PONE-D-22-28769HIGH PREVALENCE AND CO-INFECTION OF HIGH-RISK HUMAN PAPILLOMAVIRUS GENOTYPES AMONG UNVACCINATED YOUNG WOMEN FROM PARAGUAYPLOS ONE

Dear Dr. Bobadilla,

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.

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Dear authors, 

the topic of the present article titled “HIGH PREVALENCE AND CO-INFECTION OF HIGH-RISK HUMAN PAPILLOMAVIRUS GENOTYPES AMONG UNVACCINATED YOUNG WOMEN FROM PARAGUAY” is very interesting, the paper and the aim falls within the scope of the journal but the article needs major improvements.

The introduction, material and method section and tables should be modified and improved. 

The manuscript should be organized better and English should be improved.

I suggest improving the manuscript with the reviewers' comments.

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Please submit your revised manuscript by Dec 26 2022 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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Andrea Giannini

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: Partly

Reviewer #3: Partly

Reviewer #4: Partly

Reviewer #5: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: 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: Yes

Reviewer #2: No

Reviewer #3: Yes

Reviewer #4: No

Reviewer #5: 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: No

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: 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: Thank you for including me in reviewing this manuscript. The article is that “HIGH PREVALENCE AND CO-INFECTION OF HIGH-RISK HUMAN PAPILLOMAVIRUS GENOTYPES AMONG UNVACCINATED YOUNG WOMEN FROM PARAGUAY”. The sample size is small. The current study does not deliver anything significant new or outbreaking in this matter; at this point, all of this, justifies rejection of the manuscript.

1. The introduction is long. Authors should abbreviate some places by combining them. Otherwise, the interest in the subject and the flow of the subject are scattered. For example, sections on vaccines and HPV can be shortened. Places that indicate the purpose of the subject can be highlighted.

2. There are minimal grammer errors. I recommend that a native English speaker review the article or use a professional language editing service.

3. ”National primary screening is mainly aimed

at women aged 30 years and above; younger women does not frequently attend health services

for screening tests.” What is this program like, how is it done?

4. You mentioned the vaccination program that started in 2013. What is this program?

5. “which was similar to that reported in other…” Is the age group of all the other studies mentioned in this paragraph the same?

6. As you know, HPV can be eliminated after a certain period of time. There may be elimination, especially under the age of 25. What do the authors think about this and about screening in the early age group?

Reviewer #2: The authors conducted an HPV prevalence and genotyping study among unvaccinated females using screening samples in women aged 18-25 years. The data are important because they establish a baseline for monitoring impact of Paraguay’s HPV vaccination program. The study appears to be internally valid and most of the analyses seem to be appropriate. Some edits could improve the readability and further focus the paper on the important information.

Although the manuscript is generally well-written, additional attention to English grammar is warranted.

Abstract:

First sentence stating “several young women remain unvaccinated” is quite confusing. Isn’t it true that at the time of this study, women in the age group included were never vaccine eligible? Quadrivalent vaccine should be moved from second sentence to first (if virologic surveillance is important, then it would be true for a vaccination program with any formulation). Methods in abstract are almost non-existent – please include some information on the recruitment procedures, including how epidemiologic data and specimen were collected and some basics of the typing assay, such as number of types detected.

Introduction:

The introduction is quite long and could be trimmed considerably to focus more on essential information needed to understand the context of the paper. Some examples are listed below.

The first two sentences of the paper suggest the main reason young women are susceptible to STIs is immaturity of genital tract. While this may be a contributor, the initiation of sexual activity and non-monogamous relationships should not be ignored. Those first two sentences could be deleted – “HPV is the most common STI” is a better start. The second paragraph is largely extraneous and most if not all of it could be deleted. Line 53 introduces the term “high-risk types,” which needs to be defined, as it was not introduced in the previous paragraph about the categories of HPV types.

The introduction really needs a more complete description of the vaccination program. There is a long paragraph on page 4 beginning with line 64 describing all the internationally approved vaccines, and easily lost at the end of this paragraph is one sentence about Paraguay: “In Paraguay, the quadrivalent Gardasil vaccine was introduced to 10-year-old girls in 2013.” The international information could be shortened, and more information on the Paraguay program added. How many doses of Gardasil do girls receive? In what setting (e.g., schools or primary care)? What has the coverage been? Doing the math, with the study conducted in 2020-21, the females included in the prevalence study had mainly not been vaccine eligible, is this correct? This needs to be spelled out for the reader. On first read, it seemed like the authors had screened out HPV-vaccinated women, which raised questions about vaccination coverage and known herd effects from HPV vaccination. This may still be an issue in the youngest women, and should be addressed in the discussion. It is not a real problem with internal validity, but it needs to be clear what exactly was done and why.

Terminology with different meanings has been used interchangeably (e.g., on page 5 “impact” in line 90, “efficacy” in line 93, and “effectiveness” in line 95). Impact is the correct term for what this study provides a baseline to assess. Similarly, throughout the paper, the authors refer frequently to vaccines “containing” different HPV types; as there is no virus in the vaccines, it may be more appropriate to say the vaccines “target” different HPV types.

Materials and methods:

Study Population:

Please provide more clarity regarding recruitment methods. Do women in this age group normally attend the LCSP for reasons other than study participation? Were women recruited from the LCSP or from other community sites? Was any incentive provided? Were women given results of the HPV testing, and if so, how were they counseled? Was the study actually conducted on “randomly selected samples”, as stated on page 5 in line 103, or were a convenience sample of women recruited to participate in this study? These details are important for replicating study methods in a future evaluation of impact.

Statistical analysis:

In the statistical analysis section on page 7 in lines 150-156, please describe the HPV type groupings, and how multiple types were accounted for. For example, if a woman had HPV 18 and HPV 58 detected, would she be included in the numerator of both types? In Figure 1, if a woman had both a HR and a LR type detected, where would she be represented?

Figure 1 is potentially misleading because it shows proportion positive for the different type groups out of 100% -- this makes it appear that the prevalence of HR-HPV is higher in older age groups, but that may not be true, and the prevalence is not shown. Consider remaking this figure so that the height of the bars represents total prevalence in each age group.

On page 7 in line 151, consider not using “qualitative variables” in the description of the methods. “Participant characteristics” would be a better descriptor.

Some analyses presented in the results (e.g. odds ratios and 95% CIs) are not described in the stastistical analysis section.

Results:

Table 1 and the corresponding text description are inconsistent. For example, the text refers to percentage who were students without employment, but the table does not include employment status. The text refers to percentage with at least high school education, but the table combines primary and secondary. Table 1 does not show results by age; age is usually one of the strongest predictors of HPV prevalence, and this basic descriptive information needs to be provided.

Consider not reporting odds ratios since this may not be the most appropriate measure of association for this study. Since HPV is not a rare outcome, odds ratios overestimate the strength of the association. Consider not reporting a measure of association at all (relying on comparing percentages with p-values) or consider using prevalence ratios instead. Regardless, the description of this measure of association should be included in the statistical analyses section.

In table 3, please specify N. HPV6 prevalence appears to be off by .1.

In the description of figure 2 on page 11 beginning with line 200, please be clear about how prevalence is determined for single and multiple infections. What is the denominator? Is it 208?

In the legend of figure 2 on page 11 in line 210, HPV 72 LR is not mentioned.

Discussion:

Generally, the discussion could be trimmed to focus on the most salient information. For example, the paragraph on multiple types that starts on page 13 with line 245 may be extraneous. The last paragraph before the conclusion beginning on page 15 with line 295 is confusing, with discussion expanding to other STIs. This paragraph may not be needed as well. A very high number of references is cited (76), and the authors should focus on those that are most important to the topic of establishing a baseline HPV prevalence in Paraguay.

On page 14 beginning with line 271, consider adding more context to the discussion of HPV 58. While HPV 58 has a high prevalence in Paraguay and in other countries worldwide, before suggesting it be given equal consideration to HPV-16, its degree of carcinogenicity should be considered. Most HPV-attributable cancers are caused by HPV-16 and 18. International studies found HPV-58 in 2.2% (de Sanjose JNCI Cancer Spectr 2018) and 4.4% (Guan et al., IJC 2012) of invasive cervical cancers, far below the contributions of the high-risk types targeted by the quadrivalent vaccine.

On page 15 in line 285, it is confusing to state that HPV 58 is not vaccine preventable (it is targeted by Gardasil-9); better to say it is not targeted by the vaccine currently in use in Paraguay’s vaccination program.

The discussion does not appear to include a paragraph on limitations of the study. It is important to at least acknowledge limitations of the study population not being representative of the population of Paraguay or Asuncion and how recruitment of a comparable population for a follow-up study to measure vaccine impact would be accomplished.

On page 16, in the conclusion in lines 308-309, consider deleting “Considering the high prevalence, multiple infections, and non-vaccine-type HPV infections“ from the second sentence. The data provide a baseline regardless of these considerations.

Reviewer #3: 1) The title the authors picked is an interesting and pioneer base line focus of interest specific to Paraguay. Authors used simple random sampling to select 208 women aged 18-25. Do you have an explanation or a local reference for the statement on line 106 "younger women..."? Secondly, what is the conclusion you have drawn from this research? Furthermore, what are your recommendations?

2) Although looking grossly sound, I have one questions regarding the result/analysis section. Why is the summation of those with History of STI and Negative test not 94; it is 91 in your case?

Overall, it is a very well written manuscript discussing a hot issue mainly concerning developing countries like Paraguay. It is a good initiative but the conclusion and recommendation needed further elaboration than short statements.

Reviewer #4: This study aimed to estimate the type-specific HPV frequency in unvaccinated women aged 18–25 years in the metropolitan area of Asuncion as a baseline for monitoring the HPV vaccination program

I have reviewed the manuscript and would like to suggest the following changes.

Discussion :

The first paragraph should mention the noteworthy findings of the study. The authors should compare and contrast these findings with other studies in subsequent paragraphs.

Also, author should add a paragraph regarding limitations and future implication of this study.

the conclusion should be a separate paragraph

the references are very numerous, it may be necessary to reduce a little

Reviewer #5: The paper was on the HIGH PREVALENCE AND CO-INFECTION OF HIGH-RISK HUMAN PAPILLOMAVIRUS GENOTYPES AMONG UNVACCINATED YOUNG WOMEN FROM PARAGUAY. The results showed important information for public health.

However, as the title was on prevalence, there were no mentioned on the estimation of sample size as N=208 was small. the demographic data showed that the participants were actually moderate risk eg. only 24.0 % used condom, 33% reported STI, 40.9% had reported 2 or more partners. These should add in the discussion, all these are risk factors for acquiring HPV. That's why the results reported quite high high risk HPV types.

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Reviewer #1: Yes: Dr. Burak Bayraktar

Reviewer #2: No

Reviewer #3: Yes: Dr. Kaleab A. Betru

Reviewer #4: No

Reviewer #5: No

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PONE-D-22-28769R1HIGH PREVALENCE AND CO-INFECTION OF HIGH-RISK HUMAN PAPILLOMAVIRUS GENOTYPES AMONG UNVACCINATED YOUNG WOMEN FROM PARAGUAYPLOS ONE

Dear Dr. Bobadilla,

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 submit your revised manuscript by 10/02/2023. 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,

Andrea Giannini

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:

Dear authors,

the manuscript it has now been evaluated by our experts and they have recommended that minor changes be made to the submission.

Please improving the manuscript with the reviewers' comments.

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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 #4: All comments have been addressed

Reviewer #6: (No Response)

Reviewer #7: (No Response)

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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 #4: Partly

Reviewer #6: Partly

Reviewer #7: Yes

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

Reviewer #1: Yes

Reviewer #4: No

Reviewer #6: Yes

Reviewer #7: Yes

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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: Yes

Reviewer #4: Yes

Reviewer #6: Yes

Reviewer #7: 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: No

Reviewer #4: Yes

Reviewer #6: Yes

Reviewer #7: Yes

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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 authors made several corrections. But as I said in my first review:The current study does not deliver anything significant new or outbreaking in this matter.

Reviewer #4: (No Response)

Reviewer #6: I read with great interest the manuscript, which falls within the aim of this Journal. In my honest opinion, the topic is interesting enough to attract the readers’ attention. Nevertheless, the authors should clarify some points and improve the discussion, as suggested below.

Authors should consider the following recommendations:

- Manuscript should be further revised in order to correct some typos and improve style.

- To date, several lines of evidence support the possibility to use specific biomarkers to identify early-stage cervical cancer and, in this way, offer a better prognosis to the patients. This point deserves to be discussed, referring to: PMID: 28918603; PMID: 35549629.

- I would recommend to stress novel pieces of evidence about high-risk HPV-negative high-grade cervical dysplasia, which seems to have more favorable outcomes than patients with documented high-risk-HPV infection (PMID: 35742340; PMID: 33514481).

Reviewer #7: I read with great interest the Manuscript titled "HIGH PREVALENCE AND CO-INFECTION OF HIGH-RISK HUMAN PAPILLOMAVIRUS GENOTYPES AMONG UNVACCINATED YOUNG WOMEN FROM PARAGUAY " which falls within the aim of the Journal.

In my honest opinion, the topic is interesting enough to attract the readers’ attention. Methodology is accurate and conclusions are supported by the data analysis. Nevertheless, authors should clarify some point and improve the discussion citing relevant and novel key articles about the topic.

-The introduction should be extended and completed. I find interesting a reference to the efforts made for the prevention and early diagnosis of gynecological cancers (see PMID: 36141217).

- Although it is a retrospective analysis, inclusion/exclusion criteria should be better clarified by extending their description.

- Discussions can be expanded and improved by citing relevant articles (I suggest authors to read and insert in references the following article PMID: 35742340).

Considered all this points, I think it could be of interest for the readers and, in my opinion, it deserves the priority to be published after minor revisions.

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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: Yes: Burak Bayraktar

Reviewer #4: No

Reviewer #6: No

Reviewer #7: Yes: Tullio Golia D'Augè

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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.

"High prevalence and co-infection of high-risk Human Papillomavirus genotypes among unvaccinated young women from Paraguay”

PONE-D-22-28769R2

Dear Dr. Bobadilla,

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,

Andrea Giannini

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

The manuscript has been modified with the comments of the reviewers. It is now ready to be published.

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 #6: All comments have been addressed

Reviewer #7: 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 #6: Yes

Reviewer #7: Yes

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

Reviewer #6: Yes

Reviewer #7: Yes

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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 #6: Yes

Reviewer #7: 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 #6: Yes

Reviewer #7: Yes

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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 #6: I carefully evaluated the revised version of this manuscript.

The authors have performed the required changes, improving significantly the quality of the paper.

Reviewer #7: Dear authors, thank you for sending me the corrected manuscript.

I read your work with great interest and pleasure. The work with the changes made after my advices and those of the other reviewers is complete and, in my opinion, ready for publication.

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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.

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Reviewer #6: No

Reviewer #7: Yes: Tullio Golia D'Augè

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