Rapid Identification of Emerging Human-Pathogenic Sporothrix Species with Rolling Circle Amplification

Autor(es)

Rodrigues, Anderson M. ^[1] ; Najafzadeh, Mohammad J. ^[2] ; de Hoog, G. Sybren ^[3] ; de Camargo, Zoilo P. ^[1]

Afiliação do(s) autor(es)

^[1] Univ Fed Sao Paulo, Dept Microbiol Immunol & Parasitol, Div Cell Biol, Sao Paulo - Brazil

^[2] Mashhad Univ Med Sci, Ghaem Hosp, Sch Med, Dept Parasitol & Mycol, Mashhad - Iran

^[3] CBS KNAW Fungal Biodivers Ctr, Cent Bur Schimmelcultures, Utrecht - Netherlands

• Tipo de documento: Artigo Científico
• Fonte: FRONTIERS IN MICROBIOLOGY; v. 6, DEC 8 2015.
• Citações Web of Science: 9

Resumo

Sporothrix infections are emerging as an important human and animal threat among otherwise healthy patients, especially in Brazil and China. Correct identification of sporotrichosis agents is beneficial for epidemiological surveillance, enabling implementation of adequate public-health policies and guiding antifungal therapy. In areas of limited resources where sporotrichosis is endemic, high throughput detection methods that are specific and sensitive are preferred over phenotypic methods that usually result in misidentification of closely related Sporothrix species. We sought to establish rolling circle amplification (RCA) as a low-cost screening tool for species-specific identification of human pathogenic Sporothrix. We developed six species specific padlock probes targeting polymorphisms in the gene encoding calmodulin. BLAST-searches revealed candidate probes that were conserved intraspecifically; no significant homology with sequences from humans, mice, plants or microorganisms outside members of Sporothrix were found. The accuracy of our RCA based assay was demonstrated through the specificity of probe-template binding to 25 S. brasiliensis, 58 S. schenokii, 5 S. globosa, 1 S. luriei, 4 S. mexicana, and 3 S. pallida samples. No cross reactivity between closely related species was evident in vitro, and padlock probes yielded 100% specificity and sensitivity down to 3 x 10(6) copies of the target sequence. RCA-based speciation matched identifications via phylogenetic analysis of the gene encoding calmodulin and the rDNA operon (kappa 1.0; 95% confidence interval 1.0-1.0), supporting its use as a reliable alternative to DNA sequencing. This method is a powerful tool for rapid identification and specific detection of medically relevant Sporothrix, and due to its robustness has potential for ecological studies. (AU)

• Processo FAPESP: 11/07350-1 - Biologia do complexo Sporothrix schenckii: Análise de marcadores moleculares e proteômicos e o papel de moléculas antigênicas e antifúngicas na esporotricose
• Beneficiário: Anderson Messias Rodrigues
• Modalidade de apoio: Bolsas no Brasil - Doutorado
• Processo FAPESP: 09/54024-2 - Biologia molecular e proteômica de fungos de interesse médico: Paracoccidioides brasiliensis e Sporothrix schenckii
• Beneficiário: Zoilo Pires de Camargo
• Modalidade de apoio: Auxílio à Pesquisa - Temático