Development of new molecular tools for the identification of medically relevant Fusarium spp.

Abstract

Recent advances in medicine have improved and prolonged lives, but they have also increased the number of individuals vulnerable to fungal diseases. Fusariosis leads to the development of onychomycosis and keratitis in immunocompetent individuals, and there is the possibility of systemic spread in immunocompromised individuals. Fusarium transmission route refers to the inhalation of fungal propagules or traumatic inoculation into the host's tissues. The main etiologic agents are inserted in the F. solani (FSSC), F. oxysporum (FOSC), and F. dimerum (FDSC) complexes. Despite their clinical relevance, diagnostic methodologies are not yet fully elucidated or have limitations. Therefore, this project aims to establish and standardize real-time quantitative PCR (qPCR) to identify Fusarium spp. of clinical importance differentiating FSSC, FOSC, and FDSC. Standardization will be based on the "Sequence Detection System - Chemistry Guide" protocol, which includes guidelines for preparing qPCR assays. Therefore, we will design complex-specific molecular primers and hydrolysis probes to detect FSSC, FOSC, and FDSC, favoring multiplex assays. qPCR performance will be assessed through amplification efficiency (E), sensitivity (Sen), specificity (Esp), detection limit (LoD), competition between targets, positive predictive value (VPP), and negative predictive value (VPN). The performance of qPCR will be compared with other standard molecular tools, such as DNA sequencing (e.g., EF-1±) using the Kappa concordance test. We will evaluate the potential for DNA detection of Fusarium spp. using a murine model of fusariosis (BALB/c). The present study will improve the diagnosis of fusariosis with the possibility of detecting species of clinical relevance in Brazil. (AU)