Ex Vivo Malaria Drug Susceptibility Assay

Chloroquine, quinine, mefloquine, desethylamodiaquine, lumefantrine, piperaquine, and pyronaridine were obtained from Sigma-Aldrich (St Quentin Fallavier, France). For the ex vivo chemosusceptibility assay, each isolate was aliquoted in 96-well plates previously treated with a gradient of antimalarial drug concentrations without culture adaptation, as previously described [37 (link)]. The plates were then incubated for 72 h at 37 °C in a controlled atmosphere with 10% O₂, 5% CO₂, and 85% N₂. An ELISA assay was then performed by targeting the HRP2 protein using the Malaria Ag Celisa kit (ref KM2159, Cellabs PTY LDT, Brookvale, Australia) to estimate parasite growth, as previously described [38 (link)].
Each batch of plates was controlled by assessing the Chloroquine-resistant P. falciparum strain W2 (MR4, Charlottesville, VA, USA) in between three and six independent experiments.