Using Next Generation Chemiluminescent Probes to Improve the Plasmodium falciparum in vitro Parasite Reduction Ratio (PRR) Assay.

Malaria remains a major global health threat, and the emergence of partial artemisinin resistance challenges current treatment regimens. Reliable antimalarial screening assays are therefore essential for identifying new drug candidates. The parasite reduction ratio (PRR) assay provides valuable pharmacodynamic insights but is limited by its labor-intensive, 14- to 28-day incubation period. We developed an optimized PRR assay protocol using the highly sensitive chemiluminescence-based lacZ/β-galSENSOR readout, reducing assay incubation duration to 7 days while maintaining informative pharmacodynamic parameters, including lag phase, parasite clearance time, parasite reduction ratio, and maximum killing effect. In contrast, the [3H]-hypoxanthine incorporation method failed to detect viable parasites reliably and consistently overestimated drug activity with the shortened protocol. This novel lacZ/β-galSENSOR PRR assay enables laboratories without access to radioactive facilities to evaluate antimalarial compounds efficiently, providing robust time-killing profiles with greater convenience, higher throughput, and lower equipment requirements than existing readout methods.