PONE-D-22-14386Colonization of intervertebral discs by Cutibacterium acnes in patients with low back pain: protocol for an analytical study with microbiological, phenotypic, genotypic, and multiomic techniquesPLOS ONE

Dear Dr. Ferreira,

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 see my comments and the reviewers' suggestions below.  Minor revisions to this very good protocol would strengthen the proposed study further.  Looking forward to seeing this work proceed.

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We look forward to receiving your revised manuscript.

Kind regards,

D. William Cameron, MD

Academic Editor

PLOS ONE

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Additional Editor Comments:

This is a well-written, well informed and well thought out protocol for a timely, and perhaps overdue study of correlation of C. acnes presence, and lumbago due to disc disease, at the point of first surgery. The reviews are well done and should be paid attention to by the authors, as their suggestions will improve the robustness of their findings.

In reading the MS, the authors should remove one double-negative in phrasing, and replace the word incidence with prevalence.

The inclusion of a SPIRIT checklist is appreciated; as this is an observational study, the authors might have used the STROBE reportage guidelines and checklist.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Does the manuscript provide a valid rationale for the proposed study, with clearly identified and justified research questions?

The research question outlined is expected to address a valid academic problem or topic and contribute to the base of knowledge in the field.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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2. Is the protocol technically sound and planned in a manner that will lead to a meaningful outcome and allow testing the stated hypotheses?

The manuscript should describe the methods in sufficient detail to prevent undisclosed flexibility in the experimental procedure or analysis pipeline, including sufficient outcome-neutral conditions (e.g. necessary controls, absence of floor or ceiling effects) to test the proposed hypotheses and a statistical power analysis where applicable. As there may be aspects of the methodology and analysis which can only be refined once the work is undertaken, authors should outline potential assumptions and explicitly describe what aspects of the proposed analyses, if any, are exploratory.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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3. Is the methodology feasible and described in sufficient detail to allow the work to be replicable?

Descriptions of methods and materials in the protocol should be reported in sufficient detail for another researcher to reproduce all experiments and analyses. The protocol should describe the appropriate controls, sample size calculations, and replication needed to ensure that the data are robust and reproducible.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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4. Have the authors described where all data underlying the findings will be made available when the study is complete?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception, at the time of publication. 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: Yes

Reviewer #2: Yes

Reviewer #3: 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

Reviewer #3: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above and, if applicable, provide comments about issues authors must address before this protocol can be accepted for publication. You may also include additional comments for the author, including concerns about research or publication ethics.

You may also provide optional suggestions and comments to authors that they might find helpful in planning their study.

(Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The central question/controversy in this area concerns the conclusion that any organisms recovered from excised tissues by culture result from skin contamination during sampling. The approach proposed to counter this argument is that proteomic or metabolomic signatures obtained from the tissues might indicate infection, as has been concluded from cited studies. Since no control tissues samples are involved in the study (for example analogous tissues from other surgical sites) reliance is placed on effective skin disinfection prior to surgery. However, viable organisms can remain deep within the skin following pre-operative surgical preparation and these could result in false positive cultures, particularly using the enrichment culture approach in broth before plating. To address this problem, the study protocol proposes swabbing the skin after preparation and before excision. The swab is plated directly onto enriched blood agar without treatment with a neutralizing solution. This omission could give a false idea of the efficacy of skin preparation since residual, active chlorhexidine will be transferred from the skin to the swab and onto the plates. A suitable neutralizer solution such as Dey-Engley broth should be included in the swab transfer tube. Even with addition of this step, there remains concern that skin surface swabs will not pick up viable organisms residing deeper within the skin after preparation and before excision.

PCR methods will be used only to identify and characterize organisms recovered by enrichment culture, no attempt being made to amplify microbial DNA directly from tissues without culture. Whilst this approach will suffer the same problem of possible skin contamination it should at least be considered since it could give some idea of levels of microbial DNA present in the tissues (not possible using the tissue enrichment culture approach).

Reviewer #2: I have read your protocol with great interest, which has the potential to provide an important insights into low virulence infections in lumbar intervertebral discs, and have following optional suggestions based on my experience:

1. Homogenization: Our previous study based on fluorescence in situ hybridization confocal scanning laser microscopy has shown that P. acnes is present deep within intervertebral disc tissue as a biofilm and, as a consequence, it is important that the biofilm is disrupted prior to culture to maximize detection and reduce the possibility of a false-negative result (Capoor 2017). With regard to biofilm disassembly, homogenization has demonstrated the ability to disrupt biofilm deep within the tissue, while sonication has shown demonstrated ability to disrupt biofilm on the surface of implants.

2. Sample size: Two meta-analyses of previous studies addressing infection of intervertebral discs reported a pooled prevalence of bacteria at 34% and 36.2%, respectively with P. acnes as the predominant species (Urquhart 2015, Ganko 2015). An appropriate sample size estimation should therefore be calculated based upon these prevalence rates [Sample size = (1.96^2 x PR (1-PR))/0.05^2); note PR = prevalence rate] (Naing 2006). To ensure that the 95% confidence interval estimate of the proportion positive cases is within 5% of the true proportion, a sample size of approximately 350 cases is necessary.

3. CFU/Gram: Disc fragments for culture should be weighed, placed into a Micro Bag (Seward) containing 4 mL of Viande-Levure medium, and homogenized with a Stomacher 80 (Seward) under aseptic conditions. 100 µL of the resultant homogenate should be used to inoculate Wilkins Chalgren Anaerobic Agar with 7% sheep’s blood and vitamin K (Hi Media Laboratories). An Anaerobic Work Station Concept 400 (Ruskinn Technology) should be used for culture; inoculated plates will be incubated for 14 days at 37 °C under an atmosphere of 80% N2, 10% CO2, and 10% H2. The same amount of the homogenate should be cultured aerobically on Columbia Blood Agar (Oxoid) for 7 days at 37°C to detect aerobic bacteria. Following incubation, bacterial colonies should be counted and the quantity of each colonial morphotype will be expressed as CFU/g of tissue using the Miles and Misra method.

4. qPCR verification of the presence of P. acnes. The human β-globin gene should be included as an internal control to allow assessment of the specimen quality and the nucleic acid extraction as well as the inhibition amplification process. Our experience shows that found that significant number of samples are eliminated due to:

a) inadequate DNA concentration (outside of range 2-60 ng/µL)

b) inadequate human DNA control (Ct of human β-globin gene outside of range 21-27)

c) inconclusive P. acnes template Ct values (32-35)

Reviewer #3: Lines 30, 121, 135: correlate refers to a particular type of analysis. Is this intended (only appropriate for 2 continuous variables), or would “link” or similar be a more appropriate word?

Line 470+ will baseline measures for patient reported outcomes be included in models for future time points?

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Reviewer #1: No

Reviewer #2: Yes: Manu Capoor

Reviewer #3: No

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Colonization of intervertebral discs by Cutibacterium acnes in patients with low back pain: protocol for an analytical study with microbiological, phenotypic, genotypic, and multiomic techniques

PONE-D-22-14386R1

Dear Dr. Ferreira,

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,

D. William Cameron, MD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Does the manuscript provide a valid rationale for the proposed study, with clearly identified and justified research questions?

The research question outlined is expected to address a valid academic problem or topic and contribute to the base of knowledge in the field.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

2. Is the protocol technically sound and planned in a manner that will lead to a meaningful outcome and allow testing the stated hypotheses?

The manuscript should describe the methods in sufficient detail to prevent undisclosed flexibility in the experimental procedure or analysis pipeline, including sufficient outcome-neutral conditions (e.g. necessary controls, absence of floor or ceiling effects) to test the proposed hypotheses and a statistical power analysis where applicable. As there may be aspects of the methodology and analysis which can only be refined once the work is undertaken, authors should outline potential assumptions and explicitly describe what aspects of the proposed analyses, if any, are exploratory.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Is the methodology feasible and described in sufficient detail to allow the work to be replicable?

Descriptions of methods and materials in the protocol should be reported in sufficient detail for another researcher to reproduce all experiments and analyses. The protocol should describe the appropriate controls, sample size calculations, and replication needed to ensure that the data are robust and reproducible.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Have the authors described where all data underlying the findings will be made available when the study is complete?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception, at the time of publication. 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: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

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

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above and, if applicable, provide comments about issues authors must address before this protocol can be accepted for publication. You may also include additional comments for the author, including concerns about research or publication ethics.

You may also provide optional suggestions and comments to authors that they might find helpful in planning their study.

(Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The issue of examining "control" tissues has been explained and dealt with satisfactorily.

The thioglycolate broths used for the skin swabs should neutralize any carry-over of chlorhexidine. Primary swabs onto the blood plates may still give false negatives but I note that this has been standard procedure in previous studies.

Reviewer #2: I read reviewer comments and your corrections and believe you did a reasonable and thoughtful job addressing these comments.

Reviewer #3: It would be useful to mention in this text that the baseline measures in the stats analysis section, or at least state a statistician will specify the analyses in a statistical analysis plan.

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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: No

Reviewer #3: No

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