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The rhinitis and granulomatous lymphadenitis was characterised histopathologically by caseous necrosis with proliferation of connective tissue, a neutrophilic and eosinophilic infiltrate, epithelioid cells, multinucleate giant cells, and irregular, moderately septate and ramified hyphae, frequently surrounded by abundant granular and eosinophilic material with radial disposition (Splendore-Hoeppli phenomenon). Lesions from the dorsal and lateral regions of the nose were collected aseptically and transported to the laboratory at room temperature. The samples were placed on to 2 per cent Sabouraud's dextrose agar and incubated at 37°C. Forty-eight hours later, the cultures demonstrated a slight growth of white colonies with submerged root-like elements. Later, it was possible to observe organisms morphologically compatible with P insidiosum following the induction of zoosporo genesis, performed as described by Santurio and others (2003). The identity of the pathogen was confirmed in all four cases through the amplification of DNA by PCR, as described by Rodrigues and others (2006). The extensive ulcerative lesions observed in the sheep resembled the cutaneous lesions described in sheep by Tabosa and others (2004), in horses by Alfaro and Mendoza (1990), in dogs by Thomas and Lewis (1998) and in cattle by Santurio and others (1998). The histopathological findings of granulomatous eosinophilic lesions with many giant cells and Splendore-Hoeppli material involving P insidiosum hyphae are consistent with those observed in previous studies involving sheep from northeastern Brazil (Tabosa and

We describe here the in vitro activities of azithromycin, clarithromycin, minocycline, or tigecycline alone and in combination with amphotericin B, itraconazole, terbinafine, voriconazole, anidulafungin, caspofungin, or micafungin against 30 isolates of the oomycete Pythium insidiosum. The assays were based on the CLSI M38-A2 technique and the checkerboard microdilution method. The main synergisms observed were through the combination of minocycline with amphotericin B (73.33%), itraconazole (70%), and micafungin (70%) and of clarithromycin with micafungin (73.33%). Pythiosis is a life-threatening disease caused by the oomycete Pythium insidiosum, which can cause infections in humans and in animals, such as horses, bovines, cats, dogs, and sheep. Clinically, the disease may manifest in cutaneous, gastrointestinal, vascular, and systemic forms and has been described in tropical and subtropical areas (1). The hyphae of P. insidiosum are morphologically similar to those of certain mucoraceous molds, but P. insidiosum is not a true fungus because it does not synthesize ergosterol, which is the target of most antifungal drugs. Despite this challenge, two cases of pythiosis in humans, one case of ocular pythiosis and one case of pleuropericarditis (2, 3), have been successfully treated using combination antifungal therapy. However, combination antifungal therapy has been ineffective in cases of vascular and disseminated human pythiosis (4).Previous studies have shown that the growth of P. insidiosum is inhibited in vitro by the glycylcycline, macrolide, and tetracycline classes of antibacterial drugs (5, 6). However, studies evaluating the antimicrobial combination of antibacterial and antifungal agents against P. insidiosum have not been performed. In this context, this study evaluated the in vitro combination of the antibacterial drugs azithromycin, clarithromycin, minocycline, or tigecycline with the antifungal drugs amphotericin B, itraconazole, voriconazole, terbinafine, anidulafungin, caspofungin, or micafungin against P. insidiosum.Twenty-eight P. insidiosum isolates obtained from Brazilian cases of equine pythiosis and the reference strains ATCC 58.637 and CBS 101555 were evaluated in this study. The identities of the clinical isolates were confirmed using PCR-based assays (7). The antibacterial drugs azithromycin (Pharma Nostra, Rio de Janeiro, Brazil), clarithromycin (Genix, Anápolis, Brazil), minocycline (Pharma Nostra), and tigecycline (Pfizer, New York, NY) and the antifungal drugs amphotericin B (Sigma-Aldrich, St. Louis, MO), itraconazole (Frangon do Brasil Farmacêutica Ltda., São Paulo, Brazil), voriconazole (Pfizer), terbinafine (Pharma Nostra), anidulafungin (Pfizer), caspofungin (Merck, Darmstadt, Germany), and micafungin (Astellas, Chuo, Japan) were obtained commercially and diluted in dimethyl sulfoxide or distilled water, as recommended, to generate stock solutions. The concentrations of the antimicrobial agents tested were 0.03 to 16 g/ml and 1 to 512 g/ml for the antibacterial and antifungal dr...

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