Design and implementation of a protocol for the detection of Legionella in clinical and environmental samples Elizabeth J. Nazarian, Dianna J. Bopp, Amy Saylors, Ronald J. Limberger, Kimberlee A. Musser ⁎ Wadsworth Center, New York State Department of Health, Albany, NY 12208, USA Received 22 February 2008; accepted 13 May 2008 Abstract Our laboratory has developed a novel real-time polymerase chain reaction (PCR) assay for the detection of Legionella pneumophila and differentiation from other Legionella spp. in clinical and environmental samples. The 23S rRNA gene was used as a target to detect all Legionella spp., and the mip gene was targeted for the specific detection of L. pneumophila in this multiplex Taqman® real-time PCR assay. The 23S rRNA gene is a novel target for Legionella testing; it detects all species and serogroups of Legionella without the contamination issues that accompany the use of the 16S rRNA gene as a target. This assay provides an analytical sensitivity of <1 colony- forming unit and a specificity of 100%. Because culture is important and provides a means for molecular typing via pulsed-field gel electrophoresis (PFGE), we developed a testing algorithm that includes both the new real-time PCR assay and culture for clinical and environmental samples and applied this algorithm during a period of 3 years. Of the 64 clinical samples received by our laboratory for Legionella testing during this period, PCR was found to be an essential diagnostic tool because only 13.3 % (2/15) clinical samples that were determined to be L. pneumophila were detected by culture during this period. Of the 276 environmental samples received for Legionella testing during this period, 140 were found to be positive for L. pneumophila. Of these 140 samples, 69.3% were detected by both PCR and culture methods, 29.3% were positive by PCR alone, and 1.4% were positive by culture methods alone. We feel these results indicate that our algorithm, including both PCR and culture, should be used for environmental samples. Among both the clinical and environmental Legionella samples identified by PCR, a subset was not suitable for culture because of issues of lengthy transport, antimicrobial treatment, or bacterial overgrowth. Samples like these are commonly submitted to our laboratory, so the use of our testing algorithm combining these methods is critical. We conclude that molecular and culture methods must be used in combination to provide the best and most comprehensive approach to laboratory detection and investigation of legionellosis. © 2008 Elsevier Inc. All rights reserved. Keywords: Legionella; Real-time PCR; Protocol 1. Introduction Whereas the bacteria of the genus Legionella are responsible for an acute self-limiting nonpneumonic illness known as Pontiac fever, a far more significant public health issue is the potentially fatal pneumonic form of legionellosis, Legionnaires' disease. Legionella pneumophila is the most common pathogenic species within the genus Legionella, accounting for 90% of all reported cases of legionellosis in the United States (Fields et al., 2002). L. pneumophila serogroup 1 accounts for the majority of these (Rantakokko-Jalava and Jalava, 2001). Other species of Legionella that have been implicated in disease include Legionella dumoffii, Legionella micdadei, Legionella bozemanii, and Legionella longbeachae; however, they occur at much lower frequencies. As natural inhabitants of aquatic environments, legionellae are ubiqui- tous. However, their presence can be particularly problematic when found in the water distribution systems of healthcare facilities. Of particular concern is the risk of nosocomial and community-acquired infection of the immune compromised, the elderly, and individuals with underlying respiratory conditions via aerosolization of water from contaminated water supplies. Available online at www.sciencedirect.com Diagnostic Microbiology and Infectious Disease 62 (2008) 125 – 132 www.elsevier.com/locate/diagmicrobio ⁎ Corresponding author. Wadsworth Center, New York State Department of Health, 120 New Scotland Avenue, Albany, NY 12208, USA. Tel.: +1-518- 474-4177; fax: +1-518-486-7971. E-mail address: musser@wadsworth.org (K.A. Musser). 0732-8893/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2008.05.004