An improved and highly sensitive microfluorimetric method for assessing susceptibility of Plasmodium falciparum to antimalarial drugs in vitro

Background: The standard in vitro protocol currently in use for drug testing against Plasmodium falciparum, based on the incorporation of the purine [H-3]-hypoxanthine, has two serious drawbacks. Firstly it is unsuitable for the testing of drugs that directly or indirectly impact on purine salvage or metabolism. Secondly, it relies on the use of expensive radiolabelled material, with added issues concerning detection, storage and waste disposal that make it unsuitable for use in many disease-endemic areas. Recently, the use of fluorochromes has been suggested as an alternative, but quenching of the fluorescence signal by the haemoglobin present in cultures of Plasmodium falciparum-infected erythrocytes severely limits the usefulness of this approach. Methods: In order to resolve this problem, a new PicoGreen(R)-based procedure has been developed which incorporates additional steps to remove the interfering haemoglobin. The 50% inhibitory concentration (IC50) values of chloroquine and pyrimethamine against P. falciparum laboratory lines 3D7 and K1 were determined using the new protocol. Results: The IC50 values of chloroquine and pyrimethamine against P. falciparum laboratory lines 3D7 and K1 determined with the new fluorescence-based protocol were statistically identical to those obtained using the traditional H-3-hypoxanthine incorporation method, and consistent with literature values. Conclusion: The new method proved to be accurate, reproducible and sensitive, and has the advantage of being non-radioactive. The improved PicoGreen(R) method has the potential to replace traditional in vitro drug resistance assay techniques.