Comparison of Agar Dilution, Microdilution, E-Test, and Disk Diffusion Methods for Testing Activity of Cefditoren against Streptococcus pneumoniae

This study evaluated the susceptibility of pneumococci to cefditoren by agar dilution and microdilution methods (both in air) and by E-test (AB Biodisk, Solna, Sweden) and disk diffusion methods (both in CO(2)). By the three MIC tests, the MICs at which 50 and 90% of isolates were inhibited (MIC(50)s and MIC(90)s) were, respectively, as follows (in micrograms per milliliter): for the 65 penicillin-susceptible strains tested, 0.016 and 0.03 (by agar dilution), 0.016 and 0.03 (by microdilution), and 0.016 and 0.03 (by E test); for the 68 penicillin-intermediate strains tested, 0.125 and 0.5 (by agar dilution), 0.125 and 0.5 (by microdilution), and 0. 25 and 0.5 (by E test); and for the 67 penicillin-resistant strains tested, 1.0 and 1.0 (by agar dilution), 0.5 and 1.0 (by microdilution), and 1.0 and 1.0 (by E test). With tentative cefditoren breakpoints (in micrograms per milliliter) of /=8.0 (resistant), all strains were susceptible to cefditoren by agar, microdilution, and E-test results; with breakpoints of /=4.0 microg/ml, 97% of strains were cefditoren susceptible by agar dilution results, 98% were susceptible by microdilution results, and 99% were susceptible by E-test results. When microdilution and E-test results were compared to those from the reference agar dilution method, 191 (95.5%) and 183 (91.5%) of strains gave essential agreement (+/-1 log(2) dilution); 8 (2.7%) minor discrepancies were found for both methods with a breakpoint of /=20 (susceptible), 17 to 19 (intermediate), and /=16 mm (susceptible). All three methods for testing the MIC of cefditoren showed excellent correlation.     

Characterization of erythromycin-resistant Streptococcus pneumoniae in Korea, and In Vitro activity of telithromycin against erythromycin-resistant Streptococcus pneumoniae

To characterize the phenotypes and genotypes of erythromycin-resistant clinical isolates of Streptococcus pneumoniae in Korea and to evaluate the in vitro activity of telithromycin against these erythromycin-resistant isolates, we tested a total of 676 isolates of S. pneumoniae collected from 1997 to 2002 in a tertiary hospital in Seoul, Republic of Korea. MICs for erythromycin and telithromycin were determined by the agar dilution method. The macrolide resistance phenotypes of erythromycin-resistant isolates were determined by the erythromycin- clindamycin-rokitamycin triple disk (ECRTD) and MIC induction tests, whereas their macrolide resistance genotypes were determined by PCR for the erm(B), erm(A), subclass erm(TR), and mef genes. To discriminate between mef(A) and mef(E), PCR-restriction fragment length polymorphism (RFLP) analyses were performed. Of the 676 S. pneumoniae isolates, 459 (67.9%) were resistant to erythromycin. Of the 459 erythromycin-resistant isolates, 343 (74.7%) were assigned to the cMLS phenotype, 48 (10.4%) to the iMcLS phenotype, 4 (0.9%) to the iMLS phenotype, and 64 (14.0%) to the M phenotype. The erm(B) gene was detected in 251 (54.6%) isolates, the mef gene was detected in 64 (14.0%), and both the erm(B) and mef genes were detected in 144 (31.4%) isolates. All of the mef genes detected were identified as mef(E). Of the 459 erythromycin- resistant isolates, all but one were susceptible to telithromycin. The MIC(50)/MIC(90) to telithromycin of isolates carrying erm(B), mef(E), and both genes was 0.06/0.5 microg/ml, 0.03/0.125 microg/ml, and 0.5/1.0 microg/ml, respectively. Although the MICs of telithromycin for the erythromycin-resistant isolates varied according to genotype, telithromycin was very active against these erythromycin-resistant S. pneumoniae.     

Influence of variations in test methods on susceptibility of Haemophilus influenzae to ampicillin, azithromycin, clarithromycin, and telithromycin

The National Committee for Clinical Laboratory Standards standard broth microdilution method for testing the susceptibility of Haemophilus influenzae to ampicillin, azithromycin, clarithromycin, and telithromycin was evaluated by altering one variable at a time. Variables that were tested included age of colony for inoculum preparation, inoculum density, test medium, incubation atmosphere, and incubation time. For the macrolide, azalide, and ketolide agents, incubation in 5 to 7% CO(2) most significantly affected the MICs, producing nearly twofold increases for clarithromycin and telithromycin and a greater than threefold increase for azithromycin. For ampicillin, a 10-fold increase in inoculum density increased the geometric mean MICs for beta-lactamase-negative strains from 1. 50 to 2.45 microg/ml. In addition, 206 H. influenzae strains were tested for their susceptibilities to the same drugs by the broth microdilution tests in two media, as well as by agar dilution tests, disk diffusion tests, and Etests, on six different agar media. The three standard methods with Haemophilus test medium (HTM) compared favorably with each other except for a high minor discrepancy rate (27%) by the disk diffusion test with ampicillin and clarithromycin. Agar dilution test MICs on the five comparative media were generally higher than those on HTM agar but were only rarely more than one twofold concentration higher. Etest MICs of azithromycin and telithromycin were more than twofold higher than agar dilution and broth microdilution MICs on HTM; ampicillin Etest MICs were nearly twofold lower. The use of media other than HTM agar appears to have a minimal effect on susceptibility test results for the ketolide, azalide, or macrolide drugs that we tested against H. influenzae.     

Assessment of metronidazole susceptibility in Helicobacter pylori: statistical validation and error rate analysis of breakpoints determined by the disk diffusion test

Metronidazole susceptibility of 100 Helicobacter pylori strains was assessed by determining the inhibition zone diameters by disk diffusion test and the MICs by agar dilution and PDM Epsilometer test (E test). Linear regression analysis was performed, allowing the definition of significant linear relations, and revealed correlations of disk diffusion results with both E-test and agar dilution results (r2 = 0.88 and 0.81, respectively). No significant differences (P = 0.84) were found between MICs defined by E test and those defined by agar dilution, taken as a standard. Reproducibility comparison between E-test and disk diffusion tests showed that they are equivalent and with good precision. Two interpretative susceptibility schemes (with or without an intermediate class) were compared by an interpretative error rate analysis method. The susceptibility classification scheme that included the intermediate category was retained, and breakpoints were assessed for diffusion assay with 5-microg metronidazole disks. Strains with inhibition zone diameters less than 16 mm were defined as resistant (MIC > 8 microg/ml), those with zone diameters equal to or greater than 16 mm but less than 21 mm were considered intermediate (4 microg/ml < MIC 相似文献   

Problems with the disk diffusion test for detection of vancomycin resistance in enterococci.

A total of 53 strains of enterococci, including recently isolated strains with high-level resistance to vancomycin, were tested for vancomycin susceptibility by broth microdilution and disk diffusion using Mueller-Hinton media with and without supplementation with 5% blood. By using currently published parameters of the National Committee for Clinical Laboratory Standards for the disk diffusion test, we found that strains for which MICs were 8 to 32 micrograms/ml were incorrectly placed in the susceptible or intermediate category, which caused both very major (1.9%) and minor (11.5%) errors. When we used newer, recently proposed breakpoints for vancomycin, we found 13.5% minor errors but no very major errors. Changing disk diffusion breakpoints to less than or equal to 14 mm for resistant [corrected] and greater than or equal to 15 mm for susceptible [corrected] would eliminate the problem for the strains with MICs of 32 micrograms/ml but not for those with MICs of 8 micrograms/ml. For those strains, it is necessary to perform an MIC test to differentiate them from strains with MICs of less than or equal to 4 micrograms/ml.     

Activity of Gatifloxacin against Haemophilus influenzae and Moraxella catarrhalis, Including Susceptibility Test Development, E-Test Comparisons, and Quality Control Guidelines for H. influenzae

In vitro antimicrobial activity and susceptibility testing interpretation criteria and quality control were studied for gatifloxacin, a new 8-methoxy fluoroquinolone, tested against Haemophilus influenzae. Moraxella catarrhalis (600 strains) and H. influenzae (1,400 strains) from the SENTRY Antimicrobial Surveillance Program in North America (Canada and the United States) were also tested against gatifloxacin and 12 other antimicrobial agents. Gatifloxacin (MIC at which 90% of the isolates are inhibited [MIC90], /=18 mm) was also suggested for H. influenzae testing. No interpretive errors were observed. Quality control guidelines for H. influenzae ATCC 49247 were determined by using the NCCLS M23-T3 (1998) study design. The results from the nine-laboratory protocol suggested the following control ranges: for broth microdilution tests, 0.004 to 0.03 microg/ml; for disk diffusion testing, 33 to 41 mm. Gatifloxacin appears to be a potent anti-Haemophilus fluoroquinolone compound with in vitro testing interpretive criteria that will produce accurate results (disk diffusion, broth microdilution, and E-test).     

Reliability of the E test for detection of ampicillin, vancomycin, and high-level aminoglycoside resistance in Enterococcus spp.

By comparison with agar dilution results, the E test was investigated for the ability to detect high-level aminoglycoside (gentamicin and streptomycin), ampicillin, and vancomycin resistance among strains representing six enterococcal species. For ampicillin and vancomycin, disk diffusion results also were obtained. No false high-level aminoglycoside resistance occurred, and no false gentamicin susceptibility was noted. With the high-range streptomycin E test (2,048 micrograms), 24% of the 38 resistant strains were falsely susceptible. However, these discordances could likely be reconciled by adjustments in incubation duration and by using broth microdilution rather than agar screen breakpoint criteria, or by using the lower-range (1,024-micrograms) strip. For ampicillin, category results obtained by E test and disk diffusion showed good agreement with agar dilution; E test MICs were generally comparable to agar dilution MICs. The E test was more sensitive than disk diffusion for detecting vancomycin-intermediate strains, but for these strains and those exhibiting low-level vancomycin resistance (MIC, 32 to 128 micrograms/ml), disk diffusion and E test inhibition zones must be interpreted with caution. Given the reliability of E test for detecting resistance to anti-enterococcal agents, the decision to use this method should be based on convenience, cost, testing frequency, and satisfaction with currently used methods.     

Reevaluation of interpretive criteria for Haemophilus influenzae by using meropenem (10-microgram), imipenem (10-microgram), and ampicillin (2- and 10-microgram) disks.

A collection of 300 Haemophilus influenzae clinical strains was used to assess in vitro susceptibility to carbapenems (meropenem, imipenem) by MIC and disk diffusion methods and to compare disk diffusion test results with two potencies of ampicillin disks (2 and 10 micrograms). The isolates included ampicillin-susceptible or- intermediate (167 strains), beta-lactamase-positive (117 strains), and beta-lactamase-negative ampicillin-resistant (BLNAR; 16 strains) organisms. Disk diffusion testing was performed with 10-micrograms meropenem disks from two manufacturers. Meropenem was highly active against H. influenzae strains (MIC50, 0.06 microgram/ml; MIC90, 0.25 microgram/ml; MIC50 and MIC90, MICs at which 50 and 90%, respectively, of strains are inhibited) and was 8- to 16-fold more potent than imipenem (MIC50, 1 microgram/ml; MIC90, 2 micrograms/ml). Five non-imipenem-susceptible strains were identified (MIC, 8 micrograms/ml), but the disk diffusion test indicated susceptibility (zone diameters, 18 to 21 mm). MIC values of meropenem, doxycycline, ceftazidime, and ceftriaxone for BLNAR strains were two- to fourfold greater than those for other strains. The performance of both meropenem disks was comparable and considered acceptable. A single susceptible interpretive zone diameter of > or = 17 mm (MIC, < = or 4 micrograms/ml) was proposed for meropenem. Testing with the 2-micrograms ampicillin disk was preferred because of an excellent correlation between MIC values and zone diameters (r = 0.94) and superior interpretive accuracy with the susceptible criteria at > or = 17 mm (MIC, < or = 1 microgram/ml) and the resistant criteria at < or = 13 mm (MIC, > or = 4 micrograms/ml). Among the BLNAR strains tested, 81.3% were miscategorized as susceptible or intermediate when the 10-micrograms ampicillin disk was used, while the 2-micrograms disk produced only minor interpretive errors (12.5%). Use of these criteria for testing H. influenzae against meropenem and ampicillin should maximize reference test and standardized disk diffusion test performance with the Haemophilus Test Medium. The imipenem disk diffusion test appears compromised and should be used with caution for detecting strains for which imipenem MICs are elevated.     

Evaluation of in vitro spectra of activity of azithromycin, clarithromycin, and erythromycin tested against strains of Neisseria gonorrhoeae by reference agar dilution, disk diffusion, and Etest methods.

The macrolide-azilide susceptibility testing (agar dilution, disk diffusion, Etest) criteria for 105 Neisseria gonorrhoeae strains were evaluated. In addition, the potencies of azithromycin, clarithromycin, and erythromycin were studied. The most active macrolide-azilide agent was azithromycin (MIC at which 90% of the isolates are inhibited [MIC90], 0.5 microgram/ml) compared with clarithromycin (MIC90, 1.5 to 2 micrograms/ml) and erythromycin (MIC90, 2 to 4 micrograms/ml). The Etest (AB Biodisk, Solna, Sweden) was observed to produce MIC results very similar to those of the reference agar dilution test (GC agar base), with 100% of the results within 1 log2 dilution step of the reference MICs. The disk diffusion test zone diameters for all three drugs correlated at an acceptable level (r = -0.81 to -0.92) with the reference agar dilution MICs. Interpretive criteria for susceptibility were proposed for azithromycin at a MIC of < or = 2 micrograms/ml and a disk diffusion test zone of > or = 25 mm. No category for resistance was proposed because of the paucity of strains for which MICs were > 2 micrograms/ml. These tentative criteria should be further validated by correlations with clinical trial data for gonococcal strains (as they emerge) that have azithromycin MICs above the proposed susceptible category range.     

Modification of interpretive breakpoints for netilmicin disk susceptibility tests with Pseudomonas aeruginosa.

Regression analysis of data correlating 30-micrograms netilmicin disk zone diameters with microdilution MICs, obtained by testing close-interval dilution steps, was performed with 77 selected strains of Pseudomonas aeruginosa, each tested in three independent laboratories. A zone of greater than or equal to 15 mm correlated with an MIC of less than or equal to 12 micrograms/ml (susceptible), and a zone of less than or equal to 12 mm correlated with an MIC of greater than 16 micrograms/ml (resistant). Additional disk tests were performed with 256 strains having known resistance mechanisms and 280 susceptible strains: the majority were appropriately categorized by these interpretive zone standards. The previously recommended standards of greater than or equal to 17 mm (MIC, less than or equal to 8.0 micrograms/ml) for the susceptible category inappropriately placed a significant number of truly susceptible P. aeruginosa strains in the intermediate category.     

Detection of fluconazole-resistant Candida strains by a disc diffusion screening test

A commercial disc diffusion test has been evaluated as a screening method for the detection of Candida species with decreased susceptibility to fluconazole. A total of 1,407 Candida strains of different species were tested, and the results were compared with the MIC results. The recently published National Committee for Clinical Laboratory Standards breakpoint criteria have been used. Isolates were classified as susceptible if the MIC for the isolates was /=64 microg/ml. All 77 resistant strains and 121 of 122 S-DD strains had fluconazole zone diameters of 相似文献   

Antipneumococcal Activities of Levofloxacin and Clarithromycin as Determined by Agar Dilution, Microdilution, E-Test, and Disk Diffusion Methodologies

The activities of levofloxacin and clarithromycin against 199 penicillin- and macrolide-susceptible and -resistant pneumococci were tested by agar and microdilution methods in air and by disk diffusion and E-test methods in air and CO₂. For levofloxacin, ≥99.0% of strains were susceptible at ≤2.0 μg/ml with zone diameters of ≥17 mm, regardless of incubation in air or CO₂. Although zone sizes were smaller and E-test MICs were higher for clarithromycin in CO₂ than those in air, category differences were minor, and susceptibility rates for clarithromycin were similar to those obtained by agar and microdilution in air (range, 76.9 to 80.9% by all methods). For clarithromycin, adjustment of breakpoints based upon distribution of results resulted in susceptibility rates which were similar by all methods (75.8 to 76.9% susceptible, 0 to 1.5% intermediate, 22.6 to 23.1% resistant). Minor discrepancies were obtained with levofloxacin for one strain (0.5%) by microdilution and two strains (1.0%) by disk diffusion in CO₂. For clarithromycin, minor discrepancies were found in three strains (1.5%) by microdilution, seven strains (3.5%) by agar dilution, four strains (2.0%) by E-test in air, six strains (3.0%) by disk diffusion in air, and five strains (2.5%) by disk diffusion in CO₂. Major discrepancies occurred with levofloxacin in one strain (0.5%) by microdilution but were not found with clarithromycin. Very major discrepancies were not seen with levofloxacin, but occurred with clarithromycin in five strains (2.5%) by microdilution, three strains (1.5%) by agar dilution, two strains (1.0%) by E-test in air, eight strains (4.0%) by disk diffusion in air, and one strain (0.5%) by disk diffusion in CO₂.     

Inaccuracy of the disk diffusion method compared with the agar dilution method for susceptibility testing of Campylobacter spp

The agar dilution method has been standardized by the CLSI for the susceptibility testing of Campylobacter species, and according to these standards, the disk diffusion method should be used only in screening for macrolide and ciprofloxacin resistance. Nevertheless, the disk diffusion test is currently widely used, since it is easy to perform in clinical microbiology laboratories. In this study, the disk diffusion method was compared to the agar dilution method by analyzing the in vitro activities of seven antimicrobial agents against 174 Campylobacter strains collected in Finland between 2003 and 2008. Recommendations of the CLSI were followed using Mueller-Hinton agar plates with 5% of sheep blood. For each strain, the disk diffusion tests were performed two to four times. Of the 33 erythromycin-resistant strains (MIC, ≥16 μg/ml), 24 (73%) constantly showed a 6-mm erythromycin inhibition zone (i.e., no inhibition), while for seven strains the inhibition zone varied from 6 to 44 mm in repeated measurements. Among the 141 erythromycin-susceptible strains (MIC, <16 μg/ml), erythromycin inhibition zones varied between 6 and 61 mm. Of the 87 ciprofloxacin-resistant strains, 47 (54%) showed 6-mm inhibition zones, while 40 strains showed inhibition zones between 6 and 60 mm. Significant differences between the repetitions were observed in the disk diffusion for all antimicrobial agents and all strains except for the macrolide-resistant strains regarding the macrolides. For 17 (10%) strains, the variation in repeated measurements was substantial. These results show that the disk diffusion method may not be a reliable tool for the susceptibility testing of Campylobacter spp. Further studies are needed to assess whether the disk diffusion test could be improved or whether all susceptibilities of campylobacters should be tested using an MIC-based method.     

Use of an oxacillin disk screening test for detection of penicillin- and ceftriaxone-resistant pneumococci

In a context of worldwide emergence of resistance among Streptococcus pneumoniae strains, early detection of strains with decreased susceptibility to beta-lactam antibiotics is important for clinicians. If the 1-microgram oxacillin disk diffusion test is used as described by the National Committee for Clinical Laboratory Standards, no interpretation is available for strains showing zone sizes of /=2.0 microgram/ml) to penicillin. For ceftriaxone, among 98 strains with no zone of inhibition in response to oxacillin, 68 had intermediate resistance (MIC, 1.0 microgram/ml), and 22 were resistant (MIC, >/=2.0 microgram/ml). To optimize the use of the disk diffusion method, we propose that the absence of a zone of inhibition around the 1-microgram oxacillin disk be regarded as an indicator of nonsusceptibility to penicillin and ceftriaxone and recommend that such strains be reported as nonsusceptible to these antimicrobial agents, pending the results of a MIC quantitation method.     

Interpretive criteria for susceptibility testing of CI-960 (PD127391, AM-1091), fleroxacin, lomefloxacin, and temafloxacin against Neisseria gonorrhoeae, including drug stability in GC agar medium.

CI-960, fleroxacin, lomefloxacin, and temafloxacin were tested against over 100 strains of Neisseria gonorrhoeae. Each organism was tested in triplicate by using agar dilution and disk diffusion methods recommended by the National Committee for Clinical Laboratory Standards. CI-960 was the most potent compound, with a MIC against 90% of the strains tested of 0.008 microgram/ml, and the least active was fleroxacin (MIC against 90% of strains, 0.12 microgram/ml). Only the susceptible interpretive category was recommended for the CI-960 tests as follows: 5-micrograms disk, greater than or equal to 39 mm (MIC correlate, less than or equal to 0.12 microgram/ml). Three interpretive categories were proposed for the other fluoroquinolones as follows: fleroxacin, 5-micrograms disk susceptible at greater than or equal to 33 mm (MIC correlate, less than or equal to 0.25 microgram/ml), intermediate at 28 to 32 mm (MIC correlate, 0.5 microgram/ml), and resistant at less than or equal to 27 mm (MIC correlate, greater than 0.5 microgram/ml); lomefloxacin, 10-micrograms disk susceptible at greater than or equal to 35 mm (MIC correlate, less than or equal to 0.12 microgram/ml), intermediate at 28 to 34 mm (MIC correlates, 0.25 to 0.5 microgram/ml), and resistant at less than or equal to 27 mm (MIC correlate, greater than 0.5 microgram/ml); and temafloxacin, 5-micrograms disk susceptible at greater than or equal to 36 mm (MIC correlate, less than or equal to 0.06 microgram/ml), intermediate at 28 to 35 mm (MIC correlates 0.12 to 0.25 microgram/ml), and resistant at less than or equal to 27 mm (greater than 0.25 microgram/ml). Interpretive agreement between disk diffusion results and the MICs was 100% for each agent, with the exception of lomefloxacin, which had a 0.9% minor error. All drugs were stable in GC agar medium for at least 21 days when stored at 2 to 5 degrees C.     

Fluconazole disk diffusion procedure for determining susceptibility of Candida species.

A simple disk diffusion test was defined for quick determination of the susceptibility of Candida species to fluconazole. The standard macrotube dilution reference method, the fluconazole E test, and a 25-microgram fluconazole disk test were all performed with each of 250 Candida species selected to provide a broad range of fluconazole MICs. All three methods were in excellent agreement. On RPMI 1640-glucose agar, isolates with inhibition zone diameters of > or = 19 mm were all susceptible (MIC, < or = 8.0 micrograms/ml) by the E test and 94% were susceptible by the macrotube method. Strains with smaller zones were either resistant, intermediate (dose-dependent susceptibility), or susceptible by the reference methods. The disk test did not adequately separate fully resistant strains from those with dose-dependent susceptibility: additional quantitative tests are needed for the few strains that are not unequivocally susceptible by the disk method.     

Screening pneumococci for penicillin resistance

Eighty-four pneumococci with various MICs of penicillin (38 with MICs of less than or equal to 0.06 micrograms/ml [susceptible], 35 with MICs of 0.12 to 1.0 micrograms/ml [relatively resistant], and 11 with MICs of greater than 1.0 micrograms/ml [resistant] ) were screened by a disk diffusion test using oxacillin and methicillin to see how well they distinguished penicillin-susceptible strains from those with decreased susceptibility to penicillin. The effects of Mueller-Hinton agar plus 5% sheep blood and Trypticase soy agar plus 5% sheep blood and two atmospheres, ambient air and a candle extinction jar (increased CO2), were compared. There were no obvious differences between the effects of the two media, but zones were generally larger in ambient air than in increased CO2. Although the oxacillin test can separate penicillin-susceptible and -resistant strains, it cannot separate penicillin-resistant from relatively penicillin-resistant strains by using the breakpoint of less than 20 mm recommended by the National Committee for Clinical Laboratory Standards. When the 20-mm breakpoint was applied to methicillin, 12% of the relatively resistant strains tested were erroneously classified as susceptible. When different breakpoints were used for methicillin, there was better separation of the two classes of penicillin-resistant isolates, but a few relatively resistant strains were still classified as susceptible. We recommend that oxacillin, not methicillin, be used as the screening agent with Mueller-Hinton sheep blood agar and ambient air incubation and that the breakpoint be less than 20 mm to indicate resistance or relative resistance.     

Relationship between in vitro susceptibility test results for chloramphenicol and production of chloramphenicol acetyltransferase by Haemophilus influenzae, Streptococcus pneumoniae, and Aerococcus species.

Haemophilus influenzae, Streptococcus pneumoniae, and Aerococcus species were tested for susceptibility to chloramphenicol by standard broth microdilution and disk-diffusion methods. MICs and zone diameter breakpoints were correlated with production of chloramphenicol acetyltransferase (CAT). A comparison of MICs and zone diameters indicated that the interpretative criteria for H. influenzae and S. pneumoniae should be an MIC of less than or equal to 4 micrograms/ml or a zone diameter greater than or equal to 25 mm for susceptible strains and an MIC of greater than or equal to 8 micrograms/ml or a zone diameter of less than or equal to 20 mm for resistant strains; for Aerococcus species, interpretative criteria should be an MIC of less than or equal to 8 micrograms/ml or a zone diameter of greater than or equal to 20 mm for susceptible strains and an MIC of greater than or equal to 32 micrograms/ml or a zone diameter of less than or equal to 12 mm for resistant strains. All but four strains of H. influenzae and one strain of S. pneumoniae that were resistant to chloramphenicol by these criteria produced CAT. For Aerococcus species, however, chloramphenicol-resistant strains were negative for CAT as determined by a commercially available disk test. When comparing susceptibility results with CAT production, thiamphenicol was a better indicator of the presence of the enzyme than chloramphenicol and may be useful in assaying resistance to chloramphenicol.     

Fluconazole and voriconazole multidisk testing of Candida species for disk test calibration and MIC estimation

Fluconazole and voriconazole MICs were determined for 114 clinical Candida isolates, including isolates of Candida albicans, Candida glabrata, Candida krusei, Candida lusitaniae, Candida parapsilosis, and Candida tropicalis. All strains were susceptible to voriconazole, and most strains were also susceptible to fluconazole, with the exception of C. glabrata and C. krusei, the latter being fully fluconazole resistant. Single-strain regression analysis (SRA) was applied to 54 strains, including American Type Culture Collection reference strains. The regression lines obtained were markedly different for the different Candida species. Using an MIC limit of susceptibility to fluconazole of < or =8 microg/ml, according to NCCLS standards, the zone breakpoint for susceptibility for the 25-microg fluconazole disk was calculated to be > or =18 mm for C. albicans and > or =22 mm for C. glabrata and C. krusei. SRA results for voriconazole were used to estimate an optimal disk content according to rational criteria. A 5-microg disk content of voriconazole gave measurable zones for a tentative resistance limit of 4 microg/ml, whereas a 2.5-microg disk gave zones at the same MIC level for only three of the species. A novel SRA modification, multidisk testing, was also applied to the two major species, C. albicans and C. glabrata, and the MIC estimates were compared with the true MICs for the isolates. There was a significant correlation between the two measurements. Our results show that disk diffusion methods might be useful for azole testing of Candida isolates. The method can be calibrated using SRA. Multidisk testing gives direct estimations of the MICs for the isolates.     

Comparison of results of voriconazole 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 testing is important for reliable resistance surveillance and for the clinical management of patients with serious infections. Our primary objective was to compare the results of voriconazole disk diffusion testing of Candida spp. performed by centers participating in the ARTEMIS program with disk diffusion and MIC results obtained by the central reference laboratory. A total of 2,934 isolates of Candida spp. were tested by CLSI 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 CLSI recommendations, as follows: susceptible (S), MIC of or=17 mm); susceptible dose dependent (SDD), MIC of 2 microg/ml (14 to 16 mm); and resistant (R), MIC of >or=4 microg/ml (or=4 microg/ml) by 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.