Dual-luciferase-based fast and sensitive detection of malaria hypnozoites for the discovery of anti-relapse compounds.

Efforts to eradicate Plasmodium vivax malaria are hampered by the presence of hypnozoites, persisting stages in the liver that can reactivate after prolonged periods of time enabling further transmission and causing renewed disease. Large-scale drug screening is needed to identify compounds with antihypnozoite activity, but current platforms rely on time-consuming high-content fluorescence imaging as read-out, limiting assay throughput. We here report an ultrafast and sensitive dual-luciferase-based method to differentiate hypnozoites from liver stage schizonts using a transgenic P. cynomolgi parasite line that contains Nanoluc driven by the constitutive hsp70 promoter, as well as firefly luciferase driven by the schizont-specific lisp2 promoter. The transgenic parasite line showed similar fitness and drug sensitivity profiles of selected compounds to wild type. We demonstrate robust bioluminescence-based detection of hypnozoites in 96-well and 384-well plate formats, setting the stage for implementation in large scale drug screens.