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

Abstract

Sporotrichosis is a chronic, cosmopolitan granulomatous mycosis that affects humans and animals. The infection is caused by the dimorphic fungi Sporothrix 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 Sporothrix sp. detection. FFPE mycological culture pellet samples of different Sporothrix species (S. chilensis, S. mexicana, S. pallida, S. globosa, S. brasiliensis and S. schenckii) were used as positive controls and clinical FFPE tissue samples of animals positive for Cryptococcus sp., Leishmania infantum and Histoplasma 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 Sporothrix. The chemical extraction protocol with the 5 μm thick paraffin section was more effective in extracting DNA from Sporothrix 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 Sporothrix sp. diagnosis in FFPE samples of cats.

Trends in Molecular Diagnostics and Genotyping Tools Applied for Emerging Sporothrix Species

“…Major samples may include pus, exudates, aspirates, and fresh or FFPE tissues. Encouraging results have been published in the past decade using specimens from human, murine or feline origins [ 64 , 89 , 100 , 112 , 113 , 147 , 165 ]. Trends in molecular diagnosis of sporotrichosis show that multiplexing is the leading strategy for the near future, allowing the detection and speciation of different agents in real-time reactions [ 89 , 113 ].…”

“…The method had a detection limit of 40 fg of Sporothrix DNA from cultures to biological samples [ 125 ]. Recently, this assay was successfully used for the genus-level diagnosis in formalin-fixed and paraffin-embedded (FFPE) samples of cats with sporotrichosis [ 165 ]. Interestingly, in experimentally infected mice, Mendoza et al [ 166 ] reported low performance of Hu et al assay [ 125 ] when compared with the diagnosis obtained by culture and direct examination [ 166 ].…”

PCR and Other Genomic Techniques Applied to Veterinary Medicine

“…Nested PCR is, therefore, a variation of PCR that involves two amplification steps, producing greater sensitivity and specificity. Luiz et al (2022) used the protocol in the diagnosis of sporotrichosis, a cosmopolitan granulomatous mycosis that affects humans and animals, in felines (LUIZ et al, 2022).…”