Skip to main content
PMC home page
Antimicrobial Agents and Chemotherapy logoLink to Antimicrobial Agents and Chemotherapy
. 1996 Sep;40(9):1998–2003. doi: 10.1128/aac.40.9.1998

Comparison of a spectrophotometric microdilution method with RPMI-2% glucose with the National Committee for Clinical Laboratory Standards reference macrodilution method M27-P for in vitro susceptibility testing of amphotericin B, flucytosine, and fluconazole against Candida albicans.

J L Rodríguez-Tudela 1, J Berenguer 1, J V Martínez-Suárez 1, R Sanchez 1
PMCID: PMC163462  PMID: 8878570

Abstract

The National Committee for Clinical Laboratory Standards has proposed a reference broth macrodilution method for in vitro antifungal susceptibility testing of yeasts (the M27-P method). This method is cumbersome and time-consuming and includes MIC endpoint determination by visual and subjective inspection of growth inhibition after 48 h of incubation. An alternative microdilution procedure was compared with the M27-P method for determination of the amphotericin B, flucytosine, and fluconazole susceptibilities of 8 American Type Culture Collection strains (6 of them were quality control or reference strains) and 50 clinical isolates of candida albicans. This microdilution method uses as culture medium RPMI 1640 supplemented with 18 g of glucose per liter (RPMI-2% glucose). Preparation of drugs, basal medium, and inocula was done by following the recommendations of the National Committee for Clinical Laboratory Standards. The MIC endpoint was calculated objectively from the turbidimetric data read at 24 h. Increased growth of C. albicans in RPMI-2% glucose and its spectrophotometric reading allowed for the rapid (24 h) and objective calculation of MIC endpoints compared with previous microdilution methods with standard RPMI 1640. Nevertheless, good agreement was shown between the M27-P method and this microdilution test. The MICs obtained for the quality control or reference strains by the microdilution method were in the ranges published for those strains. For clinical isolates, the percentages of agreement were 100% for amphotericin B and fluconazole and 98.1% for flucytosine. These data suggest that this microdilution method may serve as a less subjective and more rapid alternative to the M27-P method for antifungal susceptibility testing of yeasts.

Full Text

The Full Text of this article is available as a PDF (185.2 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Anaissie E. J., Bodey G. P., Rinaldi M. G. Emerging fungal pathogens. Eur J Clin Microbiol Infect Dis. 1989 Apr;8(4):323–330. doi: 10.1007/BF01963467. [DOI] [PubMed] [Google Scholar]
  2. Anaissie E., Paetznick V., Bodey G. P. Fluconazole susceptibility testing of Candida albicans: microtiter method that is independent of inoculum size, temperature, and time of reading. Antimicrob Agents Chemother. 1991 Aug;35(8):1641–1646. doi: 10.1128/aac.35.8.1641. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Banerjee S. N., Emori T. G., Culver D. H., Gaynes R. P., Jarvis W. R., Horan T., Edwards J. R., Tolson J., Henderson T., Martone W. J. Secular trends in nosocomial primary bloodstream infections in the United States, 1980-1989. National Nosocomial Infections Surveillance System. Am J Med. 1991 Sep 16;91(3B):86S–89S. doi: 10.1016/0002-9343(91)90349-3. [DOI] [PubMed] [Google Scholar]
  4. Beck-Sagué C., Jarvis W. R. Secular trends in the epidemiology of nosocomial fungal infections in the United States, 1980-1990. National Nosocomial Infections Surveillance System. J Infect Dis. 1993 May;167(5):1247–1251. doi: 10.1093/infdis/167.5.1247. [DOI] [PubMed] [Google Scholar]
  5. Espinel-Ingroff A., Kerkering T. M., Goldson P. R., Shadomy S. Comparison study of broth macrodilution and microdilution antifungal susceptibility tests. J Clin Microbiol. 1991 Jun;29(6):1089–1094. doi: 10.1128/jcm.29.6.1089-1094.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Espinel-Ingroff A., Kish C. W., Jr, Kerkering T. M., Fromtling R. A., Bartizal K., Galgiani J. N., Villareal K., Pfaller M. A., Gerarden T., Rinaldi M. G. Collaborative comparison of broth macrodilution and microdilution antifungal susceptibility tests. J Clin Microbiol. 1992 Dec;30(12):3138–3145. doi: 10.1128/jcm.30.12.3138-3145.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Espinel-Ingroff A., Rodríguez-Tudela J. L., Martínez-Suárez J. V. Comparison of two alternative microdilution procedures with the National Committee for Clinical Laboratory Standards reference macrodilution method M27-P for in vitro testing of fluconazole-resistant and -susceptible isolates of Candida albicans. J Clin Microbiol. 1995 Dec;33(12):3154–3158. doi: 10.1128/jcm.33.12.3154-3158.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Galgiani J. N., Stevens D. A. Antimicrobial susceptibility testing of yeasts: a turbidimetric technique independent of inoculum size. Antimicrob Agents Chemother. 1976 Oct;10(4):721–728. doi: 10.1128/aac.10.4.721. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Martinez-Suarez J. V., Rodriguez-Tudela J. L. Patterns of in vitro activity of itraconazole and imidazole antifungal agents against Candida albicans with decreased susceptibility to fluconazole from Spain. Antimicrob Agents Chemother. 1995 Jul;39(7):1512–1516. doi: 10.1128/aac.39.7.1512. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Odds F. C., Vranckx L., Woestenborghs F. Antifungal susceptibility testing of yeasts: evaluation of technical variables for test automation. Antimicrob Agents Chemother. 1995 Sep;39(9):2051–2060. doi: 10.1128/aac.39.9.2051. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Pfaller M. A., Bale M., Buschelman B., Lancaster M., Espinel-Ingroff A., Rex J. H., Rinaldi M. G., Cooper C. R., McGinnis M. R. Quality control guidelines for National Committee for Clinical Laboratory Standards recommended broth macrodilution testing of amphotericin B, fluconazole, and flucytosine. J Clin Microbiol. 1995 May;33(5):1104–1107. doi: 10.1128/jcm.33.5.1104-1107.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Pfaller M. A., 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. doi: 10.1128/jcm.32.2.506-509.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Pfaller M. A., Messer S. A., Coffmann S. Comparison of visual and spectrophotometric methods of MIC endpoint determinations by using broth microdilution methods to test five antifungal agents, including the new triazole D0870. J Clin Microbiol. 1995 May;33(5):1094–1097. doi: 10.1128/jcm.33.5.1094-1097.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Polanco A. M., Rodriguez-Tudela J. L., Baquero F., Sanchez-Sousa A., Martinez-Suarez J. V. Improved method of determining the susceptibility of Candida albicans to fluconazole. J Antimicrob Chemother. 1995 Jan;35(1):155–159. doi: 10.1093/jac/35.1.155. [DOI] [PubMed] [Google Scholar]
  15. Rex J. H., Pfaller M. A., Rinaldi M. G., Polak A., Galgiani J. N. Antifungal susceptibility testing. Clin Microbiol Rev. 1993 Oct;6(4):367–381. doi: 10.1128/cmr.6.4.367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Rodriguez-Tudela J. L., Martinez-Suarez J. V. Improved medium for fluconazole susceptibility testing of Candida albicans. Antimicrob Agents Chemother. 1994 Jan;38(1):45–48. doi: 10.1128/aac.38.1.45. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Rodriguez-Tudela J. L., Martinez-Suárez J. V. Defining conditions for microbroth antifungal susceptibility tests: influence of RPMI and RPMI-2% glucose on the selection of endpoint criteria. J Antimicrob Chemother. 1995 Jun;35(6):739–749. doi: 10.1093/jac/35.6.739. [DOI] [PubMed] [Google Scholar]
  18. Rodríguez-Tudela J. L., Martínez-Suárez J. V., Dronda F., Laguna F., Chaves F., Valencia E. Correlation of in-vitro susceptibility test results with clinical response: a study of azole therapy in AIDS patients. J Antimicrob Chemother. 1995 Jun;35(6):793–804. doi: 10.1093/jac/35.6.793. [DOI] [PubMed] [Google Scholar]
  19. Rodríguez-Tudela J. L., Martínez-Suárez J. V. Pruebas de sensibilidad a los antifúngicos para levaduras a partir del estándar propuesto por los NCCLS en 1992. Enferm Infecc Microbiol Clin. 1995 Nov;13(9):559–568. [PubMed] [Google Scholar]
  20. Ryley J. F., Wilson R. G., Barrett-Bee K. J. Azole resistance in Candida albicans. Sabouraudia. 1984;22(1):53–63. [PubMed] [Google Scholar]

Articles from Antimicrobial Agents and Chemotherapy are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES