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J Clin Microbiol. Mar 1995; 33(3): 661–667.
PMCID: PMC228010

Susceptibility testing of Candida albicans and Aspergillus species by a simple microtiter menadione-augmented 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay.

Abstract

We describe a simple microtiter method for determining the susceptibility of Candida albicans and hyphal forms of Aspergillus fumigatus against antifungal agents. The assay measures mitochondrial respiration by determining reduction of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) to formazan, a process that is enhanced in the presence of menadione. C. albicans or conidial suspensions of A. fumigatus are seeded into microtiter plates. Hyphal outgrowth of Aspergillus spp. was achieved by a 12 to 14-h culture at 30 degrees C. Antifungal agents (amphotericin B, fluconazole, itraconazole) were added to the cultures for 24 h. Thereafter, incubations were continued for 3 h in the presence of MTT plus 0.1 mM menadione. Formazan formation was quantified photometrically after extraction of the formazan with acid isopropanol. Well-defined dose-response curves reflecting impairment of mitochondrial function by the antifungal agents were obtained. With C. albicans, the results correlated excellently with the MIC determinations performed according to the standard macrodilution procedure. In confirmation of a recent report, it was found that fluconazole was unable to exert its fungistatic action on a sensitive C. albicans strain in the presence of serum. The presented method can easily be integrated in the standard repertoire of a diagnostic microbiology laboratory and should prove useful as a means to assess the antifungal action of various agents on yeasts and filamentous fungi in the presence and absence of serum proteins or body fluids.

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Selected References

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  • Baker JH, Goodpasture HC, Kuhns HR, Jr, Rinaldi MG. Fungemia caused by an amphotericin B-resistant isolate of Sporothrix schenckii. Successful treatment with itraconazole. Arch Pathol Lab Med. 1989 Nov;113(11):1279–1281. [PubMed]
  • Boken DJ, Swindells S, Rinaldi MG. Fluconazole-resistant Candida albicans. Clin Infect Dis. 1993 Dec;17(6):1018–1021. [PubMed]
  • Büchner T, Roos N. Antifungal treatment strategy in leukemia patients. Ann Hematol. 1992 Oct;65(4):153–161. [PubMed]
  • Cameron ML, Schell WA, Bruch S, Bartlett JA, Waskin HA, Perfect JR. Correlation of in vitro fluconazole resistance of Candida isolates in relation to therapy and symptoms of individuals seropositive for human immunodeficiency virus type 1. Antimicrob Agents Chemother. 1993 Nov;37(11):2449–2453. [PMC free article] [PubMed]
  • Conly J, Rennie R, Johnson J, Farah S, Hellman L. Disseminated candidiasis due to amphotericin B-resistant Candida albicans. J Infect Dis. 1992 Apr;165(4):761–764. [PubMed]
  • Dan M, Priel I. Failure of fluconazole therapy for sternal osteomyelitis due to Candida albicans. Clin Infect Dis. 1994 Jan;18(1):126–127. [PubMed]
  • Denning DW, Hanson LH, Perlman AM, Stevens DA. In vitro susceptibility and synergy studies of Aspergillus species to conventional and new agents. Diagn Microbiol Infect Dis. 1992 Jan;15(1):21–34. [PubMed]
  • Dermoumi H. In vitro susceptibility of fungal isolates of clinically important specimens to itraconazole, fluconazole and amphotericin B. Chemotherapy. 1994 Mar-Apr;40(2):92–98. [PubMed]
  • Espinel-Ingroff A, Kerkering TM. Spectrophotometric method of inoculum preparation for the in vitro susceptibility testing of filamentous fungi. J Clin Microbiol. 1991 Feb;29(2):393–394. [PMC free article] [PubMed]
  • Fraser IS, Denning DW. Empiric amphotericin B therapy: the need for a reappraisal. Blood Rev. 1993 Dec;7(4):208–214. [PubMed]
  • Fromtling RA, Galgiani JN, Pfaller MA, Espinel-Ingroff A, Bartizal KF, Bartlett MS, Body BA, Frey C, Hall G, Roberts GD, et al. Multicenter evaluation of a broth macrodilution antifungal susceptibility test for yeasts. Antimicrob Agents Chemother. 1993 Jan;37(1):39–45. [PMC free article] [PubMed]
  • Galgiani JN. Susceptibility testing of fungi: current status of the standardization process. Antimicrob Agents Chemother. 1993 Dec;37(12):2517–2521. [PMC free article] [PubMed]
  • Garn H, Krause H, Enzmann V, Drössler K. An improved MTT assay using the electron-coupling agent menadione. J Immunol Methods. 1994 Feb 10;168(2):253–256. [PubMed]
  • Gokhale PC, Barapatre RJ, Advani SH, Kshirsagar NA, Pandya SK. Pharmacokinetics and tolerance of liposomal amphotericin B in patients. J Antimicrob Chemother. 1993 Jul;32(1):133–139. [PubMed]
  • Goldberg SL, Geha DJ, Marshall WF, Inwards DJ, Hoagland HC. Successful treatment of simultaneous pulmonary Pseudallescheria boydii and Aspergillus terreus infection with oral itraconazole. Clin Infect Dis. 1993 Jun;16(6):803–805. [PubMed]
  • Graybill JR. Future directions of antifungal chemotherapy. Clin Infect Dis. 1992 Mar;14 (Suppl 1):S170–S181. [PubMed]
  • Hahn YH, Ahearn DG, Wilson LA. Comparative efficacy of amphotericin B, clotrimazole and itraconazole against Aspergillus spp. An in vitro study. Mycopathologia. 1993 Sep;123(3):135–140. [PubMed]
  • Kujath P, Lerch K, Kochendörfer P, Boos C. Comparative study of the efficacy of fluconazole versus amphotericin B/flucytosine in surgical patients with systemic mycoses. Infection. 1993 Nov-Dec;21(6):376–382. [PubMed]
  • Levitz SM, Diamond RD. A rapid colorimetric assay of fungal viability with the tetrazolium salt MTT. J Infect Dis. 1985 Nov;152(5):938–945. [PubMed]
  • Martin E, Maier F, Bhakdi S. Antagonistic effects of fluconazole and 5-fluorocytosine on candidacidal action of amphotericin B in human serum. Antimicrob Agents Chemother. 1994 Jun;38(6):1331–1338. [PMC free article] [PubMed]
  • Martin E, Schlasius U, Bhakdi S. Flow cytometric assay for estimating fungicidal activity of amphotericin B in human serum. Med Microbiol Immunol. 1992;181(3):117–126. [PubMed]
  • Martin E, Stüben A, Görz A, Weller U, Bhakdi S. Novel aspect of amphotericin B action: accumulation in human monocytes potentiates killing of phagocytosed Candida albicans. Antimicrob Agents Chemother. 1994 Jan;38(1):13–22. [PMC free article] [PubMed]
  • Meyer RD. Current role of therapy with amphotericin B. Clin Infect Dis. 1992 Mar;14 (Suppl 1):S154–S160. [PubMed]
  • Mills W, Chopra R, Linch DC, Goldstone AH. Liposomal amphotericin B in the treatment of fungal infections in neutropenic patients: a single-centre experience of 133 episodes in 116 patients. Br J Haematol. 1994 Apr;86(4):754–760. [PubMed]
  • Moreau P, Milpied N, Fayette N, Ramée JF, Harousseau JL. Reduced renal toxicity and improved clinical tolerance of amphotericin B mixed with intralipid compared with conventional amphotericin B in neutropenic patients. J Antimicrob Chemother. 1992 Oct;30(4):535–541. [PubMed]
  • Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983 Dec 16;65(1-2):55–63. [PubMed]
  • Musial CE, Cockerill FR, 3rd, Roberts GD. Fungal infections of the immunocompromised host: clinical and laboratory aspects. Clin Microbiol Rev. 1988 Oct;1(4):349–364. [PMC free article] [PubMed]
  • Pfaller MA, Barry AL. Evaluation of a novel colorimetric broth microdilution method for antifungal susceptibility testing of yeast isolates. J Clin Microbiol. 1994 Aug;32(8):1992–1996. [PMC free article] [PubMed]
  • Pfaller MA, Dupont B, Kobayashi GS, Müller J, Rinaldi MG, Espinel-Ingroff A, Shadomy S, Troke PF, Walsh TJ, Warnock DW. Standardized susceptibility testing of fluconazole: an international collaborative study. Antimicrob Agents Chemother. 1992 Sep;36(9):1805–1809. [PMC free article] [PubMed]
  • Pfaller MA, Grant C, Morthland V, Rhine-Chalberg J. Comparative evaluation of alternative methods for broth dilution susceptibility testing of fluconazole against Candida albicans. J Clin Microbiol. 1994 Feb;32(2):506–509. [PMC free article] [PubMed]
  • Pfaller MA, Rinaldi MG. Antifungal susceptibility testing. Current state of technology, limitations, and standardization. Infect Dis Clin North Am. 1993 Jun;7(2):435–444. [PubMed]
  • Powderly WG, Kobayashi GS, Herzig GP, Medoff G. Amphotericin B-resistant yeast infection in severely immunocompromised patients. Am J Med. 1988 May;84(5):826–832. [PubMed]
  • Ralph ED, Khazindar AM, Barber KR, Grant CW. Comparative in vitro effects of liposomal amphotericin B, amphotericin B-deoxycholate, and free amphotericin B against fungal strains determined by using MIC and minimal lethal concentration susceptibility studies and time-kill curves. Antimicrob Agents Chemother. 1991 Jan;35(1):188–191. [PMC free article] [PubMed]
  • Redding S, Smith J, Farinacci G, Rinaldi M, Fothergill A, Rhine-Chalberg J, Pfaller M. Resistance of Candida albicans to fluconazole during treatment of oropharyngeal candidiasis in a patient with AIDS: documentation by in vitro susceptibility testing and DNA subtype analysis. Clin Infect Dis. 1994 Feb;18(2):240–242. [PubMed]
  • Rex JH, Bennett JE, Gallin JI, Malech HL, DeCarlo ES, Melnick DA. In vivo interferon-gamma therapy augments the in vitro ability of chronic granulomatous disease neutrophils to damage Aspergillus hyphae. J Infect Dis. 1991 Apr;163(4):849–852. [PubMed]
  • Rex JH, Bennett JE, Gallin JI, Malech HL, Melnick DA. Normal and deficient neutrophils can cooperate to damage Aspergillus fumigatus hyphae. J Infect Dis. 1990 Aug;162(2):523–528. [PubMed]
  • Rex JH, Pfaller MA, Rinaldi MG, Polak A, Galgiani JN. Antifungal susceptibility testing. Clin Microbiol Rev. 1993 Oct;6(4):367–381. [PMC free article] [PubMed]
  • Roilides E, Uhlig K, Venzon D, Pizzo PA, Walsh TJ. Enhancement of oxidative response and damage caused by human neutrophils to Aspergillus fumigatus hyphae by granulocyte colony-stimulating factor and gamma interferon. Infect Immun. 1993 Apr;61(4):1185–1193. [PMC free article] [PubMed]
  • Roilides E, Uhlig K, Venzon D, Pizzo PA, Walsh TJ. Prevention of corticosteroid-induced suppression of human polymorphonuclear leukocyte-induced damage of Aspergillus fumigatus hyphae by granulocyte colony-stimulating factor and gamma interferon. Infect Immun. 1993 Nov;61(11):4870–4877. [PMC free article] [PubMed]
  • Sekhon AS, Padhye AA, Garg AK, Hamir Z. Effects of culture media on the in vitro susceptibility of selected opportunistic fungi to fluconazole and itraconazole. Chemotherapy. 1992;38(3):169–173. [PubMed]
  • Swerdloff JN, Filler SG, Edwards JE., Jr Severe candidal infections in neutropenic patients. Clin Infect Dis. 1993 Nov;17 (Suppl 2):S457–S467. [PubMed]
  • Tellier R, Krajden M, Grigoriew GA, Campbell I. Innovative endpoint determination system for antifungal susceptibility testing of yeasts. Antimicrob Agents Chemother. 1992 Aug;36(8):1619–1625. [PMC free article] [PubMed]
  • Tollemar J, Andersson S, Ringdén O, Tydén G. A retrospective clinical comparison between antifungal treatment with liposomal amphotericin B (AmBisome) and conventional amphotericin B in transplant recipients. Mycoses. 1992 Sep-Oct;35(9-10):215–220. [PubMed]
  • Tollemar J, Ringdén O, Andersson S, Sundberg B, Ljungman P, Tydén G. Randomized double-blind study of liposomal amphotericin B (Ambisome) prophylaxis of invasive fungal infections in bone marrow transplant recipients. Bone Marrow Transplant. 1993 Dec;12(6):577–582. [PubMed]
  • vanden Bossche H, Marichal P, Odds FC, Le Jeune L, Coene MC. Characterization of an azole-resistant Candida glabrata isolate. Antimicrob Agents Chemother. 1992 Dec;36(12):2602–2610. [PMC free article] [PubMed]
  • Yamada H, Tsuda T, Watanabe T, Ohashi M, Murakami K, Mochizuki H. In vitro and in vivo antifungal activities of D0870, a new triazole agent. Antimicrob Agents Chemother. 1993 Nov;37(11):2412–2417. [PMC free article] [PubMed]

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