Identification and validation of Rhizopus-specific glucoamylase as a novel biomarker for diagnosing mucormycosis.

Abstract

Mucormycosis is a life-threatening invasive fungal infection. Current diagnosis relies on insensitive techniques with prolonged turnaround times, underscoring the need for rapid non-invasive diagnostic tools. The study aimed to identify the antigens of Rhizopus arrhizus that can facilitate serodiagnosis of mucormycosis and assess their diagnostic potential in patients. Two-stage systematic study combining antigen discovery and clinical validation. Using immunoproteomics, we identified immunoreactive protein spots in R. arrhizus secretory extract using pooled sera from six mucormycosis patients. We performed LC-MS/MS for characterizing the most reactive spot. Next, we developed an indirect ELISA using purified protein and evaluated its diagnostic performance in 92 mucormycosis cases and 41 controls (other mold infections [n=22], diabetes mellitus alone [n=19]). Participants were randomly divided 1:1 into derivation and validation cohorts. ROC curve analysis identified optimal cutoff values. Immunoproteomics identified three immunoreactive protein spots (70, 45, and 30 kDa) in crude extract. The most reactive 70 kDa spot was identified as Rhizopus-specific glucoamylase (RSG) by LC-MS/MS and confirmed with specific anti-Rhizopus glucoamylase antibody. In the derivation cohort (46 cases, 11 controls), anti-RSG IgG demonstrated area under ROC curve of 0.933. In the validation cohort, anti-RSG IgG achieved 76.1% sensitivity (95% CI: 61.2-87.4%) and 100% specificity (95% CI: 71.5-100%). Besides, the assay detected antibodies in 85.1% of culture-negative but microscopy-positive cases and in 87.5% of mixed fungal infection cases. RSG is a potential biomarker for diagnosing mucormycosis. Anti-RSG IgG shows strong diagnostic promise and merits prospective multicenter clinical validation.