Int. J. Hyg. Environ.-Health 211 (2008) 403–411 Real-time PCR assay for the detection and quantification of Legionella pneumophila in environmental water samples: Utility for daily practice Florent Morio a , Ste´phane Corvec a,Ã , Nathalie Caroff b , Florence Le Gallou a , Henri Drugeon a , Alain Reynaud b a Laboratoire de Bacteriologie-Virologie-Hygiene Hospitaliere, Institut de Biologie des Hopitaux de Nantes, 9 quai Moncousu, 44093 Nantes Cedex 01, France b Laboratoire de Microbiologie, Faculte´de Pharmacie, Universite´de Nantes, France Received 5 October 2006; received in revised form 24 May 2007; accepted 26 June 2007 Abstract We developed a quantitative real-time PCR assay targeting the mip gene of Legionella pneumophila for a prospective study from September 2004 to April 2005. It was compared with a standard culture method (French guideline AFNOR T90-431), analysing 120 water samples collected to monitor the risk related to Legionellae at Nantes hospital and to investigate a case of legionellosis acquired from hospital environment. Samples from six distinct water distribution systems were analysed by DNA extraction, amplification and detection with specific primers and FRET probes. The detection limit was 100 genomic units of L. pneumophila per liter (GU/l), the positivity threshold about 600 GU/l and the quantification limit 800 GU/l. PCR results were divided into three groups: negative (n ¼ 63), positive but non-quantifiable (n ¼ 22) or positive (n ¼ 35). PCR showed higher sensitivity than culture, whereas four culture-positive samples appeared negative by PCR (PCR inhibitor detected for two of them). Although no correlation was observed between both methods and real-time PCR cannot substitute for the reference method, it represents an interesting complement. Its sensitivity, reproducibility and rapidity appear particularly interesting in epidemic contexts in order to identify the source of contamination or to evaluate critical points of contamination in water distribution systems. r 2007 Elsevier GmbH. All rights reserved. Keywords: Legionella pneumophila; Real-time PCR; Standard culture method; Environmental water samples; Quantification Introduction Legionella pneumophila, first recognised in 1977 (Fraser et al., 1977), is a significant cause of acquired pneumonia (Yu et al., 2002). About 90% of the cases of legionellosis are due to this species and more than 80% of them to serogroup 1 (Doleans et al., 2004; Cooke, 2004; Yu et al., 2002). The hospital water supplies often contain Legionella species, a potential source of severe nosocomial infec- tions, especially for immunocompromised patients. In France, from 2001 to 2005, cases increased from 801 to 1527, partially related to improvement of diagnosis methods, better approach and increased reporting of the disease (Campese et al., 2006; Den Boer et al., 2006). ARTICLE IN PRESS www.elsevier.de/ijheh 1438-4639/$ - see front matter r 2007 Elsevier GmbH. All rights reserved. doi:10.1016/j.ijheh.2007.06.002 Ã Corresponding author. Tel.: +33 240 083955; fax: +33 240 083829. E-mail address: stephane.corvec@chu-nantes.fr (S. Corvec).