No abstract

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 first time the 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 pyrimethamine resistance locus in 38 P. vivax populations to look for evidence of positive selection pressure in human patients. The S58R (AGA)/S117N (AAC) double mutation was most common, being detected in 10/38 populations. Single mutation S117N (AAC), I173L (CTT) and S58R (AGA) SNPs were detected in 8/38, 2/38 and 1/38 populations, respectively. The F57L/I (TTA/ATA) and T61M (ATG) SNPs were not detected in any population examined. Although both soft and hard selective sweeps have occurred with striking differences between populations, there was a predominance of hard sweeps. A single resistance haplotype was present at high frequency in 9/14 populations, providing a strong evidence for the single emergence of these mutations. In contrast, 5/14 populations 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 (AGA)/S117N (AAC) and S117N (AAC) mutations arose multiple times from a single origin and spread to multiple different cities in the Punjab province through gene flow. Interestingly, the I173L (CTT) 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 exiting and new antimicrobial drugs and their combinations, control the movement of infected patients and mosquito vector control strategies.

No abstract

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 first time the 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 pyrimethamine resistance locus in 38 P. vivax populations to look for evidence of positive selection pressure in human patients. The S58R (AGA)/S117N (AAC) double mutation was most common, being detected in 10/38 populations. Single mutation S117N (AAC), I173L (CTT) and S58R (AGA) SNPs were detected in 8/38, 2/38 and 1/38 populations, respectively. The F57L/I (TTA/ATA) and T61M (ATG) SNPs were not detected in any population examined. Although both soft and hard selective sweeps have occurred with striking differences between populations, there was a predominance of hard sweeps. A single resistance haplotype was present at high frequency in 9/14 populations, providing a strong evidence for the single emergence of these mutations. In contrast, 5/14 populations 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 (AGA)/S117N (AAC) and S117N (AAC) mutations arose multiple times from a single origin and spread to multiple different cities in the Punjab province through gene flow. Interestingly, the I173L (CTT) 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 exiting and new antimicrobial drugs and their combinations, control the movement of infected patients and mosquito vector control strategies.

Pyrimethamine was first introduced for the treatment of malaria in Asia and Africa during the early 1980s, replacing chloroquine, and has become the first line of drugs in many countries. In recent years, development of pyrimethamine resistance in Plasmodium vivax has become a barrier to effective malaria control strategies. Here, we describe the use of meta-barcoded deep amplicon sequencing technology to assess the evolutionary origin of pyrimethamine resistance by analysing the flanking region of dihydrofolate reductase (dhfr) locus. The genetic modelling suggests that 58R and 173L single mutants and 58R/117N double mutants are present on a single lineage; suggesting a single origin of these mutations. The triple mutants (57L/58R/117N, 58R/61M/117N and 58R/117N/173L) share the lineage of 58R/117N, suggesting a common origin. In contrast, the 117N mutant is present on two separate lineages suggesting that there are multiple origins of this mutation. We characterised the allele frequency of the P. vivax dhfr locus. Our results support the view that the single mutation of 117N and double mutations of 58R/117N arise commonly, whereas the single mutation of 173L and triple mutations of 57L/58R/117N, 58R/61M/117N and 58R/117N/173L are less common. Our work will help to inform mitigation strategies for pyrimethamine resistance in P. vivax.