Development and evaluation of a multiplex qPCR assay for rapid diagnostics of emerging sporotrichosis

Autor(es)

Della Terra, Paula Portella ^{[1, 2]} ; Gonsales, Fernanda Fidelis ^[3] ; de Carvalho, Jamile Ambrosio ^{[1, 2]} ; Hagen, Ferry ^{[4, 5, 6]} ; Kano, Rui ^[7] ; Bonifaz, Alexandro ^[8] ; de Camargo, Zoilo Pires ^{[1, 2]} ; Rodrigues, Anderson Messias ^{[1, 2]}

Afiliação do(s) autor(es)

^[1] Fed Univ Sao Paulo UNIFESP, Dept Med, Discipline Infect Dis, Sao Paulo - Brazil

^[2] Fed Univ Sao Paulo UNIFESP, Lab Emerging Fungal Pathogens, Dept Microbiol Immunol & Parasitol, Discipline Cellular Biol, BR-04023062 Sao Paulo - Brazil

^[3] Univ Sao Paulo, Fac Anim Sci & Food Engn, Pirassununga - Brazil

^[4] Univ Med Ctr Utrecht, Dept Med Microbiol, Utrecht - Netherlands

^[5] Westerdijk Fungal Biodivers Inst, Dept Med Mycol, Utrecht - Netherlands

^[6] Jining 1 Peoples Hosp, Med Mycol Lab, Jining, Shandong - Peoples R China

^[7] Nihon Univ, Coll Bioresource Sci, Dept Vet Dermatol, Fujisawa, Kanagawa - Japan

^[8] Hosp Gen Mexico City, Dermatol Serv, Dept Mycol, Mexico City, DF - Mexico

• Tipo de documento: Artigo Científico
• Fonte: TRANSBOUNDARY AND EMERGING DISEASES; NOV 2021.
• Citações Web of Science: 0

Resumo

Sporothrix schenckii and related species are the agents of human and animal sporotrichosis. Routine diagnoses using classical mycological approaches are unspecific due to overlapping phenotypes. As the frequency and prevalence of sporotrichosis increases worldwide, developing specific, sensitive and cost-effective diagnostic tools is essential to understand the distribution patterns, map-affected areas and promote specific public health strategies to mitigate future outbreaks. Polymorphisms among the beta-tubulin gene were exploited to speciate S. brasiliensis, S. schenckii and S. globosa in a one-tube multiplex probe-based qPCR assay. A panel of 84 Sporothrix revealed 100% specificity (AUC = 1.000, 95% CI = 0.971-1.000, p < .0001) without cross-reacting with other medically relevant fungi, human, feline or murine DNA. Speciation via multiplex qPCR matched phylogenetic identification (Kappa = 1.0; 95% CI = 1.0-1.0; very good agreement), supporting its use as a reliable alternative to DNA sequencing. Remarkably, the lower limit of detection was 3 copies of the target for all species. As a proof of concept, we used swabs of wound exudate of 70 cats suspected of sporotrichosis to reveal an overwhelming occurrence of S. brasiliensis in 69 specimens (sensitivity = 98.57%; 95%CI: 92.3-100.0 and specificity = 100%; 95% CI = 78.2-100). In comparison to culture, qPCR showed a larger area under the curve (AUC = 0.993 +/- 0.007; 95% CI = 0.944-1.000; p < .0001; Youden's index = 0.9857), supporting that qPCR is an essential tool for accurately detect Sporothrix DNA directly from clinical samples, thus accelerating the diagnosis of sporotrichosis. Moreover, our multiplex qPCR system has the potential to increase diagnostic capacity in Sporothrix-affected areas, helping the local animal health agent or veterinarian to quickly identify and isolate new cases, which will likely benefit thousands of patients infected every year worldwide. (AU)

• Processo FAPESP: 18/21460-3 - Estudo de diferentes preparações antigênicas de Paracoccidioides lutzii para a padronização do teste de ELISA como auxílio no diagnóstico da paracoccidioidomicose por P. lutzii
• Beneficiário: Zoilo Pires de Camargo
• Modalidade de apoio: Auxílio à Pesquisa - Regular
• Processo FAPESP: 17/27265-5 - Epidemiologia molecular e perspectivas genômicas na evolução e propagação de patógenos fúngicos emergentes
• Beneficiário: Anderson Messias Rodrigues
• Modalidade de apoio: Auxílio à Pesquisa - Jovens Pesquisadores