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
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Abstract |
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Introduction |
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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
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Materials and methods |
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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 1280.06 mg/L for fluconazole and 320.01 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
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Results |
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Discussion |
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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.
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Notes |
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References |
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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, 108994.[ISI][Medline]
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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, 12802.[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, 21720.[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