The St Paul’s Hospital laboratory-developed PJ real-time-PCR assay was adapted from one previously published (19 (link)). Briefly, 400 μL of BAL fluid in phosphate-buffered saline was extracted with 10 μL of internal amplification control according to the total nucleic acid isolation blood protocol using a Roche Magna Pure Compact instrument (Roche Molecular Diagnostics, USA) and eluted in 100 μL. Respiratory samples containing thick mucus were pretreated with proteinase K (25 μL 1 g/mL) and sodium dodecyl sulfate then lysed using zirconium beads (150 μL 2.4 mm) and silica beads (50 μL 0.1 mm) (Biospec Products, USA) before DNA extraction. Primers and fluorescence resonance evergy transfer probes were previously described, and were designed to detect a 166-base pair region of the cdc2 gene of PJ (19 (link)). PCR was performed on 5 μL of DNA extract using the LightCycler-FastStart DNA Master Hybridization Probes kit (Roche Diagnostics, Canada) in the presence of 4 mmol/L MgCl2 using the Light Cycler 2.0 (Roche Diagnostics, Canada). Melting curve analysis was performed at the end of the cdc2 PCR amplification to confirm positives. All primers and probes were synthesized by Metabion (Germany).