Molecular marker of Plasmodium falciparum resistance to chloroquine: Implications for malaria control and anti-malarial immunity

Abstract

Malaria is a major public health problem in tropical countries and is becoming a threat to global health. Resistance of Plasmodium falciparum to chloroquine, the cheapest, safest and most widely used antimalarial drug is now widespread. The basic mechanisms involved are just beginning to be understood. Three genes, pfcrt, cg2 and pfmdr 1 encoding PfCRT, CG2 and Pgh1, respectively, have been associated with chloroquine resistance in laboratory lines of Plasmodium falciparum. The role of these genes in field chloroquine resistance is not known. We conducted prospective efficacy trials in Mali (West Africa) to collect rigorously characterized resistant and sensitive isolates of Plasmodium falciparum. PCR based molecular methods were developed to detect gene polymorphisms and assess their selection by chloroquine and association with treatment outcome. We show that resistance-conferring polymorphisms of all three genes were selected in vivo. However, chloroquine resistant parasites carrying wild-type polymorphisms were found for all genes but pfcrt. The presence of PfCRT K76T mutation was the most strongly associated with clinical chloroquine resistance (OR, 18.8; 95% CI 6.5-58.3). The data confirm PfCRT's principal role in chloroqine resistance and demonstrate that the K76T mutation is the best available molecular marker of chloroquine resistance. To assess PfCRT K76T as a tool for monitoring chloroquine resistance, we conducted similar surveys in three sites with different malaria epidemiological characteristics and calculated genotype-resistance indices (GRI) for each site. Although the prevalence of the PfCRT 76T was consistently higher than clinical chloroquine resistance, GRIs adjusted for age were between 1.6 and 2.7. This GRI model should permit the determination of rates of clinical chloroquine resistance in large regions based on simple molecular surveys of prevalence of PfCRT 76T, without the need for lengthy and expensive in vivo studies. Because most patients cleared PfCRT K76T mutant parasites we hypothesized that host factors accounted for this phenomenon. We show that the ability to clear these mutant parasites is strongly associated with age, which is the only accepted marker of protective immunity in malaria. We propose the clearance of PfCRT mutant parasites as a model system for the investigation of antimalarial immunity.