A novel approach for feline sporotrichosis pathogen detection based on loop-mediated isothermal amplification

BACKGROUND: Sporotrichosis is a chronic, mycotic infection caused by fungi of the genus Sporothrix. Zoonotic sporotrichosis occurs mainly through S. brasiliensis transmission, resulting from the organism's traumatic introduction via scratches or bites, or contact with exudate from contaminated cats. The loop-mediated isothermal amplification (LAMP) assay is a viable molecular alternative for detecting Sporothrix in veterinary low-resource settings. HYPOTHESIS/OBJECTIVES: To develop a LAMP method for Sporothrix identification using fungal isolates and clinical samples of domestic cats (Felis catus). MATERIALS AND METHODS: DNA samples were collected from Sporothrix isolates and clinical samples of cats positive for sporotrichosis. Six LAMP primers were designed to amplify the 28S ribosomal RNA region of S. schenckii and S. brasiliensis. Colorimetric assay and agarose gel electrophoresis were used to analyse the LAMP amplification. Isolated samples were sequenced using the Sanger technique, employing the amplification of genetic material by conventional PCR with the external primers of LAMP. RESULTS: A sensitivity of 96.77% for isolated Sporothrix samples was found using the LAMP method, confirmed by Sanger sequencing. The detection limit of LAMP was between 1 and 10 pg of Sporothrix DNA according to the sample matrix. LAMP showed a sensitivity of 100% using blood samples, 77.78% using intranasal swabs and 92.31% and 100% using swab and adhesive tape samples of cutaneous lesions, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: These findings support a simple and quick LAMP-based screening tool for detecting Sporothrix in isolated and clinical samples. This accessible test can aid in disease management when standard culture analysis is unavailable or impractical.

Authors: S. Amadei, J. Campos, A. Bertão-Santos, A. Frentzel, H. Ávila, F. S. Monti and M. R. Farias Title: A novel approach for feline sporotrichosis pathogen detection based on loop-mediated isothermal amplification Full source: Journal, Year,Vol Volume, Iss Issue, pp Pages
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Abstract	Source
BACKGROUND: Sporotrichosis is a chronic, mycotic infection caused by fungi of the genus Sporothrix. Zoonotic sporotrichosis occurs mainly through S. brasiliensis transmission, resulting from the organism's traumatic introduction via scratches or bites, or contact with exudate from contaminated cats. The loop-mediated isothermal amplification (LAMP) assay is a viable molecular alternative for detecting Sporothrix in veterinary low-resource settings. HYPOTHESIS/OBJECTIVES: To develop a LAMP method for Sporothrix identification using fungal isolates and clinical samples of domestic cats (Felis catus). MATERIALS AND METHODS: DNA samples were collected from Sporothrix isolates and clinical samples of cats positive for sporotrichosis. Six LAMP primers were designed to amplify the 28S ribosomal RNA region of S. schenckii and S. brasiliensis. Colorimetric assay and agarose gel electrophoresis were used to analyse the LAMP amplification. Isolated samples were sequenced using the Sanger technique, employing the amplification of genetic material by conventional PCR with the external primers of LAMP. RESULTS: A sensitivity of 96.77% for isolated Sporothrix samples was found using the LAMP method, confirmed by Sanger sequencing. The detection limit of LAMP was between 1 and 10 pg of Sporothrix DNA according to the sample matrix. LAMP showed a sensitivity of 100% using blood samples, 77.78% using intranasal swabs and 92.31% and 100% using swab and adhesive tape samples of cutaneous lesions, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: These findings support a simple and quick LAMP-based screening tool for detecting Sporothrix in isolated and clinical samples. This accessible test can aid in disease management when standard culture analysis is unavailable or impractical.