Persistent and Circulating Plasmodium falciparum dhfr and dhps Mutations in Busia County, Western Kenya

Malaria in pregnancy remains a major driver of poor maternal and neonatal health outcomes in sub-Saharan Africa. For decades, intermittent preventive treatment in pregnancy (IPTp), with sulphadoxine-pyrimethamine (SP), has mitigated malaria-associated health risks, but concerns have been raised regarding accumulated Plasmodium falciparum dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) mutations on the efficacy of SP. Western Kenya, including Busia County, is a high malaria transmission setting where molecular surveillance of dhfr and dhps mutations remains limited. This study assessed the prevalence and haplotype structure of dhfr and dhps mutations in P. falciparum isolates from Busia County, Kenya. A total of 66 samples of P. falciparum isolates collected from patients attending Matayos Sub-County Hospital between November 2024 and January 2025 were analysed. PCR amplification and Sanger sequencing targeted dhfr codons C50R, N51I, C59R, S108N/T, I164L, and dhps codons I431V, S436A/F, A437G, K540E, A581G, and A613S/T to determine mutation frequencies, haplotypes, and combined dhps and dhfr haplotype profiles. High frequencies of dhfr and dhps mutations were observed across the parasite isolates. The most common dhfr substitutions included N51I (85.2%) and C59R (75.4%), while S108N (32.8%) and S108T (19.7%) were detected at lower frequencies. Dhfr haplotypes identified included N51I + C59R, N51I + C59R + S108N, and a N51I + C59R + S108T + I164L variant. The I164L mutation was detected at a frequency of 18.0% and was observed exclusively on a non-canonical S108T background (19.7%). Dhps haplotypes were dominated by A437G (92.3%), K540E (40%) alone, and the A437G + K540E double mutant. Combined dhfr and dhps haplotype analyses revealed circulation of classical dhfr triple-mutant (N51I + C59R + S108N) backgrounds with dhps A437G. Quintuple haplotypes (dhfr N51I + C59R + S108T + I164L with dhps A437G) and rare complex haplotypes incorporating both I164L and K540E or I164L and S436F were also detected. These findings indicate the persistence and circulation of both canonical and non-canonical dhfr and dhps haplotypes in P. falciparum isolates from Busia County. This study highlights the need for continuous molecular and phenotypic surveillance to clarify the functional and epidemiological significance of parasites carrying S108T and I164L mutations, and to inform IPT policy.