In vitro sensitivity of Malassezia furfur isolates from HIV-positive and negative patients to antifungal agents / Sensibilidad in vitro a antifúngicos de aislamientos de Malassezia furfur de pacientes positivos y negativos para HIV

Introduction. Malassezia is a lipophilic and lipid-dependent yeast genus belonging to the skin microbiota of humans and other animals. However, due to dysbiosis processes or other factors in the host, this yeast can cause different pathologies, ranging from skin diseases, such as seborrheic dermatitis, to fungemia. Isolation of Malassezia furfur has been reported in HIV-positive patients with or without skin lesions. Due to its opportunistic nature and its variable resistance to antifungal compounds, it is relevant to know the Malassezia sensitivity profiles. Objective. To determine the sensitivity to different antifungal agents, of clinical isolates of M. furfur obtained from HIV-positive or negative patients, with or without seborrheic dermatitis. Materials and methods. Assessment of isolates sensitivity to itraconazole, voriconazole, fluconazole, and amphotericin B was performed by two techniques: (1) Broth microdilution using Clinical and Laboratory Standards Institute (CLSI) protocol M27-A3 with modifications; and (2) agar tests using Etest®. Results. Isolates obtained from HIV patients showed an increase in the minimum inhibitory concentration of fluconazole, voriconazole, and amphotericin B, compared with those of non-HIV patients. Itraconazole was the antifungal with the lowest minimum inhibitory concentration (MIC) in most isolates. Conclusion. We observed differences in the sensitivity profiles of M. furfur isolates according to the context of the patient. High MIC of antifungals like fluconazole, commonly used for treating pathologies caused by Malassezia, were identified.

Introducción. Malassezia es un género de levaduras lipofílicas que dependen de los lípidos y hacen parte de la microbiota de la piel de humanos y otros animales. No obstante, debido a procesos de disbiosis u otros factores en el huésped, esta levadura puede llegar a causar diferentes enfermedades: desde cutáneas (como dermatitis seborreica) hasta fungemias. Se han reportado aislamientos de Malassezia furfur en pacientes positivos para HIV, con lesiones cutáneas o sin ellas. Por su carácter oportunista y sensibilidad variable a los compuestos antifúngicos, es relevante conocer los perfiles de sensibilidad. Objetivo. Determinar la sensibilidad a diferentes antifúngicos de aislamientos clínicos de M. furfur obtenidos de pacientes positivos o negativos para HIV, con dermatitis seborreica o sin ella. Materiales y métodos. La sensibilidad de los aislamientos a itraconazol, voriconazol, fluconazol y anfotericina B, se determinó mediante dos técnicas: microdilución en caldo según el protocolo M27-A3 del Clinical & Laboratory Standards Institute (CLSI), con modificaciones, y pruebas en agar mediante Etest®. Resultados. Los aislamientos obtenidos de pacientes con HIV mostraron aumento de la concentración inhibitoria mínima a fluconazol, voriconazol y anfotericina B, en comparación con los de pacientes sin HIV. Por otro lado, al evaluar la mayoría de los aislamientos, el itraconazol fue el antifúngico con la menor concentración inhibitoria mínima. Conclusión. Se evidencian diferencias en los perfiles de sensibilidad de los aislamientos de M. furfur, según el contexto del paciente, y elevadas concentraciones inhibitorias mínimas de antifúngicos como el fluconazol, usados comúnmente para el tratamiento de las enfermedades causadas por Malassezia spp.

Refractory feline sporotrichosis: a comparative analysis on the clinical, histopathological, and cytopathological aspects / Esporotricose felina refratária: uma análise comparativa sobre os aspectos clínico, histopatológico e citopatológico

Sporotrichosis is a chronic fungal infection caused by Sporothrix species. The occurrence of cases that are resistant to long-term treatment, especially in the nasal planum of cats, emphasizes the importance of studying its pathogenesis. The purpose of this study was to analyze and compare the inflammatory process of cutaneous lesions of feline refractory sporotrichosis to clinical aspects through cytopathological and histopathological examination. Moreover, the study included 13 cats with cutaneous lesions that had been resistant to itraconazole treatment for more than a year. Cutaneous lesions samples were collected for cytopathological, histopathological, and fungal culture analyses. Tissue fragments were processed and stained with hematoxylin-eosin (HE) and Grocott methenamine silver (GMS). Further, two clinical presentations had the highest occurrence: the localized cutaneous form in animals with good general condition and stable disease (n=9, 69.2%) and the disseminated cutaneous form in cats with poor general condition (n=4, 30.8%). In cats with refractory sporotrichosis, the nasal planum (84.6%) was the most common location of lesions. In the cytopathological study, cats with fewer than two lesions and in good general condition (n=9, 69.2%) showed absence or mild yeast intensity (up to 5 yeasts per field), lower intensity of macrophages and neutrophils, and higher intensity of epithelioid cells, lymphocytes, plasma cells, and eosinophils. On the other hand, (n=4, 30.8%) of the cats with disseminated sporotrichosis and a poor general condition had a marked intensity of yeasts, which were mostly phagocytosed by an increased number of macrophages and neutrophils. Of those animals with good general condition, the majority (n=6, 66.7%) had higher eosinophil intensity. In histopathology, malformed suppurative granuloma was the predominant type (n=9, 69.2%) in feline sporotrichosis lesions, followed by well-formed granulomas (n=4, 30.8%). Malformed granulomas showed mild to moderate fungal intensity (55.6%) in animals with good general condition and localized lesions while marked fungal intensity (44.4%) in cats with the disseminated form of the disease and poor general condition. Well-formed granulomas (n=4, 30.7%) had mild to moderate intensity of fungal load, and 75% of the animals with this type of granuloma had only one lesion and were in good general condition. Long-term itraconazole treatment in these cats with refractory sporotrichosis can keep the infection under control and localized lesions stable; however, fungus reactivation can occur, resulting in an exuberant and inefficient immune response.(AU)

A esporotricose é uma infecção fúngica crônica causada por espécies do gênero Sporothrix. A ocorrência de casos refratários ao tratamento de longo prazo, especialmente na região nasal de gatos, alerta para a importância do estudo de sua patogênese. Este estudo teve como objetivo analisar o processo inflamatório das lesões cutâneas de esporotricose felina refratária ao tratamento, por meio de exames citopatológico e histopalógico, e comparar com aspectos clínicos. Treze gatos com lesões cutâneas refratárias ao tratamento com Itraconazol por mais de um ano foram incluídos no estudo. Amostras de lesões cutâneas foram coletadas para análises citopatológicas, histopatológicas e cultura fúngica. Fragmentos de tecidos foram processados e corados pela hematoxilina-eosina (HE) e Prata Metenamina de Grocott (GMS). Duas apresentações clínicas tiveram maior ocorrência: a forma localizada cutânea em animais com bom estado geral e doença estável (n=9, 69,2%); e a forma disseminada cutânea em gatos com estado geral ruim (n=4, 30,8%). A região nasal (84,6%) foi a localização mais frequente das lesões nos gatos com esporotricose refratária ao tratamento. No estudo citopatológico, felinos com menos de duas lesões, e em bom estado geral, (n=9, 69,2%) revelaram ausência ou leve intensidade de leveduras (até 5 leveduras por campo), menor intensidade de macrófagos e neutrófilos, e maior intensidade de células epitelióides, linfócitos, plasmócitos e eosinófilos. Enquanto, (n=4, 30,8%) dos felinos que apresentavam a forma disseminada da esporotricose associada a um estado geral ruim, revelaram acentuada intensidade de leveduras em sua maioria fagocitadas por acentuado número de macrófagos e neutrófilos. Dos animais com bom estado geral, a maioria (n=6, 66,7%) apresentava maior intensidade de eosinófilos. Na histopatologia, o granuloma supurativo mal formado foi o tipo predominante (n=9, 69,2%) nas lesões de esporotricose felina, e (n=4, 30,8%) foram de granulomas bem formados. Granulomas mal formados apresentaram leve a moderada intensidade fúngica (55,6%) nos animais com bom estado geral e lesões localizadas, e acentuada intensidade fúngica (44,4%) nos gatos com a forma disseminada da doença e estado geral ruim. Granulomas bem formados (n=4, 30,7%) apresentaram leve a moderada intensidade de carga fúngica, sendo 75% dos animais com esse tipo de granuloma com apenas uma lesão e bom estadogeral. O tratamento de longo prazo com itraconazol, nesses gatos com esporotricose refratária, pode manter a infecção controlada e lesões localizadas estáveis de forma temporária, contudo a reativação do fungo pode ocorrer levando a uma resposta imunológica exuberante e ineficiente.(AU)

Refractory feline sporotrichosis: a comparative analysis on the clinical, histopathological, and cytopathological aspects / Esporotricose felina refratária: uma análise comparativa sobre os aspectos clínico, histopatológico e citopatológico

Sporotrichosis is a chronic fungal infection caused by Sporothrix species. The occurrence of cases that are resistant to long-term treatment, especially in the nasal planum of cats, emphasizes the importance of studying its pathogenesis. The purpose of this study was to analyze and compare the inflammatory process of cutaneous lesions of feline refractory sporotrichosis to clinical aspects through cytopathological and histopathological examination. Moreover, the study included 13 cats with cutaneous lesions that had been resistant to itraconazole treatment for more than a year. Cutaneous lesions samples were collected for cytopathological, histopathological, and fungal culture analyses. Tissue fragments were processed and stained with hematoxylin-eosin (HE) and Grocott methenamine silver (GMS). Further, two clinical presentations had the highest occurrence: the localized cutaneous form in animals with good general condition and stable disease (n=9, 69.2%) and the disseminated cutaneous form in cats with poor general condition (n=4, 30.8%). In cats with refractory sporotrichosis, the nasal planum (84.6%) was the most common location of lesions. In the cytopathological study, cats with fewer than two lesions and in good general condition (n=9, 69.2%) showed absence or mild yeast intensity (up to 5 yeasts per field), lower intensity of macrophages and neutrophils, and higher intensity of epithelioid cells, lymphocytes, plasma cells, and eosinophils. On the other hand, (n=4, 30.8%) of the cats with disseminated sporotrichosis and a poor general condition had a marked intensity of yeasts, which were mostly phagocytosed by an increased number of macrophages and neutrophils. Of those animals with good general condition, the majority (n=6, 66.7%) had higher eosinophil intensity. In histopathology, malformed suppurative granuloma was the predominant type (n=9, 69.2%) in feline sporotrichosis lesions, followed by well-formed granulomas (n=4, 30.8%). Malformed granulomas showed mild to moderate fungal intensity (55.6%) in animals with good general condition and localized lesions while marked fungal intensity (44.4%) in cats with the disseminated form of the disease and poor general condition. Well-formed granulomas (n=4, 30.7%) had mild to moderate intensity of fungal load, and 75% of the animals with this type of granuloma had only one lesion and were in good general condition. Long-term itraconazole treatment in these cats with refractory sporotrichosis can keep the infection under control and localized lesions stable; however, fungus reactivation can occur, resulting in an exuberant and inefficient immune response.

A esporotricose é uma infecção fúngica crônica causada por espécies do gênero Sporothrix. A ocorrência de casos refratários ao tratamento de longo prazo, especialmente na região nasal de gatos, alerta para a importância do estudo de sua patogênese. Este estudo teve como objetivo analisar o processo inflamatório das lesões cutâneas de esporotricose felina refratária ao tratamento, por meio de exames citopatológico e histopalógico, e comparar com aspectos clínicos. Treze gatos com lesões cutâneas refratárias ao tratamento com Itraconazol por mais de um ano foram incluídos no estudo. Amostras de lesões cutâneas foram coletadas para análises citopatológicas, histopatológicas e cultura fúngica. Fragmentos de tecidos foram processados e corados pela hematoxilina-eosina (HE) e Prata Metenamina de Grocott (GMS). Duas apresentações clínicas tiveram maior ocorrência: a forma localizada cutânea em animais com bom estado geral e doença estável (n=9, 69,2%); e a forma disseminada cutânea em gatos com estado geral ruim (n=4, 30,8%). A região nasal (84,6%) foi a localização mais frequente das lesões nos gatos com esporotricose refratária ao tratamento. No estudo citopatológico, felinos com menos de duas lesões, e em bom estado geral, (n=9, 69,2%) revelaram ausência ou leve intensidade de leveduras (até 5 leveduras por campo), menor intensidade de macrófagos e neutrófilos, e maior intensidade de células epitelióides, linfócitos, plasmócitos e eosinófilos. Enquanto, (n=4, 30,8%) dos felinos que apresentavam a forma disseminada da esporotricose associada a um estado geral ruim, revelaram acentuada intensidade de leveduras em sua maioria fagocitadas por acentuado número de macrófagos e neutrófilos. Dos animais com bom estado geral, a maioria (n=6, 66,7%) apresentava maior intensidade de eosinófilos. Na histopatologia, o granuloma supurativo mal formado foi o tipo predominante (n=9, 69,2%) nas lesões de esporotricose felina, e (n=4, 30,8%) foram de granulomas bem formados. Granulomas mal formados apresentaram leve a moderada intensidade fúngica (55,6%) nos animais com bom estado geral e lesões localizadas, e acentuada intensidade fúngica (44,4%) nos gatos com a forma disseminada da doença e estado geral ruim. Granulomas bem formados (n=4, 30,7%) apresentaram leve a moderada intensidade de carga fúngica, sendo 75% dos animais com esse tipo de granuloma com apenas uma lesão e bom estadogeral. O tratamento de longo prazo com itraconazol, nesses gatos com esporotricose refratária, pode manter a infecção controlada e lesões localizadas estáveis de forma temporária, contudo a reativação do fungo pode ocorrer levando a uma resposta imunológica exuberante e ineficiente.

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.

A Ten-year Retrospective Study of Invasive Candidiasis in a Tertiary Hospital in Beijing / 生物医学与环境科学（英文）

Objective@#This study aimed to evaluate the epidemiological, clinical and mycological characteristics of invasive candidiasis (IC) in China.@*Methods@#A ten-year retrospective study including 183 IC episodes was conducted in a tertiary hospital in Beijing, China.@*Results@#The overall incidence of IC from 2010-2019 was 0.261 episodes per 1,000 discharges. Candidemia (71.0%) was the major infective pattern; 70.3% of the patients tested positive for @*Conclusion@#The incidence of IC has declined in the recent five years.

Antifungal susceptibility profile of Candida clinical isolates from 22 hospitals of São Paulo State, Brazil

This study aimed to evaluate the frequency of cryptic Candida species from candidemia cases in 22 public hospitals in São Paulo State, Brazil, and their antifungal susceptibility profiles. During 2017 and 2018, 144 isolates were molecularly identified as 14 species; C. parapsilosis (32.6%), C. albicans (27.7%), C. tropicalis (14.6%), C. glabrata (9.7%), C. krusei (2.8%), C. orthopsilosis (2.8%), C. haemulonii var. vulnera (2.1%), C. haemulonii (1.4%), C. metapsilosis (1.4%), C. dubliniensis (1.4%), C. guilliermondii (1.4%), C. duobushaemulonii (0.7%), C. kefyr (0.7%), and C. pelliculosa (0.7%). Poor susceptibility to fluconazole was identified in 6.4% of C. parapsilosis isolates (0.12 to >64 µg/mL), 50% of C. guilliermondii (64 µg/mL), 66.6% of C. haemulonii var. vulnera (16-32 µg/mL), and C. duobushaemulonii strain (MIC 64 µg/mL). Our results corroborated the emergence of C. glabrata in Brazilian cases of candidemia as previously reported. Importantly, we observed a large proportion of non-wild type C. glabrata isolates to voriconazole (28.6%; <0.015 to 4 µg/mL) all of which were also resistant to fluconazole (28.6%). Of note, C. haemulonii, a multidrug resistant species, has emerged in the Southeast region of Brazil. Our findings suggested a possible epidemiologic change in the region with an increase in fluconazole-resistant species causing candidemia. We stress the relevance of routine accurate identification to properly manage therapy and monitor epidemiologic trends.

Candida auris osteomielitis: Case report / Osteomielitis por Candida auris: reporte de caso

Abstract Introduction: Candida auris is an opportunistic yeast associated with multiple infections, which was first reported in 2009 in Tokyo, Japan. Provided that it has great antifungal resistance to azoles and amphotericin B, its treatment options are limited, and therefore an empiric therapy using echinocandins such as micafungin should be considered. Case presentation: A rare case of a 48-year-old male patient with osteomyelitis caused by C. auris was reported in the city of Popayán, Colombia. The patient had a history of femoral head fracture, paraplegia due to firearm-related injury and neurogenic bladder, and reported having experienced abundant purulent foul-smelling secretions through trochanteric right ulcer for 15 days. MRI images revealed myositis and bone intensity alterations, which allowed diagnosing him with osteomyelitis. Due to repeated isolations of C. haemulonii in several bone samples, antifungal management was initiated. However, since no improvement in the patient's condition was observed, a culture was sent to the Colombian National Institute of Health to identify the pathogen considering the repeated isolations of C. haemulonii and its apparent resistance to antifungals. C. auris was finally confirmed as the pathogen. Conclusion: Osteomyelitis by C. auris is a rare entity, which must be considered when treating patients with predisposing risk factors such as long hospital stays, bearing in mind that this is an inpatient-associated opportunistic infection.

Resumen Introducción. Candida auris es una levadura oportunista asociada a múltiples infecciones que, en 2009, fue descrita por primera vez en Tokio, Japón. Dado que tiene una gran resistencia antifúngica a los azoles y a la anfotericina B, su manejo es limitado, por lo que se debe considerar iniciar un tratamiento empírico con equinocandinas como la micafungina. Presentación de caso. Caso inusual de osteomielitis por C. auris en un hombre de 48 años de Popayán, Colombia, con antecedentes de fractura de cabeza de fémur, paraplejia por herida con arma de fuego y vejiga neurogénica. El paciente tenía cuadro clínico de 15 días de evolución consistente en salida abundante de líquido purulento fétido en úlcera derecha por presión trocantérica. Mediante resonancia magnética se identificaron miositis y alteraciones de intensidad ósea, por lo que fue diagnosticado con osteomielitis. Debido a la identificación de aislamientos repetidos de Candida haemulonii en varias muestras óseas, se inició manejo antifúngico; sin embargo, ya que no se observó ninguna mejora en la condición del paciente, el cultivo fue enviado al Instituto Nacional de Salud para confirmar la identificación del patógeno debido a aislamientos repetidos de C. haemulonii y su aparente resistencia a los antifúngicos. Finalmente, el patógeno identificado fue C. auris. Conclusión. La osteomielitis por C. auris es una entidad inusual cuyo diagnóstico debe ser considerado en pacientes con factores de riesgo predisponente, como aquellos con larga estancia hospitalaria, ya que esta es una infección oportunista asociada a pacientes hospitalizados.

Azoles de antes y ahora: una revisión / Azoles of then and now: a review

Resumen Los azoles son fármacos que inhiben la enzima 14α-esteroldemetilasa, impidiendo la unión de ergosterol; esto altera la estructura y función de la pared celular fúngica. Especialmente el grupo de los triazoles: fluconazol, itraconazol, voriconazol, posaconazol e isavuconazol, son una alternativa farmacológica para el tratamiento de la enfermedad fúngica invasora causada por Aspergillus spp, Candida spp, Cryptococcus spp, patógenos emergentes como los Mucorales, y de micosis endémicas como las ocasionadas por Histoplasma spp y Coccidioides spp. Los efectos adversos de los triazoles son menos frecuentes comparados con los ocasionados por anfotericina B, un antifúngico de uso común para estas micosis. Los principales efectos adversos de los triazoles son hepáticos, gastrointestinales y cardiovasculares como la prolongación del intervalo QT. Las interacciones farmacológicas son usuales y se presentan con moléculas que usan sustratos del citocromo CYP3A4, lo que incluye anti-retrovirales, anti-tuberculosos e inmunomoduladores. En este trabajo se revisan la historia, características farmacológicas y los ensayos clínicos que evidencian su eficacia clínica en los diferentes escenarios clínicos.

Abstract The azoles are drugs that inhibit the 14α-sterol-demethylase enzyme preventing the binding of ergosterol, altering the functionality and structure of the fungal cell wall. Especially the group of triazoles: fluconazole, itraconazole, voriconazole, posaconazole and isavuconazole, are a pharmacological alternative for the treatment of the invasive fungal disease, caused by Aspergillus spp, Candida spp, Cryptococcus spp, by emerging pathogens for example, the Mucoral and finally of endemic mycosis as those caused by Histoplasma spp. and Coccidioides spp. The adverse effects of the triazoles are less frequent compared to those caused by amphotericin B, the main ones being hepatics, gastrointestinals and cardiovasculars, such as the prolongation of the QT interval. The pharmacological interactions are common and occur with molecules that use the substrates of the CYP3A4 cytochrome, for example: antiretroviral, anti-tuberculous and immunomodulators. The history, pharmacological characteristics and clinical trials are reviewed.

Análisis cuantitativo de la expresión de genes de resistencia a fluconazol en cepas de Candida albicans aisladas al ingreso de adultos mayores a una unidad de cuidados intensivos de Manizales, Colombia / Quantitative analysis of the expression of fluconazole-resistant genes in strains of Candida albicans isolated from elderly people at their admission in an intensive care unit in Manizales, Colombia

Introducción. Las infecciones oportunistas asociadas con Candida albicans han tenido gran repercusión en la salud pública por la mortalidad que generan en determinados grupos poblacionales. Aunque existen tratamientos farmacológicos disponibles, es evidente el aumento de la resistencia desarrollada por el agente patógeno, por lo que la determinación de los mecanismos de resistencia de las cepas presentes en las áreas hospitalarias es importante, ya que permitiría plantear mejores esquemas de tratamiento. Objetivo. Analizar la expresión de los genes ERG11, CDR1 y MDR1 en cepas de C. albicans aisladas de adultos mayores a su ingreso en la unidad de cuidados intensivos del Hospital Santa Sofía de Manizales, Colombia. Materiales y métodos. Se seleccionaron 29 muestras (21 resistentes y 8 sensibles) y se conformaron dos grupos de trabajo, uno de muestras con exposición al fluconazol y el otro sin esta. El ARN extraído se cuantificó mediante reacción en cadena de la polimerasa con transcriptasa inversa en tiempo real (RT-qPCR). Resultados. Se encontraron diferencias significativas en la expresión del gen MDR1 en el grupo de cepas de C. albicans resistentes. Dos de las cepas resistentes (104 y 62-2) expuestas al antifúngico presentaron valores muy elevados en la expresión de este gen. La expresión del ERG11 y del CDR1 no fue significativa en los grupos estudiados. Conclusión. El aumento de sobreexpresión del gen MDR1 indica que este puede ser el responsable de la resistencia; sin embargo, algunas cepas resistentes no sobreexpresaron los genes analizados, lo que indica que puede haber otros genes involucrados en la resistencia de las cepas estudiadas.

Introduction: Opportunistic infections associated with Candida albicans have had a great impact on public health due to the mortality they generate in certain population groups. Although pharmacological treatments are available, the resistance developed by the pathogen has become increasingly evident. For this reason, determining the mechanisms of resistance associated with the strains found in different hospital areas is important since it would help improving treatment plans. Objective: To analyze the expression of ERG11, CDR1, and MDR1 genes in strains of C. albicans isolated from elderly patients at admittance in the intensive care unit of Hospital Santa Sofía in Manizales, Colombia. Materials and methods: A total of 29 samples (21 resistant and 8 sensitive) were selected and distributed in two working groups: with and without exposure to fluconazole. The extracted RNA was quantified by real-time reverse transcription polymerase chain reaction (RT-qPCR). Results: Significant differences were found in the expression of the MDR1 gene in the group of resistant C. albicans strains. Two of the resistant strains (104 and 62-2) exposed to the antifungal showed very high values in the expression of this gene. The expression of ERG11 and CDR1 was not significant among the groups studied. Conclusion: The increased overexpression of the MDR1 gene indicates that it may be responsible for the resistance. However, some resistant strains did not overexpress any of the genes analyzed, which indicates that there may be other genes involved in the resistance of the strains under study.

Tolerance of dark septate endophytic fungi (DSE) to 1 agrochemicals in vitro / Tolerancia de hongos endofíticos septados oscuros a agroquímicos in vitro

Abstract Dark septate endophytes (DSE) are a heterogeneous group of fungi, mostly belonging to the Phylum Ascomycota, that are involved in a mutualistic symbiosis with plant roots. The aim of this study is to evaluate the behavior of two strains of DSE isolated from wheat roots of two cropping areas in the province of Buenos Aires, Argentina, against some agrochemicals. Of all the isolates obtained, two strains were identified as Alternaria alternata and Cochliobolus sp. These DSE were found to be tolerant to glyphosate, carbendazim and cypermethrin when evaluated at the recommended agronomic dose (AD), 2 AD and, in some cases, 10 AD. This work contributes to the study of the biology of this group of fungi and their tolerance in the presence of xenobiotics widely used in agriculture.© 2019 Asociaci´on Argentina de Microbiolog´ıa. Published by Elsevier Espa˜na, S.L.U. This is an open access article under the CC BY-NC-ND license (https://creativecommons.org/licenses/bync-nd/4.0/).

Resumen Los endófitos septados oscuros (DSE) son un grupo heterogéneo de hongos que participan de una simbiosis mutualista con raíces de plantas, perteneciendo principalmente al Phylum Ascomycota. El objetivo de este estudio fue aislar DSE de raíces de trigo proveniente de dos áreas de cultivo de la provincia de Buenos Aires y evaluar el comportamiento de dos cepas de DSE aisladas de raíces de trigo frente a algunos agroquímicos en dos áreas de cultivo de la provincia de Buenos Aires. De todos los aislamientos obtenidos se seleccionaron dos cepas que se identificaron como Alternaria alternata y Cochliobolus sp. Se encontró que estos DSE son tolerantes al glifosato, el carbendazim y la cipermetrina, evaluados a las dosis agronómicas recomendadas (AD), a 2x AD y, en algunos casos, a 10x AD. Este trabajo contribuye al conocimiento de la biología de este grupo de hongos y su tolerancia a xenobióticos ampliamente utilizados en la agricultura.

Antifungal susceptibility profile of Aspergillus fumigatus isolates from avian lungs / Perfil de suscetibilidade antifúngica de isolados de Aspergillus fumigatus provenientes de pulmões de aves

Susceptibility testing is essential to inform the correct management of Aspergillus infections. In this study we present antifungal susceptibility profile of A. fumigatus isolates recovered from lungs of birds with and without aspergillosis. Fifty three isolates were tested for their antifungal susceptibility to voriconazole (VRC), itraconazole (ITZ), amphotericin (AMB) and caspofungin (CSP) using the M38-A2 broth microdilution reference method. Five isolates were resistant to more than one antifungal drug (CSP + AMB, VRC + ITZ and AMB + ITZ). Fifteen (28%) isolates with susceptible increased exposure (I) to ITZ were sensible to VRC. Resistance to AMB (>2µg/mL) was observed in only four isolates. Eleven (21%) A. fumigatus present resistance to ITZ (13%) and VRC (8%). Fungal isolation from respiratory samples has been regarded as being of limited usefulness in the ante mortem diagnosis of aspergillosis in birds. However, the results suggest that the detection and antifungal susceptibility profile may be helpful for monitoring of therapy for avian species and where antifungal resistance might be emerging and what conditions are associated to the event.(AU)

Os testes de suscetibilidade são essenciais para informar o correto manejo das infecções por Aspergillus. Neste estudo apresentamos o perfil antifúngico de isolados de A. fumigatus provenientes de pulmões de aves com e sem aspergilose. Cinqüenta e três isolados foram testados quanto à susceptibilidade antifúngica ao voriconazol (VRC), itraconazol (ITZ), anfotericina B (AMB) e caspofungina (CSP) pelo método de referência de microdiluição do caldo M38-A2. Cinco isolados foram resistentes a mais de um antifúngico (CSP + AMB, VRC + ITZ e AMB + ITZ). Quinze (28%) isolados suscetíveis - com exposição aumentada (I) ao ITZ foram sensíveis ao VRC. A resistência ao AMB (>2µg/mL) foi observada em apenas quatro isolados. Onze (21%) A. fumigatus apresentaram resistência a ITZ (13%) e VRC (8%). O isolamento de fungos de amostras respiratórias tem sido considerado de utilidade limitada no diagnóstico ante mortem de aspergilose em aves. No entanto, os resultados sugerem que a detecção e o perfil de suscetibilidade a antifúngicos podem ser úteis para o monitoramento da terapia de espécies aviárias, assim como a emergência da resistência antifúngica e quais condições podem estar associadas ao evento.(AU)

Relationship between antifungal susceptibility profile and virulence factors in Candida albicans isolated from nail specimens

Abstract INTRODUCTION: The aim of this study was to evaluate some virulence factors in Candida albicans isolates from patients with onychomycosis and determine the correlation between these factors and the antifungal resistance profile. METHODS: Seventy species of C. albicans were confirmed using polymerase chain reaction amplification of the HWP1 gene. According to the Clinical & Laboratory Standards Institute guidelines, the susceptibility profile of four antifungal agents was investigated, and the production of aspartyl protease, phospholipase, haemolysin, and biofilm was determined. The correlation between these profiles was also investigated. RESULTS: The isolates indicated different levels of resistance and production of virulence factors. Significant correlations were observed between the minimum inhibitory concentration (MIC) of fluconazole/itraconazole and biofilm production, between phospholipase production and fluconazole/itraconazole MIC, and between fluconazole MIC and hemolytic activity in C. albicans isolates. The results also showed significant correlations between phospholipase activity and biofilm production. CONCLUSIONS: Our findings will contribute to a better understanding of the pathogenesis of C. albicans and characterize the relationship between virulence factors and antifungal resistance, which may suggest new therapeutic strategies considering the possible involvement of the virulence mechanism in the effectiveness of treatment.

Human fusariosis: An emerging infection that is difficult to treat

Abstract Fusarium spp. has been associated with a broad spectrum of emerging infections collectively termed fusariosis. This review includes articles published between 2005 and 2018 that describe the characteristics, clinical management, incidence, and emergence of these fungal infections. Fusarium solani and F. oxysporum are globally distributed and represent the most common complexes. Few therapeutic options exist due to intrinsic resistance, especially for the treatment of invasive fusariosis. Therefore, the use of drug combinations could be an important alternative for systemic antifungal resistance. Increase in the number of case reports on invasive fusariosis between 2005 and 2018 is evidence of the emergence of this fungal infection.

Eficácia antimicrobiana da terapia fotodinâmica no tratamento da candidíase oral em pessoas vivendo com HIV/Aids / Antimicrobial efficacy of photodynamic therapy on the treatment of oral candidiasis in people living with HIV/Aids / Eficacia antimicrobiana de la terapia fotodinámica en el tratamiento de la candidiasis bucal en personas viviendo con VIH/Sida

Objetivo: avaliar a eficácia antimicrobiana da terapia fotodinâmica no tratamento da candidíase oral em pessoas vivendo com HIV/aids. Método: estudo experimental, qualitativo e descritivo com 18 pessoas vivendo com HIV/aids que manifestavam a candidíase oral, maiores de 18 anos, que estavam em tratamento no Hospital Universitário Gaffrée e Guinle. Este grupo subdividiu-se em um grupo controle, composto por sete pessoas, que recebeu tratamento com a terapia fotodinâmica e antifúngicos, e um grupo experimental, com 11, que recebeu apenas a terapia fotodinâmica. A evolução do tratamento de cada participante foi acompanhada por registros fotográficos em duas consultas, inicial e final. Esta pesquisa foi aprovada pelo Comitê de Ética em Pesquisa do hospital, parecer número 2.431.107. Resultados: a maioria dos participantes apresentou melhora clínica, ainda que discreta, e em apenas um houve piora clínica. Conclusão: a terapia fotodinâmica antimicrobiana pode ser eficaz no tratamento da candidíase oral em pessoas vivendo com HIV/Aids

Objective: evaluate the antimicrobial efficacy of photodynamic therapy in the treatment of oral candidiasis in people living with HIV/ aids. Method: experimental, qualitative and descriptive study with 18 people living with HIV/aids who presented oral candidiasis, over 18 years of age, who were being treated at the Gaffrée and Guinle University Hospital. This group was subdivided into a control group, composed of seven people, who received treatment with photodynamic and antifungal therapy, and an experimental group, with 11, who received only the photodynamic therapy. The evolution of the treatment of each participant was followed by photographic registers in two appointments, initial and final. This research was approved by the Research Ethics Committee from the hospital, dictum number 2.431.107. Results: most of the participants showed clinical improvement, albeit discrete, and in only one there was clinical worsening. Conclusion: antimicrobial photodynamic therapy may be effective in the treatment of oral candidiasis in people living with HIV/Aids

Objetivo: evaluar la eficacia antimicrobiana de la terapia fotodinámica en el tratamiento de la candidiasis bucal en personas que viven con VIH/sida. Método: estudio experimental, cualitativo y descriptivo con 18 personas viviendo con VIH/sida que manifestaban la candidiasis bucal, mayores de 18 años, que estaban en tratamiento en el Hospital Universitario Gaffrée y Guinle. Este grupo se subdividió en grupo control, compuesto por siete personas, que recibió tratamiento con la terapia fotodinámica y antifúngicos, y un grupo experimental, con 11, que recibió sólo la terapia fotodinámica. La evolución del tratamiento de cada participante fue acompañada por registros fotográficos en dos consultas, inicial y final. La investigación fue aprobada por el Comité de Ética en Investigación del lugar, dictamen número 2.431.107. Resultados: la mayoría de los participantes presentó mejoría clínica, aunque discreta, y en apenas uno hubo empeoramiento clínico. Conclusión: la terapia fotodinámica antimicrobiana puede ser eficaz en el tratamiento de la candidiasis bucal en personas que viven con el VIH/Sida

Análise fenotípica de isolados de Cryptococcus neoformans coletados no estado de Roraima, norte do Brasil / Phenotypic analysis of Cryptococcus neoformans isolates collected in the state of Roraima, northern Brazil

As espécies Cryptococcus neoformans e Cryptococcus gatti representam patógenos fúngicos ambientais encapsulados que afetam não apenas indivíduos imunocomprometidos, mas também indivíduos aparentemente imunocompetentes. No Brasil, a criptococose permanece sendo uma das infecções fúngicas sistêmicas mais importantes. Em humanos, a fagocitose de Cryptococcus hipocapsular por macrófagos foi associada com longo prazo de sobrevida do paciente. Com base no papel fundamental da cápsula criptocócica na doença, analisamos a diversidade de estruturas capsulares em 23 isolados de excretas de pombos, coletados nos municípios de Boa Vista, Bonfim e Pacaraima, no estado de Roraima (Norte do Brasil). Todos os isolados foram identificados como C. neoformans (genótipo VNI) por espectrometria de massa MALDI-TOF. Através de uma combinação de microscopia de fluorescência, citometria de fluxo, ELISA e métodos espectrofotométricos, cada isolado foi fenotipicamente caracterizado, o que incluiu medidas de taxas de crescimento a 30 e 37ºC, pigmentação, tamanho do corpo celular, dimensões capsulares, reatividade sorológica, produção de urease e capacidade de secreção de glucuronoxilomanana (GXM), o principal componente capsular de C. neoformans. Foi também avaliada a sensibilidade aos antifúngicos fluconazol e AmB. Com exceção da melanização, uma grande diversidade foi observada considerando todos os parâmetros testados em nosso estudo. Vale ressaltar que isolados hiper e hipo produtores de GXM foram identificados, além de isolados com perfis de hiper e hiporreatividade com o anticorpo monoclonal reconhecedor do polissacarídeo.

As dimensões capsulares também foram altamente variáveis nos isolados analisados. A produção de GXM extracelular foi correlacionada positivamente com dimensões capsulares, atividade da urease e tamanho da célula. Inesperadamente, as concentrações de GXM não se correlacionaram com a reatividade sorológica com a cápsula criptocócica. Os testes de sensibilidade aos antifúngicos revelaram alta sensibilidade dos isolados frente a AmB. Foram observados valores de susceptibilidade superiores para o fluconazol (8.0 µg/mL) em dois isolados (151A1 e 176A1) em comparação com a cepa padrão H99 / ATCC 208821 (2.0 µg/mL). Estes resultados revelam uma alta diversidade na capacidade de C. neoformans ambientais em produzir componentes capsulares, o que pode afetar o curso da criptococose humana. (AU)

Chemical and cytotoxic analyses of three varieties of Brazilian propolis (green propolis, jataí propolis and brown propolis) and its anti-Sporothrix brasiliensis in vitro activity / [Análises químicas e citotóxicas de três variedades de própolis brasileiras (própolis verde, própolis jataí e própolis marrom) e sua atividade in vitro anti-Sporothrix brasiliensis]

In this study, we described the antifungal activity of three Brazilian propolis extracts: brown, green and from jataí bees against Sporothrix brasiliensis. The extracts were obtained from ethanolic extraction and their chemical composition was determined by high-performance liquid chromatography coupled to mass spectrometry. The cellular toxicity was measured in MDBK (Madin-Darby Bovine Kidney) cells and quantified by the MTT assay (3- (4,5 dimethylthiazol-2yl -2,5-diphenyl-2H bromine tetrazolato). For antifungal activity, the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) were determined by broth microdilution. The results showed that cell toxicity was not observed at lower concentrations (0.097 to 0.39μg/ml) for all extracts in comparison to cell control. Among the chemical compounds identified, caffeic acid, p-coumaric acid, chlorogenic acid, ferulic acid and rutin were quantified. In antifungal activity, green and jataí did not exhibit activity against the isolates (MIC and MFC greater than 0.78mg/ml). However, all isolates of S. brasiliensis were sensitive to brown propolis (MIC of 0.09 to 0.78mg/ml), including the standard strain (P<0.001). Among the Brazilian propolis studied, the brown propolis showed activity against the S. brasiliensis isolates and more studies should be undertaken in order to evaluate its promising use in the treatment of sporotrichosis.(AU)

Neste estudo, descreveu-se a atividade antifúngica de três extratos de própolis brasileiras: marrom, verde e de abelhas jataí (Tetragonisca angustula), contra Sporothrix brasiliensis. Os extratos foram obtidos de extração etanólica, e a sua composição química foi determinada por cromatografia líquida de alta eficiência, acoplada à espectrometria de massa. A toxicidade celular foi medida em células MDBK (Madin-Darby Bovine Kidney), avaliada por observação microscópica e quantificada pelo ensaio MTT (3- (4,5-dimetiltiazol-2-ilo -2,5-difenil-2H bromo tetrazolato). Para a atividade antifúngica, determinou-se a concentração inibitória mínima (CIM) e a concentração fungicida mínima (CFM) por meio de microdiluição em caldo. Os resultados mostraram que a toxicidade celular não foi observada em concentrações menores (0,097 a 0,39μg/ml). Entre os compostos químicos identificados, foram quantificados o ácido cafeico, ácido p-cumárico, ácido clorogênico, ácido ferúlico e a rutina. Na atividade antifúngica, as própolis verde e jataí não apresentaram atividade contra os isolados (CIM e CFM maior que 0,78mg/ml), porém todos os isolados de S. brasiliensis foram sensíveis à própolis marrom (CIM de 0,09 a 0,78mg/ml), incluindo a cepa padrão (P<0,001). Entre as própolis brasileiras estudadas, a marrom mostrou atividade contra S. brasiliensis, e mais estudos devem ser realizados para avaliar seu uso promissor no tratamento da esporotricose.(AU)

Fungicidas agrícolas azólicos e pressão seletiva de fenótipos resistentes em Aspergillus fumigatus / Agricultural azole fungicides and selective pressure of resistant phenotypes in Aspergillus fumigatus

A utilização de fungicidas azólicos pode estar ligada a resistência aos fármacos utilizados no tratamento da aspergilose invasiva. Em cepas de Aspergillus fumigatus, foram descritas mutações no gene cyp51A resultando em menor afinidade azol-alvo e resistência antifúngica. Mecanismos de resistência aos triazóis já foram descritos em isolados ambientais de áreas agrícolas e em pacientes sob tratamento. A similaridade dos compostos azólicos, clínicos e agrícolas, e o mesmo mecanismo de ação na célula fúngica podem contribuir para a seleção de cepas com resistência cruzada no hospedeiro e no meio ambiente. O gene cyp51A codifica a enzima-chave na produção de ergosterol da membrana celular-14-demetilase, alvo de todos os compostos azólicos. Mutações no gene cyp51A resultam na produção de enzima com alterações em sua conformação tridimensional levando à menor afinidade azol-alvo e resistência antifúngica. A indução de tais mutações foi atribuída à exposição extensiva das cepas ambientais a agrotóxicos fungicidas triazólicos. Estudos em diversos países sugerem que esses produtos exercem pressão seletiva em fenótipos resistentes de A. fumigatus O objetivo deste estudo foi avaliar a hipótese da origem ambiental de mecanismos de resistência a triazólicos, em 2 isolados clínicos e 2 ambientais de A. fumigatus, modulada pela exposição a um fungicida agrícola. Modelo de horta experimental, parte orgânica e parte exposta a agrotóxico triazólico, foi construída e inoculada com os 4 isolados, todos selvagens não mutantes de A. fumigatus. Ao longo de um ano, 11 pulverizações e recuperações mensais dos 4 isolados a partir de amostras de solo permitiram monitorar possível emergência de resistência aos triazólicos: difenoconazol, metconazol, tebuconazol, itraconazol, voriconazol e posaconazol. A busca da resistência foi feita pela determinação de MIC, tanto de fungicidas quanto de fármacos triazólicos e pesquisa de mutações gênicas no gene cyp51A e sua região promotora. O gene cyp51A sequenciado para um desses isolados não apresentou mutações. Conclui-se que a pulverização de difenoconazol não resultou em alteração de MIC de triazóis nas cepas expostas indicando que não houve seleção de fenótipos resistentes. Outra possibilidade é que, de fato, o tempo de exposição, ou volume/concentração de fungicida não foram suficientes para pressão seletiva. (AU)

Clinical resistance to triazoles, drugs used for invasive aspergillosis, is a growing problem and its relationship with environmental issues is under discussion. The triazolics are also used to control phytopathogenic fungi and the same problem of increasing resistance is present. The relationship between resistance in clinical and environmental strains is based on the same mode of action of the compounds, so that exposure to one triazole compound may select another resistant strain, characterizing "crossresistance". In environmental strains of the main etiological agent of aspergillosis-Aspergillus fumigatus, classified as azole resistant, mutations associated with the cyp51A gene have been described. This gene encodes the key enzyme in the production of cell membrane ergosterol-14-demethylase, target of all azole compounds. Mutations in the cyp51A gene result in enzyme production with changes in its three-dimensional conformation leading to lower target azole affinity and antifungal resistance. The induction of such mutations was attributed to the extensive exposure of environmental strains to triazole fungicide pesticides. Studies in several countries suggest that these products exert selective pressure on resistant A. fumigatus phenotypes. The objective of this study was to evaluate the hypothesis of the environmental origin of triazole resistance mechanisms in 2 clinical isolates and 2 environmental isolates of A. fumigatus, modulated by exposure to an agricultural fungicide. Experimental garden model, organic part and part exposed to triazolic pesticide, was built and inoculated with the 4 isolates, all non-mutant wild A. fumigatus. Over a year, 11 sprayings and monthly recoveries of the 4 isolates from soil samples allowed monitoring of possible emergence of resistance to triazoles: difenoconazole, metconazole, tebuconazole, itraconazole, voriconazole and posaconazole. The search for resistance was made by the search for gene mutations that, so far, has not been found. MIC determination of both fungicides and triazole drugs against isolates, exposed and recovered by the 5th month. The cyp51A gene sequenced for one of these isolates showed no mutations. By microsatellite technique, the same isolate was not similar to the seeded isolate. It was concluded that difenoconazole spraying did not result in alteration of triazole MIC in the exposed strains indicating that there was no selection of resistant phenotypes. On the other hand, it was not possible to correlate the inoculated strain with the recovered strain and evaluated for MIC. Therefore, other recovered strains should be compared to inoculated strains for more grounded conclusion. Another possibility is that, in fact, exposure time, or fungicide volume / concentration was not sufficient for selective pressure. (AU)

A 12-year study of fungal infections in Rio Grande do Sul, southern Brazil

Background: The number of fungal infections has increased in recent years in Rio Grande do Sul (RS), Brazil. Epidemiological studies are important for proper control of infections. Aims: To evaluate the etiology of fungal infections in patients in RS, from 2003 to 2015. Methods: This is a retrospective and longitudinal study carried out at Mycology Department of Central Laboratory of RS; 13,707 samples were evaluated. The variables sex, age, site of infection, and etiologic agent were analyzed. Susceptibility of Candida to fluconazole was tested in samples collected in 2015from 51 outpatients. Results: Of the 13,707 samples, 840 cases (6.12%) of fungal infections were found and included in the analyses; female gender accounted for the 55.9% of the cases. The main fungus was Candida albicans (450 cases, 53.38%; p < 0.001). Onychomycosis was the most frequent infection in superficial mycoses. Systemic mycoses accounted for 54.05% of the cases, from which 68.8% occurred in males, mainly HIVpositive (33.11%), and the main etiologic agent in these cases was Cryptococcus neoformans (73.13%). Among 51 samples tested for susceptibility to fluconazole, 78.43% of Candida isolates were susceptible; 5.88% were susceptible in a dose-dependent manner, and 15.69% were resistant. Conclusions: C. albicans is a common cause of fungal infections in RS, accounting for half of the cases;resistance to antifungals was found in non-hospitalized patients. In addition, women seem to be moresusceptible to fungal infections than men, however men show more systemic mycoses than women. Thenails are the most common site of infection. (AU)