Plasmodium Ovale Spp Dhfr Mutations Associated with Reduced Susceptibility to Pyrimethamine in Sub-Saharan Africa: a Retrospective Genetic Epidemiology and Functional Study

Background Mutations in the Plasmodium falciparum dhfr gene confer resistance to pyrimethamine, which is widely used for malaria chemoprevention in Africa. We aimed to evaluate the frequency and evolution of dhfr mutations Plasmodium ovale spp in Africa and their functional consequences, which are incompletely characterised. Methods We analysed dhfr mutations and their frequencies in P ovale spp isolates collected between Feb 1, 2004, Aug 31, 2023, from the French National Malaria Reference Centre collection and from fi eld studies in Benin, Gabon, Kenya. Genetic patterns of positive selection were investigated. Full-length recombinant wild-type and mutant DHFR enzymes from both P ovale curtisi and P ovale wallikeri were expressed in bacteria to test whether the most common mutations reduced pyrimethamine susceptibility. Findings We included 518 P ovale spp samples (314 P ovale curtisi and 204 P ovale wallikeri ). In P ovale curtisi Ala15Ser-Ser58Arg was the most common dhfr mutation (39%; 124 of 314 samples). In P ovale wallikeri , mutations were less frequent, with Phe57Leu-Ser58Arg reaching 17% (34 of 204 samples). These two mutants were the most prevalent in central and east Africa and were fi xed in Kenyan isolates. We detected six and four other non-synonymous mutations, representing 8% (24 isolates) and 2% ( fi ve isolates) of the P ovale curtisi P ovale wallikeri isolates, respectively. Whole-genome sequencing and microsatellite analyses revealed reduced genetic diversity around the mutant pocdhfr and powdhfr genes. The mutant DHFR proteins showed structural changes at the pyrimethamine binding site in-silico, con fi rmed by a 4-times increase in pyrimethamine half-maximal inhibitory concentration in an Escherichia coli growth assay for the Phe57Leu-Ser58Arg mutant 50-times increase for the Ala15Ser-Ser58Arg mutant, compared with the wild-type counterparts. Interpretation The widespread use of sulfadoxine - pyrimethamine for malaria chemoprevention might have exerted fortuitous selection pressure for dhfr mutations in P ovale spp. This calls for closer monitoring of dhfr and dhps mutations in P ovale spp.