In Vitro Activity of Antifolate and Polymorphism in Dihydrofolate Reductase of Plasmodium falciparum Isolates from the Kenyan Coast: Emergence of Parasites with Ile-164-Leu Mutation

<jats:title>ABSTRACT</jats:title> <jats:p>We have analyzed the activities of the antifolates pyrimethamine (PM), chlorcycloguanil (CCG), WR99210, trimethoprim (TMP), methotrexate (MTX), and trimetrexate (TMX) against Kenyan <jats:italic>Plasmodium falciparum</jats:italic> isolates adapted in vitro for long-term culture. We have also assessed the relationship between these drug activities and mutations in dihydrofolate reductase (<jats:italic>dhfr</jats:italic>), a domain of the gene associated with antifolate resistance. As expected, WR99210 was the most potent drug, with a median 50% inhibitory concentration (IC<jats:sub>50</jats:sub>) of &lt;0.075 nM, followed by TMX, with a median IC<jats:sub>50</jats:sub> of 30 nM. The median IC<jats:sub>50</jats:sub> of CCG was 37.80 nM, and that of MTX was 83.60 nM. PM and TMP were the least active drugs, with median IC<jats:sub>50</jats:sub>s of 733.26 nM and 29,656.04 nM, respectively. We analyzed parasite <jats:italic>dhfr</jats:italic> genotypes by the PCR-enzyme restriction technique. No wild-type <jats:italic>dhfr</jats:italic> parasite was found. Twenty-four of 33 parasites were triple mutants (mutations at codons 108, 51, and 59), and only 8/33 were double mutants (mutations at codons 108 and 51 or at codons 108 and 59). IC<jats:sub>50</jats:sub>s were 2.1-fold (PM) and 3.6-fold (TMP) higher in triple than in double mutants, though these differences were not statistically significant. Interestingly, we have identified a parasite harboring a mutation at codon 164 (Ile-164-Leu) in addition to mutations at codons 108, 51, and 59. This quadruple mutant parasite had the highest TMP IC<jats:sub>50</jats:sub> and was in the upper 10th percentile against PM and CCG. We confirmed the presence of this mutation by sequencing. Thus, TMX and MTX are potent against <jats:italic>P. falciparum</jats:italic>, and quadruple mutants are now emerging in Africa.</jats:p>