TB Diagnostics at the Point of Care

摘要 结核病在全球十大死因中排名第一，现在排在艾滋病毒之前， 一个传染病杀手也许，最令人担忧的是耐多药结核病（MDR-1）的全球流行。 结核病和广泛耐药结核病：5%以上的新结核病病例是耐多药结核病，10%的新结核病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例数量从2010年的58个国家增加到105个国家 2015年对防止结核病传播的关注推动了结核病诊断市场，预计该市场将达到 到2024年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，用于检测广泛耐药结核病的培养需要6 到16周。唯一的商业替代品是需要熟练技术人员进行的测试 20个手动步骤，通常包括前期培养步骤和PCR以提高灵敏度。其中一 步骤是将扩增的DNA从PCR管转移到需要目视判读的线性探针条上。的 转移步骤易受PCR污染影响;目视判读易受错误读数影响; 并且复杂的工作流程容易出错。 开发广泛耐药结核病的分子检测本来就很复杂，主要有三个原因： （通常是痰液）是高度粘稠和异质的，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耐药性， 以及（3）市场对成本敏感，因为受感染者往往很贫穷。 今年，Akonni获得了Frost & Sullivan颁发的2017年北美新产品创新奖。 我们正在开发的测试包括从痰液中提取DNA的自动化方法和 a-胶片诊断，其由印刷在常规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。我们将这种薄膜层压到额外的柔性薄膜层上， 形成腔室和通道的压敏粘合剂-不需要泵、阀或特殊的 镀层.然后将所得的膜上实验室组件用于整合的PCR/杂交，并且包括一个膜上实验室。 废物室吸收剂用于最终限制所有液体，以防止PCR污染。的 这种装配策略的承诺是，制造吞吐量可以从每分钟一个零件增加到每分钟一个零件， 利用机器人技术以每秒一份的速度印刷报纸的生产方式：卷到卷 制造业 在SBIR项目的第1和第2阶段，我们开发并提供了一个全自动的样本到答案 系统，其中包括实验室的一个电影消耗品，我们的合作者在墨西哥和演示 结核分枝杆菌异烟肼和利福平耐药菌株的样本-应答基因分型 在与加州大学圣地亚哥医学院的一个单独的R 01项目中，我们开发了一种 模块化（非自动化）系统和使用我们的玻璃上的凝胶滴阵列的广泛耐药结核检测。对于第2B阶段，我们 建议：（1）进一步发展薄膜实验室组件，使其能够以卷到卷的方式生产， 卷轴制造，（2）将广泛耐药结核检测转移到我们的胶片上实验室设备，以及（3）将其与我们的 用于全自动工作流程的样品到答案仪器。我们建议评估该系统， 在R 01项目下收集的回顾性广泛耐药结核分枝杆菌标本。

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.