Evaluation of the Etest and disk diffusion methods for determining susceptibilities of 235 bloodstream isolates of Candida glabrata to fluconazole and voriconazole

The performances of the Etest and the disk diffusion methods for testing of the susceptibilities of 235 Candida glabrata isolates to fluconazole and voriconazole were compared with that of the National Committee for Clinical Laboratory Standards (NCCLS) approved standard broth microdilution (BMD) method. The NCCLS method used RPMI 1640 broth medium, and MICs were read after incubation for 48 h at 35 degrees C. Etest MICs were determined with RPMI 1640 agar containing 2% glucose (RPG agar) and with Mueller-Hinton agar containing 2% glucose and 0.5 microg of methylene blue per ml (MBE agar) and were read after incubation for 48 h at 35 degrees C. Disk diffusion testing was performed with MBE agar, 25-microg fluconazole disks, and 1- microg voriconazole disks and by incubation at 35 degrees C for 24 h. Overall agreements between the Etest and the BMD MICs obtained with RPG and MBE agars were 91 and 96%, respectively, for fluconazole and 93 and 95%, respectively, for voriconazole. Categorical agreements between the agar-based methods and BMD were 52.3 to 64.7% with fluconazole and 94.8 to 97.4% with voriconazole. The vast majority of the discrepancies by the disk diffusion and Etest methods with fluconazole were minor errors. The agar-based methods performed well in identifying isolates with resistance to fluconazole and decreased susceptibility to voriconazole.     

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.     

Evaluation of the Etest method for determining voriconazole susceptibilities of 312 clinical isolates of Candida species by using three different agar media

Performance of the Etest for voriconazole susceptibility testing of 312 isolates of Candida spp. was assessed against that of 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. Etest MICs were determined with RPMI agar containing 2% glucose (RPG), Casitone agar (CAS), and antibiotic medium 3 (AM3) agar and were read after incubation for 48 h at 35 degrees C. The Candida spp. isolates included C. albicans (n = 174), C. glabrata (n = 55), C. tropicalis (n = 31), C. parapsilosis (n = 39), C. krusei (n = 5), C. lusitaniae (n = 2), and C. guilliermondii (n = 6). The Etest results obtained using RPG correlated well with the reference MICs. Overall agreement ranged from 91% for C. glabrata to 100% for C. tropicalis, C. parapsilosis, C. guilliermondii, C. krusei, and C. lusitaniae. When CAS was used, agreement ranged from 80% for C. krusei to 100% for C. parapsilosis, C. guilliermondii, and C. lusitaniae. With AM3, agreement ranged from 58% for C. glabrata to 100% for C. lusitaniae and C. guilliermondii. The Etest method using RPG appears to be a useful method for determining voriconazole susceptibilities of Candida species.     

Evaluation of Etest method for determining fluconazole and voriconazole MICs for 279 clinical isolates of Candida species infrequently isolated from blood

The performance of Etest in fluconazole and voriconazole testing of 279 isolates of uncommon Candida spp. was assessed in comparison with the National Committee for Clinical Laboratory Standards (NCCLS)-approved standard broth microdilution (BMD) 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 include Candida krusei, C. lusitaniae, C. guilliermondii, C. kefyr, C. rugosa, C. lipolytica, C. pelliculosa, C. dubliniensis, C. famata, C. zeylanoides, C. inconspicua, and C. norvegensis. Overall agreement between Etest and BMD MICs was 96% for fluconazole and 95% for voriconazole. Where a discrepancy was observed between Etest and the reference method, the Etest tended to give lower values with both fluconazole and voriconazole. The Etest method using RPMI agar appears to be a useful method for determining fluconazole and voriconazole susceptibilities of uncommon species of Candida.     

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.     

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.     

Evaluation of Etest Method for Determining Caspofungin (MK-0991) Susceptibilities of 726 Clinical Isolates of Candida Species

The performance of the Etest for testing the susceptibilities to caspofungin (MK-0991) of 726 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 726 isolates with RPMI agar containing 2% glucose (RPG) and were read after incubation for 48 h at 35 degrees C. The Candida isolates included Candida albicans (n = 486), Candida glabrata (n = 96), Candida tropicalis (n = 51), Candida parapsilosis (n = 47), Candida krusei (n = 11), Candida lusitaniae (n = 2), and Candida guilliermondii (n = 33). In addition, a subset of 314 isolates were also tested by Etest using Casitone agar (CAS) and antibiotic medium 3 agar (AM3). The Etest results obtained using RPG correlated well with reference MICs. Overall agreement was 94% with RPG, 82% with CAS, and 79% with AM3. When RPG was used, agreement ranged from 79% for C. parapsilosis to 100% for C. krusei, C. lusitaniae, and C. guilliermondii. When CAS was used, agreement ranged from 0% for C. lusitaniae to 100% for C. glabrata. With AM3, agreement ranged from 0% for C. lusitaniae to 100% for C. guilliermondii. All three media supported growth of each of the Candida species. Etest results were easy to read, with sharp zones of inhibition. In most instances (75%) where a discrepancy was observed between the Etest and the reference method, the Etest MIC was lower. The Etest method using RPG appears to be useful for determining caspofungin susceptibilities of Candida species.     

Evaluation of the Etest Method for Determining Fluconazole Susceptibilities of 402 Clinical Yeast Isolates by Using Three Different Agar Media

The performance of the Etest for fluconazole susceptibility testing of 402 yeast isolates 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°C. Etest MICs were determined with RPMI agar containing 2% glucose (RPG), Casitone agar (CAS), and Mueller-Hinton agar (MHA) and were read after incubation for 48 h at 35°C. The yeast isolates included Candida albicans (n = 161), Candida glabrata (n = 41), Candida tropicalis (n = 35), Candida parapsilosis (n = 29), Candida krusei (n = 32), Candida lusitaniae (n = 31), Candida species (n = 19), Cryptococcus neoformans (n = 40), and miscellaneous yeast species (n = 14). The Etest results correlated well with reference MICs. Overall agreement was 94% with RPG, 97% with CAS, and 53% with MHA. When RPG was used, agreement ranged from 89% for Candida spp. to 100% for C. krusei. When CAS was utilized, agreement ranged from 93% for Cryptococcus neoformans to 100% for C. tropicalis, C. parapsilosis, C. lusitaniae, Candida spp., and miscellaneous yeast species. With MHA, agreement ranged from 17% for C. parapsilosis to 90% for C. krusei. Both RPG and CAS supported growth of all yeast species, whereas growth on MHA was comparatively weaker. Etest results were somewhat easier to read on CAS. The Etest method using either RPG or CAS, but not MHA, appears to be a viable alternative to the NCCLS reference method for determining fluconazole susceptibilities of yeasts.     

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 new colorimetric assay with the NCCLS broth microdilution method (M-27A) for antifungal drug MIC determination

We evaluated a new microtiter assay for antifungal susceptibility testing based on a colorimetric reaction to monitor fungal substrate utilization. This new method (rapid susceptibility assay [RSA]) provides quantitative endpoint readings in less than 8 h compared with visual determination of MIC by the National Committee for Clinical Laboratory Standards (NCCLS) broth microdilution method, which requires a minimum of 48 h of incubation. In this study, we tested clinical isolates from each of the following species: Candida albicans (20 isolates), C. glabrata (20 isolates), C. krusei (19 isolates), C. tropicalis (19 isolates), and C. parapsilosis (28 isolates). RSA and NCCLS broth dilution methods were used to determine the MICs of amphotericin B, fluconazole, itraconazole, and 5-flucytosine for all 106 isolates. RPMI 1640 medium buffered with morpholinopropanesulfonic acid was used for both methods; however, glucose and inoculum concentrations in the RSA were modified. RSA MICs were determined as the lowest drug concentration that prevented glucose consumption by the organism after 6 h of incubation. MICs obtained from the RSA were compared with those obtained from the NCCLS M-27A method read at 24 and 48 h. MIC pairs were considered in agreement when the difference between the pairs was within 2 twofold dilutions. For the 106 isolates tested, amphotericin B and 5-flucytosine demonstrated the highest agreement in MICs between the two methods (100 and 98%, respectively), whereas fluconazole and itraconazole produced less favorable MIC agreement (63.2 and 61.3%, respectively). The azole MIC differences between the two methods were significantly reduced when lower inocula were used with a prolonged incubation time. This preliminary comparison suggests that this rapid procedure may be a reliable tool for the in vitro determination of MICs of amphotericin B and 5-flucytosine and warrants further evaluation.     

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.     

Evaluation of the etest method using Mueller-Hinton agar with glucose and methylene blue for determining amphotericin B MICs for 4,936 clinical isolates of Candida species

The performance of the Etest using Mueller-Hinton agar supplemented with glucose (2%) and methylene blue (0.5 microg/ml) (MH-GMB) for amphotericin B susceptibility testing of 4,936 isolates of Candida spp. was assessed against that of Etest using RPMI agar with 2% glucose (RPG). MICs were determined by Etest in both media for all 4,936 isolates and were read after incubation for 48 h at 35 degrees C. The Candida isolates included C. albicans (n = 2,728), C. glabrata (n = 722), C. parapsilosis (n = 666), C. tropicalis (n = 528), C. krusei (n = 143), C. lusitaniae (n = 54), C. guilliermondii (n = 39), C. pelliculosa (n = 17), C. kefyr (n = 15), C. rugosa (n = 11), C. dubliniensis (n = 5), C. zeylanoides (n = 4), C. lipolytica (n = 3), and C. famata (n = 1). The Etest results with MH-GMB correlated well with those with RPG. Overall agreement was 92.9%, and agreements for individual species were as follows: C. lusitaniae, 98.1%; C. albicans, 95.1%; C. glabrata, 94.3%; C. krusei, 91.6%; C. parapsilosis, 86.6%; and C. tropicalis, 86.4%. The Etest method using MH-GMB appears to be a useful method for determining amphotericin B susceptibilities of Candida species.     

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.     

Improved detection of amphotericin B-resistant isolates of Candida lusitaniae by Etest

Both intrinsic and acquired resistance to amphotericin B have been documented for Candida lusitaniae. Amphotericin B remains the drug of choice for many critical fungal infections, and the detection of resistance is essential to monitor treatment effectively. The limitations of the National Committee for Clinical Laboratory Standards (NCCLS) reference methodology for detection of amphotericin B resistance are well documented, and several alternative methods have been proposed. Etest assays with RPMI and antibiotic medium 3 (AM3) agar were compared to the NCCLS M27-A broth macrodilution method using AM3 for amphotericin B resistance testing with 49 clinical isolates of C. lusitaniae. The panel included nine isolates with known or presumed resistance to amphotericin B on the basis of in vivo and/or in vitro data. The distribution of amphotericin B MICs by Etest with RPMI ranged from 0. 032 to 16 microg/ml and was bimodal. All of the putatively resistant isolates were inhibited by amphotericin B at >/=0.38 microg/ml and could be categorized as resistant using this breakpoint. Etest with AM3 yielded a broader amphotericin B MIC range (0.047 to 32 microg/ml), and there were six putatively resistant isolates for which MICs were >1 microg/ml. The separation of putatively susceptible and resistant isolates was less obvious. Broth macrodilution with AM3 generated a unimodal distribution of MICs (ranging from 0.032 to 2 microg/ml) and failed to discriminate most of the putatively resistant isolates at both 24 and 48 h. Etest using RPMI and, to a lesser extent, using AM3 provided better discrimination between amphotericin B-resistant and -susceptible isolates of C. lusitaniae.     

Correlation of Neo-Sensitabs tablet diffusion assay results on three different agar media with CLSI broth microdilution M27-A2 and disk diffusion M44-A results for testing susceptibilities of Candida spp. and Cryptococcus neoformans to amphotericin B, caspofungin, fluconazole, itraconazole, and voriconazole

We compared the Neo-Sensitabs tablet assay to both reference M27-A2 broth microdilution and M44-A disk diffusion methods for testing susceptibilities of 110 isolates of Candida spp. and Cryptococcus neoformans to amphotericin B, caspofungin, fluconazole, itraconazole, and voriconazole. Neo-Sensitabs assay inhibition zone diameters in millimeters on three agars (Mueller-Hinton agar supplemented with 2% dextrose and 0.5 microg/ml methylene blue [MGM], Shadomy [SHA], and RPMI 1640 [RPMI, 2% dextrose]) were obtained at 24 to 72 h. The correlation coefficient of Neo-Sensitabs results with MICs was similar to that of the disk method for most of the five agents on MGM (R, 0.80 to 0.89 versus 0.76 to 0.89, respectively). Overall, superior correlation was observed at 24 h for most agents. The exception was amphotericin B (R values of 0.68 and 0.5 for disk and tablet, respectively, at 48 h versus 0.68 and 0.48, respectively, at 24 h). In general, Neo-Sensitabs results were less consistent on SHA and RPMI agars. Although agreement by breakpoint category of Neo-Sensitabs and disk results with CLSI method M27-A2 was also similar on MGM (92.7 to 98.2% versus 95.5 to 100%, respectively), the Neo-Sensitabs method failed to identify two of the six isolates with high amphotericin B MICs. These data suggest the potential value of the Neo-Sensitabs assay for testing at least four of the five agents against yeasts evaluated in the clinical laboratory.     

Microdilution susceptibility testing of amphotericin B, itraconazole, and voriconazole against clinical isolates of Aspergillus and Fusarium species

We compared the activities of amphotericin B, itraconazole, and voriconazole against clinical Aspergillus (n = 82) and Fusarium (n = 22) isolates by a microdilution method adopted from the National Committee for Clinical Laboratory Standards (NCCLS-M27A). RPMI 1640 (RPMI), RPMI 1640 supplemented to 2% glucose (RPMI-2), and antibiotic medium 3 supplemented to 2% glucose (AM3) were used as test media. MICs were determined after 24, 48, and 72 h. A narrow range of amphotericin B MICs was observed for Aspergillus isolates, with minor variations among species. MICs for Fusarium isolates were higher than those for Aspergillus isolates. MICs of itraconazole were prominently high for two previously defined itraconazole-resistant Aspergillus fumigatus isolates and Fusarium solani. Voriconazole showed good in vitro activity against itraconazole-resistant isolates, but the MICs of voriconazole for F. solani were high. RPMI was the most efficient medium for detection of itraconazole-resistant isolates, followed by RPMI-2. While the significance remains unclear, AM3 lowered the MICs, particularly those of amphotericin B.     

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.     

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.     

Fluconazole Susceptibility Testing of Cryptococcus neoformans: Comparison of Two Broth Microdilution Methods and Clinical Correlates among Isolates from Ugandan AIDS Patients

We compared the yeast nitrogen base (YNB) broth microdilution method with the National Committee for Clinical Laboratory Standards (NCCLS) M27-A microdilution reference method for measuring the in vitro susceptibility of Cryptococcus neoformans isolates to fluconazole. A total of 149 isolates of C. neoformans var. neoformans from Ugandan AIDS patients was tested by both methods. An overall agreement of 88% between the two microdilution methods was observed. All isolates grew well in both RPMI 1640 and YNB media, and MICs could be read after 48 h of incubation by both methods. The range of fluconazole MICs obtained with the YNB method was broader than that obtained with the NCCLS method. The extended range of MICs provided by the YNB method may be of clinical value, as it appears that the clinical outcome may be better among patients infected with strains inhibited by lower concentrations of fluconazole as determined by the YNB method. The YNB method appears to be a viable option for testing C. neoformans against fluconazole.     

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.