Comparison of a spectrophotometric microdilution method with RPMI-2% glucose with the National Committee for Clinical Laboratory Standards reference macrodilution method M27-P for in vitro susceptibility testing of amphotericin B, flucytosine, and fluconazole against 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 visual and subjective inspection of growth inhibition after 48 h of incubation. An alternative microdilution procedure was compared with the M27-P method for determination of the amphotericin B, flucytosine, and fluconazole susceptibilities of 8 American Type Culture Collection strains (6 of them were quality control or reference strains) and 50 clinical isolates of candida albicans. This microdilution method uses as culture medium RPMI 1640 supplemented with 18 g of glucose per liter (RPMI-2% glucose). Preparation of drugs, basal medium, and inocula was done by following the recommendations of the National Committee for Clinical Laboratory Standards. The MIC endpoint was calculated objectively from the turbidimetric data read at 24 h. Increased growth of C. albicans in RPMI-2% glucose and its spectrophotometric reading allowed for the rapid (24 h) and objective calculation of MIC endpoints compared with previous microdilution methods with standard RPMI 1640. Nevertheless, good agreement was shown between the M27-P method and this microdilution test. The MICs obtained for the quality control or reference strains by the microdilution method were in the ranges published for those strains. For clinical isolates, the percentages of agreement were 100% for amphotericin B and fluconazole and 98.1% for flucytosine. These data suggest that this microdilution method may serve as a less subjective and more rapid alternative to the M27-P method for antifungal susceptibility testing of yeasts.     

Comparison of the Sensititre YeastOne colorimetric microdilution panel and the NCCLS broth microdilution method for antifungal susceptibility testing against Candida species

We evaluated the commercially prepared Sensititre YeastOne colorimetric antifungal panel to determine the susceptibility of 170 Candida spp isolates to amphotericin B, fluconazole, itraconazole, and flucytosine. The NCCLS reference microdilution method (M27-A document) was used as reference method. The YeastOne panel was performed according to the manufacturer's instructions. For the colorimetric method, MICs were determined at 24 h of incubation. MICs for the NCCLS reference method were read at 48 h of incubation. The overall agreement within +/-2 dilutions by both methods was calculated against the four antifungal agents. This agreement was 92.9, 68.2, 77.6 and 80% for amphotericin B, fluconazole, itraconazole, and flucytosine, respectively. Thirteen isolates (7.6%) showed very major discrepancies for fluconazole and 12 (7%) for itraconazole. We found that the reading of MIC with the YeastOne panel was somewhat easier than the reading of reference MIC, although the determination of endpoint was sometimes difficult, especially for azoles, because the trailing effect appeared in a high percentage of isolates.     

Broth microdilution and E-test for determining fluoroquinolone activity against Streptococcus pneumoniae

OBJECTIVE: To compare broth microdilution and E-test minimum inhibitory concentrations (MICs) of 4 fluoroquinolones against Streptococcus pneumoniae and to determine the effect of these in vitro MIC methods on the calculation of AUC00-24/MIC ratios. METHODS: Levofloxacin, gatifloxacin, moxifloxacin, and gemifloxacin MICs were determined by broth microdilution (incubated in air) and E-test (incubated in CO2) for 100 clinical isolates of S. pneumoniae. MIC50, MIC90, and geometric mean MIC were calculated. Steady-state serum concentration-time profiles were simulated for once-daily, oral dosing of levofloxacin 500 mg, gatifloxacin 400 mg, moxifloxacin 400 mg, and gemifloxacin 320 mg. After correcting for protein binding, AUC0-24 of unbound drug was calculated for each regimen, and AUC0-24/MIC ratios were calculated using MIC data from both in vitro methods. Differences in MICs between methods were determined for each agent using the paired t-test (after logarithmic transformation of MICs) and the Wilcoxon signed-rank test. Differences in AUC0-24/MIC ratios were also determined using the paired t-test and the Wilcoxon signed-rank test. The level of significance for all analyses was p < 0.05. RESULTS: Broth microdilution and E-test MICs were within +/- 1 log2 dilution for 94%, 93%, 61%, and 35% of the isolates for levofloxacin, gatifloxacin, moxifloxacin, and gemifloxacin, respectively. Broth microdilution MICs were significantly lower than E-test MICs for all 4 agents (p < 0.001). However, a categorical change in susceptibility was seen for only 1 isolate with gatifloxacin and moxifloxacin (intermediate by broth microdilution, resistant by E-test). AUC0-24/MIC ratios were significantly higher for each regimen when MICs were determined by broth microdilution compared with E-test (p < 0.001). CONCLUSIONS: There is a significant difference in the activity of the newer fluoroquinolones against S. pneumoniae when MICs are determined by broth microdilution and E-test. When evaluating fluoroquinolone activity and pharmacodynamics against this organism, clinicians must be aware that MIC testing methodology may have a significant impact on the results.     

Bactericidal activity of DU-6859a compared to activities of three quinolones, three beta-lactams, clindamycin, and metronidazole against anaerobes as determined by time-kill methodology.

The activities of DU-6859a, ciprofloxacin, levofloxacin, sparfloxacin, piperacillin, piperacillin-tazobactam, imipenem, clindamycin, and metronidazole against 11 anaerobes were tested by the broth microdilution and time-kill methods. DU-6859a was the most active drug tested (broth microdilution MICs, 0.06 to 0.5 microg/ml), followed by imipenem (MICs, 0.002 to 4.0 microg/ml). Broth macrodilution MICs were within 3 (but usually 1) dilutions of the broth microdilution MICs. All compounds were bactericidal at the MIC after 48 h; after 24 h, 90% killing was shown for all strains when the compounds were used at four times the MIC. DU-6859a at < or = 0.5 microg/ml was bactericidal after 48 h.     

Activity of retapamulin against Streptococcus pyogenes and Staphylococcus aureus evaluated by agar dilution, microdilution, E-test, and disk diffusion methodologies

The in vitro activity of retapamulin against 106 Staphylococcus aureus isolates and 109 Streptococcus pyogenes isolates was evaluated by the agar dilution, broth microdilution, E-test, and disk diffusion methodologies. Where possible, the tests were performed by using the CLSI methodology. The results of agar dilution, broth microdilution, and E-test (all with incubation in ambient air) for S. aureus yielded similar MICs, in the range of 0.03 to 0.25 microg/ml. These values corresponded to zone diameters between 25 and 33 mm by the use of a 2-microg retapamulin disk. Overall, 99% of the agar dilution results and 95% of E-test results for S. aureus were within +/-1 dilution of the microdilution results. For S. pyogenes, the MICs obtained by the agar and broth microdilution methods (both after incubation in ambient air) were in the range of 0.008 to 0.03 microg/ml, and E-test MICs (with incubation in ambient air) were 0.016 to 0.06 microg/ml. For S. pyogenes, 100% of the agar dilution MIC results were within +/-1 dilution of the broth microdilution results. E-test MICs (after incubation in ambient air) were within +/-1 and +/-2 dilutions of the broth microdilution results for 76% and 99% of the isolates, respectively. E-test MICs for S. pyogenes strains in CO(2) were up to 4 dilutions higher than those in ambient air. Therefore, it is recommended that when retapamulin MICs are determined by E-test, incubation be done in ambient air and not in CO(2), due to the adverse effect of CO(2) on the activity of this compound. Diffusion zones (with incubation in CO(2)) for S. pyogenes were 18 to 24 mm. Retapamulin MICs for all strains by all methods (with incubation in ambient air) were < or =0.25 microg/ml. These results demonstrate that S. pyogenes (including macrolide-resistant strains) and S. aureus (including methicillin-resistant and vancomycin-nonsusceptible strains) are inhibited by very low concentrations of retapamulin and that all four testing methods are satisfactory for use for susceptibility testing.     

Comparison of BSAC agar dilution and NCCLS broth microdilution MIC methods for in vitro susceptibility testing of Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis: the BSAC Respiratory Resistance Surveillance Programme

OBJECTIVE: The aim of this study was to establish the degree of comparability between the NCCLS broth microdilution and BSAC agar dilution MIC methods of antimicrobial susceptibility testing. METHODS: Six hundred and sixty-one clinical isolates of Streptococcus pneumoniae, 936 Haemophilus influenzae and 421 Moraxella catarrhalis, collected in the winter of 1999-2000 by 20 laboratories in the UK and Eire from patients with presumed community-acquired lower respiratory infections, were tested by the two methods. MIC agreement was defined as excellent (good) if results were within +/- 1 doubling dilution for > or =90% (> or =80%) of isolates and within +/- 2 doubling dilutions for > or =95%. Isolates were categorized as susceptible, intermediate or resistant using the breakpoints appropriate to the testing method. RESULTS: MIC agreement was good or excellent in 27 of 36 organism-agent combinations. Agreement was less for M. catarrhalis than for other species, and lower in all three species for cefaclor and trimethoprim than for other antimicrobials. Discrepancies in categorization occurred only occasionally, and were generally explained by differences in breakpoints rather than in measured MICs. One exception was S. pneumoniae with penicillin. Despite excellent MIC agreement and identical breakpoints, 9% of these had minor discrepancies, mainly because 7% of isolates were found intermediate by the BSAC method but resistant by the NCCLS method. CONCLUSION: There is generally very good agreement between the MICs obtained by the BSAC agar dilution and NCCLS broth microdilution methods in this population of isolates, comparable to the level of agreement achieved between different laboratories using a single method. Breakpoint differences contribute to most of the discrepancies in susceptibility categorization.     

Susceptibility testing of Campylobacter fetus subsp. jejuni, using broth microdilution panels.

Twenty-five isolates of Campylobacter fetus subsp. jejuni were tested by broth microdilution panels (Sensititre; GIBCO Diagnostics, Chagrin Falls, Ohio) and the minimal inhibitory concentrations (MICs) were compared with the corresponding MICs obtained by the standard agar dilution technique. Microdilution panels designed for testing gram-positive organisms were used so that erythromycin, the antibiotic of choice for this organism, could be included. The correlation with agar dilution was relatively poor when Mueller-Hinton broth was used; the MICs that were within one twofold dilution of the corresponding agar dilution MIC ranged from 15% with tetracycline to 75% with ampicillin. The overall agreement for all antibiotics tested was 48%. The correlation improved significantly, however, to an overall agreement of 87% when Wilkins-Chalgren broth was substituted in the broth microdilution procedure. Our results indicate that the broth microdilution test is an accurate method for testing this organism, provided than an appropriate medium is used.     

Flow cytometry antifungal susceptibility testing of pathogenic yeasts other than Candida albicans and comparison with the NCCLS broth microdilution test

Candida species other than Candida albicans frequently cause nosocomial infections in immunocompromised patients. Some of these pathogens have either variable susceptibility patterns or intrinsic resistance against common azoles. The availability of a rapid and reproducible susceptibility-testing method is likely to help in the selection of an appropriate regimen for therapy. A flow cytometry (FC) method was used in the present study for susceptibility testing of Candida glabrata, Candida guilliermondii, Candida krusei, Candida lusitaniae, Candida parapsilosis, Candida tropicalis, and Cryptococcus neoformans based on accumulation of the DNA binding dye propidium iodide (PI). The results were compared with MIC results obtained for amphotericin B and fluconazole using the NCCLS broth microdilution method (M27-A). For FC, the yeast inoculum was prepared spectrophotometrically, the drugs were diluted in either RPMI 1640 or yeast nitrogen base containing 1% dextrose, and yeast samples and drug dilutions were incubated with amphotericin B and fluconazole, respectively, for 4 to 6 h. Sodium deoxycholate and PI were added at the end of incubation, and fluorescence was measured with a FACScan flow cytometer (Becton Dickinson). The lowest drug concentration that showed a 50% increase in mean channel fluorescence compared to that of the growth control was designated the MIC. All tests were repeated once. The MICs obtained by FC for all yeast isolates except C. lusitaniae were in very good agreement (within 1 dilution) of the results of the NCCLS broth microdilution method. Paired t test values were not statistically significant (P = 0.377 for amphotericin B; P = 0.383 for fluconazole). Exceptionally, C. lusitaniae isolates showed higher MICs (2 dilutions or more) than in the corresponding NCCLS broth microdilution method for amphotericin B. Overall, FC antifungal susceptibility testing provided rapid, reproducible results that were statistically comparable to those obtained with the NCCLS method.     

Protective effect of trovafloxacin, ciprofloxacin and ampicillin against Streptococcus pneumoniae in a murine sepsis model

The Etest strip is a promising tool of broad application in clinical microbiology. The method provides MIC readings and is easier to perform than broth microdilution. We carried out a study to compare the MICs of fluconazole and itraconazole obtained by the Etest with those obtained by broth microdilution, performed according to the guidelines of the NCCLS document M27-A, with 402 clinical isolates (360 Candida albicans, 17 Candida tropicalis, nine Candida krusei, nine Candida glabrata and seven Candida parapsilosis) and seven control isolates. The agreement between MICs by the two methods (at +/- 2 dilutions) was 74.5% for fluconazole and 61.4% for itraconazole. These results suggest that further development is necessary to standardize the medium and incubation conditions before introduction of the Etest as a routine method in the clinical microbiology laboratory for fluconazole and itraconazole susceptibility testing.     

Comparison of the Sensititre YeastOne colorimetric antifungal panel and Etest with the NCCLS M38-A method to determine the activity of amphotericin B and itraconazole against clinical isolates of Aspergillus spp

OBJECTIVE: The evaluation of the Sensititre YeastOne and Etest methods for determining susceptibility to amphotericin B and itraconazole by comparing the MICs obtained by these methods at different times of reading with those of the M38-A broth microdilution method. METHODS: Sixty-three clinical isolates of Aspergillus spp. (23 Aspergillus flavus, 24 Aspergillus fumigatus, nine Aspergillus niger, three Aspergillus glaucus, two Aspergillus terreus and two Aspergillus flavipes) were assayed. Two itraconazole-resistant strains (NCPF7100 and 7099) were also included. RESULTS: Itraconazole MICs for the two resistant strains were >4 mg/L by the three methods. The overall agreement (+/-2 log2) between M38-A (48 h) and colorimetric (48 h) method was 93.4% for amphotericin B and 90.2% for itraconazole. By the Etest, the best agreement with M38-A was obtained when readings were made at 24 h: 88.5% for amphotericin B and 67.2% for itraconazole. Etest MICs were higher for all species except A. niger. CONCLUSIONS: The colorimetric method appears to be a suitable alternative procedure for antifungal susceptibility testing of Aspergillus spp. and is able to detect resistance to itraconazole. The range of MICs for amphotericin B by Etest is wider and for some strains is >16 mg/L, suggesting that this method could be useful for detecting resistant strains as occur in yeasts.     

Clinical evaluation of a dried commercially-prepared microdilution panel for antifungal susceptibility testing

A commercially-prepared dried broth microdilution panel (Sensititre) was compared with a reference microdilution method for antifungal susceptibility testing of two reference yeast strains and 98 clinical isolates of Candida spp. The antifungal agents tested include 5-fluorocytosine (5FC), fluconazole, itraconazole, and D0870. Microdilution testing was performed according to National Committee for Clinical Laboratory Standards (NCCLS) recommendations. Minimum inhibitory concentration (MIC) endpoints were read visually after 48 hours of incubation and were assessed independently for each microdilution panel. The MICs for the reference strains were within published control limits for both reference and Sensititre microdilution panels. Discrepancies among MIC endpoints of no more than two dilutions (two wells) were used to calculate the percent agreement. An acceptable level of agreement between Sensititre and reference panels was observed for all antifungal agents when tested against the 98 clinical isolates. Agreement ranged from 83% for itraconazole to 93% for 5FC. The Sensititre dried microdilution panel appears to be a viable alternative to inhouse prepared microdilution panels and to the NCCLS macrodilution reference method.     

Comparative antimicrobial activity of gatifloxacin tested against Streptococcus spp. including quality control guidelines and etest method validation. Quality Control Study Group.

Gatifloxacin (formerly AM-1155 or CG 5501) is a new 8-methoxy fluoroquinolone with enhanced activity against Gram-positive cocci, especially Streptococcus pneumoniae and other streptococci. Recent clinical strains (599 isolates) were tested against gatifloxacin, three comparison fluoroquinolones, and penicillin by the reference broth microdilution, Etest (AB BIODISK, Solna, Sweden) and standardized disk diffusion methods (5 micrograms gatifloxacin disk). Gatifloxacin (MIC90, 0.5 microgram/ml) activity was generally comparable to that of trovafloxacin (MIC90, 0.25 microgram/ml), or sparfloxacin (MIC90, 0.5 microgram/ ml) and markedly superior to ofloxacin (MIC90, 2-4 micrograms/ml) against the streptococci. Rates of penicillin non-susceptibility were 41.9, 38.0, and 16.2% for S. pneumoniae (301 strains), viridans group streptococci (150 strains), and beta-haemolytic streptococci (148 strains). Etest results correlated well (95.7-100.0% +/- one log2 dilution) with the reference MIC results, but Etest tended to have elevated gatifloxacin MIC results compared to the broth microdilution method for the highly resistant isolates (MICs, > 2 micrograms/ml). Gatifloxacin disk zone diameters correlate well to reference MICs for all streptococci and proposed interpretive criteria (susceptible at < or = 1 microgram/ml or > or = 18 mm, and resistant at > or = 4 micrograms/ml or < or = 14 mm) did not produce discords between method results (absolute agreement). A nine laboratory quality control (QC) study conforming to the National Committee for Clinical Laboratory Standards (NCCLS) Guideline M23-T3 studied S. pneumoniae ATCC 49619 and gatifloxacin. Proposed ranges for QC of NCCLS tests were 0.12-0.5 microgram/ml for the broth microdilution test and 24-31 mm for the disk diffusion method. These reported results indicate that gatifloxacin was a potent fluoroquinolone with extensive activity against streptococcal isolates. In vitro test methods to measure this activity appear accurate and comparable; and QC guidelines have been established for routine clinical laboratory use pending approval by the NCCLS and the Food and Drug Administration (FDA).     

Comparison of Etest and a tablet diffusion test with the NCCLS broth microdilution method for fluconazole and amphotericin B susceptibility testing of Candida isolates

Three methods were compared for the susceptibility testing of yeast isolates to fluconazole and amphotericin B: two fagar diffusion methods (Etest and a tablet diffusion test) and the National Committee for Clinical Laboratory Standards (NCCLS) broth microdilution method. Given as MIC(50)s (range), fluconazole endpoints were: for the 24 h broth microdilution test, 0.25 mg/L (0.06-32 mg/L); for the Etest, 0.38 mg/L (0.064-24 mg/L); and for the NCCLS broth microdilution test, 2 mg/L (0.06->or=64 mg/L). With breakpoints of <3 mg/L for susceptible and >16 mg/L for resistant, the Etest and the 24 h microdilution test classified the isolates in agreement with the classification obtained by the NCCLS method. Results obtained by Etest were in closer NCCLS method than those obtained with the tablet test. Amphotericin B endpoints were lower for the 24 h microdilution and Etests than MICs obtained by the NCCLS broth microdilution method. Reproducibility was high for all tests; however, disadvantages of both diffusion tests were microcolonies in the inhibition zone and dependence on stringent standardization of inoculum.     

Pitfalls of polymyxin antimicrobial susceptibility testing of Pseudomonas aeruginosa isolated from cystic fibrosis patients

OBJECTIVES AND METHODS: With their potent activity against Gram-negative bacteria, the polymyxins are important alternative antibiotics for cystic fibrosis (CF) patients. A retrospective evaluation of polymyxin activity against 6001 Pseudomonas aeruginosa, 150 Achromobacter xylosoxidans and 506 Stenotrophomonas maltophilia CF isolates was initiated. In addition, we looked at how polymyxin susceptibility testing was affected by the testing method (agar dilution versus microdilution), the agent (polymyxin E versus polymyxin B), incubation time (24 h versus 48 h) and by different interpretative criteria (German DIN, French FSM, British BSAC). RESULTS: Polymyxin B exhibited reasonable activity against P. aeruginosa (MIC(90)< or =2 mg/L), whereas it was less active against A. xylosoxidans (MIC(90)< or =16 mg/L) and S. maltophilia (MIC(90)< or =16 mg/L). During 2000-2002, polymyxin B resistance in P. aeruginosa, S. maltophilia and A. xylosoxidans was found to be 6.7%, 17.0% and 29.9% (corresponding to 12.4%, 20.7% and 35.4% of infected patients), respectively. When the agar dilution method was used, polymyxin E exhibited higher MICs than polymyxin B. The microdilution method produced lower polymyxin MICs than the agar dilution method. Therefore, the microdilution MICs after prolonged incubation (48 h) and the agar dilution MICs of polymyxin B correlated best (AUC of 0.93, r(2) of 0.44 and s of 0.83). CONCLUSIONS: Polymyxin resistance among common CF pathogens is not rare, thus underlining the necessity of accurate susceptibility testing. When compared with the agar dilution method, it was found that the microdilution method is a valid, rapid and cost effective alternative for the determination of polymyxin activity. The performance of the microdilution method was most reliable after prolonged incubation (48 h) at a susceptibility breakpoint of < or =4 mg/L according to the BSAC guidelines (specificity 91%, sensitivity 89%, 1.5% very major errors).     

In vitro antifungal activity of FK463, a new water-soluble echinocandin-like lipopeptide

The antifungal activity of FK463 against 72 recent clinical isolates of Candida albicans (24), Candida glabrata (17), Candida tropicalis (11), Candida krusei (8) and Candida parapsilosis (12) was compared with those of amphotericin B, fluconazole and itraconazole by means of a broth microdilution method specified by the National Committee for Clinical Laboratory Standards (NCCLS) document M27-A. The lowest drug concentration at which 90% of the population was inhibited (MIC(90)) of FK463 against C. albicans, C. glabrata, C. tropicalis, C. krusei and C. parapsilosis was 0.0156, 0. 0156, 0.0313, 0.125 and 1 mg/L, respectively. FK463 exhibited broad-spectrum activity against clinically important Candida spp. (MIC range < or =0.0039-2 mg/L), and its MICs for such fungi were lower than those of other antifungal agents tested. The minimum fungicidal concentrations for Candida spp. did not differ by more than two-fold from the MICs. Results from pre-clinical evaluations performed to date indicate that FK463 should be a potent parenteral antifungal agent.     

In vitro activities of beta-lactam-beta-lactamase inhibitor combinations against Stenotrophomonas maltophilia: correlation between methods for testing inhibitory activity, time-kill curves, and bactericidal activity.

The activities of ampicillin, ampicillin-sulbactam, amoxicillin, amoxicillin-clavulanic acid, ticarcillin, ticarcillin-clavulanic acid, piperacillin, piperacillin-tazobactam, aztreonam, and aztreonam-clavulanic against Stenotrophomonas maltophilia strains for which the MICs of penicillins and commercially available beta-lactam-beta-lactamase inhibitor combinations were higher than the breakpoints usually recommended for Pseudomonas aeruginosa in commercially available broth microdilution methods were tested by the agar diffusion, agar dilution, and broth microdilution methods. Time-kill curve studies were performed when discrepancies between these methods were observed. The MICs obtained by the commercially available broth microdilution method, the agar dilution method, and the broth microdilution method were almost identical. Twenty-five percent of the strains tested showed inhibition diameters of > or =15 mm for ticarcillin-clavulanic acid, and 43.7% of the strains tested showed inhibition diameters of > or =18 mm for piperacillin-tazobactam by the agar diffusion method. The time-kill curves for these strains confirmed the results obtained by dilution methods. Aztreonam-clavulanic acid (2:1) at concentrations of < or =16 microg/ml inhibited all of these strains (MIC range, 1 to 16 microg/ml). The time-kill curves confirmed this activity. The addition of piperacillin to this combination did not modify the MICs. The combination aztreonam-clavulanic acid-ticarcillin was two- to fourfold more active than aztreonam-clavulanic acid alone. We studied the inhibitory and bactericidal activities of the two most active combinations (aztreonam-clavulanic acid and aztreonam-clavulanic acid-ticarcillin) against the standard inoculum and 10 and 50 times the standard inoculum. Inoculum modifications did not modify the MICs. Both combinations showed good bactericidal activity against the standard inoculum. With 10 times the standard inoculum, minimum bactericidal concentration (MBC) results were heterogeneous (for 55% of the strains, MBCs were between the MIC and 4-fold the MIC, and for 45% of the strains MBCs were between 8- and >32-fold the MIC). With 50 times the standard inoculum, MBCs were at least 32-fold the MICs for all the strains tested.     

Improved medium for fluconazole susceptibility testing of Candida albicans.

We have compared fluconazole susceptibilities of 92 clinical isolates of Candida albicans by broth microdilution in two different media: standard RPMI 1640 (RPMI) and the same medium supplemented with 18 g of glucose per liter (RPMI-glucose). Preparation of media, drugs, and inocula, as well as incubation conditions, followed the preliminary recommendations of the National Committee for Clinical Laboratory Standards (Villanova, Pa.) antifungal agent working group for broth macrodilution tests with antifungal agents, adapted to microdilution. Microtiter plates were agitated for 5 min before spectrophotometric readings were performed with an automatic plate reader set at 405 mm. The MIC endpoint was defined as an inhibitory concentration calculated from the turbidimetric data as a function of the turbidity in the drug-free control wells. The mean absorbances in the drug-free wells in RPMI and RPMI-glucose were, respectively, 0.38 (41.6% transmission) and 0.99 (10.2% transmission) (P < 0.001; Student's t test). Despite the increased growth in RPMI-glucose, 98.9% of the C. albicans strains tested for fluconazole susceptibility yielded similar MICs (+/- 1 dilution) in both media. Moreover, strains with decreased susceptibility to fluconazole displaying similar MICs in both media are easier to detect in RPMI-glucose because of the greater differences between turbidimetric readings in wells with grown or fluconazole-inhibited cultures. This objective turbidimetric method, with an easy-to-read improved medium (RPMI with glucose), together with previous experience of the National Committee for Clinical Laboratory Standards antifungal agent subcommittee, could overcome some of the present problems associated with lack of reproducibility of azole susceptibility testing.     

In vitro susceptibility of Candida species to five antifungal agents in a German university hospital assessed by the reference broth microdilution method and Etest

OBJECTIVES: The aim of this study is to evaluate the susceptibilities of Candida spp. to the common antifungal agents in a German university hospital. Since quick results of in vitro testing are desirable, Etest and the CLSI broth microdilution (BMD) method (reference method) were compared, focusing on the validity of early readings. METHODS: A total of 512 Candida spp. isolates, including 174 from primarily sterile sites, were collected in the clinical routine. The yeasts were differentiated by CHROMagar and verified by API 20C AUX if necessary. In vitro susceptibilities to amphotericin B, flucytosine, fluconazole, voriconazole and caspofungin were determined using the BMD method described in the CLSI (formerly NCCLS) M27-A2 document and Etest. MICs were noted after 24 and 48 h of incubation. RESULTS: The most frequently isolated species was Candida albicans. Among the non-albicans species, Candida glabrata was the most prevalent, followed by Candida tropicalis, Candida parapsilosis and Candida krusei. MICs (mg/L) at which 90% of the strains were inhibited were 1 for amphotericin B, 32 for flucytosine, 8 for fluconazole, 0.25 for voriconazole and 1 for caspofungin. Susceptibility to fluconazole was 85.0% for C. glabrata and 5.3% for C. krusei, almost all other isolates were susceptible in over 90% except very rare species. The 48 h MIC values of Etest and BMD were in agreement (no more than 2 log(2) dilutions) in 88.7% to 98.1% with categorical agreement rates of 91.6% to 98.2%, depending on the antifungal agent. Comparison of the 24 h MICs of both BMD and Etest with the 48 h MICs of the reference method showed categorical agreement in 94.9% to 99.2%. For caspofungin, however, a comparison of the categorical agreement was not possible due to the lack of interpretive breakpoints. The order of frequency and the resistance patterns of the isolates from primarily sterile sites and those of isolates from non-sterile sites did not differ. CONCLUSIONS: No alarming resistances against the agents tested were found; however, owing to the relatively high frequency of C. glabrata with elevated fluconazole MICs, this species and, to a certain extent, C. krusei must be taken into consideration when choosing antifungal agents for calculated therapy. Etest is a reliable method for the susceptibility testing of Candida spp. and the 24 h readings of both Etest and BMD can serve as helpful preliminary results in most cases.     

Quantitation of Candida albicans ergosterol content improves the correlation between in vitro antifungal susceptibility test results and in vivo outcome after fluconazole treatment in a murine model of invasive candidiasis

MIC end point determination for the most commonly prescribed azole antifungal drug, fluconazole, can be complicated by "trailing" growth of the organism during susceptibility testing by the National Committee for Clinical Laboratory Standards approved M27-A broth macrodilution method and its modified broth microdilution format. To address this problem, we previously developed the sterol quantitation method (SQM) for in vitro determination of fluconazole susceptibility, which measures cellular ergosterol content rather than growth inhibition after exposure to fluconazole. To determine if SQM MICs of fluconazole correlated better with in vivo outcome than M27-A MICs, we used a murine model of invasive candidiasis and analyzed the capacity of fluconazole to treat infections caused by C. albicans isolates which were trailers (M27-A MICs at 24 and 48 h, /=64 microg/ml, respectively; SQM MIC, /=64 microg/ml; SQM MIC, 54 microg/ml). Compared with the untreated controls, fluconazole therapy increased the survival of mice infected with a sensitive isolate and both trailing isolates but did not increase the survival of mice infected with a resistant isolate. These results indicate that the SQM is more predictive of in vivo outcome than the M27-A method for isolates that give unclear MIC end points due to trailing growth in fluconazole.     

In vitro activities of voriconazole (UK-109,496) against fluconazole-susceptible and -resistant Candida albicans isolates from oral cavities of patients with human immunodeficiency virus infection.

The susceptibility of Candida albicans to a new antifungal triazole, voriconazole (UK-109,496), was investigated in 105 isolates obtained from the oral cavities of patients with human immunodeficiency virus (HIV) infection to study this drug's activity against fluconazole-susceptible and -resistant isolates. MICs were determined by a broth microdilution technique according to document M27-T from the National Committee for Clinical Laboratory Standards and by using a broth microdilution technique and a synthetic high-resolution medium. These antifungal susceptibility testing methods showed high levels of agreement (93% for fluconazole and 86% for voriconazole). Data from in vitro studies showed that voriconazole has good activity against fluconazole-susceptible and -resistant C. albicans isolates; the MICs at which 90% of all isolates were inhibited were 0.19 to 0.39 microgram/ml. We found that for isolates for which fluconazole MICs were high, voriconazole MICs were proportionally higher than those for fluconazole-susceptible C.albicans (P < 0.001). Pretreatment isolates from six patients with fluconazole-refractory esophageal candidiasis were included in the study. For these isolates the MICs were < or = 0.39 microgram/ml, and all patients responded to voriconazole. These results suggest that voriconazole is effective even in the treatment of fluconazole-refractory esophageal candidiasis and should be studied further to determine its clinical relevance in patients with HIV infection.