Qualification of a 21-valent pneumococcal urine antigen detection assay and development of clinical positivity cutoffs

<b>Aim:</b> Serotype-specific assays detecting pneumococcal polysaccharides in bodily fluids are needed to understand the pneumococcal serotype distribution in non-bacteremic pneumonia. <b>Methods:</b> We developed a urine antigen detection assay and using urine samples from adult outpatients without pneumonia developed positivity cutoffs for both a previously published 15-valent and the new 21-valent assay. Clinical sensitivity was confirmed with samples from patients with invasive pneumococcal disease. <b>Results:</b> Total assay precision ranged from 7.6 to 17.8% coefficient of variation while accuracy ranged between 80 and 150% recovery, except for three serotypes where recoveries ranged from 32 to 60%. Clinical sensitivity was 86.4% and specificity was 96.5% across all 30 serotypes. <b>Conclusion:</b> The assay could potentially assess serotype-distribution in non-infected and infected participants with pneumococcal disease. A 21-valent serotype-specific urine antigen detection assay was developed to measure capsular polysaccharides and fragments thereof from <i>Streptococcus pneumoniae</i> (Pneumococcus) Pneumococcal polysaccharides are captured from urine samples using a multiplexed, sandwich Luminex immunoassay with each bead representing a distinct serotype. Assay qualification determined assay performance: total assay precision ranged from 7.6 to 17.8% coefficient of variation while accuracy ranged between 80 and 150% recovery, except for three serotypes where recoveries ranged from 32 to 60%. Low spike recoveries of 15A, 23A and 35B were mostly due to potential urine interference. Assay specificity was assessed against bacterial lysates from 61 pneumococcal serotypes and 11 non-pneumococcal bacteria. A novel method was developed to determine clinical positivity cut-offs based on nonparametric tolerance intervals combined with assay LLOQs and further enhancement based on serotype prevalence. The new cutoffs were then determined using a cutoff strategy, primarily based on a statistical method called nonparametric tolerance interval (NTI). The extended strategy considered using both the NTI method and the values of the low LOQs of the assays to jointly determine the cutoffs. The cutoffs were then refined by utilizing negative control urine samples from adult outpatients without pneumonia (same data previously used) and urine samples from patients with community acquired pneumonia with invasive pneumococcal diseases (positive blood cultures obtained from a sterile site). Clinical sensitivity was 86.4% and specificity was 96.5% across 30 pneumococcal serotypes measured by a previously published 15-valent and this 21-valent assay.