In vitro characterization of Trichophyton rubrum and T. mentagrophytes biofilms.

Dermatophytes are fungi responsible for a disease known as dermatophytosis. Biofilms are sessile microbial communities surrounded by extracellular polymeric substances (EPS) with increased resistance to antimicrobial agents and host defenses. This paper describes, for the first time, the characteristics of Trichophyton rubrum and T. mentagrophytes biofilms. Biofilm formation was analyzed by light microscopy, scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) as well as by staining with crystal violet and safranin. Metabolic activity was determined using the XTT reduction assay. Both species were able to form mature biofilms in 72 h. T. rubrum biofilm produced more biomass and EPS and was denser than T. mentagrophytes biofilm. The SEM results demonstrated a coordinated network of hyphae in all directions, embedded within EPS in some areas. Research and characterization of biofilms formed by dermatophytes may contribute to the search of new drugs for the treatment of these mycoses and might inform future revisions with respect to the dose and duration of treatment of currently available antifungals.

Cryptococcosis: epidemiology, fungal resistance, and new alternatives for treatment.

Cryptococcosis is an important systemic mycosis and the third most prevalent disease in human immunodeficiency virus (HIV)-positive individuals. The incidence of cryptococcosis is high among the 25 million people with HIV/acquired immunodeficiency syndrome (AIDS), with recent estimates indicating that there are one million cases of cryptococcal meningitis globally per year in AIDS patients. In Cryptococcus neoformans, resistance to azoles may be associated with alterations in the target enzyme encoded by the gene ERG11, lanosterol 14α-demethylase. These alterations are obtained through mutations, or by overexpressing the gene encoding. In addition, C. gattii and C. neoformans present a heteroresistance phenotype, which may be related to increased virulence. Other species beyond C. neoformans and C. gattii, such as C. laurentii, have been diagnosed mainly in patients with immunosuppression. Infections of C. albidus have been isolated in cats and marine mammals. Recent evidence suggests that the majority of infections produced by this pathogen are associated with biofilm growth, which is also related with increased resistance to antifungal agents. Therefore, there is a great need to search for alternative antifungal agents for these fungi. The search for new molecules is currently occurring from nanoparticle drugs of plant peptide origin. This article presents a brief review of the literature regarding the epidemiology of cryptococcosis, as well as fungal resistance and new alternatives for treatment.

Microplate alamarBlue assay for Paracoccidioides susceptibility testing.

CLSI method M27-A3 is not available for use with dimorphic fungi, such as those of the Paracoccidioides genus. In this study, we developed a microdilution method and added the alamarBlue reagent to test the responses of Paracoccidioides brasiliensis and Paracoccidioides lutzii against amphotericin B and itraconazole antifungals. The test proved to be sensitive, practical, and inexpensive and can be used to monitor the activity of low-growth microorganisms and their response to various drugs.

Candida species: current epidemiology, pathogenicity, biofilm formation, natural antifungal products and new therapeutic options.

The incidence of fungal infections has increased significantly, so contributing to morbidity and mortality. This is caused by an increase in antimicrobial resistance and the restricted number of antifungal drugs, which retain many side effects. Candida species are major human fungal pathogens that cause both mucosal and deep tissue infections. Recent evidence suggests that the majority of infections produced by this pathogen are associated with biofilm growth. Biofilms are biological communities with a high degree of organization, in which micro-organisms form structured, coordinated and functional communities. These biological communities are embedded in a self-created extracellular matrix. Biofilm production is also associated with a high level of antimicrobial resistance of the associated organisms. The ability of Candida species to form drug-resistant biofilms is an important factor in their contribution to human disease. The study of plants as an alternative to other forms of drug discovery has attracted great attention because, according to the World Health Organization, these would be the best sources for obtaining a wide variety of drugs and could benefit a large population. Furthermore, silver nanoparticles, antibodies and photodynamic inactivation have also been used with good results. This article presents a brief review of the literature regarding the epidemiology of Candida species, as well as their pathogenicity and ability to form biofilms, the antifungal activity of natural products and other therapeutic options.

Adhesion of Histoplasma capsulatum to pneumocytes and biofilm formation on an abiotic surface.

The pathogenic fungus, Histoplasma capsulatum, causes the respiratory and systemic disease 'histoplasmosis'. This disease is primarily acquired via inhalation of aerosolized microconidia or hyphal fragments of H. capsulatum. Evolution of this respiratory disease depends on the ability of H. capsulatum yeasts to survive and replicate within alveolar macrophages. It is known that adhesion to host cells is the first step in colonization and biofilm formation. Some microorganisms become attached to biological and non-biological surfaces due to the formation of biofilms. Based on the importance of biofilms and their persistence on host tissues and cell surfaces, the present study was designed to investigate biofilm formation by H. capsulatum yeasts, as well as their ability to adhere to pneumocyte cells. H. capsulatum biofilm assays were performed in vitro using two different clinical strains of the fungus and biofilms were characterized using scanning electron microscopy. The biofilms were measured using a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium-hydroxide (XTT) reduction assay. The results showed that both the H. capsulatum strains tested were very efficient at adhering to host cells and forming biofilm. Therefore, this is a possible survival strategy adopted by this fungus.

A new fibrin sealant from Crotalus durissus terrificus venom: applications in medicine.

Fibrin sealant, a widely available tissue adhesive, has been used since 1940 in a variety of clinical applications. Commercially available fibrin sealant products are synthesized from bovine thrombin and human fibrinogen, which may transmit infectious diseases, and recipients may also develop antibodies against bovine thrombin. Bearing these disadvantages in mind, a new fibrin sealant was developed in 1989 by a group of researchers from the Center for the Study of Venoms and Venomous Animals, in Sao Paulo State, Brazil. The main purpose was to produce an adhesive fibrin without using human blood, to avoid transmitting infectious diseases. The components of this novel sealant were extracted from large animals and a serine proteinase extracted from Crotalus durissus terrificus snake venom. The applicability of this sealant was tested in animals and humans with beneficial results. The new fibrin sealant can be a useful tool clinically due to its flexibility and diversity of applications. This sealant is a biological and biodegradable product that (1) does not produce adverse reactions, (1) contains no human blood, (3) has a good adhesive capacity, (4) gives no transmission of infectious diseases, and (5) may be used as an adjuvant in conventional suture procedures. The effectiveness of this new fibrin sealant is reviewed and its development and employment are described.

Occurrence of fungi in water used at a haemodialysis centre.

AIMS: The aim of this study was to identify and determine the diversity, occurrence and distribution of fungi in water used at a haemodialysis centre. METHODS AND RESULTS: Samples in the hydraulic circuit for the distribution of the water, dialysate samples and samples of sterilization solution from dialysers were collected over a 3-month period, and 500 ml of each sample was filtered through membranes. All together 116 isolates of fungi were recovered from 89% of all water samples collected inside the haemodialysis unit, with prevalence of moulds in tap water samples and of yeasts in dialysate samples. Fusarium spp. was the most abundant genus found, whereas Candida parapsilosis was the predominant yeast species. CONCLUSIONS: This study demonstrated that various fungi were present in the water system. These data suggest the inclusion of the detection and quantification of fungi in the water of haemodialysis. SIGNIFICANCE AND IMPACT OF THE STUDY: The recovery of fungi from aqueous haemodialysis environments implies a potential risk for haemodialysis patients and indicates the need for continuous maintenance and monitoring. Further studies on fungi in haemodialysis water systems are required to investigate the organism ability to persist, their role in biofilm formation and their clinical significance.

Induction and secretion of elastinolytic and proteolytic activity in cultures of Paracoccidioides brasiliensis

P. brasiliensis parasitizes various human tissues and proteinases exported by this fungus may allow it to metabolize and invade host tissues. The influence of the culture medium on the production of proteinases by P. brasiliensis isolates was studied and the export of these enzymes was followed as a function of culture time. The fungus was grown in neopeptone, BSA, elastin orcollagen medium. The culture medium was assayed for azocollytic, elastinolytic and caseinolytic activity. Proteolytic activity was also analysed by electrophoresis of the culture medium on gelatin and casein substrate gels. P. brasiliensis expressed relatively high levels of azocoll, elastin and casein degrading activity in all types of medium, except in neopeptone medium. Generally, expression of azocollytic activity peaked during the third week of culture and caseinolytic activity during the fourth week of culture. Azocollytic activity was highest at pH 4.0 and caseinolytic activity at pH 8.0. Elastinolytic activity was also highest at pH 8.0. This activity, as well as the others, may provide the fungus with a source of carbon and nitrogen and may alsobe responsible for the invasion of host tissues, such as pulmonary elastic fiber, by P. brasiliensis.

Epithelial cells treated with genistein inhibit adhesion and endocytosis of Paracoccidioides brasiliensis.

Paracoccidioidomycosis is caused by Paracoccidioides brasiliensis, which although not formally considered an intracellular pathogen, can be internalized by epithelial cells in vitro and in vivo. The mechanisms used by P. brasiliensis to adhere to and invade non-professional phagocytes have not been identified. The signal-transduction networks, involving protein tyrosine kinase (PTK) and protein phosphatase activities, can modulate crucial events during fungal infections. In this study, the involvement of PTK has been investigated in P. brasiliensis adherence and invasion in mammalian epithelial cells. A significant inhibition of the fungal invasion occurred after the pre-treatment of the epithelial cells with genistein, a specific tyrosine kinase inhibitor, indicating that the tyrosine kinase pathway is involved in P. brasiliensis internalization. In contrast, when the fungus was treated, a slight (not significant) inhibition of PTK was observed, suggesting that PTK might not be the fungus' transduction signal pathway during the invasion process of epithelial cells. An intense PTK immunofluorescence labeling was observed in the periphery of the P. brasiliensis infected cells, little PTK labeling was found in both uninfected cells and yeast cells, at later infection times (8 and 24 h). Moreover, when the epithelial cells were treated with genistein and infected with P. brasiliensis, no labeling was observed, suggesting the importance of the PTK in the infectious process. These results suggest that PTK pathway participates in the transduction signal during the initial events of the adhesion and invasion processes of P. brasiliensis to mammalian epithelial cells.

Use of fibrin glue derived from snake venom in the repair of deep corneal ulcers: experimental study in dogs (Canis familiaris, Linnaeus, 1758)

Fibrin glue has been researched as an alternative method for tissue synthesis and is known for its capability to promote hemostasis at the application site, good approximation of wound edges and fast healing. The current study consisted in the application of fibrin glue derived from snake venom as treatment for experimental corneal ulcers. Twenty-one dogs had their corneas experimentally prepared through lamellar keratectomy (of standardized diameter and depth). Animals were divided into seven groups of three animals each. Six experimental groups were periodically evaluated and collection was carried out on the 1st, 3rd, 7th, 15th, 30th and 60th post-operative days, whereas one control group was evaluated throughout the experiment. Analyses consisted in the clinical evolution and in the histopathological study of all operated on eyes. Results indicated that fibrin glue was efficient in repairing keratectomy wounds in dogs and contributed to an earlier healing phenomenon, avoiding edema formation and keeping corneal clearness. The use of fibrin glue derived from snake venom showed to be easy to apply, feasible with animal models and of low cost, avoiding the lesion progress and allowing fast and appropriate corneal healing.

Comparative study of disk diffusion and microdilution methods for evaluation of antifungal activity of natural compounds against medical yeasts Candida spp and Cryptococcus sp / Estudo comparativo entre os métodos de difusão em disco e de microdiluição para avaliação de atividade antifúngica de produtos naturais contra as leveduras de interesse médico Candida spp e Cryptococcus sp

Antifungal activity of natural products has been tested by adapting methods designed for synthetic drugs. Inthis study, two methods for the determination of antifungal activity of natural products, agar diffusionand broth microdilution, the CLSI reference methods for synthetic drugs, are compared and discussed. Themicrodilution method was more sensitive. The minimal inhibitory concentrations (MIC) of crude extracts,fractions and pure substances from different species of the plant families Piperaceae, Rubiaceae, Clusiaceae, Fabaceae and Lauraceae, from the Biota project, were determined. Antifungal activities against Candida albicans, C.krusei, C.parapsilosis and Cryptococcus neoformans were produced by several samples

Atividade antifúngica de produtos naturais foi determinada após algumas adaptações de métodos preconizados para fármacos sintéticos. Neste estudo foram comparados e discutidos os métodos para determinação de atividade antifúngica de produtos naturais por duas metodologias, difusão em ágar e microdiluição em caldo, segundo método preconizado pelo CLSI para fármacos sintéticos. A concentração mínima inibitória foi determinada de extratos brutos, frações e de substâncias puras de diferentes espécies de plantas das famílias Piperaceae, Rubiaceae, Clusiaceae, Fabaceae and Lauraceae do projeto Biota. Vários apresentaram atividade antifúngica para as levedurasCandida albicans, C.krusei, C.parapsilosis and Cryptococcus neoformans.

Interação de Paracoccidioides brasiliensis com células endoteliais / Interaction of Paracoccidioides brasilienses with endothelial cells

A paracoccidioidomicose apresenta um amplo espectro de manifestações clínicas e Paracoccidioides brasiliensis, seu agente etiológico, pode atingir vários tecidos com ênfase ao pulmão. A migração de fungos patogênicos através da camada de células endoteliais é considerada pré-requisito para a invasão de múltiplos órgãos e sua disseminação. No presente estudo verificou-se a adesão de P. brasiliensis às células endoteliais in vitro e se esta adesão poderia representar um mecanismo para a disseminação do fungo. Para tanto, além da técnica convencional de microscopia ótica, uma outra metodologia foi desenvolvida, emblocando os cordões umbilicais em parafina, no intuito de detectar o fungo presente no material (in vivo). Experimento de migração de P. brasiliensis através da monocamada de células endoteliais também foi realizado, e nos poços sem células, a migração de células leveduriformes foi maior em menor período de tempo. Os fungos conseguiram passar através da monocamada, quando comparados com o controle sem as células, mas com redução em torno de 30%. Isso mostra que a monocamada foi parcialmente impediente para o fungo, mas que este foi capaz de migrar através dessas células. Em nossos experimentos com estas células, houve grande dificuldade de se encontrar P. brasiliensis aderido ao tapete celular nos períodos de tempo padronizados. Sugere-se com esses resultados que o fungo atravessa as células endoteliais de uma maneira muito rápida, que não pode ser detectada através do cultivo in vitro. Portanto, P. brasiliensis teria capacidade de atravessar rapidamente as células endoteliais e provavelmente alcançar tecidos mais profundos

Cryptococcosis outbreak in psittacine birds in Brazil.

An outbreak of cryptococcosis occurred in a breeding aviary in São Paulo, Brazil. Seven psittacine birds (of species Charmosyna papou, Lorius lory, Trichoglossus goldiei, Psittacula krameri and Psittacus erithacus) died of disseminated cryptococcosis. Incoordination, progressive paralysis and difficulty in flying were seen in five birds, whereas superficial lesions coincident with respiratory alterations were seen in two birds. Encapsulated yeasts suggestive of Cryptococcus sp. were seen in faecal smears stained with India ink in two cases. Histological examination of the birds showed cryptococcal cells in various tissues, including the beak, choana, sinus, lungs, air sacs, heart, liver, spleen, kidneys, intestines and central nervous system. High titres of cryptococcal antigen were observed in the serum of an affected bird. In this case, titres increased during treatment and the bird eventually died. Yeasts were isolated from the nasal mass, faeces and liver of one bird. Cryptococcus neoformans var. gattii serovar B was identified based on biochemical, physiological and serological tests. These strains were resistant (minimum inhibitory concentration 64 microg/ml) to fluconazole. This is the first report of C. neoformans var. gattii occurring in psittacine birds in Brazil.

Epidemiologia das dermatofitoses em instituições públicas da região de Araraquara - SP / Epidemiology of the dermatophytosis in public institutions of the region of Araraquara city - SP

As dermatofitoses são processos infecciosos de pele, pêlo e unhas muito comuns no mundo inteiro. Com o intuito de avaliar a epidemiologia das infecções causadas por estes fungos em Instituições Públicas de Araraquara, 105 amostras de indivíduos com suspeita clínica de dermatofitose foram examinadas no Laboratório de Micologia da Faculdade de Ciências Farmacêuticas, de agosto a dezembro de 2001 e, destas, 47 foram positivas para dermatófitos. Trichophyton rubrum foi a espécie prevalente (56,9%), seguida por Microsporum canis (17%), T. tonsurans (10,6%), T. mentagrophytes (8,5%) e Epidermophyton floccosum (4,3%). T. rubrum foi mais frequente nas lesões dos interdígitos (81,5%) e M. canis foi o principal envolvido nas lesões do couro cabeludo (58,3%). Portanto, houve predomínio de fungos antropofílicos e zoofílicos, respectivamente, dado este que está de acordo com as estatísticas dos estados brasileiros da região Sudeste e Sul, bem como de outras regiões do mundo em que estes fungos foram os mais frequentes isolados de tinha dos pés e do couro cabeludo. Neste estudo, foi também verificada elevada percentagem de T. tonsurans (41,7%) em tinha do couro cabeludo, dado este inédito na região Sudeste

Microbiota fúngica de um prédio com laboratórios da região de Araraquara - SP / Environment fungi of laboratory buildings in Araraquara - SP region

De setembro de 2000 a janeiro de 2001, foram coletadas amostras de 21 ambientes internos de um prédio com laboratórios didáticos, de pesquisa e de atendimento à comunidade, bem como da área externa, em Araraquara, estado de São Paulo. Foi utilizado aparelho que funciona de acordo com o princípio descrito por Andersen, o MAS-100R (MERCK), de simples estágio, que utiliza placas de Petri de 90mm de diâmetro contendo meio de agar Sabouraud-cloranfenicol. Após cinco dias de incubação à temperatura de 25ºC, as colônias foram contadas, reisoladas e identificadas resultando na descrição de 21 taxa. Clodophialophora spp. foi o fungo mais isolado, tanto em ambientes internos como externos, seguido pelos gêneros Penicillium spp. e Mycelia sterilia. De acordo com resolução nº 9, de janeiro de 2003 da ANVISA, fungos considerados inaceitáveis foram encontrados em nove ambientes internos e um dos ambientes apresentou quantidade de fungos (em unidades formadoras de colônia por meio cúbico - UFC/m3) acima do limite aceitável. Entre os fungos isolados, 16 foram relatados como fungos oportunistas, nove relacionados a doenças de plantas e sete associados a problemas alérgicos

Epidemiologia de candidíase hospitalar: importância da identificação específica / Epidemiology of nosocomial candidiasis: the importance of specific identification

Infecções fúngicas sistêmicas são hoje importante causa de morbidade e mortalidade em pacientes imunossuprimidos ou com outras condições predisponentes. Candida albicans e as não-albicans são importante causa de infecções nosocomiais e vários destes agentes são menos suscetíveis às drogas antifúngicas, principalmente os azólicos, um fato que tem significado no tratamento destes pacientes. O moderno laboratório de micologia tem importante papel no esclarecimento destas infecções, incluindo sua detecção , identificação e a sensibilidade a drogas antifúngicas, bem como a análise epidemiológica. Neste estudo, foi comparada a distribuição de espécies de Candida relacionadas a fungemias e outras fontes, em quatro hospitais do Estado de São Paulo. Das 40 leveduras identificadas, C. albicans, C. parapsilosis e C. tropicalis foram isoladas, respectivamente, em 35 por cento, 50 por cento e 15 por cento, revelando uma tendência de ser maior a freqüência de espécies não-albicans. As fungemias foram causadas por C. parapsilosis (45,4 por cento). C. albicans (36,4 por cento) e C. tropicalis (18,2 por cento), o que revela um aumento de espécies não-albicans em relação a séries históricas. As três diferentes espécies foram incluídas em 6,3 e 4 biótipos diferentes, respectivamente para C.albicans, C.parapsilosis e C.tropicalis. Este estudo enfatiza a importância da avaliação de espécies de Candida especialmente em centros hospitalares com pacientes de risco.

Surgical adhesives

The authors have performed a literature review of surgical adhesives, such as cyanoacrylate, collagen gelatin, and fibrin glue. They have included different types of commercial and non-commercial fibrin sealants and have reported on the different components in these adhesives, such as fibrinogen, cryoprecipitate, bovine thrombin, and thrombin-like fraction of snake venom.

An evaluation by rat colon anastomosis of the efficacy of fibrin glue derived from snake venom

The objective of this study was to evaluate the efficacy of fibrin adhesive made up of snake venom and bubaline fibrinogen by rat colon anastomosis. Eighty rats were randomly assigned into 2 experimental groups: GI control (anastomosis with extramucous interrupted suture) and GII (repair suture + fibrin glue). The animals were studied at the following 4 times: T0 - preoperative - T1 - 7th day postoperative, T2 - 14th day postoperative, and T3 - 21th day postoperative. The macroscopic characteristics of the intestinal segment open and closed anastomosis and the bursting strength of the anastomosed segments were observed at each of the above times. The results showed that the anastomosed segments coapted and there was no difference in the bursting strength values between the 2 groups. There was a decrease in the bursting strength values up until de 7th day postoperative in both groups with its progressive increase at the other times. Although important experimental studies using large animals are needed for a better evaluation of tissue repair processes, this adhesive may become a valuable tool for use in anastomosis.

The evaluation of clotting time in bovine thrombin, reptilase©, and thrombin-like fraction of Crotalus durissus terrificus venom using bovine, equine, ovine, bubaline and human cryoprecipitates

The objective of this study was to evaluate the effects of the thrombin-like fraction of Crotalus durissus terrificus venom, Reptilase©, and bovine thrombin of fibrinogen polls on bovine, equine, ovine, bubaline and human cryoprecipitates. The authors also made a comparative study between animal and human cryoprecipitates to see if there was any possibility of future use in medicine. Fibrinogen levels in cryoprecipitate were studied using 48 blood samples obtained as follows: 12 samples from humans, 9 from bovine, 10 from equine, 10 from ovine and 7 from bubaline. The results obtained showed average levels of 375.50 mg per cent for humans, 218.33 mg per cent for bovine, 240.80 mg per cent for equine, 267.70 mg per cent for ovine and 664.00 mg per cent for bubaline. Upon the formation of pools of human and animals fibrinogens, the following results were obtained: 435 mg per cent for humans, 444 mg per cent for bovine, 337 mg per cent por equine, 390 mg per cent for ovine and 530 mg per cent for bubaline. Statistical analysis (using the analysis of variance for entirely randomized experiment for the calculation of F statistics) demonstrated that the bubaline fibrinogen level was higher than that of human, and both were higher than those of ovine, equine, and bovine. Clotting times were determined using different dilutions of bovine thrombin, thrombin-like fraction of Crotalus durissus terrificus venom, and Reptilase©. Comparing these clotting times, results for human and bovine were found to be very similar, whereas using equine, ovine and bubaline the results above a dilution of 1:3 were markedly different. The results obtained permitted the following conclusions to be drawn show that: 1) bovine thrombin presented better interactivity with fibrinogen extracted both from human and bovine cryoprecipitates; 2) there was similar behavior when bovine thrombin was substituted for Reptilase© and for the thrombin-like fraction of Crotalus durissus terrificus venom; 3) cryoprecipitate from bovine can, in special circumstances, substitute human cryoprecipitate in medical practice; 4) human and bovine cryoprecipitates can be used with both Reptilase© and Crotalus durissus terrificus fractions using a dilution up to 1:5; 5) the use of bovine cryoprecipitate can be recomended using either bovine thrombin, Reptilase©, or thrombin-like fraction of Crotalus durissus terrificus venom.