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.
Highlights
Sporotrichosis is a chronic cosmopolitan granulomatous mycosis, caused by the thermodimorphic fungus of the genus Sporothrix, which affects humans and a wide variety of animals, especially cats and dogs [1,2,3,4]
S. schenckii and S. globosa are the species generally associated with the classical transmission route, while the species S. brasiliensis is associated with zoonotic sporotrichosis transmitted by cats [11, 15,16,17,18] in Brazil, mainly in Rio de Janeiro [19, 20], and in Argentina [21] and Paraguay [22]
The comparison of the DNA extraction protocols in the formalin fixed and paraffin-embedded tissues (FFPE) mycological culture pellet samples based on the concentration and purity of the DNA using both paraffin sectioning protocols (5 and 16 μm) is shown in the Table 1A
Summary
Sporotrichosis is a chronic cosmopolitan granulomatous mycosis, caused by the thermodimorphic fungus of the genus Sporothrix, which affects humans and a wide variety of animals, especially cats and dogs [1,2,3,4]. S. schenckii and S. globosa are the species generally associated with the classical (non-zoonotic) transmission route, while the species S. brasiliensis is associated with zoonotic sporotrichosis transmitted by cats [11, 15,16,17,18] in Brazil, mainly in Rio de Janeiro [19, 20], and in Argentina [21] and Paraguay [22]. Cats are the animal species most affected by sporotrichosis caused by S. brasiliensis. This disease can lead to death of cats and its main clinical signs in these animals are multiple skin nodules and ulcers, mucosal lesions, enlarged lymph nodes and respiratory signs [20]. The transmission of S. brasiliensis from an infected cat to humans, cats and dogs occurs through the bite, scratch, or contact with the exudate of cutaneous lesions [22]
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