Allergenic extracts from Metarhizium canisopliae: obtainment and characterization.

Metarhizium anisopliae is used as a biopesticide for insects that damage agricultural plantations like sugar cane and forage plants. In a previous study the sensitization to this fungus of asthmatic patients coming from sugar cane areas was showed. The aims of this work were: to compare crude extracts obtained with Tris-HCl and Coca liquid from several growth phases of M. anisopliae concerning the total content of proteins and their electrophoretic analysis profile; to evaluate in vivo allergic sensitization in Balb/c mice and allergic patients from a sugar cane area, and to characterize the allergenic fractions in the sera of patients positive for the prick test by means of Western-blotting. The extract obtained with Coca liquid on the 16th day was the one that presented the greatest number of proteic fractions, including all those present in the other extracts. Twelve fractions were verified in this extract with approximate molecular weights from 94 to 14 kDa. The allergenicity of the extract obtained on the 16th day was proven by the production of IgE antibodies in Balb/c mice, with titres of 200. Prick tests carried out with the extract of the 16th day in 79 atopic individuals (from sugar cane area), 35 atopic individuals (from urban area) and 11 non- atopic individuals showed respective positivity of 29%, 9% and 0%. The allergenic characterization in vitro was performed by means of Western blotting, and the fractions that reacted with the positive individuals' sera were those of approximate molecular weights of 67 kDa (95%); 20 kDa (55%); 94 kDa (36%); 34 and 36 kDa (23%); 43 and 48 kDa (14%); 16 kDa (9%) and 54kDa (5%). It was concluded that the crude allergenic extract, obtained with Coca liquid from the 16th day growth of Metarhizium anisopliae, contains allergenic fractions and can be used in diagnostic screening tests.

Biosynthesis of chondroitinase and hyaluronidase by different strains of Paracoccidioides brasiliensis.

The biosynthesis of chondroitinase and hyaluronidase by different isolates of Paracoccidioides brasiliensis was investigated in 20 strains isolated from patients (17 strains), a penguin (Pygocelis adeliae, one strain), an armadillo (Dasypus novemcinctus, one strain) and the environment (dog food, one strain). All the P. brasiliensis isolates studied had the ability to produce chondroitinase and hyaluronidase, although differences in colony morphology and enzyme production were detected among them. These results suggest that further investigations should be carried out in the clinical field in order to clarify the potential role of P. brasiliensis enzyme production in the pathogenesis of paracoccidioidomycosis.

Library fungi at the University of São Paulo and their relationship with respiratory allergy.

In this study, we investigated the fungi most frequently found in the air and the books of 28 libraries at the University of São Paulo, Brazil. In a second phase, 314 librarians were questioned about the presence of asthmatic or rhinitic symptoms, and the relationship with the site of work. Forty-nine percent of them reported this type of symptoms and 80% related them to the place of work. In the third stage, librarians underwent intracutaneous tests against the 20 fungi most frequently isolated in libraries. Eighteen librarians presented positive tests, 12 of whom reported rhinitis and 6 of whom were asymptomatic. In 19.5% of symptomatic patients wheals > 15 mm were observed after 6 h, while 9% of the asymptomatic patients presented similar wheals. Fourteen librarians with positive tests underwent tests with each of the 20 fungi constituting the pool. The airborne fungi isolated in libraries are likely to be found anywhere in the city of São Paulo, but they are likely to be present in higher concentrations in libraries, subsequently producing respiratory allergies.

Characterization of allergenic fractions from Drechslera monoceras.

Drechslera monoceras, a fungus of the Deuteromycota phylum, is fairly frequent in Brazil, and is spread through the atmosphere. In previous studies done in the city of Sao Paulo, it was found that in relation to 42 other fungi extracts, the crude extract of this fungi demonstrated a more intense cutaneous reaction in patients with respiratory allergies. Biochemical, antigenic and allergenic evaluations were carried out at various growth stages of this fungus. Based on these facts, the purpose of this research was the fractionation and allergenic characterization of the allergenic extract of D. monoceras to be used in diagnosis and immunotherapy in patients with positive cutaneous reaction to this fungus. In the city of Sao Paulo, 13 of 248 patients with respiratory allergy (asthma and/or rhinitis) showed positive reaction following cutaneous tests (skin prick tests). The crude extract of D. monoceras was fractionated by SDS-PAGE. The visible fractions were then separated by electroelution to be inoculated into BALB/c mice to evaluate the production of IgE antibody. The IgE content was detected by passive cutaneous anaphylaxis test in Wistar rats, and two fractions of approximate molecular weights of 14.4 and 36 KDa reacted to the test. The in vitro allergenic characterization was carried out by Western blotting, and three fractions of approximate molecular weights of 14.4, 36 and 60 KDa were positive. It was concluded that the extract of D. monoceras has at least three allergenic determinants, which can be used for diagnosis and immunotherapy in patients with respiratory allergy to this fungi.

Candidin in immediate hypersensitivity. Comparison of two antigens.

One hundred outpatients in a Clinic of Allergy were submitted to intradermic tests with two types of candidins. The main focus of the research was the comparison of two antigens obtained from the same strains of Candida albicans: one a suspension of yeast cells and the other, a polysaccharide. The readings, taken 20 minutes after the intradermic injections, with positive results were considered as hypersensitivity of the immediate type. Positive results were obtained in 74% of the patients with the yeast cell antigen and in 73% with the polysaccharide antigen. This research mainly deals with the advantages that can be obtained by using the polysaccharide antigen in intradermic tests for evaluating hypersensitivity of the immediate type.

Comparative performance of two air samplers for monitoring airborne fungal propagules.

Many studies have attempted to evaluate the importance of airborne fungi in the development of invasive fungal infection, especially for immunocompromised hosts. Several kinds of instruments are available to quantitate fungal propagule levels in air. We compared the performance of the most frequently used air sampler, the Andersen sampler with six stages, with a portable one, the Reuter centrifugal sampler (RCS). A total of 84 samples were analyzed, 42 with each sampler. Twenty-eight different fungal genera were identified in samples analyzed with the Andersen instrument. In samples obtained with the RCS only seven different fungal genera were identified. The three most frequently isolated genera in samples analyzed with both devices were Penicillium, Aspergillus and Cladophialophora. In areas supplied with a high efficiency particulate air filter, fungal spore levels were usually lower when compared to areas without these filters. There was a significant correlation between total fungal propagule measurements taken with both devices on each sampling occasion (Pearson coefficient = 0.50). However, the Andersen device recovered a broader spectrum of fungi. We conclude that the RCS can be used for quantitative estimates of airborne microbiological concentrations. For qualitative studies, however, this device cannot be recommended.

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

The utilization of the fluorescent method (fluorescein diacetate DF and ethidium bromide BE), to verify the viability of fungal cells, was studied in 40 samples of liquor, from patients with neurocryptococcosis. For removing leukocytes and red blood cells, which produce interfering fluorescence, good results were obtained with 0.3% saponin solution. After processing of liquor, 0.1 ml aliquots of resulting suspension were mixed to equal volumes of fresh DF-BE solution. The best incubation period for staining was 30 minutes, resulting in good differentiation between viable (green fluorescence) and non viable (red fluorescence) cells.

Candidin: comparison of two antigens for cutaneous delayed hypersensitivity testing.

A candidin, which is a suspension of killed yeast cells, is commonly used for intradermal tests of delayed hypersensitivity, to evaluate the immunological cellular competence of the patient, when the test is applied along with other similar tests. When working with a cellular antigen, the histopathology of positive skin tests reveals a cellular infiltrate which not only presents a characteristic hypersensitivity reaction but also a neutrophilic abscess in the central part. This research presents the results of a comparison between the yeast cell suspension and the polysaccharide antigens, both obtained from the same strains of Candida albicans. The results obtained by skin tests in one hundred individuals were 61.0% with the polysaccharide antigen and 69.0% with the yeast cell suspension antigen. Concordant results concerning the two antigens were observed in 82.0% of the individuals. The discussion section presents an assumption to explain the differences of positivity obtained with the two antigens. We conclude that the polysaccharide antigen can be utilized in the intradermal test of delayed hypersensitivity to Candida albicans.

Evaluation of the allergenicity of spore and mycelia extracts of Pisolithus tinctorius.

The antigenic and allergenic chemical analysis of spore and mycelia extracts of Pisolithus tinctorius was carried out. The spores were collected from basidiocarps in plantations of Eucalyptus spp and the mycelia from culture in MNM medium. With basis on the fungus growth curve, the mycelia masses were obtained after 10, 20, 30, and 40 days of incubation, which correspond, respectively, to the beginning, middle and end of the log phase, and beginning of the decline phase. The mycelia masses, together with the spores, were submitted to the action of three extractors (Coca, Tris-HCl, and ammonium bicarbonate). The contents of carbohydrates and proteins were determined. The SDS-PAGE electrophoretical analysis revealed separate fractions in these extracts, besides common fractions, in function of cultivation time and extraction methods. The selected extracts for the allergic tests were the ones with the highest number of fractions. The prick-tests were conducted in 374 patients--rural workers, eucalyptus plantation workers, and college students. The positivity to the "prick test" with the antigenic extract of P. tinctorius was, respectively, 3.78%, 28.20% and 6.40%. Most prick-test positive patients (82.75%) also presented symptoms of respiratory allergy (asthma and rhinitis). There was no reactivity difference when the spore and mycelia extracts were employed. The analysis of the positive patients' sera revealed the presence of IgE specific to the P. tinctorius antigens. Since Pisolithus tinctorius is found as mycorrhiza of Eucalyptus spp, and this plant is used in reforestation in most countries, the importance of that fungus should be regarded as a possible cause of respiratory allergies, especially in occupationally exposed workers.

Leveduras isoladas do pelame de cães sadios que vivem em regime domiciliar / Yeasts isolated from the haircoat of healthy dogs that live indoor

Potentially pathogenic yeasts from the haircoat of dogs that live indoor and visit regularly an esthetic service were collected by the carpet technique applied on the fur. The microorganisms were isolated on Sabouraud dextrose agar supplied with chloramphenicol and identified by their morphological and biochemistry characteristics. Candida albicans was identificated in 95.2% of the canine population, followed by Rhodotorula mucilaginosa (4.8%). It is known that C. albicans is uncommon on health skin. By the other hand, the high frequency of this agent in this study may represent exposure and risk for opportunistic infections in dogs and other susceptible animals.

Trichophyton rubrum isolated from aids and human immunodeficiency virus-infected patients in São Paulo, Brazil: antifungal susceptibility and extracellular enzyme production.

BACKGROUND: In order to identify intraspecific variations in Trichophyton rubrum and to correlate them to the immunological status of the host, sixty strains isolated from AIDS, HIV-positive and HIV-negative patients were compared for the production of extracellular enzymes and for their susceptibility to several antifungal drugs. METHODS: The isolates were tested for their ability to secrete keratinases, proteinases, phospholipases, lipases and DNases. Likewise, we investigated their susceptibility to amphotericin B, ketoconazole, ciclopiroxolamine, griseofulvin, miconazole and tolnaftate. RESULTS: Variations in the Minimal Inhibitory Concentration (MIC80)) values were observed for all antifungals tested, but they were similarly distributed among the three clinical groups. Griseofulvin showed the most prominent differences among the three groups of isolates. Regarding enzyme secretion, all samples secreted keratinases and DNases, while none secreted phospholipases. Proteinases and lipases were secreted by some of them. CONCLUSIONS: The differences among isolates of the three groups were not statistically significant and therefore could not be ascribed to a given clinical status. Intraspecific variations similarly occurred in each group, irrespective of the immunological status of the patients.

Cutaneous positivity in patients with respiratory allergies to 42 allergenic extracts of airborne fungi isolated in São Paulo, Brazil.

With the purpose of measuring the frequency of allergies of the respiratory tract to airborne fungi in São Paulo, Brazil, 201 patients with bronchial asthma and/or allergic rhinitis were submitted to intradermal testings with allergenic extracts of 42 fungus genera isolated from the air in São Paulo. The extracts of these fungi were prepared according to Coca's method and then standardized by the weight by the volume method. At first, the patients were submitted to intradermal testing with the total polyvalent (TP) extract, getting positive reaction in 70 of them (34.8%). The patients with positive reaction to the TP were tested with the 42 individual extracts and 74.3% of them reacted positively to one or more extracts. These results led us to the conclusion that the most frequent fungi in the air of São Paulo were not the ones which cause the highest number of positive intradermal reactions. Therefore, the convenience in employing as many allergens as possible in intradermal testings for diagnosis should be reinforced.

Systematic isolation of the genus Alternaria in the atmosphere of the "Grande Sao Paulo" area (Brazil).

The genus Alternaria has been isolated from the atmosphere of several countries and its spores are considered as having a strong allergenic power. The description of the genus isolation with a relative frequency was noted in the following countries: Mexico, the United States of America, Canada, Great Britain, Spain, South Africa, Denmark and Argentina. Many Brazilian authors have pointed out the rare incidence of this genus excluding Faria (5) who recorded in the city of Belo Horizonte, State of Minas Gerais, a frequency of 21.1%. Several authors have failed to detect Alternaria sp. spores in the atmosphere of the city of Sao Paulo. As we were studying in our laboratory the airborne fungi of a region called Greater Sao Paulo, where the collections were made in some boroughs of the main city and its neighborhood, we began to make findings of the Alternaria sp. This gave us the opportunity of studying its incidence in that area during a period of one year. We chose 11 areas of this region where we made weekly plate expositions performing 429 trials in all. The technique of collection was the usual one with the exposition of the Petri dishes containing Sabouraud Agar distributed 72 hours before. The collection was made from September 1973 to August 1974. The exposition period was of 5 minutes duration in the open air or in a protected place when it rained. The exposition height was 1 meter from the ground. The identification was made through the colony macroscopic morphology and through the Riddell technique. The Alternaria sp. was isolated in the mentioned area through all the months of the year with its highest frequency in August (35.8%) and its lowest in December 3.7%). The average relative frequency obtained was of 17%. The highest isolation frequency was attained in Winter, the lowest in Spring; in Summer and Autumn, it remained at a medium level. In relation to the studied areas, the Alternaria sp. was isolated in all of them with a minimum frequency of 8.3% in Campos Eliseos and with a maximum frequency of 28.9% in Osasco. The authors would like to emphasize this unusual finding because in the region studied the Alternaria sp. was previously considered absent.

Teaching of microbiology in Brazil: progress or stagnation?

There is a severe shortage of trained microbiologists in Brazil which is hampering the development and expansion of microbiology/biotechnology research, the development of the biotechnology industries and environmental control agencies. Even some medical areas are affected. The reason for this situation lies in the deficient teaching of microbiology, where undergraduate courses in the biological sciences contain little or no microbiology, and in the low number of professionals that are produced. There are a few post-graduates courses in microbiology but these have high costs and little capacity for expansion. There is a need for incentives for the establishment of more undergraduate university courses, more practical training in specific areas, courses for retraining or updating of professionals already at work and specific programmes by the funding agencies.

Production of proteinase and phospholipase by Paracoccidioides brasiliensis.

We have investigated the production of proteinase and phospholipase by 20 different isolates of Paracoccidioides brasiliensis. Isolates were grown in Bacto-peptone, Dextrose, pH 5.5, agar slants, at 27 degrees C for 30 days, and cultures were transferred onto Petri dishes containing basis medium and bovine serum albumin fraction V and sterile egg yolk as substrates for enzyme production, and incubated at 27 degrees C. After 30 days net enzyme activity was visualized and quantitatively evaluated, measuring a ratio between colony diameter and diameter of the transparent (proteinase) or white (phospholipase) ring zone surrounding it. Results demonstrated that all isolates had the ability to produce proteinase and phospholipase, even though variability in enzyme production was noted among different isolates of P. brasiliensis.

Dimorphism of Scopulariopsis brevicaulis: morphogenesis of the mould to yeast phase.

The dimorphism of Scopulariopsis brevicaulus was induced in malt agar (Oxoid), at 37 degrees C and 5-10% rate of CO2. Six cultures of S. brevicaulus were converted from hyphae to yeast-like cells by the following processes: One was that the hyphae formed intercalar and terminal chlamydoconidia-like cells. These cells increased in size, gained double contour and gave rise to budding cells. The other process was via conidia that assumed yeast characteristics. So, a transformation cycle M----Y was proposed. The yeast-like form presented slow reversion to the mould phase, standing, many times, in transition forms. A high concentration of sugars, peptone, temperature of cultivation of 37 degrees C and 5-10% rate of CO2 seem to be favourable factors for the maintenance of the yeast phase. Ethanol, E.D.T.A., potassium nitrate, temperature of 25 degrees C and aerobiosis are considered favourable factors to conversion of the mould phase.

Evaluation of a fluorescent method (fluorescein diacetate and ethidium bromide solution) in the study of the viability Cryptococcus neoformans strains.

The effectiveness of the fluorescent viability test (fluorescein diacetate-FDA and ethidium bromide-EB-solution) compared to the plaque counting test (Miles & Misra M & M) was performed on 10 samples of Cryptococcus neoformans cultivated in Sabouraud dextrose agar at 25 degrees C. The optimum incubation period of 50 minutes was determined. Growth curves of the fungal strains studied based on the mean cell number were drawn for both the FDA & EB and M & M methods. The statistical evaluation (Student's T test) of the average sum of the viable cell counts showed that the FDA-EB method is more sensitive than the M & M test for the studied species. The growth curves of the samples usually followed a homogeneous pattern comparable to other non-dimorphic fungi.

Phenotypic characterization of Trichophyton rubrum isolates from two geographic locations in Brazil.

To characterize possible Trichophyton rubrum phenotypes, which circulate in two Brazilian localities, we tested 53 isolates of this dermatophyte for their ability to assimilate several carbon sources, for keratinase, proteinase, phospholipase, lipase and desoxiribonuclease (DNase) secretions, and for their susceptibility to the antifungals fluconazole, ketoconazole and itraconazole. For each method, the isolates were submitted to similarity analysis and the methods were evaluated for their discriminatory indexes. None of the isolates were capable of assimilating arabinose, dulcitol, lactose, melibiose, ribose and xylose, while all of the isolates assimilated maltose, sucrose and sorbitol. However, adonitol, cellobiose, dextrin, erythritol, fructose, galactose, inulin, mannitol, mannose, raffinose, rhamnose and trehalose were assimilated by some isolates but not by others. All isolates secreted keratinase and DNase, while none secreted phospholipase. Proteinase and lipase were secreted only by some isolates. All but four isolates were resistant to fluconazole, most of them were sensitive to ketoconazole and all were sensitive to itraconazole. Carbohydrate assimilation was the method that presented the highest discriminatory index, and also the method that displayed the largest number of biotypes. Taken together, these data suggest that significant phenotypic variations exist among T. rubrum isolates. They seem to occur independently from their geographic origins.

Postharvest and stored corn in Brazil: mycoflora interaction, abiotic factors and mycotoxin occurrence.

The mycoflora of 130 samples of postharvest and stored corn was analysed throughout one year. The sample originated from Riberirão Preto, State of São Paulo, Brazil. The influences of abiotic factors (moisture content, relative humidity, temperature, rainfall) and mycotoxin occurrence were also verified. The isolation of the fungi was performed with Potato Dextrose Agar. Fungi were identified by using standard techniques. The determination of mycotoxins (aflatoxins, ochratoxin A, sterigmatocystin, zearalenone, deoxynivalenol and fumonisin B1) was carried out by thin-layer chromatography. The Fusarium spp. was the most dominant fungi (83.8%) followed by Penicillium spp. (55.3%), Aspergillus spp. (40.7%) and 11 other filamentous fungi. All of these were isolated from grains with moisture contents of 12.3-17.8%, an average temperature of 18.4-24.1 degrees C, a relative humidity between 64.0 and 97.5% and rainfall of up to 337 mm. With regard to the number of colony forming units (cfu), Fusarium spp. was the main contaminant, varying from 545 x 10(3) to 1.5 x 10(3). The Simple linear correlation analysis showed significant positive correlation of the Fusarium genus with moisture content of grains, and a significant negative correlation in relation to the minimum and medium temperatures, rainfall and relative humidity. The linear correlation of the Penicillium and Aspergillus genera with the abiotic factors was not significant. In the samples analysed only one contained aflatoxin B1.

Phenotypic characterization of Microsporum canis isolated from cats and dogs.

To characterize strains of Microsporum canis that infect dogs and cats in São Paulo city, 30 isolates of this dermatophyte were tested for their ability to assimilate carbon and nitrogen sources, for proteinase and phospholipase secretion, for susceptibility to yeast killer toxins, and for susceptibility to the antifungals fluconazole, ketoconazole, itraconazole, 5-fluorocytosine and amphotericin B, in E test. All samples assimilated the nitrogen sources asparagine, ammonium sulphate, urea and sodium nitrate, as well as the carbon sources inulin, mannitol, trehalose, meso-erythritol, maltose, mannose, sorbitol, cellobiose, fructose and dextrin. Not all the samples assimilated adonitol, galactose, arabinose, rhamnose, raffinose, melibiose, ribose and sucrose, and none of them was capable of growing with dulcitol, lactose, or xylose as the only carbon source. Proteinase and phospholipase secretion was observed for most isolates. In the test of yeast killer toxin, 10 types could be identified, with four types exclusively observed in isolates from dogs and two types exclusively observed in isolates from cats. In the E test, all isolates were found to be resistant to the fluconazole and 5-fluorocytosine, while they were variably sensitive to amphotericin B, ketoconazole and itraconazole. When the data were submitted to the qualitative analysis in the matrix distance program FITOPAC, the similarity of the isolates could be assessed.