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.

**研究目的：** 目前亟需能够在体液中检测肺炎链球菌多糖的血清型特异性检测方法，以明确非菌血症性肺炎患者体内的肺炎链球菌血清型分布特征。**研究方法：** 本研究开发了一款尿液抗原检测方法，并针对已发表的15价检测方法与本研究新开发的21价检测方法，招募无肺炎症状的成人门诊患者收集尿液样本，以此确立两种检测方法的阳性临界值。本研究通过侵袭性肺炎链球菌病患者的临床样本验证了该检测方法的临床灵敏度。**研究结果：** 总检测精密度的变异系数（coefficient of variation）介于7.6%至17.8%之间，检测回收率的准确度区间为80%至150%；仅3种血清型的加标回收率为32%至60%。针对全部30种覆盖的血清型，该检测方法的临床灵敏度为86.4%，特异性（specificity）为96.5%。**研究结论：** 本研究所开发的检测方法有望用于评估肺炎链球菌感染与未感染受试者体内的血清型分布情况。本研究成功构建了一款21价血清型特异性尿液抗原检测方法，可用于捕获并检测肺炎链球菌（*Streptococcus pneumoniae*，Pneumococcus）的荚膜多糖（capsular polysaccharides）及其降解片段。该检测采用多重夹心Luminex免疫测定法（multiplexed, sandwich Luminex immunoassay），通过绑定于微球的特异性抗体捕获尿液样本中的肺炎链球菌多糖，每一类微球对应一种独特的血清型。该检测方法的性能通过多项指标进行评估：总检测精密度的变异系数介于7.6%至17.8%之间，检测回收率的准确度区间为80%至150%，仅血清型15A、23A与35B的加标回收率为32%至60%，该三类血清型的低回收率主要源于尿液样本的潜在基质干扰。本研究针对61种肺炎链球菌血清型与11种非肺炎链球菌细菌的裂解液开展了检测特异性评价。关于阳性临界值的确立：本研究开发了一种基于非参数耐受区间（nonparametric tolerance interval, NTI）结合检测方法最低定量限（lower limit of quantitation, LLOQ）的临床阳性临界值确定策略，并进一步结合血清型流行率对该策略进行优化。最终的临界值确立方案主要采用非参数耐受区间统计方法，同时结合检测方法的低定量限数值共同确定临界值。随后，利用无肺炎症状的成人门诊患者尿液样本（即此前使用的同一批队列数据）与社区获得性肺炎合并侵袭性肺炎链球菌病患者（经无菌部位采血获得阳性血培养结果）的尿液样本对临界值进行了精细调整。针对已发表的15价检测方法与本研究21价检测方法所覆盖的全部30种肺炎链球菌血清型，最终检测结果显示临床灵敏度为86.4%，特异性为96.5%。