PONE-D-25-43925

Comparison of self-collected vaginal swabs and first-void urine for detection of human papillomavirus in sexually active girls and women in three South Asian countries

PLOS One

Dear Dr. Telele,

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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Thank you for submitting your paper to PLOS One for potential publication.

This is relevant and important study in the era of cervical cancer elimination and WHO 2030 goals.

Please address the comments from Reviewer 1 and 9 fully, having a response for every comment. Please also pay attention to the feedback from reviewers 2, 4, 6, 8, and 11.

These are critical issues to address:

1. The novelty of the study must be made clear by the authors since this is not the first paper to report the comparison of FVU and self collected samples.

2. The study participants have different characteristics, from all three sites, please explain the rationale and how bias would be avoided. This is includes age, marital status etc.

In some instances the first void urine was not collected however this is not accounted for in the analysis and the title, this may compromise the results.

3. How was consistency in sample collection and recruitment ensured throughout the three sites?

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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

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

Reviewer #6: Yes

Reviewer #7: Yes

Reviewer #8: Yes

Reviewer #9: Yes

Reviewer #10: Yes

Reviewer #11: 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

Reviewer #6: Yes

Reviewer #7: Yes

Reviewer #8: Yes

Reviewer #9: Yes

Reviewer #10: Yes

Reviewer #11: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: No

Reviewer #5: Yes

Reviewer #6: Yes

Reviewer #7: Yes

Reviewer #8: Yes

Reviewer #9: No

Reviewer #10: Yes

Reviewer #11: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

Reviewer #6: Yes

Reviewer #7: Yes

Reviewer #8: Yes

Reviewer #9: Yes

Reviewer #10: Yes

Reviewer #11: No

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5. Review Comments to the Author

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Reviewer #1: The article entitled, “Comparison of self-collected vaginal swabs and first-void urine for detection of human papillomavirus in sexually active girls and women in three South Asian countries,” by Telele et al., describes a cross-sectional study wherein the authors compared two non-invasive sampling methods for HPV DNA detection. The authors collected 753 samples from sexually active females ages 15–25 in Bangladesh, Pakistan, and Nepal using two methods, i.e., self-collected vaginal swabs (SCVS) and first-void urine (FVU). The authors used a harmonized protocol not only to assess HPV prevalence but also to determine concordance between the two sample types.

Though the study appears to be scientifically sound, and the manuscript is well organized and prepared, there are few changes required to strengthen the objectives and outcomes of the study. The manuscript can be accepted for publication in the PLoS One after replies to the following comments are addressed satisfactorily:

1. The authors cited similar studies that have compared self-collected vs urine samples for HPV. The authors should mention with clarity what is the uniqueness/novelty of the present study.

2. In the introduction section, instead of over emphasizing the HPV background and self-sampling the authors should focus on the research question and what knowledge gap is being filled by carrying out the present study.

3. The authors should provide the justification of cohort chosen especially the reason for age of 15-25 years and if / how it is related to HPV vaccination. Why do authors have not included broader age range? The reasoning behind the narrative should be provided in the manuscript as it would strengthen the manuscript.

4. The recruitment strategies detailed in the present study differed by country. Such as, the authors carried clinical based sampling in Nepal; demographic surveillance in Pakistan and sampling of married women in Bangladesh. The impact of recruitment strategy can be on the results / observations of the study. The authors should provide a clarification on the approach.

5. In discussion section, the authors could provide information on comparing the practical advantages between the two methods of sampling. They should also discuss how the present study outcomes can have influence on screening strategies. The authors can summarize the major findings/takeaways of the present study in conclusion.

6. As the samples were self-collected, the authors should acknowledge the limitations of the method because of absence of clinician / trained personnel collected samples as it can impact the outcome of the result. The authors should mention other limitations too.

7. The authors should provide the probable reasons for the observations that why Nepal had higher HPV prevalence than other two countries. Further, authors should mention if they have any plans for future studies to follow up or some other objectives.

8. The authors should ensure consistency in use of HPV-related terms. Such as at some places for High Risk ‘hr’ is used while at other places ‘HR’ is used. Authors should check the manuscript to make sure about uniformity.

9. Authors should make sure that all web references have accessed date and formatted as per PLoS One requirements. Also, make sure that all the references are cited and citations provided meet the PLoS One requirements.

Reviewer #2: This is an automated report for PONE-D-25-43925. This report was solicited by the PLOS One editorial team and provided by ScreenIT.

ScreenIT is an independent group of scientists developing automated tools that analyze academic papers. A set of automated tools screened your submitted manuscript and provided the report below. Each tool was created by your academic colleagues with the goal of helping authors. The tools look for factors that are important for transparency, rigor and reproducibility, and we hope that the report might help you to improve reporting in your manuscript. Within the report you will find links to more information about the items that the tools check. These links include helpful papers, websites, or videos that explain why the item is important. While our screening tools aim to improve and maintain quality standards they may, on occasion, miss nuances specific to your study type or flag something incorrectly. Each tool has limitations that are described on the ScreenIT website. The tools screen the main file for the paper; they are not able to screen supplements stored in separate files. Please note that the Academic Editor had access to these comments while making a decision on your manuscript. The Academic Editor may ask that issues flagged in this report be addressed. If you would like to provide feedback on the ScreenIT tool, please email the team at ScreenIt@bih-charite.de. If you have questions or concerns about the review process, please contact the PLOS One office at plosone@plos.org.

Reviewer #3: Excellent work! This is a very important piece of research regarding Human Papilloma Virus infection in a young sexually active population. The methods used are rigorous and reproducable. They serve as the template for future work on this topic.

Reviewer #4: This manuscript presents a valuable multi-country study evaluating the concordance between self-collected vaginal swabs (SCVS) and first-void urine (FVU) for HPV detection among young women in Bangladesh, Nepal, and Pakistan. The study addresses a critical public health need in resource-limited settings. The methodology is robust, the sample size is adequate, and the manuscript is well-written. The harmonized protocol across three countries is a significant strength.

However, I have a few minor comments and queries that, if addressed, would further strengthen the manuscript.

Specific Comments:

Sample Validity: The results state (Page 12, Line 196) that "No inadequate test results were reported from either sample type." Given the nature of self-collection and the large sample size (N=753 pairs), a 0% invalid rate is exceptionally evident of high-quality supervision or robust DNA extraction methods. Could the authors briefly clarify in the Methods section if any participants were excluded prior to the final N=753 count due to sample insufficiency, or if the "UCM" buffer and extraction protocol truly yielded 100% valid DNA for all collected samples? This detail speaks to the feasibility of this method in real-world surveillance.

Data Availability: The authors have indicated that data is available "upon reasonable request" due to ethical restrictions. Please ensure this complies with PLOS ONE’s strict data availability policy. If the data contains sensitive patient information that prevents public deposition, the specific ethical restrictions and the contact information for a non-author institutional body (e.g., an Ethics Committee) should be clearly stated in the final declaration, rather than just the authors' contact.

Cycle Threshold (Ct) Analysis: The comparison of Ct values between concordant and discordant samples is a very insightful addition (Figure 1). It clearly demonstrates that discordance is driven by low viral loads. It would be beneficial to briefly discuss the clinical implication of this: are the "missed" infections in discordant cases likely to be clinically significant (e.g., persistent infections) or transient low-load infections?

Demographics: The study population varies slightly by country (e.g., married women in Bangladesh/Pakistan vs. sexually active women in Nepal). While the authors acknowledge this in the limitations, a brief sentence in the Discussion on whether marital status (and implied sexual behavior differences) might impact the vaginal microbiome or viral load—and thus detection concordance—would be interesting, though not mandatory.

Logistics: The conclusion emphasizes the potential for these methods in "resource-limited settings." However, the methods describe a cold chain (2-8^{\circ}C transport, -20^{\circ}C storage). In the Discussion, could the authors comment on the stability of samples in the UCM buffer at ambient temperature? This is crucial for true utility in remote areas without cold chain infrastructure.

Conclusion:

The study is technically sound and provides important data for HPV surveillance strategies in South Asia. I recommend publication after these minor points are addressed.

Reviewer #5: This is a very important study for public health in South Asia. Comparing self-collected vaginal swabs and urine for HPV testing is a great idea, especially for reaching women in resource-limited settings.

I really appreciate that you collected data from three different countries (Bangladesh, Nepal, and Pakistan). The results are clear and promising—showing that both methods work well for detecting high-risk HPV. This will be very helpful for future vaccination monitoring programs.

I have no changes to request. Congratulations on a successful multi-country study.

Reviewer #6: Introduction

* Your final aim statement says “sexually active girls and women aged 15–25 years” (line 85), but your Methods section indicates 18–25 in Pakistan and Nepal and “ever-married ≤25” in Bangladesh. Ensure the aim statement aligns with the enrolled age ranges ("18–25 years” overall, or “≤25 years"), maybe with a brief note about country-specific eligibility.

* You have said that “Pakistan has not yet introduced the HPV vaccine” (lines 54–55). That is no longer accurate: Pakistan launched its first HPV vaccine rollout/campaign beginning September 15, 2025, with plans to integrate it into routine immunization thereafter.

* The reader will want a tighter argument for why concordance between SCVS and FVU matters in your specific context. I recommend adding 1–2 sentences on whether your primary use case is population-based surveillance, screening uptake, or post-vaccine monitoring, and why urine might be preferable in South Asia (acceptability, cultural norms, logistics, clinic capacity).

* The introduction can be precise. Consider trimming 1–2 sentences on virology/classification, and use that space to sharpen the study rationale.

Methodology and Results

* The target populations and recruitment strategies vary (Bangladesh: “ever-married ≤25” with stratified systematic sampling; Pakistan: married 18–25; Nepal: sexually active 18–25). This affects both prevalence estimates and potentially concordance if underlying risk factors differ. Please provide baseline participant characteristics by country (age distribution, marital status, parity, education, contraceptive use, recent sexual activity, etc.) and discuss how these differences may influence outcomes.

* Self-collection procedures require a little more detail, such as the order of collection and assistance/observation, FVU timing....

* The high overall agreement/negative agreement (Table 4) likely reflects low prevalence and a large proportion of negative pairs (as your negative agreement is >99%). This is expected but can overstate “equivalence.” Mention this issue in the discussion or emphasize positive concordance and directional discordance.

* Table 3 text: “full discordance ranged from 8.4% (38 HPV types)” appears to be a typo; it should likely be 28 HPV types.

Reviewer #7: Excellent paper and an important topic. This paper discusses an important and emerging topic. It helps inform our current knowledge and will prove beneficial for the medical community. The methodology is rigorous.

Reviewer #8: Thanks for your manuscript.

Comments

The inclusion criteria and clinical background of the study population are not clearly defined. It is not fully explained whether participants were attending for routine screening, symptomatic evaluation, abnormal cytology follow-up, or other clinical reasons. Because the sample is hospital-based, this may introduce selection bias and limit generalizability to the broader population. The authors should clearly describe the recruitment setting and clinical indications for participation.

The cross-sectional nature of the study limits causal interpretation. Some parts of the discussion imply transmission directionality or risk relationships between cervical and anal HPV infection. However, cross-sectional data cannot establish temporality or causation. The manuscript should revise the language to focus strictly on associations rather than inferred transmission pathways.

Potential confounding variables are not adequately addressed. Important factors such as sexual behavior, number of sexual partners, smoking status, contraceptive use, immunosuppression, and HIV status could influence HPV prevalence. Without adjustment for these variables, observed associations may be partially confounded. If such data were not collected, this should be clearly acknowledged as a limitation.

The manuscript does not clearly report whether any samples were excluded due to insufficient DNA, invalid results, or missing data. A participant flow diagram summarizing eligible, excluded, and analyzed cases would improve transparency and align with reporting standards.

Reviewer #9: General comments

This study compares self-collected vaginal swabs with first-void urine samples for HPV detection among sexually active girls and women in three South Asian countries. The question is both practical and relevant, particularly in settings where expanding access to cervical cancer screening remains a challenge. Overall, the study appears thoughtfully designed, and the main findings are generally supported by the data presented. A few areas, however, would benefit from additional clarification to improve transparency and reporting.

Major Comments

1. Study Design and Recruitment

It would be helpful to provide more detail about how participants were recruited (e.g., consecutive vs convenience sampling) and to clarify the inclusion and exclusion criteria more explicitly. Please also indicate whether HPV vaccination status was recorded and considered in the analysis. In addition, clarifying whether the order of sample collection (swab vs urine) was standardized would help readers assess potential procedural effects.

2. Laboratory Procedures

Additional detail regarding the HPV assay would improve reproducibility, including its validation status, limit of detection, and any internal quality controls used. Please also clarify whether laboratory personnel were blinded to specimen type and whether quality assurance procedures were consistent across the participating countries.

3.Statistical Methods

The statistical analysis would benefit from clearer reporting. Please specify whether a sample size or power calculation was performed. Confidence intervals should be consistently reported for measures such as sensitivity, specificity, and agreement statistics. If not already included, stratified analyses by country or age group could help explore potential variability across sites.

4.Interpretation of Findings

The conclusions are generally supported by the results; however, where equivalence between sample types is implied, the language may need to be slightly moderated unless a predefined non-inferiority framework was applied. Expanding briefly on possible reasons for discordant results between specimen types would also strengthen the discussion.

5. Data Availability

The Data Availability Statement indicates that data are available upon reasonable request. Given PLOS ONE’s data-sharing policy, please clarify whether the dataset can be deposited in a public repository or provide a more detailed explanation of any restrictions that prevent open access.

Minor Comments

The manuscript is generally well written and clear, with only minor language refinements needed.

Please ensure consistent terminology (e.g., “first-void urine” throughout).

Tables should clearly specify denominators when percentages are reported.

Overall, this is an important and relevant study. With the clarifications above, the manuscript would be strengthened and better aligned with reporting standards.

Reviewer #10: The manuscript is well structured, all of the data was provided, statistical analysis was performed appropriately. the manuscript and data was supporting the conclusion and all of the required details were shared.

Reviewer #11: General Comments:

- This manuscript reports original, multicountry research comparing self-collected vaginal swabs (SCVS) and first-void urine (FVU) for HPV detection among young women in South Asia. The study addresses an important public health question in low- and middle-income countries and contributes valuable evidence for HPV surveillance and screening strategies.

- Minor language editing could further enhance readability.

Abstract

- The abstract is well structured and clearly summarizes the background, objectives, methods, key findings, and conclusions. The study design, sample size, laboratory methods, and main statistical approaches are appropriately described. The results are presented quantitatively and support the stated conclusions. The abstract accurately reflects the content of the manuscript and meets PLOS ONE standards for clarity and completeness

Introduction

- Provides a comprehensive overview of HPV epidemiology, cervical cancer burden, and screening challenges in South Asia. The authors effectively contextualize the need for self-sampling approaches and cite relevant international and regional literature. The rationale for comparing SCVS and FVU is well justified, and the research objectives are clearly stated.

- Lines 44–60 in the Introduction contain detailed country-specific descriptions of cervical cancer burden in Bangladesh, Pakistan, and Nepal. While this information is relevant, this section could be streamlined to improve conciseness and flow. Given that the study focuses on three countries, the authors may consider briefly explaining the rationale for selecting these specific settings (e.g., representativeness, programmatic relevance, epidemiological burden, or participation in the GLOBE-HPV project). In addition, the epidemiological background could be summarized more succinctly by presenting key indicators—such as HPV prevalence, cervical cancer incidence, and mortality rates—for each country, rather than providing extended narrative descriptions. Condensing this section would enhance readability while maintaining its contextual value, and would allow readers to more quickly understand the public health relevance of the selected study sites.

Materials and Methods

The manuscript states that in Bangladesh and Pakistan, participants were required to be married, whereas in Nepal eligibility was based on being sexually active regardless of marital status.

Could the authors please clarify the rationale for this difference in inclusion criteria across countries?

Specifically:

- Was marital status used as a proxy for sexual activity in Bangladesh and Pakistan due to sociocultural or ethical considerations?

- Were there regulatory or institutional constraints that prevented recruitment of unmarried sexually active women in those settings?

- How might this difference affect comparability of HPV prevalence and concordance estimates across the three countries?

A brief explanation in the Methods section, and possibly a note in the Limitations section addressing potential selection bias or differences in sexual behavior patterns, would strengthen transparency and interpretation of cross-country comparisons.

Sample Size

Could the authors please clarify:

- Whether a formal sample size or power calculation was performed for this concordance analysis?

- If so, which assumptions were used (expected HPV prevalence, anticipated agreement/Kappa value, precision, alpha level)?

- Whether the target of approximately 250 participants per country was based on logistical considerations, cohort design, or statistical requirements of the GLOBE-HPV project?

Results

- The manuscript reports no inadequate test results; however, additional clarification on data completeness (e.g., missing demographic or behavioral variables) would improve transparency.

- The description of discordant cases could be expanded, for example by indicating whether discordance was more frequent in specific subgroups (country, age range, or HPV type categories).

Discussion

The Discussion section is generally well written and provides an appropriate interpretation of the study findings in relation to existing literature. The authors successfully contextualize their results within prior studies on self-sampling for HPV detection and highlight the relevance for surveillance in low- and middle-income settings.

However, several aspects could be strengthened:

- Balance between interpretation and speculation Some explanations regarding differences in sensitivity and concordance between SCVS and FVU appear speculative and would benefit from stronger empirical support or clearer citation of supporting evidence.

- Cross-country comparability Given the differences in inclusion criteria and recruitment strategies across countries, the authors should more explicitly discuss how these variations may influence the comparability of findings and interpretation of pooled analyses.

- Public health and implementation implications While the potential use of SCVS and FVU in surveillance programs is mentioned, further discussion on operational feasibility, cost considerations, and integration into existing screening systems in South Asia would enhance the practical relevance of the findings.

- Limitations and potential biases The limitations section could be expanded to more clearly address potential selection bias, reporting bias related to self-sampling, and variability in laboratory processing across sites.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Muhammad Saqib

Reviewer #4: Yes: Hussein Mussa Muafa

Reviewer #5: Yes: Ganesh Praneeth Roy Avula

Reviewer #6: No

Reviewer #7: Yes: Muhammad Saqib

Reviewer #8: No

Reviewer #9: No

Reviewer #10: Yes: Reenam Mushtaq

Reviewer #11: Yes: Jessica Marsigit

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Comparison of self-collected vaginal swabs and first-void urine for detection of human papillomavirus in sexually active girls and women in three South Asian countries

PONE-D-25-43925R1

Dear Dr. Telele

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,

Ramokone Lisbeth Lebelo, Ph.D

Academic Editor

PLOS One