Multiplex clinical viral detection in formalin-fixed paraffin-embedded tissue samples

福尔马林固定石蜡包埋组织样本中的多重临床病毒检测

• 批准号: 8824269
• 负责人: Benjamin Pinsky
• 金额: $ 25.89万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-05-01 至 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8824269
• 关键词: Accounting; Acute; Affect; Base Sequence; Biological Assay; Biopsy; Blinded; Calibration; Cessation of life; Clinical; Complex; Custom; Cytomegalovirus; Detection; Development; Diagnosis; Diagnostic; Dideoxy Chain Termination DNA Sequencing; Discrimination; Disease; Etiology; Evaluation; Exclusion; Formalin; Genomic DNA; Genomic Segment; Hematopoietic Stem Cell Transplantation; Histologic; Human; Immunohistochemistry; Immunosuppression; In Situ Hybridization; Infection; Inflammatory Bowel Diseases; Laboratories; Libraries; Liver; Lung; Methods; Morbidity - disease rate; Nucleic Acids; Nucleic acid sequencing; Opportunistic Infections; Organ; Organ Transplantation; Outcome; Paraffin Embedding; Pathologic; Patients; Physicians; Process; RNA; Reaction; Reading; Research; Running; Sampling; Sensitivity and Specificity; Sequence Analysis; Serological; Services; Small Intestines; Solid; Source; Specificity; Specimen; Surgical Pathology; Technology; Testing; Time; Tissue Sample; Tissues; Transplant Recipients; Transplantation; Treatment outcome; University Hospitals; Validation; Viral; Viral Genome; Viral Load result; Virus; Virus Diseases; abstracting; base; clinically relevant; cost; diagnostic assay; genomic RNA; graft vs host disease; human DNA; improved; interest; mortality; preconditioning; public health relevance; success; targeted sequencing; viral detection

DESCRIPTION (provided by applicant): Multiplex clinical viral detection in formalin-fixed paraffin-embedded tissue samples Abstract: Transplantation is increasingly common and successful; however, high levels of immunosuppression remain a near-universal precondition for success. As such, solid organ and hematopoietic stem cell transplantation, as well as other conditions associated with immunosuppression, remain complicated by high rates of opportunistic infections. Of these, viral etiologies are the most difficult to diagnose due to substantial clinical/histologic overlap with non-viral processes as well as a lack of assays capable of detecting viruses in affected tissues. Indeed, virus diagnostics examining affected tissue directly are currently limited to immunohistochemistry and in situ hybridization, both of which suffer from severely limited clinical and analytic sensitivity/accuracy. As such, physicians often must rely on single-analyte serologic or singleplex nucleic acid assays of questionable applicability that are associated with substantial detection bias. There is thus an urgent need for multiplexed tissue-based viral diagnostics. Here we propose to develop a highly specific, highly sensitive method to simultaneously detect and quantify 55 clinically relevant viruses within formalin-fixed paraffin-embedded (FFPE) biopsy tissue, which is the standard specimen for histopathologic analysis. Our approach utilizes complementary long padlock probes (cLPPs) to selectively capture and quantitatively amplify defined genomic regions from clinically relevant viruses with very low off-target reactivity. Subsequently, reciprocal paired-end sequencing (Illumina MiSeq) will produce an accurate and high coverage representation of the targeted viral repertoire from which species/strain identity is assigned. Moreover, quantitative cLPP target capture allows viral copy number to be determined by simple read counting and normalization to internal human calibration targets, allowing discrimination between pathologic (high copy) and bystander (low copy) viruses. We will determine our methods' capture efficiency and accuracy, false discovery rate, and analytical sensitivity by using 100 well- characterized commercial and clinical viral isolates currently available in our laboratory. Additionally, clinical diagnostic feasibility will be established using 100 FFPE biopsies from various clinical contexts with either objective evidence for or clinical suspicion of viral infection in comparison to 100 similar biopsis with established non-viral etiologies (GVHD, rejection, inflammatory bowel disease, etc.). Overall, this technology will significantly improve our ability to diagnose and treat viral infectin in transplant patients while simultaneously reducing turnaround time and cost.

描述（由申请人提供）：福尔马林固定石蜡包裹的组织样品中的多重临床病毒检测摘要：移植越来越普遍且成功；然而，高水平的免疫抑制仍然是成功的近乎宇宙前提。因此，固体器官和造血干细胞移植以及与免疫抑制相关的其他疾病仍然因机会性感染率高而变得复杂。其中，由于与非病毒过程的实质性临床/组织学重叠以及缺乏能够检测受影响组织病毒的测定方法，病毒病因是最难诊断的。实际上，目前直接检查受影响组织的病毒诊断技术仅限于免疫组织化学和原位杂交，这两者都受到严重限制的临床和分析敏感性/准确性。因此，医生通常必须依靠单分析物血清学或单膜核酸测定法，其可疑适用性与实质性检测偏置相关。因此迫切需要 基于多重组织的病毒诊断。在这里，我们建议开发一种高度特异性，高度敏感的方法，以同时检测和量化福尔马林固定石蜡包含（FFPE）活检组织中的55个临床相关病毒，这是组织病理学分析的标准标本。我们的方法利用互补的长挂锁探针（CLPP）选择性地捕获和定量放大临床相关病毒的基因组区域，其脱靶反应性非常低。随后，相互配对的末端测序（Illumina Miseq）将产生靶向病毒库的准确且高的覆盖范围表示，从中分配了物种/应变身份。此外，定量CLPP目标捕获允许病毒拷贝数通过简单的读取计数和标准化与内部人类校准目标确定，从而可以区分病理学（高复制）和旁观者（低复制）病毒。我们将通过使用我们实验室中当前可用的100种特征性的商业和临床病毒分离株来确定方法的捕获效率和准确性，错误发现率以及分析敏感性。此外，与100个具有既定的非病毒病因（GVHD，抑制，炎症性的肠子病等）相比，将使用来自各种临床环境的100个FFPE活检来建立临床诊断可行性。总体而言，这项技术将显着提高我们诊断和治疗移植患者病毒感染素的能力，同时减少周转时间和成本。