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...
This study shows the possibility of using real-time PCR to monitor L. pneumophila proliferations in cooling systems and the importance to adapt nucleic acid extraction and purification protocols to raw waters.
A new method was developed for the rapid and sensitive detection of viable Legionella pneumophila. The method combines specific immunofluorescence (IF) staining using monoclonal antibodies with a bacterial viability marker (ChemChrome V6 cellular esterase activity marker) by means of solid-phase cytometry (SPC). IF methods were applied to the detection and enumeration of both the total and viable L. pneumophila cells in water samples. The sensitivity of the IF methods coupled to SPC was 34 cells liter ؊1 , and the reproducibility was good, with the coefficient of variation generally falling below 30%. IF methods were applied to the enumeration of total and viable L. pneumophila cells in 46 domestic hot water samples as well as in cooling tower water and natural water samples, such as thermal spring water and freshwater samples. Comparison with standard plate counts showed that (i) the total direct counts were always higher than the plate counts and (ii) the viable counts were higher than or close to the plate counts. With domestic hot waters, when the IF assay was combined with the viability test, SPC detected up to 3.4 ؋ 10 3 viable but nonculturable L. pneumophila cells per liter. These direct IF methods could be a powerful tool for high-frequency monitoring of domestic hot waters or for investigating the occurrence of viable L. pneumophila in both man-made water systems and environmental water samples.
Aims: To test Fountain FlowTM Cytometry (FFC) for the rapid and sensitive detection of Naegleria lovaniensis amoebae (an analogue for Naegleria fowleri) in natural river waters. Methods and Results: Samples were incubated with one of two fluorescent labels to facilitate detection: ChemChrome V6, a viability indicator, and an R‐phycoerytherin (RPE) immunolabel to detect N. lovaniensis specifically. The resulting aqueous sample was passed as a stream in front of a light‐emitting diode, which excited the fluorescent labels. The fluorescence was detected with a digital camera as the sample flowed toward the imager. Detections of N. lovaniensis were made in inoculated samples of natural water from eight rivers in France and the United States. FFC enumeration yielded results that are consistent with other counting methods: solid‐phase cytometry, flow cytometry, and hemocytometry, down to concentrations of 0·06 amoebae ml−1, using a flow rate of 15 ml min−1. Conclusions: This study supports the efficacy of using FFC for the detection of viable protozoa in natural waters and indicates that use of RPE illuminated at 530 nm and detected at 585 nm provides a satisfactory means of attenuating background. Significance and Impact of the Study: Because of the severe global public health issues with drinking water and sanitation, there is an urgent need to develop a technique for the real‐time detection of viable pathogens in environmental samples at low concentrations. FFC addresses this need.
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.
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