Diagnosis of invasive aspergillosis using bronchoalveolar lavage in haematology patients: influence of bronchoalveolar lavage human DNA content on real-time PCR performance

  • E. Fréalle
  • K. Decrucq
  • F. Botterel
  • B. Bouchindhomme
  • D. Camus
  • E. Dei-Cas
  • J. M. Costa
  • I. Yakoub-Agha
  • S. Bretagne
  • L. Delhaes
Article

Abstract

In order to improve invasive pulmonary aspergillosis (IPA) diagnosis, a real-time polymerase chain reaction (PCR) assay detecting Aspergillus spp. was developed. Its detection limit reached 2–20 conidia. The retrospective evaluation on 64 bronchoalveolar lavage (BAL) fluids from 57 patients at risk for IPA, including 20 probable and five proven IPA patients, revealed a 88% or 38% sensitivity in direct examination (DE)/culture-positive or culture-negative BAL, respectively, whereas galactomannan (GM) sensitivity reached 88% or 58%, respectively. Influence on the Aspergillus-PCR yield of BAL fluid volume, cellular count and DNA content (evaluated by human β-globin quantification) was assessed. Significantly higher β-globin levels were detected in Aspergillus PCR-positive (median 5,112 pg/μl) than negative (median 1,332 pg/μl) BAL fluids, suggesting that the β-globin level could reflect BAL yields and DNA extraction. Using β-globin for the interpretation of fungal PCR could improve the negative predictive value of this test.

Notes

Acknowledgements

This work was supported by grants from the Lille 2 University Hospital of Lille and the Faculty of Medicine (Lille 2 University), and was developed in the framework of the EA 3609 scientific project (French Research Office). It was presented at the 3rd Trends in Medical Mycology (TIMM 2007), Turin, Italy, October 2007. We thank the members of the Department of Parasitology-Mycology of Lille 2 University Hospital: Fabienne Soula for her valuable help in the clinical data collection; Michèle Wauquier and Filoména Naji for their technical assistance; Boualem Sendid, Nadine François, Stéphanie Delbart, Pascale Daelman and Laurence Dumortier for their involvement in the routine mycological tests. We are grateful to Anthony Pinon (research assistant, Lille Pasteur Institute, France) for the statistical analysis.

References

  1. 1.
    Pagano L, Girmenia C, Mele L, Ricci P, Tosti ME, Nosari A et al (2001) Infections caused by filamentous fungi in patients with hematologic malignancies. A report of 391 cases by GIMEMA Infection Program. Haematologica 86(8):862–870PubMedGoogle Scholar
  2. 2.
    Ascioglu S, Rex JH, de Pauw B, Bennett JE, Bille J, Crokaert F et al (2002) Defining opportunistic invasive fungal infections in immunocompromised patients with cancer and hematopoietic stem cell transplants: an international consensus. Clin Infect Dis 34(1):7–14. doi: 10.1086/323335 PubMedCrossRefGoogle Scholar
  3. 3.
    Hope WW, Walsh TJ, Denning DW (2005) Laboratory diagnosis of invasive aspergillosis. Lancet Infect Dis 5(10):609–622. doi: 10.1016/S1473-3099(05)70238-3 PubMedCrossRefGoogle Scholar
  4. 4.
    Verdaguer V, Walsh TJ, Hope W, Cortez KJ (2007) Galactomannan antigen detection in the diagnosis of invasive aspergillosis. Expert Rev Mol Diagn 7(1):21–32. doi: 10.1586/14737159.7.1.21 PubMedCrossRefGoogle Scholar
  5. 5.
    Tarrand JJ, Lichterfeld M, Warraich I, Luna M, Han XY, May GS et al (2003) Diagnosis of invasive septate mold infections. A correlation of microbiological culture and histologic or cytologic examination. Am J Clin Pathol 119(6):854–858. doi: 10.1309/EXBVYAUPENBM285Y PubMedCrossRefGoogle Scholar
  6. 6.
    Aquino VR, Goldani LZ, Pasqualotto AC (2007) Update on the contribution of galactomannan for the diagnosis of invasive aspergillosis. Mycopathologia 163(4):191–202. doi: 10.1007/s11046-007-9010-2 PubMedCrossRefGoogle Scholar
  7. 7.
    Mennink-Kersten MA, Donnelly JP, Verweij PE (2004) Detection of circulating galactomannan for the diagnosis and management of invasive aspergillosis. Lancet Infect Dis 4(6):349–357. doi: 10.1016/S1473-3099(04)01045-X PubMedCrossRefGoogle Scholar
  8. 8.
    Tanriover MD, Metan G, Altun B, Hascelik G, Uzun O (2005) False positivity for Aspergillus antigenemia related to the administration of piperacillin/tazobactam. Eur J Intern Med 16(7):489–491. doi: 10.1016/j.ejim.2005.04.007 PubMedCrossRefGoogle Scholar
  9. 9.
    Dalle F, Charles PE, Blanc K, Caillot D, Chavanet P, Dromer F et al (2005) Cryptococcus neoformans Galactoxylomannan contains an epitope(s) that is cross-reactive with Aspergillus Galactomannan. J Clin Microbiol 43(6):2929–2931. doi: 10.1128/JCM.43.6.2929-2931.2005 PubMedCrossRefGoogle Scholar
  10. 10.
    Becker MJ, Lugtenburg EJ, Cornelissen JJ, Van Der Schee C, Hoogsteden HC, De Marie S (2003) Galactomannan detection in computerized tomography-based broncho-alveolar lavage fluid and serum in haematological patients at risk for invasive pulmonary aspergillosis. Br J Haematol 121(3):448–457. doi: 10.1046/j.1365-2141.2003.04308.x PubMedCrossRefGoogle Scholar
  11. 11.
    Costa C, Costa JM, Desterke C, Botterel F, Cordonnier C, Bretagne S (2002) Real-time PCR coupled with automated DNA extraction and detection of galactomannan antigen in serum by enzyme-linked immunosorbent assay for diagnosis of invasive aspergillosis. J Clin Microbiol 40(6):2224–2227. doi: 10.1128/JCM.40.6.2224-2227.2002 PubMedCrossRefGoogle Scholar
  12. 12.
    Einsele H, Hebart H, Roller G, Löffler J, Rothenhofer I, Müller CA et al (1997) Detection and identification of fungal pathogens in blood by using molecular probes. J Clin Microbiol 35(6):1353–1360PubMedGoogle Scholar
  13. 13.
    Ferrer C, Colom F, Frasés S, Mulet E, Abad JL, Alió JL (2001) Detection and identification of fungal pathogens by PCR and by ITS2 and 5.8S ribosomal DNA typing in ocular infections. J Clin Microbiol 39(8):2873–2879. doi: 10.1128/JCM.39.8.2873-2879.2001 PubMedCrossRefGoogle Scholar
  14. 14.
    Halliday C, Wu QX, James G, Sorrell T (2005) Development of a nested qualitative real-time PCR assay to detect Aspergillus species DNA in clinical specimens. J Clin Microbiol 43(10):5366–5368. doi: 10.1128/JCM.43.10.5366-5368.2005 PubMedCrossRefGoogle Scholar
  15. 15.
    Skladny H, Buchheidt D, Baust C, Krieg-Schneider F, Seifarth W, Leib-Mösch C et al (1999) Specific detection of Aspergillus species in blood and bronchoalveolar lavage samples of immunocompromised patients by two-step PCR. J Clin Microbiol 37(12):3865–3871PubMedGoogle Scholar
  16. 16.
    Spiess B, Buchheidt D, Baust C, Skladny H, Seifarth W, Zeilfelder U et al (2003) Development of a LightCycler PCR assay for detection and quantification of Aspergillus fumigatus DNA in clinical samples from neutropenic patients. J Clin Microbiol 41(5):1811–1818. doi: 10.1128/JCM.41.5.1811-1818.2003 PubMedCrossRefGoogle Scholar
  17. 17.
    Bretagne S, Costa JM, Marmorat-Khuong A, Poron F, Cordonnier C, Vidaud M et al (1995) Detection of Aspergillus species DNA in bronchoalveolar lavage samples by competitive PCR. J Clin Microbiol 33(5):1164–1168PubMedGoogle Scholar
  18. 18.
    Buchheidt D, Baust C, Skladny H, Ritter J, Suedhoff T, Baldus M et al (2001) Detection of Aspergillus species in blood and bronchoalveolar lavage samples from immunocompromised patients by means of 2-step polymerase chain reaction: clinical results. Clin Infect Dis 33(4):428–435. doi: 10.1086/321887 PubMedCrossRefGoogle Scholar
  19. 19.
    Raad I, Hanna H, Huaringa A, Sumoza D, Hachem R, Albitar M (2002) Diagnosis of invasive pulmonary aspergillosis using polymerase chain reaction-based detection of Aspergillus in BAL. Chest 121(4):1171–1176. doi: 10.1378/chest.121.4.1171 PubMedCrossRefGoogle Scholar
  20. 20.
    Rantakokko-Jalava K, Laaksonen S, Issakainen J, Vauras J, Nikoskelainen J, Viljanen MK et al (2003) Semiquantitative detection by real-time PCR of Aspergillus fumigatus in bronchoalveolar lavage fluids and tissue biopsy specimens from patients with invasive aspergillosis. J Clin Microbiol 41(9):4304–4311. doi: 10.1128/JCM.41.9.4304-4311.2003 PubMedCrossRefGoogle Scholar
  21. 21.
    Sanguinetti M, Posteraro B, Pagano L, Pagliari G, Fianchi L, Mele L et al (2003) Comparison of real-time PCR, conventional PCR, and galactomannan antigen detection by enzyme-linked immunosorbent assay using bronchoalveolar lavage fluid samples from hematology patients for diagnosis of invasive pulmonary aspergillosis. J Clin Microbiol 41(8):3922–3925. doi: 10.1128/JCM.41.8.3922-3925.2003 PubMedCrossRefGoogle Scholar
  22. 22.
    Verweij PE, Latgé JP, Rijs AJ, Melchers WJ, De Pauw BE, Hoogkamp-Korstanje JA et al (1995) Comparison of antigen detection and PCR assay using bronchoalveolar lavage fluid for diagnosing invasive pulmonary aspergillosis in patients receiving treatment for hematological malignancies. J Clin Microbiol 33(12):3150–3153PubMedGoogle Scholar
  23. 23.
    Tuon FF (2007) A systematic literature review on the diagnosis of invasive aspergillosis using polymerase chain reaction (PCR) from bronchoalveolar lavage clinical samples. Rev Iberoam Micol 24(2):89–94PubMedCrossRefGoogle Scholar
  24. 24.
    Papanicolaou Society of Cytopathology Task Force on Standard Practice (1999) Guidelines of the papanicolaou society of cytopathology for the examination of cytologic specimens obtained from the respiratory tract. Diagn Cytopathol 21(1):61–69. doi: 10.1002/(SICI)1097-0339(199907)21:1<61::AID-DC17>3.0.CO;2-O CrossRefGoogle Scholar
  25. 25.
    Chamberlain DW, Braude AC, Rebuck AS (1987) A critical evaluation of bronchoalveolar lavage. Criteria for identifying unsatisfactory specimens. Acta Cytol 31(5):599–605PubMedGoogle Scholar
  26. 26.
    Musher B, Fredricks D, Leisenring W, Balajee SA, Smith C, Marr KA (2004) Aspergillus galactomannan enzyme immunoassay and quantitative PCR for diagnosis of invasive aspergillosis with bronchoalveolar lavage fluid. J Clin Microbiol 42(12):5517–5522. doi: 10.1128/JCM.42.12.5517-5522.2004 PubMedCrossRefGoogle Scholar
  27. 27.
    Durand-Joly I, Chabé M, Soula F, Delhaes L, Camus D, Dei-Cas E (2005) Molecular diagnosis of Pneumocystis pneumonia. FEMS Immunol Med Microbiol 45(3):405–410. doi: 10.1016/j.femsim.2005.06.006 PubMedCrossRefGoogle Scholar
  28. 28.
    Bolehovska R, Pliskova L, Buchta V, Cerman J, Hamal P (2006) Detection of Aspergillus spp. in biological samples by real-time PCR. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 150(2):245–248PubMedGoogle Scholar
  29. 29.
    Millon L, Piarroux R, Deconinck E, Bulabois CE, Grenouillet F, Rohrlich P et al (2005) Use of real-time PCR to process the first galactomannan-positive serum sample in diagnosing invasive aspergillosis. J Clin Microbiol 43(10):5097–5101. doi: 10.1128/JCM.43.10.5097-5101.2005 PubMedCrossRefGoogle Scholar
  30. 30.
    Martín Juan J, Valenzuela Mateos F, Soto Campos G, Segado Soriano A, Rodríguez Panadero F, Castillo Gómez J (1996) Quality and selection of samples of bronchoalveolar lavage (BAL) in diffuse pneumopathies. Arch Bronconeumol 32(7):332–340PubMedGoogle Scholar
  31. 31.
    Griffiths LJ, Anyim M, Doffman SR, Wilks M, Millar MR, Agrawal SG (2006) Comparison of DNA extraction methods for Aspergillus fumigatus using real-time PCR. J Med Microbiol 55(Pt 9):1187–1191. doi: 10.1099/jmm.0.46510-0 PubMedCrossRefGoogle Scholar
  32. 32.
    Löffler J, Hebart H, Schumacher U, Reitze H, Einsele H (1997) Comparison of different methods for extraction of DNA of fungal pathogens from cultures and blood. J Clin Microbiol 35(12):3311–3312PubMedGoogle Scholar
  33. 33.
    Khot PD, Ko DL, Hackman RC, Fredricks DN (2008) Development and optimization of quantitative PCR for the diagnosis of invasive aspergillosis with bronchoalveolar lavage fluid. BMC Infect Dis 8(1):73. doi: 10.1186/1471-2334-8-73 PubMedCrossRefGoogle Scholar
  34. 34.
    Löffler J, Henke N, Hebart H, Schmidt D, Hagmeyer L, Schumacher U et al (2000) Quantification of fungal DNA by using fluorescence resonance energy transfer and the light cycler system. J Clin Microbiol 38(2):586–590Google Scholar
  35. 35.
    Francesconi A, Kasai M, Harrington SM, Beveridge MG, Petraitiene R, Petraitis V et al (2008) Automated and manual methods of DNA extraction for Aspergillus fumigatus and Rhizopus oryzae analyzed by quantitative real-time PCR. J Clin Microbiol 46(6):1978–1984PubMedCrossRefGoogle Scholar
  36. 36.
    Bretagne S, Costa JM (2005) Towards a molecular diagnosis of invasive aspergillosis and disseminated candidosis. FEMS Immunol Med Microbiol 45(3):361–368. doi: 10.1016/j.femsim.2005.05.012 PubMedCrossRefGoogle Scholar
  37. 37.
    Francesconi A, Kasai M, Petraitiene R, Petraitis V, Kelaher AM, Schaufele R et al (2006) Characterization and comparison of galactomannan enzyme immunoassay and quantitative real-time PCR assay for detection of Aspergillus fumigatus in bronchoalveolar lavage fluid from experimental invasive pulmonary aspergillosis. J Clin Microbiol 44(7):2475–2480. doi: 10.1128/JCM.02693-05 PubMedCrossRefGoogle Scholar
  38. 38.
    Vallor AC, Kirkpatrick WR, Najvar LK, Bocanegra R, Kinney MC, Fothergill AW et al (2008) Assessment of Aspergillus fumigatus burden in pulmonary tissue of guinea pigs by quantitative PCR, galactomannan enzyme immunoassay, and quantitative culture. Antimicrob Agents Chemother 52(7):2593–2598. doi: 10.1128/AAC.00276-08 PubMedCrossRefGoogle Scholar
  39. 39.
    Meersseman W, Lagrou K, Maertens J, Wilmer A, Hermans G, Vanderschueren S et al (2008) Galactomannan in bronchoalveolar lavage fluid: a tool for diagnosing aspergillosis in intensive care unit patients. Am J Respir Crit Care Med 177(1):27–34. doi: 10.1164/rccm.200704-606OC PubMedCrossRefGoogle Scholar
  40. 40.
    Lachnik J, Ackermann B, Bohrssen A, Maass S, Diephaus C, Puncken A et al (2002) Rapid-cycle PCR and fluorimetry for detection of mycobacteria. J Clin Microbiol 40(9):3364–3373. doi:doi: 10.1128/JCM.40.9.3364-3373.2002 PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • E. Fréalle
    • 1
  • K. Decrucq
    • 1
  • F. Botterel
    • 2
  • B. Bouchindhomme
    • 3
  • D. Camus
    • 1
  • E. Dei-Cas
    • 1
  • J. M. Costa
    • 2
    • 4
  • I. Yakoub-Agha
    • 5
  • S. Bretagne
    • 2
  • L. Delhaes
    • 1
  1. 1.EA 3609, Département de Parasitologie-Mycologie, Faculté de Médecine, Pôle de MicrobiologieCHRU de Lille, Université de Lille 2, Laboratoire d’Ecologie du Parasitisme, Institut Pasteur de LilleLilleFrance
  2. 2.Laboratoire de Parasitologie MycologieHôpital Henri Mondor APHP et Université Paris 12CréteilFrance
  3. 3.Département de PathologieCHRU de Lille, Université de Lille 2LilleFrance
  4. 4.Laboratoire Pasteur-CerbaCergy-PontoiseFrance
  5. 5.EA 2686, Service des Maladies du SangCHRU de Lille, Université de Lille 2LilleFrance

Personalised recommendations