In vitro antimalarial susceptibility testing using parasite survival assays

The parasite survival rate (% S) of a subset of parasite isolates after DHA or PPQ exposure was measured using parasite survival assays, as previously described (18, 19), because standard IC₅₀ assays fail to accurately capture parasite susceptibility to these compounds. Briefly, parasites were exposed to a pharmacologically relevant drug dose (700-nM DHA for 6 h or 200-nM PPQ for 48 h) in 48-well plates in a modular incubation chamber under standard culture conditions and gassed with 5% O₂, 5% CO₂, and 90% N₂. The drug was then removed, and parasites were allowed to recover and proliferate in normal growth media for 66 h in ring-stage survival assay (RSA) for DHA or 24 h in piperaquine survival assay (PSA) for PPQ. Percent parasite survival was determined by examining and counting viable parasites in drug-treated versus untreated wells using light microscopy (see Fig. 1). Parasites showing a survival rate of ≥10% were deemed drug resistant based on previously defined thresholds (18, 19, 22).