Clinical and Experimental Mycology Research Group (GREMEC), Institut Municipal d'Investigació Mèdica (IMIM), Autonomous University of Barcelona, Avda Dr Aiguader 80, E-08003 Barcelona, Spain
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Abstract |
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Introduction |
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We evaluated the reliability of the results obtained by the Alamar colorimetric method compared with the results generated by the reference microdilution method for 42 clinical isolates of C. neoformans.
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Materials and methods |
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A total of 42 clinical isolates of C. neoformans var. neoformans (serotypes A, D and AD) obtained from AIDS patients during the initial diagnosis of cryptococcal infection were tested. Quality control was ensured by testing Candida albicans ATCC 90028, Candida krusei ATCC 6258, Candida parapsilosis ATCC 22019 and Candida neoformans ATCC 90112 strains. Before testing, all isolates were subcultured on to Sabouraud dextrose agar (bioMérieux, Marcy l'Etoile, France) to ensure optimal growth characteristics. Stock suspensions were prepared in sterile phosphate-buffered saline (PBS) and adjusted to give a final inoculum concentration of 1 x 1065 x 106 cells/mL.
Colorimetric method
The Alamar colorimetric method was performed according to the manufacturer's instructions. Working suspensions were prepared by adding 20 µL of stock yeast suspension to 11 mL of RPMI 1640 broth (American Biorganics, Niagara Falls, NY, USA) buffered to pH 7.0 with 0.165 M morpholinepropanesulphonic acid (MOPS). This resulted in a final inoculum of 1.5 x 1038 x 103 cells/mL. The final concentrations of the antifungal agents were 0.044 mg/L of amphotericin B, 0.12256 mg/L of fluconazole, 0.0464 mg/L of 5-flucytosine and 0.00816 mg/L of itraconazole and ketoconazole. The wells were reconstituted by the addition of 100 µL of the inoculum suspension. After incubation at 35°C for 48 h for Candida spp. and 72 h for C. neoformans, MICs were determined by observing the lowest antifungal concentration preventing the development of a red colour (first blue well).
Reference microbroth method
The microdilution method was performed according to the recommendations of M27-A7 using RPMI 1640 medium and 2% glucose in MOPS buffered to pH 7. Dimethyl sulphoxide (DMSO) was used as solvent for the antifungals, the stock solutions were prepared at 100 x the highest concentration to be tested. The final concentration was prepared from the antifungal stock solution in RPMI plus 2% glucose. The antifungal agents were dispensed in sterile, individually wrapped polystyrene round bottom assay plates. The stock solutions of antifungal agents were dispersed in the assay medium to obtain appropriate concentrations in wells 110 in each row; drug-free medium was dispensed in wells 11 and 12. Well 12 served as sterility control and well 11 as growth control. The antifungal concentrations were 0.0316 mg/L of amphotericin B (Squibb, Princeton, NJ, USA), ketoconazole and itraconazole (Janssen Biotech, Beerse, Belgium), 0.12564 mg/L of fluconazole (Pfizer Inc, New York, USA) and 5-flucytosine (La-Roche Laboratory Inc., Nutley, NJ, USA). The yeast inoculum was adjusted to 0.5 McFarland standard. A working suspension was made at 1:100 dilution followed by a 1:20 dilution of a stock suspension with RPMI 1640 plus 2% glucose.
The inoculated plates were incubated for 72 h at 35°C and readings were taken daily. Absorbance was determined spectrophotometrically at 420 nm after agitation of the plates. The MIC endpoint was defined as the lowest drug concentration exhibiting approximately 80% (or more) reduction of growth compared with the control well. For amphotericin B the MIC was defined as the lowest concentration giving 100% inhibition (optically clear).
Analysis of results
Discrepancies between MIC endpoints of no more than two dilutions were used to calculate the percentage agreement between the Alamar colorimetric method and the NCCLS reference microdilution method. The Student's t test for paired data was used for statistical analysis. Statistical significance was set at P < 0.05. The SPSS computer program was used for analysis of data.
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Results |
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Discussion |
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Testing of the susceptibility of C. neoformans to different drugs has been extensively studied with a variety of culture media, although the number of published reports of clinical resistance to amphotericin B in C. neoformans is surprisingly low. Lozano-Chiu et al.6 have shown that both substitution of RPMI 1640 for antibiotic medium 3 in the microdilution variant of the M27-A method and use of the Etest agar diffusion methodology permits detection of amphotericin B-resistant Candida isolates. Only antibiotic medium 3, however, permitted consistent detection of amphotericin B-resistant C. neoformans. Because RPMI 1640 is the medium used in the colorimetric method, this may be the reason for the low number of resistant isolates found in the present study. Although all isolates were susceptible to amphotericin B, MIC ranges were lower for the Alamar test than for the reference microdilution method in which the RPMI 1640 medium was also used. Similar findings were obtained with 5-flucytosine, MICs varying between 0.06 and 4 mg/L for the Alamar test and between 0.5 and 16 mg/L for the reference method (33% of isolates showed MIC values 8 mg/L). These differences were even greater for MIC90.
With amphotericin and itraconazole the distribution of MICs for both methods was largely different. It should be noted, however, that no discrepancies in resistance or sensitivity of C. neoformans strains to these two antifungal agents were observed. In contrast, MIC values of fluconazole and 5-flucytosine obtained with the Alamar test and the reference method were quite similar, but with the important drawback that some strains apparently sensitive to fluconazole and 5-flucytosine with the Alamar test were resistant to the agents when tested with the reference method.
If the MIC breakpoints for fluconazole resistance in C. albicans mucosal infections (64 mg/L)14 are applied, the number of C. neoformans strains classified as resistant is low. Davey et al.15 have reported an incidence of 5.6%, so that it may be expected that the 2.3% found in the present study with the reference method is more close to the actual situation than the total absence of resistance observed with the colorimetric micromethod.
The degree of agreement of the two methods in terms of absolute values is very low when concentrations are identical (924%), increasing to 2456% when there is a difference of one dilution, to more than 70% for two dilutions and between 92.5100% for three dilutions. Therefore, when different methods are used, identical MIC values should not be expected; what is necessary is that changes in absolute values do not cause a change in scoring of an isolate as resistant or susceptible, and vice versa, as has occurred, especially with fluconazole.
In summary, the Alamar colorimetric method is a useful tool in the study of MICs of the five systemic antifungal agents available for systemic treatment of fungal infections. With C. neoformans however, the distribution of MICs of fluconazole and 5-flucytosine does not provide sufficiently concordant results in a low but not negligible number of isolates.
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Notes |
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References |
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Received 21 June 1999; returned 7 November 1999; revised 25 November 1999; accepted 30 November 1999