Temporal Trends in Artemisinin Partial Resistance and Other Antimalarial Drug Mutations in Plasmodium falciparum from Kagera Region, Northwestern Tanzania, 2021-2023.

Artemisinin-based combination therapies (ACTs) remain the cornerstone of malaria treatment, yet the emergence of artemisinin partial resistance (ART-R) in Africa threatens their efficacy. ART-R is primarily associated with mutations in the Plasmodium falciparum kelch13 (K13) gene, notably R561H, which has been linked to delayed parasite clearance in East Africa. We conducted longitudinal molecular surveillance in Tanzania's Kagera region from 2021 to 2023 to characterize temporal and spatial trends in ART-R and other antimalarial resistance markers. Using molecular inversion probes targeting key antimalarial resistance genes, we genotyped 2,826 isolates from seven districts. The WHO-validated K13 mutation R561H persisted in border districts of Karagwe and Kyerwa, with prevalence ranging from 14-26%, and appeared for the first time in Muleba (5.0%) and Bukoba rural district (0.7%) in 2023, indicating eastward spread toward Lake Victoria. Regional average prevalence of R561H rose from 5.5% in 2021 to 11.3% in 2022, then stabilized at 6.9% in 2023. Additional validated (A675V) and candidate (V568G, P441L) mutations were detected at low frequencies, suggesting ongoing diversification of the parasite population under local selection pressures. Partner-drug resistance markers showed minimal change: MDR1 N86Y remained near fixation, while CRT K76T declined from 5.9% (2021) to 2.4% (2023). Antifolate resistance was entrenched, with early DHFR and DHPS mutations near fixation and high-level resistance markers (DHFR I164L and DHPS A581G) exhibiting marked spatial heterogeneity, peaking at 38.1% and 48.1%, respectively, in eastern districts. These findings reveal micro-geographic heterogeneity in resistance and ongoing spread, emphasizing the need for district-level surveillance to detect emerging hotspots and guide interventions. Sustained molecular monitoring is critical to inform treatment policy, preserve ACT efficacy, and mitigate the risk of widespread resistance in East Africa. Artemisinin-based combination therapies (ACTs) remain the cornerstone of malaria treatment, yet the emergence of artemisinin partial resistance (ART-R) in Africa threatens their efficacy. ART-R is primarily associated with mutations in the Plasmodium falciparum kelch13 (K13) gene, notably R561H, which has been linked to delayed parasite clearance in East Africa. We conducted longitudinal molecular surveillance in Tanzania's Kagera region from 2021 to 2023 to characterize temporal and spatial trends in ART-R and other antimalarial resistance markers. Using molecular inversion probes targeting key antimalarial resistance genes, we genotyped 2,826 isolates from seven districts. The WHO-validated K13 mutation R561H persisted in border districts of Karagwe and Kyerwa, with prevalence ranging from 14-26%, and appeared for the first time in Muleba (5.0%) and Bukoba rural district (0.7%) in 2023, indicating eastward spread toward Lake Victoria. Regional average prevalence of R561H rose from 5.5% in 2021 to 11.3% in 2022, then stabilized at 6.9% in 2023. Additional validated (A675V) and candidate (V568G, P441L) mutations were detected at low frequencies, suggesting ongoing diversification of the parasite population under local selection pressures. Partner-drug resistance markers showed minimal change: MDR1 N86Y remained near fixation, while CRT K76T declined from 5.9% (2021) to 2.4% (2023). Antifolate resistance was entrenched, with early DHFR and DHPS mutations near fixation and high-level resistance markers (DHFR I164L and DHPS A581G) exhibiting marked spatial heterogeneity, peaking at 38.1% and 48.1%, respectively, in eastern districts. These findings reveal micro-geographic heterogeneity in resistance and ongoing spread, emphasizing the need for district-level surveillance to detect emerging hotspots and guide interventions. Sustained molecular monitoring is critical to inform treatment policy, preserve ACT efficacy, and mitigate the risk of widespread resistance in East Africa.