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Published in: Malaria Journal 1/2010

Open Access 01-12-2010 | Methodology

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

Authors: Séverine Chevalley, Agnès Coste, Alexandrine Lopez, Bernard Pipy, Alexis Valentin

Published in: Malaria Journal | Issue 1/2010

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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 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.
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Literature
1.
go back to reference Azas N, Rathelot P, Djekou S, Delmas F, Gellis A, Di Giorgio C, Vanelle P, Timon-David P: Antiparasitic activity of highly conjugated pyrimidine-2,4-dione derivatives. Farmaco. 2003, 58: 1263-1270. 10.1016/j.farmac.2003.07.009.CrossRefPubMed Azas N, Rathelot P, Djekou S, Delmas F, Gellis A, Di Giorgio C, Vanelle P, Timon-David P: Antiparasitic activity of highly conjugated pyrimidine-2,4-dione derivatives. Farmaco. 2003, 58: 1263-1270. 10.1016/j.farmac.2003.07.009.CrossRefPubMed
2.
go back to reference Wyatt CR, Goff W, Davis WC: A flow cytometric method for assessing viability of intraerythrocytic hemoparasites. J Immunol Meth. 1991, 140: 23-30. 10.1016/0022-1759(91)90122-V.CrossRef Wyatt CR, Goff W, Davis WC: A flow cytometric method for assessing viability of intraerythrocytic hemoparasites. J Immunol Meth. 1991, 140: 23-30. 10.1016/0022-1759(91)90122-V.CrossRef
3.
go back to reference Contreras CE, Rivas MA, Dominguez J, Charris J, Palacios M, Bianco NE, Blanca I: Stage-specific activity of potential antimalarial compounds measured in vitro by flow cytometry in comparison to optical microscopy and hypoxanthine uptake. Memorias do Instituto Oswaldo Cruz. 2004, 99: 179-184.CrossRefPubMed Contreras CE, Rivas MA, Dominguez J, Charris J, Palacios M, Bianco NE, Blanca I: Stage-specific activity of potential antimalarial compounds measured in vitro by flow cytometry in comparison to optical microscopy and hypoxanthine uptake. Memorias do Instituto Oswaldo Cruz. 2004, 99: 179-184.CrossRefPubMed
4.
go back to reference Bhakdi SC, Sratongno P, Chimma P, Rungruang T, Chuncharunee A, Neumann HP, Malasit P, Pattanapanyasat K: Re-evaluating acridine orange for rapid flow cytometric enumeration of parasitemia in malaria-infected rodents. Cytometry. 2007, 71: 662-667. 10.1002/cyto.a.20406.CrossRefPubMed Bhakdi SC, Sratongno P, Chimma P, Rungruang T, Chuncharunee A, Neumann HP, Malasit P, Pattanapanyasat K: Re-evaluating acridine orange for rapid flow cytometric enumeration of parasitemia in malaria-infected rodents. Cytometry. 2007, 71: 662-667. 10.1002/cyto.a.20406.CrossRefPubMed
5.
go back to reference Li Q, Gerena L, Xie L, Zhang J, Kyle D, Milhous W: Development and validation of flow cytometric measurement for parasitemia in cultures of P. falciparum vitally stained with YOYO-1. Cytometry A. 2007, 71: 297-307.CrossRefPubMed Li Q, Gerena L, Xie L, Zhang J, Kyle D, Milhous W: Development and validation of flow cytometric measurement for parasitemia in cultures of P. falciparum vitally stained with YOYO-1. Cytometry A. 2007, 71: 297-307.CrossRefPubMed
6.
go back to reference Bianco AE, Battye FL, Brown GV: Plasmodium falciparum: rapid quantification of parasitemia in fixed malaria cultures by flow cytometry. Expl Parasitol. 1986, 62: 275-282. 10.1016/0014-4894(86)90032-9.CrossRef Bianco AE, Battye FL, Brown GV: Plasmodium falciparum: rapid quantification of parasitemia in fixed malaria cultures by flow cytometry. Expl Parasitol. 1986, 62: 275-282. 10.1016/0014-4894(86)90032-9.CrossRef
7.
go back to reference van Vianen PH, van Engen A, Thaithong S, Keur van der M, Tanke HJ, Kaay van der HJ, Mons B, Janse CJ: Flow cytometric screening of blood samples for malaria parasites. Cytometry. 1993, 14: 276-280. 10.1002/cyto.990140307.CrossRefPubMed van Vianen PH, van Engen A, Thaithong S, Keur van der M, Tanke HJ, Kaay van der HJ, Mons B, Janse CJ: Flow cytometric screening of blood samples for malaria parasites. Cytometry. 1993, 14: 276-280. 10.1002/cyto.990140307.CrossRefPubMed
8.
go back to reference Corbett Y, Herrera L, Gonzalez J, Cubilla L, Capson TL, Coley PD, Kursar TA, Romero LI, Ortega-Barria E: A novel DNA-based microfluorimetric method to evaluate antimalarial drug activity. Am J Trop Med Hyg. 2004, 70: 119-124.PubMed Corbett Y, Herrera L, Gonzalez J, Cubilla L, Capson TL, Coley PD, Kursar TA, Romero LI, Ortega-Barria E: A novel DNA-based microfluorimetric method to evaluate antimalarial drug activity. Am J Trop Med Hyg. 2004, 70: 119-124.PubMed
9.
go back to reference Heyde van der HC, Elloso MM, Waa vande J, Schell K, Weidanz WP: Use of hydroethidine and flow cytometry to assess the effects of leukocytes on the malarial parasite Plasmodium falciparum. Clin Diagn Lab Immunol. 1995, 2: 417-425.PubMedCentralPubMed Heyde van der HC, Elloso MM, Waa vande J, Schell K, Weidanz WP: Use of hydroethidine and flow cytometry to assess the effects of leukocytes on the malarial parasite Plasmodium falciparum. Clin Diagn Lab Immunol. 1995, 2: 417-425.PubMedCentralPubMed
10.
go back to reference Jouin H, Daher W, Khalife J, Ricard I, Puijalon OM, Capron M, Dive D: Double staining of Plasmodium falciparum nucleic acids with hydroethidine and thiazole orange for cell cycle stage analysis by flow cytometry. Cytometry. 2004, 57: 34-38. 10.1002/cyto.a.10110.CrossRefPubMed Jouin H, Daher W, Khalife J, Ricard I, Puijalon OM, Capron M, Dive D: Double staining of Plasmodium falciparum nucleic acids with hydroethidine and thiazole orange for cell cycle stage analysis by flow cytometry. Cytometry. 2004, 57: 34-38. 10.1002/cyto.a.10110.CrossRefPubMed
11.
go back to reference Grimberg BT, Erickson JJ, Sramkoski RM, Jacobberger JW, Zimmerman PA: Monitoring Plasmodium falciparum growth and development by UV flow cytometry using an optimized Hoechst-thiazole orange staining strategy. Cytometry. 2008, 73: 546-554. 10.1002/cyto.a.20541.PubMedCentralCrossRefPubMed Grimberg BT, Erickson JJ, Sramkoski RM, Jacobberger JW, Zimmerman PA: Monitoring Plasmodium falciparum growth and development by UV flow cytometry using an optimized Hoechst-thiazole orange staining strategy. Cytometry. 2008, 73: 546-554. 10.1002/cyto.a.20541.PubMedCentralCrossRefPubMed
12.
go back to reference Trager W, Jensen JB: Human malaria parasites in continuous culture. Science. 1976, 193: 673-675. 10.1126/science.781840.CrossRefPubMed Trager W, Jensen JB: Human malaria parasites in continuous culture. Science. 1976, 193: 673-675. 10.1126/science.781840.CrossRefPubMed
13.
go back to reference Lambros C, Vanderberg JP: Synchronization of Plasmodium falciparum erythrocytic stages in culture. J Parasitol. 1979, 65: 418-420. 10.2307/3280287.CrossRefPubMed Lambros C, Vanderberg JP: Synchronization of Plasmodium falciparum erythrocytic stages in culture. J Parasitol. 1979, 65: 418-420. 10.2307/3280287.CrossRefPubMed
14.
go back to reference Ifediba T, Vanderberg JP: Complete in vitro maturation of Plasmodium falciparum gametocytes. Nature. 1981, 294: 364-366. 10.1038/294364a0.CrossRefPubMed Ifediba T, Vanderberg JP: Complete in vitro maturation of Plasmodium falciparum gametocytes. Nature. 1981, 294: 364-366. 10.1038/294364a0.CrossRefPubMed
15.
go back to reference Benoit-Vical F, Lelievre J, Berry A, Deymier C, Dechy-Cabaret O, Cazelles J, Loup C, Robert A, Magnaval JF, Meunier B: Trioxaquines are new antimalarial agents active on all erythrocytic forms, including gametocytes. Antimicrob Agents Chemother. 2007, 51: 1463-1472. 10.1128/AAC.00967-06.PubMedCentralCrossRefPubMed Benoit-Vical F, Lelievre J, Berry A, Deymier C, Dechy-Cabaret O, Cazelles J, Loup C, Robert A, Magnaval JF, Meunier B: Trioxaquines are new antimalarial agents active on all erythrocytic forms, including gametocytes. Antimicrob Agents Chemother. 2007, 51: 1463-1472. 10.1128/AAC.00967-06.PubMedCentralCrossRefPubMed
16.
go back to reference Ribaut C, Berry A, Chevalley S, Reybier K, Morlais I, Parzy D, Nepveu F, Benoit-Vical F, Valentin A: Concentration and purification by magnetic separation of the erythrocytic stages of all human Plasmodium species. Malar J. 2008, 7: 45-10.1186/1475-2875-7-45.PubMedCentralCrossRefPubMed Ribaut C, Berry A, Chevalley S, Reybier K, Morlais I, Parzy D, Nepveu F, Benoit-Vical F, Valentin A: Concentration and purification by magnetic separation of the erythrocytic stages of all human Plasmodium species. Malar J. 2008, 7: 45-10.1186/1475-2875-7-45.PubMedCentralCrossRefPubMed
17.
go back to reference Trang DT, Huy NT, Kariu T, Tajima K, Kamei K: One-step concentration of malarial parasite-infected red blood cells and removal of contaminating white blood cells. Malar J. 2004, 3: 7-10.1186/1475-2875-3-7.PubMedCentralCrossRefPubMed Trang DT, Huy NT, Kariu T, Tajima K, Kamei K: One-step concentration of malarial parasite-infected red blood cells and removal of contaminating white blood cells. Malar J. 2004, 3: 7-10.1186/1475-2875-3-7.PubMedCentralCrossRefPubMed
18.
go back to reference Kawamoto F, Alejo-Blanco R, Fleck SL, Kawamoto Y, Sinden RE: Possible roles of Ca2+ and cGMP as mediators of the exflagellation of Plasmodium berghei and Plasmodium falciparum. Mol Biochem Parasitol. 1990, 42: 101-108. 10.1016/0166-6851(90)90117-5.CrossRefPubMed Kawamoto F, Alejo-Blanco R, Fleck SL, Kawamoto Y, Sinden RE: Possible roles of Ca2+ and cGMP as mediators of the exflagellation of Plasmodium berghei and Plasmodium falciparum. Mol Biochem Parasitol. 1990, 42: 101-108. 10.1016/0166-6851(90)90117-5.CrossRefPubMed
19.
go back to reference Arai M, Billker O, Morris HR, Panico M, Delcroix M, Dixon D, Ley SV, Sinden RE: Both mosquito-derived xanthurenic acid and a host blood-derived factor regulate gametogenesis of Plasmodium in the midgut of the mosquito. Mol Biochem Parasitol. 2001, 116: 17-24. 10.1016/S0166-6851(01)00299-7.CrossRefPubMed Arai M, Billker O, Morris HR, Panico M, Delcroix M, Dixon D, Ley SV, Sinden RE: Both mosquito-derived xanthurenic acid and a host blood-derived factor regulate gametogenesis of Plasmodium in the midgut of the mosquito. Mol Biochem Parasitol. 2001, 116: 17-24. 10.1016/S0166-6851(01)00299-7.CrossRefPubMed
20.
go back to reference Bhattacharyya MK, Kumar N: Effect of xanthurenic acid on infectivity of Plasmodium falciparum to Anopheles stephensi. Int J Parasitol. 2001, 31: 1129-1133. 10.1016/S0020-7519(01)00222-3.CrossRefPubMed Bhattacharyya MK, Kumar N: Effect of xanthurenic acid on infectivity of Plasmodium falciparum to Anopheles stephensi. Int J Parasitol. 2001, 31: 1129-1133. 10.1016/S0020-7519(01)00222-3.CrossRefPubMed
21.
go back to reference Pace T, Olivieri A, Sanchez M, Albanesi V, Picci L, Siden Kiamos I, Janse CJ, Waters AP, Pizzi E, Ponzi M: Set regulation in asexual and sexual Plasmodium parasites reveals a novel mechanism of stage-specific expression. Mol Microbiol. 2006, 60: 870-882. 10.1111/j.1365-2958.2006.05141.x.CrossRefPubMed Pace T, Olivieri A, Sanchez M, Albanesi V, Picci L, Siden Kiamos I, Janse CJ, Waters AP, Pizzi E, Ponzi M: Set regulation in asexual and sexual Plasmodium parasites reveals a novel mechanism of stage-specific expression. Mol Microbiol. 2006, 60: 870-882. 10.1111/j.1365-2958.2006.05141.x.CrossRefPubMed
22.
go back to reference Staalsoe T, Giha HA, Dodoo D, Theander TG, Hviid L: Detection of antibodies to variant antigens on Plasmodium falciparum-infected erythrocytes by flow cytometry. Cytometry. 1999, 35: 329-336. 10.1002/(SICI)1097-0320(19990401)35:4<329::AID-CYTO5>3.0.CO;2-Y.CrossRefPubMed Staalsoe T, Giha HA, Dodoo D, Theander TG, Hviid L: Detection of antibodies to variant antigens on Plasmodium falciparum-infected erythrocytes by flow cytometry. Cytometry. 1999, 35: 329-336. 10.1002/(SICI)1097-0320(19990401)35:4<329::AID-CYTO5>3.0.CO;2-Y.CrossRefPubMed
23.
go back to reference Billker O, Lindo V, Panico M, Etienne AE, Paxton T, Dell A, Rogers M, Sinden RE, Morris HR: Identification of xanthurenic acid as the putative inducer of malaria development in the mosquito. Nature. 1998, 392: 289-292. 10.1038/32667.CrossRefPubMed Billker O, Lindo V, Panico M, Etienne AE, Paxton T, Dell A, Rogers M, Sinden RE, Morris HR: Identification of xanthurenic acid as the putative inducer of malaria development in the mosquito. Nature. 1998, 392: 289-292. 10.1038/32667.CrossRefPubMed
24.
go back to reference Garcia GE, Wirtz RA, Barr JR, Woolfitt A, Rosenberg R: Xanthurenic acid induces gametogenesis in Plasmodium, the malaria parasite. J Biol Chem. 1998, 273: 12003-12005. 10.1074/jbc.273.20.12003.CrossRefPubMed Garcia GE, Wirtz RA, Barr JR, Woolfitt A, Rosenberg R: Xanthurenic acid induces gametogenesis in Plasmodium, the malaria parasite. J Biol Chem. 1998, 273: 12003-12005. 10.1074/jbc.273.20.12003.CrossRefPubMed
25.
go back to reference Talman AM, Domarle O, McKenzie FE, Ariey F, Robert V: Gametocytogenesis: the puberty of Plasmodium falciparum. Malar J. 2004, 3: 24-10.1186/1475-2875-3-24.PubMedCentralCrossRefPubMed Talman AM, Domarle O, McKenzie FE, Ariey F, Robert V: Gametocytogenesis: the puberty of Plasmodium falciparum. Malar J. 2004, 3: 24-10.1186/1475-2875-3-24.PubMedCentralCrossRefPubMed
26.
go back to reference Sall C, Yapi AD, Desbois N, Chevalley S, Chezal JM, Tan K, Teulade JC, Valentin A, Blache Y: Design, synthesis, and biological activities of conformationally restricted analogs of primaquine with a 1,10-phenanthroline framework. Bioorganic & Medicinal Chemistry Letters. 2008, 18: 4666-4669.CrossRef Sall C, Yapi AD, Desbois N, Chevalley S, Chezal JM, Tan K, Teulade JC, Valentin A, Blache Y: Design, synthesis, and biological activities of conformationally restricted analogs of primaquine with a 1,10-phenanthroline framework. Bioorganic & Medicinal Chemistry Letters. 2008, 18: 4666-4669.CrossRef
27.
go back to reference Khan SM, Franke-Fayard B, Mair GR, Lasonder E, Janse CJ, Mann M, Waters AP: Proteome analysis of separated male and female gametocytes reveals novel sex-specific Plasmodium biology. Cell. 2005, 121: 675-687. 10.1016/j.cell.2005.03.027.CrossRefPubMed Khan SM, Franke-Fayard B, Mair GR, Lasonder E, Janse CJ, Mann M, Waters AP: Proteome analysis of separated male and female gametocytes reveals novel sex-specific Plasmodium biology. Cell. 2005, 121: 675-687. 10.1016/j.cell.2005.03.027.CrossRefPubMed
28.
go back to reference Dixon MW, Peatey CL, Gardiner DL, Trenholme KR: A green fluorescent protein-based assay for determining gametocyte production in Plasmodium falciparum. Mol Biochem Parasitol. 2009, 163: 123-126. 10.1016/j.molbiopara.2008.10.004.CrossRefPubMed Dixon MW, Peatey CL, Gardiner DL, Trenholme KR: A green fluorescent protein-based assay for determining gametocyte production in Plasmodium falciparum. Mol Biochem Parasitol. 2009, 163: 123-126. 10.1016/j.molbiopara.2008.10.004.CrossRefPubMed
Metadata
Title
Flow cytometry for the evaluation of anti-plasmodial activity of drugs on Plasmodium falciparum gametocytes
Authors
Séverine Chevalley
Agnès Coste
Alexandrine Lopez
Bernard Pipy
Alexis Valentin
Publication date
01-12-2010
Publisher
BioMed Central
Published in
Malaria Journal / Issue 1/2010
Electronic ISSN: 1475-2875
DOI
https://doi.org/10.1186/1475-2875-9-49

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