Genomic Evidence of Pyrimethamine Resistance and Selective Sweeps in Plasmodium Vivax from Pakistan

Abstract

Background: Resistance to antifolate drugs such as pyrimethamine threatens malaria elimination in South and Southeast Asia. Mutations in the dihydrofolate reductase (dhfr) gene of Plasmodium vivax reduce drug efficacy by altering enzyme binding, leading to treatment failure. Despite P. vivax causing most malaria cases in Pakistan, genomic data on antifolate resistance remain scarce. This study examines dhfr polymorphisms in P. vivax isolates from Punjab, Pakistan, using deep amplicon sequencing to assess mutation prevalence, genetic diversity, and signatures of positive selection, providing critical insights into the molecular evolution and regional spread of pyrimethamine-resistant P. vivax lineages.

Methods: A total of 38 P. vivax–positive blood samples collected during a cross-sectional survey of malaria-endemic districts were analysed using deep amplicon sequencing of the dhfr locus. After quality filtering, single-nucleotide polymorphisms (SNPs) associated with antifolate resistance were quantified. Haplotype reconstruction and genetic diversity analyses were performed using DnaSP and Network 4.6.1, while neutrality and selection pressures were assessed through Tajima’s D and Fay & Wu’s H statistics.

Results: Three major nonsynonymous dhfr mutations—S58R, S117N, and I173L—were identified, with the S58R/S117N double mutant being most prevalent (26%). Allele frequencies ranged from 2.1% to 100%, indicating differential local selection intensity. Fourteen isolates exhibited strong signals of positive directional selection (Tajima’s D = −2.1), consistent with ongoing selective sweeps driven by antifolate exposure. Forty-two unique dhfr haplotypes were detected, nine dominated by resistant alleles (>95% frequency) characteristic of hard sweeps, and five showing multiple coexisting haplotypes suggestive of soft sweeps. The distribution of resistant haplotypes across multiple districts indicates gene flow and regional transmission of resistant lineages.

Conclusion: This genomic analysis provides compelling evidence of widespread pyrimethamine resistance and adaptive evolution within P. vivax populations in Pakistan. The predominance of double- and triple-dhfr mutant haplotypes reflects sustained antifolate pressure and the emerging fixation of resistant alleles. Integrating genomic surveillance into national malaria control frameworks is imperative for early detection of resistance trends, refinement of treatment policies, and prevention of further spread of antifolate-resistant P. vivax strains.