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Antimicrobial Agents and Chemotherapy, March 2001, p. 966-968, Vol. 45, No. 3
Department of Bacteriology, University of
Göttingen, Göttingen,1 and
Institute for Hygiene and Microbiology,2
Department of Pediatrics,3 and
Institute for Molecular Biology of
Infection,4 University of Würzburg,
Würzburg, Germany
Received 25 March 2000/Returned for modification 21 October
2000/Accepted 30 November 2000
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
( 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,
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.3.966-968.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Synergism of Voriconazole and Terbinafine against Candida
albicans Isolates from Human Immunodeficiency Virus-Infected
Patients with Oropharyngeal Candidiasis
ABSTRACT
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FIC) 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.
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
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.
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ACKNOWLEDGMENTS |
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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).
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FOOTNOTES |
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* Corresponding author. Mailing address for Michael Weig: Department of Bacteriology, University of Göttingen, Kreuzbergring 57, D-37075 Göttingen, Germany. Phone: 49-551397099. Fax: 49-551395861. E-mail: mweig{at}gwdg.de. Mailing address for F.-M. C. Müller: Department of Pediatrics, University of Würzburg, Josef-Schneider-Straße 2, D-97080 Würzburg, Germany. Phone: 49-931201-5831. Fax: 49-931201-5833. E-mail: fmmueller{at}mail.uni-wuerzburg.de.
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Antimicrobial Agents and Chemotherapy, March 2001, p. 966-968, Vol. 45, No. 3
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.3.966-968.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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