The results showed that caspofungin has limited fungistatic activity against P. insidiosum. This work is the first study to analyse the susceptibility of this oomycete to inhibitors of beta-glucans of the cellular wall.

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 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 ␤-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 ϫ 10 3 to 3 ϫ 10 3 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 l 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 4ϫ concentrate of 50 l of drug A plus 50 l of drug B and 100 l of the inoculum, resulting in a final 1ϫ drug concentration of each compound....

We evaluated the in vitro activities of voriconazole, itraconazole, and terbinafine against 30 clinical isolates of Pythium insidiosum using a checkerboard macrodilution method. The combined activity of terbinafine plus itraconazole or plus voriconazole was synergic against 17% of the strains. Antagonism was not observed.Pythium insidiosum is classified in the kingdom Stramenopila, class Oomycetes (3). It causes pythiosis, a disease mainly diagnosed in horses, dogs, and humans (14). Human pythiosis was first documented in 1985 (3). Since then, several cases have been reported, with high rates of morbidity and mortality (12). It is found mostly in Thailand, and two factors contribute to importance of pythiosis in that country: the prevalence of ␤-thalassemia and the presence of large flooded areas used for agriculture (18). Combinations of antifungal agents have been poorly studied in medical mycology, and their activities against P. insidiosum are almost unknown (17).The aim of the present study was to investigate the in vitro activity of terbinafine (TRB) combined with itraconazole (ITC) and of TRB combined with voriconazole (VRC) against 30 isolates of Pythium insidiosum from animal pythiosis by using a macrodilution methodology based on the M38-A technique (10).This study included 28 Brazilian P. insidiosum strains obtained from animal pythiosis (horses, dogs, and sheep) and two standard strains (ATCC 58637 and CBS 101555). The identities of the isolates were confirmed by a PCR-based assay (13). The inocula consisted of P. insidiosum zoospores obtained as previously described (11). These were counted in a hemacytometer and diluted in RPMI 1640 broth containing L-glutamine and buffered to pH 7.0 with 0.165 M morpholinepropanesulfonic acid, yielding a final concentration of 2 ϫ 10 3 to 3 ϫ 10 3 zoospores/ml. Candida parapsilosis (ATCC 22019) and Aspergillus flavus (ATCC 204304) were used as quality control organisms (10).The antifungal agents tested were TRB (Novartis) at 1 to 64 mg/liter, ITC (Sigma Pharma) at 0.125 to 32 mg/liter, and VRC (Pfizer) at 0.125 to 32 mg/liter. The interactions of the combinations (TRB-ITC and TRB-VRC) were evaluated by using the checkerboard technique according to the broth macrodilution design (2). The range of drug concentrations for use in the checkerboard assay was the same used in individual tests. Aliquots (50 l) containing different concentrations of each antifungal agent (seven of TRB and nine of triazole agents) were placed in tubes to provide 63 drug combinations; 0.9 ml of inoculum was added to each tube. The interactions were interpreted as synergistic (fractional inhibitory concentration index [FICI] Յ 0.5), indifferent (0.5 Ͻ FICI Յ 4), or antagonistic (FICI Ͼ 4) based on the respective FICI (5), using the following formula: FICI ϭ (MIC A in combination/MIC A) ϩ (MIC B in combination/MIC B). Off-scale MICs were converted to the next higher dilution for calculation purposes.MIC-1 and MIC-0 were used as the reading criteria for TRB and were determined as the lowest dru...

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Pythium insidiosum is a fungus-like organism present in subtropical and tropical areas, such as Brazil, known to infect humans and various animal species. P. insidiosum is the etiological agent of pythiosis, an emerging and granulomatous disease characterized mainly by cutaneous and subcutaneous lesions in horses, the principal species affected. Accurate diagnosis of pythiosis and identification of its causal agent by microbiological and serological tests can be often difficult and inconclusive principally for horses and humans. The aim of this study was to evaluate the application of the previously described P. insidiosum-specific nested polymerase chain reaction (PCR) assay to directly detect P. insidiosum DNA in clinical and experimental lesions. Universal fungal primers (ITS1 and ITS4) were used during the first-round of PCR to amplify ITS1, 5.8s, and ITS2. A second-round of PCR was conducted with P. insidiosum-specific primers (PI1 and PI2) to amplify a variable region within this ITS1. In this study, a total of 21 equine clinical samples (kunkers) and 28 specimens from experimentally infected rabbits were analyzed by nested PCR. The first-round of PCR generated 800-base pair products, and the second-round produced 105-base pair amplicons for each P. insidiosum-specific sample; no amplicons were generated in negative control samples. Our results suggest that nested PCR is an important and efficient tool for diagnosis of both endemic (horse samples) and experimental (rabbit samples) pythiosis.