In vitro activity of drugs against Pythium insidiosum. Federal de Santa Maria (UFSM), Santa Maria, RS, Brasil.

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1 AAC Accepts, published online ahead of print on 16 March 2009 Antimicrob. Agents Chemother. doi: /aac Copyright 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved Title: Running title: In Vitro Activity of Terbinafine Combined with Caspofungin and Azoles Against Pythium insidiosum. In vitro activity of drugs against Pythium insidiosum Authors: Ayrton S. Cavalheiro a,c, Grazieli Maboni c, Maria I. de Azevedo c, Juliana S. Argenta c, Daniela I. B. Pereira c, Tatiana B. Spader b,c, Sydney H. Alves a,b,c, Janio M. Santurio a,c Institution address: Departamento de Microbiologia e Parasitologia, Universidade Federal de Santa Maria (UFSM), Santa Maria, RS, Brasil. Affiliations: a Programa de Pós-Graduação em Farmacologia, Universidade Federal de Santa Maria (UFSM), Santa Maria, RS, Brasil. b Programa de Pós-Graduação em Ciências Farmacêuticas, UFSM, Santa Maria, RS, Brasil. c Laboratório de Pesquisas Micológicas (LAPEMI), Departamento de Microbiologia e Parasitologia, UFSM, Santa Maria, RS, Brasil In this text we evaluated the in vitro antifungal activities of terbinafine combined with caspofungin, miconazole, ketoconazole, and fluconazole against 17 P. insidiosum strains 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 combinations of terbinafine plus caspofungin or terbinafine plus fluconazole may have significant therapeutic potential for treatment of pythiosis Corresponding author: Prof Dr Janio Morais Santurio, Campus UFSM, Prédio 20, sala 4139, Santa Maria RS, Brazil. Phone/Fax: santurio@smail.ufsm.br

2 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 granulomatous (8). In humans, the infection occurs as ophthalmic, subcutaneous, and systemic forms, which are frequently associated with α- 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 combined with caspofungin, miconazole, ketoconazole, and fluconazole 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 and CBS ) were tested. All strains were maintained in corn meal 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, which were diluted in RPMI 1640 containing L- glutamine and buffered to ph 7.0 with 0.165M MOPS to obtain a final concentration range of zoospores/ml (10). 2

3 The combinations of terbinafine (Novartis) + caspofungin (Merck) (TRB-CAS), terbinafine + miconazole (Labware) (TRB-MNZ), terbinafine + ketoconazole (Janssen) (TRB-KTC), and terbinafine + fluconazole (Pfizer) (TRB-FLC) 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 4 concentrate of 50 µl of drug A plus 50 µl of drug B and 100 µl of the inoculum, resulting in a final 1 drug concentration of each compound. The microplates were incubated at 37 C for 24 h. 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 FICI (fractional inhibitory concentration index), were interpreted as the following: FICI 0.5 = synergism, FICI > 0.5 to 4 = indifferent, or FICI > 4 = antagonism. FICIs were obtained using the formula: FICI = (MIC A in combination / MIC A) + (MIC B in combination / MIC B). 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 or no antifungal activity. The combinations of terbinafine plus fluconazole (TRB-FLC) and terbinafine plus caspofungin (TRB-CAS) both exhibited synergistic effects against seven (41.2%) P. insidiosum strains. The combination of terbinafine plus ketoconazole was also synergistic against five (29.4%) isolates, while the combination of terbinafine plus miconazole (TRB-MNZ) exhibited synergistic effects against only two (11.8%) isolates (Table 2). Antagonistic effects were not indicated. 3

4 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, which 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 non-existent. The first report on the potential synergistic effects of terbinafine plus itraconazole was 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 terbinafine 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 terbinafine plus fluconazole and terbinafine plus caspofungin were synergistic against 41.2% of P. insidiosum strains. Additionally, synergistic effects were indicated with combinations of terbinafine plus ketoconazole and terbinafine plus miconazole, albeit to a lower extent (against 29.4% and 11.8% of strains, respectively). To our knowledge, the synergistic effects of these antifungals 4

5 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 experimentally investigated, since 48.5% of the combined MICs were lower than the serum concentrations (C max ) achieved by the respective agents, which indicates the potential therapeutic utility of our results References 1. Argenta, J. S., J. M. Santurio, S. H. Alves, D. I. B. Pereira, A. S. Cavalheiro, A. Spanamberg, and L. Ferreiro In vitro activities of voriconazole, itraconazole, and terbinafine alone or in combination against Pythium insidiosum isolates from Brazil. Antimicrob. Agents Chemother. 52: Clinical and Laboratory Standard Institute / NCCLS Reference method for broth dilution antifungal susceptibility testing of filamentous fungi. Approved standard. CLSI document M38-A2. CLSI, Wayne, Pensylvania. 3. Cuenca-Estrella, M Combinations of antifungal agents in therapy: what value are they? J. Antimicrob. Chemother. 54: Grooters, A.M. and M.K. Gee Development of a nested polymerase chain reaction assay for the detection and identification of Pythium insidiosum. J. Vet. Intern. Med. 16:

6 Imwidthaya, P. 1994b. Human pythiosis in Thailand. Postgrad. Med. J. 70: Johnson, M. D., C. MacDougall, L. Ostrosky-Zeichner, J. R. Perfect, and J. H. Rex Combination antifungal therapy. Antimicrob. Agents Chemother. 48: Krajaejun, T., B. Sathapatayavongs, R. Pracharktam, P. Nitiyanant, P. Leelachaikul, W. Wanachiwanawin, A. Chaiprasert, P. Assanasen, M. Saipetch, P. Mootsikapun, P. Chetchotisakd, A. Lekhakula, W. Mitarnun, S. Kalnauwakul, K. Supparatpinyo, R. Chaiwarith, S. Chiewchanvit, N. Tananuvat, S. Srisiri, C. Suankratay, W. Kulwichit, M. Wongsaisuwan, and S. Somkaew Clinical and epidemiological analyses of human pythiosis in Thailand. Clin. Infect. Dis. 43: Leal, A. B. M., A. T. Leal, J. M. Santurio, G. D. Kommers, and J. B. Catto Pitiose eqüina no Pantanal brasileiro: aspectos clínico-patológicos de casos típicos e atípicos. Pesq. Vet. Bras. 21: Mendoza, L., and L. Ajello Infections caused by the oomycetous pathogen Pythium insidiosum. J. Mycol. Med. 6: Pereira, D. I. B, J. M. Santurio, S. H. Alves, J. S. Argenta, L. Pötter, A. Spanamberg, and L. Ferreiro Caspofungin in vitro and in vivo activity against Brazilian Pythium insidiosum strains isolated from animals. J. Antimicrob. Chemother. 60: Schurko, A. M., L. Mendoza, C. A. Levesque, N. L. Desaulniers, A. W. de Cock, and G. R. Klaussen A molecular phylogeny of Pythium insidiosum. Mycol. Res. 107:

7 Sekhon, A. S., A. A. Padhye, and A. K. Garg In vitro sensitivity of Penicillium marneffei and Pythium insidiosum to various antifungal agents. Eur. J. Epidemiol. 8: Shenep, J. L., B. K. English, L. Kaufman, T. A. Pearson, J. W. Thompson, R. A. Kaufman, G. Frisch, and M. G. Rinaldi Successful medical therapy for deeply invasive facial infection due to Pythium insidiosum in a child. Clin. Infect. Dis. 27: Vitale, R.G., J. Afeltra, E. Dannaoui Antifungal Combinations, p In E.J. Ernest and P.D. Rogers (ed), Methods in Molecular Medicine, vol. 118: Methods and Protocols. Humana Press Inc., Totowa, N.J

8 Table 1. In vitro activity of terbinafine, caspofungin and azoles against isolates of Pythium insidiosum (mg/l). * Drugs MIC range a GM b MIC 50 c MIC 90 d Terbinafine Caspofungin Miconazole Ketoconazole Fluconazole * n=17 a Range between the lowest and highest minimum inhibitory concentration for all isolates. b Geometric Means of MICs. c Minimum inhibitory concentration of drug capable of inhibiting the growth of 50% of isolates. d Minimum inhibitory concentration of drug capable of inhibiting the growth of 90% of isolates

9 Table 2. In vitro activity of terbinafine combined with fluconazole, miconazole, ketoconazole or caspofungin against Pythium insidiosum. * Terbinafine and Fluconazole Terbinafine and Miconazole Terbinafine and Ketoconazole Terbinafine and Caspofungin MIC of combination (mg/liter) MIC of combination (mg/liter) MIC of combination (mg/liter) MIC of combination (mg/liter) Isolate a TRB FLC FICI (interpret.) b TRB MNZ FICI (interpret.) TRB KTC FICI (interpret.) TRB CAS FICI (interpret.) LAPEMI ( S ) ( S ) ( S ) ( S ) LAPEMI ( I ) ( I ) ( I ) ( I ) LAPEMI ( I ) ( I ) ( S ) ( S ) LAPEMI ( I ) ( I ) ( I ) ( I ) LAPEMI ( S ) ( I ) ( I ) ( I ) LAPEMI ( I ) ( I ) ( I ) ( S ) LAPEMI ( I ) ( I ) ( I ) ( I ) LAPEMI ( S ) ( I ) ( S ) ( S ) LAPEMI ( S ) ( S ) ( I ) ( I ) LAPEMI ( I ) ( I ) ( S ) ( S ) LAPEMI ( I ) ( I ) ( I ) ( I ) LAPEMI ( S ) ( I ) ( I ) ( I ) LAPEMI ( I ) ( I ) ( I ) ( I ) LAPEMI ( I ) ( I ) ( I ) ( S ) LAPEMI ( S ) ( I ) ( S ) ( S ) ATCC ( I ) ( I ) ( I ) ( I ) CBS ( S ) ( I ) ( I ) ( I ) * n=17. a LAPEMI, Laboratório de Pesquisas Micológicas; ATCC, American Type Culture Collection; CBS, Centraalbureau voor Schimmelcultures. b Interpretations: S, synergistic; I, indifferent. 2