a Department of Clinical Sciences and Administration, University of Houston College of Pharmacy, Texas Medical Center, 1441 Moursund Street #423, Houston, TX 77030; b Department of Internal Medicine, Division of Infectious Diseases, c Department of Pathology and d Division of Clinical and Administrative Pharmacy, University of Iowa Hospitals and Clinics, Iowa City, IA 52242, USA
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Abstract |
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Introduction |
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Chequerboard testing and timekill studies are generally carried out under the assumption that the agents tested in combination possess similar doseresponse curves and a comparable timecourse of activity.1 This assumption may be problematic, however, for polyeneazole combinations. Previous in vitro studies carried out in our laboratories have demonstrated that the onset of activity for azoles against Candida and Cryptococcus species is considerably slower than the fungicidal activity exerted by amphotericin B (AMB).2,3 Additionally, several investigators have documented the importance of FLU pre-exposure with in vitro test methodology for the detection of antagonism between AMB and FLU.46 Because the rapid onset of AMB activity may pre-empt the contribution of azole antifungal activity in the test system, traditional techniques of assessing antifungal combinations, which rely on simultaneous administration of drugs, may fail to detect synergic or antagonistic antifungal interactions.
More recently, the Etest has been investigated as a method for antifungal susceptibility testing and has demonstrated good correlation with broth macrodilution and microdilution testing methods.711 Moreover, Etest has been used successfully to test drug combinations against mycobacterium and aerobic Gram-negative bacilli.12,13 If an Etest method could be standardized for testing antifungal combinations, it would represent an ideal testing methodology for the clinical microbiology laboratory.
The objectives of this study were three-fold: (i) to examine the utility of Etest for testing antifungal combinations against Candida species; (ii) to compare agreement between chequerboard, timekill and Etest methods for the detection of synergy or antagonism of antifungal combinations against Candida species; and (iii) to compare the results of chequerboard, timekill and Etest methods before and after pre-exposure of test isolates to FLU.
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Materials and methods |
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Six isolates obtained from the Clinical Microbiology Laboratory at the University of Iowa College of Medicine were selected. Three C. albicans isolates (ATCC CA 90028, CA 1378.33 and CA 20096.0097), one C. glabrata isolate (ATCC CG 582), one C. krusei isolate (ATCC CK 6258) and one C. tropicalis isolate (ATCC CT 2697) were used for all test procedures.
Antifungal agents
FLU (Pfizer, New York, NY), AMB and flucytosine (5FC) (Sigma, St Louis, MO, USA) were used to prepare stock solutions in RPMI 1640 (Sigma) buffered to a pH of 7.0 with 0.165 M morpholinepropanesulphonic acid (MOPS) according to NCCLS recommendations.14
Antifungal susceptibility testing
The MIC of each antifungal was determined by broth microdilution and Etest using methods described by the NCCLS and the Etest manufacturer (AB Biodisk, Solna, Sweden).14 Broth-microdilution MICs were determined in RPMI 1640 medium buffered to a pH of 7.0 with MOPS. The starting inoculum was c. 0.5 x 102 to 2.5 x 103 cfu/mL.
Etest MICs were determined with FLU (0.016256 mg/L), AMB (0.00232 mg/L) and 5FC (0.00232 mg/L) strips provided by the manufacturer (AB Biodisk). Solidified (1.5%) 150 mm RPMI + MOPS agar plates (Remel, Lenexa, KS, USA) served as the test medium. A standardized cell suspension (0.5 McFarland) in sterile 0.85% NaCl was prepared by transferring 34 colonies from a 24 h culture on potato dextrose agar (PDA) (Remel). Plates were then inoculated by pouring 5 mL of a 1:4 dilution of this standardized cell suspension on to the agar. After allowing 12 min for the suspension to achieve a uniform distribution, excess moisture was aspirated with a vacuum pipette and the plate was allowed to dry at room temperature (15 min). Antifungal strips were then placed on to the inoculated agar using sterile technique.
All tests were incubated at 35°C in a dark, moist chamber. MICs were recorded at 24 and 48 h. The susceptibility endpoint for broth-based testing of AMB was defined as the lowest concentration of drug that resulted in complete inhibition of visible growth. Broth-based susceptibility endpoints for FLU and 5FC were defined as the lowest concentration of drug that resulted in an 80% reduction of visual growth compared with the growth of the control. Similarly, Etest susceptibility endpoints for FLU and 5FC were read at the intersection of the scale of the strip and the first discernible growth-inhibition ellipse according to the recommendations of the manufacturer. The Etest susceptibility endpoint for AMB was read at the intersection of the scale of the strip with the first completely clear ellipse.
Chequerboard microdilution studies
Combinations of AMB + FLU, AMB + 5FC and FLU + 5FC were tested in duplicate using previously described methods.15 The concentration of each antifungal agent tested ranged from 1/32 to 4 x MIC. The initial inoculum was prepared as described for broth-microdilution susceptibility testing. Readings were determined visually and spectrophotometrically (80% reduction) at 490 nm. MIC endpoints were determined as described for broth-microdilution tests. Both on- and off-scale MICs were included in the analysis. High off-scale MICs were converted to the next two-fold dilution, whereas low off-scale MICs were left unchanged. Trays were incubated at 35°C in a dark, moist chamber. Results were read at 24 and 48 h.
To evaluate the interaction of antifungals, the fractional inhibitory concentration (FIC) was calculated for each combination.1,16 The FIC was calculated for each agent by dividing the inhibitory concentration of each antifungal when used in combination by its MIC. FIC values were then added together to define the interaction of the combination. Synergy was defined as an FIC of 0.5, and additivity was defined as an FIC of >0.5 but <1. Indifference was defined as an FIC of
1 but <4, whereas antagonism was defined as an FIC of
4.
Timekill studies
Timekill studies of each antifungal combination were carried out by a method that we have described previously.17 Earlier studies in our laboratory have documented a lack of antifungal carryover with these methods with the range of antifungal concentrations tested in this study. The lower limit of accuracy with this method is 50 cfu/mL. Both a fungicidal high concentration (HC) 2.5 mg/L (24 x MIC) and a fungistatic low concentration (LC) 0.25 mg/L (0.250.5 x MIC) of AMB alone and in combination were tested. One millilitre of a standardized cell suspension (0.5 McFarland) was transferred to sterile tubes containing 9 mL of growth medium (RPMI 1640 + MOPS) plus the following antifungal concentrations: (i) control (no drug); (ii) LC-AMB; (iii) HC-AMB; (iv) FLU 20 mg/L; (v) 5FC 50 mg/L; (vi) LC-AMB + FLU; (vii) HC-AMB + FLU; (viii) LC-AMB + 5FC; (ix) HC-AMB + 5FC; (x) FLU + 5FC. Tubes were incubated at 35°C for 24 h on an orbital shaker. At predetermined timepoints (0, 4, 8, 12 and 24 h), samples were aseptically removed and serially diluted, and 30 µL aliquots were plated on to PDA for colony-count determination. All plates were incubated for 48 h at 35°C. All testing was carried out in duplicate.
Antifungal interaction tested by timekill methods was defined using established criteria. Briefly, a 2 log10 increase in killing as measured by viable cfu/mL at 24 h between the combination and the most active constituent was defined as synergy, and a <2 but >1 log10 increase was defined as additive. A decrease in killing from the least active agent of <2 log10 cfu/mL was defined as indifference, whereas a decrease in killing of >2 log10 cfu was defined as antagonism.16
Etest studies
A modification of previously reported methods for testing antibacterial combinations against Mycobacterium spp. with Etest was used to test antifungal combinations (Fig-ure 1).12,13 Test medium and inoculum preparation were as described previously for susceptibility testing. Antifungal interactions were defined by criteria recommended by the manufacturer (AB Biodisk). Synergy was defined as a decrease of 3 dilutions in the resultant MIC. Additivity was defined as a decrease of
2 but <3 and indifference as a decrease of <2 dilutions in the MIC. Antagonism was defined as an increase of
3 dilutions of the MIC for the antifungal combination. To facilitate comparisons with the chequerboard method, an FIC index was calculated from Etest MIC data that were rounded up to the nearest two-fold dilution. All testing was carried out in duplicate.
FLU pre-exposure
To evaluate the influence of FLU pre-exposure, each test isolate was prepared as follows. One millilitre of a standardized cell suspension (3.0 McFarland) was added to a culture vial containing 9 mL of growth medium with FLU (20 mg/L). The cell suspension was then incubated for 12 h at 35°C on an orbital shaker. Following the incubation period, the cell suspension was pelleted and washed in sterile saline twice by centrifugation (2800 rpm x 10 min x 3) and resuspended in warm, drug-free growth medium. The resultant suspension was then re-standardized to 1 x 105 to 5 x 105 cfu/ml (0.5 McFarland turbidity) and used for chequerboard, timekill and Etest testing.
Analysis
All plates and trays were read at 24 and 48 h by a single investigator, and results were confirmed by a second investigator. The frequency of synergy, additivity, indifference and antagonism were tabulated and compared for each isolatedrug combination before and after FLU pre-exposure. For purposes of comparison, the timekill test was considered to be the reference method.
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Results |
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The median MICs determined by broth microdilution and Etest are presented in Table 1. In general, there was good agreement between the Etest and broth-microdilution methods.
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Results presented in Tables 2 and 3 demonstrate that agreement between the Etest and the chequerboard methods was poor. Antifungal synergy was detected by the chequerboard method in six of 18 (33%) tests with or without FLU pre-exposure, whereas synergy was not detected by Etest (0 of 18). Combinations that included 5FC were most likely to exhibit synergic activity by chequerboard testing. Antagonism was not detected for any of the antifungal combinations tested by chequerboard dilution, but it was noted in three of 18 (17%) isolates tested by Etest (all isolates susceptible or susceptible dose-dependent to FLU).
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Discussion |
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AMB and azole antifungals possess a different timecourse of activity; therefore, we speculated that preexposure of the Candida isolates to FLU (thus ensuring the full antifungal effect of FLU at the time that AMB or 5FC was introduced into the test system) might change the results of the chequerboard tests. Interestingly, FLU pre-exposure did not increase the frequency of antagonism noted by the chequerboard method. This lack of antagonism could be explained in two ways. First, broth-microdilution antifungal susceptibility testing of AMB in RPMI medium frequently demonstrates a clustering (narrow distribution) of MICs.14,18 Thus, the method is not well suited for discriminating between susceptible and resistant isolates. Therefore, the broth-microdilution method (on which chequerboard testing is based) may not be able to discern a synergic or antagonistic interaction with AMB combinations. Secondly, because AMBazole antagonism appears to be one-sided (the activity of the azole appears to persist despite the abrogation of AMB fungidal activity), detecting a four-dilution change in the MIC of AMB, which would be necessary to detect antagonism (an FIC index of 4), seems unlikely. Whether or not antagonism could be detected by chequerboard testing in other media (i.e. antibiotic medium #3) is a question currently under investigation.
Another interesting finding in this study was the influence of FLU susceptibility on the development of AMBFLU antagonism detected in vitro. With the exception of C. glabrata (CG 582), antagonism was noted by timekill and Etest methods for isolates that were FLU susceptible or susceptible dose-dependent (CA 90028, CA 1378.33 and CT 2697). Antagonism, however, was absent or less pronounced for the FLU-resistant C. albicans isolate (CA 20096.097) or for C. krusei (CK 6258). These findings are consistent with the theory that AMB antagonism develops secondary to azole-mediated depletion of ergosterol binding sites. Further studies are required, however, before this finding can be validated.
Antifungal synergy in this study was seen only with 5FC combinations. However, the fact that synergy was largely detected by the chequerboard method and not corroborated by Etest or timekill studies raises the question of a testing artefact seen only with chequerboard dilution. Broth-microdilution susceptibility testing of 5FC alone is often difficult to interpret because of trailing phenomena seen with 5FC against Candida spp. In the presence of a second agent, the trailing seen with 5FC is often absent or decreased. Therefore, it is possible that the visual/spectrophotometric endpoint (80% reduction) used to determine the MIC of 5FC alone is less appropriate when the drug is used in combination.
The results of the Etest studies indicate that this method could be an acceptable alternative to timekill studies with antifungal agents. The technique employed for testing the antifungal combination was simple to use, time-efficient and yielded reproducible results. Unlike the findings with timekill studies, FLU pre-exposure produced no discernible effect on the Etest results. It is possible that pre-exposure for 1 h with an azole in agar-based medium is sufficient for detecting polyeneazole antagonism. Unlike previous Etest work carried out with antibacterials, we opted not to place the Etest strips simultaneously on the agar at 90° angles.12 AMB and FLU exhibit different diffusion characteristics through agar, resulting in dissimilar ellipse patterns. Moreover, the growth endpoints used to measure the MIC by Etest are different for the two agents. Therefore, we felt interpretation of separate ellipses at 90° angles would be difficult. It may be possible to test antifungal combinations by incorporating azoles or 5FC into agar, but this would be time-consuming and the advantage of testing continuous gradients of antifungal concentrations would be lost.
In summary, our results indicate that Etest and timekill methods correlate well for the testing of antifungal combinations. Chequerboard testing for antifungals, particularly those involving AMB in RPMI media, should be interpreted with caution, as this technique appears to display poor sensitivity for detecting changes in the activity of AMB. On the basis of our work, Etest appeared to be a good method for the in vitro testing of antifungal combinations, although more prolonged pre-exposure studies are required. Further studies utilizing different growth media, testing of novel antifungal combinations, synergy testing of azole-resistant yeast and the evaluation of antifungal combinations against moulds such as Aspergillus should be pursued with Etest methodology. Although the concentrations used in our studies reflect antifungal concentrations that can be achieved in human patients, the in vivo significance of our findings remains to be determined. Therefore, our in vitro results cannot be extrapolated to the clinical setting until better in vivo correlative data are available.
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Acknowledgements |
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Notes |
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References |
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Received 16 January 2001; returned 17 July 2001; revised 28 August 2001; accepted 31 October 2001