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Commercial Molecular Tests for Fungal Diagnosis from a Practical Point of View

  • Michaela LacknerEmail author
  • Cornelia Lass-Flörl
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1508)

Abstract

The increasing interest in molecular diagnostics is a result of tremendously improved knowledge on fungal infections in the past 20 years and the rapid development of new methods, in particular polymerase chain reaction. High expectations have been placed on molecular diagnostics, and the number of laboratories now using the relevant technology is rapidly increasing—resulting in an obvious need for standardization and definition of laboratory organization. In the past 10 years, multiple new molecular tools were marketed for the detection of DNA, antibodies, cell wall components, or other antigens. In contrast to classical culture methods, molecular methods do not detect a viable organisms, but only molecules which indicate its presence; this can be nucleic acids, cell components (antigens), or antibodies (Fig. 1). In this chapter, an overview is provided on commercially available detection tools, their strength and how to use them. A main focus is laid on providing tips and tricks that make daily life easier. We try to focus and mention methodical details which are not highlighted in the manufacturer’s instructions of these test kits, but are based on our personal experience in the laboratory. Important to keep in mind is that molecular tools cannot replace culture, microscopy, or a critical view on patients’ clinical history, signs, and symptoms, but provide a valuable add on tool. Diagnosis should not be based solely on a molecular test, but molecular tools might deliver an important piece of information that helps matching the diagnostic puzzle to a diagnosis, in particular as few tests are in vitro diagnostic tests (IVD) or only part of the whole test carries the IVD certificate (e.g., DNA extraction is often not included). Please be aware that the authors do not claim to provide a complete overview on all commercially available diagnostic assays being currently marketed for fungal detection, as those are subject to constant change. A main focus is put on commonly used panfungal assays and pathogen-specific assays, including Aspergillus-specific, Candida-specific, Cryptococcus specific, Histoplasma-specific, and Pneumocystis-specific assays. Assays are categorized according to their underlying principle in either antigen-detecting or antibody-detecting or DNA-detecting (Fig. 1). Other non-DNA-detecting nucleic acid methods such as FISH and PNA FISH are not summarized in this chapter and an overview on test performance, common false positives, and the clinical evaluation of commercial tests in studies is provided already in a previous book series by Javier Yugueros Marcos and David H. Pincus (Marcos and Pincus, Methods Mol Biol 968:25–54, 2013).

Key words

Real-time PCR Rt-PCR Polymerase chain reaction ELISA Antigen test Antibody test Candida Aspergillus Cryptococcus Fungi Pneumocystis 

Notes

Acknowledgement

We are thankful to the tips and tricks and remarks provided by staff of molecular routine laboratory Alexander Engl, Michaela Mayer, Raphaela Löffler, and Wolfgang Mutschlechner.

References

  1. 1.
    Harrison E, Stalhberger T, Whelan R et al (2010) Aspergillus DNA contamination in blood collection tubes. Diagn Microbiol Infect Dis 67:392–394CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Alanio AB, S. (2014) Diagnosis of Pneumocystis jirovecii pneumonia: role of β-D-glucan detection and PCR. Curr Fungal Infect Rep 8:322–330Google Scholar
  3. 3.
    Neumaier M, Braun A, Wagener C (1998) Fundamentals of quality assessment of molecular amplification methods in clinical diagnostics. International Federation of Clinical Chemistry Scientific Division Committee on Molecular Biology Techniques. Clin Chem 44:12–26PubMedGoogle Scholar
  4. 4.
    LIFE, Cheshire, UK. http://www.life-worldwide.org/. Accessed 7 Oct 2015
  5. 5.
    Anonymous (2013) UK Standards for Microbiological Investigations: Good Laboratory Practice when Performing Molecular Amplification Assays. In: Standard Unit MS, Public Health England (ed) Public Health England (PHE) National Health Services (NHS), London, pp 1–15Google Scholar
  6. 6.
    Baxter CG, Denning DW, Jones AM et al (2013) Performance of two Aspergillus IgG EIA assays compared with the precipitin test in chronic and allergic aspergillosis. Clin Microbiol Infect 19:e197–e204CrossRefPubMedGoogle Scholar
  7. 7.
    Beutler E, Gelbart T, Kuhl W (1990) Interference of heparin with the polymerase chain reaction. Biotechniques 9:166PubMedGoogle Scholar
  8. 8.
    Chen SC, Kontoyiannis DP (2010) New molecular and surrogate biomarker-based tests in the diagnosis of bacterial and fungal infection in febrile neutropenic patients. Curr Opin Infect Dis 23:567–577CrossRefPubMedGoogle Scholar
  9. 9.
    Chong L (2001) Molecular cloning—a laboratory manual, 3rd edition. Science 292:446CrossRefGoogle Scholar
  10. 10.
    Chumpitazi BF, Lebeau B, Faure-Cognet O et al (2014) Characteristic and clinical relevance of Candida mannan test in the diagnosis of probable invasive candidiasis. Med Mycol 52:462–471CrossRefPubMedGoogle Scholar
  11. 11.
    Cimino GD, Metchette KC, Tessman JW et al (1991) Post-PCR sterilization: a method to control carryover contamination for the polymerase chain reaction. Nucleic Acids Res 19:99–107CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Fairfax MR, Metcalf MA, Cone RW (1991) Slow inactivation of dry PCR templates by UV light. PCR Methods Appl 1:142–143CrossRefPubMedGoogle Scholar
  13. 13.
    Hauser PM, Bille J, Lass-Flörl C et al (2011) Multicenter, prospective clinical evaluation of respiratory samples from subjects at risk for Pneumocystis jirovecii infection by use of a commercial real-time PCR assay. J Clin Microbiol 49:1872–1878CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Irinyi L, Lackner M, De Hoog GS, Meyer W (2015) DNA barcoding of fungi causing infections in humans and animals. Fungal Biol. In press. Doi:  10.1016/j.funbio.2015.04.007
  15. 15.
    Marcos JY, Pincus DH (2013) Fungal diagnostics: review of commercially available methods. Methods Mol Biol 968:25–54CrossRefPubMedGoogle Scholar
  16. 16.
    Montesinos I, Brancart F, Schepers K et al (2015) Comparison of 2 real-time PCR assays for diagnosis of Pneumocystis jirovecii pneumonia in human immunodeficiency virus (HIV) and non-HIV immunocompromised patients. Diagn Microbiol Infect Dis 82:143–147CrossRefPubMedGoogle Scholar
  17. 17.
    Gillespie DH, Cuddy KK, Kolbe T et al (1994) Dissolve and capture: a strategy for analysing mRNA in blood. Nature 367:390–391CrossRefPubMedGoogle Scholar
  18. 18.
    Morris A, Lundgren JD, Masur H et al (2004) Current epidemiology of Pneumocystis pneumonia. Emerg Infect Dis 10:1713–1720CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Thornton CR (2010) Detection of invasive aspergillosis. Adv Appl Microbiol 70:187–216CrossRefPubMedGoogle Scholar
  20. 20.
    Verdaguer V, Walsh TJ, Hope W et al (2007) Galactomannan antigen detection in the diagnosis of invasive aspergillosis. Expert Rev Mol Diagn 7:21–32CrossRefPubMedGoogle Scholar
  21. 21.
    Williams DA, Kiiza T, Kwizera R et al (2015) Evaluation of fingerstick cryptococcal antigen lateral flow assay in HIV-infected persons: a diagnostic accuracy study. Clin Infect Dis. In press. doi:  10.1093/cid/civ263

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  1. 1.Division of Hygiene and Medical MicrobiologyMedical University of InnsbruckInnsbruckAustria

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