Simple and inexpensive fluorescence-based technique for high-throughput antimalarial drug screening

Abstract

Radioisotopic assays involve expense, multistep protocols, equipment, and radioactivity safety requirements which are problematic in high-throughput drug testing. This study reports an alternative, simple, robust, inexpensive, one-step fluorescence assay for use in antimalarial drug screening. Parasite growth is determined by using SYBR Green I, a dye with marked fluorescence enhancement upon contact with Plasmodium DNA. A side-by-side comparison of this fluorescence assay and a standard radioisotopic method was performed by testing known antimalarial agents against Plasmodium falciparum strain D6. Both assay methods were used to determine the effective concentration of drug that resulted in a 50% reduction in the observed counts (EC(50)) after 48 h of parasite growth in the presence of each drug. The EC(50)s of chloroquine, quinine, mefloquine, artemisinin, and 3,6-bis-epsilon-(N,N-diethylamino)-amyloxyxanthone were similar or identical by both techniques. The results obtained with this new fluorescence assay suggest that it may be an ideal method for high-throughput antimalarial drug screening.

FIG. 1.

Comparison of test drug dose-response curves determined by the fluorescence (A) and radioisotope (B) assays. Values are normalized by use of the upper and the lower plateaus of the best-fit curve as 100 and 0% responses, respectively, and are plotted as the means ± standard errors of the means for triplicate wells.

FIG. 2.

Relationship between parasitemia and measured fluorescence. Values are plotted as the means ± standard errors of the means for triplicate wells after subtraction of the background fluorescence for nonparasitized erythrocytes.