In vitro susceptibility of Cryptococcus neoformans isolates to five antifungal drugs using a colorimetric system and the reference microbroth method

O. López-Jodra, J. M. Torres-Rodríguez*, R. Méndez-Vásquez, E. Ribas-Forcadell, Y. Morera-López, T. Baró-Tomás and C. Alia-Aponte

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


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Minimum inhibitory concentrations (MICs) of amphotericin B, 5-flucytosine, fluconazole, itraconazole and ketoconazole were determined against 42 clinical isolates of Cryptococcus neoformans var. neoformans using the Alamar YeastOne colorimetric method and the NCCLS reference microdilution method. No strains with resistance to amphotericin B, itraconazole or ketoconazole were detected with either method. Using the reference method, the MICs of fluconazole were >= 64 mg/L, whereas using the colorimetric method all MICs were >=16 mg/L. The MIC values of 5-flucytosine were also higher using the reference method (8–16 mg/L for 32% of isolates) compared with the colorimetric method. The percentage of agreement between the methods, using a difference of two dilutions, was 70.7% for itraconazole, 73.2% for amphotericin B, 80% for fluconazole, 88% for 5-flucytosine and 95% for ketoconazole. Overall, we conclude that for fluconazole and 5-flucytosine, in a low but not insignificant number of isolates, results with the two methods are discordant, some isolates being found sensitive with the colorimetric test, but resistant with the reference method.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Strains of Cryptococcus neoformans resistant to antifungal agents including flucytosine,1,2 fluconazole3,4 and amphotericin B5,6 have been shown to account for failure or relapse during azole treatment of cryptococcosis. This makes antifungal susceptibility testing even more important in selecting and monitoring antifungal chemotherapy. Although standardized methods for broth macrodilution and microdilution testing of yeasts have been developed by the National Committee for Clinical Laboratory Standards (NCCLS),7 the availability of commercially prepared microdilution antifungal panels has been shown to enhance the ability to detect resistant isolates.8 Recently, a dried microdilution panel that incorporates an oxidation– reduction indicator (Alamar Bioscience Inc., Sacramento, CA, USA) has become available. Alamar YeastOne (Sensititre-Alamar YeastOne, Westlake, OH, USA) is a ready-to-use product; the 96-well microtitre plate has a colorimetric agent incorporated with the dried drugs. This produces a change from blue to pink in response to chemical reduction in the growth medium caused by the growing organisms in the inoculated wells. The first blue well in a row corresponds to the MIC of that drug. The antifungals included in the kit are amphotericin B, 5-flucytosine, fluconazole, itraconazole and ketoconazole. The Alamar colorimetric technique has demonstrated excellent agreement with the reference method when testing Gram-negative bacteria9 as well as for the susceptibility testing of yeasts10 and a high degree of intra- and interlaboratory reproducibility.10,11

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.


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

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 106–5 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 103–8 x 103 cells/mL. The final concentrations of the antifungal agents were 0.04–4 mg/L of amphotericin B, 0.12–256 mg/L of fluconazole, 0.04–64 mg/L of 5-flucytosine and 0.008–16 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 1–10 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 0.03–16 mg/L of amphotericin B (Squibb, Princeton, NJ, USA), ketoconazole and itraconazole (Janssen Biotech, Beerse, Belgium), 0.125–64 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.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
After 48 h incubation, MIC for Candida spp. were within the expected ranges in both the reference microdilution method and the Alamar colorimetric method. By the Alamar colorimetric method, 40% of C. neoformans isolates failed to produce any colour change in the growth control well after 48 h incubation, so readings were performed after 72 h incubation. Because the MIC scale is two dilutions (0.016 and 0.008 µg/L) lower than that of the reference method, values <=0.03 mg/L were considered equivalent. Table IGo summarizes MIC ranges of the five antifungals tested against 42 C. neoformans isolates determined by the reference microdilution method and the Alamar colorimetric method. The results are reported as MIC ranges, MIC50 and MIC90, respectively. The highest MIC values were found for fluconazole and the lowest for ketoconazole. The levels of agreement between the two methods for C. neoformans isolates are given in Table IIGo. Maximum and minimum agreements were found for ketoconazole (95.1%) and itraconazole (70.7%), respectively.


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Table I. MIC distribution in 42 isolates of Cryptococcus neoformans against five antifungal drugs with the microbroth reference method and the Alamar-Blue colorimetric test (data in percentages)
 

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Table II. Agreement and differences (%) between the colorimetric and reference microdilution methods for five antifungal drugs: amphotericin B, fluconazole, itraconazole, ketoconazole and 5-flucytosine against C. neoformans isolates
 
There were statistically significant differences between the methods in both the range and distribution of MICs for amphotericin B and 5-flucytosine, with a wider range and significantly higher mean MIC values for the microdilution reference method as compared with the Alamar colorimetric technique (amphotericin B, 0.143 versus 0.026 mg/L, P < 0.005; 5-flucytosine, 6.316 versus 2.06 mg/L, P < 0.0005). In respect of the three azole compunds, lower mean MIC values (P = 0.08) were obtained for fluconazole with the Alamar colorimetric method than with the reference method, in which MIC values >=64 mg/L were obtained in 2.3% of strains. Differences between the three azole agents were greater for MIC90 than MIC50. All compounds showed identical MIC50 values.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
There are only a few studies on the susceptibility of C. neoformans, so it is interesting to have data available on isolates from different sources and geographical distributions.12 To compare results, however, it is necessary to use a similar and standardized methodology. Although the reference methods provide objective and comparable results, the availability of automated plate-reading technology simplifies the procedures including MIC endpoint determination and avoids many manipulations of the standardized reference methods.13 In isolates of Candida spp., the recently introduced colorimetric micromethod has shown a good reproducibility and correlation with macrodilution and microdilution methods.11 In a study similar to ours, Pfaller & Barry10 compared the NCCLS reference method and the Alamar colorimetric technique in 600 clinical yeast isolates. The collection, however, included only four C. neoformans isolates. These authors found a 100% agreement between both methods in MIC50 of amphotericin B, fluconazole and 5-flucytosine. It should be noted that all four C. neoformans isolates were susceptible to all antifungal agents with MIC ranges 0.25–1.0 mg/L.

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 (9–24%), increasing to 24–56% when there is a difference of one dilution, to more than 70% for two dilutions and between 92.5–100% 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.


    Notes
 
* Corresponding author. Tel: +34-93-2211009; Fax: +34-93-2213237; E-mail: jmtorres{at}imim.es Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Block, E. R., Jennings, A. E. & Bennett, J. E. (1973). 5-Fluorocytosine resistance in Cryptococcus neoformans. Antimicrobial Agents and Chemotherapy 3, 649–56.[ISI][Medline]

2 . Douhet, E., Alayse, A. M. & Improvis, L. (1976). Phenotypes de résistance à la 5-fluorocitosine chez Cryptococcus neoformans. Bulletin Société Française de Mycologie Médicale 5, 17–20.

3 . Paugam, A., Dupouy-Camet, J., Blanche, P., Gangneux, J. P., Tourte-Schaefer, C. & Sicard, D. (1994). Increased fluconazole resistance of Cryptococcus neoformans isolated from a patient with AIDS and recurrent meningitis. Clinical Infectious Diseases 19, 975–6.[ISI][Medline]

4 . Chryssanthou, E., Grönfors, C. & Khanna, N. (1997). Comparison of broth macrodilution, broth microdilution and E-test susceptibility tests of Cryptococcus neoformans for fluconazole. Mycoses 40, 423–7.[ISI][Medline]

5 . Powderly, W. G., Keath, E. J., Sokol-Anderson, M., Robinson, K., Kitz, D., Little, J. R. & Kobayashi, G. (1992). Amphotericin B-resistant Cryptococcus neoformans in a patient with AIDS. Infectious Diseases in Clinical Practice 1, 314–6.

6 . Lozano-Chiu, M., Paetznick, V. L., Ghannoum, M. A. & Rex, J. H. (1998). Detection of resistance to amphotericin B among Cryptococcus neoformans cinical isolates: performances of three different media assessed by using E-test and National Committee for Clinical Laboratory Standards M27-A methodologies. Journal of Clinical Microbiology 36, 2817–22.[Abstract/Free Full Text]

7 . National Committee for Clinical Laboratory Standards. (1997). Reference Method for Broth Dilution Antifungal Susceptibility Testing for Yeasts: Approved Standard M27-A. NCCLS, Wayne, PA.

8 . To, W.-K., Fothergill, A. W. & Rinaldi, M. G. (1995). Comparative evaluation of macrodilution and Alamar colorimetric microdilution broth methods for antifungal susceptibility testing of yeast isolates. Journal of Clinical Microbiology 33, 2660–4.[Abstract]

9 . Baker, C. N., Barnerjee, S. N. & Tenover F. C. (1994). Evaluation of Alamar colorimetric MIC method for antimicrobial susceptibility testing of gram-negative bacteria. Journal of Clinical Microbiology 32, 1261–7.[Abstract]

10 . Pfaller, M. A. & Barry, A. L. (1994). Evaluation of a novel colorimetric broth microdilution method for antifungal susceptibility testing of yeast isolates. Journal of Clinical Microbiology 32, 1992–6.[Abstract]

11 . Pfaller, M. A., Messer, S. A., Hollis, R. J., Espinel-Ingroff, A., Ghannoum, M. A., Plavan, H. et al. (1998). Multisite reproducibility of MIC results by the sensititre YeastOne colorimetric antifungal susceptibility panel. Diagnostic Microbiology and Infectious Disease 31, 543–7.[ISI][Medline]

12 . Franzot, S. P. & Hamdan, J. S. (1996). In vitro susceptibilities of clinical and environmental isolates of Cryptococcus neoformans to five antifungal drugs. Antimicrobial Agents and Chemotherapy 40, 822–4.[Abstract]

13 . Ghannoum, M., Ibrahim, A. S., Fu., Y., Shafiq, M. C., Edwards, J. E. & Criddle, R. S. (1992). Susceptibility testing of Cryptococcus neoformans: a microdilution technique. Journal of Clinical Microbiology 30, 2881–6.[Abstract]

14 . Rex, J. H., Pfaller, M. A., Galgiani, J. N., Bartlett, M. S., Espinell-Ingroff, A., Ghannoum, M. A. et al. (1997). Development of interpretative breakpoints for antifungal susceptibility testing: conceptual framework and analysis of in vitro–in vivo correlation data for fluconazole, itraconazole, and candida infections. Clinical Infectious Diseases 24, 235–47.[ISI][Medline]

15 . 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]

Received 21 June 1999; returned 7 November 1999; revised 25 November 1999; accepted 30 November 1999