TB Diagnostics at the Point of Care

摘要 结核病位居全球十大死因之列，现在排在艾滋病毒之前，使其成为 一名传染病杀手。也许，最令人担忧的是全球多药耐药结核病(MDR- 结核病)和广泛耐药结核病(XDR-TB)：超过5%的新增结核病病例是耐多药结核病，10%的新病例是耐多药结核病 这些人患有广泛耐药结核病。登记的广泛耐药结核病病例从2010年的58个国家增加到105个国家 在2015年。对预防结核病传播的担忧推动了结核病诊断市场的发展，预计将达到 到2024年达到31亿美元。 诊断广泛耐药结核病的主要方法仍然是培养。而且，检测广泛耐药结核病的培养需要6 到16周。唯一商业上可用的替代方案是一种需要熟练技术人员执行的测试 20个手动步骤，通常包括预先培养步骤和聚合酶链式反应以提高敏感性。这其中的一个 步骤是将扩增的DNA从聚合酶链式反应试管转移到需要视觉解释的线探针条带上。这个 转移步骤容易受到聚合酶链式反应的污染；视觉解释容易受到错误读数的影响； 而且复杂的工作流程很容易出错。 发展广泛耐药结核病的分子检测本身就很复杂，主要有三个原因：(1)样本类型 (通常是痰)是高度粘性和异质性的，因此提取和纯化微生物DNA 提出了重大挑战，(2)有数百种基因突变赋予结核病抗药性， (3)市场对成本敏感，因为感染者往往是贫困的。 今年，Akonni获得了Frost和Sullivan颁发的2017年度北美新产品创新奖。 我们正在开发的测试包括一种从痰中提取DNA的自动方法和一种 A-胶片诊断，由印刷在传统胶片上的数百个分子凝胶滴组成。这部电影 成本比标准微阵列玻璃低350倍。我们将这种薄膜层叠成额外的柔性薄膜， 形成腔室和通道的压敏粘合剂-不需要泵、阀门或特殊的 涂层。最终得到的Lab-on-a-Film组件然后用于集成的PCR/杂交，并包括 废料室，用于最终限制所有液体，以防止多氯联苯污染。这个 这一装配策略的承诺是，制造产能可以从每分钟一个零件增加 使用机器人技术以每秒一个零件的速度使用报纸印刷生产方法：卷筒到卷筒 制造业。 在此SBIR项目的第一阶段和第二阶段，我们开发并交付了全自动的样本到答案 系统，其中包括Lab-on-a-Film消耗品，并向我们在墨西哥的合作者展示了 结核分枝杆菌异烟肼和利福平耐药株的样本对答案基因分型。 在与加州大学圣地亚哥分校医学院的一个单独的R01项目下，我们开发了一个 模块化(非自动化)系统和使用我们在玻璃上的凝胶滴阵列的广泛耐药结核检测。对于2B阶段，我们 建议：(1)进一步开发胶片上的实验室组件，使其能够以卷轴到胶片的方式生产 卷轴制造，(2)将XDR-TB检测转移到我们的Lab-on-a-Film设备，(3)将其与我们的 用于全自动化工作流程的样本到答案工具。我们建议用以下指标来评估该系统 在R01项目下收集的回顾性广泛耐药结核痰标本。

ABSTRACT Tuberculosis ranks in the top ten worldwide cause of death and now ranks ahead of HIV, making it the number one infectious disease killer. Perhaps, most alarming is the global prevalence of multidrug-resistant TB (MDR- TB) and extensively drug-resistant TB (XDR-TB): more than 5% of new TB cases are MDR-TB and 10% of these have XDR-TB. And, the number of enrolled XDR-TB cases has grown from 58 countries in 2010 to 105 in 2015. The concern to prevent the spread of TB has fueled a TB diagnostics market that is expected to reach $3.1 billion by 2024. The predominant method for diagnosing XDR-TB is still culture. And, culture for XDR-TB detection requires 6 to 16 weeks. The only commercially-available alternative is a test that requires a skilled technician to perform 20 manual steps, which often include an upfront culture step and PCR to improve sensitivity. One of these steps is to transfer amplified DNA from a PCR tube to a line probe strip that requires visual interpretation. The transfer step is susceptible to PCR contamination; the visual interpretation is susceptible to incorrect readings; and the complex workflow is susceptible to mistakes. Developing molecular tests for XDR-TB are inherently complex for primarily three reasons: (1) the sample type (typically sputum) is highly viscous and heterogenous, and thus extracting and purifying microbial DNA presents significant challenges, (2) there are hundreds of genetic mutations that confer TB-drug resistance, and (3) the market is cost sensitive because those infected are often impoverished. This year, Akonni received a 2017 North American New Product Innovation Award from Frost and Sullivan. The test that we are developing includes an automated method of extracting DNA from sputum and a Lab-on- a-Film diagnostic, which consists of hundreds of molecular gel drops printed on a conventional film. This film costs 350X less than standard microarray glass. We laminate this film to additional layers of flexible films and pressure-sensitive adhesives that form chambers and channels – without need for pumps, valves, or special coatings. The resulting Lab-on-a-Film assembly is then used for integrated PCR/hybridization and includes a waste chamber absorbent for eventual confinement of all liquids in order to prevent PCR contamination. The promise of this assembly strategy is that manufacturing throughput can increase from one part per minute using robotics to one part per second using the newspaper printing production method: reel-to-reel manufacturing. During Phases 1 and 2 of this SBIR project, we developed and delivered a fully-automated sample-to-answer system, which included the Lab-on-a-Film consumable, to our collaborator in Mexico and demonstrated sample-to-answer genotyping of both isoniazid and rifampin resistant strains of Mycobacterium tuberculosis. Under a separate R01 project with University of California, San Diego Medical School, we developed a modular (non-automated) system and an XDR-TB assay using our gel drop arrays on glass. For Phase 2B, we propose to: (1) further the development of the Lab-on-a-Film assembly so that it can be produced with reel-to- reel manufacturing, (2) transfer the XDR-TB assay to our Lab-on-a-Film device, and (3) couple it with our sample-to-answer instrument for a fully-automated workflow. We propose to evaluate this system with retrospective XDR-TB sputa specimens collected under the R01 project.