Differentiating Dyes: A Spectroscopic Investigation into the Composition of Scarlet Bloodroot (Haemodorum coccineum R.Br.) Rhizome.

Haemodorum coccineum, commonly known as scarlet bloodroot, is a plant native to New Guinea and the northern most parts of Australia. The highly coloured H. coccineum is used by communities in Larrakia country for dyeing garments and occasionally to treat snake bites. Previous studies into H. coccineum have focused on its taxonomic classification, with this being the first evaluation of the chemical composition of the plant. Haemodoraceae plants are reported to contain phenylphenalenones (PhPs), which are highly conjugated polycyclic oxygenated aromatic hydrocarbons. We report the characterisation of 20 compounds extracted from the rhizome of H. coccineum: four sugars and 16 compounds belonging to the PhP family. The compounds include five aglycones and seven glycosylated compounds, of which four contain malonate esters in their structures. Characterisation of these compounds was achieved through 1D and 2D NMR, MS analysis and comparison to the known phytochemistry of other species from the Haemodorum genus. Preliminary anti-microbial activity of the crude extract shows significant inhibition of the growth of both gram-positive and gram-negative bacteria, but no activity against Candida albicans.

Implementation and operational feasibility of SAMBA I HIV-1 semi-quantitative viral load testing at the point-of-care in rural settings in Malawi and Uganda.

OBJECTIVE: We monitored a large-scale implementation of the Simple Amplification-Based Assay semi-quantitative viral load test for HIV-1 version I (SAMBA I Viral Load = SAMBA I VL) within Médecins Sans Frontières' HIV programmes in Malawi and Uganda, to assess its performance and operational feasibility. METHODS: Descriptive analysis of routine programme data between August 2013 and December 2016. The dataset included samples collected for VL monitoring and tested using SAMBA I VL in five HIV clinics in Malawi (four peripheral health centres and one district hospital), and one HIV clinic in a regional referral hospital in Uganda. SAMBA I VL was used for VL testing in patients who had been receiving ART for between 6 months and ten years, to determine whether plasma VL was above or below 1000 copies/mL of HIV-1, reflecting ART failure or efficacy. Randomly selected samples were quantified with commercial VL assays. SAMBA I instruments and test performance, site throughput, and delays in communicating results to clinicians and patients were monitored. RESULTS: Between August 2013 and December 2016 a total of 60 889 patient samples were analysed with SAMBA I VL. Overall, 0.23% of initial SAMBA I VL results were invalid; this was reduced to 0.04% after repeating the test once. Global test failure, including instrument failure, was 1.34%. Concordance with reference quantitative testing of VL was 2620/2727, 96.0% (1338/1382, 96.8% in Malawi; 1282/1345, 95.3% in Uganda). For Chiradzulu peripheral health centres and Arua Hospital HIV clinic, where testing was performed on-site, same-day results were communicated to clinicians for between 91% and 97% of samples. Same-day clinical review was obtained for 84.7% across the whole set of samples tested. CONCLUSIONS: SAMBA I VL testing is feasible for monitoring cohorts of 1000 to 5000 ART-experienced patients. Same-day results can be used to inform rapid clinical decision-making at rural and remote health facilities, potentially reducing time available for development of resistance and conceivably helping to reduce morbidity and mortality.

OBJECTIF: Nous avons suivi une mise en œuvre à grande échelle du test de la charge virale semi-quantitative du VIH -1 basé sur de Test de Simple Amplification version I (SAMBA I Viral Load = SAMBA I VL) au sein des programmes VIH de Médecins Sans Frontières au Malawi et en Ouganda, afin d'évaluer sa performance et sa faisabilité opérationnelle. MÉTHODES: Analyse descriptive des données du programme de routine entre août 2013 et décembre 2016. L'ensemble des données comprenait des échantillons collectés pour le suivi de la CV et testés à l'aide de SAMBA I VL dans cinq cliniques VIH au Malawi (quatre centres de santé périphériques et un hôpital de district), et une clinique VIH dans un hôpital régional de référence en Ouganda. SAMBA I VL a été utilisé pour le test de la CV chez les patients qui recevaient l'ART depuis 6 mois à dix ans, afin de déterminer si la CV plasmatique était supérieure ou inférieure à 1000 copies/ml de VIH-1, reflétant l'échec ou l'efficacité de l'ART. Des échantillons sélectionnés aléatoirement ont été quantifiés avec des tests de CV commerciaux. Les instruments de SAMBA I et les performances des tests, le débit du site et les délais dans la communication des résultats aux cliniciens et aux patients ont été suivis. RÉSULTATS: Entre août 2013 et décembre 2016, un total de 60.889 échantillons de patients ont été analysés avec SAMBA I VL. Dans l'ensemble, 0,23% des résultats initiaux de SAMBA I VL étaient invalides; ceux-ci ont été été réduits à 0,04% après avoir répété le test une fois. L'échec global du test, y compris l'échec de l'instrument, était de 1,34%. La concordance avec les tests quantitatifs de référence de la CV était de 2620/2727; 96,0% (1338/1382; 96,8% au Malawi; 1282/1345; 95,3% en Ouganda). Pour les centres de santé périphériques de Chiradzulu et la clinique VIH de l'hôpital d'Arua, où les tests ont été effectués sur place, les résultats ont été communiqués le jour même aux cliniciens pour entre 91% et 97% des échantillons. Un examen clinique le jour même a été obtenu pour 84,7% de l'ensemble des échantillons testés. CONCLUSIONS: Le test SAMBA I VL est réalisable pour le suivi de cohortes de 1.000 à 5.000 patients déjà sous ART. Les résultats le jour même peuvent être utilisés pour éclairer la prise de décision clinique rapide dans les établissements de santé ruraux et éloignés, réduisant potentiellement le temps disponible pour le développement de la résistance et contribuant éventuellement à réduire la morbidité et la mortalité.

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.

Developing a culturally appropriate illustrated tool for the self-collection of anorectal specimens for the testing of sexually transmitted infections: lessons from Papua New Guinea.

BACKGROUND: Papua New Guinea (PNG) has a high prevalence of sexually transmitted infections (STIs). There is increasing evidence that anorectal STIs are important in terms of the dual epidemics of HIV and STIs in this setting. At the time of this study, anorectal STI testing was not possible, and there was no mechanism for self-collection of anorectal specimen among at risk 'key populations'. This paper documents the development of a culturally appropriate tool that has been used to facilitate self-collection of anorectal specimens with key populations in PNG. METHODS: This qualitative study involved four focus groups conducted with a purposive sample of 35 participants, including female sex workers, men who have sex with men and transgender women in Port Moresby and Goroka in 2015. During focus groups, participants reviewed and provided critical feedback for the adaption of a previously piloted and published pictorial anorectal specimen collection tool for use with key populations in PNG. RESULTS: The final instruction tools are presented in English language and Tok Pisin. To develop these, participants feedback resulted in six key areas of the existing instruction document being modified to ensure it was appropriate for use in PNG. These included translating complex words for sexual health issues (i.e. 'STIs', 'anorectal STIs', 'anus', 'anal sex'), biomedical instruments (i.e. 'specimen bottle', 'specimen packet' and 'swab'), and aspects of the clinical procedure (i.e. inserting the swab 3-4 cm into the anus to collect a specimen). The visual identity of the graphics was redesigned to localise the images for use in PNG. CONCLUSIONS: This paper describes the development of a culturally and linguistically appropriate tool for a biomedical and clinical intervention with key populations in PNG based around self-collection of anorectal specimens for molecular STI testing. The final tools have been used to facilitate the self-collection of anorectal specimens following a clear clinical protocol during a large bio-behavioural survey in PNG.

Acceptability of testing for anorectal sexually transmitted infections and self-collected anal swabs in female sex workers, men who have sex with men and transgender women in Papua New Guinea.

BACKGROUND: Papua New Guinea (PNG) has some of the highest prevalence of urogenital sexually transmitted infections (STIs) in Pacific Asia, but to date, anorectal STI prevalence data do not exist, and diagnosis of anorectal STIs does not occur. The purpose of this study was to document the acceptability of anorectal STI testing and self-collection of anorectal swabs for testing among populations at risk of anorectal STIs, in advance of a large bio-behavioural survey during which this approach to specimen collection was planned among key populations in PNG. METHODS: Four focus groups were conducted, collecting data from a purposive sample of 35 members of two civil society groups representing female sex workers, men who have sex with men and transgender women in Port Moresby and Goroka. RESULTS: All participants were in favour of anorectal STI testing in PNG. Reasons given for willingness to undertake anorectal STI testing included that anal sex is practised; that anorectal STIs are not perceived to exist; there are self-reported experiences of anorectal symptoms indicative of anorectal STIs; that anorectal STI testing will enhance personal health; and that anorectal STI testing is not currently available in PNG. All participants were confident they could obtain self-collected specimens, although several stated that support from trained health workers should be available for community members who may not feel comfortable with self-collection. CONCLUSIONS: This qualitative research is the first study of acceptability of anorectal STI testing and specimen self-collection procedures in PNG, and Pacific Asia more broadly. Our qualitative findings show support for anorectal STI testing including the use of self-collected swabs among key populations in PNG. Study findings informed the inclusion of anorectal STI testing in a large bio-behavioural survey to be used to estimate anorectal STI prevalence among key populations in PNG for the first time.

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.

Sulphadoxine-pyrimethamine plus azithromycin for the prevention of low birthweight in Papua New Guinea: a randomised controlled trial.

BACKGROUND: Intermittent preventive treatment in pregnancy has not been evaluated outside of Africa. Low birthweight (LBW, <2,500 g) is common in Papua New Guinea (PNG) and contributing factors include malaria and reproductive tract infections. METHODS: From November 2009 to February 2013, we conducted a parallel group, randomised controlled trial in pregnant women (≤ 26 gestational weeks) in PNG. Sulphadoxine-pyrimethamine (1,500/75 mg) plus azithromycin (1 g twice daily for 2 days) (SPAZ) monthly from second trimester (intervention) was compared against sulphadoxine-pyrimethamine and chloroquine (450 to 600 mg, daily for three days) (SPCQ) given once, followed by SPCQ placebo (control). Women were assigned to treatment (1:1) using a randomisation sequence with block sizes of 32. Participants were blinded to assignments. The primary outcome was LBW. Analysis was by intention-to-treat. RESULTS: Of 2,793 women randomised, 2,021 (72.4%) were included in the primary outcome analysis (SPCQ: 1,008; SPAZ: 1,013). The prevalence of LBW was 15.1% (305/2,021). SPAZ reduced LBW (risk ratio [RR]: 0.74, 95% CI: 0.60-0.91, P = 0.005; absolute risk reduction (ARR): 4.5%, 95% CI: 1.4-7.6; number needed to treat: 22), and preterm delivery (0.62, 95% CI: 0.43-0.89, P = 0.010), and increased mean birthweight (41.9 g, 95% CI: 0.2-83.6, P = 0.049). SPAZ reduced maternal parasitaemia (RR: 0.57, 95% CI: 0.35-0.95, P = 0.029) and active placental malaria (0.68, 95% CI: 0.47-0.98, P = 0.037), and reduced carriage of gonorrhoea (0.66, 95% CI: 0.44-0.99, P = 0.041) at second visit. There were no treatment-related serious adverse events (SAEs), and the number of SAEs (intervention 13.1% [181/1,378], control 12.7% [174/1,374], P = 0.712) and AEs (intervention 10.5% [144/1,378], control 10.8% [149/1,374], P = 0.737) was similar. A major limitation of the study was the high loss to follow-up for birthweight. CONCLUSIONS: SPAZ was efficacious and safe in reducing LBW, possibly acting through multiple mechanisms including the effect on malaria and on sexually transmitted infections. The efficacy of SPAZ in the presence of resistant parasites and the contribution of AZ to bacterial antibiotic resistance require further study. The ability of SPAZ to improve pregnancy outcomes warrants further evaluation. TRIAL REGISTRATION: ClinicalTrials.gov NCT01136850 (06 April 2010).

Distinct patterns of diversity, population structure and evolution in the AMA1 genes of sympatric Plasmodium falciparum and Plasmodium vivax populations of Papua New Guinea from an area of similarly high transmission.

BACKGROUND: As Plasmodium falciparum and Plasmodium vivax co-exist in most malaria-endemic regions outside sub-Saharan Africa, malaria control strategies in these areas must target both species in order to succeed. Population genetic analyses can predict the effectiveness of interventions including vaccines, by providing insight into patterns of diversity and evolution. The aim of this study was to investigate the population genetics of leading malaria vaccine candidate AMA1 in sympatric P. falciparum and P. vivax populations of Papua New Guinea (PNG), an area of similarly high prevalence (Pf = 22.3 to 38.8%, Pv = 15.3 to 31.8%). METHODS: A total of 72 Pfama1 and 102 Pvama1 sequences were collected from two distinct areas, Madang and Wosera, on the highly endemic PNG north coast. RESULTS: Despite a greater number of polymorphic sites in the AMA1 genes of P. falciparum (Madang = 52; Wosera = 56) compared to P. vivax (Madang = 36, Wosera = 34), the number of AMA1 haplotypes, haplotype diversity (Hd) and recombination (R) was far lower for P. falciparum (Madang = 12, Wosera = 20; Hd ≤0.92, R ≤45.8) than for P. vivax (Madang = 50, Wosera = 38; Hd = 0.99, R = ≤70.9). Balancing selection was detected only within domain I of AMA1 for P. vivax, and in both domains I and III for P. falciparum. CONCLUSIONS: Higher diversity in the genes encoding P. vivax AMA1 than in P. falciparum AMA1 in this highly endemic area has important implications for development of AMA1-based vaccines in PNG and beyond. These results also suggest a smaller effective population size of P. falciparum compared to P. vivax, a finding that warrants further investigation. Differing patterns of selection on the AMA1 genes indicate that critical antigenic sites may differ between the species, highlighting the need for independent investigations of these two leading vaccine candidates.

Formulation and Biomedical Activity of Oil-in-Water Nanoemulsion Combining Tinospora smilacina Water Extract and Calophyllum inophyllum Seeds Oil.

Introduction: Tinospora smilacina is a native plant used in traditional medicine by First Nations peoples in Australia to treat inflammation. In our previous study, an optimised Calophyllum inophyllum seed oil (CSO) nanoemulsion (NE) showed improved biomedical activities such as antimicrobial, antioxidant activity, cell viability and in vitro wound healing efficacy compared to CSO. Methods: In this study, a stable NE formulation combining T. smilacina water extract (TSWE) and CSO in a nanoemulsion (CTNE) was prepared to integrate the bioactive compounds in both native plants and improve wound healing efficacy. D-optimal mixture design was used to optimise the physicochemical characteristics of the CTNE, including droplet size and polydispersity index (PDI). Cell viability and in vitro wound healing studies were done in the presence of CTNE, TSWE and CSO against a clone of baby hamster kidney fibroblasts (BHK-21 cell clone BSR-T7/5). Results: The optimised CTNE had a 24 ± 5 nm particle size and 0.21± 0.02 PDI value and was stable after four weeks each at 4 °C and room temperature. According to the results, incorporating TSWE into CTNE improved its antioxidant activity, cell viability, and ability to promote wound healing. The study also revealed that TSWE has >6% higher antioxidant activity than CSO. While CTNE did not significantly impact mammalian cell viability, it exhibited wound-healing properties in the BSR cell line during in vitro testing. These findings suggest that adding TSWE may enhance CTNE's potential as a wound-healing treatment. Conclusion: This is the first study demonstrating NE formulation in which two different plant extracts were used in the aqueous and oil phases with improved biomedical activities.

Seroprevalence and prevention of hepatitis B, measles and rubella among healthcare workers in Dili, Timor-Leste.

Introduction: The World Health Organisation recommends that healthcare workers (HCWs) are immune to measles and rubella, and those at risk of exposure are offered the hepatitis B vaccine. No formal programme for occupational assessment and provision of vaccinations to HCWs currently exists in Timor-Leste. Methods: A cross-sectional study was undertaken to determine the seroprevalence of hepatitis B, measles and rubella among HCWs in Dili, Timor-Leste. All patient-facing employees at three healthcare institutions during April-June 2021 were invited to participate. Epidemiological data were collected by interview-questionnaire and a serum sample was collected by phlebotomy and analysed at the National Health Laboratory. Participants were contacted to discuss their results. Relevant vaccines were offered to seronegative individuals and those with active hepatitis B infection were referred for further assessment and management in a hepatology clinic as per national guidelines. Results: Three-hundred-and-twenty-four HCWs were included (representing 51.3% of all eligible HCWs working at the three participating institutions). Sixteen (4.9%; 95% CI: 2.8-7.9%) had active hepatitis B infection, 121 (37.3%; 95% CI: 32.1-42.9%) had evidence of previous (cleared) hepatitis B infection, 134 (41.4%; 95% CI: 35.9-46.9%) were hepatitis B seronegative, and 53 (16.4%; 95% CI: 12.5-20.8%) had been vaccinated. Two-hundred-and-sixty-seven (82.4%; 95% CI: 77.8-86.4%) and 306 (94.4%; 95% CI: 91.4-96.7%) individuals exhibited antibodies to measles and rubella, respectively. Interpretation: There are significant immunity gaps and a high prevalence of hepatitis B infection among HCWs in Dili Municipality, Timor-Leste. Routine occupational assessment and targeted vaccination of this group would be beneficial and should include all types of HCWs. This study provided an opportunity to develop a programme for the occupational assessment and vaccination of HCWs and forms the template for a national guideline. Funding: This work was supported by the Department of Foreign Affairs and Trade, Australian Government [Complex Grant Agreement Number 75889].

The COVID-19 laboratory response in Timor-Leste; a story of collaboration.

Timor-Leste is a small nation of 1.3 million people which shares a land border with Indonesia and is 550 km from Darwin, Australia. It is one of the poorest nations in Asia. The National Health Laboratory (NHL) and its network of smaller laboratories in Timor-Leste had limited capacity to perform molecular diagnostic testing before the coronavirus disease 2019 (COVID-19) pandemic began. With the support of international development partners, the NHL rapidly expanded its molecular testing service. From March 2020 to February 2022, over 200,000 molecular tests were performed; COVID-19 testing sites were established in hospital and community health center laboratories and all 13 municipalities, and the number of scientists and technicians at the molecular diagnostic laboratory at the NHL increased from five to 28 between 2019 and 2022. Molecular diagnostic testing for COVID-19 was successfully established at the NHL and in the municipalities. The molecular diagnostic laboratory at NHL is now equipped to respond to not only large-scale COVID-19 testing but also laboratory detection of other infectious diseases, preparing Timor-Leste for future outbreaks or pandemics.

Optimisation of Calophyllum inophyllum seed oil nanoemulsion as a potential wound healing agent.

BACKGROUND: Efficient delivery systems of Calophyllum inophyllum seed oil (CSO) in the form of nanoemulsion were optimised to enhance its stability and ensure its therapeutic efficiency as a potential agent for various biomedical applications. METHOD: Response Surface Methodology (RSM) was used to determine the effects of independent variables (oil, surfactant, water percentage and homogenisation time) on physicochemical characteristics, including droplet size, polydispersity index and turbidity. RESULTS: The optimised CSO nanoemulsion (CSONE) has a 46.68 nm particle size, 0.15 Polydispersity index value and 1.16 turbidity. After 4 weeks of storage at 5 ± 1 °C and 25 ± 1 °C, the CSONE was physically stable. The optimised CSO nanoemulsion showed enhancement in cell viability and wound healing in baby hamster kidney a clone BHK-21 (BSR) cells as compared to the CSO. The wound healing property of CSONE was higher than CSO. CONCLUSION: Thus, our in vitro wound healing results demonstrated that CSO in the nanoemulsion form can promote wound healing by enhancing the proliferation and migration of epidermal cells. The coarse emulsion of Calophyllum inophyllum seed oil nano emulsion was prepared using high shear homogeniser techniques. The optimised CSONE with the droplet size of 46.68 nm was prepared from a mixture of CSO, Tween 80, and high pure water (HPW), then used for the biological investigation. The in vitro cell monolayer scratch assay revealed that CSONE in the lowest concentration of CSO resulted in 100% wound closure after 48 hrs. The optimised CSO nanoemulsion was found to be a promising and effective approach in the treatment of wounds by boosting the proliferation and migration of epidermal cells.

Sexual and reproductive health needs and practices of female sex workers in Papua New Guinea: findings from a biobehavioral survey Kauntim mi tu ('Count me too').

BACKGROUND: Little research has explored the sexual and reproductive health (SRH) experience of female sex workers (FSW), including girls aged < 18 years who are commercially sexually exploited (CSE), in Papua New Guinea (PNG). This paper describes the SRH history of FSW and CSE girls and factors associated with their use of moderately or highly effective contraceptive methods in three settings in PNG. METHODS: From 2016 to 2017, respondent-driven sampling (RDS) surveys were conducted among FSW and CSE girls in Port Moresby, Lae, and Mt. Hagen. FSW and CSE girls who were born female, aged ≥12 years, sold or exchanged vaginal sex in the past 6 months, spoke English or Tok Pisin, and had a valid RDS study coupon were eligible to participate. Interviews were conducted face-to-face and participants were offered rapid routine HIV and syphilis testing. Survey logistic regression procedures were used to identify factors associated with the use of moderately or highly effective contraceptive methods. Weighted data analysis was conducted. RESULTS: A total of 2901 FSW and CSE girls (Port Moresby, 673; Lae, 709; and Mt. Hagen, 709) were enrolled. The proportion using moderately or highly effective contraceptive methods was 37.7% in Port Moresby, 30.9% in Lae, and 26.5% in Mt. Hagen. After adjusting for covariates, factors significantly associated with the use of moderately or highly effective contraceptive methods in Port Moresby were being age 20-24, being married, being divorced or separated, having one or more dependent children, being away from home for more than 1 month in the last 6 months, and having tested HIV negative. No factors were significantly associated in Lae or Mt. Hagen. ANC attendance amongst FSW and CSE girls who gave birth in last 3 years was highest in Port Moresby at 91.2%. HIV testing was inconsistently and inadequately offered at ANC across the three cities. CONCLUSIONS: Kauntim mi tu provides much-needed insight into the SRH experiences of FSW and CSE girls in PNG, where their use of moderately or highly effective contraceptive methods is low. We hope to shed light on the complicated reality they face due to illegality of sex work and multitude of complex healthcare experiences.

Integrated serological surveillance of acute febrile illness in the context of a lymphatic filariasis survey in Timor-Leste: a pilot study using dried blood spots.

BACKGROUND: Acute febrile illnesses (AFIs), including dengue, scrub typhus and leptospirosis, cause significant morbidity and mortality in Southeast Asia. Serological surveillance can be used to investigate the force and distribution of infections. Dried blood spot (DBS) samples are an attractive alternative to serum because they are easier to collect and transport and require less cold storage. We conducted a pilot study to determine the feasibility of integrating serological surveillance for dengue, scrub typhus and leptospirosis into a population-representative lymphatic filariasis seroprevalence survey in Timor-Leste using DBSs. METHODS: A total of 272 DBSs were collected from healthy community participants. DBSs were analysed at the National Health Laboratory using commercially available enzyme-linked immunosorbent assays. To validate assays for DBSs, 20 anonymised serum samples of unknown serostatus were used to create dried serum spots (DSSs). These were analysed with optical densities compared with those of serum. Where low variance was observed (dengue assay) the published kit cut-offs for serum were applied to the analysis of DBSs. For the other assays (scrub typhus and leptospirosis), index values (IVs) were calculated and cut-offs were determined to be at 2 standard deviations (SDs) above the mean. RESULTS: Of the 272 samples analysed, 19 (7.0% [95% confidence interval {CI} 4.3 to 10.7]) were positive for dengue immunoglobulin G (IgG), 11 (4.0% [95% CI 2.1 to 7.1]) were positive for scrub typhus IgG and 16 (5.9% [95% CI 3.4 to 9.4%]) were positive for leptospira IgG. CONCLUSIONS: While dengue seroprevalence was lower than in nearby countries, results represent the first evidence of scrub typhus and leptospirosis transmission in Timor-Leste. Integrated programmes of serological surveillance could greatly improve our understanding of infectious disease epidemiology in remote areas and would incur minimal additional fieldwork costs. However, when planning such studies, the choice of assays, their validation for DBSs and the laboratory infrastructure and technical expertise at the proposed location of analysis must be considered.

Serological surveillance of healthcare workers to evaluate natural infection- and vaccine-derived immunity to SARS-CoV-2 during an outbreak in Dili, Timor-Leste.

Background Serosurveillance can be used to investigate the extent and distribution of immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within a population. Characterisation of humoral immune responses gives insight into whether immunity is infection- or vaccine-derived. Methods A longitudinal study of health care workers (HCWs) in Dili, Timor-Leste, was conducted during vaccine rollout (ChAdOx1) and a concurrent SARS-CoV-2 outbreak. Results A total of 324 HCWs were included at baseline (April-May 2021). Out of those, 32 (9.9%) were seropositive for anti-nucleocapsid protein (anti-N) IgG antibodies, indicating a significant sub-clinical infection among HCWs early in the local outbreak. Follow-up was conducted in 157 (48.5%) participants (July-September 2021), by which time there had been high uptake of vaccination (91.7%), and 86.0% were seropositive for anti-spike protein antibodies. Acquisition of anti-N antibodies was observed in partially vaccinated HCWs (30/76, 39.5%), indicating some post-dose-1 infections. Discussion Serosurveillance of HCWs may provide early warning of SARS-CoV-2 outbreaks and should be considered in non-endemic settings, particularly where there is limited availability/uptake of testing for acute infection. Characterisation of humoral immune responses may be used to assess vaccine impact and coverage. Such studies should be considered in national and international efforts to investigate and mitigate against future emerging pathogens.

Population genetic analysis of the Plasmodium falciparum 6-cys protein Pf38 in Papua New Guinea reveals domain-specific balancing selection.

BACKGROUND: The Plasmodium falciparum merozoite surface protein Pf38 is targeted by antibodies of malaria immune adults and has been shown to be under balancing (immune) selection in a Gambian parasite population, indicating potential as a malaria vaccine candidate. This study explores the population genetics of Pf38 in Papua New Guinea, to determine the extent and geographic distribution of diversity and to measure selective pressure along the length of the gene. METHODS: Using samples collected during community-based cross-sectional surveys in the Mugil and Wosera regions, the Pf38 genes of 59 P. falciparum isolates were amplified and sequenced. These sequences, along with previously sequenced Gambian and laboratory isolates, were then subjected to an array of population genetic analyses, examining polymorphisms, haplotype diversity and balancing selection. In addition to whole-gene analysis, the two 6-cys domains were considered separately, to investigate domain specific polymorphism and selection. RESULTS: Nineteen polymorphic sites were identified in the Pf 38 gene. Of these, 13 were found in the Gambia, 10 in Mugil and 8 in Wosera. Notably, the majority of common polymorphisms were confined to domain I. Although only moderate levels of nucleotide diversity were observed, the haplotype diversity was high in all populations, suggesting extensive recombination. Analyses of the full-length sequence provided only modest evidence for balancing selection. However, there was a strong contrast between domain I, which showed strong evidence for positive balancing selection, and domain II which was neutral. Analyses of the geographic distribution of Pf38 haplotypes showed that four haplotypes accounted for the majority of sequences found world-wide, but there were many more haplotypes unique to the African than the PNG populations. CONCLUSION: This study confirmed previous findings that Pf38 is a polymorphic gene under balancing selection. However, analysing polymorphism and selection across the length of the gene painted a considerably different picture. Domain I is highly polymorphic and the target of significant balancing selection. In contrast, domain II is relatively conserved and does not show evidence of immune selective pressure. The findings have implications for future population genetic studies on vaccine candidates, showing that the biological context must also be considered as a framework for analysis.

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.

Multilocus haplotypes reveal variable levels of diversity and population structure of Plasmodium falciparum in Papua New Guinea, a region of intense perennial transmission.

BACKGROUND: The South West Pacific nation of Papua New Guinea has intense year round transmission of Plasmodium falciparum on the coast and in the low-lying inland areas. Local heterogeneity in the epidemiology of malaria suggests that parasites from multiple locations will need to be surveyed to define the population biology of P. falciparum in the region. This study describes the population genetics of P. falciparum in thirteen villages spread over four distinct catchment areas of Papua New Guinea. METHODS: Ten microsatellite loci were genotyped in 318 P. falciparum isolates from the parasite populations of two inland catchment areas, namely Wosera (number of villages (n) = 7) and Utu (n = 1) and; and two coastal catchments, Malala (n = 3) and Mugil (n = 3). Analysis of the resultant multilocus haplotypes was done at different spatial scales (2-336 km) to define the genetic diversity (allelic richness and expected heterozygosity), linkage disequilibrium and population structure throughout the study area. RESULTS: Although genetic diversity was high in all parasite populations, it was also variable with a lower allelic richness and expected heterozygosity for inland populations compared to those from the more accessible coast. This variability was not correlated with two proxy measures of transmission intensity, the infection prevalence and the proportion multiple infections. Random associations among the microsatellite loci were observed in all four catchments showing that a substantial degree of out-crossing occurs in the region. Moderate to very high levels of population structure were found but the amount of genetic differentiation (FST) did not correlate with geographic distance suggesting that parasite populations are fragmented. Population structure was also identified between villages within the Malala area, with the haplotypes of one parasite population clustering with the neighbouring catchment of Mugil. CONCLUSION: The observed population genetics of P. falciparum in this region is likely to be a consequence of the high transmission intensity combined with the isolation of human and vector populations, especially those located inland and migration of parasites via human movement into coastal populations. The variable genetic diversity and population structure of P. falciparum has important implications for malaria control strategies and warrants further fine scale sampling throughout Papua New Guinea.

Point-of-care viral load monitoring: outcomes from a decentralized HIV programme in Malawi.

INTRODUCTION: Routinely monitoring the HIV viral load (VL) of people living with HIV (PLHIV) on anti-retroviral therapy (ART) facilitates intensive adherence counselling and faster ART regimen switch when treatment failure is indicated. Yet standard VL-testing in centralized laboratories can be time-intensive and logistically difficult in low-resource settings. This paper evaluates the outcomes of the first four years of routine VL-monitoring using Point-of-Care technology, implemented by Médecins Sans Frontières (MSF) in rural clinics in Malawi. METHODS: We conducted a retrospective cohort analysis of patients eligible for routine VL- testing between 2013 and 2017 in four decentralized ART-clinics and the district hospital in Chiradzulu, Malawi. We assessed VL-testing coverage and the treatment failure cascade (from suspected failure (first VL>1000 copies/mL) to VL suppression post regimen switch). We used descriptive statistics and multivariate logistic regression to assess factors associated with suspected failure. RESULTS AND DISCUSSION: Among 21,400 eligible patients, VL-testing coverage was 85% and VL suppression was found in 89% of those tested. In the decentralized clinics, 88% of test results were reviewed on the same day as blood collection, whereas in the district hospital the median turnaround-time for results was 85 days. Among first-line ART patients with suspected failure (N = 1544), 30% suppressed (VL<1000 copies/mL), 35% were treatment failures (confirmed by subsequent VL-testing) and 35% had incomplete VL follow-up. Among treatment failures, 80% (N = 540) were switched to a second-line regimen, with a higher switching rate in the decentralized clinics than in the district hospital (86% vs. 67%, p < 0.01) and a shorter median time-to-switch (6.8 months vs. 9.7 months, p < 0.01). Similarly, the post-switch VL-testing rate was markedly higher in the decentralized clinics (61% vs. 26%, p < 0.01). Overall, 79% of patients with a post-switch VL-test were suppressed. CONCLUSIONS: Viral load testing at the point-of-care in Chiradzulu, Malawi achieved high coverage and good drug regimen switch rates among those identified as treatment failures. In decentralized clinics, same-day test results and shorter time-to-switch illustrated the game-changing potential of POC-based VL-testing. Nevertheless, gaps were identified along all steps of the failure cascade. Regular staff training, continuous monitoring and creating demand are essential to the success of routine VL-testing.

Wave 2 strains of atypical Vibrio cholerae El Tor caused the 2009-2011 cholera outbreak in Papua New Guinea.

Vibrio cholerae is the causative agent of cholera, a globally important human disease for at least 200 years. In 2009-2011, the first recorded cholera outbreak in Papua New Guinea (PNG) occurred. We conducted genetic and phenotypic characterization of 21 isolates of V. cholerae, with whole-genome sequencing conducted on 2 representative isolates. The PNG outbreak was caused by an atypical El Tor strain harbouring a tandem repeat of the CTX prophage on chromosome II. Whole-genome sequence data, prophage structural analysis and the absence of the SXT integrative conjugative element was indicative that the PNG isolates were most closely related to strains previously isolated in South-East and East Asia with affiliations to global wave 2 strains. This finding suggests that the cholera outbreak in PNG was caused by an exotic (non-endemic) strain of V. cholerae that originated in South-East Asia.