A case of sporotrichosis caused by different Sporothrix brasiliensis strains: mycological, molecular, and virulence analyses

BACKGROUND Sporotrichosis is a subcutaneous mycosis caused by dimorphic pathogenic fungi belonging to the Sporothrix genus. Pathogenic Sporothrix species typically produce melanin, which is known to be a virulence factor. OBJECTIVES The aim of this study was to perform phenotypic, genotypic, and virulence analyses of two distinct Sporothrix brasiliensis strains isolated from the same lesion on a patient from Rio de Janeiro. METHODS AND FINDINGS Genotypic analyses by partial sequencing of the calmodulin, β-tubulin, and chitin synthase genes, as well as polymerase chain reaction (PCR)-fingerprinting by T3B, M13, and GACA, showed that the isolates were very similar but not identical. Both isolates had similar phenotypic characteristics and effectively produced melanin in their yeast forms, accounting for their ability of causing disease in a murine sporotrichosis model. Remarkably, isolate B was albino in its environmental form but caused more severe disease than the pigmented A isolate. CONCLUSIONS These findings indicate that the patient was infected by two genetically and biologically distinct S. brasiliensis that vary in their production of melanin in their environmental forms. The results underscore the importance of characterizing phenotypically different isolates found in the same clinical specimen or patient.

Fungal infection control by garlic extracts (Allium sativum L) and modulation of peritoneal macrophages activity in murine model of sporotrichosis / Controle da infecção fúngica pelos extratos de alho (Allium sativum L) e modulação da atividade de macrófagos peritoneais em modelo murino de esporotricose

Abstract Garlic (Allium sativum L.) is grown all over the world as seasoning and medicinal vegetable since 3,000 BC. Allicin is the main component of garlic, being attributed to it the most of its biological activities, such as bactericidal, antifungal and antiviral actions. However, other compounds of garlic present antioxidant, hypocholesterolemic, vasodilator activities, protective action against different types of cancer, and immunomodulatory. Fungal infections are important causes of morbidity and mortality in people mainly in immunosuppressed ones. Sporothrix schenckii, the causing agent of Sporotrichosis (most common subcutaneous mycosis in Latin America), is dimorphic fungus, of saprophytic life in soil or plants, infecting people and animals mainly through skin injuries and bruises. The main of this work was to evaluate the influence of garlic consuming on immune modulation of healthy and infected Swiss mice in induced way by S. schenckii, since these animals functioning of peritoneal macrophages as well as the nitric oxide and cytokines' production (IL-1β, IL-10 and IL-12) and to evaluate the antifungal potential of garlic with S. schenckii through minimum inhibitory concentration test and colony-forming units. The results showed that garlic offers antifungal potential with S. schenckii. The oral taking of garlic extracts influences the releasing of cytokines by macrophages, regular consuming shows anti-inflammatory effect, and its acute use may take to an inflammatory response. Mice that consumed garlic responded more effectively to fight against the infection.

Resumo O alho (Allium sativum L.) é cultivado em todo o mundo como hortaliça condimentar e medicinal desde 3.000 a. C. A alicina é o principal componente do alho, sendo atribuída a ela a maior parte das suas atividades biológicas, dentre elas as ações bactericida, antifúngica e antiviral. Porém, outros compostos do alho apresentam atividade antioxidante, hipocolesterolemiante, vasodilatadora, ação protetora contra diversos tipos de câncer e imunomoduladora. As infecções por fungos são causas importantes de morbidade e mortalidade no homem principalmente em indivíduos imunossuprimidos. O Sporothrix schenckii, agente causal da esporotricose (micose subcutânea mais comum na América Latina), é fungo dimórfico, de vida saprofítica no solo ou em vegetais, infectando homens e os animais principalmente através de lesões e arranhões na pele. O objetivo desse trabalho foi avaliar a influência do consumo de alho na imunomodulação de camundongos Swiss saudáveis e infectados de forma induzida por S. schenckii, a partir do estado funcional dos macrófagos peritoneais desses animais quanto à produção de óxido nítrico e das citocinas (IL-1β, IL-10 e IL-12) e avaliar o potencial antifúngico do alho frente ao S. schenckii por meio de teste de concentração inibitória mínima e unidades formadoras de colônia. Os resultados demonstraram que o alho apresenta potencial antifúngico frente S. schenckii. A administração oral de extratos de alho influencia a liberação de citocinas por macrófagos, o consumo regular apresenta efeito anti-inflamatório, e seu uso agudo pode gerar uma resposta inflamatória. Camundongos que consumiram alho responderam de forma mais efetiva no combate da infecção.

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.

Cytotoxicity and anti-Sporothrix brasiliensis activity of the Origanum majorana Linn. oil

Abstract The study aimed to evaluate the anti-Sporothrix sp. activity of the essential oil of Origanum majorana Linn. (marjoram), its chemical analysis, and its cytotoxic activity. A total of 18 fungal isolates of Sporothrix brasiliensis (n: 17) from humans, dogs and cats, and a standard strain of Sporothrix schenckii (n: 1) were tested using the broth microdilution technique (Clinical and Laboratory Standard Institute - CLSI M27-A3) and the results were expressed in minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC). The MIC50 and MIC90 of itraconazole against S. brasiliensis were 2 µg/mL and 8 µg/mL, respectively, and the MFC50 and MFC90 were 2 µg/mL and >16 µg/mL, respectively, with three S. brasiliensis isolates resistant to antifungal. S. schenckii was sensitive at MIC of 1 µg/mL and MFC of 8 µg/mL. For the oil of O. majorana L., all isolates were susceptible to MIC of ≤2.25-9 mg/mL and MFC of ≤2.25-18 mg/mL. The MIC50 and MIC90 were ≤2.25 mg/mL and 4.5 mg/mL, respectively, and the MFC50/90 values were twice more than the MIC. Twenty-two compounds were identified by gas chromatography with a flame ionization detector (CG-FID) and 1,8-cineole and 4-terpineol were the majority. Through the colorimetric (MTT) assay, the toxicity was observed in 70-80% of VERO cells between 0.078 and 5 mg/mL. For the first time, the study demonstrated the satisfactory in vitro anti-Sporothrix sp. activity of marjoram oil and further studies are needed to ensure its safe and effective use.

Adamantylidene-substituted alkylphosphocholine TCAN26 is more active against Sporothrix schenckii than miltefosine

Sporotrichosis is the most frequent subcutaneous mycosis in the world and its increasing incidence has led to the search for new therapeutic options for its treatment. In this study, we demonstrated that three structural analogues of miltefosine (TCAN26, TC19, and TC70) showed inhibitory activity against Sporothrix schenckii sensu stricto and that TCAN26 was more active in vitro than miltefosine against several isolates. Scanning electron microscopy showed that S. schenckii exposure to TCAN26 resulted in cells that were slightly more elongated than untreated cells. Transmission electron microscopy showed that TCAN26 treatment induced loss of the regular cytoplasmic electron-density and altered the cell envelope (disruption of the cell membrane and cell wall, and increased cell wall thickness). Additionally, TCAN26 concentrations required to kill S. schenckii cells were lower than concentrations that were cytotoxic in mammalian cells, and TCAN26 was more selective than miltefosine. Thus, the adamantylidene-substituted alkylphosphocholine TCAN26 is a promising molecule for the development of novel antifungal compounds, although further investigations are required to elucidate the mode of action of TCAN26 in S. schenckii cells.

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.

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.

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.

Suscetibilidade in vitro de isolados de Sporothrix schenckii frente à terbinafina e itraconazol / In vitro susceptibility of isolates of Sporothrix schenckii to terbinafine and itraconazole

O estudo objetiva determinar a atividade in vitro da terbinafina e itraconazol através da técnica de microdiluição em caldo (NCCLSM27-A2) adaptado para um fungo dimórfico frente a 12 isolados de Sporothrix schenckii, sendo seis de esporotricose felina, três de esporotricose humana, um isolado de cão e dois isolados humanos provenientes do Instituto Oswaldo Cruz (IOC). O inóculo e as concentrações antifúngicas foram distribuídas em microplacas, as quais foram incubadas a 35°C por cinco dias, quando foi realizada a leitura da concentração inibitória mínima. A concentração inibitória mínima para a terbinafina variou de 0,055µg/ml a 0,109µg/ml e para o itraconazol de 0,219µg/ml a 1,75µg/ml, sendo que para ambos os fármacos as CIMs entre os isolados do IOC foi de 0,875µg/ml. O estudo demonstrou uma alta suscetibilidade do Sporothrix schenckii frente à terbinafina, necessitando mais estudos que correlacionem os testes in vitro frente ao fármaco com a resposta clínica em pacientes com esporotricose.

The study objective was to determine the in vitro activity of terbinafine and itraconazole through the microdilution technique in broth (NCCLSM27-A2), adapted for dimorphic fungus, in relation to 12 isolates of Sporothrix schenckii. Six were from feline sporotrichosis, three from human sporotrichosis, one from a dog and two from human isolates originating from Instituto Oswaldo Cruz. The inoculum and antifungal concentrates were distributed on microplates that were incubated at 35°C for five days. Minimum inhibitory concentration readings were made at the end of this period. The MIC for terbinafine ranged from 0.055µg/ml to 0.109µg/ml, and the MIC for itraconazole ranged from 0.219µg/ml to 1.75µg/ml. For both drugs, the MIC from the isolates from IOC was 0.875µg/ml. The present study demonstrates the high susceptibility of Sporothrix schenckii to terbinafine. Further studies to correlate the in vitro susceptibility tests with the clinical response of patients with sporotrichosis are needed.

In vitro susceptibility pattern of Sporothrix schenckii strains isolated from three centers in India.

BACKGROUND & OBJECTIVES: With the availability of more number of antifungal agents in recent years, drugs other than saturated solution of potassium iodide (SSKI) are being increasingly used to treat sporotrichosis. It was therefore considered pertinent to evaluate in vitro antifungal susceptibility pattern of Sporothrix schenckii strains isolated at three centers in India against five commonly used antifungal agents. METHODS: Agar dilution method was used to evaluate 50 clinical isolates (25 from north, 17 from east and 8 from south India) both in its yeast and mycelial forms against amphotericin-B, 5-fluorocytosine, ketoconazole, fluconazole and itraconazole. RESULTS: No resistance was observed in the yeast form of S. schenckii against amphotericin B and azoles. However, 54 per cent strains in the yeast form were resistant to 5-fluorocytosine. None of the strains was susceptible to amphotericin B and ketoconazole, 56 and 10 per cent strains in the mycelial form were susceptible to itraconazole and fluconazole respectively. No significant difference was observed in the antifungal susceptibility pattern among the strains isolated from these three regions in India. INTERPRETATION & CONCLUSION: Clinical isolates of S. schenckii from three regions of India had a more or less uniform antifungal susceptibility pattern. Itraconazole had the best in vitro susceptibility results against the clinical isolates of S. schenckii and has the potential to replace SSKI.

Itraconazol no tratamento da esporotricose: relato de 3 casos / Itraconsazole in the treatment of sporothricosis: report of three case

O itraconazol é um composto triazólico primariamente fungistático que age alterando a permeabilidade da célula fúngica. Tem mostrado maior atividade contra o Sporothrix schnckii quando comparado a outros antifúngicos. Os autores descrevem três casos de esporotricose na forma cutâneo-linfática tratados com itraconazol na dose de 100 mg por dia, via oral, durante três meses aproximadamente, com completa cicatrizaçäo das lesöes