In France, the clinical distribution of Legionella species and serogroups does not correspond to their environmental distribution. Legionella pneumophila serogroup 1 is more prevalent among clinical isolates (95.4%) than in the environment (28.2%), whereas L. anisa is more frequent in the environment (13.8%) than in the clinical setting (0.8%).

Quantitative Legionella PCRs targeting the 16S rRNA gene (specific for the genus Legionella) and the mip gene (specific for the species Legionella pneumophila) were applied to a total of 223 hot water system samples (131 in one laboratory and 92 in another laboratory) and 37 cooling tower samples (all in the same laboratory). The PCR results were compared with those of conventional culture. 16S rRNA gene PCR results were nonquantifiable for 2.8% of cooling tower samples and up to 39.1% of hot water system samples, and this was highly predictive of Legionella CFU counts below 250/liter. PCR cutoff values for identifying hot water system samples containing >10 3 CFU/liter legionellae were determined separately in each laboratory. The cutoffs differed widely between the laboratories and had sensitivities from 87.7 to 92.9% and specificities from 77.3 to 96.5%. The best specificity was obtained with mip PCR. PCR cutoffs could not be determined for cooling tower samples, as the results were highly variable and often high for culture-negative samples. Thus, quantitative Legionella PCR appears to be applicable to samples from hot water systems, but the positivity cutoff has to be determined in each laboratory.

We evaluated a ready-to-use real-time quantitative Legionella pneumophila PCR assay system by testing 136 hot-water-system samples collected from 55 sites as well as 49 cooling tower samples collected from 20 different sites, in parallel with the standard culture method. The PCR assay was reproducible and suitable for routine quantification of L. pneumophila. An acceptable correlation between PCR and culture results was obtained for sanitary hot-water samples but not for cooling tower samples. We also monitored the same L. pneumophilacontaminated cooling tower for 13 months by analyzing 104 serial samples. The culture and PCR results were extremely variable over time, but the curves were similar. The differences between the PCR and culture results did not change over time and were not affected by regular biocide treatment. This ready-to-use PCR assay for L. pneumophila quantification could permit more timely disinfection of cooling towers.Legionellosis can be acquired by inhalation of Legionella pneumophila bacteria dispersed by environmental sources, such as hot-water systems and cooling towers. Legionellosis outbreaks are often associated with high mortality rates (15 to 20%) (10). Legionella pneumophila serogroup 1 is responsible for up to 80% of cases (9,11,30). The density of Legionella cells in water is theoretically associated with the risk of legionellosis (5, 15): cell densities above 10 4 to 10 5 CFU per liter of water have been shown to represent a potential health risk to humans (20,23). Environmental Legionella monitoring is recommended in several countries (7,16), and regular treatment of cooling tower installations is obligatory in France (7).In France, conventional culture is the only approved technique for the detection and quantification of Legionella in water samples (2). However, definitive culture results take 10 days to obtain and may have decreased sensitivities due to Legionella growth characteristics (3, 6). Several authors have developed real-time-PCR-based methods for rapid detection of Legionella in water samples (3,12,27,29). However, Joly et al. recently reported that quantitative real-time PCR is influenced by the type of water sample and that the results may be laboratory dependent (12). Several commercial real-time PCR kits are currently available, such as the iQ-check real-time PCR kit (Bio-Rad, France), the Aqua Screen Lp-qDual kit (Minerva Biolabs, Germany), and the GeneDisc Legionella kit (GeneSystems, France). The main differences between these kits are based on the degrees of standardization of the three critical steps: DNA extraction, PCR preparation, and data analysis.In this study, we compared a standardized real-time quantitative PCR assay system with the conventional culture method. The PCR system, developed by GeneSystems (Bruz, France), is the first ready-to-use PCR instrument dedicated to routine Legionella detection in water samples that includes a dedicated filtration unit and DNA extraction instrument. The two methods were both applied to 136 hot-water-system sam...