We designed a multiplex real time PCR for rapid, sensitive and specific detection of Streptococcus pneumoniae, Legionella pneumophila, Chlamydophila pneumoniae and Mycoplasma pneumoniae. The study cases consisted of 129 patients with community acquired pneumonia (CAP). Bacteriological techniques were implemented for detection of the cultivable organisms. DNA were extracted from sputa, throat swabs, bronchoalveolar lavages and tracheal aspirates and used as templates in real time PCR. The primers and probes were designed for cbpA (S. pneumoniae), p1adhesin (M. pneumoniae), mip (L. pneumophila) and ompA (C. pneumoniae). After optimization of real time PCR for every organism, the experiments were continued in multiplex in a single tube. Of 129 CAP specimens, the positive cultures included 14 (10.85%) for S. pneumoniae, 9 (6.98%) for L. pneumophila and 3 (2.33%) for M. pneumoniae. Four specimens (3.10%) were positive for C. pneumoniae by real time PCR. The sensitivity of our real time PCR was 100% for all selected bacteria. The specificity of the test was 98.26%, 98.34%, 100% and 100% for S. pneumoniae, L. pneumophila, M. pneumoniae and C. pneumoniae, respectively. This is the first report on the use of multiplex real time PCR for detection of CAP patients in the Middle East. The method covers more than 90% of the bacterial pathogens causing CAP.

The aim of this study was to develop a real time polymerase chain reaction (PCR) for quantitative detection of Streptococcus pneumoniae from clinical respiratory specimens. Initially, 184 respiratory specimens from patients with community acquired pneumonia (CAP) (n = 129) and 55 cases with hospital associated pneumonia (HAP) were bacteriologically investigated. To check the colonization status among the healthy individuals, 32 preschool and 31 adults were screened in parallel. All specimens were cultured on selective culture media to isolate S. pneumoniae, Legionella spp. and Mycoplasma spp. A 166 bp fragment corresponding to cbp A gene of S. pneumoniae was amplified from clinical specimens using Taqman probe real time PCR. Culture showed 14, but real time PCR showed 15 specimens as being positive for S. pneumoniae. The specificity and sensitivity of real time PCR was 99.14% and 100 respectively. Co-infections of S. pneumoniae with Legionella pneumophila, Chlamydophila pneumoniae, Mycoplasma pneumoniae and Staphylococcus aureus were observed in 5 cases (35.72%). S. pneumoniae was counted <10 3 cfu/ml from the co-infected cases. Using real time PCR, a cutoff of 10 3 cfu/ml is introduced to differentiate colonization from infection in respiratory tract. This is the first report on the prevalence CAP with S. pneumoniae in Iran (12.40%).