A rtemisinin-based combination therapies (ACTs) have contributed greatly to the global decline of illness and death from malaria (1). However, the novel emergence of artemisinin resistance in eastern Africa has threatened the effectiveness of these breakthrough treatments (2-4). To avert potential disaster resulting from increased resistant malaria cases, the nature and extent of this resistance in Africa urgently needs to be characterized.Artemisinin resistance is conferred by some Plasmodium falciparum kelch 13 (K13) gene mutations, only a few of which are validated markers of resistance, defined by both in vitro resistance and delayed parasite clearance in treated patients. For candidate markers, only parasite clearance applies (1). In Rwanda, K13 mutations have increased over the past decade. K13 R561H, a validated marker associated with delayed parasite clearance, was recently observed in >10% of P. falciparum-positive samples (2,3,5). In neighboring Uganda, artemisinin resistance conferred by another mutation, K13 A675V, has recently been reported (4). We document in vitro artemisinin resistance in 3 P. falciparum patient isolates from Rwanda carrying K13 R561H, A675V, and C469F mutations.
The StudyWe recruited malaria patients in Huye District, Rwanda, during September-December 2019 and documented patient characteristics and consent, ethical clearance, and K13 variants elsewhere (2). Within 6 hours of sample collection, we cryopreserved all 66 P. falciparum isolates in ethylenediaminetetraacetic acid by washing the red blood cell pellet, adding freezing solution (3% sorbitol, 28% glycerol, 0.65% NaCl), and freezing at −80°C. Eight of the 66 isolates carried nonsynonymous K13 mutations (2). We successfully thawed and culture-adapted 4 of the isolates in which we identified K13 mutations: R561H, the current prevalent mutation in Rwanda; A675V, found in 11% of P. falciparum samples in Uganda; C469F, another candidate marker; and V555A, which is of unknown significance.We conducted a 0-3-h postinvasion ring-stage susceptibility assay (RSA) with the active metabolite dihydroartemisinin (6). We exposed ring stages to a 6-h pulse of 700 nmol/L dihydroartemisinin and cultured exposed and nonexposed isolates in vitro in triplicate for 72 h. We counted parasite density per ≥10,000 red blood cells on Giemsa-stained thin blood films and calculated the means of triplicates. Dividing parasite density in dihydroartemisinin-exposed cultures by the density in nonexposed cultures provided the RSA survival rate. We considered results if 72-h growth rates exceeded 1.5× rates in the nonexposed controls and had >3 successful independent triplicate experiments per isolate. We