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...

Free-living Naegleria fowleri amoebae cause primary amoebic meningoencephalitis (PAM). Because of the apparent conflict between their ubiquity and the rarity of cases observed, we sought to develop a model characterizing the risk of PAM after swimming as a function of the concentration of N. fowleri. The probability of death from PAM as a function of the number of amoebae inhaled is modeled according to results obtained from animals infected with amoeba strains. The calculation of the probability of inhaling one or more amoebae while swimming is based on a double hypothesis: that the distribution of amoebae in the water follows a Poisson distribution and that the mean quantity of water inhaled while swimming is 10 ml. The risk of PAM for a given concentration of amoebae is then obtained by summing the following products: the probability of inhaling n amoebae ؋ the probability of PAM associated with inhaling these n amoebae. We chose the lognormal model to assess the risk of PAM because it yielded the best analysis of the studentized residuals. Nonetheless, the levels of risk thereby obtained cannot be applied to humans without correction, because they are substantially greater than those indicated by available epidemiologic data. The curve was thus adjusted by a factor calculated with the least-squares method. This provides the PAM risk in humans as a function of the N. fowleri concentration in the river. For example, the risk is 8.5 ؋ 10 ؊8 at a concentration of 10 N. fowleri amoebae per liter.

Naegleria fowleri, a free-living amoeba, is the causative agent of primary amoebic meningoencephalitis, a fatal human disease of the central nervous system often contracted after swimming in fresh water. Identifying sites contaminated by N. fowleri is important in order to prevent the disease. An Enzyme-Linked ImmunoSorbent Assay (ELISA) has been developed for the specific identification of N. fawleri in primary cultures of environmental water samples. Of 939 samples isolated from artificially heated river water and screened by ELISA, 283 were positive. These results were subsequently confirmed by isoelectric focusing, the established reference method. A sensitivity of 97.4% and a specificity of 97% were obtained. These results indicate that this ELISA method is reliable and can be considered as a powerful tool for the detection of N. fowleri in environmental water samples.