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Journal of Clinical Microbiology, November 2001, p. 4181-4183, Vol. 39, No. 11
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.11.4181-4183.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Trends in Antifungal Susceptibility among Swedish Candida Species Bloodstream Isolates from 1994 to 1998: Comparison of the E-test and the Sensititre YeastOne Colorimetric Antifungal Panel with the NCCLS M27-A Reference Method

Department of Clinical Microbiology, Karolinska Hospital, Stockholm, Sweden

Received 16 April 2001/Returned for modification 30 July 2001/Accepted 30 August 2001

	ABSTRACT

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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.

	TEXT

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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 AntimycoticsMethodology. 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).

View this table: [in this window] [in a new window]   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

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.

View this table: [in this window] [in a new window]   TABLE 2.   Percent agreement of the E-test and YeastOne methods with the NCCLS reference macrodilution method

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.

View this table: [in this window] [in a new window]   TABLE 3.   Categorization of Candida species within the established breakpoints for resistance to fluconazole, itraconazole, and flucytosine

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.

	FOOTNOTES

^* Mailing address: Department of Clinical Microbiology, L202 Karolinska Hospital, S-171 76 Stockholm, Sweden. Phone: (46) 851773566. Fax: (46) 8308099. E-mail: Erja.Chryssanthou{at}ks.se.

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