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Background   Plasmodium vivax  is the most prevalent malaria species in Pakistan, with a distribution that coincides with  Plasmodium falciparum  in many parts of the country. Both species are likely exposed to drug pressure from a number of anti-malarials including chloroquine, sulphadoxine-pyrimethamine (SP), and artemisinin combination therapy, yet little is known regarding the effects of drug pressure on parasite genes associated with drug resistance. The aims of this study were to determine the prevalence of polymorphisms in the SP resistance-associated genes  pvdhfr ,  pvdhps  and chloroquine resistance-associated gene  pvmdr1  in  P. vivax  isolates collected from across the country.    Methods  In 2011, 801 microscopically confirmed malaria-parasite positive filter paper blood samples were collected at 14 sites representing four provinces and the capital city of Islamabad. Species-specific polymerase chain reaction (PCR) was used to identify human  Plasmodium  species infection. PCR-positive  P. vivax  isolates were subjected to sequencing of  pvdhfr ,  pvdhps  and  pvmdr1  and to real-time PCR analysis to assess  pvmdr1  copy number variation.    Results  Of the 801 samples, 536 were determined to be  P. vivax , 128 were  P. falciparum , 43 were mixed  vivax / falciparum  infections and 94 were PCR-negative for  Plasmodium  infection. Of PCR-positive  P. vivax  samples, 372 were selected for sequence analysis. 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 copies of the  pvmdr1  gene.    Conclusions  The prevalence of mutations associated with SP resistance in  P. vivax  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 the clinical relevance of this observation. These findings will serve as a baseline for further monitoring of drug-resistant  P. vivax  malaria in Pakistan.

Prevalence and patterns of antifolate and chloroquine drug resistance markers in Plasmodium vivax across Pakistan