Cell-free DNA as a versatile analyte for the monitoring of sepsis

脓毒症是一种致命的疾病，由对感染的免疫反应失调引起， 仅在美国，每年就有300名患者。早期和准确诊断脓毒症和脓毒症的感染， 这对于指导治疗选择和预防严重器官损伤和死亡至关重要，但诊断选择仍然存在 有限公司该提案通过开发微创血液透析解决了这一未满足的医疗需求。 基于循环cfDNA的多组学分析的测试，以同时告知宿主反应，定量器官 损伤和确定脓毒症潜在感染 我们最近的试点工作为这一建议的可行性提供了重要支持。首先，我们发现， 循环无细胞DNA（cfDNA）的宏基因组测序可用于检测广泛的病毒和 血液中的细菌病原体为了克服环境污染的挑战， 宏基因组cfDNA测序的特异性我们已经开发了Coffee-seq，一种宏基因组测序， 对环境污染具有鲁棒性的测定。Coffee-seq的核心思想是标记DNA， 在样品制备之前，使用可通过DNA测序记录的标记物对样品进行标记。任何污染 然后，在标记后引入样品中的DNA可以被生物信息学识别和去除。我们 原理证明工作表明，Coffee-seq可以减少由于污染而产生的噪音， 到三个数量级。因此，Coffee-seq显著提高了宏基因组的特异性。 测序测定。第二，我们已经开发了cfDNA测定来告知对宿主组织和器官的损伤。这 该检测采用cfDNA甲基化标记的全基因组分析，其具有细胞、组织和器官类型特异性 以追踪它们的起源组织，并量化组织特异性损伤。我们已经证明了这一点的实用性 鉴定与尿路感染、COVID-19和移植物抗宿主病相关的宿主组织损伤的试验， 干细胞移植的常见并发症 我们有两个目标：在目标1中，我们将测试Coffee-seq的效用，以确定感染潜在的脓毒症与高 灵敏度和特异性。在目标2中，我们将研究cfDNA宏基因组测序测定法用于检测CfDNA的用途。 告知败血症相关器官功能障碍。这是一项探索性研究，其测试了cfDNA作为多功能免疫调节剂的效用。 分析物以监测脓毒症。我们将通过对300名生物医学专家的回顾性研究来测试这一高度转化的概念。 库存样本（Cornell Biobank of Critical Illness），包括来自败血症患者的120份样本， 血培养和180份来自非传染性危重病患者的样本。成功实施 这项研究将为识别脓毒症感染、分类脓毒症亚型、监测脓毒症的发生和发展提供新的途径。 脓毒症相关的器官损伤，并指导治疗决策。

Sepsis is a deadly condition caused by a dysregulated immune response to infection which affects 700,000 patients per year in the US alone. Early and accurate diagnosis of sepsis, and infection underlying sepsis is critical to inform treatment choices and to prevent serious organ injury and death, but diagnostic options remain limited. This proposal addresses this unmet medical need with the development of a minimally invasive blood test based on a multi-omics profiling of circulating cfDNA to simultaneously inform host responses, quantify organ injury and identify infection underlying sepsis Our recent pilot work provides significant support for the feasibility of this proposal. First, we have found that metagenomic sequencing of circulating cell-free DNA (cfDNA) can be used to detect a broad range of viral and bacterial pathogens in blood. To overcome the challenge of environmental contamination which limits the specificity of metagenomic cfDNA sequencing we have developed Coffee-seq, a metagenomic sequencing assay that is robust against environmental contamination. The core idea of Coffee-seq is to tag the DNA in the sample prior to sample preparation with a label that can be recorded by DNA sequencing. Any contaminating DNA that is introduced in the sample after tagging can then be bioinformatically identified and removed. Our proof-of-principle work demonstrates that Coffee-seq leads to a reduction of noise due to contamination by up to three orders of magnitude. Coffee-seq thereby dramatically improves the specificity of metagenomic sequencing assays. Second, we have developed cfDNA assays to inform injury to host tissues and organs. This test employs genome-wide profiling of methylation marks of cfDNA that are cell, tissue and organ-type specific to trace their tissues-of-origin, and to quantify tissue-specific injury. We have demonstrated the utility of this assay to identify host-tissue injury related to urinary tract infection, COVID-19, and Graft-Versus Host Disease, a frequent complication of stem cell transplantation. We have two aims: In Aim 1, we will test the utility of Coffee-seq to identify infection underlying sepsis with high sensitivity and specificity. In Aim 2, we will investigate the utility of a cfDNA metagenomic sequencing assay to inform sepsis related organ dysfunction. This is an exploratory study that tests the utility of cfDNA as a versatile analyte to monitor sepsis. We will test this highly translational concept with a retrospective study of 300 bio- banked samples (Cornell Biobank of Critical Illness), including 120 samples from septic patients with positive blood culture, and 180 samples from patients with non-infectious critical illness. Successful implementation of this study will lead to new avenues to identify infection underlying sepsis, to classify sepsis subtypes, to monitor sepsis-related organ damage, and to guide treatment decisions.