Simultaneous detection of pathogens in clinical samples from patients with community-acquired pneumonia by real-time PCR with pathogen-specific molecular beacon probes

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.

FIG. 1.

Correlation of C_T values derived from the real-time PCR assay and culture count results for S. pneumoniae (n = 100). Negative (−), no colonies observed; +, <10³/sample (1 to 24 CFU per plate); ++, 10³ to <10⁴/sample (25 to 240 CFU per plate); +++, ≥10⁴/sample (≥241 CFU per plate). Each square represents the mean ± standard deviation.

FIG. 2.

Correlation of C_T values derived from the real-time PCR assay compared to culture count results for H. influenzae (n = 100). Negative (−), no colonies observed; +, <10³/sample (1 to 24 CFU per plate); ++, 10³ to <10⁴/sample (25 to 240 CFU per plate); +++, ≥10⁴/sample (≥241 CFU per plate). Each square represents the mean ± standard deviation.