Skip to main content

Advertisement

SpringerLink
Log in

Susceptibility Screening of Hyphae-Forming Fungi with a New, Easy, and Fast Inoculum Preparation Method

  • Published:
Mycopathologia Aims and scope Submit manuscript

Abstract

In vitro susceptibility testing of clinically important fungi becomes more and more essential due to the rising number of fungal infections in patients with impaired immune system. Existing standardized microbroth dilution methods for in vitro testing of molds (CLSI, EUCAST) are not intended for routine testing. These methods are very time-consuming and dependent on sporulating of hyphomycetes. In this multicentre study, a new (independent of sporulation) inoculum preparation method (containing a mixture of vegetative cells, hyphae, and conidia) was evaluated. Minimal inhibitory concentrations (MIC) of amphotericin B, posaconazole, and voriconazole of 180 molds were determined with two different culture media (YST and RPMI 1640) according to the DIN (Deutsches Institut für Normung) microdilution assay. 24 and 48 h MIC of quality control strains, tested per each test run, prepared with the new inoculum method were in the range of DIN. YST and RPMI 1640 media showed similar MIC distributions for all molds tested. MIC readings at 48 versus 24 h yield 1 log2 higher MIC values and more than 90 % of the MICs read at 24 and 48 h were within ±2 log2 dilution. MIC end point reading (log2 MIC-RPMI 1640−log2 MIC-YST) of both media demonstrated a tendency to slightly lower MICs with RPMI 1640 medium. This study reports the results of a new, time–saving, and easy-to-perform method for inoculum preparation for routine susceptibility testing that can be applied for all types of spore-/non-spore and hyphae-forming fungi.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Richardson MD. Changing patterns and trends in systemic fungal infections. J Antimicrob Chemother. 2005;56:5–11.

    Article  Google Scholar 

  2. Wingard JR. The changing face of invasive fungal infections in hematopoietic cell transplant recipients. Curr Opin Oncol. 2005;17:89–92.

    Article  PubMed  Google Scholar 

  3. Alcazar-Fuoli L, Rodriguez-Tudela J, Mellado E. Antifungal drug resistance in molds: clinical and microbiological factors. Curr Fung Infect Rep. 2008;2:36–42.

    Article  Google Scholar 

  4. Kanafani Z, Perfect J. Resistance to antifungal agents: mechanisms and clinical impact. Clin Infect Dis. 2008;46:120–8.

    Article  PubMed  Google Scholar 

  5. Arendrup MC, Mavridou E, Mortensen KL, et al. Development of azole resistance in Aspergillus fumigatus during azole therapy associated with change in virulence. PLoS ONE. 2010;5:e10080.

    Article  PubMed  Google Scholar 

  6. Denning DW, Venkateswarlu K, Oakley KL, et al. Itraconazole resistance in Aspergillus fumigatus. Antimicrob Agents Chemother. 1997;41:1364–8.

    PubMed  CAS  Google Scholar 

  7. Howard S, Webster I, Moore BC, et al. Multi-azole resistance in Aspergillus fumigatus. Int J Antimicrob Agents. 2006;28:450–3.

    Article  PubMed  CAS  Google Scholar 

  8. Howard SJ, Arendrup MC. Acquired antifungal drug resistance in Aspergillus fumigatus: epidemiology and detection. Med Mycol 2010.

  9. Howard S, Cerar D, Anderson M, et al. Frequency and evolution of azole resistance in Aspergillus fumigatus associated with treatment failure. Emerg Infect Dis. 2009;15:1068–76.

    Article  PubMed  CAS  Google Scholar 

  10. Lass- Florl C, Perkhofer S. In vitro susceptibility testing in Aspergillus species. Mycoses. 2008;51:437–46.

    Article  PubMed  Google Scholar 

  11. Nascimento AM, Goldman GH, Park S, et al. Multiple resistance mechanisms among Aspergillus fumigatus mutants with high-level resistance to itraconazole. Antimicrob Agents Chemother. 2003;47:1719–26.

    Article  PubMed  CAS  Google Scholar 

  12. Verweij PE, Mellado E, Melchers WJG. Multiple-triazole-resistant aspergillosis. N Engl J Med. 2007;356:1481–3.

    Article  PubMed  CAS  Google Scholar 

  13. Warris A, Weemaes C, Verweij PE. Multidrug resistance in Aspergillus fumigatus. N Engl J Med. 2002;347:2173–4.

    Article  PubMed  Google Scholar 

  14. Manavathu EK, Alangaden GJ, Chandrasekar PH. In vitro isolation and antifungal susceptibility of amphotericn B-resistant mutants of Aspergillus fumigatus. J Antimicrob Chemother. 1998;41:615–9.

    Article  PubMed  CAS  Google Scholar 

  15. Manavathu EK, Cutright JL, Loebenberg D, Chandrasekar PH. A comparative study of the in vitro susceptibilities of clinical and laboratory-selected resistant isolates of Aspergillus spp. to amphotericin B, itraconazole, voriconazole and posaconazole (SCH 56592). J Antimicrob Chemother. 2000;46:229–34.

    Article  PubMed  CAS  Google Scholar 

  16. Dannaoui E, Borel E, Monier M, et al. Acquired itraconazole resistance in Aspergillus fumigatus. J Antimicrob Chemother. 2001;47:333–40.

    Article  PubMed  CAS  Google Scholar 

  17. van Leer-Buter C, Takes RP, Hebeda KM, Melchers WJ, Verweij PE. Aspergillosis—and a misleading sensitivity result. Lancet. 2007;370:102.

    Article  PubMed  Google Scholar 

  18. Snelders E, van der Lee H, Kuijper J, et al. Emergence of azole resistance in Aspergillus fumigatus and spread of a single resistance mechanism. Plos Med. 2009;5:e 219.

    Google Scholar 

  19. van der Linden J, Jansen R, Bresters D, et al. Azole-resistant central nervous system aspergillosis. Clin Infect Dis. 2009;48:1111–3.

    Article  PubMed  Google Scholar 

  20. Hodiamont C, Dolman K, Ten Berge I, et al. Multiple-azole-resistant Aspergillus fumigatus osteomyelitis in a patient with chronic granulomatous disease successfully treated with long-term oral posaconazole and surgery. Med Mycol. 2009;47:217–20.

    Article  PubMed  CAS  Google Scholar 

  21. Balajee SA, Weaver M, Imhof A, Gribskov J, Marr KA. Aspergillus fumigatus variant with decreased susceptibility to multiple antifungals. Antimicrob Agents Chemother. 2004;48:1197–203.

    Article  PubMed  CAS  Google Scholar 

  22. Pfaller MA, Diekema DJ. Rare and emerging opportunistic fungal pathogens: concern for resistance beyond Candida albicans and Aspergillus fumigatus. J Clin Microbiol. 2004;42:4419–31.

    Article  PubMed  CAS  Google Scholar 

  23. Lass-Florl C. In vitro susceptibility testing in Aspergillus species: an update. Future Microbiol. 2010;5:789–99.

    Article  PubMed  Google Scholar 

  24. Cuenca-Estrella M, Rodriguez-Tudela JL. The current role of the reference procedures by CLSI and EUCAST in the detection of resistance to antifungal agents in vitro. Expert Rev Anti Infect Ther. 2010;8:267–76.

    Article  PubMed  CAS  Google Scholar 

  25. Arenderup M, Perkhofer S, Howard SJ, et al. Establishing in vitro-in vivo correlations for Aspergillus fumigatus: the challenge of azoles versus echinocandins. Antimicrob Agents Chemother. 2008;52:3504–11.

    Article  Google Scholar 

  26. EUCAST DEFINITIVE DOCUMENT E.DEF 9.1. Method for the determination of broth dilution minimum inhibitory concentrations of antifungal agents for conidia forming moulds 2012.

  27. Deutsches Institut für Normung. DIN 58940-81. Medical microbiology-susceptibility testing of microbial pathogens to antimicrobial agents-Part 84. Berlin: DIN, Beth Verlag;2002.

  28. Manavathu EK, Cutright J, Chandrasekar PH. Comparative study of susceptibilities of germinated and ungerminated conidia of Aspergillus fumigatus to various antifungal agents. J Clin Microbiol. 1999;37:858–61.

    PubMed  CAS  Google Scholar 

  29. Bowman JC, Hicks PS, Kurtz MB, et al. The antifungal echinocandin caspofungin acetate kills growing cells of Aspergillus fumigatus in vitro. Antimicrob Agents Chemother. 2002;46:3001–12.

    Article  PubMed  CAS  Google Scholar 

  30. Antachopoulos C, Meletiadis J, Sein T, Roilides E, Walsh TJ. Comparative in vitro pharmacodynamics of caspofungin, micafungin, and anidulafungin against germinated and nongerminated Aspergillus conidia. Antimicrob Agents Chemother. 2008;52:321–8.

    Article  PubMed  CAS  Google Scholar 

  31. Pfaller MA, Messer SA, Boyken L, et al. In vitro survey of triazole cross-resistance among more than 700 clinical isolates of Aspergillus species. J Clin Microbiol. 2008;46:2568–72.

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  33. Rocha EM, Garcia-Effron G, Park S, Perlin DS. A Ser678Pro substitution in Fks1p confers resistance to echinocandin drugs in Aspergillus fumigatus. Antimicrob Agents Chemother. 2007;51:4174–6.

    Article  PubMed  CAS  Google Scholar 

  34. Perlin D. Resistance to echinocandin-class antifungal drugs. Drug Resist Update. 2007;10:121–30.

    Article  CAS  Google Scholar 

  35. Rodriguez-Tudela JL, Alcazar-Fuoli L, Alastruey-Izquiderdo A, et al. Time of incubation for antifungal susceptibility testing of Aspergillus fumigatus: can MIC values be obtained at 24 hours? Antimicrob Agents Chemother. 2007;51:4502–4.

    Article  PubMed  CAS  Google Scholar 

  36. Arendrup MC, Perkhofer S, Howard SJ, et al. Establishing in vitro-in vivo correlations for Aspergillus fumigatus: the challenge of azoles versus echinocandins. Antimicrob Agents Chemother. 2008;52:3504–11.

    Article  PubMed  CAS  Google Scholar 

  37. Pfaller MA, Boyken L, Hollis RJ, et al. Wild-type minimum effective concentration distributions and epidemiologic cutoff values for caspofungin and Aspergillus spp. as determined by clinical and laboratory standards institute broth microdilution methods. Diagn Microbiol Infect Dis. 2010;67:56–60.

    Article  PubMed  CAS  Google Scholar 

  38. Rodriguez-Tudela J, Cazar-Fuoli L, Mellado E, et al. Epidemiological cutoffs and cross-resistance to azole drugs in Aspergillus fumigatus. Antimicrob Agents Chemother. 2008;52:2468–72.

    Article  PubMed  CAS  Google Scholar 

  39. Arendrup MC, Garcia-Effron G, Lass-Florl C, et al. Echinocandin susceptibility testing of Candida species: comparison of EUCAST EDef 7.1, CLSI M27–A3, Etest, disk diffusion, and agar dilution methods with RPMI and isosensitest media. Antimicrob Agents Chemother. 2010;54:426–39.

    Article  PubMed  CAS  Google Scholar 

  40. Arendrup M, Kahlmeter G, Rodriguez-Tudela JL, Donnelly JP. Breakpoints for susceptibility testing should not divide wild-type distributions of important target species. Antimicrob Agents Chemother. 2009;53:1628–9.

    Article  PubMed  CAS  Google Scholar 

  41. Pfaller M, Diekema DJ, Ghannoum A, et al. Wild type MIC distribution and epidemiological cutoff values for Aspergillus fumigatus and three triazoles as determined by the clinical and laboratory standards institute broth microdilution methods. J Clin Microbiol. 2009;47:3142–6.

    Article  PubMed  CAS  Google Scholar 

  42. Kahlmeter G, Brown DF, Goldstein FW, et al. European harmonization of MIC breakpoints for antimicrobial susceptibility testing of bacteria. J Antimicrob Chemother. 2003;52:145–8.

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

Free of charge was provided the antifungal agents anidulafungin, fluconazole, and voriconazole by Pfizer GmbH (Germany), and MSD Sharp & Dohme GmbH (Germany), posaconazole by Essex Pharma GmbH (Germany) and miconazole by Astellas Pharma GmbH (Germany). This work was supported by a grant from Pfizer GmbH (Germany) for purchasing and manufacturing of culture media, ready-to-use microdilution trays with the eight antifungal agents and for transport materials of collected strains. The opinions expressed in this paper are those of the authors and do not necessarily represent those of the pharmaceutical companies.

Author information

Authors and Affiliations

  1. MBS, Munich, Germany

    Arno Schmalreck

  2. Division of Hygiene and Medical Microbiology, Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria

    Birgit Willinger

  3. Dermatology Out-patient Department, Department of Internal Medicine, Helios Clinic Bad Saarow, Bad Saarow, Germany

    Viktor Czaika

  4. Institute of Medical Microbiology, University Hospital Münster, Münster, Germany

    Wolfgang Fegeler & Karsten Becker

  5. Division of Hygiene and Medical Microbiology, Department of Hygiene, Microbiology and Social Medicine, Innsbruck Medical University, Fritz Pregl Str. 3/3, 6020, Innsbruck, Tirol, Austria

    Gerhard Blum & Cornelia Lass-Flörl

Authors
  1. Arno Schmalreck
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Birgit Willinger
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Viktor Czaika
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Wolfgang Fegeler
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Karsten Becker
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Gerhard Blum
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Cornelia Lass-Flörl
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Gerhard Blum.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Schmalreck, A., Willinger, B., Czaika, V. et al. Susceptibility Screening of Hyphae-Forming Fungi with a New, Easy, and Fast Inoculum Preparation Method. Mycopathologia 174, 467–474 (2012). https://doi.org/10.1007/s11046-012-9570-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11046-012-9570-7

Keywords

Search

Navigation