G. Del Negro

IgG, IgM and IgA antibody response for the diagnosis and follow-up of paracoccidioidomycosis: comparison of counterimmunoelectrophoresis and complement fixation.

IgG, IgM and IgA antibodies to GP43 (glycoprotein fraction of Paracoccidioides brasiliensis) were measured by ELISA in 63 samples from 23 patients with paracoccidioidomycosis before and twice after chemotherapy was started. Antibodies against P. brasiliensis were detected by indirect immunofluorescence (IF) (IgG, IgM and IgA isotypes), counterimmunoelectrophoresis (CIE) and complement fixation. Two control groups composed of 19 healthy individuals and 12 patients with other diseases (six with histoplasmosis, three with tuberculosis and three with other mycoses). The highest efficiency percentages were found with IgG and IgA-ELISA (100%), IgG-IF (96.2%), CIE (94.4%) and the lowest with CF (75.9%). Highest positive and negative predictive values (100%) were observed for IgG and IgA ELISA. IgG and IgM-ELISA antibodies are more often found in patients with acute than chronic disease (P = 0.01). Four to six months after treatment follow-up showed decreased levels of IgG and IgM-ELISA for acute cases and decreased titres of CIE for chronic cases in relation to pretreatment levels. This study suggests that IgG-ELISA anti-GP43 represents a good marker to monitor clinical response to therapy.

Comprometimento da medula óssea e eosinofilia na paracoccidioidomicose

Sao descritos 3 casos de paracoccidioidomicose com a forma aguda da doenca, nos quais formas leveduriformes de Paracoccidioides brasiliensis foram visualizadas ao exame direto de medula ossea, sendo a cultura tambem positiva em um caso. Salienta-se o acometimento do sistema fagocitico-mononuclear e a ausencia de resposta as provas cutâneas de hipersensibilidade tardia a antigenos microbianos e de P. brasiliensis em todos, bem como a gravidade do quadro clinico e lesoes osseas generalizadas em um caso, com 20.260 eosinofilos/mm3 no sangue periferico. Os autores discutem o possivel papel do eosinofilo na interacao hospedeiro-parasita na paracoccidioidomicose, sugerindo que a ativacao de subpopulacao TH 2 e o aumento de secrecao de IL 5 e de GM-CSF possam estar relacionados a grande eosinofilia presente no caso mais grave

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.

Activation of human complement system in paracoccidioidomycosis

Plasma samples of 14 patients with paracoccidioidomycosis were analysed for components that represent activation of the complement system. Most patients (12/13) showed significant titres of complement-fixing antibodies and 14/14 had increased C4d/C4 ratios. There was no conclusive correlation between these two immunological indices, however. Factor B values of patients were similar to normal donors and fragment Ba was not detected in any of the patients. These results indicate a classical complement pathway activation in the plasma of patients with paracoccidioidomycosis.

Alteraçöes hematológicas na leptospirose

30 cases of leptospirosis admitted to the Clinica de Doenças Infecciosas e Parasitárias do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo were studied for blood count alterations. 16 patients (53.3%) had a normal white blood cell count at the moment of admission. 12 patients (40%) presented a high white blood cell count and 2 (6.6%) had a low count. 29 patients (96.6%) had a high proportion of neutrophils and 25 patients (83.3%) presented a high number of immature forms. 24 patients had anemia. Thrombocytopenia was present in 26 patients (86.6%). The most characteristic changes found in bone marrow aspirate were the alterations of M:E ratio (myeloid-erythroid ratio) due to relative and/or absolute hyperplasia of the myeloid series, and/or relative and/or absolute hypoplasia of the erythroid series; erythropoiesis was predominantly micro-erythroblastic in many patients; mild to moderate plasmocytosis was found; and, regarding the interstitial series, increased macrophagic activity was noted. There was no direct correlation between the number of megakaryocytes and the blood platelet count, but there was a direct correlation between the bone marrow platelet production and blood platelet count. We believe that it is very difficult to have a good idea of the dynamic mechanisms that lead to medullar platelet production in the presence of platelet consumption, through a random test of the bone marrow.

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.

Dual candidemia detected by nested polymerase chain reaction in two critically ill children

The use of improved microbiological procedures associated with molecular techniques has increased the identification of Candida bloodstream infections, even if the isolation of more than one species by culture methods remains uncommon. We report the cases of two children presenting with severe gastrointestinal disorders and other risk factors that contribute to Candida infections. In the first patient, C. albicans DNA was initially detected by a nested-amplification and C. tropicalis was found later during hospitalization, while blood cultures were persistently negative. In the second child, there was amplification of C. albicans and C. glabrata DNA in the same samples, but blood cultures yielded only C. albicans. Both patients received antifungal therapy but had unfavorable outcomes. These two cases illustrate that PCR was more successful than culture methods in detecting Candida in the bloodstream of high risk children, and was also able to detect the presence of more than one species in the same patient that might impact therapy when the fungi are resistant to azole compounds.

Is the S405F mutation in Candida albicans ERG11 gene sufficient to confer resistance to fluconazole

Fluconazole resistance in Candida albicans has been favored by the indiscriminate use of the fluconazole (FLZ) in clinical practice. Until now, many different mechanisms of FLZ resistance were described and may occur simultaneously in resistant isolates [2]. One of these mechanisms is caused by missense mutations in ERG11 gene leading to a decrease in the affinity of the FLZ for its enzyme target, 14a-demethylase [2]. To date, more than 140 different missense mutations have been detected in ERG11 gene of clinical isolates of C. albicans. Among then, S405F is one of the most known mutations associated to FLZ resistance and responsible to an increase of 4 (four) fold in minimal inhibition concentration (MIC) values when cloned into a Saccharomyces cerevisae strain [1,4,5]. In addition, this mutation is often found associated with other mutations, as Y132H, in resistant isolates [1,4,5]. The ERG11 gene from 32 C. albicans clinical isolates was amplified by PCR with previously described primers [3] and sequenced in both strands. FLZ susceptibility was evaluated

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.