Optimisation of a flow cytometry protocol for detection of Cryptosporidium parvum in hospital tap water and human stools
Abstract number: 1733_56
Barbosa J., Costa-de-Oliveira S., Gonçalves Rodrigues A., Pina-Vaz C.
Cryptosporidium parvum is a waterborne agent, causing diarrhoea in immunocompromised patients. Its diagnosis is based upon microscopic detection of oocysts in faeces following alcohol-acid staining. This conventional procedure presents low sensibility and specificity, being highly dependent of the observe expertise. Our main objective was to optimise a specific Flow Cytometric (FC) protocol for detection of C. parvum in water and stools and to establish its sensibility limit. C. parvum oocysts (Waterborne Inc, USA) were used for protocol optimisation. FC analysis was performed using oocyst suspensions stained with different concentrations of a specific monoclonal antibody conjugated with R-phycoerytrin (Crypt-a-Glo, Waterbone). Serial concentrations (2×105, 2×104, 2×103, 2×102 oocysts/mL) were later stained with an optimised antibody concentration and analysed by FC. Specificity and the sensibility limit of the method were established using both prokaryotic (Escherichia coli, Sthaphylococcus aureus) and eukaryotic microorganisms (Candida albicans, Giardia lamblia cysts). FC analysis was repeated according of the optimised conditions, using human stools and hospital tap water, simulating clinical and environmental settings. Several procedures were also assayed to reduce the loss of oocysts and improve its detection: different filters (gauze, paper filters), centrifugation time and velocity, and distinct flotation solutions (NaCl, ZnSO4.7H2O). As the antibody concentration decreased, a decline of peak intensity was registered. The optimal concentration of antibody was 3.0 mg/mL for 105 oocysts/mL. We established a threshold of detection of 2×103 oocysts/mL. The staining procedure was shown to be specific, no cross-reaction occurring with bacteria, fungi or parasites. However, a decrease in staining was seen especially when Giardia was present, but it did not interfere with the result. Bellow threshold limit, fluorescence was not enough to allow the discrimination of oocysts. Interference of debris was more frequently observed in faeces than in water samples. A prolonged incubation of faeces in a ZnSO4 solution followed by centrifugation for 10 minutes allowed a clear separation of oocysts from debris. With the use of specific antibodies, a distinct cellular population corresponding to oocysts could be represented in the FC histogram. This study describes the first optimised FC protocol for detection of C. parvum in hospital tap water and human faeces.
|Session name:||European Society of Clinical Microbiology and Infectious Diseases|
|Location:||ICC, Munich, Germany|
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