Development and evaluation of a real-time PCR assay for detection of Pneumocystis jirovecii DNA in bronchoalveolar lavage fluid of HIV-infected patients.

摘要

BACKGROUND: Pneumocystis pneumonia (PCP) is conventionally diagnosed by identifying Pneumocystis jirovecii in lower respiratory tract samples using cytochemical stains. Molecular diagnosis of PCP is potentially more sensitive. METHODS: A study was undertaken to use an extensively optimised real-time polymerase chain reaction (PCR) using primers designed to hybridise with the P. jirovecii heat shock protein 70 (HSP70) gene to quantify P. jirovecii DNA in bronchoalveolar lavage (BAL) fluid from HIV-infected patients with and without PCP, and to compare this assay with conventional PCR targeting the P. jirovecii mitochondrial large subunit rRNA gene sequence (mt LSU rRNA). RESULTS: Sixty-one patients had 62 episodes of PCP (defined by detection of P. jirovecii in BAL fluid by cytochemical stains and typical clinical presentation). Quantifiable HSP70 DNA was detected in 61/62 (range approximately 13-18,608 copies/reaction; median approximately 332) and was detectable but below the limit of quantification (approximately 5 copies/reaction) in 1/62. Seventy-one other patients had 74 episodes with alternative diagnoses. Quantifiable HSP70 DNA was detectable in 6/74 (8%) episodes (range approximately 6-590 copies/reaction; median approximately 14) and detectable but below the limit of quantification in 34/74 (46%). Receiver-operator curve analysis (cut-off >10 copies/reaction) showed a clinical sensitivity of 98% (95% 91% to 100%) and specificity of 96% (95% CI 87% to 99%) for diagnosis of PCP. By contrast, clinical sensitivity of mt LSU rRNA PCR was 97% (95% CI 89% to 99%) and specificity was 68% (95% CI 56% to 78%). CONCLUSION: The HSP70 real-time PCR assay detects P. jirovecii DNA in BAL fluid and may have a diagnostic application. Quantification of P. jirovecii DNA by real-time PCR may also discriminate between colonisation with P. jirovecii and infection.