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

Abstract

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 <i>Plasmodium falciparum</i> dihydrofolate reductase (<i>dhfr</i>) and dihydropteroate synthase (<i>dhps</i>) mutations on the efficacy of SP. Western Kenya, including Busia County, is a high malaria transmission setting where molecular surveillance of <i>dhfr</i> and <i>dhps</i> mutations remains limited. This study assessed the prevalence and haplotype structure of <i>dhfr</i> and <i>dhps</i> mutations in <i>P. falciparum</i> isolates from Busia County, Kenya. A total of 66 samples of <i>P. falciparum</i> isolates collected from patients attending Matayos Sub-County Hospital between November 2024 and January 2025 were analysed. PCR amplification and Sanger sequencing targeted <i>dhfr</i> codons C50R, N51I, C59R, S108N/T, I164L, and <i>dhps</i> codons I431V, S436A/F, A437G, K540E, A581G, and A613S/T to determine mutation frequencies, haplotypes, and combined <i>dhps</i> and <i>dhfr</i> haplotype profiles. High frequencies of <i>dhfr</i> and <i>dhps</i> mutations were observed across the parasite isolates. The most common <i>dhfr</i> substitutions included N51I (85.2%) and C59R (75.4%), while S108N (32.8%) and S108T (19.7%) were detected at lower frequencies. <i>Dhfr</i> 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%). <i>Dhps</i> haplotypes were dominated by A437G (92.3%), K540E (40%) alone, and the A437G + K540E double mutant. Combined <i>dhfr</i> and <i>dhps</i> haplotype analyses revealed circulation of classical <i>dhfr</i> triple-mutant (N51I + C59R + S108N) backgrounds with dhps A437G. Quintuple haplotypes (<i>dhfr</i> N51I + C59R + S108T + I164L with <i>dhps</i> 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 <i>dhfr</i> and <i>dhps</i> haplotypes in <i>P. falciparum</i> 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.