Development and evaluation of a real-time PCR assay for detection of Pneumocystis jirovecii DNA in bronchoalveolar lavage fluid of HIV-infected patients.
Thorax 2007 Aug 10; In press
Huggett J JF, Taylor M MS, Kocjan G G, Evans H HE, Morris-Jones S S, Gant V V, Novak T T, Costello A AM, Zumla A A, Miller R RF
University College London, United Kingdom.
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. Objectives and METHODS: To use an extensively optimized 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 ~13-18608 [median ~332] copies/reaction and detectable, below the limit of quantification (~5 copies/reaction,