Detection of Sporothrix brasiliensis in Human Tissue by Direct PCR Using Species-Specific Primers and NESTED PCR in a New Epidemic Area of Sporotrichosis

Abstract

Sporotrichosis is a fungal disease caused by the infection of Sporothrix spp. The reference standard method for the diagnosis of sporotrichosis is still the mycological culture from clinical samples. Molecular methods have been developed to facilitate the diagnosis of the disease. The aim of the study was to compare two Polymerase Chain Reaction (PCR) techniques, using species-specific primers and nested PCR, to diagnose human sporotrichosis directly from tissues of patients treated at a public hospital in Pernambuco, Brazil. Tissue fragments samples were subjected to DNA extraction followed by PCR using species-specific primers for S. brasiliensis and S. schenckii and nested PCR. Positive DNA bands for S. brasiliensis were amplified in both methods. Our findings suggest that PCR is an important tool for the rapid and accurate diagnosis of sporotrichosis in humans. Histological analysis of the same number of patients, who had a tissue fragment analyzed by species-specific primers or nested PCR, showed yeast-like cells by periodic acid of Shiff staining in only 10%. Considering hematoxylin and eosin staining, only one case was diagnosed as deep mycosis and the other cases had nonspecific diagnoses. The present work compared different molecular tools for the diagnosis of sporotrichosis from clinical samples. As far as we know, the comparison of these methodologies is pioneering and has the advantage of improving diagnosis and early detection in patients with sporotrichosis caused by Sporothrix sensu lato, especially in new endemic areas, as well as the possibility of implementing these tools in the diagnostic routine.

Keywords: Biopsy; In situ; Molecular diagnosis; Nested PCR; Sporotrichosis.