High-level artemisinin-resistance with quinine co-resistance emerges in P. falciparum malaria under in vivo artesunate pressure

P. falciparum was subjected to discontinuous in vivo artemisinin drug pressure by capitalizing on a novel model that allows for long-lasting, high-parasite loads. Intravenous artesunate was administered, using either single flash-doses or a 2-day regimen, to P. falciparum-infected humanized NOD/SCID IL-2Rγ^−/−immunocompromised mice, with progressive dose increments as parasites recovered. The parasite’s response to artemisinins and other available anti-malarial compounds was characterized in vivo and in vitro.

Artemisinin resistance evolved very rapidly up to extreme, near-lethal doses of artesunate (240 mg/kg), an increase of > 3000-fold in the effective in vivo dose, far above resistance levels reported from the field. Artemisinin resistance selection was reproducible, occurring in 80% and 41% of mice treated with flash-dose and 2-day regimens, respectively, and the resistance phenotype was stable. Measuring in vitro sensitivity proved inappropriate as an early marker of resistance, as IC₅₀ remained stable despite in vivo resistance up to 30 mg/kg (ART-S: 10.7 nM (95% CI 10.2–11.2) vs. ART-R₃₀: 11.5 nM (6.6–16.9), F = 0.525, p = 0.47). However, when in vivo resistance strengthened further, IC₅₀ increased 10-fold (ART-R₂₄₀ 100.3 nM (92.9–118.4), F = 304.8, p < 0.0001), reaching a level much higher than ever seen in clinical samples. Artemisinin resistance in this African P. falciparum strain was not associated with mutations in kelch-13, casting doubt over the universality of this genetic marker for resistance screening. Remarkably, despite exclusive exposure to artesunate, full resistance to quinine, the only other drug sufficiently fast-acting to deal with severe malaria, evolved independently in two parasite lines exposed to different artesunate regimens in vivo, and was confirmed in vitro.