Nilma Maciel Garcia

The sensitivity, specificity and efficiency values of some serological tests used in the diagnosis of paracoccidioidomycosis

This work reports on the results of double immunodiffusion (ID), counterimmunoelectrophoresis (CIE), complement fixation (CF) and indirect immunofluorescence (IIF) techniques in the serodiagnosis of paracoccidioidomycosis. The study was undertaken on four groups of individuals: 46 patients with untreated paracoccidioidomycosis, 22 patients with other deep mycoses, 30 with other infectious diseases (tuberculosis and cutaneous leishmaniasis) and 47 blood donors as negative controls. Data were obtained using Paracoccidioides brasiliensis antigens, i.e., a yeast culture filtrate for ID, CIE and CF, and a yeast cell suspension for IIF. The sensitivity, specificity and efficiency values were measured according to GALEN & GAMBINO. The gel precipitation tests (ID and CIE) showed the greatest sensitivity (91.3 and 95.6%, respectively), maximum specificity (100%) and the highest efficiency values when compared to the CF and IIF tests.

Lack of reactivity of paracoccidioidomycosis sera in the double immunodiffusion test with the gp43 antigen: report of two cases

Sera from two patients with chronic active paracoccidioidomycosis yielded negative double immunodiffusion results with a culture filtrate antigen from Paracoccidioides brasiliensis routinely used in our laboratory. Complement fixation tests were positive for both sera using a polysaccharide-rich antigen. This study reports the results of a more extensive serological investigation of these two sera. Both a somatic antigen and a saline extract from the fungus yielded positive results in the double immunodiffusion. However, the immunodominant 43 kDa glycoprotein antigen showed negative results, although it was recognized by both sera in the Western blot assay. The value of the double immunodiffusion as a single serological test in paracoccidioidomycosis diagnosis is discussed.

Immunochemical study of a Paracoccidioides brasiliensis polysaccharide-like antigen

The polysaccharide antigen from P. brasiliensis has been largely employed in serologic tests ,as well as in skin tests, to evaluate cellular immunity. SDS-PAGE analysis of this antigen has revealed a variability in the number of bands exhibited by isolates SN, 265, 339, 113, and 18 (7 to 16 bands). The antigens obtained from isolates 2, PTL, 192 and Adel showed two or three bands. Glycoprotein analysis demonstrated a broad region between 50 and 90 kDa. Major bands of 48 and 30 kDa were present in almost all antigens. Optimal complement fixing dilution appears to be unaffected by the number of bands presented by different antigens. The immunoblot analysis revealed that the 90 and 30 kDa bands were mainly recognized by sera from paracoccidioidomycosis patients. Bands of high molecular weight were also recognized by most of the sera studied. Sera from histoplasmosis recognized the 94 kDa band. In conclusion, although the isolates exhibit quantitative variability in the number of fractions, it is possible to use only one or two samples given the greatest frequency of reactivity is seen in the 30 and 90 kDa fractions.

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.

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

The purpose of this work is obtaining exocellular antigens H and M from 4 H. capsulatum strains using NGTA medium (neopeptone, glucose, thiamine and asparagine) for periods of 1,2 and 3 months, at 36oC and continuously shaken. The exocellular antigens were evaluated by double immunodiffusion test against H. capsulatum rabbit antiserum, 7 histoplasmosis sera, 4 paracoccidioidomycosis sera and a reference antigen and antibody furnished by C.D.C. (Atlanta - USA). Except for the exocellular antigen from strain B.679 with 1 month of culture, all exocellular antigens obtained from the strains B.679, 58 and O187 showed the H and M bands. The A.811 strain demonstrated only the fraction H. All the exocellular antigens reacted positively with sera from histoplasmosis patients, except those obtained from strains 58 and B.679 with 1 month of culture. With regard to paracoc-cidioidomycosis patients sera, the exocellular antigens from strains 58 and O187 did not cross-react with them.The purpose of this work is obtaining exocellular antigens H and M from 4 H. capsulatum strains using NGTA medium (neopeptone, glucose, thiamine and asparagine) for periods of 1, 2 and 3 months, at 36 degrees C and continuously shaken. The exocellular antigens were evaluated by double immunodiffusion test against H. capsulatum rabbit antiserum, 7 histoplasmosis sera, 4 paracoccidioidomycosis sera and a reference antigen and antibody furnished by C.D.C. (Atlanta--USA). Except for the exocellular antigen from strain B.679 with 1 month of culture, all exocellular antigens obtained from the strains B.679, 58 and O187 showed the H and M bands. The A.811 strain demonstrated only the fraction H. All the exocellular antigens reacted positively with sera from histoplasmosis patients, except those obtained from strains 58 and B.679 with 1 month of culture. With regard to paracoccidioidomycosis patients sera, the exocellular antigens from strains 58 and O187 did not cross-react with them.O presente trabalho teve como objetivo a producao de exoantigenos 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 periodos de cultivo de 1, 2 e 3 meses, a 36oC, sob agitacao constante (50 v.p.m.). Os antigenos brutos foram avaliados contra anti-soro e antigeno de Histoplasma capsulatum de referencia (Center for Disease Control), 4 soros de pacientes portadores de paracoccidioidomicose, 7 de histoplasmose e soro hiperimune anti-H. capsulatum produzido em coelhos, atraves da reacao de imunodifusao dupla. Verificou-se que, com excecao de B-679 com 1 mes de crescimento, todos os demais exoantigenos apresentaram as fracoes H e M de precipitacao. Os exoantigenos obtidos de A-811 apresentaram so a banda H. Excetuando-se os exoantigenos 58 e B-679 com 1 mes de crescimento, todos os demais exoantigenos reagiram contra soros de pacientes com histoplasmose. Em relacao aos soros de pacientes com paracoccidioidomicose, somente os exoantigenos 58 e O187 nao apresentaram reacao cruzada. Todos os exoantigenos reagiram frente ao soro hiperimune de coelho anti-H. capsulatum. Para obtencao de exoantigenos de H. capsulatum, sugerimos que as amostras sejam cultivadas sob as condicoes anteriormente descritas, adotando-se o periodo de 3 meses de crescimento, utilizando-se exoantigenos de referencia como controles da reacao.

Histoplasma capsulatum exoantigens obtained in neopeptone, glucose, thiamine and asparagine medium (NGTA).

The purpose of this work is obtaining exocellular antigens H and M from 4 H. capsulatum strains using NGTA medium (neopeptone, glucose, thiamine and asparagine) for periods of 1,2 and 3 months, at 36oC and continuously shaken. The exocellular antigens were evaluated by double immunodiffusion test against H. capsulatum rabbit antiserum, 7 histoplasmosis sera, 4 paracoccidioidomycosis sera and a reference antigen and antibody furnished by C.D.C. (Atlanta - USA). Except for the exocellular antigen from strain B.679 with 1 month of culture, all exocellular antigens obtained from the strains B.679, 58 and O187 showed the H and M bands. The A.811 strain demonstrated only the fraction H. All the exocellular antigens reacted positively with sera from histoplasmosis patients, except those obtained from strains 58 and B.679 with 1 month of culture. With regard to paracoc-cidioidomycosis patients sera, the exocellular antigens from strains 58 and O187 did not cross-react with them.The purpose of this work is obtaining exocellular antigens H and M from 4 H. capsulatum strains using NGTA medium (neopeptone, glucose, thiamine and asparagine) for periods of 1, 2 and 3 months, at 36 degrees C and continuously shaken. The exocellular antigens were evaluated by double immunodiffusion test against H. capsulatum rabbit antiserum, 7 histoplasmosis sera, 4 paracoccidioidomycosis sera and a reference antigen and antibody furnished by C.D.C. (Atlanta--USA). Except for the exocellular antigen from strain B.679 with 1 month of culture, all exocellular antigens obtained from the strains B.679, 58 and O187 showed the H and M bands. The A.811 strain demonstrated only the fraction H. All the exocellular antigens reacted positively with sera from histoplasmosis patients, except those obtained from strains 58 and B.679 with 1 month of culture. With regard to paracoccidioidomycosis patients sera, the exocellular antigens from strains 58 and O187 did not cross-react with them.O presente trabalho teve como objetivo a producao de exoantigenos 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 periodos de cultivo de 1, 2 e 3 meses, a 36oC, sob agitacao constante (50 v.p.m.). Os antigenos brutos foram avaliados contra anti-soro e antigeno de Histoplasma capsulatum de referencia (Center for Disease Control), 4 soros de pacientes portadores de paracoccidioidomicose, 7 de histoplasmose e soro hiperimune anti-H. capsulatum produzido em coelhos, atraves da reacao de imunodifusao dupla. Verificou-se que, com excecao de B-679 com 1 mes de crescimento, todos os demais exoantigenos apresentaram as fracoes H e M de precipitacao. Os exoantigenos obtidos de A-811 apresentaram so a banda H. Excetuando-se os exoantigenos 58 e B-679 com 1 mes de crescimento, todos os demais exoantigenos reagiram contra soros de pacientes com histoplasmose. Em relacao aos soros de pacientes com paracoccidioidomicose, somente os exoantigenos 58 e O187 nao apresentaram reacao cruzada. Todos os exoantigenos reagiram frente ao soro hiperimune de coelho anti-H. capsulatum. Para obtencao de exoantigenos de H. capsulatum, sugerimos que as amostras sejam cultivadas sob as condicoes anteriormente descritas, adotando-se o periodo de 3 meses de crescimento, utilizando-se exoantigenos de referencia como controles da reacao.

Histoplasma capsulatum exocellular antigens. Obtention in neopeptone, glucose, thiamine and asparagine medium (NGTA)

The purpose of this work is obtaining exocellular antigens H and M from 4 H. capsulatum strains using NGTA medium (neopeptone, glucose, thiamine and asparagine) for periods of 1,2 and 3 months, at 36oC and continuously shaken. The exocellular antigens were evaluated by double immunodiffusion test against H. capsulatum rabbit antiserum, 7 histoplasmosis sera, 4 paracoccidioidomycosis sera and a reference antigen and antibody furnished by C.D.C. (Atlanta - USA). Except for the exocellular antigen from strain B.679 with 1 month of culture, all exocellular antigens obtained from the strains B.679, 58 and O187 showed the H and M bands. The A.811 strain demonstrated only the fraction H. All the exocellular antigens reacted positively with sera from histoplasmosis patients, except those obtained from strains 58 and B.679 with 1 month of culture. With regard to paracoc-cidioidomycosis patients sera, the exocellular antigens from strains 58 and O187 did not cross-react with them.The purpose of this work is obtaining exocellular antigens H and M from 4 H. capsulatum strains using NGTA medium (neopeptone, glucose, thiamine and asparagine) for periods of 1, 2 and 3 months, at 36 degrees C and continuously shaken. The exocellular antigens were evaluated by double immunodiffusion test against H. capsulatum rabbit antiserum, 7 histoplasmosis sera, 4 paracoccidioidomycosis sera and a reference antigen and antibody furnished by C.D.C. (Atlanta--USA). Except for the exocellular antigen from strain B.679 with 1 month of culture, all exocellular antigens obtained from the strains B.679, 58 and O187 showed the H and M bands. The A.811 strain demonstrated only the fraction H. All the exocellular antigens reacted positively with sera from histoplasmosis patients, except those obtained from strains 58 and B.679 with 1 month of culture. With regard to paracoccidioidomycosis patients sera, the exocellular antigens from strains 58 and O187 did not cross-react with them.O presente trabalho teve como objetivo a producao de exoantigenos 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 periodos de cultivo de 1, 2 e 3 meses, a 36oC, sob agitacao constante (50 v.p.m.). Os antigenos brutos foram avaliados contra anti-soro e antigeno de Histoplasma capsulatum de referencia (Center for Disease Control), 4 soros de pacientes portadores de paracoccidioidomicose, 7 de histoplasmose e soro hiperimune anti-H. capsulatum produzido em coelhos, atraves da reacao de imunodifusao dupla. Verificou-se que, com excecao de B-679 com 1 mes de crescimento, todos os demais exoantigenos apresentaram as fracoes H e M de precipitacao. Os exoantigenos obtidos de A-811 apresentaram so a banda H. Excetuando-se os exoantigenos 58 e B-679 com 1 mes de crescimento, todos os demais exoantigenos reagiram contra soros de pacientes com histoplasmose. Em relacao aos soros de pacientes com paracoccidioidomicose, somente os exoantigenos 58 e O187 nao apresentaram reacao cruzada. Todos os exoantigenos reagiram frente ao soro hiperimune de coelho anti-H. capsulatum. Para obtencao de exoantigenos de H. capsulatum, sugerimos que as amostras sejam cultivadas sob as condicoes anteriormente descritas, adotando-se o periodo de 3 meses de crescimento, utilizando-se exoantigenos de referencia como controles da reacao.