MOLECULAR EPIDEMIOLOGY AND DRUG RESISTANCE ANALYSIS OF MALARIA PARASITIC SPECIES

Malaria is a devastating disease for the world and it is also endemic in Pakistan.This study was undertaken to determine the current epidemiology and antimalarial resistance of Plasmodium species, across Pakistan.This malariometric population survey was conducted in 2011 using blood samples collected from 801 febrile patients of all ages in four provinces and the capital city of Islamabad. Microscopically confirmed Plasmodium-positive blood samples were later reconfirmed by PCR. Of the 707 PCR-positive samples, Plasmodium falciparum infection was found in 128, Plasmodium vivax in 536 and mixed infection of P. falciparum plus P. vivax in 43 isolates.Ninety-four microscopy-positive samples were PCR-negative and results were reconfirmed by repeating PCR.None of the isolates was positive for Plasmodium malariae or Plasmodium ovale DNA. Although microscopy is gold standard for malaria diagnosis but in our study PCR showed diagnostic sensitivity and specificity over microscopy in malaria speciation.Limited numbers of studies have been carried out in Pakistan to establish the efficacy for treatment of P. falciparum and P. vivax with chloroquine (CQ) and sulfadoxine/pyrimethamine (SP), as CQ plus primaquine is recommended for P. vivax treatment and SP in combination with artesunate to P. falciparum. So we investigated molecular resistance markers in the pfcrt, pfmdr1, pfdhfr and pfdhps genes and determined the origin of chloroquine-resistant in P. falciparum parasites across several sites in Pakistan. Pyrosequencing, microsatellite typing and real-time PCR techniques were used for molecular characterization.The pfcrt K76T mutation was found in all samples as part of the SVMNT haplotype. Microsatellites flanking pfcrt showed high similarity to the signature found in India and Papua New Guinea. Pfmdr1 N86Y was found in 20% of samples and all samples carried a one copy of the pfmdr1 gene.The pfdhfr double mutation C59R+S108N was present in 87% of samples while the pfdhfr triple mutant (N51I+C59R+S108N) was not detected. Pfdhps A437G was found in 60% of samples. Pure pfdhps K540E was rare, at 4%, but mixed genotype 540K/E was found in 77% of samples. Similarly, pure pfdhps A581G was found in 4% of the isolates while mixed 581A/G was found in 39% of samples. Plasmodium vivax is the most common human malaria species in Pakistan.This study presents pattern of polymorphisms in the pvdhfr, pvdhps and pvmdr1 genes conferring drug resistance in P. vivax among Pakistani isolates. Out of 579 PCR-confirmed (536 P. vivax and 43 P. vivax/P.falciparum mixed infection) samples, 372 isolates were randomly selected for further molecular characterization by sequencing and real-time PCR. Seventy six of the isolates (23%) were double mutant at positions S58R and S117N in pvdhfr. Additionally, two mutations at positions N50I and S93H were observed in 55 (15%) and 24 (7%) of samples, respectively.Three 18 base pair insertion-deletions (indels) were observed in pvdhfr, with two insertions at different nucleotide positions in 36 isolates and deletions in 10. Ninety-two percent of samples contained the pvdhps (S382/A383G/K512/A553/V585) SAKAV wild type haplotype. For pvmdr1, all isolates were wild type at position Y976F and 335 (98%) carried the mutation at codon F1076L.All isolates harboured single copy of the pvmdr1 gene. Malaria epidemiological results of this study indicate that malaria infections in Pakistan are largely attributed by P. vivax then P. falciparum and mixed species (P. vivax + P. falciparum) infections are also prevalent. In addition, regional variation in the prevalence and species composition of malaria is high in this region.The P. falciparum drug resistance results suggest an emerging problem of multi-drug resistant in Pakistan.The chloroquine resistance genotype has reached complete fixation in the population, with a microsatellite pattern indicative of a selective sweep. Moreover, the incidence of mutations in both pfdhfr and pfdhps, albeit without the presence of the pfdhfr triple mutant, indicates that therapeutic efficacy trials are warranted to assess whether SP remains efficacious drug in combination with artesunate for the treatment of P. falciparum. P. vivax molecular studies showed that the prevalence of mutations associated with sulphadoxine-pyrimethamine resistance is low in Pakistan.The high prevalence of P. vivax mutant pvmdr1 codon F1076L indicates that efficacy of chloroquine plus primaquine could be in danger of being compromised, but further studies are required to assess its clinical relevance.These findings will serve as a baseline for further monitoring of drug-resistant P. vivax malaria in Pakistan.