Countrywide survey shows very high prevalence of Plasmodium falciparum multilocus resistance genotypes in Cambodia

Abstract

Cambodia is located in an area of resistance to multiple antimalarials and has been the first country to implement the systematic use of an artesunate-mefloquine combination as first-line treatment for Plasmodium falciparum malaria. Little is known, however, about the prevalence of resistance mutations within the natural parasite populations, impeding rational drug policy in this context. Using direct sequencing of PCR products, we have analyzed sequence polymorphism of the dihydrofolate reductase-thymidylate synthase, dihydropteroate synthetase, and multidrug resistance 1 genes in a large number of clinical P. falciparum isolates collected in various areas of Cambodia. This highlighted a 100% prevalence of haplotypes with multiple mutations in the target genes of antifolates after more than a decade without use of antifolates for malaria therapy. A high prevalence of mutations in Pfmdr1, including mutations associated with decreased in vitro susceptibility to mefloquine and quinine, was also observed. In addition, novel, low-frequency mutations were detected in Pfmdr1. Our findings show an alarming rate of multilocus resistance genotypes in Cambodia, requiring diligent surveillance and imposing limitations on possible future drug combinations.

FIG. 1.

Map of Cambodia with location of the sampling sites.

FIG.2.

Sequence polymorphism in the Pfdhfr-ts, Pfdhps, and Pfmdr1 genes and resulting amino acid changes in clinical P. falciparum isolates from Cambodia as deduced by a PCR and direct sequencing strategy. Filled boxes show the position of the observed single nucleotide polymorphism for each haplotype. Empty boxes indicate a wild-type sequence at that position. The prevalence of the various mutations and haplotypes is indicated, as well as the in vitro responses of isolates to pyrimethamine (PYR), chloroquine (CQ), mefloquine (MF), quinine (QN), and artesunate (AS). In vitro responses are expressed as the GMIC₅₀. (a) Pfdhfr-ts sequence polymorphism and its association with in vitro pyrimethamine resistance in 173 P. falciparum isolates from six different sites in Cambodia: Rattanakiri (n = 25), Snoul (n = 43), Kampong Speu (n = 21), Samlot (n = 17), Sampovloum (n = 62), and Anlong Veng (n = 5). (b) Pfdhps sequence polymorphism in 53 P. falciparum isolates from Rattanakiri (n = 2), Snoul (n = 26), Kampong Speu (n = 2), Sampovloum (n = 13), Samlot (n = 7), and Anlong Veng (n = 3). (c) Pfmdr1 sequence polymorphism and its association with in vitro susceptibility to a panel of antimalarials in 66 P. falciparum isolates from Rattanakiri (n = 2), Snoul (n = 21), Kampong Speu (n = 6), Sampovloum (n = 20), Samlot (n = 15), and Anlong Veng (n = 2). *, new mutations. The boxed GMIC₅₀s indicate values above the threshold for in vitro resistance (16). In panel a, 95% confidence intervals observed for pyrimethamie were 5.07 to 14.0 μM (n = 26) and 3.06 to 11.8 μM (n = 27) for the ACIRNVI and ACIRNVL Pfdhfr-ts haplotype, respectively. In panel c, confidence interval values observed for the NEYSNVD (wild type), NEFSNVD, and NEFSDVD Pfmdr1 haplotypes were 69.0 to 288.2 nM (n = 6), 82.5 to 263.4 nM (n = 12), and 25.8 to 230.2 (n = 4), respectively, for CQ; 9.6 to 33.2 nM (n = 7), 27.2 to 81.6 nM (n = 13), and 16.9 to 55.6 nM (n = 5), respectively, for mefloquine; 33.0 to 187.8 nM (n = 7), 78.3 to 253.1 nM (n = 11), and 66.5 to 491.7 nM (n = 3), respectively, for quinine; and finally 0.56 to 1.65 nM (n = 7), 2.22 to 4.66 nM (n = 13), and 0.74 to 9.6 nM (n = 5), respectively, for artesunate. nd, no data.

FIG. 3.

Synopsis of multilocus genotypes in P. falciparum isolates from Cambodia. *, mutations in the Pfcrt gene have been reported elsewhere (8, 17).