Detection of Legionella species in respiratory specimens using PCR with sequencing confirmation

Legionella spp. are a common cause of community-acquired respiratory tract infections and an occasional cause of nosocomial pneumonia. A PCR method fo r the detection of legionellae in respiratory samples was evaluated and was compared to culture. The procedure can be performed in 6 to 8 h with a com mercially available DNA extraction kit (Qiagen, Valencia, Calif,) and by PC R with gel detection. PCR is performed with primers previously determined t o amplify a 386-bp product within the 16S rRNA gene of Legionella pneumophi la, We can specifically detect the clinically significant Legionella specie s including L. pneumophila, L. micdadei, L. longbeachae, L. bozemanii, L. f eeleii, and L. dumoffii. The assay detects 10 fg (approximately two organis ms) of Legionella DNA in each PCR, Of 212 clinical specimens examined by cu lture, 100% of the culture-positive samples (31 of 31) were positive by thi s assay. By gel detection of amplification products, 12 of 181 culture-nega tive samples were positive for Legionella species by PCR, resulting in 93% specificity. Four of the 12 samples with discrepant results (culture negati ve, PCR positive) were confirmed to be positive for Legionella species by s equencing of the amplicons, The legionella-specific PCR assay that is descr ibed demonstrates high sensitivity and high specificity for routine detecti on of legionellae in respiratory samples.