Bacteriology Development of a multiplex real-time quantitative PCR assay to detect Chlamydia pneumoniae, Legionella pneumophila and Mycoplasma pneumoniae in respiratory tract secretions Martine Welti a , Katia Jaton a, *, Martin Altwegg b , Roland Sahli a , Aline Wenger a , Jacques Bille a a Institute of Microbiology, University Hospital of Lausanne, Lausanne, Switzerland b Department of Medical Microbiology, University of Zu ¨rich, Zu ¨rich, Switzerland Received 25 April 2002; accepted 20 August 2002 Abstract Atypical pathogens such as Chlamydia pneumoniae, Legionella pneumophila and Mycoplasma pneumoniae are an important cause of community-acquired pneumonia. The available detection methods (culture and serology) either lack sensitivity or give only a retrospective diagnosis. In order to improve their detection and quantification in respiratory samples, a real-time multiplex PCR, performed in two separate reactions, was developed for these three pathogens. The comparison of multiplex real-time and conventional PCR assay on 73 respiratory specimens showed an overall agreement of 98.3%, corresponding to 95.8%, 100% and 100% agreement for C. pneumoniae, L. pneumophila and M. pneumoniae, respectively. Clinical application of this multiplex real-time PCR was done on 40 respiratory samples from 38 patients with respiratory tract infections. Of 19 serology-positive patients, 14 were confirmed by the multiplex real-time PCR to be infected by either one of the three pathogens. All samples from serology-negative patients were negative with the multiplex real-time PCR. © 2003 Elsevier Science Inc. All rights reserved. 1. Introduction Community-acquired pneumonia (CAP) is a frequent cause of medical consultation in both hospital emergency departments and general practices. Most etiologic studies identified Streptococcus pneumoniae as the primary cause of CAP (Bochud et al., 2001; Marrie et al., 1996; Menendez et al., 1999). However, during the last decade with diagnos- tic improvements, agents of atypical pneumonias, such as Chlamydia pneumoniae, Legionella pneumophila and My- coplasma pneumoniae have emerged as an important cause of respiratory tract infections, accounting for between 15 to 50% of CAP (Marrie et al., 1996; Menendez et al., 1999; Sopena et al., 1999). Since these organisms are not susceptible to -lactam antibiotics, which are often used for empiric treatment of lower respiratory tract infections, and because no clinical or laboratory findings can be considered specific of infection by these pathogens, a microbiologic diagnosis is essential to select an appropriate treatment. Moreover, the delay re- quired to obtain results is critical for the selection of a diagnostic test for acute infections and a helpful test to guide initial antibiotic treatments should be available ideally within 4 – 6 h, or prior to the administration of a second dose of antibiotics. For Legionella pneumophila infection, two tests, albeit lacking sensitivity, fulfill this condition: direct fluorescent antibody screening and detection of L. pneumophila urinary antigen specific for serogroup 1. Culture may require 3–7 days for identification and serologic methods give only a retrospective diagnosis and are relatively insensitive (Wa- terer et al., 2001). For the detection of C. pneumoniae, an obligate intracel- lular organism, cell cultures require long incubation times, are difficult and insensitive. Because the prevalence of C. pneumoniae specific antibodies is high in the general pop- ulation, and because IgM antibodies are often absent in reinfection, serology results are difficult to interpret (Birke- baek et al., 2000; Dowell et al., 2001; Hammerschlag, 2001). * Corresponding author. Tel.: +41-21-314-40-76; fax: +41-21-314- 40-60. E-mail address: Katia.Jaton-Ogay@chuv.hospvd.ch (K. Jaton). Diagnostic Microbiology and Infectious Disease www.elsevier.com/locate/diagmicrobio 45 (2003) 85–95 0732-8893/03/$ – see front matter © 2003 Elsevier Science Inc. All rights reserved. PII: S0732-8893(02)00484-4