The rapid spread of
antimalarial drug resistance in Plasmodium falciparum over the past few decades has necessitated intensive monitoring of such resistance for an effective
malaria control strategy. P. falciparum
dihydropteroate synthase (Pfdhps) and P. falciparum
dihydrofolate reductase (Pfdhfr) genes act as molecular markers for resistance against the
antimalarial drugs sulphadoxine and
pyrimethamine, respectively. Resistance to
pyrimethamine which is used as a partner
drug in
artemisinin combination
therapy (ACT) is associated with several mutations in the Pfdhfr gene, namely A16V, N51I, C59R, S108N/T and I164L. Therefore, routine monitoring of Pfdhfr-
drug-resistant alleles in a population may help in effective drug resistance management. Allele-specific PCR (ASPCR) is one of the commonly used methods for molecular genotyping of these alleles. In this study, we genotyped 55 samples of P. falciparum for allele discrimination at four
codons of Pfdhfr (N51, C59, S108 and I164) by ASPCR using published methods and by Sanger's
DNA sequencing method. We found that the ASPCR identified a significantly higher number of mutant alleles as compared to the
DNA sequencing method. Such discrepancies arise due to the non-specificity of some of the allele-specific primer sets and due to the lack of sensitivity of Sanger's
DNA sequencing method to detect minor alleles present in multiple clone
infections. This study reveals the need of a highly specific and sensitive method for genotyping and detecting minor
drug-resistant alleles present in multiple clonal
infections.