Genetic models suggest single and multiple origins of dihydrofolate reductase mutations in Plasmodium vivax

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.