, Volume 179, Issue 5–6, pp 359–371 | Cite as

Comparison of Different In Vitro Tests to Detect Cryptococcus neoformans Not Susceptible to Amphotericin B

  • Susana Córdoba
  • Walter Vivot
  • Wanda Szusz
  • Guillermina Isla
  • Graciela Davel


Infections due to Cryptococcus neoformans cause severe disease, mostly in AIDS patients. The antifungal drug recommended for the initial treatment of these infections is amphotericin B with or without flucytosine, but treatment failure occurs, associated with high mortality. Thus, antifungal susceptibility testing is needed. However, the in vitro susceptibility tests available for C. neoformans are not useful to detect isolates that are not susceptible to antifungal agents such as amphotericin B. The aims of the present study were: (1) to determine and compare the in vitro activity of amphotericin B against C. neoformans clinical isolates by using different dilution and diffusion methods; (2) to evaluate the concordance among the methods used and the reference method; (3) to evaluate which method could be the best to correlate with the clinical outcome. The reference method EDef 7.2 from the European Committee on Antimicrobial Susceptibility Testing and commercial Etest strips were used to determine the minimal inhibitory concentration against amphotericin B. curves, minimal fungicidal concentration, and a disk diffusion method were also developed to evaluate the cidal activity of amphotericin B. The time–kill curve assay showed correlation (p < 0.05) with clinical outcome, whereas EDef 7.2, minimal fungicidal concentration, Etest, and disk diffusion showed no correlation (p > 0.05). Thus, the time–kill curve assay could be a potential tool to guide a more efficient treatment when amphotericin B is used.


Cryptococcus neoformans Time–kill curves Amphotericin B Resistance 



We thank Alicia Trovero from the Instituto Nacional de Producción de Biológicos, ANLIS “Dr. C. G Malbrán” (Buenos Aires, Argentina), for her collaboration with serotype determinations.

Conflict of interest


Ethical standard

This study did not need an ethics committee approval because it did not include direct contact, personal information, or clinical samples of patients, just isolates.


  1. 1.
    Negroni R, Helou SH, Lopez Daneri G, Robles AM, Arechavala A, Bianchi MH. Interrupción exitosa de la profilaxis secundaria antifúngica en la criptococosis asociada al SIDA. Rev Argent Microbiol. 2004;36:113–7.PubMedGoogle Scholar
  2. 2.
    Negroni R. Criptococosis. In: Benetucci J, editor. SIDA y enfermedades asociadas. Diagnóstico, clínica y tratamiento. 3rd ed. Buenos Aires: FUNDAI; 2008. p. 332–6.Google Scholar
  3. 3.
    Perfect JR, Dismukes WE, Dromer F, et al. Clinical practice guidelines for the management of cryptococcal disease: 2010 update by the Infectious Diseases Society of America. Clin Infect Dis. 2010;50:291–322.CrossRefPubMedGoogle Scholar
  4. 4.
    Clinical and Laboratory Standards Institute. Reference method for broth dilution antifungal susceptibility testing of yeasts. Approved standard. CLSI document M27-A3. 3rd ed. Wayne: Clinical and Laboratory Standards Institute; 2008. p. 19087–1898.Google Scholar
  5. 5.
    Arendrup MC, Cuenca-Estrella M, Lass-Flörl C, Hope W and the Subcommittee on Antifungal Susceptibility Testing (AFST) of the ESCMID European Committee for Antimicrobial Susceptibility Testing (EUCAST). Eucast Definitive Document EDef 7.2 Revision. Method for the determination of broth dilution minimum Inhibitory concentrations of antifungal agents for yeasts. (2012).
  6. 6.
    Rodríguez-Tudela JL, Martín-Diez F, Cuenca-Estrella M, Rodero L, Carpintero Y, Gorgojo B. Influence of shaking of antifungal susceptibility testing of Cryptococcus neoformans: a comparison of NCCLS Standard M27-A medium, buffered yeast nitrogen base, and RPMI-2 % glucose. Antimicrob Agent Chemoter. 2000;44:400–4.CrossRefGoogle Scholar
  7. 7.
    Zaragoza O, Mesa-Arango AC, Gómez-López A, Bernal-Martínez L, Rodríguez-Tudela JL, Cuenca-Estrella M. Process analysis of variables for standardization of antifungal susceptibility testing of nonfermentative yeasts. Antimicrob Agents Chemother. 2011;55:1563–70.CrossRefPubMedCentralPubMedGoogle Scholar
  8. 8.
    Govender N, Patel J, van Wyk M, Chiller T, Lockhart SR, GERMS-AS. Trends in antifungal drug susceptibility of cryptococcus neoformans isolates obtained through population-based surveillance in South Africa in 2002–2003 and 2007–2008. Antimicrob Agents Chemother. 2011;55:2606–11.CrossRefPubMedCentralPubMedGoogle Scholar
  9. 9.
    Pfaller MA, Castanheira M, Diekema DJ, Messer SA, Jones RN. Wild-type MIC distributions and epidemiologic cutoff values for fluconazole, posaconazole, and voriconazole when testing Cryptococcus neoformans as determined by the CLSI broth microdilution method. Diagn Microbiol Infect Dis. 2011;71:252–9.CrossRefPubMedGoogle Scholar
  10. 10.
    Espinel-Ingroff A, Aller AI, Canton E, et al. Cryptococcus neoformansCryptococcus gattii species complex: an international study of wild-type susceptibility endpoint distributions and epidemiological cutoff values for fluconazole, itraconazole, posaconazole and voriconazole. Antimicrob Agents Chemother. 2012;56:5898–906.CrossRefPubMedCentralPubMedGoogle Scholar
  11. 11.
    Espinel-Ingroff A, Chowdhary A, Cuenca-Estrella M, et al. Cryptococcus neoformansCryptococcus gattii species complex: an international study of wild-type susceptibility endpoint distributions and epidemiological cutoff values for amphotericin B and flucytosine. Antimicrob Agents Chemother. 2012;56:3107–13.CrossRefPubMedCentralPubMedGoogle Scholar
  12. 12.
    Li M, Liao Y, Chen M, Pan W, Weng L. Antifungal susceptibilities of Cryptococcus species complex isolates form AIDS and non-AIDS patients in southeast China. Braz Infect Dis. 2012;16:175–9.CrossRefGoogle Scholar
  13. 13.
    Dannaoui E, Abdul M, Arpin M, et al. Results obtained with various antifungal susceptibility testing methods do not predict early clinical outcome in patients with cryptococcosis. Antimicrob Agents Chemother. 2006;50:2464–70.CrossRefPubMedCentralPubMedGoogle Scholar
  14. 14.
    Arechavala AI, Ochiuzzi ME, Borgnia MD, Santiso GM. Fluconazole and amphotericin B susceptibility testing of Cryptococcus neoformans: results of minimal inhibitory concentrations against 265 isolates from HIV-positive patients before and after two or more months of antifungal therapy. Rev Iberoam Micol. 2009;26:194–7.CrossRefPubMedGoogle Scholar
  15. 15.
    Larsen RA, Bauer M, Pitisuttithum P, et al. Correlation of susceptibility of Cryptococcus neoformans to amphotericin B with clinical outcome. Antimicrob Agents Chemother. 2011;55:5624–30.CrossRefPubMedCentralPubMedGoogle Scholar
  16. 16.
    Lee Ch, Chang TY, Liu JW, et al. Correlation of anti-fungal susceptibility with clinical outcomes in patients with cryptococcal meningitis. BMC Infect Dis. 2012;12:361.CrossRefPubMedCentralPubMedGoogle Scholar
  17. 17.
    Rex JH, Chester R, Cooper JR, et al. Detection of amphotericin B-resistant candida isolates in a broth-based system. Antimicrob Agents Chemother. 1995;39:906–9.CrossRefPubMedCentralPubMedGoogle Scholar
  18. 18.
    Rodero L, Córdoba S, Cahn P, et al. Timed-kill curves for Cryptococcus neoformans isolated from patients with AIDS. Med Mycol. 2000;38:201–7.CrossRefPubMedGoogle Scholar
  19. 19.
    Córdoba S, Afeltra J, Vitale RG. Evaluation of the in vitro activity of amphotericin B by time kill curves methodology against Cryptococcus neoformans encapsulated and capsulate-deficient strains. Diagn Microbiol Infect Dis. 2011;71:260–2.CrossRefPubMedGoogle Scholar
  20. 20.
    National Committee for Clinical Laboratory Standards. Methods for determining bactericidal activity of antimicrobial agents: approved guideline. NCCLS document M26-A. Wayne, 1999.Google Scholar
  21. 21.
    Pfaller MA, Sheehan DJ, Rex JH. Determination of fungicidal activities against yeasts and molds: lessons learned from bactericidal testing and the need for standardization. Clin Microbiol Rev. 2004;17:268–80.CrossRefPubMedCentralPubMedGoogle Scholar
  22. 22.
    Mouton JW, Vinks AA. Pharmacokinetic/pharmacodynamic modelling of antibacterials in vitro and in vivo using bacterial growth and kill kinetics: the minimum inhibitory concentration versus stationary concentration. Clin Pharmacokinet. 2005;44:201–10.CrossRefPubMedGoogle Scholar
  23. 23.
    Klepser MA, Wolfe EJ, Jones RN, Nightingale CH, Pfaller MA. Antifungal pharmacodynamic characteristics of fluconazole and amphotericin B tested against Candida albicans. Antimicrob Agents Chemother. 1997;42:1207–12.Google Scholar
  24. 24.
    Klepser ME, Ernst EJ, Lewis RE, Ernst ME, Pfaller MA. Influence of test conditions on antifungal time-kill curve results: proposal for standardized methods. Antimicrob Agents Chemother. 1998;42:1207–12.PubMedCentralPubMedGoogle Scholar
  25. 25.
    Burgess DS, Hastings RW. A comparison of dynamic characteristics of fluconazole, itraconazole, and amphotericin B against Cryptococcus neoformans using time-kill methodology. Diagn Microbiol Infect Dis. 2000;38:87–93.CrossRefPubMedGoogle Scholar
  26. 26.
    Ernst EJ, Yodoi K, Roling EE, Klepser ME. Rates and extents of antifungal activities of amphotericin B, flucytosine, fluconazole, and voriconazole against Candida lusitaniae determined by microdilution, Etest, and time-kill methods. Antimicrob Agents Chemother. 2002;46:578–81.CrossRefPubMedCentralPubMedGoogle Scholar
  27. 27.
    Sandoval-Denis M, Pastor FJ, Capilla J, Sutton DA, Fothergill AW, Guarro J. In Vitro pharmacodynamics and in vivo efficacy of fluconazole, amphotericin B and caspofungina in a murine infection by Candida lusitaniae. Int J Antimicrob Agents. 2014;43:161–4.CrossRefPubMedGoogle Scholar
  28. 28.
    Pappalardo MCSM, Szeszs MW, Martins MA, et al. Susceptibility of clinical isolates of Cryptococcus neoformans to amphotericin B using time-kill methodology. Diagn Microbiol Infect Dis. 2009;64:146–51.CrossRefPubMedGoogle Scholar
  29. 29.
    Kurtzman CP, Fell JW. The yeast, a taxonomic study. 5th ed. Amsterdam: Elsevier; 2011.Google Scholar
  30. 30.
    Ikeda R, Shinoda T, Fukazawa Y, Kaufman L. Antigenic characterization of Cryptococcus neoformans serotypes and its application to serotyping of clinical isolates. J Clin Microbiol. 1982;16:22–9.PubMedCentralPubMedGoogle Scholar
  31. 31.
    Neo-Sensitabs user’s guide; A/S Rosco Diagnostica, Taastrup, Denmark. 2007.
  32. 32.
    Maxwell MJ, Messer SA, Hollis RJ, Diekema DJ, Pfaller MA. Evaluation of Etest method for determining voriconazole and amphotericin B MICs for 162 clinical isolates of Cryptococcus neoformans. J Clin Microbiol. 2003;41:97–9.CrossRefPubMedCentralPubMedGoogle Scholar
  33. 33.
    Cuenca-Estrella M, Gomez-Lopez A, Alastruey-Izquierdo A, et al. Comparison of the Vitek 2 antifungal susceptibility system with the Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) broth microdilution reference methods and with the Sensititre YeastOne and Etest techniques for in vitro detection of antifungal resistance in yeast isolates. J Clin Microbiol. 2010;48:1782–6.CrossRefPubMedCentralPubMedGoogle Scholar
  34. 34.
    Dias ALT, Matsumoto FE, Melhem MSC, et al. Comparative anlysis of Etest and broth microdilution method (AFST-EUCAST) for trends in antifungal drug susceptibility testing of Brazilian Cryptococcus neoformans isolates. J Med Microbiol. 2006;55:1693–9.CrossRefPubMedGoogle Scholar
  35. 35.
    Sow D, Clement Tine R, Sylla K, et al. Cryptococcal meningitis in Senegal: epidemiology, laboratory findings, therapeutic and outcome of cases diagnosed from 2004 to 2011. Mycopathologia. 2013;176:443–9.CrossRefPubMedGoogle Scholar
  36. 36.
    Meda J, Kalluvya S, Downs JA, et al. Cryptococcal meningitis management in Tanzania with strict schedule of serial lumber punctures using intravenous tubing sets: an operational research study. J Acquir Immune Defic Syndr. 2014;66(2):e31–6. doi: 10.1097/QAI.0000000000000147.PubMedGoogle Scholar
  37. 37.
    Ochiuzzi ME, Santiso GM, Arechavala AI. Correlation of Etest and neo-sensitabs diffusion assays on Mueller–Hinton-methylene blue agar with broth microdilution reference method (CLSI-M27-A2) for testing susceptibilities of Cryptococcus neoformans to amphotericin B and fluconazole. Med Mycol. 2010;48:893–6.CrossRefPubMedGoogle Scholar
  38. 38.
    Dannaoui E, Paugam A, Develoux M, et al. Comparison of antifungal MICs for yeasts obtained using the EUCAST method in a reference laboratory and the Etest in nine different hospital laboratories. Clin Microbiol Infect. 2010;16:863–9.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  • Susana Córdoba
    • 1
  • Walter Vivot
    • 1
  • Wanda Szusz
    • 1
  • Guillermina Isla
    • 1
  • Graciela Davel
    • 1
  1. 1.Departamento MicologiaInstituto Nacional de Enfermedades Infecciosas, “Dr. C. G. Malbrán”Buenos AiresArgentina

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