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Cryptococcosis is a life-threatening infection in humans and animals caused by encapsulated yeasts of the genus Cryptococcus. Cryptococcus neoformans and Cryptococcus gattii are the main agents of this mycosis. Until 2002 C. gattii was classified as a variety of C. neoformans but now is accepted as an independent species. The laccase (phenoloxydase) enzyme produced by these yeasts is considered one of the main pathogenic factors for its ability to induce melanin from dihydroxyphenolic compounds. The vast majority of the studies in laccase and melanin synthesis have been developed using isolates of C. neoformans. The main objective of this study was to evaluate laccase activity in strains of C. gattii, serotype B isolated from immunocompetent goats that died of lung and disseminated cryptococcosis, in several outbreaks occurring in Spain. The laccase activities of these isolates were compared with those of other strains of C. gattii and C. neoformans. After fungal cell rupture, the supernatant of each isolate was analyzed for its laccase activity using as substrate an L-dopa 20 mM solution. The degree of enzymatic activity was assessed according to its absorbance at 450 nm and scored using Enzymatic Units (EU). The maximum values were observed in three strains of C. gattii from goats (EU > 12). The smallest values were observed in one environmental isolate of C. gattii serotype C (EU = 0.7). The highest recorded value for C. neoformans was 6.3 EU in a serotype A isolate from one human case of meningitis. C. gattii serotype B obtained from goats showed different degrees of laccase activity, being the highest in those isolated from severe outbreaks of cryptococcosis. This enzyme appears to represent a major, though nonexclusive, pathogenic factor for Cryptococcus gattii.

Three hundred and ten Cryptococcus neoformans strains isolated from AIDS patients in five different countries (151 from Brazil, 23 from Italy, 28 from Spain, 104 from Thailand and four from Turkey) were tested by the API-ZYM kit to detect their extracellular enzymatic activity. The enzymes esterase (C4) (no 3), esterase lipase (C8) (no 4), leucine arylamidase (no 6) and acid phosphatase (no 11) were commonly positive in most of the strains (more than 95%). These enzymes could be considered a useful tool not only for C. neoformans identification, but in particular for their possible relationship to new C. neoformans virulence factors and also for epidemiological research. Interestingly, it is also the high positive percentage of alpha-glucosidase and beta-glucosidase detected in all isolates. The serotype A was the most predominant serotype in all countries, except for Italy where the serotype D was predominant. Further studies are needed to draw a clear correlation between the API-ZYM profile and serotype.

In screening for antifungal substances, a new macrolide, eushearilide (1), was isolated from Eupenicillium shearii IFM54447. The structure of 1 was established to be 24-membered macrolide having a non-conjugated diene and a choline phosphate ester moetiy on the basis of detailed investigation of NMR, UV, IR and MS spectral data. Compound 1 showed antifungal activity against various fungi and yeasts, including human pathogens Aspergillus fumigatus, Trichophyton spp. and Candida spp.

One hundred and fifty-one Cryptococcus neoformans strains isolated from AIDS patients in Brazil and maintained in the Adolfo Lutz Institute (São Paulo, Brazil) were tested for phospholipase, protease and other extracellular enzymatic activities and their serotypes determined. Production of extracellular phospholipase and protease was tested by the agar plate methods. Determination of extracellular enzyme profile of the strains was performed by using the API-ZYM kit system, which can test 19 different enzymes. The serotypes were determined by cell agglutination using the Crypto-check method. Among the 151 strains, 147 were identified as serotype A and four strains were serotype AD. Production of extracellular phospholipase and protease was extensive and observable at early stages of incubation. All of the tested strains were positive for the production of both enzymes. In the API-ZYM tests, more than 90 % of the 151 tested strains were positive for esterase C4 (No. 3), esterase lipase C8 (No. 4), leucine arylamidase (No. 6), phosphatase acid (No. 11), naphtol-AS-BI-phosphohydrolase (No. 12), alpha-glucosidase (No. 16) and beta-glucosidase (No. 17). Differences in enzymatic activities between the Brazilian strains and strains isolated in other countries were observed. The phospholipase, protease and other enzyme activities may play a role in host tissue invasion by C. neoformans.

Melanin production is one of the most important criteria for rapid identification of Cryptococcus neoformans. Most of the media described in the literature for identifying C. neoformans are very complex; they contain many organic or inorganic compounds and are difficult to prepare and store. The new minimal synthetic caffeic acid medium described in this paper is simpler to prepare, convenient and constitutes an interesting new medium for the rapid identification of C. neoformans isolates.

Twenty-six Candida dubliniensis and 27 Candida albicans oral strains isolated from patients infected by the human immunodeficiency virus (HIV) were tested for germ tube production and 21 extracellular enzymatic activities. Assessment of the enzymatic profile was performed by using the API-ZYM commercial kit system (bioMerieux, France), which tests 19 different enzymes. Protease activity was expressed during the first days of incubation by 100% of the strains studied and resulted higher than phospholipase activity in the C. dubliniensis and C. albicans strains tested. The API-ZYM profile of the C. dubliniensis and C. albicans strains differs with respect to the number and percentage of the enzymes considered, as well as with the intensity of the substrate metabolized by the strains, in particular for the enzymes n 8 (cystine-arylamidase), n 12 (naphtol-AS-BI-phosphohydrolase) and n 16 (alpha-glucosidase). These enzymes may be useful to differentiate C. dubliniensis and C. albicans together with other phenotypic characteristics proposed in the literature. No relationship among protease, phospholipase and other extracellular enzymatic activities was observed in C. dubliniensis. The average percentage of strains filamentation after 4 h was between 32 and 42%.

Twenty-seven Candida albicans strains and 26 Candida dubliniensis strains, isolated from HIV patients, were tested for their adherence to buccal and vaginal epithelial cells. Both species showed important levels of adhesion to buccal and vaginal epithelial cells, although C. albicans showed the highest levels of adhesion. These results suggest that both Candida species are well adapted, in terms of adhesion capability, to the oral and vaginal environment.

Nineteen Cryptococcus neoformans strains isolated from AIDS patients and 16 from bird droppings were tested for their extracellular activity. Typical enzymatic activity that was different from other medically important yeasts was found. The results obtained may indicate that there are new extracellular enzymatic activities that imply a relationship between C. neoformans and its virulence. A correlation among the different enzymatic activities was also investigated and according to the results obtained no relationship was observed among any of the recorded extracellular enzymatic activities. Research on C. neoformans extracellular enzymatic activity is useful not only to better understand its metabolism but in particular to establish a possible relationship between its virulence and pathogenicity.

One-hundred and thirteen Candida albicans strains isolated from patients infected by the human immunodeficiency virus (HIV) and twenty five from HIV-negative individuals were studied. The C. albicans strains isolated from different sites of the body were tested for germ-tube (GT), phospholipase production and serotype. The results obtained indicate that the serotype A was predominant in all the groups except for the vaginal strains. No correlation was observed between phospholipase activity and serotype distribution. Germ tube (GT) production was higher among the serotype B strains. A positive correlation between GT induction and phospholipase activity was observed only for the isolates from the oral cavity. It is possible that the correlation between phospholipase activity and high GT production in C. albicans strains can facilitate the penetration through the mucosa.

Determination of the enzymatic profile of 41 Cryptococcus neoformans strains, 20 isolated from AIDS patients and 21 from bird droppings, was performed by using the API-ZYM commercial kit system (Bio-Mérieux, France), which tests 19 different kinds of enzymes. All the strains showed positive enzymatic activity to the esterase (C4) (n. 3). On the contrary, alkaline phosphatase (n. 2), cystine arylamidase (n. 8), trypsin (n. 9), chymotripsin (n. 10), alpha-galactosidase (n. 13), beta-glucuronidase (n. 15), alpha-mannosidase (n. 19), alpha-fucosidase (n. 20) were negative in all the strains. The other 10 enzymes (n. 4,5,6,7,11,12,14,16,17,18) were distributed among the strains in different positive percentages. From the results of each enzymatic profile obtained, the 20 AIDS strains were grouped into 15 types, while the 21 bird dropping strains were grouped into 14 types. Interestingly, only one enzyme profile type occurred in the strains isolated from the AIDS patients and from the bird droppings. These results suggest that the API ZYM system is useful in discriminating between the AIDS strains and the bird dropping strains.

Sixty-six oral strains of Candida albicans, which had been consecutively isolated from 22 normal, young females in three isolation trials at intervals of one to three weeks, were biotyped by their susceptibility to boric acid, cetrimide, silver nitrate, sodium periodate and sodium selenite. The 66 isolates were grouped into 13 resistogram types. An identical biotype strain was found three times and twice in seven and six each of the 22 subjects in the three isolation trials, respectively. In the remaining nine subjects, different strains were found at the three trials. These results suggested that certain strains tended to persist in the oral cavity of the normal subjects although changes in the biotype of oral C. albicans strains occurred to a certain extent.