The online version of this article (doi:10.1186/1475-2875-11-235) contains supplementary material, which is available to authorized users.
Prajakta S Jogdand, Susheel K Singh contributed equally to this work.
The authors declare that they have no competing interests.
PSJ, SS and MT conceived the study. PSJ, SKS, MC and MD performed the laboratory work and the statistical analysis. PSJ, SKS, SS and MT wrote the manuscript. All authors have read the manuscript and agree with its contents.
Functional in vitro assays could provide insights into the efficacy of malaria vaccine candidates. For estimating the anti-parasite effect induced by a vaccine candidate, an accurate determination of live parasite count is an essential component of most in vitro bioassays. Although traditionally parasites are counted microscopically, a faster, more accurate and less subjective method for counting parasites is desirable. In this study mitochondrial dye (Mitotracker Red CMXRos) was used for obtaining reliable live parasite counts through flow cytometry.
Both asynchronous and tightly synchronized asexual blood stage cultures of Plasmodium falciparum were stained with CMXRos and subjected to detection by flow cytometry and fluorescence microscopy. The parasite counts obtained by flow cytometry were compared to standard microscopic counts obtained through examination of Giemsa-stained thin smears. A comparison of the ability of CMXRos to stain live and compromised parasites (induced by either medium starvation or by anti-malarial drug treatment) was carried out. Finally, parasite counts obtained by CMXRos staining through flow cytometry were used to determine specific growth inhibition index (SGI) in an antibody-dependent cellular inhibition (ADCI) assay.
Mitotracker Red CMXRos can reliably detect live intra-erythrocytic stages of P. falciparum. Comparison between staining of live with compromised parasites shows that CMXRos predominantly stains live parasites with functional mitochondria. Parasite counts obtained by CMXRos staining and flow cytometry were highly reproducible and can reliably determine the ability of IgG from hyper-immune individuals to inhibit parasite growth in presence of monocytes in ADCI assay. Further, a dose-dependent parasite growth inhibitory effect could be detected for both total IgG purified from hyper-immune sera and affinity purified IgGs against the N-terminal non-repeat region of GLURP in ADCI assays coupled with determination of parasite counts through CMXRos staining and flow cytometry.
A flow cytometry method based on CMXRos staining for detection of live parasite populations has been optimized. This is a rapid and sensitive method with high inter-assay reproducibility which can reliably determine the anti-parasite effect mediated by antibodies in functional in vitro assays such as ADCI assay.
Druilhe P, Sabchareon A, Bouharoun-Tayoun H, Oeuvray C, Perignon JL: In vivo veritas: lessons from immunoglobulin-transfer experiments in malaria patients. Ann Trop Med Parasitol. 1997, 91 (Supp): 37-53. CrossRef
Bouharoun-Tayoun H, Attanath P, Sabchareon A, Chongsuphajaisiddhi T, Druilhe P: Antibodies that protect humans against Plasmodium falciparum blood stages do not on their own inhibit parasite growth and invasion in vitro, but act in cooperation with monocytes. J Exp Med. 1990, 172: 1633-1641. CrossRefPubMed
Shi YP, Udhayakumar V, Oloo AJ, Nahlen BL, Lal AA: Differential effect and interaction of monocytes, hyperimmune sera, and immunoglobulin G on the growth of asexual stage Plasmodium falciparum parasites. AmJTrop Med Hyg. 1999, 60: 135-141.
Oeuvray C, Bouharoun-Tayoun H, Gras-Masse H, Bottius E, Kaidoh T, Aikawa M, Filgueira MC, Tartar A, Druilhe P: Merozoite surface protein-3: a malaria protein inducing antibodies that promote Plasmodium falciparum killing by cooperation with blood monocytes. Blood. 1994, 84: 1594-1602. PubMed
Hermsen CC, Verhage DF, Telgt DS, Teelen K, Bousema JT, Roestenberg M, Bolad A, Berzins K, Corradin G, Leroy O, Theisen M, Sauerwein RW: Glutamate-rich protein (GLURP) induces antibodies that inhibit in vitro growth of Plasmodium falciparum in a phase 1 malaria vaccine trial. Vaccine. 2007, 25: 2930-2940. CrossRefPubMed
Belard S, Issifou S, Hounkpatin AB, Schaumburg F, Ngoa UA, Esen M, Fendel R, de Salazar PM, Murbeth RE, Milligan P, Imbault N, Imoukhuede EB, Theisen M, Jepsen S, Noor RA, Okech B, Kremsner PG, Mordmuller B: A randomized controlled phase Ib trial of the malaria vaccine candidate GMZ2 in African children. PLoS One. 2011, 6: e22525- PubMedCentralCrossRefPubMed
Mordmuller B, Szywon K, Greutelaers B, Esen M, Mewono L, Treut C, Murbeth RE, Chilengi R, Noor R, Kilama WL, Imoukhuede EB, Imbault N, Leroy O, Theisen M, Jepsen S, Milligan P, Fendel R, Kremsner PG, Issifou S: Safety and immunogenicity of the malaria vaccine candidate GMZ2 in malaria-exposed, adult individuals from Lambarene, Gabon. Vaccine. 2010, 28: 6698-6703. PubMedCentralCrossRefPubMed
Makler MT, Hinrichs DJ: Measurement of the lactate dehydrogenase activity of Plasmodium falciparum as an assessment of parasitemia. AmJTrop Med Hyg. 1993, 48: 205-210.
Maguire JD, Lederman ER, Barcus MJ, O'Meara WA, Jordon RG, Duong S, Muth S, Sismadi P, Bangs MJ, Prescott WR, Baird JK, Wongsrichanalai C: Production and validation of durable, high quality standardized malaria microscopy slides for teaching, testing and quality assurance during an era of declining diagnostic proficiency. Malar J. 2006, 5: 92- PubMedCentralCrossRefPubMed
van Vianen PH, Thaithong S, Reinders PP, van Engen A, van der Keur M, Tanke HJ, van der Kaay HJ, Mons B: Automated flow cytometric analysis of drug susceptibility of malaria parasites. AmJTrop Med Hyg. 1990, 43: 602-607.
Jimenez-Diaz MB, Mulet T, Gomez V, Viera S, Alvarez A, Garuti H, Vazquez Y, Fernandez A, Ibanez J, Jimenez M, Gargallo-Viola D, Angulo-Barturen I: Quantitative measurement of Plasmodium-infected erythrocytes in murine models of malaria by flow cytometry using bidimensional assessment of SYTO-16 fluorescence. Cytometry A. 2009, 75: 225-235. CrossRefPubMed
Pratt-Riccio LR, Bianco C, Totino PR, Perce-Da-Silva Dde S, Silva LA, Riccio EK, Ennes-Vidal V, Neves-Ferreira AG, Perales J, Da Rocha SL, Dias-Da-Silva F, Ferreira-da-Cruz MF, Daniel-Ribeiro CT, De Oliveira-Ferreira J, Theisen M, Carvalho LJ, Banic DM: Antibodies against the Plasmodium falciparum glutamate-rich protein from naturally exposed individuals living in a Brazilian malaria-endemic area can inhibit in vitro parasite growth. Mem Inst Oswaldo Cruz. 2011, 106 (Suppl 1): 34-43. CrossRefPubMed
Jensen JB: Concentration from continuous culture of erythrocytes infected with trophozoites and schizonts of Plasmodium falciparum. AmJTrop Med Hyg. 1978, 27: 1274-1276.
Lousada-Dietrich S, Jogdand PS, Jepsen S, Pinto VV, Ditlev SB, Christiansen M, Larsen SO, Fox CB, Raman VS, Howard RF, Vedvick TS, Ireton G, Carter D, Reed SG, Theisen M: A synthetic TLR4 agonist formulated in an emulsion enhances humoral and Type 1 cellular immune responses against GMZ2–a GLURP-MSP3 fusion protein malaria vaccine candidate. Vaccine. 2011, 29: 3284-3292. CrossRefPubMed
- Flow cytometric readout based on Mitotracker Red CMXRos staining of live asexual blood stage malarial parasites reliably assesses antibody dependent cellular inhibition
Prajakta S Jogdand
Susheel K Singh
Morten H Dziegiel
- BioMed Central
Neu im Fachgebiet Innere Medizin
Meistgelesene Bücher aus der Inneren Medizin
Mail Icon II