmaniasis “AND “Leishmania RNA virus OR “leishmaniasis, cutaneous” OR mucocutaneous, prevalence.”

Was this combinations of search terms: “cutaneous leishmaniasis “AND (“Leishmania RNA virus OR “leishmaniasis, cutaneous” OR mucocutaneous, prevalence.”)?

Surely RNA virus was always in the search strategy? In which case I would have expectd Leishmania RNA virus AND (“cutaneous leishmaniasis “ “OR “leishmaniasis, cutaneous” OR mucocutaneous, prevalence.”)

Is this correct? Please clarify

‘The articles were independently reviewed by two investigators (EY, EN)’. What did you do if you disagreed?

Discussion: ‘The detection of the LRV from the lesion biopsies would be much easier and help to run more samples in short period of time in areas with poor laboratory setup.’ I don’t understand this sentence. Please clarify.

Reviewer #2: the authors can discuss the article titled "Global status of synchronizing Leishmania RNA virus in Leishmania parasites: A systematic review with meta‐analysis" because are similar and to discuss limitations of the present study, discuss some similar finding to, argue that more studies need to be carried out to reach more reliable results and at the same time to emphasize that there is a gap in this area of ​​knowledge.

Reviewer #3: The MS is relevant to show the prevalence of LRN in the isolates of different species of Leishmania. However, the main concern that is the correlation of the presence of the virus with the disease development is not clearly presented based on the supporting data.

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Reviewer #1: Yes: Dr Richard Weller

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

Dear Mr Shita,

Thank you very much for submitting your manuscript "Prevalence of Leishmania RNA virus in Leishmania parasites in patients with tegumentary leishmaniasis: a 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. 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.

The manuscript was revised after some changes made by the authirs. The answers provided by the authors, in relation to the comments of the first review, covered all the questions raised. Only one additional suggestion, made by reviewer 2, should be taken into account. As described below:

TOPIC 2.1. Search strategy and eligibility criteria is not clear yet , after text modifications; maybe a table with the word combination strategy used in the present work will be necessary.

Improvement oh this topic is very important for other groups would reproduce the work or use the same estrategy to other studies , this is important for reproducibility of the work and increases the chances of the work being cited.

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,

José Angelo Lauletta Lindoso

Guest Editor

PLOS Neglected Tropical Diseases

Fabiano Oliveira

Deputy Editor

PLOS Neglected Tropical Diseases

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

The manuscript was revised after some changes made by the authirs. The answers provided by the authors, in relation to the comments of the first review, covered all the questions raised. Only one additional suggestion, made by reviewer 2, should be taken into account. As described below:

TOPIC 2.1. Search strategy and eligibility criteria is not clear yet , after text modifications; maybe a table with the word combination strategy used in the present work will be necessary.

Improvement oh this topic is very important for other groups would reproduce the work or use the same estrategy to other studies , this is important for reproducibility of the work and increases the chances of the work being cited.

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: Objectives, design and analysis all clearly described. Sample size is a function of the relatively sparse number of studies.

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? NO; BECAUSE THE TOPIC 2.1. Search strategy and eligibility criteria is not clear yet maybe a table for combination word estrategy will be necessary , modified in order to make it clearer

-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? NO

-Were correct statistical analysis used to support conclusions? YES

-Are there concerns about ethical or regulatory requirements being met? NO

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

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: Results clearly presented. Good tables and figures.

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

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

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: Conclusions and limitations all supported.

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

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

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: Much Improved manuscript which reads very clearly.

Reviewer #2: "Minor Revision"

TOPIC 2.1. Search strategy and eligibility criteria is not clear yet maybe a table for combination word estrategy will be necessary , modified in order to make it clearer

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

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: A well presented paper

Reviewer #2: the study was made with heterogeneus and low quantity of data but in the other hand the authors get all avialible information to do the work

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

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: Dr Richard Weller

Reviewer #2: Yes: Eduardo Milton Ramos Sanchez

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 Mr Shita,

We are pleased to inform you that your manuscript 'Prevalence of Leishmania RNA virus in Leishmania parasites in patients with tegumentary leishmaniasis: a 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,

José Angelo Lauletta Lindoso

Guest Editor

PLOS Neglected Tropical Diseases

Fabiano Oliveira

Deputy Editor

PLOS Neglected Tropical Diseases

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

The authors reviewed the manuscripts and accepted the suggestions of reviewer 2. The authors included the word search strategy to carry out this systematic review and meta-analysis. Therefore, I consider that the article should be accepted for publication.

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