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ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, May 2009, p. 2136–2138
0066-4804/09/$08.00ϩ0 doi:10.1128/AAC.01506-08Copyright 2009, American Society for Microbiology. All Rights Reserved.
In Vitro Activity of Terbinafine Combined with Caspofungin and
Azoles against Pythium insidiosum
Ayrton S. Cavalheiro,1,2 Grazieli Maboni,2 Maria I. de Azevedo,2
Juliana S. Argenta,2 Daniela I. B. Pereira,2 Tatiana B. Spader,2,3
Sydney H. Alves,1,2,3 and Janio M. Santurio1,2*
˜o em Farmacologia, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil
1; Programa de
˜o em Cieˆncias Farmaceˆuticas, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil
´gicas (LAPEMI), Departamento de Microbiologia e Parasitologia,
Universidade Federal de Santa Maria, Santa Maria, RS, Brazil
Received 11 November 2008/Returned for modification 18 December 2008/Accepted 4 March 2009
In this text we evaluated the in vitro antifungal activities of terbinafine combined with caspofungin,
miconazole, ketoconazole, and fluconazole against 17 Pythium insidiosum strains by using the microdilution
checkerboard method. Synergistic interactions were observed with terbinafine combined with caspofungin
(41.2% of the strains), fluconazole (41.2%), ketoconazole (29.4%), and miconazole (11.8%). No antagonistic
effects were observed. The combination of terbinafine plus caspofungin or terbinafine plus fluconazole may
have significant therapeutic potential for treatment of pythiosis.
Pythiosis is a life-threatening infectious disease in humans
to obtain a final concentration range of 2 ϫ 103 to 3 ϫ 103
and animals that is caused by the aquatic oomycete Pythium
(9). Horses are the most frequently infected ani-
The combinations of TRB (Novartis) plus CAS (Merck),
mals, and equine pythiosis typically involves ulcerative granu-
TRB plus MNZ (Labware), TRB plus ketoconazole (Janssen),
lomas (8). In humans, the infection occurs as ophthalmic, sub-
and TRB plus FLC (Pfizer) were evaluated using the checker-
cutaneous, and systemic forms, which are frequently associated
board technique, according to the broth microdilution design
with ␣- and ␤-thalassemia (5, 7). Pythiosis therapy, which is
(2, 14). In the individual tests, 100 l of each drug concentra-
based on amphotericin B or azoles, has been ineffective or
tion was plated in microplate wells and an equal volume of the
controversial, and the associated prognosis for human and
inoculum was added to each well. In the combination tests, the
equine pythiosis is poor (5, 7, 8, 9, 12). Therefore, surgical
antifungals were plated at a 4ϫ concentrate of 50 l of drug A
procedures, including amputation, are often effective, but dis-
plus 50 l of drug B and 100 l of the inoculum, resulting in a
ease reoccurrence rates are unfortunately high (7).
final 1ϫ drug concentration of each compound. The micro-
Combinations of antifungal agents against pythiosis have not
plates were incubated at 37°C for 24 h. The MIC was defined
been thoroughly studied, and therefore, such in vitro combi-
as the lowest drug concentration at which there was 100%
natory activities against P
require attention (1, 6).
inhibition of fungal growth by visual readings. The tests were
The purpose of this study was to investigate the in vitro
performed in duplicate, and the assay was repeated when dis-
activity of terbinafine (TRB) combined with caspofungin
parate values were obtained. The interactions, based on the
(CAS), miconazole (MNZ), ketoconazole, and fluconazole
respective fractional inhibitory concentration index (FICI),
(FLC) against 17 strains of Pythium insidiosum
were interpreted as the following: FICI Յ 0.5, synergism; FICI Ͼ
0.5 to Յ4, indifference; FICI Ͼ 4, antagonism. FICIs were ob-
A total of 15 Brazilian P. insidiosum
strains isolated from
tained using the formula FICI ϭ (MIC of drug A in combination/
equines with pythiosis and two standard strains (ATCC 58637
MIC of drug A alone) ϩ (MIC of drug B in combination/MIC of
and CBS 101555) were tested. All strains were maintained in
cornmeal agar, and strain identification was confirmed by aPCR-based assay (4).
The susceptibility of the P. insidiosum
strains to the antifun-
TABLE 1. In vitro activities of TRB, CAS, and azoles against
gal agents was tested by microdilution, based on protocol
M38-A2 (2). The inoculum consisted of P. insidiosum
spores obtained following zoosporogenesis. Cell numbers of
zoospores were counted on a hemocytometer; zoospores were
diluted in RPMI 1640 containing L-glutamine and buffered to
pH 7.0 with 0.165 M MOPS (morpholinepropanesulfonic acid)
* Corresponding author. Mailing address: Campus UFSM, Pre
20, sala 4139, 97105-900 Santa Maria, RS, Brazil. Phone and fax: 55 55
Range between the lowest and highest MICs for all isolates.
32208906. E-mail: firstname.lastname@example.org.
ᰔ Published ahead of print on 16 March 2009.
MIC of drug capable of inhibiting the growth of 90% of isolates.
IN VITRO ACTIVITY OF DRUGS AGAINST PYTHIUM INSIDIOSUM
TABLE 2. In vitro activity of TRB combined with FLC, MNZ, ketoconazole, or CAS against Pythium insidiosumc
LAPEMI, Laborato´rio de Pesquisas Micolo´gicas; ATCC, American Type Culture Collection; CBS, Centraalbureau voor Schimmelcultures.b
interpret., interpretation; S, synergistic; I, indifferent.c n
The in vitro activities of individual antifungal agents against
either itraconazole or voriconazole was synergistic against 17%
are shown in Table 1. In general, the patterns of
of the strains tested and no antagonistic effects were observed.
susceptibility demonstrated that individual drugs had only
In this study, we demonstrated significant synergistic effects, as
the combinations of TRB plus FLC and TRB plus CAS were
The combinations of TRB plus FLC and TRB plus CAS
synergistic against 41.2% of P. insidiosum
both exhibited synergistic effects against seven (41.2%) P. in-
synergistic effects were indicated with combinations of TRB
strains. The combination of TRB plus ketoconazole
plus ketoconazole and TRB plus MNZ, albeit to a lower extent
was also synergistic against five (29.4%) isolates, while the
(against 29.4% and 11.8% of strains, respectively). To our
combination of TRB plus MNZ exhibited synergistic effects
knowledge, the synergistic effects of these antifungals against
against only two (11.8%) isolates (Table 2). Antagonistic ef-
are being reported for the first time in this study,
and antagonistic effects of these combination antifungal treat-
The use of combination therapy in the treatment of pythiosis
could be an alternative to monotherapy (3), but such applica-
However, a concern for the use of combination antifungal
tion would require further investigation. Herein, we examined
therapy in treating P. insidiosum
infection is the great variation
the in vitro activities of selected antifungal agents singularly or
in susceptibility among the different strains, which may be
in combination against P. insidiosum
related to the genetic variability of the strains tested (11).
Our results are difficult to interpret, since only a few previ-
However, our in vitro results demonstrate that combination
ous studies investigating the susceptibility of P. insidiosum
antifungal therapy may be an alternative in the treatment of P.
been reported, and those studies were performed using differ-
. In vivo studies must be further investigated exper-
ent experimental techniques (1, 13). In addition, the break-
imentally, since 48.5% of the combined MICs were lower than
points for susceptibility tests with antifungal agents against P.
the serum concentrations achieved by the respective agents,
are not defined (2). Therefore, these results suggest
which indicates the potential therapeutic utility of our results.
relatively weak antifungal activity of the individual agents,which is in accordance with the well-known therapeutic failures
This study was supported by CNPq (the National Council for Sci-
in pythiosis treatment. In contrast, the results obtained utiliz-
entific and Technological Development of Brazil) and by Laborato
ing drug combinations, which are based on FICIs, can be in-
´gicas, Universidade Federal de Santa Maria, Rio
terpreted with more confidence for activity.
Studies focusing on the use of combination therapy against
are almost nonexistent. The first report on the
1. Argenta, J. S., J. M. Santurio, S. H. Alves, D. I. B. Pereira, A. S. Cavalheiro,
potential synergistic effects of TRB plus itraconazole was that
A. Spanamberg, and L. Ferreiro.
2008. In vitro activities of voriconazole,
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2. Clinical and Laboratory Standards Institute.
2008. Reference method for
broth dilution antifungal susceptibility testing of filamentous fungi. Ap-
in a child with this combination therapy.
proved standard. CLSI document M38-A2. Clinical and Laboratory Stan-
Argenta et al. (1) reported that the combination of TRB and
3. Cuenca-Estrella, M.
2004. Combinations of antifungal agents in therapy:
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1996. Infections caused by the oomycetous
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