PONE-D-24-17510

Oncogenic human papillomavirus and anal microbiota in men who have sex with men and are living with HIV in Northern Taiwan

PLOS ONE

Dear Dr. Chu,

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 note that some of the comments (Reviewer 1)  were provided in a separate attachment and the attachment should be accompanying the email as per the note at the end..

Critically, additional clarity in the methods section is necessary and the data should be presented more concisely. Some of the statistical methodology might also be suboptimal.

Please submit your revised manuscript by Aug 23 2024 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.

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Ivan Sabol

Academic Editor

PLOS ONE

Journal requirements:   

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

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2. We noticed you have some minor occurrence of overlapping text with the following previous publication(s), which needs to be addressed:

https://academic.oup.com/jid/article/224/7/1247/6129308?login=false

In your revision ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text outside the methods section. Further consideration is dependent on these concerns being addressed.

Additional Editor Comments:

Please also take a look these more technical issues in addition to the reviewer’s comments:

Page 4 Line 79 listing inclusion and exclusion criteria for invitation to participate explicitly might be helpful. Currently the Line 79 implies MSM was an inclusion criteria but 9 participants failed this (P8L166)?

P6 L123– 124 how many indexes were used in one run? If all 291 samples were assessed by 16rRNA seq this means that batches were made. Was batch effect investigated and how was it addressed?

P6 L127 which flow cell and reagents (cartridge) were used (v2/v3)?

P7 L129 are the raw sequences deposited in a sequence repository?

P7 L138 was some preprocessing / filtering done on the pyloseq object or during preceeding steps? Ie normalization or filtering of taxa?

P7 L140-144 last two paragraphs lack version and or references for the packages used.

P7 L150 testing every combination with t-test is inappropriate without multiple testing correction. Why was Deseq not used since phyloseq is supposed to be analysed with deseq for differential abundance http://joey711.github.io/phyloseq-extensions/DESeq2.html?

P7 L151 6 groups for analysis seems excessive? Possibly this level of detail is appropriate for the supplement tables. It is difficult to confirm but the Figure 1 groups appear to be not mutually exclusive (Groups 3 and 6 appear to have the most participants and those 2 groups do not specify the CD4 cell counts unlike all other groups where the cutoff of 5000 cells was employed.)

P8 L165 it might be more informative if failed cytology and failed HPV exclusions were shown separately

P8 Table 1 “educational year” is worded sub optimally

P8 Table 1 it might be necessary to specify what smoking and drinking represent? Were some guidelines placed in the questionnaire that might be important for interpreting what “yes” answers mean?

P9 Table 1Was the normality of numerical variables tested? Can all reliably be summarized with mean(SD)?

P9 Table 1 were there missing answers in the questionnaires?

P8-9 Table 1 contains excessive white space.

P10 data regarding HPV/cytology and other laboratory measures should be provided in a table for the groups (plus total) discussed instead of text only.

P10 L195 the materials and methods do not adequately introduce the alpha and beta diversity metrics used in the results section. Figure 1 shows some numbers but no context is provided what 500 or 1000 means

P11 L199-201 using t-test for the pairwise comparison of 6 groups is inappropriate. If the data is normally distributed then an ANOVA test with suitable post test should be done.

P11 L201/201 total number of participants in each group should be shown.

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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: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: I Don't Know

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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: Yes

Reviewer #2: Yes

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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 manuscript by Chu et al describes the results of a study that performed 16S analysis in anal samples collected from MSM-LWH, characterized in terms of cytology and HPV infection. This is a quite underexplored field of research. The study seems to be well designed and well conducted, although histology is missing and only cytology results are available.

Reviewer #2: In this study “Oncogenic human papillomavirus and anal microbiota in men who have sex with men and are living with HIV in Northern Taiwan”, HPV DNA tests and citology results in anal samples collected Jun 2018 and Dec 2021 in 291 persons living with HIV (PLWH), mainly MSM were analyzed and 16S sequencing was performed to characterize the anal microbiome. Data on the interaction of HPV, HIV and resident commensal bacteria are needed and may be useful in determining the relative risk of lesion progression to anal cancer precursors. Despite the merits of the study, a clearer presentation of the results, additional information and more in-depth discussion are needed in a revised version.

Specific points

Method:

A paragraph on the Bioinformatic analysis methods should be added to explain the tests that are listed in Results: e.g. UniFrac, PcoA on GuniFrac, Non-metric Multidimensional Scaling (NMDs), Bray-Curtis distance (VST), Adonis tests and the Betadisper test.

The paragraph HPV genotyping should be renamed to HPV detection and it should be more clearly stated that individual results for HPV 16 and HPV 18 are given, along with a simultaneous, pooled result for the other oncogenic HPVs.

Was high-resolution anoscopy (HRA) performed after an ASCUS+ result?

Results

The presentation of results should be improved. For example, the distribution of samples in the six groups could be presented in a table.

The individual results for HPV 16, HPV 18 and other oncogenic HPVs should be reported in a table or text and whether positivity was statistically associated with ASCUS+ results. Moreover, how many patients were diagnosed with anal SIL should be reported.

Discussion:

Lines 281-282: “The interaction between microbiota, HPV acquisition and persistence, and the development of cervical cancer in women has been researched in detail.” I would suggest to modify this sentence in: the vaginal microbiota has been studied in detail, but the interactions with HPV acquisition and persistence and cervical cancer risk are still not fully understood. Accordingly, I would suggest to shorten the description of the vaginal microbiota community state types (CSTs) that are well known (lines 283-295) and to focus on studies (if any) about the anal mucosa microbiota in women.

Lines 306-308: “In the pathophysiology of infectious diseases, pathogenic colonization and infection involve dynamic interactions between microbes and the microbiome, host, and environmental factors [39]. This biology is also applicable to PLWH who are MSM” The sentences “This biology is also applicable to PLWH who are MSM” should be rephrased, e.g. in This biology also applies to the anal mucosal environment of PLWH who are MSM.

At line 322: “homogeneity of the dispersion also differed between the groups...” This should be rephrased for a better comprehension of this concept.

At lines 323-325: “Enterobacteriaceae, Proteus, Morganellaceae, Mycoplasma, Sutterella, Ruminococcus, and Bacilli were significantly abundant in PLWH whose CD4+ T cell counts were < 500 cells/µL and who had anal cytology yielding ASCUS or higher grades with detectable HPV 16/18 (group 1).” As this sentence implies a comparison, other group(s) should be mentioned.

At lines 328-331: “HIV infection also frequently correlates with increased tissue and circulating measures of inflammation, such as CD14, IL-6, and CD38+HLA-DR+CD8+ T cells in the gut [41], as well as increases in traditionally pathogenic bacteria such as Enterobacteriaceae, including Morganellaceae and Proteus [42,43].” The sentence should be reworded to more clearly relate the results of the references mentioned here [41-43] to the results of this study.

The same applies to the sentences at lines 342-343: “Our findings are consistent with those of Ron et al., who described Ruminococcus-predicting HSIL [40]. In contrast, a decreased abundance of Ruminococcus was previously noted in the guts of PLWH [46].”

and to the sentences at lines 353-354 (limitations): “Third, previous studies have mentioned the contribution of Fusobacterium and Sneathia to HPV-related lesions [22,39], which could not be proven in this study” Ref 39 (Pathogens, microbiome and the host: emergence of the ecological Koch's postulates) is a review; hence, the specific study reporting about Fusobacterium and/or Sneathia should be specified and discussed.

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Reviewer #1: No

Reviewer #2: No

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Attachments

Attachment

Submitted filename: gabri.DOCX

PONE-D-24-17510R1Oncogenic human papillomavirus and anal microbiota in men who have sex with men and are living with HIV in Northern TaiwanPLOS ONE

Dear Dr. Chu,

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 reviewer comments were sufficiently addressed. However, PLOS One publication criteria emphasize the need for high standard and rigor in data analysis and statistics.Therefore the problems with potentially inappropriately performed statistics and uncertain p values derived from this analysis must be addressed.

Please submit your revised manuscript by Nov 01 2024 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: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Ivan Sabol

Academic Editor

PLOS ONE

Journal Requirements:

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.

Additional Editor Comments:

The material and methods revisions are appreciated and improved the clarity of the manuscript

Some small methodological problems remain

P7 Line 143-144 grammatically incorrect and possibly misplaced. Batch effect would be compensated after trimming and denoising.

Page 8 Line 173 statistical analysis section

The authors reply “Line 170: We used the Wilcoxon test, considering it as a better alternative to the ttest when the normal distribution of the differences between paired individuals cannot be assumed.“

However, there are several issues with this revision.

1)The revision deleted the mention of t-test from Statistical analysis section at line 177 leaving only chi-square test mentioned

2) The Figure 2 p values, the Figure 2 legend, or the text at P12 L227 remains unchanged suggesting that it still shows the old t-test individual p values. The test used to obtain the p values is undeclared and inconsistent with the paragraph Statistical analysis

3) The comparisons between the six groups presented at Figure 2 cannot be considered paired individuals and Wilcoxon test cannot be considered as appropriate for these comparisons

P12 Table 2 footer would benefit from listing the 12 other HPV types as a footnote

Supplementary table 1 was uploaded twice. (once as Table_S1.docx and once as S1_table.docx) on pages 40 and 41 of the combined PDF

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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 #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 #2: Yes

**********

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

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 #2: 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 #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 #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 #2: No

**********

[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.

Oncogenic human papillomavirus and anal microbiota in men who have sex with men and are living with HIV in Northern Taiwan

PONE-D-24-17510R2

Dear Dr. Chu,

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.

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Kind regards,

Ivan Sabol

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: