Abstract
A comparative evaluation of the NCCLS macrodilution method, the E-test, and the Sensititre YeastOne Colorimetric Antifungal Panel for the susceptibility testing of fluconazole, itraconazole, amphotericin B, and flucytosine was conducted with 233 blood isolates of Candida species collected between 1994 and 1998 in Sweden. Antifungal susceptibility profiles of Candida albicans and non-C. albicans Candida species remained essentially unchanged within the 5-year study period. The overall agreement rates for the E-test and the NCCLS MICs and for the YeastOne and the NCCLS MICs were ≥86 and ≥87%, respectively, within ±1 dilution for fluconazole, amphotericin B, and flucytosine, and ≥66 and ≥57%, respectively, for itraconazole. The E-test and the YeastOne panels are equivalent, and both are convenient methods for routine use.
Non-Candida albicans Candida species are frequently isolated from bloodstream infections, although C. albicans remains the most common species (12). Susceptibility to antifungal drugs varies among different species of Candida, which highlights the importance of species identification and antifungal MIC determination (7, 11, 14). With the advent of the NCCLS reference method for antifungal susceptibility testing, it is now possible to compare and evaluate alternative, easier-to-perform methods (6). The commercially available E-test and Sensititre YeastOne antifungal panel have both demonstrated good agreement with the NCCLS method in previous studies (2, 3, 4, 16).
Here we present the first nationwide retrospective epidemiological survey of antifungal susceptibility patterns of Candida species isolated from blood cultures, initiated in 1998 by the Swedish Reference Group for Antimycotics—Methodology. Moreover, we compared the NCCLS procedure with the E-test and the YeastOne antifungal panel in order to evaluate these commercial methods for routine testing of antifungal agents.
Clinical isolates.
Candida species blood isolates collected between 1994 and 1998 were requested from 15 Swedish microbiological laboratories. A total of 499 Candida species isolates (C. albicans, n = 371; non-C. albicans Candida spp., n = 128) were received, and 233 isolates were selected for the study. They comprised C. albicans (n = 123), C. glabrata (n = 52), C. parapsilosis (n = 33), C. tropicalis (n = 11), C. krusei (n = 9), and C. lusitaniae (n = 5). Isolates were identified by standard methods and stored frozen at −70°C until use. Prior to antifungal testing, each isolate was subcultured twice on Sabouraud's dextrose agar (Oxoid). C. krusei ATCC 6258 and C. parapsilosis ATCC 22019 were used as controls.
Susceptibility testing.
Broth macrodilution testing was performed in accordance with the NCCLS M27-A guidelines (6). Antifungal agents were obtained from their respective manufacturers. The final drug concentration ranges were 0.125 to 64 μg/liter for fluconazole, 0.0313 to 64 μg/liter for flucytosine, 0.0313 to 16 μg/liter for itraconazole, and 0.125 to 4 mg/liter for amphotericin B. The E-tests (AB Biodisk, Stockholm, Sweden) were performed in accordance with the manufacturer's instructions. The MIC endpoints were determined after 48 h of incubation at 35°C. Sensititre YeastOne test panels (kindly supplied by AccuMed International Ltd., East Grinstead, United Kingdom) were processed in accordance with the manufacturer's instructions. The plates were incubated at 35°C, and the MICs were read after 24 h if the growth control well was red; otherwise, they were read after 48 h.
Data analysis.
Both on-scale and off-scale MICs were included in the analysis. The low off-scale MICs were left unchanged, and the high off-scale MICs were converted to the next highest concentration.
I studied the antifungal susceptibility patterns of 233 Candida sp. blood isolates cultured between 1994 and 1998 in Sweden. With the NCCLS method, yearly MICs for 50% of the C. albicans and non-C. albicans Candida species studied (MIC50s) remained constant within the 5-year study period (data not shown). Equivalent MIC50s and MIC90s were obtained by all three methods for C. albicans isolates. Compared to those obtained by the NCCLS method, the flucytosine MIC90s and itraconazole MIC50s obtained by the E-test were higher for non-C. albicans isolates. On the other hand, the YeastOne method produced lower fluconazole, itraconazole, and flucytosine MIC50s and MIC90s for non-C. albicans isolates (Table 1).
TABLE 1.
In vitro susceptibilities of Candida blood isolates cultured between 1994 and 1998 to fluconazole, itraconazole, flucytosine, and amphotericin B by the NCCLS macrodilution, E-test, and Sensititre YeastOne methodsa
| Species (no. of isolates) and antifungal drug | NCCLS method
|
E-test
|
YeastOne
|
||||||
|---|---|---|---|---|---|---|---|---|---|
| MIC range | MIC50 | MIC90 | MIC range | MIC50 | MIC90 | MIC range | MIC50 | MIC90 | |
| C. albicans(n = 123) | |||||||||
| Fluconazole | ≤0.125–1 | 0.25 | 0.5 | 0.032–0.5 | 0.25 | 0.25 | <0.125–1 | 0.25 | 0.5 |
| Itraconazole | ≤0.0313–1 | 0.0625 | 0.125 | 0.008–0.5 | 0.125 | 0.25 | <0.008–0.125 | 0.0313 | 0.0625 |
| Amphotericin B | ≤0.125–1 | 0.5 | 0.5 | 0.064–0.5 | 0.5 | 0.5 | 0.016–0.5 | 0.5 | 0.5 |
| Flucytosine | ≤0.0313–>32 | 0.0625 | 0.25 | 0.008–>32 | 0.064 | 0.25 | <0.03–>64 | 0.0625 | 0.125 |
| Non-C. albicans Candida spp. (n = 110) | |||||||||
| Fluconazole | ≤0.125–>64 | 16 | >64 | 0.25–>256 | 16 | >256 | <0.125–256 | 8 | 32 |
| Itraconazole | ≤0.0313–>16 | 0.5 | >16 | 0.016–>32 | 2 | >32 | 0.016–>16 | 0.125 | 1 |
| Amphotericin B | 0.25–1 | 0.5 | 1 | 0.125–1 | 0.5 | 1 | 0.0625–1 | 0.5 | 0.5 |
| Flucytosine | 0.0313–>32 | 0.0625 | 4 | 0.008–>32 | 0.032 | >32 | <0.03–64 | <0.03 | 0.5 |
All values are in micrograms per milliliter.
The overall agreement between the MICs obtained by the E-test and NCCLS macrodilution methods was ≥86% within ±1 dilution for fluconazole, amphotericin B, and flucytosine and ≥66% for itraconazole (Table 2). The overall agreement between the MICs obtained by the YeastOne and NCCLS methods was ≥87% for fluconazole, amphotericin B, and flucytosine and ≥57% for itraconazole. The discrepancies between the YeastOne panel and the NCCLS macrodilution method results consisted mainly of lower itraconazole MICs, which were observed for 187 isolates. E-tests, on the other hand, gave higher itraconazole MICs for 119 isolates.
TABLE 2.
Percent agreement of the E-test and YeastOne methods with the NCCLS reference macrodilution method
| Species (no. of isolates) and antifungal drug | % Agreement
|
|
|---|---|---|
| E-test vs NCCLS method | YeastOne vs NCCLS method | |
| C. albicans (123) | ||
| Fluconazole | 85 | 93 |
| Itraconazole | 72 | 70 |
| Amphotericin B | 97 | 97 |
| Flucytosine | 89 | 94 |
| Non-C. albicans Candida spp. (110) | ||
| Fluconazole | 77 | 78 |
| Itraconazole | 60 | 44 |
| Amphotericin B | 86 | 82 |
| Flucytosine | 84 | 75 |
The categorization of Candida species within the established breakpoints of resistance for fluconazole (MIC, ≥64 mg/liter), itraconazole (MIC ≥1 mg/liter), and flucytosine (MIC ≥32 mg/liter), obtained by the three methods, is given in Table 3. Resistance to fluconazole, itraconazole, and flucytosine was almost entirely accounted for by C. glabrata, C. krusei, and, to a minor extent, C. parapsilosis isolates. Of 233 isolates, 15% were resistant to fluconazole by the NCCLS method, 12 were resistant by the E-test method, and 9% were resistant by the YeastOne method. Itraconazole resistance was found in 23% of the isolates by the NCCLS method, in 28% by the E-test method, and in 13% by the YeastOne method.
TABLE 3.
Categorization of Candida species within the established breakpoints for resistance to fluconazole, itraconazole, and flucytosine
| Species (no. of isolates) and antifungal agent | % of isolates resistant by:
|
||
|---|---|---|---|
| NCCLS method | E-test | YeastOne | |
| C. albicans (123) | |||
| Fluconazole | 0 | 0 | 0 |
| Itraconazole | 1 | 0 | 0 |
| Flucytosine | 1 | 2 | 2 |
| C. glabrata (52) | |||
| Fluconazole | 40 | 25 | 13 |
| Itraconazole | 92 | 98 | 58 |
| Flucytosine | 0 | 0 | 0 |
| C. parapsilosis (33) | |||
| Fluconazole | 15 | 15 | 15 |
| Itraconazole | 3 | 15 | 0 |
| Flucytosine | 12 | 15 | 15 |
| C. tropicalis (11) | |||
| Fluconazole | 0 | 0 | 0 |
| Itraconazole | 0 | 9 | 0 |
| Flucytosine | 9 | 9 | 9 |
| C. krusei (9) | |||
| Fluconazole | 100a | 100a | 100a |
| Itraconazole | 33 | 100 | 0 |
| Flucytosine | 0 | 100 | 0 |
| C. lusitaniae (5) | |||
| Fluconazole | 0 | 0 | 0 |
| Itraconazole | 0 | 0 | 0 |
| Flucytosine | 0 | 0 | 0 |
C. krusei isolates were considered to be intrinsically resistant to fluconazole regardless of the MIC (for six isolates, the MIC was 32 μg/ml, and for three isolates, the MIC was ≥64 μg/ml by the NCCLS method).
I found essentially unchanged antifungal susceptibility profiles of 233 Swedish C. albicans and non-C. albicans Candida sp. bloodstream isolates within the 5-year study period. Constant fluconazole susceptibility among Candida isolates other than C. glabrata and C. krusei was recently also reported in the United States (9). Conversely, Baran et al. found a trend toward slightly increasing fluconazole MICs (1). Our MIC50s for C. albicans agree with those reported in North and South America (7, 10). Aside from C. glabrata, C. krusei, and, to a minor extent, C. parapsilosis, there were almost no fluconazole-, itraconazole-, and flucytosine-resistant isolates in Sweden. This has also been reported in other countries in Europe (8) and in North and Latin America (9, 10). Azole resistance among Swedish C. glabrata isolates was considerably more frequent than the 6.7% fluconazole and 32.8% itraconazole resistance recently reported in the United States (10), while no fluconazole resistance was found among Latin American and Canadian isolates (14).
The performance of both the E-test and the YeastOne panel was comparable to that of the NCCLS reference for C. albicans (2, 3, 5). In general, the E-test tended to give higher MIC50s of flucytosine and itraconazole among non-C. albicans Candida isolates. Similar findings have previously been reported for C. krusei and C. tropicalis isolates, respectively (2, 5). The YeastOne method gave lower fluconazole, itraconazole, and flucytosine MICs, also observed for C. glabrata, C. tropicalis (3), and C. albicans (itraconazole) (4), than the NCCLS method. To et al. previously reported lower amphotericin B, fluconazole, and flucytosine MICs, when comparing the susceptibilities of some Candida species by the Alamar blue method with those obtained with the NCCLS macrodilution method (15), thereby supporting the findings reported here. The reasons for these species-specific discrepancies between the methods tested are not known.
Overall, the agreement between the E-test and NCCLS methods and between the YeastOne panel and the NCCLS method was good (2, 3, 4, 13). However, we found lower agreement for itraconazole, which was in accord with a recent report (5).
No amphotericin B-resistant isolates were identified, although the E-test is claimed to be superior for the detection of less-susceptible isolates (17). The major discrepancy between the non-NCCLS methods concerned the itraconazole resistance of C. glabrata, C. krusei, and C. parapsilosis isolates, which appeared to be resistant by the E-test method but susceptible by the YeastOne method. Both the E-test and YeastOne methods misclassified some C. glabrata isolates that were fluconazole resistant by the NCCLS method as susceptible. This is in contrast to one multicenter study of the E-test method, in which azole-susceptible isolates appeared to be resistant (18).
This is the first comparison of the NCCLS broth macrodilution, E-test, and YeastOne methods for susceptibility testing of Candida species. The E-test is equivalent to the YeastOne panels, and both are simple and convenient methods for routine use. However, because of inconsistency, the results of azole susceptibility testing of C. glabrata, C. krusei, and C. parapsilosis isolates should be confirmed by a reference method.
REFERENCES
- 1.Baran J, Jr, Klauber E, Barczak J, Riederer K, Khatib R. Trends in antifungal susceptibility among Candida sp. urinary isolates from 1994 and 1998. J Clin Microbiol. 2000;38:870–871. doi: 10.1128/jcm.38.2.870-871.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Chen S C, O'Donnell M L, Gordon S, Gilbert G L. Antifungal susceptibility testing using the E test: comparison with the broth macrodilution technique. J Antimicrob Chemother. 1996;37:265–273. doi: 10.1093/jac/37.2.265. [DOI] [PubMed] [Google Scholar]
- 3.Davey K G, Szekely A, Johnson E M, Warnock D W. Comparison of a new commercial colorimetric microdilution method with a standard method for in-vitro susceptibility testing of Candida spp. and Cryptococcus neoformans. J Antimicrob Chemother. 1998;42:439–444. doi: 10.1093/jac/42.4.439. [DOI] [PubMed] [Google Scholar]
- 4.Espinel-Ingroff A, Pfaller M, Messer S A, Knapp C C, Killian S, Norris H A, Ghannoum M A. Multicenter comparison of the Sensititre YeastOne colorimetric antifungal panel with the National Committee for Clinical Laboratory Standards M27-A reference method for testing clinical isolates of common and emerging Candida spp., Cryptococcus spp., and other yeasts and yeast-like organisms. J Clin Microbiol. 1999;37:591–595. doi: 10.1128/jcm.37.3.591-595.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Martín-Mazuelos E, Gutiérrez M J, Aller A I, Bernal S, Martínez M A, Montero O, Quindós G. A comparative evaluation of Etest and broth microdilution methods for fluconazole and itraconazole susceptibility testing of Candida spp. J Antimicrob Chemother. 1999;43:477–481. doi: 10.1093/jac/43.4.477. [DOI] [PubMed] [Google Scholar]
- 6.National Committee for Clinical Laboratory Standards. Reference method for broth dilution antifungal susceptibility testing of yeasts. Approved standard M27-A, vol. 17, no. 9. Wayne, Pa: National Committee for Clinical Laboratory Standards; 1997. [Google Scholar]
- 7.Pfaller M A, Jones R N, Doern G V, Sader H S, Hollis R J, Messer S A.The SENTRY Participant Group. International surveillance of bloodstream infections due to Candida species: frequency of occurrence and antifungal susceptibilities of isolates collected in 1997 in the United States, Canada, and South America for the SENTRY program J Clin Microbiol 1998361886–1889. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Pfaller M A, Jones R N, Doern G V, Fluit A C, Verhoef J, Sader H, Messer S A, Houston A, Coffman S, Hollis R J for The SENTRY Participant Group (Europe) International surveillance of blood stream infections due to Candida species in the European SENTRY program: species distribution and antifungal susceptibility including the investigational triazole and echinocandin agents. Diagn Microbiol Infect Dis. 1999;35:19–25. doi: 10.1016/s0732-8893(99)00046-2. [DOI] [PubMed] [Google Scholar]
- 9.Pfaller M A, Messer S A, Hollis R J, Jones R N, Doern G V, Brandt M E, Hajjeh R A. Trends in species distribution and susceptibility to fluconazole among blood stream isolates of Candida species in the United States. Diagn Microbiol Infect Dis. 1999;33:217–222. doi: 10.1016/s0732-8893(98)00160-6. [DOI] [PubMed] [Google Scholar]
- 10.Pfaller M A, Jones R N, Doern G V, Sader H S, Messer S A, Houston A S, Coffman R, Hollis J The SENTRY Participant Group. Bloodstream infections due to Candida species: SENTRY antimicrobial surveillance program in North America and Latin America, 1997–1998. Antimicrob Agents Chemother. 2000;44:747–751. doi: 10.1128/aac.44.3.747-751.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Sandven P, Bevanger L, Digranes A, Gaustad P, Haukland H, Steinbakk M The Norwegian Yeast Study Group. Constant low rate of fungemia in Norway, 1991 to 1996. J Clin Microbiol. 1998;36:3455–3459. doi: 10.1128/jcm.36.12.3455-3459.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Sandven P. Epidemiology of candidemia. Rev Iberoam Micol. 2000;17:73–81. [PubMed] [Google Scholar]
- 13.Sewell D L, Pfaller M A, Barry A L. Comparison of broth macrodilution, broth microdilution, and E test antifungal susceptibility tests for fluconazole. J Clin Microbiol. 1994;32:2099–2102. doi: 10.1128/jcm.32.9.2099-2102.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.St-Germain G, Lavardière M, Pelletier R, Bourgault A-M, Libman M, Lemieux C, Noel G. Prevalence and antifungal susceptibility of 442 Candida isolates from blood and other normally sterile sites: results of a 2-year (1996 to 1998) multicenter surveillance study in Quebec, Canada. J Clin Microbiol. 2001;39:949–953. doi: 10.1128/JCM.39.3.949-953.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.To W-K A W, Fothergill, Rinaldi M G. Comparative evaluation of macrodilution and Alamar colorimetric microdilution broth methods for antifungal susceptibility testing of yeast isolates. J Clin Microbiol. 1995;33:2660–2664. doi: 10.1128/jcm.33.10.2660-2664.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.van Eldere J, Joosten L, Verhaeghe A, Surmont I. Fluconazole and amphotericin B antifungal susceptibility testing by National Committee for Clinical Laboratory Standards broth macrodilution method compared with E-test and semiautomated broth microdilution test. J Clin Microbiol. 1996;34:842–847. doi: 10.1128/jcm.34.4.842-847.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Wanger A, Mills K, Nelson P W, Rex J H. Comparison of E test and National Committee for Clinical Laboratory Standards broth macrodilution method for antifungal susceptibility testing: enhanced ability to detect amphotericin B-resistant Candida isolates. Antimicrob Agents Chemother. 1995;39:2520–2522. doi: 10.1128/aac.39.11.2520. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Warnock D W, Johnson E M, Rogers T R on behalf of The BSAC Working Party on Antifungal Chemotherapy. Multi-centre evaluation of the E test method for antifungal drug susceptibility testing of Candida spp. and Cryptococcus neoformans. J Antimicrob Chemother. 1998;42:321–331. doi: 10.1093/jac/42.3.321. [DOI] [PubMed] [Google Scholar]