Dear Dr Campillo,

Thank you very much for submitting your manuscript "Association between arterial stiffness and Loa loa microfilaremia in a rural area of the Republic of Congo: a population-based cross-sectional study (the MorLo project)" 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,

Subash Babu

Academic Editor

PLOS Neglected Tropical Diseases

Francesca Tamarozzi

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: In their manuscript, the authors report the results of a study examining arterial stiffness, ankle brachial index and pulse pressure in a large cohort of patients living in an area endemic for loiasis in an effort to explain the previously described association of microfilaremic loiasis with increased mortality. The objectives are clearly stated and the study design is appropriate. Specific comments follow:

1) A better description of the amicrofilaremic controls is needed. Were they infected (i.e., history of eye worm or Calabar swellings), uninfected, or both?

2) The description of the “reference populations” is unclear. Per the explanation in the methods, the difference between reference populations is only the definition of hypertension. As such, the assignment to normal or abnormal PWV group should be identical since this is an independent measure (i.e., why are there differences in line items other than arterial pressure?).

3) Although the authors state that the study was approved by the Congolese Ethics Committee, there is no mention of whether the participants provided informed consent.

Reviewer #2: Please see the comments

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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 matches the analysis plan.

The relationship between Loa loa microfilaremia and PWV is not significant in Table 1 and demonstrated in Table 2 only for the highest mf load where >10,000 mf/mL is associated with a higher PWV. Although the Loa loa microfilaremia is associated with PAD, the way the Loa loa data is displayed in Table 3 is very difficult to follow and the logistic regression only showed a relationship in the low microfilaremia group. This seems at odds with the PWV data. The PP data, in contrast, would seem to support a relationship, but no data is given broken down by microfilarial load.

In table 2 and supplementary table 6, there are two sets of data for each variable for each reference dataset. What is the difference between the two sets?

Reviewer #2: Please see the comments

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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: Based on the findings (see comment in results section), the conclusion provided in the abstract that high Loa loa microfilarial load is related to cardiovascular deterioration seems overreaching.

There are some additional confounders that should be addressed in the discussion. 1) The authors point out that there no association between lipid levels and MFD. This does not exclude a role for the influence of genetic factors playing a shared role in the level of circulating microfilariae in an individual and their risk of cardiovascular disease. 2) Eosinophilia is common in loiasis, and eosinophil-platelet interactions and release of eosinophil extracellular traps have been reported to promote atherosclerosis (Marx et al. Blood 2019 among others). The potential role of eosinophils should be mentioned.

The public health relevance is not directly addressed and could be better highlighted in the discussion.

Reviewer #2: Please see the comments

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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: Although they bite horses, the common name for Chrysops is deerfly. Horsefly typically refers to the genus Tabanus.

If p=0.073 is at “the borderline of significance” (line 263), why does p= 0.066 indicate a lack of significance (line 278). Since neither p value is <0.05, it would be more correct to indicate that neither is significant.

On line 263, the authors state that the data in the supplemental table is similar to the data in Table 1; however, creatinine elevation is significantly different in the normal and abnormal PWV groups in the supplemental table.

There are many minor English errors that should be corrected.

Reviewer #2: (No Response)

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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: This is a novel study performed in a large cohort of patients in an area endemic for loiasis. Although the results are intriguing, the lack of sufficient information about the control population and lack of clarity in some of the data tables make it difficult to assess the overall significance of the results.

Reviewer #2: Association between arterial stiffness and Loa loa microfilaremia in a rural area of the

Republic of Congo: a population-based cross-sectional study

Jérémy T. Campillo et al examined is there any association between the density of L. loa microfilariae (mfs) in the blood with cardiovascular disease. They found that high L. loa MFD and cardiovascular health negative association which is deterioration.

Kindly mention about the consent details from study individuals under the ethical statement.

Study exclusion criteria’s should be included.

Sample size and the power calculation can be mentioned under statistical section.

Authors should include the methodology how they excluded the other helminth infection.

Flowchart for the study can be included

Did authors check for the post anthelminthic treatment effect on these cardiovascular markers. This data will improve the manuscript immensely

Did authors check for other confounding factors like Diabetes mellitus, lipid profile hypertension, etc…

Are these individuals are on treatment for cardiovascular disease? Do they have any impact on treatment?

Authors could show the comparison/ alteration of cardiac markers like NT-proBNP and high-sensitivity cardiac troponin T (hs-cTnT).

The above mentioned cardiac markers can be correlated with arterial stiffness and MFD

Discussion can be improved. Discussion regarding malaria and its impact is missing.

Study limitation and implication can be included at the end of the discussion.

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

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: Yes: Anuradha Rajamanickam

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: Arterial stiffness and Loa loa microfilaremia Comments PLOSNTDs.docx

Dear Dr Campillo,

Thank you very much for submitting your manuscript "Association betweenarterial stiffness and Loa loa microfilaremiain a rural area of the Republic of Congo:a population-based cross-sectional study (the MorLo project)" 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,

Subash Babu

Academic Editor

PLOS Neglected Tropical Diseases

Francesca Tamarozzi

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 objectives are clearly stated and the study design is appropriate. Specific comments follow:

1) Although Table 1 helps in terms of clarifying the populations in the study, it is hard to follow and could be simplified. For example, out of range definitions and the definitions of the healthy cohorts (together with an explanation for why two separate definitions were used) would be better placed in the methods text describing the study populations.

2) Table 1 indicates 69 additional people were added. Were these microfilaremic, not microfilaremia or both?

Reviewer #2: Yes

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

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 authors have addressed most of the reviewers' comments; however, the two different sets of data in Table 2 and Supplementary Table 6 remain unexplained. Is the data in the left PWV >90th percentile "yes" and the data on the right "no"?

Reviewer #2: 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: The authors have adequately addressed the reviewers' comments.

Reviewer #2: 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: The auhtors have made the necessary corrections.

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: In their manuscript, the authors report the results of a study examining arterial stiffness, ankle brachial index and pulse pressure in a large cohort of patients living in an area endemic for loiasis in an effort to explain the previously described association of microfilaremic loiasis with increased mortality.

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: No

Reviewer #2: Yes: Anuradha Rajamanickam

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 Dr Campillo,

We are pleased to inform you that your manuscript 'Association between arterial stiffness and Loa loa microfilaremia in a rural area of the Republic of Congo: a population-based cross-sectional study (the MorLo project)' 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,

Subash Babu

Academic Editor

PLOS Neglected Tropical Diseases

Francesca Tamarozzi

Section Editor

PLOS Neglected Tropical Diseases

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