Rapid Detection and Enumeration of Naegleria fowleri in Surface Waters by Solid-Phase Cytometry

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 Rphycoerythrin 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.The free-living amoeba Naegleria fowleri (3, 16), found in diverse freshwater environments, produces a rapidly fatal primary amoebic meningoencephalitis after exposure to contaminated water (7,11,12). N. fowleri infects mostly young and healthy people swimming in contaminated water. Symptoms occur in a few days, followed by a dramatic clinical course and death. Therefore, risk prevention is essential and necessitates environmental monitoring using a rapid and accurate assay to distinguish pathogenic N. fowleri from other free-living amoeba in water samples.Current methods for detection and enumeration of Naegleria species are based on culture techniques (8) followed by identification using monoclonal antibodies (19,21), PCR (10,20), or enzyme electrophoresis (15). Additionally, isolates are tested for pathogenicity in mice. These methods are timeconsuming, and novel methods are being developed to increase the sensitivity and rapidity of detection and thus reduce the amount of time required to obtain results. The main challenges for the development of an assay are to provide tools for the real-time monitoring of the pathogen in the aquatic environment which are highly quantitative and sensitive.Epifluorescence microscopy and flow cytometry are commonly used for the detection and enumeration of cells after fluorescent staining (1, 6). However, none of these techniques can be applied to the detection of low concentrations of pathogens in the aquatic environment because of their low quantitative sensitivity (10). The ChemScan system (Chemunex, Ivry, France) is a recently developed solid-phase cytometer that uses fluorescent labeling of microorganisms after concentration of organisms by filtration on a membrane in combination with an automated detection and counting system (13, 23). Solid-phase cytometry is the only technique that allows the accurate enumeration of rare events (down to one cell on a filtration membrane), providing the same sensitivity as traditional culture methods (10). This sy...

Rapid Detection and Enumeration of Legionella pneumophila in Hot Water Systems by Solid-Phase Cytometry

“…Briefly, light is provided by an air-cooled argon laser emitting at 488 nm, and the fluorescence emission is collected in the Aeromonas hydrophila The device is able to differentiate between labeled organisms and fluorescent particles present in the sample based on the optical and electronic characteristics of the signals generated (18). When this discrimination procedure is not used, all fluorescent events detected, including the background signal, are counted by the cytometer and can be manually distinguished by using an epifluorescence microscope (Olympus BX41) fitted on a motorized stage driven by the computer of the cytometer, as described elsewhere (5,18,20). Data obtained from manual microscope validation of bacteria were used to optimize the setting of the instrument and the automatic discrimination procedure.…”

“…The recent development of solid-phase cytometry (ChemScanRDI; Chemunex, Ivry-sur-Seine, France) has been seen as a great stride forward in the field of quality control, since it represents a rapid and sensitive device for the enumeration of low concentrations of fluorescence-labeled cells in water samples (15). This system can be applied to the detection of pathogenic microorganisms when combined with specific labeling using FISH or IF (2,20,21).…”

Primary amoebic meningoencephalitis due to <i>Naegleria fowleri</i>

“…Patient's survival depends on an initial awareness of the organism and laboratory confirmation by growing it in a liquid axenic media or 1.5% non-nutrient agar plates, flagellation test, exposition to higher temperature, biochemical and molecular methods. 11,12 In the present case, the clinical and epidemiologic history demonstrated that the patient contracted PAM seven days after swimming in pond water during the unusually hot summer. Laboratory investigations confirmed the clinical suspicion.…”

Meningoencephalitis by Naegleria fowleri: epidemiological study in Anzoategui state, Venezuela

“…However, the real incidence of this infection in this country is unknown due to a lack of experience in its diagnosis 13 and only clinical cases caused by N. fowleri and B. mandrillaris 3 10 15 have been described.…”