Two Decades of Molecular Surveillance in Kenya Reveal Shifting Plasmodium falciparum Drug Resistance Mutations Linked to Frontline Drug Changes

Malaria control has faced repeated challenges due to the emergence and spread of antimalarial resistance. In the present study, temporal trends in Plasmodium falciparum resistance mutations were analyzed using molecular inversion probe sequencing of 625 samples from 11 Kenyan sites (1998-2021), spanning multiple frontline treatments. After the phased adoption of artemether-lumefantrine as first-line treatment of uncomplicated malaria in 2006, a rapid increase in multidrug resistance protein 1 N86 and D1246 alleles associated with reduced lumefantrine susceptibility was observed, along with an accelerated decline in the chloroquine resistance transporter CVIET haplotype associated with chloroquine and amodiaquine resistance. Sulfadoxine-pyrimethamine (SP) resistance mutations (dihydrofolate reductase [DHFR] 51I, 59R, 108N; dihydropteroate synthase 437G, 540E) reached near-fixation after the introduction of SP as the first-line therapy for uncomplicated malaria in 1998 and persisted at near-fixation through 2021. The high-level pyrimethamine resistance mutation DHFR 164L emerged and expanded after 2012. Furthermore, mutations associated with reduced artemisinin efficacy were identified in ubiquitin-specific protease 1, adaptor protein complex 2, mu subunit, and sarcoplasmic/endoplasmic reticulum calcium adenosine triphosphatase. Although no validated kelch13 (K13) artemisinin partial resistance mutations were detected, several novel K13 propeller domain missense mutations, including 473F and 569S, were observed after the adoption of artemisinin-based combination therapies. These findings reveal the rapid, dynamic evolution of drug resistance in the context of shifting antimalarial drug pressures and underscore the need for sustained genomic surveillance in malaria-endemic regions to inform adaptive treatment strategies. The persistence of SP resistance and the emergence of markers associated with reduced susceptibility to artemisinins and partner drugs raise concerns about the long-term efficacy of current antimalarials for treatment, seasonal malaria chemoprevention, and intermittent preventive interventions.