2.5. Standard in vitro antimalarial susceptibility testing

In vitro antimalarial susceptibility of three multiclonal isolates and their component parasite haplotypes were measured against chloroquine (CQ), mefloquine (MFQ) and piperaquine (PPQ) using a standardized SYBR® Green Antimalarial Assay (Hartwig et al., 2013). CQ and MFQ were both purchased from Sigma Aldrich while PPQ was obtained from the Worldwide Antimalarial Resistance Network (https://www.wwarn.org/). Drugs were prepared as 10 mM stock solutions in distilled water (CQ) or 70% ethanol (MFQ and PPQ) and were filter-sterilized. Drug assays were run in duplicate at 0.5% parasitemia and 1.5% hematocrit in 96-well plates and were incubated at 37 °C for 72 h. Each drug assay run included at least one of the three clonal P. falciparum laboratory lines 3D7 (MRA-102), DD2 (MRA-150) or K1 (MRA-159) as a control. Following the incubation step, parasites were lysed by freeze-thaw, transferred to a new 96-well detection plate and then labeled with a DNA-fluorescent dye SYBR® Green I (Molecular Probes, USA). The detection plate was incubated in the dark for 45 min and fluorescence was measured at excitation and emission wavelengths of 490 and 540 nm respectively using the SpectraMax fluorescence microplate reader (Molecular Devices, USA). Dose-response curves and IC₅₀ values were determined from log-transformed fluorescence counts at each serial dilution of the antimalarial drug using GraphPad Prism v7.0 (GraphPad, USA). An unpaired t-test was used to assess whether any two IC₅₀ values are significantly different. Mean IC₅₀s for ≥3 independent assays were compared using one-way analysis of variance (One-way ANOVA). Both statistical tests were implemented in GraphPad Prism v7.0 with the level of significance set at p < 0.05.