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.     

Proposed quality control guidelines for antimicrobial susceptibility tests using tilmicosin.

Quality control guidelines for tilmicosin, a novel veterinary-use-only macrolide, were developed in a multi-laboratory study according to established National Committee for Clinical Laboratory Standards (NCCLS) procedures (M23-T2). Tilmicosin was incorporated into Sensititre plates for broth microdilution endpoint testing and into two lots of 15-micrograms disks for Kirby-Bauer agar disk diffusion testing. One common lot and five unique lots of Mueller-Hinton media were used. (Broth was cation adjusted, and agar was supplemented with 5% defibrinated sheep blood.) Bacteria used for reference strains included Pasteurella haemolytica 128K, Pasteurella multocida ATCC 43137, and Staphylococcus aureus ATCC 29213 (microdilution) and ATCC 25923 (disk). Replicate tests were conducted. Disk diffusion and broth microdilution quality control ranges are proposed.     

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.     

Proposed MIC quality control guidelines for National Committee for Clinical Laboratory Standards susceptibility tests using seven veterinary antimicrobial agents: ceftiofur, enrofloxacin, florfenicol, penicillin G-novobiocin, pirlimycin, premafloxacin, and spectinomycin.

The present multicenter study proposes broth microdilution quality control (QC) ranges for the antimicrobial agents ceftiofur, enrofloxacin, florfenicol, penicillin G-novobiocin, pirlimycin, premafloxacin, and spectinomycin, which are used in veterinary practice. Six separate laboratories tested replicates of National Committee for Clinical Laboratory Standards (NCCLS)-recommended QC organisms (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 29213, and Enterococcus faecalis ATCC 29212) on medium lots both common and unique to all laboratories. The proposed ranges were within 3 or 4 log2 dilution steps of the modal MICs for all organism-antimicrobial agent pairs, depending on their MIC distributions. With > or = 94.7% of all MIC results being within the proposed QC ranges, all combinations tested comply with NCCLS guidelines and all have been accepted by the NCCLS subcommittee developing susceptibility testing procedures for veterinary laboratories.     

Collaborative study of the NCCLS and flow cytometry methods for antifungal susceptibility testing of Candida albicans

One hundred clinical isolates of Candida albicans were tested for amphotericin B and fluconazole susceptibilities by the National Committee for Clinical Laboratory Standards (NCCLS) broth microdilution test at center 1 (C1). The same isolates were tested blinded at center 2 (C2) by NCCLS and flow cytometry (FC) methods. The agreement between NCCLS and FC methods ranged from 96 to 99%.     

Quality control guidelines for disk diffusion and broth microdilution antimicrobial susceptibility tests with seven drugs for veterinary applications

This multicenter study proposes antimicrobial susceptibility (MIC and disk diffusion methods) quality control (QC) parameters for seven compounds utilized in veterinary health. Alexomycin, apramycin, tiamulin, tilmicosin, and tylosin were tested by broth microdilution against various National Committee for Clinical Laboratory Standards (NCCLS)-recommended QC organisms (Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, Streptococcus pneumoniae ATCC 49619, Escherichia coli ATCC 25922, and Pseudomonas aeruginosa ATCC 27853). In addition, disk diffusion zone diameter QC limits were determined for apramycin, enrofloxacin, and premafloxacin by using E. coli ATCC 25922, P. aeruginosa ATCC 27853, and S. aureus ATCC 25923. The results from five or six participating laboratories produced >/=99.0% of MICs and >/=95.0% of the zone diameters within suggested guidelines. The NCCLS Subcommittee for Veterinary Antimicrobial Susceptibility Testing has recently approved these ranges for publication in the next M31 document.     

Flow cytometry susceptibility testing for the antifungal caspofungin

Rapid antifungal susceptibility testing for the antifungal agent caspofungin can be performed using flow cytometry (FC). An FC procedure using acridine orange provided minimum inhibitory concentration (MIC) results within 7 to 9 h which were compared with results obtained using the NCCLS M27-A2 protocol. To evaluate the consistency of this method, susceptibility testing using caspofungin was performed using 73 isolates of eight different species of Candida from various clinical samples in Central California. Macrotiter or microdilution tests were performed according to the NCCLS M27-A2 protocol, and the MICs were compared to those provided by our flow cytometry method. All isolates tested had results within the sensitive interpretive category, and 90% of the results compared within 1 dilution, showing excellent agreement between the methods. The MIC at which 50% of the isolates tested were inhibited (MIC50) and the MIC90 of caspofungin for all eight Candida species were within 1 dilution. This flow cytometer 7-h protocol for testing the antifungal susceptibility of Candida species to caspofungin provided results equivalent to those obtained with the M27-A2 protocol.     

Quality control guidelines for cefdinir, cefepime, cefetamet, cefmetazole, cefpodoxime, cefprozil, and clinafloxacin (CI-960) for various National Committee for Clinical Laboratory Standards susceptibility testing methods. Quality Control Study Group.

Several multilaboratory studies to determine quality control (QC) ranges for a variety of National Committee for Clinical Laboratory Standards (NCCLS) susceptibility tests are summarized. Replicate testing used multiple lots of media and antimicrobial disks in accordance with NCCLS recommendations, including the appropriate medium modifications for tests with Haemophilus spp. and Neisseria gonorrhoeae. QC ranges for MIC and disk diffusion testing of N. gonorrhoeae ATCC 49226 were proposed for cefepime, cefetamet, cefmetazole, and cefpodoxime. Disk diffusion QC ranges for Haemophilus influenzae ATCC 49247 or ATCC 49766 were recommended with cefepime, cefetamet (10- and 30-microgram disks), cefmetazole, cefpodoxime, and cefprozil. Disk diffusion QC ranges for Staphylococcus aureus ATCC 25923 and Escherichia coli ATCC 25922 with cefdinir and clinafloxacin and those for Pseudomonas aeruginosa ATCC 27853 with clinafloxacin were also proposed.     

Multicenter comparative evaluation of six commercial systems and the national committee for clinical laboratory standards m27-a broth microdilution method for fluconazole susceptibility testing of Candida species

Fluconazole susceptibility among 800 clinical Candida isolates (60% C. albicans) and two control strains (C. krusei ATCC 6258 and C. parapsilosis ATCC 22019) was tested with the NCCLS M27-A method (gold standard) and six commercial products (Candifast, disk, Etest, Fungitest, Integral System Yeasts, and Sensititre YeastOne). Results were classified as susceptible, susceptible-dose dependent, or resistant using M27-A breakpoints or, for Fungitest, Integral System Yeasts, and Candifast, as susceptible, intermediate, or resistant, according to the manufacturers' instructions. Concordance with NCCLS M27-A results was analyzed with the chi(2) test. Intra- and interlaboratory reproducibility was also evaluated. NCCLS M27-A (90.1%), Etest (93.1%), Sensititre YeastOne (93.1%), disk (96.7%), Fungitest (92.6%), Integral System Yeasts (40.6%), and Candifast (6.0%) classified the indicated percentages of C. albicans isolates as susceptible. Among non-C. albicans strains, the percentages of susceptible isolates were as follows: NCCLS M27-A, 74.0%; Etest, 83.8%; Sensititre YeastOne, 64.1%; disk, 60.6%; Fungitest, 76.6%; Integral System Yeasts, 28.3%; and Candifast, 27.4%. All methods except Candifast and Integral System Yeasts showed good agreement with NCCLS M27-A results for both C albicans and non-C. albicans isolates. Intralaboratory reproducibility was excellent for NCCLS M27-A, Etest, Sensititre YeastOne, disk, and Fungitest (88 to 91%). Similar results emerged from the interlaboratory reproducibility evaluation. Our findings indicate that some commercial methods can be useful for fluconazole susceptibility testing of clinical Candida isolates. Those characterized by a lack of medium standardization and/or objective interpretative criteria should be avoided. Particular caution is necessary when testing is being done for clinical and epidemiological purposes.     

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.     

Novel Fluorescent Broth Microdilution Method for Fluconazole Susceptibility Testing of Candida albicans

A comparative evaluation of the reference National Committee for Clinical Laboratory Standards (NCCLS) broth microdilution method with a novel fluorescent carboxyfluorescein diacetate (CFDA)-modified microdilution method for the susceptibility testing of fluconazole was conducted with 68 Candida strains, including 53 Candida albicans, 5 Candida tropicalis, 5 Candida glabrata, and 5 Candida parapsilosis strains. We found trailing endpoints and discordant fluconazole MICs of < 8 microg/ml at 24 h and of > or =64 microg/ml at 48 h for 12 of the C. albicans strains. These strains satisfy the definition of the low-high MIC phenotype. All 12 low-high phenotype strains were correctly shown to be susceptible at 48 h with the CFDA-modified microdilution method. For the 41 non-low-high phenotype C. albicans strains, the CFDA-modified microdilution method yielded 97.6% (40 of 41 strains) agreement within +/-1 dilution at 24 h compared with the reference method and 92.7% (38 of 41 strains) agreement within +/-1 dilution at 48 h compared with the reference method. The five strains each from C. tropicalis, C. glabrata, and C. parapsilosis that were tested showed 100% agreement within +/-2 dilutions for the two methods being evaluated.     

Comparison of the Antifungal Susceptibility Testing Subcommittee of the European Committee on Antibiotic Susceptibility Testing proposed standard and the E-test with the NCCLS broth microdilution method for voriconazole and caspofungin susceptibility testing of yeast species

The proposed standard of the Antifungal Susceptibility Testing Subcommittee of the European Committee on Antibiotic Susceptibility Testing (AFST-EUCAST) and the E-test procedures were compared with the NCCLS reference broth microdilution method for voriconazole and caspofungin susceptibility testing of 102 clinical Candida species and Saccharomyces cerevisiae isolates. The voriconazole MIC at which 50% of strains were inhibited (MIC(50)) was < or =0.125 mg/liter for all yeast species except for Candida glabrata and Candida krusei, which yielded MIC(50) values of 0.25 to 1 mg/liter depending on the method. Caspofungin exhibited in vitro activity (MIC(50) of < or =0.125 to 2 mg/liter) against all yeast species except for Candida guilliermondii. The agreements between MICs within +/-2 dilutions obtained by the NCCLS method and the EUCAST standard were 97% for voriconazole and 96% for caspofungin. Intraclass correlation coefficients were statistically significant (P < 0.05). The agreements between voriconazole MICs provided by the E-test and the NCCLS and between the E-test and the AFST-EUCAST method were 100 and 90%, respectively. Because of lower caspofungin MICs provided by the E-test, the agreement was slightly poorer with the NCCLS method (89%) than with the AFST-EUCAST procedure (94%). Both the EUCAST and the E-test procedures can be reliable techniques for susceptibility testing of yeasts to voriconazole and caspofungin.     

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.     

Quality control limits for voriconazole disk susceptibility tests on Mueller-Hinton agar with glucose and methylene blue

An international collaborative study was performed in order to propose quality control limits for voriconazole disk diffusion tests on Mueller-Hinton agar with 2% glucose and 0.5 micro g of methylene blue per ml. The supplement may be added to the agar before autoclaving, or Mueller-Hinton agar plates may be flooded with a glucose-methylene blue solution. Replicate tests on both types of agar plates with 1- micro g voriconazole disks generated data to propose zone size limits for tests of Candida parapsilosis ATCC 22019 (28 to 37 mm), Candida albicans ATCC 90028 (31 to 42 mm), and Candida krusei ATCC 6258 (16 to 25 mm). Candida tropicalis ATCC 750 was not useful for this purpose.     

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

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

Comparison of results of fluconazole disk diffusion testing for Candida species with results from a central reference laboratory in the ARTEMIS global antifungal surveillance program

The accuracy of antifungal susceptibility tests is important for accurate resistance surveillance and for the clinical management of patients with serious infections. Our main objective was to compare the results of fluconazole disk diffusion testing of Candida spp. performed by ARTEMIS participating centers with disk diffusion and MIC results obtained by the central reference laboratory. A total of 2,949 isolates of Candida spp. were tested by NCCLS disk diffusion and reference broth microdilution methods in the central reference laboratory. These results were compared to the results of disk diffusion testing performed in the 54 participating centers. All tests were performed and interpreted following NCCLS recommendations. Overall categorical agreement between participant disk diffusion test results and reference laboratory MIC results was 87.4%, with 0.2% very major errors (VME) and 3.3% major errors (ME). The categorical agreement between the disk diffusion test results obtained in the reference laboratory with the MIC test results was similar: 92.8%. Likewise, good agreement was observed between participant disk diffusion test results and reference laboratory disk diffusion test results: 90.4%, 0.4% VME, and 3.4% ME. The disk diffusion test was especially reliable in detecting those isolates of Candida spp. that were characterized as resistant by reference MIC testing. External quality assurance data obtained by surveillance programs such as the ARTEMIS Global Antifungal Surveillance Program ensure the generation of useful surveillance data and result in the continued improvement of antifungal susceptibility testing practices.     

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

The performance of the Etest for testing the susceptibilities to caspofungin (MK-0991) of 726 isolates of Candida spp. was assessed against the National Committee for Clinical Laboratory Standards (NCCLS) microdilution broth method. The NCCLS method employed RPMI 1640 broth medium, and MICs were read after incubation for 48 h at 35 degrees C. MICs were determined by Etest for all 726 isolates with RPMI agar containing 2% glucose (RPG) and were read after incubation for 48 h at 35 degrees C. The Candida isolates included Candida albicans (n = 486), Candida glabrata (n = 96), Candida tropicalis (n = 51), Candida parapsilosis (n = 47), Candida krusei (n = 11), Candida lusitaniae (n = 2), and Candida guilliermondii (n = 33). In addition, a subset of 314 isolates were also tested by Etest using Casitone agar (CAS) and antibiotic medium 3 agar (AM3). The Etest results obtained using RPG correlated well with reference MICs. Overall agreement was 94% with RPG, 82% with CAS, and 79% with AM3. When RPG was used, agreement ranged from 79% for C. parapsilosis to 100% for C. krusei, C. lusitaniae, and C. guilliermondii. When CAS was used, agreement ranged from 0% for C. lusitaniae to 100% for C. glabrata. With AM3, agreement ranged from 0% for C. lusitaniae to 100% for C. guilliermondii. All three media supported growth of each of the Candida species. Etest results were easy to read, with sharp zones of inhibition. In most instances (75%) where a discrepancy was observed between the Etest and the reference method, the Etest MIC was lower. The Etest method using RPG appears to be useful for determining caspofungin susceptibilities of Candida species.     

Boric acid susceptibility testing of non-C. albicans Candida and Saccharomyces cerevisiae: comparison of three methods.

To establish the best method for boric acid susceptibility testing, we compared two agar dilution methods (high and low inoculum) and a standard broth microdilution method (from the National Commitee for Clinical Laboratory Standards document NCCLS M-27A). Saccharomyces cerevisiae (37) and non-C. albicans Candida (39) isolates, as well as one isolate of Trichosporon sp., were included. All were isolated from female workers with vulvovaginitis. Good agreement within a fourfold dilution range was found between the three methods, and only the broth microdilution method versus the agar dilution method with high inoculum showed significant discrepancies. Reading results was easier with the broth microdilution method than with the agar dilution methods because of partial growth inhibition in the latter. In conclusion, broth microdilution is a suitable method for testing yeast susceptibility to boric acid.     

Comparative evaluation of PASCO and national committee for clinical laboratory standards M27-A broth microdilution methods for antifungal drug susceptibility testing of yeasts

The PASCO antifungal susceptibility test system, developed in collaboration with a commercial company, is a broth microdilution assay which is faster and easier to use than the reference broth microdilution test performed according to the National Committee for Clinical Laboratory Standards (NCCLS) document M27-A guidelines. Advantages of the PASCO system include the system's inclusion of quality-controlled, premade antifungal panels containing 10, twofold serial dilutions of drugs and a one-step inoculation system whereby all wells are simultaneously inoculated in a single step. For the prototype panel, we chose eight antifungal agents for in vitro testing (amphotericin B, flucytosine, fluconazole, ketoconazole, itraconazole, clotrimazole, miconazole, and terconazole) and compared the results with those of the NCCLS method for testing 74 yeast isolates (14 Candida albicans, 10 Candida glabrata, 10 Candida tropicalis, 10 Candida krusei, 10 Candida dubliniensis, 10 Candida parapsilosis, and 10 Cryptococcus neoformans isolates). The overall agreements between the methods were 91% for fluconazole, 89% for amphotericin B and ketoconazole, 85% for itraconazole, 80% for flucytosine, 77% for terconazole, 66% for miconazole, and 53% for clotrimazole. In contrast to the M27-A reference method, the PASCO method classified as resistant seven itraconazole-susceptible isolates (9%), two fluconazole-susceptible isolates (3%), and three flucytosine-susceptible isolates (4%), representing 12 major errors. In addition, it classified two fluconazole-resistant isolates (3%) and one flucytosine-resistant isolate (1%) as susceptible, representing three very major errors. Overall, the agreement between the methods was greater than or equal to 80% for four of the seven species tested (C. dubliniensis, C. glabrata, C. krusei, and C. neoformans). The lowest agreement between methods was observed for miconazole and clotrimazole and for C. krusei isolates tested against terconazole. When the data for miconazole and clotrimazole were removed from the analysis, agreement was >/=80% for all seven species tested. Therefore, the PASCO method is a suitable alternative procedure for the testing of the antifungal susceptibilities of the medically important Candida spp. and C. neoformans against a range of antifungal agents with the exceptions only of miconazole and clotrimazole and of terconazole against C. krusei isolates.     

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.