Molecular epidemiology of malaria in Yaounde, Cameroon. VI. Sequence variations in the Plasmodium falciparum dihydrofolate reductase-thymidylate synthase gene and in vitro resistance to pyrimethamine and cycloguanil

Pyrimethamine and cycloguanil, the major human metabolite of proguanil, are inhibitors of dihydrofolate reductase that play a key role in the treatmen t and prevention of chloroquine-resistant Plasmodium falciparum infections in sub-Saharan Africa. Resistance to these antifolate drugs has emerged in some areas of Africa. Earlier molecular studies have demonstrated that poin t mutations at key positions of the dihydrofolate reductase-thymidylate syn thase gene are strongly associated with antifolate resistance. However, whe ther the same or distinct mutations are involved in the development of resi stance to both pyrimethamine and cycloguanil has not been well established in naturally occurring P. falciparum isolates. In this study, the in vitro responses to both antifolate drugs were measured in 42 Cameroonian isolates and compared with the complete sequence of the dihydrofolate reductase dom ain of the gene (from 34 of 42 isolates) to analyze the genotype that may d istinguish between pyrimethamine and cycloguanil resistance. The wild-type profile (n = 11 isolates) was associated with low 50% inhibitory concentrat ions (IC(50)s) ranging from 0.32 to 21.4 nanamole for pyrimethamine and 0.6 0-6.40 nM for cycloguanil. Mutant isolates had at least one amino acid subs titution, Asn-108. Only three mutant codons were observed among the antifol ate-resistant isolates: Ile-51, Arg-59, and Asn-108. The increasing number of point mutations was associated with an increasing level of pyrimethamine IC50 and, to a much lesser extent, cycloguanil IC50. These results support a partial cross-resistance between pyrimethamine and cycloguanil that is b ased on similar amino acid substitutions in dihydrofolate reductase and sug gest that two or three mutations, including at least Asn-108, may be necess ary for cycloguanil resistance, whereas a single Asn-108 mutation is suffic ient for pyrimethamine resistance.