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Mycopathologia

, Volume 178, Issue 1–2, pp 53–61 | Cite as

The Application of Laser Microdissection in Molecular Detection and Identification of Aspergillus fumigatus from Murine Model of Acute Invasive Pulmonary Aspergillosis

  • Chong Wang
  • Ping Zhan
  • Le Wang
  • Rong Zeng
  • Yongnian Shen
  • Guixia Lv
  • Dongmei Li
  • Shuwen Deng
  • Weida Liu
Article

Abstract

Invasive aspergillosis (IA) is a major concern in patients with severe immune deficiency. As antifungal susceptibility varies in different fungal pathogens, accurate and timely identification of species is becoming imperative for guidance of therapy and reducing high mortality rates in patients with IA. But, in fact, the diagnosis is challenging and new validated techniques are required for the detection and identification of clinically relevant isolates. The laser capture microdissection (LCM) system enables analysis of cytologically and/or phenotypically defined cell types from heterogeneous tissue and has been used in diagnosis and fungal species identification in pulmonary aspergillosis of white storks. To establish the experimental foundation for clinical application of the system, we microdissected and collected Blankophor-stained single hyphal strands from tissue cryosections of murine model of invasive pulmonary aspergillosis (IPA) with A. fumigatus by LCM, subsequently processed for DNA extraction, PCR sequencing, and species molecular identification. The sensitivity of LCM–PCR sequencing was 89 % (89/100), and the specificity was 100 %. Moreover, the positive predictive value and negative predictive value were 100 and 78.43 %, respectively. The result approved that the LCM-based methods had the potential for accurately diagnosis and rapidly identification fungal pathogens of IPA.

Keywords

Invasive aspergillosis Laser capture microdissection PCR Murine model 

Notes

Acknowledgments

This investigation was supported by Youth Innovative Project (2012x13) from Peking Union Medical College, by Major National Science and Technology Projects (2013ZX10004612-005) from Ministry of Science and Technology of P.R.C., by Jiangsu Provincial Special Program of Medical Science (BL2012003) from Science and Technology Department of Jiangsu Province, and by key project of the ministry of health, clinical disciplines (2010–2012) from the ministry of health of P.R.C.

Conflict of interest

The authors declare that they have no conflict of interests.

References

  1. 1.
    Steinbach WJ, Marr KA, Anaissie EJ, et al. Clinical epidemiology of 960 patients with invasive aspergillosis from the PATH Alliance registry. J Infect. 2012;65(5):453–64.PubMedCrossRefGoogle Scholar
  2. 2.
    Pagano L, Caira M, Candoni A, et al. The epidemiology of fungal infections in patients with hematologic malignancies: the SEIFEM-2004 study. Haematologica. 2006;91(8):1068–75.PubMedGoogle Scholar
  3. 3.
    Pagano L, Caira M, Nosari A, et al. Fungal infections in recipients of hematopoietic stem cell transplants: results of the SEIFEM B-2004 study—Sorveglianza Epidemiologica Infezioni Fungine Nelle Emopatie Maligne. Clin Infect Dis. 2007;45(9):1161–70.PubMedCrossRefGoogle Scholar
  4. 4.
    Meersseman W, Vandecasteele SJ, Wilmer A, Verbeken E, Peetermans WE, Van Wijngaerden E. Invasive aspergillosis in critically ill patients without malignancy. Am J Respir Crit Care Med. 2004;170(6):621–5.PubMedCrossRefGoogle Scholar
  5. 5.
    Fourneret-Vivier A, Lebeau B, Mallaret MR, et al. Hospital-wide prospective mandatory surveillance of invasive aspergillosis in a French teaching hospital (2000–2002). J Hosp Infect. 2006;62(1):22–8.PubMedCrossRefGoogle Scholar
  6. 6.
    Cornillet A, Camus C, Nimubona S, et al. Comparison of epidemiological, clinical, and biological features of invasive aspergillosis in neutropenic and nonneutropenic patients: a 6-year survey. Clin Infect Dis. 2006;43(5):577–84.PubMedCrossRefGoogle Scholar
  7. 7.
    Von EM, Roos N, Schulten R, Hesse M, Zuhlsdorf M, van de Loo J. Pulmonary aspergillosis: early diagnosis improves survival. Respiration. 1995;62(6):341–7.CrossRefGoogle Scholar
  8. 8.
    Greene RE, Schlamm HT, Oestmann JW, et al. Imaging findings in acute invasive pulmonary aspergillosis: clinical significance of the halo sign. Clin Infect Dis. 2007;44(3):373–9.PubMedCrossRefGoogle Scholar
  9. 9.
    Lass-Florl C. In vitro susceptibility testing in Aspergillus species: an update. Futur Microbiol. 2010;5(5):789–99.CrossRefGoogle Scholar
  10. 10.
    Sutton DA, Sanche SE, Revankar SG, Fothergill AW, Rinaldi MG. In vitro amphotericin B resistance in clinical isolates of Aspergillus terreus, with a head-to-head comparison to voriconazole. J Clin Microbiol. 1999;37(7):2343–5.PubMedCentralPubMedGoogle Scholar
  11. 11.
    Kontoyiannis DP, Lewis RE, May GS, Osherov N, Rinaldi MG. Aspergillus nidulans is frequently resistant to amphotericin B. Mycoses. 2002;45(9–10):406–7.PubMedCrossRefGoogle Scholar
  12. 12.
    De Pauw B, Walsh TJ, Donnelly JP, et al. Revised definitions of invasive fungal disease from the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) Consensus Group. Clin Infect Dis. 2008;46(12):1813–21.PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    Marr KA, Laverdiere M, Gugel A, Leisenring W. Antifungal therapy decreases sensitivity of the Aspergillus galactomannan enzyme immunoassay. Clin Infect Dis. 2005;40(12):1762–9.PubMedCrossRefGoogle Scholar
  14. 14.
    Sulahian A, Touratier S, Ribaud P. False positive test for Aspergillus antigenemia related to concomitant administration of piperacillin and tazobactam. N Engl J Med. 2003;349(24):2366–7.PubMedCrossRefGoogle Scholar
  15. 15.
    Walsh TJ, Shoham S, Petraitiene R, et al. Detection of galactomannan antigenemia in patients receiving piperacillin-tazobactam and correlations between in vitro, in vivo, and clinical properties of the drug-antigen interaction. J Clin Microbiol. 2004;42(10):4744–8.PubMedCentralPubMedCrossRefGoogle Scholar
  16. 16.
    Koo S, Bryar JM, Page JH, Baden LR, Marty FM. Diagnostic performance of the (1 → 3)-beta-D-glucan assay for invasive fungal disease. Clin Infect Dis. 2009;49(11):1650–9.PubMedCrossRefGoogle Scholar
  17. 17.
    Hardak E, Yigla M, Avivi I, Fruchter O, Sprecher H, Oren I. Impact of PCR-based diagnosis of invasive pulmonary aspergillosis on clinical outcome. Bone Marrow Transpl. 2009;44(9):595–9.CrossRefGoogle Scholar
  18. 18.
    Bretagne S, Costa JM, Marmorat-Khuong A, et al. Detection of Aspergillus species DNA in bronchoalveolar lavage samples by competitive PCR. J Clin Microbiol. 1995;33(5):1164–8.PubMedCentralPubMedGoogle Scholar
  19. 19.
    Olias P, Jacobsen ID, Gruber AD. Fungal species identification from avian lung specimens by single hypha laser microdissection and PCR product sequencing. Med Mycol. 2011;49(1):56–61.PubMedCrossRefGoogle Scholar
  20. 20.
    Ma Y, Qiao J, Liu W, et al. The sho1 sensor regulates growth, morphology, and oxidant adaptation in Aspergillus fumigatus but is not essential for development of invasive pulmonary aspergillosis. Infect Immun. 2008;76(4):1695–701.PubMedCentralPubMedCrossRefGoogle Scholar
  21. 21.
    Mayr A, Lass-Florl C. Epidemiology and antifungal resistance in invasive Aspergillosis according to primary disease: review of the literature. Eur J Med Res. 2011;16(4):153–7.PubMedCentralPubMedCrossRefGoogle Scholar
  22. 22.
    Lortholary O, Gangneux JP, Sitbon K, et al. Epidemiological trends in invasive aspergillosis in France: the SAIF network (2005–2007). Clin Microbiol Infect. 2011;17(12):1882–9.PubMedCrossRefGoogle Scholar
  23. 23.
    Burgos A, Zaoutis TE, Dvorak CC, et al. Pediatric invasive aspergillosis: a multicenter retrospective analysis of 139 contemporary cases. Pediatrics. 2008;121(5):e1286–94.PubMedCrossRefGoogle Scholar
  24. 24.
    Reichenberger F, Habicht J, Matt P, et al. Diagnostic yield of bronchoscopy in histologically proven invasive pulmonary aspergillosis. Bone Marrow Transpl. 1999;24(11):1195–9.CrossRefGoogle Scholar
  25. 25.
    Tarrand JJ, Lichterfeld M, Warraich I, et al. Diagnosis of invasive septate mold infections. A correlation of microbiological culture and histologic or cytologic examination. Am J Clin Pathol. 2003;119(6):854–8.PubMedCrossRefGoogle Scholar
  26. 26.
    Rickerts V, Mousset S, Lambrecht E, et al. Comparison of histopathological analysis, culture, and polymerase chain reaction assays to detect invasive mold infections from biopsy specimens. Clin Infect Dis. 2007;44(8):1078–83.PubMedCrossRefGoogle Scholar
  27. 27.
    Shah AA, Hazen KC. Diagnostic accuracy of histopathologic and cytopathologic examination of Aspergillus species. Am J Clin Pathol. 2013;139(1):55–61.PubMedCrossRefGoogle Scholar
  28. 28.
    Sangoi AR, Rogers WM, Longacre TA, Montoya JG, Baron EJ, Banaei N. Challenges and pitfalls of morphologic identification of fungal infections in histologic and cytologic specimens: a ten-year retrospective review at a single institution. Am J Clin Pathol. 2009;131(3):364–75.PubMedCrossRefGoogle Scholar
  29. 29.
    Pasqualotto AC, Xavier MO, Sanchez LB, et al. Diagnosis of invasive aspergillosis in lung transplant recipients by detection of galactomannan in the bronchoalveolar lavage fluid. Transplantation. 2010;90(3):306–11.PubMedCrossRefGoogle Scholar
  30. 30.
    Clancy CJ, Jaber RA, Leather HL, et al. Bronchoalveolar lavage galactomannan in diagnosis of invasive pulmonary aspergillosis among solid-organ transplant recipients. J Clin Microbiol. 2007;45(6):1759–65.PubMedCentralPubMedCrossRefGoogle Scholar
  31. 31.
    Alexander BD, Smith PB, Davis RD, Perfect JR, Reller LB. The (1,3){beta}-D-glucan test as an aid to early diagnosis of invasive fungal infections following lung transplantation. J Clin Microbiol. 2010;48(11):4083–8.PubMedCentralPubMedCrossRefGoogle Scholar
  32. 32.
    Emmert-Buck MR, Bonner RF, Smith PD, et al. Laser capture microdissection. Science. 1996;274(5289):998–1001.PubMedCrossRefGoogle Scholar
  33. 33.
    Espina V, Heiby M, Pierobon M, Liotta LA. Laser capture microdissection technology. Expert Rev Mol Diagn. 2007;7(5):647–57.PubMedCrossRefGoogle Scholar
  34. 34.
    van der Marel J, Quint WG, Schiffman M, et al. Molecular mapping of high-grade cervical intraepithelial neoplasia shows etiological dominance of HPV16. Int J Cancer. 2012;131(6):E946–53.PubMedCentralPubMedCrossRefGoogle Scholar
  35. 35.
    Wang K, Mahalingam G, Hoover SE, et al. Diverse herpes simplex virus type 1 thymidine kinase mutants in individual human neurons and Ganglia. J Virol. 2007;81(13):6817–26.PubMedCentralPubMedCrossRefGoogle Scholar
  36. 36.
    Selva E, Hofman V, Berto F, et al. The value of polymerase chain reaction detection of Mycobacterium tuberculosis in granulomas isolated by laser capture microdissection. Pathology. 2004;36(1):77–81.PubMedCrossRefGoogle Scholar
  37. 37.
    Dannaoui E, Schwarz P, Slany M, et al. Molecular detection and identification of zygomycetes species from paraffin-embedded tissues in a murine model of disseminated zygomycosis: a collaborative European Society of Clinical Microbiology and Infectious Diseases (ESCMID) Fungal Infection Study Group (EFISG) evaluation. J Clin Microbiol. 2010;48(6):2043–6.PubMedCentralPubMedCrossRefGoogle Scholar
  38. 38.
    Woo PC, Leung SY, To KK, et al. Internal transcribed spacer region sequence heterogeneity in Rhizopus microsporus: implications for molecular diagnosis in clinical microbiology laboratories. J Clin Microbiol. 2010;48(1):208–14.PubMedCentralPubMedCrossRefGoogle Scholar
  39. 39.
    Ferns RB, Fletcher H, Bradley S, Mackinnon S, Hunt C, Tedder RS. The prospective evaluation of a nested polymerase chain reaction assay for the early detection of Aspergillus infection in patients with leukaemia or undergoing allograft treatment. Br J Haematol. 2002;119(3):720–5.PubMedCrossRefGoogle Scholar
  40. 40.
    Li Y, Gao L, Ding Y, et al. Establishment and application of real-time quantitative PCR for diagnosing invasive aspergillosis via the blood in hematological patients: targeting a specific sequence of Aspergillus 28S-ITS2. BMC Infect Dis. 2013;13:255.PubMedCentralPubMedCrossRefGoogle Scholar
  41. 41.
    Torelli R, Sanguinetti M, Moody A, et al. Diagnosis of invasive aspergillosis by a commercial real-time PCR assay for Aspergillus DNA in bronchoalveolar lavage fluid samples from high-risk patients compared to a galactomannan enzyme immunoassay. J Clin Microbiol. 2011;49(12):4273–8.PubMedCentralPubMedCrossRefGoogle Scholar
  42. 42.
    Jones ME, Fox AJ, Barnes AJ, et al. PCR-ELISA for the early diagnosis of invasive pulmonary aspergillus infection in neutropenic patients. J Clin Pathol. 1998;51(9):652–6.PubMedCentralPubMedCrossRefGoogle Scholar
  43. 43.
    White PL, Parr C, Thornton C, Barnes RA. Evaluation of real-time PCR, galactomannan enzyme-linked immunosorbent assay (ELISA), and a novel lateral-flow device for diagnosis of invasive aspergillosis. J Clin Microbiol. 2013;51(5):1510–6.PubMedCentralPubMedCrossRefGoogle Scholar
  44. 44.
    Hadrich I, Mary C, Makni F, et al. Comparison of PCR-ELISA and Real-Time PCR for invasive aspergillosis diagnosis in patients with hematological malignancies. Med Mycol. 2011;49(5):489–94.PubMedGoogle Scholar
  45. 45.
    Nosari A, Anghilieri M, Carrafiello G, et al. Utility of percutaneous lung biopsy for diagnosing filamentous fungal infections in hematologic malignancies. Haematologica. 2003;88(12):1405–9.PubMedGoogle Scholar
  46. 46.
    Lass-Florl C, Griff K, Mayr A, et al. Epidemiology and outcome of infections due to Aspergillus terreus: 10-year single centre experience. Br J Haematol. 2005;131(2):201–7.PubMedCrossRefGoogle Scholar
  47. 47.
    Marr KA, Carter RA, Crippa F, Wald A, Corey L. Epidemiology and outcome of mould infections in hematopoietic stem cell transplant recipients. Clin Infect Dis. 2002;34(7):909–17.PubMedCrossRefGoogle Scholar
  48. 48.
    Singh N, Husain S. Aspergillus infections after lung transplantation: clinical differences in type of transplant and implications for management. J Heart Lung Transpl. 2003;22(3):258–66.CrossRefGoogle Scholar
  49. 49.
    Orzechowski XM, Pasqualotto AC, Uchoa SMP, Bittencourt SC, Peixoto CJJ, Severo LC. Invasive pulmonary aspergillosis due to a mixed infection caused by Aspergillus flavus and Aspergillus fumigatus. Rev Iberoam Micol. 2008;25(3):176–8.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  • Chong Wang
    • 1
    • 2
  • Ping Zhan
    • 1
    • 2
  • Le Wang
    • 1
    • 2
  • Rong Zeng
    • 1
    • 2
  • Yongnian Shen
    • 1
    • 2
  • Guixia Lv
    • 1
    • 2
  • Dongmei Li
    • 3
  • Shuwen Deng
    • 4
  • Weida Liu
    • 2
    • 1
  1. 1.Department of Mycology, Institute of DermatologyChinese Academy of Medical Science and Peking Union Medical CollegeNanjingPeople’s Republic of China
  2. 2.Jiangsu Key Laboratory of Molecular Biology for Skin Diseases and STIsNanjingPeople’s Republic of China
  3. 3.Department of Microbiology and ImmunologyGeorgetown University Medical CenterWashingtonUSA
  4. 4.Shanghai Institute of Medical Mycology, Changzheng HospitalSecond Military Medical UniversityShanghaiPeople’s Republic of China

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