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.

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.

High prevalence of pulmonary tuberculosis among female sex workers, men who have sex with men, and transgender women in Papua New Guinea.

BACKGROUND: Papua New Guinea (PNG) has a tuberculosis (TB) case notification rate of 333 cases per 100,000 population in 2016 and is one of the 14 countries classified by the World Health Organization (WHO) as "high-burden" for TB, multi-drug-resistant TB (MDR-TB), and TB/HIV. HIV epidemic is mixed with a higher prevalence among key populations, female sex workers (FSW), men who have sex with men (MSM), and transgender women (TGW). METHODS: We conducted a cross-sectional HIV biobehavioral survey (BBS) using respondent-driven sampling method among FSW, MSM, and TGW in Port Moresby, Lae, and Mt. Hagen (2016-2017). As part of the study, participants were screened for the four symptoms suggestive of TB infection using the WHO TB screening algorithm. Sputum and venous whole blood samples were collected and tested for pulmonary TB and HIV infection, respectively. Pulmonary TB testing was performed using GeneXpert®MTB/RIF molecular point-of-care test, and HIV testing was done following the PNG national HIV testing algorithm. All data discussed are weighted unless otherwise mentioned. RESULTS: Among FSW, 72.6%, 52.0%, and 52.9% in Port Moresby, Lae, and Mt. Hagen, respectively, experienced at least one symptom suggestive of TB infection. Among MSM and TGW, 69% and 52.6% in Port Moresby and Lae, respectively, experienced at least one symptom suggestive of TB infection. Based on GeneXpert®MTB/RIF results, the estimated TB prevalence rate among FSW was 1200, 700, and 200 per 100,000 in Port Moresby, Lae, and Mt. Hagen, respectively. Among MSM and TGW, the estimated TB prevalence rate was 1000 and 1200 per 100,000 in Port Moresby and Lae, respectively. Co-prevalence of TB/HIV among FSW was 0.1% in Port Moresby and 0.2% in Lae. There were no co-prevalent cases among FSW in Mt. Hagen or among MSM and TGW in Port Moresby and Lae. CONCLUSIONS: Key populations have a higher estimated rate of pulmonary TB than the national rate of pulmonary and extra-pulmonary TB combined. This showed that screening key populations for TB should be integrated into HIV programs regardless of HIV status in PNG's national TB response.

Gaps in HIV Testing and Treatment Among Female Sex Workers in Lae and Mt. Hagen, Papua New Guinea.

We conducted biobehavioral surveys among female sex workers (FSW) in Lae and Mt. Hagen, Papua New Guinea (January-December 2017). Respondent-driven sampling was used to recruit FSW aged ≥ 12 years, who were assigned female sex at birth, who spoke English or Tok Pisin, and who sold or exchanged sex for money, goods, or services in the last 6 months. When adjusted for viral suppression, 48.9% of FSW Lae and 61.9% in Mt. Hagen were aware of their HIV positive status. Of these women, 95.3% in Lae and 98.9% in Mt. Hagen were on antiretroviral therapy, and of these, 83.5% in Lae and 87.0% in Mt. Hagen had suppressed viral load. Renewed efforts are needed to increase HIV testing among FSW and provide support to FSW on treatment in both cities to attain viral suppression.

Factors associated with HIV and syphilis infection among female sex workers in three cities in Papua New Guinea: findings from Kauntim mi tu, a biobehavioral survey.

Background In this paper, factors associated with HIV and syphilis infection in three cities in Papua New Guinea are explored. METHODS: Respondent-driven sampling surveys among FSW in Port Moresby, Lae, and Mt. Hagen (2016-17) were conducted. FSW who were aged ≥12 years, who were born female, who spoke English or Tok Pisin and who had sold or exchanged vaginal sex in the past 6 months were eligible to participate. Participants were interviewed face-to-face and offered rapid HIV and syphilis testing. Survey logistic procedures were used to identify factors associated with HIV and syphilis infection, including modern contraception use, physical violence and having a casual male partner. Weighted data analysis was conducted. RESULTS: Overall, 2901 FSW (Port Moresby, 673; Lae, 709; and Mt. Hagen, 709) were enrolled in the study. HIV prevalence was 15.2% in Port Moresby, 11.9% in Lae and 19.6% in Mt. Hagen. Factors associated with HIV varied by city; for example, use of modern contraception in Port Moresby, experiences of physical violence in Lae and ever having tested for HIV in Mt. Hagen. No one variable was associated with HIV in all cities. Prevalence of syphilis infection was 7.1%, 7.0%, and 3.0% in Port Moresby, Lae, and Mt. Hagen, respectively. Factors associated with syphilis infection also varied by city and were only significant in Lae. CONCLUSION: The different factors associated with HIV and syphilis infection in each city highlight the complex HIV and syphilis epidemics among FSW and the importance of conducting surveys in multiple locations and developing local interventions.

Confidential, accessible point-of-care sexual health services to support the participation of key populations in biobehavioural surveys: Lessons for Papua New Guinea and other settings where reach of key populations is limited.

To achieve the UNAIDS 90-90-90 targets at a national level, many countries must accelerate service coverage among key populations. To do this, key population programs have adopted methods similar to those used in respondent-driven sampling (RDS) to expand reach. A deeper understanding of factors from RDS surveys that enhance health service engagement can improve key population programs. To understand the in-depth lives of key populations, acceptance of expanded point-of-care biological testing and determine drivers of participation in RDS surveys, we conducted semi-structured interviews with 111 key population participants (12-65 years) were purposefully selected from six biobehavioral surveys (BBS) in three cities in Papua New Guinea. Key populations were female sex workers, men who have sex with men, and transgender women. Four reasons motivated individuals to participate in the BBS: peer referrals; private, confidential, and stigma-free study facilities; "one-stop shop" services that provided multiple tests and with same-day results, sexually transmitted infection treatment, and referrals; and the desire to know ones' health status. Biobehavioral surveys, and programs offering key population services can incorporate the approach we used to facilitate key population engagement in the HIV cascade.

Considerable distance to reach 90-90-90 targets among female sex workers, men who have sex with men and transgender women in Port Moresby, Papua New Guinea: findings from a cross-sectional respondent-driven sampling survey.

OBJECTIVE: To characterise the Joint United Nations Programme on HIV/AIDS 90-90-90 cascade among female sex workers (FSW) and men who have sex with men (MSM)/transgender women (TGW) in Port Moresby, Papua New Guinea (PNG). METHODS: We conducted respondent-driven sampling surveys among FSW and MSM/TGW in Port Moresby, PNG from June to October 2016. All participants spoke English or Tok Pisin and were aged >12 years. FSW had to be born female and sell/exchange sex with a male in the past 6 months. MSM/TGW had to be born male and have oral/anal sex with another male-born person in the past 6 months. Participants were interviewed and offered rapid HIV diagnostic and viral load testing. HIV viral suppression (VS) was defined as <1000 copies/mL. RESULTS: We recruited 674 FSW and 400 MSM/TGW; HIV prevalence was 15.2% (95% CI 11.7 to 18.8) and 8.5% (95% CI 5.0 to 11.9), respectively. Among FSW living with HIV, 39.0% (95% CI 26.6 to 51.4) self-reported having been diagnosed; of them 79.6% (95% CI 62.7 to 96.5) self-reported being on antiretroviral therapy (ART), and 54.1% (95% CI 31.8 to 76.4) achieved VS. Among MSM/TGW living with HIV, 24.4% (95% CI 4.7 to 44.1) self-reported having been diagnosed; of them 43.9% (95% CI 33.6 to 54.8) self-reported being on ART, and 86.1% (95% CI 71.1 to 93.9) achieved VS. CONCLUSIONS: ART use among those aware of their HIV status is encouraging. However, the generally low awareness of infection status among FSW and MSM/TGW with HIV and the low VS among FSW on ART indicate an urgent need for innovative strategies to increase testing uptake and ART adherence among these populations. Monitoring drug resistance may be warranted.

One size does not fit all: HIV prevalence and correlates of risk for men who have sex with men, transgender women in multiple cities in Papua New Guinea.

BACKGROUND: Biobehavioral data about men who have sex with men (MSM) and transgender women (TGW) in Papua New Guinea (PNG) are limited to those who sell sex. Information about those MSM and TGW who do not sell sex is necessary to guide HIV prevention and treatment efforts. METHODS: We conducted respondent-driven sampling (RDS) surveys among MSM and TGW in Port Moresby, Lae, and Mt. Hagen, PNG from in 2016 and 2017. Eligibility criteria was: aged > 12 years, born male, could speak English or Tok Pisin and had oral or anal sex with another person born male in the past 6 months. Participants were interviewed face-to-face and offered rapid HIV testing. Weighted data analysis was conducted using RDS-Analyst (v. 0.62). RESULTS: We enrolled 400 participants in Port Moresby, 352 in Lae, and 111 in Mt. Hagen. In the last six months, 73.2% of MSM/TGW in Port Moresby, 77.9% in Lae, and 75.9% in Mt. Hagen, had a concurrent sexual partnership. Upwards of 70% of MSM/TGW in all three cities had sex with a woman in the same period. Less than half of MSM/TGW had ever tested for HIV. HIV prevalence among MSM/TGW was 8.5% in Port Moresby and 6.9% in Lae. Among participants in Mt. Hagen it was 1.3%. HIV was associated with not having sex with a woman in the last six months and sexually transmitted disease symptoms in the last 12 months in Port Moresby and Lae. In Port Moresby, it was also associated with an uncut foreskin, and in Lae with earning income in the formal sector and being unable to rely on other MSM or TGW to accompany them to healthcare services. CONCLUSIONS: The large proportion of MSM and TGW with concurrent sexual partnerships, combined with the low testing coverage, indicates strong potential for the spread of HIV. The different correlates of HIV in Port Moresby and Lae highlight the importance of conducting surveys in multiple locations and using data to develop locally appropriate interventions even within a country.

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.