Sequence analysis of pfcrt and pfmdr1 genes and its association with chloroquine resistance in Southeast Indian Plasmodium falciparum isolates

Abstract

Background: Due to the widespread resistance of Plasmodium falciparum to chloroquine drug, artemisinin-based combination therapy (ACT) has been recommended as the first-line treatment. This study aims to evaluate the extent of chloroquine resistance in P. falciparum infection after the introduction of ACT. This study was carried out based on the mutation analysis in P. falciparum chloroquine resistant transporter (pfcrt) and P. falciparum multidrug resistance 1 (pfmdr1) genes. Identification of these molecular markers plays a significant role in monitoring and assessment of drug resistance as well as in designing an effective antimalarial drug policy in India.

Methods: Sixty blood samples were collected from patients infected with P. falciparum from JIPMER, Puducherry and MKCG Medical College, Odisha. Polymerase chain reaction-restriction fragment length polymorphism was performed, targeting the point mutation of K76T in pfcrt and N86Y in pfmdr1 gene. The PCR products were sequenced, genotyped and further analysed for amino acid changes in these codons.

Results: The frequency of pfcrt mutation at 76th position was dominant for mutant T allele with 56.7% and wild type K, 43.3%. Majority of pfmdr1 86 allele were wild type, with N (90%) and mutant, Y (10%). Additionally, we found three haplotypes for CQ resistance, SVMNT, CVIET and CVIKT in association with the pfcrt gene. However, a poorly studied SNP in pfmdr1 gene (Y184F) associated with CQ resistance showed high frequency (70%) in P. falciparum isolates.

Conclusions: The point mutation K76T of pfcrt is high in P. falciparum suggesting a sustained high CQ resistance even after five years of the introduction of ACTs for antimalarial therapy. The present study suggests a strong association of CQ resistance with pfcrt T76, but not with the pfmdr1 Y86 mutation. However, sequence analysis showed that Y184F mutation on pfmdr1 gene was found to be associated with high resistance. Also, a new pfcrt haplotype 'CVIKT' associated with CQ resistance was found to be present in Indian strains of P. falciparum. The data obtained from this study helps in continuous monitoring of drug resistance in malaria and also suggests the need for careful usage of CQ in Plasmodium vivax malarial treatment.

Keywords: AQ, amodiaquine; CQ, chloroquine; Chloroquine; Drug resistance; K76T, lysine residue replaced by threonine at 76th position; LD, linkage disequilibrium; N86Y, asparagine replaced by tyrosine at 86th position; Plasmodium falciparum; SNP, single nucleotide polymorphism; Y184F, tyrosine replaced by phenylalanine at 184th position; pfcrt; pfcrt, Plasmodium falciparum chloroquine resistance transporter; pfmdr1; pfmdr1, Plasmodium falciparum multidrug resistance 1.

Fig. 1

Distribution of pfcrt and pfmdr1 alleles P. falciparum isolates collected from Puducherry and Odisha, India. K76T mutation in the pfcrt gene showed higher frequency associated with CQ-resistance. However, in the pfmdr1 gene, the Y184F mutation showed a higher distribution of frequency.

Fig. 2

Linkage disequilibrium plot between the SNPs pairs of pfcrt and pfmdr1 gene in Indian P.falciparum. The intergenic and intragenic association between the genes were illustrated using the LD plot for isolates collected from (a) Puducherry and (b) Odisha regions. The strength of LD between the SNPs is determined by the association of statistical significance by calculating the r² values and represented by the darkness of the boxes.