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.

The effect of the Xpert HIV-1 Qual test on early infant diagnosis of HIV in Myanmar and Papua New Guinea: a pragmatic, cluster-randomised, stepped-wedge, open-label trial.

BACKGROUND: Despite proven benefits for child health, coverage of early infant diagnosis of HIV remains suboptimal in many settings. We aimed to assess the effect of a point-of-care early infant diagnosis test on time-to-results communication for infants vertically exposed to HIV. METHODS: This pragmatic, cluster-randomised, stepped-wedge, open-label trial assessed the effect of the Xpert HIV-1 Qual early infant diagnosis test (Cepheid) on time-to-results communication, compared with standard care laboratory-based testing of dried blood spots using PCR. Hospitals were the unit of randomisation for one-way crossover from control to intervention phase. Each site had between 1 month and 10 months of control phase before transitioning to the intervention, with a total of 33 hospital-months in the control phase and 45 hospital-months in the intervention phase. We enrolled infants vertically exposed to HIV at six public hospitals: four in Myanmar and two in Papua New Guinea. Infants had to have mothers with confirmed HIV infection, be younger than 28 days, and required HIV testing to be eligible for enrolment. Health-care facilities providing prevention of vertical transmission services were eligible for participation. The primary outcome was communication of early infant diagnosis results to the infant's caregiver by 3 months of age, assessed by intention to treat. This completed trial was registered with the Australian and New Zealand Clinical Trials Registry, 12616000734460. FINDINGS: In Myanmar, recruitment took place between Oct 1, 2016, and June 30, 2018; in Papua New Guinea, recruitment was between Dec 1, 2016, and Aug 31, 2018. A total of 393 caregiver-infant pairs were enrolled in the study across both countries. Independent of study time, the Xpert test reduced time to early infant diagnosis results communication by 60%, compared with the standard of care (adjusted time ratio 0·40, 95% CI 0·29-0·53, p<0·0001). In the control phase, two (2%) of 102 study participants received an early infant diagnosis test result by 3 months of age compared with 214 (74%) of 291 in the intervention phase. No safety and adverse events were reported related to the diagnostic testing intervention. INTERPRETATION: This study reinforces the importance of scaling up point-of-care early infant diagnosis testing in resource-constrained and low HIV-prevalence settings, typical of the UNICEF East Asia and Pacific region. FUNDING: National Health and Medical Research Council of Australia.

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

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

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

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

Association between visual inspection of the cervix with acetic acid examination and high-risk human papillomavirus infection, Chlamydia trachomatis, Neisseria gonorrhoeae and Trichomonas vaginalis in Papua New Guinea.

BACKGROUND: Papua New Guinea (PNG) has among the highest estimated burdens of cervical cancer globally but currently has no national cervical screening program. Visual inspection of the cervix with acetic acid (VIA) is a low-cost screening strategy endorsed by the World Health Organization that has been adopted in many low-resource settings but not previously evaluated in PNG. AIM: To evaluate the association between VIA examination findings and high-risk HPV (hrHPV) infection; and the impact of concomitant genital Chlamydia trachomatis, Neisseria gonorrhoeae and Trichomonas vaginalis on the interpretation of VIA findings. METHODS: A prospective clinical cohort study among women aged 30-59 years attending Well Woman Clinics in PNG. Main outcome measures were VIA examination findings and laboratory-confirmed hrHPV, C. trachomatis, N. gonorrhoeae and T. vaginalis. RESULTS: A total of 614 women were enrolled, of whom 87.5% (537/614) underwent VIA, and 12.5% (77/614) did not due to pre-existing cervicitis or inability to visualise the transformation zone. Among the 537 women who underwent VIA, 21.6% were VIA positive, 63.7% VIA negative, and 14.7% had indeterminate findings. The prevalence of hrHPV infection (n = 614) was 14.7%; C. trachomatis, 7.5%; N. gonorrhoeae, 8.0%; and T. vaginalis, 15.0%. VIA positive women were more likely to have HPV16 (odds ratio: 5.0; 95%CI: 1.6-15.6; P = 0.006) but there was no association between HPV18/45, all hrHPV types (combined), C. trachomatis, N. gonorrhoeae or T. vaginalis. CONCLUSIONS: VIA positivity was associated with HPV16, but not with other hrHPV infections, nor with genital C. trachomatis, N. gonorrhoeae or T. vaginalis in this setting.

Performance of syndromic management for the detection and treatment of genital Chlamydia trachomatis, Neisseria gonorrhoeae and Trichomonas vaginalis among women attending antenatal, well woman and sexual health clinics in Papua New Guinea: a cross-sectional study.

OBJECTIVE: Papua New Guinea (PNG) has among the highest estimated prevalences of genital Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG) and Trichomonas vaginalis (TV) of any country in the Asia-Pacific region. Diagnosis and treatment of these infections have relied on the WHO-endorsed syndromic management strategy that uses clinical presentation without laboratory confirmation to make treatment decisions. We evaluated the performance of this strategy in clinical settings in PNG. DESIGN: Women attending antenatal (ANC), well woman (WWC) and sexual health (SHC) clinics in four provinces were invited to participate, completed a face-to-face interview and clinical examination, and provided genital specimens for laboratory testing. We estimated the performance characteristics of syndromic diagnoses against combined laboratory diagnoses. RESULTS: 1764 women were enrolled (ANC=765; WWC=614; SHC=385). The prevalences of CT, NG and TV were highest among women attending ANC and SHC. Among antenatal women, syndromic diagnosis of sexually transmitted infection had low sensitivity (9%-21%) and positive predictive value (PPV) (7%-37%), but high specificity (76%-89%) and moderate negative predictive value (NPV) (55%-86%) for the combined endpoint of laboratory-confirmed CT, NG or TV. Among women attending WWC and SHC, 'vaginal discharge syndrome' had moderate to high sensitivity (72%-78%) and NPV (62%-94%), but low specificity (26%-33%) and PPV (8%-38%). 'Lower abdominal pain syndrome' had low sensitivity (26%-41%) and PPV (8%-23%) but moderate specificity (66%-68%) and high NPV (74%-93%) among women attending WWC, and moderate-high sensitivity (67%-79%) and NPV (62%-86%) but low specificity (26%-28%) and PPV (14%-33%) among SHC attendees. CONCLUSION: The performance of syndromic management for the detection and treatment of genital chlamydia, gonorrhoea and trichomonas was poor among women in different clinical settings in PNG. New diagnostic strategies are needed to control these infections and to prevent their adverse health outcomes in PNG and other high-burden countries.

Dorsal longitudinal foreskin cut is associated with reduced risk of HIV, syphilis and genital herpes in men: a cross-sectional study in Papua New Guinea.

INTRODUCTION: Various forms of penile foreskin cutting are practised in Papua New Guinea. In the context of an ecological association observed between HIV infection and the dorsal longitudinal foreskin cut, we undertook an investigation of this relationship at the individual level. METHODS: We conducted a cross-sectional study among men attending voluntary confidential HIV counselling and testing clinics. Following informed consent, participants had a face-to-face interview and an examination to categorize foreskin status. HIV testing was conducted on site and relevant specimens collected for laboratory-based Herpes simplex type-2 (HSV-2), syphilis, Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and Trichomonas vaginalis (TV) testing. RESULTS: Overall, 1073 men were enrolled: 646 (60.2%) were uncut; 339 (31.6%) had a full dorsal longitudinal cut; 72 (6.7%) a partial dorsal longitudinal cut; and 14 (1.3%) were circumcised. Overall, the prevalence of HIV was 12.3%; HSV-2, 33.6%; active syphilis, 12.1%; CT, 13.4%; NG, 14.1%; and TV 7.6%. Compared with uncut men, men with a full dorsal longitudinal cut were significantly less likely to have HIV (adjusted odds ratio [adjOR] 0.25, 95%CI: 0.12, 0.51); HSV-2 (adjOR 0.60, 95%CI: 0.41, 0.87); or active syphilis (adjOR 0.55, 95%CI: 0.31, 0.96). This apparent protective effect was restricted to men cut prior to sexual debut. There was no difference between cut and uncut men for CT, NG or TV. CONCLUSION: In this large cross-sectional study, men with a dorsal longitudinal foreskin cut were significantly less likely to have HIV, HSV-2 and syphilis compared with uncut men, despite still having a complete (albeit morphologically altered) foreskin. The protective effect of the dorsal cut suggests that the mechanism by which male circumcision works is not simply due to the removal of the inner foreskin and its more easily accessible HIV target cells. Exposure of the penile glans and inner foreskin appear to be key mechanisms by which male circumcision confers protection.Further research in this unique setting will help improve our understanding of the fundamental immunohistologic mechanisms by which male circumcision provides protection, and may lead to new biomedical prevention strategies at the mucosal level.

Comparative performance of the Kalon and HerpeSelect enzyme-linked immunosorbant assays to determine the prevalence of herpes simplex virus type 2 in Papua New Guinea.

UNLABELLED: Background Infection with herpes simplex virus type 2 (HSV-2) is common worldwide and an important risk factor for HIV infection. Aetiological diagnosis of HSV-2 is typically determined with the use of commercially available type-specific enzyme-linked immunosorbent assays (ELISAs). This study aimed to determine the prevalence of HSV-2 among people attending sexual health clinics in the Highlands of Papua New Guinea. The study also aimed to compare the performance of two type-specific ELISA assays, the Kalon and HerpeSelect glycoprotein G2 assays, in this context. METHODS: Participants were recruited as part of a longitudinal sexual health study. Participants attended four appointments over a 12-month period and had blood taken for HSV-2 serology at each time point. Both the Kalon and HerpeSelect assays were performed as per manufacturer's instructions. RESULTS: A total of 132 participants were tested for HSV-2 using the Kalon and HerpeSelect ELISAs. HSV-2 prevalence was 52% (95% CI, 43-60) and 61% (95% CI, 52-69) with Kalon and HerpeSelect assays respectively. There was high concordance (87%, ?=0.75, P<0.001, n=115) between the two assays at the manufacturer recommended index value cut-offs. For participants with discordant results at baseline, (n=16), three sero-conversions were observed over the 12-month period when sequential sera was tested. CONCLUSIONS: A high HSV-2 prevalence was observed in this clinic-based population. Our longitudinal data indicate the higher prevalence of HSV-2 detected with the HerpeSelect ELISA was likely due to false positives rather than a higher sensitivity in the early stages of infection.

Presence of HIV drug resistance in antiretroviral therapy-naive and -experienced patients from Papua New Guinea.

OBJECTIVES: The optimal benefits of antiretroviral therapy (ART) can be compromised by the emergence of HIV drug resistance (HIVDR) resulting in treatment failure. ART was introduced in Papua New Guinea (PNG) in 2004, yet biological data on HIVDR are lacking. The aim of the study was to investigate levels of HIVDR in ART-naive and -experienced patients in PNG. METHODS: We recruited, interviewed and collected blood from 108 ART-naive and 102 ART-experienced patients from two Highlands provinces of PNG. Dried blood spots were tested for HIVDR from all patients with detectable plasma viral load of ≥200 copies/mL using established in-house assays. RESULTS: The PCR amplification success was 90.6% (n = 96) and 66.7% (n = 12) using dried blood spots from ART-naive and -experienced patients, respectively. Transmitted drug resistance was detected in 2.1% (n = 2) of samples from ART-naive patients; acquired drug resistance was detected in 50% (n = 6) of samples from ART-experienced individuals. CONCLUSIONS: Our data showed that transmitted drug resistance in PNG is low and acquired drug resistance is higher with 12.7% of the ART-experienced patients failing treatment. As ART access is rapidly expanding in PNG, monitoring of drug resistance is paramount for early detection of treatment failure.

High prevalence and incidence of HIV, sexually transmissible infections and penile foreskin cutting among sexual health clinic attendees in Papua New Guinea.

UNLABELLED: Background Papua New Guinea (PNG) has one of the highest prevalences of HIV and sexually transmissible infections (STIs) in the Asia-Pacific region, and one of the highest burdens of maternal syphilis and cervical cancer globally. Despite this disease burden, only limited clinical research in sexual and reproductive health has been conducted in PNG. METHODS: A longitudinal clinical cohort study was conducted at two sexual health clinics. Participants completed a behavioural interview, clinical assessment and genital examination at baseline, and at 12, 24 and 50 weeks, including specimen collection for STI diagnostics. RESULTS: In total, 154 people attended a screening visit. Reattendance at 12, 24 and 50-weeks was 87%, 78% and 80% respectively. At baseline, HIV prevalence was 3.3%; chlamydia (Chlamydia trachomatis), 29.2%; gonorrhoea (Neisseria gonorrhoeae), 22.1%; Trichomonas vaginalis 15.6%; herpes simplex type-2 (HSV-2), 46.1%; active syphilis, 11.7%. Multiple infections were common particularly among women. The incidence of chlamydia was 27 per 100 person-years (PY); gonorrhoea, 15 out of 100 PY; T. vaginalis, 29 out of 100 PY; HSV-2, 12 out of 100 PY; syphilis, 8 out of 100 PY. No incident HIV cases were recorded. At baseline, 39% of men in Mt Hagen and 65% in Port Moresby had a penile foreskin cut, with a dorsal slit being the most common. Two men underwent penile cutting during the follow-up period. CONCLUSIONS: The prevalence and incidence of STIs, HIV and penile cutting were high among sexual health clinic attendees. High retention figures suggest that this population may be suitable for future interventions research and clinical trials.