Cell-Free DNA in Peritoneal Fluid as a Novel and Versatile Analyte for Monitoring Peritonitis

腹膜液中的游离 DNA 作为监测腹膜炎的新型多功能分析物

• 批准号: 10288893
• 负责人: Iwijn De Vlaminck
• 金额: $ 23.34万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-15 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10288893
• 关键词: Affect; Antibiotic Therapy; Bioinformatics; Biological Assay; Biomass; Catheters; Cell Culture Techniques; Cells; Clinical; Complication; Country; Culture-independent methods; Cytosine; DNA; DNA sequencing; Data; Detection; Development; Diagnosis; Diagnostic; Dialysis patients; Dialysis procedure; End stage renal failure; Environmental Pollution; Etiology; Excision; Gold; Hemodialysis; Human; Immune; Immune response; Infection; Knowledge; Lead; Liquid substance; Metagenomics; Methylation; Modality; Monitor; Morbidity - disease rate; New York City; Noise; Organism; Patients; Pattern; Peritoneal; Peritoneal Dialysis; Peritoneal Fluid; Peritonitis; Population; Preparation; Publishing; Research Design; Sampling; Specificity; Specimen; Techniques; Time; Tissues; Uracil; Visit; base; bisulfite; bisulfite sequencing; cell free DNA; diagnosis standard; genome-wide; improved; insight; metagenomic sequencing; microbial; mortality; novel; organ injury; pathogen; personalized medicine; precision medicine; programs; prospective; recruit; response; response to injury; screening; tissue injury; whole genome

PROJECT SUMMARY Peritonitis is a common complication in patients with end stage renal disease on peritoneal dialysis (PD) and is associated with significant morbidity and mortality in this population. Diagnosing the etiology for peritonitis can be challenging with approximately 25% of peritonitis cases being culture-negative. Currently, conventional cell culture is the gold standard for diagnosing the etiology, but this technique is limited to the detection of culturable organisms. Culture-independent methods based on metagenomic sequencing are promising techniques to screen infection in biofluids. However, the specificity of metagenomic sequencing is limited by two factors. First, contamination with microbial DNA introduced during sample preparations leads to background noise that can be significant for samples with an inherent low biomass. Second, conventional metagenomic sequencing assays do not inform about the host’s response to infection. The overall objective of this application is to apply precision medicine approaches to monitor PD patients with culture-negative peritonitis and culture-positive peritonitis. We will develop and apply a metagenomic cell-free DNA sequencing assay that is insensitive to environmental contamination and that informs the host’s response to infection. This is achieved by sequencing of cell-free DNA in peritoneal fluid after bisulfite conversion of unmethylated cytosines to uracils. Bisulfite conversion will be performed directly on the biofluid prior to DNA isolation, thereby tagging any microbial and human cell-free DNA that is intrinsic to the sample. Any contaminating environmental DNA introduced in the sample in downstream steps will not be bisulfite converted, making it straightforward to bio-informatically identify and remove any contaminant sequences. In addition to making this assay robust against contamination, bisulfite conversion of the host DNA will enable quantification of the host injury response to infection through genome-wide profiling of methylation marks in cfDNA. In this study, we will recruit PD patients at the time of suspected peritonitis: 50 PD patients with culture-positive peritonitis and 25 PD patients with culture-negative peritonitis. We will also recruit 40 PD patients with no evidence of clinical peritonitis. In Aim 1, we will determine the utility of whole genome bisulfite sequencing for monitoring PD patients with suspected peritonitis. In Aim 2, we will investigate the host-pathogen response during culture-positive peritonitis and culture-negative peritonitis. Our study will lead to the development of a metagenomic assay that is insensitive to environmental microbial contamination and informs the host’s response to infection, which can be more broadly applicable to profiling of other low biomass specimens. In addition, our study will lead to new avenues for personalized assessment and management of suspected peritonitis which cause significant morbidity and mortality in PD patients.

项目概要 腹膜炎是终末期肾病腹膜透析（PD）患者的常见并发症， 与该人群的显着发病率和死亡率相关。诊断腹膜炎的病因可以 大约 25% 的腹膜炎病例培养呈阴性，因此具有挑战性。目前，传统电池 培养是诊断病因的金标准，但该技术仅限于检测可培养的细菌 有机体。基于宏基因组测序的独立于培养物的方法是有前途的技术 筛查生物体液中的感染。然而，宏基因组测序的特异性受到两个因素的限制。第一的， 样品制备过程中引入的微生物 DNA 污染会导致背景噪音 对于固有低生物量的样品具有重要意义。二、常规宏基因组测序检测 不要告知宿主对感染的反应。 该应用程序的总体目标是应用精准医学方法来监测 PD 患者 培养阴性腹膜炎和培养阳性腹膜炎。我们将开发并应用宏基因组无细胞 DNA 测序分析对环境污染不敏感，并可告知宿主的反应 到感染。这是通过对亚硫酸氢盐转化后腹膜液中的无细胞 DNA 进行测序来实现的。 未甲基化的胞嘧啶变为尿嘧啶。亚硫酸氢盐转化将在 DNA 之前直接在生物流体上进行 分离，从而标记样品固有的任何微生物和人类无细胞 DNA。任何 在下游步骤中引入样品的污染环境 DNA 不会被亚硫酸氢盐转化， 使得生物信息学识别和去除任何污染物序列变得简单。此外 宿主 DNA 的亚硫酸氢盐转化可实现定量分析，从而使该测定具有抗污染能力 通过 cfDNA 中甲基化标记的全基因组分析来了解宿主对感染的损伤反应。 在本研究中，我们将招募疑似腹膜炎时的PD患者：50名培养阳性的PD患者 腹膜炎和 25 名培养阴性腹膜炎的 PD 患者。我们还将招募 40 名没有症状的 PD 患者 临床腹膜炎的证据。在目标 1 中，我们将确定全基因组亚硫酸氢盐测序的实用性 监测疑似腹膜炎的腹膜透析患者。在目标 2 中，我们将研究宿主-病原体反应 在培养阳性腹膜炎和培养阴性腹膜炎期间。我们的研究将导致开发 对环境微生物污染不敏感并告知宿主反应的宏基因组检测 感染，这可以更广泛地应用于其他低生物量样本的分析。此外，我们的 研究将为疑似腹膜炎的个性化评估和管理带来新的途径 导致 PD 患者显着的发病率和死亡率。