Susceptibility of Cryptococcus neoformans by the NCCLS microdilution and Etest methods using five defined media

Michael A. Petroua,* and David C. Shansonb

a Department of Infectious Diseases and Microbiology, Imperial College School of Medicine, Hammersmith Campus, Du Cane Road, London W12 0NN; b Department of Microbiology, OMNILABS, 27 Harley Street, London W1N 1DA, UK


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The susceptibility of 12 isolates of Cryptococcus neoformans to amphotericin B, 5-fluorocytosine, fluconazole, itraconazole and ketoconazole was tested using the NCCLS and Etest methods with yeast nitrogen base (YNB) pH 5.6 and pH 7.0, RPMI MOPS pH 7.0 with and without added glucose (2%) and RPMI buffered with phosphate buffer to pH 7.0. Some isolates yielded poor growth in RPMI MOPS after 72 h. Tests indicated that YNB pH 5.6 was the best medium for 5-fluorocytosine but was unsuitable for ketoconazole. In conclusion, YNB pH 7.0 or RPMI MOPS with 2% glucose can be used with either method.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The National Committee for Clinical Laboratory Standards (NCCLS) proposed a macrodilution method, and then approved both a macrodilution and a microdilution method for the in vitro susceptibility testing of yeasts, with RPMI 1640 medium buffered with MOPS to pH 7.0.1 Many studies have compared these methods, whereas others compared both with the Etest, and found good correlation.2–4 One observation from these studies was that the RPMI medium as proposed does not adequately support the growth of many important yeast pathogens and the incubation of Cryptococcus neoformans isolates had to be extended to 72 h. Increasing the glucose concentration to 2% improved the growth of many isolates;5 however, one study highlighted the inadequacy of RPMI for MIC determination of yeasts, and C. neoformans isolates were specifically excluded because of low levels of growth in this medium.6

Undefined media such as casitone agar have been proposed, particularly for amphotericin B, whereas others have used yeast nitrogen base (YNB), either at an acid or neutral pH.7,8 Five media have been recommended for testing five drugs by the Etest manufacturers during recent years. The use of undefined media in a laboratory where 5-fluorocytosine MIC results are reported to clinicians is not desirable, neither is the use of more than one medium per isolate.

This study was designed to evaluate and compare five defined media by the NCCLS and Etest methods, for both nutritional adequacy and time period to MIC interpretation for the fastidious yeast C. neoformans


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Organisms

Ten clinical isolates of C. neoformans var. neoformans from AIDS patients and two isolates from the National Collection of Pathogenic Fungi (NCPF, Bristol, UK), C. neoformans var. neoformans NCPF 3379 and C. neoformans var. gattii NCPF 3216, were used.

Inoculum

All isolates were passaged twice on Sabouraoud dextrose agar (Sanofi, Guildford, UK) before the inoculum was prepared to a 1 McFarland standard in sterile saline from a 48 h culture at 36 ± 1°C.

Drugs

Pure substances of amphotericin and 5-fluorocytosine (Sigma, Gillingham, Dorset, UK), fluconazole (Pfizer, Sandwich, UK), itraconazole and ketoconazole (Janssen, Beerse, Belgium) were prepared as 10 mg/L stock solutions: 5-fluorocytosine was dissolved in sterile distilled water and the other drugs were prepared in dimethylsulphoxide. The Etest strips for all five drugs (Cambridge Diagnostics, Cambridge, UK) contained drug concentrations ranging from 256 to 0.016 mg/L for fluconazole and from 32 to 0.002 mg/L for the other four drugs. All stock solutions and strips were stored at –70°C until use.

Assay media

RPMI 1640 with l-glutamine (Sigma) buffered with MOPS with or without added 2% glucose, RPMI buffered with monobasic phosphate buffer and YNB pH 5.6 and pH 7.0 were prepared as described before9 with the exception that all were prepared at double strength.

Growth rates

These were performed in flat bottom microtitre plates (Sterilin, Stone, UK) in 200 µL volumes with eight cultures per isolate. The media were dispensed in 100 µL volumes and were inoculated with the same inoculum in sterile distilled water to 5 x 104 cfu/mL. All plates were incubated at 36 ± 1°C in a moist chamber. After shaking the plates for 30 s, the optical density (OD) was recorded every 2 h for the first 12 h and at regular time intervals thereafter for 72 h with the aid of a Dynatech MR7000 spectrophotometer at a wavelength of 530 nm. The graphs of log OD against time were plotted for each isolate and the growth rate was calculated from the exponential phase whereas the lag phase was calculated from the time axis.

NCCLS susceptibility testing

This method was performed in microtitre plates with the same volumes and inocula as above with final drug concentration of 128–0.06 mg/L for fluconazole and 32–0.01 mg/L for the other drugs. The plates were incubated at 36 ± 1°C, the ODs were recorded after 24, 48 and 72 h incubation as above and the results were interpreted according to the NCCLS guidelines. Inoculum size, purity and viability were confirmed for each isolate in each medium by spreading 10 µL from one of the drug-free controls on Sabouraud plates. Purity of all wells and the MIC was confirmed by examining the plates with an inverted microscope. All experiments were performed in duplicate.

Etest susceptibility testing

The two-fold concentrated media were added to equal volumes of sterile agar (Technical No. 3, Oxoid, Basingstoke, UK) final agar concentration 1.5%, and were poured into 150 mm round Petri dishes in 60 mL volumes and allowed to set. The plates were inoculated and left to dry completely before the strips were applied, and the results recorded after 24, 48 and 72 h incubation according to the manufacturer's guidelines.9


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The mean ODs, growth rates and lag phases achieved by the isolates tested in the five media are summarized in Table IGo. Comparable growth rates can be seen with both YNB media and RPMI 2% glucose and the MIC results could be interpreted after 48 h.


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Table I. Optical density, relative growth rates and lag phase for the 12 isolates of Cryptococcus neoformans tested
 
The MIC ranges, MIC50s and MIC90s obtained by both methods are summarized in Table IIGo. With the exception of RPMI buffered with phosphate the MICs obtained for fluconazole with each medium tested by the NCCLS method were comparable (± 1 dilution), whereas those obtained with the Etest method were generally higher.


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Table II. In vitro activity of the five antifungal drugs tested in the five media in terms of MIC range, MIC50 and MIC90
 
The MICs obtained for itraconazole for YNB media and RPMI MOPS were in perfect agreement with either method and in RPMI 2% glucose they were within one dilution. For ketoconazole the MICs obtained with YNB pH 5.6 were 4–13 times higher than those with the other four media which were in very good agreement (± 1 dilution) by either method.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
With the exception of amphotericin B, the drugs tested in this study are fungistatic. Fungi will grow for a period in vitro, during which they will exhaust all reserves before a fungistatic drug can achieve full or noticeable inhibition. This results in the production of turbidity, which does not change significantly with prolonged incubation in liquid media and the formation of microcolonies on solid media. In our study the MIC of the antifungals tested did not increase by more than one dilution with prolonged incubation to 72 h. The incubation time in nutritionally poor media, such as RPMI MOPS, had to be extended to 72 h not only to achieve appreciable growth but also to increase the differential between partially inhibited growth and full growth. Interestingly, only the activity of ketoconazole appeared to be adversely affected by acid pH. In agreement with others we report close correlation between the NCCLS and Etest methods.3,4

The use of antifungals at twice the final concentration has been reported previously; if the medium is also prepared at a two-fold concentration there are many advantages, including preparation of smaller volumes, use of identical inoculum preparations in different media and, in cases where the plates are frozen, medium does not need to be stored, thus avoiding the possibility of wastage or mixing of different batches.

The Etest strip manufacturers in their lastest guidelines recommend RPMI MOPS with 2% glucose. Our study supports their recommendations and adds that for the routine MIC testing of C. neoformans the choice between RPMI 2% glucose and YNB pH 7.0 can be left to individual laboratories, as both support growth, neither interferes with drug activity and the MICs can be interpreted within 2 days.


    Notes
 
* Correspondence address. Mycology, Department of Investigative Diseases, Imperial College School of Medicine, Hammersmith Campus, Du Cane Road, London W12 0NN, UK. Tel: + 44-20-8383-5820; Fax: + 44-20-8383-5824; E-mail m.petrou{at}ic.ac.uk Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . National Committee for Clinical Laboratory Standards. (1997). Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts—Approved Standard M27-A. NCCLS, Villanova, PA.

2 . Espinel-Ingroff, A., Kerkering, T. M., Goldson, P. R. & Shadomy, S. (1991). Comparison study of broth macrodilution and microdilution antifungal susceptibility tests. Journal of Clinical Microbiology 29, 1089–94.[ISI][Medline]

3 . Sewell, D. L., Pfaller, M. A. & Barry, A. L. (1994). Comparison of broth macrodilution, broth microdilution, and Etest antifungal susceptibility tests for fluconazole. Journal of Clinical Microbiology 32, 2099–102.[Abstract]

4 . Chen, S. C. A., O'Donnell, M. L., Gordon, S. & Gilbert, G. L. (1996). Antifungal susceptibility testing using the Etest: comparison with the broth macrodilution technique. Journal of Antimicrobial Chemotherapy 37, 265–73.[Abstract]

5 . Rodriguez-Tudela, J. & Martinez-Suárez, J. V. (1995). Defining conditions for microbroth antifungal susceptibility tests: influence of RPMI and RPMI-2% glucose on the selection of endpoint criteria. Journal of Antimicrobial Chemotherapy 35, 739–49.[Abstract]

6 . Odds, F. C., Vranckx, L. & Woestenborghs, F. (1995). Antifungal susceptibility testing of yeasts: evaluation of technical variables for test automation. Antimicrobial Agents and Chemotherapy 39, 2051– 60.

7 . Sanati, H., Messer, S. A., Pfaller, M. A., Witt, M., Larsen, R., Espinel-Ingroff, A. et al. (1996). Multicenter evaluation of broth microdilution method for susceptibility testing of Cryptococcus neoformans against fluconazole. Journal of Clinical Microbiology 34, 1280–2.[Abstract]

8 . Davey, K. G., Johnson, E. M., Holmes, A. D., Szekely, A. & Warnock, D. W. (1998). In-vitro susceptibility of Cryptococcus neoformans isolates to fluconazole and itraconazole. Journal of Antimicrobial Chemotherapy 42, 217–20.[Abstract]

9 . Anonymous. (1994). Etest Technical Guide No. 4b: Antifungal Susceptibility Testing of Yeasts. AB Biodisk, Solna, Sweden.

Received 18 November 1999; returned 25 April 2000; revised 5 June 2000; accepted 17 July 2000