Quercetin and rutin as potential agents antifungal against Cryptococcus spp / Quercetina e rutina: potenciais agentes para terapia antifúngica

Abstract Amphotericin B is a fungicidal substance that is treatment of choice for most systemic fungal infections affecting as cryptococcosis the immunocompromised patients. However, severe side effects have limited the utility of this drug. The aim of this study was to evaluate the antifungal effect of the combination of amphotericin B with quercetin or rutin and as a protective of citotoxic effect. The antifungal activity to amphotericin B, quercetin and rutin alone and in combination was determined in Candida sp and Cryptococcus neoformans strains. Cytotoxicity test on erythrocytes was performed by spectrophotometric absorbance of hemoglobin. The amphotericin B MIC was reduced when used in combination with quercetin or rutin to C. neoformans ATCC strain and reduced when combined with rutin to a clinical isolate of C. neoformans. In addition, the combination of quercetin with amphotericin B may reduce the toxicity of amphotericin B to red blood cells. Our results suggest that quercetin and rutin are potential agents to combine with amphotericin B in order to reduce the amphotericin dose to lessen side effects and improve antifungal efficacy.

Resumo A anfotericina B é uma substância fungicida e é o tratamento de escolha para a maioria das infecções fúngicas sistêmicas que afetam os pacientes imunocomprometidos, como a criptococose. No entanto, as severas reações adversas têm limitado a utilização desta droga. O objetivo deste estudo foi avaliar o efeito antifúngico e o potencial efeito protetor de citotoxicidade da combinação de anfotericina B com quercetina ou rutina. A atividade antifúngica de anfotericina B, quercetina e rutina, isoladamente e em combinação foi determinada em cepas de Candida sp e Cryptococcus neoformans. O teste de citotoxicidade em eritrócitos foi realizado por espectrofotometria, através da determinação da absorbância da hemoglobina. A concentração inibitória mínima da anfotericina B foi reduzida quando utilizada em combinação com a quercetina e rutina em C. neoformans ATCC e reduzida quando combinados com rutina em um isolado clínico de C. neoformans. Além disso, a combinação de quercetina com anfotericina B pode reduzir a toxicidade da droga em eritrócitos. Os resultados sugerem que quercetina e rutina são potenciais agentes para combinação com anfotericina B, a fim de reduzir a dose de anfotericina, diminuindo os efeitos colaterais e melhorando sua eficácia antifúngica.

THE COPPER INTERFERENCE WITH THE MELANOGENESIS OF Cryptococcus neoformans / A interferência de cobre na melanogênese de Cryptococcus neoformans

Melanin is a pigment produced by laccase, a phenoloxydase enzyme, and is related to the virulence of Cryptococcus neoformans as it is also considered an adaption mechanism to environmental conditions and protection against UV radiation, phagocytic system attack and antifungal drugs. Laccase synthesis is stimulated by several factors, including copper metabolism. The current study shows C. neoformans strains with higher melanization intensity when grown in L-dopa medium supplemented with different concentrations of copper sulfate. This increase shows that melanization rates may be enhanced in the presence of copper ions and may also enhance the virulence of C. neoformans in infected patients that present increasing copper concentrations in serum, such as those with HIV. The virulence of these strains may also be increased in the environment, where this metal is available as CuSO4 in algicidal and fungicidal compounds.

A melanina é um pigmento produzido pela enzima lacase, uma fenoloxidase, e está associada à virulência de Cryptococcus neoformans sendo considerada mecanismo de adaptação às condições ambientais e proteção contra a radiação UV, ataque do sistema fagocítico e antifúngicos. A lacase tem sua síntese estimulada por diversos fatores, incluindo o metabolismo de cobre. Este estudo mostra linhagens de C. neoformans com maior intensidade de melanização quando cultivadas em meio L-dopa suplementado com diferentes concentrações de sulfato de cobre. Este aumento demonstra que as taxas de melanização podem ser aumentadas na presença de íons cobre e também aumentar a virulência de C. neoformans em pacientes infectados que apresentam aumento nas concentrações séricas de íons cobre tais como pacientes com HIV. A virulência destas linhagens também pode ser incrementada no meio ambiente, onde este metal está disponível como CuSO4 em compostos algicidas e fungicidas.

Techniques for the detection of pathogenic Cryptococcus species in wood decay substrata and the evaluation of viability in stored samples

In this study, we evaluated several techniques for the detection of the yeast form of Cryptococcus in decaying wood and measured the viability of these fungi in environmental samples stored in the laboratory. Samples were collected from a tree known to be positive for Cryptococcus and were each inoculated on 10 Niger seed agar (NSA) plates. The conventional technique (CT) yielded a greater number of positive samples and indicated a higher fungal density [in colony forming units per gram of wood (CFU.g-1)] compared to the humid swab technique (ST). However, the difference in positive and false negative results between the CT-ST was not significant. The threshold of detection for the CT was 0.05.10³ CFU.g-1, while the threshold for the ST was greater than 0.1.10³ CFU-1. No colonies were recovered using the dry swab technique. We also determined the viability of Cryptococcus in wood samples stored for 45 days at 25ºC using the CT and ST and found that samples not only continued to yield a positive response, but also exhibited an increase in CFU.g-1, suggesting that Cryptococcus is able to grow in stored environmental samples. The ST.1, in which samples collected with swabs were immediately plated on NSA medium, was more efficient and less laborious than either the CT or ST and required approximately 10 min to perform; however, additional studies are needed to validate this technique.

Morfología atípica de Cryptococcus neoformans en tejidos autopsiados / Atypical morphology of Cryptococcus neoformans in post-mortem tissues

Cryptococcus neoformans es el agente de la Blastomicosis Europea, Torulosis o Cryptococcosis. Causa enfermedad en pacientes inmunocomprometidos. Estos hongos viven en distintos ambientes, penetran a los pulmones por inhalación y por diseminación hematógena, pueden comprometer el cerebro, meninges y otros órganos. En los tejidos invadidos y en su habitat natural se presenta con igual morfología, como levaduras gemantes globosas a ovales, de 4 a 7 u de diámetro aunque pueden verse células de 2 a 15 u, rodeadas de una gran cápsula de mucopolisacáridos que duplica o triplica el diámetro de las células. Típicamente son células simples o con un brote, en ciertas lesiones pueden encontrarse cadenas cortas de 2 a 3 células. Las levaduras están ampliamente separadas por espacios ocupados por sus cápsulas. El objetivo de este trabajo es informar la morfología atípica de un aislamiento de C. neoformans en tejidos de órganos: hígado, bazo, riñón y cerebro obtenidos por autopsia de una persona de sexo masculino de 33 años de edad. La Identificación de C. neoformans fue realizada por sus caracteres morfológicos, cultivos y fenotípicos. En hígado, bazo y riñón se observaron abundantes levaduras grmantes con gran cápsula. Algunas levaduras estaban unidas a las células madres por una base ancha, lo cual no es característica de la especie. Presentaba marcada asociación entre células formando estructuras caprichosas y muy desarrolladas con escasas formas típicas del hongo y pseudomicelios. En cerebro fue más frecuente la observación de elementos gemantes simples.

C. neoformans is the agent of the Cryptococcosis, European Blastomycosis or Torulosis. This fungus can cause disease in normal individuals and in inmunocompromised patients. Infection follows inhalation, but shows a remarkable propensity to spread hematogenously to the brain and meninges and even other organs. C. neoformans is an encapsulated budding yeasts, globular to oval in shape, 4 to 7 u in diameter not including capsules, but cells from 2 to 15 u may occur. The yeasts are surrounded by a large mucopolysaccharide capsule which duplicates or triplicates the cell diameter. Cells are typically single or with one bud, but may be found in short chains of 2 to 3 cells. The yeasts are clearly separated by spaces occupied by capsules. The aim of this work is to communicate the atypical morphology presented by a C. neoformans strain isolated from liver, spleen, kidney and brain tissue. Tissues were obtained through autopsy of a 33-year-old male patient without accurate cause of death. The identification of C. neoformans was made by its morphological, culture and phenotypical characteristics. In liver, spleen and kidney were observed budding yeast abundant with large capsule and numerous buds. The buds were linked to mather cells by a wide base, which is not characteristic of the species. Showed a strong association between cells, without letting go of the parent cell, were capricious and highly developed structures. Presented few typical forms of the fungus and pseudomycelia rudimentary. In brain was more often observed budding simple elements.

Terbinafine inhibits Cryptococcus neoformans growth and modulates fungal morphology

Cryptococcus neoformans is an encapsulated fungus that causes cryptococcosis. Central nervous system infection is the most common clinical presentation followed by pulmonary, skin and eye manifestations. Cryptococcosis is primarily treated with amphotericin B (AMB), fluconazole (FLC) and itraconazole (ITC). In the present work, we evaluated the in vitro effect of terbinafine (TRB), an antifungal not commonly used to treat cryptococcosis. We specifically examined the effects of TRB, either alone or in conjunction with AMB, FLC and ITC, on clinical C. neoformans isolates, including some isolates resistant to AMB and ITC. Broth microdilution assays showed that TRB was the most effective drug in vitro. Antifungal combinations demonstrated synergism of TRB with AMB, FLC and ITC. The drug concentrations used for the combination formulations were as much as 32 and 16-fold lower than the minimum inhibitory concentration (MIC) values of FLC and AMB alone, respectively. In addition, calcofluor white staining revealed the presence of true septa in hyphae structures that were generated after drug treatment. Ultrastructural analyses demonstrated several alterations in response to drug treatment, such as cell wall alterations, plasma membrane detachment, presence of several cytoplasmic vacuoles and mitochondrial swelling. Therefore, we believe that the use of TRB alone or in combination with AMB and azoles should be explored as an alternative treatment for cryptococcosis patients who do not respond to standard therapies.

Criptococcose cutânea primária em paciente imunocompetente / Primay cutaneous cryptococcosis in an immunocompetent patient

Os autores relatam caso de Criptococcose cutânea primária, causada pelo Cryptococcus neoformans, em paciente imunocompetente, fazendeiro que desenvolveu extensivas lesões, no antebraço, após injúria provocada por galináceo, quando fazia limpeza de seu celeiro. Tratamento oral com fluconazol resultou em cura total. A literatura relata raridade de criptococcose cutânea primária em imunocompetentes e sua relativa frequência em imunodeprimidos.

The authors report a primary cutaneous cryptococcosis, caused by Cryptococcus neoformans in immunocompetent patient, a farmer who developed extensive lesions at the site of an injury caused by one of the chickens on his right forearm, while he was cleaning out his barn. Oral treatment with fluconazole was totally successful. A review of the literature showed the rarity of cutaneous cryptococcosis in immunocompetent patients and in contrast, that skin lesions frequently occur in immunocompromised patients.

Different culture media containing methyldopa for melanin production by Cryptococcus species / Avaliação de diferentes meios de cultura contendo metildopa para a produção de melanina por espécies de Cryptococcus

INTRODUCTION: Melanin production by species of Cryptococcus is widely used to characterize C. neoformans complex in mycology laboratories. This study aims to test the efficacy of methyldopa from pharmaceutical tablet as a substrate for melanin production, to compare the production of melanin using different agar base added with methyldopa, and to compare the melanin produced in those media with that produced in Niger seed agar and sunflower seed agar by C. neoformans, C. laurentii, and C. albidus. Two isolates of each species, C. neoformans, C. laurentii, and C. albidus, and one of Candida albicans were used to experimentally detect conditions for melanin production. METHODS: The following media were tested: Mueller-Hinton agar (MHA), brain and heart infusion agar (BHIA), blood agar base (BAB), and minimal medium agar (MMA), all added with methyldopa, and the media Niger seed agar (NSA) and sunflower seed agar (SSA). RESULTS: All isolates grew in most of the culture media after 24h. Strains planted on media BAB and BHIA showed growth only after 48h. All isolates produced melanin in MMA, MHA, SSA, and NSA media. CONCLUSIONS: Methyldopa in the form pharmaceutical tablet can be used as a substrate for melanin production by Cryptococcus species; minimal medium plus methyldopa was more efficient than the BAB, MHA, and BHIA in the melanin production; and NSA and SSA, followed by MMA added with methyldopa, were more efficient than other media studied for melanin production by all strains studied.

INTRODUÇÃO: A produção de melanina por espécies de Cryptococcus é uma característica amplamente utilizada em laboratórios de micologia para caracterização do complexoC. neoformans. O objetivo deste estudo foi verificar a eficácia da metildopa na forma farmacêutica de comprimido, como substrato para a produção de melanina por Cryptococcus, comparar diferentes bases de meios de cultura acrescidas de metildopa para produção de melanina e comparar o pigmento produzido nestes meios com o produzido em ágar Níger e ágar girassol por C. neoformans, C. laurentii e C. albidus. MÉTODOS: Foram testados dois isolados de cada uma das espécies, C. neoformans, C.laurentii e C.albidus, e um de C. albicans para avaliar a produção de melanina nos meios de cultura ágar Müeller-Hinton (MH), ágar brain heart infusion (BHI), ágar base sangue (BS), meio mínimo (MM), todos acrescidos de metildopa, e ainda ágar girassol e ágar Níger. RESULTADOS: Todos os isolados cresceram na maioria dos meios após 24h. O crescimento nos meios BS e BHI somente ocorreu após 48h. Todos os isolados produziram melanina nos meios MM, MH, girassol e Niger. CONCLUSÕES: A metildopa de origem farmacêutica pode ser utilizada como substrato para a produção de melanina por espécies de Cryptococcus; o MM acrescido de metildopa mostrou-se mais eficiente na produção de melanina do que os meios BS, MH e BHI; ágar girassol e ágar Níger seguidos de MM acrescido de metildopa foram os mais eficientes na produção de melanina pelos isolados estudados.

Desarrollo de Cryptococcus neoformans en medios de cultivo empleados para diagnóstico bacteriológico / Growth of Cryptococcus neoformans in culture media for bacteriologic diagnosis

Se evaluó la capacidad de desarrollo de una cepa de Cryptococcus neoformans en cinco medios de cultivo (sin antibióticos antibacterianos) empleados habitualmente para el diagnóstico bacteriológico: agar sangre de carnero (ASC), chocolate, cisteína lactosa deficiente en electrolitos (CLDE), eosina-azul de metileno y Salmonella-Shigella (SS). Una suspensión de la cepa Cn 003, de la colección de la Cátedra de Micología, fue sembrada en la superficie de estos medios contenidos en placas de Petri que se incubaron a 28 ºC y 37 ºC, durante una semana. Excepto en el SS, C. neoformans desarrolló colonias evidenciables a simple vista en los medios evaluados. En ASC y agar chocolate, a 37 ºC desarrollaron colonias mucoides mientras que a 28 ºC, éstas fueron pastosas o cremosas. En todos los medios y a las temperaturas de incubación empleadas, el tamaño de las colonias fue menor que el alcanzado en el medio de Sabouraud, empleado como control. La falta de reconocimiento de las colonias de Cryptococcus neoformans cuando desarrollan en los medios habitualmente empleados en el diagnóstico bacteriológico puede provocar la falta de diagnóstico de la criptococosis, cuando ésta no es sospechada o bien cuando las colonias desarrollan a partir de muestras no habituales.

The ability of C. neoformans to grow in five culture media usually employed in bacteriologic diagnosis (blood agar, chocolate agar, CLDE, eosin-metilen blue and Salmonella-Shigella agars) was evaluated. A Cn 003 strain suspension, obtained from our collection was sown in these media contained in Petri dishes and incubated at 28 ºC and 37 ºC during one week. Except in SS agar, C. neoformans developed visually observed colonies. In ASC and chocolate agar, mucous colonies developed at 37 ºC, whereas at 28 ºC, they were pasty and creamy. The size of the colonies was, in all the bacteriological media and temperatures employed, smaller than that observed on Sabouraud agar, used as control. No recognition of the Cryptococcus neoformans colonies developed in culture media usually employed for the bacteriologic diagnosis can cause a lack of cryptococcosis' diagnosis, when this disease is not suspected or develops from unusual samples.

Actividad de fosfolipasa y susceptibilidad in vitro a fluconazol en aislamientos clínicos y aviarios de Cryptococcus neoformans / Phospholipase activity and in vitro susceptibility to fluconazol in clinical and aviars isolates of Cryptococcus neoformans

Se detectó la actividad de fosfolipasa en 19 cepas clínicas y 17 aviarias de C. neoformans var. neoformans, usando Agar Sabouraud con yema de huevo, incubándose a 37ºC por 5 días. Se determinó el índice Pz estableciéndose los siguientes rangos: Pz muy alto (0.9-1), alto (0.89-0.80), bajo (0.79-0.70) y muy bajo (<0.69). El 84 por ciento de las cepas clínicas presentaron índice Pz muy bajo, el 5 por ciento bajo y 11 por ciento muy alto. Mientras en las cepas aviarias el 82 por ciento presentaron índice muy bajo y un 18 por ciento muy alto. Los valores de Pz promedio fueron muy bajos en todos los aislamientos, sin existir diferencias significativas (p > 0,05) entre las cepas clínicas y aviarias, lo que implica una alta actividad enzimática. La susceptibilidad in vitro a Fluconazol se realizó por el método de difusión con discos y el 89,5 por ciento de las cepas clínicas fueron sensibles y el 10,5 por ciento resistentes, mientras en las cepas aviarias, el 59 por ciento fueron sensibles, 29 por ciento sensible dosis dependiente y un12 por ciento resistentes.

Phospholipase activity was determined to 19 clinical and 17 aviars trains of C. neoformans var. neoformans, incubating the yeast for 5 days at 37º C on Sabouraud Agar supplemented with egg yolk. Pz values were determined and the following ranges were established: very high (0.9-1), high (0.89-0.80), low (0.79-0.70) and very low (<0.69). The 84 percent of the clinical isolates showed Pz values very low (5 percent) and 11 percent very high. On the other hand, the 82 percent of the aviars strains presented Pz values very low and 18 percent very high. Average Pz values were very low in all isolates , there were no statically significant differences (p>0.05) implying a high enzymatic activity. Susceptibility in vitro testing to Fluconazole was performed by a disk diffusion method (M44-A).The 89.5 percent of the clinical isolates were susceptible and 10.5 percent resistant, while in avian strains, 59 por ciento were susceptible, 29 percent susceptible dose dependent and 12 percent resistant.

Estudio de una cepa de Cryptococcus Neoformans pobremente capsulada, en un paciente con SIDA / Fungal meningitis (Cryptococcus neoformans) in a male patient with AIDS

Se estudió la cepa de Cryptococcus neoformans var. neoformans aislada de LCR de un paciente con SIDA. Con el material se realizaron exámenes directos con tinta china, cultivos y prueba de aglutinación de partículas de látex sensibilizadas. La cepa aislada se identificó mediante auxanograma de hidratos de carbono, pruebas de ureasa y de Salkin. Con el fin de estimular la producción de la cápsula se la sembró en agar chocolate y se la inoculó en ratones Rockland por vías intracraneal e intraperitoneal. Se observó que la cepa era pobremente capsulada en los exámenes directos y pobremente capsulada en los cultivos. Se observó el mayor tamaño de su cápsula en el examen directo del cerebro del ratón. La presentación de cepas pobremente capsuladas en pacientes inmunosuprimidos ya fue observada por otros autores. Se postula la posibilidad que, en estos pacientes, el grado de inmunodeficiencia pueda afectar la infectividad y patogenicidad de estas variedades de C. neoformans

Estudio comparativo de dos sistemas de hemocultivos en micosis sistémicas asociadas al SIDA / Two blood culture systems for the detection of fungemia in AIDS patients: a comparative study

Se realizó un estudio comparativo de dos métodos de hemocultivos para hongos en pacientes VIH positivos que presentaban fiebre prolongada o una micosis sistémica confirmada. La técnica convencional consistió en sembrar 5-10 ml de sangre venosa en frascos con caldo tripteina-soya y polianetol sulfonato de sodio como anticoagulante. A los 7 y 14 días de incubación a 37-C se realizaron repiques en medios de agar-miel de Sabouraud y agar-infusión de cerebro y corazón. El método de centrifugación-lisis se llevó a cabo con 5 a 10 ml de sangre venosa heparinizada, se separó el plasma por centrifugación: el sedimento fue tratado con una solución de saponina al 0.5%, después de obtenida la lisis, la muestra fue nuevamente centrifugada y el material restante se sembró en agar-miel de Sabouraud y agar-infusión de cerebro y corazón, la incubación se realizó a 28-C y 37-C. Fueron estudiadas 288 muestras, que se procesaron simultáneamente por los dos sistemas. La técnica convencional permitió el aislamiento de Cryptococcus neoformans en el 7.9%de los materiales, en tanto que el Histoplasma capsulatum, fue aislado en el 1%. El sistema de centrifugación-lisis recuperó el C. neoformans en el 7.3%de los hemocultivos y el H. capsulatum en el 3.8%. El lapso medio para el aislamiento e identificación de ambos hongos disminuyó 7 días, con el método de centrifugación-lisis. Esta última técnica resultó ser más simple, más rápida y más sensible que el procedimiento convencional. Se comprobaron fungemias en el 86.2%de los casos de criptococosis y en el 70.5%de las histoplasmosis asociadas al SIDA

Método fluorescente (diacetato de fluoresceina e brometo de etidio) para o estudo da viabilidade de Cryptococcus neoformans em líquor / Fluorescent method (fluorescein diacetate and ethidium bromide) to study the viability of Criptococcus neoformans in liquor

Padronizou-se método de fluorescência (soluçäo de diacetato de fluoresccína DF e brometo de etídio BE) para análise de viabilidade de células fúngicas, em 40 amostras de líquor, provenientes de casos comprovados de neurocriptococose. A utilizaçäo de soluçäo aquosa de saponina a 0,3% eliminou fluorescências interferentes emitidas por hemácias e leucócitos. Após o processamento dos materiais biológicos, foram retiradas alíquotas de 0,1ml das supensöes obtidas e misturadas a volumes iguais da soluçäo DF-BE preparada pouco antes do uso. O tempo de coloraçäo ideal foi de 30 minutos, resultando perfeita direnciaçäo entre microrganismos viáveis (fluorescência verde) e näo viáveis (fluorescência vermelha)