Low-cost portable system for the rapid detection and drug resistance profiling of Tuberculosis in low resource environments.

结核病是当今世界主要的公共卫生挑战之一，据估计 2013年有150万人死亡。更令人担忧的是，耐多药结核病(MDR)的发病率正在迅速增加，特别是在资源匮乏的环境中。 这个项目的目标是开发一种廉价的便携式系统和方法，使用它可以检测痰中低负荷结核分枝杆菌(Mtb)的存在 12种药物制剂的样品和药敏试验(DST)：均在样品采集后3天内完成。 建议的方法包括(A)在标准去污过程中，使用一套表面带有氨基多糖部分的超顺磁性纳米颗粒(MNP)，将2-10ml痰样本中的~1000 Mtb细胞全部收集到一小块(200 Ul)缓冲液(生长介质)中；(B)将200ul的悬液分配到16个微孔中，每个微孔包含不同配方的冻干生长介质和/或感兴趣的药物；(C)使用微通道阻抗谱(m-EIS)监测每个孔中的细胞。M-EIS实时显示细胞在抗生素存在下是否生长、死亡或呈现静止状态。前两个步骤(使用MNPs预浓缩并装入微流控通道)将花费不到1小时，死亡/生长/停滞的监测将需要3天。 在第一阶段，我们将通过(1)验证我们涉及MNPs的样品制备/预浓缩技术的有效性，(2)建立m-EIS方法在候选药物制剂存在的情况下实时记录分支杆菌的死亡、增殖或统计的能力，以及(3)设计包含16个孔的原型盒，并使用3D打印(立体平版印刷)来制造原型盒，从而展示上述方法的概念验证。此外，我们将(4)提出我们将在第二阶段建造的自动化系统(一次性和非一次性硬件)的详细设计(电气、机械和流体)。 能够在3天内检测到低负荷分枝杆菌的存在，并了解特定菌株的耐药性情况，将是我们抗击结核病(特别是结核病)传播的努力的“游戏规则改变者”。耐多药结核病)。它将为患者带来更好的结果(更低的死亡率，更快的康复)，也将带来更好的公共健康(更少向他人传播，更少在人群中培养抗药性)。

Tuberculosis (TB) is one of the major public-health challenges in the world today, with an estimated 1.5 million deaths in 2013. More alarmingly, the incidence of multi-drug resistant (MDR) TB is rapidly increasing, especially in low resource environments. The goal of this project is to develop an inexpensive portable system and method using which it will be possible to both detect the presence of low loads of Mycobacterium tuberculosis (Mtb) in sputum samples AND perform Drug Susceptibility Testing (DST) for 12 drug formulations: all within 3 days of sample collection. The proposed approach involves (a) collecting all the ~1000 Mtb cells present in 2-10 ml of sputum sample into a small (200 ul) of buffer (growth medium) during the standard decontamination process using a set of super-paramagnetic nanoparticles (MNPs) functionalized on the surface with amino-polysaccharide moieties that bind to bacterial cells (b) dispensing the 200ul suspension into 16 micro-wells, each containing different formulations of freeze-dried growth media and/or drugs of interest (c)monitor cells in each well using micro-channel Electrical Impedance Spectroscopy (m-EIS). m-EIS reveals in real time if cells are growing, dying, or rendered static in the presence of antibiotic. The first 2 steps (pre-concentration using MNPs and loading into microfluidic channel) will take less than 1 hour, and the monitoring of death/growth/stasis will take 3 days. In Phase I, we will demonstrate proof-of-concept for the above approach by (1) verifying the efficacy of our sample preparation / pre-concentration technique involving MNPs, (2) establishing the ability of the m-EIS method to record in real time, the death, proliferation, or statis of mycobacteria in the presence of candidate drug formulations, and (3) designing a prototype cassette containing 16 wells, and fabricating the same using 3D printing (stereolithography). In addition, we will (4) propose a detailed design (electrical, mechanical and fluidic) of the automated system (disposables and non- disposable hardware) that we will build in Phase II. Being able to detect the presence of low loads of mycobacteria and gaining knowledge about the drug resistance profile of a particular isolate in 3 days will be a “game changer” in our efforts to combat the spread of TB (esp. MDR TB). It will lead to better outcomes for patients (less mortality, quicker recovery), and also better public health (less transmission to others and less fostering of drug resistance in populations).