Comparative study of broth macrodilution and microdilution techniques for in vitro antifungal susceptibility testing of yeasts by using the National Committee for Clinical Laboratory Standards' proposed standard.

A comparative study of broth macro- and microdilution methods for susceptibility testing of fluconazole, itraconazole, flucytosine, and amphotericin B was conducted with 273 yeasts. The clinical isolates included 100 Candida albicans, 28 Candida tropicalis, 25 Candida parapsilosis, 15 Candida lusitaniae, 15 Candida krusei, 50 Cryptococcus neoformans var. neoformans, 25 Torulopsis (Candida) glabrata, and 15 Trichosporon beigelii strains. Both methods were performed according to the National Committee for Clinical Laboratory Standards' (NCCLS) recommendations (document M27-P). For fluconazole, itraconazole, and flucytosine, the endpoint was the tube that showed 80% growth inhibition compared with the growth control for the macrodilution method and the well with slightly hazy turbidity (score 1) compared with the growth control for the microdilution method. For amphotericin B, the endpoint was the tube and/or well in which there was absence of growth. For the reference macrodilution method, the MICs were determined after 48 h of incubation for Candida spp., T. glabrata, and T. beigelii and after 72 h for C. neoformans var. neoformans. For the microdilution method, either the first-day MICs (24 h for all isolates other than C. neoformans and 48 h for C. neoformans var. neoformans) or the second-day MICs (48 and 72 h, respectively) were evaluated. The agreement within one doubling dilution of the macrodilution reference for all drugs was higher with the second-day MICs than with the first-day MICs for the microdilution test for most of the tested strains. General agreement was 92% for fluconazole, 85.7% for itraconazole, 98.3% for flucytosine, and 96.4% for amphotericin B. For C. neoformans var. neoformans and T. beigelii, the agreement of the first-day reading was higher than that of the second-day reading for fluconazole (94 versus 92%, respectively, for C. neoformans var. neoformans, and 86.7 versus 80%, respectively, for T. beigelii). Our studies indicate that the microdilution technique performed following the NCCLS guidelines with a second-day reading is a valid alternative method for testing fluconazole, itraconazole, flucytosine, and amphotericin B against these eight species of yeasts.     

Emergence of fluconazole-resistant strains of Candida albicans in patients with recurrent oropharyngeal candidosis and human immunodeficiency virus infection.

After repeated use of fluconazole for therapy of oropharyngeal candidosis, the emergence of in vitro fluconazole-resistant Candida albicans isolates (MIC, > or = 25 micrograms/ml) together with oral candidosis unresponsive to oral dosages of up to 400 mg of fluconazole were observed in patients with human immunodeficiency virus (HIV) infection. Antifungal susceptibility testing was done by broth microdilution and agar dilution techniques on C. albicans isolates recovered from a cohort of patients with symptomatic HIV infection who were treated repeatedly with fluconazole for oropharyngeal candidosis. In vitro findings did show a gradual increase in the MICs for C. albicans isolates recovered from selected patients with repeated episodes of oropharyngeal candidosis. Primary resistance of C. albicans to fluconazole was not seen. Cross-resistance in vitro occurred between fluconazole and other azoles (ketoconazole, itraconazole), but to a lesser extent. The results of the study suggest that the development of clinical resistance to fluconazole could be clearly correlated to in vitro resistance to fluconazole. Itraconazole may still serve as an effective antifungal agent in patients with HIV infection and oropharyngeal candidosis nonresponsive to fluconazole.     

Multisite reproducibility of the Etest MIC method for antifungal susceptibility testing of yeast isolates.

A multicenter study was performed to establish the interlaboratory reproducibility of Etest, to provide an additional comparison of Etest MICs with reference broth macrodilution MICs, and to develop some tentative quality control (QC) guidelines for using Etest for antifungal susceptibility testing of Candida spp. Two QC strains, Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258, were tested by Etest against amphotericin B, fluconazole, flucytosine, itraconazole, and ketoconazole in each of four laboratories. The QC strains were tested 20 times each against the five antifungal agents by using a common lot of RPMI agar. A total of 80 MICs per drug per strain were generated during the study. Overall, 98 to 100% of the MICs fell within a 3 log2 dilution range for the respective yeast-antifungal agent combinations. The level of agreement of Etest MICs with broth macrodilution MICs was 86 to 100% with amphotericin B (C. krusei and C. parapsilosis), itraconazole (C. krusei and C. parapsilosis), flucytosine (C. parapsilosis), and fluconazole (C. parapsilosis). A lower level of agreement was observed with ketoconazole (C. krusei and C. parapsilosis). Although all participants reported identical Etest MICs, the MICs of flucytosine and fluconazole when tested against C. krusei fell well above the upper limits of the reference range for this strain. The tentative QC limits for the two QC strains and five antifungal agents when tested by the Etest methodology are the same as the QC limits when tested by the reference broth macrodilution method for amphotericin B and C. krusei, itraconazole and C. krusei, flucytosine and C. parapsilosis, fluconazole and C. parapsilosis, and itraconazole and C. parapsilosis. The Etest QC ranges are 1 dilution broader (4-dilution range) than the reference macrodilution method QC ranges for ketoconazole and C. krusei, amphotericin B and C. parapsilosis, and ketoconazole and C. parapsilosis.     

Evaluation of a novel colorimetric broth microdilution method for antifungal susceptibility testing of yeast isolates.

A comparative evaluation of two broth microdilution methods for antifungal susceptibility testing of 600 clinical yeast isolates (Candida spp., Torulopsis glabrata, and Cryptococcus neoformans) against amphotericin B, fluconazole, and flucytosine (5FC) was conducted. Microdilution testing was performed according to National Committee for Clinical Laboratory Standards (NCCLS) recommendations (NCCLS document M27-P). By using the growth control for comparison, reference microdilution MIC endpoints for amphotericin B were scored as the lowest concentration at which a score of 0 (complete absence of growth) was observed, and those for 5FC and fluconazole were scored at the lowest concentration at which a score of 2 (prominent decrease in turbidity) (MIC-2) was observed. The second microdilution method employed a colorimetric endpoint using an oxidation-reduction indicator (Alamar Biosciences, Inc., Sacramento, Calif.) and was assessed independently of the reference microdilution MICs. The MICs for the two microdilution test systems were read after 24 and 48 h of incubation. Excellent agreement between the reference and colorimetric microdilution MICs was observed. Overall agreement was > or = 95% for all three drugs at 24 h. At 48 h, agreement was > or = 98% for amphotericin B and 5FC but dropped to 84% for fluconazole. Given these results it appears that the colorimetric microdilution approach to antifungal susceptibility testing may be viable alternative to the NCCLS reference method for testing yeasts.     

Comparison of visual and spectrophotometric methods of MIC endpoint determinations by using broth microdilution methods to test five antifungal agents, including the new triazole D0870.

A study to compare three different methods for reading MIC endpoints tested by the broth microdilution modification of the National Committee for Clinical Laboratory Standards (Villanova, Pa.) reference method was conducted. MICs of amphotericin B, flucytosine, fluconazole, itraconazole, and a new triazole, D0870, were determined for five reference yeast strains and 100 clinical isolates of Candida spp. MICs were read visually according to National Committee for Clinical Laboratory Standards guidelines from microdilution trays that had been (VS) and had not been (V) shaken. MICs were also determined spectrophotometrically (SP) at 492 nm. SP endpoints were determined as the concentrations resulting in a > or = 50% inhibition of growth (flucytosine and azoles) and a > or = 90% inhibition of growth (amphotericin B) relative to control growth. The five reference strains were tested nine times each against all five antifungal agents, and the MIC results for each reading method were compared with a 3-log2 dilution reference range determined by the macrodilution (M27-P) method. Overall, 84 to 100% of the MICs determined by V, 93 to 100% of those determined by VS, and 89 to 100% of those determined by SP fell within the 3-log2 dilution reference range for each reference strain and antifungal agent. Reproducibility was 99% for V and SP and 98% for VS. Agreement among the three methods of reading ranged from 97 to 99%. Excellent agreement among reading methods was also observed for all antifungal agents when tested against 100 clinical isolates.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro activities of voriconazole and five licensed antifungal agents against Candida dubliniensis: comparison of CLSI M27-A2, Sensititre YeastOne, disk diffusion, and Etest methods

We compared the in vitro activity of six antifungal agents against 62 isolates of Candida dubliniensis by the Clinical Laboratory Standards Institute (CLSI [formerly National Committee for the Clinical Laboratory Standards]) M27-A2, Sensititre YeastOne, disk diffusion, and Etest methods and we studied the effect of the time of reading. For the azoles, voriconazole was the most potent in vitro followed by fluconazole, ketoconazole, and itraconazole. All the isolates were susceptible to amphotericin B and flucytosine. The highest rate of resistance was obtained against itraconazole with a high number of isolates defined as susceptible dose-dependent. At 24 hr, 100% of the isolates were susceptible to ketoconazole, amphotericin B, and flucytosine, 98% susceptible to voriconazole and fluconazole, and 95% for itraconazole. At 48 hr, 100% of the isolates remained susceptible for flucytosine and amphotericin B, 95% for voriconazole, 93% for fluconazole, 90% for ketoconazole, and 82% for itraconazole. The agreement between the CLSI and the other methods was better at 24 than 48 hr.     

Variations in fluconazole susceptibility and electrophoretic karyotype among oral isolates of Candida albicans from patients with AIDS and oral candidiasis.

DNA subtyping by pulsed-field gel electrophoresis and in vitro susceptibility testing were used to study strain variation and fluconazole resistance in Candida albicans isolates from patients with AIDS undergoing azole (fluconazole and clotrimazole) therapy for oropharyngeal candidiasis. A total of 29 patients suffered 71 episodes of oropharyngeal candidiasis. Overall, 121 isolates of C. albicans recovered throughout the course of treatment of each infection were available for further characterization. DNA subtyping revealed a total of 61 different DNA subtypes. In vitro susceptibility testing of the 121 isolates by using proposed standard methods of the National Committee for Clinical Laboratory Standards revealed MICs of fluconazole ranging from < or = 0.125 to > 64 micrograms/ml. The MIC for 50% of isolates tested was 0.25 microgram/ml, and the MIC for 90% of isolates tested was 8.0 micrograms/ml. MICs were > or = 64 micrograms/ml for only 7.4% of the isolates tested. The majority (62%) of the patients with oropharyngeal candidiasis and undergoing azole therapy were infected or colonized with more than one DNA subtype, and the introduction or selection of strains with a more resistant DNA subtype during the course of fluconazole therapy was not uncommon. With one exception, this did not appear to have an adverse effect on clinical outcome. In contrast, for patients with AIDS and oropharyngeal candidiasis infected with a single DNA subtype of C. albicans, an increase in fluconazole MICs for the infecting strain was rarely demonstrated over the course of therapy.     

Comparison of Etest and National Committee for Clinical Laboratory Standards broth macrodilution method for azole antifungal susceptibility testing.

The use of Etest strips for antimicrobial susceptibility testing is a new and promising method with broad applications in microbiology. Since these strips contain a predefined continuous gradient of a drug, it is possible to obtain a reproducible, quantitative MIC reading. We performed a prospective and double-blinded study to compare the Etest and National Committee for Clinical Laboratory Standards (Villanova, Pa.) broth macrodilution methods for determining the MICs of fluconazole, itraconazole, and ketoconazole for 100 clinical isolates (25 Candida albicans, 25 Cryptococcus neoformans var. neoformans, 20 Torulopsis [Candida] glabrata, 15 Candida tropicalis, and 15 Candida parapsilosis). The Etest method was performed according to the manufacturer's instructions, and the reference method was performed according to National Committee for Clinical Laboratory Standards document M27-P guidelines. Despite differences between results for some species-drug combinations, Etest and macrobroth MICs were, in general, in good agreement. The MIC agreement rates for the two methods, within +/- 1 dilution, were 71% for ketoconazole, 80% for fluconazole, and 84% for itraconazole. According to our data, Etest has potential utility as an alternative method.     

Use of fatty acid RPMI 1640 media for testing susceptibilities of eight Malassezia species to the new triazole posaconazole and to six established antifungal agents by a modified NCCLS M27-A2 microdilution method and Etest

A novel formulation of RPMI 1640 medium for susceptibility testing of Malassezia yeasts by broth microdilution (BMD) and Etest is proposed. A modification of the NCCLS M27-A2 BMD method was used to test 53 isolates of Malassezia furfur (12 isolates), M. sympodialis (8 isolates), M. slooffiae (4 isolates), M. globosa (22 isolates), M. obtusa (2 isolates), M. restricta (2 isolates), M. pachydermatis (1 isolates), and M. dermatis (2 isolates) against amphotericin B, ketoconazole, itraconazole, fluconazole, voriconazole, terbinafine, and posaconazole by BMD and Etest. RPMI and antibiotic medium 3 (AM3) were supplemented with glucose, bile salts, a mixture of fatty acids, and n-octadecanoate fatty acids and Tween 20. M. furfur ATCC 14521 and M. globosa ATCC 96807 were used as quality control strains. Depending on the species, MICs were read after 48 or 72 h of incubation at 32 degrees C. Low azole and terbinafine MICs were recorded for all Malassezia species, whereas amphotericin B displayed higher MICs (>/=16 microg/ml) against M. furfur, M. restricta, M. globosa, and M. slooffiae strains, which were AM3 confirmed. Agreement of the two methods was 84 to 97%, and intraclass correlation coefficients were statistically significant (P < 0.001). Because of higher amphotericin B MICs provided by Etest for strains also displaying high BMD MICs (>/=1 microg/ml), agreement was poorer. The proposed media are used for the first time and can support optimum growth of eight Malassezia species for recording concordant BMD and Etest MICs.     

Comparison of a 2,3-Bis(2-Methoxy-4-Nitro-5-Sulfophenyl)-5-[(Phenylamino)Carbonyl]-2H-Tetrazolium Hydroxide (XTT) Colorimetric Method with the Standardized National Committee for Clinical Laboratory Standards Method of Testing Clinical Yeast Isolates for Susceptibility to Antifungal Agents

MICs for clinical Candida and Cryptococcus isolates were determined by a method incorporating the colorimetric indicator 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT), and the results were compared with MICs obtained by the National Committee for Clinical Laboratory Standards approved standard method (M27-A). One hundred percent of all isolates demonstrated agreement within 2 dilutions between the MICs of amphotericin B, fluconazole, itraconazole, ketoconazole, and flucytosine obtained by the two methods. These data suggest that an XTT-based method could provide a useful means for the determination of antifungal susceptibility of yeasts.     

Comparison study of broth macrodilution and microdilution antifungal susceptibility tests.

An evaluation of broth dilution antifungal susceptibility tests was performed by determining both the micro- and macrodilution MICs of amphotericin B, flucytosine, fluconazole, ketoconazole, and cilofungin against 38 isolates of Candida albicans, Candida lusitaniae, Candida parapsilosis, Candida tropicalis, Cryptococcus neoformans, and Torulopsis glabrata. The following preliminary antifungal working group recommendations of the National Committee for Clinical Laboratory Standards for broth macrodilution tests with antifungal agents were used: inocula standardized to 1 x 10(4) to 5 x 10(4) CFU/ml with a spectrophotometer, RPMI 1640 medium buffered with morpholinopropanesulfonic acid (pH 7.0), incubation at 35 degrees C for 24 to 48 h, and an additive drug dilution procedure. Broth microdilution MICs were higher (two or more dilutions) than broth macrodilution MICs for all isolates tested with amphotericin B and for most isolates tested with ketoconazole, fluconazole, and cilofungin. MICs of flucytosine were the same by both techniques or lower by the broth microdilution test except in tests with C. neoformans. However, the only statistically significant differences between the two tests were observed with amphotericin B against all isolates (P = 0.01 to 0.07), ketoconazole against C. neoformans (P = 0.01 to 0.02), and cilofungin against C. albicans (P = 0.05 to 0.14). Tests performed with less dense inocula (1 x 10(3) to 5 x 10(3] produced similar results.     

Interpretation of Trailing Endpoints in Antifungal Susceptibility Testing by the National Committee for Clinical Laboratory Standards Method

Trailing endpoints remain a problem in antifungal susceptibility testing using the National Committee for Clinical Laboratory Standards (NCCLS) method. For isolates for which trailing endpoints are found, MICs of ≤1 μg/ml at 24 h and of >64 μg/ml at 48 h are usually observed. In a study of human immunodeficiency virus (HIV)-infected patients with oropharyngeal candidiasis, we identified three patients with multiple serial isolates for which trailing endpoints were observed with fluconazole. At 24 h, MICs were generally ≤1 μg/ml by both broth macro- and microdilution methods. However, at 48 h, MICs were >64 μg/ml, while the organism remained susceptible by agar dilution testing with fluconazole. Most episodes of oropharyngeal candidiasis with trailing-endpoint isolates responded to doses of fluconazole as low as 100 mg/day. Two patients had both susceptible and trailing-endpoint isolates by NCCLS broth macro- and microdilution testing; these isolates were found to be the same strain by pulsed-field gel electrophoresis using restriction fragment length polymorphisms. Another patient had two different strains, one for which trailing endpoints were observed and one which was susceptible at 48 h. Trailing endpoints may be seen with selected isolates of a strain or may be a characteristic finding for most or all isolates of a strain. In addition, with isolates for which trailing endpoints are observed, reading the endpoint for the NCCLS method at 24 h may be more appropriate.     

Interlaboratory evaluation of Etest method for testing antifungal susceptibilities of pathogenic yeasts to five antifungal agents by using Casitone agar and solidified RPMI 1640 medium with 2% glucose.

An interlaboratory evaluation (two centers) of the Etest method was conducted for testing the antifungal susceptibilities of yeasts. The MICs of amphotericin B, fluconazole, flucytosine, itraconazole, and ketoconazole were determined for 83 isolates of Candida spp., Cryptococcus neoformans, and Torulopsis glabrata. Two buffered (phosphate buffer) culture media were evaluated: solidified RPMI 1640 medium with 2% glucose and Casitone agar. MIC endpoints were determined after both 24 and 48 h of incubation at 35 degrees C. Analysis of 3,420 MICs demonstrated higher interlaboratory agreement (percentage of MIC pairs within a 2-dilution range) with Casitone medium than with RPMI 1640 medium when testing amphotericin B (84 to 90% versus 1 to 4%), itraconazole (87% versus 63 to 74%), and ketoconazole (94 to 96% versus 88 to 90%). In contrast, better interlaboratory reproducibility was determined between fluconazole MIC pairs when RPMI 1640 medium rather than Casitone medium was used (96 to 98% versus 77 to 90%). Comparison of the flucytosine MICs obtained with RPMI 1640 medium revealed greater than 80% reproducibility. The study suggests the potential value of the Etest as a convenient alternative method for testing the susceptibilities of yeasts. It also indicates the need for further optimization of medium formulations and MIC endpoint criteria to improve interlaboratory agreement.     

Interlaboratory evaluation of the Etest for antifungal susceptibility testing of dermatophytes.

A three-site interlaboratory reproducibility evaluation of the Etest concentration gradient strip method for testing antifungal susceptibilities was conducted using 30 strains of dermatophytes exposed to strips loaded with ketoconazole (KTZ), itraconazole (ITZ), amphotericin B (AMB) and fluconazole (FCZ). Etest minimal inhibitory concentrations were compared with those obtained using a broth microdilution method. All isolates produced clearly detectable growth at 28 degrees C within 72-96 h for reading with the Etest method. The highest interlaboratory agreement between Etest and the microdilution method was shown with FCZ (94%), and the lowest was seen with KTZ (60%). Overall, agreement between the Etest and microdilution method was variable. It was excellent for AMB (97%), good for ITZ (80%) and KTZ (77%), and low for fluconazole (27%).     

Quality control guidelines for National Committee for Clinical Laboratory Standards--recommended broth macrodilution testing of ketoconazole and itraconazole.

Ketoconazole and itraconazole were tested in a multilaboratory study to establish quality control (QC) guidelines for yeast antifungal susceptibility testing. Two isolates that had been previously identified as QC isolates for amphotericin B, fluconazole, and flucytosine (Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258) were tested in accordance with the National Committee for Clinical Laboratory Standards M27-P guidelines. Each isolate was tested 20 times with the two antifungal agents in the five laboratories by using a lot of RPMI 1640 unique to each laboratory as well as a lot common to all five laboratories, thus generating 200 MICs per drug per organism. Overall, 96 to 99% of the MICs for each drug fell within the desired 3-log2 dilution range (mode +/- 1 log2 dilution). By using these data, 3-log2 dilution QC ranges encompassing 98% of the observed MICs for three of the organism-drug combinations and 94% of the observed MICs for the fourth combination were established. These QC ranges are 0.064 to 0.25 micrograms/ml for both ketoconazole and itraconazole against C. parapsilosis ATCC 22019 and 0.125 to 0.5 micrograms/ml for both ketoconazole and itraconazole against C. krusei ATCC 6258.     

Fluconazole- and itraconazole-resistant Candida albicans strains from AIDS patients: multilocus enzyme electrophoresis analysis and antifungal susceptibilities.

Multilocus enzyme electrophoresis and in vitro susceptibility testing with a broth microdilution method were used to analyze Candida albicans strain diversity in four AIDS patients with recurrent oropharyngeal candidiasis who successively developed clinical resistance to fluconazole (FCZ) and itraconazole (ITZ). One to ten colonies per sample were randomly chosen from oral washings collected before the initial FCZ treatment and just before every other antifungal treatment; a total of 98 isolates were analyzed. Multilocus enzyme electrophoresis analysis revealed 14 different electrophoretic types (ETs). Statistical analysis of genetic distances showed that C. albicans isolates clustered into five subpopulations (I to V). In each subpopulation, isolates are closely related, and genetic distances between subpopulations I to IV are short. In contrast, subpopulation V, which contained isolates typed as ET8 and ET14, is strongly divergent from the others; these isolates may represent atypical C. albicans isolates. Only one patient was infected with a single strain during the course of azole therapy; for the three remaining patients, variants of the same strain and different strains were concurrently isolated. Clinical FCZ resistance was clearly correlated with in vitro data for three patients. Moreover, MICs of ITZ increased during FCZ therapy, and MICs of ITZ which were > or = 1.56 micrograms/ml were found when clinical ITZ resistance occurred; isolates from subpopulation V showed the highest MICs of ITZ. Because of the emergence of clinical ITZ resistance after clinical FCZ resistance, the feasibility of long-term azole therapy for mucosal candidiasis in AIDS patients is questioned.     

Candida nivariensis isolated from an Indonesian human immunodeficiency virus-infected patient suffering from oropharyngeal candidiasis

Candida nivariensis was isolated from an Indonesian human immunodeficiency virus-infected patient who suffered from oropharyngeal candidiasis and was identified with molecular tools. Our isolate demonstrated low MICs to amphotericin B, flucytosine, posaconazole, caspofungin, and isavuconazole and was susceptible to fluconazole, itraconazole, and voriconazole.     

Evaluation of Etest for direct antifungal susceptibility testing of yeasts in positive blood cultures

The performance of the Etest (AB BIODISK, Solna, Sweden) for direct antifungal susceptibility testing of yeasts in positive blood cultures was compared with that of the macrodilution method for determining the MICs of five antifungal agents. Culture broths with blood from bottles positive for yeasts were inoculated directly onto plates for susceptibility testing with the Etest, and the MICs were read after 24 and 48 h of incubation. A total of 141 positive blood cultures (72 cultures of Candida albicans, 31 of Candida tropicalis, 14 of Candida glabrata, 11 of Candida parapsilosis, 3 of Candida krusei, and 3 of Cryptococcus neoformans, 4 miscellaneous yeast species, and 3 mixed cultures) were tested, and the rates of MIC agreement (+/-1 log(2) dilution) between the direct Etest (at 24 and 48 h, respectively) and macrodilution methods were as follows: amphotericin B, 81.8 and 93.5%; flucytosine, 84.8 and 87.7%; fluconazole, 89.4 and 85.5%; itraconazole, 69.7 and 63.8%; ketoconazole, 87.9 and 79.0%. By a large-sample t test, the difference in log(2) dilution between the direct Etest and the macrodilution method was found to be small (P < 0.05). The lone exceptions were ketoconazole at 48 h of incubation and itraconazole at both 24 and 48 h of incubation (P > 0.05). By Tukey's multiple comparisons, the difference between the direct Etest (48 h) and reference methods among different species was found to be less than 1 log(2) dilution. When the MICs were translated into interpretive susceptibility, the minor errors caused by the direct Etest (at 24 and 48 h, respectively) were as follows: flucytosine, 2.3 and 1.4%; fluconazole, 3.0 and 3.6%; itraconazole, 21.2 and 21.3%. Itraconazole also produced an additional 3.0 and 3.6% major errors as determined by the direct Etest at 24 and 48 h, respectively. It was concluded that, except for itraconazole, the Etest method was feasible for direct susceptibility testing of blood cultures positive for yeasts. The method is simple, and the results could be read between 24 and 48 h after direct inoculation, whenever the inhibition zones were discernible.     

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

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 > or =86 and > or =87%, respectively, within +/-1 dilution for fluconazole, amphotericin B, and flucytosine, and > or =66 and > or =57%, respectively, for itraconazole. The E-test and the YeastOne panels are equivalent, and both are convenient methods for routine use.     

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

National Committee for Clinical Laboratory Standards (NCCLS) standard guidelines are available for the antifungal susceptibility testing of common Candida spp. and Cryptococcus neoformans, but NCCLS methods may not be the most efficient and convenient procedures for use in the clinical laboratory. MICs of amphotericin B, fluconazole, flucytosine, itraconazole, and ketoconazole were determined by the commercially prepared Sensititre YeastOne Colorimetric Antifungal Panel and by the NCCLS M27-A broth microdilution method for 1,176 clinical isolates of yeasts and yeast-like organisms, including Blastoschizomyces capitatus, Cryptococcus spp., 14 common and emerging species of Candida, Hansenula anomala, Rhodotorula spp., Saccharomyces cerevisiae, Sporobolomyces salmonicolor, and Trichosporon beigelii. Colorimetric MICs of amphotericin B corresponded to the first blue well (no growth), and MICs of the other agents corresponded to the first purple or blue well. Three comparisons of MIC pairs by the two methods were evaluated to obtain percentages of agreement: 24- and 48-h MICs and 24-h colorimetric versus 48-h reference MICs. The best performance of the YeastOne panel was with 24-h MICs (92 to 100%) with the azoles and flucytosine for all the species tested, with the exception of C. albicans (87 to 90%). For amphotericin B, the best agreement between the methods was with 48-h MIC pairs (92 to 99%) for most of the species tested. The exception was for isolates of C. neoformans (76%). These data suggest the potential value of the YeastOne panel for use in the clinical laboratory.