Comparative Analysis of HPV Detection Efficiency: Evaluating Cobas 8800 Performance in Vaginal Self-Sampling versus Clinician-Collected Samples at a Regional Thai Hospital

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This article titled HPV Ct Analysis at Chomburi Hospital: Comparing Cobas 8800 Performance in Self-Sapling Versus Clinician Collected Methods in Thailand is about an interesting and current issue.

Nevertheless, I personally believe that the article has some important shortcomings.

First of all, it is not clear how cytology is performed in patients who did Self sampling . It seems that the cytology is performed from the device provided for the self-test and it is widely known that these systems are not suitable for cytology. In the text it is only specified that patients with HPV non 16/18 are cytologically screened by a clinician. If this is the case, the results obtained would be worthless, as they could never be compared with samples taken by a clinician.

The authors assume that the self-collection sample is equivalent to that taken by a clinician, but the reality is that the self-collection also represents vaginal flora, so the HPV detected could be from the vagina rather than the cervix. In lines 221 to 223 the authors state that self-collected has higher positive rates, but this may be due to transient vaginal viruses.

They also assume that the Ct value is equivalent to viral load but viral load depends on the number of cells present in the sample, which can cause variability in viral load. In order to compare the two types of samples with each other, a comparison of the betaglobin gene should be made.

Lines 274-278 should be in the material and methods section.

The discussion should be very summarize and not constantly repeat the same thing and delete paragraphs 4.5 and 4.6.

The self-collected sample is widely described as a good sample for HPV screening and detection, but if positive, follow-up should be done with a cytology taken by a clinician to assess whether or not there is a lesion and the extent of the lesion. So the objective of this study does not make much sense. At least the whole part related to epithelial lesions should be ruled out.

 

Author Response

Response to reviewers 1

We would like to express our sincere gratitude to Reviewer 1 for their insightful comments and suggestions regarding our manuscript titled HPV Ct Analysis at Chomburi Hospital: Comparing Cobas 8800 Performance in Self-Sapling Versus Clinician Collected Methods in Thailand." We have carefully considered each point and have made corresponding revisions to enhance the clarity and quality of our manuscript.

This article titled HPV Ct Analysis at Chomburi Hospital: Comparing Cobas 8800 Performance in Self-Sapling Versus Clinician Collected Methods in Thailand is about an interesting and current issue.

Nevertheless, I personally believe that the article has some important shortcomings.

Reviewer 1

Q1: First of all, it is not clear how cytology is performed in patients who did Self sampling. It seems that the cytology is performed from the device provided for the self-test and it is widely known that these systems are not suitable for cytology. In the text it is only specified that patients with HPV non 16/18 are cytologically screened by a clinician. If this is the case, the results obtained would be worthless, as they could never be compared with samples taken by a clinician.

Response1: regarding cytological analysis in patients who did self-sampling, it is important to clarify the methodology used for cytological evaluation in our study. Cytology was not performed directly from the self-sampling devices. Instead, when a patient's self-collected sample tested positive for hrHPV, particularly for non-16/18 HPV types, these patients were invited to undergo a subsequent clinician-collected sampling specifically for cytological analysis. This procedure ensured that all cytological assessments were conducted on samples collected by a clinician using the standard method suitable for cytological evaluation.

This two-step process—initial HPV detection via self-sampling followed by clinician-collected samples for cytology in cases of positive HPV results—allows for effective risk stratification while maintaining the integrity and comparability of cytological outcomes. The results from self-sampling were thus used primarily for HPV screening, and the follow-up clinician-collected samples provided the necessary material for reliable cytological evaluation. This approach addresses the limitations of self-sampling for cytology and ensures that the cytological results are robust and comparable across all samples.

Q2: The authors assume that the self-collection sample is equivalent to that taken by a clinician, but the reality is that the self-collection also represents vaginal flora, so the HPV detected could be from the vagina rather than the cervix. In lines 221 to 223 the authors state that self-collected has higher positive rates, but this may be due to transient vaginal viruses.

Response 2: In response to the reviewer's comment regarding the equivalency of self-collected and clinician-collected samples for HPV detection, it is acknowledged that self-sampling can indeed capture vaginal flora, which may contribute to the detection of HPV not localized specifically to the cervix. The potential for detecting transient vaginal HPV infections in self-samples is a valid consideration and has been highlighted in other studies as well.

Our study observes higher positivity rates in self-collected samples, which could indeed reflect the broader capture of HPV, including potentially transient types from the vaginal environment. However, it's crucial to note that the primary goal of initial HPV screening, particularly in a high-throughput public health context, is to identify individuals at potential risk, warranting further diagnostic follow-up.

The observation that self-collected samples often yield higher Ct values—indicative of a lower viral load—supports the hypothesis that some of the detected HPV may be less clinically relevant or represent transient infections. Despite this, the higher incidence of abnormal cytological findings in the self-sampled group, which underwent further clinician-collected cytological evaluation, underscores the utility of self-sampling in identifying individuals who may require more detailed examination.

The higher rates of positive HPV findings in self-samples are considered alongside their diagnostic follow-up, where clinician-collected samples provide a more targeted and clinically relevant cytological assessment. This two-tiered approach ensures that those initially screened via self-sampling, and found to be at potential risk, are subsequently verified through more precise clinician-collected cytology, addressing concerns regarding the specificity of initial HPV findings.

Our methodology, therefore, leverages the accessibility and broader screening potential of self-sampling while relying on clinician-collected samples for definitive diagnostic procedures like cytology and colposcopy. This approach helps to balance the broad reach of self-sampling for population screening with the need for accurate diagnostic confirmation in individuals identified as at-risk.

Q 3: They also assume that the Ct value is equivalent to viral load but viral load depends on the number of cells present in the sample, which can cause variability in viral load. In order to compare the two types of samples with each other, a comparison of the beta globin gene should be made.

Response 3:

Thank you for your constructive comments regarding the use of Ct values as indicators of viral load. It is important to clarify that in our study, the Ct values were specific to HPV testing and did not include beta-globin normalization due to the limitations in data collection and retention policies at the facility. The data available to us only encompassed the Ct values for HPV, without corresponding beta-globin values, which are typically used to normalize for the amount of cellular material present in the samples.

 

This limitation is particularly significant given that the hospital's protocol involves deleting all Ct value data every three months for privacy and data management reasons. As a result, reanalysis of the samples to include beta-globin as a normalization factor was not feasible within the scope of our current analysis.

 

The discussion of viral load in our manuscript is based on the assumption that the Ct values, albeit unnormalized, provide a rough comparative basis for understanding the relative quantities of HPV present in the samples. We acknowledge that this approach has its limitations and that the results should be interpreted with caution. We appreciate your suggestion and agree that future studies should incorporate beta-globin normalization to ensure more accurate and reliable interpretations of viral load.

 

If the exclusion of beta-globin normalization is viewed as a significant oversight that might undermine the validity of our findings, we are open to revising the discussion to better reflect the limitations and the speculative nature of our conclusions regarding viral load. Your feedback is invaluable, and we are committed to addressing these issues comprehensively in our manuscript revisions.

Q4: Lines 274-278 should be in the material and methods section.

Response 4: Lines 274-278 have been moved to the material and methods section Line 84-89.

Q5: The discussion should be very summarized and not constantly repeat the same thing and delete paragraphs 4.5 and 4.6.

Response 5:

Thank you for your feedback regarding the discussion section of our manuscript. We have taken your suggestions into consideration and significantly condensed the discussion to avoid redundancy and enhance clarity. We've also removed paragraphs 4.5 and 4.6 as recommended, ensuring the content is more focused and succinct.

 

The revised discussion now succinctly addresses the key findings and their implications, maintaining a streamlined narrative that directly supports the conclusions drawn from our data. This revision is aimed at improving readability and ensuring that each point is clearly articulated without unnecessary repetition.

 

We appreciate the opportunity to refine our manuscript and believe these changes will make the discussion more effective and aligned with the journal's standards. Thank you for guiding these improvements.

Q6: The self-collected sample is widely described as a good sample for HPV screening and detection, but if positive, follow-up should be done with a cytology taken by a clinician to assess whether or not there is a lesion and the extent of the lesion. So the objective of this study does not make much sense. At least the whole part related to epithelial lesions should be ruled out.

Response 6: Thank you for your comment, which raises an important aspect of HPV screening protocols. In this study, while self-collected samples were primarily utilized to increase screening accessibility and detect HPV presence, clinician-collected samples were indeed used for detailed cytological evaluations whenever HPV positivity was identified. The study acknowledges the limitation of self-sampling primarily in the detection phase, not diagnostic specificity for lesion assessment.

The focus on cytological outcomes from clinician-collected samples post-HPV detection in self-samples aligns with current best practices, where a positive HPV test, particularly for high-risk types, prompts a more thorough examination using clinician-collected cytology. This step is critical for accurately determining the presence and extent of potential lesions.

This study aims to highlight not just the efficacy of self-sampling in broadening screening outreach but also underscores the necessity of follow-up clinical assessments to ascertain the precise oncogenic potential of detected HPV infections. The findings intend to reinforce the model where self-sampling serves as a preliminary screening tool, with subsequent diagnostic steps managed clinically to ensure comprehensive care and management. This dual-step approach allows for broader screening coverage while maintaining the diagnostic accuracy needed for effective clinical management.

Reviewer 2 Report

Comments and Suggestions for Authors

The authors of HPV Ct Value Analysis at Chonburi Hospital: Comparing Cobas 8800 Performance in Self-Sampling Versus Clinician Collected Methods in Thailand paper analysed 1,541 self-collected and 1,398 clinician collected samples, and they concluded that clinician collection remains more accurate, impacting future national screening policies.

 

Ø  Cobas 8800 assay is a very sensitive one.

 

Ø  Which means efforts should be performed in teaching and educating women in selfsampling.

 

Ø  Samples collected by clinicians using a cervical brush, then placed into ThinPrep PreservCyt Solution (Preservative solution).

 

Ø  Samples collected from the cervix by self-sampling used FLOQ Swab to collect cervical cells, then immersed in Roche Cell Collection Medium.

 

Ø  Maybe the sampling device and medium could explain the different results? Even I know from my experience that both are qualitative.

 

Ø  Do you have any realistic plan to optimise the women education in your country, regarding self sampling procedure?

Comments on the Quality of English Language

Minor editing of English language required

Author Response

We would like to express our sincere gratitude to Reviewer 2 for their insightful comments and suggestions regarding our manuscript titled HPV Ct Value Analysis at Chonburi Hospital: Comparing Cobas 8800 Performance in Self-Sampling Versus Clinician Collected Methods in Thailand. We have carefully considered each point and have made corresponding revisions to enhance the clarity and quality of our manuscript.

Reviewer 2

The authors of HPV Ct Value Analysis at Chonburi Hospital: Comparing Cobas 8800 Performance in Self-Sampling Versus Clinician Collected Methods in Thailand paper analysed 1,541 self-collected and 1,398 clinician collected samples, and they concluded that clinician collection remains more accurate, impacting future national screening policies.

Response 1: The findings from the "HPV Ct Value Analysis at Chonburi Hospital: Comparing Cobas 8800 Performance in Self-Sampling Versus Clinician Collected Methods in Thailand" study provide crucial insights into the efficiency of HPV detection methods. With a significant sample size of 1,541 self-collected and 1,398 clinician-collected samples, the study's conclusion that clinician-collected samples offer greater accuracy is a valuable point for consideration in shaping future national screening policies. This underscores the need for ongoing evaluation and potential enhancement of self-sampling techniques to ensure they meet the high standards of accuracy required for effective cervical cancer prevention and control strategies.

Reviewer comment: Cobas 8800 assay is a very sensitive one.

Response 2: The Cobas 8800 assay, indeed, is renowned for its high sensitivity, which allows for accurate and reliable detection of HPV DNA. This capability ensures that even low concentrations of the virus can be identified, making it an invaluable tool in the early detection and management of HPV-related conditions. Such sensitivity is critical for effective screening programs aimed at preventing cervical cancer by catching infections before they lead to more severe outcomes.

Reviewer comment: Which means efforts should be performed in teaching and educating women in self-sampling.

Response 3: Absolutely, the findings emphasize the importance of enhancing education and training programs for women on proper self-sampling techniques. By improving the reliability and accuracy of self-collected samples through focused educational efforts, we can maximize the effectiveness of cervical cancer screening programs. This approach not only supports the empowerment of women in managing their own health but also ensures that self-sampling can be a viable alternative in diverse healthcare settings, potentially increasing screening uptake and early detection of cervical cancer.

Reviewer comment:  Samples collected by clinicians using a cervical brush, then placed into ThinPrep PreservCyt Solution (Preservative solution).

Response 4: Line 102-104 has been changed following reviewer’s comment.

Ø Samples collected from the cervix by self-sampling used FLOQ Swab to collect cervical cells, then immersed in Roche Cell Collection Medium.

Response 5: Line 108-111 has been changed following reviewer’s comment.

Ø Maybe the sampling device and medium could explain the different results? Even I know from my experience that both are qualitative.

Response 6: Indeed, the type of sampling device and medium used can significantly influence the results of HPV testing. While both self-sampling and clinician-collected methods are qualitative, differences in the efficacy of sample collection devices and preservation mediums can affect the quality and integrity of the samples, leading to variability in Ct values and HPV detection rates. It's essential to consider these factors when interpreting the differences observed between self-collected and clinician-collected samples. Ensuring that both methods use optimally designed devices and mediums can help minimize discrepancies and improve the overall reliability of HPV screening outcomes.

Ø Do you have any realistic plan to optimise the women education in your country, regarding self-sampling procedure?

Response 7: Yes, there are several actionable strategies that could be implemented to optimize education about self-sampling for HPV testing among women in Thailand:

Public Health Campaigns: Launch extensive public health campaigns using various media platforms, including television, radio, social media, and print, to raise awareness about the importance of HPV screening and the availability of self-sampling kits. These campaigns can include instructions on how to properly collect samples to ensure accuracy.

Community Workshops: Organize community-based workshops in collaboration with local health centers and community leaders to educate women on the correct technique for self-sampling. These workshops could provide hands-on demonstrations and educational materials in multiple languages, considering the diverse linguistic landscape of the country.

 

Healthcare Provider Training: Train healthcare providers to educate patients during regular health visits. Ensuring that doctors, nurses, and community health workers are knowledgeable about self-sampling and can confidently instruct women will help improve trust and uptake of the method.

Inclusion in National Screening Programs: Integrate self-sampling education into the national cervical cancer screening program guidelines. This would formalize the process, making it an official part of health policy, which can increase credibility and acceptance among the population.

School-Based Health Education: Integrate health education into the school curriculum to educate young women early about HPV, cervical cancer, and the importance of screening. This early education can empower young women with knowledge and reduce stigma around HPV and related screenings.

Collaboration with NGOs and Women’s Groups: Partner with NGOs and women’s health groups that have a strong presence and trust within communities. These organizations can help disseminate information effectively and provide peer support.

Feedback Mechanism: Establish a feedback mechanism where women can share their experiences and suggestions about the self-sampling process. This feedback can be used to continuously improve educational materials and strategies.

Mobile Health Units: Utilize mobile health units to reach remote areas where women might have limited access to healthcare facilities. These units can provide education and distribute self-sampling kits.

By implementing these strategies, the education on self-sampling in Thailand can be significantly enhanced, potentially increasing its acceptance and effectiveness in the broader public health strategy against cervical cancer.

 

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors, 

I want to personally congratulate you for your wonderful work. Apart from some minor english errors, the concept of this study is correct and similar to one of my previous works. Thus, I am supporting the publishing of this manuscript after minor English revisions. Good luck! 

Comments on the Quality of English Language

Minor spelling errors were encountered. 

Author Response

Dear Reviewer 3,

We are immensely grateful for your encouraging comments and your support for the publication of our manuscript titled "[Manuscript Title]." It is truly rewarding to receive positive feedback from an esteemed colleague who has also explored similar research themes. Your acknowledgment of the concept and relevance of our study further motivates our team.

Regarding your suggestion for minor English revisions: We have carefully reviewed the manuscript and addressed all English language issues to enhance the clarity and readability of the text. We believe these revisions have refined the manuscript substantially.

Thank you once again for your constructive feedback and support. We are excited about the prospect of our work contributing to the ongoing scholarly dialogue in our field and hope it meets the journal’s standards for publication.

Reviewer 4 Report

Comments and Suggestions for Authors

This submission represents the revised version of a study assessing vaginal self-sampling implementation in a Thai setting. The authors utilized the Cobas 8800 system, a potent & validated molecular platform. The study’s results largely corroborate those of previous older work conducted in China (Duan et al PLoS One 2020, Song et al J Med Screen 2020) and in Europe (Inturrisi et al Lancet Regional Health-Europe 2021).

Despite the Discussion and Conclusions sections are in line with current literature and the overall merit of the article, this submission is hampered by the unclear Material and Methods section, a certain attitude on the role of cytology in self sampling material and perhaps an excessively lengthy Discussion section.

Notably, in East Asia, cytology in self sampling material is highly regarded among physicians and women, for various reasons (Yabusaki et al Asian Pac J Cancer Prev 2024). However, in a global perspective, cytology does not represent an established reliable approach in self sampling material in the context of cervical cancer prevention, mainly because of its problematic sensitivity (Song re Loopik BJOG 2020), which is also evidenced in this paper. This should be taken into account by the authors, given this manuscript is submitted in a Journal with international impact.

ABSTRACT:

It is unusual to name the exact setting in an Abstract; the authors might consider “Regional Thai Hospital” instead of “Chonburi Hospital”.

Lines 24-25: “This work underscores the need for robust sample collection methods to enhance the efficacy of cervical cancer screening programs”. The need for optimization of self-sampling assays and techniques is also a major issue and ongoing need; this clearly emerges from this study.

INTRODUCTION:

Line 39: “829,096 (45.9%) underwent screening” was this clinician-obtained molecular screening, cytology screening, self sampling or otherwise? Please specify.

MATERIALS AND METHODS:

Despite this being a revised version of the manuscript, the Section “2.1. Cervical cancer screening guidelines in Thailand” is disappointingly vague and unclear.

It might help to split the first paragraph (lines 64-89) in three distinct entities: 1) general information about cervical sample collection in Thailand, 2) pathway and subsequent management for vaginal self samples and 3) pathway and subsequent management in clinician-obtained cervical samples.

Line 76: “LBC is conducted” can be clarified to “reflex LBC in the same vial”.

Lines 80-81: “In the case of HPV self-sampling, if a sample collected from the cervix is deemed inadequate, women are instructed to provide a new sample”: a) inadequate for cytology, presumably? (indeed at the very high rate of 73,52%) and b) provide a new sample by which means, self sampling or clinician obtained? In rows 111-112 the sample is obtained by a clinician, however.

The diagram in Figure 1 is not correctly designed with the green boxes in the 2nd row essentially being part of the 3rd row.

Lines 80-81 & Figure 1: “In the case of HPV self-sampling, if a sample collected from the cervix is deemed inadequate” The authors probably mean inadequate for cytologic assessment; this should be clarified. Besides, even using specially designed SS devices, vaginal self sampling material is collected from the vagina and fornix and NOT from the cervix.

Line 84 & Lines 203-204: The classification "CIN1-3*" lacks correlation with current international nomenclature systems; otherwise please provide valid citation.

Figure 1: Cytology triage of hrOther specimens with a 75% inadequate cytology rate is clearly not cost-effective!!

Line 103: Hologic and not Halogic

RESULTS:
3.1. Analysis of Ct Values for HPV Detection across Various Age Groups

Lines 175-177: This finding has major importance. The poorer performance of SS especially in older ages could be attributed to anatomic changes in the accessibility/position of the cervical transformation zone.

3.2. Analysis of Ct Values for HPV Detection across Various hrHPV types

Lines 186-188 : The notably higher mean Ct value for HPV 16 in self samples , than in clinician-collected samples indeed represents a cause of concern for SS overall performance in the detection of the most prevalent and oncogenic HPV genotype.

Lines 198-200: “The substantial proportion of non 16,18 types indicates a prevalent risk that could be pivotal in shaping targeted screening and vaccination strategies within the Thai healthcare framework” is also particularly insightful.

3.3. Comparative Analysis of HPV Detection and Cytological Outcomes in Self-Collected vs. Clinician-Collected Samples

Lines 211-212: “The study encompassed 282 self-collected and 963 clinician-collected samples”, could perhaps be amended for clarity reasons to “The study encompassed 282 self-collected and 963 clinician-collected samples adequate for reflex cytology in the same sample”

Lines 234-236: “This suggests that a significant proportion of self-collected samples may not have met the cytological criteria necessary for a valid analysis” This remark, together with the low overall cytology’s sensitivity in self sampling material (ranging from 25-30% in most studies) underscores cytology’s poor cost-effectiveness and overall performance as a SS triage strategy

Lines 264-266: Totally substantiated

3.4. Histological Findings and Ct Value Analysis in hrHPV-Positive Samples

Line 313-314: “suggesting that Ct values alone may not be a strong discriminator for CIN2 or CIN3 risk assessment” Extremely insightful, in line with the literature. Inturrisi and colleagues also found higher mean real-time PCR Ct values (indicating lower viral load) in self-samples versus clinician samples, and this difference was greater by underlying disease status (Inturrisi F et al Lancet Reg Health Eur 2021)

DISCUSSION:

Lines 346-347: Optimization of SS assays is equally important (Arbyn et al CEBP 2023, Inturrisi F et al Lancet Reg Health Eur 2021, Colonetti et al EJOG 2024).

For clarity, a paragraph should be necessarily added stressing the cytology does not represent a globally adopted standard triage approach in hrHPV(+)ve self samples (Teghavi K et al Front. Oncol. 2023 ) Cytology performs very poorly in self sampling material and better triage options (methylation etc) using the same vial are feasible in settings were a new cytology specimen health obtained by a care provider during a new appointment is not the preset triage policy of hrHPV(+)ve self samples.

REFERENCES:

Reference No6 is not downloadable

 

Comments on the Quality of English Language

Minor language polishing might be required.

Author Response

Dear Reviewer,

Thank you for your thoughtful and constructive comments. We appreciate your insights, which have prompted further refinements to our manuscript. We have carefully addressed each of your concerns and provided detailed responses to the points you raised. For clarity and to ensure that we comprehensively address all the issues, we have detailed our responses in the attached file.

We believe these revisions have significantly strengthened the paper, enhancing its clarity, and addressing the potential shortcomings you've identified. We hope that our responses and revisions meet your approval and look forward to your feedback.

Thank you once again for your valuable input, which is instrumental in improving the quality of our research.

Best regards,

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

In my opinion, the study continues to have shortcomings since self-testing cannot be evaluated with the pathology results.

Author Response

Comment1:In my opinion, the study continues to have shortcomings since self-testing cannot be evaluated with the pathology results.

Response 1:Thank you for your valuable feedback. We acknowledge the challenges associated with evaluating self-collected samples using pathology results, which is a limitation in the current study framework. Self-sampling primarily serves as a broad-reaching, initial screening tool aimed at increasing participation rates, especially in underserved or hard-to-reach populations. It is not a standalone diagnostic measure but part of a larger screening continuum that ideally leads to clinician-reviewed diagnostic follow-ups where necessary.

Our study aims to demonstrate the feasibility and potential effectiveness of self-sampling in expanding coverage and accessibility of cervical cancer screening, recognizing that positive results should be followed by more definitive clinician-administered tests to confirm any pathological findings. This stepwise approach ensures that while self-sampling can significantly increase screening uptake, it remains integrated within a comprehensive diagnostic protocol that includes follow-up with healthcare providers for accurate diagnosis and appropriate treatment planning.

We appreciate your insight and will consider detailing these points more explicitly in our discussion to clarify the role and limitations of self-sampling in the context of a complete cervical cancer screening and diagnostic pathway.

Reviewer 4 Report

Comments and Suggestions for Authors

The authors undertook a huge effort in this revision, adopting several point of the previous review. As a result, the new version is more reader-friendly, provides more sound messages together with a potential for citations.

Lines 85-87: Apparently, for some individuals, self sampling was obligatory “or if the participant prefers not to perform another self-sampling, we then REQUIRE a new self-collected sample. This sample is again obtained through self-sampling”; please clarify

Some linguistic suggestions (among others):

Line 2: “vaginal self sampling” since SS is also feasible in urine

Line 58: “delve deeply” instead of “extend”

Line 67: “participates in” instead of “part of “

Line 95: “lesions” or “disease” or “dysplasias” instead of “conditions “

Line 99: “referred” instead of “escalated”

Line 139: “comparing Ct values” instead of “assessing Ct value”

Line 376: “Teghavi et al” instead of “Teghavi K et al”

Comments on the Quality of English Language

Some language polishing would be necessary.

Author Response

We greatly appreciate the time and effort you have invested in reviewing our manuscript and providing valuable feedback. We have carefully considered your comments and suggestions from the second round of review and have revised the manuscript accordingly. Below is a summary of the changes made and our responses to your specific comments:

1. Line 2: As suggested, we have revised "self sampling" to be more specific. Now referred to as "Vaginal Self-Sampling," to clarify the method used.

2. Line 58: We have replaced "extend" with "delve deeply" to better capture the intent of our analysis.

3. Line 95: We have changed "conditions" to "lesions" to better describe the medical specifics of HPV effects.

4. Line 99 (now Line 94): "Escalated" has been updated to "referred," reflecting a more appropriate term for the clinical process described.

5. Line 139 (now Line 134): Updated from "assessing Ct value" to "comparing Ct values" to correctly reflect the comparative analysis conducted.

6. Line 376 (now Line 375): We have corrected the citation format from "Teghavi K et al" to "Teghavi et al."

Each of these changes has been implemented to enhance the clarity, accuracy, and quality of the manuscript, ensuring it meets the high standards of your journal. We have attached a revised version of the manuscript with all changes highlighted for your review.

Thank you once again for your constructive feedback, which has been instrumental in refining our manuscript. We believe that the revisions made address your concerns and hope that the manuscript is now suitable for publication.

Looking forward to your favorable response.

Author Response File: Author Response.pdf