Pre-Vaccination Human Papillomavirus Genotypes and HPV16 Variants among Women Aged 25 Years or Less with Cervical Cancer.

BACKGROUND: In 2007, Australia introduced a national human papillomavirus (HPV) vaccination program. In 2017, the onset of cervical screening changed from 18 to 25 years of age, utilising human papillomavirus (HPV) nucleic acid testing. The objective of the study is to describe the HPV genotypes and HPV16 variants in biopsies from women ≤ 25 years of age with cervical carcinoma (CC) (cases), compared with those aged >25 years (controls), in a pre-vaccination cohort. METHODS: HPV genotyping of archival paraffin blocks (n = 96) was performed using the INNO-LiPA HPV Genotyping assay. HPV16-positive samples were analysed for variants by type-specific PCR spanning L1, E2 and E6 regions. RESULTS: HPV16 was the commonest genotype in cases (54.5%, 12/22) and controls (66.7%, 46/69) (p = 0.30), followed by HPV18 (36.3%, 8/22 vs. 17.3% 12/69, respectively) (p = 0.08). Furthermore, 90% (20/22) of cases and 84.1% (58/69) of controls were positive for HPV16 or 18 (p = 0.42); 100% (22/22) of cases and 95.7% (66/69) of controls had at least one genotype targeted by the nonavalent vaccine (p = 0.3). The majority of HPV16 variants (87.3%, 48/55) were of European lineage. The proportion of unique nucleotide substitutions was significantly higher in cases (83.3%, 10/12) compared with controls (34.1%, 15/44), (p < 0.003, χ2, OR 9.7, 95%CI 1.7-97.7). CONCLUSIONS: Virological factors may account for the differences in CCs observed in younger compared with older women. All CCs in young women in this study had preventable 9vHPV types, which is important messaging for health provider adherence to new cervical screening guidelines.

Performance of Human Papillomavirus Attribution Algorithms to Predict Causative Genotypes in Anal High-Grade Lesions.

BACKGROUND: Gay and bisexual men (GBM) are at increased risk of human papillomavirus (HPV)-associated anal high-grade squamous intraepithelial lesions (HSILs). Understanding the fractions of HSILs attributable to HPV genotypes is important to inform potential impacts of screening and vaccination strategies. However, multiple infections are common, making attribution of causative types difficult. Algorithms developed for predicting HSIL-causative genotype fractions have never been compared with a reference standard in GBM. METHOD: Samples were from the Study of the Prevention of Anal Cancer. Baseline HPV genotypes detected in anal swab samples (160 participants) were compared with HPV genotypes in anal HSILs (222 lesions) determined by laser capture microdissection (LCM). Five algorithms were compared: proportional, hierarchical, maximum, minimum, and maximum likelihood estimation. RESULTS: All algorithms predicted HPV-16 as the most common HSIL-causative genotype, and proportions differed from LCM detection (37.8%) by algorithm (with differences of -6.1%, +20.9%, -20.4%, +2.9%, and +2.2% respectively). Fractions predicted using the proportional method showed a strong positive correlation with LCM, overall (R = 0.73 and P = .002), and by human immunodeficiency virus (HIV) status (HIV positive, R = 0.74 and P = .001; HIV-negative, R = 0.68 and P = .005). CONCLUSIONS: Algorithms produced a range of inaccurate estimates of HSIL attribution, with the proportional algorithm performing best. The high occurrence of multiple HPV infections means that these algorithms may be of limited use in GBM.

Point-of-care HPV DNA testing of self-collected specimens and same-day thermal ablation for the early detection and treatment of cervical pre-cancer in women in Papua New Guinea: a prospective, single-arm intervention trial (HPV-STAT).

BACKGROUND: WHO recommends human papillomavirus (HPV) testing and same-day treatment for cervical screening in low-income and middle-income countries (LMICs); however, few published data exist on the validity of the strategy. We aimed to evaluate the clinical performance, treatment completion rates, adverse events profile, and acceptability of a fully integrated strategy, comprising point-of-care HPV DNA testing of self-collected specimens and same-day thermal ablation, for screening of cervical cancer in women in Papua New Guinea. METHODS: HPV-STAT was a large-scale, prospective, single-arm intervention trial conducted at two clinical sites in Papua New Guinea. Cervical screening clinics with an on-site consultant gynaecologist were selected in consultation with national and provincial health authorities, church health services, and local stakeholders. Eligible participants were women aged 30-59 years attending cervical screening services at the two clinics, who were willing to comply with study procedures and able to provide written informed consent. Women self-collected vaginal specimens for point-of-care GeneXpert testing (Cepheid, Sunnyvale, CA, USA) for oncogenic HPV types. Women testing positive for HPV underwent pelvic examination followed by same-day thermal ablation or referral for gynaecology review. All HPV-positive women and a 15% random sample of HPV-negative women provided a clinician-collected cervical specimen for liquid-based cytology. The primary outcome was clinical performance (ie, sensitivity, specificity, positive predictive value [PPV], and negative predictive value [NPV]) of the strategy for the detection of high-grade squamous intraepithelial lesion (HSIL) or worse. This trial is registered with ISRCTN, ISRCTN13476702. FINDINGS: Between June 5, 2018, and Jan 6, 2020, we recruited 4285 women, 3638 (84·9%) of whom tested negative for HPV and 647 (15·1%) tested positive for one or more oncogenic HPV type. Sensitivity of the algorithm to detect HSIL or worse was 85·4% (95% CI 81·0-89·6), with specificity 89·6% (88·6-90·6), PPV 35·2% (31·6-39·0), and NPV 98·9% (98·6-99·2). Among HPV-positive women, 602 (93·0%) received same-day thermal ablation and 42 (6·5%) were referred for gynaecology review, 37 (88·1%) of whom attended. Acceptability was high among both HPV-positive and HPV-negative women. Among the 329 HPV-positive women who attended a 3-month follow-up visit, 51 (15·5%) reported mild adverse symptoms that resolved in all cases by the follow-up visit. There were no serious adverse events. INTERPRETATION: We conducted the first real-world evaluation of a fully integrated point-of-care HPV self-collect, test, and treat strategy for same-day cervical screening in a LMIC and found it to be effective, acceptable, and safe when implemented at scale in primary health-care facilities in Papua New Guinea. Our findings support the introduction and scale-up of HPV screening and treatment for the control and elimination of cervical cancer in LMICs, as recommended by WHO. FUNDING: Australian National Health and Medical Research Council.

Gene methylation of CADM1 and MAL identified as a biomarker of high grade anal intraepithelial neoplasia.

Human papillomavirus (HPV) is detected in up to 96% of anal squamous cell cancers, where screening programs needed. However, the best methodology is still undetermined. Host DNA methylation markers CADM1, MAL and miR124 have been identified in cervical disease, but not anal disease. Anal swabs varying by disease grade were assessed for DNA methylation of CADM1, MAL and miR124-2. Each marker was compared across disease grades, stratified by HPV and HIV status. Receiver operating characteristic curves identified the predictive value of significant gene candidates. CADM1 methylation was significantly higher in high-grade squamous intraepithelial lesions (HSIL) compared with low-grade (LSIL) (p = 0.005) or normal (p < 0.001) samples with 67.2% correctly identified as HSIL. MAL methylation was significantly (p = 0.002) increased in HSIL compared with LSIL in HIV positive participants with 79.8% correctly indicated as HSIL. Gene miR124-2, showed no difference between disease grades. Biomarkers with established diagnostic value in cervical disease have limited utility in the prediction of anal disease, with CADM1 identified as a marker with screening potential in a gay and bisexual men (GBM) population and MAL in HIV positive GBM population. New markers specific to the anal mucosa are required to improve triage of high-risk individuals.

Postnatal Cytomegalovirus Infection of Preterm and Very-low-birth-weight Infants Through Maternal Breast Milk: Does It Matter?

BACKGROUND: Postnatal infection with cytomegalovirus (CMV) in very-preterm and very-low-birth-weight infants, transmitted through breast milk (BM), is potentially associated with adverse outcomes. This study aimed to investigate the incidence and clinical significance of postnatal CMV infection in a tertiary neonatal intensive care unit. METHODS: Infants of CMV-seropositive mothers born in a neonatal intensive care unit in Melbourne, Australia, were observed for 14 weeks from birth in a prospective cohort study. Maternal BM and infant urine were tested weekly for CMV by culture and polymerase chain reaction, respectively. Clinical and laboratory data were collected and analyzed in relation to the infants' CMV infection status. RESULTS: Data from 65 infants of 56 CMV-seropositive mothers were available for analysis. Of these mothers, 88% (49/56) shed CMV in their BM. Of the 58 infants exposed to CMV-positive BM, 27 (47%) became urine polymerase chain reaction CMV-positive. There was no significant difference in gestational age, birth weight, incidence of bronchopulmonary dysplasia, or necrotizing enterocolitis between the CMV-positive and CMV-negative groups. However, CMV-positive infants had a longer length of hospital stay and more episodes of prolonged neutropenia. Of the CMV-positive infants, 30% (8/27) remained asymptomatic, 48% (13/27) had symptoms categorized as mild and 22% (6/27) as severe. CONCLUSIONS: About half of preterm and very-low-birth-weight infants exposed to CMV-positive BM become infected, and a fifth develop significant clinical symptoms. Future studies should address the maternal and neonatal factors that determine the risk of mother-to-infant CMV transmission, as well as those leading to clinical deterioration and long-term sequelae.

Human Papillomavirus Antibody Levels Following Vaccination or Natural Infection Among Young Men Who Have Sex With Men.

BACKGROUND: Australia introduced a school-based gender-neutral human papillomavirus (HPV) vaccination program for girls and boys aged 12-13 years in 2013. We examined HPV type-specific antibody levels in unvaccinated young men who have sex with men (MSM) with natural infection and compared these with levels in those vaccinated against HPV. METHODS: Serum specimens at baseline were collected from MSM aged 16-20 years in the HYPER1 (Human Papillomavirus in Young People Epidemiological Research) and HYPER2 studies, conducted in 2010-2013 and 2017-2019, respectively. Merck's 4-plex HPV competitive Luminex Immunoassay was used to quantify HPV6-, HPV11-, HPV16-, and HPV18-specific antibodies. We compared antibody levels for each HPV genotype between unvaccinated men (HYPER1) and vaccinated men (HYPER2) using the Mann-Whitney U test. RESULTS: There were 200 unvaccinated men and 127 vaccinated men included in the analysis. Median antibody levels among vaccinated men were significantly higher than levels among unvaccinated men for HPV6 (223 milli-Merck units per milliliter [mMU/mL] vs 48 mMU/mL, P < .0001), HPV11 (163 mMU/mL vs 21 mMU/mL, P < .0001), HPV16 (888 mMU/mL vs 72 mMU/mL, P < .0001), and HPV18 (161 mMU/mL vs 20 mMU/mL, P < .0001). Antibody levels did not change over time for up to 66 months for all 4 genotypes among vaccinated men. CONCLUSIONS: Among young MSM vaccinated with the quadrivalent HPV vaccine, antibody levels for HPV6, HPV11, HPV16, and HPV18 were significantly higher than those in unvaccinated MSM following natural infection. Antibody levels following vaccination appeared to remain stable over time. CLINICAL TRIALS REGISTRATION: NCT01422356 for HYPER1 and NCT03000933 for HYPER2.

Accuracy of Self-reported Human Papillomavirus Vaccination Status Among Gay and Bisexual Adolescent Males: Cross-sectional Study.

BACKGROUND: Men who have sex with men are a risk group for anal human papillomavirus (HPV) and anal cancer. Australia introduced a universal school-based HPV vaccination program in 2013. Self-reported HPV vaccination status has been widely used in clinical and research settings, but its accuracy is understudied. OBJECTIVE: We aimed to examine the accuracy of self-reported HPV vaccination status among gay and bisexual adolescent males. METHODS: We included 192 gay and bisexual males aged 16-20 years from the Human Papillomavirus in Young People Epidemiological Research 2 (HYPER2) study in Melbourne, Australia. All participants had been eligible for the universal school-based HPV vaccination program implemented in 2013 and were asked to self-report their HPV vaccination status. Written informed consent was obtained to verify their HPV vaccination status using records at the National HPV Vaccination Program Register and the Australian Immunisation Register. We calculated the sensitivity, specificity, positive predictive value, and negative predictive value of self-reported HPV vaccination status. RESULTS: The median age of the 192 males was 19 (IQR 18-20) years. There were 128 males (67%) who had HPV vaccination records documented on either registry. Self-reported HPV vaccination had a sensitivity of 47.7% (95% CI 38.8%-56.7%; 61/128), a specificity of 85.9% (95% CI 75.0%-93.4%; 55/64), a positive predictive value of 87.1% (95% CI 77.0%-93.9%; 61/70), and a negative predictive value of 45.1% (95% CI 36.1%-54.3%; 55/122). CONCLUSIONS: Self-reported HPV vaccination status among Australian gay and bisexual adolescent males underestimates actual vaccination and may be inaccurate for clinical and research purposes.

Absence of human papillomavirus in nasopharyngeal swabs from infants in a population at high risk of human papillomavirus infection.

Maternal urogenital human papillomavirus (HPV) infection may place neonates at risk of HPV acquisition and subsequently lower respiratory infections as HPV can influence development of immunity. The respiratory HPV prevalence is not known in remote-dwelling Aboriginal infants, who are at high risk of respiratory infection and where the population prevalence of urogenital HPV in women is high. These data are necessary to inform HPV vaccination regimens. A retrospective analysis using PCR specific for HPV was performed on 64 stored nasopharyngeal swabs from remote-dwelling Aboriginal infants < 6 months of age, with and without hospitalised pneumonia. HPV DNA was not detected in any specimen. Despite the negative result, we cannot exclude a role for HPV in respiratory infections affecting infants in this population; however, our data do not support HPV as an important contributor to acute respiratory infection in remote-dwelling Aboriginal children.

Prevalence of human papillomavirus in young men who have sex with men after the implementation of gender-neutral HPV vaccination: a repeated cross-sectional study.

BACKGROUND: Anal infection with high-risk human papillomavirus (HPV) genotypes 16 and 18 and anal cancer are overrepresented in men who have sex with men (MSM). This study investigated HPV prevalence in young MSM before and after the implementation of a school-based quadrivalent HPV (genotypes 6, 11, 16, and 18) vaccination programme for boys in Australia in 2013. METHODS: In this repeated cross-sectional study, MSM aged 16-20 years were recruited from two successive birth cohorts via sexual health clinics and the community in Melbourne, Australia. The first cohort was before the implementation of gender-neutral vaccination (HYPER1 study, done in 2010-12, NCT01422356), and the second was the post-vaccination cohort (HYPER2 study, done in 2017-18, NCT03000933). Men who self-identified as being same-sex attracted were enrolled, and those recruited via the HYPER2 study had to be resident in Australia since 2013 to ensure eligibility. Study procedures were done in the Melbourne Sexual Health Centre. A clinician-collected anal swab and self-collected penile swab and oral rinse were tested for 28 HPV genotypes, and data on demographics and sexual health practices were collected via questionnaires. Only assessable samples were included in the analyses. We compared anatomical site-specific prevalence of HPV genotypes between cohorts by calculating the prevalence ratio, adjusting for age, circumcision, and sex with women. Herd protection was also assessed, by calculating the adjusted prevalence ratios by vaccination status. FINDINGS: 400 MSM, 200 per cohort, were included in the study. In both cohorts, the median number of lifetime male partners was ten (IQR 5-25). The prevalence of any anal quadrivalent vaccine-preventable HPV genotype was higher in the pre-vaccination cohort (54 [28%] of 193) than in the post-vaccination cohort (14 [7%] of 193; adjusted prevalence ratio [PR] 0·24, 95% CI 0·14-0·42), largely driven by decreases in HPV6, followed by HPV11, 16, and 18. Nevertheless, there was also a significant reduction in anal HPV16 and 18 in the post-vaccination cohort from the pre-vaccination cohort (0·31, 0·14-0·68). The prevalence of any penile quadrivalent vaccine-preventable HPV genotype was also higher in the pre-vaccination cohort (21 [12%] of 177) than in the post-vaccination cohort (11 [6%] of 179; 0·48, 0·24-0·97), driven by decreases in HPV 6 and 11, but not by 16 and 18. The prevalence of any oral quadrivalent vaccine-preventable HPV genotype was higher in the pre-vaccination cohort (seven [4%] of 200) than in the post-vaccination cohort (one [1%] of 199; 0·10, 0·01-0·97); there were no cases of oral HPV6 or 11 detected in HYPER2. Comparing the pre-vaccinated cohort with the 149 confirmed vaccinated men from HYPER2 showed a reduction in any quadrivalent vaccine-preventable HPV genotype for anal (0·09, 0·03-0·25) and penile (0·18, 0·05-0·59) infection but not for oral infection (0·17, 0·03-1·08). INTERPRETATION: A reduction in anal, penile, and oral quadrivalent vaccine-targeted genotypes occurred in young MSM following the implementation of a school-based gender-neutral HPV vaccination programme. The fall in anal HPV16 and 18 may lead to a reduction in the incidence of anal cancer. FUNDING: Merck and the Australian Government Department of Health.

HPV16/18 prevalence in high-grade cervical lesions in an Australian population offered catch-up HPV vaccination.

OBJECTIVES: Using laser capture microdissection (LCM) and sensitive human papillomavirus (HPV) genotyping, we aimed to determine the distribution of vaccine-preventable types in cervical intraepithelial neoplasia grade 3 (CIN3) lesions and adenocarcinoma in situ (AIS) in young women in Victoria, Australia, offered catch-up HPV vaccination, as a baseline for ongoing vaccine impact monitoring. We also compared findings with available pre-vaccination estimates from women with HPV detected on concurrently-collected cytology samples. METHODS: Consecutive histologically-confirmed CIN3/AIS biopsies were collected between May 2011 and December 2014 from vaccine-eligible women (born after 30th June 1981). Genotypes present in whole tissue sections (WTS) were determined by a sensitive reverse hybridisation assay; RHA kit HPV SPF10-LiPA25, v1 (Labo Bio-medical Products). Where multiple genotypes were detected, lesions were isolated using LCM and genotyped. Cervical cytology samples from a pre-vaccine cohort had been previously collected and genotyped using HPV Linear Array HPV Genotyping Test (Roche Diagnostics). Mixed-genotype detections in this cohort were resolved to single-lesion-attributable genotypes using hierarchical attribution. RESULTS: Overall, 213 and 530 cases were included from pre- and post-vaccine time-periods, respectively. In 18-25 year-olds, the proportion of HPV16/18-positive CIN3/AIS decreased significantly over time from 69% in 2001-2005 (pre-vaccine), to 62% in 2011-2012 (post-vaccine), to 47% in 2013-2014 (p-trend = 0.004). There was no significant change in HPV16/18 in 26-32 year-olds (p-trend = 0.15). In 2013/14, nonavalent vaccine types accounted for 80% of CIN3/AIS in 18-25 year old women and 90% in 26-32 year old women. CONCLUSION: Four to 8 years following implementation of HPV vaccination in Australia, approximately 70% of CIN3/AIS in young women was due to HPV16/18. Our data, despite some limitations due to change in methods between pre- and post-vaccine periods, suggests that for vaccine-eligible women aged 18-25 at the time of biopsy, the proportion of HPV16/18-attributable CIN3/AIS lesions is significantly declining post-vaccination.

Assessment of attribution algorithms for resolving CIN3-related HPV genotype prevalence in mixed-genotype biopsy specimens using laser capture microdissection as the reference standard.

To make accurate determinations regarding potential and actual impact of HPV vaccine programs, precise estimates of genotype-specific contributions to disease are required for pre- and post-vaccine populations. Definitive determination of lesion-specific genotypes, particularly where multiple genotypes are detected in a sample, can be technically demanding and resource intensive; therefore, most prevalence studies use mathematical algorithms to adjust for multiple genotype detections. There are currently several algorithms, which can produce genotype estimates within a wide range of variability. The use of these for cervical cytology samples has recently been assessed for accuracy against a definitive reference standard, but none have yet been assessed for multiple-genotype-containing whole biopsy specimens. Using laser capture microdissection (LCM) on biopsy samples, lesion-specific genotype prevalence data were generated for a cohort of 516 young Australian women (aged 18-32 years) with cervical intraepithelial neoplasia grade 3 or adenocarcinoma in situ. Using whole tissue section genotype data from the same cohort, including 71 (13.7%) with multiple genotypes, lesion-associated genotype prevalence was estimated using four different attribution algorithms. The proportion of lesions attributable to HPV16 and HPV18 by LCM were 58.4% and 5%, respectively; hierarchical, proportional, single type/minimum and any type/maximum attribution estimates were comparable across genotypes. For analyses utilising whole tissue biopsy cervical specimens, attribution estimates are appropriate for estimating the proportional contribution of individual genotypes to lesions in a population.

Prevalence of human papillomavirus in teenage heterosexual males following the implementation of female and male school-based vaccination in Australia: 2014-2017.

BACKGROUND: Australia introduced a school-based human papillomavirus (HPV) vaccination program for females aged 12-13 years in 2007, with a three-year catch-up to age 26; and for boys aged 12-13 from 2013, with a two-year catch-up to age 15. This study aimed to compare the prevalence of penile HPV between teenage heterosexual males in cohorts eligible or non-eligible for the school-based male vaccination program. METHODS: Between 2014 and 2017, sexually active heterosexual males aged 17-19 were recruited from sexual health centres and community sources across Australia. Males provided a self-collected penile swab for 37 HPV genotypes using Roche Linear Array and completed a questionnaire. We calculated adjusted prevalence ratios (aPR) of HPV between males in two periods: 2014-2015 (preceding implementation of school-based male vaccination) and 2016-2017 (eligible for school-based male vaccination). Self-reported vaccine doses were confirmed with doses reported to the National HPV Vaccination Program Register. RESULTS: Overall, 152 males were recruited in 2014-2015 and 146 in 2016-2017. Numbers of female sex partners and condom use did not differ between the two periods. The prevalence of quadrivalent vaccine-preventable [4vHPV] genotypes (6/11/16/18) was low in both periods (2.6% [2014-15] versus 0.7% [2016-17]; p = 0.371; aPR 0.28 [95% CI: 0.03-2.62]). Compared with men in 2014-2015, men in 2016-2017 had a lower prevalence of any of the 37 HPV genotypes tested (21.7% versus 11.6%; aPR 0.62 [95% CI: 0.36-1.07]) and any of the 13 high-risk genotypes tested (15.8% versus 7.5%; aPR 0.59 [95% CI: 0.30-1.19]). Prevalence of low-risk HPV genotypes did not differ between the two periods. Of the males recruited in 2016-2017, 55% had received ≥1 vaccine dose. CONCLUSION: The prevalence of 4vHPV genotypes among teenage heterosexual males in both cohorts was low, presumably due to herd protection from the female-only vaccination program. Further studies are required to determine the impact of universal HPV vaccination on HPV prevalence in males.

Performance of clinical screening algorithms comprising point-of-care HPV-DNA testing using self-collected vaginal specimens, and visual inspection of the cervix with acetic acid, for the detection of underlying high-grade squamous intraepithelial lesions in Papua New Guinea.

The performance of different clinical screening algorithms comprising point-of-care HPV-DNA testing using self-collected vaginal ('V') specimens, and visual inspection of the cervix with acetic acid (VIA) was evaluated in Papua New Guinea. Women aged 30-59 years provided V specimens that were tested at point-of-care using the Xpert HPV Test (Cepheid, Sunnyvale, CA). A clinician-collected cervical ('C') specimen was then collected for point-of-care Xpert testing, and liquid-based cytology (LBC). Following this, VIA examination was conducted, blind to HPV test results, and ablative cervical cryotherapy provided if indicated. Detection of high-grade squamous intraepithelial lesion (HSIL) by LBC was the reference standard used to evaluate clinical screening algorithms. Of 1005 women, 36 had HSIL+. Xpert HPV Test performance using V specimens (sensitivity 91.7%, specificity 87.0%, PPV 34.0%, NPV 99.3%) was superior to VIA examination alone (51.5%, 81.4%, 17.5%, 95.6% respectively) in predicting underlying HSIL+. A screening algorithm comprising V specimen HPV testing followed by VIA examination had low sensitivity (45.5%) but comparable specificity, PPV and NPV to HPV testing alone (96.3%, 45.5%, 96.3% respectively). A 'test-and-treat' screening algorithm based on point-of-care HPV testing of V specimens had superior performance compared with either VIA examination alone, or a combined screening algorithm comprising HPV testing plus VIA.

Development of a pilot proficiency program for human papillomavirus DNA detection.

Human papillomavirus (HPV) DNA testing has become routine in many diagnostic laboratories, particularly with changes from cervical cytology to HPV DNA as primary screening as of 1 December 2017 in Australia. External quality assurance (EQA) is essential for assessment of laboratory performance once HPV testing is implemented. The aim of this study was to develop a pilot program to evaluate and determine stability of material that could be utilised in an ongoing external quality assurance program (EQAP). Two sample types were evaluated: cells in PreservCyt solution (ThinPrep) from stored clinical specimens and HPV-seeded swabs. Two panels sent 5 months apart were distributed to 18 Australian and two New Zealand laboratories (participants) for testing by Hybrid Capture 2 (HC2) or alternative molecular methods. Participants were given 1 month to test specimens. Eight ThinPrep specimens in Panel 1 were reported correctly by 73% (11/15) of HC2 participants and 40% (2/5) of participants performing alternative methods. Of eight dry swab specimens, 58% (23/40) and 78% (25/32) were correctly identified by HC2 and alternative methods, respectively. Panel 2 included four ThinPrep and two swab specimens. ThinPrep specimens were reported correctly by 100% (60/60) of participants utilising HC2 and 95% (19/20) utilising alternative methods. Dry swab specimens were reported correctly by 89% (25/28) of participants utilising HC2 and 100% (10/10) of participants utilising alternative methods. These results indicate that both specimen types are suitable for utilisation in an EQAP and outline some issues of EQAP for ongoing assessment of HPV molecular methods in diagnostic molecular laboratories.

HLA and KIR Associations of Cervical Neoplasia.

Background: Cervical cancer is the fourth most common cancer in women, and we recently reported human leukocyte antigen (HLA) alleles showing strong associations with cervical neoplasia risk and protection. HLA ligands are recognized by killer immunoglobulin-like receptors (KIRs) expressed on a range of immune cell subsets, governing their proinflammatory activity. We hypothesized that the inheritance of particular HLA-KIR combinations would increase cervical neoplasia risk. Methods: Here, we used HLA and KIR dosages imputed from single-nucleotide polymorphism genotype data from 2143 cervical neoplasia cases and 13858 healthy controls of European decent. Results: The following 4 novel HLA alleles were identified in association with cervical neoplasia, owing to their linkage disequilibrium with known cervical neoplasia-associated HLA-DRB1 alleles: HLA-DRB3*9901 (odds ratio [OR], 1.24; P = 2.49 × 10-9), HLA-DRB5*0101 (OR, 1.29; P = 2.26 × 10-8), HLA-DRB5*9901 (OR, 0.77; P = 1.90 × 10-9), and HLA-DRB3*0301 (OR, 0.63; P = 4.06 × 10-5). We also found that homozygosity of HLA-C1 group alleles is a protective factor for human papillomavirus type 16 (HPV16)-related cervical neoplasia (C1/C1; OR, 0.79; P = .005). This protective association was restricted to carriers of either KIR2DL2 (OR, 0.67; P = .00045) or KIR2DS2 (OR, 0.69; P = .0006). Conclusions: Our findings suggest that HLA-C1 group alleles play a role in protecting against HPV16-related cervical neoplasia, mainly through a KIR-mediated mechanism.

Follow-up study of HPV58 variants in women with incident HPV58 infection from a Colombian cohort.

Certain variants of human papillomavirus (HPV)type 58 are associated with an increased risk of high grade squamous intraepithelial lesions and cervical cancer. However, little is known about the persistence of HPV58 E6/E7 variants in women with incident HPV58 infections. The aim of the present study was to evaluate the presence and persistence of HPV58 E6/E7 variants in 71 women with incident HPV58 infection throughout their follow-up. These women belonged to a cohort examined in a longitudinal study of 1,610 Colombian women, who were HPV-negative and had normal baseline cytology. E6/E7 DNA regions of HPV58-positive samples were amplified and sequenced using automated direct sequencing. A total of 639 samples were analyzed from the 71 women, and 117 samples (18.3%) were HPV58-positive. HPV58 E6/E7 variants were detected in 85.5% of the samples. The T307/A694/G744/A761 variant was identified in 88% of the samples, the T307/G744 variant was identified in 9% of samples and the T187/T307/A367/G744/G793/T798/A801/T840/C852 was identified in 3% of the samples. Overall, 50% of the HPV58 infections were present after 1 year of follow-up and all infections were cleared after 7 years. Women who had first sexual intercourse at >15 years of age had a lower clearance rate than those who had sexual intercourse for the first time at ≤15 years of age [hazard ratio (HR)=0.29; 95% confidence interval (CI)=0.09-0.92]. Likewise, parous women had a higher clearance rate than nulliparous women (HR=3.43, 95% CI=1.23-9.60). There was no difference in clearance rates between HPV58 E6/E7 variants. In conclusion, HPV58 variants were not associated with persistence of the infection in this group of women.

Cellular Immune Responses 6 Years Following 1, 2, or 3 Doses of Quadrivalent HPV Vaccine in Fijian Girls and Subsequent Responses to a Dose of Bivalent HPV Vaccine.

BACKGROUND: This study examined the cellular immunity of 0, 1, 2, and 3 doses of Gardasil vaccine (4vHPV) in girls after 6 years and their responses to a subsequent dose of Cervarix vaccine (2vHPV). METHODS: A subset of girls (n = 59) who previously received 0, 1, 2, or 3 doses of 4vHPV 6 years earlier were randomly selected from a cohort study of Fijian girls (age 15-19 years). Blood was collected before and 28 days after a dose of 2vHPV. The HPV16- and HPV18-specific cellular immune response was determined by IFNγ-ELISPOT and by measurement of cytokines in peripheral blood mononuclear cell supernatants. RESULTS: Six years after 4vHPV vaccination, HPV18-specific responses were significantly lower in the 1- (1D) or 2-dose (2D) recipients compared with 3-dose recipients (2D: IFNγ-ELISPOT: P = .008; cytokines, IFNγ: P = .002; IL-2: P = .022; TNFα: P = .016; IL-10: P = .018; 1D: IL-2: P = .031; IL-10: P = .014). These differences were no longer significant post-2vHPV. No significant differences in HPV16 responses (except IL-2, P < .05) were observed between the 2- or 1-dose recipients and 3-dose recipients. CONCLUSIONS: These data suggest that cellular immunity following reduced-dose schedules was detectable after 6 years, although the responses were variable between HPV types and dosage groups. The clinical significance of this is unknown. Further studies on the impact of reduced dose schedules are needed, particularly in high-disease burden settings.

Decline in prevalence of human papillomavirus infection following vaccination among Australian Indigenous women, a population at higher risk of cervical cancer: The VIP-I study.

BACKGROUND: Cervical cancer occurrence and mortality are strongly correlated with socioeconomic disadvantage, largely due to unequal access to screening and treatment. Universal human papillomavirus (HPV) vaccination provides the opportunity to greatly reduce this global health disparity. Australian Indigenous women have substantially higher rates of cervical cancer than non-Indigenous women, primarily due to under-screening. We investigated HPV infection rates in Indigenous women 7 years after implementation of the national HPV vaccination program. METHODS: We used a repeat cross-sectional design, with the baseline being provided by an HPV prevalence survey among Indigenous women attending clinics for cervical cytology screening, prior to the start of the vaccination program in 2007. We returned to clinics in four locations during 2014-15, and invited women aged 18-26 years attending for screening to provide a cervical specimen for HPV testing, as well as to complete a short questionnaire and consent to allow access of their records in the National HPV Vaccination Program Register. We used well-established laboratory methods to test specimens for specific HPV genotypes. RESULTS: A total of 142 women were recruited at participating sites and compared to 155 who had been recruited at the same locations in the 2007 pre-vaccine survey. The two groups were identical in regard to age, with the more recent group having a higher proportion of hormonal contraception users, and a lower proportion of smokers. The proportion found to have any HPV type fell from 58 to 36% with the decline being entirely due to reductions in vaccine types, which fell by 94% from 24 to 1.4%. CONCLUSION: Australia's national HPV vaccination program appears to be successfully protecting a very high proportion of Indigenous women against vaccine targeted HPV types, who have in the past been at elevated risk of cervical cancer.

Final analysis of a study assessing genital human papillomavirus genoprevalence in young Australian women, following eight years of a national vaccination program.

OBJECTIVES: The VACCINE [Vaccine Against Cervical Cancer Impact and Effectiveness] study evaluated the prevalence of quadrivalent vaccine-targeted human papillomavirus (HPV) genotypes (HPV 6, 11, 16, 18) amongst young women of vaccine-eligible age. METHODS: Between October 2011 - June 2015, women aged 18-25 years from Victoria, Australia, were recruited through targeted advertising on the social networking website Facebook. Participants completed an online questionnaire and provided a self-collected vaginal swab for HPV DNA detection and genotyping (Linear Array HPV genotyping assay). Self-reported HPV vaccination details were verified with the National HPV Vaccination Program Register (NHVPR). RESULTS: Of 1223 who agreed to participate, 916 (74.9%) completed the survey and, for 1007 (82.3%) sexually-active participants, 744 (73.9%) returned the self-collected swab, of which 737 contained detectable DNA. 184/737 (25.0%) were positive for HPV. Vaccine-targeted HPV genotypes were detected in only 13 (1.7%) women: 11 HPV 16 (six vaccinated after sexual debut, five unvaccinated) and two HPV 6. Prevalence of any of HPV 31/33/45 collectively was 2.9%, varying significantly by vaccination status (fully 2.0%, unvaccinated 6.8%; p = 0.01). Vaccination rates among the sexually-active cohort were high, with 65.6%, 71.6% and 74.2% of participants having received three, at least two or at least one dose of vaccine, respectively. Of women self-reporting HPV vaccination, the NHVPR confirmed one or more doses were received in 90%. Strong associations were observed between vaccination status, age, language spoken at home and country of birth, as well as between HPV detection and the number of male sexual partners. CONCLUSION: Surveillance five to eight years' post-initiation of a national HPV vaccination program demonstrated a consistent and very low prevalence of vaccine-related HPV genotypes and some evidence of cross protection against related types amongst vaccine-eligible women from Victoria, Australia.