Optimizing the HRP-2 in vitro malaria drug susceptibility assay using a reference clone to improve comparisons of Plasmodium falciparum field isolates

Abstract

Apparent emerging artemisinin-resistant Plasmodium falciparum malaria in Southeast Asia requires development of practical tools to monitor for resistant parasites. Although in vitro anti-malarial susceptibility tests are widely used, uncertainties remain regarding interpretation of P. falciparum field isolate values. Performance parameters of the W2 P. falciparum clone (considered artemisinin “sensitive”) were evaluated as a reference for the HRP-2 immediate ex vivo assay. Variability in W2 IC₅₀s was assessed, including intra- and inter-assay variability among and between technicians in multiple experiments, over five freeze-thaw cycles, over five months of continuous culture, and before and after transport of drug-coated plates to remote field sites. Nominal drug plate concentrations of artesunate (AS) and dihydroartemisinin (DHA) were verified by LC-MS analysis. Plasmodium falciparum field isolate IC₅₀s for DHA from subjects in an artemisinin-resistant area in Cambodia were compared with W2 susceptibility. Plate drug concentrations and day-to-day technical assay performance among technicians were important sources of variability for W2 IC₅₀s within and between assays. Freeze-thaw cycles, long-term continuous culture, and transport to and from remote sites had less influence. Despite variability in W2 susceptibility, the median IC₅₀s for DHA for Cambodian field isolates were higher (p ex vivo HRP-2 assay from areas of artemisinin resistance. Methods to increase the reproducibility of plate coating may improve overall assay interpretability and utility.