title: 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.
authors.affiliation: Department of Microbiology, Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), Puducherry 605006, India.
authors.forename: Hiasindh Ashmi
authors.initials: HA
authors.lastname: Antony
authors.affiliation: Department of Microbiology, Maharaja Krishna Chandra Gajapati Medical College (MKCG Medical College), Odisha 760004, India.
authors.forename: Sindhusuta
authors.initials: S
authors.lastname: Das
authors.affiliation: Department of Microbiology, Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), Puducherry 605006, India.
authors.forename: Subhash Chandra
authors.initials: SC
authors.lastname: Parija
authors.affiliation: Department of Microbiology, Maharaja Krishna Chandra Gajapati Medical College (MKCG Medical College), Odisha 760004, India.
authors.forename: Sanghamitra
authors.initials: S
authors.lastname: Padhi
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