AUTHOR=Luiz Raul Leal Faria , Menezes Rodrigo Caldas , Pereira Sandro Antonio , Oliveira Raquel de Vasconcellos Carvalhaes de , Oliveira Manoel Marques Evangelista 

TITLE=Nested PCR for the Diagnosis of Feline Sporotrichosis From Formalin-Fixed and Paraffin-Embedded Samples Using Different DNA Extraction Protocols

JOURNAL=Frontiers in Veterinary Science

VOLUME=8

YEAR=2022

URL=https://www.frontiersin.org/journals/veterinary-science/articles/10.3389/fvets.2021.755897

DOI=10.3389/fvets.2021.755897

ISSN=2297-1769

ABSTRACT=<p>Sporotrichosis is a chronic, cosmopolitan granulomatous mycosis that affects humans and animals. The infection is caused by the dimorphic fungi <italic>Sporothrix</italic> sp. The aims of the present study were to evaluate, standardize and validate a nested PCR technique using two DNA purification kits for the extraction of DNA from formalin fixed and paraffin-embedded tissues (FFPE) for <italic>Sporothrix</italic> sp. detection. FFPE mycological culture pellet samples of different <italic>Sporothrix</italic> species (<italic>S. chilensis, S. mexicana, S. pallida, S. globosa, S. brasiliensis and S. schenckii</italic>) were used as positive controls and clinical FFPE tissue samples of animals positive for <italic>Cryptococcus</italic> sp., <italic>Leishmania infantum</italic> and <italic>Histoplasma</italic> sp. were used as negative controls. Ten clinical FFPE skin samples from cats with sporotrichosis were used to validate the nested PCR. These samples were cut into two distinct paraffin sectioning protocols (5 and 16 μm thick). The paraffin sections were subjected to two different DNA extraction kits (chemical and thermal extractions). A nested PCR was performed on the extracted DNA to identify the genus <italic>Sporothrix</italic>. The chemical extraction protocol with the 5 μm thick paraffin section was more effective in extracting DNA from <italic>Sporothrix</italic> sp. from FFPE samples and the nested PCR technique showed the highest sensitivities (100% in the positive controls and of 50% in the skin samples of cats) and specificity (100%). Therefore, the nested PCR using this protocol has great potential to be applied in <italic>Sporothrix</italic> sp. diagnosis in FFPE samples of cats.</p>