Detection of gp43 and ITS1-5.8S-ITS2 Ribosomal RNA Genes of Paracoccidioides brasiliensis in Paraffin-embedded Tissue

Paracoccidioidomycosis (PCM) is a deep mycosis caused by the thermo-dependent dimorphic fungus Paracoccidioides brasiliensis and is prevalent in Latin American countries. An increase in PCM has been reported in recent years and the disease is now recognized as one of the imported fungal infections in Japan. To date, more than 15 cases of PCM have been reported in our country, and five of them were diagnosed by clinical and histopathological findings without mycological study. We applied 2 nested polymerase chain reaction (PCR) amplification methods for detecting P. brasiliensis genes from paraffin-embedded tissue specimens. Successfully amplified were: a 473 base pairs fragment of gp43 gene of P. brasiliensis (located from 741st to 1, 213rd base), and a 418 base pairs fragment of 5.8S ribosomal RNA gene of P. brasilienisis which included internal transcribed spacers (ITS) 1 and 2 (located from 131st at ITS1 to 195th at ITS2) in paraffin-embedded murine tissues infected with P. brasiliensis yeast cells. The authenticity of the PCR products was confirmed by nucleotide sequence analysis. These results indicate that the two nested PCR methods may be useful for diagnosis of PCM.