Critical comparison of molecular genotyping methods for detection of drug-resistant Plasmodium falciparum

Abstract

We have critically evaluated 3 techniques for the detection of mutations conferring drug resistance of Plasmodium falciparum, using samples containing known numbers of well-characterized parasites and artificial mixtures of these parasites at known proportions. We compared the sensitivity and specificity of mutation-specific polymerase chain reaction (MS-PCR), polymerase chain reaction followed by restriction enzyme digestion at polymorphic sites (PCR/RFLP), and a dot-blot/probe hybridization technique, for detection of point mutations at nucleotide 323 of the P. falciparum dihydrofolate reductase gene (dhfr) that confer resistance to the antimalarial drug pyrimethamine. We have also investigated the benefits in terms of sensitivity and reproducibility of the incorporation of radiolabelled nucleotides into the PCR/RFLP assay. We found that MS-PCR was very sensitive--at least 10 parasites could be detected in a sample--but non-specific amplification resulted in erroneous typing of some samples. PCR/RFLP was less sensitive; 10 parasites per sample could not always be detected, but the technique was specific. The addition of radiolabelled nucleotides to the assay did not markedly improve the sensitivity but the results were easier to read and there was less subjectivity in scoring the results. The dot-blot/probe hybridization technique was specific and sensitive, with similar levels of specificity and sensitivity to PCR/RFLP. On balance, the dot-blot/probe hybridization technique seems best suited to large-scale epidemiological surveys of genes associated with antimalarial drug resistance.

Genotyping of Plasmodium falciparum Pyrimethamine Resistance by Matrix-Assisted Laser Desorption-Ionization Time-of-Flight Mass Spectrometry

“…Mutations at codon positions 51, 59, and 108 of the P. falciparum pfdhfr gene were reproducibly and consistently identified. No mutations were found at position pfdhfr 16 , a finding corresponding to the previous observation that variants at that position are rare (1,13). A biplexed assay for the detection of more than one variant was feasible only for the simultaneous detection of pfdhfr 16 and pfdhfr 108 variants.…”

“…3). Since the mutant variant pfdhfr 16 was not detected in our study group, one peak only resulted for the nucleotide at codon position 16 (m/z 5,699; wild-type strain; Fig. 3).…”

“…For variants of pfdhfr 16 /pfdhfr 108 , a biplex reaction was established to simultaneously detect SNPs. The design and orientation of extension primers (sense or antisense primer) was dependent on the sequence adjacent to the SNP under question.…”

“…20 ng) in H 2 O for reactions to detect pfdhfr 51 and pfdhfr 59 variants. For the reactions established to identify pfdhfr 16 and pfdhfr 108 variants, ddATP was used instead of dATP. …”

“…For the pfdhfr 16 and pfdhfr 108 primer extension reactions, an initial denaturation was performed at 94°C for 1 min, followed by 49 cycles of denaturation at 94°C for 15 s, annealing at 54°C for 1 min, and extension and/or elongation at 72°C for 30 s. For pfdhfr 51 and pfdhfr 59 reactions, the annealing temperature was 52°C in 49 cycles. After the extension reaction, the single-stranded primer extension products were purified either manually or automatically (Genesis Workstation 200; Tecan, Durham, N.C.) by using a Magnetic Bead DNA Purification Kit 1000 (GenoPure Oligo; Bruker Daltonics, Bremen, Germany).…”