Circulating Bacteriophages for the Diagnosis of Sepsis

项目摘要 脓毒症患者细菌病原体的快速诊断对早期治疗决策至关重要。 延迟诊断会导致开出不必要的抗生素处方的比率很高，患者的预后也会更差。 一种用于改善脓毒症诊断的方法是循环游离DNA(CfFNA)。这里, 血清中存在的细菌DNA被用来识别微生物病原体，并为抗生素治疗决策提供信息。 不幸的是，虽然cfdna方法很好地识别了一些败血症病原体，但cfdna方法做得很差。 在区分细菌感染和细菌定植方面。在大量背景信号的设置中 存在于肠道、皮肤或肺部的相关细菌中，现有的方法往往将定植混淆为 感染。同样，将感染误解为殖民也可能损害敏感性。我们建议 这种缺乏分辨率的存在是因为cfDNA诊断只在物种水平上识别细菌。他们是 无法提供对细菌菌株动态的洞察，这不利于感染。 为了解决这个问题，我们已经确定了一种新的方法来改善cfdna在脓毒症中的表现。 噬菌体--细菌产生的病毒。因为噬菌体对其特定的 对于宿主菌株，对独特噬菌体的量化可以提供对细菌种群动态的洞察 应变水平。我们的初步数据显示，从中国采集的cfDNA中存在噬菌体序列 败血症患者。使用之前收集和测序的cfDNA数据，我们发现我们可以诊断 使用无法诊断的独特噬菌体序列的铜绿假单胞菌感染 单独的细菌序列。有可能将这种方法扩展到与其他细菌病原体一起工作。 然而，首先我们必须开发强大的计算管道来研究cfdna中的噬菌体，就像现有的 算法设计用于处理人类和细菌的DNA。 CfDNA测序的另一个问题是速度。为了使cfDNA有助于识别脓毒症病原体， 及时交付结果至关重要。为此，较新的纳米孔技术在更长的时间内具有优势 耗费Illumina测序方法。然而，噬菌体cfDNA的纳米孔测序的效用是 未经测试。我们必须证明，噬菌体的纳米孔测序既准确又及时。 我们的假设是，噬菌体cfDNA可以为脓毒症的细菌发病机制提供洞察力。至 测试这一点，在这笔拨款的R21部分，我们将开发计算协议来研究现有的噬菌体 脓毒症cfDNA数据集。然后，在这些研究的R33部分，我们将开发快速测序方案 现有脓毒症生物仓库样本中噬菌体cfDNA的特征 综上所述，这些研究将产生所需的工具和概念框架，以调查 脓毒症中的细菌菌株。此外，这些研究将为大规模、前瞻性的人类队列研究奠定基础。 目的探讨噬菌体cfDNA在脓毒症诊断中的价值。

Project Summary The rapid diagnosis of bacterial pathogens in septic patients is critical for early treatment decision making. Delayed diagnoses lead to a high rate of unnecessary antibiotic prescriptions and worse patient outcomes. One approach that has been used to improve sepsis diagnoses is circulating free DNA (cfFNA). Here, bacterial DNA present in serum is used to identify microbial pathogens and inform antibiotic treatment decisions. Unfortunately, while cfDNA approaches are good at identifying some sepsis pathogens, cfDNA does poorly at distinguishing bacterial infection from bacterial colonization. In settings where substantial background signal exists from related bacteria present in the gut, skin or lungs, existing approaches often confuse colonization as infection. Similarly, sensitivity can also be compromised by misinterpreting infection as colonization. We propose that this lack of resolution exists because cfDNA diagnostics only identify bacteria at the species level. They are unable to provide insight into the bacterial strain dynamics that underly infection. To address this, we have identified a novel approach for improving the performance of cfDNA in sepsis using bacteriophage –viruses produced by bacteria. Because bacteriophages are exquisitely specific to their particular host strain, the quantification of unique phages can provide insights into bacterial population dynamics at the strain level. Our preliminary data reveal that bacteriophage sequences are present in cfDNA collected from individuals with sepsis. Using previously collected and sequenced cfDNA data, we find that we can diagnose Pseudomonas aeruginosa infections using unique phage sequences that were not possible to diagnose with bacterial sequences alone. It may be possible to extend this approach to work with other bacterial pathogens. However, first we must develop robust computational pipelines for studying phages in cfDNA as the existing algorithms are designed to work with human and bacterial DNA. A further issue with cfDNA sequencing is speed. For cfDNA to be helpful in identifying sepsis pathogens, delivering timely results is critical. To this end, newer nanopore technologies have advantages over more time consuming illumina sequencing methods. However, the utility of nanopore sequencing for phage cfDNA is untested. We must demonstrate that nanopore sequencing of phages is both accurate and timely. Our hypothesis is that bacteriophage cfDNA can provide insight into the bacterial pathogenesis of sepsis. To test this, in the R21 portion of this grant we will develop computational protocols for studying phages in existing sepsis cfDNA datasets. Then, in the R33 portion of these studies we will develop rapid sequencing protocols for characterizing phage cfDNA in existing sepsis biorepository samples Together, these studies will generate the tools and conceptual frameworks needed to investigate the role of bacterial strains in sepsis. Moreover, these studies will set the stage for large, prospective human cohort studies to test the value of phage cfDNA in sepsis diagnosis.