Reliability of antimalarial sensitivity tests depends on drug mechanisms of action

Abstract

In vitro antimalarial activity tests play a pivotal role in malaria drug research or for monitoring drug resistance in field isolates. We applied two isotopic tests, two enzyme-linked immunosorbent assays (ELISA) and the SYBR green I fluorescence-based assay, to test artesunate and chloroquine, the metabolic inhibitors atovaquone and pyrimethamine, our fast-acting choline analog T3/SAR97276, and doxycycline, which has a delayed death profile. Isotopic tests based on hypoxanthine and ethanolamine incorporation are the most reliable tests provided when they are applied after one full 48-h parasite cycle. The SYBR green assay, which measures the DNA content, usually requires 72 h of incubation to obtain reliable results. When delayed death is suspected, specific protocols are required with increasing incubation times up to 96 h. In contrast, both ELISA tests used (pLDH and HRP2) appear to be problematic, leading to disappointing and even erroneous results for molecules that do not share an artesunatelike profile. The reliability of these tests is linked to the mode of action of the drug, and the conditions required to get informative results are hard to predict. Our results suggest some minimal conditions to apply these tests that should give rise to a standard 50% inhibitory concentration, regardless of the mechanism of action of the compounds, and highlight that the most commonly used in vitro antimalarial activity tests do not have the same potential. Some of them might not detect the antimalarial potential of new classes of compounds with innovative modes of action, which subsequently could become promising new antimalarial drugs.

FIG. 1.

Effect of adding [³H]hypoxanthine and [³H]ethanolamine early on in vitro T3/SAR97276 and artesunate sensitivity tests. (A) The experimental setups are summarized. T3/SAR97276 (B and D) or artesunate (C and E) were added at the indicated concentrations to synchronized P. falciparum parasites at the ring stage. [³H]hypoxanthine (B and C) or [³H]ethanolamine (D and E) was added at time zero (▪ and dashed line), 24 h (▴ and dashed line), or 48 h (•) for the indicated times, and then the reactions were stopped by freezing. Incorporations are expressed as means ± the standard error of the mean (SEM) (n > 3) and expressed as a percentage of the controls.

FIG. 2.

Time course of P. falciparum growth inhibition by T3/SAR97276. The drug T3/SAR97276 was added at 40 nM (A) or 80 nM (B) to synchronized cultures at the ring (6 h) (▪), trophozoite (24 h) (▴), or schizont (36 h) (•) stages. After incubation for the indicated times, cells were washed and resuspended in fresh medium. Parasite viability was monitored by adding [³H]hypoxanthine at 52 h until 76 h. Hypoxanthine incorporations are expressed as means ± the SEM (n ≥ 3) and expressed as a percentage of the controls.

FIG. 3.

Giemsa-stained smears of T3/SAR97276-treated and untreated cultures. T3/SAR97276 was added at 80 nM at the early ring stage. The morphology of parasites in treated and untreated cultures is shown. Morphological changes are clearly observed at 30 h with an absence of schizont formation and invasion at 48 h (T_48h). From 34 h, the parasites were blocked at the trophozoite/schizont stage, and then they collapsed and disappeared (T_72h).