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The efficiency of PCR-based diagnostic assays can be impacted by the quality of DNA template, and anal samples can be particularly problematic due to the presence of faecal contaminants. Here, we compared the Quick-DNA Viral Kit (Zymo, Zymo Research, CA) and MagNA Pure 96 DNA and Viral NA Small Volume Kit (MP96, Roche) for use of the Seegene Anyplex II HPV28 assay (Anyplex28, Seegene) with anal samples. A total of 94 anal samples extracted using the MP96 and Zymo kits were tested via the Anyplex28, which detects high-risk human papillomavirus (HR-HPV, Panel A) and low-risk (LR-HPV, Panel B) HPV types. Testing the HR-HPV types (Panel A), 86 (91.5%) MP96 and 84 (89.4%) Zymo samples were deemed assessable. Overall agreement between the two methods was 87/94 (92.6%, 95% CI: 85.3-97.0) with the Kappa value of 0.678 (0.5-0.9). Of the 87 assessable samples, 50 (57.5%) were concordant, 34 (39.1%) partially concordant, and 10 (11.5%)discordant. In conclusion, the Anyplex28 produces comparable HPV genotyping results when using DNA extracts from either of these two methods.

Global methylation analysis of gene promoters is promising for detection of high-grade squamous intraepithelial lesions or worse (HSIL+) in high-risk human papillomavirus (hrHPV)-positive women. However, diagnostic performance of methylation data at individual CpG-sites is limited. We explored methylation for predicting HSIL+ in self- and clinician-collected samples from Papua New Guinea. Methylation of EPB41L3 (1-6 CpG-sites), hTERT (1-10 CpG-sites) and FAM19A4 (1-5 CpG-sites) was assessed through pyrosequencing from 44 HPV+ samples (4 cancers, 19 HSIL, 4 low-grade squamous intraepithelial lesions (LSIL), 17 normal). New primers were designed for FAM19A4 directed to the first exon region not explored previously. In clinician-collected samples, methylation at CpG-sites 4 and 5 of EPB41L3 were the best HSIL predictors (AUC >0.83) and CpG-site 4 for cancer (0.925). Combination of EPB41L3 sites 2/4 plus FAM19A4 site 1 were the best HSIL+ markers [100% sensitivity, 63.2% specificity]. Methylation at CpG-site 5 of FAM19A4 was the best HSIL predictor (0.67) in self-collected samples, and CpG-sites 1 and 3 of FAM19A4 for cancer (0.77). Combined, FAM19A4 site 1 plus HPV 16/18 detection yielded sensitivity of 82.6% and specificity of 61.9%. In conclusion, methylation at individual CpG-sites of EPB41L3 and FAM19A4 outperformed global analysis and improved HSIL+ detection, warranting further investigation.

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.

BACKGROUND: The transition of Australia's National Cervical Screening Program from cytology to a molecular test for human papillomavirus (HPV) (locally referred to as the 'Renewal'), including a longer five-year interval and older age at commencement, significantly impacted all sectors of program delivery. The Renewal had major implications for the roles and requirements of pathology laboratories providing services for the Program. This study aimed to understand the early impacts of the Renewal and its implementation on the pathology sector. METHODS: Semi-structured qualitative interviews were conducted with key stakeholders (N = 49) involved in the STakeholder Opinions of Renewal Implementation and Experiences Study (STORIES), 11-20 months after the program transition. A subset of interviews (N = 24) that discussed the pathology sector were analysed using inductive thematic analysis. RESULTS: Four overarching themes were identified: implementation enablers, challenges, missed opportunities, and possible improvements. Participants believed that the decision to transition to primary HPV screening was highly acceptable and evidence-based, but faced challenges due to impacts on laboratory infrastructure, resources, staffing, and finances. These challenges were compounded by unfamiliarity with new information technology (IT) systems and the new National Cancer Screening Register ('Register') not being fully functional by the date of the program transition. The limited availability of self-collection and lack of standardised fields in pathology forms were identified as missed opportunities to improve equity in the Program. To improve implementation processes, participants suggested increased pathology sector involvement in planning was needed, along with more timely and transparent communication from the Government, and clearer clinical management guidelines. CONCLUSION: The transition to primary HPV screening had a significant and multifaceted impact on the Australian p

Mycoplasma genitalium is a sexually transmitted infection with increasing concerns around antimicrobial resistance. Droplet digital PCR (ddPCR) is a rapid quantification method with high precision that may be useful for absolute quantitation of bacteria in samples. This study aimed to develop a ddPCR assay for the quantification of M. genitalium. ddPCR targeting the gene mgpB was established and analysed using the QX100 ddPCR system. The assay was evaluated against quantitated DNA standards, and then in comparison to an established quantitative PCR performed on the Lightcycler 480 II. DNA template of increasing complexity was used, including synthetic double stranded DNA, DNA extracts from laboratory-cultured M. genitalium strains (n = 17) and DNA from M. genitalium-positive clinical samples (n = 21). There was a strong correlation between ddPCR concentration estimates and measured DNA standards (r2 = 0.997), and between ddPCR and qPCR quantitation for different templates (r2 ranging from 0.953 to 0.997). ddPCR reliably detected template in a range from <10 copies per reaction to >104 copies per reaction and demonstrated linearity over dilution series. Concentration estimates by ddPCR were reproducibly less than those determine by qPCR. ddPCR demonstrated precise and reproducible quantitation of M. genitalium with a variety of templates.

The COVID-19 pandemic has exacerbated the existing challenges to achieving the WHO target of eliminating cervical cancer as a public health problem by working towards the target of fewer than four cases per 100 000 women. We reviewed the literature to identify potential recovery strategies to support cervical cancer prevention programs in lower-and middle-income countries (LMICs) following COVID-19 disruptions and the extent to which strategies have been implemented. Utilising the WHO health systems framework, we mapped these recovery strategies against the six building blocks to examine their reach across the health system. Most recovery strategies were focused on service delivery, while leadership and governance played a pivotal role in the continuity of cervical cancer prevention programs during the pandemic. Leadership and governance were the drivers for outcomes in the building blocks of health information systems, financing and critical support in operationalising service delivery strategies. In the aftermath of the COVID-19 pandemic with strained health resources and economies, stakeholders would significantly influence the coverage and sustainability of cervical cancer prevention programs. The support from multisectoral stakeholders would accelerate the recovery of cervical cancer prevention programs. To achieve the WHO target by 2030, we call for future studies to understand the barriers and facilitators from the perspectives of stakeholders in order to support the decision-making processes and information required to implement recovery strategies in LMICs.

In Australia's HPV-based cervical screening program, we previously showed that risk of histological high-grade abnormality at 1 year post screening decreased with age in women with oncogenic HPV. In this study, we followed 878 HPV16/18 positive women aged 55 years and over for up to 3 years post screening test, to determine the proportion with histological high-grade abnormality (HGA, incorporating high-grade squamous intraepithelial abnormality (HSIL), adenocarcinoma in situ (AIS), squamous cell carcinoma (SCC) and adenocarcinoma) and to correlate risk of HGA with liquid-based cytology result and with prior screening history. HGA was detected in 7.8% at 1 year and 10.0% at 3 years, with no significant difference (P = .136), despite the number of women with follow-up information significantly increasing from 82.9% to 91.0% (P < .0001). The proportion of HPV16/18 positive women with HGA at 3 years was highest in those with an HSIL cytology result (79.0%) and lowest in those with negative cytology (6.2%). Women with an adequate screening history had fewer HGA than such women with inadequate prior screening (6.6% vs 16.0%, P = .001) or with a history of an abnormality (6.6% vs 14.4%, P = .001). HPV16/18 infection in women over 55 years may have a different natural history from that in younger women, in whom HGA are more common after HPV16/18 detection. In HPV-based cervical screening programs, management algorithms for screen-detected abnormalities based on risk stratification should include factors such as age, screening history and index cytology result, so that women receive appropriate investigation and follow-up.

The quantity of the human papillomavirus (HPV) is associated with disease outcome. We designed an accurate and precise digital PCR assay for quantitating HPV in anal samples, a sample type that is typically problematic due to the presence of PCR inhibitors.

BACKGROUND: Although single nucleotide polymorphisms (SNPs) in Mycoplasma genitalium parC contribute to fluoroquinolone treatment failure, data are limited for the homologous gene, gyrA. This study investigated the prevalence of gyrA SNPs and their contribution to fluoroquinolone failure. METHODS: Samples from 411 patients (male and female) undergoing treatment for M. genitalium infection (Melbourne Sexual Health Centre, March 2019-February 2020) were analyzed by Sanger sequencing (gyrA and parC). For patients treated with moxifloxacin (n = 194), the association between SNPs and microbiologic treatment outcome was analyzed. RESULTS: The most common parC SNP was G248T/S83I (21.1% of samples), followed by D87N (2.3%). The most common gyrA SNP was G285A/M95I (7.1%). Dual parC/gyrA SNPs were found in 8.6% of cases. One third of infections harboring parC G248T/S83I SNP had a concurrent SNP in gyrA conferring M95I. SNPs in gyrA cooccurred with parC S83I variations. Treatment failure was higher in patients with parC S83I/gyrA dual SNPs when compared with infections with single S83I SNP alone from analysis of (1) 194 cases in this study (81.2% vs 45.8%, P = .047), and (2) pooled analysis of a larger population of 535 cases (80.6% vs 43.2%; P = .0027), indicating a strong additive effect. CONCLUSIONS: Compared with parC S83I SNP alone, M. genitalium infections with dual mutations affecting parC/gyrA had twice the likelihood of failing moxifloxacin. Although antimicrobial resistance varies by region globally, these data indicate that gyrA should be considered as a target for future resistance assays in Australasia. We propose a strategy for the next generation of resistance-guided therapy incorporating parC and gyrA testing.

BACKGROUND: As of mid-2021, Australia's only nationwide coronavirus disease 2019 (COVID-19) epidemic occurred in the first 6 months of the pandemic. Subsequently, there has been limited transmission in most states and territories. Understanding community spread during the first wave was hampered by initial limitations on testing and surveillance. To characterize the prevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific antibody seroprevalence generated during this time, we undertook Australia's largest national SARS-CoV-2 serosurvey. METHODS: Between June 19 and August 6, 2020, residual specimens were sampled from people undergoing general pathology testing (all ages), women attending antenatal screening (20-39 years), and blood donors (20-69 years) based on the Australian population's age and geographic distributions. Specimens were tested by Wantai total SARS-CoV-2-antibody assay. Seroprevalence estimates adjusted for test performance were produced. The SARS-CoV-2 antibody-positive specimens were characterized with microneutralization assays. RESULTS: Of 11 317 specimens (5132 general pathology; 2972 antenatal; 3213 blood-donors), 71 were positive for SARS-CoV-2-specific antibodies. Seroprevalence estimates were 0.47% (95% credible interval [CrI], 0.04%-0.89%), 0.25% (CrI, 0.03%-0.54%), and 0.23% (CrI, 0.04%-0.54%), respectively. No seropositive specimens had neutralizing antibodies. CONCLUSIONS: Australia's seroprevalence was extremely low (<0.5%) after the only national COVID-19 wave thus far. These data and the subsequent limited community transmission highlight the population's naivety to SARS-CoV-2 and the urgency of increasing vaccine-derived protection.

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.

Prevalence, trends, and treatment outcome estimates were generated for parC variants in macrolide-resistant Mycoplasma genitalium. Among 539 cases, the most common amino acid change was S83I, which increased from 13% in 2012 to 2013, to 23% in 2019 to 2020 (Ptrend = 0.046). From 381 moxifloxacin treatments, failure occurred in 58.7% (95% confidence interval [CI], 46.7 to 69.9) of cases with S83I. Other changes affecting S83 or D87 were uncommon and minor contributors to failure. The absence of S83I was highly predictive of moxifloxacin cure (96.4%; 95% CI, 93.7 to 98.2), highlighting diagnostic potential.

Doxycycline targets the 16S rRNA and is widely used for the treatment of sexually transmitted infections. While it is not highly effective at eradicating Mycoplasma genitalium infections, it can reduce organism load. The aim of this study was to investigate the association between single nucleotide polymorphisms (SNPs) in the 16S rRNA gene of M. genitalium and change in organism load. M. genitalium samples were collected from 56 men prior to commencing doxycycline and at a median of 13 of 14 doses. These were sequenced for the 16S rRNA, and the association between 16S rRNA SNPs and change in organism load was determined. 16S rRNA sequences were available for 52/56 (92.9%) M. genitalium-infected men, of which 20 (38.5%) had an undetectable load, 26 (50.0%) had a decrease in M. genitalium load (median change of 105-fold), and 6 (11.5%) had an increase in load (median change of 5-fold). The most common SNPs identified were A742G (10/52 [19.2%]), GG960-961TT/C (7/52 [13.5%]), and C1435T (28/52 [53.8%]) (M. genitalium numbering). None were associated with a change in organism load (P = 0.76, 0.16, and 0.98, respectively). Using pooled published data from 28 isolates, no clear relationship between the SNPs and doxycycline MIC was identified. In conclusion, the low efficacy of doxycycline against M. genitalium does not appear to be due to variation in the 16S rRNA gene.

BACKGROUND: In December 2017, the Australian National Cervical Screening Program transitioned from 2-yearly cytology-based to 5-yearly human papillomavirus (HPV)-based cervical screening, including a vaginal self-collection option. Until July 2022, this option was restricted to under- or never-screened people aged 30 years and older who refused a speculum exam. We investigated the perspectives and experiences of stakeholders involved in, or affected by, the initial implementation of the restricted self-collection pathway. METHODS: Semi-structured interviews were conducted with 49 stakeholders as part of the STakeholder Opinions of Renewal Implementation and Experiences Study. All interviews were audio recorded and transcribed. Data were thematically analysed and coded to the Conceptual Framework for Implementation Outcomes. RESULTS: Stakeholders viewed the introduction of self-collection as an exciting opportunity to provide under-screened people with an alternative to a speculum examination. Adoption in clinical practice, however, was impacted by a lack of clear communication and promotion to providers, and the limited number of laboratories accredited to process self-collected samples. Primary care providers tasked with communicating and offering self-collection described confusion about the availability, participant eligibility, pathology processes, and clinical management processes for self-collection. Regulatory delay in developing an agreed protocol to approve laboratory processing of self-collected swabs, and consequently initially having one laboratory nationally accredited to process samples, led to missed opportunities and misinformation regarding the pathway's availability. CONCLUSIONS: Whilst the introduction of self-collection was welcomed, clear communication from Government regarding setbacks in implementation and how to overcome these in practice were needed. As Australia moves to a policy of providing everyone eligible for screening the choice of se

Rapidly identifying and isolating people with acute SARS-CoV-2 infection has been a core strategy to contain COVID-19 in Australia, but a proportion of infections go undetected. We estimated SARS-CoV-2 specific antibody prevalence (seroprevalence) among blood donors in metropolitan Melbourne following a COVID-19 outbreak in the city between June and September 2020. The aim was to determine the extent of infection spread and whether seroprevalence varied demographically in proportion to reported cases of infection. The design involved stratified sampling of residual specimens from blood donors (aged 20-69 years) in three postcode groups defined by low (<3 cases/1,000 population), medium (3-7 cases/1,000 population) and high (>7 cases/1,000 population) COVID-19 incidence based on case notification data. All specimens were tested using the Wantai SARS-CoV-2 total antibody assay. Seroprevalence was estimated with adjustment for test sensitivity and specificity for the Melbourne metropolitan blood donor and residential populations, using multilevel regression and poststratification. Overall, 4,799 specimens were collected between 23 November and 17 December 2020. Seroprevalence for blood donors was 0.87% (90% credible interval: 0.25-1.49%). The highest estimates, of 1.13% (0.25-2.15%) and 1.11% (0.28-1.95%), respectively, were observed among donors living in the lowest socioeconomic areas (Quintiles 1 and 2) and lowest at 0.69% (0.14-1.39%) among donors living in the highest socioeconomic areas (Quintile 5). When extrapolated to the Melbourne residential population, overall seroprevalence was 0.90% (0.26-1.51%), with estimates by demography groups similar to those for the blood donors. The results suggest a lack of extensive community transmission and good COVID-19 case ascertainment based on routine testing during Victoria's second epidemic wave. Residual blood donor samples provide a practical epidemiological tool for estimating seroprevalence and information on popula

In order to achieve the global elimination of cervical cancer as a public health problem, close surveillance of progress in public health and clinical activities and outcomes across the three pillars of vaccination, screening and treatment will be required. Surveillance should ideally occur within an integrated system that is planned, funded, and regularly evaluated to ensure it is providing timely, accurate and relevant feedback for action. In this paper, we conceptualise the main public health surveillance objectives as process and outcome measures in each of the three pillars. Process measures include coverage/participation measures for vaccination, screening and treatment alongside the ongoing assessment of the quality and reach of these programs and activities. Outcome measures related to the natural history of human papillomavirus (HPV) infection include HPV infection prevalence, precursor cervical lesions and cervical cancers (including stage at diagnosis, cancer incidence and mortality). These outcome measures can be used for monitoring the effectiveness of the three core activities in the short, medium and long term to assess whether these interventions are effectively reducing their occurrence. We discuss possible methods for the surveillance of these measures in the context of country capacity, drawing from examples in Australia, the USA and in low and middle income countries.

Background: Substantial declines in genital warts have been observed in countries with quadrivalent/nonavalent human papillomavirus (q/n HPV) vaccination programmes, with Australia showing the most pronounced and long-term reductions. No study has assessed progress towards elimination of genital warts in a nation-wide sample of patients, and migrants' contribution to population-level control of genital warts. We assessed Australia's progress towards genital warts elimination by examining trends in diagnoses in Australian- and overseas-born patients of sexual health clinics (SHCs) across Australia. Methods: A cross-sectional trend analysis of new genital warts diagnoses among first-time patients of 34 SHCs, between 2004 and 2018, was performed. Rate ratios (RR) were calculated using Poisson regression models, for comparing trends in proportions of new genital warts diagnoses in Australian- and overseas-born patients during the pre-vaccination era (2004-2007) and the vaccination era (2008-2018), and by 2018 relative to 2004-2007. Findings: A total of 439,957 new patients (Australian-born: 230,230; overseas-born: 209,727) were seen at SHCs, 6•4% were diagnosed with genital warts (Australian-born: 7•1%; overseas-born: 5•6%). By 2018, there had been a 64% reduction in the proportion of all SHC patients with a genital warts diagnosis relative to 2004-2007 (RR: 0•36, 95% CI: 0•35-0•38). The decline was more pronounced at 72% (RR: 0•28, 95% CI: 0 •27-0•30) among Australian-born patients, with the greatest reduction in women and men aged <21 years, at 98% (RR: 0•02, 95% CI: 0•01-0•03) and 92% (RR: 0•08, 95% CI: 0•06-0•11), respectively. By 2018, there was a 49% reduction in the proportion of overseas-born patients diagnosed with genital warts (RR: 0•51, 95% CI:0•48-0•54), and a 21% reduction in overseas-born patients from countries with no or bivalent HPV (bHPV) vaccination programme (RR: 0•79, 95% CI: 0•71-0•90). Interpretation: The substantial reductions in Australian-born

Introduction. Mycoplasma genitalium is a sexually transmitted pathogen with increasing resistance to first- and second-line antimicrobials. The 'near-patient test' ResistancePlus MG FleXible (SpeeDx) detects M. genitalium plus four macrolide resistance mutations (MRMs), facilitating same-day patient follow up.Hypothesis/Gap Statement. This assay has not been assessed on freshly collected samples.Aim. Our goal was to evaluate the performance of the ResistancePlus MG FleXible test against the standard of care open platform test.Methods. ResistancePlus MG FleXible (analysed on the Cepheid GeneXpert platform) was evaluated on freshly collected samples and compared to the standard of care open platform test ResistancePlus MG (SpeeDx) analysed on the LightCycler 480 II (Roche).Results. For 270 valid tests, ResistancePlus MG FleXible yielded a high positive per cent agreement (PPA) of 94.1% [96/102; 95 % confidence interval (CI): 87.6-97.8 %] and negative per cent agreement (NPA) of 95.2% (160/168; 95 % CI: 90.8-97.9%) for M. genitalium detection compared to the reference assay (kappa for test concordance of 0.89; 95 % CI: 0.83-0.95). Performance was similar across different sample types. For the detection of MRMs, ResistancePlus MG FleXible had a PPA of 97.1% (66/68; 95% CI: 89.8-99.6) and NPA of 78.6% (22/28; 95 % CI: 59.0-91.7), with test comparison kappa of 0.79 (95 % CI: 0.65-0.93). Notably, of six discordant results (i.e. determined to be wild type by the reference assay), five were positive for MRMs by Sanger sequencing, indicating that the ResistancePlus MG FleXible assay has an improved performance for mutation detection.Conclusion. ResistancePlus MG FleXible had comparable test performance for M. genitalium detection as the open platform assay, with improved detection of MRMs. The ResistancePlus MG FleXible 'near-patient' assay can deliver a rapid result to expedite appropriate treatment.

OBJECTIVES: To estimate SARS-CoV-2-specific antibody seroprevalence after the first epidemic wave of coronavirus disease 2019 (COVID-19) in Sydney. SETTING, PARTICIPANTS: People of any age who had provided blood for testing at selected diagnostic pathology services (general pathology); pregnant women aged 20-39 years who had received routine antenatal screening; and Australian Red Cross Lifeblood plasmapheresis donors aged 20-69 years. DESIGN: Cross-sectional study; testing of de-identified residual blood specimens collected during 20 April - 2 June 2020. MAIN OUTCOME MEASURE: Estimated proportions of people seropositive for anti-SARS-CoV-2-specific IgG, adjusted for test sensitivity and specificity. RESULTS: Thirty-eight of 5339 specimens were IgG-positive (general pathology, 19 of 3231; antenatal screening, 7 of 560; plasmapheresis donors, 12 of 1548); there were no clear patterns by age group, sex, or location of residence. Adjusted estimated seroprevalence among people who had had general pathology blood tests (all ages) was 0.15% (95% credible interval [CrI], 0.04-0.41%), and 0.29% (95% CrI, 0.04-0.75%) for plasmapheresis donors (20-69 years). Among 20-39-year-old people, the age group common to all three collection groups, adjusted estimated seroprevalence was 0.24% (95% CrI, 0.04-0.80%) for the general pathology group, 0.79% (95% CrI, 0.04-1.88%) for the antenatal screening group, and 0.69% (95% CrI, 0.04-1.59%) for plasmapheresis donors. CONCLUSIONS: Estimated SARS-CoV-2 seroprevalence was below 1%, indicating that community transmission was low during the first COVID-19 epidemic wave in Sydney. These findings suggest that early control of the spread of COVID-19 was successful, but efforts to reduce further transmission remain important.

Introduction. Increasing levels of antibiotic resistance are complicating treatment for the sexually transmitted pathogen Mycoplasma genitalium. Resistance to fluoroquinolones is associated with mutations in the parC gene. Although the precise mutations conferring resistance are not fully understood, the single nucleotide polymorphism (SNP) G248T/S83I is most implicated.Aim. To evaluate the performance of the MG+parC(beta2) assay (SpeeDx, Australia), which detects single nucleotide polymorphisms (SNPs) in the parC gene at amino acid position S83 (A247C/S83R, G248T/S83I, G248A/S83N) and D87 (G259A/D87N, G259T/D87Y, G259C/D87H).Methods. Clinical samples were analysed by MG+parC(beta2) assay and results compared to Sanger sequencing. Sensitivity, specificity, and predictive value for treatment failure were calculated.Results. From analysis of 205 samples, the MG+parC(beta2) assay performed with a high sensitivity 98.2% (95% CI:90.3-100) and specificity 99.3% (95% CI:96.3-100) for parC SNP detection with a kappa of 0.97 (95% CI:0.94-1.00). The predictive value of G248T/S83I detection (the most common SNP, prevalence of 13% in the study population) was analysed with respect to treatment failure (patients received sequential doxycycline-moxifloxacin). The positive-predictive-value for moxifloxacin failure after detection of S83I was only 44% (95% CI:24.4-65.1), while negative-predictive-value was high at 96.9% (95% CI:92.7-99.0), suggesting that other SNPs are contributing to resistance.Conclusion. MG+parC(beta2) performed with high concordance compared to Sanger sequencing. Such qPCR assays can assist in understanding causes of treatment failure, inform the development of diagnostic assays, and can be applied to surveillance of mutations in populations. Due to an incomplete understanding of the basis for fluoroquinolone resistance, such tests do not appear to be ready for clinical application.

Introduction. Failure of fluoroquinolones, the principal treatment option for macrolide-resistant Mycoplasma genitalium infections, has recently emerged. This is of particular concern for men who have sex with men (MSM), who have high proportions of macrolide-resistant M. genitalium infections. Treatment failure with moxifloxacin is likely the result of single nucleotide polymorphisms (SNPs) in parC, whilst concurrent gyrA mutations may play a role.Gap Statement. The levels of fluoroquinolone resistance and dual-class (i.e. macrolide and fluoroquinolone) resistance in M. genitalium among asymptomatic MSM is unknown.Aim. To (i) determine the proportion of fluoroquinolone resistance and dual-class resistance in M. genitalium infections among asymptomatic MSM, (ii) explore any clinical and behavioural associations with fluoroquinolone resistance, and (iii) determine the distribution of antibiotic resistance among M. genitalium mgpB sequence types (STs).Methodology. M. genitalium positive samples (N=94) were obtained from 1001 asymptomatic MSM enrolled in a study at Melbourne Sexual Health Centre (Carlton, Australia) between August 2016 and September 2017. Sanger sequencing was performed to determine the proportion of M. genitalium infections with SNPs in parC that have previously been associated with failure of moxifloxacin (corresponding to amino changes S83I, D83R, D87Y and D87N) and in gyrA (corresponding to amino acid changes M95I, D99N, D99Y and D99G). Associations between clinical/behavioural factors and parC SNPs were examined. Strain typing was performed by sequencing a portion of the mgpB gene.Results. The proportion of MSM with infections harbouring parC and gyrA SNPs was 13.0 % [95 % confidence interval (CI): 6.8-23.2 %] and 4.7 % (95 % CI: 1.1-13.4 %), respectively; dual-class resistance was 13.0 %. No significant clinical/behavioural associations were found. Antibiotic resistance was not restricted to specific mgpB STs.Conclusion. One in eight (13 %) of as

Background: Substantial declines in genital warts (GW) have been observed in countries with quadrivalent HPV vaccination programmes, with Australia showing the highest reductions due to early commencement and high vaccination coverage. There is a real potential to achieve GW elimination; however, no GW elimination definition exists. Taking Australia as a case study, we aimed to reach expert consensus on a proposed GW elimination definition using a modified Delphi process. Method: We used modelling and epidemiological data to estimate the expected number of new GW cases, from pre-vaccination (baseline) in 2006 to the year 2060 in Australian heterosexuals, men who have sex with men (MSM), and newly arrived international travellers and migrants. We used these data and the literature, to develop a questionnaire containing ten elimination-related items, each with 9-point Likert scales (1—strongly disagree; 9—strongly agree). The survey was completed by 18 experts who participated in a full day face-to-face modified Delphi study, in which individuals and then small groups discussed and scored each item. The process was repeated online for items where consensus (≥70% agreement) was not initially achieved. Median and coefficient of variation (COV) were used to describe the central tendency and variability of responses, respectively. Findings: There was a 95% participation rate in the face-to-face session, and 84% response rate in the final online round. The median item score ranged between 7.0 and 9.0 and the COV was ≤0.30 on all items. Consensus was reached that at ≥80% HPV vaccination coverage, GW will be eliminated as a public health problem in Australia by 2060. During this time period there will be a 95% reduction in population-level incidence compared with baseline, equivalent to <1 GW case per 10,000 population. The reductions will occur most rapidly in Australian heterosexuals, with 73%, 90% and 97% relative reductions by years 2021, 2030 and 2060, respectively. The

Background: Mycoplasma genitalium is now recognised as an important bacterial sexually transmitted infection. We summarised data from studies of mutations associated with macrolide and fluoroquinolone resistance in M genitalium to establish the prevalence of resistance. We also investigated temporal trends in resistance and aimed to establish the association between resistance and geographical location. Methods: In this systematic review and meta-analysis, we searched PubMed, Embase, and MEDLINE for studies that included data for the prevalence of mutations associated with macrolide and fluoroquinolone resistance in M genitalium published in any language up to Jan 7, 2019. We defined prevalence as the proportion of M genitalium samples positive for key mutations associated with azithromycin resistance (23S rRNA gene, position 2058 or 2059) or moxifloxacin resistance (S83R, S83I, D87N, or D87Y in parC), or both, among all M genitalium samples that were successfully characterised. We used random-effects meta-analyses to calculate summary estimates of prevalence. Subgroup and meta-regression analyses by WHO region and time period were done. This study was registered with PROSPERO, number CRD42016050370. Results: Overall, 59 studies from 21 countries met the inclusion criteria for our study: 57 studies of macrolide resistance (8966 samples), 25 of fluoroquinolone resistance (4003 samples), and 22 of dual resistance to macrolides and fluoroquinolones (3280 samples). The summary prevalence of mutations associated with macrolide resistance among M genitalium samples was 35·5% (95% CI 28·8–42·5); prevalence increased from 10·0% (95% CI 2·6–20·1%) before 2010, to 51·4% (40·3–62·4%) in 2016–17 (p<0·0001). Prevalence of mutations associated with macrolide resistance was significantly greater in samples in the WHO Western Pacific and Americas regions than in those from the WHO European region. The overall prevalence of mutations associated with fluoroquinolone resistance in M g

Australia's new HPV-based cervical screening program is based on an algorithm that incorporates reflex cytology to guide decisions about further follow-up with colposcopy and, if indicated, biopsy. We reviewed results for 2300 women referred directly for colposcopy after their first positive HPV screening test, to determine the proportion that had underlying histological high-grade abnormality (HGA). Overall, HGA was detected in 24.3% of women. Among HPV16/18 positive women, 18.0% had HGA, increasing from 6.6% among women with negative cytology to 79.7% among women with high-grade squamous lesion or worse, or any glandular lesion on cytology (HSIL+; P-trend < .001). For this latter group, the proportion with HGA was higher among HPV16/18 positive women than among those positive for other oncogenic types (68.8%; P = .029). Among women with ASC-H cytology, 51.8% had HGA, with no difference between HPV groups (P = .314). In analyses by age-groups, detection of HGA was highest, at 36.4%, among women younger than 35 years, then decreased significantly to 5.9%, among women aged 65 to 74 years (P-trend < .001). The relationship of decreasing HGA detection with increasing age was strong for women with negative cytology, and those with ASC-H cytology (P-trend < .001 for each). For women with HSIL+ cytology, detection of HGA was high and stable, regardless of age (P-trend = .211). This report describes the first follow-up colposcopy findings in Australia's new HPV-based cervical screening program. The results demonstrate the additional value of reflex cytology in managing HPV positive women and suggest that further refinement of the risk-based algorithm to account for age may be warranted.

Background: More than 10 years have elapsed since human papillomavirus (HPV) vaccination was implemented. We did a systematic review and meta-analysis of the population-level impact of vaccinating girls and women against human papillomavirus on HPV infections, anogenital wart diagnoses, and cervical intraepithelial neoplasia grade 2+ (CIN2+) to summarise the most recent evidence about the effectiveness of HPV vaccines in real-world settings and to quantify the impact of multiple age-cohort vaccination. Methods: In this updated systematic review and meta-analysis, we used the same search strategy as in our previous paper. We searched MEDLINE and Embase for studies published between Feb 1, 2014, and Oct 11, 2018. Studies were eligible if they compared the frequency (prevalence or incidence) of at least one HPV-related endpoint (genital HPV infections, anogenital wart diagnoses, or histologically confirmed CIN2+) between pre-vaccination and post-vaccination periods among the general population and if they used the same population sources and recruitment methods before and after vaccination. Our primary assessment was the relative risk (RR) comparing the frequency (prevalence or incidence) of HPV-related endpoints between the pre-vaccination and post-vaccination periods. We stratified all analyses by sex, age, and years since introduction of HPV vaccination. We used random-effects models to estimate pooled relative risks. Findings: We identified 1702 potentially eligible articles for this systematic review and meta-analysis, and included 65 articles in 14 high-income countries: 23 for HPV infection, 29 for anogenital warts, and 13 for CIN2+. After 5–8 years of vaccination, the prevalence of HPV 16 and 18 decreased significantly by 83% (RR 0·17, 95% CI 0·11–0·25) among girls aged 13–19 years, and decreased significantly by 66% (RR 0·34, 95% CI 0·23–0·49) among women aged 20–24 years. The prevalence of HPV 31, 33, and 45 decreased significantly by 54% (RR 0·46, 95% CI 0·3

OBJECTIVES: To report human papillomavirus (HPV) testing patterns and rates of oncogenic HPV-positivity for specimens submitted during the first 6 months after the National Cervical Screening Program switched from cytology- to primary HPV-based screening. DESIGN, PARTICIPANTS: Retrospective cross-sectional review of 195 606 specimens submitted for HPV testing, 1 December 2017 - 31 May 2018. SETTING: Large community-based general pathology laboratory in metropolitan Sydney. MAIN OUTCOME MEASURES: Prevalence of oncogenic HPV types (all, HPV16/18, non-HPV16/18) by reason for HPV test (primary screening, non-screening); for oncogenic HPV-positive women in the age band recommended for primary HPV screening (25-74 years), prevalence of cytologic abnormality and rates of 12-month follow-up and colposcopy recommendations. RESULTS: 195 606 samples were received: 157 700 (80.6%) for primary screening, 37 906 (19.4%) for non-screening tests. Oncogenic HPV was detected in 8.1% of screening tests (95% CI, 7.9-8.2%) and 20.9% of non-screening tests (95% CI, 20.5-21.3%); 35.5% (95% CI, 34.7-36.4%) of women of recommended screening age with positive oncogenic HPV screening test results also had a cytologic abnormality. The proportion of HPV16/18-positive samples with high grade abnormality was 15.3% (95% CI, 14.2-16.6%); for samples positive for other oncogenic HPV types, the proportion was 6.3% (95% CI, 5.8-6.8%). Repeat HPV testing after 12 months was recommended for 5.4% (95% CI, 5.3-5.5%) and direct colposcopy for 2.6% (95% CI, 2.5-2.7%) of screened women aged 25-74 years. CONCLUSIONS: High grade cytologic abnormalities were more common in women positive for HPV16/18, supporting their higher risk classification. Colposcopy referral rates were higher than during primary cytology-based testing, as predicted by clinical trial and modelling data. The prevalence of HPV was much higher in non-screening than in primary screening samples. Our findings indicate the renewed program is pe

AIMS: Mycoplasma genitalium causes a common, sexually transmitted bacterial infection. This study assessed the detection of M. genitalium in stored urine samples to understand the impact of sample storage on M. genitalium detection. METHODS: Aliquots of M. genitalium-positive urine (n = 20 patients) were stored at either room temperature (22°C) or 4°C, without a preservative. At weekly intervals, samples were tested using the commercial test ResistancePlus MG® (SpeeDx® , Australia). We report the analysis at 1 week, an acceptable collection-to-test turnaround time, with further analysis over 5 weeks to illustrate degradation trends. RESULTS: After storing at 4°C, the proportion of specimens that remained positive for M. genitalium was 100% after 1 week and 95% after 4 weeks. Storage at 22°C led to more rapid decline in detection in the first 4 weeks, with 95% detected after 1 week and 85% at 2 weeks onwards. At 5 weeks, samples stored at both temperatures had an 85% M. genitalium detection rate, with increase in crossing points (Cq) of 0·72 (95% confidence interval (CI) 0·01-1·43; P-trend = 0·027) at 4°C, and 1·75 ((95% CI 0·79-2·71), P-trend <0·001) at 22°C. CONCLUSIONS: Urine samples stored without preservative, and unfrozen, retained high M. genitalium detection levels over the short term (up to 5 weeks). To minimize degradation, storing at 4°C is recommended. SIGNIFICANCE AND IMPACT OF THE STUDY: There is little known about the stability of clinical samples for M. genitalium detection. This study found that a high proportion (85-100%) of samples are still suitable for M. genitalium detection after storage for up to 5 weeks.

Background. In Australia, high uptake of the quadrivalent human papillomavirus (4vHPV) vaccine has led to reductions in the prevalence of human papillomavirus (HPV) genotypes 6, 11, 16, and 18 in women and girls aged ≥25 years.We evaluated the impact of the program impact on HPV prevalence in unvaccinated male subjects. Methods. Sexually active heterosexual male subjects aged 16-35 years were recruited in 2014-2016. Participants provided a selfcollected penile swab sample for HPV genotyping (Roche Linear Array) and completed a demographic and risk factor questionnaire. Results. The prevalence of 4vHPV genotypes among 511 unvaccinated male subjects was significantly lower in those aged ≥25 than in those aged <25 years: 3.1% (95% confidence interval, 1.5%-5.7%) versus 13.7% (8.9%-20.1%), respectively (P < .001); adjusted prevalence ratio, 0.22 (.09-.51; P < .001). By contrast, the prevalence of high-risk HPV genotypes other than 16 and 18 remained the same across age groups: 16.8% (95% confidence interval, 12.6%-21.9%) in men aged ≤25 years and 17.9% (12.4%- 25.0%) in those aged >25 years (P = .76); adjusted prevalence ratio, 0.98, (.57-1.37; P = .58). Conclusions. A 78% lower prevalence of 4vHPV genotypes was observed among younger male subjects. These data suggest that unvaccinated men may have benefited from herd protection as much as women from a female-only HPV vaccination program with high coverage.

OBJECTIVE: HPV causes ~90% of anal cancer and HPV16 is the type most commonly associated with anal cancer. Gay and bisexual men (GBM) are at greatly increased risk. We investigated patterns of vaccine-preventable anal HPV in older GBM. METHODS: The Study of the Prevention of Anal Cancer (SPANC) is an ongoing, prospective cohort study of HIV-positive and HIV-negative Australian GBM. Participants completed questionnaires and underwent an anal swab for HPV genotyping using Roche Linear Array. We analysed baseline data from SPANC by HPV type, mean number of types, stratified by age and HIV status. RESULTS: Anal HPV results from 606 (98.2%) of 617 participants (median age 49 years, 35.7% HIV-positive) showed 525 (86.7%) had ≥1 HPV type and 178 (29.4%) had HPV16. Over one third of participants (214, 35.3%) had no nonavalent vaccine-preventable types detected. Two (0.3%) participants had all quadrivalent types and none had all nonavalent vaccine types. HIV-positive participants (p<0.001) and younger participants (p=0.059) were more likely to have more vaccine-preventable HPV types detected. CONCLUSION: Anal HPV was highly prevalent in this largely community-based GBM cohort. Vaccine-preventable HPV16 was detected in approximately one third of participants. These findings suggest that the potential efficacy of HPV vaccination of older GBM should be explored.

BACKGROUND: Anal intraepithelial neoplasia grade 2 (AIN2) and AIN grade 3 (AIN3) are commonly grouped together as high grade squamous intraepithelial lesions (HSIL). We assessed risk factors for HSIL-AIN2 and HSIL-AIN3 in a cohort of homosexual men. METHODS: At the baseline visit in the Study for the Prevention of Anal Cancer (SPANC), all men completed a questionnaire and underwent anal swabbing for cytology and HPV genotyping, followed by high resolution anoscopy. RESULTS: Composite-HSIL prevalence was 47% and 32% among 220 HIV-positive and 396 HIV-negative men, respectively. HSIL-AIN3 (37.7% versus 24.7%; p<0.001), but not HSIL-AIN2 (9.5% versus 7.6%; p=0.395) was more common in HIV-positive men. Recent receptive anal partners (p-trend=0.045), and increasing number of high-risk (HR)-HPV types (p-trend<0.001) were associated with HSIL-AIN2. Lifetime receptive partners (p-trend<0.001), HIV status (OR 1.74; 95% CI: 1.05-2.87) and HPV16 (OR 3.00; 95% CI: 1.56-5.75) were associated with HSIL-AIN3. HPV16 was the most common HR-HPV type detected in men with HSIL-AIN3, both HIV-negative (61.1%) and HIV-positive (54.9%). HPV16 was less commonly detected in men with HSIL-AIN2. CONCLUSIONS: Grouping HSIL-AIN2 and HSIL-AIN3 as HSIL may mask considerable heterogeneity in anal cancer risk. Given the strong link between HPV16 and anal cancer, men with HSIL-AIN3 and HPV16 are likely to be at greatest risk of cancer.

Incidence and mortality rates of anal cancer are increasing globally. More than 90% of anal squamous cell carcinomas (ASCC) are associated with human papillomavirus (HPV). Studies on HPV-related anogenital lesions have shown that patterns of methylation of viral and cellular DNA targets could potentially be developed as disease biomarkers. Lesion-specific DNA isolated from formalin-fixed paraffin-embedded (FFPE) tissues from existing or prospective patient cohorts may constitute a valuable resource for methylation analysis. However, low concentrations of DNA make these samples technically challenging to analyse using existing methods. We therefore set out to develop a sensitive and reproducible nested PCR-pyrosequencing based method to accurately quantify methylation at 10 CpG sites within the E2BS1, E2BS2,3,4 and Sp1 binding sites in the viral upstream regulatory region of HPV16 genome. Methylation analyses using primary and nested PCR-pyrosequencing on 52 FFPE tissue [26 paired whole tissue sections (WTS) and laser capture microdissected (LCM) tissues] from patients with anal squamous intraepithelial lesions was performed. Using nested PCR, methylation results were obtained for the E2BS1, E2BS2,3,4 and Sp1 binding sites in 86.4% of the WTS and 81.8% of the LCM samples. Methylation patterns were strongly correlated within median values of matched pairs of WTS and LCM sections, but overall methylation was higher in LCM samples at different CpG sites. High grade lesions showed low methylation levels in the E2BS1 and E2BS2 regions, with increased methylation detected in the E2BS,3,4/Sp1 regions, showing the highest methylation at CpG site 37. The method developed is highly sensitive in samples with low amounts of DNA and demonstrated to be suitable for archival samples. Our data shows a possible role of specific methylation in the HPV16 URR for detection of HSIL.

BACKGROUND: Human papillomavirus (HPV) vaccination targets high-risk HPV16/18 that cause 70% of all cancers of the cervix. In Australia there is a fully-funded, school-based National HPV Vaccination Program which has achieved vaccine initiation rate of 82% among age-eligible females. Improving HPV vaccination rates is important in the prevention of morbidity and mortality associated with HPV-related disease. This study aimed to identify factors and barriers associated with uptake of the HPV vaccine in the Australian Program. METHODS: Between 2011 and 2014, females aged 18-25 years, living in Victoria, Australia who were offered HPV vaccination between 2007 and 2009 as part of the National HPV Vaccination Program, living in Victoria, Australia were recruited into a a young women's study examining effectiveness of the Australian National HPV Vaccination Program. Overall, 668 participants completed the recruitment survey, which collected data of participants' demographics and HPV knowledge. In 2015 these participants were invited to complete an additional supplementary survey on parental demographics and attitudes towards vaccinations. RESULTS: In 2015, 417 participants completed the supplementary survey (62% response rate). Overall, 19% of participants were unvaccinated. In multivariate analyses, HPV vaccination was significantly associated with their being born in Australia (p<0.001), having completed childhood vaccinations (p<0.001) and their parents being main decision-makers for participants' HPV vaccination (p<0.001). The main reason reported for HPV non-vaccination was parental concern about vaccine safety (43%). Compared with HPV-vaccinated participants, those unvaccinated were significantly more likely to be opposed to all vaccines, including HPV vaccines (p<0.001) and were less likely to consider vaccinating their own children with all vaccines (p = 0.033), including HPV vaccines (p<0.001). Overall, 61% of unvaccinated participants reported that a recommendat

Introduction: Anal squamous cell carcinoma is preceded by persistent infection with high-risk human papillomavirus (HPV) and the cancer precursor, highgrade squamous intraepithelial lesion (HSIL). Detection of specific HPV genotypes and HPV-related biomarkers may be an option for primary anal screening. However, more data on the natural history of HPV-related anal lesions are required. The outcomes from this study will enhance our understanding of the clinical and biological behaviour of HPV-related anal lesions and inform the development of future HPV genotype and/or biomarker screening tests. Methods and analysis: HIV-negative and HIV-positive men who have sex with men, aged 35 years and over, recruited from community-based settings in Sydney, Australia, attend 6 clinic visits over 3 years. At the first 5 visits, participants undergo a digital anorectal examination, an anal swab for HPV genotyping and anal cytology, and high-resolution anoscopy with directed biopsy of any visible abnormalities that are suggestive of any abnormality suspicious of SIL. Tissue sections from participants diagnosed with histologically confirmed HSIL at the baseline clinic visit will undergo laser capture microdissection, HPV detection and genotyping, and quantitation of CpG methylation in baseline and followup biopsies. Histological and cytological findings in combination with HPV genotyping data will be used to identify persistent HSIL. HSIL will be stratified as nonpersistent and persistent based on their status at 12 months. The performance of HPV genotype and methylation status in predicting disease persistence at 12 months will be assessed, along with associations with HIV status and other covariates such as age. Ethics and dissemination: The St Vincent's Hospital Ethics Committee granted ethics approval for the study. Written informed consent is obtained from all individuals before any study-specific procedures are performed. Findings from this study will be disseminated to parti

INTRODUCTION: Anal squamous cell carcinoma is preceded by persistent infection with high-risk human papillomavirus (HPV) and the cancer precursor, high-grade squamous intraepithelial lesion (HSIL). Detection of specific HPV genotypes and HPV-related biomarkers may be an option for primary anal screening. However, more data on the natural history of HPV-related anal lesions are required. The outcomes from this study will enhance our understanding of the clinical and biological behaviour of HPV-related anal lesions and inform the development of future HPV genotype and/or biomarker screening tests. METHODS AND ANALYSIS: HIV-negative and HIV-positive men who have sex with men, aged 35 years and over, recruited from community-based settings in Sydney, Australia, attend 6 clinic visits over 3 years. At the first 5 visits, participants undergo a digital anorectal examination, an anal swab for HPV genotyping and anal cytology, and high-resolution anoscopy with directed biopsy of any visible abnormalities that are suggestive of any abnormality suspicious of SIL. Tissue sections from participants diagnosed with histologically confirmed HSIL at the baseline clinic visit will undergo laser capture microdissection, HPV detection and genotyping, and quantitation of CpG methylation in baseline and follow-up biopsies. Histological and cytological findings in combination with HPV genotyping data will be used to identify persistent HSIL. HSIL will be stratified as non-persistent and persistent based on their status at 12 months. The performance of HPV genotype and methylation status in predicting disease persistence at 12 months will be assessed, along with associations with HIV status and other covariates such as age. ETHICS AND DISSEMINATION: The St Vincent's Hospital Ethics Committee granted ethics approval for the study. Written informed consent is obtained from all individuals before any study-specific procedures are performed. Findings from this study will be disseminated to pa

BACKGROUND: The incidence of human papillomavirus (HPV)-associated anal cancer is increasing in men who have sex with men (MSM). Screening for the presumed cancer precursor, high-grade anal squamous intraepithelial lesions (HSIL) in a manner analogous to cervical cancer screening has been proposed. Uncertainty remains regarding anal HPV natural history and the role of anal cytology and high-resolution anoscopy (HRA) as screening tests. Well-designed cohort studies are required to address these issues. METHODS/DESIGN: The SPANC study is a prospective study of the epidemiology of low-risk and high-risk anal HPV infection and related cytological and histological abnormalities in HIV-negative and HIV-positive homosexual men aged 35 years and over. The study aims to recruit 600 men from community-based settings in Sydney, Australia. There are six study visits over three years. At the first five visits men undergo a digital ano-rectal examination (DARE), an anal "Papanicolaou" (Pap) test for HPV detection, genotyping and anal cytology, followed by HRA and directed biopsy of any visible abnormalities. The men also complete a behavioural questionnaire before each visit. Questions include a detailed history of sexual behaviour, of anal symptoms, possible anal cancer risk factors and validated quality of life and psychosocial questions. Questionnaires are also completed 2 weeks and 3 months following the provision of test results and include questions on participant experience during the procedure and post-procedure symptoms, including pain and bleeding in addition to quality of life/ psychosocial outcomes. DISCUSSION: Recruitment for the study began in September 2010 and will conclude in mid-2015, with follow up continuing to 2018. Thus far, over 350 men have been recruited from a variety of community-based settings and are broadly representative of the target screening population. The SPANC study is one of only a small number of cohort studies globally to perform HPV, cytol

Incidence and mortality rates of anal cancer are increasing globally. More than 90% of anal squamous cell carcinomas (ASCC) are associated with human papillomavirus (HPV). Studies on HPV-related anogenital lesions have shown that patterns of methylation of viral and cellular DNA targets could potentially be developed as disease biomarkers. Lesion-specific DNA isolated from formalin-fixed paraffin-embedded (FFPE) tissues from existing or prospective patient cohorts may constitute a valuable resource for methylation analysis. However, low concentrations of DNA make these samples technically challenging to analyse using existing methods. We therefore set out to develop a sensitive and reproducible nested PCR-pyrosequencing based method to accurately quantify methylation at 10 CpG sites within the E2BS1, E2BS2,3,4 and Sp1 binding sites in the viral upstream regulatory region of HPV16 genome. Methylation analyses using primary and nested PCR-pyrosequencing on 52 FFPE tissue [26 paired whole tissue sections (WTS) and laser capture microdissected (LCM) tissues] from patients with anal squamous intraepithelial lesions was performed. Using nested PCR, methylation results were obtained for the E2BS1, E2BS2,3,4 and Sp1 binding sites in 86.4% of the WTS and 81.8% of the LCM samples. Methylation patterns were strongly correlated within median values of matched pairs of WTS and LCM sections, but overall methylation was higher in LCM samples at different CpG sites. High grade lesions showed low methylation levels in the E2BS1 and E2BS2 regions, with increased methylation detected in the E2BS,3,4/Sp1 regions, showing the highest methylation at CpG site 37. The method developed is highly sensitive in samples with low amounts of DNA and demonstrated to be suitable for archival samples. Our data shows a possible role of specific methylation in the HPV16 URR for detection of HSIL.