Integrated Real-Time PCR for Detection and Monitoring of Legionella pneumophila in Water Systems

Abstract

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...

Specific Real-Time PCR for Simultaneous Detection and Identification of Legionella pneumophila Serogroup 1 in Water and Clinical Samples

“…Many studies have shown that qPCR for the detection of Legionella species in the environment is a promising method that could be used instead of culture (3,4,17,33,39,40). However, in contrast to culture, available qPCR methods do not allow the identification of L. pneumophila Sg1, the L. pneumophila serogroup responsible for more than 85% of human infections.…”

“…Thus, research into the development of molecular tools to improve the detection of Legionella in water, especially quantitative real-time PCR (qPCR) methods, is very active (3,17,33,38,39,40). Indeed, real-time PCR allows rapid detection and quantification of DNA with high sensitivity and specificity.…”

Viability PCR, a Culture-Independent Method for Rapid and Selective Quantification of Viable Legionella pneumophila Cells in Environmental Water Samples

“…qPCR is an alternative tool that offers rapid, sensitive, and specific detection of Legionella bacteria in environmental water samples (4,5,12,26,65,68). PCR results can be obtained in hours instead of days, and VBNC Legionella cells can also be detected (12,26).…”

Detection of <i>Legionella</i> spp. in Natural and Man-made Water Systems Using Standard Guidelines

“…This method allows the isolation and the quantification of cultivable Legionella species from environmental and clinic samples but it does have limitations [13,59]. The cultivation method has also the disadvantage of being a very time-consuming technique, requiring selective media and several days of incubation (7 to 10 days) to obtain results [25,59].…”

“…These organisms may be a cause of nosocomial and community-acquired infections, principally in immunocompromised patients [10][11][12]. Legionellosis outbreaks are often associated with high mortality rates [13].…”