, Volume 174, Issue 5–6, pp 467–474 | Cite as

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

  • Arno Schmalreck
  • Birgit Willinger
  • Viktor Czaika
  • Wolfgang Fegeler
  • Karsten Becker
  • Gerhard Blum
  • Cornelia Lass-Flörl


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.


Susceptibility testing New inoculum preparation method MIC distribution Multicentre study Hyphomycetes 



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.


  1. 1.
    Richardson MD. Changing patterns and trends in systemic fungal infections. J Antimicrob Chemother. 2005;56:5–11.CrossRefGoogle Scholar
  2. 2.
    Wingard JR. The changing face of invasive fungal infections in hematopoietic cell transplant recipients. Curr Opin Oncol. 2005;17:89–92.PubMedCrossRefGoogle Scholar
  3. 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.CrossRefGoogle Scholar
  4. 4.
    Kanafani Z, Perfect J. Resistance to antifungal agents: mechanisms and clinical impact. Clin Infect Dis. 2008;46:120–8.PubMedCrossRefGoogle Scholar
  5. 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.PubMedCrossRefGoogle Scholar
  6. 6.
    Denning DW, Venkateswarlu K, Oakley KL, et al. Itraconazole resistance in Aspergillus fumigatus. Antimicrob Agents Chemother. 1997;41:1364–8.PubMedGoogle Scholar
  7. 7.
    Howard S, Webster I, Moore BC, et al. Multi-azole resistance in Aspergillus fumigatus. Int J Antimicrob Agents. 2006;28:450–3.PubMedCrossRefGoogle Scholar
  8. 8.
    Howard SJ, Arendrup MC. Acquired antifungal drug resistance in Aspergillus fumigatus: epidemiology and detection. Med Mycol 2010.Google Scholar
  9. 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.PubMedCrossRefGoogle Scholar
  10. 10.
    Lass- Florl C, Perkhofer S. In vitro susceptibility testing in Aspergillus species. Mycoses. 2008;51:437–46.PubMedCrossRefGoogle Scholar
  11. 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.PubMedCrossRefGoogle Scholar
  12. 12.
    Verweij PE, Mellado E, Melchers WJG. Multiple-triazole-resistant aspergillosis. N Engl J Med. 2007;356:1481–3.PubMedCrossRefGoogle Scholar
  13. 13.
    Warris A, Weemaes C, Verweij PE. Multidrug resistance in Aspergillus fumigatus. N Engl J Med. 2002;347:2173–4.PubMedCrossRefGoogle Scholar
  14. 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.PubMedCrossRefGoogle Scholar
  15. 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.PubMedCrossRefGoogle Scholar
  16. 16.
    Dannaoui E, Borel E, Monier M, et al. Acquired itraconazole resistance in Aspergillus fumigatus. J Antimicrob Chemother. 2001;47:333–40.PubMedCrossRefGoogle Scholar
  17. 17.
    van Leer-Buter C, Takes RP, Hebeda KM, Melchers WJ, Verweij PE. Aspergillosis—and a misleading sensitivity result. Lancet. 2007;370:102.PubMedCrossRefGoogle Scholar
  18. 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. 19.
    van der Linden J, Jansen R, Bresters D, et al. Azole-resistant central nervous system aspergillosis. Clin Infect Dis. 2009;48:1111–3.PubMedCrossRefGoogle Scholar
  20. 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.PubMedCrossRefGoogle Scholar
  21. 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.PubMedCrossRefGoogle Scholar
  22. 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.PubMedCrossRefGoogle Scholar
  23. 23.
    Lass-Florl C. In vitro susceptibility testing in Aspergillus species: an update. Future Microbiol. 2010;5:789–99.PubMedCrossRefGoogle Scholar
  24. 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.PubMedCrossRefGoogle Scholar
  25. 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.CrossRefGoogle Scholar
  26. 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.Google Scholar
  27. 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.Google Scholar
  28. 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.PubMedGoogle Scholar
  29. 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.PubMedCrossRefGoogle Scholar
  30. 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.PubMedCrossRefGoogle Scholar
  31. 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.PubMedCrossRefGoogle Scholar
  32. 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.PubMedCrossRefGoogle Scholar
  33. 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.PubMedCrossRefGoogle Scholar
  34. 34.
    Perlin D. Resistance to echinocandin-class antifungal drugs. Drug Resist Update. 2007;10:121–30.CrossRefGoogle Scholar
  35. 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.PubMedCrossRefGoogle Scholar
  36. 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.PubMedCrossRefGoogle Scholar
  37. 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.PubMedCrossRefGoogle Scholar
  38. 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.PubMedCrossRefGoogle Scholar
  39. 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.PubMedCrossRefGoogle Scholar
  40. 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.PubMedCrossRefGoogle Scholar
  41. 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.PubMedCrossRefGoogle Scholar
  42. 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.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  • Arno Schmalreck
    • 1
  • Birgit Willinger
    • 2
  • Viktor Czaika
    • 3
  • Wolfgang Fegeler
    • 4
  • Karsten Becker
    • 4
  • Gerhard Blum
    • 5
  • Cornelia Lass-Flörl
    • 5
  1. 1.MBSMunichGermany
  2. 2.Division of Hygiene and Medical Microbiology, Department of Laboratory MedicineMedical University of ViennaViennaAustria
  3. 3.Dermatology Out-patient Department, Department of Internal MedicineHelios Clinic Bad SaarowBad SaarowGermany
  4. 4.Institute of Medical MicrobiologyUniversity Hospital MünsterMünsterGermany
  5. 5.Division of Hygiene and Medical Microbiology, Department of Hygiene, Microbiology and Social MedicineInnsbruck Medical UniversityInnsbruckAustria

Personalised recommendations