DEVELOPMENT AND COMPARISON OF QUANTITATIVE ASSAYS FOR THE DIHYDROPTEROATE SYNTHETASE CODON-540 MUTATION ASSOCIATED WITH SULFADOXINE RESISTANCE IN PLASMODIUM-FALCIPARUM

A point mutation in codon 540 of the dihydropteroate synthetase (dhps) gene affecting sulfadoxine resistance has previously been found in pa rasites from patients with Plasmodium falciparum infection. Here, we i nvestigated 4 methods of identifying this mutation in clinical specime ns and established a reliable quantitative assay to estimate the perce ntage of resistant type in mixed infections. A diagnostic PCR assay ba sed on allele-specific amplification was developed, which clearly type d the clinical specimens examined. The mutation in codon 540 introduce s an additional FokI cleavage site which provided a second method to d ifferentiate mutant from wild type, where the former gives rise to 2 c haracteristic fragments of 538 and 326 bp that are absent from the lat ter. To calibrate quantitatively the ratio of alleles in mixed samples , we constructed artificial mixes containing 2 plasmid DNAs, one carry ing the mutation and the other a wild-type insert. When P-32-labelling was employed, the allele-specific PCR assay could detect the level of resistant type in a mixture down to 0.1-1 %, while for the restrictio n enzyme/PCR analysis, the figure was approximately 10 %. Furthermore, neither fluorescent dye-labelled terminator nor dye-labelled primer c ycle sequencing was able to detect the mutant allele if it was present at less than 20-30 %. We conclude that the allele-specific PCR assay is the most sensitive method of detecting the codon 540 mutation in P. falciparum dhps, and the method of choice for estimating the composit ion of mixed samples.