Strep-CaDI: A fast, accurate, and sensitive point-of-care test for Group A Streptococcus

摘要 目前对A群链球菌的快速抗原检测缺乏敏感性，导致1000多万人不必要 每年向儿童提供抗生素治疗。当患者出现在诊所时所使用的当前诊断方法 喉咙痛的患者包括咽喉拭子和快速链球菌检测。抗生素处方中快速检测结果呈阳性，但应 由于现有快速链球菌免疫分析的灵敏度较低，医生通常会开抗生素作为预防措施，而 在快速检测为阴性后等待培养结果。据估计，这些剂量中高达70%是不必要的， 导致了抗菌素耐药性的上升。而ELISA和分子(以聚合酶链式反应为基础)的检测方法有所改进 据报道，敏感性可以减少不必要的抗生素治疗，这些测试依赖于实验室仪器和 基础设施庞大，不适合医疗机构和乡村诊所，无法实施 作为用于远程医疗目的的家庭测试协议。因此，迫切需要一种改进的、超灵敏的、尚未得到满足的 快速链球菌检测，可减少不必要的抗生素使用，遏制抗菌素耐药性的增加，以及 保护儿童的肠道微生物群。此外，如果可以优化改进的快速链球菌检测以实现稳健性 易用性和敏感度，它可以在家中执行，支持远程医疗协议，并减少需要 对于可能被感染的个人来说，暴露一线医护人员。这个项目的结果将是一个证明- 使用机载试剂进行超灵敏快速链球菌检测的原理演示，只需简单的拭子插入步骤 以比目前的试纸法低10-100倍的检出限启动完成。提高的敏感度将 通过使用毛细管驱动免疫分析(CADI)技术实现，在该技术中，我们能够集成所有步骤 在一个简单的微流控设备上进行高灵敏的实验室执行的ELISA(洗涤、标记和放大) 不需要仪器或训练有素的人员。这是同类产品中首创的微流控护理点(POC)设备 将通过以下目标实现。首先，将演示链球菌快速链球菌检测的概念证明 用抗体筛选和检测方法超灵敏地检测添加缓冲液中的化脓性链球菌抗原 参数优化。其次，细菌提取方法及相应的CADI参数和装置设计 将被优化用于使用添加了完整细菌的临床样本。最终的优化设计将与 目前的市场解决方案用于比较分析灵敏度(LOD)、结果时间和复杂性(结果的步骤)。最后，为了 增加批准在家中使用的可能性，并提供相对于当前解决方案的明显市场优势，我们将 原型(通过建模、3D打印和测试)能够将CADI操作简化为简单的 棉签-插入-读取机构，启动化验完成并将所有试剂与操作员隔离。一次 实现了所需的功能，外壳和免疫分析将由未经培训的个人进行测试。反馈来自 这些试验将用于住房设计的迭代。完成这三个目标将产生一个原型快速链球菌 简单试纸检测的易用性和实验室酶联免疫吸附试验的敏感性。

SUMMARY The current rapid antigen detection tests for group A strep lack sensitivity and result in upwards of 10 million unnecessary antibiotic treatments provided to children each year. Current diagnostic methods utilized when a patient presents at a clinic with a sore throat include a throat swab and rapid strep test. A positive rapid test results in antibiotic prescription, but due to the low sensitivity of existing rapid strep immunoassays, physicians often prescribe antibiotics as a precaution while waiting for culture results after a negative rapid test. It is estimated that up to 70% of these doses are unnecessary, contributing to the rise in antimicrobial resistance. While ELISAs and molecular (PCR-based) assays with improved sensitivity have been reported to reduce unnecessary antibiotic treatment, these tests rely on laboratory instrumentation and significant infrastructure, making them unsuitable for point-of-care settings and rural clinics, and impossible to implement as at-home testing protocols for telemedicine purposes. Thus, there is an urgent unmet need for an improved, ultrasensitive rapid strep test that could reduce the unnecessary use of antibiotics, stemming the increase in antimicrobial resistance and preserving the gut microbiome of children. Furthermore, if an improved rapid strep test could be optimized for robustness and ease of use, as well as sensitivity, it could be performed at home enabling telemedicine protocols and reducing the need for a potentially infected individual to expose frontline healthcare workers. The outcome of this project will be a proof-of- principle demonstration of an ultrasensitive rapid strep test with onboard reagents that requires a simple swab insertion step to initiate to completion with limits of detection 10-100x lower than current dipstick methods. Improved sensitivity will be achieved through use of the Capillary-Driven Immunoassay (CaDI) technology in which we are able to integrate all steps of highly sensitive, laboratory-performed ELISAs (washing, labeling, and amplification) on a simple microfluidic device without requiring instrumentation or highly trained personnel. This first-in-its-class microfluidic point-of-care (POC) device will be realized through the following aims. First, proof of concept for a Strep-CaDI rapid strep test will be demonstrated for the ultrasensitive detection of Streptococcus pyogenes antigen in spiked buffer through antibody screening and assay parameter optimization. Secondly, the bacterial extraction method and corresponding CaDI parameters and device design will be optimized for use with clinical samples spiked with whole bacteria. The final, optimized design will be compared to current market solutions to compare assay sensitivity (LOD), time to result, and complexity (steps to result). Lastly, to increase the likelihood of approval for at-home use and to provide a clear market advantage over current solutions, we will prototype (through modeling, 3D printing, and testing) an assay housing capable of simplifying CaDI operation to a simple swab->insert-> read mechanism which initiates the assay to completion and isolates all reagents from the operator. Once the required functionality is achieved, the housing and immunoassay will be tested by untrained individuals. Feedback from these trials will be used for housing design iterations. Completion of these three aims will result in a prototype rapid strep test with the ease-of-use of a simple dipstick test and the sensitivity of a laboratory ELISA.