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Real-Time PCR Assay for the Diagnosis of Pneumocystis jirovecii Pneumonia

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PCR Detection of Microbial Pathogens

Part of the book series: Methods in Molecular Biology ((MIMB,volume 943))

Abstract

Pneumocystis jirovecii is a common cause of life-threatening pneumonia among immunocompromised patients. Since P. jirovecii cannot be cultured, specific identification of it depends on examining respiratory specimens. In the last decade, PCR has been developed which allows the detection of very low levels of P. jirovecii not detectable by routine histochemical staining. We have shown that the direct immunofluorescence assay can be replaced by a real-time PCR assay given its feasibility, sensitivity, and specificity, for the detection of P. jirovecii. A negative PCR, performed on a LightCycler System®, enables a diagnosis of Pneumocystis jirovecii pneumonia (PjP) to be excluded, and the semiquantitative result with the application of some cutoff values can have a role in distinguishing between colonized or subclinically infected patients and PjP patients.

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References

  1. Cisneros JM, Munoz P, Torre-Cisneros J, Gurgui M, Rodriguez-Hernandez MJ, Aguado JM et al (1998) Pneumonia after heart transplantation: a multi-institutional study. Spanish Transplantation Infection Study Group. Clin Infect Dis 27:324–331

    Article  PubMed  CAS  Google Scholar 

  2. Huang L, Morris A, Limper AH, Beck JM (2006) An Official ATS Workshop Summary: Recent advances and future directions in Pneumocystis pneumonia (PCP). Proc Am Thorac Soc 3:655–664

    Article  PubMed  Google Scholar 

  3. Sepkowitz KA (2002) Opportunistic infections in patients with and patients without Acquired Immunodeficiency Syndrome. Clin Infect Dis 34:1098–1107

    Article  PubMed  Google Scholar 

  4. Wakefield AE (2002) Pneumocystis carinii. Br Med Bull 61:175–188

    Article  PubMed  CAS  Google Scholar 

  5. DeLorenzo LJ, Huang CT, Maguire GP, Stone DJ (1987) Roentgenographic patterns of Pneumocystis carinii pneumonia in 104 patients with AIDS. Chest 91:323–327

    Article  PubMed  CAS  Google Scholar 

  6. Huang L, Stansell J, Osmond D, Turner J, Shafer KP, Fulkerson W et al (1999) Performance of an algorithm to detect Pneumocystis carinii pneumonia in symptomatic HIV-infected persons. Pulmonary Complications of HIV Infection Study Group. Chest 115:1025–1032

    Article  PubMed  CAS  Google Scholar 

  7. Dei-Cas E (2000) Pneumocystis infections: the iceberg? Med Mycol 38(Suppl 1):23–32

    PubMed  Google Scholar 

  8. Thomas CF, Limper AH (2004) Pneumocystis pneumonia. N Engl J Med 350:2487–2498

    Article  PubMed  CAS  Google Scholar 

  9. Broaddus C, Dake MD, Stulbarg MS, Blumenfeld W, Hadley WK, Golden JA et al (1985) Bronchoalveolar lavage and transbronchial biopsy for the diagnosis of pulmonary infections in the acquired immunodeficiency syndrome. Ann Intern Med 102:747–752

    PubMed  CAS  Google Scholar 

  10. Huang L, Hecht FM, Stansell JD, Montanti R, Hadley WK, Hopewell PC (1995) Suspected Pneumocystis carinii pneumonia with a negative induced sputum examination. Is early bronchoscopy useful? Am J Respir Crit Care Med 151:1866–1871

    PubMed  CAS  Google Scholar 

  11. Calderon EJ, Regordan C, Medrano FJ, Ollero M, Varela JM (1996) Pneumocystis carinii infection in patients with chronic bronchial ­disease. Lancet 347:977

    Article  PubMed  CAS  Google Scholar 

  12. Helweg-Larsen J, Jensen JS, Dohn B, Benfield TL, Lundgren B (2002) Detection of Pneumocystis DNA in samples from patients suspected of bacterial pneumonia-a case-control study. BMC Infect Dis 2:28

    Article  PubMed  Google Scholar 

  13. Maskell NA, Waine DJ, Lindley A, Pepperell JC, Wakefield AE, Miller RF et al (2003) Asymptomatic carriage of Pneumocystis jiroveci in subjects undergoing bronchoscopy: a prospective study. Thorax 58:594–597

    Article  PubMed  CAS  Google Scholar 

  14. Miller RF, Ambrose HE, Wakefield AE (2001) Pneumocystis carinii f. sp. hominis DNA in immunocompetent health care workers in ­contact with patients with P. carinii pneumonia. J Clin Microbiol 39:3877–3882

    Article  PubMed  CAS  Google Scholar 

  15. Nevez G, Raccurt C, Jounieaux V, Dei-Cas E, Mazars E (1999) Pneumocystosis versus pulmonary Pneumocystis carinii colonization in HIV-negative and HIV-positive patients. AIDS 13:535–536

    Article  PubMed  CAS  Google Scholar 

  16. Fillaux J, Malvy S, Alvarez M, Fabre R, Cassaing S, Marchou B et al (2008) Accuracy of a routine real-time PCR assay for the diagnosis of Pneumocystis jirovecii pneumonia. J Microbiol Methods 75:258–261

    Article  PubMed  CAS  Google Scholar 

  17. Larsen HH, Masur H, Kovacs JA, Gill VJ, Silcott VA, Kogulan P et al (2002) Development and evaluation of a quantitative, touch-down, real-time PCR assay for diagnosing Pneumocystis carinii pneumonia. J Clin Microbiol 40:490–494

    Article  PubMed  CAS  Google Scholar 

  18. Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA et al (1985) (1992) Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Biotechnology 24:476–480

    Google Scholar 

  19. Rennard SI, Ghafouri M, Thompson AB, Linder J, Vaughan W, Jones K et al (1990) Fractional processing of sequential bronchoalveolar lavage to separate bronchial and alveolar samples. Am Rev Respir Dis 141:208–217

    PubMed  CAS  Google Scholar 

  20. Bustin SA (2000) Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol 25:169–193

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

We acknowledge S. Chalmeton, E. Duthu, and S. Gisquet for their technical support.

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Correspondence to Judith Fillaux .

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Fillaux, J., Berry, A. (2013). Real-Time PCR Assay for the Diagnosis of Pneumocystis jirovecii Pneumonia. In: Wilks, M. (eds) PCR Detection of Microbial Pathogens. Methods in Molecular Biology, vol 943. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60327-353-4_11

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  • DOI: https://doi.org/10.1007/978-1-60327-353-4_11

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-60327-352-7

  • Online ISBN: 978-1-60327-353-4

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