Molecular Diagnosis of Pathogenic Sporothrix Species
Anderson Messias Rodrigues1, G Sybren de Hoog2, Zoilo Pires de Camargo1
1Departamento de Microbiologia, Imunologia e Parasitologia, Disciplina de Biologia Celular, Universidade Federal de São Paulo (UNIFESP), São Paulo, São Paulo, Brazil.
Background:
Sporotrichosis is a chronic (sub)cutaneous infection caused by thermodimorphic fungi in the order, Ophiostomatales. These fungi are characterized by major differences in routes of transmission, host predilections, species virulence, and susceptibilities to antifungals. Sporothrix species emerge in the form of outbreaks. Large zoonoses and sapronoses are ongoing in Brazil and China, respectively. Current diagnostic methods based on morphology and physiology are inaccurate due to closely related phenotypes with overlapping components between pathogenic and non-pathogenic Sporothrix. There is a critical need for new diagnostic tools that are specific, sensitive, and cost-effective.
Methodology:
We developed a panel of novel markers, based on calmodulin (CAL) gene sequences, for the large-scale diagnosis and epidemiology of clinically relevant members of the Sporothrix genus, and its relative, Ophiostoma. We identified specific PCR-based markers for S. brasiliensis, S. schenckii, S. globosa, S. mexicana, S. pallida, and O. stenoceras. We employed a murine model of disseminated sporotrichosis to optimize a PCR assay for detecting Sporothrix in clinical specimens.
Results:
Primer-BLAST searches revealed candidate sequences that were conserved within a single species. Species-specific primers showed no significant homology with human, mouse, or microorganisms outside the Sporothrix genus. The detection limit was 10-100 fg of DNA in a single round of PCR for identifying S. brasiliensis, S. schenckii, S. globosa, S. mexicana, and S. pallida. A simple, direct PCR assay, with conidia as a source of DNA, was effective for rapid, low-cost genotyping. Samples from a murine model of disseminated sporotrichosis confirmed the feasibility of detecting S. brasiliensis and S. schenckii DNA in spleen, liver, lungs, heart, brain, kidney, tail, and feces of infected animals.
Conclusions:
This PCR-based method could successfully detect and identify a single species in samples from cultures and from clinical specimens. The method proved to be simple, high throughput, sensitive, and accurate for diagnosing sporotrichosis.
Insights
A new PCR-based diagnostic tool accurately identifies Sporothrix fungal species, crucial for diagnosing sporotrichosis. This method offers a sensitive, rapid, and cost-effective solution for clinical specimens.
Area of Science:
- Medical Mycology
- Molecular Diagnostics
- Infectious Diseases
Background:
- Sporotrichosis is a chronic fungal infection caused by Sporothrix species, presenting diagnostic challenges due to phenotypic similarities between pathogenic and non-pathogenic strains.
- Outbreaks of sporotrichosis, including zoonotic and sapronotic forms, highlight the need for accurate epidemiological tracking.
- Current diagnostic methods lack specificity and sensitivity, necessitating novel approaches for reliable identification.
Purpose of the Study:
- To develop and validate novel, specific molecular markers for the large-scale diagnosis and epidemiology of clinically relevant Sporothrix species.
- To establish a sensitive, accurate, and cost-effective PCR-based assay for identifying Sporothrix in clinical samples.
Main Methods:
- Novel markers were designed based on calmodulin (CAL) gene sequences for specific PCR detection of Sporothrix species (S. brasiliensis, S. schenckii, S. globosa, S. mexicana, S. pallida) and Ophiostoma stenoceras.
- A murine model of disseminated sporotrichosis was utilized to optimize the PCR assay for detecting Sporothrix DNA in various clinical specimens.
- Species-specific primers were validated using Primer-BLAST to ensure accuracy and minimize off-target amplification.
Main Results:
- The developed PCR assay demonstrated high specificity, with primers showing no significant homology to non-target organisms.
- The detection limit for target species was as low as 10-100 fg of DNA in a single PCR round.
- The assay successfully identified Sporothrix DNA in various tissues and feces from a murine model, confirming its feasibility for clinical specimens.
Conclusions:
- A novel PCR-based method enables accurate, sensitive, and high-throughput detection and identification of single Sporothrix species.
- This diagnostic approach is simple, cost-effective, and suitable for both cultured samples and direct clinical specimens.
- The developed assay represents a significant advancement for the diagnosis of sporotrichosis, addressing the limitations of current methods.
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