Antimalarial Drug Resistance Assay

The ring-stage survival assays from 0 to 3 h were carried out as previously described with minor modifications [28 (link)]. Briefly, parasites were grown to 40 ml cultures at 5% hematocrit and schizonts were synchronized using a MACS magnet column (Miltenyi Biotec) into 10 ml cultures. Once schizonts had burst and merozoites had reinvaded, 2 ml of culture was transferred to 6 wells of 12-well cell culture cluster plates (Corning) at 2% hematocrit and 2% parasitaemia. Half of the wells were treated with 700 nM dihydroartemisinin (DHA) (Sigma) and the other half of the wells were treated with dimethyl sulfoxide (DMSO) (ThermoFisher). After 6 h all cultures were washed with ICM three times and transferred to a new plate to ensure complete removal of the drug. After an additional 66 h, slides were made on all cultures and 5000 RBCs were counted per culture. Proliferation was measured by the percent parasitaemia in the DHA treated culture over the percent parasitaemia in DMSO treated cultures. Two biological replicates each with three technical replicates were carried out for NHP4026, NHP4076, NHP4333, NHP1337, and NF54. Parasites are considered resistant if the percent proliferation is greater than 5% [13 (link)].