Skip to main content
Top
Published in: European Journal of Clinical Microbiology & Infectious Diseases 3/2009

01-03-2009 | Article

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

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

Published in: European Journal of Clinical Microbiology & Infectious Diseases | Issue 3/2009

Login to get access

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.
Literature
1.
go back to reference 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):862–870PubMed 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):862–870PubMed
2.
go back to reference 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 PubMedCrossRef 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 PubMedCrossRef
5.
go back to reference 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 PubMedCrossRef 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​ PubMedCrossRef
10.
go back to reference 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 PubMedCrossRef 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 PubMedCrossRef
11.
go back to reference 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 PubMedCrossRef 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 PubMedCrossRef
12.
go back to reference 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):1353–1360PubMed 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):1353–1360PubMed
15.
go back to reference 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):3865–3871PubMed 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):3865–3871PubMed
16.
go back to reference 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 PubMedCrossRef 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 PubMedCrossRef
17.
go back to reference 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):1164–1168PubMed 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):1164–1168PubMed
18.
go back to reference 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 PubMedCrossRef 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 PubMedCrossRef
19.
20.
go back to reference 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 PubMedCrossRef 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 PubMedCrossRef
21.
go back to reference 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 PubMedCrossRef 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 PubMedCrossRef
22.
go back to reference 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):3150–3153PubMed 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):3150–3153PubMed
23.
go back to reference 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):89–94PubMedCrossRef 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):89–94PubMedCrossRef
25.
go back to reference Chamberlain DW, Braude AC, Rebuck AS (1987) A critical evaluation of bronchoalveolar lavage. Criteria for identifying unsatisfactory specimens. Acta Cytol 31(5):599–605PubMed Chamberlain DW, Braude AC, Rebuck AS (1987) A critical evaluation of bronchoalveolar lavage. Criteria for identifying unsatisfactory specimens. Acta Cytol 31(5):599–605PubMed
26.
28.
go back to reference 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):245–248PubMed 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):245–248PubMed
30.
go back to reference 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):332–340PubMed 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):332–340PubMed
31.
32.
go back to reference 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):3311–3312PubMed 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):3311–3312PubMed
33.
go back to reference 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 PubMedCrossRef 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 PubMedCrossRef
34.
go back to reference 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):586–590 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):586–590
35.
go back to reference 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):1978–1984PubMedCrossRef 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):1978–1984PubMedCrossRef
37.
go back to reference 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 PubMedCrossRef 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 PubMedCrossRef
38.
go back to reference 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 PubMedCrossRef 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 PubMedCrossRef
39.
go back to reference 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 PubMedCrossRef 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 PubMedCrossRef
Metadata
Title
Diagnosis of invasive aspergillosis using bronchoalveolar lavage in haematology patients: influence of bronchoalveolar lavage human DNA content on real-time PCR performance
Authors
E. Fréalle
K. Decrucq
F. Botterel
B. Bouchindhomme
D. Camus
E. Dei-Cas
J. M. Costa
I. Yakoub-Agha
S. Bretagne
L. Delhaes
Publication date
01-03-2009
Publisher
Springer-Verlag
Published in
European Journal of Clinical Microbiology & Infectious Diseases / Issue 3/2009
Print ISSN: 0934-9723
Electronic ISSN: 1435-4373
DOI
https://doi.org/10.1007/s10096-008-0616-1

Other articles of this Issue 3/2009

European Journal of Clinical Microbiology & Infectious Diseases 3/2009 Go to the issue