Abstract
BackgroundThe activity of promising anti-malarial drugs against Plasmodium gametocytes is hard to evaluate even in vitro. This is because visual examination of stained smears, which is commonly used, is not totally convenient. In the current study, flow cytometry has been used to study the effect of established anti-malarial drugs against sexual stages obtained from W2 strain of Plasmodium falciparum. Gametocytes were treated for 48 h with different drug concentrations and the gametocytaemia was then determined by flow cytometry and compared with visual estimation by microscopy.Results and conclusionsInitially gametocytaemia was evaluated either using light microscopy or flow cytometry. A direct correlation (r2 = 0.9986) was obtained. Two distinct peaks were observed on cytometry histograms and were attributed to gametocyte populations. The activities of established anti-malarial compounds were then measured by flow cytometry and the results were equivalent to those obtained using light microscopy. Primaquine and artemisinin had IC50 of 17.6 μM and 1.0 μM, respectively.Gametocyte sex was apparently distinguishable by flow cytometry as evaluated after induction of exflagellation by xanthurenic acid. These data form the basis of further studies for developing new methods in drug discovery to decrease malaria transmission.
Highlights
The activity of promising anti-malarial drugs against Plasmodium gametocytes is hard to evaluate even in vitro
The interaction between ethidium and parasite nucleic acids resulted in a red fluorescence emission allowing discrimination between uninfected and infected erythrocytes independently of the parasitic stage
flow cytometry and optical microscopy (FCA), in combination with magnetic enrichment, has here been shown to be useful to estimate the inhibitory concentrations of known drugs against P. falciparum gametocytes and should be useful to evaluate promising anti-gametocyte drugs
Summary
The activity of promising anti-malarial drugs against Plasmodium gametocytes is hard to evaluate even in vitro. This is because visual examination of stained smears, which is commonly used, is not totally convenient. The most used methods are light microscopy, which enables visual quantification of parasitized red blood cells, and radioactive methods that evaluate the viability of parasites by tracking the incorporation of 3H-hypoxanthine into their nucleic acids. Flow cytometry has been proposed to assess the viability of intra-erythrocytic stages of Plasmodium using DNA fluorescent stains [1,2]. Some authors have proposed to combine thiazole orange with HE [10] or with Hoechst 33342 [11] to stain nucleic acids in order to differentiate intra-erythrocytic stages of P. falciparum
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