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

The Plasmodium falciparum dihydrofolate reductase (PfDHFR) inhibitor pyrimethamine is combined with sulfadoxine for malaria chemoprevention, and proguanil, which is metabolized to the PfDHFR inhibitor cycloguanil, is combined with atovaquone for chemoprophylaxis in travelers. However, drug resistance, mediated by PfDHFR mutations, challenges the efficacies of these inhibitors. P218 is a novel PfDHFR inhibitor designed to overcome this challenge. However, data are available only for a small number of P. falciparum laboratory strains. We studied ex vivo susceptibilities to P218 and other PfDHFR inhibitors of 559 P. falciparum isolates collected in Tororo and Busia districts, Uganda from 2016-2020. 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 383 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 to proguanil, which does not directly inhibit PfDHFR. Importantly, differences in P218 susceptibilities between haplotypes were modest, with susceptibility to quadruple mutant (51I/59R/108N/164L) parasites (median IC50 1.4 nM for mixed isolates and 5.7 nM for pure mutants at position 164) at a level expected to maintain excellent treatment or preventive efficacy for the compound. Previous studies reported duplication of the GTP cyclohydrolyse I (pfgchI) gene in mutant (particularly 164L) parasites. For 58 Ugandan isolates with various genotypes and drug susceptibilities, associations were not seen between pfgchI copy number and I164L genotype or susceptibility to PfDHFR inhibitors. Overall, PfDHFR mutations associated with high level resistance to pyrimethamine and cycloguanil were common in Ugandan isolates; activity of P218 decreased with increasing mutations, but the compound retained potent activity against all Ugandan isolates, warranting continued research on PfDHFR inhibitors.