Population-level shift in ex vivo artemisinin susceptibility in Plasmodium falciparum : comparison of 2014–2015 and 2022–2024 surveys in northern Uganda

BACKGROUND: Artemisinin (ART) partial resistance has been reported in Southeast Asia and East Africa, but its population-level phenotypic dynamics remain poorly understood. We aimed to evaluate population-level changes in ART susceptibility in Plasmodium falciparum in northern Uganda using an ex vivo quantitative ring-stage survival assay (qRSA), which assesses parasite survival across seven serial dihydroartemisinin (DHA) concentrations. METHODS: We conducted repeated cross-sectional surveys in 2014, 2015, 2022, and 2024 in Gulu, northern Uganda. Ex vivo qRSA was used to compare parasite survival across DHA concentrations before and after the known emergence of ART partial resistance. qRSA resistance level was operationally defined as the highest DHA concentration at which survival rate (SR) was ≥1%. RESULTS: 323 participants with uncomplicated symptomatic P. falciparum monoinfection were enrolled. qRSA data were obtained from 156 samples. After the known emergence of ART partial resistance, low-level parasite survival persisted toward progressively higher DHA concentrations, resulting in a shift toward higher qRSA resistance level. All pure kelch13 A675V mutants were classified at the highest qRSA resistance level (700 nM), whereas pure C469Y mutants were distributed across qRSA resistance levels from 87.5 to 700 nM. A similar, but less robust, shift was observed among kelch13 wild-type isolates, accompanied by increased SRs, particularly at 700 nM. CONCLUSIONS: These findings suggest that the population-level shift in ART susceptibility was characterized by persistence of low-level survival toward higher DHA concentrations. Multi-concentration RSA may provide additional insights into early changes in ART susceptibility that are not captured by conventional binary resistance classifications.