Decreased susceptibility to dihydrofolate reductase inhibitors associated with genetic polymorphisms in Ugandan Plasmodium falciparum isolates

Background The Plasmodium falciparum dihydrofolate reductase (PfDHFR) inhibitors pyrimethamine and cycloguanil (the active metabolite of proguanil) have important roles in malaria chemoprevention, but drug resistance challenges their efficacies. A new compound, P218, was designed to overcome resistance, but drug susceptibility data for P. falciparum field isolates are limited. Methods We studied ex vivo PfDHFR inhibitor susceptibilities of 559 isolates from Tororo and Busia districts, Uganda from 2016-2020, sequenced 383 isolates, and assessed associations between genotypes and drug susceptibility phenotypes. Results Median IC50’s were 42,100 nM for pyrimethamine, 1,200 nM for cycloguanil, 13,000 nM for proguanil, and 0.6 nM for P218. Among sequenced isolates, three PfDHFR mutations, 51I (100%), 59R (93.7%), and 108N (100%), were very common, as previously seen in Uganda, and another mutation, 164L (12.8%), had moderate prevalence. Increasing numbers of mutations were associated with decreasing susceptibility to pyrimethamine, cycloguanil, and P218, but not proguanil, which does not act directly against PfDHFR. Differences in P218 susceptibilities were modest, with median IC50 1.4 nM for parasites with mixed genotype at position 164 and 5.7 nM for pure quadruple mutant (51I/59R/108N/164L) parasites. Conclusion Resistance-mediating PfDHFR mutations were common in Ugandan isolates, but P218 retained excellent activity against mutant parasites.