Detection of Galactofuranose-Containing Antigens Associated With Aspergillus Extracellular Vesicles in Urine: The Biological Basis of a New Diagnostic for Aspergillosis.

BACKGROUND: Establishing diagnosis of pulmonary infections caused by Aspergillus spp., invasive aspergillosis (IA), is difficult. We developed a urinary assay (MycoMEIA-Aspergillus) that deploys novel monoclonal antibodies (mAbs). We describe the biology of urinary antigens and performance as an aid to diagnose IA. METHODS: Urine samples from people with IA were screened with β-D-galactofuranose (Galf)-specific mAbs for optimal design. Epitope specificity of mAbs was characterized using synthesized Galf-oligomers. Antigens recognized by Galf-specific mAb476 were characterized by immunoprecipitation followed by mass spectrometry and immunogold transmission electron microscopy (ITEM). Assay performance was determined with 920 urine samples from 310 people. RESULTS: Combining N-glycan-recognizing mAb476 with a galactomannan (GM)-specific mAb optimized performance across samples. Urine samples from people with IA contained multiple mAb476-reactive Aspergillus proteins, with prominent localization on or within Aspergillus extracellular vesicles (EVs). MycoMEIA receiver operating characteristic curves demonstrated area under curve of 0.97 (95% CI .93-1, P < .0001). Per-subject sensitivity was 91.2% (95% CI 76%-98%) and specificity was 89.2% (95% CI 82%-94%), with performance aligned diagnosis. CONCLUSIONS: The new diagnostic assay detects free and EV-associated Galf-containing glycans enabling urine testing for IA. Further analyses of the design-locked assay are needed for confirmation.