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.
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DS and ML acknowledge the award of Senior Research Fellowship by University Grants Commission, Government of India. The work was supported by intramural grant of the National Institute of Malaria Research (NIMR/VBC/2010/162). CLD was supported by National Institute of Allergy and Infectious Diseases, National Institutes of Health (NIH) grant U19AI089676. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or National Institute of Malaria Research.
Compliance with ethical standards
The study has been approved by the Institutional Ethics Committee (IEC) of National Institute of Malaria Research with approval number ECR/NIMR/EC/2012/42.
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Sharma, D., Lather, M., Dykes, C.L. et al. Disagreement in genotyping results of drug resistance alleles of the Plasmodium falciparum dihydrofolate reductase (Pfdhfr) gene by allele-specific PCR (ASPCR) assays and Sanger sequencing. Parasitol Res 115, 323–328 (2016). https://doi.org/10.1007/s00436-015-4750-2
- Plasmodium falciparum
- Pfdhfr gene
- Allele-specific PCR