Quantification of Plasmodium falciparum HRP-2 as an alternative method to [3H]hypoxanthine incorporation to measure the parasite reduction ratio in vitro

Abstract

In the absence of a highly efficacious vaccine, chemotherapy remains the cornerstone to control malaria morbidity and mortality. The threat of the emergence of parasites resistant to artemisinin-based combination therapies highlights the need for new antimalarial drugs ideally with superior properties. The killing rate reflects the speed of action of antimalarial drugs that can be measured in vitro through the parasite reduction ratio (PRR) assay to shortlist interesting candidates. As a standard, the in vitro PRR assay is performed by measuring [3H]hypoxanthine incorporation of Plasmodium falciparum. This methodology is restricted to specialized laboratories due to the handling of radioactive material. In this work, we describe the sandwich enzyme-linked immunosorbent assay to detect P. falciparum histidine-rich protein 2 (HRP-2) as an alternative methodology to assess the PRR. We first validated the methodology with established antimalarial drugs (artesunate, chloroquine, pyrimethamine, and atovaquone) by comparing our results to previous results of the [3H]hypoxanthine incorporation readout provided by an expert lab and subsequently assessed the speed of action of four new antimalarial candidates (compound 22, chlorotonil A, boromycin, and ivermectin). The HRP-2 PRR assay obtained comparable results to the hypoxanthine incorporation readout in terms of parasite growth rate over time, lag phase, and parasite clearance time. In addition, parasite growth following drug exposure was quantified after 7, 14, 21, and 28 days of recovery time. In conclusion, the PRR assay based on HRP-2 is similar to [3H]hypoxanthine in determining a drug´s parasite-killing rate and can be widely used in all research laboratories.