RAPID DETECTION OF PYRIMETHAMINE SUSCEPTIBILITY OF PLASMODIUM-FALCIPARUM BY RESTRICTION-ENDONUCLEASE DIGESTION OF THE DIHYDROFOLATE-REDUCTASE GENE

A rapid and simple method to detect pyrimethamine susceptibility of Pl asmodium falciparum by analyzing DNA from whole blood is presented. Sa mples from cultured isolates and from patients infected with P. falcip arum were spotted onto filter paper disks, dried, and stored for subse quent analyses. After extracting the P. falciparum DNA using Chelex-10 0 ion-chelating resin (Bio-Rad, Richmond, CA), the polymerase chain re action (PCR) was used to amplify the dihydrofolate reductase (dhfr) ge ne. The PCR product of 727 basepairs was digested with the Alu I restr iction endonuclease to detect whether the isolates were sensitive or r esistant to the antimalarial drugs pyrimethamine and cycloguanil. This restriction endonuclease digests only DNA from pyrimethamine-sensitiv e parasites because the recognition locus of Alu I is changed by mutat ions giving rise to pyrimethamine and cycloguanil resistance. This met hod is simple and sensitive and could therefore be used to study the e pidemiology of pyrimethamine resistance in P. falciparum. The DHFR gen e of pyrimethamine-resistant clones from Vietnamese patients showed th ree amino acid changes that were previously found in pyrimethamine-res istant isolates. Two other clones, T9/94 and T9/96, originally isolate d from a single malaria patient from Thailand, had different DHFR gene sequences. The nucleotide sequence of the DHFR gene from T9/96 was id entical to the wild-type DHFR sequence, whereas T9/94 possessed amino acid substitutions at positions 16 and 108 that have been described in cycloguanil-resistant parasites.