title: Development and comparison of quantitative assays for the dihydropteroate synthetase codon 540 mutation associated with sulfadoxine resistance in Plasmodium falciparum.
abstract: A point mutation in codon 540 of the dihydropteroate synthetase (dhps) gene affecting sulfadoxine resistance has previously been found in parasites from patients with Plasmodium falciparum infection. Here, we investigated 4 methods of identifying this mutation in clinical specimens and established a reliable quantitative assay to estimate the percentage of resistant type in mixed infections. A diagnostic PCR assay based on allele-specific amplification was developed, which clearly typed the clinical specimens examined. The mutation in codon 540 introduces an additional FokI cleavage site which provided a second method to differentiate mutant from wild type, where the former gives rise to 2 characteristic fragments of 538 and 326 bp that are absent from the latter. To calibrate quantitatively the ratio of alleles in mixed samples, we constructed artificial mixes containing 2 plasmid DNAs, one carrying the mutation and the other a wild-type insert. When 32P-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 restriction enzyme/PCR analysis, the figure was approximately 10%. Furthermore, neither fluorescent dye-labelled terminator nor dye-labelled primer cycle 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 composition of mixed samples.
authors.affiliation: Department of Biomolecular Sciences, University of Manchester Institute of Science and Technology, UK.
authors.forename: M F
authors.initials: MF
authors.lastname: Shaio
authors.forename: P
authors.initials: P
authors.lastname: Wang
authors.forename: C S
authors.initials: CS
authors.lastname: Lee
authors.forename: P F
authors.initials: PF
authors.lastname: Sims
authors.forename: J E
authors.initials: JE
authors.lastname: Hyde
mesh_terms: D000818:Animals; D000962:Antimalarials / Q000494:pharmacology*; D003062:Codon / Q000737:chemistry / Q000235:genetics*; D017931:DNA Primers; D016054:DNA, Protozoan / Q000032:analysis; D004094:Dihydropteroate Synthase / Q000235:genetics*; D004351:Drug Resistance / Q000235:genetics; D004587:Electrophoresis, Agar Gel; D005456:Fluorescent Dyes; D006801:Humans; D016778:Malaria, Falciparum / Q000469:parasitology; D010963:Plasmodium falciparum / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D017354:Point Mutation / Q000235:genetics*; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D015183:Restriction Mapping; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D013413:Sulfadoxine / Q000494:pharmacology*
chemical_list: D000962:Antimalarials; D003062:Codon; D017931:DNA Primers; D016054:DNA, Protozoan; D005456:Fluorescent Dyes; D013413:Sulfadoxine; D004094:Dihydropteroate Synthase