Fig. 1.

In vitro drug assays. Standard drug assay. In the standard IC₅₀ assay, ring-stage parasites are treated with a gradient of drug concentrations, and readout is done at 72 h (by hypoxanthine incorporation, SYBR Green I staining, or HRP2 ELISA). IC₅₀ and IC₉₀ values are calculated based on the plotted sigmoid curve. PPQ (piperaquine) assay. For some PPQ-resistant parasites, drug assay is done using a much wider range of drug concentrations. In addition to the IC₅₀ and IC₉₀ values, area under the curve (AUC) is calculated for the second peak at PPQ concentrations of 0.1–10 μM. RSA (ring survival assay). Synchronized early ring-stage (0–3 h) parasites are treated with 700 nM DHA for 6 h, the drug is washed off, parasites are incubated for another 66 h, and surviving parasites are counted microscopically or by flow cytometry. A survival rate of 1% is used as the threshold for ART resistance. PSA (piperaquine survival assay). Synchronized ring-stage parasites are treated with 200 nM PPQ for 48 h, the drug is washed off, and parasites are incubated for an additional 24 h. Surviving parasites are counted by microscopy, and a survival rate of 10% is used as the cutoff value for PPQ resistance. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)