Diagnóstico de Esporotricose: Reações de Detecção (PCR) e Diferenciação (PCR- RFLP) de Espécies de Sporothrix spp.

Abstract

Sporotrichosis is an emerging subcutaneous mycosis of significant relevance in the Amazon region, with a marked increase in cases among humans and felines. Rapid and accurate identification of the etiological agents is essential for clinical management and epidemiological surveillance. This study aimed to enhance the molecular diagnosis of sporotrichosis by evaluating different DNA extraction methods and comparing the sensitivity of three target regions of ribosomal DNA (ITS, ITS/LSU, and D1/D2 of 28S) for PCR amplification. Three DNA extraction protocols (phenol-chloroform, silica columns, and salt precipitation) were applied to isolates of Sporothrix brasiliensis, S. schenckii, a regional isolate of Sporothrix spp., as well as Candida albicans and Aspergillus niger as external controls. The phenol-chloroform method yielded the highest DNA yield and purity. The primers ITS1–ITS4 were the most sensitive, detecting as little as 0.0002 ng of DNA, followed by NL1–NL4 (0.002 ng) and ITS5–NL4 (0.02 ng). An in silico analysis evaluated five restriction enzymes (HaeIII, MspI, HinfI, DdeI, and RsaI), but the fragmentation profiles generated by the different Sporothrix species showed very similar sizes, with differences of less than 20 bp, making practical differentiation by PCR-RFLP challenging. Experimentally, PCR-RFLP allowed for the distinction of Sporothrix isolates from unrelated fungi (C. albicans and A. niger), but it was not effective in differentiating between S. brasiliensis and S. schenckii. Phylogenetic analysis based on the ITS region confirmed that all clinical isolates from Manaus belonged to the S. brasiliensis clade. It is concluded that the combination of phenol-chloroform extraction and PCR with ITS1–ITS4 primers represents a sensitive and accessible alternative for the diagnosis of sporotrichosis in the Amazon region.