Selective sweep and phylogenetic models for the emergence and spread of pyrimethamine resistance mutations in Plasmodium vivax

Pyrimethamine resistance is a major concern for the control of human haemoprotozoa, especially Plasmodium species. Currently, there is little understanding of how pyrimethamine resistance developed in Plasmodium vivax in the natural field conditions. Here, we present for the first time evidence of positive selection pressure on a dihydrofolate reductase locus and its consequences on the emergence and the spread of pyrimethamine resistance in P. vivax in the Punjab province of Pakistan. First, we examined the dihydrofolate reductase locus in 38 P. vivax isolates to look for evidence of positive selection pressure in human patients. The S58R (AG(A)under bar )/S117N (A(A)under barC) double mutation was most common, being detected in 10/38 isolates. Single mutation S117N (A(A)under barC), I173L ((C)under barTT) and S58R (AG(A)under bar) SNPs were detected in 8/38, 2/38 and 1/38 isolates, respectively. The F57L/I (TT(A)under bar/(A)under barT(A)under bar) and T61M (A(T)under barG) SNPs were not detected in any isolates examined. Although both soft and hard selective sweeps have occurred with striking differences between isolates, there was a predominance of hard sweeps. A single resistance haplotype was present at high frequency in 9/14 isolates, providing a strong evidence for single emergence of resistance by the single mutation, characteristics of hard selective sweeps. In contrast, 5/14 isolates carried multiple resistance haplotypes at high frequencies, providing an evidence of the emergence of resistance by recurrent mutations, characteristics of soft selective sweeps. Our phylogenetic relationship analysis suggests that S58R (AG(A)under bar)/S117N (A(A)under barC) and S117N (A(A)under barC) mutations arose multiple times from a single origin and spread to multiple different cities in the Punjab province through gene flow. Interestingly, the I173L ((C)under barTT) mutation was present on a single haplotype, suggesting that it arises rarely and has not spread between cities. Our work shows the need for responsible use of existing and new antimicrobial drugs and their combinations, control the movement of infected patients and mosquito vector control strategies.