Development of methods to analyse Plasmodium falciparum single nucleotide polymorphisms in PfCRT (А > C), PfMDR1 (A > T) and PfDHFR (G > A) genes that determine resistance to quinoline, diamino-pyrimidine and sulfonamide groups of antimalarial drugs

Abstract

BACKGROUND: The drug resistance of tropical malaria pathogens to mefloquine, chloroquine, pyrimethamine and their derivatives is associated with three single nucleotide polymorphisms: K76T (A403627C), S1034C (A960989T) and S108N (G748410A). These mutations are linked to changes in the structure of the PfCRT and PfMDR1 genes of Plasmodium falciparum.
 AIM: Develop methods for identifying single nucleotide polymorphisms that are suitable for early diagnosis of drug-resistant forms of tropical malaria.
 RESULTS: A method was developed based on restriction fragment length analysis using ApoI endonuclease to detect the K76T polymorphism (A403627C). The criterion for determining parasite resistance to chloroquine was the appearance of a single 145 bp band on the electropherogram. The genotype of the pathogens remained unchanged and their drug sensitivity was preserved, as indicated by the separation of two fragments of 98 and 47 bp.
 A system for detecting S108N (G748410A) was developed using the Bse1I endonuclease. The appearance of a single 507 bp band on the electropherogram indicated the mutant genotype of the pathogens, while the appearance of two fragments (323 and 184 bp) indicated an unchanged genotype and preservation of drug sensitivity of plasmodiae.
 To identify the A T polymorphism in the PfMDR1 gene at position 960989, polymerase chain reaction technology will be used with two allele-specific primers. One primer will detect the wild-type allele, and the other will detect the mutant genotype. The amplifiable fragment of the PfMDR1 gene contains sequences of the 1034th codon. Depending on the P. falciparum genotype, 261 bp fragments will be obtained with one of the allele-specific primers.
 CONCLUSION: Criteria for assessing drug resistance of P. falciparum were developed based on the analysis of obtained data. Haplotypes K76T (band 145 bp) and S1034C (band 262 bp with the direct primer S1034C-F2) serve as indicators of the relative resistance of pathogens to chloroquine, mefloquine, and their derivatives. Positive results of examination for haplotype S108N (bands 323 and 184 bp) should be considered as a sign of decreased sensitivity to pyrimethamine. The developed methods can be used in clinical practice and for epidemiological monitoring.