Abstract
The synergism of voriconazole (VRC) and terbinafine was studied by using 39 genotypically defined clinical Candida albicans isolates that were cross-resistant to fluconazole and VRC and serial isolates that gradually developed azole resistance. Synergy was noticed in 100% (eight of eight) of the strains that were resistant to VRC. Antagonism was not observed.
The emergence of azole resistance in Candida albicans has been associated with broad prophylactic use and long-term treatment with fluconazole (FLC) (12). Voriconazole (VRC; UK-109, 496) is a new potent broad-spectrum triazole antifungal agent. Data on the effectiveness of VRC against clinical isolates with decreased susceptibility to FLC are limited (3). In a recent study we have reported on the development of cross-resistance to VRC and FLC in human immunodeficiency virus (HIV)-infected children who were never treated with VRC (7). Combination therapy in which the synergy of different antifungals is taken advantage of is a promising novel approach in the therapy of candidiasis caused by strains resistant to conventional antifungal agents.
The combination of VRC with terbinafine (TRB) was tested for synergy in 31 paired and serial C. albicans isolates obtained from the oral mucosae of 10 HIV-infected children with therapy-resistant symptomatic oropharyngeal candidiasis (OPC). Eight paired isolates were obtained from four adult AIDS patients with recurrent OPC (4), and C. albicans strains 8621 (6) and 230 (M. Weig and F. M. C. Müller, Abstr. 40th Intersci. Conf. Antimicrob. Agents Chemother., abstr. 930, p. 367, 2000) served as control organisms. The control strains were clinical istolates from patients who had not received antifungal treatment.
Polyethylene glycol 400 was used to solubilize TRB reagent-grade powder (Novartis, Vienna, Austria), whereas dimethyl sulfoxide (Merck, Darmstadt, Germany) was used to solubilize VRC (Pfizer, Sandwich, United Kingdom). Stock solutions of FLC (Pfizer), TRB, and VRC were prepared in high-resolution (HR) antifungal assay medium (Oxoid, Wesel, Germany). A serial twofold dilution of the agents was performed with the appropriate diluent. The final concentrations of the antifungal compounds were 0.125 to 64 μg/ml for FLC, 0.007 to 16 μg/ml for VRC, and 0.06 to 8 μg/ml for TRB. Broth microdilution testing was done by the NCCLS M27-A reference method (1). The C. albicans inoculum size ranged between 0.5 × 103 and 2.5 × 103 CFU/ml. MIC endpoints were determined visually and spectrophotometrically at 24 and 48 h. The MIC was defined as the lowest concentration of a compound in which a prominent decrease in turbidity was observed (50% inhibition of growth relative to that of the control) (10, 11).
Drug interactions were determined by a checkerboard microdilution method which included the determination of the MIC of each drug alone (5). The reproducibilities of the MICs were ensured by repetitive testing. In vitro interactions were calculated algebraically and were interpreted as synergistic, indifferent, or antagonistic. The calculation was done on the basis of the fractional inhibitory concentration (FIC) index, depending on whether the antifungal activity of the combination was greater than, equivalent to, or less than the activities of the individual agents, respectively. The summation (ΣFIC) was interpreted as synergistic if the FIC index was ≤0.5, indifferent if the FIC index was >0.5 but ≤4, and antagonistic if the FIC index was >4.
The significance of the reduction in the geometric mean TRB and VRC MICs when the substances were given in combination compared to the MICs of the substances when they were given alone were determined by the paired rank test, a nonparametric test for the dependent two-sample problem (8). A P value of <0.01 was considered significant.
The TRB MICs for the 39 clinical C. albicans isolates ranged from 1.0 to >8.0 μg/ml (geometric mean, 4.59 μg/ml; median, 4 μg/ml; MIC at which 50% of isolates are inhibited [MIC50], 4 μg/ml; MIC90, 8 μ/ml), FLC MICs ranged from 0.5 to >64 μg/ml (geometric mean, 27.8 μg/ml; median, 16 μg/ml; MIC50, 16 μg/ml; MIC90, ≥64 μg/ml), and VRC MICs ranged from ≤0.007 to >16 μg/ml (geometric mean, 2.9; median, 0.25 μg/ml; MIC50, 0.25 μg/ml; MIC90, 16 μg/ml) (Table 1). When TRB and VRC were given in combination, significant reductions in the geometric mean TRB MICs (4.59 to 0.63 μg/ml [P < 0.00001, paired rank test]) and VRC MICs (2.87 to 0.09 μg/ml [P < 0.00001]) for the clinical isolates were observed. For the combination, the MIC50s and MIC90s were reduced from 4 and 8 to 0.25 and 2 μg/ml, respectively, for TRB and from 0.25 and 16 to 0.03 and 0.25 μg/ml, respectively, for VRC. Fifty-nine percent (23 of 39) of the interactions were synergistic, and 41% (16 of 39) of the interactions were additive, while antagonistic effects were not observed. For all strains (eight of eight) that were resistant to VCZ (MICs, >1 μg/ml), synergistic effects were detected when VRC and TRB were tested in combination. The median MICs for VRC-resistant strains dropped from 16 to 0.03 μg/ml when VRC was tested in combination with TRB with these isolates. For 69% (9 of 13) of the strains that were resistant to FLC (MICs ≥64 μg/ml), synergistic effects were detected when VRC was combined with TRB.
TABLE 1.
Interaction of VRC and TRB against 39 paired and serial C. albicans isolates from HIV-infected patients with OPC
| Patient no. | Isolate | MIC (μg/ml) of the following drugs:
|
ΣFIC index for TRB-VRC | Interpretationa | |||
|---|---|---|---|---|---|---|---|
| FLC | TRB | VRC | TRB-VRC | ||||
| 1 | mb4389 | >64 | >8 | >16 | 0.25/0.03 | 0.03 | Syn |
| mb6860 | >64 | 8 | 16 | 0.25/0.015 | 0.03 | Syn | |
| mb1307 | >64 | 4 | 16 | 0.25/0.015 | 0.06 | Syn | |
| mb8808 | >64 | 8 | 16 | 0.25/0.03 | 0.03 | Syn | |
| mb8456 | >64 | 8 | 4 | 0.25/0.125 | 0.06 | Syn | |
| mb4544 | 32 | 8 | 1 | 4.0/0.5 | 1.00 | In | |
| mb8577 | 32 | >8 | 1 | 4.0/0.5 | 1.00 | In | |
| 2 | mb2771 | 2 | 2 | 0.015 | 0.125/≤0.007 | 0.53 | In |
| mb8586 | 2 | 1 | ≤0.007 | ≤0.06/≤0.007 | 1.06 | In | |
| mb6935 | 8 | 2 | 0.06 | 0.5/≤0.007 | 0.37 | Syn | |
| mb4657 | 8 | 2 | 0.06 | 0.5/≤0.007 | 0.37 | Syn | |
| mb2421 | >64 | 1 | 0.25 | 0.5/0.125 | 1.00 | In | |
| 3 | mb1873 | 8 | 8 | 0.25 | ≤0.06/0.25 | 1.01 | In |
| mb5550 | 32 | 8 | 0.5 | 0.5/0.25 | 0.56 | In | |
| 4 | mb1672 | 2 | 4 | 16 | 0.125/0.015 | 0.03 | Syn |
| mb8781 | >64 | 8 | 4 | 0.25/0.06 | 0.05 | Syn | |
| mb5330 | >64 | 8 | 16 | 0.125/0.25 | 0.03 | Syn | |
| 5 | mb4074 | 0.5 | 2 | 0.06 | ≤0.06/0.015 | 0.25 | Syn |
| mb74080 | 2 | 2 | 0.015 | 0.5/≤0.007 | 0.72 | In | |
| 6 | mb1447 | 2 | 2 | 0.06 | ≤0.06/0.06 | 1.03 | In |
| mb2771 | 4 | 2 | 0.125 | ≤0.06/0.06 | 0.51 | In | |
| mb7040 | 8 | 4 | 0.125 | 0.25/0.03 | 0.49 | Syn | |
| 7 | mb9329 | 16 | 8 | 0.25 | 2/0.125 | 0.75 | In |
| mb8292 | >64 | 8 | 0.5 | 2/0.25 | 0.75 | In | |
| 8 | mb2143 | 0.5 | 2 | 0.25 | ≤0.06/≤0.007 | 0.06 | Syn |
| mb4821 | 2 | 2 | ≤0.007 | ≤0.06/≤0.007 | 1.03 | In | |
| 9 | mb4256 | 16 | 8 | 0.5 | 2/0.06 | 0.37 | Syn |
| mb3961 | 64 | 8 | 1 | 0.5/0.25 | 0.32 | Syn | |
| 10 | mb4738 | 16 | 1 | 0.125 | 0.25/0.015 | 0.37 | Syn |
| mb5645 | 16 | 4 | 0.125 | 0.25/0.015 | 0.18 | Syn | |
| mb4753 | 1 | 2 | 0.03 | 0.25/≤0.007 | 0.36 | Syn | |
| 11 | B3 | 0.5 | 1 | ≤0.007 | ≤0.06/≤0.007 | 1.06 | In |
| B4 | 32 | 8 | 0.125 | 2/0.015 | 0.37 | Syn | |
| 12 | F2 | 8 | 1 | 0.03 | 0.25/≤0.007 | 0.48 | Syn |
| F5 | >64 | 8 | 0.25 | 1/0.03 | 0.25 | Syn | |
| 13 | G2 | 0.5 | 2 | 0.015 | ≤0.06/≤0.007 | 0.50 | Syn |
| G5 | >64 | 4 | 0.125 | 0.5/0.06 | 0.61 | In | |
| 14 | Gu4 | 4 | 2 | 0.03 | 0.5/≤0.007 | 0.48 | Syn |
| Gu5 | >64 | >8 | 1 | 0.125/0.5 | 0.52 | In | |
| Control strains | 8621 | 0.5 | 2 | 0.03 | ≤0.06/≤0.007 | 0.26 | Syn |
| 230 | ≤0.125 | 2 | 0.03 | 0.125/≤0.007 | 0.36 | Syn | |
Syn, synergistic; In, indifferent.
In our study we have tested VRC in combination with the potent allylamine TRB for synergistic interactions against clinical C. albicans isolates that were cross-resistant to FLC and VRC and serial isolates that gradually developed azole resistance. Although preliminary in vivo data indicate that oral TRB monotherapy (250 mg/day) of AIDS-associated OPC is not an effective regimen (9), Barchiesi and coworkers (2) were able to demonstrate that the combination of TRB with itraconazole or with FLC results in a significant synergistic effect against C. albicans. The mechanism of synergy seems to be explained by the blockage of ergosterol biosynthesis at different levels. Our data indicate a clear enhancement of the in vitro activity of VRC when it is given in combination with TRB. This effect seems to be more prominent in VRC-resistant strains than in VRC-susceptible strains (synergistic effects were detected for 100% of the VRC-resistant isolates and 48% of the VRC-susceptible strains). All isolates in our study were genotypically characterized by random amplified polymorphic DNA analysis, interrepeat PCR, and electrophoretic karyotype analysis for genetic alterations (F. M. Müller et al., Abstr. 98th Gen. Meet. Am. Soc. Microbiol., abstr. F-49, p. 261, 1998). For all except one of the patients, consecutively obtained strains were found to be isogenic by all three biotyping methods. For patient 1 the first five isolates were isogenic, whereas two further isolates showed a different karyotype (Table 1, strains mb4544 and mb8577). Therefore, the loss of a synergistic effect against the serial C. albicans isolates from this patient seems to be explained by the replacement of a nonisogenic strain rather than the development of effective mechanisms of resistance against the combination.
In conclusion, we were able to demonstrate an effective synergism between VRC and TRB in vitro against clinical isolates of C. albicans from HIV-infected patients. The in vivo efficacy and the clinical utility of the synergistic effects presented here for the treatment of infections caused by azole-cross-resistant C. albicans strains require further clinical investigation.
Acknowledgments
We thank Conni Heeg and Birgit Hofmann for expert technical assistance, and we acknowledge M. Munzel for statistical advice.
F.-M. C. Müller was supported by a grant from the Bundesministerium für Bildung und Forschung (BMBF).
REFERENCES
- 1.Barchiesi F, Colombo A L, McGough D A, Rinaldi M G. Comparative study of broth macrodilution and microdilution techniques for in vitro antifungal susceptibility testing of yeasts by using the National Committee for Clinical Laboratory Standards' proposed standard. J Clin Microbiol. 1994;32:2494–2500. doi: 10.1128/jcm.32.10.2494-2500.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Barchiesi F, Falconi D F, Scalise G. In vitro activities of terbinafine in combination with fluconazole and itraconazole against isolates of Candida albicans with reduced susceptibility to azoles. Antimicrob Agents Chemother. 1997;41:1812–1814. doi: 10.1128/aac.41.8.1812. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Cuenca E M, Diaz-Guerra T M, Mellado E, Monzon A, Rodriguez-Tudela J L. Comparative in vitro activity of voriconazole and itraconazole against fluconazole-susceptible and fluconazole-resistant clinical isolates of Candida species from Spain. Eur J Clin Microbiol Infect Dis. 1999;18:432–435. doi: 10.1007/s100960050313. [DOI] [PubMed] [Google Scholar]
- 4.Franz R, Kelly S L, Lamb D C, Kelly D E, Ruhnke M, Morschhauser J. Multiple molecular mechanisms contribute to a stepwise development of fluconazole resistance in clinical Candida albicans strains. Antimicrob Agents Chemother. 1998;42:3065–3072. doi: 10.1128/aac.42.12.3065. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Moody J A. Synergism testing: broth microdilution checkerboard and broth macrodilution methods. In: Isenberg H D, editor. Clinical microbiology procedures handbook. Washington, D.C.: American Society for Microbiology; 1992. pp. 1–28. [Google Scholar]
- 6.Müller F M C, Kasai M, Francesconi A, Brillante B, Roden M, Peter J, Chanock S J, Walsh T J. Transmission of an azole-resistant isogenic strain of Candida albicans among human immunodeficiency virus-infected family members with oropharyngeal candidiasis. J Clin Microbiol. 1999;37:3405–3408. doi: 10.1128/jcm.37.10.3405-3408.1999. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Müller F M C, Weig M, Peter J, Walsh T J. Azole cross-resistance to ketoconazole, fluconazole, itraconazole and voriconazole in clinical Candida albicans isolates from HIV-infected children with oropharyngeal candidiasis. J Antimicrob Chemother. 2000;46:338–340. doi: 10.1093/jac/46.2.338. [DOI] [PubMed] [Google Scholar]
- 8.Munzel U. Nonparametric methods for paired samples. Stat Neerland. 1999;53:277–286. [Google Scholar]
- 9.Nandwani R, Parnell A, Youle M, Lacey C J, Evans E G, Midgley J, Cartledge J, Hawkins D A. Use of terbinafine in HIV-positive subjects: pilot studies in onychomycosis and oral candidiasis. Br J Dermatol. 1996;134(Suppl. 46):22–24. doi: 10.1111/j.1365-2133.1996.tb15655.x. [DOI] [PubMed] [Google Scholar]
- 10.National Committee for Clinical Laboratory Standards. Reference method for broth dilution antifungal susceptibility testing for yeasts: proposed standard. Document M27-A. Wayne, Pa: National Committee for Clinical Laboratory Standards; 2000. [Google Scholar]
- 11.Pfaller M A, Messer S A, Coffmann S. 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. 1995;33:1094–1097. doi: 10.1128/jcm.33.5.1094-1097.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Sanguineti A, Carmichael J K, Campbell K. Fluconazole-resistant Candida albicans after long-term suppressive therapy. Arch Intern Med. 1993;153:1122–1124. [PubMed] [Google Scholar]