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: 10428638
• 负责人: Iwijn De Vlaminck
• 金额: $ 24.71万
• 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 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10428638
• 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.

项目摘要 腹膜炎是腹膜透析（PD）末期肾脏疾病患者的常见并发症，IS 与该人群的发病率和死亡率的显着相关。诊断腹膜炎的病因 大约25％的腹膜炎病例是培养性阴性的挑战。目前，传统单元格 培养是诊断学的黄金标准，但该技术仅限于对文化的检测 有机体。基于元基因组测序的培养无关方法是有希望的技术 生物流体中的筛查感染。然而，元基因组测序的特异性受两个因素的限制。第一的， 样品制备过程中引入的微生物DNA的污染导致背景噪声可能是 对于继承低生物质的样品的重要性。第二，常规的宏基因组测序测定 不要告知宿主对感染的反应。 该应用的总体目的是采用精确医学方法来监视PD患者 培养性阴性腹膜炎和培养阳性腹膜炎。我们将开发并应用无元基因组细胞 对环境污染不敏感的DNA测序测定法，并告知主机的反应 感染。这是通过在亚硫酸盐转化后的腹膜液中无细胞DNA测序来实现的 未甲基化的胞嘧啶对尿嘧啶。 Bisulfite转化将直接在DNA之前的生物流体上进行 分离，从而标记样品固有的任何微生物和无细胞的DNA。任何 样品中引入的污染环境DNA在下游步骤中不会被硫甘硫酸盐转化， 使生物信息直接识别并删除任何污染物序列变得直接。此外 使此测定可靠地抵抗污染，宿主DNA的Bisulfite转换将实现定量 通过CFDNA中甲基化标记的全基因组分析对感染的宿主损伤反应。 在这项研究中，我们将在怀疑腹膜炎时招募PD患者：50名PD患者培养阳性患者 腹膜炎和25例培养性阴性腹膜炎的PD患者。我们还将招募40名PD患者 临床腹膜炎的证据。在AIM 1中，我们将确定整个基因组Bisulfite测序的实用性 监测可疑腹膜炎的PD患者。在AIM 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中。我们的研究将导致发展 对环境微生物污染不敏感并告知宿主的反应的宏基因组评估 感染，这可能更广泛地适用于其他低生物质标本的分析。另外，我们的 研究将导致新的途径，用于对可疑腹膜炎进行个性化评估和管理，这 在PD患者中引起明显的发病率和死亡率。