title: Unambiguous determination of Plasmodium vivax reticulocyte invasion by flow cytometry.
abstract: The invasion of CD71+ reticulocytes by Plasmodium vivax is a crucial yet poorly characterised event. The application of flow cytometry to ex vivo invasion assays promises to facilitate the quantitative analysis of P. vivax reticulocyte invasion. However, current protocols suffer from a low level of sensitivity due to the absence of a particular design for P. vivax cell tropism. Importantly, merozoite invasion into contaminating red blood cells from the schizont inoculum (auto-invasion) may confound the analysis. Here we present a stable two-color flow cytometry assay for the accurate quantification of P. vivax merozoite invasion into intracellularly labelled CD71+ reticulocytes. Various enzymatic treatments, antibodies and invasion inhibitory molecules were used to successfully demonstrate the utility of this method. Fluorescent labelling of red blood cells did not affect the invasion and early intra-erythrocytic development of P. vivax. Importantly, this portable field assay allows for the economic usage of limited biological material (parasites and reticulocytes) and the intracellular labeling of the target cells reduces the need for highly purified schizont inoculums. This assay will facilitate the study of P. vivax merozoite biology and the testing of vaccine candidates against vivax malaria.
authors.affiliation: Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, National University Health System, 5 Science Drive 2, Blk MD4, Level 3, Singapore 117597, Singapore; Singapore Immunology Network (SIgN), A(∗)STAR, 8A Biomedical Grove, Singapore 138648, Singapore.
authors.forename: Jee-Sun
authors.initials: JS
authors.lastname: Cho
authors.affiliation: Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, National University Health System, 5 Science Drive 2, Blk MD4, Level 3, Singapore 117597, Singapore.
authors.forename: Bruce
authors.initials: B
authors.lastname: Russell
authors.affiliation: Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, National University Health System, 5 Science Drive 2, Blk MD4, Level 3, Singapore 117597, Singapore.
authors.forename: Varakorn
authors.initials: V
authors.lastname: Kosaisavee
authors.affiliation: Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, National University Health System, 5 Science Drive 2, Blk MD4, Level 3, Singapore 117597, Singapore.
authors.forename: Rou
authors.initials: R
authors.lastname: Zhang
authors.affiliation: INSERM, UMR_S1134, INTS 6, rue Alexandre Cabanel, 75739 Paris, France; Institut National de la Transfusion Sanguine, F-15013 Paris, France; Universite Paris 7-Denis Diderot, Sorbonne Paris cite, F-15-13 Paris, France.
authors.forename: Yves
authors.initials: Y
authors.lastname: Colin
authors.affiliation: INSERM, UMR_S1134, INTS 6, rue Alexandre Cabanel, 75739 Paris, France; Institut National de la Transfusion Sanguine, F-15013 Paris, France; Universite Paris 7-Denis Diderot, Sorbonne Paris cite, F-15-13 Paris, France.
authors.forename: Olivier
authors.initials: O
authors.lastname: Bertrand
authors.affiliation: Pillar of Engineering Product Development, Singapore University of Technology & Design, 8 Somapah Road, Singapore 487372, Singapore.
authors.forename: Rajesh
authors.initials: R
authors.lastname: Chandramohanadas
authors.affiliation: Shoklo Malaria Research Unit, Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, 68/30 Bantung Road, Mae Sot 63110, Thailand; Centre for Tropical Medicine, Nuffield Department of Medicine, University of Oxford, Old Road, Oxford OX3 7LJ, United Kingdom.
authors.forename: Cindy S
authors.initials: CS
authors.lastname: Chu
authors.affiliation: Shoklo Malaria Research Unit, Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, 68/30 Bantung Road, Mae Sot 63110, Thailand; Centre for Tropical Medicine, Nuffield Department of Medicine, University of Oxford, Old Road, Oxford OX3 7LJ, United Kingdom.
authors.forename: Francois
authors.initials: F
authors.lastname: Nosten
authors.affiliation: Singapore Immunology Network (SIgN), A(∗)STAR, 8A Biomedical Grove, Singapore 138648, Singapore.
authors.forename: Laurent
authors.initials: L
authors.lastname: Renia
authors.affiliation: Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, National University Health System, 5 Science Drive 2, Blk MD4, Level 3, Singapore 117597, Singapore; Singapore Immunology Network (SIgN), A(∗)STAR, 8A Biomedical Grove, Singapore 138648, Singapore. Electronic address: micbjbm@nus.edu.sg.
authors.forename: Benoit
authors.initials: B
authors.lastname: Malleret
mesh_terms: D015703:Antigens, CD / Q000276:immunology; D001431:Bacteriological Techniques / Q000191:economics / Q000379:methods; D001483:Base Sequence; D004912:Erythrocytes / Q000469:parasitology; D005434:Flow Cytometry / Q000379:methods*; D057166:High-Throughput Screening Assays / Q000379:methods; D006801:Humans; D016780:Malaria, Vivax / Q000097:blood / Q000469:parasitology*; D010966:Plasmodium vivax / Q000166:cytology / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D011990:Receptors, Transferrin / Q000276:immunology; D012156:Reticulocytes / Q000276:immunology / Q000469:parasitology*
chemical_list: D015703:Antigens, CD; C068126:CD71 antigen; D011990:Receptors, Transferrin
keywords: CD71; DARC; Flow cytometry; Invasion; Plasmodium vivax; Reticulocytes; Sugar mimetics