Multiple Origins of Mutations in the mdr1 Gene--A Putative Marker of Chloroquine Resistance in P. vivax.

BACKGROUND: Chloroquine 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. OBJECTIVE: In 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. METHODS: We 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. RESULTS: SNP 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. CONCLUSIONS: Our 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.

Immunoglobulin G antibodies to merozoite surface antigens are associated with recovery from chloroquine-resistant Plasmodium falciparum in Gambian children.

We examined the hypothesis that recovery from uncomplicated malaria in patients carrying drug-resistant Plasmodium falciparum is a measure of acquired functional immunity and may therefore be associated with humoral responses to candidate vaccine antigens. Gambian children with malaria were treated with chloroquine in 28-day trials, and recovery was defined primarily as the absence of severe clinical malaria at any time and absence of parasitemia with fever after 3 days. Plasma samples from these children were assayed by enzyme-linked immunosorbent assay for immunoglobulin G (IgG) to recombinant merozoite antigens: apical membrane antigen 1 (AMA-1) and the 19-kDa C-terminal region of merozoite surface protein 1 (MSP-1(19)), including antigenic variants of MSP-1(19) with double and triple substitutions. Antigen-specific IgG was more frequent in children who recovered, particularly that for MSP-1(19) (age-adjusted odds ratios: 0.32 [95% confidence interval, 0.05, 1.87; P = 0.168] for AMA-1, 0.19 [0.03, 1.11; P = 0.019] for recombinant MSP-1(19), 0.24 [0.04, 1.31; P = 0.032] for the recombinant MSP-1(19) double variant, and 0.18 [0.03, 0.97; P = 0.013] for the triple variant). IgG titers to MSP-1(19) and to the triple variant were higher in plasma samples taken 7 days after chloroquine treatment from children who carried resistant parasites but recovered and remained parasite free. Moreover, in children who were parasitemic on day 14 or day 28, there was an age-independent relationship between parasite density and IgG to both MSP-1(19) and the triple variant (coefficients of -0.550 and -0.590 and P values of 0.002 and 0.001, respectively). The results validate the use of this approach to identify antigens that are associated with protection from malaria.