Tuberculosis Second Line Drug Susceptibility Chip

描述（由申请人提供）：该提案涉及 RFA-AI-10-003“生物防御伙伴关系”，并将学术研究人员、行业、耐多药结核病临床医生、BSL3 实验室和现场站点聚集在一起，为多重和广泛耐药结核病开发更快、定量的二线药物敏感性测试 (DST)。耐药结核病（C 类威胁）的出现导致了这种高度传播且在某些情况下无法治愈的疾病的不祥局面。多个 Tb DST 平台正在使用中，但都有很大的限制，特别是周转时间。我们认为，我们必须坚持使用基于表型培养的方法，因为直接分子检测对于二线药物的定义仍然不明确，但我们已经证明，可以通过早期的 3 天定量 PCR 读数来测量药物环境中 DNA 扩增，从而改进现有的液体方法。通过使用高通量小体积微量滴定板，我们的方法可以产生准确的快速定性结果和类似于 MIC 的定量“抑制指数”——这是一种临床上需要但很少用于结核病的测量方法。由于 MDR/XDR Tb 的生物安全对于实验室人员来说至关重要，因此我们将把这种检测方法转变为一种新颖设计、封闭系统、一次性、支持 qPCR 的微流控芯片。该微流控室可以精确计量进入每个孔的杆菌数量，最大限度地减少重复性差的区域，并且还可以通过 qPCR 之外的其他参数测量原位生长，包括新发明的 DNA 检测技术（我们称之为风车）。因此，qPCR 检测开发和芯片项目将从彼此的成功中受益，但并不相互依赖。目标是改进二线药物的 DST 平台，更快地产生信息，以更好地治疗和减少 MDR/XDR Tb 的传播。叙述 MDR 和 XDR Tb 的药物敏感性测试进展缓慢且充满技术困难。该提案将开发一种基于 PCR 的快速定量诊断方法，以在 3 天内以微孔板形式检测 Tb 对二线药物的敏感性或耐药性。该检测将采用封闭系统一次性芯片，以增强实验室人员的生物安全性，并允许通过我们新的“风车”方法进行 DNA 定量。耐多药结核病临床医生将立即使用结果来更好地治疗和减少耐多药/广泛耐药结核病的传播。

DESCRIPTION (provided by applicant): This proposal addresses RFA-AI-10-003 "Partnerships for Biodefense" and brings together academic researchers, industry, MDR TB clinicians, BSL3 Labs, and field sites to develop a faster, quantitative, second line drug susceptibility test (DST) for multiple and extensively drug-resistant tuberculosis. The emergence of drug resistant TB, a Category C threat, has led the ominous situation of a highly transmissible and in some instances untreatable disease. Several Tb DST platforms are in use yet all have significant constraints, particularly turnaround time. We feel that we must persevere with a phenotypic culture based method, since direct molecular testing will remain poorly defined for second line drugs, but we have shown that can improve existing liquid methods with an earlier 3 day quantitative PCR-based readout that measures DNA amplification in the setting of drug. By using high-throughput small-volume microtiter plates, our method can yield both an accurate rapid qualitative result and a quantitative "inhibition index" akin to the MIC - a measurement that is clinically needed but rarely available for Tb. Because biosafety of MDR/XDR Tb for laboratory personnel is of utmost importance, we will then transform this assay into a novel-design, closed- system, disposable, qPCR-capable microfluidic chip. This microfluidic chamber can accurately meter the number of bacilli fed into each well, minimizing one area of poor reproducibility, and can also measure growth in situ by other parameters beyond qPCR, including a newly-invented DNA detection technique we term pinwheel. Thus the qPCR assay development and chip project will benefit from but are not tied to each other's success. The goal is an improved DST platform for second line drugs that yields faster information to better treat and decrease transmission of MDR/XDR Tb. NARRATIVE Drug susceptibility testing for MDR and XDR Tb is slow and fraught with technical difficulties. This proposal will develop a rapid, quantitative PCR-based diagnostic to detect susceptibility or resistance of Tb to second line drugs in a microplate format within 3 days. The assay will be adapted to a closed-system disposable chip to enhance biosafety for lab personnel and permit DNA quantification by our new 'pinwheel' approach. The results will be immediately usable by MDR Tb clinicians to better treat and decrease transmission of MDR/XDR Tb.