RESPIRATORY INFECTION

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

J F Huggett¹, M S Taylor², G Kocjan³, H E Evans⁴, S Morris-Jones⁵, V Gant⁵, T Novak¹, A M Costello⁶, A Zumla¹, R F Miller^4,7

¹ Centre for Infectious Diseases and International Health, Windeyer Institute for Medical Sciences, University College London, London, UK
² Welcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK
³ Department of Histopathology, University College London, London, UK
⁴ Centre for Sexual Health and HIV Research, Department of Primary Care and Population Sciences, University College London, London, UK
⁵ Department of Microbiology, University College London Hospitals NHS Foundation Trust, Windeyer Institute for Medical Sciences, London, UK
⁶ International Perinatal Care Unit, Institute of Child Health, University College London, London, UK
⁷ Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, UK

Professor R F Miller, Centre for Sexual Health and HIV Research, Department of Primary Care and Population Sciences, University College London, London WC1E 6JB, UK; rmiller{at}gum.ucl.ac.uk

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 13–18 608 copies/reaction; median 332) and was detectable but below the limit of quantification (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 6–590 copies/reaction; median 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.

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