Identification and analysis of dihydrofolate reductase alleles from Plasmodium falciparum present at low frequency in polyclonal patient samples

As resistance to chloroquine spreads in sub-Saharan Africa, pyrimethamine p lus sulfadoxine (PSD) is increasingly used as a first-line treatment for fa lciparum malaria. Populations of Plasmodium falciparum (Pf) resistant to PS D have been selected quickly in other regions. The resistance is strongly c orrelated with point mutations in dihydrofolate reductase (DHFR) and dihydr opteroate synthase (DHPS), the two targets of the drug. It is critical to i dentify drug-resistant Pf-DHFR alleles that are present at a low frequency in these populations since alleles that confer drug resistance will be quic kly selected by PSD use. It is difficult to identify these rare alleles by standard molecular techniques. We have designed a yeast expression system t hat facilitates the identification and rapid analysis of Pf-DHFR alleles th at confer PSD resistance, even when they are present at very low frequency in polyclonal patient samples. We analyzed samples from patients in Kilifi, Kenya collected between 1992 and 1995. We determined the prevalence of the drug-sensitive and drug-resistant alleles in patient samples analyzed in p arallel by an allele-specific enzyme digestion (ASED) assay. We identified a pyrimethamine-resistant allele (S108N) present at a frequency of < 1% in a sample that was scored as only S108 by ASED. In addition, a novel pyrimet hamine-resistant allele (I164M) was isolated twice, once each from two diff erent patient samples. This approach will allow determination of the preval ence of Pf-DHFR alleles that confer pyrimethamine resistance in particular regions, and the rapid identification of novel alleles that confer drug res istance.