A health disparity exists for African American (AA) women with systemic lupus erythematosus (SLE) who have increased prevalence of human papilloma virus (HPV) infection and cervical neoplasia. We used a self-sampling brush to obtain cervical cells to assess cytology, HPV infection, and vaginal cytokine production in AA women with SLE.Thirty AA women with SLE ages 18–50 years consented to participate. Clinical information was obtained by review of records and patient interviews, and surveys administered to assess cervical health history, knowledge of HPV, and satisfaction with the self-sampling brush. Vaginal samples were analyzed for cytology, HPV DNA and RNA, and vaginal cytokine RNA.Our cohort  had moderate/severe SLE and were on immunosuppressives. The majority had history of abnormal pap smears (63%) with prevalent risk factors for HPV infection: multiple sex partners (9.5 ± 7), not vaccinated for HPV (83.3%), smoking (26.7%), and not using condoms (73.3%). Most were aware of HPV causing cervical cancer (70%) but were unaware of other HPV-related diseases. Most preferred self-sampling over traditional pap smear (80%). Abnormal cytology was detected in 13.3%. HPV DNA was detected in 70%, with half showing multiple types, and all showing active infection (+RNA). HPV-infected samples demonstrated RNA expression of multiple cytokines with no specific/ consistent pattern.Our high-risk cohort lacked knowledge about HPV-related diseases and were not employing strategies to reduce their risk with vaccination and condoms. This study highlights the need for cervical health education, increased monitoring, and intervention in these high-risk women. Consequently, this group of women has poor access to health care due to barriers in insurance, resources, education, and social support.6African American (AA) women bear significant disease burdens for both systemic lupus erythematosus (SLE) and cervical cancer with higher proportions for presentation of cervical cancer at later stages, as well as increased morbidity and mortality for both diseases.Current guidelines by the American College of Obstetricians and Gynecologists (ACOG) and the United States Preventive Services Task Force (USPSTF) for cervical cancer screening are inadequate to monitor these high-risk women, and there are no specific recommendations for women with SLE such as exists for women with HIV. Pap smear testing requires an invasive examination by a trained health care professional in an office setting. Underserved populations with poor resources may have social and economic barriers that prevent compliance with this type of preventive health care.Little is known about the biology and genetics of human papilloma virus (HPV) infection and related cervical disease in women with SLE. We sought to pilot a novel methodology using a self-sampling brush to obtain cervical cells for HPV DNA and RNA, RNA for vaginal cytokines, and cervical cytopathology to understand this disease process better and develop a means to make cervical monitoring more accessible and less costly to these high-risk, underserved women.7–10 Exclusion criteria: current pregnancy, and not meeting inclusion criteria. Clinical information was obtained by records review and interviewing the patient, and surveys administered to assess cervical health information, knowledge of HPV, and satisfaction with using the self-sampling brush.Thirty AA women with SLE recruited from the Rheumatology clinics consented to participate in the study. Inclusion criteria: AA women with SLE and meeting American College of Rheumatology (ACR), Systemic Lupus International Collaborating Clinics (SLICC), and 2019 European Alliance of Associations for Rheumatology (EULAR)/ACR classification criteria, ages 18–50 years, able to consent and obtain vaginal self-sampling.®) was provided by the manufacturer.11 There were no serious adverse events from the device in this study.Vaginal self-sampling by the participant was performed after instruction and the brush immersed in an RNA-stable ThinPrep vial. Hologic ThinPrep automated processor was used for slide preparation and cytology was read by a board-certified cytopathologist. The vaginal self-sampling device .Cervical specimens were divided for separate DNA and RNA isolations. Genomic DNA was extracted using a standard lysis/Proteinase K/isopropanol precipitation method, followed by ethanol wash. RNA extraction was performed using the ROCHE High Pure Viral RNA Kit and frozen at −80°C for further analysis. HPV DNA detection and typing was carried out by competitive multiplex polymerase chain reaction (PCR) to amplify the E6 region of HPV, followed by probe-specific single base extension to discriminate between naturally occurring HPV present in the sample and the synthetic competitors included in the reaction. Matrix-assisted laser desorption/ionization time of flight mass spectroscopy allows separation of products on a matrix-loaded silicon chip array.® Green One-Step RT-PCR Kit  and run on the CFX96 qPCR machine (Bio-Rad).The concentration of RNA extracted from the samples as detailed above was determined using the NanoDrop One spectrophotometer  and purity of the RNA was assessed through the A260/A280 ratio. A custom-developed human cytokine 96-well panel assay  was used to detect the mRNA for 12 cytokines  using the iTaq™ Universal SYBR−ΔΔCt method as described.13 Assay data were then analyzed using the delta-delta Ct method with Bio-Rad CFX Manager 3.1 software. A heatmap was plotted using the ComplexHeatmap R package utilizing the ddCt values obtained from reverse transcription (RT)-PCR for the various cytokines. The 2−ΔΔCt values for each measured cytokine was Z-score normalized across samples for the heatmaps.Values were normalized to glyceraldehyde 3-phosphate dehydrogenase and calculated with 2Z-score for a sample indicates high expression in that sample while a negative Z-score indicates low expression in that sample. Additionally, the expression level was compared between the HPV-positive and the HPV-negative groups for each cytokine using the Mann–Whitney analysis. Boxplots were generated using the ggpubr package in R.For each cytokine, a positive 14For cervical cytology, the cells from the brush system were resuspended in 30 mL of RNA-stable ThinPrep provided by Hologic (ThinPrep System). The specimen was processed using the Hologic ThinPrep T2000 or T5000 automated Processor to prepare a microscope slide for cytopathological examination by a certified histotechnologist and board-certified cytopathologist. An interpretation of the smear was rendered using the Bethesda System for Reporting Cervical Cytology 3rd Edition.t-test and nonparametric testing to determine any significance. Data for RT-PCR were generated by Bio-Rad CFX manager 3.1 software and the cytokine gene expression fold-change was calculated using the formula 2−ΔΔCt and the Prism 9 Software . The heatmap was plotted using the ComplexHeatmap R package program and Boxplots were generated using the ggpubr package in R.Descriptive analysis was performed for the clinical features, survey results, and HPV DNA and RNA results. For the cytokine analyses, cytokine heat mapping was used with statistical analysis using machine learning to determine any correlations between cytokines and clinical and HPV variables using Student's www.irb.org).Written consent was obtained from each participant in the study.This study was approved by the Institutional Review Board (IRB) of Ascension St. John Hospital in Detroit, Michigan , and disease duration a mean of 14.5 years (±9.9). These women had moderate-to-severe disease, 40% of which had lupus nephritis and 16.7% neuropsychiatric lupus. All were treated with chronic immunosuppressives with 96.7% on corticosteroids, 96.7% on hydroxychloroquine, and 80% on nonsteroid immunosuppressives .Most of the women had adverse cervical health  and reprPrior HPV test results were documented in the records in only 43.4%, of which eight were positive. Risk factors for acquiring HPV infection were prevalent in this cohort with history of sexually transmitted diseases other than HPV in 70%, not being vaccinated for HPV in 83.3%, multiple lifetime sex partners (mean = 9.5 ± 7), history of cigarette smoking (26.7%), early age of first sexual intercourse (mean = 16.5 years ±3.2 years), and not using condoms (73.3%). Pregnancy histories showed a mean of 2.3 pregnancies (±2.5) with a mean of 1.7 liveborn infants (±1.4).Adverse pregnancy outcomes were prevalent in these women with 12 having history of miscarriages and 2 having stillbirths but there was no apparent association with sample positivity for HPV. Nine patients had voluntary interruption of pregnancies. Most of our patients had insurance that reflected poor socioeconomics or disability by insurance type. Of the 30 patients, 17 (56.7%) had Medicaid insurance, 9 (30%) had Medicare insurance (through disability), and only 4 (13.3%) had commercial insurance.The women in general lacked knowledge of HPV-related oral and genital diseases other than cervical cancer . Most of15 All HPV-positive samples showed active infection (+RNA). Most of the types identified were the well-described high-risk types and some newly identified as potential high-risk types, 73 and 90. HPV 16 and 18 were identified in only eight samples (26.7%), with 3 showing type 18, 6 showing type 16, with one sample containing both 16 and 18 types. Only five women were vaccinated for HPV, and of those, three were positive for HPV, including types not covered by the bivalent and quadrivalent vaccines: one tested positive for HPV 52, one for HPV 90, and one for both HPV 31 and 56, types not included in the bivalent or quadrivalent vaccines, but neither had HPV 16 or HPV 18 that are included in both vaccines.Most of the samples were positive for HPV DNA 73.3% (21/30) with about half of these (10/21 samples) being positive for multiple types . This isThe nonavalent vaccine was implemented in 2016, which was after the women in our study would have been eligible to receive this vaccine. Of note, of the vaccinated women showing +HPV, only HPV 90 is not included in the nonavalent HPV vaccine.16 All four samples with abnormal cytology were positive for multiple HPV types. In addition, 53% (16/30) showed other non-neoplastic abnormalities on cytology, such as bacterial vaginosis, trichomonas, parakeratosis, keratosis, and presence of fungi. All women were notified of the research study cytology findings and HPV testing and strongly advised to see their gynecologist for a gynecological exam with a pap smear and HPV testing. Most patients were not aware when notified that they had an HPV infection.Cytology was performed on all samples using standard techniques applied to samples in the outpatient setting as detailed under methods. Cell samples obtained from the brush showed preservation of morphology, but quality indicators were generally absent. Abnormal cytology was detected in 13.3% (4/30) with three showing low-grade squamous intraepithelial lesion and one showing ASCUS. This is above the expected population rate of 5.1%.p = 0.042). Two cytokines, IL-12B (p = 0.073) and IL-13 (p = 0.099), also showed a trend of higher expression in HPV-positive samples compared with HPV-negative samples. However, these were not statistically significant at p = 0.05 significance threshold.The cytokine RNA analyses showed that HPV-negative samples had low gene expression for all cytokines tested . In cont1–3 The immune suppression in SLE likely creates a facilitative environment for persistence of HPV infection and subsequent malignant transformation. Just having SLE as a diagnosis was shown to be associated with increased prevalence of premalignant lesions even though these patients had fewer classical risk factors. SLE patients also are exposed to long-term immunosuppression, which has been shown to increase the chance of presenting with more premalignant lesions.17To our knowledge, this is the first study to assess HPV DNA and RNA and vaginal RNA cytokine profiles in AA women with SLE, who are high risk for HPV infection and cervical cancer. In addition, we used a novel self-sampling technique to collect cervical cells and showed that cell morphology is preserved, and cytology can be determined using the standard technique for processing and reading pap smears in the outpatient setting. HPV infection is a serious health issue for women with SLE as they have a higher incidence of genital infection and cervical neoplasia.19Cervical cancer screening remains problematic for these women as current screening guidelines are inadequate for these women. Current guidelines recommend initial screening at age 21 years, then every 3 years from age 21–29 without HPV cotesting. For ages 30–65, screening either every 5 years along with HPV cotesting, cytology alone every 3 years, or FDA-approved high-risk HPV testing alone every 5 years.20 There are no specific screening guidelines for women with SLE who are immunosuppressed similarly to those with HIV.For the HIV population, who are considered at increased risk for cervical cancer, pap smear screening is recommended in the first year following diagnosis of HIV (regardless of age), then yearly thereafter for 3 years. After that, if three consecutive pap smears are normal, then pap smear testing can be done every 3 years. HPV testing should be done following the diagnosis of HIV. If HPV cotesting is available, then cotesting can be done at the time of diagnosis of HIV, and if positive for HPV 16 or 18, then colposcopy should be performed.16 Thus, although the cytology using the brush sampling may be less sensitive than the traditional pap smear, a less sensitive test done more frequently is better than no testing.Although most of the samples lacked quality indicators for cytology, we did detect abnormal findings in 13.3%, which is more than twice the expected population rate of 5.6%.22 In women with SLE followed for 3 years, about half (48.5%) of incident infections persisted ≥6 months and only 14.7% of these were cleared during the study period.23 More studies now support the concept of HPV latency with HPV remaining dormant expressing a low copy number in infected stem cells of the basal cervical cell layer with intermittent reactivation.24–28Newly acquired HPV infections generally clear or become undetectable in 90% within 2 years, which was previously thought to signal viral clearance from the cervix.24 Reactivation of dormant HPV infections can occur in situations where host immune surveillance in the cervical microenvironment is perturbed, such as with aging, prolonged contraception, smoking, or immunosuppression.29–31 Increased redetection of HPV after clearance has been reported in women with HIV, organ transplant recipients, and patients on immunosuppressive drugs.33 In addition, women who are abstinent show redetection of HPV.34Local cell-mediated immunity maintains immune control of HPV infection. Continuous surveillance by resident T memory cells in the cervical epithelium downregulates HPV gene expression and keeps the HPV dormant.35 Although natural infection does not generally result in protective antibodies, HPV vaccination boosts humoral immunity and may protect against reactivation of latent infection.36 Our cohort showed that the majority of women had active HPV infections that they were not aware of and half of them were infected with multiple HPV types. In addition, risk factors for acquiring HPV infections were prevalent in our cohort. The technique we used to detect HPV DNA and RNA is very sensitive and can detect small quantities of HPV DNA, which is not normally detected using commercial testing and would be missed in the normal outpatient setting.HPV has been shown to be latent and not actively being transcribed in dysplastic/neoplastic cervical tissue in women with SLE.+ T lymphocytes and macrophages, which maintain local immune surveillance and cell-mediated immunity. Studies suggest that the HPV virus may alter the local cytokine environment to suppress the immune system and evade immune surveillance.38 In addition, aging and immune suppression may result in cytokine microenvironment changes that facilitate reactivation of latent infection or persistence and neoplastic transformation.40The role of cytokines in the microenvironment of the cervix is an evolving area of study. The cytokine microenvironment may promote latency, persistence, and progression to cervical cancer. Cytokines in the cervix are predominantly secreted by CD441 It is postulated that a decrease in T helper type 1 response or cell-mediated immunity and an increase in T helper type 2 response or humoral immunity is associated with neoplastic transformation, but studies are not consistent42–44 with one study showing increased intralesional interferon gamma to be associated with oncogenic potential.45HPV infection is associated with increased expression of proinflammatory cytokines as well as upregulation of immunosuppressive cytokines and regulatory T cells.We showed a trend of cytokine up in HPV-positive samples. Only expression of IL-2 was statistically significant in HPV-positive samples compared with HPV-negative samples. For the rest of the cytokines, although there were shown associations with HPV infection, they were not statistically significant, likely due to the small sample size.The limitations of our study were that the sample size was small, and we could not achieve normal distribution of data for statistical purposes. In addition, most of the cytology specimens lacked quality indicators so would be less sensitive to detect cytologic abnormalities, which were likely underestimated in this study. In addition, our HPV DNA and RNA testing in this research setting was much more sensitive than the commercial Clinical Laboratory Improvement Amendments (CLIA)-approved laboratory testing done in the outpatient setting so we could not officially report the results to the medical record.46Cervical cancer is a preventable disease and risk can be reduced by simple measures such as condom use and HPV vaccination. Awareness, education, and access to testing are key to engage high-risk women to reduce their cervical cancer risk. The women in our study were not aware of the spectrum of HPV-related diseases and were not using simple health prevention measures to protect themselves. HPV vaccination rates in AA adults are 10% lower than Caucasians  but in our cohort, the rate was much lower at 16.7% indicating that being a woman of color is not the sole determinant of poor vaccine uptake.36 The quadrivalent HPV vaccine has been shown to be safe and immunogenic in women with SLE.47In view of mounting evidence that HPV infections become latent rather than clear, particularly in immunosuppressed populations, preventing infection remains the best option for addressing the increased rates of HPV-related disease and cervical cancer in AA women with SLE. In addition to prevention of infection, HPV vaccination has been associated with lower redetection rates in previously infected women indicating that the vaccine may function to maintain the dormancy of HPV.48HPV vaccine uptake is low in women with SLE, however, despite the increased risk for HPV infection in this population.In conclusion, risk factors for HPV infection were prevalent in our cohort, yet these women were not engaging in these simple prevention strategies. Primary care doctors are likely unaware of this increased risk of HPV infection and cervical neoplasia in AA women with SLE, and thus may not be advocating for close cervical monitoring and HPV vaccination in these patients. AA women with SLE are also likely unaware of their increased risk for HPV infection and cervical neoplasia and not advocating for themselves to request more frequent testing, nor adhering to regular pap smear and HPV testing with the current guidelines. Awareness, education, and access to testing are key to engage high-risk women to reduce their cervical cancer risk. The self-sampling brush has the potential to make cancer screening more accessible as well as allow more frequent cervical monitoring at lower cost. This also raises the question as to whether screening should be done earlier than 21 years of age for those who are sexually active and on aggressive immunosuppression.We have just touched the surface of this problem, which needs further studies to better delineate the biology of HPV infection in AA women with SLE. However, it is clear that we need educate and empower these high-risk women to advocate for themselves and take control of their cervical health.


This study aimed to assess human papillomavirus (HPV) vaccine effectiveness (VE) against both vaccine-type and nonvaccine-type high-risk HPV (hrHPV) infection, and duration of protection in United States. The study population was female participants aged 18–35 years with an HPV vaccination history and genital testing for HPV from the National Health and Nutrition Examination Survey, 2007–2016. Participants vaccinated before sexual debut were assessed against 13 nonvaccine-type hrHPV infection including 31/33/35/39/45/51/52/56/58/59/68/73/82. Multivariable logistic regression was used to estimate VE overall, by age at diagnosis, time since vaccination and lifetime sexual partners. A total of 3866 women were included in the analysis, with 23.3% (95% CI 21.3%–25.4%) having been vaccinated (≥1 dose). VE against vaccine-type HPV18/16/11/6 infection was 58% overall, which was mainly driven by those aged 18–22 years (VE = 64%) and 23–27 years (65%). Among participants aged 18–22 years vaccinated before sexual debut, the VE was 47% (23%–64%) against 13 nonvaccine-type hrHPV and 61% (95% CI 36%–77%) against 5 selected nonvaccine-type hrHPV35/39/52/58/59. Both direct effectiveness and cross-protection maintained effective for 5–10 years post vaccination. We also found the prevalence of ever diagnosed cervical cancer among vaccinated was significantly lower  than that among unvaccinated participants . These findings highlight the potential of significant reduction of cervical cancer following the universal HPV vaccination programme. Human papillomavirus (HPV) infection is the most common sexually transmitted infection in the United States (US) with estimated 42.5 million persons infected with some type of HPV and half of new infections occurring before age 24 years . AlthougSince 2006, HPV vaccine has been recommended in US for adolescent girls at age 11–12 years, with catch-up vaccination recommended through age 26 years . FollowiBased on real-world data from other countries, the vaccine effectiveness (VE) against vaccine-type HPV infection ranged between 73% and 100% among girls or younger women aged between 12 and 21 years . To our As the US has implemented the universal HPV vaccination programme for more than one decade, it is expected that such programme might show some VE against nonvaccine-type hrHPV infection. By using real-world data from NHANES from 2007 to 2016, this study aims to assess the effectiveness of quadrivalent HPV vaccine against genital vaccine-type HPV infection and its cross-protection against nonvaccine-type hrHPV infections among the noninstitutional women in US.NHANES datasets from 2007 to 2016 were used for this study, including 5 surveys of 2-years . The surFor this study, female participants aged between 18 and 35 years were included in the analysis from the NHANES survey for years 2007–2016; 35 years was the oldest observed age for individuals who were vaccinated through 26 years of age (the oldest recommended age for vaccination in the United States) [HPV vaccination history for participants was categorised into two groups (vaccinated for ≥1 dose of HPV vaccine and unvaccinated for no doses reported). The immunisation history from questionnaire data was also used to obtain age at receiving first vaccine dose, which was used to calculate time since vaccination (categorised into two groups: ≤4 years and between >4 and ≤10 years). Time since vaccination was calculated by subtracting age at interview from age at receipt of first vaccine dose. As first HPV vaccine was only available since year 2006, the calculated duration was considered valid only if it was ≤10 years otherwise categorised as unknown. In addition, although the survey did not have age at receiving first vaccine dose for participants in 2007–2010, vaccinated participants in this period would not be possible to have received their vaccine more than 4 years and therefore they were categorised into ≤4 years group for the analysis of time since vaccination.Sexual behaviour from questionnaire data was also used for this study. Vaccinated participants were categorised by sexual debut according to age at receipt of first vaccine dose and age at having first sexual intercourse. Participants were considered as vaccinated before sexual debut if age at receiving first vaccine dose was equal or younger than age at initiating sexual intercourse, otherwise considered as vaccinated after sexual debut. For vaccinated participants who have not had any sexual intercourse at interview, they were considered as vaccinated prior to sexual debut. However, participants aged 18–19 years in NHANES 2007–2008 were not available for their sexual behaviour from the public available data. Therefore, all participants in this age range from 2007–2008 were categorised as unknown for the analysis by sexual debut as well as other variables from sexual behaviour questionnaire for this age group.Medical conditions from questionnaire data were used to identify cervical cancer. Participants were considered as having cervical cancer if ever being told by doctors or other health professionals had cervical cancer. The age of cervical cancer diagnosis was also recorded.Laboratory data were used to ascertain testing results by using self-collected cervicovaginal sample. Extracted DNA was evaluated for the presence of 37 HPV genotypes using PGMY09/11 polymerase chain reaction and Roche Linear Array . All samAll analyses were conducted with weighted data as previously . WeightsP < 0.1, known associations with HPV infection , or of interest for this study (HPV vaccination and survey cycle) were evaluated in multivariate modelling. Backward elimination (P < 0.05) was used to select the adjusted multivariate models, retaining covariates related to HPV infection or variables of interest to this study, regardless of statistical significance.The prevalence of HPV vaccine, vaccine-type HPV16/18/6/11 and nonvaccine-type hrHPV (HPV31/33/35/39/45/51/52/56/58/59/68/73/82) infections were calculated from 2007 to 2016 for all participants and by sociodemographic information, health condition and healthcare seeking behaviour. Multivariate log-linear regression model and calculated PR were then used to assess the association of HPV vaccine with vaccine-type HPV infections and nonvaccine-type hrHPV infections by adjusting the potential characteristic variables. Characteristics with a univariate VE against HPV infection was calculated as (1-adjusted PR) × 100% as previously , 28. We For the analysis of cross-protection against nonvaccine-type hrHPV infection, the study population was restricted to participants aged 18–22 years who have not received any HPV vaccine or had received their first vaccine dose prior to sexual debut. The VE against all 13 nonvaccine type hrHPV infection and selected nonvaccine-type hrHPV was investigated by time since vaccination and lifetime sexual partners.P value of <0.05 was considered statistically significant.All the analysis were conducted in R (version 4.1.3) using the survey package. A two-sided Because this study used deidentified, publicly available data, it did not meet the definition of human subjects research of the institutional review board, and therefore ethical review and informed consent were not required.From 2007 to 2016, a total of 25 516 female participants were enrolled in NHANES, of whom 3866 aged 18–35 years completed HPV testing and survey questionnaires for HPV vaccination were included in this study .Fig. 1.P = 0.001) in those aged 18–22 years and 65%  among those aged 23–27 years, while no effect was found among those aged 28–35 years. In the analysis according to time since vaccination, the VE increased with year since vaccination from 42% among those vaccinated within 4 years to 76% among those vaccinated 5–10 years ago. As for the analysis by vaccination in relation to sexual debut, the VE was 88% among participants received HPV vaccine prior to sexual debut and 55% among participants received vaccine after sexual debut. While VE was not found for those with ≤2 lifetime sexual partner, it was 63% for participants with ≥3 sexual partners  in participants aged 18–22 years and no effect was found in other age groups .Table 2We therefore further restricted our sample to participants aged 18–22 years for the subgroup analysis of cross-protection, including 742 unvaccinated and 225 vaccinated prior to sexual debut . The VE We also evaluated the type-specific VE for the individual nonvaccine-type hrHPV infection . Some inThis study has also tried to investigate the impact of HPV vaccination on cervical cancer from all included participants. We found the prevalence of cervical cancer ever diagnosed among vaccinated was significantly lower  than that among unvaccinated participants . Of the 4 women with a history of cervical cancer diagnosis, 3 of them received HPV vaccine after the diagnosis of cervical cancer and the other one received HPV vaccine in the same year of diagnosis of cervical cancer.In this study, we reported the overall VE against vaccine-type HPV infection was 58% among women aged 18 to 35 years from 2007 to 2016 based on the NHANES datasets, which was mainly found among participants aged 18–22 and 23–27 years. While cross-protection was not found against 13 nonvaccine-type hrHPV among all participants, substantial effectiveness was found among participants aged 18–22 years for the nonvaccine-type hrHPV infection overall and particularly for the following 5 individual types HPV 35/38/52/58/59. Both direct protection and cross-protection maintained effectiveness after 5–10 years of vaccination.Consistent with previous studies , the quaWhile a previous study reported that vaccinated young adult women (age 20–26 years) had a higher prevalence of nonvaccine-type hrHPV infection than unvaccinated women based on NHANES data, it did not exclude the impact of baseline HPV infection on this . After rTo our knowledge, cross-protection was mainly reported against HPV31/35/45/52 from bivalent HPV vaccine based on real-world data in Japan, Netherlands, Scotland and on clinical trial data in Costa Rica –22, 31. vs 0.46%). However, due to the limited number of participants with cervical cancer, we did not conduct some VE analysis in this study. We are expected to perform the analysis to confirm the effect on cervical cancer with larger sample size in the future rounds of survey.In our study, the VE against nonvaccine-type hrHPV and vaccine-type HPV was similar or even higher among women who were vaccinated 5–10 years ago compared to women who were vaccinated within 4 years. These findings are consistent with those from Japan and Netherland, where high VE against the vaccine-types HPV16/18 and against HPV31/33/45 was observed up to 7 years after vaccination , 21. GivWhile the HPV vaccination has shown strong protection against both vaccine-type HPV and nonvaccine-type hrHPV infection and some potential impact on reducing cervical cancer in US, globally only 15% of girls in the target age for HPV vaccination are fully protected in 2019 . Out of The strength of this study includes using large nationally representative samples from NHANES datasets with decreased selection bias. This provided comprehensive surveys and laboratory testing results in a consistent manner from 2007 to 2016. Several limitations are present in this study. One of the limitations is that only self-reported vaccination information is available from NHANSE. It is therefore recall bias could exist, which might have resulted in misclassification . HoweverIn conclusion, quadrivalent HPV vaccine does not only show substantial effectiveness against genital vaccine-type HPV among women aged 18–35 years, but also against nonvaccine-type hrHPV infections among those aged 18–22 years who were vaccinated before sexual debut. Such protection could last for 5–10 years following the receipt of HPV vaccine. These findings highlight the potential of significant reduction of cervical cancer following the universal HPV vaccination programme.


For a HPV vaccination program to be successful, it is important that the target population has sufficient knowledge about HPV and HPV vaccines. The aim of this study was to evaluate HPV-related knowledge levels among students at a university in northern Turkey, to assess vaccination willingness, and to determine factors associated with HPV-related knowledge.This cross-sectional study was conducted on 824 (93.1%) students studying at 16 different faculties. The study population was identified through the proportional stratified sampling method. Data were collected through a questionnaire including socio-demographic characteristics and the HPV Knowledge Scale. Multiple linear regression analysis was performed to identify factors that may be associated with knowledge scores.p < 0.05). The mean HPV knowledge score was quite low (6.74 ± 7.13 out of 29 points). The studying in a field related to health sciences, being a senior student, intending to be vaccinated, being a woman, having had sex were found to be associated with high knowledge levels (p < 0.05).43.6% of the students had never heard of HPV, 50.6% of them had never heard of HPV screening tests or HPV vaccination. Only 2.7% of the students were vaccinated against HPV and 15.7% were willing to receive HPV vaccination. Awareness of HPV and willingness to receive vaccination were found to be higher among women, while previous experience with sexual intercourse was higher in men (Educational programmes should be developed to increase university students' knowledge about HPV and the HPV vaccine.The online version contains supplementary material available at 10.1186/s12905-023-02455-4. Human Papillomavirus (HPV) infection is one of the most frequently encountered sexually transmitted infections . It is eIn addition, in Islamic countries with a traditional culture such as Turkey, it may result in significant psychological consequences due to the sexually transmitted nature of the virus , 7. In sWith the development of vaccines against critical HPV types (types 16 and 18), vaccination has become the primary preventative measure . With thReceiving HPV vaccination before sexual activity and before the first exposure to HPV is crucial , 17. AltIn order to carry out a successful HPV vaccination program, the target population should have adequate knowledge about HPV and HPV vaccines. Therefore, determining the level of HPV-related knowledge in the university student population can guide interventions and policies aimed at eliminating HPV-related cervical cancer. In this study, we aimed to evaluate knowledge and perceptions about HPV and HPV vaccines among students studying at Giresun University, to assess their willingness to be vaccinated, and to investigate factors associated with HPV-related knowledge.n = 824).This cross-sectional study was conducted in Giresun University and included undergraduate students between February 2022 and April 2022. No special policies or news about HPV or HPV vaccination were released by the Ministry of Health or other organizations during the study period. The study population was to be selected from an overall pool of 9443 students enrolled in 16 faculties of the university. The pilot study was conducted in 20 participants to evaluate the comprehensiveness and language of the questionnaire. A target sample size of 885 students was determined using 50% prevalence with 5% error margin, 95% confidence interval, design effect 2 and 20% non-response rate. In sample selection, students were stratified according to their faculty first and then according to their grade level (years) in each faculty. The proportional stratified sample selection method was employed to select subjects in proportion for each faculty in the student pool. After the faculties were stratified according to grade level, simple random selection was made from each class list. The inclusion criteria for the study are as follows: to be a current student at the relevant university, to be native speakers to complete the questionnaire form in full. Participants filled out the questionnaire in-person with the self-report method. A total of 885 questionnaires were handed out, and the response rate was 93.1% , which is a 35-item scale developed by Waller et al. to measure knowledge about HPV, HPV vaccination and screening tests. It includes three subscales concerning general information regarding HPV infection/treatment, HPV screening, and HPV vaccination, while there is also an independent 6-item subscale . The TurAll steps of the study conformed to the Declaration of Helsinki and the study protocol was approved by the Institutional Review Board of Ordu University . Additionally, permission to conduct the study on university students was obtained from the university administration.Before completing the questionnaires, the participants were well informed about the purpose and contents of the study. Participants were asked to sign informed voluntary consent forms. To prevent response bias, participants were guaranteed anonymity and their personal information was kept strictly confidential. After data collection was completed, an informative online booklet about HPV and HPV vaccines containing the correct answers to the questions used to measure their knowledge levels was sent to the students via their university e-mail addresses.p < 0.05, and 95% confidence intervals were calculated.Study data were evaluated using the SPSS 24.0 statistics program. Since much of the literature relies on data from studies conducted on females, many analyses were stratified according to sex. For descriptive analyses, numeric variables were described with mean and standard deviation, categorical variables were described as absolute and relative (%) frequencies. Normal distribution of data was evaluated with the Kolmogorov–Smirnov test, histograms, and skewness and kurtosis coefficients. According to these results, the independent samples t-test and Kruskal Wallis tests were used to evaluate relationships between HPV knowledge levels and independent variables. Multiple linear regression analysis was performed to evaluate factors independently associated with HPV knowledge scores. The aim was to eliminate the effect of confounding factors with multiple regression analysis. Variables that were found to be related with HPV knowledge scores in univariate analyses were included in the regression model. Of note, 33 data identified by their Mahalanobis values before regression were not included in the analysis. Significance level was accepted as A total of 824 university students participated in the study. Table p < 0.05)  Table .Table 2MFigure p < 0.05) (Table The average mean score of the students’ HPV-KS was 6.74 ± 7.13 (highest score possible: 29 points). General HPV knowledge mean score was 4.68 ± 4.42, HPV screening test knowledge mean score was 0.80 ± 1.38, and HPV vaccination knowledge mean score was 1.25 ± 1.92 points. HPV knowledge scale total score and subscale mean scores were significantly higher in women (p < 0.05) ; together with the relevant variables, it explained 42% of the change in HPV knowledge (adjusted R2 = 0.418). According to the regression analysis, being a female, studying health sciences, being a 4th year student, to have had sex previously, having visited a gynecologist / urologist within the last year, and being willing to receive HPV vaccination were identified as factors that were independently associated with higher levels of HPV knowledge (p < 0.05) (Table To clarify parameters independently associated with HPV knowledge levels, we created a multiple linear regression model by including variables that were found to be statistically significant in univariate analyses. The model generated was significant (F = 19.70, This study evaluated the HPV and HPV vaccination-related knowledge levels of Turkish university students, who are the target population for HPV vaccination due to the estimated age of first sexual experience.Approximately one-fourth of the students in the study group stated that they had sexual intercourse before, but men had almost three times greater frequency than that of women. In our study, the average age of first sexual intercourse was 19, but this value was higher among women. The age at first sexual intercourse differs with respect to countries and cultural contexts. The average age of sexual intercourse is around 19 in some western European countries, 17 in China and 16 in the United States. However, in these studies, no sex-based difference was observed in terms of the age of first sexual experience –23. DespIn our study group, 44% of participants had never heard of HPV, 51% had never heard of HPV screening tests or HPV vaccines. These rates were lower in women than in men. In a study conducted with university students in China, 49% of them stated that they had never heard of HPV and 51% of them stated that they had never heard about the HPV vaccine, and this rate was found to be significantly lower in women than in men similar to our study . AnotherVaccination rates against HPV are very low in the study group. Only 2.7% were vaccinated against HPV. In many European countries, almost 80% of the target population has been vaccinated . In a stIn the present study, while 16% of participants were willing to be vaccinated, 40% were undecided about HPV vaccination, and 44% did not want to be vaccinated. While the desire to be vaccinated was higher in women, indecision was similar for both sexes. There are a wide variety of behavioral approaches from country to country to improve HPV vaccination intent. In a study conducted in Hong Kong, 13.3% of individuals were vaccinated against HPV and 69.6% of students stated that they planned to be vaccinated . A similAlthough almost half of the students included in this study were aware about HPV, HPV screening tests and HPV vaccination; however, their knowledge scores were unfortunately quite low. The level of knowledge about HPV was lower compared to studies conducted in developed countries , 29, 34.In our study, knowledge scores on HPV, HPV screening and HPV vaccine were found to be higher in women. In the literature, similar studies conducted with the participation of students from countries with different levels of development have shown that women generally have better knowledge and attitudes towards HPV than men , 26, 30.This study also showed that students studying in non-health-related programs have less knowledge about HPV than students studying in health-related faculties/vocational schools. Studies conducted on students in the field of health sciences have shown that they have more awareness about HPV and HPV vaccines , 41. A sWillingness to receive the HPV vaccination was significantly associated with knowledge scores for HPV for both male and female participants. This study shows that the level of knowledge is higher among those who are willing to be vaccinated. This is consistent with previous studies that found an association between greater knowledge of HPV and HPV vaccines and intention to vaccinate , 45–47. Most studies on HPV and HPV vaccination in Turkey generally focus only on women, and few studies have analyzed HPV-related knowledge in both males and females. This study has the advantages of obtaining a good sample size with appropriate sample selection, lack of refusal in participation, and that the selected group represented students who originated from the great majority of the country’s provinces. In addition, a scale with proven validity and reliability was used to evaluate the level of knowledge. However, there are some limitations of our study. First, the population of this study is not representative of the general youth population in Turkey, as it consists of university students. Second, we should take caution when interpreting the findings of our study, because a drawback of the cross-sectional study design is the lack of causality assessment.Even among university students, who are a more educated group in Turkey, knowledge of HPV and HPV vaccination is at a very low level. In addition, HPV vaccination rates and vaccination intent appear to be very low. Despite having sexual experience at an earlier age, the knowledge level of men about HPV and HPV vaccine is lower. Students who have higher knowledge about HPV and HPV vaccine have a higher willingness to vaccinate.The results of this study show that there is a need for education programs on HPV infection and vaccination. Studies should be conducted on the effectiveness of HPV-related educational interventions on willingness to be vaccinated. Before inclusion of HPV vaccination in the national immunization program, it is necessary to carry out health promotion studies and policies in order to improve the knowledge and attitudes regarding HPV and HPV vaccination, particularly for young men. The results of these studies should be taken into account when developing interventions and policies to eradicate HPV-related cervical cancer in countries where the HPV vaccine is not included in the national immunization program.Additional file 1: Supplementary Table S1.


Patients are compelled to become more involved in shared decision making with healthcare professionals in the self-management of chronic disease and general adherence to treatment. Therefore, it is valuable to be able to identify patients with low functional health literacy so they can be given special instructions about the management of chronic disease and medications. However, time spent by both patients and clinicians is a concern when introducing a screening instrument in the clinical setting, which raises the need for short instruments for assessing health literacy that can be used by patients without the involvement of healthcare personnel. This paper describes the development of a short version of the full-length Danish TOFHLA (DS-TOFHLA) that is easily applicable in the clinical context and where the use does not require a trained interviewer.Data were collected as a part of a large-scale telehomecare project (TeleCare North), which was a randomized controlled trial that included 1225 patients with chronic obstructive pulmonary disease. The DS-TOFHLA was developed solely using an algorithm-based selection of variables and multiple linear regression. A multiple linear regression model was developed using an exhaustive search strategy.The exhaustive search showed that the number of items in the full-length TOFHLA could be reduced from 17 numeracy items and 50 reading comprehension items to 20 reading comprehension items while maintaining a correlation of r = 0.90 between the scores from full-length and short versions. A generic model-based approach was developed, which is suitable for development of short versions of the TOFHLA in other languages, including the original American version.This study demonstrated how a generic model-based approach could be applied in the development of a short version of the TOFHLA, thereby reducing the 67 items to 20 items in the short version. Furthermore, this study showed that the inclusion of numeracy items was not necessary. The development of the DS-TOFHLA presents an opportunity to reliably identify patients with inadequate functional health literacy in approximately 5 minutes without involvement of healthcare personnel. The approach may be used in the development of short versions of any scaling questionnaire. Healthcare systems all over the world are developing in a way that compels patients to become more active in the management of their own health and disease–a development that changes the role of modern patients and the skills needed to navigate the healthcare system. Demographic changes resulting in more elderly people have led to increases in the burden of chronic diseases and put pressure on increasingly scarce healthcare resources . One strThe core elements in HL are obtaining, understanding, and applying health-related information. Don Nutbeam has described these three elements as functional , interactive  and critical HL  . The numA recent review  aimed toA full-length Danish version of the full-length American TOFHLA  was developed according to acknowledged guidelines with assessment of face validity  content validity , internal consistency  etc. and has proven accurate in assessing functional health literacy (FHL) in previous studies –17. LikeThis paper aims to use machine learning to develop a short version of the D-TOFHLA  that is easily applicable in the Danish clinical context and that does not require the involvement of an interviewer . This paper seeks to describe a generic model-based approach applied in the development of the short version of the D-TOFHLA that can also be used in the development of short versions of the TOFHLA in other languages or, in general, to develop short versions of full-length screening instruments.The development of the DS-TOFHLA was based on the D-TOFHLA ; the D-TMotivated by a need to reduce the administration time and create an easier-to-use screening instrument, a short version of the original full-length American TOFHLA was designed: the S-TOFHLA . Due to The development of the DS-TOFHLA was based solely on an algorithm-based selection of variables and multiple linear regression (MLR). The classical method to obtain an unbiased evaluation, when building and testing models, is to have separate training and testing datasets, which can be accomplished by splitting a given dataset into a training set and a test set. In smaller datasets K-fold cross-validation is often used, which makes it possible to use almost the whole dataset for both training and testing while still avoiding bias. The present study used a special form of K-fold cross-validation, the Leave-one-out cross-validation (LOOCV), where K is set to the number of samples (K = N). LOOCV can be particularly useful when working with small datasets  because it allows for a more reliable estimate of the model’s performance. With a small dataset, there is a higher risk of overfitting, which occurs when a model is too complex and fits the training data too closely, resulting in poor generalization to new data. LOOCV helps to mitigate this risk by repeatedly training the model on slightly different subsets of the data, allowing for a more robust evaluation of its performance. LOOCV also ensures the best possible use of the dataset  . We perfThe quality goal in the development of the DS-TOFHLA was expressed by two conditions. First, Pearson’s correlation coefficient between the DS-TOFHLA score  and the D-TOFHLA score should be at least 0.9 (r ≥ 0.9 being indicative of a very strong correlation). Second, if possible, the model should not contain numeracy items to eliminate the involvement of an interviewer.The trial has been presented to the Regional Ethical Committee for Medical Research in the North Denmark. The committee determined that no ethical approval was necessary.The selection of items for the DS-TOFHLA was based on data from a previous large study that used the D-TOFHLA ,17. DataThe D-TOFHLA is created as whole sentences, where one or more words are missing, and the participant is asked to select word/words that best complete a sentence. Therefore, before modelling the DS-TOFHLA, the 50 reading comprehension items in the D-TOFHLA were grouped into meaningful sets of items (items that create a whole sentence) to ensure that the intended meaning were maintained. This resulted in 19 sets: 5 sets with 1 item, 7 sets with 2 items, 1 set with 3 items, 3 sets with 4 items, 2 sets with 5 items, and 1 set with 6 items.1, C2,…, Cn are the scores (1 for correct and 0 for incorrect) for the reading comprehension items included in the DS-TOFHLA (from the D-TOFHLA), and b0,b1,…, bn are the regression coefficients that are adjusted to fit the model.The DS-TOFHLA was based on the following MLR equation:The model was developed using an exhaustive search strategy: for every model size , all possible combinations of sets of comprehension items were tested. For each model, the root mean square error (RMS error or RMSE) between the DS-TOFHLA MLR and the D-TOFHLA score was used as the model fit criterion. After minimizing the RMSE by adjusting the regression coefficients for each model, the model with the highest Pearson’s correlation coefficient was identified.The internal consistency of the DS-TOFHLA was determined by using Cronbach’s alpha coefficient. An instrument is considered reliable if the Cronbach’s alpha exceeds a value of 0.7 . Item toBecause of the DS-TOFHLA being based on a MLR that was used to predict the D-TOFHLA, the scoring system for the DS-TOFHLA was assumed to be similar to that of the D-TOFHLA: inadequate level: 0–59 points, marginal level: 60–74 points, adequate level: 75–100 points .To assess the predicted scores of the DS-TOFHLA, a confusion matrix was used to illustrate the relation between the FHLs in the DS-TOFHLA and those in the D-TOFHLA.The accuracy for predicting the three FHLs was calculated. In addition, the ability of the DS-TOFHLA to correctly detect ‘low FHL’ was assessed in accordance with the procedure described by Parker et al. .The development of the S-TOFHLA was based on subjective decisions combined with some linear regression modelling, the latter being subjectively adapted to facilitate easy scoring ,21. The For comparison, a subset of the items in the D-TOFHLA was selected in accordance with the subset used in the S-TOFHLA  and, using the same weighting as in the S-TOFHLA (2 and 7 respectively), a Danish mirror version of the S-TOFHLA was constructed. Thus, the ‘D-36-4-TOFHLA’ was based on 40 items from the D-TOFHLA combined in the following MLR equation :Pearson’s correlation coefficient between the D-36-4-TOFHLA score and the D-TOFHLA score, for the 158 COPD patients recruited from the TeleCare North cohort, was calculated.Most studies using the S-TOFHLA, have chosen to omit the numeracy items . Even thUsing the same 158 COPD patients, Pearson’s correlation coefficient between the D-36-0-TOFHLA score and the D-TOFHLA score was calculated.The basic demographic characteristics of the 158 participants can be found in The exhaustive search showed that the number of items in the D-TOFHLA could be reduced to 20 reading comprehension items and that there was no need for numeracy items. The sets of reading comprehension items were item 2–3, item 13–14, item 18–21, item 23–25, item 37–41, and item 42–45, each set corresponding to a sentence in the Danish TOFHLA leading to the following regression model:S1 Appendix. The maximum time for administration could be reduced from 22 minutes (10 minutes for numeracy items and 12 minutes for comprehension items) to 5 minutes (12*20/50 minutes).An English version of the DS-TOFHLA is presented in The internal consistency measured by Cronbach’s alpha was 0.885. This indicated that the reliability of the DS-TOFHLA was acceptable (>0.7 as set by Houser ). Item tThe accuracy of the prediction of the inadequate level (lowest level)  was 92%. The accuracy of the prediction of the marginal level (middle level)  was 80%. The accuracy of the prediction of the adequate level (highest level)  was 88%, which can also be expressed as the accuracy of the prediction of ‘low FHL’ as defined by Parker et al. .To enable a comparison with the performance of the S-TOFHLA, the relation between the D-36-4-TOFHLA (the Danish mirror version of S-TOFHLA) score and the D-TOFHLA score, for the 158 COPD patients recruited from the TeleCare North cohort, is illustrated in The aim of this study was to investigate the use of machine learning to develop a short test of FHL in adults (the DS-TOFHLA) that can be used in the development of short versions of the TOFHLA in various languages, including the original version of the American TOFHLA in English. In addition to investigating the machine learning approach, this study also addressed the problem that, to the authors’ knowledge, there are no efficient, suitable, and objective screening instruments for assessing HL in a clinical setting in Denmark and other non-English speaking countries.A review has shown that most studies using the S-TOFHLA chose to omit the numeracy items to simplify the test and make it usable in a clinical setting . In our For comparison, an assessment of the performance of the S-TOFHLA was performed using Danish mirror versions of the short American versions. Both the S-TOFHLA and the Prose S-TOFHLA (without numeracy items) was assessed, the latter being the most widely used . PearsonThe DS-TOFHLA was developed solely using an algorithm-based selection of variables and MLRs. A major strength of this method was that the design principles were founded on objective algorithm-based decisions, and the MLR used for development selected the items from the D-TOFHLA that led to the most accurate predictions of the level of FHL. The reading comprehension items in both the D-TOFHLA and the American TOFHLA are ordered by increasing difficulty in readability level and thus, it is reasonable to assume that the more difficult items are most accurate in predicting the functional level of HL. In this regard, it should be noted that only 4 of 20 items selected for the DS-TOFHLA by the algorithm herein were from the first and easiest part of the reading comprehension items. The 36 items assessing reading comprehension in the S-TOFHLA is primarily from the first and middle part (lowest difficulty) and none from the latter and most difficult part. In line with this, the regression model presents quite different weights to the selected items for the DS-TOFHLA, e.g., C42 = 0 and C43 = 6. In comparison, the development of the American S-TOFHLA seems based on a subjective decision to include the first 36 items, without explicitly considering if these items contribute to the most accurate prediction of the FHL or considering the ascending difficulty in readability.The accuracy of the prediction of the three levels was ranged from 80%-92%; the middle level had the lowest accuracy, which can be explained by the fact that this level is defined by a relatively narrow range of 60–74 points. The prediction of the lowest and highest levels was a one-sided classification, whereas the prediction of the middle layer was two-sided. It should be noted that the prediction of ‘low FHL’ , as defined by Parker et al , had an During the development of a novel questionnaire, it is customary to adhere to established guidelines for scale development and validation. The DS-TOFHLA was based on a predictive MLR and should not be regarded as a de novo questionnaire, and it, therefore, should not go through the same rigorous evaluation. Instead, the focus should be on developing a short questionnaire with the best possible predictions of the validated full-length questionnaire. Likewise, it makes sense to develop the prediction model in the DS-TOFHLA based on the same data that was used to develop and validate the full-length Danish TOFHLA ,17. HoweThis study demonstrated how a generic model-based approach could be applied in the development of a short version of the TOFHLA, thereby reducing the 67 items in the full-length version to 20 items. Furthermore, this study showed that the inclusion of numeracy items was not necessary to meet the chosen quality goal of a Pearson’s correlation coefficient ≥0.9, resulting in a short version of TOFHLA where the use does not require a trained interviewer. The work was based on Danish data and a validated Danish full-length version of TOFHLA. The generic model-based approach used herein may also be used in the development of short versions of the TOFHLA in other languages and in the development of short versions of any scaling questionnaire.S1 Appendix(PDF)Click here for additional data file.


Anelloviridae, Genomoviridae) and double-stranded DNA viruses . We identified 110 unique, complete HPV genomes within two genera (Alphapapillomavirus and Gammapapillomavirus) representing 40 HPV types and 12 species. Of the 40 HPV types identified, 35 showed positive co-infection patterns with at least one other type, mainly HPV-16. HPV-35, a high-risk genotype currently not targeted by available vaccines, was the most prevalent HPV type identified in this cohort. Bacterial taxa commonly associated with bacterial vaginosis also correlated with the presence of HPV. Bacterial vaginosis, rather than HPV, was associated with increased genital inflammation. This study lays the foundation for future work characterizing the vaginal virome and its role in women’s health.The interaction between cervicovaginal virome, bacteriome and genital inflammation has not been extensively investigated. We assessed the vaginal DNA virome from 33 South African adolescents (15–19 years old) using shotgun DNA sequencing of purified virions. We present analyses of eukaryote-infecting DNA viruses, with a focus on human papillomavirus (HPV) genomes and relate these to the vaginal bacterial microbiota (assessed by 16S rRNA gene sequencing) and cytokines (assessed by Luminex). The DNA virome included single-stranded ( Adenoviridae, Herpesviridae, Papillomaviridae and Polyomaviridae) and single-stranded (ss) DNA viruses (families Anelloviridae) [Alloherpesviridae, Iridoviridae, Marseilleviridae, Mimiviridae, Phycodnaviridae and Poxviridae families have been identified in the vaginal samples of pregnant people, and those with reproductive disorders [.Various eukaryote-infecting DNA viruses have been identified in the genital tracts of generally healthy, asymptomatic women of reproductive age by shotgun metagenomic sequencing, including double-stranded (ds) DNA viruses (families viridae) ,2. Additisorders ,4,5.Alphapapillomavirus were the most common DNA viruses detected in the lower reproductive tract, with 38% of the participants being infected with at least one Alphapapillomavirus. Longitudinal sampling showed the presence of many HPVs over multiple time points, suggesting that up to 50% of these papillomaviruses established productive infections [To date, more than 220 human papillomavirus (HPV) types have been identified, including at least 50 types that preferentially infect the genital mucosa . In the fections . Similarfections , confirmHigh-risk HPV types have been well described to cause high-grade cervical intraepithelial neoplasia (CIN) and cancer , and morLactobacillus spp. is replaced by a range of diverse anaerobes [Lactobacillus spp. or Lactobacillus iners had up to five times higher odds of being infected with any HPV compared with women with vaginal microbiomes dominated by L. crispatus [Bacterial vaginosis (BV) is a vaginal condition in women of reproductive age where the optimal microbiota dominated by naerobes . Meta-annaerobes . Similarrispatus . In thesHere, we used an untargeted approach to characterize eukaryotic DNA viruses in the FGT of South African adolescents to capture the FGT virus landscape in this population. Our emphasis lies on HPV genotypes, including their co-infection patterns and association with cervicovaginal bacteriomes and cytokines.®; MSD Pty Ltd., Johannesburg, South Africa), and combined oral contraceptive pills . The participants were seen at enrollment, 4 months, and 8 months. For this exploratory viral metagenome analysis, we included single time point samples from 33 participants, with samples from two longitudinal time points included for participants who experienced a change in BV status throughout the study.The participants of this sub-study were recruited through a parent study, UChoose , which was conducted between 2015–2017 and enrolled cis-gender females aged 15–19 years. None of the participants had received an HPV vaccine, as the roll out of HPV vaccinations in the public sector in South Africa only commenced in 2014 and targeted younger girls . The stu®) placed over the cervix for half an hour to measure genital cytokines by Luminex, and a clinician collected a vulvovaginal swab for sexually transmitted infection (STI) testing  by multiplex PCR [® fluid and vaginal swabs were stored at −80 °C until further processing.At all study visits, rapid HIV and pregnancy testing were performed, and if positive, the participant was counselled and referred for management, and no further mucosal samples were collected. Detailed interviewer-assisted questionnaires assessing medical/health history, sexual behavior, menstrual cycle, contraceptive use, intravaginal practices, and antibiotic use were completed at all visits. Venous blood was obtained for herpes simplex virus 2 (HSV-2) serological testing. Cervicovaginal secretions were collected using a disposable menstrual cup , included 13 cytokines: interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, IL-23, IL-33, IL-17A, IL-17F, IL-21, IL-22, IL-25, IL-31, interferon (IFN)-γ, and soluble CD40 ligand. Specimens from six participants were run across all plates (inter-plate controls), and samples from six participants were duplicated on each set of plates (intraplate controls) for quality control measures. Spearman’s rank test was used to measure intra-assay and inter-assay correlation coefficients to determine assay reliability and reproducibility. Data were collected using a Bio-plex Suspension Array reader, as described before ,21.https://www.ebi.ac.uk/ (accessed on 15 December 2022) under project number PRJEB30774.DNA extraction followed by amplification, sequencing and bioinformatics analysis of the V4 region of the 16S rRNA gene using 515F and 806R primers from vaginal lateral wall swabs were described previously in detail . AmplicoTM TempliPhi kit  and stored at −80 °C until library preparation.The genital tract virome was determined by shotgun sequencing of an aliquot (450 μL) from the vaginal lateral wall swab buffer solution. Virus-like particles (VLPs) in the vaginal swabs were resuspended by the addition of 1 mL SM buffer, 0.1 M NaCl, 50 mM Tris/HCl (pH 7.4), and 10 mM MgSO4, to the sample. This step was followed by filtering the sample through a 0.2 µm filter  to separate VLPs from bacteria, human cells, and larger particles. Viral DNA was extracted from the resulting filtrate using the High Pure Viral Nucleic acid extraction kit  following the manufacturer’s protocol. The extracted viral DNA was subjected to rolling circle amplification using the IllustraDe novo assembled contigs >500 nucleotides in length were identified via alignment against a viral protein RefSeq sequence database (downloaded from NCBI) using BLASTx [https://www.ncbi.nlm.nih.gov/orffinder/ (accessed 7 January 2022) and refined and annotated with data from PaVE [n = 65) identified via viral metagenome sequencing are deposited in GenBank , and raw reads have been deposited in SRA under BioProject PRJNA881266.Library preparation and quantitation were performed by the University of Washington (UW) Northwest Genomics Center (NWGC), and the resulting libraries were sequenced in multiplex mode on a NovaSeq6000 S4 flow cell, generating ~30 Gb per sample. The raw reads were trimmed using Trimmomatic 0.39 , and theg BLASTx . Circularom PaVE . The HPVn = 45) have been deposited in GenBank (OP970997- OP971041). Raw metagenomic reads have been deposited in SRA under BioProject PRJNA767784.We leveraged previously generated lateral vaginal wall shotgun metagenome sequencing data  from paAlphapapillomavirus or Gammapapillomavirus genus, we assembled a dataset of known L1 sequences of all HPV types in these two genera, as well as those in this study and a subset of those from the genera Betapapillomavirus, Nupapillomavirus, Mupapillomavirus and avian-infecting papillomaviruses. This dataset was translated and aligned using MAFFT v7.113 [CenoteTaker2 , coupledT v7.113 . The aliT v7.113 , and a mT v7.113  with appT v7.113 .p values and 95% confidence intervals were used to assess statistical significance.All downstream analyses were conducted in Rstudio using R version 4.2.2  unless o® as hormonal contraceptives , Megasphaera, L. iners, Prevotella spp. , Fanyhessa vaginae, Sneathia, and Aerococcus christensenii (community state type IV (CST-IV), L. crispatus (CST-I) or L. iners (CST-III). For a subset of eight participants that experienced a change in BV status, the vaginal viromes of longitudinal samples were sequenced from visits both with and without BV, 4 months apart. As expected, participants had a more diverse bacterial microbiota (CST-IV) when diagnosed with BV, and most vaginal microbiotas were dominated by Lactobacillus spp. (CST-I or -III) with a lower diversity in the absence of BV. A higher proportion of women were using oral contraceptive pills during episodes of BV, but no other evaluated demographic or biological variable differed by BV status  with a median age of sexual debut of 15 years (IQR 14–16 years). Most of the sampling was performed when participants were using injectables, oral contraceptive pills or Nuvaringceptives . The preceptives . About hV status .Anelloviridae and Genomoviridae; and dsDNA viruses that share sequence similarities to Adenoviridae, Alloherpesviridae, Herpesviridae, in the subfamilies Alphaherpesvirinae, Betaherpesvirinae, and Gammaherpesvirinae, Marseilleviridae, Mimiviridae, Polyomaviridae, Poxviridae in the subfamilies Chordopoxvirinae and Entomopoxvirinae. We also observed sequences with similarities to Retroviridae, primarily human endogenous retroviruses. Finally, we identified complete genomes in the viral families Anelloviridae (n = 3), Genomoviridae (n = 1), and Polyomaviridae (n = 2), which have been reported by Jimoh et al. [To identify eukaryote-infecting DNA viruses in the vaginal samples, we characterized the viral taxonomy of contigs longer than 500 nucleotides using BLASTx and the NCBI Refseq viral protein database. Sequences from 11 eukaryote-infecting virus families were detected . We idenh et al. .Papillomaviridae (subfamily Firstpapillomavirinae) family, which are all HPVs. We assembled 65 full genomes from the viral metagenomics dataset and leveraged our total metagenome dataset from a subset of participants from the same cohort [Alphapapillomavirus (n = 104) and Gammapapillomavirus (n = 6)  .The overall prevalence of any HPV in this group of women was 60.6% (20/33), with 54.5% (18/33) being infected with high-risk HPV types. Most of the women infected with any HPV were infected with multiple HPV types . The most frequent HPV types detected were HPV-35 (high-risk), found in six women, followed by HPV-16 and -53 (both high-risk) and HPV-81 and -90 (both low-risk). HPV-16, -53, -81, and -90 were each detected in four women. Interestingly, one woman had HPV-214, which was only recently described in a penile swab from a study conducted in Cape Town, South Africa . HPV typp < 0.05) (n = 6) had significantly more other HPV types  5–12) compared to samples where HPV-16 was not present . Low-risk HPV-61 and high-risk HPV-69 co-occurred in all samples in which the given genotypes were detected (p = 0.0008). This was also true for the co-occurrence of HPV-39 and -68  and HPV-40 and -53 . These data suggest that HPV genotypes frequently co-occur and that the co-occurrence between specific HPV genotypes might be more common than for others.We next evaluated HPV co-infection patterns using a model that employs combinatorics to determine the probability that the observed frequency of HPV genotype co-occurrence is significantly greater than expected (positive association), significantly less than expected (negative association), or approximately equal to expected (random association) a. Of the < 0.05) a. In agrp = 0.031). The proportion of women with high-risk HPV types was not statistically different between women with BV  compared to those without BV . Twelve of the 39 HPV genotypes included in this analysis (30.7%) only occurred in women with BV, including high-risk types HPV-35 (present in 5/19 (26.3%) of women with BV), HPV-26 (n = 1) and HPV-31 (n = 1), and low-risk genotypes HPV-40 (n = 3), HPV-6, -42, -74, -81, -91 , HPV-84, -108 and -214  with unknown risk association . There were two main clusters of bacteria that best distinguished HPV status: one dominated by L. crispatus (with a lower abundance in women infected with HPV) and one comprised of Coriobacteriaceae, Prevotella spp. , Parvimonas micra and Dialister micraerophilus (with a higher abundance in women infected with HPV). Most of the latter clusters were highly abundant in CST-IV in this cohort   c. These p = 0.638). The same observations were made on genotype (median 2 (IQR 0–4) vs. 3 (IQR 1–4), p = 0.678) and species (median 2 (IQR 0–3) vs. 2 (IQR 1–5), p = 0.674) level. This suggests that while there were associations between the presence of HPV and some BV-associated taxa, and for some women episodes of BV coincide with the acquisition of new HPV genotypes, this pattern is not universal.We sought to investigate this relationship in more detail in a subset of eight participants who experienced a change in BV status longitudinally over 4 months . This anPreviously, in a study of South African women at high risk of HIV acquisition, infection with any HPV type was associated with increases in several chemokines, growth/hematopoietic factors, pro-inflammatory, adaptive and regulatory cytokines, including those found to be associated with HIV risk . SimilarAlphapapillomovirus 6  was generally associated with lower cytokines concentrations. In univariate analyses, infection with HPV-35 was associated with a significantly higher concentration of six of the thirteen cytokines measured, including IL-1β (median 131.65 pg/mL (IQR 109.08–214.31) vs. 7.15 pg/mL (0.61–59.33), p = 0.0009), TNF-α (median 20.12 pg/mL (IQR 6.21–22.94) vs. 0.68 pg/mL (0.16–1.93), p = 0.0009), IL-33 (median 24.24 pg/mL (IQR 11.70–25.02) vs. 5.85 pg/mL (IQR 1.68–9.86), p = 0.012), IL-17A (median 3.80 pg/mL (IQR 2.74–4.50) vs. 1.24 pg/mL (IQR 0.82–2.82), p = 0.037), IL-6 (median 4.24 pg/mL (IQR 2.96–17.26) vs. 0.95 (IQR 0.47–2.73), p = 0.043) and IL-17F (median 22.23 pg/mL (IQR 11.33–23.08) vs. 3.40 pg/mL (IQR 0.34–8.89), p = 0.048), with IL-1β and TNF-α remaining significant after adjustment for multiple comparisons . As BV has been shown to increase genital inflammation, we adjusted for BV status in mixed effect logistic regressions that included PID as a random variable. In these models, only TNF-α showed a significant association with the presence of HPV-35 .Here, we compared cervicovaginal cytokine levels from women infected with a given HPV genotype to those without that specific genotype in univariate analyses a. Infectp = 0.006), IL-17F , IL-25 , IL-33  and TNF-α . This suggests that the presence of BV has a greater impact on cytokine levels in the lower FGT than the presence of HPV in this group of women.To further dissect the relationship between HPV and genital cytokines, we compared cervicovaginal cytokine levels from women according to HPV status, including infections with any HPV, multiple HPVs, high-risk, or low-risk HPVs versus those without any HPV infection b. We foup-values after adjustment for multiple comparisons using a false discovery rate step-down procedure [We controlled for BV and included PID as a random variable, as some participants had repeated measurements. rocedure  are showOur exploratory study describes the eukaryote-infecting DNA virome in young women from Sub-Saharan Africa, with an emphasis on HPV genomes. Using a metagenome sequencing approach, we assembled 110 complete HPV genomes across 40 HPV genotypes. We have made these publicly available to expand the current databases.This untargeted approach for HPV typing allowed us to capture a broader snapshot of the FGT HPV landscape, as we were not limited to HPV types included in commercial arrays. While most HPV types detected are included in currently available commercial assays like the HPV Direct Flow CHIP , more recently described HPV genotypes like HPV-108, -214, -215 or -mSD2 would not have been detected using commercial arrays. The risks associated with infections with these HPV genotypes are still unknown and will be difficult to assess if only commercial arrays are used to conduct HPV typing.HPV-35 was the most common HPV type detected, with a prevalence of 15% in this small cohort of asymptomatic, young South African women. Studies in Sub-Saharan African populations have reported an HPV-35 prevalence of up to 40% among women with cervical intraepithelial neoplasia (CIN) . HPV-35 Lactobacillus spp. and a higher abundance of Prevotella buccalis and P. timonensis, Parvimonas micra and Dialister micraerophilus, which were highly abundant in women with CST-IV in this cohort. This supports findings from a recent study that has described that HPV infection alters the vaginal microbiome through the down-regulation of host mucosal innate peptides used by Lactobacillus spp. as amino acid sources [Lactobacillus-dominant state, e.g., by using live biotherapeutic products, could be leveraged as a treatment strategy for women with HPV. While larger studies in diverse populations are warranted, previous studies have suggested that the administration of Lactobacillus probiotics may favor HPV clearance [A meta-analysis has suggested a causal link between vaginal dysbiosis and HPV risk . There w sources . Larger learance ,56,57.Following the adjusting for BV status, we found that having any HPV type or a high-risk HPV type was not associated with cytokine levels in this small cohort of women. Only infection with HPV-35 was associated with significantly higher concentrations of TNF-α after adjusting for BV status. In contrast, having BV was associated with higher levels of many cytokines that overlap substantially with cytokines associated with HIV risk. While HPV infection has been found to increase the risk of HIV acquisition in Sub-Sharan African women , and an The sample size of this exploratory study was small, thereby limiting our ability to make strong conclusions regarding associations between vaginal microbiota, cytokines, and the presence of HPV genotypes. Nonetheless, we analyzed longitudinal, matched data for a subset of participants, which strengthened our approach. While we focused on DNA viruses here, we plan to include RNA viruses in subsequent studies and to assess bacteriophages. The effect of other clinical parameters on the vaginal microbiota, such as diet or vaginal insertion practices, should be considered in future studies to be carried out in different populations. Strengths of this study included using a viral metagenomic sequencing approach to capture the HPV landscape in a more unbiased way and to identify full HPV genomes from Sub-Saharan Africa, which we have made publicly available.This viral metagenome sequencing study has identified 110 unique HPV genomes from healthy, asymptomatic South African women. Amongst the most common high-risk HPV types were genotypes that are not currently targeted by available vaccines. Describing the HPV landscape helps inform HPV vaccination strategies and should be conducted on a larger scale in Sub-Saharan African women.