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Antimicrobial Agents and Chemotherapy, February 2000, p. 470-472, Vol. 44, No. 2
Department of Medical Microbiology,
University Hospital Nijmegen,1 and
Department of Medical Microbiology, Canisius Wilhelmina
Hospital,2 Nijmegen, The Netherlands, and
Unidad de Micología, Centro Nacional de
Microbiología, Instituto de Salud Carlos III, Majadahonda
(Madrid), Spain3
Received 19 July 1999/Returned for modification 25 October
1999/Accepted 10 November 1999
In order to develop new approaches for the chemotherapy of invasive
infections caused by Scedosporium prolificans, the in vitro
interaction between itraconazole and terbinafine against 20 clinical
isolates was studied using a checkerboard microdilution method.
Itraconazole and terbinafine alone were inactive against most isolates,
but the combination was synergistic against 95 and 85% of isolates
after 48 and 72 h of incubation, respectively. Antagonism was not
observed. The MICs obtained with the terbinafine-itraconazole combination were within levels that can be achieved in plasma.
Invasive infections caused by
Scedosporium species are uncommon but are generally fatal in
immunocompromised patients, especially when they are caused by
Scedosporium prolificans (2, 13). Treatment
includes surgical debridement, if possible, and antifungal chemotherapy, although the optimal choice and duration of therapy are
unknown. Azoles, such as miconazole and itraconazole, have been used
with some success for the treatment of invasive infections with
Scedosporium apiospermum (6), but treatment
failures have also been reported (15). S. prolificans is considered multiresistant since low in vitro
activities have been reported for amphotericin B, flucytosine, and the
azoles (3). Even novel antifungal agents such as the
triazoles voriconazole, posaconazole (SCH56592), and Syn-2869 (5,
9) and the echinocandins LY303366 and caspofungin (MK-0991) show
limited or no in vitro activity against this fungus (4, 5).
We have previously reported a patient with pulmonary pseudallescheriosis who failed itraconazole therapy but responded after
treatment was changed to terbinafine (15). The S. apiospermum isolate was resistant to either drug alone but was
susceptible in vitro to the terbinafine-itraconazole combination
(N. S. Ryder and I. Leitner, Abstr. 38th Intersci. Conf.
Antimicrob. Agents Chemother., abstr. J-155, 1998). In the present
study we investigated whether terbinafine and itraconazole act
synergistically against 20 clinical S. prolificans isolates
to determine if this combination is a potentially useful combination in
the treatment of these infections.
All isolates were obtained from clinical specimens (3) and
subcultured on Sabouraud glucose agar (SAB) plates with 0.5% chloramphenicol and incubated at room temperature for 7 days. They were
then subcultured again on SAB plates and incubated for another 5 to 7 days at 37°C, and spores were collected. Paecilomyces variotii (ATCC 22319) was used as a quality control strain, and all isolates were tested in duplicate.
MICs were determined by a broth microdilution method according to
National Committee for Clinical Laboratory Standards guidelines (proposed standard M38-P) (11). Briefly, a suspension of
spores was adjusted with a spectrophotometer (Spectronic 20D; Milton Roy, Rochester, N.Y.) to 68 to 70% transmission at a wavelength of 530 nm and diluted 10-fold to yield a final inoculum of 1 × 104 to 5 × 104 CFU/ml. The
spectrophotometer transmissions were verified by enumeration of
colonies per milliliter of serial dilution on SAB plates that were
incubated at 35°C for 48 h. These cultures showed that the final
inoculum varied between 1.5 × 104 and 3.5 × 104 CFU/ml. Terbinafine (Novartis, Basel, Switzerland) and
itraconazole (Janssen Research Foundation, Beerse, Belgium) were tested
in RPMI 1640 medium with L-glutamine and without
bicarbonate (GIBCO BRL, Life Technologies, Breda, The Netherlands),
buffered to pH 7.0 with 0.165 M MOPS (morpholinepropanesulfonic acid).
The final concentrations ranged from 0.5 to 32 µg/ml for itraconazole
and 0.06 to 64 µg/ml for terbinafine. Growth was graded on a scale of
0 to 4 as follows: 4 indicated no reduction in growth, 3 indicated a
25% reduction of growth, 2 indicated a 50% reduction of growth, 1 indicated a 75% reduction of growth, and 0 indicated an optically clear well. The MIC was defined as the lowest concentration of antifungal compound that inhibited growth by 50% or more.
A two-dimensional, two-agent broth microdilution checkerboard technique
was used to study the interactions between itraconazole and
terbinafine. Serial twofold dilutions of itraconazole and terbinafine,
alone and in combination, were tested against final inocula of 1 × 104 to 5 × 104 CFU/ml. In order to
obtain an exact percentage of growth for each well, the dye
3-(4,5-dimethyl-2-thiazyl)-2,5 diphenyl-2H-tetrazolium bromide (MTT;
Sigma Chemical, St. Louis, MO.) was added together with the inoculum to
each well at a final concentration of 0.1 mg/ml (8). After
48 or 72 h of incubation, the content of each well was removed and
200 µl of isopropanol containing 5% HCl (1 M) was added. After 30 min of incubation at room temperature and gentle agitation, the optical
density (OD) was measured with a spectrophotometer (MS2 reader,
Titertekplus; ICN Biomedical Ltd., Basingstoke, United Kingdom) at 550 nm (8). The OD of the blank, to which a conidium-free
inoculum had been added, was subtracted from the OD values. The
percentage of growth for each well was calculated by comparing the OD
of a well with that of the drug-free control. For each
itraconazole-terbinafine combination the summation of the fractional
inhibitory concentration ( The MICs of terbinafine and itraconazole based on 50% reduction of
growth for P. variotii were 0.125 and 0.25 µg/ml,
respectively. Itraconazole was inactive in vitro against most isolates,
with the MIC at which 90% of the isolates were inhibited being >32 µg/ml after both 48 and 72 h of incubation (Table
1). An attempt was made to establish the
exact MIC of itraconazole by an agar dilution method. Serial dilutions
ranging from 32 to 512 µg of itraconazole per ml were made in RPMI
1640 agar. The growth of none of the S. prolificans isolates
was inhibited by any of these concentrations after 48 h of
incubation. Therefore, a MIC of 64 µg/ml was chosen for calculations
for those isolates that grew in the wells that contained the highest
concentration of itraconazole. The MIC of terbinafine at which 90% of
the isolates were inhibited was 2 µg/ml after 48 h but increased
to 64 µg/ml after 72 h. Synergism was found for 19 of 20 (95%)
of the S. prolificans isolates after 48 h and for 17 of
20 (85%) of the isolates after 72 h of incubation (Table 1). For
three isolates the effect of the combination appeared to be indifferent
after 72 h of incubation, and antagonism was not observed.
Although drug interactions in vitro are difficult to assess, we believe
that the observed synergism is significant for several reasons. (i)
Despite the fact that we selected a stringent criterion for the
definition of synergism, almost all isolates showed synergism after
both 48 and 72 h of incubation. (ii) Because the MIC of
itraconazole was set at 64 µg/ml for most isolates, the calculated
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Copyright © 2000, American Society for Microbiology. All rights reserved.
In Vitro Interaction of Terbinafine with
Itraconazole against Clinical Isolates of Scedosporium
prolificans
ABSTRACT
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FIC) was calculated as follows: (MIC of
itraconazole plus terbinafine/MIC of itraconazole) + (MIC of
itraconazole plus terbinafine/MIC of terbinafine). The interpretation
of the FIC was as follows: the synergistic effect was 
0.5, the
indifferent effect was >0.5 but 4, and the antagonistic effect was
>4 (7). The results of experiments with the two agents
alone and in combination were analyzed separately as well as together
by calculating the mean ODs. Since all analyses yielded identical
results, we present the results based on the mean OD from both experiments.
FIC underestimates the actual level of synergism. Calculations with
higher MICs of single drugs would have resulted in even lower FIC.
(iii) Itraconazole and terbinafine block different steps of the same
pathway of fungal ergosterol biosynthesis, which supports the
possibility of synergistic action. The classic example of proven
synergism in this respect is the combination of trimethoprim with
sulfonamides, which also interacts with consecutive steps of a common
pathway. Furthermore, the combination of terbinafine with azoles has
been shown to be synergistic in vitro for other fungi, including
Candida albicans (1), Candida
glabrata, and Cryptococcus neoformans (A. W. Fothergill, I. Leitner, J. G. Meingassner, N. S. Ryder, and
M. G. Rinaldi, Abstr. 36th Intersci. Conf. Antimicrob. Agents
Chemother., abstr. E53, 1996), and for Pythium insidiosum (14). Synergism has been reported for combinations of
amphotericin B with azoles against S. apiospermum but the
synergistic activity was less pronounced than for the
terbinafine-itraconazole combination and occurred only for a limited
number of isolates (16). In another study, the effect of
amphotericin B combined with terbinafine was indifferent against
S. apiospermum, but terbinafine with fluconazole was
synergistic against the same isolate (Ryder and Leitner, 38th ICAAC,
abstr. J-155). Terbinafine appears to interact synergistically with the
class of azole antifungal drugs.
TABLE 1.
MICs and FICs of itraconazole and terbinafine alone
and in combination against S. prolificans after 48 and
72 h of incubation
The MICs of the terbinafine-itraconazole combination are within the range that can be achieved in blood. The achievable maximum concentrations of terbinafine are approximately 1.7 µg/ml within 2 h of oral administration of a dose of 500 mg (10). Levels in serum above 3.0 µg/ml can be achieved with itraconazole (12), and even higher levels may be achieved with the oral solution or the intravenous formulation that is now undergoing clinical evaluation.
Invasive infections caused by S. prolificans are generally fatal, and at present there is no antifungal regimen that has been shown to be effective. The in vitro synergism of itraconazole and terbinafine that we demonstrated may prove effective for the treatment of these infections. Animal-model and clinical studies are warranted to further elucidate the potential of terbinafine-itraconazole combination therapy in difficult-to-treat infections by filamentous fungi.
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ACKNOWLEDGMENTS |
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This work was supported by a grant from Novartis and the EC-TMR-EUROFUNG network (ERBFMXR-CT970145).
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Medical Microbiology, University Hospital Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. Phone: 31-24-3614356. Fax: 31-24-3540216. E-mail: p.verweij{at}mmb.azn.nl.
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