In vitro susceptibilities of clinical yeast isolates to the new antifungal eberconazole compared with their susceptibilities to clotrimazole and ketoconazole

Article abstract of DOI:10.1128/aac.43.5.1258

The antifungal activity of eberconazole, a new imidazole derivative, against 124 clinical isolates of Candida comprising eight different species and to 34 isolates of Cryptococcus neoformans was compared to those od clotrimazole and ketoconazole. MICs of eberconazole, determined by the National Committee for Clinical Laboratory Standards standardized microbroth method, were equal to or lower than those of other azoles, especially for Candida krusei and Candida glabrata, which are usually resistant to triazoles.

Full text of DOI:10.1128/aac.43.5.1258

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, May 1999, p. 1258–1259
0066-4804/99/$04.00ϩ0
Copyright © 1999, American Society for Microbiology. All Rights Reserved.
Vol. 43, No. 5
In Vitro Susceptibilities of Clinical Yeast Isolates to the New
Antifungal Eberconazole Compared with Their Susceptibilities
to Clotrimazole and Ketoconazole
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JOSEP M. TORRES-RODRIGUEZ, * RAUL MENDEZ, OLGA LOPEZ-JODRA, YOLANDA MORERA,
MATEU ESPASA,1 TERESA JIMENEZ,1 AND CARME LAGUNAS2
Experimental and Clinical Mycology Research Group (GREMEC), IMIM, Autonomous University of Barcelona,1
and Department of Microbiology, Laboratories Salvat,2 Barcelona, Spain
Received 17 September 1998/Returned for modification 20 October 1998/Accepted 1 March 1999
The antifungal activity of eberconazole, a new imidazole derivative, against 124 clinical isolates of Candida
comprising eight different species and to 34 isolates of Cryptococcus neoformans was compared to those of clo-
trimazole and ketoconazole. MICs of eberconazole, determined by the National Committee for Clinical Lab-
oratory Standards standardized microbroth method, were equal to or lower than those of other azoles,
especially for Candida krusei and Candida glabrata, which are usually resistant to triazoles.
Eberconazole (1-(2,4-dichloro-10,11-dihydro-5H-dibenzo-
[a,d]cyclohepten-5-yl)-1H-imidazole) is a new antifungal imid-
azole derivative developed by Salvat Laboratories (Barcelona,
Spain). In preliminary studies, eberconazole showed an excel-
lent antifungal in vitro activity (3) against yeasts of the genus
Candida and against dermatophytes and therefore has been
developed as a topical antimycotic for the treatment of superfi-
cial fungal infections. It is currently in phase III clinical trials (1).
The first studies to determine its MICs were carried out
between 1988 and 1989 using a nonstandardized micromethod
with Sabouraud broth medium. In these studies, it was found
that MICs for 116 yeast isolates were between 0.6 and 5.0 ␮g/
ml, similar to or lower than that of clotrimazole, which was
used as the reference (11).
The aim of the present study was to verify these earlier
results and to compare the activity of eberconazole with those
of clotrimazole and ketoconazole in clinical isolates of yeasts,
using the micromethod standardized by the National Commit-
tee for Clinical Laboratory Standards (NCCLS).
A total of 124 isolates of Candida species were selected for
the study: C. albicans (47 isolates, including 12 with MICs of
fluconazole of Ͼ64 ␮g/ml), C. tropicalis (17 isolates), C. para-
psilosis (14 isolates), C. krusei (13 isolates), C. glabrata (17 iso-
lates), C. guilliermondii (7 isolates), C. famata (5 isolates), and
C. dubliniensis (4 isolates). Thirty-four isolates of Cryptococcus
neoformans were also included. All the strains were isolated
from clinical samples, mainly from AIDS patients with oropha-
ryngeal candidosis, and on a smaller scale from patients with
candiduria, vaginal candidosis, and onyxis. Cryptococcus neofor-
mans was isolated from AIDS patients with meningoencepha-
litis caused by this fungus.
Roig-Farma (Barcelona, Spain) and clotrimazole was from
Sigma (St. Louis, Mo.).
MICs were determined by the NCCLS microbroth dilution
method M27-A (5) with the following modification: RPMI 1640
medium supplemented with glucose (18 g/liter) was used (10).
Antifungal agents were distributed in the wells of microtiter
plates, giving final drug concentrations of between 0.03 and 16
␮g/ml.
Microdilution plates were incubated in a moist chamber at
35°C. The incubation times were 48 h for Candida species and
72 h for Cryptococcus neoformans isolates. Readings were made
both visually and spectrophotometrically at 414 nm with an
automatic reader (Multiscan MS; Labsystems, Barcelona, Spain).
The MIC endpoints were defined as the lowest drug concen-
trations in the wells which resulted in an 80% reduction of
fungal growth compared to the drug-free control (2).
The MIC range, the geometric mean (GM) MIC, and the
MICs at which 90% and 50% of isolates are inhibited were
determined (Table 1). Variance analysis and the Wilcoxon test
were applied; a P value of Ͻ0.05 was considered to be statis-
tically significant. The data were analyzed with SPSS software.
MICs of ketoconazole for the reference ATCC strains were
within the acceptable quality control range described by the
NCCLS (5). MICs ranged from 0.03 to 2 ␮g/ml for ebercon-
azole and from 0.03 to 4 ␮g/ml for ketoconazole and clotrim-
azole.
Only MICs for C. glabrata and Cryptococcus neoformans
presented normally distributed values. Statistically significant
differences were noted for C. tropicalis and C. krusei when all
three antifungal agents were compared together. MICs of eber-
conazole for C. krusei and C. glabrata were lower than those of
the other two azoles, and the differences between eberconazole
and ketoconazole were statistically significant. For C. tropicalis,
there was a significant difference between the MICs of keto-
conazole and those of the other two agents. MICs for Crypto-
coccus neoformans were lower for ketoconazole (GM MIC,
0.035 ␮g/ml) than for clotrimazole (GM MIC, 0.042 ␮g/ml)
and eberconazole (GM MIC, 0.162 ␮g/ml).
Isolates were stored at Ϫ40°C in skim milk, when required,
and they were subcultured on Sabouraud agar with chlor-
amphenicol plus gentamicin. Eight reference strains from
the American Type Culture Collection (ATCC) were included
as quality controls (5).
Eberconazole was provided by Salvat S.A. Laboratories (Bar-
celona, Spain) as standard powder. Ketoconazole was from
In a study performed before reference methods were pub-
lished, we used a microdilution method with Sabouraud broth
to compare the activity of eberconazole (WAS 2160) against
yeasts and dermatophytes to those of clotrimazole and bifon-
azole (11). Bifonazole presented the lowest activity against
* Corresponding author. Mailing address: Experimental and Clinical
Mycology Research Group, IMIM, Autonomous University of Barce-
lona, Avda. Dr. Aiguader 80, Barcelona 08003, Spain. Phone: 34-93-
221-1009. Fax: 34-93-221-3237. E-mail: jmtorres@imim.es.
1258

VOL. 43, 1999
NOTES
1259
TABLE 1. GM MICs, MIC ranges, MIC50s, and MIC90sa of eberconazole, ketoconazole, and clotrimazole for 124 isolates of Candida species
Result (␮g/ml)
Isolate (n)
Eberconazole
Range MIC50 MIC90 GM MIC
0.03–1
Clotrimazole
Range MIC50 MIC90 GM MIC
0.03–2
Ketoconazole
GM MIC
Range
MIC50 MIC90
Candida albicans (35)
Candida albicans (12)b
Candida tropicalis (17)
Candida parapsilosis (14)
Candida krusei (13)
0.05
0.03
0.27
0.05
0.04
0.1
0.08
0.046
0.03
0.03
0.03
0.27
0.03
0.03
0.06
0.03
0.03
0.03
0.5
0.08
0.03
0.16
0.03
0.08
0.4
0.08
0.03
0.125
0.03
0.03
0.5
0.03
0.03
1.0
0.04
0.03
0.06
0.043
0.42
0.4
0.03–1
0.03
0.03
0.03
0.03
0.03
0.5
0.03
0.03
0.25
0.125
1.0
0.03
0.03–2
0.03
2.0
0.125
0.125
0.05
0.5
0.03
0.03–2
0.03–1
0.03–0.25
0.03–2
0.03–4
0.03–0.06
0.03
0.03–1
0.03–0.06
0.03–0.5
0.5–4.0
0.03
0.5
0.03–0.25
0.03–2
0.03–0.5
0.03–0.125
0.03
Candida glabrata (17)
Candida guilliermondii (7)
Candida famata (5)
2.0
1.0
2.0
0.07
0.061
0.03
0.03–0.25
0.03
0.25
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.03
0.06
0.03
0.03–0.125 0.03
Candida dubliniensis (4)
0.03
0.03
0.03
All isolates (124)
0.064
0.079
0.068
a MIC50 and MIC90, the MICs at which 50% and 90% of the isolates, respectively, are inhibited.
b Isolates with MICs of fluconazole of Ͼ64 ␮g/ml.
Candida sp., and that of eberconazole was slightly greater than
that of clotrimazole. This difference was especially evident with
C. glabrata, which, as in this study, showed a high level of sus-
ceptibility to eberconazole. This antifungal agent showed great
activity against C. glabrata and C. krusei, even higher than keto-
conazole, which is especially relevant given that these two spe-
cies have shown a high frequency of intrinsic resistance to flu-
conazole (8) and, on a smaller scale, to itraconazole (6, 9).
The in vitro activity of every new antifungal agent gives us an
approximate idea of its therapeutic indications, which will be
verified later in animal models and clinical trials. The MIC
distributions of the studied yeasts showed that eberconazole’s
activity is comparable to that of ketoconazole but higher than
that of clotrimazole against C. albicans, which still is the most
common pathogenic clinical yeast isolate (7). Eberconazole also
demonstrated a marked activity against C. parapsilosis, one of
the most important species in skin and nail disorders (4).
In spite of the primary topical indication of eberconazole, it
was considered relevant to include isolates of Cryptococcus
neoformans because there is no information in the literature
about the susceptibility of this species to the dichloroimidaz-
oles and because other derivatives of them could be developed
for systemic administration. This yeast was less sensitive to eber-
conazole than to ketoconazole and clotrimazole, but low MICs
(GM MIC, 0.162 ␮g/ml) were observed.
such as C. krusei and C. glabrata and also fluconazole-resistant
C. albicans, are sensitive to eberconazole.
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