BackgroundAccurate 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.MethodsThe 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.ResultsThe 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.ConclusionsThe 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.

Abstract. Plasmodium falciparum and Plasmodium vivax have varying transmission dynamics that are informed by molecular epidemiology. This study aimed to determine the complexity of infection and genetic diversity of P. vivax and P. falciparum throughout Papua New Guinea (PNG) to evaluate transmission dynamics across the country. In 2008-2009, a nationwide malaria indicator survey collected 8,936 samples from all 16 endemic provinces of PNG. Of these, 892 positive P. vivax samples were genotyped at PvMS16 and PvmspF3, and 758 positive P. falciparum samples were genotyped at Pfmsp2. The data were analyzed for multiplicity of infection (MOI) and genetic diversity. Overall, P. vivax had higher polyclonality (71%) and mean MOI (2.32) than P. falciparum (20%, 1.39). These measures were significantly associated with prevalence for P. falciparum but not for P. vivax. The genetic diversity of P. vivax (PvMS16: expected heterozygosity = 0.95, 0.85-0.98; PvMsp1F3: 0.78, 0.66-0.89) was higher and less variable than that of P. falciparum (Pfmsp2: 0.89, 0.65-0.97). Significant associations of MOI with allelic richness (rho = 0.69, P = 0.009) and expected heterozygosity (rho = 0.87, P < 0.001) were observed for P. falciparum. Conversely, genetic diversity was not correlated with polyclonality nor mean MOI for P. vivax. The results demonstrate higher complexity of infection and genetic diversity of P. vivax across the country. Although P. falciparum shows a strong association of these parameters with prevalence, a lack of association was observed for P. vivax and is consistent with higher potential for outcrossing of this species.

A three-arm randomized trial conducted among infants in Papua New Guinea estimates the preventive effect against malaria episodes of intermittent preventive treatment, in an area where children are exposed to both falciparum and vivax malaria.

The molecular force of blood-stage infection (molFOB) is a quantitative surrogate metric for malaria transmission at population level and for exposure at individual level. Relationships between molFOB, parasite prevalence and clinical incidence were assessed in a treatment-to-reinfection cohort, where P.vivax (Pv) hypnozoites were eliminated in half the children by primaquine (PQ). Discounting relapses, children acquired equal numbers of new P. falciparum (Pf) and Pv blood-stage infections/year (Pf-molFOB = 0–18, Pv-molFOB = 0–23) resulting in comparable spatial and temporal patterns in incidence and prevalence of infections. Including relapses, Pv-molFOB increased >3 fold (relative to PQ-treated children) showing greater heterogeneity at individual (Pv-molFOB = 0–36) and village levels. Pf- and Pv-molFOB were strongly associated with clinical episode risk. Yearly Pf clinical incidence rate (IR = 0.28) was higher than for Pv (IR = 0.12) despite lower Pf-molFOB. These relationships between molFOB, clinical incidence and parasite prevalence reveal a comparable decline in Pf and Pv transmission that is normally hidden by the high burden of Pv relapses.Clinical trial registration: ClinicalTrials.gov NCT02143934

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BackgroundReports of severe cases and increasing levels of drug resistance highlight the importance of improved Plasmodium vivax case management. Whereas monitoring P. vivax resistance to anti-malarial drug by in vivo and in vitro tests remain challenging, molecular markers of resistance represent a valuable tool for high-scale analysis and surveillance studies. A new high-throughput assay for detecting the most relevant markers related to P. vivax drug resistance was developed and assessed on Papua New Guinea (PNG) patient isolates.MethodsPvdhfr, pvdhps and pvmdr1 fragments were amplified by multiplex nested PCR. Then, PCR products were processed through an LDR-FMA (ligase detection reaction - fluorescent microsphere assay). 23 SNPs, including pvdhfr 57-58-61 and 173, pvdhps 382-383, 553, 647 and pvmdr1 976, were simultaneously screened in 366 PNG P. vivax samples.ResultsGenotyping was successful in 95.4% of the samples for at least one gene. The coexistence of multiple distinct haplotypes in the parasite population necessitated the introduction of a computer-assisted approach to data analysis. Whereas 73.1% of patients were infected with at least one wild-type genotype at codons 57, 58 and 61 of pvdhfr, a triple mutant genotype was detected in 65.6% of the patients, often associated with the 117T mutation. Only one patient carried the 173L mutation. The mutant 647P pvdhps genotype allele was approaching genetic fixation (99.3%), whereas 35.1% of patients were infected with parasites carrying the pvmdr1 976F mutant allele.ConclusionsThe LDR-FMA described here allows a discriminant genotyping of resistance alleles in the pvdhfr, pvdhps, and pvmdr1 genes and can be used in large-scale surveillance studies.

BackgroundDrug resistance remains a major obstacle to malaria treatment and control. It can arise and spread rapidly, and vary substantially even at sub-national level. National malaria programmes require cost-effective and timely ways of characterizing drug-resistance at multiple sites within their countries.MethodsAn improved multiplexed post-PCR ligase detection reaction—fluorescent microsphere assay (LDR-FMA) was used to simultaneously determine the presence of mutations in chloroquine resistance transporter (crt), multidrug resistance 1 (mdr1), dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes in Plasmodium falciparum (n = 727) and Plasmodium vivax (n = 574) isolates collected in 2006 from cross-sectional community population surveys in two geographically distinct regions (Madang and East Sepik) of Papua New Guinea (PNG) where strong regional differences in in vivo aminoquinoline and antifolate therapeutic efficacy had previously been observed. Data were compared to those of a follow-up survey conducted in 2010.ResultsDespite some very low parasite densities, the assay successfully amplified all P. falciparum and P. vivax loci in 77 and 69 % of samples, respectively. In 2006, prevalences of pfdhfr (59R-108 N) double mutation/wild type pfdhps haplotype, pfcrt SVMNT haplotype (72S-76T double mutation), and 86Y pfmdr1 mutation all exceeded 90 %. For P. vivax, 65 % carried at least two pvdhfr mutations, 97 % the 647P pvdhps mutation and 54 % the 976F pvmdr1 mutation. Prevalence of mutant haplotypes was higher in Madang than East Sepik for pfcrt SVMNT (97.4 vs 83.3 %, p = 0.001), pfdhfr (59R-108 N) (100 vs 90.6 %, p = 0.001), pvdhfr haplotypes (75.8 vs 47.6 %, p = 0.001) and pvmdr1 976F (71.2 vs 26.2 %, p < 0.001). Data from a subsequent Madang survey in 2010 showed that the prevalence of pfdhps mutations increased significantly from <5 % to >30 % (p < 0.001) as did the prevalence of pvdhfr mutant haplotypes (from 75.8 to 97.4 %, p = 0.012).ConclusionsThis LDR-FMA multiplex platform shows feasibility for low-cost, high-throughput, rapid characterization of a broad range of drug-resistance markers in low parasitaemia infections. Significant geographical differences in mutation prevalence correlate with previous genotyping surveys and in vivo trials and may reflect variable drug pressure and differences in health-care access in these two PNG populations.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-015-0879-9) contains supplementary material, which is available to authorized users.

Intermittent preventive treatment of infants (IPTi) reduces early childhood malaria-related morbidity. While genotypic drug resistance markers have proven useful in predicting the efficacy of antimalarial drugs in case management, there are few equivalent data relating to their protective efficacy when used as IPTi. The present data from an IPTi trial in Papua New Guinea demonstrate how these markers can predict protective efficacy of IPTi for both Plasmodium falciparum and Plasmodium vivax. Intermittent preventive treatment of infants (IPTi) has reduced early childhood malaria-related morbidity in many settings (1,2). Although its precise mechanism of action has been debated, it is now generally agreed that the intermittent short courses of slowly eliminated antimalarial drugs provide protection due to prolonged prophylactic activity (2). Therefore, the pharmacokinetic and pharmacodynamic properties that determine a drug's suitability for IPTi may differ from those important for therapeutic efficacy in case management.Genotypic drug resistance markers correlate well with in vivo therapeutic response in the case management of acute infections. Mutations of Plasmodium falciparum and Plasmodium vivax dihydrofolate reductase (pfdhfr and pvdhfr) and dihydropteroate synthase (pfdhps and pvdhps) genes are associated with resistance to pyrimethamine and sulfadoxine, respectively (3-6). Parasite resistance to 4-aminoquinolines is associated with mutations in the chloroquine resistance transporter gene (pfcrt) (7) and multidrug resistance gene 1 (pfmdr1) in P. falciparum infections and with P. vivax multidrug resistance gene 1 (pvmdr1) mutations in some studies of vivax malaria (8, 9) but not others (10).IPTi effectiveness is likely to be highly dependent on drug resistance patterns in local parasite populations. Current IPTi regimens utilize either 4-aminoquinoline or antifolate drugs (2), and so existing genotypic resistance markers should predict protective efficacy in a given setting without the need for large expensive field-based efficacy studies. However, data directly relating drug resistance genotypes to IPTi efficacy is limited to African IPTi trials of sulfadoxine-pyrimethamine (SP) dual therapy, which have demonstrated that the pfdhfr triple mutant genotype predicts reduced efficacy (11,12). No studies have examined the relationship between IPTi efficacy and genotypic markers in P. vivax infections nor between 4-aminoquinoline resistance markers and IPTi efficacy in falciparum malaria. In addition, there are concerns that the high burden of antimalarial drug use through IPTi will drive the selection and spread of parasite drug resistance, with a consequent reduction in public health benefit (13). Serial monitoring of validated genotypic markers could help identify this phenomenon at an early stage (12).In Papua New Guinea (PNG), an IPTi study of 1,121 infants conducted from 2006 to 2010 showed that amodiaquine plus SP (AQ-SP) reduced P. falciparum and P. vivax incidence by 35% and 23%, respectively, compared ...