Antonio Martins de Siqueira

Oral yeasts in patients with cancer of the mouth, before and during radiotherapy

The yeasts of patients with oral cancer has been studied before and during Xr-therapy. Gram and PAS smears revealed an increase of yeast-like structures, during treatment, from 56% to 66% of the cases. Before radiotherapy oral yeasts were isolated from 56% of the patients with cancer represented by Candida albicans (30%); C. tropicalis (12%); C. glabrata and C. krusei (4%), besides six other different species (2%). During radiotherapy yeasts were isolated in 72% of the cases, as follow: C. albicans (36%); C. tropicalis (16%); Rhodotorula rubra (6%); C. kefyr; C. krusei and Pichia farinosa (4%), besides other nine species (2%). C. albicans serotype A represented 93% of the isolated samples, before treatment and 88,8% during Xr-therapy.

The "in vitro" antifungal activity evaluation of propolis G12 ethanol extract on Cryptococcus neoformans

Cryptococcosis is a worldwide disease caused by the etiological agent Cryptococcus neoformans. It affects mainly immunocompromised humans. It is relatively rare in animals only affecting those that have received prolonged antibiotic therapy. The propolis is a resin that can present several biological properties, including antibacterial, antifungal and antiviral activities. The standard strain C. neoformans ATTC 90112 was used to the antifungal evaluation. The tests were realized with propolis ethanol extract (PEE) G12 in concentrations from 0.1 to 1.6 mg mL-1. The evaluation of MIC and MFC were done according to DUARTE (2002)5. The inhibitory effect of PEE G12 on the fungal growing was seen at the concentration of 0.2 mg mL-1 and 1.6 mg mL-1 was considered a fungicidal one.

Serotype and mating type characterization of Cryptococcus neoformans by Multiplex PCR

Cryptococcus neoformans is an encapsulated yeast, etiological agent of cryptococcosis. The species is commonly associated with pigeon droppings and plant materials. The aim of the present work was to verify the presence of the yeast in pigeon droppings, and to identify the isolates obtained in serotypes and mating types (MAT). Ten samples of pigeon droppings were collected in the rural area of the city of Alfenas, Brazil. Samples were inoculated in agar Niger medium for fungal isolation and 22 isolates with characteristics of C. neoformans were obtained. The serotypes and MAT were determined by multiplex PCR using specific primers. Serotypes were also determined by using the Kit Crypto Check. Among the 22 samples evaluated, eight were identified as C. neoformans by classic identification tests. These samples were characterized as serotype A by the Kit Crypto check and as serotype A MAT alpha by the multiplex PCR. The present study reinforces the evidence that pigeon droppings are a reservoir for C. neoformans and confirms the prevalence of C. neoformans var. grubii (A alpha) among environmental isolates. It also demonstrates that multiplex PCR is an acceptable alternative for serotype analysis because it reduces the costs for each reaction and analyses serotype and MAT simultaneously.

Isolation of Cryptococcus neoformans from hollows of living trees in the city of Alfenas, MG, Brazil

Cryptococcus neoformans is an encapsulated yeast, aetiological agent of cryptococcosis, commonly associated with pigeon droppings and plant materials. The species has also been associated with tree hollows. The aim of the present work was to verify the presence of the yeast in hollows of living trees and identify the isolates obtained in varieties and serotypes. Three samples were collected from 18 trees of five different species totalling 54 samples. Wood samples were collected by scraping the surface of the trunks and the inner face of the hollows. Samples were inoculated on to agar Niger medium for fungal isolation. The serotypes were determined by PCR using specific primers. Among the 54 samples evaluated, two were positive for the presence of C. n. var. neoformans (serotype A and MATα). The trees belonged to Caesalpinia peltophoroides and Anadenanthera peregrina species. The results of this study suggest that decayed wood obtained from hollows of C. peltophoroides and A. peregrina can be used as natural habitat for C. n. var. neoformans.

Comportamento de amostras ambientais e clínicas de Cryptococcus neoformans frente a fungicidas de uso agronômico e ao fluconazol

The activity of azole fungicides for agronomical use (epoxiconazole, difenoconazole and cyproconazole) was evaluated in comparison with the therapeutic antifungal agent fluconazole, on 23 environmental samples of Cryptococcus neoformans var neoformans isolated from pigeon feces that were collected from farms with agricultural practices using azole compounds, and on 11 clinical samples isolated from patients with cryptococcosis. Sensitivity tests were performed using the agar dilution technique. The minimum inhibitory concentration capable of inhibiting 50% of the environmental isolates (MIC 50) was 6.00 microg/ml to epoxiconazole, 1.00 microg/ml for difenoconazole, 2.00 microg/ml for cyproconazole and 64.00 microg/ml for fluconazole. Among the clinical isolates the MIC 50 values were 2.00 microg/ml, 0.38 microg/ml, 1.00 microg/ml and 16.00 microg/ml for epoxiconazole, difenoconazole, cyproconazole and fluconazole, respectively. The MIC 50 values for environmental isolates were greater than the MIC 50 values for clinical isolates. In our study, in relation to the same antifungal agent, the environmental samples presented significantly different behaviour in relation to the clinical samples (p<0.05). Differences in the MIC values (p<0.05) presented by fluconazole and the other antifungal agents for agronomical use, both in the environmental isolates and in the clinical isolates, were also observed.

Evaluation of laccases and melanization in clinical and environmental Cryptococcus neoformans samples by non-denaturing PAGE.

The increased incidence of infections caused by the opportunistic pathogen Cryptococcus neoformans, which mainly affects immunocompromised patients but can also infect immunocompetent individuals, has needed additional studies on this micro-organisms pathogenicity and factors related to virulence, such as enzyme production, for a better understanding of the aetiology of cryptococcosis. The aim of this study was to verify the applicability of non-denaturing PAGE for analysis of laccases by quantification of the amount of melanin pigment produced by clinical and environmental strains of C. neoformans. After incubation of the gel with the substrate L-dopa, strains produced melanin spots of a bright brown to black colour. Quantification of these spots was performed by densitometry analysis and the amount of melanin produced was calculated and compared among the strains. All strains showed laccase activity. Serotype B strains showed a higher melanin intensity than serotype A strains. Over half of the clinical strains (56.2%) showed the lowest melanin intensities, suggesting that melanin production may not be the main virulence factor against host defence. The clinical strain ICB 88 revealed two melanin spots on the gel, indicating the presence of two laccase isoforms. The environmental strains showed the highest values of melanin intensity, which may be related to previous exposure to environmental stress conditions.

Superoxide dismutase in Cryptococcus neoformans varieties gattii, grubi, and neoformans

Some clear dissimilarities occur among the varieties of Cryptococcus neoformans but there are few studies about the differences among individual yeast antioxidant enzymes. The total superoxide dismutase (SOD) activities and the copper, zinc-depend SOD (Cu,ZnSOD) and manganese-dependent SOD (MnSOD) isoenzymes of five reference C. neoformans strains belonged to A, B, C, AD and D serotypes (Table I) and other nine C. neoformans isolates (Table II) were determined. There were significant differences (p < 0.01 and p < 0.05) in total SOD activity among the varietie gattii (serotype C) and the other varieties. Cu,ZnSOD showed difference (p < 0.05) between A and D serotypes. These results point out a variety and serotype-independent SOD activity in C. neoformans reference strains and the other isolates that were evaluated.

Histoplasmose disseminada em transplantado renal

Os Autores registram caso de histoplasmose generalizada em paciente transplantado com rim de doador nao aparentado. Alem da infeccao fungica diagnosticada sorologicamente e pela histopatologia, a autopsia revelou cirrose hepatica macro e micronodular, de provavel etiologia viral (virus B), hepatocarcinoma, deplecao linfoide do baco e glomerulopatia de transplante. Revendo a literatura sobre o assunto, chegam a conclusao de que, provavelmente, com a imunodepressao medicamentosa, as lesoes pulmonares por reinfeccao endogena foram as primeiras a aparecer sob a forma de uma histoplasmose pulmonar cronica.