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

• Type: Article in International Scientific Journal
• Date: 2008

Authors

barbosa, j (Author) Other; costa-de-oliveira, s (Author) FMUP; rodrigues, ag (Author) FMUP; pina-vaz, c (Author) FMUP

Journal

Title: Travel Medicine and Infectious Disease

Vol. 6

Pages: 234-239

ISSN: 1477-8939

Publisher: Elsevier

Indexing

ISI Web of Knowledge

Scopus - 23 Citations

Pubmed / Medline

Scientific classification

CORDIS: Health sciences

Other information

Authenticus ID: P-007-M9M

DOI: 10.1016/j.tmaid.2008.01.004

Abstract (EN): Giardia lamblia is responsible for causing diarrhoeal diseases in humans. Infection occurs by fecal-oral route and is considered an important water pathogen, since many water surfaces are infected by cysts. Most studies involve cyst concentration procedures, followed by conventional microscopy, but are often tedious and influenced by fatigue. Our main objective was to optimize a specific flow cytometric (FC) protocol for detection of G. lamblia, to establish its sensibility limit and also the cyst viability. G. lamblia cysts (Waterborne, Inc., USA) were used for protocol optimization. FC analysis was performed using cyst suspensions stained with serial concentrations of a fluorescein-labelled mouse monoclonal antibody (Giardia-a-Glo, Waterborne). Serial concentrations (2×105, 1×105, 2×104, 1×104, 2×103, 1×103, 2×102 and 1×102 cysts/ml) were stained with the optimized antibody concentration and analysed by FC. Specificity and sensibility limit of the method were established using both prokaryotic (Escherichia coli, Staphylococcus aureus) and eukaryotic microorganisms (Candida albicans, Cryptosporidium parvum oocysts). Dead cysts were stained with 5.0 ¿g/ml of propidium iodide (PI, Sigma), with and without the specific fluorescent antibody. As the antibody concentration decreased, a decline of peak intensity was registered; 1.5 ¿g/ml was considered as the optimal antibody concentration, yielding a histogram clearly separated. We established a threshold of detection of 2×102 cysts/ml. Below threshold limit fluorescence was not enough to allow the discrimination of cysts. The staining procedure was shown to be specific, no cross-reaction occurring with bacteria, fungi or parasites. When using both antibody and PI, we could distinguish the viable cyst. With the use of specific antibodies, a distinct cellular population corresponding to cysts could be represented in the FC histogram.

Language: English

Type (Professor's evaluation): Scientific

No. of pages: 6