Qualification and clinical validation of an Immunodiagnostic Assay for detecting 11 additional S. Pneumoniae Serotype- specific Polysaccharides in Human Urine

BACKGROUND: Identifying Streptococcus pneumoniae (Sp) serotypes by urinary antigen detection assay (UAD) is the most sensitive way to evaluate the epidemiology of non-bacteremic community acquired pneumonia (CAP). We first described a UAD to detect the Sp serotypes 1,-3,-4,-5,-6A,-6B,-7F,-9V,-14,-18C,-19A,-19F,-23F covered by the licensed 13-valent Sp conjugate vaccine PCV13. To assess the substantial remaining pneumococcal disease burden after introduction of several pneumococcal vaccines, a UAD-2 assay was developed to detect 11 additional serotypes (-2,-8,-9N,-10A,-11A,-12F,-15B,-17F,-20,-22F,-33F) in individuals with radiographically-confirmed CAP. METHODS: UAD-2 specificity was achieved by capturing pneumococcal polysaccharides with serotype-specific monoclonal antibodies using Luminex technology. Assay qualification assessed accuracy, precision, sample linearity. Serotype positivity was based on cutoffs determined by non-parametric statistical evaluation of urine samples from individuals without pneumococcal disease. Sensitivity and specificity of the positivity cutoffs were assessed in a clinical validation using urine samples obtained from a large study that measured the proportion of radiographically-confirmed CAP caused by Sp serotypes in hospitalized US adults. RESULTS: The UAD-2 was shown to be specific and reproducible. Clinical validation demonstrated assay sensitivity and specificity of 92.2% and 95.9% against a gold standard of bacteremic pneumonia. Additionally, the UAD-2 assay identified a Sp serotype in 3.72% of non-bacteremic CAP cases obtained from hospitalized US adults. When combined with bacteremic CAP cases, the percent of pneumonias with a UAD-2 serotype was 4.33%. CONCLUSIONS: The qualified/clinically validated UAD-2 method has applicability in understanding the epidemiology of non-bacteremic Sp CAP and utility to assess vaccine efficacy of future pneumococcal conjugate vaccines that are under development.