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Identification and Validation of Rhizopus-Specific Glucoamylase as a Novel Biomarker for Diagnosing Mucormycosis
Hansraj Choudhary1,2, Harsimran Kaur1, Shreya Singh3
1Department of Medical Microbiology, Postgraduate Institute of Medical Education & Research (PGIMER), Chandigarh, India.
Background:
Mucormycosis is a life-threatening invasive fungal infection. Current diagnosis relies on insensitive techniques with prolonged turnaround times, underscoring the need for rapid noninvasive diagnostic tools. The current study aimed to identify the antigens of Rhizopus arrhizus that can facilitate serodiagnosis of mucormycosis and assess their diagnostic potential in patients.
Methods:
This was a 2-stage systematic study combining antigen discovery and clinical validation. Using immunoproteomics, we identified immunoreactive protein spots in R. arrhizus secretory extract, using pooled serum samples from 6 patients with mucormycosis. We performed liquid chromatography-tandem mass spectrometry (LC-MS/MS) to characterize the most reactive spot. Next, we developed an indirect enzyme-linked immunosorbent assay using purified protein and evaluated its diagnostic performance in 92 patients with mucormycosis and 41 controls (22 with other mold infections and 19 with diabetes mellitus alone). Participants were randomly divided 1:1 into derivation and validation cohorts. Receiver operating characteristic curve analysis identified optimal cutoff values.
Results:
Immunoproteomics identified 3 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 patients with mucormycosis and 11 controls), the area under the receiver operating characteristic curve for anti-RSG immunoglobulin (Ig) G was 0.933. In the validation cohort, anti-RSG IgG achieved 76.1% sensitivity (95% confidence interval, 61.2%-87.4%) and 100% specificity (71.5%-100%). The assay also detected antibodies in 85.1% of culture-negative but microscopy-positive cases and in 87.5% of mixed fungal infection cases.
Conclusions:
RSG is a potential biomarker for diagnosing mucormycosis. Anti-RSG IgG shows strong diagnostic promise and merits prospective multicenter clinical validation.
