Detection and counting of Cryptosporidium parvum in HCT-8 cells by flowcytometry.
The objective of the present study was to evaluate flowcytometry analysis (FCA) as a tool for rapidly and objectively estimating the percentage of cells infected with Cryptosporidium parvum in an in vitro model. We compared the results to those obtained with immunofluorescence assay (IFA) and evaluated the intra-assay variability of both assays and the inter-assay variability of IFA. Human ileocecal adenocarcinoma cells (HCT-8) were infected with different doses of excysted oocysts. After 24 hours, cells were analysed by FCA and by IFA using a monoclonal antibody that recognises a C. parvum antigenic protein and a lectin that binds with glycoproteins present in the parasitophorous vacuoles. The coefficient of variability in terms of the percentage of infected cells was lower for FCA (i.e., 13-14%) than for IFA (i.e., 27-38% when performed by a single operator and 19-22% when performed by three operators), suggesting that FCA is more accurate, in that it is not subject to operator expertise. FCA also has the advantage of allowing the entire culture to be examined, thus avoiding problems with heterogeneity among microscopic fields. In light of these results, this method could also be used to test new anti-Cryptosporidium drugs.
Mele
R
R
Department of Infectious, Parasitic and Immunomediated Diseases, Istituto Superiore di Sanità, 00161 Rome, Italy.
Gomez
Morales M
MM
Tosini
F
F
Pozio
E
E
D000818:Animals; D000911:Antibodies, Monoclonal; D000953:Antigens, Protozoan / Q000276:immunology; D003457:Cryptosporidiosis / Q000175:diagnosis; D016785:Cryptosporidium parvum / Q000254:growth & development / Q000276:immunology / Q000302:isolation & purification*; D005434:Flow Cytometry / Q000379:methods*; D019084:Fluorescent Antibody Technique, Indirect / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured / Q000469:parasitology*
D000911:Antibodies, Monoclonal; D000953:Antigens, Protozoan