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

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

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.     

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

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

Rapid flow cytometric susceptibility testing of Candida albicans.

A rapid flow cytometric assay for in vitro antifungal drug susceptibility testing was developed by adapting the proposed reference method for broth macrodilution testing of yeasts. Membrane permeability changes caused by the antifungal agent were measured by flow cytometry using propidium iodide, a nucleic acid-binding fluorochrome largely excluded by the intact cell membrane. We determined the in vitro susceptibility of 31 Candida albicans isolates and two quality control strains (Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258) to amphotericin B and fluconazole. Amphotericin B MICs ranged from 0.03 to 2.0 microg/ml, while fluconazole MICs ranged from 0.125 to 128 microg/ml. This method results in clear-cut endpoints that were reproducible. Four-hour incubation was required for fluconazole, whereas a 2-h incubation was sufficient for amphotericin B to provide MICs comparable to the reference macrodilution method developed by the National Committee for Clinical Laboratory Standards Subcommittee on Antifungal Susceptibility Tests. Results of these studies show that flow cytometry provides a rapid and sensitive in vitro method for antifungal susceptibility testing of C. albicans.     

Fluconazole and amphotericin B antifungal susceptibility testing by National Committee for Clinical Laboratory Standards broth macrodilution method compared with E-test and semiautomated broth microdilution test.

A comparative study of fluconazole and amphotericin B susceptibility testing was performed with 68 clinical Candida species isolates and three test methods. The methods used were an agar diffusion method (E-test) and two broth dilution methods, the National Committee for Clinical Laboratory Standards (NCCLS) reference broth macrodilution method and an in-house-prepared semiautomated broth microdilution method based on the Bioscreen turbidometer. In the microdilution method, growth of the yeasts was measured continuously by the automatic turbidometer (Bioscreen), which permitted precise and objective determination of endpoints. MIC endpoints were read after 24 h for the microdilution method and the E-test. Amphotericin B susceptibility testing with the NCCLS method and the E-test yielded comparable results in 89% of the tests, meaning that the endpoints obtained were identical or differed by no more than 2 twofold dilutions. The NCCLS and broth microdilution tests scored 97% comparable results, and the E-test and the broth microdilution test yielded 90% comparable results. Fluconazole susceptibility testing produced 96% comparable results with the NCCLS test and the E-test, 100% comparable results with the NCCLS and the microdilution methods, and 98.5% comparable results with the microdilution method and the E-test. We conclude that the E-test and the Bioscreen microdilution method are valuable alternatives to the NCCLS reference method for routine susceptibility testing of Candida species with fluconazole and amphotericin B.     

Comparison of two alternative microdilution procedures with the National Committee for Clinical Laboratory Standards reference macrodilution method M27-P for in vitro testing of fluconazole-resistant and -susceptible isolates of Candida albicans.

The National Committee for Clinical Laboratory Standards has proposed a reference broth macrodilution method for in vitro antifungal susceptibility testing of yeasts (the M27-P method). This method is cumbersome and time-consuming and includes MIC endpoint determination by the visual and subjective inspection of growth inhibition after 48 h of incubation. Two alternative microdilution procedures for MIC endpoint determination, a spectrophotometric MIC endpoint test that evaluates 80% growth inhibition by the drug and a colorimetric method with an oxidation-reduction indicator (Alamar Blue), were compared with the M27-P method for fluconazole susceptibility testing of 45 susceptible and resistant isolates of Candida albicans. The spectrophotometric method was performed with RPMI 1640 medium with 2% glucose, and the other two tests were performed with plain RPMI 1640 medium. All tests were incubated at 35 degrees C. Excellent agreement was demonstrated between the M27-P method and both 24-h microdilution tests (97.7%) as well as between the two microdilution tests (95.5%). Also, there was agreement in the detection in vivo of fluconazole resistance by the three methods. These preliminary data indicate that both microdilution methods may serve as less subjective alternatives to the M27-P method for the determination of fluconazole MIC endpoints.     

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.     

In vitro antifungal susceptibility testing of Candida blood isolates and evaluation of the E-test method.

Fungal infections have dramatically increased in recent years, along with the increase of drug-resistant isolates in immunocompromised patients. Ninety eight Candida species obtained from blood cultures at the Tri-Service General Hospital, Taiwan, from 1998 to 2000 were studied. These included 50 Candida albicans, 13 Candida glabrata, 24 Candida tropicalis and 11 Candida parapsilosis isolates. To investigate their susceptibility to commonly used antifungal drugs, minimum inhibitory concentrations (MIC) of amphotericin B, fluconazole, flucytosine, and ketoconazole were determined. Both the National Committee for Clinical Laboratory Standards reference broth macrodilution method and E-test were used in parallel. Ninety five isolates (95/98, 96.94%) were susceptible to amphotericin B at a concentration < or = 1 microg/mL. All isolates (100%, 98/98) were susceptible to flucytosine. Approximately 30% of these Candida isolates were resistant to fluconazole. The MIC for 90% of isolates (MIC90) values for both methods for these isolates were 0.5 microg/mL for amphotericin B, 32 microg/mL for fluconazole, 0.25 microg/mL for flucytosine (0.125 microg/mL by E-test method), and 4 microg/mL for ketoconazole. MIC for 50% of isolates (MIC50) values for these agents were 0.25, 2, 0.06, and 0.06 microg/mL, respectively. The essential agreement of MIC values within 2 dilutions for the 2 methods was 99.0% for amphotericin B, 90.8% for ketoconazole, 92.9% for fluconazole, and 91.8% for flucytosine. This study showed that E-test has equivalent performance to the broth macrodilution method and can be used as an alternative MIC technique for antifungal susceptibility testing.     

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

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

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

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

Comparison of 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.     

Quantitation of Ergosterol Content: Novel Method for Determination of Fluconazole Susceptibility of Candida albicans

MIC end points for the most commonly prescribed azole antifungal drug, fluconazole, can be difficult to determine because its fungistatic nature can lead to excessive "trailing" of growth during susceptibility testing by National Committee for Clinical Laboratory Standards broth macrodilution and microdilution methods. To overcome this ambiguity, and because fluconazole acts by inhibiting ergosterol biosynthesis, we developed a novel method to differentiate fluconazole-susceptible from fluconazole-resistant isolates by quantitating ergosterol production in cells grown in 0, 1, 4, 16, or 64 microg of fluconazole per ml. Ergosterol was isolated from whole yeast cells by saponification, followed by extraction of nonsaponifiable lipids with heptane. Ergosterol was identified by its unique spectrophotometric absorbance profile between 240 and 300 nm. We used this sterol quantitation method (SQM) to test 38 isolates with broth microdilution end points of /=64 microg/ml (resistant) and 10 isolates with trailing end points by the broth microdilution method. No significant differences in mean ergosterol content were observed between any of the isolates grown in the absence of fluconazole. However, 18 susceptible isolates showed a mean reduction in ergosterol content of 72% after exposure to 1 microg of fluconazole/ml, an 84% reduction after exposure to 4 microg/ml, and 95 and 100% reductions after exposure to 16 and 64 microg of fluconazole/ml, respectively. Ten SDD isolates showed mean ergosterol reductions of 38, 57, 73, and 99% after exposure to 1, 4, 16, and 64 microg of fluconazole/ml, respectively. In contrast, 10 resistant isolates showed mean reductions in ergosterol content of only 25, 38, 53, and 84% after exposure to the same concentrations of fluconazole. The MIC of fluconazole, by using the SQM, was defined as the lowest concentration of the drug which resulted in 80% or greater inhibition of overall mean ergosterol biosynthesis compared to that in the drug-free control. Of 38 isolates which gave clear end points by the broth microdilution method, the SQM MIC was within 2 dilutions of the broth microdilution MIC for 33 (87%). The SQM also discriminated between resistant and highly resistant isolates and was particularly useful for discerning the fluconazole susceptibilities of 10 additional isolates which gave equivocal end points by the broth microdilution method due to trailing growth. In contrast to the broth microdilution method, the SQM determined trailing isolates to be susceptible rather than resistant, indicating that the SQM may predict clinical outcome more accurately. The SQM may provide a means to enhance current methods of fluconazole susceptibility testing and may provide a better correlation of in vitro with in vivo results, particularly for isolates with trailing end points.     

Quality control limits for broth microdilution susceptibility tests of ten antifungal agents

Broth microdilution susceptibility tests of Candida species have now been standardized by the National Committee for Clinical Laboratory Standards (NCCLS). An eight-laboratory collaborative study was carried out in order to document reproducibility of tests of Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258 by the NCCLS method. Replicate broth microdilution tests were used to define control limits for 24- and 48-h MICs of amphotericin B, flucytosine, fluconazole, voriconazole, ketoconazole, itraconazole, caspofungin (MK 0991), ravuconazole (BMS 207147), posaconazole (SCH 56592), and LY 303366.     

Multicenter comparison of the VITEK 2 yeast susceptibility test with the CLSI broth microdilution reference method for testing fluconazole against Candida spp

A fully automated commercial antifungal susceptibility test system (VITEK 2 yeast susceptibility test; bioMerieux, Inc., Hazelwood, Mo.) was compared in three different laboratories with Clinical and Laboratory Standards Institute (CLSI) reference broth microdilution (BMD) method by testing two quality control strains and a total of 426 isolates of Candida spp. (103 to 135 clinical isolates in each laboratory plus 80 challenge isolates in one laboratory) against fluconazole. Reference BMD MIC endpoints were established after 24 and 48 h of incubation. VITEK 2 endpoints were determined spectrophotometrically after 10 to 26 h of incubation (mean, 13 h). Excellent essential agreement (within two dilutions) between the VITEK 2 and the 24- and 48-h BMD MICs was observed. The overall agreement values were 97.9 and 93.7%, respectively. Both intra- and interlaboratory agreement was 100%. The overall categorical agreement between VITEK 2 and BMD was 97.2% at the 24-h BMD time point and 88.3% at the 48-h BMD time point. Decreased categorical agreement at 48 h was attributed to trailing growth observed with Candida glabrata. The VITEK 2 system reliably detected fluconazole resistance among Candida spp. and demonstrated excellent quantitative and qualitative agreement with the reference BMD method.     

Use of a colorimetric system for yeast susceptibility testing.

We examined the reliability and accuracy of a colorimetric assay using Alamar Blue reagent in the performance of susceptibility tests for Candida albicans. We compared the broth macrodilution method recommended by the National Committee for Clinical Laboratory Standards (NCCLS) with a macrodilution method modified with the Alamar reagent and a microdilution method modified with the Alamar reagent. The MICs of fluconazole and itraconazole for 97 isolates of C. albicans and 3 control isolates were tested. For fluconazole, the Alamar-modified broth macrodilution method yielded 94% (91 of 97) concordance within 2 dilutions compared with the NCCLS method, while the microdilution method yielded 95% (92 of 97) concordance. With Alamar-modified methods for itraconazole, broth macrodilution yielded 97% (94 of 97) concordance within 2 dilutions. MICs obtained by the microdilution method, although tightly nested, were shifted to a higher value when compared with those obtained by the NCCLS method; there was only 77% (75 of 97) concordance within 2 dilutions but 97% concordance (94 of 97) within 3 dilutions. Tests by all methods with quality control strains showed excellent reproducibilities. For fluconazole, the methods modified with the Alamar reagent yielded clear endpoints and excellent correlation for the broth macrodilution and microdilution methods. For itraconazole, the methods modified with the Alamar reagent yielded clear endpoints and were reproducible, but higher MICs were obtained by the microdilution methods compared with those obtained by the NCCLS methods.     

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.     

Multisite reproducibility of colorimetric broth microdilution method for antifungal susceptibility testing of yeast isolates.

MICs of fluconazole and amphotericin B were determined independently for 100 coded yeast isolates by each of six laboratories to determine reproducibility of results by using a colorimetric oxidation-reduction-based broth microdilution test. In addition, each site tested five quality control isolates on at least four different occasions during the study. Results agreed within a three-dilution range (mode +/- 1 log2 dilution) for 96.2% of fluconazole tests and 92.7% of amphotericin B tests. Agreement among tests with the quality control isolates was 99.4% with fluconazole and 98.6% with amphotericin B. These results indicate that the colorimetric microdilution method is reproducible among laboratories.     

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.     

Multicenter comparison of the VITEK 2 antifungal susceptibility test with the CLSI broth microdilution reference method for testing amphotericin B, flucytosine, and voriconazole against Candida spp

A fully automated commercial antifungal susceptibility test system (VITEK 2; bioMérieux, Inc., Hazelwood, MO) was compared in three different laboratories with the Clinical and Laboratory Standards Institute (formerly the NCCLS) reference broth microdilution method (BMD) by testing 2 quality control strains, 10 reproducibility strains, and 426 isolates of Candida spp. against amphotericin B, flucytosine, and voriconazole. Reference BMD MIC endpoints were established after 24 and 48 h of incubation. VITEK 2 system MIC endpoints were determined spectrophotometrically after 9.1 to 27.1 h of incubation (mean, 12 to 14 h). Excellent essential agreement (within 2 dilutions) between the VITEK 2 system and the 24- and 48-h BMD MICs was observed for all three antifungal agents: amphotericin B, 99.1% and 97%, respectively; flucytosine, 99.1% and 98.8%, respectively; and voriconazole, 96.7% and 96%, respectively. Both intra- and interlaboratory agreements were >98% for all three drugs. The overall categorical agreements between the VITEK 2 system and BMD for flucytosine and voriconazole were 98.1 to 98.6% at the 24-h BMD time point and 96.9 to 97.4% at the 48-h BMD time point. The VITEK 2 system reliably detected flucytosine and voriconazole resistance among Candida spp. and demonstrated excellent quantitative and qualitative agreement with the reference BMD method.     

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.