Extracellular matrix protein laminin binds specifically to yeast forms of Paracoccidioides brasiliensis and enhances adhesion of the fungus to the surface of epithelial Madin-Darby canine kidney cells in vitro. Immunoblotting of fungal extracts showed that the gp43 glycoprotein is responsible for adhesion. This was confirmed by binding assays using purified gp43, with a Kd of 3.7 nM. The coating of P. brasiliensis yeast forms with laminin before injection into hamster testicles enhanced the fungus virulence, resulting in a faster and more severe granulomatous disease. These results indicate that interaction of fungi with extracellular matrix elements may constitute a basis for the evolution of fungal infection toward regional spreading and dissemination.

In this study, exoantigens produced from two Paracoccidioides brasiliensis strains isolated in two different geographical areas were compared in terms of sensitivity and specificity in relation to paracoccidioidomycosis (PCM) diagnosis. Exoantigens from P. brasiliensis 550B (Ag 550B) isolated in the central-west region of Brazil (Mato Grosso State) and exoantigen produced from P. brasiliensis B-339 (Ag B-339) used in reference laboratories were compared by immunodiffusion (ID) tests. When Ag 550B was used in ID test against sera of patients from Mato Grosso and São Paulo, positivity was 92.3% and 41.3%, respectively. On the other hand, when Ag B-339 was tested with the same sera, positivity was 26.2% and 100%, respectively. These results suggest that differences in the antigenic composition probably related to phylogenetic peculiarities in P. brasiliensis isolates from the central-western region of Brazil should be considered in the diagnosis of PCM.Paracoccidioidomycosis (PCM) has long been recognised as a public health problem in many areas of Latin America, especially Brazil, Colombia, Venezuela and Argentina (Brummer E et al., Clin Microbiol Rev 1993; 6: 89-117). The only causal agent of the disease is the thermodimorphic fungus Paracoccidioides brasiliensis. Brazil has sustained the largest epidemiological indicators since 1908 when the first case of PCM was described by Adolpho Lutz (Lutz A. Brasil -Medico 1908; 22: 121). As PCM is not a compulsory notification disease, the real incidence of the disease in Brazil is unknown. Mato Grosso State contributes significantly to these statistics (Hahn RC et al., XXII Congresso Brasileiro de Microbiologia, 2003, Florianópolis. Resumos do Congresso Brasileiro de Microbiologia, DOC & Knowledge Management, São Paulo, 2003.As the control of PCM remains limited to the treatment of patients, precise clinical and laboratorial diagnoses are considered of prime importance to enable adequate therapeutic measures as early as possible. The Ôgold standardsÕ for PCM diagnosis are isolation of the fungus by culture and positive identification of multi-budding and birefringent yeast cells by direct examination of biological fluids or biopsy specimens (Brummer E et al., Clin Microbiol Rev 1993; 6: 89-117). Serological diagnosis relies on the detection of specific antibodies, mainly by immunodiffusion (ID) (Camargo ZP et al., Rev Iberoam Micol 2000; 17: 41-48; Del Negro GMB et al., J Med Microbiol 2000; 49: 37-46; Restrepo A et al., Appl Microbiol 1974; 28: 138-44); methods for detecting circulating antigens (Marques da Silva SH et al., J Clin Microbiol 2003; 41: 3675-80; Marques da Silva SH et al., J Clin Microbiol 2004; 42: 4480-6; Marques da Silva SH et al., J Clin Microbiol 2005; 43: 4680-3; Marques da Silva SH

ABSTRACT

A review of 400 clinical records of paracoccidioidomycosis (PCM) patients, 93 with the acute/subacute (AF) and 307 with the chronic form (CF), attended from 1977 to 2011, selected as to the schedule of release for study by the Office of Medical Records at the University Hospital of the Faculdade de Medicina de Botucatu – São Paulo State University – UNESP, was performed to detect cases in relapse. The control of cure was performed by clinical and serological evaluation using the double agar gel immunodiffusion test (DID). In the diagnosis of relapse, DID, enzyme-linked immunosorbent assay (ELISA) and immunoblotting assay (IBgp70 and IBgp43) were evaluated. Out of 400 patients, 21 (5.2%) went through relapse, 18 of them were male and 3 were female, 6∶1 male/female ratio. Out of the 21 patients in relapse, 15 (4.8%) showed the CF, and 6 (6.4%) the AF (p>0.05). The sensitivity of DID and ELISA before treatment was the same (76.1%). DID presented higher sensitivity in pre-treatment (80%) than at relapse (45%; p = 0.017), while ELISA showed the same sensitivity (80% vs 65%; p = 0.125). The serological methods for identifying PCM patients in relapse showed low rates of sensitivity, from 12.5% in IBgp70 to 65.0% in IBgp43 identification and 68.8% in ELISA. The sensitivity of ELISA in diagnosing PCM relapse showed a strong tendency to be higher than DID (p = 0.06) and is equal to IBgp43 (p = 0.11). In sum, prevalence of relapse was not high in PCM patients whose treatment duration was based on immunological parameters. However, the used methods for serological diagnosis present low sensitivity. While more accurate serological methods are not available, we pay special attention to the mycological and histopathological diagnosis of PCM relapse. Hence, direct mycological, cytopathological, and histopathological examinations and isolation in culture for P. brasiliensis must be appropriately and routinely performed when the hypothesis of relapse is considered.

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.