Analysis-only detection of Giardia by combining immunomagnetic separation and two-color flow cytometry.

Abstract

Giardia is a protozoan parasite of concern to water utilities. Giardia detection relies on cyst isolation and confirmation with the use of fluorescence microscopy. It is of interest to develop a flow cytometric (FCM) method that reliably detects one cyst in 10 L of water. To date all available antibodies have targeted the same epitope on the cyst wall. To achieve a reliable method, two independent probes are required. Giardia cysts were spiked into a backwash water sample with and without prior hybridization to peptide nucleic acid (PNA) probes. Immunomagnetic separation (IMS) as a pre-enrichment step was compared with filtration of the water sample. Cysts were recovered with two-color FCM. Those cysts hybridized with PNA and fluorescein isothiocyanate (FITC) were dual stained with monoclonal antibody (mAb) conjugated to phycoerythrin (PE); those not hybridized to PNA were dual stained with mAb-FITC and mAb-PE. A fourfold increase in fluorescent signal intensity was obtained when combining the mAb-PE and PNA probe compared with two-color antibody staining. When combined with IMS, Giardia was successfully identified by FCM, with no false positives detected. Analysis-only FCM detection of Giardia in water is feasible. Further method development incorporating PNA probe hybridization after IMS is necessary.