Metagenomic profiling of urinary cell-free DNA to monitor urinary tract infection after kidney transplantation

项目总结 在美国，每年有超过1.5万名患者接受挽救生命的肾脏移植手术。然而，频繁的 移植后并发症限制了肾移植接受者的前景。尿路感染(UTI)， 包括多药耐药感染在内，肾移植后的发病率高得惊人。 至少20%的肾移植受者在移植后的前3个月经历了严重的尿路感染。 尿路感染与尿毒症的发展、移植物功能降低、移植物丢失和死亡有关。 目前诊断尿路感染的金标准是细菌培养，这从根本上受到其能力的限制。 只检测可培养的细菌。此外，细菌培养不能告知混合感染，共生 微生物区系，或宿主对感染的反应。我们的团队开发了新的生化和遗传方法 可以从根本上改变尿路感染诊断和移植监测的范式。 这项提议的目标是开发和应用精确医学方法来监测尿路感染 肾移植。我们将利用(1)新开发的技术进行元基因组测序 尿中cfDNA的超短片段，(2)新的元基因组宿主反应测量，(3)技术 对抗性决定因素进行生物信息学分类，以及(4)快速进行cfDNA的新方法 基于牛津大学的cfDNA中表观遗传标记的元基因组测序和直接图谱分析 纳米孔技术。 在第一个目标中，我们将调查尿液cfdna的元基因组测序作为监测工具的应用。 尿路感染和描述尿路微生物组。这一概念得到了广泛的试点数据的支持。在第二个目标中 我们将研究一种cfDNA元基因组测序分析，它可以告知宿主因 感染。第二个目标也得到了试点数据的显著结果的支持。在第三个目标中，我们将 研究新的纳米孔测序技术和算法的性能，这些技术和算法将使 成本，更快的周转时间，以及更大的cfDNA元基因组测序分析的实用性。 总而言之，这些检测方法将为监测尿路感染提供新的途径。 耐药性，描述尿液微生物群，并检查宿主与病原体的相互作用。 每年有超过15,000名患者接受肾移植，其中许多患者都会出现并发症 来自UTI的。在普通人群中，尿路感染是最常见的医疗问题之一，有1.5亿人 全球每年都会受到影响。这些研究的成功实施将提供新的途径 监测细菌性尿路感染，从而解决肾移植的紧急医疗需求，并通过 延期，对公众的健康有很大的好处。

PROJECT SUMMARY More than 15,000 patients receive life-saving kidney transplants in the US every year. Nevertheless, frequent post-transplant complications limit the outlook for kidney transplant recipients. Urinary tract infections (UTIs), including multi-drug antibiotic-resistant infections, occur at an alarmingly high rate after kidney transplantation. At least 20% of kidney transplant recipients experience a serious UTI in the first 3 months after transplantation. UTI has been associated with development of urosepsis, decreasing graft function, graft loss, and death. The current gold standard for diagnosis of UTI is bacterial culture, which is fundamentally limited by its ability to detect only culturable bacteria. Furthermore, bacterial culture does not inform about co-infections, commensal microbiota, or the host response to infection. New biochemical and genetic approaches developed by our groups can fundamentally change the paradigm for UTI diagnosis and transplant monitoring. The goal of this proposal is to develop and apply precision medicine approaches for the monitoring of UTIs after kidney transplantation. We will utilize (1) newly developed technologies for the metagenomic sequencing of ultrashort fragments of cfDNA in urine, (2) new metagenomic host-response measurements, (3) technologies to perform bioinformatic sorting of resistance determinants, and (4) new approaches to perform rapid cfDNA metagenomic sequencing and direct profiling of epigenetic markers comprised within cfDNA based on Oxford Nanopore Technologies. In the first Aim, we will investigate the utility of metagenomic sequencing of urinary cfDNA as a tool to monitor UTIs and to profile the urinary microbiome. This concept is supported by extensive pilot data. In the second Aim we will investigate a cfDNA metagenomic sequencing assay that informs the degree of host injury due to infection. This second Aim is also supported by significant results from pilot data. In the third Aim, we will investigate the performance of novel, Nanopore sequencing technologies and algorithms that will enable a lower cost, faster turnaround time, and greater utility of cfDNA metagenomic sequencing assays. Together, these assays will provide new avenues to monitor infections of the urinary tract, profile antibiotic resistance, describe the urinary microbiome, and examine host-pathogen interactions. More than 15,000 patients receive kidney transplants each year, and many of these patients suffer complications from UTI. In the general population, UTI is one of the most common medical problems, with 150 million persons affected per year worldwide. Successful implementation of these studies will provide new avenues for the monitoring of bacterial UTI and will thereby address an acute medical need in kidney transplantation, and by extension, a substantial health benefit to the general public.