Tipagem molecular de isolados clínicos e ambientais de Cryptococcus neoformans

As micoses estão incluídas entre as doenças infecciosas mais ubíquas em todo o mundo, afectando todos os estratos sociais e todos os grupos etários numa variedade de manifestações superficiais, cutâneas, subcutâneas e sistémicas. Um diagnóstico rápido e eficaz destas doenças, com uma correcta identificação da espécie fúngica responsável pela infecção, é essencial para um planeamento do tratamento mais eficaz para o doente infectado. Tem-se registado um aumento da incidência das infecções fúngicas também em consequência do aumento do número de casos de doentes imunodeprimidos, devido particularmente à crescente utilização de terapêuticas imunossupressivas, procedimentos médicos invasivos, prescrição de tratamentos prolongados, entre outros aspectos. O aparecimento da epidemia da Imunodeficiência Humana no início da década de 80, causada pelo vírus VIH, contribuiu também decisivamente para o aumento das infecções fúngicas oportunistas. A Criptococose é uma infecção fúngica, predominantemente oportunista e com uma distribuição epidemiológica mundial, causada por leveduras encapsuladas do género Cryptococcus. A espécie clinicamente mais relevante é Cryptococcus neoformans, cujas estirpes têm sido tradicionalmente classificadas em cinco serotipos relacionados com os antigénios da respectiva cápsula polissacárida: A (C. neoformans var. grubii); D (C. neoformans var. neoformans); B e C (actualmente reconhecidos como uma espécie distinta mas filogeneticamente próxima, C. gattii); e AD (estirpes híbridas). O presente trabalho teve como principal objectivo determinar retrospectivamente os tipos moleculares de uma colecção alargada de estirpes de C. neoformans, isoladas e mantidas durante os últimos 18 anos no Laboratório de Micologia do IHMT/UNL. A maioria das estirpes foi isolada de doentes imunodeprimidos com criptococose, existindo também algumas estirpes de origem ambiental. Foi utilizada a técnica de PCR-RFLP do gene URA5 para diferenciar as estirpes de Cryptococcus neoformans, tendo sido detectados quatro tipos moleculares: VN1 e VN2 (relacionados com C. neoformans var. grubii, serotipo A); VN3 (relacionado com as estirpes híbridas de serotipo AD); e VN4 (relacionado com C. neoformans var. neoformans, serotipo D). Não foram encontrados entre os isolados de origem clínica perfis de restrição correspondentes aos tipos moleculares VG1, VG2, VG3 e VG4 (relacionados com C. gattii, serotipos B e C). O tipo molecular VN1 foi o mais abundante entre os isolados de origem clínica (45% dos isolados), seguindo-se o grupo de estirpes híbridas do tipo molecular VN3 (31%). Os tipos moleculares menos abundantes entre os isolados foram o VN2 (12%) e VN4 (12%). A estandardização do método de tipagem molecular utilizado neste trabalho permite comparar os resultados obtidos com os de outros estudos epidemiológicos semelhantes, realizados noutras regiões do globo e publicados em anos recentes, contribuindo para um conhecimento melhorado da epidemiologia global deste importante fungo patogénico. Em Portugal obteve-se uma percentagem mais elevada de isolados dos grupos moleculares VN1 e VN3 em relação a outros países da Europa e América Latina, em que os tipos mais abundantes são VN1 e VN2. Nestas mesmas regiões, o tipo molecular VN4, relacionado com as estirpes de C. neoformans var. neoformans do serotipo D, é muito raro. Esta variedade é mais comum em zonas mediterrâneas e está muito associada a casos clínicos de infecções cutâneas associadas a infecções do Sistema Nervoso Central. Este tipo molecular parece ser também significativamente mais abundante no nosso país.

Projecto solvência II - Modelação do risco de subscrição numa companhia de seguros não vida

Dissertação apresentada na Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para obtenção do grau de Mestre em Matemática e Aplicações - Actuariado, Estatística e Investigação Operacional

CRYPTOSPORIDIOSIS IN PEDIATRIC PATIENTS

Cryptosporidium was detected in 21 (3.8%) individual stool samples collected from 553 pediatric patients hospitalized in our center employing a Telemann concentration technique (formalinethercentrifugation) and stained with the modified Kinyoun method. The mean age of populations with Cryptosporidiosis (16 boys and 5 girls) was 11 months; 15 months for girls and 6.5 for boys. Ages of 81% of them were less than 19 months. Seventysix per cent of patients lived on the outskirts of Buenos Aires and 71% lacked pretreated running water at home. In 62% of the cases parasitological diagnoses coincided with warm seasons. At diagnosis mucous (63%) or watery (36%) diarrhea was presented in 90% of the patients with a median of 5 (38) bowel movements per day. Fever was presented in 66% of patients while abdominal pain and vomits in 60% and 52%, respectively. The median time from hospitalization up to parasitologic diagnosis was 20 days. Concomitant diseases observed were malnutrition, acute leukemia, bronchiolitis, HIV infection, anemia, celiac disease, myelofibrosis, vitelline sac tumor, neutropenia, osteosarcoma and dehydration. Cryptosporidiosis in our environment seems to occur more frequently in children younger than 18 months of age; who present diarrhea; are immunodeficient; come from a low socioeconomical background; and who live in poor sanitary conditions with no potable running water.

Occurrence of Cryptosporidium oocysts and Giardia cysts in raw water from the Atibaia river, Campinas, Brazil

Cryptosporidium parvum and Giardia duodenalis are waterborne parasites that have caused several outbreaks of gastrointestinal disease associated with drinking water. Due to the lack of studies about the occurrence of these protozoa in water in the Southeast of Brazil, an investigation was conducted to verify the presence of cysts and oocysts in superficial raw water of the Atibaia River. The water samples were submitted to membrane filtration (3.0 mum) and elution was processed by (1) scraping and rinsing of membrane (RM method) and (2) acetone-dissolution (ADM method). Microbiologic and chemical parameters were analyzed. Aliquots of the pellets were examined by immunofluorescence (Merifluor, Meridian Diagnostics, Cincinnati, Ohio). All water samples were positive for Cryptosporidium and Giardia, in spite of the high turbidity. Higher recovery rates occurred in samples treated by the RM method than by the ADM technique. The goal for future work is the assessment of viability of cysts and oocysts to determine the public health significance of this finding.

Serotype, mating type and ploidy of Cryptococcus neoformans strains isolated from patients in Brazil

Serotype, mating type and ploidy of 84 strains of Cryptococcus neoformans isolated from 61 AIDS and 23 non-AIDS patients admitted in a tertiary teaching hospital in São Paulo, Brazil were examined. Among 61 strains isolated from AIDS patients, 60 strains were var. grubii (serotype A). Only one strain was var. gattii (serotype B). No var. neoformans (serotype D) was found. Among 23 strains isolated from non-AIDS patients, 15 were var. grubii (serotype A) and the remaining 8 were var. gattii, all of which were serotype B. Seventy-three of the 75 serotype A strains were the heterothallic alpha type (MATalpha) and the remaining 2 were untypable (asexual). Most of the MATalpha strains (69/73) were haploid and the remaining 4 strains were diploid. Similarly, both of the 2 asexual strains among the 75 serotype A strains were haploid. There were no alpha-mating type (MATalpha) strains among the 84 isolates. All of the 8 var. gattii strains were serotype B and haploid. Among a total of 84 strains tested, neither serotype AD nor serotype D were found. Neither triploid nor tetraploid were found. These results suggest that the serological, sexual and ploidy characteristics in C. neoformans strains isolated from AIDS patients in São Paulo were rather simple, whereas strains isolated from non-AIDS patients presented serotype A and B with predominance of serotype A.

Modeling Vegetable Fractals by means of Fractional-order Equations

This paper characterizes four ‘fractal vegetables’: (i) cauliflower (brassica oleracea var. Botrytis); (ii) broccoli (brassica oleracea var. italica); (iii) round cabbage (brassica oleracea var. capitata) and (iv) Brussels sprout (brassica oleracea var. gemmifera), by means of electrical impedance spectroscopy and fractional calculus tools. Experimental data is approximated using fractional-order models and the corresponding parameters are determined with a genetic algorithm. The Havriliak-Negami five-parameter model fits well into the data, demonstrating that classical formulae can constitute simple and reliable models to characterize biological structures.

Persistence of Vectobac WDG and Metoprag S-2G against Aedes aegypti larvae using a semi-field bioassay in Rio de Janeiro, Brazil

Persistence of Bacillus thuringiensis var. israelensis (Vectobac WDG) and methoprene (Metoprag S-2G) was evaluated against Aedes aegypti late third instar larvae of the Rockefeller strain in a semi-field bioassay. Tests were performed in Rio de Janeiro, using containers made of plastic, iron, concrete and asbestos, placed in a shaded area. The formulations used were 0.2 g of Vectobac-WDG and 1g of Metoprag S-2G per 100 liters of water in house storage containers. Vectobac WDG was tested twice, in March and in April/May, 2002. In March (temperature ranging from 21.5 to 39.3 ºC), 70-100% mortality was observed by the 7th day and declined abruptly thereafter. No significant differences were observed among the container types. In April/May (18.6 to 34.8 ºC) mortality was higher than 70% to 30-36 days in all cases, except in the iron container (40% mortality on the 12th day). Metoprag S-2G was evaluated in April/May, 2002, and induced mortality higher than 70% up to 15 days in the plastic and iron containers and only seven days in the concrete container. In the asbestos container, maximal mortality was achieved on day one post-treatment (66%). Our results point to a low persistence of both formulations in the weather conditions of Rio de Janeiro.

Environmental straits of Cryptococcus neoformans variety grubii in the city of Santos, SP, Brazil

This study involved a total of 116 samples, 79 taken from pigeon droppings and 37 of atmospheric air taken close to accumulations of excrement. Cryptococcus neoformans var. grubii was isolated from 11 (13.9%) of these samples. Other species of Cryptococcus were also isolated from these samples, such as C. albidus (12.6%) and C. laurentii (8.9%). C. neoformans was not isolated from the air samples, though C. albidus (5.4%) was. All the strains of C. neoformans were found to belong to the A serotype (C. neoformans var. grubii). In regard to the studies with the antifungal agents 5-fluorocytosine, fluconazole, itraconazole, amphotericin B and voriconazole, by means of the microdilution method (EUCAST), we point out that one sample demonstrated resistance to fluconazole, this being especially significant because this is an environmental strain.

Mediastinal histoplasmosis: report of the first two Brazilian cases of mediastinal granuloma

This report documents the first two Brazilian cases of mediastinal granuloma due to histoplasmosis, presenting selected aspects on the diagnosis. Tissue samples revealing histoplasmosis were obtained from each of the patients by mediastinoscopy and thoracotomy. In the second patient, a subcarinal calcified mass eroded into the bronchial tree, leading to secondary bilateral aspiration pneumonitis one week after thoracotomy. Although rare, histoplasmosis should be included in the differential diagnosis of mediastinal granuloma, specially if there are calcifications greater than 10 mm in dimension.

Characterization of Cryptococcus neoformans isolated from urban environmental sources in Goiânia, Goiás State, Brazil

Cryptococcus neoformans is an opportunistic fungal pathogen that causes meningoencephalitis as the most frequent clinical presentation in immunocompromised patients, mainly in people infected by HIV. This fungus is an environmental encapsulated yeast, commonly found in soil enriched with avian droppings and plant material. A total of 290 samples of pigeon and the other avian droppings, soil, ornamental trees and vegetable material associated with Eucalyptus trees were collected to study environmental sources of Cryptococcus species in Goiânia, Goiás State. The determination of varieties, serotypes and the susceptibility in vitro to fluconazole, itraconazole and amphotericin B of C. neoformans isolates were performed. C. neoformans var. grubii (serotype A) was found in 20.3% (36/177) of pigeon dropping samples and in 14.3% (5/35) of samples of Eucalyptus. None of the environmental isolates of C. neoformans showed in vitro resistance to three antifungal agents. The knowledge of major route for human cryptococcal infection (inhalation of infectious particles from saprophytic sources) and a total of 60 C. neoformans isolates obtained from AIDS patients with cryptococcal meningitis between October 2001 and April 2002 justify the study of the habitats of these yeasts as probable sources of cryptococcosis in this city.

Antifungal susceptibilities of clinical and environmental isolates of Cryptococcus neoformans in goiânia city, Goiás, Brasil

We evaluated the antifungal activities of amphotericin B, fluconazole, itraconazole and voriconazole in 70 Cryptococcus neoformans strains obtained from cerebrospinal fluid from AIDS patients and 40 C. neoformans strains isolated from the environment. Four clinical isolates were identified as C. neoformans var. gattii. The susceptibility test was done using a broth microdilution method according to NCCLS M27-A2. Range minimal inhibitory concentrations (MICs) for C. neoformans clinical isolates were 0.06-1.0 µg/mL for amphotericin B, 0.125-8 µg/mL for fluconazole, 0.03-0.5 µg/mL for itraconazole and 0.03-0.25 µg/mL for voriconazole. C. neoformans environmental isolates showed range MICs 0.015-0.125 µg/mL, 0.25-2.0 µg/mL, 0.007-0.125 µg/mL and 0.03-0.25 µg/mL for amphotericin B, fluconazole, itraconazole and voriconazole respectively. The MICs results obtained from clinical and environmental isolates showed similar pattern of susceptibility and no resistance has been found in our isolates.

Cryptococcus neoformans strains isolated from church towers in Rio de Janeiro City, RJ, Brazil

Cryptococcosis has been a significant cause of morbidity and mortality in patients with Aids. Many reservoirs of the agent Cryptococcus neoformans have been reported, but the ecology of this yeast must be elucidated in order to establish surveillance programs and to prevent infections. The objective of this study was to evaluate the presence of C. neoformans in Rio de Janeiro City, RJ, Brazil. Ten churches were selected for sampling and detection of the yeast collecting pigeon dropping, air samples from church towers and neighboring areas during one year. The data demonstrated that C. neoformans has been present in every church selected and was present in 37.8% of 219 pigeon dropping samples. As well as, the yeast was isolated from soil, insects, eggs, pigeon nests and feathers. Fifteen air samples (4.9%) were positive. The growth on C.G.B. medium showed that all strains belonged to C. neoformans var. neoformans, with 98.8% of the strains belonging to serotype A.

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 (Aalpha) 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.

Genotypic identification of Cryptosporidium spp. isolated from hiv-infected patients and immunocompetent children of São Paulo, Brazil

Cryptosporidium isolates identified in fourteen stool samples, collected from five HIV-infected patients and nine immunocompetent children, living in the Sate of São Paulo, Brazil, were submitted to a molecular analysis using a nested PCR followed of restriction fragment length polymorphism (RFLP), for genetic characterization. The analysis was based on digestion with RsaI restriction enzyme of a DNA fragment amplified from the Cryptosporidium oocyst wall protein (COWP) gene. Based on this analysis, four samples were identified as Cryptosporidium parvum, eight as Cryptosporidium hominis and two presented a profile that correspondedto Cryptosporidium meleagridis when compared to the standards used in the analysis. The use of molecular methods can be helpful to identify source of infections and risk factors related to Cryptosporidium infection in our communities.