Obtenção de exoantígenos de Histoplasma capsulatum em meio de neopeptona, glicose, tiamina e asparagina (NGTA)

O presente trabalho teve como objetivo a produção de exoantígenos H e M das amostras 58, B-679, A-811 e O187 de Histoplasma capsulatum, utilizando o meio NGTA (neopeptona, glicose, tiamina e asparagina) em períodos de cultivo de 1, 2 e 3 meses, a 36ºC, sob agitação constante (50 v.p.m.). Os antígenos brutos foram avaliados contra anti-soro e antígeno de Histoplasma capsulatum de referência (Center for Disease Control), 4 soros de pacientes portadores de paracoccidioidomicose, 7 de histoplasmose e soro hiperimune anti-H. capsulatum produzido em coelhos, através da reação de imunodifusão dupla. Verificou-se que, com exceção de B-679 com 1 mês de crescimento, todos os demais exoantígenos apresentaram as frações H e M de precipitação. Os exoantígenos obtidos de A-811 apresentaram só a banda H. Excetuando-se os exoantígenos 58 e B-679 com 1 mês de crescimento, todos os demais exoantígenos reagiram contra soros de pacientes com histoplasmose. Em relação aos soros de pacientes com paracoccidioidomicose, somente os exoantígenos 58 e O187 não apresentaram reação cruzada. Todos os exoantígenos reagiram frente ao soro hiperimune de coelho anti-H. capsulatum. Para obtenção de exoantígenos de H. capsulatum, sugerimos que as amostras sejam cultivadas sob as condições anteriormente descritas, adotando-se o período de 3 meses de crescimento, utilizando-se exoantígenos de referência como controles da reação.

Paracoccidioides brasiliensis, nova amostra isolada de fezes de um pinguim (Pygoscelis adeliae)

Os Autores apresentam os resultados obtidos com a amostra "pinguim" de Paracoccidioides, isolada por GEZUELE et al. (1989) na Antártica uruguaia. Das fezes de um desses animais, foi isolado um fungo considerado, recentemente, como nova espécie de Paracoccididoides - P. antarclicus. Os exames micológico e imunoquímico demonstraram tratar-se de Paracoccidioides brasiliensis, inclusive com a verificação da presença da glicoproteína 43 kDa pelos métodos de imunodifusão dupla, SDS-PAGE e imunoeletroforese. A possibilidade de se tratar de uma variedade do Paracoccididoides brasiliensis somente poderá ser confirmada através de outros estudos baseados na chamada taxonomía molecular, incluindo cariotipagem. Os Autores registram o significado epidemiológico deste achado, sugerindo uma revisão nos conhecimentos do nicho ecológico do P. brasiliensis.

Paracoccidioides brasiliensis: a mycologic and immunochemical study of a sample isolated from an armadillo (Dasipus novencinctus)

A sample of P. brasiliensis isolated from the spleen and the liver of an armadillo (Dasipus novencinctus) has been analysed under a mycological and immunochemical viewpoint. The armadillo was captured in an area of Tucuruí (State of Pará, Brazil), the animal being already established as an enzootic reservoir of P. brasiliensis at that region of the country. This sample maintained in the fungal collection of the Tropical Medicine Institute of São Paulo (Brazil) numbered 135, has got all the characteristics of P. brasiliensis, with a strong antigenic power and low virulence for guinea-pigs and Wistar rats. The specific exoantigen of P. brasiliensis - the glycoprotein with a molecular weight of 43 kDa - was easily demonstrated with double immunodiffusion, immunoelectrophoresis, SDS-PAGE and immunobloting techniques.

Paracoccidioides brasiliensis: A MYCOLOGIC AND IMMUNOCHEMICAL STUDY OF TWO STRAINS

The authors conducted a mycologic, immunochemical and molecular biology study on two strains of Paracoccidioides brasiliensis, one of them, called IBIÁ, isolated from soil in the municipality of IBIÁ (Minas Gerais) by Silva-Vergara et al. (l996,1998)20,21, and the other, BAT, cultivated from a human case of paracoccidioidomycosis in Ribeirão Preto (São Paulo/Brazil) by Freitas da Silva (l996)6. Both strains showed cotton-like (M) and yeast-like (Y) forms and were pathogenic for testicularly inoculated guinea pigs, producing granulomatous and/or suppurative orchitis. Immunochemically was demonstrated the presence of gp43 by double immunodiffusion, immunoelectrophoresis and immunoblotting.

Atypical disseminated cutaneous histoplasmosis in an immunocompetent child, caused by an "aberrant" variant of Histoplasma capsulatum var. capsulatum

A case of atypical disseminated cutaneous histoplasmosis in a five-year old, otherwise healthy child, native and resident in São Paulo metropolitan area is reported. Cutaneous lesions were clinically atypical. Histologic examination disclosed a granulomatous reaction but no fungal structures could be demonstrated by specific staining nor by immunohistochemical reaction. The fungus was isolated from biopsy material on two different occasions, confirming diagnosis of an unusual fungal infection. The fungus, originally thought to be a Sepedonium sp. due to the large sized, hyaline or brownish colored tuberculated macroconidia and to lack of dimorphism (yeast form at 37 °C) produce H and M antigens, visualized by the immunodiffusion with rabbit anti-Histoplasma capsulatum hyperimmune serum. Patient’s serum sample was non reactive with H. capsulatum antigen by immunodiffusion, counterimmunoelectrophoresis and complement fixation tests, and immunoenzymatic assay failed to detect the specific circulating antigen. This serum was tested negative by double immunodiffusion when antigen obtained from one of the isolated samples was used. Both cultures were sent to Dr. Leo Kaufman, Ph.D. (Mycoses Immunodiagnostic Laboratory, CDC-Atlanta/USA), who identified them as H. capsulatum by the exoantigen and gen-probe tests. Both clinic and mycologic characteristics of the present case were atypical, suggesting the fungus isolated is an “aberrant variant” of H. capsulatum var. capsulatum, as described by SUTTON et al. in 199719. Treatment with itraconazole 100 mg/day led to cure within 90 days

Clinical and epidemiological features of definitive and presumed loxoscelism in São Paulo, Brazil

A retrospective study analysed 359 proven or presume cases of loxoscelism seen at the Hospital Vital Brazil, Instituto Butantan, São Paulo, Brazil, between 1985 and 1996. The spider was identified in 14%. The bites occurred predominantly in the urban areas (73%) between September and February. Patients > 14 years were commonest inflicted (92%) and 41% were bitten while getting dressed. Only 11% sought medical care within the first 12 hours post bite. Cutaneous loxoscelism was the commonest form presenting (96%); commonest manifestations were: pain (76%), erythema (72%), edema with enduration (66%), ecchymosis (39%). Skin necrosis occurred in 53% of patients, most frequently seen on trunk, tigh and upper arm, and when patients seek medical care more than 72 hours after bite. Local infection was detected in 12 patients (3%). Hemolysis was confirmed in 4 cases (1.1%). Generalised cutaneous rash, fever and headache were also observed in 48% of the total of patients. None of them had acute renal failure or died. Treatment usually involved antivenom administration (66%), being associated with corticosteroids (47%) or dapsone (30%). Presumptive diagnosis of loxoscelism may be established based on clinical and epidemiological findings. Further investigations are required to prove the value of antivenom and other treatment schedules.

Detection of EBV-DNA in serum samples of an immunosuppressed child during a three years follow-up: association of clinical and PCR data with active infection

Twenty-four whole blood and serum samples were drawn from an eight year-old heart transplant child during a 36 months follow-up. EBV serology was positive for VCA-IgM and IgG, and negative for EBNA-IgG at the age of five years old when the child presented with signs and symptoms suggestive of acute infectious mononucleosis. After 14 months, serological parameters were: positive VCA-IgG, EBNA-IgG and negative VCA-IgM. This serological pattern has been maintained since then even during episodes suggestive of EBV reactivation. PCR amplified a specific DNA fragment from the EBV gp220 (detection limit of 100 viral copies). All twenty-four whole blood samples yielded positive results by PCR, while 12 out of 24 serum samples were positive. We aimed at analyzing whether detection of EBV-DNA in serum samples by PCR was associated with overt disease as stated by the need of antiviral treatment and hospitalization. Statistical analysis showed agreement between the two parameters evidenced by the Kappa test (value 0.750; p < 0.001). We concluded that detection of EBV-DNA in serum samples of immunosuppressed patients might be used as a laboratory marker of active EBV disease when a Real-Time PCR or another quantitative method is not available.

Detection of Bartonella henselae DNA in clinical samples including peripheral blood of immune competent and immune compromised patients by three nested amplifications

Bacteria of the genus Bartonella are emerging pathogens detected in lymph node biopsies and aspirates probably caused by increased concentration of bacteria. Twenty-three samples of 18 patients with clinical, laboratory and/or epidemiological data suggesting bartonellosis were subjected to three nested amplifications targeting a fragment of the 60-kDa heat shock protein (HSP), the internal transcribed spacer 16S-23S rRNA (ITS) and the cell division (FtsZ) of Bartonella henselae, in order to improve detection in clinical samples. In the first amplification 01, 04 and 05 samples, were positive by HSP (4.3%), FtsZ (17.4%) and ITS (21.7%), respectively. After the second round six positive samples were identified by nested-HSP (26%), eight by nested-ITS (34.8%) and 18 by nested-FtsZ (78.2%), corresponding to 10 peripheral blood samples, five lymph node biopsies, two skin biopsies and one lymph node aspirate. The nested-FtsZ was more sensitive than nested-HSP and nested-ITS (p < 0.0001), enabling the detection of Bartonella henselae DNA in 15 of 18 patients (83.3%). In this study, three nested-PCR that should be specific for Bartonella henselae amplification were developed, but only the nested-FtsZ did not amplify DNA from Bartonella quintana. We conclude that nested amplifications increased detection of B. henselae DNA, and that the nested-FtsZ was the most sensitive and the only specific to B. henselae in different biological samples. As all samples detected by nested-HSP and nested-ITS, were also by nested-FtsZ, we infer that in our series infections were caused by Bartonella henselae. The high number of positive blood samples draws attention to the use of this biological material in the investigation of bartonellosis, regardless of the immune status of patients. This fact is important in the case of critically ill patients and young children to avoid more invasive procedures such as lymph nodes biopsies and aspirates.

Viability and molecular authentication of Coccidioides spp. isolates from the Instituto de Medicina Tropical de São Paulo culture collection, Brazil

Coccidioidomycosis is an emerging fungal disease in Brazil; adequate maintenance and authentication of Coccidioides isolates are essential for research into genetic diversity of the environmental organisms, as well as for understanding the human disease. Seventeen Coccidioides isolates maintained under mineral oil since 1975 in the Instituto de Medicina Tropical de São Paulo (IMTSP) culture collection, Brazil, were evaluated with respect to their viability, morphological characteristics and genetic features in order to authenticate these fungal cultures. Only five isolates were viable after almost 30 years, showing typical morphological characteristics, and sequencing analysis using Coi-F and Coi-R primers revealed 99% identity with Coccidioides genera. These five isolates were then preserved in liquid nitrogen and sterile water, and remained viable after two years of storage under these conditions, maintaining the same features.

Identification and differentiation of Candida species from pediatric patients by random amplified polymorphic DNA

Thirty-four Candida isolates were analyzed by random amplified polymorphic DNA using the primer OPG-10:24 Candida albicans; 4 Candida tropicalis; 2 Candida parapsilosis; 2 Candida dubliniensis; 1 Candida glabrata and 1 Candida krusei. The UPGMA-Pearson correlation coefficient was used to calculate the genetic distance between the different Candida groupings. Samples were classified as identical (correlation of 100%); highly related samples (90%); moderately related samples (80%) and unrelated samples (< 70%). The results showed that the RAPD proposed was capable of classifying the isolates coherently (such that same species were in the same dendrogram), except for two isolates of Candida parapsilosis and the positive control (Netherlands, 1973), probably because they are now recognized as three different species. Concerning the only fluconazole-resistant Candida tropicalis isolate with a genotype that was different to the others, the data were insufficient to affirm that the only difference was the sensitivity to fluconazole. We concluded that the Random Amplified Polymorphic DNA proposed might be used to confirm Candida species identified by microbiological methods.