Partial Alpha-Tubulin Gene Amplification by Nested PCR in Fresh Biological Samples and Formalin-Fixed, Paraffin-Embedded Tissues for Paracoccidioides spp. Identification.

Paracoccidioidomycosis is an important deep mycosis in Latin American countries. Its causative agents have been reclassified into five distinct species based on genetic differences: Paracoccidioides brasiliensis sensu stricto, Paracoccidioides americana, Paracoccidioides restrepiensis, Paracoccidioides venezuelensis and Paracoccidioides lutzii. In this study, we propose a new method, based on a nested PCR for partial alpha-tubulin gene amplification, as a tool for diagnosis and species differentiation directly from biological samples. The method could amplify the DNA of the five cultivable Paracoccidioides spp. without amplifying DNA from other fungal species such as Mucor fragilis, Histoplasma capsulatum, Aspergillus fumigatus, Candida albicans, Sporothrix brasiliensis, and Cryptococcus neoformans. The nested PCR detected Paracoccidioides spp. from fresh and formalin-fixed and paraffin-embedded (FFPE) tissues of experimentally infected animals. Amplicons obtained from fresh tissues were successfully used in traditional downstream methods, such as RFLP, for Paracoccidioides spp. identification.However, amplicons from FFPE tissues exhibited several artifacts induced by formalin, which interfered with the RFLP results. DNA sequencing of these nested PCR products revealed G > T/T > G, A > T/T > A, T > C/C > T, and G > A/A > G base changes, affecting the sequence targets for enzymatic digestion.