All careHPV‐positive women, plus a random sample of careHPV‐negative women, were recalled for colposcopy and biopsy. Self‐samples also underwent GP5+/6+ polymerase chain reaction (PCR)‐based HR‐HPV DNA detection and genotyping. Cross‐sectional screening indices were estimated against histological high‐grade squamous intraepithelial lesions or worse (hHSIL+), including imputation of hHSIL+ in women without colposcopy.In April/May 2016, 2590 women aged 30–60 years were screened across rural Bhutan by providing a self‐collected sample for careHPV and 14.8% by GP5+/6+ PCR. Twenty‐two cases of hHSIL+ were histologically diagnosed, including one invasive cancer; an additional 7 hHSIL+ were imputed in women without colposcopy. HR‐HPV testing by GP5+/6+ showed higher sensitivity for hHSIL+  than careHPV . Negative predictive value was also slightly higher for GP5+/6+  than careHPV . Specificity, however, was lower for GP5+/6+  than careHPV , as was positive predictive value . Of 377 HR‐HPV‐positive women by GP5+/6+, 173 (45.9%) were careHPV‐positive, including 54.7% HPV16‐positive and 30.2% HPV18‐positive women.HR‐HPV positivity was 10.2% by The final REACH‐Bhutan results show that screening for cervical cancer with self‐collection of samples and HR‐HPV testing, in addition to our previous report of achieving high participation, can also perform well to detect women with hHSIL+. The final results of REACH‐Bhutan study show that community‐based screening for cervical cancer with self‐collection of samples and HR‐HPV DNA testing can perform well as the initial step to detect and treat women with histologically proven high‐grade cervical lesions HSIL+, even in remote low‐resource settings. The design and implementation of the REACH‐Bhutan study, as well as the determinants of screening participation  and HPV positivity , have been reported previously, and showed that HPV‐based cervical cancer screening using self‐collection can achieve high coverage in rural Bhutan.careHPV on self‐collected samples was assessed against the gold standard of colposcopy and histologically proven high‐grade squamous epithelial lesions or worse (hHSIL+). Ascertainment bias was addressed by random biopsies among a subset of careHPV‐negative women and validation with a clinically validated HR‐HPV test that has shown good performance on self‐collected samples.High‐risk HPV (HR‐HPV)‐based screening has the advantage of extended screening intervals, self‐sampling, and test automation,22.1The design of the REACH‐Bhutan study has been previously described in detail.2.2careHPV universal collection medium (UCM) medium using a careBrush, as detailed previously.careHPV platform  according to the manufacturer's instructions. The careHPV test is a signal amplification, rapid batch diagnostic test designed for the detection of the DNA of 13 HR‐HPV types  and HPV66.careHPV results were given to each BHU to arrange colposcopy for all careHPV‐positive women, as well as for a random subset of careHPV‐negative women .Each participant provided a self‐collected cervicovaginal sample into 2.3careHPV‐positive women (and the randomly selected subset of careHPV‐negative women) were invited to attend a follow‐up visit. Colposcopy was used to take biopsies from all suspicious areas in women with abnormal colposcopic findings or, in the absence of a specific suspicious area, randomly from 12 o'clock of the squamocolumnar junction. Treatment of colposcopy‐detected lesions was performed according to local protocols, primarily using cryotherapy on‐site (n = 119) or referral to closest hospitals for loop electrosurgical excision procedure (n = 214).Mobile teams with a portable colposcope and cryotherapy equipment visited BHUs , at which time all Histological reading of biopsies was first performed at the Department of Pathology at JDWNRH (T.T.), with a second expert reading (T.M.D.), both blinded to individual level HPV status (even if pathologists knew that most biopsies were obtained from HR‐HPV‐positive women). Results were reported according to LAST criteria 2.4careHPV UCM medium were shipped to the Department of Pathology at Amsterdam UMC location Vrije Universiteit Amsterdam, the Netherlands, where DNA was extracted using magnetic beads on a robotic system. The presence of human DNA in all specimens was confirmed by β‐globin PCR analysis as quality control for the extraction procedure and subsequent PCR. HR‐HPV positivity was assessed by GP5+/6+−mediated PCRVials containing cellular material in 2.5careHPV and GP5+/6+ results who underwent colposcopy. This approach is valid under the assumption that lack of colposcopy was independent of the underlying hHSIL+ status, given the same combination of careHPV and GP5+/6+ test results (“missing at random”). All analyses were performed using STATA version 14.Standard screening indices of accuracy, including sensitivity, specificity, positive predictive value, negative predictive value, and their 95% confidence intervals (CI) were calculated for hHSIL+. Corrected indices were calculated after imputation of missing data for women who did not attend colposcopy.3careHPV in the REACH‐Bhutan study, 2547 with a valid HR‐HPV GP5+/6+ PCR‐EIA test result were included in the current analysis   than careHPV (75.9%), but 95% CIs overlapped. Relative sensitivity of GP5+/6+ versus careHPV+ for hHSIL+ was 1.18 (95% CI = 0.93–1.50) p = 0.172. Negative predictive value was also slightly higher for GP5+/6+ PCR‐EIA (99.9%) than careHPV (99.7%), albeit with overlapping 95% CIs. Specificity, however, was lower for GP5+/6+ PCR‐EIA (86.1%) than careHPV (90.6%), relative specificity = 0.95 (95% CI = 0.93–0.97) p < 0.001, as was positive predictive value (6.9% vs. 8.5%).Table careHPV are also shown according to GP5+/6+−genotype in Table careHPV. Fifty‐five percent of HPV16‐positive samples were careHPV‐positive. This proportion ranged from 15.6% of HPVX samples up to 100% of the few samples positive for HPV35, 39, and 68. Of note, careHPV positivity among GP5+/6+ PCR‐EIA‐positive samples varied strongly by optical density which may be used as a semiquantitative surrogate for HR‐HPV viral load, being 20.6%, 36.5%, and 80.8% for tertiles of low, medium, and high optical densities, respectively.Type‐specific HR‐HPV prevalence, according to GP5+/6+ PCR‐based genotyping, is described in Table 4careHPV testing.In addition to achieving high acceptability in rural Bhutan as we previously reported, these final REACH‐Bhutan results show that HPV‐based screening for cervical cancer with self‐collection of vaginal samples can achieve high performance in detecting women with hHSIL+. Our report adds to several existing assessments of HPV testing in self‐collected samples in underserved populations in low‐ or middle‐resource settings,careHPV was associated with a slightly lower sensitivity than the reference PCR‐based HPV test, GP5+/6+ PCR‐EIA, the performance of self‐collected careHPV to detect hHSIL+ in our study, with its 76% sensitivity and 91% specificity, is consistent with that estimated by a 2017 meta‐analysis of four careHPV evaluations (74% sensitivity and 88% specificity),careHPV screening in China (73% sensitivity and 97% specificity).Although careHPV has slightly lower sensitivity and higher specificity against cervical intraepithelial neoplasia grade 2 or worse (CIN2+) when performed on self‐collected versus clinician‐collected samples in the same study,careHPV and HC2 may be bigger for self‐collected than clinician‐collected samples.careHPV on self‐collected samples has been shown to remain more sensitive than either Pap testsPrevious studies have shown that, similar to other signal amplification methods, for instance Hybrid Capture 2 ,careHPV testing on clinician‐collected cervical specimens, which has also shown greater cross‐sectional sensitivity for CIN2+ than either VIA,careHPV in rural ChinacareHPV has been associated with slightly lower sensitivity to detect CIN2+ versus HC2There is a wider literature describing the performance of careHPV was also associated with lower sensitivity than GP5+/6+ PCR‐EIA. Indeed, we retested all REACH‐Bhutan samples with a reference PCR test for a number of reasons. Firstly, to improve the imputation of CIN2+ among careHPV‐negative women (see below). In addition, by performing full HR‐HPV genotyping on GP5+/6+ PCR‐EIA‐positive samples, we were able to investigate careHPV sensitivity according to HR‐HPV genotype. Overall, we found 46% of GP5+/6+ PCR‐based HR‐HPV‐positives to be careHPV‐positive from the same sample. This compares to 55% of HR‐HPV positives from the only previous comparison of careHPV with a PCR assay according to genotype .careHPV, which compares with 46% of HPV16 and 49% of HPV18 INNO‐LiPA–positive samples, respectively.careHPV‐positive, suggesting that these samples contain infections not easily identified by either assay. The one HPVX with hHSIL was investigated and found to be HPV66‐positive which is not considered as a HR‐HPV type. Furthermore, careHPV positivity was strongly related to the strength of the semiquantitative GP5+/6+ PCR‐EIA optical density signal, a surrogate for HR‐HPV viral load. Of note, the distribution of HR‐HPV types in the REACH‐Bhutan sample of unvaccinated rural women aged 30–60 years, characterized by a strong predominance of HPV16, followed by HPV18 and HPV59, is comparable with that reported in an urban sample of unvaccinated women in the Bhutanese capital, Thimphu.In the REACH‐Bhutan study on self‐collected samples, careHPV‐negative women, by submitting all biopsies to specialist review according to recommended LAST criteria,careHPV‐negative women that were randomly recalled for colposcopy (n = 83), and the number of observed hHSIL+ with discordant careHPV and GP5+/6+ PCR‐EIA results was small, and so the accuracy of imputation of hHSIL+ prevalence to careHPV‐negative women without colposcopy is a limitation of our study.hHSIL is considered the gold standard diagnosis for cervical precancerous lesions. However, verification bias may occur in histology when biopsies are not taken from all screened women. Here, we tried to overcome this issue by taking biopsies from a proportion of careHPV‐positive women did not attend colposcopy and undergo a biopsy. This low rate of loss to follow‐up is expected to be related to the high trust of Bhutanese population in their public health system, and the close interaction with local health workers. Of course, the loss to follow‐up in this well‐supported and punctual research study may be less than in a more widespread government campaign in Bhutan, if the follow‐up of screen‐positive women is not carefully planned and supported. Similarly, our present findings cannot necessarily be expected to be representative of other settings outside Bhutan. Indeed, this number of 4% compares with 25%–30% loss to follow‐up in some other experiences of recalling HPV‐positive women in low/medium‐income countries.A number of successes in the implementation of this screening initiative should be highlighted. Firstly, we previously reported on the high acceptability and feasibility of self‐collected samples in the rural Bhutanese population.careHPV testing, as might be expected, it being the first experience in Bhutan. For instance, as previously reported,careHPV runs, even after validation of the initial training course. Indeed, lab implementation problems for careHPV were first noted for REACH‐Bhutan,careHPV contamination issues.careHPV have been reported without any mention of laboratory issues, including in Africa,On the contrary, we also encountered certain technical challenges when implementing https://www.moh.gov.bt/hspd/health‐flagship) to shift their entire national cytology‐based cervical cancer screening program to primary testing for HPV DNA.Overall, the findings of the REACH‐Bhutan study highlight the potential for cervical cancer screening programs in low‐resource settings based on self‐collection of samples for HR‐HPV DNA testing. This is an approach that has been strongly endorsed by latest WHO guidelines for screening and treatment of cervical precancer lesions for cervical cancer prevention.Gary Clifford: Conceptualization ; formal analysis ; funding acquisition ; methodology ; project administration ; supervision ; writing – original draft ; writing – review and editing . Iacopo Baussano: Conceptualization ; funding acquisition ; methodology ; supervision ; writing – review and editing . DaniÃ«lle A.M. Heideman: Data curation ; formal analysis ; validation ; writing – review and editing . Sangay Tshering: Data curation ; writing – review and editing . Tashi Choden: Data curation ; writing – review and editing . Fulvio Lazzarato: Data curation ; software ; writing – review and editing . Vanessa Tenet: Data curation ; formal analysis ; software ; visualization ; writing – review and editing . Silvia Franceschi: Conceptualization ; funding acquisition ; methodology ; supervision ; writing – review and editing . Teresa Darragh: Data curation ; validation ; writing – review and editing . Tashi Tobgay: Data curation ; writing – review and editing . Ugyen Tshomo: Conceptualization ; funding acquisition ; methodology ; supervision ; writing – review and editing .This work was supported by the Bill and Melinda Gates Foundation (grant number OPP1053353).D.A.M.H. is minority shareholder of Self‐screen B.V., a spin‐off company of VUmc; Self‐screen B.V. develops, manufactures and licenses high‐risk HPV and methylation marker assays for cervical cancer screening and holds patents on these tests. The other authors declare no conflicts of interest.The present study had the approval of both the Research Ethical Board of the Bhutan Ministry of Health and the IARC Ethics Committee.Where authors are identified as personnel of the International Agency for Research on Cancer/World Health Organization, the authors alone are responsible for the views expressed in this article and they do not necessarily represent the decisions, policy, or views of the International Agency for Research on Cancer/World Health Organization.Table S1.Table S2.Click here for additional data file.


HPV-related diseases are mainly represented by cancers. Furthermore, real world data with respect to the effects of primary and secondary preventive strategies are lacking. Therefore, the aim of this study is to assess the effectiveness of preventive strategies in accordance with Italian HPV-related hospital admissions. From 2008 to 2018, there was a decrease (APC = −3.8%) in all HPV-related diseases. The increase in cervical cancer screening adherence was related to a decrease in invasive cervical cancer and an increase in HPV vaccine coverage, which was found to arise from a decrease in “in situ” cervical cancer. In this study, the need to improve the acceptance of preventive strategies for HPV-related diseases, as well as the homogenous information furnished by all healthcare workers involved in their promotion  is highlighted.p < 0.001), as well as between HPV vaccination coverage and in situ cervical cancer . These results indicate the positive impact of HPV vaccination coverage and cervical cancer screening on hospitalizations due to cervical cancer. Indeed, HPV vaccination also resulted in a positive impact on the decrease in hospitalization rates due to other HPV-related diseases.Human papillomavirus (HPV)-related diseases are still a challenge for public health. Some studies have shown the effects of preventive strategies on them, but studies at the national level are few in number. Therefore, a descriptive study through hospital discharge records (HDRs) was conducted in Italy between 2008 and 2018. Overall, 670,367 hospitalizations due to HPV-related diseases occurred among Italian subjects. In addition, a significant decrease in hospitalization rates for cervical cancer  = −3.8%, 95% CI = −4.2, −3.5); vulval and vaginal cancer ; oropharyngeal cancer; and genital warts  was observed during the study period. Furthermore, strong inverse correlations were found between screening adherence and invasive cervical cancer (r = −0.9,  Human papillomavirus (HPV) is still the main infection that occurs via sexual activities ,3,4,5,6.Cervical cancer affects nearly 7–35/100,000 females worldwide per year . MoreoveCurrently, when estimating Italian data, the HPV-related disease burden has remained a challenge . FurtherOther HPV-related oncological diseases had a variable trend throughout Italy with regards to both genders. Considering oropharyngeal cancer, the Italian trend regarding the hospitalization rate was nearly four times higher among men  . Penile The most efficient prevention strategies for cervical cancer are HPV vaccination and screening ,8,17,18.According to the World Health Organization, cervical cancer could be eliminated by 2030, if nearly 90% of female adolescents are vaccinated against HPV, if 70% of females aged between 35 and 45 years old perform cervical cancer screening, and finally if 90% of female patients receive healthcare assistance for either pre-cancerous lesions or cervical cancer at any stage. It is expected that reaching these goals will reduce new cases of cervical cancer to lower than 4/100,000 . In ordeThe main aim of this study is to assess hospitalization rates for HPV-related diseases in Italy. The second aim is to focus on the impact of prevention measures on hospitalization rate reductions for cervical cancer in the Italian setting.In this descriptive study, the hospitalization trends due to HPV-related diseases in Italy between 2008 and 2018, excluding the administrative region of Sicily, were evaluated. The source of the data was the Italian hospital discharge records (HDRs) database, which is a medical recording system for each admission to Italian hospitals since 1991 . The maiAccording to the Italian Statistics Institutes, an annual average of 54,992,245 inhabitants have lived in Italy (excluding Sicily) in the study period with a percentage of 51.5% females . Among tThe identification of HPV-related hospital admissions was in accordance with the following codes of ICD9-CM, as reported in other studies ,34: condIn order to increase the accuracy of cervical cancer identification, as per the inclusion criteria, the following interventions codes were also considered: cervix conization 67.2), cervical lesion cauterization (67.32), and cervical lesion cryosurgery ) to 2018 (4 [IQR = 2–7]) . This was strongly inversely correlated with hospitalization rates due to invasive cervical cancer , as shown in During the period 2008–2017, cervical screening uptake among females aged 25–64 years old was 79.6%, with significant increases observed during the period . This trend was strongly inversely correlated with hospitalization rates due to in situ cervical cancer , as shown in HPV vaccination between 2009 and 2018 had a coverage of 67.2% with significant increases observed during the period , cervical cancer was the HPV-related disease with the highest hospitalization rate . The Italian AAPC for cervical cancer was milder in comparison to the Sicilian data, in which the hospitalization rate for cervical cancer had a statistically significant AAPC = −9.9%, [This study showed the hospitalization trends of HPV-related diseases from 2008 to 2018 in Italy. Despite its significant decrease along the whole period with an AAPC = −3.8 . This was similar to the Sicilian data and may be an indirect effect of the preventive strategies. Indeed, vaccine availability was available to people born after 1996, and the older females did not have the benefit of HPV vaccination. In addition, these older females could have received the diagnosis of cervical cancer later, when it began to be symptomatic, and could have had other diseases or complications, thus resulting in extending the length of their hospital stay [The females’ median age and length of hospital stay slightly increased during the study period  . AnotherA globally observed reduction in hospitalization rates due to in situ and invasive cervical cancer was detected in most of the age classes, even if the rate was higher among those older than 75 years old for invasive cancer, and those aged 15–24 for in situ cancer. In addition, the significant increase in HPV vaccination coverage among females aged 12–21 years old and adherence to cervical cancer screening among females aged 25–64 years old seem to reduce the hospitalization rates due to in situ and invasive cervical cancer, respectively. This could suggest an impact of HPV vaccination and cervical cancer screening on cervical cancer reduction ,17. FurtAccording to the newest meta-analysis data, whatever vaccine valence was undertaken against HPV, nearly 100% of cervical lesions and infections due to some HPV serotypes (such as 16 and 18) can be avoided, thus showing an almost decennial antibody protection in several trial studies . This sup < 0.001). Nevertheless, these low trends may be attributed to HPV vaccination [Hospitalization rates due to oropharyngeal cancer in Italy decreased constantly during the period 2008–2018 (APC = −4.0%, cination . Indeed,cination . On the cination ,58. TherPenile cancer had the lowest hospitalization rate in Italy during the years 2008–2018 because of its rarity, as is shown in the European data . HoweverThis study had some limitations. HDRs could underestimate the real trend of these HPV-related diseases because some of them, especially the less severe ones such as cervical cancer, may have been treated in an outpatient setting, whose clinical and administrative sources of data were not available and not included in this study. In detail, precancerous cervical lesions are more frequently treated in outpatient settings during colposcopy, which is an outpatient procedure, according to national laws ,61,62. FCervical cancer is the HPV-related disease with the highest impact on women’s health. HPV vaccination and cervical cancer screening in Italy are associated with a reduction in hospitalization rates due to in situ and invasive cervical cancer. Additionally, other HPV-related diseases reported in this study decreased globally. Further studies should confirm our findings investigating, at an individual level, the effectiveness of preventive strategies. While waiting for these results, our analyses provide evidence-based data to support adherence to HPV preventive strategies and thus achieve the elimination of HPV.


Methods: 185 women were enrolled in the LILT  regional project. A self-sampling kit, including a dry FLOQSwab® (Copan), instructions for use, and a satisfaction questionnaire, were supplied for each woman and sent by mail to the laboratory. The HPV-DNA test was carried out using the Anyplex™ II HPV HR (Seegene) kit. To evaluate the specimen’s stability, 185 dry vaginal swabs were eluted in eNat®, a lyses-based molecular medium and tested for HPV detection at two different time points (<6 days and 1 month after elution). The Cohen’s Kappa coefficients and McNemar test were used to assess the agreement of HPV-DNA at different times. Results: We found high agreement in terms of HPV-DNA results among the samples tested at two different time points . Moreover, most of the women found it easy to use self-collection devices and the pictorial instructions clear to understand. Approximately half of the enrolled women declared preferring self-sampling to clinician-collected methods. Conclusion: Our results display the high reliability and accuracy of HPV-DNA tests using dry vaginal self-collection FLOQSwabs® devices eluted in 2 mL of molecular medium. The analysis of the questionnaire showed a high acceptability of self-collection among women, although a high percentage preferred standard collection devices. Overall, our preliminary results support the adoption of self-collection in screening programs, even though further analyses should be performed to optimize and standardize protocols for HPV tests on self-samples, and educational campaigns are needed to adequately inform and increase responsiveness in a target population.Background: Given the diagnostic accuracy of HPV-DNA tests in terms of self-collected samples, in order to implement self-sampling in cervical screening programs, the standardization of the pre-analytical phase, including decisions concerning the choice of medium, the volume of elution, and storage conditions, are necessary, in addition to understanding the potential factors involved in acceptability by women. On this basis, we carried out a cross-sectional study to assess (i) the stability of dry vaginal self-collected samples stored at room temperature for up to 4 weeks after elution in 2 mL of eNat Cervical cancer is the fourth most common cause of cancer among women worldwide, with more than 604,000 new diagnoses and over 341,000 deaths in 2020 and the third most common cause of cancer in women aged 15 to 44 years [Human Papillomavirus (HPV) infection is known as the main cause of cervical cancer, with HPV-16 and -18 accounting for approximately 70% of cervical cancer. However, only a small fraction of HPV infections are persistent, whereas the majority resolve spontaneously . VirtualThe global strategy for cervical cancer elimination by the World Health Organization (WHO) recommends primary and secondary preventive interventions, including vaccination and screening: vaccination for 90% of girls globally, 70% of screened women aged 25–64 years, and treatment of 90% of cervical disease by 2030 [Currently, cervical screening shows relevant geographical variations in terms of organization and coverage. Despite the available screening modalities, a recent review revealed that two in three women aged 30–49 years have never been screened for cervical cancer globally. Moreover, a different distribution was reported by income level, with coverage being nine times higher in high-income countries than in low-income countries  . The disThe screening strategies used in Italy slightly vary among regional organizations: on average, ~77% of women aged 25–64 years participate in cervical screening through organized programs or spontaneous initiatives. Socio-economical and geographic differences were registered at the national level: the participation is higher among socio-economically advantaged women, associated with economic status and educational level, higher in those who are married or cohabiting and lower in foreign women. Moreover, a north–south gradient is observed in the screening uptake, with a percentage ranging from 91% in North Italy to ~65% in the south. A significant fraction of women (~25%) report that they have never undergone cervical screening or are screened regularly, and the main reason is the low individual risk perception [In this scenario, it has been emerged the need to implement knowledge about HPV-related diseases and prevention systems, with health education intervention, and, contextually, considering new screening strategies, particularly addressed to non-adherence women and women with prior treatment for cervical high-grade lesions to reduce the risk to develop HPV-related extra-cervical cancers ,8.The good analytical performance of HPV-PCR testing in self-collected samples, along with the high acceptability by women due to its ease of use, safety, privacy, and convenience, supports its adoption in regular screening programs as a complementary choice ,10,11,12To answer the still open questions related to the pre-analytical phase and laboratory processing of specimens, a preliminary study based on cervical screening by vaginal self-collection was conducted in the Sardinian region . Volunteer women were enrolled within the regional LILT  project, a non-profit organization aimed at health promotion and prevention at national and regional levels, in collaboration with the Department of Medicine, Surgery and Pharmacy of University of Sassari , member of the Global HPV Laboratory Network by WHO for HPV-DNA testing. Self-collection kits were provided to the recruited women, aimed to assess (i) the pre-analytical performance of dry vaginal self-samples eluted in 2 mL of molecular medium and analyzed using a HPV-DNA PCR test at two different time points, (ii) the acceptability of self-collection devices in relation to the enrolled women.The results obtained will provide crucial information for the implementation of screening based on self-sampling, addressing the gaps regarding the pre-analytical phase and the preparedness of the target women.A cross-sectional study was conducted between March 2021 and June 2022 in Sardinia .The inclusion criteria for enrolled women were: (i) age from 25 to 64 years; (ii) women who did not participate in cervical screening during the last year; (iii) women with no prior treatment for cervical cancer; and (iv) no pregnant women. Staff involved in each regional LILT center promoted the initiative through the available communications channels, such as social communities, media, newsletters, or individual contacts. Volunteer women who met the selection criteria were recruited for the study and invited to pick up the self-sampling kit and to sign the written informed consent at the LILT territorial office.All of the performed procedures were in accordance with the ethical standards of the local Ethical Committee and with the Helsinki declaration. All women were informed by qualified health workers about the study objectives.® 552C.80 , a flocked swab that consists of a molded plastic applicator stick with a regular and shaped tip. The tip of the applicator is coated with short Nylon® fibers that are arranged in a perpendicular fashion. The samples collected and questionnaires were returned to each territorial LILT center and delivered to the reference laboratory through the healthcare practitioner.The self-sampling kit provided to the enrolled women contained a dry vaginal swab with illustrative instructions for self-collection, directly provided by the self-collection device manufacture’s and a questionnaire to collect demographic data and specified questions to assess the perceptions within the study population about vaginal self-sampling and the device’s ease of use. Vaginal self-sampling was collected using dry FLOQSwabs® were first soaked in 2 mL of eNat®  medium, a lysis-based molecular medium, and then vortexed for at least 30 s. Subsequently, the swab was removed and discarded. Lastly, the eluted samples were analyzed for HPV-PCR at two different time points in order to verify the stability of media stored at room temperature for up to 4 weeks.At the Molecular Epidemiology Laboratory, all the dry vaginal FLOQSwabs® medium is a commercially available guanidine thiocyanate-based molecular medium that stabilizes nucleic acids  at room temperature for over a month, which degrades proteins and inactivates microbial infectivity through the lysis of the cell membranes. The inactivation is due to the synergistic interaction of guanidine thiocyanate and detergents. Dry vaginal self-samples were resuspended in 2 mL of eNat medium and processed as follows.The eNat®  medium tube was used for DNA extraction by the Seegene AdvanSure™ E3 SYSTEM [A 200 µL aliquot of eNat3 SYSTEM . This au® medium was stored at room temperature for ~30 days and, subsequently, subjected to a second DNA extraction and HPV testing.In order to assess the stability of the medium over time, a second 200 µL aliquot of eNat® Inc., Seoul, Republic of Korea) kit using real-time PCR [All HPV-DNA PCR tests were carried out using Anyplex™ II HPV HR  and clinical  variables were summarized with median and interquartile range (IQR) for quantitative variables, and with absolute and relative (percentage) frequencies for qualitative variables. Shapiro–Wilk test was used to assess the normality of the data distribution. The McNemar test was used to evaluate the differences between HPV-DNA results at different time points. Cohen’s Kappa coefficients were calculated to assess HPV positivity agreement. The statistical significance threshold was set at <0.05, and data analysis was carried out through STATA version 17.A total of 185 females were enrolled during the study period, with a median (IQR) age of 41 (34–51) years. Most women (39.1%) had a high level of education, nearly 60% were married, and 24% were in menopause. Over ninety-five percent reported use of contraceptives during their lifetime, whereas 23% never used a condom . Twenty-In this study, 37.2% were smokers, and ~8% of women reported a history of previous sexually transmitted infections (STIs).Overall, 17.3% (32/185) reported positivity for at least one high-risk HPV genotype. The most common HPV genotype detected was HPV-56 (16.3%), followed by HPV-18, -31, -51 (12.2%) and HPV-52 (10.2%) . The maj® (n=185) samples were stored at room temperature for a median (IQR) of 19 (13–32) days before resuspension in 2 mL of eNat® medium. The first HPV-DNA test (t0) was carried out after a median (IQR) of 3 (2–6) days after elution, with a positivity of 17.3% (32/185). The second HPV-DNA test (t1) was carried out after a median (IQR) of 35 (35–39) days after the first analysis (t0). The results at t1 confirmed the positivity at t0 for most samples , whereas all of the samples that tested negative at first analysis were confirmed at t1 point. The results for HPV-positive specimens at t0 and t1 are reported in Dry vaginal FLOQSwabp-value: 0.32), with an agreement of 99.5%   .Regarding the acceptability questionnaire, the majority of women found the instruction of self-collection clear and understandable and the collection easy to perform, while 97% of women answered positively to the question “Did you find the self-collection instruction clear and understandable?”, whereas 95% of “yes” answers were registered for the second question “Did you find self-collection easy to perform?”. Conversely, 45% of participants declared to not prefer vaginal self-sampling to clinician collection .No significant differences were recorded in the questionnaires based on the different demographic  and epidemiological  variables. However, although below the limits of statistical significance, a higher level of self-sampling acceptance (3rd question) was observed among women younger than 41 years old and those with an advanced level of education (percentage of acceptability higher than 60%). Moreover, a slight geographic difference was found in the level of acceptability among the female respondents, with a higher preference for self-collection among women living distant from the principal regional screening centers (69% vs. 45% of “yes” answers to the third question). No difference in the questionnaire results was found between “non-adherence”, or not regularly receiving screening, to screening programs and those who regularly underwent pap smears.® (up to ~30 days) and eluted in 2 mL of eNat® molecular medium, suggesting a pre-analytical protocol for the workflow of self-sampling.Vaginal self-collection is a safe and reliable method to involve women in cervical screening programs, showing similar diagnostic accuracy to clinician-collected samples ,18. DespAlthough several collection devices have been validated in recent years for use in vaginal self-collection, the designated population, climatic and geographical conditions, and logistical infrastructures are essential elements to consider prior to adoption . Dry vag® medium, as highlighted by the high agreement in terms HPV testing at different time points, addresses one of the current needs for the implementation of self-collection in cervical cancer screening programs, particularly those related to transport and stability of specimens. Moreover, the reduced volume of eNat® medium (2 mL) makes it possible to increase the concentration of the sample, avoiding the risk of ‘invalid’ or false negative results and allowing safe transport. In fact, recent studies demonstrate that the resuspension of dry vaginal swab in 5 mL or less may maximize the detection of the HPV genome [The stability of dry vaginal self-samples in eNatV genome .In addition to elution volume, another relevant feature is the composition of the media. Until now, cytology alcohol liquid-based transport medium, usually employed for the transport and storage of cervical samples, has several disadvantages  . The use® medium allows the inactivation of microorganisms in clinical samples and, contextually, reduces the risk of exposure to infectious agents for laboratory workers [Besides preserving and stabilizing the nuclei acid for molecular analysis for up to one month, the eNat workers ,22.The questionnaire results showed that the majority of participants considered the self-collection procedures easy to perform and that the instructions provided were clear and understandable. Our results confirmed previous data about women’s acceptability of self-collection. Women mainly appreciate the “easiness”, “rapidity” and “comfort” of the device, as well as the possibility of overcoming the embarrassment and emotions involved with a visit to the gynecologist . All theThese preliminary results strongly support the implementation of primary and secondary preventive procedures against cervical cancer. Recognizing the diagnostic accuracy of molecular testing for HPV detection carried out on vaginal self-collection compared with clinician-collection samples, as previously demonstrated ; the resAlthough this represents the first study on self-report-based screening in our territory, it showed several limitations, mainly related to the small sample size and the monocentric nature of the study, as well as the recognized weaknesses of self-reported questionnaires. Moreover, as this project was carried out within the regional LILT program, consequently, the modality of enrollment did not allow us to apply the results to the general population, particularly those related to the acceptability questionnaire.® medium for the storage of vaginal specimens until one month strongly supports its potential use in cervical screening programs, especially in geographically disadvantaged areas. Further real-life research and large sample sizes are required for the standardization of screening activities based on self-collection and to improve women’s confidence.Overall, the adoption of dry vaginal self-collection devices used in screening could be worthwhile scaling up, not only in rural or remote regions, which register the lowest adherence to preventive programs and the greatest limitations in healthcare services, but also as a support to organized screening program. The stability of 2 mL eNat® self-collected eluted in 2 mL of eNat® molecular medium. Moreover, our results highlight that the acceptability of self-sampling decidedly depends on individual beliefs and knowledge and requires adequate health literacy intervention both for women and healthcare providers to ensure informed decision making and provide adequate support during the management of follow-ups.The high accuracy of HPV-DNA testing in self-collected samples has been evaluated in numerous studies, although several issues, mainly related to the processing, storage, and handling of the specimens, slow down its adoption in organized screenings. The development of standard pre-analytical protocols is needed to implement this modality into a national screening program. The present study demonstrated the reliability and stability over time of HPV-DNA testing carried out with dry vaginal FLOQSwabs


Nicotine is a known toxin, but its relationship with cervicovaginal high-risk human papillomavirus (HR-HPV) infection is uncertain. This study aimed to investigate whether tobacco exposure is associated with elevated cervicovaginal HR-HPV infection in US women, and if the strength of this association varies with the degree of exposure. Cross-sectional data from the 2011–2016 National Health and Nutrition Examination Survey (NHANES), which included a nationally representative sample of US women, were used for the study. Out of 12436 women aged 18–59 who participated in the interview, 3833 were ultimately enrolled in this study. Weighted logistic regression was used to estimate the link between tobacco exposure and cervicovaginal HR-HPV infection. The mean age of participants was 38.6 (SD 12.1) years, and non-Hispanic White individuals accounted for 37.3% of the sample. Those with any tobacco exposure tended to be younger (mean age 37.7 [SD 12.4] years vs 40.3 [11.2] years), non-Hispanic Black (27.8% vs. 15.1%), lower educated (41.8% vs. 29.4%), and have lower family income (39.9% vs. 23.5%). After adjustment, the odds of having HR-HPV infection were 1.32  for those exposed to tobacco, remaining significant in multiple sensitivity analyses and across subgroups. This study, based on a nationally representative sample from the United States, suggests that tobacco exposure is a risk factor for elevated HR-HPV infection in women, highlighting the need for further research into reducing this modifiable risk factor.The online version contains supplementary material available at 10.1007/s11356-023-30175-7. Developing countries may face an even more challenging situation due to their low socio-economic status and poor health conditions. Therefore, it is critical to explore the underlying factors associated with HR-HPV to prevent or treat it.As one of the most widespread sexually transmitted diseases, human papillomavirus (HPV) infection poses a significant public health challenge for women across the globe. Persistent infection with high-risk oncogenic HPV (HR-HPV) may give rise to severe cancers like oropharyngeal, penile, vulvovaginal, anal, and, notably, cervical cancer  is a cross-sectional study conducted every two years to assess the health and nutritional status of non-institutionalized individuals in the United States. Administered by the Centers for Disease Control and Prevention, NHANES is a nationally representative, stratified multistage probability survey. The survey involves a mobile examination center (MEC) that conducts home visits, screenings, and laboratory tests to collect detailed demographic and health data  of the National Health and Nutrition Examination Survey. Since public records for genital HPV infection are only available for this age group, our analysis included individuals who were 18 years or older but younger than 59 years at the time of participation and completed the required tests. Those who tested positive for pregnancy, reported kidney disease, or had a serum creatinine level greater than 2 mg/dl were excluded from the study due to their possible impact on nicotine metabolism . BMI was calculated based on participants' weight in kilograms and height in meters. Alcohol consumption was indicated by drinking at least 12 alcoholic beverages per year, including liquor, beer, wine, wine coolers, and other alcoholic beverages. Participants with neither private nor public insurance were coded as having no insurance. Vaginal deliveries referred to the number of deliveries during the interview. Age of first sexual activity was defined as the age at which participants had their first oral, anal, or vaginal intercourse. Lifetime sexual partners referred to the number of men with whom participants had vaginal, anal, or oral intercourse throughout their lives. HPV vaccine status was determined by whether participants had received either of the two available HPV vaccines, Cervarix and Gardasil.According to the literature  values were used to define continuous variables that were normally distributed, while median values and interquartile ranges (IQR) were used for non-normally distributed variables. Categorical variables were expressed as proportions (%). For group comparisons, either the t-test , Mann–Whitney test (for skewed distributions), or χ2 test  were used as appropriate. To assess the association between tobacco exposure and HR-HPV infection, multivariable logistic regression was used to calculate the odds ratios (OR) and 95% confidence intervals (95% CIs). Age, race/ethnicity, education, marital status, family income, BMI, alcohol consumption, insurance status, age of first sexual activity, lifetime sexual partners, HPV vaccine status, and vaginal deliveries were all included in the final model.Furthermore, sensitivity analyses were conducted using cotinine as a continuous variable, with the limit of detection of cotinine as a threshold for exposure, and smoking status classified as none (cotinine < 0.05 ng/ml and no self-reported exposure), passive (cotinine 0.05–10 ng/ml or self-reported household smoke exposure), or active (cotinine > 10 ng/ml or self-reported current smoker). The effect of the natural logarithm of cotinine on HR-HPV infection odds ratio, after adjusting for confounders, was examined.Moreover, following the adjustment of variables in Model 3, a restricted cubic spline (RCS) regression with four knots at the 5th, 35th, 65th, and 95th percentiles of the natural logarithm of cotinine was performed to investigate the relationship between cotinine and HR-HPV infection in a dose–response fashion.Subgroup analyses were conducted to explore potential modifications of the relationship between tobacco exposure and HR-HPV infection. The following variables were investigated: age (< 25 vs ≥ 25 years), education level (high school graduate or some college or above), marital status , family income (low vs. medium or high), BMI (< 25 vs. ≥ 25 kg/m2), alcohol consumption, health insurance, first age (< 17 vs. ≥ 17 years), lifetime sexual partners (< 5 vs. ≥ 5), and HPV vaccine status.http://www.R-project.org, The R Foundation) and Free Statistics software versions 1.71, with appropriate sampling weights to account for the complex survey design. Two-sided tests were used for all P values, and results were considered statistically significant if P < 0.05.No a priori statistical power estimations were conducted because the sample size was determined solely based on the available information. Statistical analysis was performed using the R-4.0.2 software package (n = 8163). Eventually, 3833 participants from NHANES conducted between 2011 and 2016 were included in the analysis. Further details regarding the selection process are presented in Fig. A total of 12,436 individuals aged 18 to 59 participated in the interview. Among them, we excluded 440 women, consisting of 192 pregnant women, 204 with kidney disease, and 44 with serum creatine levels > 2 mg/L. Additionally, those missing data on HR-HPV status, tobacco exposure, serum cotinine, education level, family income, BMI, alcohol consumption, health insurance, first age, lifetime sexual partners, or HPV vaccine status were further excluded  years, and 37.3% were non-Hispanic White. Table 2.1 yearsTable Figure After categorizing study subjects based on the lower detection limit of cotinine (0.015 ng/ml), 68.7% were identified as tobacco exposed. Results from Table In this extensive cross-sectional study of American women, we found a significant link between tobacco exposure and HR-HPV infection that remained significant even after adjusting for various factors, including age, race/ethnicity, education, marital status, PIR, BMI, alcohol use, health insurance, age at first sexual intercourse, number of lifetime sexual partners, HPV vaccination status, and vaginal deliveries. The association was similar across various subgroups of participants. Moreover, comparable associations were observed in sensitivity analyses that utilized different definitions of smoking status.The current literature on the association between tobacco exposure and HPV infection, especially HR-HPV, has yielded inconsistent results. Smoking has been linked to an increased risk of cervical dysplasia Below is the link to the electronic supplementary material.


Infection of epithelial cells with high-risk HPV (HR-HPV) types, followed by expression of virus oncogenic proteins , leads to genomic imbalance, suppression of tumor inhibitors, and induction of oncogenes. Low-risk HPV (LR-HPV) may slow the rate at which cervical cancer spreads to an invasive stage since co-infection with LR-HPV is linked to a decreased risk of future invasive cancer than infection with HR-HPV alone. We then propose that cancer-progressing changes may be distinguished through identifying the functional differences between LR-HPV and HR-HPV. Lentiviral strategies were followed to establish HaCaT cells with constitutive expression of HPV oncogenes. RNAseq experiments were designed to analyze the transcriptome modulations caused by each of the E5, E6, and E7 oncogenes of HPV-16 and HPV-84 in HaCaT cells. We identified enhanced RNA degradation, spliceosome, and RNA polymerase pathways related to mRNA processing. ATTS  was discovered to be more prevalent in cells with HPV-16E5 than HPV-84E5. In HPV-16E6-infected cells, ATTS gain was significantly higher than ATTS loss. Cells with HPV-16E7 had more isoforms with intron retention (IR) than those with HPV-84E7. We identified switches in ADAM10, CLSPN, and RNPS1 that led to greater expression of the coding isoforms in HR-HPV. The results of this work highlight differences between LR-HPV and HR-HPV in mRNA processing. Moreover, crucial cervical cancer-related switch events were detected. Viral infection accounts for approximately 12–20% of all cancers . CurrentA crucial stage in the post-transcriptional control of gene expression is alternative splicing (AS) of mRNAs. This mechanism significantly modifies cell function and proteome diversity through regulating the expression of various isoforms at particular times and in specific cell types. In this sense, through altering the mRNA processing machinery, cancer cells can gain advantages . TherefoAlthough there has been some research on the alternative splicing processes by HR-HPV types, modulation of AS through the proteins encoded with LR-HPVs receives far less attention. Splicing events generated by E5, E6, and E7 oncoproteins of HPV-84 and HPV-16 were examined for the first time to highlight the differences between HR- and LR-HPVs; these modulations were described in eight distinct splicing mechanisms and individual isoform switch events.To determine potential KEGG pathways and gene sets linked with HR- and LR-HPV types, the expression data of the HaCaT transduced cells with the oncogenes of HPV-16 and HPV-84 were subjected to GSEA analysis. Over-represented pathways with normalized gene set enrichment scores (NES) ≤ 1 are illustrated in Genome-wide alternative splicing was investigated in the eight previously identified categories, and the results make it quite evident that not all alternative splicing events were equally used . In all gains for each AS type (as opposed to loss). It is evident from this plot that the overall trend in the usage of alternative splicing is the same in the two HPV types. However, there are some variances. We can, indeed, see that ATTS has a much more significant skew in HPV-16E6 compared to HPV-84E6.In According to coding potential, identified domains, intron retention, sensitivity to non-sense mediated decay (NMD), and ORF length, isoform switch effects were predicted and characterized . The celThe general overlaps in isoform switches for all three oncogenes from HPV-16 and HPV-84 are illustrated in We discovered three events in the genes ADAM10, RNPS1, and CLSPN that were previously reported to play a role in the progression or prognosis of cervical cancer. The switch plots of these three genes are illustrated in The maturation, processing, and alternative splicing of mRNAs are necessary for regulating gene expression and preserving proteome diversity. The development of cervical cancer is thought to be significantly influenced via HPV-mediated alternative splicing, and these alterations can be utilized as diagnostic biomarkers and targets for therapeutic strategies . On the RNA degradation aids ribonucleotide recycling, while also performing surveillance through eliminating aberrant RNA that might produce damaging proteins . There aThe enrichment of mRNA processing pathways piqued our interest in further investigating mRNA processing activities in our cell models. Following the pipeline defined using IsoformSwitchAnalyzeR, we identified eight alternative splicing events in a number of significant isoforms. Notably, ATTS is more common in cells carrying HPV-16E5 than in cells harboring HPV-84E5. ATTS, on the other hand, is demonstrated to have the most notable alteration in cells with E6, with fewer isoforms with ATTS being detected in HPV-16 compared to HPV-84. Moreover, in HPV-16E6, ATTS gain is significantly higher than ATTS loss. Generally, using ATTS produces transcripts with different 3′ ends or even transcripts with different coding regions . HoweverA further finding in our study concerns E7-containing cells where HPV-16 has a more significant number of isoforms with IR than HPV-84. IR appears to be necessary at several phases of cell differentiation and development. Intron-retaining transcripts can be detained in the nucleus (a process known as intron detention) and destroyed via nuclear degradation mechanisms. As introns often include in-frame premature termination codons, when preserved in the mature mRNA transcript and exported to the cytoplasm, they can be identified via the cytoplasmic surveillance machinery and eliminated via nonsense-mediated decay (NMD). Furthermore, the intron retention mechanism may yield non-coding RNA(s) implicated in controlling oncogenes and tumor suppressor genes. . Except In our study, we found a more significant number of isoforms with domain loss, sensitivity to NMD, and shorter or longer ORFs, primarily mediated by high-risk HPV16 oncogenes. It is now well understood that viral proteins influence the cellular splicing machinery, producing RNA isoforms with carcinogenic properties ,18. FurtThese genes have previously been linked to cervical cancer. The proteolytic cleavage of NKG2D ligands (NKG2DL) on the cancer cell surface via ADAM10 can impair the recognition of cancer cells by T or NK cells . In cervg rpm for 10min to remove excess DMSO (dimethyl sulfoxide), suspended in fresh medium, and cultured in 75 cm2 culture flasks. Cell lines were grown in Invitrogen’s GIBCO™ DMEM (Dulbecco’s Modified Eagle Medium) medium with L-glutamine (584 mg/L), sodium pyruvate (110 mg/L), and D-glucose (4.5 g/L). The medium was supplemented with penicillin (100 U/mL), streptomycin (100 μg/mL), and 10% fetal bovine serum. The cultures were maintained at a confluence of 70–80%; upon reaching this level, the cells were detached with 0.25% trypsin (EDTA 380 mg/L) and counted to make the necessary dilutions for the experiments. Culture passages were created according to the cell duplication rate. Cell cultures were maintained in a humidified incubator at 37 °C in a 5% CO2 atmosphere.Briefly, E5, E6, and E7 were amplified using specific primers from genomic DNA extracted from cervical biopsies of women infected with HPV16 or 84. The amplified ORFs were subcloned into the lentiviral expression vector pLVX-Puro and verified via sequencing. Lentiviral particles were produced using the acquired plasmids pLVX-16E5, pLVX-16E6, pLVX-16E7, pLVX-84E5, pLVX-84E6, and pLVX-84E7. In the following step, HaCaT cells were individually infected with the lentiviral particles containing each viral gene. Transduced cells were selected through puromycin addition for three weeks. Transduced HaCaT cells were named HAC16E5, HAC16E6, HAC16E7, HAC84E5, HAC84E6, HAC84E7, and HACPLVX . HaCaT c6 cells of each cell line in three replicates using the PureLinkTM RNA Mini Kit extraction system . Cells were washed two times with PBS, and cell lysis was performed. The lysate obtained was column purified, and the RNA was eluted in water. The RNA concentration and integrity were evaluated with the Agilent 2100 bioanalyzer using RNA nano chips. RT-qPCR confirmed the expression of E5, E6, and E7 through specific primers. Sequencing of total mRNA was performed using the Illumina Nova-Seq 6000 platform with a 150 bp paired-end method, where most reads have a Phred quality score greater than 35.Total RNA was obtained from 5 × 10https://usegalaxy.org/, accessed on 5 April 2021 [https://www.gsea-msigdb.org/, accessed on 21 December 2021 [Data quality was verified using the FastQC v.0.11.9 tool  on the Gril 2021 . Reads wril 2021  tool. Maril 2021 . The overil 2021  input fiber 2021 ,44, and ber 2021 . The numFor accurate reconstruction of all expressed isoforms of each gene, and to estimate the relative abundance of those isoforms, the StringTie v2.2.0 transcript assembler was used . For thiIsoformSwitchAnalyzeR was used to detect and visualize the mechanisms of alternative splicing and individual isoform switch events . In the Our findings point to key differences produced by the oncogenes E5, E6, and E7 of HPV-16 and HPV-84. We focused primarily on mRNA processing pathways, where we discovered specific enhanced splicing processes and isoform switch events in HPV-16 that may favor cancer progression. These changes, in addition to providing information on biological processes implicated in cervical cancer pathways, may be beneficial for identifying prospective biomarkers and therapeutic targets. Nonetheless, only large prospective studies may determine the actual clinical and diagnostic utility of these alterations in cervical cancer.


An intercomparison of the included assays demonstrated an overall agreement ranging from 85.9 to 100.0% (κ = 0.42 to 1.00) when using the manufacturer’s guidelines and 92.9 to 100.0% (κ = 0.60 to 1.00) with the adapted range. For all assays, highly significant, strongly positive Pearson correlations were shown between the qC values of positive test results. This study thereby shows high concordance between results of the included HPV assays on mocked self-samples. Based on these findings, we imply that the novel Allplex HPV28 assay demonstrates a comparable performance to those of available qPCR HPV assays, potentially providing opportunities for the simplification and standardization of future large-scale testing.Infection with high-risk human papillomavirus (hrHPV) is well recognized as the main cause of cervical cancer. The recently developed Seegene Allplex HPV28 assay is a novel quantitative PCR (qPCR) assay designed to separately detect and quantify 28 distinct HPV genotypes in a fully automated and user-friendly manner. This study evaluated and compared the performance of this new assay with the performance of the Roche Cobas 4800, the Abbott RealTime high-risk HPV, and the Seegene Anyplex II HPV28 assays. A total of 114 mocked self-samples, i.e., semicervical samples collected by gynecologists using the Viba-Brush, were analyzed with all four HPV assays. Agreement in terms of detecting and genotyping HPV was assessed by the mean of the Cohen’s kappa (κ) coefficient. Results of all four HPV assays agreed in 85.9% of the cases when using the Abbott RealTime manufacturer’s recommended quantification cycle (IMPORTANCE This study proves that the novel Allplex HPV28 assay has a good diagnostic performance in comparison with the well-known, validated, and frequently used Roche Cobas 4800, Abbott RealTime, and Anyplex II HPV28 assays. According to our experience, the novel Allplex HPV28 assay had a user-friendly and automated workflow with short hands-on time, had an open platform which facilitates the use of add-on assays, and provided quick and easy-to-interpret results. Together with its ability to detect and quantify 28 HPV genotypes, the Allplex HPV28 assay could therefore potentially provide opportunities for the simplification and standardization of future diagnostic testing programs. As of today, it has been proven that most of these cervical malignancies are associated with an infection by the human papillomavirus (HPV), the most common sexually transmitted pathogen worldwide cancerous lesions . To accoqC) values for 28 distinct genotypes at once.Over the past years, over 100 different HPV assays have been developed, and they widely vary in the number and type of target HPV genotypes, their working mechanism, and their clinical utility , 10. Amo12–Interestingly, considering the novelty of the Allplex 28 HPV assay, no published studies have compared this assay with commercially available HPV assays. Therefore, in this study, we compared the diagnostic performance of the Allplex HPV28, the Anyplex II HPV28, the Abbott RealTime HR HPV, and the Roche Cobas 4800 HPV assay for their ability to detect and genotype hrHPV in mocked self-samples. In n = 33, 29%) or married , 19 (17%) were living together or in a relationship, 18 (16%) were divorced, and 10 (9%) were widowed. One-third of women  had a history of smoking, 20 (18%) were smoking during the study, and 75 (67%) had taken hormonal contraceptives for a period of over 5 years. Most women  were diagnosed with normal cytology, 20 (18%) with LSILs, 26 (23%) with HSILs, 11 (10%) with ASC-US, 17 (15%) with ASC-H, and 10 (9%) with cervical cancer.An overview of the clinical and sociodemographic characteristics of the study population is shown in In qC cutoff for positivity . The HPV assays hereafter agreed in 91.2%, with 7.0% of all cases testing negative and 84.2% testing positive  or with the Roche Cobas assay only .sitivity , seven spositive . All samqC cutoff for positivity (<32.00) (qC cutoff range for positivity (32.00 to 36.00)   and the o 36.00) . The oveo 36.00) . When us to 1.00 . While fqC values of different HPV assays are shown in qC values of the different HPV assays were demonstrated to be strongly and positively correlated  and statistically significant .Pearson correlations between the In this study, we aimed to compare the performance of a new HPV assay, i.e., the Seegene Allplex HPV28 assay, and the Roche Cobas 4800 HPV, Abbott RealTime HR HPV, and Anyplex II HPV28 assays. Earlier studies evaluated and compared the Roche Cobas, Abbott RealTime, and Anyplex II HPV28 assays to each other , 14, 17 n = 17) of ASC-H cervical cytology, the Anyplex and Allplex HPV28 assays both missed one case and the Abbott RealTime missed three.Overall, HPV DNA positivity rates within our study ranged between 78.9% and 92.9% (mean positivity rate of 87.3%), with the highest positivity rate detected for the Roche Cobas HPV assay. These findings are in line with previous research from Cho and colleagues 2019), who analyzed vaginal self-samples with the Abbott RealTime, Roche Cobas, and Anyplex II HPV28 assays and hereby also reported the highest positivity rate for the Roche Cobas assay (mean positivity rate of 87.4%) 19, who a, the posqC cutoff for positivity (<32.00). Of all samples, 11 samples (9.7%) tested positive with all HPV assays, except for the Abbott RealTime assay. Slightly lower than the research of Park and colleagues , as described by earlier research of Kim and colleagues , with results reported as negative indicating qC values of 32.37 and 32.47. Of the 11 samples that initially showed discrepancies for the Abbott RealTime assay, 7 (63.6%) could be explained by a similar phenomenon as the one explained above . For the four remaining discordances, as well as for the five samples testing positive with the Roche Cobas assay but negative with all other HPV assays, no NDBA cases were observed. However, as shown by multiple previous studies, the Roche Cobas 4800 assay is known for having a relatively high sensitivity compared with other HPV assays  phenomenon for the Abbott RealTime assay, i.e., the event in which the assay reports samples to be negative despite showing an amplification curve with a lleagues , also oclleagues  suggesteV assays , 13, 17.V assays . Overalltegories , socioectegories , or typetegories .According to our experience, the novel Allplex HPV28 assay had a user-friendly and automated workflow with a short hands-on time, had an open platform which facilitates the use of add-on assays, and provided quick and easy-to-interpret results. Notably, our study included two assays that display individual results for HPV16 and HPV18, along with a pooled result for the other 12 hrHPV genotypes  and two assays that are able to detect and differentiate all 14 distinct hrHPV genotypes (Anyplex and Allplex HPV28). Consequently, it should be remarked that results found within our study refer to the agreement in the presence of hrHPV genotypes in general , rather than of specific genotypes. Considering that discrepancies within specific hrHPV genotypes could still occur without being displayed in our test results, as demonstrated in Table S1 for the Anyplex and Allplex HPV 28, we therefore recommend future studies to include extended genotyping techniques to strengthen the precision of assay comparison. This recommendation could be of particular interest for the further implementation of extended hrHPV genotyping in clinical settings, which may aid in the improved risk assessment of cervical lesions in those with abnormal cytology and associated referral for colposcopy. Lastly, due to the unavailability of the Hologic ThinPrep medium in Ecuador, brushes collected here were instead rinsed in Roche Cell Collection medium. As this origin-specific use of preservation medium results in a minimal chance of performance bias because of potential increased or decreased compatibility with a specific HPV assay, we acknowledge this as a limitation of our study.Our study proved that the novel Allplex HPV28 assay has a good diagnostic performance in comparison with the well-known, validated, and frequently used Roche Cobas, Abbott RealTime, and Anyplex II HPV28 assays. Together with its ability to detect and quantify 28 HPV genotypes, the Allplex HPV28 assay could therefore potentially provide great opportunities for the simplification and standardization of future diagnostic testing programs. Nevertheless, further research on the safety, efficiency, and cost-effectiveness of newly developed HPV DNA assays for testing on self-collected specimens remains essential to support their implementation in practice and thereby improve future cervical cancer screening programs in all settings.https://elevate-hpv.com), an acronym for “EarLy dEtection of cerVical cAncer in hard-to-reach populations of women through portable and point-of-care HPV TEsting.” Within this project, a prospective collection of cervicovaginal samples (liquid-based cytology) from women was performed at the University Hospital of Ghent (Belgium), Instituto Portugues de Oncologia de Lisboa , and Barretos Cancer Hospital (Brazil) and the Sociedad de Lucha contra el Càncer (SOLCA), Vicente corral Moscoso, Asociacion Pro Bienestar De La Familia Ecuatoriana (APROFE), and Jose Carrasco Hospitals (Ecuador).The current cross-sectional study was part of the ELEVATE project  they were aged 25 to 65 years, (ii) they were sexually active and not pregnant, (iii) had no history of hysterectomy, and (iv) had never been treated for cervical lesions or cancer. Women were excluded from the study if they (i) were menstruating at the time of their visit or (ii) had sexual intercourse with vaginal penetration within the last 48 h before their visit. Study participants were informed on the study goals and procedures by their attending gynecologist, and all participants provided written informed consent for the use of their samples for HPV DNA detection. Participants were not financially rewarded.From each of the included ELEVATE participants, two mocked self-samples were collected by a gynecologist. To achieve this collection, the gynecologists held two Viba-Brush self-testing devices  together for sampling and attempted on mimicking self-collected specimens by swabbing over and around the cervix rather than going endocervical and thereby also touching a part of the more superficial vaginal canal. In Belgium, Portugal, and Brazil, the brushes were each rinsed in Hologic ThinPrep pap test medium . Due to the unavailability of Hologic ThinPrep pap test medium in Ecuador, the brushes here were rinsed in Roche cell collection medium . Hereafter, one of the samples was sent to Barretos Cancer Hospital, Brazil, for analysis via the Roche Cobas HPV assay and the second one of the pair was sent to Ghent University Hospital, Belgium, for further HPV testing. From this ELEVATE cohort, a selection of 114 participants, from whom cervical cytology data were available, was made for the current study. This selection included a balanced representation of the different sampling locations  and cervical cytology categories . On this selection, additional HPV testing via the Abbott RealTime HR HPV assay, the Anyplex II HPV28 detection assay, and the Allplex HPV28 detection assay was performed at Ghent University Hospital, Belgium. Samples were stored at an optimum temperature of 2 to 8°C in between HPV testing.qC value of 32.00 as the positivity cutoff for HPV16, HPV18, and other hrHPVs, after sample preparation and extraction on an m2000sp instrument (Abbott Molecular Inc.). The Roche Cobas HPV assay was performed on the Cobas 4800 system (Roche Diagnostics), using a qC cutoff value of 40.50 for HPV16 positivity and 40.00 for HPV18 and other hrHPV positivity. Both the Anyplex II HPV28 and Allplex HPV28 detection assays were performed using a CFX96 real-time thermocycler . In the Anyplex II HPV28 detection assay, the HPV load was categorized as either high (+++), medium (++), or low (+), if the qC value was ≤31.00, between 31.00 and 40.00, or ≥41.00, respectively; in the Allplex HPV28 detection assay, a qC cutoff value of 42.00 was used for positivity for all hrHPV genotypes. All assays and procedures (including the qC cutoffs) were executed following the manufacturers’ instructions. While the Abbott RealTime HR HPV and Roche Cobas HPV assay provide individual results for HPV16 and HPV18 along with a pooled result for the other 12 hrHPV genotypes, the Anyplex II HPV28 and Allplex HPV28 detection assays are able to simultaneously detect, differentiate, and quantify 28 distinct HPV genotypes, including low-risk HPV genotypes  the Abbott RealTime HR HPV assay , (ii) the Roche Cobas 4800 HPV assay , (iii) the Anyplex II HPV28 detection assay , and (iv) the Allplex HPV28 detection assay (Seegene Inc.). All four assays use a combination of multiple primers and probes to amplify and detect a conserved L1 region of different HPV genotypes . Moreoveenotypes . To enabqC cutoff for positivity. However, as described in earlier research  (NDBA [not detected but amplified]). Therefore, in the current study, concordance and agreement between the different HPV assays was determined both by using the Abbott RealTime manufacturer’s recommended qC cutoff for positivity (<32.00) and by using a less stringent qC range for positivity on NDBA samples (32.00 to 36.00). To examine the correlation between the HPV loads detected by different HPV assays (except for the semiquantitative Anyplex II HPV28), Pearson correlation coefficients for the qC values were calculated. For negative samples, an arbitrary qC value of 45.0 was used. P values of less than 0.05 were considered to be statistically significant.All results were statistically analyzed and visualized using the Prism 9 software . Per HPV assay, the HPV DNA-positive cases and cases of coinfection were determined for different cervical cytology types. Notwithstanding that both the Anyplex and Allplex HPV28 assays can detect the 14 hrHPV genotypes individually, the Roche Cobas and Abbott RealTime assays can only distinguish HPV16, HPV18, and other hrHPV genotypes. Accordingly, to allow for a comparison, we clustered all hrHPV genotypes except for HPV16 and HPV18 within the Anyplex and Allplex HPV28 results as well. Consequently, samples were considered coinfected only when they were infected with HPV16 and/or HPV18 and at least one other hrHPV genotype. Coinfection rates were calculated by dividing the number of coinfected cases by the number of cases positive for HPV DNA. As no reference standard assay for the use of mocked self-samples was available, concordance in terms of detecting and genotyping  HPV among the four different HPV assays was assessed by the positive percent agreement, the negative percent agreement, and the overall percent agreement with Cohen’s kappa score calculation. Within these head-to-head comparisons, either the Roche Cobas 4800 assay or the Abbott RealTime HR HPV assay was used as the nonreference standard. Two assays were negatively agreeing if they both demonstrated a negative result for all hrHPV genotypes, were positively agreeing if both detected at least one hrHPV genotype, and were overall agreeing if they both generated the same result (HPV16/HPV18/other hrHPV genotypes). A kappa statistic (κ) of 0.81 to 1.00 was interpreted as an almost perfect agreement, 0.61 to 0.80 as a good agreement, 0.41 to 0.60 as a moderate agreement, 0.21 to 0.40 as a fair agreement, 0.00 to 0.20 as a minimal agreement, and < 0.00 as a poor agreement between the different tests . For allEthical approval was obtained by the ethical committee of Ghent University Hospital (reference 2019/1687), the institutional review board (IRB) committee of Cuenca University (reference 2018-074EO-I-Ext#1), the ethical committee for Health of Instituto Portugues de Oncologia de Lisboa Francisco Gentil (reference UIC/1267 and 290/021), and the National Research Ethics Commission of Brazil (reference 16983119.7.0000.5437). Samples were collected in accordance with guidelines and regulations of the Helsinki Declaration, and all data were handled according to General Data Protection Regulations (GDPR).


People with HIV (PWH) and their sexual partners have increased risk of human papillomavirus (HPV) infection. Despite recommended HPV vaccination for PWH aged 18–26 years, vaccination rates among PWH remain low. This qualitative study used the Information–Motivation–Behavioral Skills (IMBS) model to identify factors influencing the decisions of PWH around promoting HPV vaccination to their sexual partners. Fourteen PWH with diverse sociodemographic characteristics participated in four focus-group discussions. Data were analyzed using thematic content analysis; codes and themes included IMBS constructs. For the information construct, the need for improved HPV education emerged as the driving factor for HPV vaccine uptake and discussing HPV vaccines with partners. Focal reasons for being unvaccinated included low knowledge of HPV risk, asymptomatic cancer-causing HPV, HPV vaccines, and vaccine eligibility. Salient factors in the motivation construct included the preventive benefits of HPV vaccination to both self and sexual partners. Salient factors in the behavioral skills construct included: accessing vaccine, low self-confidence and skills for promoting vaccination, relationships with sexual partners, partners’ vaccine hesitancy, and stigma. Race/ethnicity impacted HPV vaccination promotion; important determinants included perceptions of HPV-related diseases as “White people’s diseases” among Black people, and discrimination against those with HPV-related diseases among the Hispanic population. Human papillomavirus (HPV) is the most common sexually transmitted infection (STI) . The CenDespite increased HPV vaccination among adolescents nationwide and in Oklahoma ,13, genePrevious HPV vaccination promotion efforts have targeted eligible individuals, parents, and healthcare providers ,19. HoweThe IMBS model has been used to promote several health behaviors ,27,28,29We conducted four focus-group discussions (FGDs) with 14 PWH recruited from the Infectious Diseases Institute of the University of Oklahoma Health Sciences Center (OUHSC) through clinic staff or patient referrals. Using purposive sampling we selected diverse PWH, including high-risk subgroups , age (≥18), urban/rural residence, ethnicity, and sexual identity to enhance generalizability and relevance of findings for high-risk and under-represented groups. Patients were excluded if they had experienced a medical adverse event due to HPV vaccination, were unable to speak or read English, or had impaired cognitive function. The study was approved by the OUHSC Institutional Review Board (IRB# 9458), and subjects gave informed consent prior to participation.FGDs were scheduled for 60–90 min and occurred in a private meeting room at OUHSC. At least two research staff members were at each FGD: one facilitator and one note taker. Discussions were voice recorded and transcribed verbatim. Participants identified themselves and others using an assigned unique identification number to maintain privacy. The interviewer(s) asked general, IMBS construct-based, open-ended questions such as “What would motivate you to engage in encouraging sexual partners to obtain HPV vaccination?” or “When and how would you talk to them about HPV vaccination?” Potential barriers to discussing HPV vaccination and obtaining HPV vaccines were also explored. One FGD conducted in Spanish was translated to English by a professional transcription and translation service provider.Qualitative data were analyzed using a deductive approach to thematic content analysis with the aid of MAXQDA version 20.4.1, VERBI software, Berlin, Germany. One researcher conducted an initial coding of all transcripts to assign themes, and two other researchers reviewed the codes and quotes. Any discrepancies were discussed to establish agreement in coding. Themes were based on the IMBS constructs; other relevant issues/topics, such as stigma, and race/ethnicity, were also identified inductively as they were found in the data.The mean age was 48 years (range: 29–65 years) at the time of study. Five identified themselves as women (35%). Among self-identified men, seven identified themselves as homosexual and two identified as heterosexual. Per self-identification, participants were of various races, including White, Asian American, Native American, and African American; two were Hispanic. All participants were HIV positive. Over half of the participants received some college education.A lot of people don’t think of themselves as being at risk and it would be great to help educate them”.In the information construct of the IMBS model, the need to improve HPV education emerged as the driving factor for HPV vaccine uptake and for talking to partners about HPV vaccines. Specifically, focal reasons for being unvaccinated included lack of knowledge about: HPV risk, asymptomatic cancer-causing HPV strains, HPV vaccines, and HPV vaccine eligibility for high-risk adults. A 65-year-old White, homosexual man spoke about the lack of knowledge regarding HPV risk factors within the general public: “Other people [who] don’t feel that they are at high risk think that things are never going to happen to them. They’re never going to get sick, or they say they’re immune to any kind of illness. So, it’s harder to motivate those people because they think that with having their partner, or not having a partner, or protecting themselves, nothing is ever going to happen to them. So, it is more difficult for me to motivate someone who isn’t high risk like me”.A 48-year old Hispanic homosexual man discussed difficulties in promoting immunocompromised individuals due to misperceptions regarding risk: “Some people without the education [are] going to think we already have it anyway, so [why] get the vaccine right? Let them know there’s more than one strain and even if they already have one strain you can prevent others which can also cause cancer”. A 52-year old Native American, homosexual man stated: “I’ve been HIV positive for many years, and I was diagnosed with HPV because I knew nothing about it, [until] my doctor said I had it”. He continued: “I wish when I was growing up we had sex education. They told you about how not to get pregnant, but they didn’t tell you about HIV and all that stuff… I’ve never had a sexually transmitted disease ever. I come out, boom, I get the big one [HIV]”. This participant also discussed his decision to have open communication with his children about sexual activity at a young age: “I talked to my sons when they were growing up. They were 3 years old and I had prophylactics laying around, I had POZ magazines laying around. They read it and I didn’t force it down their throats. I just gave them the option so that they know what they’re getting into”.A 41-year old White, heterosexual woman stated: “Yes, [I would encourage my sexual partners to have the HPV vaccines] to protect myself. I’m more at risk…I think everyone thinks in themselves first, and then in others”. A 52-year old White, Asian American, homosexual man spoke about being non-monogamous: “I don’t have a significant other right now, but if I did have someone, I would want them protected and then in turn that would protect myself.” He also stated: “If you love somebody you want them to get it done because the research says that it can prevent cancer and other things for their own health, right?” A 41-year old White, heterosexual woman relayed her experience with HIV and a motivation to protect her partner: “Mostly loving them and caring about them. So they won’t have to go through some of the stuff we had to go through. Especially from a female perspective and having to do procedures that I had to have done due to [HIV], and I think it’s important that the person you’re with is vaccinated because they may have a different strain and then that can cause more abnormal cells and more procedures or, you know, potential cancer”.Salient factors in the motivation construct included HPV vaccination-afforded prevention of HPV infection/transmission and cancer for both self and sexual partners. A 48-year-old Hispanic, homosexual man stated: “X. [a friend] told me she wasn’t able to get it from her primary doctor yet, because [it’s] not available… Only the county health department has it… I guess it’s early stages [of] availability”.Salient factors related to the behavioral skills construct included the unavailability or inaccessibility of vaccine, lack of self-confidence and skills to talk about HPV vaccination with sexual partners, the relationship with sexual partners, and partners’ vaccine hesitancy. A 45-year-old White, heterosexual man reported: “I think the people that are already on food stamps or Soonercare [Oklahoma’s state Medicaid agency] or [of low socioeconomic status] or high risk should automatically be able to get it without worrying about cost. Because I can honestly say that I think if there’s a cost associated with it, most people probably won’t do it because of that”.Several participants also listed associated cost of and transportation issues regarding having the HPV vaccination as barriers. A 41-year old White, heterosexual woman stated: “I think that this will be better received after an initial talk with a caseworker or case manager with partners available. I myself as a partner have a certain level of understanding of what HPV is”. She also reported emotional relationships with sexual partners as a barrier: “I’m married and that makes it easier for us to communicate. But, at that moment, I couldn’t hear you because I was emotional and mad at you. So, I think a lot of times a neutral third party can always get the conversation started”. However, a 65-year-old White, homosexual man felt bringing a third party would hinder the conversation: “My concern with bringing extra people into the conversation is that you may also be inhibiting the person that needs to hear the message from their partner directly, they may feel embarrassed. It’s inappropriate to share that information publicly”.Some patients reported low self-efficacy in talking to their sexual partner(s) about HPV vaccination. A 35-year-old African American, heterosexual woman stated: “Well, my stable partner would be the one that I have that comfortability with. It depends if I feel comfortable with them. I don’t know how he would react either, but I think I could tell him. As I said, if it’s with casual relationships, that trust would be a barrier”.A 48-year old Hispanic homosexual man stated his preference for promotion to regular partners over casual partners: “Some people are opposed to vaccines in any form. Unfortunately, that’s just a barrier that sometimes we just can’t do anything about right now… I have several friends that are like that. They don’t get their kids vaccinated and things like [that] that deal with the government conspiracy. Like my cousin’s wife she is a chiropractor and she feels like they’re not necessary. She believes in like holistic medicine and things like that”.Several participants addressed vaccine hesitancy as a barrier. A 41-year-old White, heterosexual woman stated: “hen I so-called “came out,” half my life was over because I had never acknowledged that I was gay. I got [HIV] positive pretty quick and we’ve always had a group called “Oklahoma’s Living Positive,” so there’s a whole range of people and the more you talk about it the better it is… I guess it’s probably the same with HPV”. A 41-year old White, heterosexual woman discussed the current vaccine eligibility recommendations for high-risk individuals and associated stigma: “[The] HPV vaccine is recommended through age 26 for all women, men who have sex with men, bisexual, transgender and people with HIV. But what about [heterosexual] men through age 26?… First of all, I feel it would cause the stigma. It’s not right. Second… that means that all the promiscuous heterosexual male and females are going to say, ‘I’m not in that group,’…but they’ve already been exposed to [their] partner”.Stigma toward sexuality-related issues central to the topic of HPV vaccines emerged as a barrier to both HPV vaccination discussion and uptake. A 52-year old Native American, homosexual man related the stigma behind their sexual orientation and HIV+ status with HPV: “WI’m always willing to participate because there’s not many heterosexual African American women who get to share their opinions about their status and how they feel about their status and research on how to get better. I want to be an advocate for myself so I won’t be left out of the study”. She also explained that many African Americans think that HPV does not affect them: “It’s been marginalized as this one thing, and African Americans think it’s a rich White person thing. Like ‘oh please girl put a condom on and I’m not going to tell you again.’ So you’ve got to appeal to every group… It touches everybody, it doesn’t matter. [People think that HPV is a] rich White people’s disease, and I don’t personally believe like that, but there are people who would be like ‘child please we are Black we don’t need that.’ And we [are] the main ones dying from stuff because we believe we don’t get that”. A 48-year-old Hispanic, homosexual man spoke about the discrimination faced in the Hispanic community: “And also, if it’s in the Hispanic community, as I said, everything is more difficult. Well, like us—well, for me—thinking about the illness, sometimes it’s not so much the illness but the discrimination against you”.Race/ethnicity also emerged as a factor affecting HPV vaccination discussion and uptake. One 35-year-old African American woman spoke about her reason for participating in the study: “This study used the IMBS model to identify salient factors influencing the decision of PWH to promote or not promote the recommended HPV vaccine to their sexual partners. Given low HPV vaccine uptake and awareness among adults, including high-risk populations and PWH, and promising data regarding the role of patient-led partner referral approaches, this is a critical area of inquiry to meet the needs of PWH ,20,21,22For the information construct, better education regarding both HPV risk and HPV vaccines were identified as key factors influencing decisions to promote HPV vaccines. These findings are consistent with previous studies examining perceptions of HPV and attitudes towards HPV vaccines in high-risk populations ,26. AmonFor the motivation construct, preventive benefits of HPV vaccination for both self and sexual partners were key, including preventing the spread and limiting the health consequences of HPV for family and loved ones. This contrasts with recent reports regarding other vaccines in the context of COVID-19, vaccine safety concerns, and increased vaccine hesitancy, which indicate emphasizing individual benefit is more efficacious than prosocial benefit when targeting vaccine hesitancy and uptake ,31,32. IFor the behavioral skill construct, lack of self-confidence and/or skills to talk about HPV vaccination with sexual partners, relationships with sexual partners, and partners’ vaccine hesitancy were key factors. In addition, issues related to procurement of the vaccine, including perceived unavailability or inaccessibility, were barriers to vaccine uptake; barriers specifically related to transportation and cost were also identified, consistent with prior studies, particularly among individuals of low socioeconomic status ,43,44,45Lack of confidence and/or skills to talk about HPV vaccination with sexual partners results in reliance on third parties to promote vaccine uptake to loved ones. Sexual minority men reportedly do not typically discuss HPV vaccination with sexual partners, and may be responsive to and reliant upon healthcare providers, who may or may not bring up vaccination ,27,46,47Stigma and race/ethnicity were also identified as barriers to HPV uptake and promotion independent of the IMBS model. Stigma regarding sexual orientation and HIV/HPV status were barriers to promotion. This was particularly concerning for specific groups recommended by the CDC to receive HPV vaccination  and the potential to reveal a vaccine recipient’s status. Such stigma limits willingness to discuss sexuality, or related health concerns, with healthcare providers, in part based on culturally insensitive healthcare ,53,54,55This study has numerous strengths, including our purposive sampling strategy to specifically target high-risk and potentially under-represented groups. It also fills a critical gap in the literature considering the recently expanded recommendations with respect to HPV vaccination . However, it is not without limitations. These include, most notably, a small sample size, as well as a final sample which was restricted to adults in Oklahoma. These factors may limit generalizability to other populations, and future work should expand upon these data.Collectively, these data indicate that among PWH, there remains a need to improve awareness of HPV vaccines, while helping overcome both financial and logistical barriers. Our data corroborate previous work highlighting the importance of healthcare providers or other third parties in facilitating HPV vaccine uptake, by overcoming stigma and/or vaccine hesitancy. However, presently, healthcare provider interactions remain insufficient to address gaps in vaccination given associated stigma, and lack of awareness among healthcare providers and non-inclusive healthcare settings. Previous evidence supporting partner referral-based interventions in PWH highlights a potential avenue for improved vaccination in this high-risk population ,62. SincTo promote HPV vaccination among PWH, it is important to address barriers, improve HPV-related education, overcome HPV-related misconceptions, and equip PWH with communication skills to discuss HPV vaccination with both their sexual partners and other members of the PWH community. This should be coupled with other approaches  for optimal impact.