Artemisinin Resistance in Africa: Plasmodium falciparum kelch13 R561H spread and emergence of other artemisinin partial resistance mutations in RwandadissertationsWernsman Young, Neeva NicolecreatorBailey, JeffreyAdvisorSimkin, AlfredReaderFairbrother, WilliamReaderBrown University. Biology and Medicine: Biotechnologysponsor2023vi, 39 p.born digitalThesis (Sc. M.)--Brown University, 2023thesesFirst detected in Rwanda, recent monitoring has detected multiple emerging Plasmodium falciparum kelch13 (K13) propeller gene mutations across East Africa. Mutations, such as R561H in Rwanda and C469Y/F, and A675V in Uganda, have been increasing and are associated with artemisinin partial resistance mainly manifesting as delayed clearance after treatment with ACTs and recrudescence. Coordinated surveillance efforts are necessary to track these K13 mutants and inform control efforts. Here we introduce a new method of collection: liquid blood drops, and apply pooled sequencing to provide a rapid initial assessment of resistance mutation allele frequency at multiple collection sites across Rwanda. Whole blood samples (n=2,713) from malaria-positive patients were collected from 20 Rwandan health centers from May to December 2022. Equal volumes of samples were pooled, stratified by site and month to generate 104 pools. DNA was extracted using magnetic beads and genotyped using molecular inversion probe targeted sequencing of key drug resistance genes. Site allele frequencies were calculated weighted by the number of samples in a given pool. From two rounds of sequencing, R561H was detected at 16 of 18 sites with an average frequency of 18.0% (0.8% to 49.1%). The higher site frequencies clustered in the center of the country near regions of high transmission. In Bugaragara, in northeast Rwanda, we found 49.1% average frequency that progressively increased monthly from October to December (0.0% to 94.1%). Notably, A675V, previously seen in Uganda, was found at 6.0% across 13 sites. Additionally, C469F, recorded in southern Uganda, was observed in 5 sites, with the highest frequency in Mushubati (10.6%), near the DRC border. Concerningly, G449A was at 1.1%, a WHO candidate resistance mutation never before reported in Africa but associated with a two-fold prolonged parasite clearance half-life in Asia. It was detected at 3 sites including Tanda (5.5%) where a large outbreak has been occurring. Overall, it appears multiple K13 mutations are rapidly expanding in Rwanda, further endangering control efforts and treatment efficacy with the potential of engendering partner drug resistance.MalariaRwandaMalaria modellingkelchPlasmodium falciparumEnglishRPB20230602