Development and validation of a new quantitative reverse transcription PCR assay for the diagnosis of human sporotrichosis

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de Macedo, Priscila Marques ; Sturny-Leclere, Aude ; Freitas, Dayvison Francis Saraiva ; Ghelfenstein-Ferreira, Theo ; Gutierrez-Galhardo, Maria Clara ; Almeida, Marcos de Abreu ; Rodrigues, Anderson Messias ; Pautet, Thierry ; Hamane, Samia ; Almeida-Paes, Rodrigo ; Zancope-Oliveira, Rosely Maria ; Alanio, Alexandre

• Document type: Journal article
• Source: Medical Mycology; v. 61, n. 7, p. 7-pg., 2023-07-06.

Abstract

Sporotrichosis is an emergent public health problem. The mycological diagnosis of this infection is based on culture, which is fastidious and may represent a biohazard for technicians. Although not widely implemented in routine diagnosis, molecular methodologies are fast, have good accuracy, and can be easily standardized, aiding in the early diagnosis of neglected mycoses. This study aimed at implementing a new pan-Sporothrix quantitative reverse transcription PCR (RT-qPCR) assay, and then validating it on clinical samples from confirmed human sporotrichosis cases. A total of 68 human samples with culture-confirmed diagnosis of sporotrichosis were collected from 64 patients followed at a Brazilian reference center for endemic mycoses. These samples were submitted to whole nucleic acid extraction, followed by an RT-qPCR protocol. The limit of detection was 244 fg, the efficiency was 2.0 (100%), and the assay could amplify the genetic material of the three major clinically relevant species of the genus Sporothrix. Among the 68 samples analyzed, 62 were positive in RT-qPCR, showing an overall sensitivity of 91.18%, which variated according to the type of biological sample: 96.72% in skin samples (n = 61) and 100% in respiratory samples (n = 3), whereas all cerebrospinal fluid specimens (n = 4) were negative. The specificity was 100% when tested in 25 samples from patients with other mycoses and tuberculosis. In addition, DNA from 93 fungal species did not yield positive results, confirming the high specificity of this test. Our RT-qPCR presented high sensitivity and specificity, representing an excellent tool for a fast and reliable diagnosis of human sporotrichosis. Lay Summary Sporotrichosis is a deep mycosis with limited laboratorial techniques for fast diagnosis. We developed an assay able to detect the genetic material of fungal agents of sporotrichosis, and validated it in human specimens from patients with this disease, obtaining high positivity and specificity. (AU)

• FAPESP's process: 17/27265-5 - Molecular epidemiology and genomic perspectives on the evolution and spread of emerging fungal pathogens
• Grantee: Anderson Messias Rodrigues
• Support Opportunities: Research Grants - Young Investigators Grants