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

项目摘要 在美国，每年有超过15，000名患者接受挽救生命的肾脏移植。然而，频繁 移植后并发症限制了肾移植受者的前景。尿路感染（UTI）， 包括多药抗药性感染，在肾移植后发生率高得惊人。 至少20%的肾移植受者在移植后的前3个月内经历严重的UTI。 UTI与尿脓毒症的发生、移植物功能下降、移植物丢失和死亡有关。 目前诊断UTI的金标准是细菌培养，其从根本上受到其能力的限制， 只检测可培养的细菌。此外，细菌培养不能提供关于合并感染的信息， 微生物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UTI诊断和移植监测的模式。 该提案的目标是开发和应用精确医学方法，用于监测UTI后， 肾移植我们将利用（1）新开发的宏基因组测序技术， 尿液中cfDNA的超短片段，（2）新的宏基因组宿主反应测量，（3） 进行抗性决定子的生物信息学分选，以及（4）进行快速cfDNA的新方法 宏基因组测序和基于Oxford的cfDNA内包含的表观遗传标记的直接分析 Nanopore Technologies. 在第一个目的中，我们将研究尿cfDNA的宏基因组测序作为监测尿cfDNA的工具的效用。 泌尿道感染和分析泌尿道微生物组。这一概念得到了大量试点数据的支持。在第二个目标 我们将研究cfDNA宏基因组测序测定，该测定告知宿主损伤的程度， 感染这第二个目标也得到了试点数据的重要结果的支持。在第三个目标中，我们将 研究新型纳米孔测序技术和算法的性能， 这意味着cfDNA宏基因组测序测定的成本、更快的周转时间和更大的实用性。 总之，这些检测将提供新的途径来监测尿路感染，分析抗生素， 耐药性，描述尿液微生物组，并检查宿主-病原体相互作用。 每年有超过15，000名患者接受肾脏移植，其中许多患者患有并发症 关于UTI在一般人群中，UTI是最常见的医疗问题之一，有1.5亿人 每年在全球范围内受到影响。这些研究的成功实施将为 监测细菌性尿路感染，从而解决肾移植中的急性医疗需求，并通过 扩展，对公众健康有很大的好处。

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.