Towards an understanding of the mechanism of pyrimethamine-sulfadoxine resistance in Plasmodium falciparum: Genotyping of dihydrofolate reductase anddihydropteroate synthase of Kenyan parasites

The antifolate combination of pyrimethamine (PM) and sulfadoxine (SD) is th e last affordable drug combination available for wide-scale treatment of fa lciparum malaria in Africa. Wherever this combination has been used, drug-r esistant parasites have been selected rapidly. A study of PM-SD effectivene ss carried out between 1997 and 1999 at Kilifi on the Kenyan coast has show n the emergence of RI and RII resistance to PM-SD (residual parasitemia 7 d ays after treatment) in 39 out of 240 (16.25%) patients. To understand the mechanism that underlies resistance to PM-SD, we have analyzed the dihydrof olate reductase (DHFR) and dihydropteroate synthase (DHPS) genotypes of 81 patients. Fifty one samples were obtained, before treatment, from patients who remained parasite free for at least 7 days after treatment. For a furth er 20 patients, samples were obtained before treatment and again when they returned to the clinic with parasites 7 days after PM-SD treatment. Ten add itional isolates were obtained from patients who were parasitemic 7 days af ter treatment but who were not sampled before treatment. More than 65% of t he isolates (30 of 46) in the initial group had wild-type or double mutant DHFR alleles, and all but 7 of the 47 (85%) had wild-type DHPS alleles. In the paired (before and after treatment) samples, the predominant combinatio ns of DHFR and DHPS alleles before treatment were of triple mutant DHFR and double mutant DHPS (41% [7 of 17]) and of double mutant DHFR and double mu tant DHPS (29% [5 of 17]). All except one of the posttreatment isolates had triple mutations in DHFR, and most of these were "pure" triple mutants. In these isolates, the combination of a triple mutant DHFR and wild type DHPS was detected in 6 of 29 cases (20.7%), the combination of a triple mutant DHFR and a single mutant (A437G) DHPS was detected in 4 of 29 cases (13.8%) , and the combination of a triple mutant DHFR and a double mutant (A437G, L 540E) DHPS was detected in 16 of 29 cases (55.2%). These results demonstrat e that the triply mutated allele of DHFR with or without mutant DHPS allele s is associated with RI and RII resistance to PM-SD. The prevalence of the triple mutant DHFR-double mutant DHPS combination may be an operationally u seful marker far predicting the effectiveness of PM-SD as a new malaria tre atment.