Monitoring intracellular replication of Chlamydophila (Chlamydia) pneumoniae in cell cultures and comparing clinical samples by real-time PCR Athos Bonanomi a , Claudia Dohm b , Zaira Rickenbach a , Martin Altwegg c , Joachim Fischer d , Daniel Gygi b , David Nadal a, * a Division of Infectious Diseases, University Children’s Hospital of Zurich, CH-8032 Zurich; b Institute of Anatomy, University of Zurich, Winterthurerstrasse 190, CH-8057 Zu ¨rich; c Institute of Medical Microbiology, University of Zurich, CH-8028 Zurich; d Division of Growth and Development, University Children’s Hospital of Zurich, CH-8032 Zurich, Switzerland Received 18 September 2002; accepted 2 December 2002 Abstract Strains of Chlamydophila pneumoniae may be associated with respiratory disease or atherosclerosis. Two real-time quantitative PCR assays targeting the species-specific genes Cpn0278 and ArgR were developed to compare the in vitro growth of respiratory strains AR39 and K6 with that of atherosclerotic strain A03 and to quantify C. pneumoniae in clinical samples. A third real-time PCR assay was designed to assess contamination with Mycoplasma spp. The assays targeting C. pneumoniae detected DNA concentrations corresponding to 10 4 to 10 -4 inclusion-forming units (IFU)/reaction and were highly specific. AR39 exhibited the longest lag phase and period of exponential growth; K6 augmented growth rates at higher inocula; and A03 grew at highest rates. Contamination with Mycoplasma spp. of AR39 and A03 unlikely accounted for growth differences between them. Numbers of IFU in C. pneumoniae-positive respiratory secretions varied within 4 to 5 orders of magnitude. The assays described may prove valuable for pathogenicity studies. © 2003 Elsevier Inc. All rights reserved. 1. Introduction The Chlamydophila are obligate intracellular Gram-neg- ative bacteria (Schachter et al., 1999). The lack of several metabolic and biosynthetic pathways renders them depen- dent on host cells for intermediates. During growth, or developmental cycle, Chlamydophila alternate between two distinct forms. In one, the bacteria are present as free ele- mentary bodies that are infectious and mediate the initial contact with susceptible target cells such as epithelial cells, endothelial cells, smooth muscle cells and macrophages. Following invasion of host cells the elementary bodies transform to metabolically active reticulate bodies. Within the reticulate bodies DNA, RNA, and protein are synthe- sized, leading to successive rounds of binary fission and increased numbers of progeny. These non-infectious retic- ulate bodies reorganize to elementary bodies that escape or are released from the host cell and can initiate a new round of infection (Wyrick 2000). The complexity of this cycle imposes hurdles to the study of pathogenesis. Three species of Chlamydophila are known to cause disorders in humans: C. trachomatis, C. psittaci, and C. pneumoniae (Schachter et al., 1999). The latter is mainly implicated in acute respiratory infections causing up to 14% of all cases of community-acquired pneumonia in children and 5% of bronchitis and sinusitis cases (Kuo et al., 1995; Principi et al., 2001). C. pneumoniae has lately received increased attention because of the association with chronic diseases including atherosclerosis, arthritis, and asthma (Berger et al., 2000; Blessing et al., 2002; Boman et al., 2002). The reasons for such diverse manifestations of C. pneumoniae infection are not known, but growth behav- ior and tissue tropism of different strains may be contrib- uting pathogenic factors. So far, the developmental cycle of C. pneumoniae has been described with respect to ultra structural characteristics and metabolic activity (Haranaga et al., 2002; Kutlin et al., 2001; Wolf et al., 2000) whereas * Corresponding author. Tel.: +41-1- 266-7562; fax: +41-1-266-7157. E-mail address: david.nadal@kispi.unizh.ch (D. Nadal). Diagnostic Microbiology and Infectious Disease www.elsevier.com/locate/diagmicrobio 46 (2003) 39 – 47 0732-8893/03/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0732-8893(02)00572-2