Molecular epidemiology of malaria in Cameroon. XIII. Analysis of pfcrt mutations and in vitro chloroquine resistance.

The key Lys76Thr amino-acid substitution in Plasmodium falciparum chloroquine-resistance transporter (PfCRT) has been shown to be a reliable marker associated with chloroquine-resistant phenotype in reference clones, but few discordant results have been observed in field isolates. To further examine the relationship between in vitro chloroquine response and pfcrt alleles, the entire exon 2 of the pfcrt gene of 157 Cameroonian isolates was sequenced. All isolates were characterized as having either Cys-72, Met-74, Asn-75, and Lys-76 (wild-type alleles), Cys-72, Ile-74, Glu-75, and Thr-76 (mutant alleles), or mixed alleles. The hypothetical threshold 50% inhibitory concentration (IC50) set at 100 nM distinguished between isolates carrying the wild-type alleles and those with mutant alleles in a large majority of cases (135 of 139 isolates with unmixed pfcrt alleles). Isolates presenting discordant results generally had IC50s within an intermediate range. In vitro chloroquine response of isolates with mixed pfcrt alleles was highly variable. Although discordant results between chloroquine-resistant phenotype and pfcrt alleles were not explained by the immediate adjacent codons, the key Lys76Thr codon may prove to be a highly reliable genetic marker for the epidemiologic monitoring of chloroquine resistance by means of molecular techniques.