Analysis of pfcrt point mutations and chloroquine susceptibility in isolates of Plasmodium falciparum

Recent transfection based studies demonstrated that cg2, a candidate gene f or chloroquine resistance in Plasmodium falciparum, was not the resistance determinant. A further analysis of the initial 36 kb locus comprising the c g2 gene led to the discovery of another gene, pfert, which was absolutely a ssociated with chloroquine resistance in forty parasite lines [Fidock DA, N omura T, Talley AT, Su XZ, Cooper R, Dzekunov SM, Ferdig MT, Ursos LMB, Sid hu ABS, Naude B, Deitsch KW, Su XZ, Wootton JC, Roepe PD, Wellems TE. Mutat ions in the P. falciparum digestive vacuole transmembrane protein PfCRT and evidence for their role in chloroquine resistance. Mol Cell 2000;6:861-71] . The aim of this study was to evaluate, in 146 unselected clinical isolate s obtained mostly from non-immune travellers returning from various endemic countries to France in years 1995-1999, the association between in vitro c hloroquine resistance and the sequence of a part of the pfert gene. For com parison, the determination of the cg2 kappa and the pfmdr1 codon 86 genotyp es were also performed on the same isolates. As determined by an isotopic s emi-microtest, 70 isolates were susceptible to chloroquine (50% inhibitory concentration < 80 nM) and 76 were resistant. The amplification of a portio n of the pfert gene spanning codons 72-76, followed by sequencing showed th ree distinct genotypes: one type associated with susceptible isolates, one type associated mostly with resistant isolates and one type found in a resi stant isolate originating from South America. Three different zones could b e defined according to the status of codon 76. For 50% inhibitory concentra tion values less than or equal to 40 nM (n = 47), all isolates: but one had K76 (wild type). For 50% inhibitory concentration values located between 4 0 and 60 nM, isolates had either K76 (n = 5) or K76T (mutant type) (n = 6). For 50% inhibitory concentration values > 60 nM (n = 88), all isolates had K76T. A lack of a strong association between the pfmdr1 N86Y mutation and in vitro chloroquine resistance was observed. Cg2 genotypes were less stron gly linked than pfert genotypes with in vitro chloroquine susceptibility in isolates located below 40 and above 60 nM. Further studies are needed to d etermine the reliability of the pfert gene as a genetic marker for chloroqu ine resistance. (C) 2001 Elsevier Science B.V. All rights reserved.