Abstract
The in vitro activities of fluconazole or voriconazole plus terbinafine were evaluated against 20 Candida isolates by the checkerboard, time-kill, and Etest methods. Synergism (C. albicans, C. glabrata, and C. tropicalis) and indifference (C. krusei) were observed. Correlation among methods was good. The Etest is a suitable method to determine drug interactions.
The checkerboard and time-kill methods to determine in vitro interactions between drugs are time-consuming and cumbersome for use in clinical laboratories. In order to find a method that facilitates synergistic studies, our aim was dual: (i) to assess the in vitro activities of voriconazole (VRC) and fluconazole (FLC) combined with terbinafine (TRB) against four Candida spp. (resistant or susceptible to FLC and/or TRB) by the checkerboard and time-kill methods and (ii) to compare the results of these methods with those obtained by an Etest-agar dilution technique.
Twenty blood isolates (Table 1) were tested. C. parapsilosis ATCC 22019 and C. krusei ATCC 6258 were included for quality control.
TABLE 1.
In vitro interaction between FLC and TRB and between VRC and TRB by the checkerboard method
| Isolate | MIC (μg/ml)
|
Lowest ΣFIC for FLC/TRB (interpretation)a | MIC (μg/ml)
|
Lowest ΣFIC for VRC/TRB (interpretation)a | ||||
|---|---|---|---|---|---|---|---|---|
| FLC | TRB | FLC/TRB | VRC | TRB | VRC/TRB | |||
| C. albicans | ||||||||
| EU-62 | >64 | 16 | 0.06/0.25 | 0.016 (S) | >2 | >16 | 0.002/0.5 | 0.016 (S) |
| EU-78 | 1 | 0.25 | 0.06/0.25 | 1.12 (I) | 0.008 | 0.5 | 0.004/0.25 | 1.00 (I) |
| EU-80 | >64 | >16 | 0.06/0.5 | 0.016 (S) | >2 | >16 | 0.002/0.5 | 0.016 (S) |
| EU-87 | 64 | 16 | 0.06/0.25 | 0.016 (S) | >2 | >16 | 0.002/0.5 | 0.016 (S) |
| EU-170 | 2 | >16 | 0.06/0.5 | 0.054 (S) | >2 | 8 | 0.03/0.5 | 0.07 (S) |
| C. glabrata | ||||||||
| EU-12 | >64 | >16 | 4/0.5 | 0.046 (S) | >2 | >16 | 0.5/0.25 | 0.132 (S) |
| EU-38 | 16 | >16 | 32/4 | 2.125 (I) | >2 | >16 | 1/4 | 0.375 (S) |
| EU-68 | 8 | >16 | 1/1 | 0.156 (S) | >2 | >16 | 1/4 | 0.375 (S) |
| EU-151 | 32 | >16 | 1/2 | 0.093 (S) | 0.5 | >16 | 0.5/0.25 | 1.007 (I) |
| EU-195 | 16 | >16 | 4/2 | 0.312 (S) | >2 | >16 | 0.25/2 | 0.125 (S) |
| C. tropicalis | ||||||||
| EU-43 | >64 | >16 | 1/0.5 | 0.023 (S) | 0.12 | >16 | 0.002/8 | 0.266 (S) |
| EU-240 | >64 | >16 | 16/2 | 0.187 (S) | >2 | >16 | 0.25/2 | 0.125 (S) |
| EU-245 | >64 | >16 | 0.5/0.25 | 0.0117 (S) | 1 | >16 | 0.03/8 | 0.282 (S) |
| EU-255 | >64 | 16 | 1/0.5 | 0.023 (S) | >2 | >16 | 0.016/8 | 0.254 (S) |
| EU-264 | 1 | >16 | 0.5/0.25 | 0.5078 (I) | 0.03 | >16 | 0.016/0.25 | 0.5078 (I) |
| C. krusei | ||||||||
| EU-123 | 64 | >16 | 64/0.25 | 1.0078 (I) | 0.5 | >16 | 0.5/0.25 | 1.0078 (I) |
| CK-1 | 64 | >16 | 64/0.25 | 1.0078 (I) | 0.5 | >16 | 0.25/0.25 | 0.5078 (I) |
| CK-2 | >64 | >16 | 64/16 | 1 (I) | 1 | >16 | 0.25/0.25 | 0.2578 (S) |
| CK-3 | 64 | >16 | 64/0.25 | 1.0078 (I) | 0.5 | >16 | 0.25/0.25 | 0.5078 (I) |
| CK-4 | 64 | >16 | 64/0.25 | 1.0078 (I) | 0.5 | >16 | 0.5/0.25 | 1.0078 (I) |
S, synergism; I, indifference.
Stock solutions of VRC, FLC (Pfizer, Barcelona, Spain), and TRB (Novartis, Barcelona, Spain) were prepared with the appropriate solvent (dimethyl sulfoxide for VRC and TRB and distilled water for FLC). The final concentrations were 0.002 to 2 μg/ml for VRC, 0.06 to 64 μg/ml for FLC, and 0.25 to 16 μg/ml for TRB. MICs of drugs alone or in combination were determined by the NCCLS M27-A2 method (12) and corresponded to the lowest concentration that showed prominent (≥50%) growth inhibition and by the Etest method as described below.
Drug interactions were assessed by the following three methods described below: broth microdilution checkerboard, time-kill, and Etest.
(i) Broth microdilution checkerboard.
The broth microdilution checkerboard method was performed by using the fractional inhibitory concentration (FIC) index, which is defined as the sum of the MIC of each drug when used in combination divided by the MIC of the drug when used alone. For computation of FIC indices, off-scale MICs were raised to the next highest MIC; synergistic and antagonistic FIC indices were defined as ≤0.5 and >4, respectively.
(ii) Time-kill studies.
One isolate of each species was selected, and tests were conducted as previously described (RPMI 1640 medium, 105-CFU/ml inoculum, and 5-ml volume) (8). The drug concentrations tested alone were as follows: VRC, 16 and 1 μg/ml; FLC, 32 and 2 μg/ml; and TRB, 8 and 2 μg/ml. For the combinationsVRC/TRB and FLC/TRB, the drug concentrations were as follows: VRC/TRB, 16/2, 1/2, and 1/8 μg/ml; and FLC/TRB, 32/2, 32/8, 2/2, and 2/8 μg/ml. At 0, 3, 6, 24, and 48 h, aliquots were removed to determine the number of CFU per milliliter. Synergy was defined as a ≥2-log10 decrease in CFU per milliliter for a combination compared to the killing with the most active drug alone and an increase of ≥2 log10 as antagonism. Experiments were conducted in duplicate and on 2 separate days.
(iii) Etest studies.
RPMI 1640 agar with 2% dextrose and 1, 2, and 8 μg of TRB per ml, prepared as described elsewhere (7), was used. For each strain, FLC or VRC Etest strips were applied to two agar plates, one with TRB (MIC of the combination) and another without it (azole MIC). Plates were inoculated following the manufacturer's instructions; MICs were obtained at 24 and 48 h. An azole MIC reduction of ≥3 dilutions in the presence of TRB was defined as synergy, and an increase of ≥3 dilutions was defined as antagonism.
MICs for the quality control strains were within the acceptable range (6, 12). Although 24-h Etest MICs were within 2 dilutions compared with those obtained by M27-A2 (48 h) for most isolates, the M27-A2 method detected resistance while Etest provided susceptible results for some C. tropicalis isolates (Tables 1 to 3); however, Etest ellipses had heavy trailing growth.
TABLE 3.
Effect of TRB concentration on VRC activity as determined by the checkerboard and Etest methods
| Strain | VRC MIC (μg/ml) with TRB concn of:
|
|||||||
|---|---|---|---|---|---|---|---|---|
| 0
|
1 μg/ml
|
2 μg/ml
|
8 μg/ml
|
|||||
| M27-A2 | Etest | Checkerboard | Etest | Checkerboard | Etest | Checkerboard | Etest | |
| C. albicans | ||||||||
| EU-62 | >2 | >32 | 0.002 | 0.002 | 0.03* | 0.004* | 0.002* | 0.008* |
| EU-78 | 0.008 | 0.008 | 0.002* | 0.008* | 0.002* | 0.004* | 0.002* | 0.008* |
| EU-80 | >2 | >32 | 0.002-0.06* | 0.002 | 0.002* | 0.002* | 0.002* | 0.016* |
| EU-87 | >2 | >32 | 0.002* | 0.016* | 0.002* | 0.016* | 0.002* | 0.012* |
| EU-170 | >2 | >32 | 0.016 | 0.016 | 0.03* | 0.012* | 0.002* | 0.012* |
| C. glabrata | ||||||||
| EU-12 | >2 | >32 | 0.5 | NDb | 0.12 | 0.5 | 0.12 | 0.5 |
| EU-38 | >2 | 16 | >2 | ND | >2 | 0.5 | 1 | 0.5 |
| EU-68 | >2 | 8 | 2 | ND | 2 | 8 | 1 | 2 |
| EU-151 | 0.5 | 2 | 2 | ND | 2 | 1 | 0.5 | 0.25 |
| EU-195 | >2 | 0.5 | 0.5 | ND | 0.25 | 0.38 | 0.12 | 0.12 |
| C. tropicalis | ||||||||
| EU-43 | 0.12 | 0.06 | 2 | ND | 0.25 | 0.12 | 0.002 | 0.12 |
| EU-240 | >2 | 0.19 | >2 | ND | 0.25 | 0.38 | 0.25 | 0.5 |
| EU-245 | 1 | 0.06 | 0.12 | ND | 0.12 | 0.12 | 0.016 | 0.047 |
| EU-255 | >2 | 0.25 | >2 | ND | 2 | 0.25 | 0.016 | 0.5 |
| EU-264 | 0.03 | 0.12 | 0.016 | ND | 0.016 | 0.06 | 0.016 | 0.19* |
| C. krusei | ||||||||
| EU-123 | 0.5 | 0.25 | 0.5 | ND | 0.5 | 0.19* | 0.5* | 0.25* |
| CK-1 | 0.5 | 0.25 | 0.25 | ND | 0.25 | 0.25* | 0.25* | 0.12* |
| CK-2 | 1 | 0.5 | 0.5 | ND | 0.5 | 0.5* | 0.5* | 0.5* |
| CK-3 | 0.5 | 0.25 | 0.25 | ND | 0.25 | 0.25* | 0.25* | 0.25* |
| CK-4 | 0.5 | 0.25 | 0.5 | ND | 0.5 | 0.25* | 0.5* | 0.25* |
Asterisks indicate the minimum drug concentration that produced 100% growth inhibition.
ND, not determined.
By checkerboard, the combination of both azoles and TRB was synergistic against four strains each of C. albicans, C. glabrata, and C. tropicalis (ΣFIC index, ≤0.5) (Table 1). Against C. albicans, MICs of FLC and VRC were 0.06 and 0.002 to 0.03 μg/ml, respectively, when combined with 0.25 to 0.5 μg of TRB per ml. Both azoles in combination with TRB inhibited the growth of C. albicans. Against C. glabrata, FLC MICs decreased to 1 to 4 μg/ml upon combination with 0.5 to 2 μg of TRB per ml. VRC MICs decreased 2 to 3 dilutions when combined with ≥0.5 μg of TRB per ml, and TRB MICs were reduced to 0.25 to 4 μg/ml in the presence of ≥0.25 μg of VRC per ml (Table 1). MICs of FLC for C. tropicalis were ≤1 μg/ml combined with TRB (≥0.25 μg/ml), except for one strain, for which the FLC MIC was 16 μg/ml in presence of 2 μg of TRB per ml. VRC MICs were ≤0.25 μg/ml when combined with ≥2 μg of TRB per ml. Against C. krusei, the interaction of both azoles with TRB was indifferent for four strains (ΣFIC index, 0.5078 to 1.01), while the combination VRC/TRB was synergistic (ΣFIC index = 0.2578) for one isolate (Table 1).
By the time-kill method, the combination of both azoles with TRB was indifferent: none of the killing curves showed more than a 1.5-log decrease in killing in either of the two independent tests made (data not shown).
Tables 2 and 3 depict the interaction of azoles with TRB by Etest. In general, Etest synergistic results were in agreement with those obtained by the checkerboard method with the same concentrations of TRB.
TABLE 2.
Effect of TRB concentration on FLC activity as determined by the checkerboard and Etest methods
| Strain | FLC MIC (μg/ml) with TRB concn ofa:
|
|||||||
|---|---|---|---|---|---|---|---|---|
| 0
|
1 μg/ml
|
2 μg/ml
|
8 μg/ml
|
|||||
| M27-A2 | Etest | Checkerboard | Etest | Checkerboard | Etest | Checkerboard | Etest | |
| C. albicans | ||||||||
| EU-62 | >64 | 64 | <0.06-1* | 0.016* | 0.06-1* | 0.06* | 0.06-1* | 0.12* |
| EU-78 | 1 | 0.25 | <0.06 | 2* | 0.06-1* | 0.12* | 0.06-1* | 0.06* |
| EU-80 | >64 | >256 | <0.06 | 0.5 | 0.06-1* | 0.25* | 0.06-1* | 1.5* |
| EU-87 | 64 | 64 | <0.06 | 0.5 | 0.06-1* | 0.25* | 0.06-1* | 0.38* |
| EU-170 | 2 | 0.5 | <0.06 | 0.75 | 0.06 | 0.25* | 0.06-1* | 0.38* |
| C. glabrata | ||||||||
| EU-12 | >64 | >256 | 4 | NDb | 4 | 1 | 1 | 0.5 |
| EU-38 | 16 | 24 | 64 | ND | 64 | 32 | 32 | 16 |
| EU-68 | 8 | 16 | 8 | ND | 8 | 8 | 0.5 | 8 |
| EU-151 | 32 | 32 | 8 | ND | 1 | 8 | 1 | 8 |
| EU-195 | 16 | 16 | 8 | ND | 4 | 16 | 4 | 2 |
| C. tropicalis | ||||||||
| EU-43 | >64 | 1 | 1 | ND | 1 | 1 | 0.5 | 1 |
| EU-240 | >64 | 12 | 32 | ND | 16 | 12 | 16 | 12 |
| EU-245 | >64 | 0.5 | 0.5 | ND | 0.5 | 0.38 | 0.12 | 0.38 |
| EU-255 | >64 | 0.75 | 1 | ND | 1 | 2 | <0.06 | 2 |
| EU-264 | 1 | 1 | 0.5 | ND | 0.5 | 1 | 0.5 | 1 |
| C. krusei | ||||||||
| EU-123 | 64 | 64 | 64 | ND | 64 | 32 | 64 | 32 |
| CK-1 | 64 | 64 | 64 | ND | 64 | 64 | 64 | 32 |
| CK-2 | >64 | >64 | >64 | ND | >64 | 128 | >64 | 64 |
| CK-3 | 64 | 64 | 64 | ND | 64 | 48 | 64 | 48 |
| CK-4 | 64 | >256 | 64 | ND | 64 | 48 | 64 | 64 |
Asterisks indicate the minimum drug concentration that produced 100% growth inhibition.
ND, not determined.
Our results by the checkerboard method confirm previous reports (4, 5, 14, 19) and extend them to C. tropicalis and C. krusei. We found that the combination of FLC or VRC with TRB was synergistic for C. albicans, C. glabrata (strain dependent), and C. tropicalis and indifferent for C. krusei by the checkerboard and Etest methods. However, by the time-kill method, the interaction was indifferent. The lowest FIC indices for the combination of both azoles and TRB were obtained at achievable concentrations in serum of 3.6 μg/ml for TRB (3, 11, 18), 30 μg/ml for FLC (9, 17, 18), and up to 5.2 μg/ml for VRC (2, 10, 16). The three methods showed good correlation for most of the species, although by the checkerboard method, the synergistic effect was stronger. The lack of agreement between the checkerboard and Etest methods for C. tropicalis could be due to the heavy trailing growth observed at 48 h by the microdilution method; agreement was good with 24-h MICs by the latter method. To our knowledge, there are no other reports describing the same drug combinations, species, and methods. It is interesting that when the TRB concentration that produced the interaction with the azole was reached, a further increase did not decrease the azole MIC. This was demonstrated by the three methods.
By using available Etest strips and incorporating subinhibitory concentrations of TRB into the RPMI agar, we were able to determine interactions between these agents. These results were similar to those obtained by the most frequently used checkerboard method (Tables 2 and 3). The Etest could be a suitable method in clinical laboratories, because both RPMI agar and Etest strips are commercially available for established agents. In addition, the incorporation of the new agent into the agar plate can be made by flooding the agar plate with the appropriate drug concentrations as recommended in the NCCLS M44-P document (13). Our methodology could also be useful to study the interaction of antifungal agents with other substances (antineoplasic, anti-inflammatory, immunosuppressive drugs, etc.) (1, 15). Further studies are needed to determine reliability of these methods and the correlation of in vitro and in vivo results.
Acknowledgments
We thank Pfizer Laboratories for financial support for Isabel Moreno, who performed the synergy testing.
REFERENCES
- 1.Afeltra, J., J. F. G. Meis, R. G. Vitale, J. W. Mouton, P. E. Verweij, and the Eurofung Network. 2002. In vitro activities of pentamidine, trimethoprim, and sulfonamides against Aspergillus species. Antimicrob. Agents Chemother. 46:2029-2031. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Andes, D., K. Marchillo, T. Stamstad, and R. Conklin. 2003. In vivo pharmacokinetics and pharmacodynamics of a new triazole, voriconazole, in a murine candidiasis model. Antimicrob. Agents Chemother. 47:3165-3169. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Balfour, J. A., and D. Faulds. 1992. Terbinafine. A review of its pharmacokinetic properties, and therapeutic potential in superficial mycosis. Drugs 43:259-284. [DOI] [PubMed] [Google Scholar]
- 4.Barchiesi, F., L. Falconi Di Francesco, and G. Scalise. 1997. In vitro activities of terbinafine in combination with fluconazole and itraconazole against isolates of Candida albicans with reduced susceptibility to azoles. Antimicrob. Agents Chemother. 41:1812-1814. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Barchiesi, F., L. Falconi Di Francesco, P. Compagnucci, D. Arzeni, A. Giacometti, and G. Scalise. 1998. In-vitro interaction of terbinafine with amphotericin B, fluconazole and itraconazole against clinical isolates of Candida albicans. J. Antimicrob. Chemother. 41:59-65. [DOI] [PubMed] [Google Scholar]
- 6.Barry, A. L., M. A. Pfaller, S. D. Brown, et al. 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]
- 7.Cantón, E., J. Pemán, M. Romero, and M. Gobernado. 2004. Utilidad del E-test y sus condiciones de ensayo en el estudio de la interacción de los antifúngicos. Estudio piloto. Rev. Esp. Quimioterap. 17:48-56. [PubMed] [Google Scholar]
- 8.Cantón, E., J. Pemán, M. Gobernado, A. Viudes, and A. Espinel-Ingroff. 2004. Killing kinetics patterns of amphotericin B against seven Candida species. Antimicrob. Agents Chemother. 48:2477-2482. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Grant, S. M., and S. P. Clissold. 1990. Fluconazole. A review of its pharmacodynamic and pharmacokinetic properties, and therapeutic potential in superficial and systemic mycoses. Drugs 39:877-916. [DOI] [PubMed] [Google Scholar]
- 10.Hoffman, H. L., and R. C. Rathbun. 2002. Review of the safety and efficacy of voriconazole. Expert Opin. Investig. Drugs 11:409-429. [DOI] [PubMed] [Google Scholar]
- 11.Kovarik, J. M., E. A. Mueller, H. Zehender, J. Denouel, H. Caplain, and L. Millerioux. 1995. Multiple-dose pharmacokinetics and distribution in tissue of terbinafine and metabolites. Antimicrob. Agents Chemother. 39:2738-2741. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.National Committee for Clinical Laboratory Standards. 2002. Reference method for broth dilution antifungal susceptibility testing of yeasts. Approved standard, 2nd ed. NCCLS document M27-A2. National Committee for Clinical Laboratory Standards, Wayne, Pa.
- 13.National Committee for Clinical Laboratory Standards. 2004. Method for antifungal disk diffusion susceptibility testing of yeasts. Approved guideline. NCCLS document M44-A. National Committee for Clinical Laboratory Standards, Wayne, Pa.
- 14.Perea, S., G. Gonzalez, A. W. Fothergill, D. A. Sutton, and M. G. Rinaldi. 2002. In vitro activities of terbinafine in combination with fluconazole, itraconazole, voriconazole, and posaconazole against clinical isolates of Candida glabrata with decreased susceptibility to azoles. J. Clin. Microbiol. 40:1831-1833. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Pina-Vaz, C., F. Sansonetty, A. G. Rodríguez, J. Martinez-De-Oliveira, A. F. Fonseca, and P. A. Mardh. 2000. Antifungal activity of ibuprofen alone and in combination with fluconazole against Candida species. J. Med. Microbiol. 49:831-840. [DOI] [PubMed] [Google Scholar]
- 16.Purkins, L., N. Wood, P. Ghahramani, K. Greenhalgh, M. J. Allen, and D. Kleinermans. 2002. Pharmacokinetics and safety of voriconazole following intravenous- to oral-dose escalation regimens. Antimicrob. Agents Chemother. 46:2546-2553. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Silling, G. 2002. Fluconazole: optimized antifungal therapy based on pharmacokinetics. Mycoses 45(Suppl. 3):39-41. [DOI] [PubMed] [Google Scholar]
- 18.Sorensen, K. N., R. A. Sobel, K. V. Clemons, L. Calderon, K. J. Howell, P. R. Irani, D. Pappagianis, P. L. Williams, and D. A. Stevens. 2000. Comparative efficacies of terbinafine and fluconazole in treatment of experimental coccidioidal meningitis in a rabbit model. Antimicrob. Agents Chemother. 44:3087-3091. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Weig, M., and F. M. Muller. 2001. Synergism of voriconazole and terbinafine against Candida albicans isolates from human immunodeficiency virus-infected patients with oropharyngeal candidiasis. Antimicrob. Agents Chemother. 45:966-968. [DOI] [PMC free article] [PubMed] [Google Scholar]