DNA methylation at individual CpG-sites of EPB41L3, HTERT and FAM19A4 are useful for detection of cervical high-grade squamous intraepithelial lesions (HSIL) or worse: Analysis of individual CpG-sites outperforms averaging.

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.

Performance of CADM1, MAL and miR124-2 methylation as triage markers for early detection of cervical cancer in self-collected and clinician-collected samples: an exploratory observational study in Papua New Guinea.

OBJECTIVE: WHO recommends human papillomavirus (HPV) testing for cervical screening, with triage of high-risk HPV (hrHPV) positive women. However, there are limitations to effective triage for low-resource, high-burden settings, such as Papua New Guinea. In this exploratory study, we assessed the performance of host methylation as triage tools for predicting high-grade squamous intraepithelial lesions (HSIL) in self-collected and clinician-collected samples. DESIGN: Exploratory observational study. SETTING: Provincial hospital, same-day cervical screen-and-treat trial, Papua New Guinea. PARTICIPANTS: 44 hrHPV+women, with paired self/clinician-collected samples (4 squamous cell carcinomas (SCC), 19 HSIL, 4 low-grade squamous intraepithelial lesions, 17 normal). PRIMARY AND SECONDARY OUTCOME MEASURES: Methylation levels of CADM1, MAL and miR124-2 analysed by methylation-specific PCRs against the clinical endpoint of HSIL or SCC (HSIL+) measured using liquid-based-cytology/p16-Ki67 stain. RESULTS: In clinician-collected samples, MAL and miR124-2 methylation levels were significantly higher with increasing grade of disease (p=0.0046 and p<0.0015, respectively). miR124-2 was the best predictor of HSIL (area under the curve, AUC 0.819) while MAL of SCC (AUC 0.856). In self-collected samples, MAL best predicted HSIL (AUC 0.595) while miR124-2 SCC (AUC 0.812). Combined miR124-2/MAL methylation yielded sensitivity and specificity for HSIL+ of 90.5% (95% CI 69.6% to 98.8%) and 70% (95% CI 45.7% to 88.1%), respectively, in clinician-collected samples, and 81.8% (95% CI 59.7% to 94.8%) and 47.6% (95% CI 25.7% to 70.2%), respectively, in self-collected samples. miR124-2/MAL plus HPV16/HPV18 improved sensitivity for HSIL+ (95.2%, 95% CI 76.2% to 99.9%) but decreased specificity (55.0%, 95% CI 31.5% to 76.9%). CONCLUSION: miR124-2/MAL methylation is a potential triage strategy for the detection of HSIL/SCC in low-income and middle-income country.

Evaluation of Seegene Anyplex II Performance for Detection of Human Papillomavirus Genotypes in Formalin-Fixed, Paraffin-Embedded Cervical Cancer Specimens.

CONTEXT.­: Detection of human papillomavirus (HPV) in formalin-fixed, paraffin-embedded (FFPE) tissues may identify the cause of lesions and has value for the development of new diagnostic assays and epidemiologic studies. Seegene Anyplex II assays are widely used for HPV screening, but their performance using FFPE samples has not been fully explored. OBJECTIVE.­: To validate Anyplex II HPV HR Detection (Anyplex II, Seegene) using FFPE samples. DESIGN.­: We used 248 stored DNA extracts from cervical cancer FFPE samples collected during 2005-2015 that tested HPV positive using the RHA kit HPV SPF10-LiPA25, v1 (SPF10, Labo Biomedical Products) HPV genotyping assay, manufacturer-validated for FFPE samples. RESULTS.­: Of the selected 248 samples, 243 were used in our analysis. Consistent with SPF10 genotyping results, Anyplex II detected all 12 oncogenic types and had an overall HPV detection rate of 86.4% (210 of 243 samples). Anyplex II and SPF10 showed very high agreement for the detection of the 2 most important oncogenic genotypes: HPV 16 (219 of 226; 96.9%; 95% CI, 93.7-98.75) and HPV 18 (221 of 226; 97.8%; 95% CI, 94.9-99.3). CONCLUSIONS.­: Overall results showed that both platforms produced comparable HPV genotyping results, indicating the suitability of Anyplex II for FFPE samples. The Anyplex II assay has the added convenience of being an efficient, single-well semiquantitative polymerase chain reaction assay. Further optimization of Anyplex II may enhance its performance using FFPE samples by improving the detection limit.

Efficacy of a synthetic peptide Chlamydia pecorum major outer membrane protein vaccine in a wild koala (Phascolarctos cinereus) population.

Chlamydiosis is a significant disease affecting Eastern Australian koala (Phascolarctos cinereus) populations, impacting individual animal welfare and fecundity and therefore influencing population dynamics. The aim of this study was to investigate the effect of a synthetic peptide vaccine based on 4 components of the Chlamydia pecorum major outer membrane protein (MOMP), over an 18-month period in a koala population severely impacted by chlamydiosis. Wild koalas were recruited into a vaccination or a placebo treatment group on a random allocation, then followed through a period of 18 months, with recapture at 6 monthly intervals. Vaccination did not alter clinical disease expression or chlamydial shedding from the ocular or urogenital sites. Vaccination did not stimulate a significant plasma anti-MOMP IgG response, when compared to the placebo group. There was no significant effect of vaccination on IFN-γ and IL-17A mRNA expression of peripheral blood lymphocytes when stimulated with rMOMP. We have demonstrated that a synthetic peptide vaccination against chlamydiosis is not an effective management tool in a koala population with a high prevalence of C. pecorum infection and related disease. The lack of antigenic response found in this study suggests that further research utilising a larger, full-length antigen is an avenue worth investigation if we are to consider vaccination as a part of a management strategy in diseased koala populations.

Reduction of Chlamydia pecorum and Koala Retrovirus subtype B expression in wild koalas vaccinated with novel peptide and peptide/recombinant protein formulations.

Koalas are an endangered species under threat of extinction from several factors, including infections agents. Chlamydia pecorum infection results in morbidity and mortality from ocular and urogenital diseases while Koala Retrovirus (KoRV) infection has been linked to increased rates of cancer and chlamydiosis. Both C. pecorum and KoRV are endemic in many wild Australian koala populations, with limited treatment options available. Fortunately, vaccines for these pathogens are under development and have generated effective immune responses in multiple trials. The current study aimed to improve vaccine formulations by testing a novel peptide version of the Chlamydia vaccine and a combination Chlamydia - KoRV vaccine. Utilising a monitored wild population in Southeast Queensland, this trial followed koalas given either a 'Chlamydia only' vaccine (utilising four peptides from the chlamydial Major Outer Membrane Protein, MOMP), a combination 'Chlamydia and KoRV' vaccine (comprised of the chlamydial peptides plus a KoRV recombinant envelope protein (rEnv)) or no treatment. Clinical observations, C. pecorum and KoRV gene expression, serum IgG, and mucosal immune gene expression were assessed over a 17-month period. Overall, both vaccine formulations resulted in a decrease in chlamydiosis mortality, with decreases in C. pecorum, CD4, CD8ß and IL-17A gene expression observed. In addition, the combination vaccine group also showed an increase in anti-KoRV IgG production that corresponded to a decrease in detected KoRV-B expression. While these results are favourable, the chlamydial peptide vaccine did not appear to outperform the established recombinant chlamydial vaccine and suggests that a combination vaccine formulated with recombinant MOMP plus KoRV rEnv could capitalize on the demonstrated benefits of both for the betterment of koalas into the future.

Long-term survival with IDH wildtype glioblastoma: first results from the ETERNITY Brain Tumor Funders' Collaborative Consortium (EORTC 1419).

BACKGROUND: Median survival with glioblastoma remains in the range of 12 months on population levels. Only few patients survive for more than 5 years. Patient and disease features associated with long-term survival remain poorly defined. METHODS: European Organization for Research and Treatment of Cancer (EORTC) 1419 (ETERNITY) is a registry study supported by the Brain Tumor Funders Collaborative in the US and the EORTC Brain Tumor Group. Patients with glioblastoma surviving at least 5 years from diagnosis were identified at 24 sites in Europe, US, and Australia. In patients with isocitrate dehydrogenase (IDH) wildtype tumours, prognostic factors were analysed using the Kaplan-Meier method and the Cox proportional hazards model. A population-based reference cohort was obtained from the Cantonal cancer registry Zurich. RESULTS: At the database lock of July 2020, 280 patients with histologically centrally confirmed glioblastoma (189 IDH wildtype, 80 IDH mutant, 11 incompletely characterised) had been registered. In the IDH wildtype population, median age was 56 years (range 24-78 years), 96 patients (50.8%) were female, 139 patients (74.3%) had tumours with O6-methylguanine DNA methyltransferase (MGMT) promoter methylation. Median overall survival was 9.9 years (95% confidence interval [95% CI] 7.9-11.9). Patients without recurrence experienced longer median survival (not reached) than patients with one or more recurrences (8.92 years) (p < 0.001) and had a high rate (48.8%) of MGMT promoter-unmethylated tumours. CONCLUSIONS: Freedom from progression is a powerful predictor of overall survival in long-term survivors with glioblastoma. Patients without relapse often have MGMT promoter-unmethylated glioblastoma and may represent a distinct subtype of glioblastoma.

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.

Comparison of four assays for human papillomavirus detection in the anal canal.

OBJECTIVE: Anal cancer is preceded by high-risk human papillomavirus (HRHPV) infection, predominantly HPV16. No HPV assay is licenced for use in anal screening. We aimed to determine the sensitivity and specificity of four anal canal swab HPV assays to predict high-grade squamous epithelial lesions (HSIL). METHODS: In a cohort of Australian HIV-positive and negative gay and bisexual men, we compared the sensitivity and specificity of detection of 13 anal HRHPV genotypes by Linear Array (LA), Cobas 4800, EuroArray, and Anyplex II HPV28 (+ and ++ cut offs), compared their ability to predict prevalent anal HSIL, and compared anal canal HRHPV detection with HRHPV isolated from HSIL using laser capture microdissection (LCM). RESULTS: A total of 475 participants had baseline results available for all four assays (166, 35.0% HIV positive), and 169 participants had a diagnosis of cytological and/or histological HSIL. The HPV16 and any HRHPV detection were highest with Anyplex II HPV28 (+) (156, 32.8% 95% CI 28.6-37.2 and 359, 75.6%, 95% CI 71.5-79.4, respectively). For detection of concurrent HSIL and HPV16, the assay sensitivity was similar, ranging from 49.1%, 95% CI 41.4-56.9 (Anyplex II HPV28 ++) to 55.0%, 95% CI 47.2-62.7 (Anyplex II HPV28 +). For concurrent HSIL and any HRHPV detection, EuroArray was more specific than Anyplex II HPV28 (+) (45.9% 95% CI 40.2-51.7 vs 36.7%, 95% CI 31.3-42.4, p = 0.021) and had comparable specificity with Anyplex II HPV28 (++) (45.9% vs 47.2%, 95% CI 41.5-53.0, p = 0.75). All assays had high sensitivities for predicting HPV16 detected on LCM (92.5-97.5%). Anyplex II HPV28 and EuroArray were significantly more sensitive than LA for lesions caused by non-HPV16 HRHPV types on LCM. DISCUSSION: Anyplex II HPV28 and EuroArray detected more non-16 HRHPV genotypes than LA. Increasing the Anyplex II HPV28 cutoff improved specificity without compromising sensitivity for detection of concurrent HSIL.

Volatile Organic Compounds in the Early Diagnosis of Non-healing Surgical Wounds: A Systematic Review.

BACKGROUND: The normal healing of surgical wounds can be disrupted by infection and/or dehiscence, leading to development of chronic, non-healing wounds (NHW). Diagnosis of NHWs is via clinical acumen and analysis of microbiology wound swabs. Volatile organic compounds (VOCs) are emitted generally by human subjects and specifically as products of bacterial metabolism and are detected in the wound area. This systematic review will assess the potential use of VOCs released by surgical wounds as a non-invasive method for identifying bacterial species and the progression to NHW. METHOD: A systematic search of studies, via PRISMA guidelines, was conducted. Of 220 papers screened, seven studies were included. Outcome data were extracted on methods for VOC analysis and wound/bacterial VOC profiles. RESULTS: The studies have shown that VOC profiles are identified by two methods: gas chromatography-mass spectrometry and electronic nose. There are VOC profiles associated with causative bacterial species, with early indications that they could be anatomically specific or could monitor treatment effects. CONCLUSION: VOC profiling of bacterial species within wounds is possible and could become a point of care test. More research is needed on specific VOC profiles to wound location and whether these profiles may predict progression to NHW.

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.

Is Chlamydia to Blame for Koala Reproductive Cysts?

A significant threat to koala populations is infection from Chlamydia, which results in disease and death. Wild koalas with Chlamydia infections are admitted to wildlife hospitals and treated with antibiotics; however, up to 50% of koalas that present to wildlife hospitals do not survive. A major contributor to high mortality is the development of reproductive cysts, resulting in female infertility and euthanasia. However, the diagnosis of reproductive disease is limited to ultrasound with no further investigations. This communication highlights reports of histological and microbiological findings, the accuracy of ultrasound to necropsy reports and other possible causes for reproductive cyst development previously reported in other hosts. Our conclusions identify a significant knowledge gap in the aetiology of koala reproductive cysts and highlight the urgent need for future investigations.

Chlamydial clinical isolates show subtle differences in persistence phenotypes and growth in vitro.

Urogenital Chlamydia trachomatis infection is the most common sexually transmitted bacterial infection throughout the world. While progress has been made to better understand how type strains develop and respond to environmental stress in vitro, very few studies have examined how clinical isolates behave under similar conditions. Here, we examined the development and persistence phenotypes of several clinical isolates, to determine how similar they are to each other, and the type strain C. trachomatis D/UW-3/Cx. The type strain was shown to produce infectious progeny at a higher magnitude than each of the clinical isolates, in each of the six tested cell lines. All chlamydial strains produced the highest number of infectious progeny at 44 h post-infection in the McCoy B murine fibroblast cell line, yet showed higher levels of infectivity in the MCF-7 human epithelial cell line. The clinical isolates were shown to be more susceptible than the type strain to the effects of penicillin and iron deprivation persistence models in the MCF-7 cell line. While subtle differences between clinical isolates were observed throughout the experiments conducted, no significant differences were identified. This study reinforces the importance of examining clinical isolates when trying to relate in vitro data to clinical outcomes, as well as the importance of considering the adaptations many type strains have to being cultured in vitro.

HIV, Immune Dysfunction, and the Natural History of Anal High-Risk Human Papillomavirus Infection in Gay and Bisexual Men.

BACKGROUND: Incidence of anal cancer is highest in gay and bisexual men (GBM). Better understanding of the natural history of anal high-risk human papillomavirus (hrHPV) infection is needed for anal cancer prevention. METHODS: The Study of the Prevention of Anal Cancer was a 3-year study of Australian GBM, aged 35 years or older. We examined incidence, clearance, and risk factors for 13 hrHPV types at baseline and 3 annual visits. RESULTS: In 525 men with ≥ 2 visits, 348 (66.3%) acquired ≥ 1 incident hrHPV infection. HPV16 incidence rates were similar, but non-16 hrHPV incidence was higher in HIV-positive (51.8/100 person years [PY]) than HIV-negative men (36.5/100 PY, P < .001). Annual clearance rates of HPV16 (13.21/100 PY, 95% confidence interval, 10.53-16.56) were lower than for other hrHPV types. hrHPV clearance rates were not associated with HIV overall but were significantly lower in those with a lower nadir CD4 (<200 cells/µL) for HPV16 (P = .015) and other hrHPV types (P = .007). CONCLUSIONS: Higher incidence of non-16 hrHPV types, coupled with lower clearance of non-16 hrHPV types in those with past impaired immune function, is consistent with the greater role of non-16 hrHPV in anal cancer in HIV-positive people. AUSTRALIA NEW ZEALAND CLINICAL TRIALS REGISTRY: ANZCTR365383.

The Natural History of Anal High-grade Squamous Intraepithelial Lesions in Gay and Bisexual Men.

BACKGROUND: Gay and bisexual men (GBM) are disproportionately affected by anal cancer. Prevention is hindered by incomplete understanding of the natural history of its precursor, anal high-grade squamous intraepithelial lesions (HSIL). METHODS: The Study of the Prevention of Anal Cancer, conducted between 2010 and 2018, enrolled human immunodeficiency virus (HIV)-positive and HIV-negative GBM aged ≥35 years. Anal cytology and high-resolution anoscopy (HRA) were performed at baseline and 3 annual visits. A composite HSIL diagnosis (cytology ± histology) was used. Cytological high-grade squamous intraepithelial lesions (cHSIL) incidence and clearance rates were calculated with 95% confidence intervals (CIs). Predictors were calculated using Cox regression with hazard ratios (HRs) and 95% CIs. RESULTS: Among 617 men, 220 (35.7%) were HIV-positive, median age 49 years. And 124 incident cHSIL cases occurred over 1097.3 person-years (PY) follow-up (11.3, 95% CI 9.5-13.5 per 100 PY). Significant bivariate predictors of higher incidence included age <45 years (HR 1.64, 95% CI 1.11-2.41), HIV positivity (HR 1.43, 95% CI .99-2.06), prior SIL diagnosis (P-trend < .001) and human papillomavirus (HPV)16 (HR 3.39, 2.38-4.84). Over 695.3 PY follow-up, 153 HSIL cleared (clearance 22.0, 95% CI 18.8-25.8 per 100 PY). Predictors were age < 45 years (HR 1.52, 1.08-2.16), anal intraepithelial neoplasia (AIN)2 rather than AIN3 (HR 1.79, 1.29-2.49), smaller lesions (HR 1.62, 1.11-2.36) and no persistent HPV16 (HR 1.72, 1.23-2.41). There was 1 progression to cancer (incidence 0.224, 95% CI .006-1.25 per 100 PY). CONCLUSION: These data strongly suggest that not all anal HSIL detected in screening requires treatment. Men with persistent HPV16 were less likely to clear HSIL and are more likely to benefit from effective HSIL treatments. CLINICAL TRIALS REGISTRATION: Australia New Zealand Clinical Trials Registry (ANZCTR365383).

Quadrivalent human papillomavirus vaccination successfully reduces the prevalence of vaccine-targeted genotypes in a young, vaccine-eligible-age sample of Australian females.

Background The prevalence of genital tract vaccine-type human papillomavirus (HPV) is on the decline due to high vaccine uptake through the national HPV immunisation program in Australia. The aim of this study was to investigate HPV vaccine coverage and factors associated with HPV in a vaccine-eligible sample of young Australian females. METHODS: Females aged 16-25 years were recruited into the Young Female Health Initiative study, a young women's health study, via Facebook advertising from 2012 to 2017. Sexually active participants were asked to provide a self-collected vaginal swab for the detection of HPV DNA; positive samples were genotyped. Self-reported HPV vaccination status was confirmed by the National HPV Vaccination Program Register. Outcomes of the study were HPV acquisition and genotype, HPV vaccination status and factors associated with HPV. RESULTS: Overall, 22.8% of samples (95% confidence interval (CI) 17.8-27.8%; n = 62/272) were positive for any HPV DNA, of which 19.1% (95% CI 14.4-23.8%; n = 52/272) were oncogenic types. HPV 16 was detected in three samples (1.1%; 95% CI -0.1%, 2.3%; two not HPV vaccinated and one vaccinated after sexual debut). Early sexual debut (<16 years) and multiple sexual partners were independently associated with an increased risk of any HPV. CONCLUSIONS: In a community sample of vaccine-eligible-age females with a high vaccine uptake, the prevalence of vaccine-related HPV genotypes is extremely low. Early sexual debut and multiple sexual partners are positively associated with HPV, underscoring the importance of vaccination at the routinely recommended age of 12-13 years for best vaccine impact.

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.

Vaccination of koalas during antibiotic treatment for Chlamydia-induced cystitis induces an improved antibody response to Chlamydia pecorum.

Chlamydia infection and disease are endemic in free-ranging koalas. Antibiotics remain the front line treatment for Chlamydia in koalas, despite their rates of treatment failure and adverse gut dysbiosis outcomes. A Chlamydia vaccine for koalas has shown promise for replacing antibiotic treatment in mild ocular Chlamydia disease. In more severe disease presentations that require antibiotic intervention, the effect of vaccinating during antibiotic use is not currently known. This study investigated whether a productive immune response could be induced by vaccinating koalas during antibiotic treatment for Chlamydia-induced cystitis. Plasma IgG antibody levels against the C. pecorum major outer membrane protein (MOMP) dropped during antibiotic treatment in both vaccinated and unvaccinated koalas. Post-treatment, IgG levels recovered. The IgG antibodies from naturally-infected, vaccinated koalas recognised a greater proportion of the MOMP protein compared to their naturally-infected, unvaccinated counterparts. Furthermore, peripheral blood mononuclear cell gene expression revealed an up-regulation in genes related to neutrophil degranulation in vaccinated koalas during the first month post-vaccination. These findings show that vaccination of koalas while they are being treated with antibiotics for cystitis can result in the generation of a productive immune response, in the form of increased and expanded IgG production and host response through neutrophil degranulation.

Antibiotic treatment of Chlamydia-induced cystitis in the koala is linked to expression of key inflammatory genes in reactive oxygen pathways.

Chlamydial-induced cystitis in the koala (Phascolarctos cinereus) is currently treated by antibiotics. However, while reducing the chlamydial load, this treatment can also lead to gastrointestinal complications and death. Development of alternative treatments, such as a therapeutic chlamydial vaccine, are hindered by the lack of detailed understanding of the innate immune response to chlamydial clearance and disease regression during antibiotic treatment. Through clinical, microbiological and transcriptomic approaches, disease regression, bacterial clearance and innate immune responses were mapped in koalas with signs of chlamydial-induced cystitis while receiving anti-chlamydial antibiotics. Significant reduction in the signs of cystitis were observed during and post antibiotic treatment. This was observed as a thinning of the bladder wall and complete reversal of urinary incontinence. Transcriptomic analysis before treatment, at the end of treatment and prior to release identified significant down-regulation of specific genes involved in 21 biological pathways. Of these, the chemokine receptor signalling and NOD-like receptor signalling pathways where identified as important markers of inflammation. Specific genes within these pathways (NCF1 and NOX2) were significantly down-regulated, suggesting a decrease in reactive oxygen species production. Through the monitoring of specific clinical and transcriptomic markers, these findings allow detailed profiling of the clinical response to therapeutic vaccination in koalas with current signs of disease. This also adds to our understanding of innate immune responses to chlamydial infections and indicates that chlamydial-induced cystitis in the koala is linked to the regulation of reactive oxygen pathways.

Changes in Endogenous and Exogenous Koala Retrovirus Subtype Expression over Time Reflect Koala Health Outcomes.

Koala retrovirus (KoRV) is unique in that it exists as both an exogenous and actively endogenizing gamma retrovirus of koalas. While nine subtypes of KoRV have been recognized, focused study of these subtypes in koalas over time and with different health outcomes has been lacking. Therefore, in this study, three wild koala cohorts were established and monitored to examine KoRV proviral and expression data from koalas that either remained healthy over time, began healthy before developing chlamydial cystitis, or presented with chlamydial cystitis and were treated with antibiotics. Deep sequencing of the proviral KoRV envelope gene revealed KoRV-A, -B, -D, and -F to be the major subtypes in this population and allowed for subtype-specific assays to be created. Quantification of KoRV transcripts revealed that KoRV-D expression mirrored the total KoRV expression levels (106 copies/ml of plasma), with KoRV-A and KoRV-F expression being ∼10-fold less and KoRV-B expression being ∼100-fold less, when detected. Strikingly, there was significantly higher expression of KoRV-D in healthy koalas than in koalas that developed chlamydial cystitis, with healthy koalas expressing a major KoRV-D/minor KoRV-A profile, whereas koalas that developed cystitis had variable KoRV expression profiles. Total anti-KoRV IgG antibody levels were found not to correlate with the expression of total KoRV or any individual KoRV subtype. Finally, KoRV expression was consistent between systemic and mucosal body sites and during antibiotic treatment. Collectively, this gives a comprehensive picture of KoRV dynamics during several important koala health states.IMPORTANCE The long-term survival of the koala is under serious threat, with this iconic marsupial being declared "vulnerable" by the Australian Government and officially listed as a threatened species. KoRV is clearly contributing to the overall health status of koalas, and research into this virus has been lacking detailed study of the multiple subtypes at both the proviral and expressed viral levels over time. By designing new subtype-specific assays and following well-defined koala cohorts over time, this study has generated a new more complete picture of KoRV and its relationship to koala health outcomes in the wild. Only by building a comprehensive picture of KoRV during both koala health and disease can we bring meaningful koala health interventions into better focus.

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.