Abstract
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.
The use of broth microdilution methods for antimicrobial susceptibility testing of bacteria in clinical microbiology laboratories has certainly been aided by the commercial development of frozen or dried panels containing an array of antimicrobial agents at concentrations appropriate for clinical testing (3). The availability of these commercially prepared panels facilitates testing of a wide variety of agents and provides broad access to this technology as well as an additional degree of standardization and quality assurance for a process that can be quite demanding technically (3).
Although a reference broth microdilution method for antifungal susceptibility testing of Candida spp. has been established since 1997 (5), commercially available broth microdilution panels providing full-range MICs for both established and newly introduced antifungal agents have been slow to develop. Several products providing broth microdilution testing of a limited number of antifungal agents, usually in a limited-range or breakpoint format, have been introduced in Europe, but in most instances their performance has been found to compare poorly with that of the NCCLS reference method (2, 11, 15).
It has been noted that one of the major barriers to the use of antifungal susceptibility testing in clinical practice is the limited availability of test results in a timely fashion (2, 11, 15; S. Mirza, L. Strausbaugh, J. Morgan, D. Jernigan, L. Liedtke, D. W. Warnock, and R. A. Hajjeh, Program Abstr. Infect. Dis. Soc. Am. 39th Ann. Meet., abstr. 834, 2001). Clearly, the availability of a validated, commercially prepared microdilution panel for antifungal susceptibility testing is desirable both for the purpose of standardization and as an aid in the clinical management of serious fungal infections (14, 15).
Presently, Trek Diagnostic Systems (Westlake, Ohio) manufactures custom broth microdilution panels for antifungal susceptibility testing. These panels are available for research purposes in a frozen or dried format either with or without the colorimetric indicator Alamar Blue (7). The 96-well panels provide full-scale MICs for as many as eight different antifungal agents and have a shelf life of approximately 6 months at −70°C for frozen panels and 24 months at ambient temperature for dried panels. The purpose of the present study was to validate the performance of the Trek frozen microdilution panel without Alamar Blue against the NCCLS reference frozen microdilution panel (5, 6). Both panels contained the licensed antifungal agents amphotericin B, flucytosine (5FC), fluconazole, and itraconazole, as well as the investigational agents posaconazole, ravuconazole, and voriconazole.
MATERIALS AND METHODS
Test organisms.
The test organisms include two American Type Culture Collection (ATCC) strains that have been established as quality control (QC) strains (Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258) by the NCCLS (1, 5). These isolates have well-defined microdilution MIC reference ranges for amphotericin B, 5FC, fluconazole, itraconazole, posaconazole, ravuconazole, and voriconazole (1). An additional 99 clinical isolates of Candida spp. were also selected for testing. The collection included 38 isolates of Candida albicans, 24 of C. glabrata, 15 of C. tropicalis, 10 of C. parapsilosis, 7 of C. krusei, and 5 of C. lusitaniae. These were all recent clinical isolates from blood or normally sterile body fluids (12, 13) and were selected to represent the clinically prevalent species and to maximize the on-scale MIC endpoints for the various antifungal agents tested. Isolates were identified by standard methods (16) and were stored as water suspensions at ambient temperature until use in the study. Prior to testing, each isolate was passaged at least twice on potato dextrose agar (Remel, Lenexa, Kans.) to ensure purity and viability.
Antifungal agents and microdilution panels.
Amphotericin B (Sigma, St. Louis, Mo.), 5FC (Sigma), fluconazole (Pfizer, Inc., New York, N.Y.), itraconazole (Janssen, Beerse, Belgium), posaconazole (Schering-Plough, Kenilworth, N.J.), ravuconazole (Bristol-Myers Squibb, Wallingford, Conn.), and voriconazole (Pfizer) were obtained as reagent grade powders from their respective manufacturers. Reference microdilution trays containing serial dilutions of the antifungal agents in MOPS (morpholinepropanesulfonic acid)-buffered RPMI 1640 medium (Sigma) were prepared in a single lot at the University of Iowa exactly as outlined in NCCLS document M27-A (5) and were stored frozen at −70°C for as long as 4 months until use in the study. Frozen panels containing the same antifungal agents were prepared by Trek and were shipped in sealed packages directly from the manufacturer. The panels were stored at −70°C for as long as 4 months until use in the study.
Antifungal susceptibility test methods.
Broth microdilution testing was performed according to NCCLS guidelines by using the spectrophotometric method of inoculum preparation, an inoculum concentration of 0.5 × 103 to 2.5 × 103 cells per ml, and RPMI 1640 medium buffered to pH 7.0 with 0.165 M MOPS (Sigma) (5). Yeast inocula (100 μl) were added to each of the wells of both reference and Trek microdilution panels. Final concentrations of the antifungal agents were 0.007 to 8 μg/ml for amphotericin B, itraconazole, posaconazole, ravuconazole, and voriconazole, 0.06 to 64 μg/ml for 5FC, and 0.12 to 128 μg/ml for fluconazole. The panels were incubated in air at 35°C and were observed for the presence or absence of growth at 48 h.
The reference and Trek microdilution wells were read after 48 h of incubation with the aid of a reading mirror; the growth in each well was compared with that in the growth control (drug-free) well. The MICs of fluconazole, itraconazole, posaconazole, ravuconazole, voriconazole, and 5FC were each read as the lowest concentration at which a prominent decease in turbidity (approximately 50% inhibition) relative to the turbidity of the growth control well was observed (1, 5). Amphotericin B MICs were read at 100% inhibition of growth (first clear well).
Reproducibility studies.
The reproducibilities of MICs obtained by using the Trek panel were determined by testing 10 isolates (3 C. glabrata, 3 C. parapsilosis, 2 C. krusei, 1 C. tropicalis, and 1 C. albicans isolate) in triplicate on each of three separate days. The isolates were selected to provide on-scale MIC endpoints for all seven of the agents tested.
Analysis of results.
The MIC results obtained with the Trek panels read at 48 h were compared with those of the reference panels read at 48 h. Both on-scale and off-scale results were included in the analysis. As with previous studies (4), the high off-scale MICs were converted to the next higher concentration and the low off-scale MICs were left unchanged. Overall, 95% of MICs were on-scale (100% for amphotericin B, 5FC, fluconazole, itraconazole, and posaconazole, 88% for voriconazole, and 78% for ravuconazole). Discrepancies among MIC endpoints of no more than 2 dilutions (two wells) were used to calculate the percent agreement. The analysis of percent agreement included both total results (on- and off-scale) and on-scale results only. Percent reproducibility was determined as the percentage of replicate MICs within ±1 log2 dilution of the modal MIC for each test isolate.
RESULTS AND DISCUSSION
Table 1 summarizes the in vitro susceptibilities of 99 clinical isolates of Candida spp. to seven antifungal agents as determined by the NCCLS reference microdilution frozen panel. A broad range of on-scale MICs was observed with each antifungal agent. In general, MICs for each antifungal agent were typical for each species of Candida (10, 12, 13); however, the higher MICs noted for the triazoles were due to the inclusion of several isolates of C. albicans and C. glabrata with decreased susceptibility to azoles (13).
TABLE 1.
Antifungal susceptibilities of clinical isolates of Candida spp. as determined by the NCCLS microdilution broth reference method
| Organism (n) | Antifungal agent | MIC (μg/ml)
|
||
|---|---|---|---|---|
| Range | 50%a | 90%a | ||
| C. albicans (38) | Amphotericin B | 0.5-1 | 0.5 | 1 |
| 5FC | 0.12-64 | 0.25 | 2 | |
| Fluconazole | 0.12-128 | 0.5 | 128 | |
| Itraconazole | 0.015-1 | 0.12 | 1 | |
| Posaconazole | 0.015-0.5 | 0.03 | 0.25 | |
| Ravuconazole | 0.007-2 | 0.015 | 0.25 | |
| Voriconazole | 0.007-4 | 0.015 | 1 | |
| C. glabrata (24) | Amphotericin B | 0.5-1 | 1 | 1 |
| 5FC | 0.06-0.25 | 0.12 | 0.12 | |
| Fluconazole | 1-128 | 8 | 32 | |
| Itraconazole | 0.12-4 | 1 | 2 | |
| Posaconazole | 0.12-2 | 0.5 | 1 | |
| Ravuconazole | 0.015-2 | 0.25 | 1 | |
| Voriconazole | 0.03-2 | 0.25 | 1 | |
| C. tropicalis (15) | Amphotericin B | 0.5-1 | 1 | 1 |
| 5FC | 0.06->64 | 0.25 | >64 | |
| Fluconazole | 0.25-2 | 1 | 2 | |
| Itraconazole | 0.06-1 | 0.25 | 0.5 | |
| Posaconazole | 0.015-0.12 | 0.06 | 0.12 | |
| Ravuconazole | 0.007-0.06 | 0.03 | 0.06 | |
| Voriconazole | 0.015-0.06 | 0.03 | 0.06 | |
| C. parapsilosis (10) | Amphotericin B | 1 | 1 | 1 |
| 5FC | 0.06-2 | 0.12 | 0.5 | |
| Fluconazole | 0.5-8 | 2 | 4 | |
| Itraconazole | 0.25-0.5 | 0.25 | 0.5 | |
| Posaconazole | 0.03-0.12 | 0.12 | 0.12 | |
| Ravuconazole | 0.007-0.06 | 0.015 | 0.06 | |
| Voriconazole | 0.015-0.12 | 0.03 | 0.06 | |
| C. krusei (7) | Amphotericin B | 1 | 1 | |
| 5FC | 16-32 | 16 | ||
| Fluconazole | 16-64 | 32 | ||
| Itraconazole | 0.5-1 | 0.5 | ||
| Posaconazole | 0.25-0.5 | 0.25 | ||
| Ravuconazole | 0.12-0.5 | 0.25 | ||
| Voriconazole | 0.25-0.5 | 0.25 | ||
| C. lusitaniae (5) | Amphotericin B | 0.5-1 | 0.5 | |
| 5FC | 0.06-0.12 | 0.06 | ||
| Fluconazole | 0.5-1 | 0.5 | ||
| Itraconazole | 0.12-0.25 | 0.25 | ||
| Posaconazole | 0.03 | 0.03 | ||
| Ravuconazole | 0.007-0.03 | 0.015 | ||
| Voriconazole | 0.007-0.016 | 0.007 | ||
50% and 90%, MICs for 50 and 90% of isolates tested, respectively.
QC determinations were performed on at least nine different occasions with each of the QC isolates recommended by the NCCLS (1, 5). MICs were within control limits for all seven agents for both the reference and Trek panels. Likewise, reproducibility studies with 10 selected isolates documented excellent reproducibility, with 100% of MICs determined with the Trek panels falling within ±1 log2 dilution of the modal MIC for each organism-antifungal-agent combination (data not shown).
The overall agreement between the reference and Trek MICs, determined either with all values or with on-scale values only, was 98% for all seven drugs (Table 2). The overall agreement (including both on- and off-scale values) for each antifungal agent and all 99 isolates ranged from 96% for ravuconazole to 100% for amphotericin B. In general, the level of agreement was not influenced by the species of Candida tested; however, lower levels of agreement (90%) between methods were observed when fluconazole and ravuconazole were tested against C. parapsilosis. Of the 15 total discrepancies encountered during the study, all were due to MICs determined with the Trek panels being higher than the reference panel MICs. Although trailing was certainly observed with all of the triazoles, it was similar in the Trek and reference panels and did not pose a significant problem in testing these clinical isolates.
TABLE 2.
Agreement between Trek frozen panel and reference microdilution MICs for seven antifungal agents tested against 99 clinical isolates of Candida species
| Organism (n) | Antifungal agent | % Agreementa calculated with:
|
|
|---|---|---|---|
| All values | On-scale values only | ||
| C. albicans (38) | Amphotericin B | 100 | 100 |
| 5FC | 97 | 97 | |
| Fluconazole | 95 | 95 | |
| Itraconazole | 97 | 97 | |
| Posaconazole | 95 | 95 | |
| Ravuconazole | 97 | 100 | |
| Voriconazole | 97 | 100 | |
| C. glabrata (24) | Amphotericin B | 100 | 100 |
| 5FC | 100 | 100 | |
| Fluconazole | 100 | 100 | |
| Itraconazole | 100 | 100 | |
| Posaconazole | 96 | 96 | |
| Ravuconazole | 92 | 92 | |
| Voriconazole | 92 | 92 | |
| C. tropicalis (15) | Amphotericin B | 100 | 100 |
| 5FC | 100 | 100 | |
| Fluconazole | 100 | 100 | |
| Itraconazole | 100 | 100 | |
| Posaconazole | 100 | 100 | |
| Ravuconazole | 100 | 100 | |
| Voriconazole | 100 | 100 | |
| C. parapsilosis (10) | Amphotericin B | 100 | 100 |
| 5FC | 100 | 100 | |
| Fluconazole | 90 | 90 | |
| Itraconazole | 100 | 100 | |
| Posaconazole | 100 | 100 | |
| Ravuconazole | 90 | 89 | |
| Voriconazole | 100 | 100 | |
| C. krusei (7) | Amphotericin B | 100 | 100 |
| 5FC | 100 | 100 | |
| Fluconazole | 100 | 100 | |
| Itraconazole | 100 | 100 | |
| Posaconazole | 100 | 100 | |
| Ravuconazole | 100 | 100 | |
| Voriconazole | 100 | 100 | |
| C. lusitaniae (5) | Amphotericin B | 100 | 100 |
| 5FC | 100 | 100 | |
| Fluconazole | 100 | 100 | |
| Itraconazole | 100 | 100 | |
| Posaconazole | 100 | 100 | |
| Ravuconazole | 100 | 100 | |
| Voriconazole | 100 | 100 | |
| All isolates (99) | Amphotericin B | 100 | 100 |
| 5FC | 99 | 99 | |
| Fluconazole | 97 | 97 | |
| Itraconazole | 99 | 99 | |
| Posaconazole | 97 | 97 | |
| Ravuconazole | 96 | 96 | |
| Voriconazole | 97 | 98 | |
| All antifungals | 98 | 98 | |
% Agreement, percentage of MICs determined by the Trek panel within 2 dilutions of the reference microdilution MICs.
The tendency for Trek panels to give MICs higher than those obtained with the reference method raises the concern that use of Trek panels may result in the reporting of false resistance and thus have an adverse clinical impact. This concern must be tempered by the facts that false-resistant results were not seen with amphotericin B and that among the remaining licensed antifungal agents (fluconazole, itraconazole, and 5 FC), only one of five discrepancies would have resulted in a susceptible-to-resistant change (major error). Likewise, among the discrepancies observed with the investigational triazoles, only one (involving posaconazole) would have resulted in a change from a potentially susceptible result (MIC, ≤1 μg/ml) to a potentially resistant result (MIC, ≥2 μg/ml). Despite these occasionally elevated MICs produced by the Trek panels, only rarely would they produce a category change from susceptible to resistant, and thus clinical use of this system is unlikely to result in significant false-resistant results.
The results of the present study demonstrate excellent performance by the Trek frozen microdilution panel for antifungal susceptibility testing of both established agents and newly introduced and investigational agents. The reproducibilities of both the Trek and reference panels and the level of agreement between them were established by testing two well-characterized QC strains as well as selected clinical isolates. Extension of these studies to include 99 clinical isolates of Candida spp. demonstrated an overall level of agreement of 98%, which exceeds that observed in other comparison studies performed in this laboratory (4, 7, 9) and indicates that the Trek panel should provide an acceptable level of performance for testing isolates of Candida spp. in a clinical laboratory setting.
The problems with trailing MIC endpoints encountered with certain strains of Candida spp. when they were tested against the triazoles will continue to affect virtually all broth-based susceptibility testing methods. Use of agitation coupled with spectrophotometric reading of MIC endpoints is one way of overcoming this problem (8). In this regard, the adaptability of a microdilution broth system is a distinct advantage.
A commercially prepared microdilution panel containing a full-range dilution series of the clinically useful systemic antifungal agents clearly offers numerous advantages to the clinical microbiology laboratory over a panel prepared in-house. Laboratories that currently perform macrodilution antifungal susceptibility testing or that make their own microdilution panels stand to reduce costs associated with production and test performance in terms of both labor and materials. Variation in the quality of panels during production, the need for extensive QC of panels prepared in-house, limited shelf life, and storage considerations are all factors that may favor the use of a commercially prepared panel. These potential advantages should be confirmed by a formal cost analysis study. As demonstrated in the present study, the seven antifungal agents used in the Trek panel were stable at −70°C for at least 4 months and produced results comparable to those obtained with the frozen reference panel.
Given these results, it appears that the Trek microdilution panel for antifungal susceptibility testing is a viable alternative to microdilution panels prepared in-house and will provide clinical laboratories with a means of performing NCCLS reference quality MIC testing on isolates of Candida spp. The ability to test several antifungal agents simultaneously in a microdilution format makes the Trek product particularly attractive for use in the busy clinical microbiology laboratory.
Acknowledgments
The excellent secretarial support of Linda Elliott is greatly appreciated.
This study was supported in part by Trek Diagnostic Systems, Schering-Plough Research Institute, Pfizer Pharmaceuticals, and Bristol-Myers Squibb.
REFERENCES
- 1.Barry, A. L., M. A. Pfaller, S. D. Brown, A. Espinel-Ingroff, M. A. Ghannoum, C. Knapp, R. P. Rennie, J. H. Rex, and M. G. Rinaldi. 2000. Quality control limits for broth microdilution susceptibility tests of ten antifungal agents. J. Clin. Microbiol. 38:3457-3459. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Hoffman, H. L., and M. A. Pfaller. 2001. In vitro antifungal susceptibility testing. Pharmacotherapy 21(8 Pt. 2):111S-123S. [DOI] [PubMed] [Google Scholar]
- 3.Jorgensen, J. H., J. D. Turnidge, and J. A. Washington. 1999. Antibacterial susceptibility tests: dilution and disk diffusion methods, p. 1526-1543. In P. R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover, and R. H. Yolken (ed.), Manual of clinical microbiology, 7th ed. ASM Press, Washington, D.C.
- 4.Messer, S. A., and M. A. Pfaller. 1996. Clinical evaluation of a dried commercially-prepared microdilution panel for antifungal susceptibility testing. Diagn. Microbiol. Infect. Dis. 25:77-81. [DOI] [PubMed] [Google Scholar]
- 5.National Committee for Clinical Laboratory Standards. 1997. Reference method for broth dilution antifungal susceptibility testing of yeast. Approved standard M27-A. National Committee for Laboratory Standards, Wayne, Pa.
- 6.National Committee for Clinical Laboratory Standards. 2001. Development of in vitro susceptibility testing criteria and quality control parameters. Approved guideline M23-A2. National Committee for Clinical Laboratory Standards, Wayne, Pa.
- 7.Pfaller, M. A., B. Buschelman, M. J. Bale, M. Lancaster, A. Espinel-Ingroff, J. H. Rex, and M. G. Rinaldi. 1994. Multicenter comparison of a colorimetric microdilution broth method with the reference macrodilution method for in vitro susceptibility testing of yeast isolates. Diagn. Microbiol. Infect. Dis. 19:9-13. [DOI] [PubMed] [Google Scholar]
- 8.Pfaller, M. A., S. A. Messer, and S. Coffman. 1995. Comparison of visual and spectrophotometric methods of MIC endpoint determinations by using broth microdilution methods to test five antifungal agents, including the new triazole D0870. J. Clin. Microbiol. 33:1094-1097. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Pfaller, M. A., S. Arikan, M. Lozano-Chiu, Y. S. Chen, S. Coffman, S. A. Messer, R. Rennie, C. Sand, T. Heffner, J. H. Rex, J. Wang, and N. Yamane. 1998. Clinical evaluation of the ASTY colorimetric microdilution panel for antifungal susceptibility testing. J. Clin. Microbiol. 36:2609-2612. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Pfaller, M. A., S. A. Messer, R. J. Hollis, R. N. Jones, G. V. Doern, M. E. Brandt, and R. A. Hajjeh. 1998. In vitro susceptibilities of Candida bloodstream isolates to the new triazole antifungal agents BMS-207147, SCH 56592, and voriconazole. Antimicrob. Agents Chemother. 42:3242-3244. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Pfaller, M. A., and W. Yu. 2001. Antifungal susceptibility testing. New technology and clinical applications. Infect. Dis. Clin. N. Am. 15:1227-1261. [DOI] [PubMed] [Google Scholar]
- 12.Pfaller, M. A., D. J. Diekema, R. N. Jones, H. S. Sader, A. C. Fluit, R. J. Hollis, S. A. Messer, and the SENTRY Participant Group. 2001. International surveillance of bloodstream infections due to Candida species: Frequency of occurrence and in vitro susceptibilities to fluconazole, ravuconazole, and voriconazole of isolates collected from 1997 through 1999 in the SENTRY antimicrobial surveillance program. J. Clin. Microbiol. 39:3254-3259. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Pfaller, M. A., S. A. Messer, R. J. Hollis, and R. N. Jones. 2001. In vitro activities of posaconazole (Sch 56592) compared with those of itraconazole and fluconazole against 3,685 clinical isolates of Candida spp. and Cryptococcus neoformans. Antimicrob. Agents Chemother. 45:2862-2864. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Rex, J. H., T. J. Walsh, J. D. Sobel, S. G. Filler, P. G. Pappas, W. E. Dismukes, and J. E. Edwards. 2000. Practice guidelines for the treatment of candidiasis. Clin. Infect. Dis. 30:662-678. [DOI] [PubMed] [Google Scholar]
- 15.Rex, J. H., M. A. Pfaller, T. J. Walsh, V. Chaturvedi, A. Espinel-Ingroff, M. A. Ghannoum, L. L. Gosey, F. C. Odds, M. G. Rinaldi, D. J. Sheehan, and D. W. Warnock. 2001. Antifungal susceptibility testing: practical aspects and current challenges. Clin. Microbiol. Rev. 14:643-658. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Warren, N. G., and K. C. Hazen. 1999. Candida, Cryptococcus, and other yeasts of medical importance, p. 1184-1199. In P. R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover, and R. H. Yolken (ed.), Manual of clinical microbiology, 7th ed. ASM Press, Washington, D.C.