title: 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.
abstract: 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.
authors.affiliation: National Institute of Malaria Research (NIMR), Sector-8, Dwarka, New Delhi, India. sharma5divya@gmail.com.
authors.forename: Divya
authors.initials: D
authors.lastname: Sharma
authors.affiliation: National Institute of Malaria Research (NIMR), Sector-8, Dwarka, New Delhi, India. manila.lather@gmail.com.
authors.forename: Manila
authors.initials: M
authors.lastname: Lather
authors.affiliation: National Institute of Malaria Research (NIMR), Sector-8, Dwarka, New Delhi, India. cherry_dyke@yahoo.com.
authors.forename: Cherry L
authors.initials: CL
authors.lastname: Dykes
authors.affiliation: Maharshi Dayanand University (MDU), Rohtak, 124001, Haryana, India. suneja_a@yahoo.co.in.
authors.forename: Amita S
authors.initials: AS
authors.lastname: Dang
authors.affiliation: National Institute of Malaria Research (NIMR), Sector-8, Dwarka, New Delhi, India. adak.mrc@gmail.com.
authors.forename: Tridibes
authors.initials: T
authors.lastname: Adak
authors.affiliation: National Institute of Malaria Research (NIMR), Sector-8, Dwarka, New Delhi, India. singh@mrcindia.org.
authors.forename: Om P
authors.initials: OP
authors.lastname: Singh
mesh_terms: D000483:Alleles; D000962:Antimalarials / Q000494:pharmacology* / Q000627:therapeutic use; D037621:Artemisinins / Q000494:pharmacology / Q000627:therapeutic use; D003062:Codon; D017931:DNA Primers / Q000235:genetics; D016054:DNA, Protozoan / Q000737:chemistry / Q000302:isolation & purification; D004094:Dihydropteroate Synthase / Q000235:genetics; D004351:Drug Resistance / Q000235:genetics; D004359:Drug Therapy, Combination; D060005:Genotyping Techniques; D006801:Humans; D016778:Malaria, Falciparum / Q000188:drug therapy; D009154:Mutation; D010963:Plasmodium falciparum / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D020641:Polymorphism, Single Nucleotide; D011739:Pyrimethamine / Q000494:pharmacology; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods* / Q000592:standards; D013413:Sulfadoxine / Q000494:pharmacology; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*
chemical_list: D000962:Antimalarials; D037621:Artemisinins; D003062:Codon; D017931:DNA Primers; D016054:DNA, Protozoan; D013413:Sulfadoxine; C031327:artemisinin; D013762:Tetrahydrofolate Dehydrogenase; D004094:Dihydropteroate Synthase; D011739:Pyrimethamine
keywords: Allele-specific PCR; Pfdhfr gene; Plasmodium falciparum; Pyrimethamine