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JOURNAL OF CLINICAL MICROBIOLOGY, May 2003, p. 2141–2143 0095-1137/03/$08.00ϩ0 DOI: 10.1128/JCM.41.5.2141–2143.2003 Copyright 2003, American Society for Microbiology. All Rights Reserved.
Detection of Fluconazole-Resistant Isolates of Candida glabrata by Susan M. Nelson1 and Charles P. Cartwright1,2* Department of Laboratory Medicine and Pathology, Hennepin County Medical Center, Minneapolis, Minnesota 55415,1 and Department of Laboratory Medicine and Pathology, University of Minnesota Medical School, Received 10 October 2002/Returned for modification 14 January 2003/Accepted 25 January 2003 The ability of a fluconazole-containing agar screen assay to accurately detect isolates of Candida glabrata
resistant to the azole antifungal agent fluconazole was evaluated on a collection of 100 clinical isolates of this
organism. Results were correlated with the MIC of fluconazole for these isolates and compared with the results
of a previously published disk diffusion-based fluconazole resistance screening test. Agar screen assay results
were in categorical agreement with MIC-based determinations for 97% (97/100) of the isolates tested. This
correlation was higher than that obtained with the disk diffusion technique, which categorized only 87%
(87/100) of isolates correctly, and suggests that the agar screening approach can effectively expedite fluconazole
susceptibility testing of C. glabrata isolates.
The relatively recent publication of approved procedures excellent 97% correlation between agar screen testing and and interpretive categories for determining the susceptibility of MIC determination for a small group of 30 isolates of C. clinical isolates of Candida spp. to antifungal agents (12, 18) glabrata. These results, and the relative lack of data evaluating provides a basis for susceptibility testing to become a valuable fluconazole disk diffusion testing on C. glabrata isolates, sug- tool for the optimization of antifungal therapy for patients with gested to us that it would be worthwhile to perform a compar- invasive yeast infections (5, 7). Unfortunately, both the ap- ative evaluation of a screening agar-based method and disk proved broth macrodilution method (12) and microdilution diffusion for the rapid detection of fluconazole resistance in a adaptations of this technique (6, 12) are relatively expensive large collection of clinical isolates of C. glabrata.
and laborious and consequently are used in only a minority of All organisms used in the study were originally isolated from clinical microbiology laboratories that routinely perform anti- clinical specimens submitted for culture to the Microbiology bacterial susceptibility testing (3). The absence of institution- Laboratory, Hennepin County Medical Center, between April specific susceptibility information is unfortunate given con- 1998 and December 2000. The clinical sources of the isolates cerns about the emergence of antifungal resistance in Candida were as follows: urine (42 isolates), blood (31), surgically col- spp. (17). Candida glabrata in particular has become a more lected tissue or aspirate (11), abscess or drainage fluid (9), and frequent isolate in many laboratories (2, 16), and the flucon- wound (7). Using standard methods (4), isolates were identi- azole MIC at which 50% of tested isolates of this organism are fied as C. glabrata. The MICs of fluconazole for isolates were inhibited is typically 8- to 16-fold higher than that seen for C. determined using the NCCLS reference broth microdilution albicans (15, 16, 19). In addition, the typical fluconazole MICs method (12). Final fluconazole concentrations tested ranged for 10 to 15% of invasive isolates of C. glabrata place them in from 0.125 to 64 ␮g/ml. Microtiter plates were incubated for the resistant category, compared with less than 1% of C. albi- 48 h at 35°C, and the MIC was read as the lowest concentration cans isolates (15, 20). Antifungal susceptibility testing of C. of fluconazole that effected a visually distinct decrease (Ͼ50%) glabrata is, therefore, of greater clinical import than for many in turbidity relative to the growth control. The fluconazole other Candida species, and techniques that facilitate the per- screen agar was formulated as described by Patterson et al.
formance of fluconazole susceptibility testing of C. glabrata in (13). To prepare the inoculum for the agar-screening assay, routine clinical microbiology laboratories need to be devel- several isolated colonies of each isolate were picked and sus- oped. Previous studies have described the use of simple disk pended in sterile saline (0.9%). The optical density of each diffusion-based techniques for detecting resistance to flucon- suspension was then adjusted until it corresponded to a 0.5 azole in Candida spp. (1, 8, 9, 10, 11, 13, 19), but with the McFarland standard, and a sterile calibrated loop (1 ␮l) was notable exception of the study of Sandven (19), these studies used to streak a set of three plates containing 0, 8, and 16 ␮g have concentrated on testing C. albicans isolates. Use of flu- of fluconazole/ml. Inoculated plates were incubated at 30°C for conazole-containing agar plates has also been reported as a a total of 48 h. Growth characteristics of individual isolates possible means of identifying fluconazole-resistant isolates of were recorded after 24 and 48 h of incubation by visually Candida spp. (15, 20), and Patterson et al. (13) reported an comparing the diameters of 15 to 20 colonies on the flucon-azole-containing plates with those on the fluconazole-freeplate. To provide for a more objective analysis of the data, * Corresponding author. Mailing address: Clinical Laboratories, results observed in the fluconazole-agar screen assay were di- MC #812, Hennepin County Medical Center, 701 Park Ave., Minne- apolis, MN 55415. Phone: (612) 347-3026. Fax: (612) 904-4229. E-mail: vided into four categories: category I (colonies indistinguish- charles.cartwright@co.hennepin.mn.us.
able in size on media with and without fluconazole); category TABLE 1. Distribution of 48-h broth microdilution fluconazole TABLE 3. Correlation between results obtained by disk diffusion MIC for C. glabrata isolates evaluated in the present study and MIC broth microdilution testing of fluconazole susceptibility No. of isolates with fluconazole MIC (␮g/ml) of: No. of isolates with disk diffusion zone sizes (mm) II (colonies visually smaller on fluconazole-containing media but with a typical colony diameter Ͼ50% of the diameter seen on fluconazole-free media); category III (colonies significantly Categories: susceptible (Յ8 ␮g/ml), susceptible-dose dependent (16 to 32 smaller on fluconazole-containing media and typical colony diameter Ͻ50% of the diameter seen on fluconazole-free me- dia); and category IV (no growth or only pinpoint colonies on fluconazole-containing media). Fluconazole disk diffusion test- study (14). Indeed, a majority of isolates with fluconazole ing was performed as described by Barry and Brown (1). Zone MICs of 16 or 32 ␮g/ml (13 of 18 [72%] at 24 h and 15 of 18 diameters were measured after 48 h of incubation at 35°C in [83%] at 48 h) were designated susceptible (category III or IV) ambient air, and breakpoints of Ն19 mm (susceptible), 15 to 18 when 16 ␮g of fluconazole/ml was added to the medium. All mm (intermediate), and Յ14 mm (resistant) were used, corre- isolates determined to be in the R category by the reference sponding (according to previously published data) to flucon- MIC method were designated category I (little or no decrease azole MICs of Յ8, 16 to 32, and Ն64 ug/ml, respectively (1).
in mean colony diameter) in the fluconazole agar screen assay The fluconazole MICs for the 100 isolates of C. glabrata irrespective of the concentration of fluconazole used or length evaluated in this study are shown in Table 1. Significant clus- of incubation. Unfortunately, under none of the tested condi- tering of isolates around the susceptible breakpoint of 8 ␮g/ml tions were isolates in the S-DD category able to be accurately was observed, with fluconazole MICs either at or within one differentiated from those isolates demonstrating outright resis- doubling dilution of this value for 71% (71/100) of the isolates.
tance to fluconazole (Table 2). The results of disk diffusion The fluconazole MICs for a total of 31 isolates were Ͼ8 ␮g/ml, testing are shown in Table 3. All 31 isolates placed in the R or with 18 (58%) of these showing fluconazole MICs in the S-DD category by MIC testing were identified as either inter- NCCLS susceptible–dose-dependent (S-DD) range and 13 mediate or resistant by disk diffusion testing. A total of 13 of (42%) testing in the resistant (R) range (Ն64 ␮g/ml). The the 69 (18.9%) isolates in the S category were also classified as results of the agar screen assay are shown in Table 2. The use resistant (7 isolates) or intermediate (6 isolates) by disk diffu- of 8 ␮g of fluconazole/ml and an incubation time of 24 h sion, resulting in an overall accuracy of 87% (87/100).
enabled the most accurate differentiation of fluconazole-sus- The results of our investigation of simple techniques for ceptible C. glabrata isolates (MIC Յ 8 ␮g/ml) from those in the screening isolates of C. glabrata for susceptibility to fluconazole S-DD and R categories. Under these conditions, all 31 isolates are in general agreement with those reported by previous in- with fluconazole MICs of Ͼ8 ␮g/ml were placed in category I vestigators (1, 11, 13, 19). Both the agar screen method that we or II (less than 50% decrease in mean colony diameter), with developed and the disk diffusion technique correctly desig- only 3 of the 69 isolates (4.3%) with fluconazole MICs of Յ8 nated resistant all 31 isolates with fluconazole MICs of Ͼ8 ␮g/ml being similarly designated. Using 8 ␮g of fluconazole/ml ␮g/ml. Neither methodology effectively discriminated between but prolonging the incubation period to 48 h resulted in a isolates that were determined to be in the S-DD category by marginal improvement in sensitivity but greatly decreased the MIC testing and those determined to be resistant outright to specificity of the assay (Table 2). The use of 16 ␮g of flucon- fluconazole. In the agar screen assay, 56% (10/18) of S-DD azole/ml did not improve differentiation of isolates with flu- isolates appeared fully resistant to fluconazole, while only 33% conazole MICs of 8 ␮g/ml or less from those with fluconazole (6/18) of S-DD isolates would have been classified as interme- MICs of 16 ␮g/ml or above, as had been reported in a previous diate by the disk diffusion assay. These results strongly suggest TABLE 2. Correlation between decrease in colony diameter observed on fluconazole-screening agar and NCCLS interpretive categorization of C. glabrata isolates based on broth microdilution fluconazole MICs No. of isolates of indicated colony size categoryb in test conditions (␮g) of fluconazole/incubation time (h) of: a Categories: susceptible (Յ8 ␮g/ml), susceptible-dose dependent (16 to 32 ␮g/ml), resistant (Ն64 ␮g/ml).
b Definitions of colony size categories are as follows: category I (no discernible difference in colony size on fluconazole-containing media); category II (colonies smaller on fluconazole-containing media but typically Ͼ50% of the size of control colonies); category III (colonies significantly smaller on fluconazole-containing media and typically Ͻ50% of the size of control colonies); and category IV (no growth or only pinpoint colonies on fluconazole-containing media).
that qualitative test methodologies cannot be used to defini- 5. Ghannoum, M. A., J. H. Rex, and J. N. Galgiani. 1996. Susceptibility testing
tively determine the level of susceptibility of C. glabrata iso- of fungi: current status of correlation of in vitro data with clinical outcome.
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lates to fluconazole and that MIC determination is necessary 6. Hacek, D. M., G. A. Noskin, K. Trakas, and L. R. Peterson. 1995. Initial use
to classify isolates as either R or S-DD. The principal goal of of a broth microdilution method suitable for in vitro testing of fungal isolates using a simple, qualitative screening assay for resistance testing in a clinical microbiology laboratory. J. Clin. Microbiol. 33:1884–1889.
is to minimize the necessity for performing laborious and costly 7. Hadley, S., J. A. Martinez, L. McDermott, B. Rapino, and D. R. Snydman.
2002. Real-time antifungal susceptibility screening aids management of in- MIC testing on all isolates. Assuming that any isolate desig- vasive yeast infections in immunocompromised patients. J. Antimicrob. Che- nated less than susceptible by the screening test would have a mother. 49:415–419.
fluconazole MIC assay performed, use of the disk diffusion 8. Kirkpatrick, W. R., T. M. Turner, A. W. Fothergill, D. I. McCarthy, S. W.
Redding, M. G. Rinaldi, and T. F. Patterson. 1998. Fluconazole disk diffu-
assay would have eliminated 66% (66/100) of the MIC tests sion susceptibility testing of Candida species. J. Clin. Microbiol. 36:3429–
performed on C. glabrata isolates. Since the fluconazole- screening agar was somewhat more successful than disk diffu- 9. Kronvall, G., and I. Karlsson. 2001. Fluconazole and voriconazole multidisk
testing of Candida species for disk test calibration and MIC estimation.
sion in identifying susceptible C. glabrata isolates (with only J. Clin. Microbiol. 39:1422–1428.
3/69 [4.3%] isolates with fluconazole MICs of Յ8 ␮g/ml being 10. Lee, S.-C., C.-P. Fung, N. Lee, L.-C. See, J.-S. Huang, C.-J. Tsai, K.-S. Chen,
misclassified using the optimal assay conditions), use of this and W.-B. Shieh. 2001. Fluconazole disk diffusion test with methylene blue-
and glucose-enriched Mueller-Hinton agar for determining susceptibility of screening method would have decreased MIC testing by 75%.
Candida species. J. Clin. Microbiol. 39:1615–1617.
An evaluation similar to that reported here was also performed 11. May, J. L., A. King, and C. A. Warren. 1997. Fluconazole disk diffusion
on clinical isolates of C. albicans. Using the agar screening testing for the routine laboratory. J. Antimicrob. Chemother. 40:511–516.
12. National Committee for Clinical Laboratory Standards. 1997. Reference
assay, 100% (47/47) of the susceptible isolates of C. albicans method for broth dilution antifungal susceptibility testing of yeasts: approved tested (fluconazole MIC range, 0.12 to 2.0 ␮g/ml) were cor- standard. NCCLS document M27-A. National Committee for Clinical Lab- rectly categorized using the same test conditions utilized for C. 13. Patterson, T. F., S. G. Revankar, W. R. Kirkpatrick, O. Dib, A. W. Fothergill,
glabrata (data not shown). These results suggest that use of the S. W. Redding, D. A. Sutton, and M. G. Rinaldi. 1996. Simple method for
agar screen assay can provide a highly accurate means of rap- detecting fluconazole-resistant yeasts with chromogenic agar. J. Clin. Micro- idly identifying fluconazole-susceptible isolates of Candida biol. 34:1794–1797.
14. Patterson, T. F., W. R. Kirkpatrick, S. G. Revankar, R. K. McAtee, A. W.
spp., with better performance than disk diffusion testing for C. Fothergill, D. I. McCarthy, and M. G. Rinaldi. 1996. Comparative evaluation
glabrata. The routine use of such a screening agar can poten- of macrodilution and chromogenic agar screening for determining flucon- tially decrease antifungal susceptibility testing costs in labora- azole susceptibility of Candida albicans. J. Clin. Microbiol. 34:3237–3239.
15. Pfaller, M. A., D. J. Diekema, R. N. Jones, H. S. Sader, A. C. Fluit, R. J.
tories currently performing macro- or microdilution MIC de- Hollis, and S. A. Messer. 2001. International surveillance of bloodstream
terminations while simultaneously increasing the spectrum of infections due to Candida species: frequency of occurrence and in vitro laboratories able to offer at least a qualitative assessment of susceptibilities to fluconazole, ravuconazole, and voriconazole of isolates collected from 1997 through 1999 in the SENTRY antimicrobial surveillance the susceptibility of Candida spp. to fluconazole.
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bilities of Candida species and distribution of species recovered from blood This research was supported in part by a grant from Pfizer Inc.
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