Diagnostic performance of a biotin-labeled 4D1 sandwich ELISA for serum antigen detection in talaromycosis

BACKGROUND: Talaromycosis, caused by the thermally dimorphic fungus Talaromyces marneffei, is a life-threatening opportunistic infection in individuals with advanced immunodeficiency, particularly people living with HIV in endemic regions of Southeast Asia. Early diagnosis is essential to reduce morbidity and mortality; however, conventional diagnosis relies mainly on fungal culture, which is time-consuming and has limited sensitivity. METHODS: We developed and clinically evaluated a direct sandwich enzyme-linked immunosorbent assay (ELISA) using a biotin-labeled monoclonal antibody (4D1) for the detection of T. marneffei cytoplasmic yeast antigen in serum. The assay employed the same monoclonal antibody for both capture and detection in a double-recognition format. Analytical performance, cross-reactivity, and clinical diagnostic accuracy were systematically evaluated. RESULTS: No cross-reactivity was observed among the tested fungal antigens under the experimental conditions. The analytical limit of detection (LOD) in pooled human serum was 19.398 μg/mL. Clinical evaluation included 79 culture-confirmed talaromycosis cases and 381 non-talaromycosis controls. At an optimized optical density cut-off of 0.268, the assay demonstrated a sensitivity of 88.61% (95% confidence interval [CI] 79.47-94.66%) and a specificity of 96.06% (95% CI 93.59-97.78%). CONCLUSIONS: The biotin-labeled 4D1 sandwich ELISA provides a rapid and accurate diagnostic method with good concordance to culture and may support improved clinical diagnosis of talaromycosis. Its potential application for treatment monitoring requires further validation.