The World Health Organization (WHO) calls for the development of a rapid, biomarker-based, non-sputum test capable of detecting all forms of tuberculosis (TB) at the point-of-care to enable immediate treatment initiation. Lipoarabinomannan (LAM) is the only WHO-endorsed TB biomarker that can be detected in urine, an easily collected sample matrix. For obtaining optimal sensitivity, we and others have shown that some form of sample pretreatment is necessary to remove background from patient urine samples. A number of systems are paper-based often destined for resource limited settings. Our current work presents incorporation of one such sample pretreatment, proteinase K (ProK) immobilized on paper (IPK) and test its performance in comparison to standard proteinase K (SPK) treatment that involves addition and deactivation at high temperature prior to performing a capture ELISA. Herein, a simple and economical method was developed for using ProK immobilized strips to pretreat urine samples. Simplification and cost reduction of the proposed pretreatment strip were achieved by using Whatman no.1 paper and by minimizing the concentration of ProK (an expensive but necessary reagent) used to pretreat the clinical samples prior to ELISA. To test the applicability of IPK, capture ELISA was carried out on either LAM-spiked urine or the clinical samples after pretreatment with ProK at 400 μg/mL for 30 minutes at room temperature. The optimal conditions and stability of the IPK were tested and validation was performed on a set of 25 previously analyzed archived clinical urine samples with known TB and HIV status. The results of IPK and SPK treated samples were in agreement showing that the urine LAM test currently under development has the potential to reach adult and pediatric patients regardless of HIV status or site of infection, and to facilitate global TB control to improve assay performance and ultimately treatment outcomes.

中文翻译：

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固定化蛋白酶 K 用于尿液预处理，提高活动性结核病的诊断准确性。

世界卫生组织 (WHO) 呼吁开发一种基于生物标志物的快速非痰检测，能够在护理点检测所有形式的结核病 (TB)，以便立即开始治疗。阿拉伯脂甘露聚糖 (LAM) 是世界卫生组织认可的唯一可在尿液（一种易于收集的样本基质）中检测到的结核病生物标志物。为了获得最佳灵敏度，我们和其他人已经证明，需要某种形式的样品预处理来去除患者尿液样品中的背景。许多系统都是基于纸张的，通常适用于资源有限的环境。我们目前的工作介绍了一种这样的样品预处理，即固定在纸上的蛋白酶 K (ProK) (IPK)，并与标准蛋白酶 K (SPK) 处理相比测试其性能，标准蛋白酶 K (SPK) 处理涉及在执行捕获 ELISA 之前在高温下添加和失活。本文开发了一种简单且经济的方法，使用 ProK 固定条预处理尿液样本。通过使用 Whatman 1 号纸并最大限度地减少 ELISA 之前用于预处理临床样品的 ProK（一种昂贵但必需的试剂）的浓度，实现了所提出的预处理条的简化和成本降低。为了测试 IPK 的适用性，对添加 LAM 的尿液或在室温下用 400 μg/mL ProK 预处理 30 分钟后的临床样品进行捕获 ELISA。对 IPK 的最佳条件和稳定性进行了测试，并对一组 25 个先前分析的已知 TB 和 HIV 状态的存档临床尿液样本进行了验证。IPK 和 SPK 处理样本的结果一致，表明目前正在开发的尿液 LAM 检测有可能覆盖成人和儿童患者，无论 HIV 状况或感染部位如何，并促进全球结核病控制，以提高检测性能，并最终治疗结果。