FREQUENCY OF THE ASN-108 AND THR-108 POINT MUTATIONS IN THE DIHYDROFOLATE-REDUCTASE GENE IN PLASMODIUM-FALCIPARUM FROM SOUTHWEST COLUMBIA

Several point mutations in the dihydrofolate reductase (DHFR) gene of Plasmodium falciparum have been correlated with in vitro anti-folate d rug resistance of laboratory and field isolates. Furthermore, two diff erent point mutations that generate amino acid substitutions at the sa me position of the enzyme have been observed in all the isolates studi ed to date. These point mutations change a serine (Ser-108) in the wil d type to an asparagine (Asn-108 mutation) or to a threonine (Thr-108 mutation). Using the polymerase chain reaction (PCR), it is possible t o identify isolates that present these mutations. We used a mutation-s pecific PCR to screen 71 samples from several geographic locations of Colombia for the Asn-108 mutation (pyrimethamine resistance). In this initial screening 53 of 71 yielded amplification product with the DHFR mutation-specific primers. We further analyzed the 18 samples that di d not amplify using a mutation-specific nested PCR. Of those 18 sample s, seven amplified with primers specific for the Thr-108 mutation (pro guanil resistance), one with the wild type (Ser-108), and 10 did not a mplify, Of these 10 samples, three were identified as P. falciparum us ing a species-specific diagnostic nested PCR base on sequences from th e small ribosomal RNA subunit gene. Overall, 51.6% of the samples ampl ified for the Asn-108 mutation, 10.9% for the Thr-108 mutation, 35.9% with the wild type specific primer, and 4.8% did not amplify with any of the DHFR primers. We observed variability in the frequency of the m utation between the different geographic location. The frequency of th e Asn-108 and Thr-108 mutations in the state of Narino was 25% each, w hile in Valle del Cauca the frequencies were 59% and 11%, respectively . These results contrast with observations in Brazil in which the Asn- 108 mutation was found in 90% of the blood samples screened.