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Tissue Diagnosis of Invasive Fungal Infections: Current Limitations and the Emerging Use of Molecular Techniques

  • Advances in Diagnosis of Invasive Fungal Infections (U Binder and A Groll, Section Editors)
  • Published:
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Abstract

Invasive fungal diseases (IFD) due to opportunistic fungi are commonly treated using empirical antifungal therapy. Therefore, a comprehensive study of organisms associated with IFD is essential to define successful empiric therapies in each setting. Current diagnostic tests, such as culture, histology and serology are suboptimal, leading to delays in the initiation of antifungal therapies and resulting in high mortality rates despite the availability of several new antifungal agents. Using molecular methods to identify fungal pathogens directly from formalin-fixed, paraffin-embedded tissues is emerging as a diagnostic approach. The goal of this molecular approach is to complement conventional diagnostic tests through the reliable detection and identification of fungal nucleic acids or antigens in tissues so as to direct antiinfective therapies and improve patient outcomes. Here we review challenges and recent advances in the identification of fungal pathogens from tissue samples by conventional and molecular methods.

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References

Papers of particular interest, published recently, have been highlighted as: • Of importance •• Of major importance

  1. Pastores SM, Dulu A, et al. Premortem clinical diagnoses and postmortem autopsy findings: discrepancies in critically ill cancer patients. Crit Care. 2007;11(2):R48.

    Article  PubMed  Google Scholar 

  2. Antinori S, Nebuloni M, et al. Trends in the postmortem diagnosis of opportunistic invasive fungal infections in patients with AIDS: a retrospective study of 1,630 autopsies performed between 1984 and 2002. Am J Clin Pathol. 2009;132(2):221–7.

    Article  PubMed  Google Scholar 

  3. Tejerina E, Esteban A, et al. Clinical diagnoses and autopsy findings: discrepancies in critically ill patients. Crit Care Med. 2012;40(3):842–6.

    Article  PubMed  Google Scholar 

  4. • Ghannoum MA, Jurevic RJ, et al. Characterization of the oral fungal microbiome (mycobiome) in healthy individuals. PLoS Pathog. 2010;6(1):e1000713. This study highlights the diversity of fungi found on mucous membranes of healthy people..

    Article  PubMed  Google Scholar 

  5. Chamilos G, Luna M, et al. Invasive fungal infections in patients with hematologic malignancies in a tertiary care cancer center: an autopsy study over a 15-year period (1989–2003). Haematologica. 2006;91(7):986–9.

    PubMed  Google Scholar 

  6. Rickerts V, Mousset S, 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.

    Article  PubMed  Google Scholar 

  7. Groll AH, Shah PM, et al. Trends in the postmortem epidemiology of invasive fungal infections at a university hospital. J Infect. 1996;33(1):23–32.

    Article  PubMed  CAS  Google Scholar 

  8. De Pauw B, Walsh TJ, 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.

    Article  PubMed  Google Scholar 

  9. ••Guarner J, Brandt ME. Histopathologic diagnosis of fungal infections in the 21st century. Clin Microbiol Rev. 2011;24(2):247–80. Authoritative review on diagnostic aspects of histopathology of invasive fungal infections..

    Article  PubMed  Google Scholar 

  10. •• Sangoi AR, Rogers WM, et al. 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. Evaluates discrepancies between histopathlogic-, and culture results. Suggest steps to improve reporting on results of histopathology of invasive fungal infections..

    Article  PubMed  Google Scholar 

  11. Kume H, Yamazaki T, et al. Epidemiology of visceral mycoses in autopsy cases in Japan: comparison of the data from 1989, 1993, 1997, 2001, 2005 and 2007 in Annual of Pathological Autopsy Cases in Japan. Med Mycol J. 2011;52(2):117–27.

    Article  PubMed  Google Scholar 

  12. Denning DW, Kibbler CC, et al. British Society for Medical Mycology proposed standards of care for patients with invasive fungal infections. Lancet Infect Dis. 2003;3(4):230–40.

    Article  PubMed  Google Scholar 

  13. Schelenz S, Barnes RA, et al. Standards of care for patients with invasive fungal infections within the United Kingdom: a national audit. J Infect. 2009;58(2):145–53.

    Article  PubMed  CAS  Google Scholar 

  14. Tarrand JJ, Han XY, et al. Aspergillus hyphae in infected tissue: evidence of physiologic adaptation and effect on culture recovery. J Clin Microbiol. 2005;43(1):382–6.

    Article  PubMed  Google Scholar 

  15. Tarrand JJ, Lichterfeld M, 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.

    Article  PubMed  Google Scholar 

  16. Riedel S, Eisinger SW, et al. Comparison of BD Bactec Plus Aerobic/F medium to VersaTREK Redox 1 blood culture medium for detection of Candida spp. in seeded blood culture specimens containing therapeutic levels of antifungal agents. J Clin Microbiol. 2011;49(4):1524–9.

    Article  PubMed  Google Scholar 

  17. Kontoyiannis DP, Chamilos G, et al. Increased culture recovery of Zygomycetes under physiologic temperature conditions. Am J Clin Pathol. 2007;127(2):208–12.

    Article  PubMed  Google Scholar 

  18. Lass-Florl C, Resch G, et al. The value of computed tomography-guided percutaneous lung biopsy for diagnosis of invasive fungal infection in immunocompromised patients. Clin Infect Dis. 2007;45(7):e101–4.

    Article  PubMed  Google Scholar 

  19. Blyth CC, Harun A, et al. Detection of occult Scedosporium species in respiratory tract specimens from patients with cystic fibrosis by use of selective media. J Clin Microbiol. 2010;48(1):314–6.

    Article  PubMed  CAS  Google Scholar 

  20. Marr KA, Seidel K, et al. Candidemia in allogeneic blood and marrow transplant recipients: evolution of risk factors after the adoption of prophylactic fluconazole. J Infect Dis. 2010;181(1):309–16.

    Article  Google Scholar 

  21. Magill SS, Swoboda SM, et al. The epidemiology of Candida colonization and invasive candidiasis in a surgical intensive care unit where fluconazole prophylaxis is utilized: follow-up to a randomized clinical trial. Ann Surg. 2009;249(4):657–65.

    Article  PubMed  Google Scholar 

  22. Purcell J, McKenna J, et al. Mixed mould species in laboratory cultures of respiratory specimens: how should they be reported, and what are the indications for susceptibility testing? J Clin Pathol. 2011;64(6):543–5.

    Article  PubMed  CAS  Google Scholar 

  23. Springer J, Loeffler J, et al. Pathogen-specific DNA enrichment does not increase sensitivity of PCR for diagnosis of invasive aspergillosis in neutropenic patients. J Clin Microbiol. 2011;49(4):1267–73.

    Article  PubMed  CAS  Google Scholar 

  24. Khot PD, Fredricks DN. PCR-based diagnosis of human fungal infections. Expert Rev Anti Infect Ther. 2009;7(10):1201–21.

    Article  PubMed  CAS  Google Scholar 

  25. Bretagne S, Costa JM, et al. Detection of Aspergillus species DNA in bronchoalveolar lavage samples by competitive PCR. J Clin Microbiol. 1995;33(5):1164–8.

    PubMed  CAS  Google Scholar 

  26. Lengerova M, Kocmanova I, et al. Detection and measurement of fungal burden in a guinea pig model of invasive pulmonary aspergillosis by novel quantitative nested real-time PCR compared with galactomannan and (1,3)-beta-D-glucan detection. J Clin Microbiol. 2012;50(3):602–8.

    Article  PubMed  CAS  Google Scholar 

  27. • Nguyen MH, Wissel MC, et al. Performance of Candida real-time polymerase chain reaction, beta-D-glucan assay, and blood cultures in the diagnosis of invasive candidiasis. Clin Infect Dis. 2012;54(9):1240–8. Suggest superior sensitivity of PCR in detecting etiologic agents of deep seated candidiasis, including mixed infections as compared with blood cultures..

    Article  PubMed  CAS  Google Scholar 

  28. Fredricks DN, Smith C, et al. Comparison of six DNA extraction methods for recovery of fungal DNA as assessed by quantitative PCR. J Clin Microbiol. 2005;43(10):5122–8.

    Article  PubMed  CAS  Google Scholar 

  29. Willinger B, Obradovic A, et al. Detection and identification of fungi from fungus balls of the maxillary sinus by molecular techniques. J Clin Microbiol. 2003;41(2):581–5.

    Article  PubMed  CAS  Google Scholar 

  30. Lau A, Chen S, et al. Development and clinical application of a panfungal PCR assay to detect and identify fungal DNA in tissue specimens. J Clin Microbiol. 2007;45(2):380–5.

    Article  PubMed  CAS  Google Scholar 

  31. Dannaoui E, Schwarz P, 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.

    Article  PubMed  CAS  Google Scholar 

  32. Gilbert MT, Haselkorn T, et al. The isolation of nucleic acids from fixed, paraffin-embedded tissues-which methods are useful when? PLoS One. 2007;2(6):e537.

    Article  PubMed  Google Scholar 

  33. Rickerts V, Khot PD, et al. Enhanced fungal DNA-extraction from formalin-fixed, paraffin-embedded tissue specimens by application of thermal energy. Med Mycol. 2012 in press.

  34. • Rickerts V, Khot PD, et al. Comparison of quantitative real time PCR with sequencing and ribosomal RNA-FISH for the identification of fungi in formalin fixed, paraffin-embedded tissue specimens. BMC Infect Dis. 2011;11:202. Explores the combined use of quantitative realtime PCR and fluorescence in situ hybridisation in an attempt to characterise the etiology of invasive fungal infections..

    Article  PubMed  Google Scholar 

  35. Hammond SP, Bialek R, et al. Molecular methods to improve diagnosis and identification of mucormycosis. J Clin Microbiol. 2011;49(6):2151–3.

    Article  PubMed  Google Scholar 

  36. Khot PD, Ko DL, et al. Sequencing and analysis of fungal rRNA operons for development of broad-range fungal PCR assays. Appl Environ Microbiol. 2009;75(6):1559–65.

    Article  PubMed  CAS  Google Scholar 

  37. Kommedal O, Karlsen B, et al. Analysis of mixed sequencing chromatograms and its application in direct 16S rRNA gene sequencing of polymicrobial samples. J Clin Microbiol. 2008;46(11):3766–71.

    Article  PubMed  CAS  Google Scholar 

  38. Fredricks DN, Schubert MM, et al. Molecular identification of an invasive gingival bacterial community. Clin Infect Dis. 2005;41(1):e1–4.

    Article  PubMed  Google Scholar 

  39. •• Al Masalma M, Armougom F, et al. The expansion of the microbiological spectrum of brain abscesses with use of multiple 16S ribosomal DNA sequencing. Clin Infect Dis. 2009;48(9):1169–78. Demonstrates an increased number of identified agents in cerebral abscesses, including bacteria not previously known to be associated with cerebral abscesses..

    Article  PubMed  Google Scholar 

  40. Vallor AC, Kirkpatrick WR, et al. Assessment of Aspergillus fumigatus burden in pulmonary tissue of guinea pigs by quantitative PCR, galactomannan enzyme immunoassay, and quantitative culture. Antimicrob Agents Chemother. 2008;52(7):2593–8.

    Article  PubMed  CAS  Google Scholar 

  41. Baddley JW, Marr KA, et al. Patterns of susceptibility of Aspergillus isolates recovered from patients enrolled in the Transplant-Associated Infection Surveillance Network. J Clin Microbiol. 2009;47(10):3271–5.

    Article  PubMed  CAS  Google Scholar 

  42. Balajee SA, Borman AM, et al. Sequence-based identification of Aspergillus, fusarium, and mucorales species in the clinical mycology laboratory: where are we and where should we go from here? J Clin Microbiol. 2009;47(4):877–84.

    Article  PubMed  CAS  Google Scholar 

  43. van der Linden JW, Snelders E, et al. Rapid diagnosis of azole-resistant aspergillosis by direct PCR using tissue specimens. J Clin Microbiol. 2010;48(4):1478–80.

    Article  PubMed  Google Scholar 

  44. DeLong EF, Wickham GS, et al. Phylogenetic stains: ribosomal RNA-based probes for the identification of single cells. Science. 1989;243(4896):1360–3.

    Article  PubMed  CAS  Google Scholar 

  45. Amann R, Fuchs BM. Single-cell identification in microbial communities by improved fluorescence in situ hybridization techniques. Nat Rev Microbiol. 2008;6(5):339–48.

    Article  PubMed  CAS  Google Scholar 

  46. Kempf VAJ, Trebesius K, et al. Fluorescent in situ hybridization allows rapid identification of microorganisms in blood cultures. J Clin Microbiol. 2000;38(2):830–8.

    PubMed  CAS  Google Scholar 

  47. Martins ML, Ferreira AS, et al. Direct and specific identification of Cryptococcus neoformans in biological samples using fluorescently labelled DNA probes. Eur J Clin Microbiol Infect Dis. 2010;29(5):571–6.

    Article  PubMed  CAS  Google Scholar 

  48. Oliveira K, Haase G, et al. Differentiation of Candida albicans and Candida dubliniensis by fluorescent in situ hybridization with peptide nucleic acid probes. J Clin Microbiol. 2001;39(11):4138–41.

    Article  PubMed  CAS  Google Scholar 

  49. Shepard JR, Addison RM, et al. Multicenter evaluation of the Candida albicans/Candida glabrata peptide nucleic acid fluorescent in situ hybridization method for simultaneous dual-color identification of C. albicans and C. glabrata directly from blood culture bottles. J Clin Microbiol. 2008;46(1):50–5.

    Article  PubMed  CAS  Google Scholar 

  50. Reller ME, Mallonee AB, et al. Use of peptide nucleic acid-fluorescence in situ hybridization for definitive, rapid identification of five common Candida species. J Clin Microbiol. 2007;45(11):3802–3.

    Article  PubMed  Google Scholar 

  51. Hayden RT, Qian X, et al. In situ hybridization for the identification of yeastlike organisms in tissue section. Diagn Mol Pathol. 2001;10(1):15–23.

    Article  PubMed  CAS  Google Scholar 

  52. Hayden RT, Qian X, et al. In situ hybridization for the identification of filamentous fungi in tissue section. Diagn Mol Pathol. 2002;11(2):119–26.

    Article  PubMed  CAS  Google Scholar 

  53. Hayden RT, Isotalo PA, et al. In situ hybridization for the differentiation of Aspergillus, Fusarium, and Pseudallescheria species in tissue section. Diagn Mol Pathol. 2003;12(1):21–6.

    Article  PubMed  CAS  Google Scholar 

  54. Montone KT, Livolsi VA, et al. Rapid in-situ hybridization for dematiaceous fungi using a broad-spectrum oligonucleotide DNA probe. Diagn Mol Pathol. 2011;20(3):180–3.

    Article  PubMed  CAS  Google Scholar 

  55. Montone KT, Feldman MD. In situ detection of aspergillus 18s ribosomal RNA Sequences using a terminally biotinylated locked nucleic acid (LNA) probe. Diagn Mol Pathol. 2009;18(4):239–42.

    Article  PubMed  CAS  Google Scholar 

  56. Montone KT. Differentiation of Fusarium from Aspergillus species by colorimetric in situ hybridization in formalin-fixed, paraffin-embedded tissue sections using dual fluorogenic-labeled LNA probes. Am J Clin Pathol. 2009;132(6):866–70.

    Article  PubMed  CAS  Google Scholar 

  57. Montone KT, Litzky LA, et al. In situ hybridization for Coccidioides immitis 5.8S ribosomal RNA sequences in formalin-fixed, paraffin-embedded pulmonary specimens using a locked nucleic acid probe: a rapid means for identification in tissue sections. Diagn Mol Pathol. 2010;19(2):99–104.

    Article  PubMed  CAS  Google Scholar 

  58. Shinozaki M, Okubo Y, et al. Identification of Fusarium species in formalin-fixed and paraffin-embedded sections by in situ hybridization using peptide nucleic acid probes. J Clin Microbiol. 2011;49(3):808–13.

    Article  PubMed  CAS  Google Scholar 

  59. Park CS, Kim J, et al. Detection of Aspergillus ribosomal RNA using biotinylated oligonucleotide probes. Diagn Mol Pathol. 1997;6(5):255–60.

    Article  PubMed  CAS  Google Scholar 

  60. Teertstra WR, Lugones LG, et al. In situ hybridisation in filamentous fungi using peptide nucleic acid probes. Fungal Genet Biol. 2004;41(12):1099–103.

    Article  PubMed  CAS  Google Scholar 

  61. Yilmaz LS, Parnerkar S, et al. mathFISH, a web tool that uses thermodynamics-based mathematical models for in silico evaluation of oligonucleotide probes for fluorescence in situ hybridization. Appl Environ Microbiol. 2011;77(3):1118–22.

    Article  PubMed  CAS  Google Scholar 

  62. Valm AM, Welch JL, et al. Systems-level analysis of microbial community organization through combinatorial labeling and spectral imaging. Proc Natl Acad Sci U S A. 2011;108(10):4152–7.

    Article  PubMed  CAS  Google Scholar 

  63. Rolston KV, Bodey GP, et al. Polymicrobial infection in patients with cancer: an underappreciated and underreported entity. Clin Infect Dis. 2007;45(2):228–33.

    Article  PubMed  Google Scholar 

  64. Klotz SA, Chasin BS, et al. Polymicrobial bloodstream infections involving Candida species: analysis of patients and review of the literature. Diagn Microbiol Infect Dis. 2007;59(4):401–6.

    Article  PubMed  CAS  Google Scholar 

  65. Klotz SA, Gaur NK, et al. Candida albicans Als proteins mediate aggregation with bacteria and yeasts. Med Mycol. 2007;45(4):363–70.

    Article  PubMed  CAS  Google Scholar 

  66. Harriott MM, Noverr MC. Candida albicans and Staphylococcus aureus form polymicrobial biofilms: effects on antimicrobial resistance. Antimicrob Agents Chemother. 2009;53(9):3914–22.

    Article  PubMed  CAS  Google Scholar 

  67. Peleg AY, Tampakakis E, et al. Prokaryote-eukaryote interactions identified by using Caenorhabditis elegans. Proc Natl Acad Sci U S A. 2008;105(38):14585–90.

    Article  PubMed  CAS  Google Scholar 

  68. Vadnerkar A, Clancy CJ, et al. Impact of mold infections in explanted lungs on outcomes of lung transplantation. Transplantation. 2010;89(2):253–60.

    Article  PubMed  Google Scholar 

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Disclosure

Dr. D. Fredricks has received grant support from the NIH, payment for lectures from Roche, and royalties from Lab 21.

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  1. Robert Koch-Institut, Nordufer 20, 13353, Berlin, Germany

    Volker Rickerts

  2. Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, Seattle, WA, 98109, USA

    David N. Fredricks

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  1. Volker Rickerts
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Correspondence to Volker Rickerts.

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Rickerts, V., Fredricks, D.N. Tissue Diagnosis of Invasive Fungal Infections: Current Limitations and the Emerging Use of Molecular Techniques. Curr Fungal Infect Rep 6, 221–228 (2012). https://doi.org/10.1007/s12281-012-0098-6

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