Amadei Steffanie, Campos Júlia, Bertão-Santos Amanda, Frentzel Alis, Ávila Hugo, Monti Fabiana S, Farias Marconi R
Integrated Program in Neuroscience, McGill University, Montreal, Canada.
Department of Veterinary Medicine, School of Medicine and Life Sciences, Pontifical Catholic University of Paraná, Curitiba, Brazil.
Vet Dermatol. 2025 Aug;36(4):474-484. doi: 10.1111/vde.13345. Epub 2025 Apr 21.
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).
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.
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.
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.
孢子丝菌病是一种由孢子丝菌属真菌引起的慢性真菌感染。人畜共患孢子丝菌病主要通过巴西孢子丝菌传播，该病原体通过抓伤、咬伤或接触受污染猫的渗出物经外伤进入人体。环介导等温扩增（LAMP）检测法是在兽医资源匮乏环境中检测孢子丝菌的一种可行的分子检测方法。
假设/目的：开发一种利用家猫（Felis catus）真菌分离株和临床样本鉴定孢子丝菌的LAMP方法。
从孢子丝菌分离株和孢子丝菌病阳性猫的临床样本中收集DNA样本。设计了6条LAMP引物，用于扩增申克孢子丝菌和巴西孢子丝菌的28S核糖体RNA区域。采用比色法和琼脂糖凝胶电泳分析LAMP扩增结果。使用桑格技术对分离样本进行测序，通过常规PCR用LAMP外部引物扩增遗传物质。
使用LAMP方法对分离的孢子丝菌样本的敏感性为96.77%，经桑格测序确认。根据样本基质，LAMP的检测限在1至10 pg孢子丝菌DNA之间。LAMP对血液样本的敏感性为100%，对鼻拭子的敏感性为77.78%，对皮肤病变拭子和胶带样本的敏感性分别为92.31%和100%。
这些发现支持了一种基于LAMP的简单快速筛查工具用于检测分离样本和临床样本中的孢子丝菌。当无法进行标准培养分析或不切实际时，这种易于获得的检测方法有助于疾病管理。
