Flow cytometry for the evaluation of anti-plasmodial activity of drugs on Plasmodium falciparum gametocytes
© Chevalley et al; licensee BioMed Central Ltd. 2010
Received: 16 September 2009
Accepted: 11 February 2010
Published: 11 February 2010
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 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.
Many different approaches have been used to measure the susceptibility of asexual stages of Plasmodium falciparum to anti-malarial drugs in vitro. 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. However, only the first method allows the quantification of gametocytes, since these cells do not multiply. Given that light microscopy is labour-intensive, subjective, and suffers from inter-operator variations, alternative methods for the counting of gametocytes are needed to increase the efficiency of drug-screening on this stage of the parasite's life-cycle.
In earlier studies, flow cytometry has been proposed to assess the viability of intra-erythrocytic stages of Plasmodium using DNA fluorescent stains [1, 2]. Many fluorescent stains have been used in flow cytometry studies: propidium iodide , acridine orange  and YOYO-1  require fixation and permeabilization of the parasites before use. Other dyes need complete lysis of erythocytes, such as Hoechst 33258 [6, 7] and Picogreen® , while hydroethine (HE) does not require lysis or fixatives. Plasmodium takes up and metabolizes HE into ethidium, a nucleic acid-binding fluorochrome . Some authors have also proposed to combine thiazole orange with HE  or with Hoechst 33342  to stain nucleic acids in order to differentiate intra-erythrocytic stages of P. falciparum.
In the present study, the discrimination, by flow cytometry, of P. falciparum asexual and sexual forms stained with HE was performed in order to validate a method for the screening of gametocytocidal drugs. First, the relative distribution of fluorescence in parasite subpopulations according to their maturity and sex was analyzed and then the gametocytocidal activity of drugs was determined. Finally, particular gametocyte preparations were treated with xanthurenic acid to follow the exflagellation process.
Flow cytometry was demonstrated to be usable to evaluate the different stages of sexual and asexual parasite populations and to assess the in vitro gametocytocidal activities of potentially anti-plasmodial drugs. Moreover, this method enables to discriminate the male and female gametocyte subpopulations.
Red blood cells and human serum were obtained from EFS, Toulouse (France); PBS, RPMI and additives were from Lonza (Belgium). All other reagents were from Sigma-Aldrich, l'Isle d'Abeau (France).
Plasmodium falciparum in vitro culture
Parasites were cultured according to Trager and Jensen , and synchronized according to Lambros . Briefly, parasites were routinely maintained in O+, human erythrocytes (parasitaemia: 0.5-4%, haematocrit: 4%) in RPMI 1640 with 25 mM HEPES, 2 mM L-glutamine and 7% human AB serum in a CO2 incubator.
Gametocyte cultures were initiated with W2-Indochina strain as described elsewhere , with modifications . Cultures were treated with 50 mM N-acetyl-D-glucosamine for 4-5 days to remove most of the asexual stages. Old (stage IV-V, 11-13-days-old) gametocyte cultures were mostly used.
Visual counting of schizonts and gametocytes was carried out on Giemsa-stained smears before and after concentration. Images were digitally recorded with a digital camera (DS Camera Control Unit DS-U2, Nikon) mounted on an optical microscope (Nikon Eclipse 80i, Nikon).
Magnetic purification was carried out by using the haemozoin paramagnetic complex property. Prior to purification, MACS® (25LD columns, Miltenyi BioTec, Germany) columns were filled with warmed (37°C) RPMI. The experimentation was performed under sterile conditions [16, 17]. The tested blood from cultures was then loaded on the columns (typically, 4 mL at 25-50% haematocrit) and warm (37°C) culture medium was then added until the eluent was apparently free of red blood cells. At this point, the columns were removed from the magnetic support after addition of 10 mL more culture medium and the eluent was recovered. It was then centrifuged (800 g, 10 min) and supernatant was discarded. The pellet was used to prepare blood smears that were Giemsa-stained and it was also analysed by flow cytometry.
In vitro tests with gametocytes
Primaquine and artemisinin (the latter being kindly provided by Pierre Fabre Laboratories) were dissolved in DMSO, while chloroquine was dissolved in RPMI. Twelve day-old gametocyte cultures were transferred to 24-well or 96-well plates, and increasing dilutions of each drug were added. All experiments were performed in triplicate. After 48 h of incubation at 37°C, thin blood smears were prepared and stained with Giemsa. The number of gametocytes per 10,000 erythrocytes was visually estimated by optical microscopy. In parallel, parasitaemia was evaluated by flow cytometry.
Cultures were washed with PBS. Pellets were resuspended in hydroethidine solution (50 μg/mL) for 20 minutes at 37°C in the dark. After washing in PBS, 105 cells were analysed with a FACScalibur cytometer (Becton Dickinson®) using the CellQuestPro® program for data analysis.
In order to obtain an homogenous film, glass cover-slips were coated overnight at room temperature with poly-L-lysine (1:100 in PBS). Parasite cultures were washed twice in PBS and the pellets were resuspended in 50 μg/mL hydroethidine (HE) for 20 minutes at 37°C in the dark. Samples were then washed twice in PBS and resuspended in 1 mL of PBS. Cells were distributed on the poly-L-lysine-coated cover-slips and incubated for 20 minutes at 37°C in the dark. After cell adhesion, the cover-slips were washed with PBS and analysed with a fluorescence microscope (ConfoCor, Zeiss LM510, Carl Zeiss).
In vitro exflagellation assay
The exflagellation of gametocytes was quantified according to Kawamoto et al . Gametocytes were highly purified on MACS® columns and immediately resuspended at 5% final haematocrit in 100 μM xanthurenic acid (XA) for 20 min at room temperature [19, 20]. Prior to flow cytometry analysis, a visual counting of gametocytes with or without exflagellation was carried out on Giemsa-stained smears.
Results and Discussion
Comparison between FCA (Flow cytometry analysis) an OM (Optical microscopy) for the determination of gametocytaemia.
+ Sch (%): M3
Conventional drugs activity against P. falciparum gametocytes.
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 hence should be useful to evaluate promising anti-gametocyte drugs. Moreover, HE-labelled viable parasites, which were the only labelled cells, while Giemsa staining did not allow the differentiation between living and dead parasites. Although these results were obtained on P. falciparum, they are close to those of other authors [21, 27] who have quantified GFP expression during intraerythrocytic development of transgenic A-SET/GFP P. berghei. They showed that the fluorescence intensity of gametocytes was comparable to that of late trophozoites, as showed in the present report. Using flow cytometry, other researchers have identified P. falciparum gametocytes well before they were morphologically distinguishable from asexual stage parasites, thanks to the use of the chimeric Pfs 16-GFP . This FACS based assay used one of the earliest known gametocyte proteins, the Pfs16 as a reporter. However, the present method can be used for non-genetically modified parasites.
The authors thank Nancy Voissiere for technical assistance and Antoine Berry and Françoise Benoit-Vical for fruitful discussions.
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