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.     

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.     

Comparative evaluation of macrodilution and alamar colorimetric microdilution broth methods for antifungal susceptibility testing of yeast isolates.

A comparative evaluation of the macrodilution method and the Alamar colorimetric method for the susceptibility testing of amphotericin B, fluconazole, and flucytosine was conducted with 134 pathogenic yeasts. The clinical isolates included 28 Candida albicans, 17 Candida tropicalis, 15 Candida parapsilosis, 12 Candida krusei, 10 Candida lusitaniae, 9 Candida guilliermondii, 18 Torulopsis glabrata, and 25 Cryptococcus neoformans isolates. The macrodilution method was performed and interpreted according to the recommendations of the National Committee for Clinical Laboratory Standards (document M27-P), and the Alamar colorimetric method was performed according to the manufacturer's instructions. For the Alamar colorimetric method, MICs were determined at 24 and 48 h of incubation for Candida species and T. glabrata and at 48 and 72 h of incubation for C. neoformans. The overall agreement within +/- 1 dilution for Candida species and T. glabrata against the three antifungal agents was generally good, with the values for amphotericin B, fluconazole, and flucytosine being 85.3, 77.9, and 86.2%, respectively, at the 24-h readings and 69.3, 65.2, and 97.2%, respectively, at the 48-h readings. Most disagreement was noted with fluconazole against C. tropicalis and T. glabrata. Our studies indicate that determination of MICs at 24 h by the Alamar colorimetric method is a valid alternate method for testing amphotericin B, fluconazole, and flucytosine against Candida species but not for testing fluconazole against C. tropicalis and T. glabrata. For flucytosine, much better agreement can be demonstrated against Candida species and T. glabrata at the 48-h readings by the Alamar method.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative evaluation of Etest and sensititre yeastone panels against the Clinical and Laboratory Standards Institute M27-A2 reference broth microdilution method for testing Candida susceptibility to seven antifungal agents

To assess their utility for antifungal susceptibility testing in our clinical laboratory, the Etest and Sensititre methods were compared with the Clinical and Laboratory Standards Institute (CLSI) M27-A2 reference broth microdilution method. Fluconazole (FL), itraconazole (I), voriconazole (V), posaconazole (P), flucytosine (FC), caspofungin (C), and amphotericin B (A) were tested with 212 Candida isolates. Reference MICs were determined after 48 h of incubation, and Etest and Sensititre MICs were determined after 24 h and 48 h of incubation. Overall, excellent essential agreement (EA) between the reference and test methods was observed for Etest (95%) and Sensititre (91%). Etest showed an >or=92% EA for MICs for all drugs tested; Sensititre showed a >or=92% EA for MICs for I, FC, A, and C but 82% for FL and 85% for V. The overall categorical agreement (CA) was 90% for Etest and 88% for Sensititre; minor errors accounted for the majority of all categorical errors for both systems. Categorical agreement was lowest for Candida glabrata and Candida tropicalis with both test systems. Etest and Sensititre provided better CA at 24 h compared to 48 h for C. glabrata; however, CA for C. glabrata was <80% for FL with both test systems despite MIC determination at 24 h. Agreement between technologists for both methods was >or=98% for each agent against all organisms tested. Overall, Etest and Sensititre methods compared favorably with the CLSI reference method for determining the susceptibility of Candida. However, further evaluation of their performance for determining the MICs of azoles, particularly for C. glabrata, is warranted.     

Evaluation of a capacitance method for direct antifungal susceptibility testing of yeasts in positive blood cultures

The feasibility of using a capacitance method (CM) for direct antifungal susceptibility testing of yeasts in positive blood cultures was evaluated. The CM used the same test conditions as those recommended by the National Committee for Clinical Laboratory Standards. After direct inoculation of positive culture broths into module wells (Bactometer; bioMérieux, Inc., Hazelwood, Mo.), the end-point determination was made by monitoring the capacitance change in the culture broths with Bactometer. The MIC of amphotericin B was the lowest concentration at which yeast growth was completely inhibited, while the MICs of ketoconazole, flucytosine, and fluconazole were the concentrations at which a >/=80% reduction in capacitance change was observed. The MICs of the four drugs against each blood isolate obtained on subculture plates were also determined by the macrodilution method. For 51 positive blood cultures tested, the percent agreement (+/-2 log(2) dilutions) between the CM and the macrodilution method were as follows: amphotericin B (98%), ketoconazole (92%), flucytosine (84%), and fluconazole (96%). The CM was further used for breakpoint susceptibility testing of fluconazole (8 and 64 microg/ml) and flucytosine (4 and 32 microg/ml) against yeasts in positive blood cultures. After testing of 74 specimens by the CM, flucytosine and fluconazole produced one (1.4%) major error and two (2.8%) minor errors, respectively. All yeasts that displayed resistance to flucytosine or fluconazole were detected within 24 h after direct inoculation of the positive broths into Bactometer. The CM may be useful for the rapid detection of antifungal resistance in positive blood cultures containing yeasts.     

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.     

Preliminary evaluation of a semisolid agar antifungal susceptibility test for yeasts and molds

This report presents a semisolid agar antifungal susceptibility (SAAS) method for the rapid susceptibility screening of yeasts and molds. The reproducibility and accuracy of the SAAS method were assessed by comparing the MICs of amphotericin B and fluconazole obtained for 10 candidate quality control (QC) American Type Culture Collection yeast strains in >/=15 replicates with those found by six independent laboratories using the National Committee for Clinical Laboratory Standards (NCCLS) M27-P broth macrodilution method (M. A. Pfaller et al., J. Clin. Microbiol. 33:1104-1107, 1995). Overall, 96% of MICs for both drugs fell within 1 log(2) dilution of the modal MIC for each strain. The MICs for amphotericin B showed 99% agreement with the NCCLS proposed QC ranges within 1 log(2) dilution. Likewise, the MICs for fluconazole at >/=75% growth reduction showed 99% agreement for seven strains. Three strains, Candida albicans ATCC 24333 and ATCC 76615 and Candida tropicalis ATCC 750, showed a less sharp fluconazole endpoint at >/=75% growth reduction, but at >50% growth reduction, the agreement was 98% within 1 log(2) dilution of the proposed range. The MIC agreement within the proposed range for the suggested QC strains Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258 was 100% for fluconazole and 100% within 1 log(2) dilution of the proposed range for amphotericin B. The SAAS method demonstrated the susceptibility or resistance of 25 clinical isolates of filamentous fungi such as Aspergillus fumigatus to amphotericin B, itraconazole, and fluconazole, usually within 48 h. Although the results are preliminary, this SAAS method is promising as a rapid and cost-effective screen and is worthy of concerted investigation.     

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.     

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.     

Comparison of the Etest and the sensititre colorimetric methods with the NCCLS proposed standard for antifungal susceptibility testing of Aspergillus species

The susceptibilities of 25 clinical isolates of Aspergillus fumigatus, A. flavus, A. terreus, A. nidulans, and A. ustus to itraconazole and amphotericin B were determined by an agar diffusion-dilution method (the Etest method) and a colorimetric broth microdilution method (the Sensititre method); and the results were compared with those obtained by the NCCLS proposed standard M-38P method for antifungal susceptibility testing of filamentous fungi. Various MIC endpoints for the three methods were determined visually by four different observers in three blinded experiments, and the reproducibilities among the observers (interobserver agreement) and among the replicates (interexperimental agreement) as well as the levels of agreement between the NCCLS, the Etest, and the Sensititre methods were calculated. High levels of reproducibility (within 1 twofold dilution) were found for the NCCLS method (>95%) with the MIC-0 endpoint (complete inhibition of growth) for both drugs and with the MIC-1 endpoint (slight growth) for itraconazole and for the Sensititre method (>90%) with all MIC endpoints, although for the latter the interexperimental agreement for itraconazole was comparatively lower (83 to 93%). The Etest method was less reproducible (67 to 87%) for both drugs. Using the recommended MIC endpoints, high levels of agreement (within one twofold dilution) between the NCCLS and the Sensititre methods for all species were found for amphotericin B (>77%) but not for itraconazole (<66%), for which the MICs by the Sensititre method were up to 3 twofold dilutions lower than the corresponding MICs by the NCCLS method. The use of the first blue well as an endpoint for the Sensititre method and 48 h of incubation improved the levels of agreement with the NCCLS method. Low levels of agreement between the NCCLS and the Etest methods using the recommended MIC endpoints were found for most species, especially after 48 h of incubation (<50%), when the MICs obtained by the Etest method were up to 9 twofold dilutions higher than the corresponding MICs obtained by the NCCLS method. Relatively better agreement was found after 24 h, although it was species dependent, with the highest levels of agreement (>82%) found for A. terreus and A. ustus for amphotericin B and A. fumigatus for both drugs. Overall, better agreement was found when MIC-0 was used as the MIC endpoint for the NCCLS method for both drugs and when the MICs by the Etest method were determined after 48 h of incubation for itraconazole and after 24 h of incubation for amphotericin B.     

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.     

Comparison of broth macrodilution, broth microdilution, and E test antifungal susceptibility tests for fluconazole.

A comparison of the E test, the broth microdilution test, and the reference broth macrodilution susceptibility test of the National Committee for Clinical Laboratory Standards for fluconazole susceptibility testing was performed with 238 clinical isolates of Candida species and Torulopsis (Candida) glabrata. An 80% inhibition endpoint MIC was determined by the reference broth macrodilution method after 48 h of incubation. The MICs obtained by the two study methods were read after 24 and 48 h of incubation. Overall, excellent agreement within 2 doubling dilutions was obtained between the broth microdilution and the broth macrodilution methods for the combined results for all species at both 24 h (93%) and 48 h (94%). The correlation of 24-h MIC endpoints between the E test and the broth macrodilution methods was 37% for T. glabrata, 56% for Candida tropicalis, 93% for Candida albicans, and 90% for other Candida species. The percent agreement at 48 h ranged from 34% for T. glabrata to 97% for Candida species other than C. albicans and C. tropicalis. These initial results support the further evaluation of the E test as an alternative method for fluconazole susceptibility testing of Candida species.     

In vitro susceptibility testing of filamentous fungi: comparison of Etest and reference microdilution methods for determining itraconazole MICs

The performance of the Etest for itraconazole susceptibility testing of 50 isolates of filamentous fungi was assessed in comparison with the National Committee for Clinical Laboratory Standards (NCCLS) proposed standard microdilution broth method. The NCCLS method employed RPMI 1640 broth medium, and MICs were read after incubation for 48 h at 35 degrees C. Etest MICs were determined with RPMI agar containing 2% glucose and with Casitone agar and were read after incubation for 24 h (Aspergillus spp. and Rhizopus spp.) and 48 h (all species except Rhizopus spp.) at 35 degrees C. The isolates included Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Aspergillus terreus, Fusarium spp., Pseudallescheria boydii, Rhizopus spp., Paecilomyces variotii, and an Acremonium sp. Overall agreement between Etest and microdilution MICs was 96% with RPMI agar and 80% with Casitone agar. The agreement was 100% for all species except Rhizopus spp. (83%) and Paecilomyces varioti (0%) with RPMI agar. When Casitone agar was used, the agreement ranged from 50% with Rhizopus spp. to 100% with Fusarium spp., P. boydii, P. varioti, and an Acremonium sp. Notably, for Aspergillus spp., the agreement between itraconazole Etest MICs read at 24 h and reference microdilution MICs read at 48 h was 100% with both RPMI and Casitone agar. Both media supported the growth of all filamentous fungi tested. Where a discrepancy was observed between Etest and the reference method, the Etest MIC was generally higher. The Etest method using RPMI agar appears to be a useful method for determining itraconazole susceptibilities of Aspergillus spp. and other filamentous fungi.     

In vitro susceptibility testing of Aspergillus spp.: comparison of Etest and reference microdilution methods for determining voriconazole and itraconazole MICs

The performance of the Etest for voriconazole and for itraconazole susceptibility testing of 376 isolates of Aspergillus spp. was assessed in comparison with the National Committee for Clinical Laboratory Standards (NCCLS) proposed standard microdilution broth method. The NCCLS method employed RPMI 1640 broth medium, and MICs were read after incubation for 48 h at 35 degrees C. Etest MICs were determined with RPMI agar containing 2% glucose and were read after incubation for 48 h at 35 degrees C. The isolates included A. fumigatus, A. flavus, A. niger, A. terreus, A. versicolor, A. glaucus, A. nidulans, A. ustus, and A. sydowii. Overall agreement percentages between the Etest and microdilution MICs were 97.6% for voriconazole and 95.8% for itraconazole. Where a discrepancy was observed between Etest and the reference method, the Etest tended to give lower values with voriconazole and higher values with itraconazole. The Etest method using RPMI agar appears to be a useful method for determining the voriconazole and itraconazole susceptibilities of Aspergillus spp.     

Influence of glucose supplementation and inoculum size on growth kinetics and antifungal susceptibility testing of Candida spp

The influences of inoculum size and glucose supplementation on the growth kinetics of 60 Candida spp. clinical isolates (Candida albicans, Candida tropicalis, Candida parapsilosis, Candida glabrata, Candida krusei, and Candida lusitaniae [10 isolates each]) are assessed. The combined influence of growth and reading method (visual or spectrophotometric) on the determination of the MICs of amphotericin B, flucytosine, fluconazole, itraconazole, ketoconazole, and voriconazole is also analyzed, and the MICs are compared with those determined by the National Committee for Clinical Laboratory Standards standard microdilution method (NCCLS document M27-A). Glucose supplementation and inoculum size had a significant influence on the growth cycles of these yeasts, and a statistically significant denser growth (optical density at 540 nm) was seen for both incubation periods, 24 and 48 h (P < 0.01). A longer exponential phase and shorter lag phase were also observed. The A540 values at 24 h of incubation with medium containing glucose and an inoculum of 10(5) CFU/ml were >0.4 U for all species, with the exception of that for C. parapsilosis (A540 = 0.26 +/- 0.025). The MICs at 24 h determined by testing with 2% glucose and an inoculum of 10(5) CFU/ml showed the strongest agreement (96.83%) with MICs determined by the reference method. MICs were not falsely elevated, and good correlation indexes were obtained. The reproducibility of results with this medium-inoculum combination was high (intraclass correlation coefficient, 0.955). The best agreement and reproducibility of results for spectrophotometric readings were achieved with endpoints of 50% growth inhibition for flucytosine and azoles and 95% for amphotericin B. Supplementation of test media with glucose and an inoculum size of 10(5) CFU/ml yielded a reproducible technique that shows elevated agreement with the reference procedures and a shorter incubation period for obtaining reliable MIC determinations. The spectrophotometric method offers an advantage over the visual method by providing a more objective and automated MIC determination.     

Comparison of antifungal MICs for yeasts obtained using the EU-CAST method in a reference laboratory and the Etest in nine different hospital laboratories

In routine laboratory practice, the determination of MICs of antifungals for yeasts often relies on the Etest, because of a good correlation with reference methods. However, this correlation was established through predesigned studies, rather than prospective testing. The surveillance programme of fungaemia (YEASTS programme), implemented since 2003, facilitated our comparison of the Etest and the EU-CAST results, obtained on a routine basis in nine different hospitals and in a reference laboratory, respectively. The analysis included 690 isolates recovered from blood culture (362 Candida albicans, 113 Candida glabrata, 69 Candida parapsilosis, 55 Candida tropicalis, 31 Cryptococcus neoformans, and 60 other yeast species) that were tested for their susceptibility to amphotericin B (n = 655), fluconazole (n = 669), itraconazole (n = 198), voriconazole (n = 588), flucytosine (n = 314), and caspofungin (n = 244). Agreement between the Etest and EU-CAST datasets was calculated and categorized on the basis of previously published breakpoints. The level of agreement at ±2 dilutions was 75% for amphotericin B and 90% for flucytosine; for the azoles, it ranged from 71% for itraconazole to 87% for voriconazole. No significant difference was observed among the yeast species, except for Cryptococcus neoformans and flucytosine, with an agreement <40. Categorical agreement ranged from 60% for itraconazole to 90% for flucytosine. Major and very major discrepancies occurred in <12% and 6%, respectively. The Etest, even when performed on a routine basis, shows a ≥71% agreement with the EU-CAST reference method.     

Comparison of three commercial assays and a modified disk diffusion assay with two broth microdilution reference assays for testing zygomycetes, Aspergillus spp., Candida spp., and Cryptococcus neoformans with posaconazole and amphotericin B

We compared posaconazole M27-A2 and M38-A MICs to Etest and YeastOne MICs for 92 zygomycetes, 126 Aspergillus isolates, 110 Candida isolates, and Cryptococcus neoformans. Reference MICs were also correlated with inhibition zone diameters in millimeters (modified M44-A disk and Neo-Sensitabs tablet methods). Etest MICs were obtained on solidified (1.5% agar) RPMI 1640 (2% dextrose), and zone diameters were obtained on supplemented (2% glucose and 0.5 microg/ml methylene blue [for all isolates]) and nonsupplemented Mueller-Hinton (MH; molds only) agar. MICs and zone diameters were obtained between 16 and 72 h. The overall agreement (% MIC pairs within a three-dilution range) between reference posaconazole and YeastOne MICs was 98 to 100% at 16 to 24 h for zygomycetes and yeasts and 99% at 24 to 48 h for Aspergillus. The overall agreement was lower between reference posaconazole and Etest MICs (94 to 97%) and by both methods with amphotericin B for all species (95 to 99.3%). For yeasts, the correlation coefficient was similar between reference posaconazole MICs and either disk (R, 0.810) or tablet (R, 0.769) zone diameter at 24 h and was superior on MH agar for molds at 16 to 48 h (R, 0.804 and 0.799 for disk and tablet, respectively). For amphotericin B, the best correlation between reference MICs and zone diameters was observed at 16 to 48 h for molds on MH agar (R, 0.736 to 0.812 and 0.765 to 0.749 for disk and tablet, respectively) and at 48 h for yeasts (R, 0.681 and 0.503 for disk and tablet, respectively). These data suggest the potential value of these alternative broth dilution and agar diffusion methods for testing posaconazole and amphotericin B in the clinical laboratory against the species evaluated.     

Evaluation of Etest Method for Determining Posaconazole MICs for 314 Clinical Isolates of Candida Species

The performance of the Etest for posaconazole (SCH 56592) susceptibility testing of 314 isolates of Candida spp. was assessed against 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 degrees C. MICs were determined by Etest for all 314 isolates with RPMI agar containing 2% glucose (RPG agar) and were read after incubation for 48 h at 35 degrees C. The Candida isolates included C. albicans (n = 174), C. glabrata (n = 57), C. tropicalis (n = 31), C. parapsilosis (n = 39), C. krusei (n = 5), C. guilliermondii (n = 6), and C. lusitaniae (n = 2). The Etest results correlated well with reference MICs. Overall agreement was 95%, and agreements for individual species were as follows: C. krusei, 100%; C. albicans, 98%; C. tropicalis, 97%; C. glabrata, 93%; C. parapsilosis, 85%; C. guilliermondii, 83%; and C. lusitaniae, 50%. The problem of trailing end points was minimized with RPG agar, and good agreement with broth dilution MICs was obtained when discernible growth within an established ellipse was ignored. The Etest method using RPG agar appears to be a useful method for determining posaconazole susceptibilities of Candida species.     

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.     

Comparative evaluation of alternative methods for broth dilution susceptibility testing of fluconazole against Candida albicans.

A comparative evaluation of methods for broth macro- and microdilution susceptibility testing of fluconazole was conducted with 119 clinical isolates of Candida albicans. Macro- and microdilution testing were performed according to National Committee for Clinical Laboratory Standards recommendations. For reference macrodilution testing, an 80% inhibition endpoint (MIC 80%) was determined after 48 h of incubation in accordance with National Committee for Clinical Laboratory Standards proposed standard M27-P. Microdilution endpoints were scored as the first tube or well in which a prominent reduction in turbidity (score 2 out of a possible 4) was observed compared with the growth control (Micro MIC-2). Alternative endpoint criteria were assessed independently of the reference MIC 80% and Micro MIC-2 values and included a colorimetric microdilution endpoint determined by using an oxidation-reduction indicator (Alamar Blue; Alamar Bio-sciences Inc., Sacramento, Calif.). The MICs for the two microdilution test systems were read after 24 and 48 h of incubation. The percentage of fluconazole MICs within 2 doubling dilutions of the macrodilution reference values was 94% for both microdilution tests read at 24 h. Agreement was slightly lower at 48 h and ranged from 91 to 93%. Comparison of Micro MIC-2 and colorimetric microdilution MICs resulted in agreements of 97 and 93% at 24 and 48 h, respectively. These results show excellent agreement among alternative methods for fluconazole susceptibility testing.