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

In order to resolve this problem, a new PicoGreen？-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.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 3H-hypoxanthine incorporation method, and consistent with literature values.The new method proved to be accurate, reproducible and sensitive, and has the advantage of being non-radioactive. The improved PicoGreen？ method has the potential to replace traditional in vitro drug resistance assay techniques.The emergence and spread of Plasmodium falciparum resistant to almost all available antimalarial drugs necessitates a constant monitoring of parasite susceptibility to antimalarial drugs, as well as the search for novel chemotherapeutic agents. Several in vitro drug sensitivity assays exist for this purpose [1-3]. In vitro drug sensitivity assays are generally based on culturing P. falciparum isolates in the presence of a range of antimalarial drug concentrations for one cycle of intraerythrocytic asexual replication or part thereof. The effect of antimalarial drugs is characterised by the inhibition of parasite growth. The most widely accepted test relies on microscopic scoring of parasitized and uninfected erythrocytes [2]. Other assessment methods involve the determination of the level of incorporation of radiolabelled precursors into parasite DNA [1,4] and colorimetric methods, which involve measurement of the levels of parasite lactate dehydrogenase (pLDH) [3,5] or histidine-rich protein II [6]. Each of these commonly-used assays has unique advantages as well as a number of known drawbacks. Parasitized cell counts using mic