title: Molecular Diagnosis of Pathogenic Sporothrix Species.
abstract: 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.
  
  
  METHODS
  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.
authors.affiliation: Departamento de Microbiologia, Imunologia e Parasitologia, Disciplina de Biologia Celular, Universidade Federal de São Paulo (UNIFESP), São Paulo, São Paulo, Brazil.
authors.forename: Anderson Messias
authors.initials: AM
authors.lastname: Rodrigues
authors.affiliation: CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands.
authors.forename: G Sybren
authors.initials: GS
authors.lastname: de Hoog
authors.affiliation: Departamento de Microbiologia, Imunologia e Parasitologia, Disciplina de Biologia Celular, Universidade Federal de São Paulo (UNIFESP), São Paulo, São Paulo, Brazil.
authors.forename: Zoilo Pires
authors.initials: ZP
authors.lastname: de Camargo
mesh_terms: D000825:Animal Structures / Q000382:microbiology; D000818:Animals; D002147:Calmodulin / Q000235:genetics; D004195:Disease Models, Animal; D004196:Disease Outbreaks; D005243:Feces / Q000382:microbiology; D008297:Male; D008807:Mice, Inbred BALB C; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013175:Sporothrix / Q000235:genetics / Q000302:isolation & purification*; D013174:Sporotrichosis / Q000175:diagnosis* / Q000382:microbiology*
chemical_list: D002147:Calmodulin