Flow cytometry for the evaluation of anti-plasmodial activity of drugs on Plasmodium falciparum gametocytes

Abstract

Background: 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. 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 conclusions: Initially 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 microM and 1.0 microM, 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.

Figure 1

Flow cytometry analysis of Plasmodium falciparum parasitized erythrocytes (W2 strain) stained with hydroethidine, asexual stages. A and B: ring-enriched population examined by flow cytometry. C and D: schizont-enriched population: schizonts were enriched by magnetic separation through a MACS column and examined by flow cytometry. E: Giemsa-stained thin smears. F, G: HE-stained parasites (magnification × 63 (F) and zoom 2.5 (G)).

Figure 2

Flow cytometry analysis of Plasmodium falciparum parasitized erythrocytes (W2 strain) stained with hydroethidine, gametocytes. Gametocytes were purified by magnetic separation through a MACS column and examined by flow cytometry (A and B). Giemsa-stained thin smears (D) and HE-stained enriched culture (magnification × 63 (E) and zoom 2.5 (F)). C: Giemsa-stained thin smears before gametocyte enrichment.

Figure 3

Effect of xanthurenic acid (XA) on gametocytaemia evaluated by flow cytometry. Experiments were performed on MACS^® enriched gametocyte culture. Percentages of each population (M2 and M3) of a gametocyte culture before or after exflagellation with 100 μM xanthurenic acid (XA) were evaluated by flow cytometry (mean data of three independent experiments ± SD, the parasitaemia level varied highly between these three experiments). As no decrease of gametocytaemia was observed in M2 population after XA treatment, this group represented potentially female gametocytes. The decrease observed in the M3 population after XA treatment corresponded to mature male gametocytes exflagellation. The residual M3 population after XA treatment was mostly immature gametocytes and schizonts.