Clinically applicable parasite viability assay for rapid assessment of antimalarial pharmacodynamic endpoints

Abstract

Evaluating the efficacy of antimalarial drugs is crucial in the fight against malaria, as the parasiticidal effectiveness of these drugs often predicts their clinical success. Parasite Reduction Ratio (PRR) assay is the current method of choice for assessing antimalarial's ability to halt parasite recovery after treatment; however, it is time-consuming and resource-intensive, making it less ideal for low-resource or clinical settings. Recent advancements in parasite viability assessment, such as use of the MitoTracker (MT) which probes stain active mitochondria in live cells, provide a faster way to distinguish live from dead parasites using the flow cytometry, providing, thus, timely insights to inform treatment outcomes in clinical trials. In this study, the accuracy of direct viability assessment (DVA) of the parasite using MT staining was compared with the previously established PRR assay to evaluate the efficacy of four reference antimalarial drugs (dihydroartemisinin, chloroquine, atovaquone, and pyrimethamine) using P. falciparum 3D7 strain. Additionally, a mathematical model was developed to estimate key parameters, such as maximum killing rate and lag phase. The model yielded comparable values for these compounds across both assays reinforcing the reliability of the DVA assay for rapidly assessing antimalarial drug efficacy. In conclusion, the DVA relies on specialized equipment and technical expertise. However, it can emerge as an alternative to the PRR, offering a faster and more clinically suited approach for studies.

Keywords: MitoTracker; Plasmodium; parasite viability; parasite reduction ratio; pharmacodynamics.

Fig 1

Experimental setups for in vitro PRR and DVA assays: a comparative analysis. P. falciparum 3D7 strain was exposed to antimalarials (10 × IC₅₀). Samples were collected at 0, 24, 48, 72, 96, and 120 h, then washed and distributed into 96-well plates—each compound was tested in triplicate, with eight replicates for the 0 h time point. For the DVA assay, plates were immediately stained with MT Deep Red FM, incubated for 1 h, and analyzed via flow cytometry. While for the PRR assay, each sample underwent fourfold serial dilutions, followed by a 14-day incubation. Parasite viability was determined through microscopy and flow cytometry based on the dilution limit.

Fig 2

Comparison of the killing patterns of the experimental DVA assay versus the experimental and literature (LT) PRR assays. The dots represent the observation data, while the line represents the model prediction. The maximum killing effect (E_max) and lag phase (LAG) are estimated parameters (Table 1). DVA, direct viability assessment assay; PRR, experimental parasite reduction ratio assay data; LT V1, parasite reduction ratio assay data from literature (8); LT V2, parasite reduction ratio assay data from literature (3); Error bars represent SEM (three independent experiments, each done in triplicate). The shaded area indicates the standard deviation.

Fig 3

Comparison of PD parameter estimates from DVA and PRR. PD parameter (lag phase and E_max) obtained from experimental DVA assays were compared to PD parameters from experimental and literature (LT V1 [8] and LT V2 [3]) PRR assays. The dashed, gray line indicates the identity of the two methods, the red line is the quadratic regression with the equation, and R² value displayed in the plot.

Fig 4

Applicability of DVA assay in clinical setting using IEV field isolates. The dots represent the observation data, while the line represents the model prediction. The maximum killing effect (E_max) and lag phase (LAG) are estimated parameters (Table 2). Error bars represent SEM (each done in triplicate). The shaded area indicates the standard deviation.

Fig 5

Optimizing gating strategies to identify infected red blood cells (iRBCs) and differentiate uninfected red blood cells (uRBCs) with SYBR Green and MitoTracker Staining. Each dot on the plot represents a single red blood cell, either infected or uninfected, that has passed through the cytometer laser. The combined data reveal two distinct populations: one representing uRBCs and the other representing iRBCs.