RESUMO
Paracoccidioidomycosis (PCM) is a systemic mycosis caused by a group of cryptic species embedded in the Paracoccidioides brasiliensis complex and Paracoccidioides lutzii. Four species were recently inferred to belong to the P. brasiliensis complex, but the high genetic diversity found in both human and environmental samples have suggested that the number of lineages may be higher. This study aimed to assess the 43-kilodalton glycoprotein genotypes (PbGP43) in paraffin-embedded samples from PCM patients to infer the phylogenetic lineages of the P. brasiliensis complex responsible for causing the infection. Formalin-fixed, paraffin-embedded (FFPE) tissue samples from patients with histopathological diagnosis of PCM were analyzed. DNAs were extracted and amplified for a region of the second exon of the PbGP43 gene. Products were sequenced and aligned with other PbGP43 sequences available. A haplotype network and the phylogenetic relationships among sequences were inferred. Amino acid substitutions were investigated regarding the potential to modify physicochemical properties in the proteins. Six phylogenetic lineages were identified as belonging to the P. brasiliensis complex. Two lineages did not group with any of the four recognized species of the complex, and, interestingly, one of them comprised only FFPE samples. A coinfection involving two lineages was found. Five parsimony-informative sites were identified and three of them showed radical non-synonymous substitutions with the potential to promote changes in the protein. This study expands the knowledge regarding the genetic diversity existing in the P. brasiliensis complex and shows the potential of FFPE samples in species identification and in detecting coinfections.
Assuntos
Paracoccidioides , Paracoccidioidomicose , Antígenos de Fungos/genética , Biópsia , Proteínas Fúngicas/genética , Genótipo , Humanos , Paracoccidioides/genética , Paracoccidioidomicose/diagnóstico , Inclusão em Parafina , FilogeniaRESUMO
Based on polymorphisms of the gp43 precursor gene, genotyping of Paracoccidioides brasiliensis was achieved in 6 out of 10 paraffin-embedded tissue samples by modifying a nested PCR procedure used in the diagnosis of the fungal infection. Nine of the samples originated in Brazil. Three sequences were identical to a previously published consensus sequence identifying the P. brasiliensis isolates as members of the formerly named species 1. In contrast, two sequences revealed substitutions identical to isolates of the phylogenetic species 2. Applying the method to a lung biopsy from a proven case of paracoccidioidomycosis diagnosed in Austria, the gp43 sequence revealed substitutions so far only described in five strains originating from Venezuela. Combining travel history and result of this new method, the most probable country of origin of the infections could be identified. Since endemic mycosis are often diagnosed by histopathology only, our method could help to extend epidemiological studies of paracoccidioidomycosis.