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

项目摘要 腹膜炎是终末期肾病患者腹膜透析（PD）的常见并发症， 与这一人群中显著的发病率和死亡率相关。诊断腹膜炎的病因可以 具有挑战性，约25%的腹膜炎病例为培养阴性。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 有机体基于宏基因组测序的非培养方法是有前途的技术， 在生物液体中检测感染然而，宏基因组测序的特异性受到两个因素的限制。第一、 在样品制备过程中引入的微生物DNA污染导致背景噪声， 这对于具有固有低生物量的样品是显著的。第二，常规宏基因组测序测定 不要告知宿主对感染的反应。 本申请的总体目标是应用精确医学方法监测PD患者， 培养阴性和培养阳性腹膜炎。我们将开发和应用一种宏基因组无细胞 对环境污染不敏感并告知宿主反应的DNA测序测定 感染这是通过对腹膜液中的无细胞DNA进行测序来实现的， 未甲基化胞嘧啶转化为尿嘧啶。在DNA转化之前，将直接对生物流体进行亚硫酸氢盐转化。 分离，从而标记样品固有的任何微生物和人无细胞DNA。任何 在下游步骤中引入样品中的污染环境DNA将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。除了 为了使该测定法对污染具有鲁棒性，宿主DNA的亚硫酸氢盐转化将能够进行定量 通过cfDNA中甲基化标记的全基因组分析，研究宿主对感染的损伤反应。 在这项研究中，我们将招募疑似腹膜炎的PD患者：50名培养阳性的PD患者， 腹膜炎和25例腹膜炎培养阴性的PD患者。我们还将招募40名没有 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PD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。我们的研究将导致发展一个 对环境微生物污染不敏感并告知宿主反应的宏基因组测定 感染，这可以更广泛地适用于其他低生物量标本的剖析。另外我们 这项研究将为疑似腹膜炎的个性化评估和管理开辟新的途径， 在PD患者中引起显著的发病率和死亡率。

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.