JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 2000, p. 3715–3717 0095-1137/00/$04.00⫹0 Copyright © 2000, American Society for Microbiology. All Rights Reserved.
Vol. 38, No. 10
Evaluation of the Etest Method for Determining Voriconazole Susceptibilities of 312 Clinical Isolates of Candida Species by Using Three Different Agar Media M. A. PFALLER,1* S. A. MESSER,1 A. HOUSTON,1 K. MILLS,2 A. BOLMSTROM,2
AND
R. N. JONES1
Department of Pathology, University of Iowa College of Medicine, Iowa City, Iowa,1 and AB BIODISK, Solna, Sweden2 Received 26 April 2000/Returned for modification 4 July 2000/Accepted 4 August 2000
Performance of the Etest for voriconazole susceptibility testing of 312 isolates of Candida spp. was assessed against that of the National Committee for Clinical Laboratory Standards (NCCLS) microdilution broth method. The NCCLS method employed RPMI 1640 broth medium, and MICs were read after incubation for 48 h at 35°C. Etest MICs were determined with RPMI agar containing 2% glucose (RPG), Casitone agar (CAS), and antibiotic medium 3 (AM3) agar and were read after incubation for 48 h at 35°C. The Candida spp. isolates included C. albicans (n ⴝ 174), C. glabrata (n ⴝ 55), C. tropicalis (n ⴝ 31), C. parapsilosis (n ⴝ 39), C. krusei (n ⴝ 5), C. lusitaniae (n ⴝ 2), and C. guilliermondii (n ⴝ 6). The Etest results obtained using RPG correlated well with the reference MICs. Overall agreement ranged from 91% for C. glabrata to 100% for C. tropicalis, C. parapsilosis, C. guilliermondii, C. krusei, and C. lusitaniae. When CAS was used, agreement ranged from 80% for C. krusei to 100% for C. parapsilosis, C. guilliermondii, and C. lusitaniae. With AM3, agreement ranged from 58% for C. glabrata to 100% for C. lusitaniae and C. guilliermondii. The Etest method using RPG appears to be a useful method for determining voriconazole susceptibilities of Candida species. The Etest stable agar gradient susceptibility testing method has been shown to be extremely flexible in testing a variety of fastidious and nonfastidious organisms, including bacteria, yeasts, and moulds (2–4, 6–9, 12–14, 18, 19; our unpublished data). The major perceived advantage of Etest for susceptibility testing of fungi is that laboratories wishing to test only one or two agents against an occasional yeast isolate may do so and generate quantitative MICs (7). Numerous studies have now been published documenting that the performance of Etest is comparable with that of reference broth dilution testing of amphotericin B (6, 9, 14, 19), fluconazole (2–4, 6, 13), itraconazole (2–4, 6, 18), and ketoconazole (2–4, 6). Notably, Etest may be the preferred method for detecting amphotericin Bresistant strains of Candida spp. and Cryptococcus neoformans (7, 9, 12, 14, 19). Presently, however, there are few or no data available describing the performance of Etest with the newer investigational triazole and echinocandin antifungals. Voriconazole is a new investigational monotriazole antifungal agent with systemic activity against a broad spectrum of pathogenic fungi including Candida spp., Cryptococcus neoformans, and Aspergillus spp. (5, 7, 10, 15, 16, 17). This agent has been tested extensively in vitro using broth dilution methods but has not been evaluated using agar-based methods such as Etest. In the present study, we evaluate the Etest for voriconazole using three agar media, RPMI, Casitone, and antibiotic medium 3 agar, by comparing the results with those obtained using the National Committee for Clinical Laboratory Standards (NCCLS) reference broth microdilution method for testing 312 clinical isolates of Candida spp.
glabrata isolates, 31 C. tropicalis isolates, 39 C. parapsilosis isolates, 5 C. krusei isolates, 6 C. guilliermondii isolates, and 2 C. lusitaniae isolates. The members of this collection were all recent clinical isolates from geographically diverse medical centers in North and Latin America. The majority were isolated from blood or normally sterile body fluids (15). The isolates were identified by standard methods (20) and were stored as suspensions in water at ambient temperature until used in the study. Prior to testing, each isolate was subcultured at least twice to Sabouraud dextrose agar (Remel, Lenexa, Kans.) to ensure optimal growth characteristics. Antifungal agents. Etest strips containing voriconazole were supplied by AB BIODISK (Solna, Sweden). Voriconazole was obtained as a reagent-grade powder from Pfizer Inc., Central Research Division (Groton, Conn.). Stock solutions were prepared in dimethyl sulfoxide and further diluted in RPMI 1640 medium buffered to pH 7.0 with 0.165 M morpholinepropanesulfonic acid (MOPS) buffer (Sigma, St. Louis, Mo.) and were dispensed into 96-well microdilution trays. Trays containing a 0.1-ml aliquot of appropriate drug solution (two times the final concentration) in each well were subjected to quality control (QC) and then sealed and stored at ⫺70°C until used in the study. The final concentration of voriconazole in the wells ranged from 0.007 to 8 g/ml. Media. Agar formulations used for the Etest were RPMI 1640 (American Biorganics, Buffalo, N.Y.) supplemented with 1.5% agar and 2% glucose (RPG) and buffered with MOPS, Casitone agar (CAS) (Difco), and antibiotic medium 3 (AM3) agar (BBL). The RPMI 1640 broth medium used for the microdilution testing was buffered with MOPS in accordance with the NCCLS M27-A method (11). Antifungal susceptibility test methods. Broth microdilution tests were performed according to NCCLS document M27-A (11). An inoculum concentration of 0.5 ⫻ 103 to 2.5 ⫻ 103 cells per ml was standardized spectrophotometrically and by quantitative plate counts. Microdilution trays were incubated in air at 35°C and read after 48 h of incubation. For voriconazole, the MIC endpoint was defined as the lowest concentration that produced a significant decrease in turbidity relative to the control (drug-free) well (10, 11). For the Etest, 90-mm-diameter plates containing agar at a depth of 4.0 mm were used. The agar surface was inoculated by using a nontoxic swab dipped in a cell suspension adjusted spectrophotometrically to the turbidity of a 0.5 McFarland standard. After excess moisture was absorbed into the agar and the surface was completely dry, an Etest strip was applied to each plate. The plates were incubated in air at 35°C and read at 48 h. The MIC was read at the lowest concentration at which the border of the elliptical inhibition zone intercepted the scale on the strip. Any growth, such as microcolonies, throughout a discernable inhibition ellipse was ignored. QC. QC was performed in accordance with NCCLS document M27-A using C. krusei ATCC 6258 and C. parapsilosis ATCC 22019 (11). QC determinations made on each day of testing were within the control limits for voriconazole established by Barry et al. (1): C. krusei ATCC 6258, 0.12 to 1.0 g/ml, and C. parapsilosis ATCC 22019, 0.03 to 0.25 g/ml. Analysis of results. Etest MICs read at 48 h on the three media were compared to reference microdilution MICs read at 48 h. The reference microdilution MIC
MATERIALS AND METHODS Test organisms. Three hundred twelve clinical isolates of Candida species were selected for testing. The collection included 174 C. albicans isolates, 55 C.
* Corresponding author. Mailing address: Medical Microbiology Division, C606 GH, Department of Pathology, University of Iowa College of Medicine, Iowa City, IA 52242. Phone: (319) 384-9566. Fax: (319) 356-4916. E-mail:
[email protected]. 3715
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TABLE 1. In vitro activity of voriconazole against 312 clinical isolates of Candida species as determined by the reference broth microdilution methoda MIC (g/ml)b
Organism
No. of isolates tested
Range
50%
90%
C. albicans C. glabrata C. tropicalis C. parapsilosis C. krusei C. lusitaniae C. guilliermondii All
174 55 31 39 5 2 6 312
0.007–⬎8.0 0.015–8.0 0.007–0.25 0.007–0.12 0.12–0.5 0.015 0.03–0.25 0.007–⬎8.0
0.007 0.25 0.03 0.015 0.25 0.015 0.06 0.015
0.03 1.0 0.06 0.06
0.25
a
Performed according to NCCLS M27-A (11). 50% and 90%, MICs at which 50 and 90% of isolates tested, respectively, are inhibited. b
and Etest MIC determinations were performed in two physically separate laboratories and the results were read independently; i.e., the testing was blind. Since the Etest scale has a continuous gradient of concentrations, the MICs between twofold dilutions were rounded to the next twofold level of the reference method for comparison (13, 14). Off-scale MICs at the upper limit were converted to the next higher concentration, and off-scale results at the lower limit were left unchanged. Discrepancies between MICs of no more than two dilutions were used to calculate the percent agreement.
RESULTS AND DISCUSSION Table 1 summarizes the in vitro susceptibilities of 312 Candida isolates to voriconazole, as determined by the reference broth microdilution method. A broad range of MICs was observed for most organism groups. In general, the voriconazole MICs obtained were similar to those reported previously for the individual Candida species (5, 10, 15, 17). Voriconazole MICs of ⬎1 g/ml were observed for only three isolates: one strain of C. albicans (MIC, ⬎8 g/ml) and two strains of C. glabrata (MICs of 2 and 8 g/ml, respectively). Table 2 summarizes the percentage of 48-h voriconazole MICs obtained by the Etest on the three agar media that were within two dilutions of the reference method result. Overall, the percent agreement was 98% with RPG, 89% with CAS, and 89% with AM3 agar. The agreement between Etest and microdilution MICs was ⱖ90% for all species with RPG agar. Slightly lower levels of agreement were observed when either CAS or AM3 agar was used; however, ⱖ80% agreement was observed on both media for all species with the exception of C. glabrata (58% agreement on AM3). When a discrepancy was TABLE 2. Agreement between Etest and reference voriconazole MICs for 312 clinical isolates of Candida species % agreement when tested ona,b:
Organism
No. of isolates tested
RPG
CAS
AM3
C. albicans C. glabrata C. tropicalis C. parapsilosis C. krusei C. lusitaniae C. guilliermondii All
174 55 31 39 5 2 6 312
99 91 100 100 100 100 100 98
88 87 87 100 80 100 100 89
97 58 90 92 80 100 100 89
a Percentage of Etest MICs (read at 48 h) determined with three different agar media that are within ⫾ two log2 dilutions of the reference microdilution MICs (RPMI broth, 48 h). b For details about the agar media, see Materials and Methods.
observed between the results obtained by the Etest and the reference method, the Etest provided a lower MIC with AM3 (79% of discrepancies) and a higher MIC with CAS (85%) and RPG (57%). These discrepancies reflect, in part, slower growth of the isolates on AM3 and improved growth on CAS and RPG agar relative to RPMI broth. The results of this study provide the first documentation of the applicability of the Etest stable agar gradient method for determining the in vitro susceptibilities of Candida species to the investigational triazole voriconazole. We found that RPMI agar with glucose (2% final concentration) supported optimal growth of all species tested and provided excellent agreement with the MICs obtained with the broth microdilution method (Table 2). As was seen with fluconazole (13), the problem of trailing end points due to partial inhibition of growth by azoles was minimized with the use of RPG agar and adherence to specific criteria for reading Etest MICs as described in the Etest package insert and technical guide for yeasts (AB BIODISK). Good agreement with broth dilution MICs was observed when discernable growth within the ellipse was ignored. Although CAS and AM3 agar did not perform as well as RPG, both media supported the growth of most of the test isolates and performed reasonably well compared to the reference method (Table 2). Trailing growth within a discernable ellipse was observed with certain strains with both of these media. In most instances, the MIC for voriconazole was underestimated relative to the broth MIC when determined on AM3 and overestimated relative to the broth MIC when determined on CAS. The influence of reduced growth on AM3 agar was most prominent with C. glabrata, resulting in MICs that were more than two dilutions lower than those of the reference method in 42% of the isolates tested. In summary, we have provided the first documentation of the ability of Etest to generate voriconazole MIC data that are comparable to those obtained by the NCCLS broth microdilution method. RPMI agar with 2% glucose may be used to determine reference quality MICs with triazole (fluconazole, itraconazole, and voriconazole), polyene (amphotericin B), and echinocandin (caspofungin [MK-0991]) Etest reagents in tests with Candida spp. and Cryptococcus neoformans (2–4, 6, 9, 13, 14; our unpublished data). This will be very attractive to laboratories because it will provide the flexibility to test only one agent or a panel of antifungal agents representing different classes of systemic antifungal agents as the clinical situation dictates. ACKNOWLEDGMENTS The excellent secretarial support of K. L. Meyer is greatly appreciated. This study was supported in part by Pfizer Pharmaceuticals and by AB BIODISK. REFERENCES 1. Barry, A. L., M. A. Pfaller, S. D. Brown, A. Espinel-Ingroff, M. A. Ghannoum, C. Knapp, R. P. Rennie, J. H. Rex, and M. G. Rinaldi. 2000. Quality control limits for broth microdilution susceptibility tests of ten antifungal agents. J. Clin. Microbiol. 38:3457–3459. 2. Chen, S. C. A., M. L. O’Donnell, S. Gordon, and G. L. Gilbert. 1996. Antifungal susceptibility testing using the Etest: comparison with the broth macrodilution technique. J. Antimicrob. Chemother. 37:265–273. 3. Colombo, A. L., F. Barchiesi, D. A. McGough, and M. G. Rinaldi. 1995. Comparison of Etest and National Committee for Clinical Laboratory Standards broth macrodilution method for azole antifungal testing. J. Clin. Microbiol. 33:535–540. 4. Colombo, A. L., F. Barchiesi, D. A. McGough, A. W. Fothergill, and M. G. Rinaldi. 1995. Evaluation of the Etest system versus a microtitre broth method for antifungal susceptibility testing of yeasts against fluconazole and itraconazole. J. Antimicrob. Chemother. 36:93–100.
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