Abstract. Genetic diversity and population structure of Plasmodium vivax parasites can predict the origin and spread of novel variants within a population enabling population specific malaria control measures. We analyzed the genetic diversity and population structure of 425 P. vivax isolates from Sri Lanka, Myanmar, and Ethiopia using 12 trinucleotide and tetranucleotide microsatellite markers. All three parasite populations were highly polymorphic with 3-44 alleles per locus. Approximately 65% were multiple-clone infections. Mean genetic diversity ( H E ) was 0.7517 in Ethiopia, 0.8450 in Myanmar, and 0.8610 in Sri Lanka. Significant linkage disequilibrium was maintained. Population structure showed two clusters (Asian and African) according to geography and ancestry. Strong clustering of outbreak isolates from Sri Lanka and Ethiopia was observed. Predictive power of ancestry using two-thirds of the isolates as a model identified 78.2% of isolates accurately as being African or Asian. Microsatellite analysis is a useful tool for mapping short-term outbreaks of malaria and for predicting ancestry.

BackgroundChloroquine combined with primaquine has been the recommended antimalarial treatment of Plasmodium vivax malaria infections for six decades but the efficacy of this treatment regimen is threatened by chloroquine resistance (CQR). Single nucleotide polymorphisms (SNPs) in the multidrug resistance gene, Pvmdr1 are putative determinants of CQR but the extent of their emergence at population level remains to be explored.ObjectiveIn this study we describe the prevalence of SNPs in the Pvmdr1 among samples collected in seven P. vivax endemic countries and we looked for molecular evidence of drug selection by characterising polymorphism at microsatellite (MS) loci flanking the Pvmdr1 gene.MethodsWe examined the prevalence of SNPs in the Pvmdr1 gene among 267 samples collected from Pakistan, Afghanistan, Sri Lanka, Nepal, Sudan, São Tomé and Ecuador. We measured and diversity in four microsatellite (MS) markers flanking the Pvmdr1 gene to look evidence of selection on mutant alleles.ResultsSNP polymorphism in the Pvmdr1 gene was largely confined to codons T958M, Y976F and F1076L. Only 2.4% of samples were wildtype at all three codons (TYF, n = 5), 13.3% (n = 28) of the samples were single mutant MYF, 63.0% of samples (n = 133) were double mutant MYL, and 21.3% (n = 45) were triple mutant MFL. Clear geographic differences in the prevalence of these Pvmdr mutation combinations were observed.Significant linkage disequilibrium (LD) between Pvmdr1 and MS alleles was found in populations sampled in Ecuador, Nepal and Sri Lanka, while significant LD between Pvmdr1 and the combined 4 MS locus haplotype was only seen in Ecuador and Sri Lanka. When combining the 5 loci, high level diversity, measured as expected heterozygosity (He), was seen in the complete sample set (He = 0.99), while He estimates for individual loci ranged from 0.00–0.93. Although Pvmdr1 haplotypes were not consistently associated with specific flanking MS alleles, there was significant differentiation between geographic sites which could indicate directional selection through local drug pressure.ConclusionsOur observations suggest that Pvmdr1 mutations emerged independently on multiple occasions even within the same population. In Sri Lanka population analysis at multiple sites showed evidence of local selection and geographical dispersal of Pvmdr1 mutations between sites.

BackgroundSulphadoxine-pyrimethamine (SP) and chloroquine (CQ) have been used in treatment of falciparum and vivax malaria in Nepal. Recently, resistance to both drugs have necessitated a change towards artemisinin combination therapy (ACT) against Plasmodium falciparum in highly endemic areas. However, SP is still used against P. falciparum infections in low endemic areas while CQ is used in suspected cases in areas with lack of diagnostic facilities. This study examines the prevalence of molecular markers of CQ and SP resistance in P. falciparum and Plasmodium vivax to determine if high levels of in vivo resistance are reflected at molecular level as well.MethodsFinger prick blood samples (n = 189) were collected from malaria positive patients from two high endemic districts and analysed for single nucleotide polymorphisms (SNPs) in the resistance related genes of P. falciparum and P. vivax for CQ (Pfcrt, Pfmdr1, Pvmdr1) and SP (Pfdhfr, Pfdhps, Pvdhfr), using various PCR-based methods.Results and discussionPositive P. vivax and P. falciparum infections were identified by PCR in 92 and 41 samples respectively. However, some of these were negative in subsequent PCRs. Based on a few P. falciparum samples, the molecular level of CQ resistance in P. falciparum was high since nearly all parasites had the Pfcrt mutant haplotypes CVIET (55%) or SVMNT (42%), though frequency of the Pfmdr1 wild type haplotype was relatively low (35%). Molecular level of SP resistance in P. falciparum was found to be high. The most prevalent Pfdhfr haplotype was double mutant CNRNI (91%), while frequency of Pfdhps double mutant SGEAA and AGEAA were 38% and 33% respectively. Combined, the frequency of quadruple mutations (CNRNI-SGEAA/AGEAA) was 63%. Based on P. vivax samples, low CQ and SP resistance were most likely due to low prevalence of Pvmdr1 Y976F mutation (5%) and absence of triple/quadruple mutations in Pvdhfr.ConclusionsBased on the limited number of samples, prevalence of CQ and SP resistance at molecular levels in the population in the study area were determined as high in P. falciparum and low in P. vivax. Therefore, CQ could still be used in the treatment of P. vivax infections, but this remains to be tested in vivo while the change to ACT for P. falciparum seems justified.

Comprehensive surveillance is needed to support sulfadoxine–pyrimethamine intermittent preventive treatment in pregnant women.