Advancing Diagnostics of Infectious Uveitis with MetagenomicAnalysis

通过宏基因组分析推进感染性葡萄膜炎的诊断

• 批准号: 10303756
• 负责人: Paulo J. M. Bispo
• 金额: $ 25.5万
• 依托单位: MASSACHUSETTS EYE AND EAR INFIRMARY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-30 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10303756
• 关键词: Affect; Age; Aqueous Humor; Area; Autoimmune Diseases; Bacteria; Biological Assay; Blinded; Blindness; Characteristics; Clinical; Clinical Management; Collection; Complex; DNA; Data; Data Analyses; Detection; Development; Diagnosis; Diagnostic; Disease; Endophthalmitis; Environmental Pollution; Etiology; Eye; Genetic Materials; Goals; Herpesviridae; Immune; Immunologic Tests; Immunologics; Individual; Infection; Infectious Agent; Inflammation; Institutes; Laboratories; Libraries; Light; Liquid substance; Mediating; Metagenomics; Modality; Modeling; Molecular Target; Nucleic Acids; Parasites; Pathogen detection; Pathogenesis; Patient Care; Patients; Performance; Polymerase Chain Reaction; Preparation; Process; Protocols documentation; Publishing; RNA; Reporting; Retinitis; Running; Sampling; Signal Transduction; Specimen; Speed; Technology; Testing; Therapeutic Trials; Time; Toxoplasma gondii; Translating; Trauma; Undifferentiated; United States; Uveitis; Validation; Viral; Virus; Vision; Work; accurate diagnosis; aqueous; base; biobank; bioinformatics pipeline; clinical Diagnosis; clinical application; clinical practice; clinically relevant; commensal microbes; cost; detection test; diagnostic accuracy; diagnostic assay; effective therapy; emerging pathogen; fungus; improved; innovation; insight; metagenomic sequencing; microbial host; next generation sequencing; novel; pathogen; personalized diagnostics; prospective; rapid diagnosis; rapid test; sample collection; socioeconomics; tool

PROJECT SUMMARY Uveitis is a sight-threatening intraocular inflammation that affects young working-age individuals, resulting in significant socioeconomic impacts. It is most often caused by infections or immune-mediated diseases. While herpesviruses and Toxoplasma gondii are the leading causes of infectious uveitis, many other infectious agents can be also involved, including viruses, bacteria, fungi and parasites. Clinical diagnosis is often complicated by overlapping findings upon examination among cases caused by different infectious etiologies, and also with observations made for those caused by autoimmune diseases and trauma. Because of that, complementary laboratory tests are essential for the etiological diagnosis and proper clinical management. The laboratory workup relies on multiple immunological tests and pathogen-targeted singleplex polymerase chain reaction (PCR) assays. This approach increases significantly the time to diagnosis, is often prohibitive due to the limited amount of intraocular fluid that can be safely obtained, and in many cases is unrevealing. As a result, patients can be treated for weeks or months using one-size-fits-all therapeutic trials that are not tailored for an individual and do not work for everyone. The use of metagenomic next-generation sequencing (mNGS) for unbiased detection of every pathogen in a clinical specimen is an emerging diagnostic modality that has the potential to significantly improve diagnostic yields in infectious uveitis. To date, there is a lack of an approach in which the appropriate steps for analytical and clinical validations are thoroughly performed for the unique intraocular fluid matrices tested from uveitic eyes. The studies proposed here are intended to fill this void and take on the challenge of developing and systematically validating an unbiased mNGS test that can be translated into clinical application for uveitis diagnosis and identification of new etiologies that currently escape diagnosis. First, we will thoroughly establish the analytical performance characteristics of a mNGS test for detection of uveitis pathogens from vitreous specimens. This will be done by using control vitreous containing varying levels of host DNA background spiked with a reference pathogen panel that will be used to model the effects of important determinants of analytical performance. Then, we will challenge this optimized mNGS assay with patient specimens for determination of relevant clinical performance characteristics to fully validate this test for clinical use. Using a combination of specimens stored in our biorepository and additional ones that will be collected prospectively for a blinded challenge study, we will determine the diagnostic accuracy of the assay using different sample types (e.g., aqueous vs. vitreous), its quantification capabilities, and will define clinical thresholds for reporting of detected pathogens. This proposal will result in the development of a systematically validated and innovative all-in-one diagnostic assay for infectious uveitis that is expected to substantially improve diagnostic yields and help to illuminate novel infectious uveitis etiologies in cases that remain undiagnosed, with great potential to for significantly changing the management of patients suffering from this debilitating disease.

项目摘要 葡萄膜炎是一种威胁视力的眼内炎症，影响年轻的工作年龄个体，导致 重大的社会经济影响。它通常由感染或免疫介导的疾病引起。而 疱疹病毒和弓形虫是感染性葡萄膜炎的主要原因， 包括病毒、细菌、真菌和寄生虫。临床诊断往往是复杂的， 不同感染病因引起的病例检查结果重叠， 对那些由自身免疫性疾病和创伤引起的人进行了观察。因此，互补 化验对病原学诊断和适当的临床治疗是必不可少的。实验室 检查依赖于多种免疫学测试和病原体靶向的单重聚合酶链反应 (PCR)分析。这种方法显著增加了诊断时间，由于有限的 可以安全获得的眼内液体量，并且在许多情况下是不可见的。因此，患者 可以使用不针对个人的一刀切的治疗试验治疗数周或数月 并不是对每个人都有效。使用宏基因组下一代测序（mNGS）进行无偏 检测临床标本中的每种病原体是一种新兴的诊断方式， 显著提高感染性葡萄膜炎诊断率。到目前为止，还没有一种方法， 对独特的眼内液进行适当的分析和临床验证步骤 从葡萄膜炎眼睛测试的基质。本文提出的研究旨在填补这一空白， 开发和系统验证可转化为临床的无偏倚mNGS测试的挑战 用于葡萄膜炎诊断和鉴定目前无法诊断的新病因。一是 彻底确立用于检测葡萄膜炎病原体的mNGS检测的分析性能特征 从玻璃体标本中分离出来。这将通过使用含有不同水平的宿主DNA的对照玻璃体来完成 背景加标参考病原体面板，将用于模拟重要的 分析性能的决定因素。然后，我们将用患者挑战这种优化的mNGS测定法。 用于确定相关临床性能特征的样本，以充分验证本试验的临床应用 使用.使用我们生物库中储存的标本和将收集的其他标本的组合 前瞻性地进行盲法激发研究，我们将使用不同的方法确定测定的诊断准确性。 样本类型（例如，水性与玻璃体），其量化能力，并将定义临床阈值 报告检测到的病原体。这一建议将导致制定一个经过系统验证和 用于感染性葡萄膜炎的创新型多合一诊断测定， 产量和帮助阐明新的感染性葡萄膜炎病因的情况下，仍然未确诊， 这可能会显著改变对患有这种使人衰弱的疾病的患者的管理。