Seroepidemiology of Paracoccidioides brasiliensis infection in horses from Rio Grande do Sul, Brazil

Paracoccidioides brasiliensis is the etiological agent of the major systemic mycosis in Brazil, called paracoccidioidomycosis. Although the Rio Grande do Sul is considered an endemic area of the disease, there are few studies on the ecology of P. brasiliensis in the state. Therefore, this study aimed to evaluate the infection of P. brasiliensis in horses from the mesoregion of Southwest Riograndense, using these animals as sentinels. Serological techniques, such as double immunodiffusion in agar gel (AGID) and indirect ELISA, were performed to detect the anti-gp43 P. brasiliensis antibody in horses from five different farms in the region of Bagé, RS, Brazil. Serology was performed in 200 Pure Blood English horses up to two years of age that were born and raised exclusively at the farms. Of these horses, 12% had anti-gp43 antibodies according to the ELISA results, with rates ranging from 0 to 30% according to the farm of origin (p < 0.001). Based on the immunodiffusion results, all equine serum samples were negative. These results indicate the presence of the fungus P. brasiliensis in the middle region of the southwestern state of Rio Grande do Sul, Brazil.

Cryptic species of Paracoccidioides brasiliensis: impact on paracoccidioidomycosis immunodiagnosis

We aimed to evaluate whether the occurrence of cryptic species of Paracoccidioides brasiliensis, S1, PS2, PS3 and Paracoccidioides lutzii, has implications in the immunodiagnosis of paracoccidioidomycosis (PCM). Small quantities of the antigen gp43 were found in culture filtrates of P. lutzii strains and this molecule appeared to be more variable within P. lutzii because the synonymous-nonsynonymous mutation rate was lower, indicating an evolutionary process different from that of the remaining genotypes. The production of gp43 also varied between isolates belonging to the same species, indicating that speciation events are important, but not sufficient to fully explain the diversity in the production of this antigen. The culture filtrate antigen AgEpm83, which was obtained from a PS3 isolate, showed large quantities of gp43 and reactivity by immunodiffusion assays, similar to the standard antigen (AgB-339) from an S1 isolate. Furthermore, AgEpm83 was capable of serologically differentiating five serum samples from patients from the Botucatu and Jundiaí regions. These patients had confirmed PCM but, were non-reactive to the standard antigen, thus demonstrating an alternative for serological diagnosis in regions in which S1 and PS2 occur. We also emphasise that it is not advisable to use a single antigen preparation to diagnose PCM, a disease that is caused by highly diverse pathogens.

Cell-free antigens from precocious Paracoccidioides brasiliensis culture induce a typical delayed-type hypersensitivity reaction

Cell-free antigens (CFAg) derived from Paracoccidioides brasiliensis have typically been used in immunodiffusion reactions for serodiagnosis or therapeutic follow-up of paracoccidioidomycosis patients. Thus, we investigated the usefulness of CFAg obtained from cultures at different ages, to evaluate cellular immunity by the footpad test, in experimental murine paracoccidioidomycosis. Male mice infected with P. brasiliensis 265 strain were challenged in the footpad with CFAg obtained from four- (4d CFAg) or 11-day-old cultures (11d CFAg). The increase in footpad swelling provoked by 4d CFAg and 11d CFAg was similar and showed significant difference in relation to control groups. However, the infiltrate pattern was strikingly different: 4d CFAg induced a predominant mononuclear infiltrate whereas 11d CFAg provoked a predominant polymophonuclear infiltrate. These different inflammatory patterns were associated with distinct electrophoretic characteristics. By comparison with 11d CFAg, 4d CFAg showed more numerous and intense bands, including a strong one of 43 kDa (gp43). These results suggest that CFAg derived from Pb 265 isolate can be used as a reagent to evaluate cellular immunity; however, the culture's age is critical because only young cultures are able to induce a typical mononuclear infiltrate. The efficacy of this new paracoccidioidin to assay the cellular immunity in infections caused by other P. brasiliensis isolates is under investigation.

Reavaliação e proposta de um protocolo de sorologia para paracoccidioidomicose / Serodiagnostic protocol for PCM

A necessidade de implantação de uma nova conduta sorodiagnóstica para Paracoccidiomicose (PCM), na região Norte do Paraná, surgiu pela falta de correlação entre as condições clínicas dos pacientes com exame ou anatomopatológico positivo para P. brasiliensis, com os resultados de reação de fixação de complemento utilizando antígeno polissacarídico comercial Pimenta Abreu (AgPA). A reavaliação foi realizada utilizando-se 43 soros com requisição de sorologia para PCM, negativos por fixação de complemento com AgPA. Os testes utilizados foram imunodifusão radial, Fixação de Complemento, Elisa e Western Blotting, utilizando exoantígeno de 7 dias (Ag7d de P. brasiliensis e glicoproteína de 43KDa de P. Brasiliensis. Não foi observada nenhuma reação positiva com AgPA mesmo em pacientes diagnosticados por exames anatomopatológicos e ou micológicos AgPA, enquanto que a positividade pós-reação de fixação de complemento utilizando Ag7d foi de 37,2% e por imunodifusão radial dupla de 25,5%. Foi realizada também análise desses soros pelo ensaio: ELISA clássico com o Ag7D, ELISA captura com a glicoproteína de 43KDa (Gp43) de P. brasiliensis e “Western blotting” com Ag7d, obtendo-se 81,4%. Os resultados aqui obtidos mostram claramente a necessidade de controle de qualidade na produção e padronização de antígeno como reagente para imunodiagnóstico.