Development of amplicon deep sequencing markers and data analysis pipeline for genotyping multi-clonal malaria infections.

BACKGROUND: Amplicon deep sequencing permits sensitive detection of minority clones and improves discriminatory power for genotyping multi-clone Plasmodium falciparum infections. New amplicon sequencing and data analysis protocols are needed for genotyping in epidemiological studies and drug efficacy trials of P. falciparum. METHODS: Targeted sequencing of molecular marker csp and novel marker cpmp was conducted in duplicate on mixtures of parasite culture strains and 37 field samples. A protocol allowing to multiplex up to 384 samples in a single sequencing run was applied. Software "HaplotypR" was developed for data analysis. RESULTS: Cpmp was highly diverse (He = 0.96) in contrast to csp (He = 0.57). Minority clones were robustly detected if their frequency was >1%. False haplotype calls owing to sequencing errors were observed below that threshold. CONCLUSIONS: To reliably detect haplotypes at very low frequencies, experiments are best performed in duplicate and should aim for coverage of >10'000 reads/amplicon. When compared to length polymorphic marker msp2, highly multiplexed amplicon sequencing displayed greater sensitivity in detecting minority clones.

Synergistic effect of IL-12 and IL-18 induces TIM3 regulation of γδ T cell function and decreases the risk of clinical malaria in children living in Papua New Guinea.

BACKGROUND: γδ T cells are important for both protective immunity and immunopathogenesis during malaria infection. However, the immunological processes determining beneficial or detrimental effects on disease outcome remain elusive. The aim of this study was to examine expression and regulatory effect of the inhibitory receptor T-cell immunoglobulin domain and mucin domain 3 (TIM3) on γδ T cells. While TIM3 expression and function on conventional αß T cells have been clearly defined, the equivalent characterization on γδ T cells and associations with disease outcomes is limited. This study investigated the functional capacity of TIM3+ γδ T cells and the underlying mechanisms contributing to TIM3 upregulation and established an association with malaria disease outcomes. METHODS: We analyzed TIM3 expression on γδ T cells in 132 children aged 5-10 years living in malaria endemic areas of Papua New Guinea. TIM3 upregulation and effector functions of TIM3+ γδ T cells were assessed following in vitro stimulation with parasite-infected erythrocytes, phosphoantigen and/or cytokines. Associations between the proportion of TIM3-expressing cells and the molecular force of infection were tested using negative binomial regression and in a Cox proportional hazards model for time to first clinical episode. Multivariable analyses to determine the association of TIM3 and IL-18 levels were conducted using general linear models. Malaria infection mouse models were utilized to experimentally investigate the relationship between repeated exposure and TIM3 upregulation. RESULTS: This study demonstrates that even in the absence of an active malaria infection, children of malaria endemic areas have an atypical population of TIM3-expressing γδ T cells (mean frequency TIM3+ of total γδ T cells 15.2% ± 12). Crucial factors required for γδ T cell TIM3 upregulation include IL-12/IL-18, and plasma IL-18 was associated with TIM3 expression (P = 0.002). Additionally, we show a relationship between TIM3 expression and infection with distinct parasite clones during repeated exposure. TIM3+ γδ T cells were functionally impaired and were associated with asymptomatic malaria infection (hazard ratio 0.54, P = 0.032). CONCLUSIONS: Collectively our data demonstrate a novel role for IL-12/IL-18 in shaping the innate immune response and provide fundamental insight into aspects of γδ T cell immunoregulation. Furthermore, we show that TIM3 represents an important γδ T cell regulatory component involved in minimizing malaria symptoms.

Strategies for understanding and reducing the Plasmodium vivax and Plasmodium ovale hypnozoite reservoir in Papua New Guinean children: a randomised placebo-controlled trial and mathematical model.

BACKGROUND: The undetectable hypnozoite reservoir for relapsing Plasmodium vivax and P. ovale malarias presents a major challenge for malaria control and elimination in endemic countries. This study aims to directly determine the contribution of relapses to the burden of P. vivax and P. ovale infection, illness, and transmission in Papua New Guinean children. METHODS AND FINDINGS: From 17 August 2009 to 20 May 2010, 524 children aged 5-10 y from East Sepik Province in Papua New Guinea (PNG) participated in a randomised double-blind placebo-controlled trial of blood- plus liver-stage drugs (chloroquine [CQ], 3 d; artemether-lumefantrine [AL], 3 d; and primaquine [PQ], 20 d, 10 mg/kg total dose) (261 children) or blood-stage drugs only (CQ, 3 d; AL, 3 d; and placebo [PL], 20 d) (263 children). Participants, study staff, and investigators were blinded to the treatment allocation. Twenty children were excluded during the treatment phase (PQ arm: 14, PL arm: 6), and 504 were followed actively for 9 mo. During the follow-up time, 18 children (PQ arm: 7, PL arm: 11) were lost to follow-up. Main primary and secondary outcome measures were time to first P. vivax infection (by qPCR), time to first clinical episode, force of infection, gametocyte positivity, and time to first P. ovale infection (by PCR). A basic stochastic transmission model was developed to estimate the potential effect of mass drug administration (MDA) for the prevention of recurrent P. vivax infections. Targeting hypnozoites through PQ treatment reduced the risk of having at least one qPCR-detectable P. vivax or P. ovale infection during 8 mo of follow-up (P. vivax: PQ arm 0.63/y versus PL arm 2.62/y, HR = 0.18 [95% CI 0.14, 0.25], p < 0.001; P. ovale: 0.06 versus 0.14, HR = 0.31 [95% CI 0.13, 0.77], p = 0.011) and the risk of having at least one clinical P. vivax episode (HR = 0.25 [95% CI 0.11, 0.61], p = 0.002). PQ also reduced the molecular force of P. vivax blood-stage infection in the first 3 mo of follow-up (PQ arm 1.90/y versus PL arm 7.75/y, incidence rate ratio [IRR] = 0.21 [95% CI 0.15, 0.28], p < 0.001). Children who received PQ were less likely to carry P. vivax gametocytes (IRR = 0.27 [95% CI 0.19, 0.38], p < 0.001). PQ had a comparable effect irrespective of the presence of P. vivax blood-stage infection at the time of treatment (p = 0.14). Modelling revealed that mass screening and treatment with highly sensitive quantitative real-time PCR, or MDA with blood-stage treatment alone, would have only a transient effect on P. vivax transmission levels, while MDA that includes liver-stage treatment is predicted to be a highly effective strategy for P. vivax elimination. The inclusion of a directly observed 20-d treatment regime maximises the efficiency of hypnozoite clearance but limits the generalisability of results to real-world MDA programmes. CONCLUSIONS: These results suggest that relapses cause approximately four of every five P. vivax infections and at least three of every five P. ovale infections in PNG children and are important in sustaining transmission. MDA campaigns combining blood- and liver-stage treatment are predicted to be a highly efficacious intervention for reducing P. vivax and P. ovale transmission. TRIAL REGISTRATION: ClinicalTrials.gov NCT02143934.

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).

Temporal changes in Plasmodium falciparum anti-malarial drug sensitivity in vitro and resistance-associated genetic mutations in isolates from Papua New Guinea.

BACKGROUND: In northern Papua New Guinea (PNG), most Plasmodium falciparum isolates proved resistant to chloroquine (CQ) in vitro between 2005 and 2007, and there was near-fixation of pfcrt K76T, pfdhfr C59R/S108N and pfmdr1 N86Y. To determine whether the subsequent introduction of artemisinin combination therapy (ACT) and reduced CQ-sulphadoxine-pyrimethamine pressure had attenuated parasite drug susceptibility and resistance-associated mutations, these parameters were re-assessed between 2011 and 2013. METHODS: A validated fluorescence-based assay was used to assess growth inhibition of 52 P. falciparum isolates from children in a clinical trial in Madang Province. Responses to CQ, lumefantrine, piperaquine, naphthoquine, pyronaridine, artesunate, dihydroartemisinin, artemether were assessed. Molecular resistance markers were detected using a multiplex PCR ligase detection reaction fluorescent microsphere assay. RESULTS: CQ resistance (in vitro concentration required for 50% parasite growth inhibition (IC50) >100 nM) was present in 19% of isolates. All piperaquine and naphthoquine IC50s were <100 nM and those for lumefantrine, pyronaridine and the artemisinin derivatives were in low nM ranges. Factor analysis of IC50s showed three groupings (lumefantrine; CQ, piperaquine, naphthoquine; pyronaridine, dihydroartemisinin, artemether, artesunate). Most isolates (96%) were monoclonal pfcrt K76T (SVMNT) mutants and most (86%) contained pfmdr1 N86Y (YYSND). No wild-type pfdhfr was found but most isolates contained wild-type (SAKAA) pfdhps. Compared with 2005-2007, the geometric mean (95% CI) CQ IC50 was lower (87 (71-107) vs 167 (141-197) nM) and there had been no change in the prevalence of pfcrt K76T or pfmdr1 mutations. There were fewer isolates of the pfdhps (SAKAA) wild-type (60 vs 100%) and pfdhfr mutations persisted. CONCLUSIONS: Reflecting less drug pressure, in vitro CQ sensitivity appears to be improving in Madang Province despite continued near-fixation of pfcrt K76T and pfmdr1 mutations. Temporal changes in IC50s for other anti-malarial drugs were inconsistent but susceptibility was preserved. Retention or increases in pfdhfr and pfdhps mutations reflect continued use of sulphadoxine-pyrimethamine in the study area including through paediatric intermittent preventive treatment. The susceptibility of local isolates to lumefantrine may be unrelated to those of other ACT partner drugs. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry ACTRN12610000913077 .

Artemisinin-naphthoquine versus artemether-lumefantrine for uncomplicated malaria in Papua New Guinean children: an open-label randomized trial.

BACKGROUND: Artemisinin combination therapies (ACTs) with broad efficacy are needed where multiple Plasmodium species are transmitted, especially in children, who bear the brunt of infection in endemic areas. In Papua New Guinea (PNG), artemether-lumefantrine is the first-line treatment for uncomplicated malaria, but it has limited efficacy against P. vivax. Artemisinin-naphthoquine should have greater activity in vivax malaria because the elimination of naphthoquine is slower than that of lumefantrine. In this study, the efficacy, tolerability, and safety of these ACTs were assessed in PNG children aged 0.5-5 y. METHODS AND FINDINGS: An open-label, randomized, parallel-group trial of artemether-lumefantrine (six doses over 3 d) and artemisinin-naphthoquine (three daily doses) was conducted between 28 March 2011 and 22 April 2013. Parasitologic outcomes were assessed without knowledge of treatment allocation. Primary endpoints were the 42-d P. falciparum PCR-corrected adequate clinical and parasitologic response (ACPR) and the P. vivax PCR-uncorrected 42-d ACPR. Non-inferiority and superiority designs were used for falciparum and vivax malaria, respectively. Because the artemisinin-naphthoquine regimen involved three doses rather than the manufacturer-specified single dose, the first 188 children underwent detailed safety monitoring. Of 2,542 febrile children screened, 267 were randomized, and 186 with falciparum and 47 with vivax malaria completed the 42-d follow-up. Both ACTs were safe and well tolerated. P. falciparum ACPRs were 97.8% and 100.0% in artemether-lumefantrine and artemisinin-naphthoquine-treated patients, respectively (difference 2.2% [95% CI -3.0% to 8.4%] versus -5.0% non-inferiority margin, p = 0.24), and P. vivax ACPRs were 30.0% and 100.0%, respectively (difference 70.0% [95% CI 40.9%-87.2%], p<0.001). Limitations included the exclusion of 11% of randomized patients with sub-threshold parasitemias on confirmatory microscopy and direct observation of only morning artemether-lumefantrine dosing. CONCLUSIONS: Artemisinin-naphthoquine is non-inferior to artemether-lumefantrine in PNG children with falciparum malaria but has greater efficacy against vivax malaria, findings with implications in similar geo-epidemiologic settings within and beyond Oceania. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry ACTRN12610000913077. Please see later in the article for the Editors' Summary.

Pharmacokinetic properties of single-dose primaquine in Papua New Guinean children: feasibility of abbreviated high-dose regimens for radical cure of vivax malaria.

Since conventional 14-day primaquine (PMQ) radical cure of vivax malaria is associated with poor compliance, and as total dose, not therapy duration, determines efficacy, a preliminary pharmacokinetic study of two doses (0.5 and 1.0 mg/kg of body weight) was conducted in 28 healthy glucose-6-phosphate dehydrogenase-normal Papua New Guinean children, aged 5 to 12 years, to facilitate development of abbreviated high-dose regimens. Dosing was with food and was directly observed, and venous blood samples were drawn during a 168-h postdose period. Detailed safety monitoring was performed for hepatorenal function and hemoglobin and methemoglobin concentrations. Plasma concentrations of PMQ and its metabolite carboxyprimaquine (CPMQ) were determined by liquid chromatography-mass spectrometry and analyzed using population pharmacokinetic methods. The derived models were used in simulations. Both single-dose regimens were well tolerated with no changes in safety parameters. The mean PMQ central volume of distribution and clearance relative to bioavailability (200 liters/70 kg and 24.6 liters/h/70 kg) were within published ranges for adults. The median predicted maximal concentrations (Cmax) for both PMQ and CPMQ after the last dose of a 1.0 mg/kg 7-day PMQ regimen were approximately double those at the end of 14 days of 0.5 mg/kg daily, while a regimen of 1.0 mg/kg twice daily resulted in a 2.38 and 3.33 times higher Cmax for PMQ and CPMQ, respectively. All predicted median Cmax concentrations were within ranges for adult high-dose studies that also showed acceptable safety and tolerability. The present pharmacokinetic data, the first for PMQ in children, show that further studies of abbreviated high-dose regimens are feasible in this age group.

Comparison of an assumed versus measured leucocyte count in parasite density calculations in Papua New Guinean children with uncomplicated malaria.

BACKGROUND: The accuracy of the World Health Organization method of estimating malaria parasite density from thick blood smears by assuming a white blood cell (WBC) count of 8,000/µL has been questioned in several studies. Since epidemiological investigations, anti-malarial efficacy trials and routine laboratory reporting in Papua New Guinea (PNG) have all relied on this approach, its validity was assessed as part of a trial of artemisinin-based combination therapy, which included blood smear microscopy and automated measurement of leucocyte densities on Days 0, 3 and 7. RESULTS: 168 children with uncomplicated malaria (median (inter-quartile range) age 44 (39-47) months) were enrolled, 80.3% with Plasmodium falciparum monoinfection, 14.9% with Plasmodium vivax monoinfection, and 4.8% with mixed P. falciparum/P. vivax infection. All responded to allocated therapy and none had a malaria-positive slide on Day 3. Consistent with a median baseline WBC density of 7.3 (6.5-7.8) × 10(9)/L, there was no significant difference in baseline parasite density between the two methods regardless of Plasmodium species. Bland Altman plots showed that, for both species, the mean difference between paired parasite densities calculated from assumed and measured WBC densities was close to zero. At parasite densities <10,000/µL by measured WBC, almost all between-method differences were within the 95% limits of agreement. Above this range, there was increasing scatter but no systematic bias. CONCLUSIONS: Diagnostic thresholds and parasite clearance assessment in most PNG children with uncomplicated malaria are relatively robust, but accurate estimates of a higher parasitaemia, as a prognostic index, requires formal WBC measurement.

Comparison of three methods for detection of gametocytes in Melanesian children treated for uncomplicated malaria.

BACKGROUND: Gametocytes are the transmission stages of Plasmodium parasites, the causative agents of malaria. As their density in the human host is typically low, they are often undetected by conventional light microscopy. Furthermore, application of RNA-based molecular detection methods for gametocyte detection remains challenging in remote field settings. In the present study, a detailed comparison of three methods, namely light microscopy, magnetic fractionation and reverse transcriptase polymerase chain reaction for detection of Plasmodium falciparum and Plasmodium vivax gametocytes was conducted. METHODS: Peripheral blood samples from 70 children aged 0.5 to five years with uncomplicated malaria who were treated with either artemether-lumefantrine or artemisinin-naphthoquine were collected from two health facilities on the north coast of Papua New Guinea. The samples were taken prior to treatment (day 0) and at pre-specified intervals during follow-up. Gametocytes were measured in each sample by three methods: i) light microscopy (LM), ii) quantitative magnetic fractionation (MF) and, iii) reverse transcriptase PCR (RTPCR). Data were analysed using censored linear regression and Bland and Altman techniques. RESULTS: MF and RTPCR were similarly sensitive and specific, and both were superior to LM. Overall, there were approximately 20% gametocyte-positive samples by LM, whereas gametocyte positivity by MF and RTPCR were both more than two-fold this level. In the subset of samples collected prior to treatment, 29% of children were positive by LM, and 85% were gametocyte positive by MF and RTPCR, respectively. CONCLUSIONS: The present study represents the first direct comparison of standard LM, MF and RTPCR for gametocyte detection in field isolates. It provides strong evidence that MF is superior to LM and can be used to detect gametocytaemic patients under field conditions with similar sensitivity and specificity as RTPCR.

Viral pathogens in children hospitalized with features of central nervous system infection in a malaria-endemic region of Papua New Guinea.

BACKGROUND: Viral central nervous system (CNS) infections are common in countries where malaria is endemic but, due to limited laboratory facilities, few studies have systematically examined the prevalence and clinical consequences of the presence of viruses in cerebrospinal fluid (CSF) from children with suspected CNS infection. METHODS: We performed a prospective study of Papua New Guinean children hospitalized with signs and symptoms of CNS infection. CSF samples from 300 children without proven bacterial/fungal meningitis were analyzed for human herpes viruses (HHV), picornaviruses, influenza, adenoviruses, flaviviruses and bacteria. RESULTS: Fifty-five children (18%) had viral (42), bacterial (20) or both viral and bacterial (7) nucleic acids (NA) identified in their CSF. Human herpes viruses accounted for 91% of all viruses found. The identification of viral or bacterial NA was not associated with any characteristic clinical features. By contrast, malaria was associated with increased identification of viral and bacterial NA and with impaired consciousness, multiple convulsions and age. Malaria was also inversely associated with an adverse outcome. Amongst children with HHV infection, those with HHV-6 and -7 were younger, were more likely have impaired consciousness and had a higher proportion of adverse outcomes than children with CMV. Dengue and enteroviral infections were infrequent. Adenoviral and influenza infections were not identified. CONCLUSION: Infections with HHV-6, HHV-7, dengue and enterovirus have the potential to cause serious CNS disease in young PNG children. However most HHVs in this malaria-endemic setting should be considered to be the result of reactivation from a latent reservoir without clinical sequelae.

Primaquine treatment for Plasmodium vivax--an essential tool for malaria control and elimination in Papua New Guinea.

Plasmodium vivax is a major cause of malarial infection and disease in young children in Papua New Guinea. Recent increase in funding for malaria control has improved accessibility to preventive measures, diagnosis and artemisinin combination therapies. Yet the current treatment and control measures are more effective against P. falciparum than against P. vivax and P. ovale due to the biological differences in the liver stage life-cycle of these parasites. P. vivax and P. ovale have a dormant liver stage called a hypnozoite. The artemisinin combination therapies, while highly effective against the blood stages of all plasmodium species causing human malarial illness, have no effect upon the hypnozoites in the liver and the stage V gametocytes of P. falciparum. Currently, primaquine is the only licensed drug shown to be effective against both the hypnozoites of P. vivax and P. ovale in the liver and the stage V gametocytes of P. falciparum. Primaquine has a high associated risk of life-threatening haemolytic anaemia when administered to glucose-6-phosphate dehydrogenase (G6PD)-deficient persons. The lack of cheap, reliable point-of-care testing for the diagnosis of G6PD deficiency remains a major obstacle to the widespread use of primaquine in clinical and public health practice. Furthermore, there is a paucity of primaquine safety and tolerability data, especially in young children with the highest P. vivax disease burden. For malaria control and elimination efforts to be effective, interventions such as mass drug administration must include primaquine. This opinion paper highlights the need to eradicate hypnozoites in the liver of the human host with primaquine treatment for radical cure of malarial illness and discusses the challenges in the use of primaquine as a public health tool for malaria control and elimination programs in countries such as Papua New Guinea.

Malaria epidemiology in Lihir Island, Papua New Guinea.

BACKGROUND: Plasmodium vivax and Plasmodium falciparum malaria remain highly endemic in the Pacific Islands including Lihir Island, Papua New Guinea. Lihir Gold Limited is conducting mining activities and funded an integrated vector control intervention within the villages surrounding the mine. The aim of this study was to assess the impact of such programme by comparing the epidemiological trends of malaria in different parts of the island. METHODS: Two cross-sectional surveys were conducted before and after the intervention (2006-2010) to determine malaria prevalence in mine-impact (MI) and non-MI areas. Incidence of malaria was estimated for the Lihir Medical Centre catchment area using island population denominators and a health-centre passive case detection ongoing from 2006-2011. RESULTS: A total of 2,264 and 1,653 children < 15 were surveyed in the cross-sectional studies. The prevalence of any malaria parasitaemia initially was 31.5% in MI areas and, 34.9% in non-MI (POR 1.17; 95 CI 0.97 - 1.39). After four years there was a significant reduction in prevalence in the MI areas (5.8%; POR 0.13, 95 CI 0.09-0.20), but reduction was less marked in non-MI areas (26.9%; POR 0.69, 95 CI 0.58-0.81).28,747 patients were included in the evaluation of incidence trends and overall malaria in local Lihirian population in MI areas declined over time, while it remained at similar high levels among migrants. The age-incidence analysis showed that for each higher age range the malaria incidence declines compared to that of the previous stratum. CONCLUSIONS: There was a substantial reduction in prevalence and incidence rates of both P. vivax and P. falciparum in the mining area following implementation of a malaria control intervention, which was not seen in the area outside the mining activities.

Ethical challenges in integrating patient-care with clinical research in a resource-limited setting: perspectives from Papua New Guinea.

BACKGROUND: In resource-limited settings where healthcare services are limited and poverty is common, it is difficult to ethically conduct clinical research without providing patient-care. Therefore, integration of patient-care with clinical research appears as an attractive way of conducting research while providing patient-care. In this article, we discuss the ethical implications of such approach with perspectives from Papua New Guinea. DISCUSSION: Considering the difficulties of providing basic healthcare services in developing countries, it may be argued that integration of clinical research with patient-care is an effective, rational and ethical way of conducting research. However, blending patient-care with clinical research may increase the risk of subordinating patient-care in favour of scientific gains; therapeutic misconception and inappropriate inducement; and the risk of causing health system failures due to limited capacity in developing countries to sustain the level of healthcare services sponsored by the research. Nevertheless, these ethical and administrative implications can be minimised if patient-care takes precedence over research; the input of local ethics committees and institutions are considered; and funding agencies acknowledge their ethical obligation when sponsoring research in resource-limited settings. SUMMARY: Although integration of patient-care with clinical research in developing countries appears as an attractive way of conducting research when resources are limited, careful planning and consideration on the ethical implications of such approach must be considered.

Relapses contribute significantly to the risk of Plasmodium vivax infection and disease in Papua New Guinean children 1-5 years of age.

BACKGROUND: Plasmodium vivax forms long-lasting hypnozoites in the liver. How much they contribute to the burden of P. vivax malaria in children living in highly endemic areas is unknown. METHODS: In this study, 433 Papua New Guinean children aged 1-5 years were Randomized to receive artesunate (7 days) plus primaquine (14 days), artesunate alone or no treatment and followed up actively for recurrent Plasmodium infections and disease for 40 weeks. RESULTS: Treatment with artesunate-primaquine reduced the risk of P. vivax episodes by 28% (P = .042) and 33% (P = .015) compared with the artesunate and control arms, respectively. A significant reduction was observed only in the first 3 months of follow-up (artesunate-primaquine vs control, -58% [P = .004]; artesunate-primaquine vs artesunate, -49% [P = .031]) with little difference thereafter. Primaquine treatment also reduced the risk of quantitative real-time polymerase chain reaction- and light microscopy-positive P. vivax reinfections by 44% (P < .001) and 67% (P < .001), respectively. Whereas primaquine treatment did not change the risk of reinfection with Plasmodium falciparum, fewer P. falciparum clinical episodes were observed in the artesunate-primaquine arm. CONCLUSIONS: Hypnozoites are an important source of P. vivax infection and contribute substantially to the high burden of P. vivax disease observed in young Papua New Guinean children. Even in highly endemic areas with a high risk of reinfection, antihypnozoite treatment should be given to all cases with parasitologically confirmed P. vivax infections.

Short-course, high-dose primaquine regimens for the treatment of liver-stage vivax malaria in children.

OBJECTIVES: To assess the pharmacokinetics, safety, and tolerability of two high-dose, short-course primaquine (PQ) regimens compared with standard care in children with Plasmodium vivax infections. METHODS: We performed an open-label pediatric dose-escalation study in Madang, Papua New Guinea (Clinicaltrials.gov NCT02364583). Children aged 5-10 years with confirmed blood-stage vivax malaria and normal glucose-6-phosphate dehydrogenase activity were allocated to one of three PQ treatment regimens in a stepwise design (group A: 0.5 mg/kg once daily for 14 days, group B: 1 mg/kg once daily for 7 days, and group C: 1 mg/kg twice daily for 3.5-days). The study assessments were completed at each treatment time point and fortnightly for 2 months after PQ administration. RESULTS: Between August 2013 and May 2018, 707 children were screened and 73 met the eligibility criteria (15, 40, and 16 allocated to groups A, B, and C, respectively). All children completed the study procedures. The three regimens were safe and generally well tolerated. The pharmacokinetic analysis indicated that an additional weight adjustment of the conventionally recommended milligram per kilogram PQ doses is not necessary to ensure the therapeutic plasma concentrations in pediatric patients. CONCLUSIONS: A novel, ultra-short 3.5-day PQ regimen has potential benefits for improving the treatment outcomes in children with vivax malaria that warrants further investigation in a large-scale clinical trial.

Tolerability and safety of primaquine in Papua New Guinean children 1 to 10 years of age.

Primaquine is currently the only drug available for radical cure of Plasmodium vivax and P. ovale liver infection stages, but limited safety data exist for children <10 years of age. Detailed daily assessments of side effects in glucose-6-phosphate dehydrogenase (G6PD)-normal children treated with 14 days of primaquine plus chloroquine (3 days; n = 252) or artesunate (7 days; n = 141) (0.5 mg/kg of body weight) showed that both treatments are well tolerated, do not lead to reductions in hemoglobin levels, and can thus safely be used in children 1 to 10 years of age.

Epidemiology of malaria in the Papua New Guinean highlands.

OBJECTIVES: To conduct an in-depth investigation of the epidemiology of malaria in the Papua New Guinea (PNG) highlands and provide a basis for evidence-based planning and monitoring of intensified malaria control activities. METHODS: Between December 2000 and July 2005, 153 household-based, rapid malaria population surveys were conducted in 112 villages throughout the central PNG highlands. The presence of malaria infections was determined by light microscopy and risk factors assessed using a structured questionnaire.The combined dataset from all individually published surveys was reanalysed. RESULTS: The prevalence of malaria infections in the different surveys ranged from 0.0% to 41.8%(median 4.3%) in non-epidemic surveys and 6.6% to 63.2% (median 21.2%, P < 0.001) during epidemics. Plasmodium falciparum was the predominant infection below 1400 m and during epidemics, Plasmodium vivax at altitudes >1600 m. Outside epidemics, prevalence decreased significantly with altitude, was reduced in people using bed nets [odds ratio (OR) = 0.8, P < 0.001] but increased in those sleeping in garden houses (OR = 1.34, P < 0.001) and travelling to highly endemic lowlands (OR = 1.80, P < 0.001). Below 1400 m, malaria was a significant source of febrile illness. At higher altitudes, malaria was only a significant source of febrile illness during epidemic outbreaks, but asymptomatic malaria infections were common in non-epidemic times. CONCLUSIONS: Malaria is once again endemic throughout the PNG highlands in areas below 1400­1500 m of altitude with a significant risk of seasonal malaria outbreaks in most area between 1400­1650 m. Ongoing control efforts are likely to result in a substantial reduction in malaria transmission and may even result in local elimination of malaria in higher lying areas.

Comparison of diagnostic methods for the detection and quantification of the four sympatric Plasmodium species in field samples from Papua New Guinea.

BACKGROUND: Accurate diagnosis of Plasmodium infections is essential for malaria morbidity and mortality reduction in tropical areas. Despite great advantages of light microscopy (LM) for malaria diagnosis, its limited sensitivity is a critical shortfall for epidemiological studies. Robust molecular diagnostics tools are thus needed. METHODS: The present study describes the development of a duplex quantitative real time PCR (qPCR) assay, which specifically detects and quantifies the four human Plasmodium species. Performance of this method was compared to PCR-ligase detection reaction-fluorescent microsphere assay (PCR_LDR_FMA), nested PCR (nPCR) and LM, using field samples collected from 452 children one to five years of age from the Sepik area in Papua New Guinea. Agreement between diagnostic methods was calcualted using kappa statistics. RESULTS: The agreement of qPCR with other molecular diagnostic methods was substantial for the detection of P. falciparum, but was moderate for the detection of P. vivax, P. malariae and P. ovale. P. falciparum and P. vivax prevalence by qPCR was 40.9% and 65.7% respectively. This compares to 43.8% and 73.2% by nPCR and 47.1% and 67.5% by PCR_LDR_FMA. P. malariae and P. ovale prevalence was 4.7% and 7.3% by qPCR, 3.3% and 3.8% by nPCR, and 7.7% and 4.4% by PCR_LDR_FMA. Prevalence by LM was lower for all four species, being 25.4% for P. falciparum, 54.9% for P. vivax, 2.4% for P. malariae and 0.0% for P. ovale. The quantification by qPCR closely correlated with microscopic quantification for P. falciparum and P. vivax samples (R2 = 0.825 and R2 = 0.505, respectively). The low prevalence of P. malariae and P. ovale did not permit a solid comparative analysis of quantification for these species. CONCLUSIONS: The qPCR assay developed proved optimal for detection of all four Plasmodium species. Densities by LM were well reflected in quantification results by qPCR, whereby congruence was better for P. falciparum than for P. vivax. This likely is a consequence of the generally lower P. vivax densities. Easy performance of the qPCR assay, a less laborious workflow and reduced risk of contamination, together with reduced costs per sample through reduced reaction volume, opens the possibility to implement qPCR in endemic settings as a suitable diagnostic tool for large epidemiological studies.

Vaccines against malaria: perspectives from Papua New Guinea.

Despite its small population and isolated location Papua New Guinea (PNG) with a malaria burden comparable to sub-Saharan Africa, its intense transmission of all four human Plasmodium species and an unrivalled combination of environmental and human variation offers unique perspectives on malaria vaccines. Building on a long history of malaria research, in this article we review past achievements, highlight current research and outline future directions in malaria vaccine research. With intensive transmission of all four species of human malaria, a full range of malaria endemicities, well described epidemiology and a demonstrated capacity to evaluate a malaria vaccine, PNG currently has the only field site that is ready to conduct proof-of-principle studies of currently available P. vivax vaccine candidates and future combined P. falciparum / P. vivax vaccines and also offers unique opportunities for P. falciparum vaccine research. PNG is thus ready to contribute significantly in the global malaria vaccine endeavor.

Longitudinal tracking and quantification of individual Plasmodium falciparum clones in complex infections.

Longitudinal tracking of individual Plasmodium falciparum strains in multi-clonal infections is essential for investigating infection dynamics of malaria. The traditional genotyping techniques did not permit tracking changes in individual clone density during persistent natural infections. Amplicon deep sequencing (Amp-Seq) offers a tool to address this knowledge gap. The sensitivity of Amp-Seq for relative quantification of clones was investigated using three molecular markers, ama1-D2, ama1-D3, and cpmp. Amp-Seq and length-polymorphism based genotyping were compared for their performance in following minority clones in longitudinal samples from Papua New Guinea. Amp-Seq markers were superior to length-polymorphic marker msp2 in detecting minority clones (sensitivity Amp-Seq: 95%, msp2: 85%). Multiplicity of infection (MOI) by Amp-Seq was 2.32 versus 1.73 for msp2. The higher sensitivity had no effect on estimates of force of infection because missed minority clones were detected in preceding or succeeding bleeds. Individual clone densities were tracked longitudinally by Amp-Seq despite MOI > 1, thus providing an additional parameter for investigating malaria infection dynamics. Amp-Seq based genotyping of longitudinal samples improves detection of minority clones and estimates of MOI. Amp-Seq permits tracking of clone density over time to study clone competition or the dynamics of specific, i.e. resistance-associated genotypes.