ABSTRACT: The accurate diagnosis of sporotrichosis and identification at the species level are critical for public health and appropriate patient management. Compared with morphological identification methods, molecular diagnostic tests are rapid and have high sensitivity and standardized operating processes. Therefore, we designed a novel multiplex real-time polymerase chain reaction (PCR) method based on the calmodulin (CAL) gene for the identification of clinically relevant Sporothrix species: S. globosa, S. schenckii s. str., and S. brasiliensis. We evaluated the assay with clinical and spiked samples and assessed its diagnostic performance by comparing the results to those of culture and species-specific PCR. Thirty-three DNA templates were used to detect assay specificity, and three plasmids were constructed to create a standard curve and determine the limits of detection (LODs). For nucleic acid detection, the sensitivity and specificity reached 100%. The LODs were 10 copies, 10 copies and 100 copies for S. globosa, S. schenckii s. str and S. brasiliensis, respectively. For the clinical samples, the positive detection rates by culture, species-specific PCR and the multiplex real-time PCR assay were 87.9% (29/33), 39.4% (13/33), and 93.9% (31/33), respectively. For the spiked samples, the positive detection rates were both 100% for S. schenckii s. str and S. brasiliensis. Based on the above results, compared with culture and other molecular diagnosis methods, the novel multiplex real-time PCR assay is effective, fast, accurate, and highly sensitive. It has a lower reaction cost and lower sample volume requirements, can detect co-infections, and allows for standardized operation and easier interpretation of results. In the future, this assay could be developed into a commercial kit for the diagnosis and identification of S. globosa, S. schenckii s. str, and S. brasiliensis.
