Borrelia antigen detection assay for urine-based diagnosis of early Lyme disease

 描述(申请人提供)：血清学测试仍然是最广泛使用的实验室工具，以支持莱姆疏螺旋体病(LB)的诊断，俗称“莱姆病”。这种方法在一些临床应用中效果很好，但在其他方面有很大的局限性。尽管进行了有效的治疗，但对致病细菌伯氏疏螺旋体的特异性IgM和/或IgG抗体反应可能会在感染后持续数十年。因此，血清学检测不能区分活动性和既往的淋巴细胞性肝病，也不能作为治疗后症状持续存在的患者的“治愈试验”。然而，血清学检测最广为人知的局限性是对早期感染的敏感度较低。尽管早期(局部性)诊断和治疗对于防止在感染后期出现严重的、有时是永久性的病变的扩散和后续发展很重要，但70%-80%的患有局限性移行性红斑(EM)的患者的血清学检测呈假阴性。当典型的“靶心”EM皮疹出现在适当的流行病学背景下时，很容易做出诊断，但这只占EM病例的10%-20%。更典型的是，EM皮损很难与其他感染性和非感染性疾病引起的皮疹区分开来；此外，在大约20%的早期LB病例中，皮疹完全不存在。值得注意的是，上述限制可能都可以通过一种可以直接检测感染性病原体的测试来解决，而不是通过检测 宿主对该代理的免疫反应。我们建议通过发现和验证早期肝病患者尿液中新的伯氏杆菌蛋白生物标志物，为开发一种新的、高精度的早期肝病检测方法奠定基础。利用研究人员先前应用于发现活动性结核病(TB)和内脏利什曼病(VL)的新诊断生物标记物的成熟的质谱学(MS)方法，我们的团队从确诊的EM患者的尿样中确定了一种有前途的候选伯氏杆菌生物标记物。我们现在建议在这一有希望的初步发现的基础上，通过对另外20名确诊为EM的成人和儿童患者的尿样进行MS检查，使我们能够选择最好的生物标记物，用于使用我们的TB和VL生物标记物工作中审查的方法开发抗原检测分析。然后，我们将通过测试大量成人和儿童患者的尿样来验证候选生物标记物的诊断实用性。我们预计，这项工作将提供坚实的概念证据，证明可以在早期肺结核患者的尿液中检测到伯氏杆菌蛋白抗原，从而为后续的检测优化和大规模验证研究奠定坚实的基础。我们预计，我们的合作将在莱姆诊断领域取得实质性进展。

 DESCRIPTION (provided by applicant): Serologic testing remains the most widely-used laboratory tool to support the diagnosis of Lyme borreliosis (LB), colloquially known as "Lyme disease." This approach works well for some clinical applications but has major limitations in others. A specific IgM and/or IgG antibody response against the causative bacterium, Borrelia burgdorferi, may persist for decades after infection despite effective treatment. Therefore, serologic testing cannot differentiate between active and past LB, and cannot be used as a "test of cure" in patients whose symptoms persist despite therapy. The most well-known limitation of serologic testing, however, is its poor sensitivity in early infection. Despite the importance of diagnosis and treatment of LB in its earliest (localized) phase to prevent dissemination and subsequent development of severe and sometimes permanent pathology later in the course of infection, serologic testing is falsely negative in 70-80% of patients who present with localized erythema migrans (EM), the rash of early LB. When a classic "bull's eye" EM rash presents in appropriate epidemiologic context, the diagnosis is readily made, but this represents only 10-20% of EM cases. More typically, EM lesions can be difficult to distinguish from rashes caused by other infectious and non-infectious diseases; moreover, a skin rash is completely absent in approximately 20% of early LB cases. Notably, the limitations described above could potentially all be addressed by a test that can directly detect the infectious agent, rather than detecting the host's immune response to that agent. We propose to lay the groundwork for development of a novel and highly accurate test for early LB by discovering and validating novel B. burgdorferi protein biomarkers in the urine of patients with early LB. Using proven mass spectrometric (MS) methods previously applied to the discovery of novel diagnostic biomarkers of active tuberculosis (TB) and visceral leishmaniasis (VL) by the investigators, our team has identified a promising candidate B. burgdorferi biomarker in urine samples from patients with confirmed EM. We now propose to build on this promising preliminary finding by performing MS on an additional 20 urine samples from adult and pediatric patients with confirmed EM, allowing us to select the top biomarker candidates for development of antigen detection assays using methods vetted in our TB and VL biomarker work. We will then validate the diagnostic utility of the biomarker candidates by testing a large panel of urine samples from adult and pediatric patients with and without confirmed early LB. We anticipate that this work will provide firm proof of concept that B. burgdorferi protein antigens can be detected in the urine of patients with early LB, thus laying a solid foundation upon which to build in subsequent assay optimization and large-scale validation studies. We anticipate that our collaboration will lead to a substantial advance in the Lyme diagnostics field.