The data obtained suggest that chiefly macrophages, also Langerhans' cells and factor XIIIa+ dermal dendrocytes, function as antigen-presenting cells in chromoblastomycosis.

BackgroundSerological tests have long been established as rapid, simple and inexpensive tools for the diagnosis and follow-up of PCM. However, different protocols and antigen preparations are used and the few attempts to standardize the routine serological methods have not succeeded.Methodology/Principal findingsWe compared the performance of six Brazilian reference centers for serological diagnosis of PCM. Each center provided 30 sera of PCM patients, with positive high, intermediate and low titers, which were defined as the “reference” titers. Each center then applied its own antigen preparation and serological routine test, either semiquantitative double immunodifusion or counterimmmunoelectrophoresis, in the 150 sera from the other five centers blindly as regard to the “reference” titers. Titers were transformed into scores: 0 (negative), 1 (healing titers), 2 (active disease, low titers) and 3 (active disease, high titers) according to each center's criteria. Major discordances were considered between scores indicating active disease and scores indicating negative or healing titers; such discordance when associated with proper clinical and other laboratorial data, may correspond to different approaches to the patient's treatment. Surprisingly, all centers exhibited a high rate of “major” discordances with a mean of 31 (20%) discordant scores. Alternatively, when the scores given by one center to their own sera were compared with the scores given to their sera by the remaining five other centers, a high rate of major discordances was also found, with a mean number of 14.8 sera in 30 presenting a discordance with at least one other center. The data also suggest that centers that used CIE and pool of isolates for antigen preparation performed better.ConclusionThere are inconsistencies among the laboratories that are strong enough to result in conflicting information regarding the patients' treatment. Renewed efforts should be promoted to improve standardization of the serological diagnosis of PCM.

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.

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.

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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.

SUMMARYChromoblastomycosis (CBM) is a chronic subcutaneous infection caused by several dematiaceous fungi. The most commonly etiological agent found in Brazil is Fonsecaea pedrosoi, which appears as thick walled, brownish colored cells with transverse and longitudinal division in the lesions, called "muriform cells". This disease is found worldwide but countries like Madagascar and Brazil have highest incidence. Diagnosis is made by clinical, direct and histopathologic examination and culture of specimens. Serological tests have been used to identify specific antibodies against Fonsecaea pedrosoi antigens, as well as immunotechniques have been used for CBM serological identification and diagnosis. In the present study double immunodiffusion (DID), counterimmunoelectrophoresis (CIE) and immunoenzymatic test (ELISA) have been used to evaluate humoral immune response in patients with CBM caused by F. pedrosoi. Metabolic antigen was used for immunoprecipitation tests (DID and CIE) while somatic antigen for ELISA. Our results demonstrated 53% sensitivity and 96% specificity for DID, while CIE presented 68% sensitivity and 90.5% specificity. ELISA demonstrated 78% sensibility and 83% specificity. Serological tests can be a useful tool to study different aspects of CBM, such as helping differential diagnosis, when culture of the pathogenic agent is impossible.

Cryptococcus neoformans is a basidiomycetous, the etiologic agent of cryptococcosis, yeast-like fungus which, following inhalation from an environmental source causes respiratory and neurological disease in humans and animals. Cryptococcal infections can be fatal if the host does not have adequate T-cell-dependent immune function. Most important predisposing factors for infection with C. neoformans are virus immunodeficiency infection, use of immunosuppressive drugs, organ and bone marrow transplantation, chronic leukemia, and lymphomas (Dismukes 1988, Velegraki, et al. 2001, Horta et al. 2002. Kwon-Chung et al. (1982) established two varieties that were recognized: var. neoformans and var. gattii. Currently we recognize 3 varieties: grubii -serotype A-, gattiiserotype B, C, -and neoformans -serotype D (Franzot et al. 1999). Most likely, var. gattii will be renamed as a novel species and called C. bacillisporus (Diaz et al. 2000).This fungus appears in yeast form, presenting spherical cells (5-100 µm in diameter) with single or multiple buds. The colonies are cream-colored, glossy, viscous, and moist, the amount of mucous being directly related to the size of the capsule. Among the various virulence factors, the most significant are capsular polysaccharides such as glucuronoxylan, galactoxilamanane, and mannoproteins (Kurtzman & Fell 1998, Lacaz et al. 2002, Chiapello et al. 2003, Ellerbroek et al. 2004.The samples of C. neoformans maintained in our Fungus Collection are especially useful for biotechnology projects, scientific research related to antifungal therapy, determination of molecular characteristics, production antigen, and instructional programs, as well as functional as reference cultures. Various preservation techniques are used: successive subculture on solid medium (with or without the addition of mineral oil); spore cultivation (in soil, sand, or silica gel); lyophilized; liquid nitrogen storage; and suspension in distilled water (Castellani 1963, 1967, Urdaneta et al. 1965, Bosmans 1974, Rodrigues et al. 1992, Cavalcanti & Cavalcanti 1994, Spencer & Spencer 1996.With the advent of molecular biology, various techniques have come to be used to analyze the genotype of the potential polymorphism of fungus samples. These include randomly amplified polymorphic DNA (RAPD), pulsed-field gel electrophoresis, (PFGE), restriction fragment length polymorphism (RFLP), and amplified fragment length polymorphism (AFLP). The cost of performing the RFLP, PFGE, and AFLP techniques is prohibitive. However, the RAPD method can be performed at a relatively low cost. This technique suits our needs perfectly, not only due to its cost-effectiveness but also to its reproducibility (Willians 1990).In our Fungus Collection, samples of C. neoformans have been maintained in successive subcultures since 1930. In the 1970s, some of these samples were selected to be stored using the lyophilization technique. The preservation method of choice in our laboratory is still successive subculture since it is considered a simple method...