Multiple local PfDHFR I164L haplotype expansions drive Plasmodium falciparum antifolate resistance in Uganda.
Mutations in the Plasmodium falciparum genes, pfdhfr and pfdhps, drive antifolate resistance and threaten malaria control in regions where sulfadoxine-pyrimethamine (SP) is the primary chemoprevention strategy. The spatial patterns and evolutionary dynamics of these mutations in high-transmission settings remain incompletely understood. Here we genotyped 11 resistance-associated mutations in pfdhfr and pfdhps in 4,725 P. falciparum isolates collected from 16 Ugandan health facilities as part of annual surveillance between 2016 and 2022. Notably, we show that the frequency of PfDHFR I164L, which confers higher pyrimethamine resistance, increased over time from 19.4% to 32.4%. Using identity-by-descent, haplotype structure, and extended haplotype homozygosity analyses, we show that PfDHFR I164L is present on multiple haplotype backgrounds and undergoes localised expansions, without detectable signatures of recent positive selection at all but one site. Our results suggest that the evolution of antifolate resistance, driven by PfDHFR I164L, is spatially heterogeneous and complex in regions that primarily use SP chemoprevention programmes.