In 2008, the UK introduced an HPV immunisation programme in girls. Population-based prevalence estimates of bivalent (HPV-16/18), quadrivalent (HPV-6/11/16/18) and 9-valent (HPV-6/11/16/18/31/33/45/52/58) vaccine types, and comparison over time, are needed to monitor impact, evaluate effectiveness and guide decision-making on vaccination strategies.The third National Survey of Sexual Attitudes and Lifestyles  in 2010-12, tested urine for HPV from 2569 sexually-experienced women aged 16–44. We report type-specific HPV prevalence and compare results with 1798 women in Natsal-2 (1999–2001) using age-adjusted prevalence ratios (APR).In Natsal-3, 4.2% of women aged 16-44y were positive for HPV‐16/18 and 2.9% for HPV-6/11. In 16–20 year olds, 4.5%, 10.8% and 20.7% had at least one bivalent, quadrivalent or 9-valent vaccine type, respectively. Three-dose vaccine coverage was 52.0% in women aged 18-20y. In this age group, HPV-16/18 prevalence was lower in Natsal-3 than Natsal-2 , however, prevalences of HPV-6/11, HPV-31/33/45 and HPV-52/58 were unchanged. HPV-16/18 prevalence was also unchanged in women aged 21-44y (APR=0.85[0.61–1.19]).These probability surveys provide evidence of the impact of the bivalent immunisation programme. Reductions were specific to HPV-16/18 and to the age group eligible for vaccination. However, substantial vaccine-preventable HPV remains. It is likely that the cost-effectiveness of using the 9-valent vaccine will be considered by the Joint Committee on Vaccination and Immunisation (JCVI) in the UK and other policy-making bodies internationally. The National Surveys of Sexual Attitudes and Lifestyles  are large probability sample surveys of the British population that include demographic and behavioural information linked to STI test results. Results from Natsal-2, using data collected in 1999–2000, provided baseline population estimates of type-specific HPV (from urine samples) in women aged 18–44 years prior to the introduction of the bivalent immunisation programme In this paper, we extend the analysis of Natsal-3 data to include estimates of a wide range of HPV types, including HPV-6/11, HPV-31/33/45 and HPV-52/58. In addition, more detailed comparison of results from the two surveys is used to evaluate bivalent vaccine effectiveness. These include effects on HPV-16/18 prevalence and non-vaccine types in the age groups targeted by the catch-up campaign, as well as examining changes in sexual behaviour in the decade between the surveys.22.1Natsal-3 was a probability sample survey of 15,162 men and women (8869 women) aged 16–74 years and resident in Britain. Interviews were carried out during 2010–2012. Participants were interviewed using computer-assisted personal interviewing, with computer-assisted self-interview for the more sensitive questions. Women eligible for the national HPV immunisation programme  were asked “Have you ever been vaccinated against cervical cancer (received HPV vaccine)?”. Further methodological details have been described elsewhere At the end of the interview, a sample of participants aged 16–44 years who reported at least one lifetime sexual partner were invited to provide a urine sample to be tested for STIs, including HPV, and 60% agreed (similar agreement in men and women) The previous Natsal survey  was carried out in 1999–2001 2.2Data were analysed using Stata (v13.1), accounting for the stratification, clustering and weighting of the sample. To account for differences in the probability of selection for and response to providing a urine sample, an additional weight, derived from a logistic regression model, was applied to the urine data for analysis, as described previously We report prevalence ) of key HPV type combinations and HPV types in women by age group . Data for comparison between Natsal-2 and Natsal-3 were available for women aged 18–44. We compare prevalence of HPV-16/18, HPV-31/33/45, HPV-52/58 and HPV-6/11 in the age group eligible for vaccination (aged 18–20 years) and the prevalence of HPV-16/18 in older women (aged 21–44 years). We present age-adjusted prevalence ratios (APR), calculated using generalised linear models with a log link function, for the association between survey and these key type combinations.Changes in sexual behaviour in men and women aged 18–44, in the decade between Natsal-2 and Natsal-3, have been previously reported in detail 2.3Natsal-3 was granted ethical approval by the Oxfordshire Research Ethics Committee A (Reference: 09/H0604/27). Natsal-2 obtained ethical approval from University College Hospital, North Thames Multicentre, and all local research ethics committees in Britain.33.1In Natsal-3, HPV results were available for 2569 sexually-experienced women aged 16–44 years. The prevalence of HPV types and type combinations, stratified by age, is shown in In total, 4.2% of 16–44 year olds were positive for bivalent vaccine types (HPV-16/18) . PrevalePrevalence of other HR-HPV types found in the 9-valent vaccine was high – 6.8% overall . Table 23.2In women aged 18–20 years, the prevalence of HPV-16/18 was lower in Natsal-3 than Natsal-2 ), whereas for older women  was similar in the two surveys, as was the prevalence of HPV-6/11. There was also no evidence for a difference in HPV-31/33/45 prevalence between Natsal-2 and Natsal-3.3.3Between Natsal-2 and Natsal-3, the proportion of sexually-experienced 18–20 year old women reporting first heterosexual intercourse before the age of 16 increased from 29.2% (24.5–34.4) to 38.8% (34.5–43.3) (p=0.005). However, there was little evidence for a change in other markers of risky sexual behaviour. For example, the number of women reporting ≥5 lifetime partners remained unchanged (39.7% (33.8–46.0) vs. 43.6% (38.9–48.3) (p=0.332)) as did the number reporting ≥1 new partner in the past year (55.0% (49.1–60.8) vs 57.7% (53.2–62.0), p=0.483).4Population-based HPV prevalence estimates, and comparison of prevalence over time, are needed to inform decisions about future policy for HPV vaccination in the UK. This paper presents prevalence estimates for a range of HPV types circulating in the British population in 2010–2012, and shows that there remains a significant amount of vaccine-preventable HPV. Even amongst 16–20 years who were eligible for the immunisation programme, 5%, 11% and 21% of women were infected with at least one of the bivalent, quadrivalent or 9-valent vaccine types, respectively. The Natsal data do nevertheless provide an early indication of population-based effectiveness of the bivalent vaccine in women in the age groups eligible for vaccination, with a ~50% reduction seen in the prevalence of HPV-16/18 in 18–20 year olds . This reduction was specific to the bivalent vaccine types and to the age group eligible for the catch-up campaign. A recent meta-analysis showed that, in countries with female vaccination coverage of at least 50%, HPV-16/18 has reduced by 68% The strength of Natsal is that the HPV data are collected together with detailed information on sexual behaviour. This allows us to examine the counterfactual – could HPV have reduced in this age group as a result of reductions in risky sexual behaviour in the decade between the surveys? We have previously reported that there has been little change in sexual risk behaviour in those aged 18–44 between the two surveys Two other UK studies have considered the population-level impact of a national HPV vaccination programme on HPV prevalence. One considered changes in HPV type-specific prevalence in a high-risk population of young women in England attending for chlamydia screening The two Natsal studies are large population-based probability sample surveys, conducted using similar methods a decade apart, which provide an opportunity to assess the impact of public health interventions on a national scale. The combination of obtaining detailed demographic and behavioural data, linked to biological specimens, provides both subjective (eg reported vaccine coverage) and objective (eg HPV results) measures of process and outcome. Each of the Natsal datasets is weighted to account for survey participation and differential urine selection probabilities and response Whilst we show reductions in the bivalent HPV vaccine types, there remains a considerable HPV burden in the population. The 9-valent HPV vaccine has been licensed for use in Europe (3-dose schedule) . If thisThese data compare HPV prevalence in the British population before and after the introduction of a national HPV vaccination programme and strengthen evidence of a population effect against the HPV types included in the vaccines. Of note, is that these are the first population-based results in the UK general population, rather than in specific groups such as those attending for chlamydia screening These largely baseline data contribute to the evaluation of the effectiveness and cost-effectiveness of the current strategy and future decisions on who should be vaccinated, vaccine choice and schedule. Future surveys will include a larger proportion of vaccinated women, vaccinated at a younger age, and should see further reductions in the HPV vaccine types and allow further consideration of changes in the closely-related HPV types. Longer-term expectations, given higher coverage with the quadrivalent vaccine, or the introduction of the 9-valent vaccine, would be a reduction in cervical cancer precursors, cervical cancer and genital warts Medical Research Council [Grant no. G0701757] and the Wellcome Trust [Grant no. 084840], with contributions from the Economic and Social Research Council and Department of Health. NF is supported by a National Institute for Health Research Academic Clinical Lectureship. SC was funded to undertake independent research supported by the National Institute for Health Research . The views expressed in this publication are those of the author(s) and not necessarily those of the NHS, the National Institute for Health Research or the Department of Health. The sponsors had no role in the study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication.The study was supported by grants from the A.M.J. has been a Governor of the Wellcome Trust since 2011.


Lactobacillus dominance decreased with the severity of cervical neoplasm, which correlated with elevated vaginal pH. Hispanic ethnicity was also associated with decreased Lactobacillus dominance. Furthermore, Sneathia was enriched in all precancerous groups, ICC, abnormal pH and Hispanic origin. Patients with ICC, but not LGD and HGD, exhibited increased genital inflammatory scores and elevated specific immune mediators. Notably, IL-36γ was significantly associated with ICC. Our study revealed local, host immune and microbial signatures associated with cervical carcinogenesis and provides an initial step to understanding the complex interplay between mucosal inflammation, HPV persistence and the VMB.While high-risk human papillomavirus (HPV) infection is a well-established risk factor for cervical cancer, there are likely other factors within the local microenvironment that contribute to cervical carcinogenesis. Here we investigated relationships between HPV, vaginal pH, vaginal microbiota (VMB) composition, level of genital immune mediators and severity of cervical neoplasm. We enrolled women with low- and high-grade cervical dysplasia , invasive cervical carcinoma (ICC), and healthy controls. HPV16, HPV45, HPV58, and HPV31 were the most prevalent in our cohort with HPV16 and HPV31 genotypes more prevalent in Hispanics. Vaginal pH was associated with ethnicity and severity of cervical neoplasm.  Notably, incident rates of cervical cancer vary among different racial and ethnic groups3. In the United States, cervical cancer is more prevalent in Hispanic and African American women, compared to other races or ethnicities3. This disproportionate burden of cervical cancer in these populations has been primarily attributed to a lack of screening and an unequal access to health care; however, other ethnic-related factors might also contribute to these health disparities.Cervical cancer is the most common human papillomavirus (HPV)-related malignancy with estimated 526,000 new cases and 239,000 deaths worldwide in 20154, suggesting that other factors in the local microenvironment in conjunction with HPV play a role in cancer progression. An epidemiologic study associated Chlamydia trachomatis with the development of ICC5. We and others hypothesize a mechanistic role of vaginal microbiota (VMB) in both HPV persistence and cervical cancer progression15.Although persistent infection with high-risk (HR) HPV genotypes is the primary cause of precancerous cervical intraepithelial neoplasia (CIN) and invasive cervical carcinoma (ICC), only a small portion of women infected with HPV progress to CIN, and if not treated, to ICC19. The VMB composition of reproductive age women is Lactobacillus-dominated (LD) with four predominant species , and this LD has been associated with vaginal health19. However, in some women, the VMB lacks a high proportion of lactobacilli and is dominated by a diverse mixture of anaerobic and microaerophilic bacteria  commonly associated with bacterial vaginosis (BV). VMB composition varies between women of different ethnic/racial groups with higher prevalence of non-Lactobacillus-dominant (NLD) VMB in African American and Hispanic women21. However, the higher frequency of NLD VMB in these populations may reflect higher rates of asymptomatic BV19, and controlling for BV eliminates these differences22.Lactobacilli protect the host against genital infections by producing lactic acid , secretion of antimicrobial compounds and competitive exclusion10. Women infected with HPV without cervical disease have been shown to exhibit more diverse VMB relative to HPV-negative women7. Two longitudinal studies also revealed that L. gasseri-dominant VMB was associated with HPV clearance, whereas enrichment in Atopobium spp. was associated with HPV persistence15. Two additional reports showed increasing VMB diversity in patients with CIN and ICC13. However, both studies had a very limited number of ICC cases and authors recommended larger cohort studies to validate their findings. In addition, these studies identified Sneathia spp. to be associated with HPV detection and/or cervical neoplasm15.Several epidemiological studies reported an association between BV and HPV infection and persistenceLactobacillus dominance, particularly L. crispatus, has been associated with low levels of genital inflammation29. Inflammatory profiles differ between BV and classical sexually transmitted diseases such as gonorrhea, chlamydia or genital herpes28. Nevertheless, chronic genital inflammation may promote carcinogenesis similar to other mucosal sites. In clinical studies, HPV infection or clearance has not been associated with increased levels of genital inflammation, but rather VMB composition31. However, one study showed increased levels of proinflammatory cytokines in patients with cervical dysplasia32.Furthermore, BV-associated bacteria have been associated with genital inflammation, specifically increased levels of proinflammatory cytokines, but not cervical immune cells. Herein, we performed a cross-sectional study in Arizona Hispanic and non-Hispanic women to identify associations between HPV genotype distribution, vaginal pH, VMB composition, levels of genital immune mediators, ethnicity and severity of cervical neoplasm. Our systematic approach allowed us to identify unique host immune and microbial signatures associated with cervical carcinogenesis in our cohort. To our knowledge this is the first report comprehensively exploring the complex interplay between HPV, VMB, genital immune mediators, disease progression and patient-specific factors such as ethnicity.P = 0.64), body mass index (BMI) (P = 0.96), HPV status  (P > 0.05), HPV risk profile (including infections with single or multiple HR genotypes as well as mixed infections with HR and LR genotypes) (P > 0.10), gravidity (P = 0.25), parity (P = 0.51) or previous loop electrosurgical excision procedure (LEEP) (P = 0.27). Forty-seven percent of patients were of Hispanic origin and 53% were of non-Hispanic origin. Hispanic ethnicity was not significantly different among the groups, ranging from 25.0% (5/20) in Ctrl HPV− to 63.0% (17/27) in HGD (P = 0.11). Two factors, pH and type of contraception, varied across the groups. The rates of hormonal contraceptive use were significantly different (P = 0.02) with the lowest rate in Ctrl HPV−  and the highest rate in ICC . However, contraception data were only available for 62% (62/100) of the women.A total of 100 premenopausal women were recruited, enrolled in the study and classified into five groups: HPV-negative controls , HPV-positive controls , low-grade dysplasia , high-grade dysplasia  and invasive cervical carcinoma . Table P = 0.01, Table 1A). Ctrl HPV− group had the lowest rate of abnormal pH . Then, percentage of patients with abnormal pH increased to 77.8% (21/27) and 72.7% (8/11) in Ctrl HPV+ and LGD, respectively. The highest rates of abnormal pH were observed in HGD and ICC, respectively, 92.6% (25/27) and 100% (9/9), which was a significant increase when compared to Ctrl HPV− group. The differences in the percentage of women with normal and abnormal vaginal pH across groups remained statistically significant after adjustment for age and BMI (P = 0.003) (Supplementary Table P = 0.006)  , but it was still significant after adjusting only for age (P = 0.03) or only for BMI (P = 0.048).Vaginal pH was collected from 94% 94/100) of the women using nitrazine paper tests. Normal vaginal pH was defined as ≤4.5, and abnormal vaginal pH was defined as >4.5. Number of patients with abnormal pH significantly varied (00 of the02) Fig. . HoweverP = 0.02)  in Hispanic compared to 47.5% (14/40) in non-Hispanic (P = 0.002), whereas percentage of HPV31 was 35.9% in Hispanic (14/39) compared to 2.5% (1/40) in non-Hispanic (P = 0.0001)  of individuals were infected with single HR HPV genotype, 44.3% (35/79) were infected with multiple HR HPV genotypes, and only 1.3% (7/79) were infected with LR HP genotypes (Table 02) Fig. . The mos02) Fig. . While C01) Fig. . These d). Moreover, increasing severity of cervical neoplasm associated with decreasing relative abundance of Lactobacillus spp. and increasing abundance of a variety of microaerophiles and anaerobes  VMB (defined as ≥80% relative abundance of Lactobacillus spp.) were significantly decreased, whereas dysbiotic non-Lactobacillus-dominant (NLD) VMB were increased in LGD (67%), HGD (56%) and ICC (80%) when compared to Ctrl HPV− (40%) or Ctrl HPV+ (32%) (P = 0.04)   and Ctrl HPV+ , whereas other BV-associated bacteria  were enriched only in HGD. To eliminate possible effect of confounders, we controlled our data for age, BMI and ethnicity. We did not adjust for pH, since increased vaginal pH directly correlated with changes in the VMB composition across our groups   of 16S rRNA gene. The VMB analysis revealed that vaginal pH, ethnicity and age were significantly correlated with β-diversity differences  Fig. . We also04) Fig. . Since sPV+ Fig. . Other BGD) Fig. . Atopobimen Fig. . This da38. Levels of seven cytokines , RANTES, and TNFα) were used to determine inflammatory scores; patients were assigned one point for each mediator when the level was in the upper quartile. Using this scoring system, ICC, but not other precancerous groups, exhibited significantly elevated inflammatory score compared to Ctrl HPV− (P < 0.05) . Pairwise comparisons demonstrated statistically significant differences between the Ctrl HPV− and ICC (P = 0.007), Ctrl HPV+ and ICC (P = 0.0003), and HGD and ICC (P = 0.0002). Similar differences were observed also after adjusting for age, BMI and ethnicity. Then, we tested various scoring systems for evaluation of evidence of genital inflammation, which defined genital inflammation as having 3/7, 4/7, 5/7, 6/7 and 7/7 cytokines in the upper quartile , Ctrl HPV+  and HGD . Evidence of genital inflammation in other groups was not significantly increased compared to Ctrl HPV−  or Sneathia presence (relative abundance ≥0.01%) . Following adjustment for age, BMI and ethnicity, MIP-1β and IL-2 were positively associated with ICC in patients with LD VMB  Fig. . As conf05) Fig. . The firPV− Fig. . Since dVMB Fig. , whereasVMB Fig. . IL-36γ VMB Fig. . On the nce Fig. . All tesPV− Fig. . IL-1β, VMB Fig. . IL-36γ,VMB Fig. . The ana4. However, the cause of persistent HPV infection (as most infections are cleared) and progression to cervical cancer remain poorly understood39. In this report, we explored associations between HPV, vaginal pH, VMB composition, levels of secreted genital immune mediators and severity of cervical neoplasm to further investigate the complex interaction between these factors in the local microenvironment.Persistent infection with HR HPV genotypes is the most important risk factor for cervical cancer precursors and ICC34. Other frequent genotypes detected included HPV45, HPV58 and HPV31. These HPV genotypes are targeted by the newest nonavalent HPV vaccine, but not the bi- and quadrivalent vaccines; however, the women in our cohort were not vaccinated. Infection with multiple genotypes were common. In addition, rates of co-infection with multiple genotypes decreased with the severity of neoplasm, in accordance with previous reports40. Interestingly, HPV16 and HPV31 were significantly more prevalent in Hispanic compared to non-Hispanic women. Others also reported racial/ethnic differences in HPV distribution among women with CIN42. In contrast to our data, Hariri et al. showed higher prevalence of HPV45 in Hispanic women and higher rates of HPV16/18-related lesions in non-Hispanic34. Notably, geographic differences in HPV distribution have been also reported42.HPV genotype distribution in our cohort was similar to larger epidemiologic studies, as expected, with HPV16 being most prevalent (65% of HPV-positive samples)43. Our data expand these findings showing positive association of abnormal pH with advanced cervical dysplasia and ICC. Walther-António et al. also reported high vaginal pH, combined with detection of Atopobium vaginae and Porphyromonas spp., to be associated with endometrial cancer44. Collectively, these data suggest that abnormal pH might be a marker of gynecologic cancer. Moreover, we also found that Hispanic women exhibited overall higher vaginal pH, in accordance with a previous report16. Elevated vaginal pH might be an additional risk factor for cervical cancer development/progression.We found that abnormal vaginal pH (>4.5) correlated with the severity of cervical neoplasm. A large population-based study also showed that elevated vaginal pH is associated with oncogenic HPV detection, multiple HPV types infections and diagnosis of LGDLactobacillus abundance, in accordance with a previous report showing elevated vaginal pH in women with diverse NLD VMB16. In our study, NLD VMB significantly (P = 0.04) increased with severity of cervical neoplasm with significantly higher rates of diverse NLD VMB in patients with advanced cervical neoplasia and invasive carcinoma. A previous report also suggested that the lactobacilli depletion is associated with advancing CIN severity, but the analysis did not reach statistical significance12. Interestingly, we also observed that Hispanic women had higher ratios of diverse NLD VMB compared to non-Hispanic women. Higher rates of diverse NLD VMB in Hispanic women are consistent with a previous report16. However, the observed disproportion can be due to higher rates of asymptomatic BV22, which has been associated with HPV detection, as well as, delayed HPV clearance10. In contrast to other studies31, HPV positivity did not significantly alter the VMB composition in women without cervical dysplasia; however, the high proportion of Hispanic women in our cohort might contribute to this finding. Overall, our study indicates that diverse NLD VMB is also associated with cervical disease severity and progression.In addition, abnormal vaginal pH (>4.5) in our cohort correlated with decreased relative Gardnerella, Atopobium, Prevotella. Megasphaera, Parvimonas, Peptostreptococcus, Anaerococcus, Sneathia)45. However, we also identified bacterial genera associated with aerobic vaginitis  or other dysbioses (i.e. Clostridium) to be enriched in these women46. Moreover, we found novel bacterial taxa, i.e. Shuttleworthia, Gemella and Olsenella, to be also associated with HPV positivity and/or cervical neoplasia.With regard to specific bacterial taxa, the majority of identified bacteria that were enriched in HPV-positive women or women with LGD, HGD or ICC are commonly associated with BV . Moreover, after controlling for known VMB confounders , our analysis revealed that precancerous LGD and HGD are enriched in Sneathia. This finding suggests that presence of Sneathia in VMB may also be a metagenomic marker for CIN progression. Lack of enrichment of Sneathia in invasive carcinoma following adjustment for covariates might be due to small ICC sample size. Alternatively, Sneathia might be outcompeted by other bacteria , having a competitive advantage in the tumor microenvironment, as hypothesized at other mucosal sites47.Interestingly, we found Prevotella amnii, originally isolated from human amniotic fluid48. Previously, Prevotella spp. have been associated with HPV infection, but not identified to the species level7. We also found Atopobium and Parvimonas spp. to be enriched with low- and high-grade precancerous lesions. However, following adjustment, we did not detect this relationship, suggesting that enrichment in Atopobium and Parvimonas was associated with one of the covariates. Interestingly, following adjustment for covariates, we also found L. iners to be enriched in HPV-positive women, as well as, women with LGD and HGD. Others also reported L. iners to be associated with higher risk of cervical dysplasia49. L. iners can dominate VMB of healthy women; nevertheless, it is also found in the majority of women diagnosed with BV50. Moreover, L. iners-dominated VMB is more likely to transition to the dysbiotic VMB51. Our findings suggest that L. iners can contribute to the changes in VMB composition, which may lead to disease progression.Other novel taxon identified in this study to be associated with cervical dysplasia included 31. We also evaluated a previously described inflammatory score system to avoid multiple comparisons38. We observed evidence of genital inflammation (defined by elevation of ≥5/7 cytokines) only in patients with ICC, but not with precancerous lesions, when compared to HPV-negative controls. Masson et al. showed that women with BV exhibited only moderately increased levels of pro-inflammatory cytokines, and decreased levels of chemotactic cytokines, whereas women with STI, such as chlamydia and gonorrhea, exhibit the highest levels of cervicovaginal cytokines25. We also did not observe an overt proinflammatory response in our STI-negative cohort, other than the cancer group. Shannon et al. used inflammatory scores and showed that only 45% women with the diverse NLD VMB demonstrate evidence of genital inflammation (defined by elevation of ≥3/7 cytokines). When we employed the same scoring system (≥3/7) for evidence of genital inflammation, we still did not observe any significant differences between precancerous groups and controls.In this study, we systematically examined levels of secreted immune mediators in the cervicovaginal microenvironment at various stages of cervical carcinogenesis. Our analysis revealed increased levels of proinflammatory and chemotactic cytokines , hematopoietic cytokines , adaptive immune response cytokines , and an anti-inflammatory cytokine (IL-10) in women with ICC, but not with precancerous lesions. We did not observe an elevated proinflammatory response in HPV-positive women, which is in accordance with a recent report showing that neither HPV infection nor clearance is associated with broad differences in cervicovaginal cytokinesLactobacillus, which are related to vaginal health, as well as, potentially pathogenic Sneathia. Several immune mediators, such as, TNFα, TNFβ, MIP-1α, GM-CSF and IL-10 were associated with cancer, but not Lactobacillus-dominance, suggesting that their elevated levels are cancer-driven. However, the associations of several other cytokines  with cancer were dependent on the VMB composition. Interestingly, IL-36γ was highly associated with cancer, irrespective of VMB composition. IL-36γ, which belongs to the IL-1 family, is a proinflammatory cytokine expressed at various mucosal sites, including the female reproductive tract, and it functions as a putative “alarmin” in damaged tissue53. Our studies using a human 3-dimensional vaginal epithelial cell model showed that IL-36γ is a driver for epithelial and immune activation following microbial insult and might play a crucial role in the host defense against invading pathogens52. However, the role of IL-36γ in cancer is poorly understood. Wang et al. revealed that IL-36γ exerted anti-tumor effects in vivo and transformed the tumor microenvironment in favor of tumor eradication54. Moreover, the study showed that tumoral expression of IL-36γ in melanoma and lung cancer decreased tumor progression54. This might suggest that increased secretion of IL-36γ in the cervicovaginal microenvironment is a defense mechanism employed by the host to limit cervical cancer progression. On the other hand, in psoriasis, IL-36γ has been shown to promote the inflammatory response through the Wnt/β-catenin pathway, which is altered in many cancers55. This may suggest that IL-36γ is contributing to cervical carcinogenesis by promoting damaging inflammation. The impact of IL-36γ on cervical and other gynecologic cancers warrants further investigation.To decipher the relationship between VMB composition and immune mediators, we explored the associations between immune mediators and A major strength of our study is a high proportion of Hispanic women, which allowed us to determine ethnic-specific factors contributing to health disparities. Compared to other reports, we systemically explored several factors , which may contribute to HPV persistence and/or disease progression. Moreover, we used sophisticated statistical analyses to adjust our microbiome and immune mediator data for known confounders. Our study has some limitations, including a relatively modest sample size and the lack of specific sexual behavior data, i.e. number of sexual partners or sexual debut age. We employed a cross-sectional study design; therefore, we focused on associations, not causation. Longitudinal studies are required to extend these findings and determine the extent to which a diverse NLD VMB or specific bacterial species are casual factors in cervical carcinogenesis or consequences of the disease.Lactobacillus dominance decreased with the severity of cervical neoplasm. We identified Sneathia spp., other BV-associated bacterial species and novel bacterial taxa to be associated with HPV infection and/or HPV-related cervical neoplasm. In addition, we found that women with ICC, but not women with precancerous lesions, exhibited evidence of genital inflammation. Notably, IL-36γ was identified as a unique immune mediator significantly associated with cervical cancer regardless of VMB composition. Overall, this study revealed unique immune and microbial signatures in the local microenvironment associated with cervical carcinogenesis in non-Hispanic and Hispanic women. Further studies, particularly those employing longitudinal designs, are required to better understand the molecular mechanisms involved in the complex role that bacterial communities and/or individual bacterial species may play in the development of cervical neoplasm and the progression to invasive disease.In conclusion, we showed that vaginal pH was associated with ethnicity and significantly increased with the severity of cervical neoplasm. We also revealed that abnormal vaginal pH, Hispanic ethnicity and the severity of cervical neoplasm was significantly associated with depletion of lactobacilli. Furthermore, Participants were consecutively recruited at three clinical sites located in Phoenix, AZ: St. Joseph’s Hospital, and Medical Center (SJHMC), University of Arizona (UA) Cancer Center and Maricopa Integrated Health System (MIHS) and screened based off eligibility criteria and approached to participate in the study. All patients provided informed written consent and all research and related activities involving human subjects were approved by the Institutional Review Boards at UA, SJHMC and MIHS and performed in accordance with federal guidelines and regulations. One hundred premenopausal, non-pregnant, women diagnosed with cervical dysplasia and ICC, as well as, healthy HPV-positive and HPV-negative women were enrolled and contributed to the study. Histology of colposcopy-directed biopsy samples was used to classify patients into groups. A two-tiered system of CIN was used: low-grade dysplasia (LGD) was defined as CIN1, and high-grade dysplasia (HGD) was defined as CIN2/3. If histology was not available , cytology was used for classification. Additionally, HPV status was determined by the Linear Array HPV Genotyping Tests , and used to subdivide healthy women into two groups: HPV-positive  and HPV-negative controls (Ctrl HPV−). Overall, patients were divided into five groups: women with LGD, women with HGD, women with ICC, including adenocarcinoma and squamous cell carcinoma, Ctrl HPV+ and Ctrl HPV−. Exclusion criteria for the study included: currently menstruating; currently on antibiotics, antifungals or antivirals or within the previous 3 months; current vaginal infection , vulvar infection, urinary tract infection or sexually transmitted infection  or within the previous 3 weeks; unusual or foul-smelling vaginal discharge; use of douching substances, vaginal applied medications and suppositories, feminine deodorant sprays, vaginal lubricants within 48 hours prior the visit; any skin condition in the genital area interfering with the study; sexual intercourse less than 48 hours prior the visit; type I or type II diabetes; hepatitis; being HIV-positive. The exclusion criteria were verified by physician’s pelvic exam, medical record and/or self-reported. The details of exclusion criteria are listed in the Supplementary Table Vaginal swabs and cervicovaginal lavages (CVL) were collected by a clinician. A speculum was inserted without lubricant and two vaginal swabs were collected. The first swab was collected by swabbing the lateral walls of the mid vagina using an eSwab collection system containing Amies transport medium . The second swab was used to measure vaginal pH using nitrazine paper, and recorded by the clinician according to the manufacturer’s instructions using a scale of 4.5–7.5. Following vaginal swab collection, CVLs were collected using 10 ml of sterile 0.9% saline solution . Following the collection, both vaginal swab and CVL were immediately placed on ice and frozen at −80 °C within 1 hour of collection.g for 10 min at 4 °C). DNA was extracted from vaginal swabs using PowerSoil DNA Isolation Kit  following the manufacturer’s instructions. Both CVL and extracted DNA were aliquoted to avoid freeze/thaw cycles and stored at −80 °C for further analysis.CVLs were thawed on ice and clarified by centrifugation  following the manufacturer’s instructions.Levels of 21 cytokines and chemokines  were determined in CVL aliquots using the Milliplex MAP Human Cytokine/Chemokine and Th17 Magnetic Bead Panels  in accordance with the manufacturer’s protocol. Level of IL-36γ was measured in CVL aliquots by enzyme-linked immunosorbent assay using Human IL-36γ ELISA kit  in accordance with manufacturer’s instructions. A four-parameter logistic regression curve fit was used to determine the concentration. All samples were assayed in duplicate.16S rRNA sequencing analysis was performed by the Second Genome Inc. . Briefly, the V4 region of bacterial 16S rRNA operon was amplified from the genomic DNA obtained from vaginal swabs and sequenced on the MiSeq platform . The samples were analyzed using USEARCH (for details see Supplementary Methods).37, was used to perform a likelihood ratio test with shrinkage to the base 2 log fold difference to determine whether an OTU had a significantly different normalized count in one of the four groups compared to the normalized count in the HPV-negative control. Subsequently, pairwise comparisons versus the HPV-negative control were performed using the Benjamini-Yekutieli false discovery adjustment (adjusted P < 0.05). A similar analysis was performed with adjustment for age, BMI , and ethnicity .The operation taxonomic units (OTUs) present at the species level were evaluated for their differential presence across the patient groups. Initially, the R package, DESeq2Lactobacillus spp. was determined by quantitative real-time PCR analysis, performed on an Applied Biosystems QuantStudio6 Flex Real Time PCR System  using DNA extracted from vaginal swabs, TaqMan Assays specific for L. crispatus, L. gasseri, L. iners, L. jensenii and panbacterial 16S rRNA genes and TaqMan Vaginal Microbiota Amplification Control and TaqMan Fast Advanced Master Mix (Life Technologies). Relative abundances were calculated using a standard curve method and the 16S rRNA gene level as an internal standard.Relative abundance of four Lactobacillus relative abundance was classified as Lactobacillus-dominant (LD) versus non-Lactobacillus-dominant (NLD) based on a cut-point of 80%. This cut-point classified roughly equal number of patients in the LD (n = 51) and NLD (n = 49) groups. Differences across patient groups were tested using Fisher’s exact test. Comparison of the abundance of the four predominant Lactobacillus species was tested using the nonparametric Kruskal-Wallis test.Differences in the demographic variables between patient groups were tested using analysis of variance model (ANOVA) for continuous variables and Fisher’s exact test for categorical variables. The difference in normal (≤4.5) versus abnormal pH levels (>4.5) between patient groups was tested using Fisher’s exact test, overall and adjusted for age and BMI using logistic regression. P < 0.05), paired tests were performed with Tukey adjustment. Comparisons were adjusted for BMI, age and ethnicity in the linear mixed effects models. The β-coefficients therefore represent the expected change in each immune mediator for that patient group relative to the HPV-negative control for two women with the same values of the clinical covariates. Preliminary analyses demonstrated that there were statistically significant interactions between Lactobacillus dominance (LD) and the patient groups on the levels of the immune mediators (P < 0.05). Thus, whether there were significant differences between the severity groups depended on LD on non-Lactobacillus-dominance (NLD). For these immune mediators, comparison between the groups was performed separately for LD and NLD. To reduce concerns about multiple comparisons, we evaluated a previously described scoring system based on the levels of 7 cytokines. We assessed various scoring systems  and computed overall P values using Fisher’s exact test (Supplementary Fig. For the immune mediator data, the values above the detection limit were substituted with the maximum observation values of the target and values below the detection limit were substituted with 0.5 of the minimum detectable concentration provided in manufacturer’s instructions. The log transformation was applied to normalize the data. The difference in the concentration between the patient groups was tested using a linear mixed effects model where group was a fixed effect and replicate was the random effect. The β-coefficient and standard error were estimated to show the direction of association. If the overall difference was significant (Supplementary InformationSupplementary Dataset


The objective was to investigate how state level strategies in South Carolina could maximize HPV vaccine uptake.An environmental scan identified barriers, facilitators, and strategies for improving HPV vaccination in South Carolina. Interviews were conducted with state leaders from relevant organizations such as public health agencies, medical associations, K-12 schools, universities, insurers, and cancer advocacy organizations. A thematic content analysis design was used. Digital interview files were transcribed, a data dictionary was created and data were coded using the data dictionary.Thirty four interviews were conducted with state leaders. Barriers to HPV vaccination included lack of HPV awareness, lack of provider recommendation, HPV vaccine concerns, lack of access and practice-level barriers. Facilitators included momentum for improving HPV vaccination, school-entry Tdap requirement, pharmacy-based HPV vaccination, state immunization registry, HEDIS measures and HPV vaccine funding. Strategies for improving HPV vaccination fell into three categories: 1) addressing lack of awareness about the importance of HPV vaccination among the public and providers; 2) advocating for policy changes around HPV vaccine coverage, vaccine education, and pharmacy-based vaccination; and 3) coordination of efforts.A statewide environmental scan generated a blueprint for action to be used to improve HPV vaccination in the state. The Advisory Committee on Immunization Practices (ACIP) recommends vaccination at ages 11–12, but only 63% of US females ages 13–17 have received the first HPV vaccine dose, and only 42% have received all three doses needed for full protection Barriers to HPV vaccination at the national level include factors related to vaccination in general and those specific to HPV vaccine. These occur at the level of the patient, provider, and health system or policy environment. At the patient level, barriers include: lack of recommendation by the provider In response to the low national uptake of HPV vaccination, the 2014 President's Cancer Panel Report identified underuse of HPV vaccines as a “serious but correctable threat to progress against cancer National Cancer Institute (NCI) to carry out environmental scans to evaluate the barriers, facilitators, and potential strategies needed to improve state-level HPV vaccination rates After publication of the 2014 report, the Hollings Cancer Center in South Carolina (SC) was one of 18 sites funded by the 22.1This was an NCI-funded project to identify contextually appropriate strategies for improving HPV vaccination uptake in SC by conducting an environmental scan of the barriers, facilitators, and strategies needed for improving HPV vaccination. Thirty-four key informant interviews were conducted with state leaders who represented diverse organizations that have potential to impact HPV vaccination policies and practices. On average, interviews lasted 40 min (range: 30–60 min). The interviews took place during January through June of 2015. Our two interviewers were a PhD-trained cancer control researcher and a gynecological oncologist; each had expertise in cancer control, HPV vaccination and qualitative interviewing. After the interview, participants completed a self-administered demographic survey. Participants were not compensated. A thematic content analysis approach was utilized 2.2The Socio-Ecological Model (SEM) was used as the conceptual model for characterizing the multiple levels of influence on HPV vaccination. The SEM was developed based on the understanding that most public health problems are too complex to be conceptualized and addressed from any single level analyses 2.3Purposive sampling was used to recruit leaders from organizations across the state who had the potential to influence statewide HPV vaccination policies and practices. To select stakeholders, the research team held a brainstorming session to create a list of key organizations in the state whose missions could help to improve HPV vaccination practice and policy; and from this list, the name and contact information for each organization's leader was identified. Participants were recruited from this list via a series of three email invitations, followed by two personal phone calls to those who had not responded to email invitation. Each of these contact attempts were spaced approximately two weeks apart.2.4Key informant interviews were the primary data source. The semi-structured interview guide, as shown in Interviews were audiotaped and transcribed. Transcripts were checked for accuracy by a second person. The data collected were highly structured based on the specific purpose of the study. Two researchers reviewed a sample of the interview transcripts independently to create a joint codebook to guide formal analysis. The codebook was used to code all transcripts, and emergent themes were added as needed, such as when themes arose from tailored interview questions with specific participants. The preliminary results were circulated to all co-authors and additional researchers for review and discussion. This iterative process strengthened the ability of the research team to optimize valid interpretations of the interviews.3The characteristics of the 34 individuals who participated in the key informant interviews are shown in 3.1The main barriers identified were lack of HPV awareness among the general public, lack of provider recommendation, concerns about HPV vaccination, lack of access among young adults and underinsured adolescents, reimbursement barriers for pharmacy administration, difficulty completing the three-dose series, and practice-level barriers .Table 2B3.1.1Participants reported a wide range of barriers related to HPV vaccine awareness among the general public. A commonly reported barrier was that there has been a total absence of systematic messaging to promote accurate information about HPV vaccination. Stakeholders made comments such as “One of the biggest barriers is lack of promotion; No big state push to say vaccinate your kids like they do for flu vaccine” and “When I drive home I see a sign for ovarian cancer about going to get yourself checked out. We don’t have anything like that for cervical cancer…nothing to tell people, hey you can avoid getting cancer.”Attempts in the state to increase awareness of HPV vaccination among the general public and parents, such as the 2016 Cervical Cancer Prevention Act, had not been supported widely, according to participants. The Cervical Cancer Prevention Act, designed to educate middle school parents about HPV vaccination and extend coverage for the vaccine, was stalled in the legislature at the time of the interviews. Concerns about lack of awareness even extended to key stakeholders, such as legislators and public health officials in the state, those with influence to modify policies and practices for addressing barriers to increase HPV vaccination. One participant explained “We have legislators that don’t support the vaccine.” Another participant described “Stronger support for the HPV vaccine is needed at the state health department.” Another commonly cited barrier was lack of HPV awareness among the general public. Parents were identified as a particular group whose general lack of awareness persisted and required intervention. As described by one participant “I teach at a college and I did a session on sexually transmitted disease; most of them hadn’t heard of the HPV vaccine.” Another participant described “There is a lot of misinformation among the parents of these children we are trying to vaccinate.” Parental lack of awareness was compounded by poor endorsement of the vaccine by health care providers. One participant explained “Frankly I think providers are a barrier. Providers not recommending vaccination due to their own personal beliefs.” Participants with children reported being left to make the decision to vaccinate by themselves and being told that sons did not need the HPV vaccine. As one participant described “My pediatrician did not bring it up. I had to request it.” Several participants suggested that confusion about the need for vaccinating males stemmed from the focus on cervical cancer in the initial awareness campaigns. One participant noted “boys are not getting the message that the vaccine is for them. The bill (in SC) is called the Cervical Cancer Prevention Act instead of HPV. You don’t think of a boy taking that—it's for cervical cancer.”3.1.2Participants perceived that factors contributing to the deficiencies in provider communication about HPV vaccine were 1) lack of awareness of HPV vaccine guidelines among some pediatricians and family practitioners, 2) providers’ limited comfort in discussing the topic, 3) perceptions about the time required to present HPV vaccination to parents and address their questions, and 4) the tendency of adolescents to visit physicians only for acute health issues making it difficult to address preventive care issues. Consistently, barriers focused on providers either not recommending HPV vaccination or providing inconsistent or ineffective recommendations. As one participant described “If you have a 16-year-old coming in with a sore throat, you could take 5 min to ask if she's having sex. But that makes them freak out. It's the same with a 9-year-old coming in with a sports injury who's not being asked if they've been vaccinated for HPV.” Another participant explained “Pediatricians are often concerned with childhood illnesses rather than a disease that could affect them later in life.” Participants felt these deficiencies in provider recommendation led to missed opportunities to increase HPV vaccination awareness among parents.3.1.3Participants widely reported parental concerns about HPV vaccination, specifically the sexually transmitted nature of HPV infection and uncertainty about safety, as barriers. Participants perceived the sexually transmitted nature of HPV as a greater barrier in the context of the conservative and religious values in SC. As one participant explained “We are in the Bible Belt, and people are not comfortable with their children being sexually active.” The sexually transmitted nature of HPV was identified by participants as a concern that permeated every level: the general public, providers, and policy makers. One participant described the parental perspective as “My child's not going to have sex so why vaccinate them for an STD.” Another participant explained “Some providers don’t feel comfortable giving clear recommendations for HPV vaccination. They have inadvertently been affected by the anti-vaccine lobby. Instead of saying you need your HPV vaccine today, they shy away.” From the policy perspective, another participant noted “We have legislators who don’t support the vaccine.” According to participants, some parents believed that giving the vaccine was tantamount to endorsing sexual activity and in particular were reluctant to vaccinate their children at the recommended age of 11–12 years old. One participant explained her thoughts about how parents perceive the vaccine, stating “HPV vaccination got a bad rap: you’re encouraging my child to have sex.” Participants who were clinicians described how anticipating such concerns caused them to avoid a strong endorsement of the vaccine or delay recommending the vaccine until patients were older. As one participant described “Some physicians, even in my practice, wait until age 15 or 16 to recommend the vaccine.” Concerns around vaccinating for a sexually transmitted virus were perceived by participants to be connected to parental resistance to interventions such as school-based vaccination.Related barriers included parental concerns about safety of vaccines in general and the perception that the HPV vaccine was new and untested. While HPV vaccines have been available for over ten years, some participants reported that uncertainty about the safety of the HPV vaccine is still an issue. One participant described “I had a talk with colleagues with children the age for the vaccine; they felt they didn’t have enough vaccine info.” Another participant explained “I think we see barriers from anti-vaccination groups who say they are concerned that there is not enough data.” A few participants felt that the African American community held a greater distrust of new vaccines and medical interventions. As one participant described “There is distrust in the AA community about medical treatments of any kinds.”3.1.4Lack of access, related to cost, was identified as a barrier to HPV vaccination among underinsured adolescents and young adults due to SC public programs and private insurers not covering the vaccine. Most children in the state had access to HPV vaccination through private insurance, Medicaid, or the federal Vaccines for Children (VFC) program. At the time of the stakeholder interviews, underinsured adolescents could obtain all ACIP recommended vaccines except HPV vaccine through the SC Vaccine Program. This is a program administered by the state health department that covers vaccinations for underinsured children who are ineligible for Medicaid, but who do not have health insurance. As one participant explained “Our state vaccine program excludes HPV vaccination, so children who are underinsured can receive any other vaccine recommended by the CDC except the HPV vaccine. This affects 2400 children every year.” Participants advised that the state health department should add HPV vaccination to the state program to extend coverage to underinsured children. As described by several participants, the health department could add the HPV vaccine within the state vaccine program based on public health authority, but has instead sought “legislative direction” several times to add HPV vaccination to the state program, which had been repeatedly denied by the legislature. Young adults in the catch-up population for HPV vaccination also faced barriers to access, mainly related to insurance coverage. At the time of the interviews, the largest insurer in the state did not cover HPV vaccination for young adults aged 18–26, though other private insurers did. As one participant explained: “Third-party reimbursement, we have to improve that. Our state health plan on campus won't cover HPV vaccine.” Participants representing university health centers indicated that HPV vaccination status was optional on matriculation immunization forms. HPV vaccination in universities is complicated by limitations in access, but these settings have been resourceful in seeking support from pharmaceutical company programs and utilizing referrals to provider settings where there may be more financial support for vaccination.3.1.5Several participants representing multiple sectors noted the importance of practice-level barriers in low HPV vaccination uptake. A commonly cited barrier was simply that adolescents did not visit the doctor unless they were sick or were in need of sports physicals. One participant stated “Most adolescents don’t come in for well visits.” Clinician participants reported that they were often busy taking care of acute care issues, which limited the amount of time available to initiate discussion about vaccination. As one participant explained “Providers may be hesitant to recommend the vaccine because they have a very limited amount of time with patients and don’t want to get caught up in a 15-min conversation about one vaccine.” Further, participants identified barriers related to the lack of infrastructure and support for prompts to initiate provider recommendation when a patient was due for HPV vaccination. While most practices used an electronic health record (EHR), few to none had invested in developing electronic prompts for HPV vaccine dose reminders. Successful practices manually identified patients, which was cumbersome and labor intensive. The lack of prompts was attributed to limitations of EHR systems. One participant stated “Most IT EHR systems do not have population management systems in them. For some, it is an add-on that needs to be purchased, so not every practice has the capability.” Another participant further explained “For some centers, staff time is the limitation to create reminder and recall templates.” Reimbursement for the cost of administering the HPV vaccination was not a problem for most practices, but may have posed a barrier for some smaller practices. As one participant explained “Some vaccines you get reimbursed less than the actual vaccine cost. Larger practices have more power to negotiate these costs.” Another participant described “Administrative fees have come up. Some clinics have to carve out vaccines because they don’t get full cost back.” Recent pharmacy legislation in SC has resulted in pharmacists being allowed to vaccinate adolescents with prescription and adults without prescription. Participants noted that this could improve access to HPV vaccination for both target populations. However, a participant from the state pharmacy association explained “An issue in the pharmacy community is that not all insurance companies will reimburse pharmacists,” which posed a barrier for utilizing pharmacy-based vaccination.3.2The main identified facilitating factors specific to our state were 1) a high degree of momentum in commitment to addressing HPV vaccination uptake, 2) the school-entry diphtheria, tetanus, and acellular pertussis (Tdap) requirement, 3) pharmacist-administered HPV vaccination, 4) the SC state immunization registry and other IT infrastructure, 6) HEDIS and other reportable quality measures, and 7) the federal VFC program and state vaccine program as funding sources for HPV vaccination that could be further leveraged. Each of these is discussed in detail below  .Table 3F3.2.1Enthusiasm about momentum at the national level was fueled by the President's Cancer Panel Report in 2014, the Cervical Cancer-Free America movement, and federal funding to address HPV vaccination efforts available through the NCI and CDC. One participant summarized this momentum saying “Momentum is going for us—this grant, state health department money, health disparities money. We have a real opportunity to tip the scale.” At a state level, Cervical Cancer Free SC, a state partner of the national advocacy organization committed to elimination of cervical cancer through vaccination, screening, and education, had made efforts to promote coordination among state players. As one participant explained “There is leverage behind cervical cancer. With Cervical Cancer Free South Carolina, the work being done at the Medical University of South Carolina, the University of South Carolina, the SC Cancer Alliance; many organizations are targeting cancer. These are wonderful resources that not every state has.” Clinician participants reported that some pediatric practices were participating in a statewide quality improvement project of the American Academy of Pediatrics to increase vaccination uptake. One participant described that SC was one of 10 states to receive a grant to focus on pediatric QI initiatives, including HPV vaccination. She explained “The project includes about 18 practices in South Carolina, with 6 of their sites focusing on process improvement to increase HPV vaccination.” Finally, at the time of the interviews, the state legislature was considering the Cervical Cancer Prevention Act, which would provide education about the HPV vaccine to parents of middle school children and add HPV vaccination to the state vaccine program. As one participant explained “We are working hard on the Cervical Cancer Prevention Act to add HPV vaccination to the state vaccine program.”3.2.2Participants reported that the Tdap vaccination requirement in SC provided a “hook” to initiate HPV vaccination. The Tdap vaccine recently became an entry requirement for middle school in SC. The state health department sent a flyer to parents noting the new Tdap requirement and promoting all adolescent vaccines recommended by the ACIP, including the HPV vaccine. One participant described “This was the first time some parents had heard of the HPV vaccine.” The Tdap requirement also offered an opportunity for providers to introduce all ACIP recommended vaccines, which was especially critical for adolescents who do not participate in well-child visits. As one participant explained “Using Tdap as the hook for HPV vaccination is the key. When children have to get the mandatory Tdap vaccine for entry to middle school, that is when you can direct them to their provider to get their HPV vaccinations.”3.2.3An opportunity exists in SC for HPV vaccination in pharmacies. Pharmacies offer increased access points for completion of the three-dose HPV vaccination series and enable young adults to be vaccinated without visiting a physician. As one participant described “The fact that pharmacists may be able to provide any vaccine with a prescription is a plus from my standpoint. We have more immunizing providers than some other states.” Participants also noted the importance of integration across systems . One participant explained “An important component is for the pharmacist to put the vaccination in the DHEC immunization registry to ensure continuity of care/recordkeeping.” One participant indicated that pharmacies have robust built-in mechanisms to transfer patient data back and forth to other entities, such as to patient medical homes.3.2.4At the health system level, participants noted the opportunity to build on recent improvements in infrastructure to support HPV vaccination. The state vaccine registry provides a new clinical tool to coordinate vaccination across multiple settings, including pharmacies. Starting in January 2017, all providers have been required by law to enter to record vaccinations in the registry 3.2.5HPV vaccination was a reportable HEDIS measure for adolescent females, which participants described as a facilitating factor for vaccination. The planned inclusion of adolescent males for the HPV vaccination measure was noted as beneficial. The HEDIS measure for HPV vaccination was reportedly important for exerting pressure on practices to measure performance and meet certain standards. As one participant explained “The 2014 National Commission for Quality Assurance (NCQA) standards put more emphasis on prevention. They (providers) must look at 2 vaccines at 2 different age groups.” Further, HEDIS measures could encourage practices to invest their limited time and energy in improving HPV vaccination rates. Participants similarly mentioned that linking reimbursement to HEDIS measures could create even more positive pressure for improvement. One participant described “Providers like to get good grades and to get paid well. You could tie vaccination rates to reimbursement. Providers will increase HPV vaccination rates just because they are being rated.”3.2.6Participants noted that the federal VFC program in SC has the opportunity to access more funds for HPV vaccines, which could be a facilitating factor. One participant explained that the number of vaccine doses available to each state is based on the number of individuals within the recommended age group for vaccination, and that “SC uses only about 2/3 of allotted doses of HPV vaccines available through the federal VFC program.” Thus, free HPV vaccine doses are available to vaccinate more children who are eligible for the VFC program (i.e. those who are uninsured or who are on Medicaid). Further, participants reported that the state health department could include HPV vaccines in the state immunization program without additional legislation and that they have funds available to do so. If this policy change were made, it would enable coverage for “underinsured” children, or those who are ineligible for Medicaid, but who do not have health insurance.3.3As shown in 3.3.1To address the lack of awareness of HPV vaccination among the general public and providers, a two-pronged educational strategy was recommended: 1) widespread public education about HPV vaccination via mass media and/or social media and 2) targeted education of healthcare providers through in-service sessions at state provider organization meetings, email listservs, and CME credit programs. Many participants noted that together parents and providers represent the driving force for HPV vaccination, and that engaging both these target groups simultaneously could be synergistic in facilitating readiness for HPV vaccination during the adolescent healthcare visit. As explained by one participant “It's two-pronged: part of it is the clinicians themselves and how they offer or inform the family that their child needs a vaccine; the other prong is the parent asking for it. Another participant noted ”Education is key to getting vaccination rates up. It needs to be across the board and include providers and parents.”3.3.2From a policy standpoint, it was recommended that organizations work together to support: 1) the Cervical Cancer Prevention Act, which was designed to enable education of middle school parents and coverage of HPV vaccines for underinsured adolescents, 2) policy change in partnership with insurers to cover the HPV vaccine among young adults in the catch-up vaccination group, and 3) legislation expanding pharmacy legislation, such as by allowing pharmacists to vaccinate adolescents without a prescription. Multiple participants cited the success in increasing flu vaccination through pharmacy-based administration. As one participant described “If pharmacists could give the vaccine (without a physician prescription for adolescents), we could reach some of those nooks and crannies in the state that are underserved by providers. We have shown a bump in flu and other vaccines since pharmacists stated vaccinating.” One stakeholder stressed that advocating for more of the major state insurers to cover pharmacy-based vaccination was a crucial step in utilizing that resource. She explained “We (state pharmacists) are trying to get listed as a provider for the VFC program.”3.3.3Purposeful coordination of efforts between state partners was also recommended as a strategy to improve HPV vaccination rates. One participant suggested “I think it would be important to invest in Cervical Cancer Free South Carolina” to coordinate these efforts. Another participant suggested “It may help to go through organizations like the American Academy of Pediatrics who endorse the vaccine.” This coordination would enable the concurrent implementation of statewide public and provider education, coupled with grassroots advocacy work to garner support for the HPV vaccine at the local community level. Universities, public health organizations, and provider organizations were noted to have the skillset needed to conduct high quality public and provider education. One participant explained “We could use assistance with wording and support for vaccination from our state partners such as the Hollings Cancer Center. We are the providers, but it could be a statewide initiative supported strongly by state partners.” Partnerships with local schools, churches, and other community organizations were recommended to reinforce the message at the local level that HPV vaccination is a safe and effective method for preventing cancer. Participants made suggestions such as “Use the school system. Get school nurses to provide information and answer parents’ questions. Parents and students already have a relationship” and “Work with Parent Teacher Associations; engage the faith community. If they approve things in the state, people may be more likely to get vaccinated.” Another participant described “Get community buy-in. We need religious leaders who understand the science and have connections to organizations like the SC Policy Council, the SC Palmetto Council, the SC Catholic Coalition and the SC Baptist Convention. That speaks volumes legislatively and in churches back home.” To improve vaccination rates at the practice level, participants advised working with organizations such as pharmaceutical companies and the South Carolina Office of Rural Health that can provide funding and/or technical support for creation of health system prompts. One participant recommended “You can work with pharma companies to help with both reminder/recalls systems and provider education. They want their vaccine out there. Pharma can help link EHR to generate reminder/recalls.” One participant described that the SC Office of Rural Health has “done a lot of work in our state to get our providers using electronic medical records. I wouldn't say its 100% but the vast majority now are using electronic health records and are attesting to meaningful use.” A few participants suggested that state agencies or provider organizations provide practices with a standard toolkit to further facilitate HPV vaccination. As one participant explained “Comprehensive educational marketing that could be like a toolkit of resources could be given to school nurses, pediatricians and family clinics to make it easier to educate parents. We need a way to catch people's attention and have the information come from somebody they trust like their healthcare providers and school nurses.” On the policy level, coordinated advocacy would help achieve needed changes. One participant recommended “Use the South Carolina Cancer Alliance for lobbying and for the advocacy piece.” Another participant explained “One thing that has worked with tobacco control is local coalitions. We should get some strong women to come out and support HPV vaccination in their communities.” Another participant added additional insight, saying “The more people understand the HPV vaccine, the more they will advocate for legislation.”4A statewide environmental scan was carried out to assess barriers, facilitators and strategies for improving HPV vaccination that are contextually specific to SC. As anticipated, the environmental scan identified many of the same types of barriers that have been previously identified nationally. For example, barriers were identified at the patient , provider , and policy  levels.More importantly, stakeholder interviews also revealed actionable information about these barriers that were contextually specific to SC. For example, lack of investment in state-level public/provider awareness campaigns was commonly identified as a problem. While vaccine cost was not identified as a common barrier among younger adolescents, lack of vaccine coverage was a barrier for “underinsured” adolescents and young adults because the state vaccine program and some insurers, respectively, did not cover vaccination for these populations at the time of this project. In addition, while pharmacists in the state could vaccinate adults without prescription and adolescents with prescription, lack of coverage for pharmacy-based vaccination by insurers and the requirement for physician prescription in order for pharmacists to vaccinate adolescents limited the potential impact of pharmacy-based vaccination. Finally, politicization of HPV vaccination related to concerns about sexuality and promiscuity had stifled efforts to make the vaccine mandatory or provide school-based vaccination or education about the vaccine. Thus, the stakeholder interviews provided a rich source of information about specific and actionable barriers to HPV vaccination.To address these barriers, the environmental scan identified facilitators that can be leveraged to improve HPV vaccination rates in SC, including demonstrated commitment from state partners, the school-entry Tdap requirement, pharmacy-based HPV vaccination, EHR infrastructure in many practices that could support reminder/recall systems, and untapped funding sources for HPV vaccination. Suggestions about strategies for improving HPV vaccination included multi-level educational efforts that combine state level awareness campaigns and engagement with grassroots organizations such as churches and schools, policy advocacy, and purposeful coordination of efforts among state stakeholders to methodically address each of the barriers to HPV vaccination.To enable comparison of our results with those from other NCI-funded HPV environmental scan projects, we identified five studies that reported environmental scan results. These studies were conducted in diverse parts of the US, including one western US region that included 10 states [42]In contrast to our study, the three environmental scans conducted in settings that served a high volume of medically underserved or multi-cultural/lingual patients identified additional needs for reading-level, language, and culturally-tailored health education materials NCI's strategic decision to fund environmental scans across the US demonstrates a strong commitment to ensuring that critical cancer prevention methods such as HPV vaccination move beyond research into communities across the country. These projects provided many NCI-designated and comprehensive cancer centers with dedicated resources to help understand and develop contextually appropriate strategies for optimizing HPV vaccination for cancer prevention. Cancer centers and health science universities are uniquely poised to facilitate coordinated statewide efforts to increase HPV vaccination for many reasons. First, these organizations often have the respect among those in their catchment areas as being their state or region's experts in residence on HPV vaccination. This was a common comment by participants in the current study. Second, these organizations may have a diverse cadre of researchers and clinicians with relevant expertise and interest to facilitate statewide efforts to increase HPV vaccination. For example, cancer centers and health science universities have professionals working in epidemiology and biological aspects of HPV vaccination, health education and behavioral sciences, dissemination and implementation, community engagement, health systems and policy and program evaluation. In addition, oncologists who care for patients with HPV-related cancers are often passionate to find ways to prevent these cancers in the future. Third, there are potential federal funding mechanisms, such as the environmental scan grant that funded the current project, that can be utilized to help support the work of NCI-affiliated cancer centers to facilitate HPV vaccination coordination efforts. Many opportunities exist for cancer centers and health science universities to help facilitate HPV vaccination through organizations such as state cancer alliances and non-profit organizations such as Cervical Cancer Free America.Since the time of this environmental scan, several accomplishments have occurred in SC related to the barriers and strategies identified by participants. The Cervical Cancer Prevention Act, which was stalled at the time of interviews, was passed in SC in April 2016 For further progress on HPV vaccination, sustained and coordinated efforts are required in SC. This environmental scan effectively evaluated the current barriers and facilitators to timely HPV vaccination in SC. This project also generated information that will be helpful in designing contextually appropriate strategies for improving HPV vaccination across the Southeast, where HPV vaccination rates are lower than many other regions of the US National Cancer Institute through grant #3P30CA138313-06S2.This project was funded by the


The mortality of cervical cancer in Longnan is as high as 39/10 million, ranking first in China.Between 2012 to 2016, 329 samples with cervicitis, cervical intraepithelial neoplasia grade 1 to 3 (CINI to III), and invasive squamous cell carcinoma (SCC) were collected. HPV genotypes were examined with a validated kit for 23 different HPV subtypes.P < 0.001) and the positivity is also higher in SCC compared to CINI (P < 0.01). The most frequently detected genotypes were HPV16 in cervicitis, HPV16, 58 and 52 in CINI and CIN II/III, and HPV16, 58 and 18 in SCC groups. HPV16 positivity in cervicitis, CINI, CIN II/III, and SCC patients were 45.46%, 46.81%, 60.32% and 78.69%, respectively. Compared to cervicitis and CINI, the odds ratios (OR) for SCC in HPV16 positive patients were 2.96  and 4.20 , respectively. In addition, the multiple infections in cervicitis, CINI, CINII/III and SCC group are 9.09%, 27.66%, 26.98% and 25.41% and HPV16 + 58 was the most common combinations.Compared to cervicitis, the HPV positivity is significantly higher in CINI, CIN II/III, and SCC (38.60%, 74.60%, 87.50% and 89.05%, These findings highlight the key role of HPV16, 58, 52 and 18 in the development of CIN and SCC in Longnan women and a fully aware of regional differences in HPV genotype distribution are tasks for cervical cancer control and prevention. Human papillomaviruses (HPV), double-stranded and non-enveloped DNA viruses (7 ~ 8 kb long), are a group of remarkably diverse DNA viruses from the Papillomaviridae family, which are causally involved in the etiology of various benign and malignant neoplastic lesions of mucosal and skin epithelium , 2. CurrCervical cancer (CC) is a major fatal malignancy among women, causing about 265,700 deaths annually world-wide. Nearly 90% of cervical cancer deaths occur in developing countries, such as China . EpidemiThe prevalence of HPV infection and the reported type-specific distribution varies greatly by geographic region and ethnicity. For example, HPV16 is slightly more prevalent in Europe and North America, HPV 31 is more prevalent in South/Central America, HPV 33 and 45 are more prevalent in Africa, and HPV 52 and 58 are more prevalent in Asia , 11–17. Longnan of Gansu Province, located in the remote areas of Northwest of China, is the high incidence areas of cervical cancer and cervical cancer mortality as high as 39/10 million, ranking first in China . This stA total of 329 Longnan patients aged 17 ~ 79 years samples were initially included in the present study between January 1, 2012, and January 30, 2016: 57 in the cervicitis group, 63 in the CINI group, 72 in the CINII/III group, and 137 in the SCC group. All the samples were obtained from patients who underwent biopsies with colposcopy or advanced operations. In all of samples, 305 were obtained at the No.1 Hospital of Longnan City as well as 24 samples from Gansu Provincial Cancer Hospital. All patients gave written informed consent for their participation. This study has been approved by the Ethics Committees of Northwest University for Nationalities prior to its start. All the samples were formalin-fixed and paraffin-embedded. All specimens were evaluated by at least 2 experienced pathologists in their respective hospital’s pathology department. Cervicitis, CINI, CINII/III and squamous cervical cancer (SCC) were diagnosed according to the standard criteria .The Human Papillomavirus Genotyping kit for 23 Types was produced by Yaneng Bioscience co., LTD , and the kit was applied for with permit number 3400994 in 2008 by the Fresh Armed State Drug Administration, China. The kit was used to perform Polymerase chain reaction (PCR) to amplify the L1 gene in conjunction with reverse dot blot (RDB) analysis to identify the HPV subtypes. This method offers a simple testing strategy involving a membrane chip that can detect infections from multiple HPV subtypes, including 18 high-risk types  and 5 low-risk types . This method had a sensitivity of 103 copies/ml and a specificity of 99%; β-globin was used as an internal positive control : 1.09–8.00, Some women were infected with 2 or 3 types of HPV simultaneously (Table Our data showed that 38.60% of patients with cervicitis, 74.60% with CINI, 87.50% with CINII∕III, and 89.05% with SCC were positive for HPV DNA. HPV infection rates were significantly higher in CINI, CINII∕III, and invasive SCC than in cervicitis patients, but they never reached 100%. In addition, in our population, HPV 16 was the dominant genotype and HPV 33, 53, 58 and 59 were the second dominant genotypes for cervicitis. HPV genotypes 16, 58, and 52 were the dominant high-risk HPV in patients with CINI and CINII∕III, however for invasive SCC, the dominant high-risk HPV were 16, 58 and 18. Besides of the genotypes suggested above, HPV 6, 11, 31, 33, 51, 56 and 59 also were detected. In some reports in which HPV16, 18, and 45, HPV16, 18 and 33 or 16, 18 and 58 were most commonly detected in cervicitis –26, CINIIn our group of CINI, CINII∕III and SCC patients, the positivity for HPV DNA are consistent with those in most other studies , 24–30. The distribution of dominant HPV genotypes showed obvious regional differences . HPV18 iOur data suggested that more patients with CINI, CIN II∕III and SCC had multiple HPV infections compared to cervicitis, however, the rates of multiple HPV infections in CIN I, CIN II/III and SCC patients showed a slightly decreasing trend with severity of lesions. Although this finding is partially consistent with the results of previous studies, in which Francois et al. and Meizhu Xiao et al. found that the rates of multiple hr-HPV infections in CINII, CINIII, and CC patients declined gradually , 39, theOur data indicated that the combination of HPV16 + 58 plays a dominant role in all cervical lesion groups, contrary to findings in the Jewish Israeli population and in Austrians, but in agreement with findings in women in Beijing, China –43. ThusOverall, as demonstrated in our study population, genotypes 16, 58, 52 and 18 are among the predominant HPV and HPV infection increased with cervical lesion, regional differences in HPV genotype distribution and the real carcinogenic HPV revealing need to be mindful in the HPV control and prevention.


HPV testing followed the manufacturers' protocols. Women with abnormal cytology were managed according to the routine recommendations. Cytology-normal/HPV-positive women were invited for repeated testing in 18 months. Screening history and histologically confirmed cervical intraepithelial neoplasia (CIN) in 2.5 years after the baseline testing were determined from the national pathology register. HPV-positive women undergoing primary screening having concordant samples were more likely to harbor high-risk infections and less likely to harbor only low-risk infections than women with discordant samples. Additionally, assay signal strengths were substantially higher in concordant samples. More than 80% of ≥CIN2 results were found for women with concordant samples, and no ≥CIN2 results were found when the infection was detected by only one assay. These patterns were similar in the referral population despite the younger age and higher number of HPV infections. HPV test result discordance identified a cluster of low-risk HPV infections that were hardly ever associated with high-grade CIN and, almost exclusively, represented false-positive screening findings.Laboratories now can choose from >100 human papillomavirus (HPV) assays for cervical screening. Our previous analysis based on the data from the Danish Horizon study, however, showed that four widely used assays, Hybrid Capture 2 (HC2), cobas, CLART, and Aptima, frequently do not detect the same HPV infections. Here, we determined the characteristics of the concordant samples  and discordant samples  in primary cervical screening at 30 to 65 years of age ( Human papillomavirus (HPV) assays will be used for cervical cancer screening  and for monitoring of the effectiveness of HPV vaccination (to determine the changes in infection epidemiology). Currently, laboratories can choose from >100 commercially available HPV assays . TherefoIn the Danish split-sample Horizon study comparing HC2 , cobas , CLART , and Aptima  HPV assays, the detection of HPV infections depended on the assay. This was particularly pronounced in women undergoing primary cervical screening at ≥30 years, where only 29% of all positive test results were concordant on all four compared assays . The remThis unexpected finding warrants an additional in-depth analysis, as it may have implications for the two intended uses of HPV assays. Hence, our aim here was to study an infected woman's likelihood of harboring high-risk HPV and high-grade CIN depending on the degree of the discordance between the HPV test results. To gain insight into the patterns across various populations, we studied two groups of women: those undergoing primary screening and those with (recent) abnormalities .The study design was described in detail previously –11. In s–in situ [AIS], HC2-positive ASCUS at ≥30 years, abnormal repeated testing following ASCUS at <30 years, low-grade squamous intraepithelial lesions [LSIL], or HC2-negative ASCUS at ≥30 years). Women with normal cytology and a positive test result on at least one HPV assay were invited, according to the study protocol, for repeated cytology and HPV testing in 18 months after the baseline. Follow-up samples with abnormal cytology or a positive HC2 test result elicited a referral for colposcopy. All colposcopies were undertaken under routine conditions either by a hospital or privately practicing gynecologists. In Denmark, it is recommended that directed biopsy specimens be taken from all suspicious areas after application of acetic acid and random biopsy specimens from four quadrants if lesions are not visible. The Hvidovre laboratory evaluated almost 90% of the high-grade CIN biopsy specimens included in the study. During the study period, between four and seven pathologists evaluated cervical cytology and gynecological histology in this laboratory. Most samples were read by one pathologist. p16 staining was not used systematically. The remaining approximately 10% of high-grade CIN biopsy specimens were evaluated in other Danish hospitals or by private pathologists. Data on the most severe histological diagnosis during follow-up were retrieved from the national Patobank in December 2013, covering the period of about 2.5 years after baseline. Follow-up was highly complete for women with abnormal baseline cytology (ca. 95%) and moderately complete (ca. 60%) for women with cytology-normal/HPV-positive test results . The instrumentation and software were used as supplied and maintained by the manufacturers. HC2 testing was undertaken on the postquot LBC material. cobas, CLART, and Aptima testing was undertaken on the original residual material, diluted approximately 1:1 in SurePath. HC2 detects 13 high-risk HPV genotypes collectively. The assay is based on hybridization of HPV DNA to a high-risk HPV RNA probe cocktail. cobas is a real-time PCR analysis detecting the 13 high-risk HPV genotypes and HPV 66. The assay separately identifies HPV 16 and HPV 18, while the remaining 12 genotypes are detected collectively. The CLART assay is a PCR-based low-density microarray detecting 35 defined genotypes, including the 13 high-risk genotypes. All genotypes are reported individually. Aptima detects E6/E7 mRNA expression of the 13 high-risk HPV types and HPV 66 collectively. HC2, cobas, and Aptima are FDA-approved assays and have also been validated according to the protocol defined by the international assay validation guidelines .n = 2,859, 56.8% of all 5,034 included women; 10 were excluded because of invalid HPV test results). The referral population included women with follow-up samples and women with screening samples showing abnormal cytology at any age, excluding women with invalid HPV test results .The primary screening population was defined as women with screening samples at 30 to 65 years of age without invalid HPV test results (TC) values of ≤40.5, ≤40.0, and ≤40.0, respectively . Aptima was considered positive when the signal-to-cutoff (S/CO) value was ≥0.5. CLART was considered positive when ≥1 high-risk HPV genotype was detected. In line with the classification by the International Agency for Research on Cancer, genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68 were considered high risk (HC2 was positive when the relative light units per cutoff (RLU/CO) value was ≥1. cobas was positive when channels 16, 18, and/or other high-risk genotypes had critical threshold  on the HC2, cobas, and Aptima assays for samples that tested positive on only one assay compared to samples that tested positive on all four assays. The 95% confidence intervals (CI) for relative proportions (RP) comparing screening and referral populations were calculated assuming lognormal distribution. Analyses were undertaken using IBM SPSS Statistics, version 19.We categorized samples by the number of positive HPV test results (one through four). For each category, we determined the proportions of samples with HPV 16 infections, with infections with the other 12 high-risk genotypes (excluding HPV 16), with only low-risk HPV infections, and without a single detected HPV genotype (from among 35 detectable by CLART); these proportions were mutually exclusive. Separately, we determined the proportions of high-risk samples with multiple infections (defined as detection of ≥2 genotypes with or without low-risk genotypes) and with abnormal cytology (≥ASCUS). Genotypes were reported as detected by CLART. Trends in the proportions of genotype detection by category were tested with the Mantel-Haenszel χThe baseline testing on the residual material was undertaken as a quality development study and did not require ethical approval, in line with Danish regulations of biomedical research. The Ethical Committee of the Danish Capital Region approved the follow-up of HPV-positive/cytology-normal women (H-4-2012-120), and women signed informed consent. The Danish Data Inspection Agency was notified of the study (notification numbers 2010-41-5594 and AHH-2015-080/I-Suite: 04109).The mean age of the 2,859 women was 42.7 years . Of these, 651 (22.8%) had at least one positive HPV test result at baseline . Only 18P < 0.001). Their samples were also more likely to contain only low-risk or no HPV genotype (P < 0.001). As this comparison could have been biased by the double role of the CLART assay , we additionally studied the concordance comparing only the HC2, cobas, and Aptima assays, with CLART used exclusively for an adjudication of the genotypes present in the sample. This comparison revealed essentially the same patterns .Women with a positive test result on only one HPV assay were less likely to have HPV 16 infections, infections with other high-risk genotypes, multiple HPV infections, and abnormal cytology than women with a positive test result on >1 HPV assay  histologically confirmed ≤CIN1, 12 (0.4%) CIN2, 50 (1.7%) ≥CIN2, and 38 (1.3%) ≥CIN3 cases . All fouThe mean age of the 885 women was 34.0 years , and 519 (58.6%) had HPV infections detected on one or more assays. Of the 519 samples with at least one positive HPV test result, 89 (17.1%) were positive on only one, and 290 (55.9%) were positive on all four assays.These HPV-positive women differed from those in the screening population. They were more likely than the screening population to be infected with HPV 16   and haveAmong the 885 women, HPV assays detected 176 (19.9%) women with histologically confirmed ≤CIN1, 40 (4.5%) with CIN2, 129 (14.6%) with ≥CIN2, and 89 (10.1%) with ≥CIN3. Among women with high-grade CIN, the concordance between the four HPV assays was similar to that in the screening population. Of the 129 women with ≥CIN2, 105 (81.3%) were positive on all four assays, as were 72 (80.9%) of the 89 women with ≥CIN3. High-grade CIN was only rarely found in women with only one positive HPV test result (2.3% of 129 women with ≥CIN2 and 3.4% of 89 women with ≥CIN3).Screening samples in which the HC2, cobas, CLART, and Aptima assays showed HPV status discordance represented a cluster with a lower risk of ≥CIN2 than samples in which all four assays detected HPV infections. Discordant samples had weaker assay signals, were less likely to contain high-risk genotypes, multiple infections, and high-grade CIN, and more likely to harbor only low-risk or no HPV genotype. These differences between weakly and strongly positive samples were also found in the referral population with a lower average age and a higher prevalence of high-risk infections, suggesting that our findings can be generalized across various populations.The study was undertaken on fresh routine samples. All samples were handled in one laboratory by the same staff. By having access to the women's screening histories, we could determine which samples were taken for screening purposes, even though this information was not registered routinely. This is one of a few studies that compared several HPV assays on primary screening samples with active follow-up of women with positive HPV test results and normal cytology. Although the follow-up with repeated testing was incomplete, it was very similar to that observed in the randomized controlled trials comparing HPV-based to cytology-based primary cervical screening . NeverthThere is no internationally agreed standard reference HPV genotyping assay. The research-only Linear Array (LA) was among the assays most frequently used for genotyping in previous studies. However, LA test results are read manually and may be prone to interobserver variability. We genotyped the infections using the CE-IVD-marked CLART assay, which interprets the test results using a built-in software algorithm. In some studies –18 but n–TC, 30.2) and samples where the assays returned different test results   .Previous screening studies showed high levels of concordance between assays in terms of the detection of high-grade CIN, which corresponds to a substantially higher likelihood of detecting these lesions when multiple assays return a positive test result . NeverthThe concordance between the assays increased with the severity of the underlying biology. The percentage of HPV-positive women testing positive on all four HPV assays increased from 22% in women with normal cytology to 68% in women with abnormal cytology and to 84% in women with ≥CIN2. The high level of concordance in detecting high-grade CIN indicates that the choice of an HPV assay for screening will have little effect on the high-risk women who should be recommended for treatment to avoid progression to cervical cancer. The likelihood that they will be detected through screening is high with any of the assays evaluated here, which is consistent with a relatively high clinical sensitivity for each assay.On the other hand, false-positive screening tests, i.e., positive HPV test results with harmless infections that do not lead to high-grade CIN, represent clinically inconsequential noise. They represent a challenge for primary HPV-based screening even in women above 30 years of age , and thiAs their infections will be detected by some but not other assays, healthy women with false-positive test results will be affected by the choice of an HPV assay for primary screening. These women will be recommended for (unnecessary) follow-up, possibly including colposcopy. This brings into focus the question of whether the management recommendations for HPV-positive women should be the same irrespective of which assay detected the infection. Studies agree in that, regardless of the assay used for primary screening, HPV-positive women should not be directly referred for colposcopy –28. For –One of the indicators for monitoring the implementation of HPV vaccination is a change in the epidemiology of HPV genotypes . Our stuIn conclusion, discordance between multiple HPV assays in detecting HPV infections identified a cluster of weakly positive infections that are less frequently associated with HPV 16 and 12 other high-risk genotypes. As these samples also hardly ever harbored high-grade CIN, they were typically associated with false positivity in screening.