Mônica Scarpelli Martinelli Vidal

Antigen distribution and antigen-presenting cells in skin biopsies of human chromoblastomycosis.

Background:  Chromoblastomycosis is a chronic, suppurative, granulomatous mycosis usually confined to skin and subcutaneous tissues. The host defense mechanisms in chromoblastomycosis have not been extensively investigated. The purpose of the present study was to determine the distribution and pathways of the fungal antigen(s) and the possible role of the different immunocompetent cells in antigen processing in skin lesions.

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.

Highly specific and sensitive, immunoblot-detected 54 kDa antigen from Fonsecaea pedrosoi

Chromoblastomycosis (CBM) is a chronic subcutaneous mycosis caused by a group of different dematiaceous fungi, first described by Rudolph in 1914. In Brazil there is a clear predominance of Fonsecaea pedrosoi. Sixty sera samples obtained from patients with F. pedrosoi-caused CBM were analysed. Sera obtained from 36 sporothricosis (SPT) patients, 34 cutaneous leishmaniasis (CL) patients and from 48 blood donors (HBD) were used as control. F. pedrosoi metabolic antigen was obtained from F. pedrosoi sample no. 884 (Instituto de Medicina Tropical de São Paulo Collection). IE reaction disclosed an anodic migrating arch, which was eluted and used as antigen. Both metabolic and eluate F. pedrosoi antigens were submitted to SDS-PAGE and two fractions, weighing approximately 54 and 66 kDa were identified. The 66-kDa fraction reacted against 43 of 60 CBM (71.7%) sera samples and was recognized by 10 SPT and eight CL sera (15.3%). No reactivity was observed against HBD sera. The 54-kDa fraction reacted against 58 of 60 CBM sera (96.7% sensitivity) and was not recognized by HBD, SPT nor CL sera (100% specificity). Such high sensitivity and specificity levels suggest this antigenic fraction is immunodominant and might prove a useful tool for further studies on F. pedrosoi-caused CBM.

Evaluation of phenotypic and genotypic alterations induced by long periods of subculturing of Cryptococcus neoformans strains.

Cryptococcus neoformans is an encapsulated fungal organism that can cause disease in apparently immunocompetent, as well as immunocompromised, hosts. Since 1930, successive subculture has been used to preserve C. neoformans isolates in our Fungus Collection. In the 1970s, some of these Fungus Collection samples were selected to be subjected to a different methods of maintenance--that of lyophilized. Our objective was to analyze C. neoformans isolates in order to make a comparative evaluation between these two methods of preservation. The overall aim of this study was to qualify the preservation technique used in our mycology laboratory since the technique used might affect the survival, stability and purity of the primary isolates in culture. The samples were analyzed using classical mycology methods and using the randomly amplified polymorphic DNA technique In the analysis of phenotypes and genotypes, the typical characteristics of C. neoformans were found to differ in relation to the different methods of preservation employed. The aim of this study was to demonstrate the importance of selecting the appropriate method of preservation for fungus collections. This selection can affect the survival and purity of the cultures, and preserve the stability of their physiological, biochemical, and genetic characteristics.

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.

Reactivity of anti-gp43 antibodies from Paracoccidioides brasiliensis antiserum with extracts from cutaneous lesions of Lobo's disease. Preliminary note.

We demonstrated through several immunochemical tests the presence of gp43 from P. brasiliensis in extracts of cutaneous lesions from Jorge Lobos disease. This glicoprotein is one of the immunodominant antigens in this species, and is used to identify it. The demonstration of gp43 tissues infected by the agent of Jorge Lobos disease is an additional evidence for classifying it in the genera Paracoccidioides, species loboi.

Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry for Differentiation of the Dimorphic Fungal Species Paracoccidioides brasiliensis and Paracoccidioides lutzii

ABSTRACT Isolates of Paracoccidioides brasiliensis and Paracoccidioides lutzii, previously characterized by molecular techniques, were identified for the first time by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). All isolates were correctly identified, with log score values of >2.0. Thus, MALDI-TOF MS is a new tool for differentiating species of the genus Paracoccidioides.

Paracoccidioides brasiliensis: A MYCOLOGIC AND IMMUNOCHEMICAL STUDY OF TWO STRAINS

Os Autores estudaram do ponto de vista micologico, imunoquimico e de sua biologia molecular, duas amostras de Paracoccidioides brasiliensis, uma isolada do solo, no municipio de IBIA (MG) por Silva-Vergara et al. (l996,1998)20,21 denominada IBIA e outra, BAT, cultivada de um caso humano de paracoccidioidomicose em Ribeirao Preto (SP) por Freitas da Silva (l996)6. Tais amostras apresentam colonias cotonosa (M) e leveduriforme (L ou Y), sendo patogenicas para cobaios inoculados por via testicular, produzindo orquite granulomatosa e/ou supurativa. Do ponto de vista imunoquimico, atraves de provas de Imunodifusao dupla, Imunoeletroforese e Western Blotting, foi demonstrada a presenca da gp43. A sequencia de nucleotideos do DNA de tais amostras, atraves do sequenciamento de 761 bases, revelou homologia de 100% com amostra padrao de P. brasiliensis, o mesmo ocorrendo com tres amostras humanas, uma isolada de fezes de pinguim e outra de racao alimentar para caes contaminada com terra. Sao discutidos varios aspectos dos resultados obtidos, comparando-os com alguns dados da literatura.The authors conducted a mycologic, immunochemical and molecular biology study on two strains of Paracoccidioides brasiliensis, one of them, called IBIA, isolated from soil in the municipality of IBIA (Minas Gerais) by Silva-Vergara et al. (1996, 1998), and the other, BAT, cultivated from a human case of paracoccidioidomycosis in Ribeirão Preto (São Paulo/Brazil) by Freitas Da Silva (1996). 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.

Does the Capsule Interfere with Performance of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry for Identification of Cryptococcus neoformans and Cryptococcus gattii?

ABSTRACT We described the impact of the capsule size for Cryptococcus neoformans and Cryptococcus gattii identification at the species level by Bruker matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). After experimental capsule size modulation, we observed that reducing the capsule size resulted in improved identification by Bruker MALDI-TOF MS across all of the reference strains analyzed.

Lymphocyte transformation assay for C neoformans antigen is not reliable for detecting cellular impairment in patients with neurocryptococcosis.

BackgroundCryptococcus neoformans causes meningitis and disseminated infection in healthy individuals, but more commonly in hosts with defective immune responses. Cell-mediated immunity is an important component of the immune response to a great variety of infections, including yeast infections. We aimed to evaluate a specific lymphocyte transformation assay to Cryptococcus neoformans in order to identify immunodeficiency associated to neurocryptococcosis (NCC) as primary cause of the mycosis.MethodsHealthy volunteers, poultry growers, and HIV-seronegative patients with neurocryptococcosis were tested for cellular immune response. Cryptococcal meningitis was diagnosed by India ink staining of cerebrospinal fluid and cryptococcal antigen test (Immunomycol-Inc, SP, Brazil). Isolated peripheral blood mononuclear cells were stimulated with C. neoformans antigen, C. albicans antigen, and pokeweed mitogen. The amount of 3H-thymidine incorporated was assessed, and the results were expressed as stimulation index (SI) and log SI, sensitivity, specificity, and cut-off value (receiver operating characteristics curve). We applied unpaired Student t tests to compare data and considered significant differences for p<0.05.ResultsThe lymphotoxin alpha showed a low capacity with all the stimuli for classifying patients as responders and non-responders. Lymphotoxin alpha stimulated by heated-killed antigen from patients with neurocryptococcosis was not affected by TCD4+ cell count, and the intensity of response did not correlate with the clinical evolution of neurocryptococcosis.ConclusionResponse to lymphocyte transformation assay should be analyzed based on a normal range and using more than one stimulator. The use of a cut-off value to classify patients with neurocryptococcosis is inadequate. Statistical analysis should be based on the log transformation of SI. A more purified antigen for evaluating specific response to C. neoformans is needed.