Evidence of selective sweeps in genes conferring resistance to chloroquine and pyrimethamine in Plasmodium falciparum isolates in India

Abstract

Treatment of Plasmodium falciparum is complicated by the emergence and spread of parasite resistance to many of the first-line drugs used to treat malaria. Antimalarial drug resistance has been associated with specific point mutations in several genes, suggesting that these single nucleotide polymorphisms can be useful in tracking the emergence of drug resistance. In India, P. falciparum infection can manifest itself as asymptomatic, mild, or severe malaria, with or without cerebral involvement. We tested whether chloroquine- and antifolate drug-resistant genotypes would be more commonly associated with cases of cerebral malaria than with cases of mild malaria in the province of Jabalpur, India, by genotyping the dhps, dhfr, pfmdr-1, and pfcrt genes using pyrosequencing, direct sequencing, and real-time PCR. Further, we used microsatellites surrounding the genes to determine the origins and spread of the drug-resistant genotypes in this area. Resistance to chloroquine was essentially fixed, with 95% of the isolates harboring the pfcrt K76T mutation. Resistant genotypes of dhfr, dhps, and pfmdr-1 were found in 94%, 17%, and 77% of the isolates, respectively. Drug-resistant genotypes were equally likely to be associated with cerebral malaria as with mild malaria. We found evidence of a selective sweep in pfcrt and, to a lesser degree, in dhfr, indicating high levels of resistance to chloroquine and evolving resistance to pyrimethamine. Microsatellites surrounding pfcrt indicate that the resistant genotypes (SVMNT) were most similar to those found in Papua New Guinea.

FIG. 1.

Percentages of samples with mutations at pfcrt (A), dhfr (B), dhps (C), and pfmdr-1 (D) by specific point mutations at each codon. wt, wild type.

FIG. 2.

Numbers of individuals with mutations at each of the genes analyzed for this study. Samples with mutations in pfcrt (C72S, K76T, A220S), dhfr (C59R, S108N), dhps (A437G, K540E, A581G), and pfmdr-1 (N86Y and Y184F) are depicted as shaded boxes. Open boxes represent ancestral wild-type codons.

FIG. 3.

Network diagram showing the relationship of Indian SVMNT pfcrt genotypes with the SVMNT, CVIET, CVMET, and CVMNT genotypes of Asia, Africa, and South America. The data for Asia, Africa, and South America are from the work of Wootton et al. (62), whereas the Indian data are from this study. Only microsatellite haplotypes that occurred two or more times are included in this analysis.

FIG. 4.

Reductions in the heterozygosity (H_e) of microsatellites surrounding pfcrt (A), dhfr (B), and dhps (C). H_e was compared between all cases with mutations (mt) and those that were wild type (wt).

FIG. 5.

Pairwise linkage disequilibrium between microsatellite loci on different chromosomes. Each box represents one comparison between polymorphic pairs of loci; nonpolymorphic pairwise comparisons are not included. Bonferroni's correction for multiple comparisons was conducted for each comparison. Filled cells represent significant values (P, 0.05), and open cells represent nonsignificant values. The location of each microsatellite locus is given at the top of the matrix (loci are named by their positions relative to dhfr, pfcrt, or dhps according to the 3D7 genome sequence from PlasmoDB, version 5.5). The filled boxes within chromosomes (Ch) 4, 7, and 8 represent the positions of dhfr, pfcrt, and dhps, respectively.