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

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 combina-tion therapies highlights the need for new antimalarial drugs ideally with superior properties. The killing rate reflects the speed of action of antimalarial drugs, which can be measured in vitro through the par-asite 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 specialised laboratories owing to the handling of radioactive material. In this work, we describe a 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 es-tablished antimalarial drugs (artesunate, chloroquine, pyrimethamine and atovaquone) by comparing our results with previous results of the [3H]hypoxanthine incorporation readout provided by an expert lab-oratory, and subsequently assessed the speed of action of four new antimalarial candidates (compound 22, chlorotonil A, boromycin and ivermectin). The HRP-2 PRR assay achieved comparable results to the [3H]hypoxanthine incorporation readout in terms of parasite growth rate over time, lag phase and par-asite 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.& COPY; 2023 Elsevier Ltd and International Society of Antimicrobial Chemotherapy. All rights reserved.

Automated summary

In the absence of an effective vaccine, chemotherapy remains crucial for controlling malaria. The emergence of drug-resistant parasites emphasizes the need for new antimalarial drugs with improved properties. A sandwich enzyme-linked immunosorbent assay that detects the presence of Plasmodium falciparum histidine-rich protein 2 (HRP-2) was developed as an alternative to assess the parasite reduction ratio (PRR). The assay showed comparable results to the traditional method of measuring [3H]hypoxanthine incorporation. Rating: 7/10.