<i>Aspergillus</i> serology for chronic pulmonary aspergillosis diagnosis: optimization of an enzyme-linked immunosorbent assay kit and assessment of a Western blot kit performance

ABSTRACT Chronic pulmonary aspergillosis (CPA) is a progressive and potentially fatal lung disease occurring in patients with underlying structural lung damage. Its diagnosis remains challenging because clinical and radiological features are often nonspecific and overlap with pre-existing respiratory conditions. Detection of anti- Aspergillus IgG antibodies is a cornerstone of diagnosis, as microscopy and culture have limited sensitivity. Several serological assays are available, including enzyme-linked immunosorbent assay (ELISA), which is highly sensitive but may lack specificity, and Western blot (WB), for which diagnostic performance data are limited. This study aimed to optimize the serological strategy for CPA diagnosis. The diagnostic performance of a commercial ELISA assay for Aspergillus -specific IgG (Platelia Aspergillus IgG, Bio-Rad) was optimized using serum samples collected at diagnosis from CPA patients and control subjects. Receiver operating characteristic (ROC) curve analysis was performed to assess alternative cut-off values. In parallel, the performance of a commercial WB assay (LDBio) was retrospectively evaluated using serum samples from five groups: CPA, Aspergillus colonization, airway contamination, negative controls with chronic respiratory disease, and healthy donors. ROC curve analysis showed that increasing the ELISA cut-off from 10 to 25 AU/mL maintained excellent sensitivity while improving specificity from 83.5% to 92.8%. The WB assay demonstrated a sensitivity of 87.0% for CPA diagnosis; however, specificity varied depending on the control group, ranging from 41.4% to 60.7%. Adjusting the ELISA threshold to 25 AU/mL significantly enhances diagnostic specificity without compromising sensitivity, whereas WB provides limited additional diagnostic value due to suboptimal specificity. IMPORTANCE Chronic pulmonary aspergillosis (CPA) is a severe and often overlooked fungal lung disease that occurs in patients with underlying structural pulmonary disorders. Diagnosis is challenging because clinical and radiological features are frequently nonspecific and overlap with pre-existing lung conditions. Detection of anti- Aspergillus IgG is central to diagnosis, but the optimal interpretation of available assays remains uncertain. In this study, we assessed the diagnostic performance of serological tests using well-defined patient groups reflecting real-world clinical practice. We show that increasing the cut-off of a widely used ELISA assay significantly improves diagnostic specificity without compromising sensitivity. In contrast, our results suggest that a commercial Western blot test provides only limited diagnostic value due to suboptimal specificity, clearly demonstrated using the different patient groups studied. These results help refine the serological strategy for CPA diagnosis and improve its clinical interpretation.