A novel cartridge-based sequencing solution for decentralized M. tuberculosis resistance detection

一种新的基于盒式测序的分散式结核分枝杆菌耐药性检测方案 利福平耐药结核病(RR-TB)是与抗菌药物有关的主要死亡原因之一 抵抗。现在全球推荐的所有含贝达奎林的口服RR-TB方案将缩短 治疗和改善结果。然而，由于增长缓慢，缺乏及时的药物敏感性数据 结核分枝杆菌感染率和对高防护性生物实验室的需要是扩大规模的主要障碍 贝达奎林和其他新的和重新调整用途的药物。Xpert MTB/RIF Ultra等商用分子结核 测试可以确定利福平的耐药性，但不能告知完整的治疗方案，因为它们是有限的 到只分析少量的遗传基因座(即“热点”)。定向下一代测序(NGS) 能够在不需要培养的情况下快速对整个基因进行测序，因此可以改变RR-TB的临床 管理层。然而，实现近患者临床应用有两个主要瓶颈：(1)复杂的， 为NGS准备临床样本的非标准化工作流程，以及(2)成本和基础设施 符合行业标准的Illumina平台的要求。与全球结核病诊断领先者Cepheid合作，我们 通过利用超声波、微流体和热循环仪的功能来解决这些障碍 现有的Xpert Ultra试剂盒，已在180多个国家和地区使用，以统一经过审查的直接样品到答案 首次使用纳米孔基因测序平台的系统。Xpert CartSeq，一款开创性的墨盒- 基于测序解决方案，适用于较低水平的医疗保健，实现了强大的DNA提取和 复杂的库准备，同时最大限度地减少用户变异性并降低技术技能要求。 通过本提案中详细介绍的NGS的开发，我们的目标是促进NGS在高等医学领域的临床应用 负担设置。

A novel cartridge-based sequencing solution for decentralized M. tuberculosis resistance detection Rifampin-resistant tuberculosis (RR-TB) is one of the principal causes of death associated with antimicrobial resistance. Newer all oral bedaquiline-containing RR-TB regimens, now recommended worldwide, will shorten treatment and improve outcomes. However, the lack of timely drug susceptibility data due to the slow growth rate of M. tuberculosis and the need for high containment biological laboratories are major barriers to scale-up of bedaquiline and other new and repurposed drugs. Xpert MTB/RIF Ultra and other commercial molecular TB tests can identify rifampin resistance, but cannot inform complete treatment regimens because they are limited to analyzing only a small number of genetic loci (i.e., “hot spots”). Targeted next-generation sequencing (NGS) is able to sequence entire genes rapidly without need for culture, and therefore could transform RR-TB clinical management. Achieving near-patient clinical application, however, has two major bottlenecks: (1) a complex, unstandardized workflow for preparing clinical samples for NGS, and (2) the cost and infrastructure requirements of industry-standard Illumina platforms. Partnering with global TB diagnostics leader Cepheid, we address these barriers by utilizing the ultrasonication, microfluidics, and thermocycler capabilities of the existing Xpert Ultra cartridge, already in use in over 180 countries, to unite a vetted direct-sample-to-answer system with a nanopore gene sequencing platform for the first time. Xpert CartSeq, a pioneering cartridge- based sequencing solution suitable for lower levels of healthcare, achieves robust DNA extraction and sophisticated library preparation while minimizing user variability and mitigating technical skill requirements. Through its development as detailed in this proposal, we aim to catalyze the clinical application of NGS in high burden settings.