Fast diagnosis of sporotrichosis caused by Sporothrix globosa, Sporothrix schenckii, and Sporothrix brasiliensis based on multiplex real-time PCR

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 .

A One Health Approach to Combatting Sporothrix brasiliensis: Narrative Review of an Emerging Zoonotic Fungal Pathogen in South America

“…These assays were evaluated using different Sporothrix species, isolates, and types of specimens, and in general, most of the protocols target the calmodulin gene (considered the reference standard for species-level identification) or nuclear ribosomal internal transcribed spacer (ITS) region. Sensitivity and specificity were consistently high across assays (>90%) [ 113 , 114 , 115 , 116 , 117 , 118 , 119 , 120 , 121 ]. However, molecular assays require advanced laboratory infrastructure, and utility is limited by the lack of commercially available kits, standardized methods, and a limited number of validation studies.…”

Can cats cause colossal contagious cutaneous carbuncles?

“…is 87.9%, 39.4%, and 93.9% in clinical samples, respectively. There was no significant difference between the culture and multiplex real-time PCR in positive detection rates [6]. Therefore, both skin biopsy In positive cultures, filamentous hyaline creamcolored colonies develop a brown/black color in several weeks on Sabouraud agar with incubation at 25 degrees Celsius.…”

A complexidade da esporotricose: médico veterinário clínico sabe diagnosticar e tratar esta doença?

“…O diagnóstico da esporotricose pode ser realizado pela observação de um conjunto de manifestações clínicas associado a testes laboratoriais incluindo exame histopatológico, detecção molecular e testes cutâneos para identificação de anticorpos, visto que o ³padrão ouro´para o diagnóstico de esporotricose consiste no isolamento microbiológico de amostras clínicas obtidas de lesões ativas, pus, secreções ou biópsias com identificação do fungo (ZHANGM et. al., 2019).…”

“…Constituindo um complexo de seis espécies, sendo no Brasil o Sporothrix brasiliensis de maior importância epidemiológica (MEGID; RIBEIRO; PAES, 2016). A S. brasiliensis é a espécie mais virulenta correlacionada a formas clínicas graves da esporotricose, por essa razão se torna de suma importância para a saúde pública e controle epidemiológico (ZHANGM et al, 2019).…”