Antifungal activities of diphenyl diselenide and ebselen alone and in combination with antifungal agents against Fusarium spp.

Herein, we describe the in vitro activity of a combination of the organoselenium compounds diphenyl diselenide and ebselen alone and in combination with amphotericin B, caspofungin, itraconazole, and voriconazole against 25 clinical isolates of Fusarium spp. For this analysis, we used the broth microdilution method based on the M38-A2 technique and checkerboard microdilution method. Diphenyl diselenide (MIC range = 4-32 µg/ml) and ebselen (MIC range = 2-8 µg/ml) showed in vitro activity against the isolates tested. The most effective combinations were (synergism rates): ebselen + amphotericin B (88%), ebselen + voriconazole (80%), diphenyl diselenide + amphotericin B (72%), and diphenyl diselenide + voriconazole (64%). Combination with caspofungin resulted in low rates of synergism: ebselen + caspofungin, 36%, and diphenyl diselenide + caspofungin, 28%; combination with itraconazole demonstrated indifferent interactions. Antagonistic effects were not observed for any of the combinations tested. Our findings suggest that the antifungal potential of diphenyl diselenide and ebselen deserves further investigation in in vivo experimental models, especially in combination with amphotericin B and voriconazole.

Sporothrix schenckii COMPLEX:SUSCEPTIBILITIES TO COMBINED ANTIFUNGAL AGENTS AND CHARACTERIZATION OF ENZYMATIC PROFILES.

Sporothrix schenckii was reclassified as a complex encompassing six cryptic species, which calls for the reassessment of clinical and epidemiological data of these new species. We evaluated the susceptibility of Sporothrix albicans(n = 1) , S. brasiliensis(n = 6) , S. globosa(n = 1), S. mexicana(n = 1) and S. schenckii(n = 36) to terbinafine (TRB) alone and in combination with itraconazole (ITZ), ketoconazole (KTZ), and voriconazole (VRZ) by a checkerboard microdilution method and determined the enzymatic profile of these species with the API-ZYM kit. Most interactions were additive (27.5%, 32.5% and 5%) or indifferent (70%, 50% and 52.5%) for TRB+KTZ, TRB+ITZ and TRB+VRZ, respectively. Antagonisms were observed in 42.5% of isolates for the TRB+VRZ combination. Based on enzymatic profiling, the Sporothrix schenckii strains were categorized into 14 biotypes. Leucine arylamidase (LA) activity was observed only for S. albicans and S. mexicana. The species S. globosa and S. Mexicana were the only species without ß-glucosidase (GS) activity. Our results may contribute to a better understanding of virulence and resistance among species of the genus Sporothrix in further studies.

Sporothrix schenckii complex: susceptibilities to combined antifungal agents and characterization of enzymatic profiles / Complexo Sporothrix schenckii: susceptibilidade à combinação de antifúngicos e caracterização dos perfis enzimáticos

SUMMARYSporothrix schenckiiwas reclassified as a complex encompassing six cryptic species, which calls for the reassessment of clinical and epidemiological data of these new species. We evaluated the susceptibility of Sporothrix albicans(n = 1) , S. brasiliensis(n = 6) , S. globosa(n = 1), S. mexicana(n = 1) and S. schenckii(n = 36) to terbinafine (TRB) alone and in combination with itraconazole (ITZ), ketoconazole (KTZ), and voriconazole (VRZ) by a checkerboard microdilution method and determined the enzymatic profile of these species with the API-ZYM kit. Most interactions were additive (27.5%, 32.5% and 5%) or indifferent (70%, 50% and 52.5%) for TRB+KTZ, TRB+ITZ and TRB+VRZ, respectively. Antagonisms were observed in 42.5% of isolates for the TRB+VRZ combination. Based on enzymatic profiling, the Sporothrix schenckiistrains were categorized into 14 biotypes. Leucine arylamidase (LA) activity was observed only for S. albicansand S. mexicana. The species S. globosaand S. mexicanawere the only species without β-glucosidase (GS) activity. Our results may contribute to a better understanding of virulence and resistance among species of the genus Sporothrixin further studies.

RESUMOAvaliou-se a susceptibilidade de Sporothrix albicans(n = 1), S. brasiliensis(n = 1), S. globosa(n = 1), S. mexicana(n = 1) e S. schenckii(n = 36) frente à terbinafina (TRB) e a TRB em combinação com itraconazol (ITZ), cetoconazol (KTZ) e voriconazol (VRZ) pelo método de microdiluição ( checkerboard); o perfil enzimático destas espécies foi também avaliado, com base no kit API-ZYM. A maioria das interações foram aditivas (27,5%, 32,5% e 5%) ou indiferentes (70%, 50% e 52,5%) para TRB+KTZ, TRB+ITZ e TRB+VRZ, respectivamente. Antagonismo foi observado em 42,5% dos isolados para a combinação TRB+VRZ. Com base nos perfis enzimáticos, as cepas de Sporothrix schenckiievidenciaram 14 biotipos distintos. A atividade da leucina arilamidase (LA) só foi observada em S. albicanse S. mexicana.As espécies S. globosae S. mexicanaforam as únicas que não evidenciaram atividade da enzima β-glucosidase (GS). Estes resultados poderão contribuir para um melhor entendimento da virulência e resistência entre as espécies do gênero Sporothrixem futuros estudos.

Synergistic effects of tacrolimus and azole antifungal compounds in fluconazole-susceptible and fluconazole-resistant Candida glabrata isolates.

In vitro interaction between tacrolimus (FK506) and four azoles (fluconazole, ketoconazole, itraconazole and voriconazole) against thirty clinical isolates of both fluconazole susceptible and -resistant Candida glabrata were evaluated by the checkerboard microdilution method. Synergistic, indifferent or antagonism interactions were found for combinations of the antifungal agents and FK506. A larger synergistic effect was observed for the combinations of FK506 with itraconazole and voriconazole (43%), followed by that of the combination with ketoconazole (37%), against fluconazole-susceptible isolates. For fluconazole-resistant C. glabrata , a higher synergistic effect was obtained from FK506 combined with ketoconazole (77%), itraconazole (73%), voriconazole (63%) and fluconazole (60%). The synergisms that we observed in vitro , notably against fluconazole-resistant C. glabrata isolates, are promising and warrant further analysis of their applications in experimental in vivo studies.

Synergistic effects of tacrolimus and azole antifungal compounds in fluconazole-susceptible and fluconazole-resistant Candida glabrata isolates

In vitro interaction between tacrolimus (FK506) and four azoles (fluconazole, ketoconazole, itraconazole and voriconazole) against thirty clinical isolates of both fluconazole susceptible and -resistant Candida glabrata were evaluated by the checkerboard microdilution method. Synergistic, indifferent or antagonism interactions were found for combinations of the antifungal agents and FK506. A larger synergistic effect was observed for the combinations of FK506 with itraconazole and voriconazole (43%), followed by that of the combination with ketoconazole (37%), against fluconazole-susceptible isolates. For fluconazole-resistant C. glabrata, a higher synergistic effect was obtained from FK506 combined with ketoconazole (77%), itraconazole (73%), voriconazole (63%) and fluconazole (60%). The synergisms that we observed in vitro, notably against fluconazole-resistant C. glabrata isolates, are promising and warrant further analysis of their applications in experimental in vivo studies.

Susceptibility variation of Malassezia pachydermatis to antifungal agents according to isolate source.

Malassezia pachydermatis is associated with dermatomycoses and otomycosis in dogs and cats. This study compared the susceptibility of M. pachydermatis isolates from sick (G1) and healthy (G2) animals to azole and polyene antifungals using the M27-A3 protocol. Isolates from G1 animals were less sensitive to amphotericin B, nystatin, fluconazole, clotrimazole and miconazole.

In vitro susceptibility of Conidiobolus lamprauges recovered from sheep to antifungal agents.

Data regarding the susceptibility of Conidiobolus lamprauges is limited and there is no consensus about the optimal treatment for infections caused by Conidiobolus spp. In this context, the objective of this study was to evaluate the in vitro susceptibility of six C. lamprauges strains isolated from sheep conidiobolomycosis to amphotericin B, ketoconazole, fluconazole, itraconazole, posaconazole, voriconazole, anidulafungin, caspofungin, micafungin, flucytosine, and terbinafine using the CLSI M38-A2 microdilution technique. Terbinafine was the most active (MIC range <0.06-0.5 µg/mL). Resistance or reduced susceptibility was observed for amphotericin B and azole and echinocandin antifungals. Additional studies are necessary to determine the therapeutic potential of terbinafine as monotherapy or in combination therapy with other antifungals.

Antifungal activities of diphenyl diselenide alone and in combination with fluconazole or amphotericin B against Candida glabrata.

Here, we evaluated combinations of diphenyl diselenide [(PhSe)2] with fluconazole and amphotericin B in a checkerboard assay against clinical Candida glabrata strains. Minimal inhibitory concentration (geometric mean) ranged from 0.25 to >64 (5.16 µg/mL) for (PhSe)2, 1 to 32 (5.04 µg/mL) for fluconazole and 0.06 to 0.5 (0.18 µg/mL) for amphotericin B. Synergistic (76.66 %) and indifferent (23.34 %) interactions were observed for (PhSe)2 + amphotericin B combination. (PhSe)2 + fluconazole combination demonstrated indifferent (50 %) and antagonistic (40 %) interactions, whereas synergistic interactions were observed in 10 % of the isolates. New experimental in vivo protocols are necessary and will promote a better understanding of the antimicrobial activity of (PhSe)2 against C. glabrata and its use as an adjuvant therapy with antifungal agents.

A simple, rapid and inexpensive screening method for the identification of Pythium insidiosum.

Growth of Pythium insidiosum mycelia around minocycline disks (30µg) did not occur within 7days of incubation at 35°C when the isolates were grown on Sabouraud, corn meal, Muller-Hinton or RPMI agar. This technique offers a simple and rapid method for the differentiation of P. insidiosum from true filamentous fungi.

Susceptibility variation of Malassezia pachydermatis to antifungal agents according to isolate source

Malassezia pachydermatisis associated with dermatomycoses and otomycosis in dogs and cats. This study compared the susceptibility of M. pachydermatis isolates from sick (G1) and healthy (G2) animals to azole and polyene antifungals using the M27-A3 protocol. Isolates from G1 animals were less sensitive to amphotericin B, nystatin, fluconazole, clotrimazole and miconazole.

In vitro susceptibility of Pythium insidiosum isolates to aminoglycoside antibiotics and tigecycline.

This study evaluated the in vitro activity of aminoglycoside antibiotics and tigecycline against Pythium insidiosum. The susceptibility tests were carried out using the broth microdilution method in accordance with the CLSI document M38-A2. MIC values for gentamicin, neomycin, paromomycin, and streptomycin ranged from 32 to 64 mg/liter, and the minimal fungicidal concentration (MFC) ranged from 32 to 128 mg/liter, which are incompatible with safe concentrations of these drugs in plasma in vivo. Tigecycline showed the lowest MIC (0.25 to 2 mg/liter) and MFC (1 to 8 mg/liter) range values. The in vitro susceptibility observed to tigecycline makes this drug a good option in future tests in vitro and in vivo for the management of pythiosis.

Diphenyl diselenide in vitro and in vivo activity against the oomycete Pythium insidiosum.

This study evaluated the in vitro activity of diphenyl diselenide against 19 Pythium insidiosum isolates and the in vivo therapeutic response of rabbits with experimentally induced pythiosis. In vitro: susceptibility tests were performed using the broth macrodilution method in accordance with the CLSI document M38-A2. The criteria for interpretation were as follows: MIC-1 and MIC-2 (inhibition of 90% and 100% of mycelium growth, respectively) and the minimum fungicide concentration (MIC-3). In vivo: twenty rabbits were divided into four groups with five animals each and treated for 40 consecutive days: groups 1 and 2 (experimentally induced pythiosis) were treated with diphenyl diselenide (10mg/kg/day) and canola oil (1 mL/kg/day), respectively; groups 3 and 4 (controls) were treated with canola oil (1 mL/kg/day) and diphenyl diselenide (10mg/kg/day), respectively. Toxicity was evaluated using biochemical and haematological parameters. In vitro susceptibility tests showed that 89.4% of isolates had a MIC-1 ≤ 0.5 µg/mL, 84.2% of isolates had a MIC-2 ≤ 1.0 µg/mL and 94.7% of isolates had a MIC-3 ≤ 2.0 µg/mL. The in vivo assay suggested that this compound has a fungistatic activity, and the biochemical and haematological parameters indicated that there was no renal, hepatic or haematological toxicity. The comparison of the unsaturated iron binding capacity levels between animals with and without pythiosis suggested the involvement of iron metabolism in the pathogenesis of pythiosis. This study demonstrated the absence of detectable toxicity caused by diphenyl diselenide and the in vitro fungicidal and in vivo fungistatic activities of this drug, which makes it an option for future therapeutic approaches in the treatment of pythiosis.

In vitro susceptibility of Pythium insidiosum to macrolides and tetracycline antibiotics.

We describe the in vitro activity of macrolides and tetracycline antibiotics against Pythium insidiosum. The MICs were determined according to CLSI procedures (visual MIC) and by a colorimetric method [3-(4,5-dimethyl-2-thiazyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT)]. The lowest geometric mean (GM) MIC (MICs in µg/ml) (0.39 and 0.7 by visual reading and colorimetric method, respectively) and MIC ranges (0.125 to 2.0) were obtained for minocycline, while the highest MICs were shown for erythromycin (GM of 7.58 and 12.25 by visual reading and colorimetric method, respectively, and MIC ranged from 2 to 32). This significant in vitro activity makes these classes of antibiotics good candidates for experimental treatment of pythiosis.

In vitro antifungal evaluation and structure-activity relationship of diphenyl diselenide and synthetic analogues.

We report on in vitro antifungal activity and the structure-activity relationship of diphenyl diselenide [(PhSe)(2) ] and its synthetic analogues, (p-Cl-C(6) H(4) Se)(2), (m-CF(3)-C(6) H(4)Se)(2) and (p-CH(3)O-C(6) H(4)Se)(2), against 116 strains of pathogenic fungi. (PhSe)(2) showed the highest inhibitory activity against Candida albicans (minimum inhibitory concentration of 4-32 µg ml(-1) ), Candida dubliniensis (2-16 µg ml(-1)), Aspergillus spp. (0.5-64 µg ml(-1)) and Fusarium spp. (2-16 µg ml(-1)). Its minimum fungicidal concentration (MFC) varied among C. albicans (4-64 µg ml(-1)), C. dubliniensis (2-32 µg ml(-1) ) and Fusarium spp. (4-64 µg ml(-1)). Antifungal activity was decreased by the introduction of functional groups to the (PhSe)(2) molecule: (PhSe)(2) >(p-CH(3)O-C(6)H(4) Se)(2) >(m-CF(3)-C(6)H(4)Se)(2) >(p-Cl-C(6) H(4)Se)(2).

Candida dubliniensis: epidemiology and phenotypic methods for identification.

Candida dubliniensis is an emerging pathogen first described in 1995, which shares many phenotypic features with Candida albicans and therefore may be misidentified in microbial laboratories. Despite various phenotypic techniques described in the literature to differentiate the two species, the correct identification of C. dubliniensis remains problematic due to phenotypic similarities between these species. Thus, as the differences between both are best characterized at genetic levels, several molecular methods have been proposed to provide a specific and rapid identification of this species. Epidemiological studies have shown that C. dubliniensis is prevalent throughout the world and it is primarily associated with oral carriage and oropharyngeal infections in patients infected with human immunodeficiency virus (HIV). However, data acquired from its isolation from other healthy and immunocompromised patients are variable, and there is still no real consensus on the epidemiological relevance of this species. In this article, we review the various phenotypic methods used in the identification of C. dubliniensis and the epidemiological impact of this new species.

Comparison of the susceptibilities of clinical isolates of Candida albicans and Candida dubliniensis to essential oils.

Here, a microdilution technique based on the M27-A2 protocol (NCCLS, 2002) was employed to compare the susceptibilities of Candida albicans and Candida dubliniensis to essential oils extracted from plants used as spices. The chemical compositions of the essential oils were defined based on the analysis of retention indices obtained by gas chromatography-mass spectroscopy. Taken together, the results showed that the activity of the compounds against the two species was similar.

Differentiation of Candida dubliniensis from Candida albicans on rosemary extract agar and oregano extract agar.

Candida dubliniensis is a recently described pathogenic species which shares many phenotypic features with Candida albicans and therefore, may be misidentified in microbiological laboratories. Because molecular methods can be onerous and unfeasible in routine mycological laboratories with restricted budgets such as those in developing countries, phenotypic techniques have been encouraged in the development of differential media for the presumptive identification of these species. We examined the colony morphology and chlamydospore production of 30 C. dubliniensis isolates and 100 C. albicans isolates on two new proposed media: rosemary (Rosmarinus officinalis) extract agar (REA) and oregano (Origanum vulgare) extract agar (OEA). These substrates are traditionally used as spices and medicinal herbs. In both of these media, all C. dubliniensis isolates (100%) showed rough colonies with peripheral hyphal fringes and abundant chlamydospores after 24 to 48 hr of incubation at 25 degrees C. In contrast, under the same conditions, all isolates of C. albicans (100%) showed smooth colonies without hyphal fringes or chlamydospores. In conclusion, REA and OEA offer a simple, rapid, and inexpensive screening media for the differentiation of C. albicans and C. dubliniensis.

Enzymatic and hemolytic activities of Candida dubliniensis strains.

Candida dubliniensis is an opportunistic yeast that has been recovered from several body sites in many populations; it is most often recovered from the oral cavities of human immunodeficiency virus-infected patients. Although extensive studies on epidemiology and phylogeny of C. dubliniensis have been performed, little is known about virulence factors such as exoenzymatic and hemolytic activities. In this study we compared proteinase, hyaluronidase, chondroitin sulphatase and hemolytic activities in 18 C. dubliniensis and 30 C. albicans strains isolated from AIDS patients. C. albicans isolates produced higher amounts of proteinase than C. dubliniensis (p < 0.05). All the tested C. dubliniensis strains expressed hyaluronidase and chondroitin sulphatase activities, but none of them were significantly different from those observed with C. albicans (p > 0.05). Hemolytic activity was affected by CaCl2; when this component was absent, we did not notice any significant difference between C. albicans and C. dubliniensis hemolytic activities. On the contrary, when we added 2.5 g% CaCl2, the hemolytic activity was reduced on C. dubliniensis and stimulated on C. albicans tested strains (p < 0.05).