Simultaneous Detection of Pathogens in Clinical Samples from Patients with Community-Acquired Pneumonia by Real-Time PCR with Pathogen-Specific Molecular Beacon Probes

Abstract

ABSTRACT In this study, real-time PCR with pathogen-specific molecular beacons (MB) and primers was evaluated for prediction of community-acquired pneumonia (CAP) causative agents, detecting six main CAP agents, Streptococcus pneumoniae , Haemophilus influenzae , Mycoplasma pneumoniae , Chlamydophila pneumoniae , Legionella pneumophila , and Streptococcus pyogenes , simultaneously. The PCR assay was evaluated for fresh clinical specimens from infants and children ( n = 389) and from adults ( n = 40). The MB probes and primers are both pathogen specific, namely, the lytA gene for S. pneumoniae , the mip gene for L. pneumophila , and 16S rRNA genes for the remaining four organisms. DNA extraction of clinical specimens was performed with a commercially available EXTRAGEN II kit, and amplification was performed with Stratagene Mx3000P. The limit of detection for these pathogens ranged from 2 copies to 18 copies. The whole process from DNA extraction to the analysis was finished in less than 2 h. The obtained sensitivity and specificity of this real-time PCR study relative to those of conventional cultures were as follows: 96.2% and 93.2% for S. pneumoniae , 95.8% and 95.4% for H. influenzae , 100% and 100% for S. pyogenes , and 100% and 95.4% for M. pneumoniae , respectively. The sensitivity and specificity for M. pneumoniae relative to those of a serologic assay were 90.2% and 97.9%, respectively. In six clinical samples of C. pneumoniae , the real-time PCR gave positive predictable values, and in those cases, elevation of the titer value was also observed. In conclusion, we demonstrated that a real-time PCR assay with pathogen-specific MB is useful in identifying CAP causative agents rapidly and in examining the clinical course of empirical chemotherapy in a timely manner, supporting conventional culture methods.