Point-of-care detection of TB and NTM pathogens with Fluorescent Deoxyribozyme Sensors and 3D-printed, battery powered device.

项目摘要 结核病是一种由结核分枝杆菌复合体(MTC)引起的传染病， 仍然是全球主要的公共卫生问题之一，新增病例约1000万例，约150万例 每年的死亡人数。2017年报告的新增病例约为7万例，巴西仍在22个高负担国家之列 这占全球所有结核病病例的80%。非结核分枝杆菌(NTM)是一个新兴的群体 引起肺结核样肺部感染的相关机会性病原体，特别是在潜在的 合并症，如艾滋病毒或囊性纤维化。NTM感染是出了名的难以治疗，50%的人 一些物种的治愈率，尽管使用口服和注射的抗生素鸡尾酒治疗方案长达一年之久 毒品。艾滋病毒合并感染进一步加剧了这种情况，这种感染促进了疾病的进展和 使结核病和非结核分支杆菌肺部感染的诊断复杂化。据估计，有350万结核病病例未被发现 这在很大程度上是因为缺乏负担得起的、有效的结核病/非结核分枝杆菌感染的现场诊断。作为一名 结果，感染患者病情加重，继续传播这些病原体。频频的 常用涂片镜检法误诊为结核的非结核分支杆菌感染 可能会导致更差的临床结果的治疗。因此，迫切需要护理点(POC)测试来 为医生提供有效治疗这些慢性衰弱患者所需的可操作数据 疾病。该项目的目标是利用DNA纳米技术的最新进展， 分子传感器和3D打印，显著提高了对结核病和NTM的POC诊断能力。我们的 平台结合了RPA等温扩增的速度和灵敏度、二进制的特异性 脱氧核酶(BiDz)传感器，以及3D打印检测设备的经济性和便携性。我们将优化 多色多路传感器检测痰标本中结核和非结核病原体的特异性。一个 使用3D打印构建的用于分析孵化和荧光检测的廉价、便携式电池供电设备 技术将受到考验。最后，这种诊断化验的“真实世界”验证将于 来自HIV+和HIV-队列的样本。如果成功，这种灵活的技术可以被用来设计PoC 其他传染病的诊断化验。这一国际合作的另一个结果将是 加强巴西的传染病研究能力和基础设施。

Project Summary Tuberculosis (TB), an infectious disease caused by species of Mycobacterium tuberculosis complex (MTC), remains one of the major public health problems worldwide, with ~10 million new cases and about ~1.5 million deaths each year. With ~70,000 new cases reported in 2017, Brazil remains among the 22 high-burden countries that account for >80% of all TB cases worldwide. Non-tuberculous mycobacteria (NTM) are an emerging group of related opportunistic pathogens that cause TB-like pulmonary infections, particularly in patients with underlying comorbidities such as HIV or cystic fibrosis. NTM infections are notoriously refractory to treatment, with <50% cure rates for some species despite year-long treatment regimens with antibiotic cocktails of oral and injectable drugs. This situation is further exacerbated by HIV co-infection which promotes disease progression and complicates diagnosis of TB and NTM pulmonary infections. An estimated 3.5 million cases of TB go undetected each year, largely due to the lack of affordable, effective point-of-care diagnostics for TB/NTM infection. As a result, infected patients develop more severe disease and continue to transmit these pathogens. The frequent misdiagnosis of NTM infections as TB by the commonly used smear microscopy method delays appropriate treatment which can result in worse clinical outcomes. Thus, point-of-care (POC) tests are urgently needed to provide physicians with actionable data required to effectively treat patients with these debilitating chronic diseases. The goal of this project is to take advantage of recent advancements in DNA nanotechnology, molecular sensors, and 3D-printing to significantly improve POC diagnostics capability for TB and NTM. Our platform combines the speed and sensitivity of RPA isothermal amplification, the specificity of binary deoxyribozyme (BiDz) sensors, and economy and portability of a 3D-printed assay device. We will optimize multi-color multiplex sensor assays for specific detection of TB and NTM pathogens from sputum samples. A cheap, portable battery-powered device for assay incubation and fluorescent detection built using 3D-printing technology will be tested. Finally, “real-world” validation of this diagnostic assay will be conducted in Brazil on samples from HIV+ and HIV- cohorts. If successful, this flexible technology could be exploited to devise POC diagnostic assay for other infectious diseases. An added outcome of this international collaboration will be enhancement of the infectious disease research capacity and infrastructure in Brazil.