Development of Molecular Diagnostic Test for Early Onset of Lyme disease

项目总结/摘要 目前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作 抗生素治疗最有可能治愈而无后遗症。诊断往往很困难 因为大多数症状通常是模糊的，只有“靶心”皮疹才能说明问题， 在大约一半的情况下可以清楚地识别。 标准类型的感染诊断测试-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仅表明接触， 不是活动性感染。这导致了治疗的显著延迟。而PCR检测B. burgdorferi 血液中的DNA有可能获得快速、准确的结果，过去的测试还不够 由于血液中细菌太少和DNA拷贝数太低，我们有 开发了一种方法，“扩大目标”，由160，000倍，从而增加了 PCR检测B的敏感性burgdorferi。我们的数据显示，我们通过以下方式成功实现了这一目标： 使用大量的血液开始（10倍），然后选择性地增加目标， 在PCR之前使用疏螺旋体定向引物（2000倍）;然后探测产物中的B。burgdorferi 在多基因座PCR平台上（8倍）。 为了确保基于这种方法的新测试的可靠性和可接受性，我们正式咨询了 FDA从一开始我们获得了Qsub（pre-IDE）批准，并获得了关于我们所做的 需要一个明确的测试。在此期间，我们将继续测试血液样本（最终从 确诊的莱姆病病例和对照受试者）与我们的基于PCR的检测，在病人 出示给医生，以证明该检测方法可以检测到B。伯氏感染，较早， 灵敏度高于FDA同时进行的两层血清学基准。如果 如果成功，在与FDA商定的数量上，该第二阶段应导致FDA 间隙 我们的目标是为早发性莱姆病提供功能性和负担得起的诊断检测， 与两级血清学相比，具有上级灵敏度和特异性。测试将允许 在血清转化或培养物呈阳性之前立即诊断， 马上就开始了。

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.