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.     

E-test method for testing susceptibilities of Aspergillus spp. to the new triazoles voriconazole and posaconazole and to established antifungal agents: comparison with NCCLS broth microdilution method

NCCLS document M38-P describes standard parameters for testing the fungistatic activities (MICs) of established agents against filamentous fungi (molds). This study evaluated the in vitro susceptibilities of 15 Aspergillus flavus isolates, 62 A. fumigatus isolates, and 10 isolates each of A. niger, A. nidulans, and A. terreus to voriconazole, posaconazole, itraconazole, and amphotericin B by the E-test and NCCLS M38-P microdilution methods. The agreement (within 3 dilutions) between methods for voriconazole was independent of the E-test incubation time (93.3 to 100% for four of five species at both incubation times). In contrast, with amphotericin B, itraconazole, and posaconazole, E-test results were more dependent on the incubation time for certain species. For A. fumigatus, posaconazole E-test MICs had better concordance with reference values after 48 h (95.2%) than after 24 h (90%), while the highest agreement for itraconazole MICs was after 24 h (90.3 versus 74.2%) of incubation. Better agreement between the methods was also obtained with 24-h E-test amphotericin B MICs for A. flavus (73.3 versus 26.7%) and A. fumigatus (96.7 versus 64.5%). E-test MICs of the four agents had the lowest percentages of agreement with reference values for A. nidulans (60 to 80%). For isolates for which high MICs were obtained for the four agents by the reference method, high MICs were also obtained by E-test at both 24 and 48 h. The utility of in vitro results of either the E-test or the NCCLS broth microdilution (M38-P) method for Aspergillus spp. needs to be established in clinical trials.     

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.     

Clinical evaluation of a frozen commercially prepared microdilution panel for antifungal susceptibility testing of seven antifungal agents, including the new triazoles posaconazole, ravuconazole, and voriconazole

A commercially prepared frozen broth microdilution panel (Trek Diagnostic Systems, Westlake, Ohio) was compared with a reference microdilution panel for antifungal susceptibility testing of two quality control (QC) strains and 99 clinical isolates of Candida spp. The antifungal agents tested included amphotericin B, flucytosine, fluconazole, itraconazole, posaconazole, ravuconazole, and voriconazole. Microdilution testing was performed according to NCCLS recommendations. MIC endpoints were read visually after 48 h of incubation and were assessed independently for each microdilution panel. The MICs for the QC strains were within published limits for both the reference and Trek microdilution panels. Discrepancies among MIC endpoints of no more than 2 dilutions were used to calculate the percent agreement. Acceptable levels of agreement between the Trek and reference panels were observed for all antifungal agents tested against the 99 clinical isolates. The overall agreement for each antifungal agent ranged from 96% for ravuconazole to 100% for amphotericin B. The Trek microdilution panel appears to be a viable alternative to frozen microdilution panels prepared in-house.     

Modification of rapid susceptibility assay for antifungal susceptibility testing of Aspergillus fumigatus

To improve objectivity and speed of current antifungal mold susceptibility testing, the yeast Rapid Susceptibility Assay (RSA) was adapted for Aspergillus species. The RSA is based on glucose utilization in the presence of an antifungal drug. Aspergillus fumigatus conidia were incubated in 0.2% glucose RPMI 1640 containing 0.03 to 16 micro g of amphotericin B or itraconazole/ml. Drug-related inhibition of glucose utilization correlated with suppression of conidial germination. Following incubation of conidia with various concentrations of antifungal drug, the percentage of residual glucose in the growth medium was determined colorimetrically and plotted against drug concentration to determine the MIC (MIC(RSA)). National Committee for Clinical Laboratory Standards (NCCLS) M38-P testing was also performed to obtain NCCLS MICs (MIC(NCCLS)) for direct comparison with MIC(RSA)s. Conidial inocula of an optical density at 530 nm (OD(530)) of 0.11 facilitated determination of amphotericin B and itraconazole MIC(RSA)s at 16 h equal to or within a single twofold dilution of MIC(NCCLS)s obtained at 48 h. Preliminary testing with a 0.11-OD(530) conidial inoculum of the slower-growing Aspergillus terreus resulted in itraconazole and amphotericin B MIC(RSA)s at 16 h equal to or within a single twofold dilution of MIC(NCCLS)s obtained at 48 h. These data indicate that the mold RSA provides a more objective and rapid method for Aspergillus spp. susceptibility testing than the NCCLS M38-P assay.     

Evaluation of Etest method for determining voriconazole and amphotericin B MICs for 162 clinical isolates of Cryptococcus neoformans

The performance of the Etest for voriconazole and amphotericin B susceptibility testing of 162 isolates of Cryptococcus neoformans was assessed against the National Committee for Clinical Laboratory Standards (NCCLS) broth microdilution method. The NCCLS method employed RPMI 1640 broth medium, and MICs were read after incubation for 72 h at 35 degrees C. MICs were determined by Etest for all 162 isolates with RPMI 1640 agar containing 2% glucose (RPG agar) and were read after incubation for 72 h at 35 degrees C. The Etest results for both voriconazole and amphotericin B correlated well with reference MICs. Agreement was 94% for voriconazole and 99% for amphotericin B. When discrepancy was noted between the results obtained by Etest and broth microdilution for voriconazole, the Etest generally provided a higher MIC. The Etest method using RPG agar appears to be a useful method for determining the susceptibility of C. neoformans to voriconazole and amphotericin B.     

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.     

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.     

Multicenter comparison of the Sensititre YeastOne colorimetric antifungal panel with the NCCLS M27-A2 reference method for testing new antifungal agents against clinical isolates of Candida spp

A multicenter (three centers) study compared MICs obtained by the Sensititre YeastOne Colorimetric Antifungal plate to reference microdilution broth (NCCLS M27-A2 document) MICs of three new triazoles (posaconazole, ravuconazole, and voriconazole) and the echinocandin caspofungin acetate for 100 isolates of Candida spp. In addition, amphotericin B and fluconazole were tested as control drugs. Colorimetric MICs of caspofungin and amphotericin B corresponded to the first blue well (no growth), and MICs of the other agents corresponded to the first slightly purple or blue well. Two comparisons of MIC pairs by the two methods were evaluated: 24-h colorimetric MICs were compared to NCCLS MICs at 24 and at 48 h. The interlaboratory reproducibility of YeastOne and reference MICs was also examined. The best performance of the YeastOne plate was with 24-h MICs (overall, 95 to 99% agreement) for all the species and antifungal agents. These results suggest the potential value of the YeastOne plate for use in the clinical laboratory for the four new antifungal agents evaluated.     

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.     

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.     

International and multicenter comparison of EUCAST and CLSI M27-A2 broth microdilution methods for testing susceptibilities of Candida spp. to fluconazole, itraconazole, posaconazole, and voriconazole

The aim of this study was to compare MICs of fluconazole, itraconazole, posaconazole, and voriconazole obtained by the European Committee on Antibiotic Susceptibility Testing (EUCAST) and CLSI (formerly NCCLS) methods in each of six centers for 15 Candida albicans (5 fluconazole-resistant and 4 susceptible-dose-dependent [S-DD] isolates), 10 C. dubliniensis, 7 C. glabrata (2 fluconazole-resistant isolates), 5 C. guilliermondii (2 fluconazole-resistant isolates), 10 C. krusei, 9 C. lusitaniae, 10 C. parapsilosis, and 5 C. tropicalis (1 fluconazole-resistant isolate) isolates. CLSI MICs were obtained visually at 24 and 48 h and spectrophotometric EUCAST MICs at 24 h. The agreement (within a 3-dilution range) between the methods was species, drug, and incubation time dependent and due to lower EUCAST than CLSI MICs: overall, 94 to 95% with fluconazole and voriconazole and 90 to 91% with posaconazole and itraconazole when EUCAST MICs were compared against 24-h CLSI results. The agreement was lower (85 to 94%) against 48-h CLSI endpoints. The overall interlaboratory reproducibility by each method was > or =92%. When the comparison was based on CLSI breakpoint categorization, the agreement was 68 to 76% for three of the four species that included fluconazole-resistant and S-DD isolates; 9% very major discrepancies (< or =8 microg/ml versus > or =64 microg/ml) were observed among fluconazole-resistant isolates and 50% with voriconazole (< or =1 microg/ml versus > or =4 microg/ml). Similar results were observed with itraconazole for seven of the eight species evaluated (28 to 77% categorical agreement). Posaconazole EUCAST MICs were also substantially lower than CLSI MIC modes (0.008 to 1 microg/ml versus 1 to > or =8 microg/ml) for some of these isolates. Therefore, the CLSI breakpoints should not be used to interpret EUCAST MIC data.     

Influence of Incubation Time, Inoculum Size, and Glucose Concentrations on Spectrophotometric Endpoint Determinations for Amphotericin B, Fluconazole, and Itraconazole

We addressed the influence of the incubation time (24 h versus 48 h), starting inoculum size (standard inoculum size, ~10³ CFU/ml, versus large inoculum size, ~10⁴ CFU/ml), and supplementation with 2% glucose of RPMI 1640 medium on the spectrophotometric determination of the MICs of amphotericin B, fluconazole, and itraconazole. We compared the MICs determined spectrophotometrically with those determined by the standard broth macrodilution method (National Committee for Clinical Laboratory Standards approved guideline M27-A). The agreement between the results of the spectrophotometric and standard methods for amphotericin B testing was 100%; this agreement was independent of the inoculum size and incubation time. On the other hand, the agreement for the results for fluconazole testing and itraconazole testing was dependent on the inoculum size and incubation time. With large inoculum size, excellent agreement can be achieved at 24 h. With standard inoculum size, acceptable agreement can be achieved only at 48 h. In contrast to previous observations, the addition of 2% glucose did not have any significant impact on the growth density at 24 h, nor did it improve the agreement with the standard method. Furthermore, supplemental glucose might falsely elevate the MIC at 48 h.     

Comparative evaluation of three antifungal susceptibility test methods for Candida albicans isolates and correlation with response to fluconazole therapy.

In vitro susceptibilities were determined for 56 Candida albicans isolates obtained from the oral cavities of 41 patients with human immunodeficiency virus infection. The agents tested included fluconazole, itraconazole, ketoconazole, flucytosine, and amphotericin B. MICs were determined by the broth microdilution technique following National Committee for Clinical Laboratory Standards document M27-P (M27-P micro), a broth microdilution technique using high-resolution medium (HR micro), and the Etest with solidified yeast-nitrogen base agar. The in vitro findings were correlated with in vivo response to fluconazole therapy for oropharyngeal candidiasis. For all C. albicans isolates from patients with oropharyngeal candidiasis not responding to fluconazole MICs were found to be > or = 6.25 micrograms/ml by the M27-P micro method and > or = 25 micrograms/ml by the HR micro method as well as the Etest. However, for several C. albicans isolates from patients who responded to fluconazole therapy MICs found to be above the suggested breakpoints of resistance. The appropriate rank order of best agreement between the M27-P micro method and HR micro method was amphotericin B > fluconazole > flucytosine > ketoconazole > itraconazole. The appropriate rank order with best agreement between the M27-P micro method and the Etest was flucytosine > amphotericin B > fluconazole > ketoconazole > or = itraconazole. It could be concluded that a good correlation between in vitro resistance and clinical failure was found with all methods. However, the test methods used in this study did not necessarily predict clinical response to therapy with fluconazole.     

Comparison of the E-test with the NCCLS M38-P method for antifungal susceptibility testing of common and emerging pathogenic filamentous fungi

The National Committee for Clinical Laboratory Standards (NCCLS) M38-P method describes standard parameters for testing the fungistatic antifungal activities (MICs) of established agents against filamentous fungi (molds). The present study evaluated the in vitro fungistatic activities of itraconazole and amphotericin B by the E-test and the NCCLS M38-P microdilution method against 186 common and emerging pathogenic molds (123 isolates of Aspergillus spp. [five species], 16 isolates of Fusarium spp. [two species], 4 Paecilomyces lilacinus isolates, 5 Rhizopus arrhizus isolates, 15 Scedosporium spp., 18 dematiaceous fungi, and 5 Trichoderma longibrachiatum isolates). The agreement between the methods for amphotericin B MICs ranged from 70% for Fusarium solani to > or =90% for most of the other species after the first reading; agreement was dependent on both the incubation time and the species being evaluated. Major discrepancies between the amphotericin B MICs determined by the E-test and the NCCLS M38-P method were demonstrated for three of the five species of Aspergillus tested and the two species of Fusarium tested. This discrepancy was more marked after 48 h of incubation; the geometric mean MICs determined by the E-test increased between 24 and 48 h from between 1.39 and 3.3 microg/ml to between 5.2 and >8 microg/ml for Aspergillus flavus, Aspergillus fumigatus, and Aspergillus nidulans. The agreement between the itraconazole MICs determined by the E-test and the NCCLS M38-P method ranged from 83.3% for A. nidulans to > or =90% for all the other species tested; the overall agreement was higher (92.7%) than that for amphotericin B (87.9%). The agreement was less dependent on the incubation time. Clinical trials need to be conducted to establish the role of the results of either the E-test or the NCCLS M38-P method in vitro for molds with the two agents as predictors of clinical outcome.     

Clinical Evaluation of the ASTY Colorimetric Microdilution Panel for Antifungal Susceptibility Testing

A method using a commercially prepared colorimetric microdilution panel (ASTY; Kyokuto Pharmaceutical Industrial Co., Ltd.) was compared in four different laboratories with the National Committee for Clinical Laboratory Standards (NCCLS) reference microdilution method by testing 802 clinical isolates of Candida spp. (C. albicans, C. glabrata, C. tropicalis, C. parapsilosis, C. krusei, C. lusitaniae, C. guilliermondii, C. lipolytica, C. rugosa, and C. zeylanoides) against amphotericin B, 5-fluorocytosine (5FC), fluconazole, and itraconazole. Reference MIC endpoints were established after 48 h of incubation, and ASTY endpoints were established after 24 and 48 h of incubation. ASTY endpoints were determined to be the time at which the color of the first well changed from red (indicating growth) to purple (indicating growth inhibition) or blue (indicating no growth). Excellent agreement (within 2 dilutions) between the reference and colorimetric MICs was observed. Overall agreement was 93% at 24 h and 96% at 48 h. Agreement ranged from 90% with itraconazole and 5FC to 96% with amphotericin B at 24 h and from 92% with itraconazole to 99% with amphotericin B and 5FC at 48 h. The ASTY colorimetric microdilution panel method appears to be comparable to the NCCLS reference method for testing the susceptibilities of Candida spp. to a variety of antifungal agents.     

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.     

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.     

Antifungal susceptibility testing method for resource constrained laboratories

Purpose: In resource-constrained laboratories of developing countries determination of antifungal susceptibility testing by NCCLS/CLSI method is not always feasible. We describe herein a simple yet comparable method for antifungal susceptibility testing. Methods: Reference MICs of 72 fungal isolates including two quality control strains were determined by NCCLS/CLSI methods against fluconazole, itraconazole, voriconazole, amphotericin B and cancidas. Dermatophytes were also tested against terbinafine. Subsequently, on selection of optimum conditions, MIC was determined for all the fungal isolates by semisolid antifungal agar susceptibility method in Brain heart infusion broth supplemented with 0.5% agar (BHIA) without oil overlay and results were compared with those obtained by reference NCCLS/CLSI methods. Results: Comparable results were obtained by NCCLS/CLSI and semisolid agar susceptibility (SAAS) methods against quality control strains. MICs for 72 isolates did not differ by more than one dilution for all drugs by SAAS. Conclusions: SAAS using BHIA without oil overlay provides a simple and reproducible method for obtaining MICs against yeast, filamentous fungi and dermatophytes in resource-constrained laboratories.     

In vitro activity of five antifungal agents against clinical isolates of Saccharomyces cerevisiae.

We evaluated the in vitro activity of fluconazole, itraconazole, ketoconazole, 5-fluorocytosine and amphotericin B against 30 clinical isolates of Saccharomyces cerevisiae by a broth microdilution method, following the NCCLS recommendation. Testing was performed either in RPMI-1640 or yeast nitrogen base (YNB). YNB supported the growth of all isolates tested, while results in RPMI-1640 were not obtained for six isolates (20%). The MIC of all three azoles in YNB were one or two dilutions higher than those obtained in RPMI-1640 (P=0.0001 for fluconazole and itraconazole, P=0.03 for ketoconazole). Elevated MICs were observed for all three azoles, while all the isolates were susceptible to 5-fluorocytosine and amphotericin B. All MIC values were confirmed by spectrophotometric reading. Six strains of S. cerevisiae isolated from the faeces and consecutive blood cultures from an AIDS patient over a 7-month period were typed by electrophoretic karyotyping (EK). EK showed the maintenance of the same karyotype over time suggesting that the faecal isolate changed from a colonizing to infection-causing strain. The relative resistance of S. cerevisiae to azole drugs as well as its ability to cause widespread infections may promote the emergence of this species as a pathogen in immunosuppressed patients.