Contrasting evolutionary trajectories of pyrimethamine resistance in human and zoonotic malaria parasites

Pyrimethamine resistance in Plasmodium falciparum is driven by mutations at dihydrofolate reductase (DHFR) residues N51I, C59R, S108N/T, and I164L, with parallel substitutions in P. vivax (N50I, S58R, S117N/T, S173L). To evaluate the influence of drug pressure on parasite evolution, we compared the dihydrofolate reductase-thymidylate synthase (dhfr-ts) genes of human and zoonotic malaria parasites exposed to different drug environments.

Blood samples were collected from patients with symptomatic malaria in Thailand (P. falciparum, n = 241; P. vivax, n = 115) and from sympatric zoonotic infections, mainly in naturally infected macaques (P. knowlesi, n = 18; P. inui, n = 64). Complete dhfr-ts sequences were determined and validated.

Among 356 human and 145 zoonotic dhfr-ts sequences, zoonotic parasites exhibited markedly higher mutation counts, haplotype diversity, and nucleotide diversity. In P. falciparum, the high prevalence of the quadruple IRNL mutant indicated strong directional selection. In P. vivax, the coexistence of wild-type alleles and mutations outside resistance-associated residues suggests incomplete fixation. In zoonotic parasites, elevated rates of synonymous versus nonsynonymous substitutions, along with an excess of rare variants, indicate that purifying selection and genetic drift act against slightly deleterious mutations, likely due to protein functional or structural constraints.

Antifolate drug pressure has driven human malaria dhfr-ts genes toward fixation, whereas zoonotic orthologues evolve under a mutation-selection-drift balance. These findings highlight both the potential utility of antifolates against zoonotic malaria and the evolutionary consequences of sustained drug pressure.