Human Papillomavirus (HPV) testing on self-collected samples allows for improved coverage rates of cervical cancer (CC) screening programs. ThinPrep®PreservCyt®  medium is widely used for the suspension of cervical and vaginal self-samples. However, this medium is costly, toxic, and flammable, involving special handling procedures which make its use difficult in screening programs, particularly in low- and middle-income countries.® (Copan SpA), an alternative non-alcohol-based suspension medium, compared to ThinPrep®PreservCyt® (HOLOGIC®) for high-risk HPV (hrHPV) detection in vaginal self-collected swabs using three different real-time polymerase chain reaction (RT-PCR) HPV assays: Anyplex™II HPV28 , Papilloplex® High Risk HPV , and HPV OncoPredict . This pilot study aimed to evaluate the analytical performance of eNat 30 women, referred to colposcopy, were enrolled in this observational, prospective pilot study and asked to collect two vaginal self-taken samples, which were suspended in 5 mL of ThinPrep®PreservCyt® or eNat®. Nucleic acids were extracted from 200 μL using Microlab Nimbus platform  and tested with the three different RT-PCR full-genotyping high-risk HPV assays. The HPV results of vaginal samples resuspended in the two different media were compared to those obtained from the reference clinician-collected cervical sample from the same woman.<k<0.796). Moreover, the discordances between vaginal self-samples collected from the same woman were found only in cases of normal cytology or low-grade cytological lesions and were generally related to low hrHPV viral loads as indicated by the quantitative HPV OncoPredict assay . hrHPV detection in vaginal self-samples suspended in both media demonstrated a substantial agreement with cervical samples with the three assays under-investigation (0.667 The study's preliminary findings demonstrated a very good agreement between cervical and vaginal self-collected samples suspended in ThinPrep®PreservCyt® and eNat®, suggesting that the latter could represent a good alternative medium in HPV screening programs based on self-collection. However, this solution is costly, toxic, and flammable, requiring special handling procedures, making its use difficult, particularly in low- and middle-income countries. This study aims to evaluate an alternative non-alcohol-based suspension medium, the eNat®  for HPV detection in vaginal self-collected samples. This study involved the enrolment of women referred to a gynaecologist for an abnormal Pap test. During their gynaecological visit, women were asked to provide two self-collected vaginal swabs, one to be suspended in the eNat® medium and the other in ThinPrep®PreservCyt® solution, prior to HPV testing. An additional sample, collected by the clinician from the cervix of the same patients, was used as gold-standard reference method to compare HPV results obtained from the two vaginal samples. All samples were tested using three different HPV assays: a commercial kit, and two other HPV tests recently developed as part of a project financed by the European Commission. Preliminary results obtained using all three evaluated HPV tests on vaginal samples suspended in the alternative eNat® medium were comparable to those obtained on vaginal and cervical samples suspended in ThinPrep®PreservCyt® solution. In conclusion, an improved and cost-effective solution for CC screening based on self-collected vaginal samples, suspended in an alternative non-flammable medium, compatible with three different innovative HPV assays, has been evaluated with the aim to favour women’s participation, particularly in low- and middle-income countries.Persistent long-term infection with Human Papillomaviruses (HPVs) is associated with the development of cervical carcinoma. Cervical cancer (CC) screening programs based on the detection of HPV can reduce its incidence and mortality. Screening programs, based on HPV testing of self-collected vaginal samples, have been shown to be more acceptable to women and can improve their participation to CC screening programs. ThinPrepGLOBOCAN 2020, a total of 604,127 new cases of cervical cancer (CC) and 341,831 CC related deaths occurred worldwide in 2020. The recent call of action proposed by the World Health Organization (WHO) to eliminate CC sets as one of the goals the screening of 70% women by the age of 35 and again by the age of 45 using high-performance assays, such as human papillomavirus (HPV) testing by 2030.According to data from.The introduction of HPV testing as an analysis tool offers the possibility of using self-sampling to increase participation in screening programs for CC prevention. HPV testing on self-samples has been reported to be similarly accurate in the detection of cervical precancerous lesions, as well as HPV testing performed on clinician-collected cervical samples. Moreover, self-sampling allows women who, for socio-cultural reasons, do not access gynaecological examination, to take part to screening programs®PreservCyt®  is an alcohol-based solution that serves as transport and liquid preservative for performing liquid-based Pap Smear on cervical samples. Therefore, HPV testing is routinely performed on the same type of sample and, for analogy, vaginal swabs for self-sampling have been usually suspended in ThinPrep®PreservCyt® solution.ThinPrep®PreservCyt® is flammable and requires special handling and additional costs for transport. These characteristics of ThinPrep®PreservCyt® make it difficult to introduce its use in self-collection-based CC screening in low to middle income countries. To overcome these problems, a non-alcohol-based medium to suspend self-collected vaginal samples that supports HPV nucleic acid stability and that is suitable for molecular HPV analysis is necessary.Because of its high percentage of methanol, ThinPrep®  is a lysing based molecular collection medium already used for nucleic acids amplification assays. Moreover, it preserves and stabilizes nucleic acids and desaturates proteins and inactivates microbial agents potentially contained in clinical samples.eNat®PreservCyt® and eNat®.This pilot study aims to evaluate the analytical concordance of HPV testing conducted on a physician-collected cervical sample (gold-standard) as compared to that performed on two dry vaginal self-collected samples eluted respectively in ThinPrepThis study was approved by The Ethics Committee of the University of Milano-Bicocca, Monza, Italy (Protocol n. 0037320/2017 and 0086409/2018). All participants gave written informed consent prior to participation.® 552C.80 device  prior to coloposcopic examination. The two vaginal self-collected samples were numbered to trace the order in which they were collected and kept dry at room temperature until analysis. During the colposcopy examination, a cervical sample was taken by the gynaecologist using an L-shaped Endo/Esocervical FLOQSwab® . Women underwent biopsy and/or conization depending on the colposcopy outcome, according to the local clinical protocol. All specimens were transported to the Laboratory of Clinical Microbiology of the Department of Medicine and Surgery, University of Milano-Bicocca, Italy, where they were processed.30 women, referred to colposcopy for a recent abnormal Pap smear reported as either low-grade intra-epithelial lesion (LSIL), high-grade intra-epithelial lesions (HSIL), atypical squamous cells of undetermined significance (ASCUS), or atypical glandular cells of undetermined significance (ACGUS), such as ASCH  and ACG , were enrolled as part of an ongoing study (from March 2020 to January 2021). Immunocompromised patients, women with autoimmune diseases or any diseases involving the immune system, including HIV infection, with a presumed or confirmed pregnancy, with a diagnosis of any malignancies, or undergoing or having finished a course of chemotherapy during the six months preceding the study were excluded from the study. After signing the written informed consent, women were provided with the vaginal collection devices as well as written instructions illustrating how to perform the self-sampling; medical and nursing staff were also available if further assistance was required by the participating women. All enrolled women autonomously collected two vaginal-self samples using FLOQSwab®PreservCyt®. In the laboratory all specimens were vortexed for 30 seconds, and subsequently 1.5 ml aliquots were dispensed in cryovials; one was used for DNA extraction and the others stored at -20°C.Clinician-collected cervical samples, obtained using the L-shaped swab, were immediately placed in 20 ml of ThinPrep®PreservCyt® and the other in 5 ml of eNat®. In order to avoid bias associated with the order of vaginal swab collection, the first vaginal swab collected from 15 women was suspended in ThinPrep®PreservCyt®, while the second in eNat®; for the remaining 15 patients the first specimen was suspended in eNat® and the second in ThinPrep®PreservCyt®. Five aliquots of 1 ml were made from each of the vaginal specimens; one was used immediately for nucleic acids extraction prior to testing and the others stored at -20°C.Vaginal self-collected swabs were transported dry to the laboratory. One swab was suspended in 5 ml of ThinPrepOne aliquot of the cervical and of each of the vaginal samples was extracted using STARMag 96x4 Universal Cartridge Kit  on Microlab Nimbus  platform, a completely automated Liquid Handling Workstation for nucleic acid extraction and polymerase chain reaction (PCR) setup of up to 72 specimens. DNA was extracted from a 200 μL volume of each sample and following extraction eluted in 100 μL of the kit elution buffer, according to the manufacturer’s instructions.® High Risk HPV  and HPV OncoPredict .Cervical and vaginal specimens were tested for HPV genotypes using three different real-time PCR full-genotyping HPV assays: Anyplex™II HPV28 , Papilloplex® High Risk HPV is able to detect and types 14 hrHPV  based on specific melting profiles. HPV OncoPredict assay detects and quantifies 12 hrHPV  and uses C-C chemokine receptor type 5 (CCR5) to detect sample’s cellularity both to evaluate sample adequacy and to allow for normalization of viral load.The first assay can identify 14 hrHPV  and 14 Low-risk HPV (lrHPV) types  in two different reaction mixes by means of real-time PCR assays. Papilloplex® High Risk HPV and HPV OncoPredict was performed using 5 μl of extracted DNA in a total volume of 20 μl.All three Real-time assays were performed using a CFX96 PCR Thermal Cycler  according to manufacturers’ instructions using 5 μl of template DNA in a total volume of 20 μl for Anyplex™II HPV28. Analysis with PapilloplexQuickCalcs 2014 software. Agreement was defined as slight (0.00<k<0.20), fair (0.20<k<0.40), moderate (0.41<k<0.60), substantial (0.61<k<0.80) and almost perfect (0.81<k<1.00) as previously reported.Patients’ age was described by median value and interquartile range . Viral load was expressed as number of viral genome copies (cp)/10,000 cells. Agreement between HPV testing results on different types of samples and different tests was evaluated with the Cohen's kappa (κ) statistics using GraphPadThe median age of the 30 women enrolled for this study was 36.5 years (interquartile range (IQ): 29.3–47). Most of the women  presented cytological alterations: low-grade intra-epithelial lesion (LSIL) was the most frequently detected  followed by the atypical squamous cells of undetermined significance (ASCUS) in 5/30 (16.7%) women. High-grade intra-epithelial lesions (HSIL) were found in 3/30 (10.0%) women, atypical glandular cells (AGC) in 1/30 (3.0%) woman and 7/30 (23.0%) women had a negative (NEG) Pap smear result.Following colposcopy examination results, 5/30 (16.7%) women underwent conization: histological result was cervical intra-epithelial neoplasia grade 1 (CIN1) for one woman, cervical intra-epithelial neoplasia grade 2 (CIN2) for two women and cervical intra-epithelial neoplasia grade 3 (CIN3) for two women.® High Risk HPV. Among vaginal self-samples suspended in ThinPrep®PreservCyt®, 20/30 (66.6%), 19/30 (63.3%) and 20/30 (66.6%) were hrHPV positive with each of the three assays, respectively; while among those suspended in eNat® 20/30 (66.6%) were found to be hrHPV positive with all three kits.17 out of 30  cervical samples were found to be hrHPV positive using Anyplex™ HPV28 detection kit, 16/30 (53.3%) with HPV OncoPredict and 15/30 (50%) with Papilloplex®PreservCyt® and eNat®. None of the enrolled women were found to be positive for HPV33 or HPV35.® or ThinPrep®PreservCyt® media and cervical samples was observed.As reported in®PreservCyt® and eNat® was demonstrated to be almost perfect with percentages of agreement of 100.0%  100.0%  and 96.7%  respectively with Anyplex™II HPV28, Papilloplex® High Risk HPV and OncoPredict.The concordance between vaginal self-samples suspended in ThinPrepData regarding HPV genotypes distribution in each sample is reported inFor all HPV DNA tests, no differences in HPV detection rate related to the order of vaginal specimens’ collection were observed.HPV OncoPredict quantification kit was used to determine HPV type specific and total viral load for all tested samples.®PreservCyt® or eNat® . Interestingly, similar viral loads were detected in both vaginal samples irrespective of the suspension medium used. Discordant results in type-specific HPV infection between the two vaginal swabs/woman were associated with either low-grade or negative cytology or with viral loads that were below 6.24E+02 cp/10,000 cells.The mean value of the total normalized viral load in cervical samples was lower than that detected in vaginal samples suspended either in ThinPrep® and ThinPrep®PreservCyt®) as compared to that detected in clinician-collected cervical samples.This pilot study compared the analytical concordance of hrHPV DNA detection in self-collected vaginal swabs resuspended in two different media . Discrepant results had been previously reported in samples with low HPV viral load. Moreover, all the observed discordances were observed in patients with low-grade or negative cytology.In this study, substantial agreement in hrHPV detection was demonstrated between cervical and vaginal self-samples with a concordance rate ranging from 83.3% to 90.0% for the different assays and suspension media; this is in agreement with previous reports®PreservCyt® and for those suspended in eNat®, was higher  than that observed in cervical samples . The higher HPV viral load detected in vaginal samples may be associated to the differences in the suspension volume of the media used for cervical and vaginal specimens (20 mL vs 5 mL). Viral load normalization based on the samples’ human cellularity should have taken into account the differences associated with suspension volumes. Statistical analysis on a larger number of samples will help to elucidate differences in normalized viral loads between the vaginal and cervical samples.The mean hrHPV viral load for self-collected vaginal swabs, eluted in ThinPrepThe main limitation of this pilot study is the restricted number of women enrolled. Future studies including a greater number of samples will be necessary to fully evaluate any potential difference in performance of HPV testing using this alternative suspension medium.®PreservCyt® or eNat®, with the second medium allowing viral inactivation and providing a good strategy to further reduce costs.In conclusion, this study demonstrated that vaginal self-sampling is a good alternative to cervical swab if the sample is collected in either ThinPrep® appeared to be the best option because of its performances, cost and the possibility to transport samples dry. To our knowledge, this is the first study that compares eNat® to ThinPrep®PreservCyt® for the suspension of vaginal samples. Future studies including a greater number of clinical samples and other alternative suspension media are necessary to better evaluate the best solution for vaginal self-samples testing.Previous studies compared different devices for vaginal self-sampling considering cost, simplicity of use and accuracy in HPV detection. FLOQSwab® as an alternative suspension medium to ThinPrep®PreservCyt® for vaginal swabs.https://doi.org/10.5281/zenodo.6077699Zenodo: Human papillomavirus (HPV) detection in vaginal self-samples: evaluation of eNatThis project contains the following files:- HPV positivity on cervical and.csv- hrHPV viral load.csv- Legend HPV positivity on cervical and vaginal self-samples with different HPV real-time assay.docx- Legend hrHPV viral load.docxCreative Commons Attribution 4.0 International license (CC-BY 4.0).Data are available under the terms of theConceptualization: Clementina Elvezia CocuzzaData curation: Chiara Giubbi, Marianna Martinelli, Clementina Elvezia Cocuzza, Ivan Vallini, Tarig Dafa'allaFormal analysis: Chiara GiubbiFunding acquisition: Clementina Elvezia Cocuzza, Paolo Romano, Winnie WuInvestigation: Chiara Giubbi, Federica Perdoni, Tarig Dafa'alla, Silvia PaganoniMethodology: Chiara Giubbi, Marianna Martinelli, Ivan Vallini, Silvia Paganoni, Santina Castriciano, Rosario MusumeciProject administration: Clementina Elvezia CocuzzaResources: Paolo Romano, Winnie Wu, Clementina CocuzzaSupervision: Clementina Elvezia CocuzzaVisualization: Chiara Giubbi, Marianna MartinelliWriting – original draft preparation: Chiara Giubbi, Marianna MartinelliWriting – review & editing: Clementina Elvezia Cocuzza, Rosario Musumeci, Tarig Dafa'alla, Winnie Wu, Santina Castriciano, Paolo Romano. The authors have addressed my minor comments satisfactorily and I have no more concerns.  I recommend that the manuscript be accepted for indexing without further revisions. Thank you for giving me the opportunity to review this manuscript.Is the study design appropriate and does the work have academic merit?YesIs the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?PartlyAre all the source data underlying the results available to ensure full reproducibility?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:NAI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Thank you to the authors for their responses. I am satisfied with these responses to my questions. The only other item I would raise is more for their own interest. The data for M0244 and M0222T12 showed that the PapilloCheck and Seegene assay produced hPV68 results in 5/6 and 6/6 specimens, however, the GeneFirst OncoPredict assay returned a HPV39 positive result for 5/6 specimens. Considering how genetically close the HPV68 and HPV39 HPV types are I might suggest a discussion with the manufacturers of that assay identifying a potential source of off-target responses (HPV39 positive result) in the presence of HPV68 (it would require more follow-up to determine whether this observation was for HPV68a and/or HPV68b).Is the study design appropriate and does the work have academic merit?YesIs the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:NAI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. et al. have compared the detection of HPV in self-collected samples transported in PreservCyt® and in Copan eNat® medium. The results show no difference in sensitivity between the two transport media.  These are useful results, which demonstrate the utility of the aqueous eNat® medium for self-collected HPV specimens.GiubbiThe weakness of this study is the low number of women (n=30). The study does not have the power to detect small differences in performance that may matter when hundreds of thousands of women are tested. The authors should point this out in their discussion.The authors compare the HPV viral loads detected in various types of specimens and conclude that vaginal specimens have a lower viral load. However, there is no statistical analysis to support this conclusion and it is possible that the small differences observed fall within the expected experimental variance.   Minor comments:Is the study design appropriate and does the work have academic merit?YesIs the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?PartlyAre all the source data underlying the results available to ensure full reproducibility?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:Molecular diagnostics, HPV molecular epidemiology, Vaccine preventable diseasesI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. The authors would like to thank Prof. Alberto Severini for his review and comments. Giubbi et al. have compared the detection of HPV in self-collected samples transported in PreservCyt® and in Copan eNat® medium. The results show no difference in sensitivity between the two transport media.  These are useful results, which demonstrate the utility of the aqueous eNat® medium for self-collected HPV specimens. Minor comments:The weakness of this study is the low number of women (n=30). The study does not have the power to detect small differences in performance that may matter when hundreds of thousands of women are tested. The authors should point this out in their discussion.Reply: We agree with the reviewer that the main limitation of this study is the number of women enrolled. As reported in the text of the manuscript, this is intended to be a pilot study aiming to perform a preliminary evaluation of a possible alternative suspension medium for vaginal-self samples. This eNAT medium by Copan has as intended use the resuspension of clinical samples for other molecular tests, such as microbiome analysis, and studies on its use for this purpose have also been previously published. However future studies on a larger number of women will be necessary to better evaluate the performance of eNAT in vaginal sample resuspension for HPV testing in order to evaluate if any difference in high risk HPV detection is related to the suspension medium.  The authors compare the HPV viral loads detected in various types of specimens and conclude that vaginal specimens have a lower viral load. However, there is no statistical analysis to support this conclusion and it is possible that the small differences observed fall within the expected experimental variance.Reply: Due to the restricted number of samples analysed no statistical analysis was performed. However, vaginal self-samples suspended in both suspension media demonstrated to have a higher viral load than cervical specimens. As reported in the text, this finding may be partially related to the different suspension volume (5 ml vs 20 ml) used for the two different sample types, but this finding needs to be confirmed by a larger study which would also allow to determine whether the difference in viral load between cervical and vaginal samples is statistically significant. et al is interesting and makes a good step forward for the development of a consistent protocol for self-collection for the purpose of PV-based cervical screening. There are a few minor comments for consideration:There may be differences in the samples stored at -20C depending on whether they were in Hologic PreservCyt or Copan eNAT. PreservCyt is alcohol based and generally does not freeze at -20C whereas it would be likely that eNAT would freeze. A freeze thaw cycle is likely to only have been experienced by the samples in eNat which may affect the sample, a freeze thaw cycle may break down DNA or alternatively it may lyse cellular material freeing more DNA . The authors need to address this issue.The authors should include a table showing the HPV results per sample (e.g. simply whether the same was positive for HPV or negative for each assay). These data are referred to in the results section and in Table 1. These data would show whether there were any trends in samples . This would also identify if there were any trends with comparison between the three specimen types.The authors need to address that there appears to be a trend (unclear whether it is significant) for lower sensitivity across all sample types for the GeneFirst assay. The Seegene assay has been clinically validated and could be utilised as a comparator assay for the purpose of determining sensitivity and specificity for the two emerging assays. The study but GiubbiIs the study design appropriate and does the work have academic merit?YesIs the work clearly and accurately presented and does it cite the current literature?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesAre the conclusions drawn adequately supported by the results?YesAre sufficient details of methods and analysis provided to allow replication by others?YesReviewer Expertise:HPV diagnosticsI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. There may be differences in the samples stored at -20C depending on whether they were in Hologic PreservCyt or Copan eNAT. PreservCyt is alcohol based and generally does not freeze at -20C whereas it would be likely that eNAT would freeze. A freeze thaw cycle is likely to only have been experienced by the samples in eNat which may affect the sample, a freeze thaw cycle may break down DNA or alternatively it may lyse cellular material freeing more DNA. The authors need to address this issue.Reply: We thank Prof. David Hawkes for this consideration related to the preanalytical process. As indicated in the “Method” section, in the paragraph on “Pre-analytical sample processing”, it was stated that processing of cervical samples was performed as follows: “In the laboratory all specimens were vortexed for 30 seconds, and subsequently 1.5 ml aliquots were dispensed in cryovials; one was used for DNA extraction and the others stored at -20°C.” Similarly, vaginal samples were processed as follows: “Five aliquots of 1 ml were made from each of the vaginal specimens; one was used for nucleic acids extraction and the others stored at -20°C.” All samples were therefore analyzed fresh and nucleic acid extraction performed on a sample aliquot prior to freezing and therefore results were not affected by a freeze thaw cycle.The authors should include a table showing the HPV results per sample (e.g. simply whether the same was positive for HPV or negative for each assay). These data are referred to in the results section and in Table 1. These data would show whether there were any trends in samples . This would also identify if there were any trends with comparison between the three specimen types.Reply: File csv reporting all results with all investigated assays for each study sample is available at the link:https://doi.org/10.5281/zenodo.6077699. In particular, you will find the results of the 3 different assays for all samples in the file entitled “HPV positivity on cervical and vaginal samples.cvs and relative file including “Legend of HPV positivity on cervical and vaginal samples.docx”. The Open Research Europe journal requires all the data to be open access but limits the number of tables/figures.However in reply to Prof. David Hawkes request we include an additional summary table reporting all the HPV results obtained from the analyzed samples. This table can be seenhere.The authors need to address that there appears to be a trend (unclear whether it is significant) for lower sensitivity across all sample types for the GeneFirst assay. The Seegene assay has been clinically validated and could be utilized as a comparator assay for the purpose of determining sensitivity and specificity for the two emerging assays.Reply: We agree with Prof. David Hawkes comment regarding the lower HPV positivity reported from GeneFirst test compared to the other two. We have reported these results in the manuscript. As you can see in the above table the discordances of HPV positivity in cervical sample are present in two samples (MO180 T18 and MO222 T12). Both samples were collected from women with a cytology result of low-grade cervical dysplasia who did not require a biopsy to be performed at colposcopy based on the clinical findings. Regarding vaginal self-samples, the discrepancy in the HPV detection were shown in only one sample (MO149 T24). In this case the associated cytology was negative for the presence of any cervical alterations.The authors would like to thank Prof. David Hawkes for his review and comments. Below we have included the response to his comments for consideration:


To explore the correlation between Heart Rate Variability Index (HRV) and poor prognosis in patients with acute decompensated heart failure (ADHF).n = 31) and the good prognosis group (n = 97). Comparative analysis of clinical data and Heart Rate Variability (HRV) parameters was executed between these two groups. Moreover, a multiple linear regression analysis was employed to identify the contributing factors associated with adverse prognoses in ADHF patients. Furthermore, the receiver operating characteristic (ROC) curve was employed to evaluate the prognostic predictive capability of HRV parameters among ADHF patients.A retrospective compilation of clinical data encompassed 128 cases of patients afflicted with acute decompensated heart failure (ADHF) who were admitted to and discharged from our hospital between April 2019 and July 2022. Subsequent to assessing their follow-up progress during the tracking period, the subjects were categorized into two cohorts: the poor prognosis group (P < 0.001), SDANN  and LF  in the poor prognosis group were significantly higher than those in the good prognosis group, and the differences were statistically significant (P < 0.05). The levels of PNN50 , HF  and LF/HF  in the poor prognosis group were significantly lower than those in the good prognosis group (P < 0.05). The results of multiple linear regression analysis indicated that SDNN, SDANN, LF, PNN50, and HF were factors influencing poor prognosis in ADHF patients (P < 0.05). The results of the ROC curve analysis indicate that the area under the curve (AUC) for predicting poor prognosis in ADHF patients using HRV parameters were as follows: SDNN , SDANN , PNN50 , LF/HF , and combined diagnosis . Among these, the combined diagnosis exhibited the highest AUC, sensitivity, and specificity for predicting poor prognosis in ADHF patients (P < 0.001).The levels of SDNN (t = 3.924, The HRV parameters of SDNN, SDANN, PNN50 and LF/HF are closely related to the prognosis of ADHF patients. The combined detection of the above HRV parameters can improve the efficacy of predicting the poor prognosis of ADHF patients. This suggests that clinical staff can identify ADHF patients at risk of poor prognosis by long-term monitoring of HRV in the future. Acute decompensated heart failure (ADHF) pertains to a clinical syndrome marked by rapid and severe cardiac functional impairment, manifesting within a brief timeframe. This condition results in an insufficient cardiac output to adequately sustain the body’s metabolic demands, ultimately causing inadequate systemic tissue and organ perfusion. Clinical manifestations of ADHF often include symptoms such as dyspnea, palpitations, chest tightness, edema, fatigue, and cough, with severe cases potentially leading to shock and posing a critical threat to patient’s life . With th(1) Inclusion criteria: Confirmed diagnosis of ADHF; age >18 years; acceptance of follow-up and completeness of case information.(2) Exclusion criteria: Severe non-cardiac complications such as malignant tumors; concurrent systemic infections or immune dysfunction; loss to follow-up or incomplete follow-up results.A retrospective analysis was performed on the clinical records of 128 patients diagnosed with ADHF, who were admitted to our medical institution and subsequently discharged alive during the period spanning from April 2019 to July 2022. All samples obtained in this study were approved by the ethics committee of the West Distrivt of Qingdao Municipal Hospital Group and abided by the ethical guidelines of the Declaration of Helsinki, and ethics committee agreed to waive informed consent.n = 31) and a good prognosis group (n = 97).All patients underwent outpatient follow-up visits and telephone follow-up for 12 months after discharge. Patients experiencing adverse events during the follow-up period  were terminated from the follow-up. Based on the follow-up outcomes, the study population was divided into a poor prognosis group  laboratory indicators: laboratory indicators such as total cholesterol, triglycerides, and urea nitrogen at the time of patient discharge were collected; (3) echocardiography: Echocardiographic results including left ventricular end-diastolic diameter (LVEDd), left ventricular posterior wall thickness (LVPWT), left ventricular ejection fraction (LVEF), etc., at the time of patient discharge were collected.Patient clinical data were collected, including (1) general information: age, gender, body mass index (BMI), smoking history, alcohol consumption history, history of hypertension, history of diabetes, medication usage, length of hospital stay, HRV data were collected before patient discharge using the Seer Light Holter monitoring system from GE Healthcare  for a 24-h three-lead dynamic electrocardiographic examination. This involved applying seven electrodes to the patient’s chest for continuous 24-h monitoring. Data collected from Holter monitoring were then statistically analyzed by our institution’s dynamic electrocardiography department professionals using the MARS ECG analysis system from GE Healthcare. The analysis included filtering ectopic beats and interference, excluding intervals exceeding 20% of adjacent RR intervals automatically, and considering only effective signals and sinus rhythm intervals exceeding 22 h.Time-domain analysis (TTDA) was utilized for HRV analysis. The criteria for time-domain assessment included: (1) SDNN: Standard deviation of all normal sinus NN intervals; (2) SDANN: Dividing the entire recording into consecutive 5-minute segments, calculating the average NN interval for each 5-minute segment, then calculating the standard deviation of the average for the entire dataset; (3) PNN50: The number of heartbeats with NN interval differences greater than 50 ms divided by the total number of NN intervals, multiplied by 100. Frequency-domain indices included high-frequency power (HF) and low-frequency power (LF).t-tests. Categorical data were represented as frequencies or percentages, and inter-group comparisons were carried out using chi-square tests. Multiple linear regression analysis was employed to identify the factors exerting influence on the poor prognosis in patients with ADHF. Receiver operating characteristic (ROC) curves were employed to evaluate the prognostic potential of HRV parameters in ADHF patients. A significance threshold of P < 0.05 was considered indicative of statistical significance.The data were subjected to analysis using SPSS version 23.0 . Continuous variables that exhibited a normal distribution were presented as mean values along with their corresponding standard deviations, and inter-group comparisons were conducted through P > 0.05), as shown in Comparison of general characteristics such as age and gender between the poor prognosis group and the good prognosis group revealed no statistically significant differences (P > 0.05), as indicated in Comparison of general characteristics including age and gender between the group with poor prognosis and the group with good prognosis did not show statistically significant distinctions (P > 0.05). Moreover, echocardiographic indices encompassing left ventricular end-diastolic diameter (LVEDd), left ventricular posterior wall thickness (LVPWT), and left ventricular ejection fraction (LVEF) also showed no statistically significant differences (P > 0.05) between the two groups, as depicted in Comparison of laboratory parameters such as total cholesterol and triglycerides between the poor prognosis group and the good prognosis group exhibited no statistically significant disparities (P < 0.05). Conversely, the poor prognosis group demonstrated significantly lower levels of PNN50, HF, and LF/HF compared to the good prognosis group, also with statistically significant differences (P < 0.05). Please refer to The poor prognosis group exhibited significantly higher levels of SDNN, SDANN, and LF compared to the good prognosis group, with statistically significant differences (P < 0.05). Please refer to A multivariate logistic regression analysis was conducted with poor prognosis in ADHF patients as the dependent variable and significant single-factor analysis variables as independent variables. The results indicated that SDNN, SDANN, PNN50, and LF/HF significantly influenced poor prognosis in ADHF patients (P < 0.001), as shown in The outcomes of the ROC curve analysis revealed the following values for the areas under the curve (AUC) in predicting the poor prognosis of ADHF patients: SDNN, SDANN, PNN50, LF/HF, and combined diagnosis had AUC values of 0.818 (95% CI [0.722–0.914]), 0.684 (95% CI [0.551–0.816]), 0.754 (95% CI [0.611–0.841]), 0.787 (95% CI [0.679–0.896]), and 0.901 (95% CI [0.832–0.970]), respectively. The AUC, sensitivity and specificity of combined diagnosis in predicting the prognosis of ADHF patients were the highest  reflects the regulation of the cardiac sinus node by the autonomic nervous system, indicating differences and fluctuations between successive heartbeats. It correlates with the activity of the autonomic nervous system and indicates the balance between sympathetic and parasympathetic nervous system activity. HRV can assess cardiovascular disease status and development and serves as an important indicator for predicting arrhythmias and sudden cardiac death . StudiesCardiac autonomic nerve regulation refers to the regulation of cardiac function through sympathetic and parasympathetic nerves. The autonomic nervous system is an autonomously controlled nervous system, including sympathetic and parasympathetic nerves. Cardiac autonomic nerve regulation maintains the function and stability of the heart through the balance between excitation and inhibition of sympathetic and parasympathetic nerves . NumerouP < 0.05), the results are similar to those of Through single factor comparison, it was found that there was no significant difference in age, sex and other clinical data between the poor prognosis group and the good prognosis group, indicating that the above clinical data were not the factors affecting the prognosis of ADHF patients in this study. SDNN, SDANN, an PNN50 are all time-domain HRV parameters. Research by The HRV parameters of SDNN, SDANN, PNN50 and LF/HF are closely related to the prognosis of ADHF patients. The combined detection of the above HRV parameters can improve the efficacy of predicting the poor prognosis of ADHF patients. This suggests that clinical healthcare professionals can determine ADHF patients at risk of poor prognosis by monitoring HRV over the long term. In the future, HRV parameters can be used to analyze the specific mechanism of poor prognosis in ADHF patients.10.7717/peerj.16377/supp-1Supplemental Information 1Click here for additional data file.


To assess risk factors for HPV infection, determine knowledge about HPV vaccines, assess willingness to receive the HPV vaccine among adolescent and early adult girls in Nigeria, we administered a structured questionnaire. We also collected samples to determine the prevalence and patterns of HPV infections.The dataset contains the responses of 205 participants from 10 randomly selected public and private secondary schools in Jos, Nigeria. The data includes information on risk factors for HPV infections such as sexual behaviours, knowledge about HPV vaccine and willingness to receive the vaccine. This is valuable information that can be compared to data from studies in other environments or to determine changes in the pattern of risk factors and HPV prevalence in this population over time. Persistent high risk Human papillomavirus (HPV) infection is a necessary cause of cervical cancer . HPV infWe collected data from 205 adolescent and early adult girls. The girls were recruited through a pre-determined aged range of 9 to 20 years old, from 10 randomly selected schools. Details of the enrolment process in the study has been published elsewhere [25.We also collected data on knowledge related to HPV infections and HPV vaccine, and willingness to receive the vaccine. Instead of socio-economic status, we used principal component analysis (PCA) to compute wealth index using data on ownership of household items and availability of household facilities such as type of house they live in, type of toilet facilities, main source of cooking fuel etc. . The queThis dataset will contribute immensely to comparisons of HPV infection among other population in Nigeria and other countries. Researchers involved in data mining and analysis of secondary data will also find the dataset useful.(data file 1 on Table 1) [The dataset Table 1)  containsTable 1) . The girTable 1) .Only 9.3% (19/186) of the girls reported history of penetrative vaginal sexual intercourse. Most of the girls  who reported sexual intercourse first experienced it at 15 years of age or less and 15.8% (3/19) experienced it before the age of 10 years. Vaginal douching practices were reported by 16.6% of the girls and most of the girls who practice douching gave ‘It is part of taking a bath’ as the main reason why they engaged in the practice. Most of the girls (89.0%) took their bath at least twice daily when menstruating and 61.4% regularly bathe twice daily. Participants also shared personal clothes (24.9%) and pants (5.4%). Some girls reported history of masturbation, use of toys for masturbation, use of condoms and knowledge about ways to prevent pregnancy.Of the 205 participants, only 5 (2.4%) had heard about HPV vaccine and only 2 (1.0%) had received the vaccine. Some 32.4% (66/205) girls had no interest in receiving HPV vaccination and the reason for their unwillingness was cost of the vaccine. Almost all the girls  would receive the vaccine if it is free or subsidized.The prevalence of any HPV infections among the participants was 13.2% (27/205) and 15 different HPV types were detected with HPV type 52 being the commonest. Participants had single or multiple HPV infections, with 5 HPV types being the highest number of types of HPV found in multiple infections.Most participants reported that they had never had any sexual contact. Discomfort about reporting sexual history among participants in this study of young and early adult girls may cause information bias . Our smaThe findings of this study have provided timely evidence-based information that policy makers can harness in the development of effective guidelines for HPV prevention, not only among adolescent and young adults, but targeting the whole population. Future research should target a varied group of teenagers and young adults for prevalence surveys and educational interventions to encourage safe sexual behaviours that reduce HPV infection. For a greater reach, such studies should include both in-school and out-of-school participants, as well as involve important stakeholders such as parents, youth groups, teachers, community leaders, and social media campaigns.


Notably, the tumor microenvironment (TME) of the HPV+ subtype has an immune landscape characterized with increased immune infiltration, higher levels of T cell activation, and higher levels of immunoregulatory stimuli compared to their HPV− counterparts. Both enveloped and nonenveloped viruses hijack the extracellular vesicle (EV) biogenesis pathway to deploy a “trojan horse” strategy with a pseudoviral envelope to enhance infectivity and evade inflammation. EVs derived from HPV-infected tumor cells could allow for the stealth transport of viral cargo to neighboring nonmalignant cellular populations or infiltrating immune cells within the TME. Furthermore, viral cargo or altered cellular cargo from HPV-associated tumor EVs (HPV-TEVs) could alter the functional state or biological responses of the recipient cellular populations, which could shape the distinctive HPV+ TME. This review will cover the impact of EVs released from HPV-infected cells on HPV-induced carcinogenesis, their role in shaping the distinctive HPV+ tumor microenvironment, and current efforts to develop a painless EV-based liquid biopsy for HPV+ cancers.Most infections with human papillomaviruses (HPVs) are self-resolving and asymptomatic. However, some infections can lead to the development of cancer at different mucosal sites, such as the cervix and the head and neck. Head and neck cancers (HNCs) are dichotomized into HPV-positive (HPV To date, there have been over 400 human papillomavirus (HPV) types identified, all with exclusive tropism for either cutaneous or mucosal epithelia . Twelve +) or HPV-negative (HPV−) [+ subtype has an immune landscape characterized with increased immune infiltration, higher levels of T cell activation, and higher levels of immunoregulatory stimuli compared to their HPV− counterparts [+ cervical cancers (CCs) [− tumor control.The number of new cases of head and neck cancers (HNCs) caused by HR HPVs are on the rise with the concomitant decline in new cases caused by non-viral factors such as smoking and excessive alcohol consumption ,7. HNCs e (HPV−) ,8,9. A nterparts ,14,15,16rs (CCs) ,18; howeAll cells, regardless of their respective domains, release extracellular vesicles (EVs) during normal physiological functions and in diseased states . The molE6 and E7 oncogenes within HPV-infected tumor cells [+ TME and lack of tumor eradication. There is evidence that HPV viral oncogenes E6 and E7 are found in EVs regardless of the integration status of the viral genome within the infected cell [The distinct immunological phenomena observed in HPV-induced TMEs could be due to the continuous expression of the or cells ,30,31,32ted cell . Furtherted cell ,34,35,36ted cell . Indeed,ted cell ,40,41,42+ TME. These potential HPV-driven biological modifications mediated by HPV-TEVs could influence tumor survival, invasiveness, metastatic dissemination, or patient outcomes and therapeutic responses. Unraveling the role of HPV-TEVs in the TME has the potential to help guide the rational design of therapies that can modulate and/or mobilize the local immune ecosystem towards an antitumor phenotype. In addition, the in-depth characterization of the multicomponent cargo of HPV-TEVs could be used in the development of EV-based liquid biopsies that are painless and could provide “real-time” insights into disease burden.EVs derived from HPV-infected tumor cells could allow for the stealth transport of viral cargo to neighboring nonmalignant cellular populations or infiltrating immune cells within the TME. Furthermore, viral cargo or altered cellular cargo from HPV-associated tumor EVs (HPV-TEVs) could alter the functional state or biological responses of the recipient cellular populations, which could contribute to the virally induced, immune-inflamed, HPV+ tumor microenvironment. In addition, we will discuss current efforts to develop an EV-based liquid biopsy for HPV+ cancers for diagnostic and/or prognostic purposes.In this review, we will provide a brief primer on the biology of EVs, examine the current evidence of EVs released from nonmalignant and malignant HPV-infected cells, their impact on HPV-induced carcinogenesis, and their role in shaping the distinctive HPVEVs are defined as membrane vesicles secreted by cells into the extracellular space that contain cytosol and molecular components from the secreting cells enclosed in a lipid bilayer . This prIn the early 1980s, studies examining the fate of the transferrin glycoprotein in maturing reticulocytes—immature red blood cells—identified a pathway by which an intermediate multivesicular body (MVB) containing multiple intraluminal vesicles (ILVs) fuses with the cell’s plasma membrane, releasing the ILVs into the extracellular space ,45. ThesEV biogenesis, specifically those of exosomes, begins with the maturation of the early endosome into late endosomes/MVBs. Once MVBs have been formed, they are mobilized and directed to the plasma membrane or the lysosome, where they may fuse membranes and release their ILVs into the extracellular space or into the lysosome for degradation, respectively. Notably, soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) appear critical in mediating MVB fusion with the plasma membrane; however, very little is understood about the regulation of the balance between EV secretion or their targeting to the lysosome for degradation . The forMolecular mechanisms that govern MVB targeting and fusion to the plasma membrane remain unclear, as is the regulation of balance between secretory and degradation pathways. Not every MVB is fated for secretion, and it appears that molecular tagging of an MVB-limiting membrane-associated protein, TSG101, tips the balance towards degradation. Indeed, it has been shown that the ISGylation of the TSG101 protein by ISG15 controls EV secretion by promoting MVB targeting to the lysosome. Furthermore, ISGylation results in aggregation of the conjugated TSG101, which appears to act as a signal to propel MVBs towards fusion with lysosomes . In contOwing to their diverse content and ability to fuse with and release within other cells their shuttled molecular cargo, EVs are important mediators of the dynamic intercellular communication network. The molecular cargo  delivered by EVs to recipient cells can alter their functional state and/or biological responses . Due to + TME is an area of current investigation.There is increasing evidence that EVs released by malignant cells are involved in intercellular communication and can induce functional alterations in recipient cellular populations within the TME and at distant sites ,52,53. T− tumors in the head and neck region, with protein profiles that resembled their respective parental tumors [E6/E7, p16, and survivin, whereas both types of EVs contained immunomodulatory molecules—OX40, OX40L, HSP70, FasL, and TGF-β. Notably, in comparison to EVs released by HPV− tumors, HPV-TEVs promoted dendritic cell (DC) maturation and sustained DC function, insinuating a potential role in antitumor immunity [+ tumors correlated with an increased abundance of regulatory T cells (Tregs) and a decreased abundance of CD4+ T cells, cancer-associated fibroblasts (CAFs), and myeloid-derived suppressor cells (MDSCs). These findings need further validation to comprehensively understand the differences in the immune landscape of viral- and carcinogen-driven HNCs; however, they provide insight into distinct EV-based effects that could contribute to the disparity in the TMEs between the two HNC subtypes.In several studies, HPV-TEVs were shown to have different protein-based cargo compared to those from HPVl tumors ,34,35,36immunity . A recen− HNC cell lines [+ and HPV− cell lines have different protein cargos, which may explain the differences in the immune landscape of these anatomically similar tumors and the observed disparity in their responses to treatment. Indeed, patients with HPV+ HNCs are more responsive to radiotherapy compared to their HPV− counterparts [+ HNCs is due to the presence of immune cells within the TME [+ HNCs were able to polarize macrophages into the antitumor M1 phenotype [HPV-TEVs isolated from in vitro HNC cell lines have been shown to be enriched with immune effector-related antigens, such as CD47 and CD276, that are not present in TEVs from HPVll lines . This isterparts ,72,73. T the TME . In a sthenotype . Further+ HNC cells were enriched with the miRNA miR-9-5p—the miRNA most often associated with HPV [+ HNCs and that a high level of miR-9-5p is correlated with a higher rate of patient survival [A recent study by Wang et al., utilizing 111 clinical samples, showed that EVs secreted from HPVwith HPV . Furtherwith HPV ,77. In asurvival .+ cervical cancer cell lines contained proangiogenic factors and had higher angiogenic potential compared to TEVs from HPV− cervical carcinomas [− counterparts [In the cervix, the TME has been shown to be a major factor in the progression of cervical cancer (CC) . An imporcinomas . Furtherrcinomas . In additerparts .+ HeLa cells were found to have a distinct miRNA signature upon silencing of the integrated HPV oncogenes E6 and E7 [E6 and E7 from HPV16 [E6 and/or E7 can modify the expression of certain miRNAs that are typically involved in tumorigenesis, such as miR-18a, miR-19a, miR-34a, and miR-590-5. Moreover, HPV-TEVs were found to contain E6 and E7 mRNAs as cargo. In addition, another study detected the presence of HPV16 E7 and other proteins, MUC16 and SIRPA, in circulating HPV-TEVs isolated from patients with aggressive oropharyngeal cancers [+ cancer cells and potentiate the effects of the tumor virus on the surrounding environment.HPV-TEVs secreted from HPV186 and E7 . The ideom HPV16 . They fo cancers . Treatme− and HPV+ cell lines on PC12 cells—rat pheochromocytoma cells extensively used in neurobiology that exhibit features of mature dopaminergic neurons [+ or HPV− cell lines induced neurite outgrowth in vitro. However, TEVs isolated from HPV− cell lines had significantly less activity compared to their HPV+ counterparts. Nevertheless, their results suggested that HPV status is not required for neurite outgrowth activity. Seeing as HPV-TEVs isolated from HPV+ cell lines had higher neurite outgrowth activity compared to their HPV− counterparts, the authors extended their studies to CC—virtually all HPV+ [An added layer of complexity to the TME is the presence of innervated tumors in some cancer types. Notably, innervated tumors have been found to be more aggressive than those that are less innervated ,87,88,89 neurons . Their rall HPV+ . In this+ HNCs [+ HNC: aldolase (ALDOA), glyceraldehye-3-phosphate dehydrogenase (GAPDH), lactate dehydrogenase A/B (LDHA and LDHB), phosphoglycerate kinase 1 (PGK1), and pyruvate kinase M1/2 (PKM). In a recent study, our group has also identified alterations in various metabolic pathways that are associated with HPV status in HNCs [+ HNCs from healthy controls [The utilization of EVs as liquid-based biomarkers of disease burden is being advanced for various cancers that include those associated with HPV infection ,93. Thei+ HNCs . Notably in HNCs . This hicontrols . + CC cell lines SiHa and HeLa was analyzed and compared to an HPV− cell line C33a [− counterparts. Notably, among the top enriched transcripts were EVC2, LUZP1, and ANKS1B, which have been implicated in the hedgehog signaling pathway, cell migration, and invasion, respectively. Furthermore, Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that the enriched transcripts found in HPV-TEVs played a role in axon guidance and tumor innervation. In addition, the HPV18 splice variant E6*I was consistently found in HPV-TEVs isolated from HeLa cells. This study suggests that the differential cargo present in HPV-TEVs, such as protumorigenic cellular RNA and viral transcripts, may have utility as clinical biomarkers of CC. These studies are prime examples of the differential multicomponent cargo present in EVs that are reflective of their COO, which allows, in this case, the ability to discriminate HPV+ carcinomas from healthy controls.In order to further elucidate the role of HPV-TEVs and their cargo in CC, RNA isolated from HPV-TEVs produced by HPVine C33a . The res+ hypopharynx cells (SCC2), HPV+ base-of-tongue cells (SCC90), and HPV− tonsil cells (SCC72 and SCC89) [+ cell lines and 19 miRNAs specifically enriched in TEVs isolated from HPV− cell lines. The enrichment of these miRNAs was specific to the HPV status of the COO and may provide utility as an EV-based biomarker for early detection and risk stratification in HNCs.Currently, there are no biomarkers available for the early detection of oral cancers, and this often leads to their detection at an advanced stage that is associated with poor survival rates. When oral cancers are detected at an early stage, the 5-year survival rate increases from 40% to 80% ,97. Therd SCC89) . Their rRecent evidence has shown that tumor cells actively release TEVs containing long-noncoding RNAs (lncRNAs) and that these molecules are differentially expressed in different cancers and have potential utility as diagnostic biomarkers ,100. In + OSCC had a demonstrated reduction in the levels of circulating HPV-DNA following treatment, with 81% having levels that were undetectable. In contrast, patients with metastatic HPV+ OSCC had no correlation between HPV-DNA levels and treatment response. This study demonstrates that both sources of liquid biopsies can be used for the detection of HPV-DNA; however, it seems that cf-DNA/RNA is a more sensitive medium compared to EV-DNA/RNA. In addition, since HPV-DNA was only correlated to treatment response in the locally advanced cohort, the utility of HPV-DNA as a dynamic biomarker to assess treatment response requires further validation.There are two main sources of DNA and/or RNA (DNA/RNA) that can be used for liquid biopsies. The first is cell-free (cf) DNA/RNA and the second is EV-associated DNA/RNA ,103. In The cervical–vaginal fluid (CVF) has the potential to be used as a noninvasive medium for CC screening. This is due to the fact that EVs are broadly present in various body fluids and are representative of their COO. Several studies have compared the use of conventional cervical cytology with liquid-based cytology (LBC); some have found that conventional cervical cytology is more sensitive and specific as a diagnostic test compared to LBC ,106, wheIn a recent study, EVs isolated from CVF from volunteers with or without an active HPV16 infection were used to explore the potential effects of miRNAs associated with HPV status on the development of CC . Their r+ OSCC patients from normal controls and those with gastroesophageal reflux disease (GERD), an inflammatory disease [+ OSCC. Their results produced a regression model that contained 11 miRNA ratios that could be useful in the clinic. Furthermore, sample permutations indicated that the cross-validated prediction accuracy of the 11-miRNA ratio signature was not due to chance alone. This work clearly identified a panel of miRNAs isolated from EVs present in blood serum that was robustly cross-validated as a biomarker for detection of HPV+ OSCC. Some HNCs, such as those of the oropharynx, are often late-stage diagnoses, which are associated with an increased mortality rate . The rea disease . They is+ or HPV− cell lines were distinct from one and other and resembled their COO. Indeed, paired cell lysates and EVs from HPV+ cells were enriched with EGFR, TP53, and HSPA1A/B mRNA, whereas those from HPV− cells were enriched with IL6, FAS, and DPP4 mRNA. Furthermore, they observed that miR-205-5p was exclusively present in HPV-TEVs, whereas miR-1972 was only detected in EVs from HPV− cells. In addition, a study by Galiveti et al. aimed to evaluate the potential of miRNAs associated with HPV-TEVs from the head and neck region and determine their potential as a liquid biopsy for early detection of HNC [In another study , the mRNn of HNC . Their r+ TME. Research initiatives that focus on viral cargo or altered cellular cargo enriched in HPV-TEVs that alter the biological responses of diverse cellular populations in the TME have the potential to help guide the rational design of therapies that can modulate and/or mobilize the local immune ecosystem towards an antitumor phenotype. In addition, the in-depth characterization of the multicomponent cargo of HPV-TEVs could be used in the development of EV-based liquid biopsies that are painless and provide “real-time” insights into disease burden.The elucidation of the impact that HPV-TEVs have on infiltrating immune cells and/or nonmalignant cellular populations could help in unraveling the origins of the virally induced, immune-inflamed, HPVHistorically, DNA tumor viruses have been utilized as tools for studies that contributed to our understanding of molecular and cancer biology of mammalian cells ,117. Lik


Squamous papillomas (SPs) of the head and neck are usually benign lesions associated with human papilloma virus (HPV) infection. However, the reported HPV detection rates vary widely, especially with respect to anatomical distribution. The etiology of SPs in the head and neck remains unclear; analyzing HPV genotypes of SPs based on anatomical sites could assist in clarifying the pathogenesis of SPs in the head and neck. Therefore, the aim of this study was to review the prevalence, subtypes, and anatomical distribution of HPV in head and neck SPs at a hospital in China; we also investigated whether p16, a marker of HPV infection in oropharyngeal carcinoma, could serve as a surrogate marker for HPV in head and neck SPs. The presence of HPV DNA of 23 types (5 low-risk HPV and 18 high-risk HPV types) was detected via real-time PCR. p16 immunohistochemistry was performed using SP sections. Age, sex, anatomical location, and HPV subtype were recorded for each case. In total, 105 SPs were identified, including 47 in the larynx, 42 in the pharynx, 6 in the external auditory canal (EAC), 5 in the oral cavity, and 5 in the nasal cavity. HPV was found in 57 (54.3%) cases, with the highest positivity rate in the larynx . Only 5/42 (11.9%) patients showed HPV positivity in the pharynx. HPV incidence was highly dependent on the anatomical site. SPs in the larynx and EAC were more likely to carry HPV than those in other anatomical sites. High-risk HPV infections were rarely associated with SPs in the head and neck region. The sensitivity and specificity of p16 immunohistochemistry for HPV infection were 88% and 96%, respectively. There may be an association between p16 and HPV infection in head and neck SPs, but further studies are needed to validate this assertion. Squamous papillomas (SPs) are histologically benign growths typically encountered from the nasal vestibule to the respiratory tract in patients visiting an otolaryngology department. SPs are precancerous lesions that can occasionally cause airway obstruction . The etiHPV genotyping for papilloma is not routinely performed in otorhinolaryngology clinics. Although usually benign, SPs can clinically show widespread growth and recurrence, and some tend to be malignant, with a possibility of HPV involvement. Analyzing the HPV genotypes of SPs based on anatomical sites could contribute to our understanding of the pathogenesis of SPs in the head and neck. Therefore, in this study, we aimed to investigate the existence, subtypes, and anatomical distribution of HPV in head and neck SPs in a Chinese population; we also aimed to establish whether p16 can serve as a surrogate marker for HPV infection in head and neck SPs.The protocols for this study were approved by the Institutional Review Board of BenQ Hospital Affiliated with Nanjing Medical University (Decision No. 2020-KL013). We obtained written informed consent from all participants in the study and the consent of parents or guardians of minors before starting the study. All of the procedures were performed in accordance with the Declaration of Helsinki and relevant policies in China.Our study comprised 105 patients diagnosed with SPs, selected from a total of 8063 patients who underwent surgical treatment at the Department of Otorhinolaryngology Head and Neck Surgery of the affiliated BenQ Hospital of Nanjing Medical University from July 2018 to December 2022. All cases diagnosed with SPs in head and neck were identified through a search of the pathology files, and lesions mostly came from mucosal membranes and the external auditory canal (EAC) skin excluding surrounding skin warts. They were reviewed from December 2020 to December 2022. The histological diagnosis was confirmed by an experienced head and neck pathologist. Inclusion criteria comprehended: (a) diagnosis of SPs; (b) anatomic site was in head and neck; (c) surgical treatment; (d) histological and clinical data available in hospital database. Final pathological diagnoses of cancer were excluded.Medical records were retrospectively evaluated for age, sex, clinical presentation, otoscopic findings, previous treatments, surgical findings, histopathology, follow-up intervals, and recurrence. Cases of recurrence were defined as the reappearance of SPs at the identical anatomic location. This was evaluated by two seasoned pathologists. Authors had not access to information that could identify individual participants during or after data collection.Surgical tissue specimens were used for HPV DNA extraction. The surgical specimens were selected from 0.2 mm tumor-tissue blocks and placed in a special cell-preservation solution in a sampling tube marked with the patient’s barcode. All the samples were stored at 4°C and tested within 48 h. After DNA extraction, HPV DNA was hybridized using a Human Papillomavirus Genotyping Kit for 23 HPV types , including 17 high-risk types  and 6 low-risk types . HPV DNA extraction, PCR amplification, hybridization, and interpretation of results were performed according to the manufacturer’s instructions.Initially identified SPs were selected for p16 immunohistochemistry . SP blocp < 0.05 was considered statistically significant.All statistical analyses were performed using SPSS software . For continuous variables, the mean and standard deviation were used for statistical analyses. The chi-square test, t-test, and rank sum test were used for hypothesis testing, and In total, 105 SPs were included in this analysis; 47 papillomas were identified in the larynx as recurrent respiratory papillomatosis, 42 were identified in the pharynx, 6 were identified in the EAC, 5 were identified in the oral cavity, and 5 were identified in the nasal cavity in both adults and children. The prevalence of SPs is high among males, as 77 (73.3%) patients diagnosed with SPs were male. The mean age at the time of surgery was 35.1 years . The mean follow-up time after primary surgical resection was 22.9 months  .The tonsils were most commonly affected by oropharyngeal papillomas. Most patients had no symptoms, and papillomas were discovered inadvertently during laryngoscopy or gastroscopy; RRP was accompanied by clinical manifestations of hoarseness and dyspnea. The clinical presentation of EAC papilloma usually involved a mass in the EAC, bleeding, aural fullness, or hearing loss. The clinical presentation of nasal papilloma was usually a mass in the nose with bleeding. The anatomic site exhibiting the most frequent recurrence of SPs in head and neck was in the larynx .p < 0.001). Only laryngeal papillomas showed combinations of subtypes. Papillomas in the EAC were produced by infection with HPV6 (100%). HPV incidence was highly dependent on the anatomical site; HPV positive SPs mainly arising in the larynx than other anatomical sites. HR-HPV infections appeared to be rare, compared with LR-HPV infections of head and neck SPs  (HPV was found in 57 (54.3%) cases of SPs, frequently associated with the EAC and larynx regions , but not the pharynx (11.9%). The larynx group showed the highest rate of HPV11; the EAC and pharynx groups showed the lowest rates of HPV11, and the difference between the larynx and pharynx groups was statistically significant. The larynx group showed a significantly higher rate of HPV6, compared with that in the other groups. The larynx group exhibited the highest HPV positivity rate, whereas the pharynx group demonstrated the lowest rate. This disparity was statistically significant. (< 0.001) .A total of 46 cases  of laryngeal papillomas were positive for HPV, 4/47 (8.5%) showed multiple HPV types. Among the cases in which various types of HPV were detected, 25/47 (53.2%) were positive for HPV11 and 17/47 (36.2%) were positive for HPV6. The present study found no statistically significant difference in the HPV positive rate between two distinct age groups: juvenile RRP  and adult RRP  .SP samples containing HPV DNA also expressed p16. We stained 105 SP samples for p16 protein. In this sample set, 57 samples were HPV positive. Of these, 50 positively expressed p16 , and onlAlthough the association between HPV infection and the occurrence and progression of laryngeal papilloma and oropharyngeal carcinoma has been well documented, few studies have considered the variability of anatomical regions in the head and neck, and ambiguities continue to exist regarding HPV infection and the occurrence of SPs in the head and neck –10. HereThe results of associations between HPV and SPs in the literature are diverse, owing to different detection methods and the wide range of prevalence . The senSPs in the pharynx are accidentally diagnosed, as most SPs are asymptomatic  and largPapillomas involving the EAC are very rare, with only a few reports describing its involvement . We confVaccination against HPV can prevent HPV infection, but only two RRP patients were vaccinated in our cases with recurrences. Given the low coverage of HPV vaccines with the large population of this country, the limited health services in developing countries, high costs, and policy limits for the HPV vaccine , we did In summary, we evaluated HPV subtypes in SPs from different regions in a sample of 105 cases. The occurrence of SPs was found to be closely related to HPV infection, especially in the larynx and EAC. Identifying the HPV DNA type can assist in predicting the risk of malignant transformation and assist doctors in determining the prognosis and personalizing follow-ups. However, this study was limited by the small sample size and limited to a single center. Further validation in a larger cohort of samples, with more HPV genotypes, and the inclusion of behaviors associated with HPV infection  is warranted.The incidence of HPV has been found to vary significantly depending on the anatomical site, with the larynx exhibiting the highest incidence. HPV infections are associated with the occurrence of SPs in the larynx and EAC, while SPs in the head and neck region are rarely associated with HR-HPV infections. There may be an association between p16 and HPV infection in head and neck SPs, but further studies are needed to validate this assertion that p16 can serve as a supplementary marker of HPV infection in head and neck SPs.S1 Dataset(XLSX)Click here for additional data file.


Head and neck cancers represent a significant source of morbidity and mortality across the world. The individual genetic makeup of each tumor can help to determine the course of treatment and can help clinicians predict prognosis. Non-invasive tools to determine the genetic status of these tumors, particularly p16 (human papillomavirus (HPV)) status could prove extremely valuable to treating clinicians and surgeons. The field of radiomics is a burgeoning area of radiology practice that aims to provide quantitative biomarkers that can be derived from radiological images and could prove useful in determining p16 status non-invasively. In this review, we summarize the current evidence for the use of radiomics to determine the HPV status of head and neck tumors.  Head and neck cancers constitute a heterogeneous group of malignancies that have a combined worldwide incidence of approximately 600,000 per year and thus represent the sixth most common form of cancer ,2. MoreoOne of the most significant risk factors for head and neck cancers, in general, is infection with a high-risk subtype of human papillomavirus (HPV) including types 16, 18, 31, and 33.  The incidence of infection by such viruses has been increasing in certain areas of the Western world, and not coincidentally, the incidence of HPV-associated oropharyngeal cancer has also been increasing over the past decade . This unThis review will specifically focus on the determination of HPV positivity as a marker for head and neck malignancies, particularly in terms of staging and management. Other subtypes of head and neck cancer have their own unique risk factors and molecular markers, such as Ebstein Barr virus-positive in nasopharyngeal carcinoma , which aHPV-positive head and neck tumors have a better prognosis when compared to HPV-negative tumors . HPV-posDue to the litany of head and neck cancers with their own distinct risk factors, molecular characteristics, and prognoses, treatment and diagnoses of such malignancies must be tailored to the individual disease, as well as the characteristics of the patients themselves. However, what has become evident over decades of clinical practice and research is the need for better tools to obtain and analyze tumor-related data that could impact management decisions. In some cases, obtaining the necessary data to guide treatment decisions is not feasible for the patient, due to the location of the tumor, or the current health status of the patient. Often what is key to guiding such decisions is the obtaining of a tissue sample to obtain genetic and histologic information about the tumor itself, an area that the budding field of radio-genomics aims to address. In other cases, information about the potential for local recurrence or distant metastasis is not available. It is these specific knowledge gaps, among others, that the field of radiomics aims to address. This review will discuss the field of radiomics, specifically the subfield of radio-genomics, and the potential to use radiomic and radio-genomic analysis to determine the HPV positivity of head and neck tumors. Radiomics: the relevant basics A focused and narrative literature review was undertaken by sourcing articles from PubMed and Google Scholar, employing keywords such as "p16 head and neck cancer" and "radiomics." The search was refined by scrutinizing article abstracts, considering the relevance of each study to the central theme of this review: the potential of radiomics in detecting p16 status in head and neck squamous cell carcinoma. Studies utilizing tissue biopsy for p16 status identification were excluded from the scope of this review.Clinical oncology has long utilized medical imaging for more than just the detection of malignancies. Computed tomography (CT), magnetic resonance imaging (MRI), and positron emission tomography (PET) scans have become key tools for personalized cancer care including: (i) the staging of the tumor to guide management decisions , (ii) raThe radiomic approach begins with the acquisition of high-quality images through any number of imaging modalities, including CT, MRI, and PET scans . Next, tThe quantitative features that have been studied in the context of radiomic extraction can be broken up into two broad groups: (i) semantic and (ii) agnostic features . SemantiOften this feature extraction process yields a high number of features, many of which prove to be redundant. It is at this point that researchers will perform several statistical measures to determine which features will prove to be statistically relevant to predict the outcome of interest. Once these statistical procedures have been performed, the statistically relevant features are then compiled into a model that can be used to discriminate between the two groups of interest. Through this process, researchers can construct predictive models that can discriminate between groups regarding a clinically relevant question, including important clinical and molecular information, such as the specific mutational status of a tumor. In the context of head and neck cancer, the HPV mutational status of the tumor can provide significant information to the treating clinician in regard to decision making. Previously, this information could only be provided through immuno-histochemical staining of biopsy tissue, or other molecular methods that required a sample of the tumor such as Next Generation Sequencing or quantitative polymerase chain reaction (qPCR). A number of publications have examined the possibility of utilizing a radiomic approach to determine this information from radiological images. This next section will examine a select group of publications that have focused on this concept. HPV, radiomics in head and neck cancerMany sub-types of head and neck cancer have their own molecular signature, in addition to common mutations seen amongst all head and neck malignancies. The presence or absence of these mutations can be key to determining prognosis and management. General mutations featured by all head and neck cancers include mutations in tumor suppressor genes such as p53, Rb, and APC, proto-oncogenes such as Myc, oncogenes such as Ras, and many more . MutatioDetermining which clinically relevant mutations are present within the tumor is a key aspect of treatment, and often requires invasive tissue sampling. However, as mentioned previously, a significant theme in modern oncology focuses on the limitations of biopsy-based assays. In the event of a patient being able to have a potentially malignant tumor biopsied, the information determined by the pathologist by examining the tissue under the microscope, while potentially useful, cannot take into account the genetic or morphological information of the entire tumor. The need to obtain this information has necessitated the development of other strategies to determine the genetic information of the entire tumor in a noninvasive fashion. The question that remains: Can a radiomic approach determine clinically meaningful genetic information? Head and neck cancer is a key area of malignancy where such an approach could be extremely useful. As previously mentioned, infection with HPV and the subsequent development of a head and neck tumor can lead to a specific subtype of malignancy, with the association of the infecting virus having drastic implications for therapy and prognosis . HPV is In terms of prognosis, a p16-positive tumor has a different staging system when compared to a p16-negative tumor. Patients with HPV-positive disease thus have a better prognosis, a better response to specific therapies, and essentially represent a completely different clinical disease compared to HPV-negative disease . TreatmeAs such, being able to determine the p16 status of a tumor non-invasively proves immensely valuable. In a 2015 study, Buch et al. set out to determine whether a combination of histogram-based values and second-order statistics could help separate p16 positive and negative tumors on CT scans . A retroFurther publications indicate the utility of radiomics-based CT analysis in clinical practice in the ability to determine the p16 status of oropharyngeal cancer. Remarkably, some of these models show significantly better predictive value when compared to radiologists. Ranjbar et al. developed a texture-analysis-based model from CT scans of oropharyngeal cancer patients and compared this model to classification attempts at HPV status by two trained neuroradiologists . A totalYu et al. found a similar conclusion, having extracted 1,683 features from the CT scans of 315 oropharyngeal cancer patients, and determined a predictive model utilizing two features, mean distribution and spherical disproportion . Mean diWhile significantly more research will need to be performed to adequately conclude that these features reflect differences in homogeneity, these publications reflect a growing consensus that these findings are accurate. Leijenaar et al. also noted significant histopathological differences may be present to differentiate between HPV positive and negative tumors, with HPV-positive tumors displaying more lobular growth, infiltrating lymphocytes, and well-differentiated cells. They note that in order to correlate radiomic features with histopathological ones, a more in-depth study would need to be performed utilizing a surgical cohort. MRI radiomics and the determination of HPV status in head and neck cancers In addition to the aforementioned studies linking CT radiomic analysis and HPV status, another publication examined the possibility of using MRI-based radiomics to determine p16 status. Ravanelli et al. looked at the diffusion-weighted MRI scans of 59 patients with oropharyngeal cancer with the intent of determining the association between HPV status and various MRI-related textural analyses, as well as apparent diffusion coefficient (ADC) . While bSuh et al. looked at the MRI scans of 60 patients with oropharyngeal squamous cell carcinoma and extracted features to develop models to determine HPV-positive status . They exThese studies, when compiled, indicate a clear possibility to utilize radiomics-based analyses to determine the HPV status of head and neck cancers. This approach could prove vital to the clinician when trying to make treatment decisions for patients. Treatment and prognostic decisions can be extremely different depending on the p16 status of the tumor. Such approaches could thus be extremely valuable in the modern-day oncology world, revolutionizing the manner in which diagnoses and treatment decisions are made by clinicians. The prospect of determining the genetic makeup of a tumor through non-invasive imaging-based techniques represents an interesting, yet significantly understudied area of investigation. While significant research has been conducted into this area, there remain large knowledge gaps that will need to be addressed before radiomics-based genetic determination can be integrated into clinical head and neck oncology practice. The clinical relevance of HPV mutations has been discussed, with the possibility to guide future diagnoses and treatments in the world of oncology. While previously these mutations were detected with invasive procedures leading to a biopsy, the prospects of determining this information non-invasively are clearly enticing both from the perspective of the patient and the clinician. Some patients can simply not tolerate a biopsy, while others have tumors that simply cannot be reached with conventional surgical techniques. These strategies can also be extremely effective in terms of patient morbidity, sparing them the discomfort associated with a biopsy. This review discussed the radiomic and radio-genomic approaches associated with determining HPV mutation status in the context of head and neck cancer. Other potential mutations remain within the scope of future research, with the goal of being able to develop radiomic signatures to determine such mutations non-invasively now within grasp. With this being said, the field of radio-genomics appears to be an extremely promising one with a bright future ahead.


Starting from gDNA, we PCR amplified the V3–V4 region of the bacterial 16S rRNA gene and applied a paired-end NGS protocol (Illumina). We report significant differences in the abundance of certain bacteria compared among different HPV-types, more particularly concerning species assigned to Lacticaseibacillus, Megasphaera and Sneathia genera. Especially for Lacticaseibacillus, we observed significant depletion in the case of HPV16, HPV18 versus hrHPVother. Overall, our results suggest that the presence or absence of specific cervicovaginal microbial genera may be linked to the observed severity in hrHPV infection, particularly in the case of HPV16, 18 types.Increasing evidence supports a role for the vaginal microbiome (VM) in the severity of HPV infection and its potential link to cervical intraepithelial neoplasia. However, a lot remains unclear regarding the precise role of certain bacteria in the context of HPV positivity and persistence of infection. Here, using next generation sequencing (NGS), we comprehensively profiled the VM in a series of 877 women who tested positive for at least one high risk HPV (hrHPV) type with the COBAS Infection with the human papillomavirus (HPV) is one of the most common sexually transmitted infections (STIs) worldwide . AlthougLactobacillus spp., a genus known for its key role in the maintenance of the cervicovaginal eubiosis by producing bacteriocins, lactic acid and hydrogen peroxide, exerting a protective effect on the cervix/vagina from pathogen infections   . The heafections . Howeversphaera) . These bsphaera) .Studies have demonstrated that abnormal changes in the composition of the vaginal microbiome, which lead to reduced mucus production and consequent clearance of pathogenic bacteria, can be associated with the late clearance of HPV infection and progression to CIN . FurtherHere, we investigated the vaginal and cervical microbiota composition and its potential role in HPV infection, employing amplicon-based Next Generation Sequencing (NGS) of cervicovaginal specimens from a large series of HPV-infected women. Our aim was to characterize in detail the resident cervical and vaginal microbial flora under conditions of HPV infection and their potential link to CIN.This is a retrospective study analyzing samples from a cohort of 877 non-pregnant women who were diagnosed with high-risk (hr) HPV infection in the context of GRECOSELF, a nationwide observational cross-sectional study aiming to suggest a way to implement HPV-DNA testing in conjunction with self-sampling for cervical cancer screening in Greece .In brief, upon written informed consent, cervico-vaginal samples were self-collected with a vaginal dry swab (Roche Molecular Systems) and then stored and transported as previously described . HPV infWomen who tested positive for at least one hrHPV type were referred for colposcopy. Overall, we acquired colposcopic data from 636/877 (72.5%) women, whereas the remainder 241/877 (27.5%) for various reasons did not comply with their colposcopy referral upon their HPV test positive result. In cases with abnormal or suspicious colposcopic findings, multiple focal biopsies, and/or endocervical curettage (ECC), were taken from the abnormal area of the cervix. ECC was performed in case of a transformation zone type III.Histopathological examination was performed by expert histopathologists. Based on the grade of CIN, cases were classified in groups, namely (from low grade to high grade CIN) CIN1 < CIN2 < CIN3 or worse (CIN3+). Women with no evidence of CIN, were classified as ‘No detectable disease (NDD)’ in terms of cervical histopathologic findings.Samples analyzed for this study included 877 self-collected vaginal samples, each corresponding to a different woman, who tested positive for either HPV16, or HPV18 types or another high-risk type, namely “HPV other” . MoreoveAll procedures were in accordance with the standards set by the 1964 Declaration of Helsinki, and were also approved by the Ethics Committees of the Aristotle University of Thessaloniki and the Centre for Research and Technology Hellas. Next Generation Sequencing, as well as clinical data were stored and handled in accordance with the ISO/IEC 27001:2013 guidelines.Starting with 12.5 ng of total DNA as template, we PCR amplified the V3–V4 region of the bacterial 16S rRNA gene using primers with overhang adapters (forward primer with overhang adapter).5’-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3′; reverse primer with overhang adapter.5’-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3′.Both the amplicon PCR, as well as the index PCR reactions were carried out using the KAPA HiFi HotStart DNA polymerase 2X ReadyΜix  and SYTO9  to monitor the amplification in real-time. Regarding thermal cycling conditions, 16S amplification was initiated by denaturation at 98°C for 20s, followed by 30 cycles of denaturation at 98°C for 20s to annealing of the primers at 55°C for 30s, and 72°C for 30s with a final extension at 72°C for 5 min.NGS libraries were prepared by direct incorporation of unique index primer pairs  to 1 μL of the adapter tailed PCR amplicons. Thermal cycling conditions included denaturation at 98°C for 2 min, followed by 10 cycles of ramping from 98°C for 15 s to 64°C for 60 s. Paired-end NGS was performed with the MiSeq reagent v3 kit (2 × 300 bp) in a MiSeq Benchtop Sequencer  according to the manufacturer’s instructions.NGS raw data were analyzed using the QIIME 2020.8.0 software . In detaMeta-analysis of the QIIME 2 results was performed using R v4.2.0. BIOM files were processed using the {rbiom} package, while alpha diversity was calculated using the {vegan} package. Relative abundance analysis was performed with DESeq2 . All ploAssessment of statistical significance was performed with the packages incorporated in the aforementioned bioinformatics tools.In total, 38,328,108 raw reads  were obtained for the 937 sequenced PCR amplicons. After quality filtering, 30,353,253 non-chimeric reads  were further processed for taxonomic assignment and meta-data analysis .p = 0.4)  bacteria associated with bacterial vaginosis, such as Gardnerella, Prevotella, Sneathia, and Megasphaera. Altogether, the dominant genera in both samples accounted for an average 57.7% of the cervicovaginal microbiome, with each bacterial genus displaying similar relative frequencies in both the self- and the physician-collected samples  . In addi samples .Taxonomic assignment was performed with the RDP classifier, and vaginal microbial composition was compared in women infected with different hrHPV types, namely HPV16, HPV18, HPV other, as well as women who exhibited precancerous cervical lesions after abnormal colposcopic findings. For the latter comparison, groups were as defined above, i.e., CIN1, CIN2, CIN3, or, in the absence of any lesions, NDD (no detectable disease).Lactobacillus-dominated vaginal flora with a median relative abundance of 60%. Besides Lactobacillus, the majority of the vaginal microbiome in HPV-infected women consisted of CST-IV bacteria common in all groups irrespective of the HPV type responsible for the infection, such as Gardnerella (median frequency 11.5%), Prevotella (median frequency 3.8%), Sneathia and Megasphaera , amongst others.Overall, 10,788 bacterial operational taxonomic units (OTUs) were identified, of which 4,860 matched with 305 different and well-characterized genera. In all groups, we observed a p = 0.6), meaning that regardless the histopathology status, all women had a similar microbial diversity.Alpha diversity was calculated at the OTU level for the different groups with the Shannon Index. No significant differences were found in terms of microbial diversity for women infected with different HPV types. In fact, although CIN2 and CIN3 groups tended to be slightly more diverse than the CIN1 or NDD groups, such comparisons did not reach statistical significance. Indeed, the CIN2 and CIN3 groups displayed a higher Shannon index , howeverp < 0.0001), supporting that, even in the context of HPV, age at infection is the dominant determinant of vaginal diversity and richness  (p < 0.0001)  . SimilarThe increasingly evident role of the cervicovaginal microbiome in HPV infection persistence and cervical cancer pathogenesis alongside the diverse applications of high-throughput sequencing methodologies in metagenomics analyses, pose an exciting challenge for the use of NGS towards identifying microbial markers that will advance our understanding of HPV-associated cervical cancer ontogeny. Identifying specific bacterial species as microbial biomarkers of high-grade and lowgrade dysplasia could conceivably assist in triaging individuals with pronounced chances of lesion regression or progression . In thatIn this context, we analyzed the vaginal and cervical microbial composition using high-throughput, amplicon-based metagenomics and investigated its relationship with HPV infection, using self- and physician-collected samples obtained from a large cohort of HPV positive women.Lactobacillus being the predominant genus, followed by anaerobic bacteria, associated with bacterial vaginosis, such as Gardnerella, Prevotella, Sneathia, Megasphaera   , allowinsphaera) . Studiessphaera) .Lacticaseibacillus. In particular, Lacticaseibacillus was significantly depleted in the case of women infected by types HPV16, HPV18 compared to cases infected by other high-risk HPV type(s). This was particularly intriguing, especially in view of published evidence that a Lacticaseibacillus species, namely Lacticaseibacillus casei LH23 strains, inhibits the expression of the E6 and E7 viral proteins, which play a crucial role in viral replication during HPV infection  can be found at: The studies involving humans were approved by Ethics Committees of the Aristotle University of Thessaloniki and the Centre for Research and Technology Hellas. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.ES: Formal analysis, Methodology, Writing – original draft. GG: Formal analysis, Methodology, Writing – original draft. NP: Formal analysis, Visualization, Writing – original draft. KP: Methodology, Writing – review & editing. KC: Investigation, Supervision, Writing – review & editing. FP: Formal analysis, Writing – review & editing. TA: Investigation, Supervision, Writing – review & editing. KS: Investigation, Supervision, Writing – review & editing.