Development and application of a modified flow cytometric procedure for rapid in vitro quantitation of malaria parasitaemia

The most lethal form of human malaria, caused by the parasite Plasmodium falciparum, adversely affects the lives of millions of people each year. In order to establish the effectiveness of therapeutics, an accurate, reproducible and convenient assay of parasitaemia is necessary. Towards this end, we modified a flow cytometric (FC) method based on thiazole orange as fluorescent intercalating dye to detect parasite DNA, by using a lower fluorochrome concentration (0.8 mu M) and micro-cultures of parasites subsequently fixed with a standard formaldehyde-based solution. A linear relationship was observed between classical microscopically determined parasitaemias and those from FC (r = 0.98), as well as between experimental FC values and parasitaemias calculated from the serial dilution of either unfixed or fixed stock cultures (r > 0.98). The applicability of the FC method was confirmed during quantitation of the extent of inhibition of parasitaemia by chloroquine treatment of Plasmodium falciparum-infected human erythrocytes. Results obtained with flow cytometric analysis in this instance correlated with those from both classical Giemsa-stained blood films and [ super(3)H]hypoxanthine incorporation (IC sub(50) = 70-76 nM). The modified flow cytometric procedure is therefore suitable for the rapid, cost-effective in vitro estimation of parasitaemias in micro-cultures and the evaluation of agents with anti-malarial potential.