Background Antimalarial drug resistance threatens global malaria control efforts. Critical resistance-mediating mutations include those in the targets for sulfadoxine-pyrimethamine, transporter proteins and propeller domain of P. falciparum Kelch-13 protein that are associated with artemisinin partial resistance.

Methods To gain insight into antimalarial drug resistance trends, we surveyed vital P. falciparum polymorphisms from 10–16 health facilities across Uganda from 2016–22. We further assessed for evidence of evolutionary selection of resistant isolates by evaluating diversity in genomic regions flanking resistance loci.

Results Five mutations in the targets of sulfadoxine (PfDHPS 437G, 540E) and pyrimethamine (PfDHFR 51I, 59R, 108N) were prevalent (80–100%). The prevalence of PfDHFR 164L and PfDHPS 581G mutations, which mediate higher level antifolate resistance, varied between sites and over time. The PfDHFR 164L mutation was most common in southwestern and central Uganda (>20–75%), with increasing prevalences from 2016–17 to 2022, and increases were also seen in other parts of the country. The PfDHPS 581G mutation was also most common in the four sites in southwestern and central Uganda, although significant changes were not detected. Mutations in PfCRT and PfMDR1, associated with aminoquinoline resistance, were increasingly uncommon. The PfCRT 76T allele was detected in western Uganda bordering the Democratic Republic of Congo. The PfMDR1 86Y mutation, which was previously very common, was absent from 2018–2022. The 1246D mutation decreased, with 0% prevalence in 2022. Four PfK13 mutations (675V, 469Y, 469F and 561H), remain highly prevalent by 2022, compared to our earlier reports. Regarding evolutionary selection, isolates with antifolate and aminoquinoline resistance-associated alleles showed similar diversity to wild-type isolates, in genomic regions flanking the resistance loci.

Conclusion These results suggest limited evidence of recent selection of antifolate resistance, consistent with stable transmission of resistant isolates; and continued recovery of aminoquinoline sensitivity in Uganda.