No abstract

Free-living amoebae (FLA) are distributed ubiquitously in aquatic environments with increasing importance in hygienic, medical and ecological relationships to man. In this study, water samples from Belgian industrial cooling circuits were quantitatively surveyed for the presence of FLA. Isolated, thermotolerant amoebae were identified morphologically as well as using the following molecular methods: enzyme-linked immunosorbent assay and isoenzyme electrophoresis and PCR. Thermophilic amoebae were present at nearly all collection sites, and the different detection methods gave similar results. Naegleria fowleri was the most frequently encountered thermotolerant species, and concentrations of thermotolerant FLA were correlated with higher temperatures.

Real-time polymerase chain reaction melting curve analysis (MCA) allows differentiation of several free-living amoebae species. Distinctive characteristics were found for Naegleria fowleri, N. lovaniensis, N. australiensis, N. gruberi, Hartmanella vermiformis, and Willaertia magna. Species specificity of the amplicons was confirmed using agarose gel electrophoresis and sequence-based approaches. Amplification efficiency ranged from 91% to 98%, indicating the quantitative potential of the assay. This MCA approach can be used for quantitative detection of free-living amoebae after cultivation but also as a culture-independent detection method.

To detect Naegleria spp, in particular Naegleria fowleri, the causative agent of human primary amoebic meningoencephalitis, a flagellation test (FT) is routinely used followed by a specific ELISA. A positive FT indicates the presence of Naegleria spp although some false negatives are likely to occur since parameters for enflagellation vary greatly. As negative FTs are not routinely screened any further for the presence of N. fowleri, this could result in an underestimation of the presence of this pathogen. Therefore, amoebae were further analysed using ELISA and standard PCR not only after a positive but also after a negative FT. In this study 39 cultures containing amoebae were tested with FT, ELISA and the two PCR assays with 11 positive for FT. These were submitted to ELISA and four confirmed as N. fowleri. PCR with the common primer-set on these 11 positive FTs revealed all as Naegleria spp. The specific PCR used on these cultures detected four positive for N. fowleri, corresponding totally with the ELISA results. The 28 negative flagellation tests were also submitted to ELISA and PCR. Of these, 11 were identified as Naegleria spp with common PCR and six as N. fowleri as well as with ELISA and the specific PCR. When the detection of Naegleria spp is based on intermediary processes, such as flagellation tests, false negatives are likely to occur leading to severe underestimations. This study has shown that amoebae taken from negative FTs can be identified as Naegleria spp and N. fowleri when using PCR and ELISA. The application of at least one of the specific N. fowleri tests is recommended for routine screening. The heterogeneous distribution of the false negative results between the different power plants suggested the presence of different genotypes.

Although traditional culture methods are appropriate for detection of Legionella species, such culture takes several days. Rapid detection (< 24 h) of individual Legionella is possible using fluorescent in situ hybridisation (FISH) on whole bacteria. Water samples were filtered and the concentrated bacteria were immediately detected (without culture) with a fluorescence microscope following appropriate labelling. The detection level was very high and quantification was possible. For the detection of all Legionella spp. the probe LEG705 was used, complementary to a 16S rRNA sequence conserved in all Legionella spp. For specific detection of L. pneumophila the probe LEGPNE1 was used. This probe is designed against a variable domain of the 16S rRNA sequence from L. pneumophila. CY3 and FLUOS labels were tested and CY3 showed clearly detectable bacteria with minimum background staining. This FISH technique is very sensitive, fast, reliable and individual bacteria are easily detected.