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 this study, we analyzed temporal trends in Plasmodium falciparum drug resistance mutations using molecular inversion probes in 642 samples collected in nine sites in Kenya from 1998 to 2021, spanning multiple changes in frontline antimalarial therapies. Following the adoption of artemether-lumefantrine (AL) in 2006, we observed a rapid expansion of MDR1 N86N, Y184F, and D1246D alleles associated with reduced lumefantrine susceptibility, alongside an accelerated decline in chloroquine resistance mutations CRT K76T and A220S. Sulphadoxine-pyrimethamine (SP) resistance mutations, including DHFR N51I, C59R, S108N and DHPS A437G, K540E, rapidly reached fixation following SP introduction in 1998 and persisted through 2021. The high-level pyrimethamine resistance mutation DHFR I164L emerged and expanded after 2012. Further, we identified mutations associated with reduced artemisinin efficacy in the upb1 , ap2mu , and atp6 genes. Although no validated Kelch 13 (K13) mutations linked to artemisinin partial resistance were detected, we observed the emergence of several previously unreported K13 propeller domain missense mutations, including C473F and A569S, after the adoption of artemisinin-based combination therapies. These findings demonstrate the rapid and dynamic evolution of drug resistance in response to shifting anti-malarial 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 artemisinin and partner drugs raise concerns about the long-term efficacy of current malaria therapies for treatment, seasonal malaria chemoprevention, and intermittent preventive interventions.