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

Jim F Huggett ¹, Martin S Taylor ², Gabrijela Kocjan ¹, Hannah E Evans ¹, Stephen Morris-Jones ³, Vanya Gant ⁴, Tanya Novak ¹, Anthony M Costello ¹, Alimuddin Zumla ¹ and Robert F Miller ^1*

¹ University College London, United Kingdom
² University of Oxford, United Kingdom
³ University Colege London Hospitals, United Kingdom
⁴ University College London Hospitals, United Kingdom

^* To whom correspondence should be addressed. E-mail: rmiller{at}gum.ucl.ac.uk.

Accepted 27 July 2007

Abstract

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, <LQ) in 1/62. Seventy-one other patients had 74 episodes with alternative diagnoses. Quantifiable HSP70 DNA was detectable in 6/74(8%) episodes; range ~6-590 [median ~14] copies/reaction and detectable but <LQ in 34/74(46%). Receiver-Operator Curve analysis (cut-off >10 copies/reaction) showed clinical sensitivity =98% (95% Confidence Interval (CI) =91-100%) and specificity =96% (95%CI =87-99%), for diagnosis of PCP. By contrast, clinical sensitivity and specificity of mt LSU rRNA PCR was 97% (95%CI =89-99%) and 68% (95 CI =56-78%), respectively.

Interpretation: 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 colonization with P. jirovecii and infection.

Keywords: PCP, bronchoalveolar lavage, diagnosis, real-time PCR

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