Optimization of a flow cytometry protocol for detection and viability assessment of Giardia lamblia

Giardia lamblia is one of the most important cause of diarrhoea in humans around the world. Transmission routes are complex including fecal—oral as well as waterborne and foodborne transmission. Detection methods in water and stools involve cyst concentration procedures, followed by conventional microscopy which is time-consuming. Higher sensibility was described with antigen detection but showing unpredictable specificity. Molecular genetic studies are complex and difficult for routine analysis. We have optimised a specific Flow Cytometric (FC) protocol for detection of G. lamblia , established its detection limit and assayed the possibility of cross-reaction with other microorganisms. Viability studies, important to evaluate the infectious risk and for treatment monitorization, were also performed by FC. A known concentration of G. lamblia cysts (for Waterborne Inc, USA and from a Giardia culture) were stained with serial concentrations of a fluorescein-labelled (FL) mouse monoclonal antibody (Giardia–a–Glo, Waterborne) and analysed by in a FACS Calibur cytometer (Becton Dickinson, Canada) at FL1 (525 nm-green). Several dilutions of the stained cysts (from 2×105 to 1×102 cysts/ml) were analysed by FC for assessment of the detection limit. Cross-reactions were investigated using both prokaryotic ( Escherichia coli , Staphylococcus aureus ) and eukaryotic microorganisms ( Candida albicans , Cryptosporidium parvum oocysts). Suspensions with dead and viable cysts were stained with 5 μg/ml of propidium iodide (PI, Sigma), associated with the specific fluorescent antibody and analysed at FL3 (670 nm-red). The optimal specific-antibody concentration was 1.5 μg/ml, yielding a clearly separated histogram from autofluorescence. A threshold of 2×102 cysts/ml was established. No cross-reaction occurred with bacteria, fungi or parasites. When using the two fluorescent probes, specific-antibody and PI (a marker of dead), viable cysts were distinguished from dead. A new detection method using FC is now available for detection of Giardia and for evaluation of its viability.