Direct detection of Pseudomonas aeruginosa from patients with healthcare associated pneumonia by real time PCR

Using oprL sequences, a TaqMan real time PCR was developed and used for quantitative detection of Pseudomonas aeruginosa from 99 broncoalveolar lavage and 11 sputum specimens collected from patients with health care associated pneumonia. All specimens were cultured on appropriate media to isolate bacteria. Twenty five specimens were positive by both methods. Polymicrobial infections were found in 13 specimens. Amplification of oprL in serial dilutions ranged from 109 CFU/ml to 102 CFU/ml. Standard curve of duplicated every dilution had slope 3.25 ± 0.1 and R2 > 0.99 with SD 0.1. Our real time PCR assay showed high sensitivity (100%) and specificity (98.85%). This technique could detect and enumerate 100 bacteria directly from clinical specimens and showed that the threshold is 103 CFU/ml in cases with clinical symptoms. Our method can be used for quantitative detection of P. aeruginosa from BAL and sputum specimens in 1 h and 10 min.