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.     

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.     

Comparison of Spectrophotometric and Visual Readings of NCCLS Method and Evaluation of a Colorimetric Method Based on Reduction of a Soluble Tetrazolium Salt, 2,3-Bis {2-Methoxy-4-Nitro-5-[(Sulfenylamino) Carbonyl]-2H- Tetrazolium-Hydroxide}, for Antifungal Susceptibility Testing of Aspergillus Species

The susceptibilities of 25 clinical isolates of various Aspergillus species (Aspergillus fumigatus, A. flavus, A. terreus, A. ustus, and A. nidulans) to itraconazole (ITC) and amphotericin B (AMB) were determined using the standard proposed by NCCLS for antifungal susceptibility testing of[filamentous fungi, a modification of this method using spectrophotometric readings, and a colorimetric method using the tetrazolium salt 2,3-bis [2-methoxy-4-nitro-5-[(sulfenylamino) carbonyl]-2H-tetrazolium-hydroxide] (XTT). Five MIC end points for ITC (MIC-0, no visible growth or 93%. Between visual and spectrophotometric readings, high levels of agreement were found for AMB (approximately 97%) and MIC-1 (approximately 92%) and MIC-2 (approximately 88%) of ITC. Poor agreement was found for MIC-0 of ITC (51% after 24 h), since the spectrophotometric readings resulted in higher MIC-0 values than the visual readings. The agreement was increased to 98% by shifting the threshold level of MIC-0 from 5 to 10% relative optical density and by establishing an optical density of greater than 0.1 for the GC as the validation criterion. No statistically significant differences were found between the NCCLS method and the XTT method, with the levels of agreement exceeding 97% for MIC-0 of AMB and 83% for MIC-0, MIC-1, and MIC-2 of ITC. The XTT method and spectrophotometric readings can increase the sensitivity and the precision, respectively, of in vitro susceptibility testing of Aspergillus species.     

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.     

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.     

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.     

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.     

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.     

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.     

Comparison of sensititre broth microdilution and agar dilution susceptibility testing techniques for meropenem to determine accuracy, reproducibility, and predictive values.

The stability, accuracy, reproducibility, and predictive value of Sensititre MIC panels containing meropenem (Merrem) were evaluated by using National Committee for Clinical Laboratory Standards (NCCLS)-recommended American Type Culture Collection (ATCC) strains and 110 selected strains of rapidly growing and fastidious aerobes and anaerobes with various degrees of susceptibility to meropenem. The NCCLS-recommended agar dilution method was used as a standard reference method. Meropenem-containing Sensititre MIC panels were monitored for their stabilities at room temperature and reproducibilities over 24 months by using six ATCC strains. Ninety-nine percent of the MICs of both meropenem and imipenem obtained for NCCLS-recommended ATCC strains were within the established ranges after 2 years. The overall agreement (+/- 1 twofold dilution) between the Sensititre and the agar dilution meropenem MICs was greater than 93%. The predictive value of meropenem MICs for indicating suspeptibility or resistance obtained by the Sensititre method was greater than 90%. No major or very major interpretive errors were observed, and only 5% of meropenem MICs were associated with minor interpretive errors. Problematic organisms were not observed. The Sensititre MIC panels containing meropenem offer a convenient and valid alternative to the NCCLS reference method for the susceptibility testing of potential pathogens likely to be recovered from mixed infections.     

Use of the sensititre colorimetric microdilution panel for antifungal susceptibility testing of dermatophytes

The Sensititre YeastOne antifungal panel was used to test 49 dermatophytes belonging to the species Epidermophyton floccosum, Microsporum gypseum, Microsporum canis, Trichophyton tonsurans, Trichophyton rubrum, and Trichophyton mentagrophytes. The MICs of four antifungals obtained with the Sensititre YeastOne antifungal panel were compared with those obtained by the reference NCCLS microdilution method. The levels of agreement between the two methods (相似文献   

Clinical evaluation of the Sensititre YeastOne colorimetric antifungal plate for antifungal susceptibility testing of the new triazoles voriconazole, posaconazole, and ravuconazole

A commercially prepared dried colorimetric microdilution panel (Sensititre YeastOne, TREK Diagnostic Systems, Cleveland, Ohio) was compared in three different laboratories with the National Committee for Clinical Laboratory Standards (NCCLS) reference microdilution method by testing two quality control strains and 300 clinical isolates of Candida spp. against fluconazole, voriconazole, posaconazole, and ravuconazole. Reference MIC endpoints were established after 48 h of incubation and YeastOne colorimetric endpoints were established after 24 h of incubation. YeastOne endpoints were determined to be the lowest concentration at which the color in the well changed from red (indicating growth) to purple (indicating growth inhibition) or blue (indicating no growth). Excellent agreement (within two dilutions) between the reference and colorimetric MICs was observed. Overall agreement was 95.4%. Agreement ranged from 92.3% with posaconazole to 98.0% with fluconazole. The YeastOne colorimetric method appears to be comparable to the NCCLS reference method for testing the susceptibility of Candida spp to the new triazoles voriconazole, posaconazole, and ravuconazole.     

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.     

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.     

Comparison of NCCLS and 3-(4,5-dimethyl-2-Thiazyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) methods of in vitro susceptibility testing of filamentous fungi and development of a new simplified method

The susceptibility of 30 clinical isolates belonging to six different species of filamentous fungi (Aspergillus fumigatus, Aspergillus flavus, Scedosporium prolificans, Scedosporium apiospermum, Fusarium solani, and Fusarium oxysporum) was tested against six antifungal drugs (miconazole, voriconazole, itraconazole, UR9825, terbinafine, and amphotericin B) with the microdilution method recommended by the National Committee for Clinical Laboratory Standards (NCCLS) (M38-P). The MICs were compared with the MICs obtained by a colorimetric method measuring the reduction of the dye 3-(4,5-dimethyl-2-thiazyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) to formazan by viable fungi. The levels of agreement between the two methods were 96 and 92% for MIC-0 (clear wells) and MIC-1 (75% growth reduction), respectively. The levels of agreement were always higher for Aspergillus spp. (97% +/- 2.5%), followed by Scedosporium spp. (87% +/- 10.3%) and Fusarium spp. (78% +/- 7.8%). The NCCLS method was more reproducible than the MTT method: 98 versus 95% for MIC-0 and 97 versus 90% for MIC-1. However, the percentage of hyphal growth as determined visually by the NCCLS method showed several discrepancies when they were compared with the percentages of MTT reduction. A new simplified assay that incorporates the dye MTT with the initial inoculum and in which the fungi are incubated with the dye for 48 h or more was developed, showing comparable levels of agreement and reproducibility with the other two methods. Furthermore, the new assay was easier to perform and more sensitive than the MTT method.     

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.     

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.     

Comparison of the Sensititre YeastOne dilution method with the Clinical Laboratory Standards Institute (CLSI) M27-A3 microbroth dilution reference method for determining MIC of eight antifungal agents on 102 yeast strains

The Clinical Laboratory Standards Institute ([CLSI] formerly NCCLS) reference broth microdilution testing method (protocol M27-A3) was compared with a commercially available methods (Sensititre YeastOne^®) by testing two quality control strains and 102 isolates of Candida sp. and Cryptococcus sp. against fluconazole, itraconazole, ketoconazole, posaconazole, voriconazole, flucytosin, amphotericin B and caspofungin. Minimal inhibitory concentrations (MIC) endpoints were determined after 24 h of incubation for Sensititre YeastOne^® and after 24 and 48 h for CLSI microdilution method. Essential agreements between methods vary from 70.6 to 92.2%. Categorical agreements vary from 94.1% for 5FC to 72.6% for AMB. Sensititre YeastOne^® reading appears to be useful for avoiding very major errors and this confirms the interest of this method for evaluating new antifungals activity in vitro.     

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.     

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.