Detection of legionellae in hospital water samples by quantitative real-time LightCycler PCR

Contamination of hospital water systems with legionellae is a well-known ca use of nosocomial legionellosis. We describe a new real-time LightCycler PC R assay for quantitative determination of legionellae in potable water samp les. Primers that amplify both a 386-bp fragment of the 16S rRNA gene from Legionella spp. and a specifically cloned fragment of the phage lambda, add ed to each sample as an internal inhibitor control, were used. The amplifie d products were detected by use of a dual-color hybridization probe assay d esign and quantified with external standards composed of Legionella pneumop hila genomic DNA. The PCR assay had a sensitivity of 1 fg of Legionella DNA (i.e., less than one Legionella organism) per assay and detected 44 Legion ella species and serogroups. Seventy-seven water samples from three hospita ls were investigated by PCR and culture. The rates of detection of legionel lae were 98.7% (76 of 77) by the PCR assay and 70.1% (54 of 77) by culture; PCR inhibitors were detected in one sample. The amounts of legionellae cal culated from the PCR results were associated with the CFU detected by cultu re (r = 0.57; P < 0.001), but PCR results were mostly higher than the cultu re results. Since L. pneumophila is the main cause of legionellosis, we fur ther developed a quantitative L. pneumophila-specific PCR assay targeting t he macrophage infectivity potentiator (mip) gene, which codes for an immuno philin of the FK506 binding protein family. All but one of the 16S rRNA gen e PCR-positive water samples were also positive in the mip gene PCR, and th e results of the two PCR assays were correlated. In conclusion, the newly d eveloped Legionella genus-specific and L. pneumophila species-specific PCR assays proved to be valuable tools for investigation of Legionella contamin ation in potable water systems.