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Comparative Study
. 2002 Jun;68(6):3102-7.
doi: 10.1128/AEM.68.6.3102-3107.2002.

Rapid detection and enumeration of Naegleria fowleri in surface waters by solid-phase cytometry

Claire Pougnard  1 , Philippe Catala, Jean-Louis Drocourt, Stephane Legastelois, Pierre Pernin, Emmanuelle Pringuez, Philippe Lebaron
Affiliations
Comparative Study

Rapid detection and enumeration of Naegleria fowleri in surface waters by solid-phase cytometry

Claire Pougnard et al. Appl Environ Microbiol. 2002 Jun.

Abstract

A new method for the rapid and accurate detection of pathogenic Naegleria fowleri amoebae in surface environmental water was developed. The method is based on an immunofluorescent assay combined with detection by solid-phase cytometry. In this study we developed and compared two protocols using different reporter systems conjugated to antibodies. The monoclonal antibody Ac5D12 was conjugated with biotin and horseradish peroxidase, and the presence of cells was revealed with streptavidin conjugated to both R-phycoerythrin and cyanine Cy5 (RPE-Cy5) and tyramide-fluorescein isothiocyanate, respectively. The RPE-Cy5 protocol was the most efficient protocol and allowed the detection of both trophozoite and cyst forms in water. The direct counts obtained by this new method were not significantly different from those obtained by the traditional culture approach, and results were provided within 3 h. The sensitivity of the quantitative method is 200 cells per liter. The limit is due only to the filtration capacity of the membrane used.

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Figures

FIG. 1.
FIG. 1.
Optical configuration of the ChemScan system. PMT, photomultiplier tube; DSP, digital signal processor; UPC, user computer.
FIG. 2.
FIG. 2.
Microscopic photographs of N. fowleri stained by immunofluorescence and two fluorochromes, FITC and RPE-Cy5. (A and B) Labeled trophozoites from axenic culture samples; (C and D) labeled cyst from monoxenic samples; (E and F) labeled flagellates from natural water samples.
FIG. 3.
FIG. 3.
Correlation between solid-phase cytometry and MPN counts of N. fowleri in natural water samples at different concentrations.
See this image and copyright information in PMC

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