Drug sensitivity testing by SYBR Green I-based assay

SYBR Green I-based in vitro and immediate ex vivo drug sensitivity assay was used to test each P. falciparum field isolate and control against a panel of five antimalarial drugs, namely, piperaquine (PPQ), dihydroartemisinin (DHA), lumefantrine (LM), artemether (ART), and chloroquine diphosphate (CQ) [25]. Drugs were prepared to the desired concentration as described earlier [25]. Freshly collected samples were subjected to immediate ex vivo assay [29, 30] while culture-adapted field isolates and reference clones that had attained 3–8% parasitemia in continuous culture were subjected to in vitro malaria SYBR Green I assay [25, 31]. Briefly, the sample parasitemia was adjusted to 1% at 2% hematocrit using complete media and uninfected O+ RBCs [25, 30] and mixed to homogeneity. 100 μl of this mixture was loaded to each well of the 96-well microtiter plates (catalog no: 167008 Nunc, Inc, Roskilde, Denmark) containing different concentrations of drug aliquots, incubated at 37°C under a 90% N₂, 5% O₂, and 5% CO₂ humidified environment and terminated 72 h later [25, 31]. Lysis buffer containing SYBR Green I dye was added and then incubated for 24 h in the dark. Readings were then done using Tecan Genios Plus® (Tecan US, Inc., Durham, NC) which gave the relative fluorescence units (RFUs). Using these readouts, the strength of inhibition of each drug, and the 50% inhibition concentration (IC₅₀) were calculated using Graphpad prism® 8.1 for Windows® software (Graphpad Software, San Diego, CA, USA) using non-linear regression analysis of the dose-response curve [25, 31].