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.
Highlights
S. brasiliensis, S. globosa, S. schenckii s. str and S. luriei make up the “pathogenic clade” of the genus Sporothrix
We designed a pair of primers based on the conserved sequence of the calmodulin gene of Sporothrix spp. and probes with different fluorescent signals based on the divergent sequences of S. globosa, S. schenckii s. str and S. brasiliensis
Through the verification of nucleic acid, clinical and spiked sample detection, the multiplex real-time polymerase chain reaction (PCR) could quickly and accurately identify the three clinically relevant species of Sporothrix spp. with high sensitivity. This new assay could be applied in epidemiology, clinical diagnosis, and experiments with sporotrichosis to control new outbreaks, reduce diagnostic and identification time, and improve test efficiency
Summary
Sporotrichosis is a subacute or chronic infectious disease caused by dimorphic fungi of Sporothrix spp., which are distributed worldwide, especially in tropical and subtropical regions[1,2]. The pathogen of sporotrichosis, S. schenckii sensu lato, was recognized as the sole agent for more than a century following its first isolation in 1898 by Benjamin Schenck[10]. Based on macroscopic characteristics and physiological and molecular aspects, S. schenckii sensu lato is considered to be a complex of several distinct species, including S. brasiliensis, S. mexicana, S. globosa, and S. schenckii s. Str, S. brasiliensis are medically important species within the Sporothrix genus. These species differ in ecology, distribution and epidemiology. S. brasiliensis, which is associated with severe clinical forms of sporotrichosis[16], is considered the most virulent species, followed by S. schenckii s. Identification at the species level is critical for public health and appropriate patient management[17,18]
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