Functional Pneumococcal Assessment Using the Multiplexed Opsonophagocytosis Assay Improves Alignment with Clinical Diagnosis of Specific Antibody Deficiency

Diagnosis of specific antibody deficiency (SAD) relies on serotype-specific IgG responses to PPSV23; however, IgG titers do not measure opsonophagocytic function and may misclassify patients due to pre-vaccine titers, variable serotype immunogenicity, and assay variability. The multiplexed opsonophagocytosis assay (MOPA) directly quantifies functional bacterial killing and may better reflect clinically meaningful antibody impairment. We evaluated how IgG versus MOPA-based interpretations aligned with clinician-diagnosed SAD.Among 60 patients evaluated for recurrent infections, 26 had paired pre- and post-PPSV23 IgG levels and MOPA results across 18 pneumococcal serotypes. SAD was defined as >70% protective serotypes for patients ≥6 years and >50% for ages 2–5. IgG protection was determined using an operational algorithm incorporating absolute pre- and post-vaccine levels. Fold-rise criteria (2–3-fold) were applied for serotypes with low pre-vaccine levels. When pre-vaccine IgG levels were high, protection was based on post-vaccine concentration alone. IgG responses were interpreted by a physician blinded to clinical history.For MOPA, opsonization indices, defined as the reciprocal serum dilution achieving 50% pneumococcal killing, were log standardized, and serotypes below a 0.1 SD Z-score threshold were classified as non-protective. Clinical SAD diagnosis was determined independently by the treating immunologist, blinded to all MOPA data. IgG- and MOPA-based SAD classifications were compared with clinician diagnosis.Patients had a median age of 10.8 years (range 2–68), with 17 females and 9 males; 5 were Hispanic and 21 non-Hispanic, 1 black, and 1 Native American. All patients presented with recurrent sinopulmonary infections, and comorbidities included atopic disease (n = 12), autoimmune or immune-dysregulatory features (n = 6), quantitative immunoglobulin abnormalities (n = 4), and defined immunodeficiencies such as Wiskott–Aldrich syndrome or memory B cell defects.Nine patients were diagnosed with SAD by the treating clinician. IgG captured 6/9 clinically confirmed SAD patients (sensitivity 0.667; specificity 0.882). Using the MOPA-derived threshold, MOPA correctly classified 8/9 clinician-diagnosed SAD cases (sensitivity 0.889; specificity 0.647).MOPA aligned more closely with clinician-identified cases of clinically meaningful antibody dysfunction, although IgG maintained higher specificity; these differences reflect operational rule sets and may indicate complementary clinical utility. Establishing serotype-specific MOPA thresholds and correlating functional responses with clinical outcomes will be essential to refine SAD diagnostic criteria.Tabular data are included as downloadable supplement files.