Tuberculosis Second Line Drug Susceptibility Chip

描述（由申请人提供）：本提案涉及RFA-AI-10-003“生物防御伙伴关系”，并将学术研究人员、工业界、耐多药结核病临床医生、BSL3实验室和现场站点聚集在一起，开发一种更快、定量的二线药敏试验（DST），用于多种和广泛耐药结核病。耐药结核病是C类威胁，它的出现导致了一种具有高度传染性、在某些情况下无法治愈的疾病的不祥局面。有几个Tb DST平台正在使用，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3天的基于pcr的定量读数来改进现有的液体方法，该方法可以测量药物环境下的DNA扩增。通过使用高通量小体积微量滴度板，我们的方法既可以产生准确的快速定性结果，也可以产生类似MIC的定量“抑制指数”——一种临床需要但很少用于结核病的测量方法。由于MDR/XDR Tb对实验室人员的生物安全性至关重要，因此我们将把这种检测方法转化为一种设计新颖、封闭系统、一次性、能够进行qpcr的微流控芯片。这种微流控室可以准确地测量每个孔中注入的杆菌数量，最大限度地减少重复性差的一个区域，并且还可以通过qPCR之外的其他参数测量原位生长，包括我们称之为风车的新发明的DNA检测技术。因此，qPCR检测的发展和芯片项目将受益于彼此的成功，但并不依赖于彼此的成功。目标是改进二线药物的DST平台，更快地提供信息，以更好地治疗和减少耐多药/广泛耐药结核病的传播。耐多药和广泛耐药结核病的药敏试验进展缓慢且充满技术困难。该提案将开发一种快速、定量的基于pcr的诊断方法，以微孔板形式在3天内检测结核病对二线药物的敏感性或耐药性。该分析将适用于封闭系统一次性芯片，以提高实验室人员的生物安全性，并允许我们的新“风车”方法进行DNA定量。研究结果将立即可供耐多药结核病临床医生使用，以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.