Development of Molecular Diagnostic Test for Early Onset of Lyme disease

项目摘要/摘要 当时还没有经过验证的检测方法来可靠地检测莱姆病感染的早期发病 当抗生素治疗有最好的机会治愈而没有后遗症时。诊断往往很困难 因为大多数症状通常是模糊的，具有说服力的“牛眼”皮疹只出现在 在大约一半的案例中可以清楚地辨认出来。 标准类型的感染诊断试验--包括培养和血清学--需要3周的时间。 感染莱姆病后的阳性结果和血清学检查仅表明接触过莱姆病 不是活动性感染。这导致了治疗的显著延误。而伯氏杆菌的聚合酶链式反应 血液中的DNA有可能得到快速、准确的结果，但过去的检测还不够 由于血液中细菌太少，DNA拷贝数太少，所以很敏感。我们有 开发了一种方法，将目标性扩大16万倍，从而增加了 伯氏杆菌聚合酶链式反应检测的敏感性。我们的数据显示，我们通过以下方式成功实现了这一目标： 使用更多的血液开始(10倍)；然后有选择地使用 聚合酶链式反应(2000倍)前扩增疏螺旋体；然后对产物进行伯氏疏螺旋体的检测 在多位点聚合酶链式反应平台上(8倍)。 为了确保基于此方法的新测试的可靠性和接受性，我们正式咨询了 从一开始就是FDA。我们获得了QSub(集成开发前)批准，并获得了关于我们 需要一个合格的测试。在这项提议期间，我们将继续检测血液样本(从最终 确认的莱姆病例和对照受试者)与患者当时的基于PCR的检测 提交给医生，以证明该检测方法可以检测到伯氏杆菌感染，更早和 比FDA同时采集的两级血清学基准更敏感。如果 成功，按照与FDA商定的数字，这一第二阶段应该会导致FDA进行测试 通行证。 我们的目标是为早发性莱姆病提供一种实用且负担得起的诊断方法 与两级血清学相比，具有更高的敏感性和特异性。这项测试将允许 在血清转换或培养呈阳性之前立即诊断，以便治疗 立刻开始了。

Project Summary/Abstract There is no validated assay to reliably detect the early onset of the Lyme disease infection at the time when antibiotic therapy has the best chance of a cure without sequelae. Diagnosis is often difficult because most symptoms are often nebulous, with the telltale “bulls-eye” rash only appearing and clearly recognizable in approximately half of cases. The standard types of infectious diagnostic tests – both culture and serology – require >3 weeks for a positive result after the onset of infection with Lyme disease and serology only indicates exposure but not active infection. This introduces a significant delay in treatment. While PCR for B. burgdorferi DNA in the blood has potential for a rapid, accurate result, past tests have not had sufficient sensitivity because of too few bacteria and low copy numbers of DNA in the blood. We have developed an approach that “enlarges the targetability”, by 160,000 fold, thereby increasing the sensitivity of PCR test for B. burgdorferi. Our data show we are successfully accomplishing this by: using a larger amount of blood to start (10 fold); then selectively multiplying the target by using Borrelia-directed primers prior to PCR (2000 fold); and then probing the product for the B. burgdorferi on a multi-loci PCR platform (8 fold). To assure reliability and acceptance of a new test based on this approach, we formally consulted with the FDA from the beginning. We obtained a Qsub (pre-IDE) approval with guidance as to what we need for a cleared test. During this proposal we will continue to test blood samples (from eventually confirmed Lyme cases and control subjects) with our PCR-based assay at the time the patient presents to the doctor in order to prove the assay can detect the B. burgdorferi infection, earlier and with greater sensitivity, than the FDA benchmark of two-tiered serology taken at the same time. If successful, at the number agreed on with the FDA, this Phase II should result in a test that has FDA clearance. Our goal is deliver a functional and affordable diagnostic assay for Early Onset Lyme Disease that has superior sensitivity and specificity compared to two-tiered serology. The test will permit immediate diagnosis well before seroconversion or cultures become positive so treatment can be started right away.