Longitudinal surveillance of kelch13 identifies C469Y, P553L, R561H and A675V mutations associated with artemisinin resistance in Western Kenya

Background Recent reports from East African countries indicate the emergence and spread of artemisinin partial resistance (ART-R), posing a significant threat to malaria control efforts in the region. The presence of critical Plasmodium falciparum kelch13 (k13) resistance markers, including C469Y , P553L and A675V , have been detected in Kenya, although their clinical significance remains unclear. This highlights an urgent need to closely monitor the prevalence of these mutations. Methods A total of 24,227 dried blood spot (DBS) samples were collected from 82 primary schools across eight counties in Western Kenya during repeated cross-sectional surveys conducted in 2019 (n = 7,941), 2022 (n = 8,086), and 2023 (n = 8,200). Initial screening was performed using a rapid diagnostic test (RDT), and DNA extraction was conducted on RDT-positive samples. These samples were further analyzed using a Pf 18S qPCR assay to quantify the Plasmodium falciparum DNA. Amplicons from malaria-positive samples were sequenced using a previously established amplicon deep sequencing pipeline to analyze mutations in Pfk13 . A total of 500, 920 and 1058 samples from 2019, 2022, and 2023, respectively were successfully processed, enabling a temporal assessment of the changes in k13 mutations in the region. Results Four mutations that have previously been associated with artemisinin resistance were found. The A675V mutation was the most prevalent, being found in all 8 counties. It was absent in 2019 and increased from 0.9% in 2022 to 5% in 2023. In contrast, the C469Y mutation declined from 4% in 2022 to 1% in 2023, maintaining a presence in 3 counties. The P553L mutation was only detected in 2022 in 1.2% of the samples across 5 counties. The R561H mutation was not detected in 2019 and 2022 but emerged at a low frequency (0.5%) in 2023 in 2 counties. Siaya and Kisumu were the only counties with all 4 validated mutations between 2022 and 2023. Conclusion The rising prevalence and geographical presence of the A675V mutation and the new detection of R561H in 2023 highlights the critical need for robust molecular surveillance systems to track the frequency and geographic spread of resistance markers. School-based sampling presents a practical and scalable approach for molecular surveillance, providing early warning signals for potential resistance hotspots. Additionally, the detection of the four WHO validated PfK13 artemisinin resistance mutations in Western Kenya underscores the urgency of conducting regular Therapeutic Efficacy Studies (TES) to assess the continued efficacy of frontline antimalarial treatments. Integrating molecular surveillance with TES will generate important data to inform national treatment policies and support the long-term effectiveness of malaria control strategies in Kenya.