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Antimicrobial Agents and Chemotherapy, May 2009, p. 2136-2138, Vol. 53, No. 5
0066-4804/09/$08.00+0     doi:10.1128/AAC.01506-08
Copyright © 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. Santurio^1,2*

Programa de Pós-Graduação em Farmacologia, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil,¹ Programa de Pós-Graduação em Ciências Farmacêuticas, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil,³ Laboratório de Pesquisas Micológicas (LAPEMI), Departamento de Microbiologia e Parasitologia, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil²

Received 11 November 2008/ Returned for modification 18 December 2008/ Accepted 4 March 2009

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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.

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Pythiosis is a life-threatening infectious disease in humans and animals that is caused by the aquatic oomycete Pythium insidiosum (9). Horses are the most frequently infected animals, and equine pythiosis typically involves ulcerative granulomas (8). In humans, the infection occurs as ophthalmic, subcutaneous, and systemic forms, which are frequently associated with - and β-thalassemia (5, 7). Pythiosis therapy, which is based on amphotericin B or azoles, has been ineffective or controversial, and the associated prognosis for human and equine pythiosis is poor (5, 7, 8, 9, 12). Therefore, surgical procedures, including amputation, are often effective, but disease reoccurrence rates are unfortunately high (7).

Combinations of antifungal agents against pythiosis have not been thoroughly studied, and therefore, such in vitro combinatory activities against P. insidiosum require attention (1, 6).

The purpose of this study was to investigate the in vitro activity of terbinafine (TRB) combined with caspofungin (CAS), miconazole (MNZ), ketoconazole, and fluconazole (FLC) against 17 strains of Pythium insidiosum isolated from animals.

A total of 15 Brazilian P. insidiosum strains isolated from equines with pythiosis and two standard strains (ATCC 58637 and CBS 101555) were tested. All strains were maintained in cornmeal agar, and strain identification was confirmed by a PCR-based assay (4).

The susceptibility of the P. insidiosum strains to the antifungal agents was tested by microdilution, based on protocol M38-A2 (2). The inoculum consisted of P. insidiosum zoospores 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) to obtain a final concentration range of 2 x 10³ to 3 x 10³ zoospores/ml (10).

The combinations of TRB (Novartis) plus CAS (Merck), TRB plus MNZ (Labware), TRB plus ketoconazole (Janssen), and TRB plus FLC (Pfizer) were evaluated using the checkerboard technique, according to the broth microdilution design (2, 14). In the individual tests, 100 µl of each drug concentration was plated in microplate wells and an equal volume of the inoculum was added to each well. In the combination tests, the antifungals were plated at a 4x concentrate of 50 µl of drug A plus 50 µl of drug B and 100 µl of the inoculum, resulting in a final 1x drug concentration of each compound. The microplates were incubated at 37°C for 24 h. The MIC was defined as the lowest drug concentration at which there was 100% inhibition of fungal growth by visual readings. The tests were performed in duplicate, and the assay was repeated when disparate values were obtained. The interactions, based on the respective fractional inhibitory concentration index (FICI), were interpreted as the following: FICI 0.5, synergism; FICI > 0.5 to 4, indifference; FICI > 4, antagonism. FICIs were obtained using the formula FICI = (MIC of drug A in combination/MIC of drug A alone) + (MIC of drug B in combination/MIC of drug B alone).

The in vitro activities of individual antifungal agents against P. insidiosum are shown in Table 1. In general, the patterns of susceptibility demonstrated that individual drugs had only weak antifungal activity or none.

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TABLE 1. In vitro activities of TRB, CAS, and azoles against isolates of Pythium insidiosum

The combinations of TRB plus FLC and TRB plus CAS both exhibited synergistic effects against seven (41.2%) P. insidiosum strains. The combination of TRB plus ketoconazole was also synergistic against five (29.4%) isolates, while the combination of TRB plus MNZ exhibited synergistic effects against only two (11.8%) isolates (Table 2). Antagonistic effects were not indicated.

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TABLE 2. In vitro activity of TRB combined with FLC, MNZ, ketoconazole, or CAS against Pythium insidiosumc

The use of combination therapy in the treatment of pythiosis could be an alternative to monotherapy (3), but such application would require further investigation. Herein, we examined the in vitro activities of selected antifungal agents singularly or in combination against P. insidiosum.

Our results are difficult to interpret, since only a few previous studies investigating the susceptibility of P. insidiosum have been reported, and those studies were performed using different experimental techniques (1, 13). In addition, the breakpoints for susceptibility tests with antifungal agents against P. insidiosum are not defined (2). Therefore, these results suggest relatively weak antifungal activity of the individual agents, which is in accordance with the well-known therapeutic failures in pythiosis treatment. In contrast, the results obtained utilizing drug combinations, which are based on FICIs, can be interpreted with more confidence for activity.

Studies focusing on the use of combination therapy against P. insidiosum are almost nonexistent. The first report on the potential synergistic effects of TRB plus itraconazole was that by Shenep et al. (13), which involved the successful treatment of deeply invasive facial infections from P. insidiosum infection in a child with this combination therapy.

Argenta et al. (1) reported that the combination of TRB and either itraconazole or voriconazole was synergistic against 17% of the strains tested and no antagonistic effects were observed. In this study, we demonstrated significant synergistic effects, as the combinations of TRB plus FLC and TRB plus CAS were synergistic against 41.2% of P. insidiosum strains. Additionally, synergistic effects were indicated with combinations of TRB plus ketoconazole and TRB plus MNZ, albeit to a lower extent (against 29.4% and 11.8% of strains, respectively). To our knowledge, the synergistic effects of these antifungals against P. insidiosum are being reported for the first time in this study, and antagonistic effects of these combination antifungal treatments were not observed.

However, a concern for the use of combination antifungal therapy in treating P. insidiosum infection is the great variation in susceptibility among the different strains, which may be related to the genetic variability of the strains tested (11). However, our in vitro results demonstrate that combination antifungal therapy may be an alternative in the treatment of P. insidiosum. In vivo studies must be further investigated experimentally, since 48.5% of the combined MICs were lower than the serum concentrations achieved by the respective agents, which indicates the potential therapeutic utility of our results.

 
This study was supported by CNPq (the National Council for Scientific and Technological Development of Brazil) and by Laboratório de Pesquisas Micológicas, Universidade Federal de Santa Maria, Rio Grande do Sul, Brazil.

 
* Corresponding author. Mailing address: Campus UFSM, Prédio 20, sala 4139, 97105-900 Santa Maria, RS, Brazil. Phone and fax: 55 55 32208906. E-mail: santurio{at}smail.ufsm.br

Published ahead of print on 16 March 2009.

Top ABSTRACT INTRODUCTION REFERENCES

 

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Antimicrobial Agents and Chemotherapy, May 2009, p. 2136-2138, Vol. 53, No. 5
0066-4804/09/$08.00+0     doi:10.1128/AAC.01506-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Cavalheiro, A. S. Articles by Santurio, J. M. Search for Related Content PubMed PubMed Citation Articles by Cavalheiro, A. S. Articles by Santurio, J. M.