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Thorax 63:154-159 doi:10.1136/thx.2007.081687
  • 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

  1. J F Huggett1,
  2. M S Taylor2,
  3. G Kocjan3,
  4. H E Evans4,
  5. S Morris-Jones5,
  6. V Gant5,
  7. T Novak1,
  8. A M Costello6,
  9. A Zumla1,
  10. R F Miller4,7
  1. 1
    Centre for Infectious Diseases and International Health, Windeyer Institute for Medical Sciences, University College London, London, UK
  2. 2
    Welcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK
  3. 3
    Department of Histopathology, University College London, London, UK
  4. 4
    Centre for Sexual Health and HIV Research, Department of Primary Care and Population Sciences, University College London, London, UK
  5. 5
    Department of Microbiology, University College London Hospitals NHS Foundation Trust, Windeyer Institute for Medical Sciences, London, UK
  6. 6
    International Perinatal Care Unit, Institute of Child Health, University College London, London, UK
  7. 7
    Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, UK
  1. 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
  • Received 22 March 2007
  • Accepted 27 July 2007
  • Published Online First 10 August 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.

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.

Footnotes

  • Competing interests: This research was supported by The Dr Hadwen Trust (JFH, TN, AZ and RFM).

  • Funding: Professor R F Miller is Co-Editor of Sexually Transmitted Infections, part of the BMJ Publishing Group.