Comparison of the Etest and microdilution method for antifungal susceptibility testing of Cryptococcus neoformans to four antifungal agents

A. I. Aller, E. Martín-Mazuelos*, M. J. Gutiérrez, S. Bernal, M. Chávez and F. J. Recio

Servicio de Microbiología, Hospital Universitario de Valme, Carretera de Cádiz s/n, E-41014 Seville, Spain


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We performed a prospective study to compare the Etest and the microdilution method (NCCLS guidelines) for determining the MICs of fluconazole, itraconazole, flucytosine and amphotericin B for 35 strains of Cryptococcus neoformans. For the microdilution method (MDM) RPMI 1640 medium with 2% glucose was used for fluconazole, itraconazole and flucytosine, and Antibiotic Medium 3 for amphotericin B. For the Etest, RPMI 1640 medium with 2% glucose and solidified with 1.5% agar was used for the four antifungal agents. Amphotericin B was also tested on Antibiotic Medium 3 solidified with 1.5% agar. Fluconazole and flucytosine MICs by the Etest showed good correlation with the broth MDM (81.1 and 89.2% agreement within two dilutions, respectively). With the tested population of itraconazole- and amphotericin B-susceptible isolates, the MIC agreement for itraconazole was 54%; amphotericin B showed the lowest agreement (8.1% on Antibiotic Medium 3 and 13.5% on RPMI).


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cryptococcosis is an infection caused by Cryptococcus neoformans, an encapsulated yeast-like fungus. The majority (75–90%) of patients with AIDS who are infected with C. neoformans develop meningitis and it is the most important neurological manifestation of AIDS. Approximately 5–10% of patients with AIDS develop cryptococosis.1,2 The high incidence of fungal infections in HIV patients has increased the use of the azoles as treatment, as well as prophylaxis, which can give rise to development of resistance. For these reasons it is necessary to develop routine methods for determining the susceptibility of C. neoformans to different antifungal agents in order to evaluate the efficacy of treatment and the evolution of the disease. The majority of efforts to develop a routine method for testing susceptibility of yeasts to antifungal agents have used different species of Candida. These methods for determining the in vitro susceptibility of Candida spp., however, cannot be extrapolated to C. neoformans, owing to the fastidious nature of this microorganism and its slow growth.3 The purpose of this study was to compare the results obtained by a reference microdilution method (MDM) with those obtained with the Etest system for antifungal susceptibility testing of C. neoformans.


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

We have studied retrospectively 35 isolates of C. neoformans from 16 HIV patients, isolated from different clinical specimens (21 CSF, 11 blood cultures, one bronchial aspirate, one lung tissue and one bone-marrow aspirate). In three cases multiple isolates were taken from the same patient, being the isolates from different clinical episodes of meningitis. Five isolates were considered putatively resistant to fluconazole. C. neoformans ATCC 90112 and C. neoformans ATCC 90113 were included as control strains.

Broth microdilution method

The four agents tested were: fluconazole (Pfizer Central Research, Sandwich, UK), itraconazole (Janssen Pharmaceutica, Titusville, NJ, USA), flucytosine (Roche Laboratories, Nutley, NJ, USA) and amphotericin B (Squibb, Princeton, NJ, USA). Testing was performed according to NCCLS guidelines (document M27-A).4 Amphotericin B, fluconazole and itraconazole were dissolved in dimethylsulphoxide, and flucytosine was dissolved in sterile distilled water. Stock solutions were diluted with RPMI 1640 (RPMI tissue culture medium supplemented with glutamine) (Sigma Chemical Co., St Louis, MO, USA) with 2% glucose, buffered to pH 7.0 with 0.156 M 3-N-morpholinopropane-sulphonic acid (MOPS; Sigma).4 For amphotericin B testing, Antibiotic Medium 3 (Difco Laboratories, Detroit, MI, USA) supplemented with 2% glucose5 was used. The final concentrations were 0.12–64 mg/L for fluconazole, 0.015–8 mg/L for itraconazole and 0.03–16 mg/L for amphotericin B and flucytosine. The inoculum size was c. 104 cfu/mL.4 Testing was performed in 96-well round-bottomed microtitre plates and were incubated at 35°C for 48 and 72 h. The MICs of fluconazole, itraconazole and flucytosine were read as the lowest concentration of the agent which inhibited growth by 80%.4 For amphotericin B the MIC was the lowest concentration of drug that completely inhibited growth.4,5 The MICs at which 50% (MIC50) and 90% (MIC90) of each of the 35 isolates tested were inhibited were determined for each drug.6

Etest method

The Etest was performed according to the manufacturer's instructions. RPMI 1640 with 2% glucose7 was buffered with potassium phosphate at pH 7.0 and 1.5% Bacto agar (Difco Laboratories) was used to prepare Etest RPMI-agar plates6,8 for fluconazole and itraconazole. For amphotericin B and flucytosine, RPMI 1640 with 2% glucose and buffered with MOPS at pH 7.0 was used. For amphotericin B, Antibiotic Medium 3 agar (1.5%) plates (Difco Laboratories) buffered to pH 7.0 with 0.165 M MOPS9 were also used. The inoculum suspensions of C. neoformans isolates matched the turbidity of no. 1 McFarland standard.6,10 The incubation time was 48–72 h. The MIC was read where the border of the elliptical inhibition zone intersected the scale on the antifungal strip.6,10

All susceptibility tests were repeated twice by each method. Essential agreement (E.A.) was defined as MIC results by Etest and reference method in exact agreement or within two dilutions. The statistical analysis was by Wilcoxon matched-pairs signed-ranks. We calculated the equivalence of the results between the two methods for every drug. Because the Etest strips contain a continuous gradient of each drug tested instead of the log2 drug dilution scheme of the broth microdilution method, the Etest MICs were elevated to the next drug concentration that matched the microdilution scheme to facilitate comparison of the results.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In Table IGo the MIC range, MIC50 and MIC90 recorded at 72 h for four antifungal agents tested by the two methods are presented. The MICs obtained after different incubation times varied by no more than one two-fold dilution. The highest MIC was used in case of disagreement. For fluconazole, itraconazole and flucytosine the MIC50 and MIC90 with the Etest were lower than those obtained with the MDM, except for MIC90 for flucytosine, which was similar (>=16 mg/L in MDM versus >=32 mg/L in Etest). For amphotericin B there was no difference between MIC range and MIC50 by the Etest on the two media studied, and there was a difference of only one dilution in the MIC90 (0.125 mg/L in Antibiotic Medium 3 versus 0.25 mg/L in RPMI).


View this table:
[in this window]
[in a new window]
 
Table I. Susceptibility of 37 isolates of C. neoformans (35 clinical isolates and two control strains) to amphotericin B, flucytosine, fluconazole and itraconazole, as determined by the microdilution method (MDM) and Etest (ET)
 
The details of the E.A. between amphotericin B, flucytosine, fluconazole and itraconazole MICs for the 37 isolates of C. neoformans by the two methods are given in Table IIGo. The antifungal agent that showed the greatest percentage of E.A. (89.2%) was flucytosine, and amphotericin B showed the lowest percentage of E.A. (8.1% for Antibiotic Medium 3 and 13.5% for RPMI). The E.A. was 81.1% for fluconazole and 54% for itraconazole. By the Wilcoxon test there was a significant correlation (P < 0.05) for fluconazole only.


View this table:
[in this window]
[in a new window]
 
Table II. Distribution of differences in Etest and MDM MICs of amphotericin B, flucytosine, fluconazole and itraconazole for 37 strains of C. neoformans (35 clinical strains and two control strains) and essential agreement (E.A.) percentages
 
With five episodes of cryptococcal infection there was evidence of clinical resistance to fluconazole during fluconazole maintenance therapy. In all but one of these cases the MIC obtained by MDM was >=8 mg/L and the MIC obtained by Etest was >=12 mg/L. In 29 of the 30 episodes with sucessful fluconazole treatment the MIC obtained by MDM was <=4 mg/L and the MIC obtained by Etest was <=4 mg/L. One of the patients, whose isolate showed clinical resistance to fluconazole maintenance therapy, showed an MIC for itraconazole >=8 mg/L by MDM and >=32 mg/L by Etest. This patient died in the relapse episode during the course of treatment with amphotericin B. For this reason the existence of clinical resistance to itraconazole could not be proved.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The present study was undertaken to compare the Etest and the MDM for the determination of the activity of four antifungal agents against C. neoformans. Apart from the MIC90 for flucytosine and fluconazole, the MIC50 and MIC90 were greater by MDM than by Etest for all antifungal agents studied. The differences were 1–2 twofold dilutions, except for the MIC50 of amphotericin B (0.12 mg/L by MDM versus <0.002 mg/L by Etest) and itraconazole (0.25 mg/L by MDM versus 0.094 mg/L by Etest). These findings disagree with those of Colombo et al.,6 who obtained a similar range by both methods for fluconazole and itraconazole. The MICs obtained by Espinel-Ingroff11 with the control strain, C. neoformans ATCC 90112, for fluconazole and flucytosine were similar (±1 dilution) to our values by the MDM, but their MICs by the Etest were greater than ours (fluconazole, 8 mg/L versus 1 mg/L; and flucytosine, 4 mg/L versus 0.25 mg/L), although these authors did not use glucose-supplemented media. Compared with Espinel-Ingroff et al.,10 we obtained a similar range of results for flucytosine by Etest (MIC range by them 1–>32 mg/L and 0.25–>32 mg/L by us).

The E.A. between methods for fluconazole was 81.1%, which is lower than that obtained by Colombo et al. (96%)6,8 and greater than that obtained by Espinel-Ingroff (70%).11 These data do not compare very well with ours, because the authors did not include 2% glucose in the medium formulation. With itraconazole, the E.A. was lower than that obtained by Colombo et al. in two studies (80 and 100%, respectively).6,8 Our E.A. for flucytosine (89.2%) was higher than that obtained by Espinel-Ingroff (60%),11 who did not include 2% glucose in the medium formulation. Previous evaluations7 of the effect of medium formulation on MICs for yeasts concluded that elevating the concentration of glucose from 0.2 to 2% optimized the growth of certain species of yeasts and this facilitated our examination of MIC endpoints. The same effect was achieved with the MICs determined by the Etest.10

Warnock et al.12 tested 18 isolates of Candida spp. and two C. neoformans against amphotericin B, flucytosine, fluconazole and itraconazole by the Etest method in a multi-centre evaluation. These authors suggest that the Etest method is suitable for routine use with Candida spp. and amphotericin B and flucytosine, and it is less reliable for the azoles. However, the two C. neoformans isolates generated a large number of discrepant results, which is in agreement with results obtained by us. In our work the greatest discrepancies were with amphotericin B (8.1% E.A on Antibiotic Medium 3 and 13.5% E.A. on RPMI). These results disagree with those obtained by Lozano-Chiu et al.,13 who evaluated the NCCLS M27-A microdilution method and the Etest agar diffusion method for the detection of resistance to amphotericin B among C. neoformans isolates. They used three different media (RPMI 1640 medium, Antibiotic Medium 3 and yeast nitrogen base) for the broth MDM and two media for the Etest method (RPMI 1640 agar and Antibiotic Medium 3 agar). For the putatively susceptible isolates Lozano-Chiu et al.13 obtained results by Etest on RPMI 1640 agar and Antibiotic Medium 3 agar similar to those obtained for Antibiotic Medium 3 by MDM, whereas the MICs for the putatively resistant isolates were noticeably increased. In our study all strains were putatively susceptible to amphotericin B and we did not find correlation between the values obtained by the two methods.

With amphotericin B, fluconazole and itraconazole the majority of MIC disagreements were associated with Etest MICs >=2 dilutions lower than the reference method. Colombo et al.8 obtained similar findings for fluconazole and itraconazole. Warnock et al.12 found that the MICs by Etest for amphotericin B were lower than the results obtained with the reference microdilution method, whereas the Etest MICs results for flucytosine, fluconazole and itraconazole were higher than those obtained with the MDM. Other authors14,15 have reported that absolute azole MICs generated by agar-based techniques tend to be lower than those produced by broth assays.

In agreement with other authors,6,10,11 we observed the growth of microcolonies around or inside of the inhibition zone when we tested fluconazole.

In the 96.6% of cases with successful fluconazole treatment the MIC values were <=4 mg/L by both methods. However, it is not possible to compare the MIC values obtained by Etest with those obtained by other authors as there are few studies that establish the predictive value of Etest in correlation to clinical outcome for C. neoformans. Moreover, the MIC breakpoint capable of accurately separating resistant from susceptible strains has not yet been established.

In conclusion, we suggest that the Etest is a useful alternative to the MDM for use in the clinical laboratory for the determination of susceptibility of C. neoformans to flucytosine and fluconazole. The isolates tested did not include any itraconazole- or amphotericin B-resistant strains, so conclusions regarding the comparative abilities of the methods to distinguish resistant from susceptible strains cannot be made. However, the observed differences between methods in MICs for susceptible strains suggest that it will be necessary to carry out further studies, including assessment of interlaboratory agreement and correlation of MICs by different methods with in vivo response.


    Notes
 
* Corresponding author. Tel: +34-95-4596352; Fax: +34-95-4596395; E-mail: micemm{at}valme.sas.cica.es Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Dismukes, W. E. (1993). Management of cryptococcosis. Clinical Infectious Diseases 17, Suppl. 2, S507–12.[ISI][Medline]

2 . Powderly, W. G. (1993). Cryptococcal meningitis and AIDS. Clinical Infectious Diseases 17, 837–42.[ISI][Medline]

3 . Ghannoum, M. A., 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]

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

5 . Rex, J. H., Cooper, C. R., Merz, W. G., Galgiani, J. N. & Anaissie, E. (1995). Detection of amphotericin B-resistant Candida isolates in a broth-based system. Antimicrobial Agents and Chemotherapy 39, 906–9.[Abstract]

6 . Colombo, A. L., Barchiesi, F., McGough, D. A. & Rinaldi, M. G. (1995). Comparison of Etest and National Committee for Clinical Laboratory Standards broth macrodilution method for azole antifungal susceptibility testing. Journal of Clinical Microbiology 33, 535–40.[Abstract]

7 . Rodriguez-Tudela, J. L. & Martínez-Suárez, J. V. (1994). Improved medium for fluconazole susceptibility testing of Candida albicans. Antimicrobial Agents and Chemotherapy 38, 45–8.[Abstract]

8 . Colombo, A. L., Barchiesi, F., McGough, D. A., Fothergill, A. W. & Rinaldi, M. G. (1995). Evaluation of the Etest system versus a microtitre broth method for antifungal susceptibility testing of yeasts against fluconazole and itraconazole. Journal of Antimicrobial Chemotherapy 36, 93–100.[Abstract]

9 . Wanger, A., Mills, K., Nelson, P. W. & Rex, J. H. (1995). Comparison of Etest and National Committee for Clinical Laboratory Standards broth macrodilution method for antifungal susceptibility testing: enhanced ability to detect amphotericin B-resistant Candida isolates. Journal of Antimicrobial Chemotherapy 39, 2520–2.

10 . Espinel-Ingroff, A., Pfaller, M., Erwin, M. E. & Jones, R. N. (1996). Interlaboratory evaluation of Etest method for testing antifungal susceptibilities of pathogenic yeast to five antifungal agents by using Casitone agar and solidified RPMI 1640 medium with 2% glucose. Journal of Clinical Microbiology 34, 848–52.[Abstract]

11 . Espinel-Ingroff, A. (1994). Etest for antifungal susceptibility testing of yeast. Diagnostic Microbiology and Infectious Diseases 19, 217–20.[ISI][Medline]

12 . Warnock, D. W., Johnson, E. M. & Rogers, R. F. (1998). Multi-centre evaluation of the Etest method for antifungal drug susceptibility testing of Candida spp. and Cryptococcus neoformans. Journal of Antimicrobial Chemotherapy 42, 321–31.[Abstract]

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

14 . Brass, C., Shainhouse, J. Z. & Stevens, D. A. (1979). Variability of agar dilution-replicator method of yeast susceptibility testing. Antimicrobial Agents and Chemotherapy 15, 763–8.[ISI][Medline]

15 . Odds, F. C. (1980). Laboratory evaluation of antifungal agents: a comparative study of five imidazole derivatives of clinical importance. Journal of Antimicrobial Chemotherapy 6, 749–61.[ISI][Medline]

Received 2 August 1999; returned 9 January 2000; revised 10 May 2000; accepted 22 August 2000