Antifolate resistance due to new and known Plasmodium falciparum dihydrofolate reductase mutations expressed in yeast.

Two new dihydrofolate reductase (DHFR) mutations were recently discovered in Plasmodium falciparum samples from an area of Bolivia with high rates of in vivo resistance to pyrimethamine-sulfadoxine: a Cys-->Arg point mutation in codon 50 and a five amino acid insertion after codon 30, termed the Bolivia repeat. We used a yeast expression system to screen these new DHFR mutants, as well as all of the other known DHFR mutant genotypes, against four antifolates: pyrimethamine, cycloguanil, chlorcycloguanil, and WR99210. The prodrug proguanil was also evaluated. The primary 108-Asn mutation, the known secondary mutations 51-Ile, 59-Arg and 164-Leu, as well as the 50-Arg mutation, all progressively enhanced pyrimethamine resistance in naturally observed combinations with one another, with the presence of 164-Leu most significantly increasing resistance. Cycloguanil and chlorcycloguanil resistance were most impacted by 164-Leu and the paired 16-Val/108-Thr. Proguanil had no effect on malaria DHFR. All DHFRs analyzed were sensitive to WR99210. The Bolivia repeat did not markedly affect drug sensitivity. We conclude that malaria DHFR can be reliably, rapidly and inexpensively analyzed in yeast for activity against a broad spectrum of antifolates. This system may be useful for initially characterizing newly discovered genotypes before proceeding to P. falciparum transfection; for large-scale geographic surveys of drug resistance; and for screening new antifolates or new antifolate combinations for their effectiveness against a large panel of DHFR mutants.

Mutations in Plasmodium falciparum dihydrofolate reductase and dihydropteroate synthase and epidemiologic patterns of pyrimethamine-sulfadoxine use and resistance.

To assess the relationship between mutations in Plasmodium falciparum dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) and clinical pyrimethamine-sulfadoxine resistance, polymerase chain reaction surveys and analyses for new mutations were conducted in four countries with increasing levels of pyrimethamine-sulfadoxine resistance: Mali, Kenya, Malawi, and Bolivia. Prevalence of mutations at DHFR codon 108 and a new mutation at DHPS 540 correlated with increased pyrimethamine-sulfadoxine resistance (P < .05). Mutations at DHFR 51, DHFR 59, and DHPS 437 correlated with resistance without achieving statistical significance. Mutations at DHFR 164 and DHPS 581 were common in Bolivia, where pyrimethamine-sulfadoxine resistance is widespread, but absent in African sites. Two new DHFR mutations, a point mutation at codon 50 and an insert at codon 30, were found only in Bolivia. DHFR and DHPS mutations occur in a progressive, stepwise fashion. Identification of specific sets of mutations causing in vivo drug failure may lead to the development of molecular surveillance methods for pyrimethamine-sulfadoxine resistance.