PONE-D-20-11864

Association between periodontal disease due to Campylobacter rectus and cerebral microbleeds in acute stroke patients

PLOS ONE

Dear Dr. Hosomi,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

The manuscript would be enhanced with the inclusion of a range of T2*-weighted MRI for white matter lesion and cerebral microbleeds. The authors should address the correlation between the severity of lesions in MRI and periodontal pathogens.  

Please submit your revised manuscript by Sep 04 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

Kind regards,

Hansel McClear Fletcher, Ph.D.

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: No

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The submission by Shiga et al. describes immunodetection of C. rectus at elevated levels in patients with cerebral microbleeds in acute stroke patients. The strengths of the manuscript are in the organization and presentation of data in the manuscript, a study that directly examines associations using human clinical data, and identification of C. rectus as on oral organism associated with microbleed in acute stroke patients. Areas of weakness include the lack of oral examination at the time of patient evaluation/serum collection, the lack of oral subgingival biofilm samples to identify the presence/absence of C. rectus in these oral samples, and reliance on antibody levels (ELISA) data only to indicate an association. Despite the clear omissions noted, this reviewer is weakly enthusiastic about the manuscript. Specific comments are as follows:

1- As identified by the authors, the strongest associated factor in acute stroke patients was hypertension and this aligns with the disease as an anticipated result. C. rectus is a significant factor in this study, but other periodontal organisms such as P. gingivalis, and A. actinomycetemcomitans have been associated with stroke previously in a study that had substantially more patients than the current study. What would be the precise limitation of enrolling fewer patients and observing differences in reported outcomes between the present study and the Pussinen et al. study that is referenced?

2- In the Methods, the number of patients should be identified, also, there is no mention of the numbers of total patients approached for exclusion in this study, or if there are any exclusion criteria.

3- There is essentially no description of the ELISA assay, determination of what type of antibodies (all, IgM, IgG, etc.) are being screed for in the sera. Further, it is not clear why pooled serum from only 5 health individuals was used to control? A similar number of control subject sera should be included in the study data, and there should be no pooling of samples as it is known that many periodontally healthy individual can have relatively different levels of circulation serum antibodies to periodontal disease-associated bacteria. This leads to potential concerns about statistical analysis of these serum antibody data. There is no description of the specific strains of each bacteria used in the present study. This must be included as it is known that organisms such as P. gingivalis have a fairly high degree of heterogeneity.

4- Page 14 (lines 209-211). These data are different than what is presented on page 10. Please clarify.

5- Page 20 (lines 291-294). With the knowledge of S. mutans associations association with CMBs, it is curious that in the present manuscript that S. mutans levels of specific antibody were not assessed in the patients examined? Why only the focus on the subgingival group? Indeed, this reviewer thinks that having these data would make for an interesting and important comparison to prior work in this area.

6- CRP was the only inflammatory marker examined. Numerous studies support that pro-inflammatory cytokines serve as important markers of overall systemic inflammation. Further, systemic inflammation is liked to IL-1 (Sobowale et al. Stroke 2016. 47:2160-67). Evaluation of the levels of other inflammatory markers would improve understanding of potential underlying mechanisms in this association, and are thus suggested.

Reviewer #2: The authors aim to evaluate the associations between periodontal pathogens with the presence or severity of cerebral microbleeds and white matter lesions in acute stroke patients. There are good numbers of observational studies that periodontal disease is highly associated with the incidence of cardioembolic and thrombotic stroke. Understanding the whether any specific types of pathogen related to periodontitis is associated with the incidence of stroke is highly crucial.

The experimental design is well organized, and the results might be impactful to the field of stroke and cerebrovascular disorders.

I have only one minor suggestion.

It is a well written and taught study, which would be impactful in the field. However, the quality/number of the figures are quite low, I will suggest improving numbers of figures including a range of descriptive T2*-weighted MRI for white matter lesion and cerebral microbleeds. It would necessary if authors have correlative illustrations between severity of lesions in MRI and of periodontal pathogens. This figure would be increase quality of presentation.

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Reviewer #1: No

Reviewer #2: No

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PONE-D-20-11864R1

Association between periodontal disease due to Campylobacter rectus and cerebral microbleeds in acute stroke patients

PLOS ONE

Dear Dr. Hosomi,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please clearly list the limitations of the study and clarify author contributions.

Please submit your revised manuscript by Oct 17 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Hansel McClear Fletcher, Ph.D.

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The revised submission PONE-D-20-11864R1 has made significant improvements from the interesting original submission. As few items are felt to require additional attention.

1- In the limitations of the study, the lack of a clinical periodontal exam of all patients included in this study should be listed as limitation #1 in the listing of limitations.

2- In the limitations of the study, the lack of a similar number of controls, and the handling of the 5 samples (as pooled) should be moved to limitation #2 in the listing of limitations described in the discussion.

3- This reviewer thanks the authors for identifying that IgG levels were determined. However, the indication of “titer” is not presented in any of the tables, rather “% positive” was used to compare between clinical groups. This reviewer suggests that the serum titer +/- error for each bacterium for each of the patient/serum samples examined be included in each of the tables reporting titer information.

Minor points:

1- Please define “WMLs” on first use in the text.

2- This reviewer thanks the authors for including the strain examination data for the various bacterial lysates in the response to reviewer comments. Please apply the strain information throughout the Methods, and presented data (all relevant Tables).

3- Table 2 and Table 3 titles. Please change “antibody” to “IgG”.

4- In all tables, please identify using symbols, those data that demonstrate significant differences between groups. Please identify and describe the symbol and test used in each of the corresponding figure legends, table descriptions, respectively.

Reviewer #2: (No Response)

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7. 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: Onder Albayram

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Association between periodontal disease due to Campylobacter rectus and cerebral microbleeds in acute stroke patients

PONE-D-20-11864R2

Dear Dr. Hosomi,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Hansel McClear Fletcher, Ph.D.

Academic Editor

PLOS ONE