DEVELOPMENT AND APPLICATION OF A MODIFIED FLOW CYTOMETRIC PROCEDURE FOR RAPID IN-VITRO QUANTITATION OF MALARIA PARASITEMIA

The most lethal form of human malaria, caused by the parasite Plasmodi um falciparum, adversely affects the lives of millions of people each year In order to establish the effectiveness of therapeutics, an accur ate, reproducible and convenient assay of parasitaemia is necessary To wards this end, we modified a flow cytometric (FC) method based on thi azole orange 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 so lution. A linear relationship was observed between classical microscop ically determined parasitemias and those from FC (r = 0.98), as well a s 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 o f Plasmodium falciparum-infected human erythrocytes. Results obtained with flow cytometric analysis in this instance correlated with those f rom both classical Giemsa-stained blood films and [H-3]hypoxanthine in corporation (IC50 = 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.