Evaluation of susceptibility and response in the surface of agents of surface mycoses (Trichophyton mentagrophytes; T. tonsurans) to antifungal drugs of interest in a medical clinic / Avaliação da susceptibilidade e resposta in vitro de agentes de micoses superficiais (Trichophyton mentagrophytes; T. tonsurans) à drogas antifúngicas de interesse em clínica médica

INTRODUCTION: The resistance of fungal species to drugs usually used in clinics is of great interest in the medical field. OBJECTIVE: To evaluate susceptibility and in vitro response of species of Trichophyton spp. to antifungal drugs of interest in clinical medicine. METHODS: 12 samples of clinical isolates from humans were used, nine of T. mentagrophytes and three of T. tonsurans. Susceptibility tests were performed according to the agar diffusion (AD) and broth microdilution (BM) methods. RESULTS: In the AD method, the species T. tonsurans presented a percentage of sensitivity of 33% in relation to amphotericin B and 66% to itraconazole, with 100% resistance to ketoconazole and fluconazole. T. mentagrophytes also showed 100% resistance to ketoconazole in this technique, with 11% sensitivity to ketoconazole, 22% to itraconazole and 22% of samples classified as sensitive dose dependent. In the MC method, the species T. tonsurans presented a sensitivity percentage of 66%, 55% and 33% in relation to ketoconazole, fluconazole and itraconazole, respectively. The T. mentagrophytes species presented sensitivity percentages of 11%, 11%, 33% and 55% for amphotericin B, itraconazole, ketoconazole and fluconazole, respectively. CONCLUSION: There was resistance in vitro of the species of T. mentagrophytes and T. tonsurans against the antifungal fluconazole and relative resistance against ketoconazole in the AD method. In BM, however, important percentages of sensitivity were observed for the two species analyzed in relation to the antifungals fluconazole and ketoconazole when compared to itraconazole and amphotericin B.

INTRODUÇÃO: A resistência de espécies fúngicas às drogas usualmente empregadas no meio clínico é motivo de grande interesse na área médica. OBJETIVO: Avaliar susceptibilidade e resposta in vitro de espécies de Trichophyton spp. a drogas antifúngicas de interesse em clínica médica. MÉTODOS: Foram utilizadas 12 amostras de isolados clínicos de humanos, sendo nove de T. mentagrophytes e três de T. tonsurans. Foram realizados testes de susceptibilidade segundo os métodos de difusão em ágar (DA) e microdiluição em caldo (MC). RESULTADOS: No método de DA, a espécie T. tonsurans apresentou percentual de sensibilidade de 33% em relação à anfotericina B e de 66% ao itraconazol, com 100% de resistência frente ao cetoconazol e ao fluconazol. A espécie T. mentagrophytes também apresentou 100% de resistência frente ao cetoconazol nesta técnica, com 11% de sensibilidade ao cetoconazol, 22% ao itraconazol e 22% das amostras classificadas como sensível dose dependente. No método de MC, a espécie T. tonsurans apresentou percentual de sensibilidade de 66%, 55% e 33% em relação ao cetoconazol, fluconazol e itraconazol, respectivamente. A espécie T. mentagrophytes apresentou percentuais de sensibilidade de 11%, 11%, 33% e 55% para anfotericina B, itraconazol, cetoconazol e fluconazol, respectivamente. CONCLUSÃO: Houve resistência in vitro das espécies do T. mentagrophytes e T. tonsurans frente ao antifúngico fluconazol e resistência relativa frente ao cetoconazol no método de DA. Na MC, no entanto, foram observados importantes percentuais de sensibilidade das duas espécies analisadas frente aos antifúngicos fluconazol e cetoconazol quando comparadas ao itraconazol e à anfotericina B.

Antifungal and Antioxidant Activities of Vernonia Chalybaea Mart. ex DC. Essential Oil and their Major Constituent β -caryophyllene

Abstract: This article describes the chemical composition of Vernonia chalybaea essential oil, and investigates its antimicrobial, antioxidant and hemolytic activities. The evaluation of the antifungal activity was performed by the broth microdilution method using strains of yeasts and dermatophytic fungi. The checkerboard technique to find antimicrobial modulatory effects was performed using ketoconazole as standard drug. The antioxidant activity was evaluated by DPPH scavenging assay and β-carotene/linoleic-acid system. The toxicity was characterized by the brine shrimp lethality test and hemolysis bioassays. The essential oil was obtained by hydrodistillation and analyzed by GC-MS method, showing to be rich in the sesquiterpenes β-caryophyllene (39.06%) and bicyclogermacrene (19.69%), and also demonstrated a relevant antifungal activity against strains of Trichophyton rubrum. In the modulatory activity assay, the essential oil of V. chalybaea and β-caryophyllene demonstrated a synergistic interaction with ketoconazole, with increasing of its antifungal action. The antioxidant activity was evidenced mainly by β-carotene/linoleic acid system, with IC50 value of 35.87 ± 0.32 µg/mL. The results suggest that V. chalybaea essential oil and β-caryophyllene are valuable natural medicinal agents with antioxidant and antimicrobial activities.

Minimal inhibitory concentration distributions and epidemiological cutoff values of five antifungal agents against Sporothrix brasiliensis

BACKGROUND Sporothrix brasiliensis is the most virulent sporotrichosis agent. This species usually responds to antifungal drugs, but therapeutic failure can occur in some patients. Antifungal susceptibility tests have been performed on this species, but no clinical breakpoints (CBPs) are available. In this situation, minimal inhibitory concentration (MIC) distributions and epidemiological cutoff values (ECVs) support the detection of identification of resistant strains. OBJECTIVES To study the MIC distributions of five antifungal drugs against S. brasiliensis and to propose tentative ECVs. METHODS MICs of amphotericin B (AMB), itraconazole (ITR), ketoconazole (KET), posaconazole (POS), and terbinafine (TRB) against 335 S. brasiliensis strains were determined by the Clinical and Laboratory Standards Institute broth microdilution method. FINDINGS The proposed ECV, in µg/mL, for AMB, ITR, KET, POS, and TRB were 4.0, 2.0, 1.0, 2.0, and 0.25, respectively. Percentages of wild-type strains in our population for the above antifungal drugs were 98.48, 95.22, 95.33, 100, and 97.67%, respectively. MAIN CONCLUSIONS These ECVs will be useful to detect strains with resistance, to define CBPs, and to elaborate specific therapeutic guidelines for S. brasiliensis. Rational use of antifungals is strongly recommended to avoid the emergence of resistant strains and ensure the therapeutic effectiveness of sporotrichosis.

[Drug susceptibility testing of clinical isolates of Candida albicans against Amphotericin B and Ketoconazole by microdilution and disk diffusion methods]

Background and Objective: Candida albicans is the normal flora of the body as opportunistic fungi. It causes candidiasis in immunocompromised condition. This study was done to drug susceptibility testing of Candida albicans isolated from patients against Amphotericin B and Ketoconazole

Methods: In this descriptive - analytic study, drug susceptibility of 30 Candida albicans isolated from patients admitted to Tehran hospitals, Iran was tested against Amphotericin B and Ketoconazole by micro dilution method in accordance with CLSI M27-A2 guideline and disk diffusion method in accordance with CLSI M44-S2 guideline. Standard isolate Candida albicans PTCC [5027] and Candida krusei PTCC [5295] were used for quality control

Results: The minimum and maximum MIC against Amphotrericin B was 0.0625 micro g.ml[-1] and 4 micro g.ml[-1], respectively. The minimum and maximum MIC against Ketoconazole was 0.5 micro g/ml[-1] and 32 micro g/ml[-1], respectively. The minimum and maximum zone diameter was 6 and 28 mm for both drugs. The results of drug susceptibility testing by two methods did not show significant differences. 25 isolates [83.3%] against ketoconazole and 2 isolates [6.7%] against Amphotericin B were resistant

Conclusion: Amphotericin B administration seems better choice in candidiasis treatment in comparision with Ketoconazole

Impacto de la clindamicina in vitro en la combinación clindamicina - ketoconazol sobre el exopolímero de biopelículas de candida spp de origen urogenital / Impact of in vitro clIndamycIn on the combInatIon of clIndamycIn and ketoconazole on exopolymer of candida spp bIofIlms of urogenItal orIgIn

La mucosa vaginal ha sido utilizada largamente para la administración de antimicrobianos destinados al tratamiento de infecciones endógenas del tracto genital inferior (IETGI) en mujeres embarazadas y no embarazadas. Candida spp elabora biopelículas (BP) y su formación es un proceso complejo que requiere que las células fúngicas establezcan múltiples interacciones con el medio. Las BP están rodeadas por un exopolímero (EPM) que puede restringir la actividad de anticuerpos, la difusión de sustancias y unirse a los antimicrobianos (AM), limitando su acción. Los antimicrobianos (AM) en general y los antimicóticos en particular (AMC) pueden tener dificultades para llegar a las células dentro del EPS. Muchas de las fórmulas que se emplean para el tratamiento empírico usan combinaciones inapropiadas con limitada o nula actividad sobre las biopelículas (BP). La presencia de moléculas que provoquen su inhibición anulando los inductores del EPM o por otro mecanismo, permitirá la actividad del AM específico. Objetivo: demostrar que la actividad de la clindamicina (CLI) en fórmula dual con ketoconazol (KET) actúa sobre BP Candida albicans (CA) y especies no albicans de Candida . (NAC). Métodos: estudiamos la actividad de clindamicina-ketoconazol (CK) sobre la adherencia y dispersión de BP de 8 aislamientos vaginales de CA y 7 de CNA. Se inocularon en 3 tubos con caldo Sabouraud y un dispositivo de vidrio para la formación de la BP según técnica ya descrita. Adherencia: Se incubaron durante 6 horas y se agregó una combinación de CK proveniente del material de óvulos, diluido convenientemente (62,5/260,4 ug/ml), a uno de los tubos de cada aislamiento tomándose como hora 0. Dispersión: esa misma dilución se agregó a otro tubo a las 16 horas. El tercer tubo quedó como testigo sin antimicrobianos. La lectura se efectuó con microscopio óptico a las 24 horas de agregada la combinación CK previa tinción con cristal violeta y se evaluaron con programas fotográficos. Por separado analizamos la actividad de CLI (62,5 ug/ml) y KET (260,4 ug/ml) con técnica similar. Seleccionamos las muestras de 7 pacientes que demostraron candidiasis vulvovaginal (CVV) y las estudiamos con la técnica de capas celulares. Se empleó la combinacion CK para el estudio de la adherencia y dispersión. Resultados: Adherencia se demostró poca influencia de CK en la adherencia con respecto a cada testigo. Dispersión: la influencia de CK se demostró en la mayoría de los aislamientos particularmente en los de CNA que mostraron una mayor presencia de EPM. Las hifas solo se observaron en 1/15 de los aislamientos de Candida spp cuando se agregó CK a las 16 horas. En las BP de las muestras clínicas no aparecieron hifas ni otro elemento micótico en 5/7 con respecto a los testigos. Conclusión: Según estos resultados el uso de una combinación de CK en BP de Candida spp, resulta en una adecuada penetración del AMC demostrada por la dispersión de la BP al cabo de 24 horas. Clindamicina no interfiere con la acción del ketoconazol sino que promovería su actividad anti-candida modificando posiblemente estructuras de superficie y la del EP por inhibición de las moléculas que facilitan la expresión del mismo. In vivo promueve la actividad inmunomoduladora que no se puede demostrar con este modelo in vitro. Su uso combinado en fórmulas duales facilitaría la actividad del AMC sobre Candida spp actuando como inhibidora o modificadora de las BP mediante la dispersión del EPM

The vaginal mucosa has been widely used for administering antimicrobial agents to treat endogenous infections of the lower genital tract in pregnant and non-pregnant women. Candida spp. elaborates biofilms, and its formation is a complex process requiring that fungal cells establish multiple interactions with the medium. Biofilms are surrounded by an exopolymer matrix that can restrict the activity of antibodies, the diffusion of substances, and be associated with antimicrobials, therefore limiting its actions. General antimicrobials and particular anti-mycotic agents can face difficulties to access the cells within the exopolymer matrix. Many formulas used for empirical treatment have improper combinations with limited or null activity on the biofilms. The presence of molecules that cause its inhibition, thus eliminating the exopolymer matrix inducers, or by other mechanism, will allow the specific antimicrobial activity. Objective: To show that the activity of clindamycin used in dual formula with ketoconazole works on Candida albicans biofilm and on non- albicans species of Candida . Methods: We studied the activity of clindamycin and ketoconazole regarding the adherence and dispersion of biofilms from eight vaginal isolates of C. albicans and 7 of non- albicans Candida . The isolates were inoculated in three tubes with Sabouraud agar and a glass device to form the biofilm according to a known technique. Adherence : Each isolate was incubated for a six-hour period and a combination of clindamycin and ketoconazole from the material of ovules was added and conveniently diluted to one of the tubes of each isolate (62.5/260.4 ug/mL), considering 0 hour. Dispersion: The same dilution was added to another tube after 16 hours. The third tube was used as a control without antimicrobials. The reading was carried out with an optical microscope after 24 hours that the clindamycin and ketoconazole combination had been added and colored with crystal violet. They were then evaluated using photographic programs. The activity of clindamycin (62.5 ug/mL) and ketoconazole (260.4 ug/mL) was analyzed alone with a similar technique. We chose vaginal samples from seven patients with vulvovaginal candidiasis and studied them through the cell layer technique. The clindamycin and ketoconazole combination was used for studying the adherence and dispersion. Results: Adherence: Little influence of clindamycin and ketoconazole was seen in adherence regarding each control. Dispersion: The clindamycin and ketoconazole influence was seen in most of the isolates, especially in those of non- albicans Candida that showed higher presence of exopolymer matrix . The hyphae were only seen in 1 of 15 isolates of Candida spp after the clindamycin and ketoconazole were added at the 16th hour. In biofilms of clinical samples, neither hyphae nor mycotic elements were seen in 5 of 7 compared with the controls. Conclusion: According to these results, the use of a clindamycin and ketoconazole combination in biofilms of Candida spp results in proper penetration of the antimicrobial agent, which is seen by the biofilm dispersion during 24 hours. Clindamycin does not interfere with the action of ketoconazole, but it would promote its anti- Candida activity and would possibly modify surface and EP structures through inhibition of the molecules that facilitate its expression. The in vivo model promotes the immunomodulatory activity that in vitro models do not. Its combined use in dual formulas would facilitate the antimicrobial activity on Candida spp, therefore working as an inhibitor or modifier of the biofilms after dispersion of the exopolymer matrix

Efeito da incorporação de agentes antifúngicos na resistência à tração e porosidade de materiais resilientes temporários para base de próteses / Effect of incorporation of antifungal agents on the ultimate tensile strength and porosity of temporary soft denture liners

Este estudo investigou a resistência à tração (ou limite de resistência à tração- LRT) e a porosidade de reembasadores resilientes temporários modificados por concentrações inibitórias mínimas (CIMs) de agentes antifúngicos para o biofilme Candida albicans (SC5314). Para os testes de LRT, corpos de prova em forma de halteres (n=7) com uma área transversal de 33 mm x 6 mm x 3 mm foram produzidos para os materiais resilientes (Trusoft e Softone) sem (controle) ou com incorporação de cinco fármacos em suas CIMs: nistatina- 0,032 g; diacetato de clorexidina- 0,064; cetoconazol- 0,128 g; miconazol- 0,256 g; itraconazol-0,256 g (grama de fármaco por grama de pó de material resiliente). Após a plastificação, as amostras foram imersas em água destilada a 37°C durante 24 h, 7 e 14 dias e, então, testadas em tensão em uma máquina universal de ensaios (EMIC DL-500 MF) a 40 mm/min. A porosidade foi mensurada por absorção de água, com base na exclusão do efeito plastificante. Inicialmente, determinou-se por isotermas de sorção, que a solução de armazenagem adequada para os corpos de prova (65 mm x 10 mm x 3,3 mm) de ambos os materiais foi o cloreto de cálcio anidro a 50% (S50). Assim, o fator de porosidade (FP) foi calculado para os grupos de estudo (n=10) formados por espécimes sem (controle) ou com incorporação de fármaco em suas CIMs (nistatina, clorexidina ou cetoconazol) após a armazenagem em água destilada ou S50 por 24 h, 7 e 14 dias. Os dados de resistência à tração (MPa) e percentagem de alongamento (%) foram submetidos à ANOVA de 3 fatores seguida pelo teste de Tukey (=0,05). Os dados de porosidade foram analisados estatisticamente por ANOVA de medidas repetidas para 4 fatores e teste de Tukey (=0,05). Ao final de 14 dias, a resistência à tração para ambos os materiais foi significativamente menor nos grupos modificados pelo miconazol e itraconazol em relação aos outros grupos (P<0,0001), que não mostraram diferenças significativas entre si (P>0,05). Após 7 e 14 dias em água, o miconazol e itraconazol adicionados a ambos os materiais resultaram em percentagens significativamente menores de alongamento em comparação com os outros fármacos e ao controle (P<0,0001), que foram semelhantes entre si (P>0,05). O cetoconazol não resultou em alterações significativas no FP para ambos os materiais resilientes em água ao longo de 14 dias (P>0,05). Em comparação aos controles, houve aumento dos FPs do Softone e Trusoft aos 14 dias de imersão em água somente após a adição de nistatina e clorexidina e de clorexidina, respectivamente (P<0,05). Ambos os materiais não apresentaram alterações significativas no FP em até 14 dias de imersão na S50, em comparação aos controles (P>0,05). Em todas as condições experimentais, os FPs do Softone e Trusoft foram significativamente menores quando imersos em S50 em comparação com a água destilada (P<0,05). Concluiu-se que a adição de nistatina, clorexidina e cetoconazol nas CIMs para o biofilme de C. albicans não resultou em efeitos deletérios na resistência à tração e na percentagem de alongamento dos materiais resilientes temporários para base de prótese até o período de 14 dias. A adição de antifúngicos nas CIMs não resultou em efeitos adversos à porosidade de ambos os materiais resilientes temporários em diferentes períodos de imersão em água, com exceção da clorexidina e nistatina no Softone e clorexidina no Trusoft aos 14 dias. Não foram observados efeitos deletérios para a porosidade de ambos os materiais resilientes modificados com as CIMs dos fármacos durante os 14 dias de imersão na S50.(AU)

This study investigated the tensile strength (ultimate tensile strength- UTS) and porosity of temporary soft denture liners modified by minimum inhibitory concentrations (MICs) of antifungal agents for Candida albicans biofilm (SC5314). For UTS tests, dumbbell-shaped specimens (n=7) with a central cross-sectional area of 33 mm x 6 mm x 3 mm were produced by resilient materials (Trusoft and Softone) without (control) or with incorporation of five drugs at MICs: nystatin- 0.032 g; chlorhexidine diacetate-0.064 g; ketoconazole- 0.128 g; miconazole- 0.256 g; itraconazole- 0.256 g (each per gram of soft liner powder). After plasticization, specimens were immersed in distilled water at 37°C for 24 h, 7 and 14 days, and then tested in tension in a universal testing machine (EMIC DL-500 MF) at 40 mm/min. The porosity was measured by water absorption, based on exclusion of the plasticizer effect. Initially, it was determined by sorption isotherms that the adequate storage solution for specimens (65 mm x 10 mm x 3.3 mm) of both materials was 50% anhydrous calcium chloride (S50). Then, the porosity factor (PF) was calculated for the study groups (n=10) formed by specimens without (control) or with drug incorporation at MICs (nystatin, chlorhexidine or ketoconazole) after storage in distilled water or S50 for 24 h, 7 and 14 days. Data of tensile strength (MPa) and elongation percentage (%) were submitted to 3-way ANOVA followed by Tukey's test (=0.05). Data of porosity were statistically analyzed by 4-way repeated measures ANOVA and Tukeys test (=0.05). At the end of 14 days, the tensile strength for both materials was significantly lower in the groups modified by miconazole and itraconazole compared to the other groups (P<0.0001), which showed no significant difference between them (P>0.05). After 7 and 14 days in water, miconazole and itraconazole added into both materials result in significant lower elongation percentages compared to the other drugs and control (P<.0001), which were similar to each other (P>0.05). Ketoconazole resulted in no significant changes in PF for both liners in water over 14 days (P>0.05). Compared to the controls, Softone and Trusoft PFs were increased at 14-day water immersion only after addition of nystatin and chlorhexidine, and chlorhexidine, respectively (P<0.05). Both materials showed no significant changes in PF in up to 14 days of S50 immersion, compared to the controls (P>0.05). In all experimental conditions, Softone and Trusoft PFs were significantly lower when immersed in S50 compared to distilled water (P<0.05). It was concluded that the addition of the nystatin, chlorhexidine and ketoconazole at MICs for C. albicans biofilm resulted in no harmful effects on the ultimate tensile strength and elongation percentage of the temporary soft denture liners up to 14-day period. The addition of antifungals at MICs resulted in no detrimental effects for the porosity of both temporary soft liners in different periods of water immersion, except for chlorhexidine and nystatin in Softone and chlorhexidine in Trusoft at 14 days. No deleterious effect was observed for the porosity of both soft liners modified by the drugs at MICs over 14 days of S50 immersion.(AU)

Antifungal susceptibility of clinical and environmental Cryptococcus neoformans and Cryptococcus gattii isolates in Jabalpur, a city of Madhya Pradesh in Central India

In this study, we present antifungal susceptibility data of clinical and environmental isolates of Central Indian Cryptococcus neoformans (Serotype A, n = 8 and n = 50 respectively) and Cryptococcus gattii (Serotype B, n = 01 and n = 04 respectively). Susceptibilities to fluconazole, itraconazole and ketoconazole were determined by using NCCLS broth micro-dilution methodology. The total number of resistant strains for fluconazole in case of C. neoformans and C. gattii showed a significant difference by using chi-square test (p < 0.05*), while considering fisher's exact p value was nonsignificant (p > 0.05). However, the total number of resistant strains for itraconazole and ketoconazole was not found statistically significant. A comparison of geometric means of clinical and environmental strains of C. gattii and C. neoformans was not found statistically significant using student ‘t’ test (p value > 0.05 NS). Though less, the antifungal data obtained in this study suggests that primary resistance among environmental and clinical isolates of C. neoformans and C. gattii against tested antifungal was present and C. gattii comparatively was less susceptible than C. neoformans var. grubii isolates to fluconazole than to itraconazole and ketoconazole. A continuous surveillance of antifungal susceptibility of clinical and environmental isolates of C. neoformans and C. gattii is desirable to monitor the emergence of any resistant strains for better management of cryptococcosis patients.

Impact of brief exposure to antifungal agents on the post-antifungal effect and hemolysin activity of oral Candida albicans

AbstractPost-antifungal effect (PAFE) of Candida and its production of hemolysin are determinants of candidal pathogenicity. Candida albicans is the foremost aetiological agent of oral candidosis, which can be treated with polyene, azole, and echinocandin antifungals. However, once administered, the intraoral concentrations of these drugs tend to be subtherapeutic and transient due to the diluent effect of saliva and cleansing effect of the oral musculature. Hence, intra-orally, Candidamay undergo a brief exposure to antifungal drugs.Objective Therefore, the PAFE and hemolysin production of oral C. albicans isolates following brief exposure to sublethal concentrations of the foregoing antifungals were evaluated.Material and Methods A total of 50 C. albicans oral isolates obtained from smokers, diabetics, asthmatics using steroid inhalers, partial denture wearers and healthy individuals were exposed to sublethal concentrations of nystatin, amphotericin B, caspofungin, ketoconazole and fluconazole for 60 min. Thereafter, the drugs were removed and the PAFE and hemolysin production were determined by previously described turbidometric and plate assays, respectively.Results Nystatin, amphotericin B, caspofungin and ketoconazole induced mean PAFE (hours) of 2.2, 2.18, 2.2 and 0.62, respectively. Fluconazole failed to produce a PAFE. Hemolysin production of these isolates was suppressed with a percentage reduction of 12.27, 13.47, 13.33, 8.53 and 4.93 following exposure to nystatin, amphotericin B, caspofungin, ketoconazole and fluconazole, respectively.Conclusions Brief exposure to sublethal concentrations of antifungal drugs appears to exert an antifungal effect by interfering with the growth as well as hemolysin production of C. albicans.

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.

Effects of the association of antifungal drugs on the antimicrobial action of endodontic sealers

This in vitro study aimed to determine the susceptibility of oral specimens and ATCC lineages of Candida albicans for five endodontic sealers, which were pure and associated with two antifungal drugs, and to analyze their effect on the physical properties. For this purpose, 30 lineages of C. albicans, collected from the oral cavity of patients assisted at the endodontics clinic of the Universidade Sagrado Coração, were analyzed. Yeasts susceptibility to the sealers was tested by diffusion on agar plates. Physical properties were evaluated according to the ADA specification no. 57. The pure versions of the Sealer 26, AH Plus, Endofill, Fillapex, and Sealapex demonstrated antifungal activity, with Endofill presenting the greatest inhibition zones. All cements, except for Endofill, had their antifungal actions enhanced by addition of ketoconazole and fluconazole (p < 0.05), and the AH Plus presented the best antifungal activity. The addition of antifungal drugs did not interfere with the setting time and flowability of the sealers. It was concluded that the addition of antifungals to endodontic sealers enhanced the antimicrobial action of most cements tested without altering their physical properties.

Comparison of the Minimum Fungicidal Concentration of Clotrimazole, Ketoconazole, Miconazole and Terbinafine Against Clinical Isolates of Dermatophytes.

Superficial fungal infections affect millions of people worldwide. Earlier most dermatophyte strains had relatively restricted geographical distribution. But currently, dermatophytosis has become one of the most common human infectious diseases worldwide. Fungal infections are common in hot and humid climate of tropical countries like India. Topical and systemic therapies are commonly used to treat dermatophyte infections.Clotrimazole is one of the most commonly used topical antifungal drugs. This study compared the minimum fungicidal concentration (MFC) of Clotrimazole with Miconazole, Ketoconazole and Terbinafine in skin dermatophytes. The study demonstrated that Clotrimazole had lower MFCs as compared to Ketoconazole and Miconazole against Trichophyton rubrum, Trichophyton mentagrophytes and Microsporum canis. Clotrimazole had comparable MFCs versus Terbinafine against Trichophyton rubrum but it had lower MFCs against Trichophyton mentagrophytes and Microsporum canis. Thus, Clotrimazole is an effective antifungal agent for dermatophytosis even today.The efficacy of Clotrimazole even against strains with intermediate resistance or resistance to the older azole anti fungal drugs reiterate the current decisions of empirical treatment with topical Clotrimazole for the management of superficial dermatophyte infections.

Comparison between two culture media for in vitro evaluation of antifungal susceptibility of the Sporothrix schenckii complex / Comparação entre dois meios de cultura para avaliação in vitro da suscetibilidade a antifúngicos do complexo Sporothrix schenckii

BACKGROUND: The standard methodology for determining the antifungal sensitivity against the Sporothrix schenckii complex recommends the use of the 1640 Roswell Park Memorial Institute culture medium (RPMI) buffered with morpholinepropanolsulfonic acid (MOPS). However, while this is a high-cost medium which requires a laborious implementation and sterilization by filtration, the Sabouraud dextrose broth is a low-cost medium, widely used in mycology, sterilized by autoclave. OBJECTIVE: To evaluate the performance of the Sabouraud dextrose broth culture medium as a substitute for the RPMI 1640-MOPS in determining the antifungal sensitivity of S. schenckii. METHODS: Forty-eight clinical isolates were evaluated against five antifungal agents: itraconazole, ketoconazole, fluconazole, amphotericin B and terbinafine, using the method of broth microdilution advocated by the M38-A2 protocol of the Clinical and Laboratory Standards Institute. RESULTS: There were no significant differences between the Minimum Inhibitory Concentrations obtained in the two culture media for all the antifungals, with the exception of the amphotericin B. Regarding this drug, the Minimum Inhibitory Concentration range obtained were wider for the Sabouraud dextrose broth than for the Roswell Park Memorial Institute morpholinepropanelsulfonic acid. CONCLUSIONS: The Sabouraud dextrose broth showed potential to be used in the in vitro evaluation of the S. schenckii complex antifungal activity.

FUNDAMENTOS: A metodologia padronizada para a determinação da sensibilidade aos antifúngicos frente ao complexo Sporothrix schenckii preconiza a utilização do meio de cultura Roswell Park Memorial Institute (RPMI) 1640 tamponado com ácido morfolinopropanosulfônico (MOPS). No entanto, este meio possui custo elevado, execução trabalhosa e esterilização por filtração. Já o caldo Sabouraud-dextrose é amplamente utilizado em micologia, de baixo custo e pode ser esterilizado por autoclavagem. OBJETIVO: Avaliar o desempenho do meio de cultura caldo Sabouraud-dextrose em substituição ao RPMI 1640-MOPS na determinação da sensibilidade de S. schenckii a antifúngicos. MÉTODO: Foram avaliados 48 isolados clínicos frente a cinco antifúngicos: itraconazol, cetoconazol, fluconazol, anfotericina B e terbinafina, utilizando a metodologia da microdiluição em caldo preconizada pelo protocolo M38-A2 do Clinical and Laboratory Standards Institute. RESULTADOS: Não houve diferenças significativas nas Concentrações Inibitórias Mínimas obtidas nos dois meios de cultura para todos os antifúngicos, com exceção da anfotericina B. Para este fármaco, foram obtidas faixas mais amplas de Concentrações Inibitórias Mínimas para caldo Sabouraud-dextrose do que para Roswell Park Memorial Institute-morfolinopropanosulfônico. CONCLUSÕES: O caldo Sabouraud-dextrose mostrou potencial para ser utilizado na avaliação in vitro da atividade antifúngica do complexo S. schenckii.

The stepwise selection for ketoconazole resistance induces upregulation of C14-demethylase (CYP51) in Leishmania amazonensis

Ketoconazole is a clinically safe antifungal agent that also inhibits the growth of Leishmania spp. A study was undertaken to determine whether Leishmania parasites are prone to becoming resistant to ketoconazole by upregulating C14-demethylase after stepwise pharmacological pressure. Leishmania amazonensis promastigotes [inhibitory concentration (IC)50 = 2 µM] were subjected to stepwise selection with ketoconazole and two resistant lines were obtained, La8 (IC50 = 8 µM) and La10 (IC50 = 10 µM). As a result, we found that the resistance level was directly proportional to the C14-demethylase mRNA expression level; we also observed that expression levels were six and 12 times higher in La8 and La10, respectively. This is the first demonstration that L. amazonensis can up-regulate C14-demethylase in response to drug pressure and this report contributes to the understanding of the mechanisms of parasite resistance.

Disturbance in hemoglobin metabolism and in vivo antimalarial activity of azole antimycotics / Alteración en el metabolismo de la hemoglobina y actividad antimalárica in vivo de azoles antimicóticos

Plasmodium parasites degrade host hemoglobin to obtain free amino acids, essential for protein synthesis. During this event, free toxic heme moieties crystallize spontaneously to produce a non-toxic pigment called hemozoin or ß-hematin. In this context, a group of azole antimycotics, clotrimazole (CTZ), ketoconazole (KTZ) and fluconazole (FCZ), were investigated for their abilities to inhibit ß-hematin synthesis (IßHS) and hemoglobin proteolysis (IHbP) in vitro. The ß-hematin synthesis was recorded by spectrophotometry at 405 nm and the hemoglobin proteolysis was determined by SDS-PAGE 12.5 percent, followed by densitometric analysis. Compounds were also assayed in vivo in a malaria murine model. CTZ and KTZ exhibited the maximal effects inhibiting both biochemical events, showing inhibition of β-hematin synthesis (IC50 values of 12.4 ± 0.9 µM and 14.4 ± 1.4 µM respectively) and inhibition of hemoglobin proteolysis (80.1 ± 2.0 percent and 55.3 ± 3.6 percent, respectively). There is a broad correlation to the in vivo results, especially CTZ, which reduced the parasitemia ( percentP) of infected-mice at 4th day post-infection significantly compared to non-treated controls (12.4 ± 3.0 percent compared to 26.6 ± 3.7 percent, p = 0.014) and prolonged the survival days post-infection. The results indicated that the inhibition of the hemoglobin metabolism by the azole antimycotics could be responsible for their antimalarial effect.

Los parásitos del género Plasmodium degradan la hemoglobina hospedera obteniendo aminoácidos libres para su síntesis proteica. Durante este evento, unidades de hemo libre tóxicas cristalizan espontáneamente formando un pigmento no tóxico denominado ß-hematina. En este trabajo, se investigó la capacidad de un grupo de azoles antimicóticos: clotrimazol (CTZ), ketoconazol (KTZ) y fluconazol (FCZ), en inhibir la síntesis de ß-hematina y la proteólisis de la globina. La síntesis de ß-hematina se registro por espectrofotometría a 405 nm y la proteólisis de la hemoglobina se determino por SDS-PAGE 15 por ciento seguido por análisis densitométrico de las bandas de hemoglobina intactas. Los compuestos fueron también ensayados in vivo en un modelo de malaria murina. CTZ y KTZ inhibieron la síntesis de ß-hematina con CI50 entre 10 y 15 µM y bloquearon la proteólisis de la hemoglobina (80.01 ± 2.04 por ciento y 55.33 ± 3.57 por ciento, respectivamente). En relación directa con los resultados encontrados in vitro, el CTZ redujo la parasitemia de ratones infectados en forma significativa, así como prolongó lo días de sobrevivencia post-infección en comparación con animales controles no tratados. Se sugiere así que la inhibición del metabolismo de la hemoglobina por los antimicóticos azólicos pudiera ser el mecanismo responsable de su actividad antimalárica.

In vitro antifungal susceptibility of Candida spp. oral isolates from HIV-positive patients and control individuals

Oropharyngeal candidiasis is the most common fungal infection among HIV-positive patients. This condition can be treated with either systemic or topical antifungal agents; treatments are usually indicated empirically on the basis of clinical data. The knowledge of in vitro antifungal susceptibility is important to determine correct therapeutic guides for the treatment of fungal infections. Therefore, the objective of this study was to determine the antifungal susceptibility profile of oral Candida isolates from HIV-positive patients and control individuals. Amphotericin B, fluconazole, flucytosine, nystatin and ketoconazole were tested according to the methodology of microdilution proposed by the Clinical and Laboratory Standards Institute (CLSI); results were recorded in values of minimal inhibitory concentration (MIC). A total of 71 Candida isolates from HIV-positive patients were examined with the following species represented: C. albicans (59), C. tropicalis (9), C. glabrata (1), C. guilliermondii (1) and C. krusei (1). A total of 15 Candida isolates were evaluated from control individuals comprised of 11 C. albicans and 4 C. tropicalis samples. Our results demonstrated that the tested antifungal agents showed good activity for most isolates from both groups; however, variability in MIC values among isolates was observed.

Sporothrix schenckii associated with armadillo hunting in Southern Brazil: epidemiological and antifungal susceptibility profiles / Sporothrix schenckii relacionado à caça ao tatu no Sul do Brasil: aspectos epidemiológicos e suscetibilidade dos isolados aos antifúngicos

INTRODUCTION: Sporotrichosis is the most common subcutaneous mycosis observed in Brazil and it is generally consequent to a little trauma caused by vegetal particles or spines which inoculate the fungi in the subcutaneous area. Although sporotrichosis had been frequently mentioned with armadillo hunting this form has not been widely reported in Brazil until now. In this study we report ten cases of sporotrichosis evolving the armadillo's hunting diagnosed in some towns located in the central and west regions of Rio Grande do Sul State. METHODS: The cases were established based on clinical and classic mycological laboratorial techniques. The susceptibility tests were conducted by microdilution technique according to M38-A2 CLSI documents. RESULTS: Ten cases of sporotrichosis associated with armadillo hunting detected in the State of Rio Grande do Sul were diagnosed by mycological methods. The susceptibility tests of Sporothrix schenckii isolates to antifungal agents itraconazole, ketoconazole and terbinafine showed that all the isolates were susceptible. CONCLUSIONS: The paper discusses some cultural aspects related to hunting of this wild animal as well as possible causes of this unexpected occurrence in southern Brazil.

INTRODUÇÃO: A esporotricose constitui-se na micose subcutânea mais frequentemente observada e, na maioria dos casos, a infecção é decorrente de pequenos traumas envolvendo fragmentos vegetais ou espinhos que inoculam o fungo no tecido subcutâneo. Embora frequentemente relacionada a caça a tatus, esta ocorrência tem sido raramente relatada no Brasil. Neste estudo relatamos dez casos envolvendo esta prática, observados em várias cidades das regiões centro e oeste do Estado do Rio Grande do Sul. MÉTODOS: o diagnóstico clínico foi confirmado pelos métodos clássicos de cultura em ágar Mycobiotic, identificação micromorfológica seguida de reversão a fase leveduriforme em ágar BHI. Os testes de suscetibilidade foram realizados pela técnica de microdiluição em caldo, de acordo com as normas estabelecidas pelo documento CLSI M38-A2 (2008). RESULTADOS: A esporotricose, decorrente de lesões causadas pela caça ao tatu foi confirmada pelo métodos microbiológicos. Os testes de suscetibilidade indicaram que todos os isolados eram sensíveis ao itraconazol, cetoconazol e terbinafina. CONCLUSÕES: O artigo discute aspectos ambientais e culturais relacionados a caça a este animal silvestre bem como àqueles relacionados a esta inesperada ocorrência.

Resposta in vitro de fungos agentes de micoses cutâneas frente aos antifúngicos sistêmicos mais utilizados na dermatologia / In vitro response of cutaneous mycosis fungal agents to the most widely used systemic antifungals in dermatology

FUNDAMENTOS - A alta frequência das micoses cutâneas justifica a necessidade de avaliar a possível contribuição da determinação do perfil de susceptibilidade aos antifúngicos in vitro. OBJETIVO - Avaliar se existe variabilidade nos isolados fúngicos quanto à susceptibilidade in vitro de fungos filamentosos, previamente isolados de micoses cutâneas, frente aos antifúngicos fluconazol, cetoconazol, itraconazol e terbinafina. MÉTODOS - Os fungos foram isolados e identificados por meio da metodologia clássica e o teste de susceptibilidade aos antifúngicos foi realizado segundo o método de microdiluição em caldo, de acordo com protocolo preconizado pelo Clinical Laboratory Standards Institute (CLSI), documento M38-A. RESULTADOS - Das 80 amostras de fungos filamentosos identificadas, o gênero Trichophyton representou 81 por cento. As quatro drogas analisadas apresentaram grande variação nos gêneros Trichophyton e Microsporum. O gênero Fusarium foi resistente a todas as drogas testadas. A terbinafina foi o antimicótico mais eficaz contra a maioria dos isolados fúngicos. CONCLUSÃO - Houve uma grande variabilidade nos perfis de resposta aos antifúngicos testados. O estabelecimento de um método-teste de referência permitirá ao clínico maior objetividade na escolha de uma terapia adequada.

BACKGROUND - The high frequency of cutaneous mycosis justify the need to evaluate the possible contribution of in vitro profile of susceptibility to antifungal agents. OBJECTIVE - To evaluate whether there is variability in in vitro susceptibility by filamentous fungi, previously isolated from cutaneous mycosis, to fluconazole, ketoconazole, itraconazole and terbinafine. METHODS - Fungi were isolated and identified by classical methods and the antifungal susceptibility test was performed using the method of broth microdilution, according to a protocol recommended by the Clinical Laboratory Standards Institute (CLSI), through M38-A document. RESULTS - Amongst the 80 filamentous fungi identified, Trichophyton genus represented 81 percent. The four examined drugs showed great variation for Trichophyton spp and Microsporum spp. Fusarium spp was resistant to all tested drugs. Terbinafine was the most effective drug against the majority of the isolated fungi. CONCLUSION - There was great variability in response profiles to the tested antifungals. The definition of a reference test method will offer higher objectivity for physicians to choose the appropriate therapy.

Alterations in growth and branching of Neurospora crassa caused by sub-inhibitory concentrations of antifungal agents / Alteraciones de crecimiento y ramificación en Neurospora crassa provocadas por concentraciones subinhibitorias de agentes antimicóticos

Six antifungal agents at subinhibitory concentrations were used for investigating their ability to affect the growth and branching in Neurospora crassa. Among the antifungals herein used, the azole agent ketoconazole at 0.5 μg/ml inhibited radial growth more than fluconazole at 5.0 μg/ml while amphotericin B at 0.05 μg/ml was more effective than nystatin at 0.05 μg/ml. Morphological alterations in hyphae were observed in the presence of griseofulvin, ketoconazole and terbinafine at the established concentrations. The antifungal agents were more effective on vegetative growth than on conidial germination. Terbinafine markedly reduced growth unit length (GU) by 54.89%, and caused mycelia to become hyperbranched. In all cases, there was a high correlation between hyphal length and number of tips (r > 0.9). All our results showed highly significant differences by ANOVA, (p < 0.001, α = 0.05). Considering that the hyphal tip is the main interface between the fungus and its environment /through which enzymes and toxins are secreted and nutrients absorbed, it would not be desirable to obtain a hyperbranched mycelia with inefficient doses of antifungal drugs.

Se investigó el efecto de seis agentes antimicóticos en concentraciones subinhibitorias sobre el crecimiento y la ramificación en Neurospora crassa. El agente azólico ketoconazol a la concentración de 0,5 μg/ml inhibió el crecimiento radial más que el fluconazol a 5,0 μg/ml, y la anfotericina B a 0,05 μg/ ml fue más eficiente que 0,05 μg/ml de nistatina, entre los agentes poliénicos usados. En presencia de griseofulvina, ketoconazol y terbinafina a las concentraciones establecidas se observaron alteraciones morfológicas en las hifas. Los agentes antimicóticos fueron más eficientes sobre el crecimiento vegetativo que sobre la germinación conidial. La terbinafina redujo marcadamente (54,89%) la longitud de la unidad de crecimiento y provocó la hiperramificación del micelio. En todos los casos, existió gran correlación entre la longitud y el número de ápices de las hifas (r > 0,9). Todos los resultados mostraron diferencias altamente significativas de acuerdo con ANOVA (p < 0,001, α = 0,05). Considerando que el ápice de la hifa es la principal interfase entre el hongo y su ambiente, a través de la cual las enzimas y las toxinas son secretadas y los nutrientes son absorbidos, un micelio hiperramificado resultante de dosis ineficientes de agentes antimicóticos sería perjudicial.

In vitro susceptibility testing of dermatophytes isolated in Goiania, Brazil, against five antifungal agents by broth microdilution method / Teste de suscetibilidade in vitro de dermatófitos isolados em Goiânia, Brasil, contra cinco agentes antifúngicos pelo método de microdiluição em caldo

The antifungal activities of fluconazole, itraconazole, ketoconazole, terbinafine and griseofulvin were tested by broth microdilution technique, against 60 dermatophytes isolated from nail or skin specimens from Goiania city patients, Brazil. In this study, the microtiter plates were incubated at 28 ºC allowing a reading of the minimal inhibitory concentration (MIC) after four days of incubation for Trichophyton mentagrophytes and five days for T. rubrum and Microsporum canis. Most of the dermatophytes had uniform patterns of susceptibility to the antifungal agents tested. Low MIC values as 0.03 µg/mL were found for 33.3 percent, 31.6 percent and 15 percent of isolates for itraconazole, ketoconazole and terbinafine, respectively.

Atividades antifúngicas de fluconazol, itraconazol, cetoconazol, terbinafina e griseofulvina foram testadas pelo método de microdiluição em caldo contra 60 isolados de dermatófitos. Os resultados mostraram que todos os isolados produziram crescimento claramente detectável a 28 ºC e a concentração inibitória mínima (CIM) foi determinada após quatro dias de incubação para Trichophyton mentagrophytes e cinco dias para T. rubrum e Microsporum canis. A maioria dos isolados teve um padrão uniforme de suscetibilidade para os agentes antifúngicos testados. Baixos valores de CIM como 0,03 µg/mL foram encontrados para 33,3 por cento, 31,6 por cento e 15 por cento dos isolados para itraconazol, cetoconazol e terbinafina, respectivamente.

[Antifungal activities of fluconazole and ketoconazole agents against Malassezia species]

Malassezia, a yeast-like fungus found in normal skin flora is known to be associated with various skin diseases, along with systemic infections. Our aim was to determine the in -vitro susceptibility of Malassezia spp. to ketoconazole and fluconazole. In this study, we identified 99 Malassezia isolates from patients with pityriasis vesicular by morphological and biochemical criteria. In vitro susceptibility testing was in macro-broth dilutions, conducted based on the National Committee for Clinical Laboratory Standards [NCCLS] M27-A proposed standard. The results were analyzed statistically by Mann-Whitney. The Malassezia isolates were identified as M. globosa [42], M. furfur [39], M. obtusa [10], M. sympodialis [6], and M. slooffiae [2]. The rate of MFC of ketoconazole against Malassezia spp. was 0.06-2 micro g/ml, while the MFC of fluconazole against Malassezia spp. was 2-64 micro g/ml. The minimal inhibitory concentration [MIC90] of ketoconazole against Malassezia spp. was 0.03-1 micro g/ml, while the minimal inhibitory concentration [MIC90] of fluconazole against Malassezia spp. was 0.5-32 micro g/ml. Although fluconazole can be an effective treatment option for pityriasis versicolor, in our study, fluconazole MICs were higher than ketoclonazole