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于早期治疗决策至关重要。 延迟诊断导致不必要的抗生素处方率高，患者预后更差。 已用于改善脓毒症诊断的一种方法是循环游离DNA（cfFNA）。在这里， 存在于血清中的细菌DNA用于鉴定微生物病原体并告知抗生素治疗决策。 不幸的是，虽然cfDNA方法在鉴定一些败血症病原体方面很好，但cfDNA的效果很差。 区分细菌感染和细菌定植的能力。在大量背景信号 存在于肠道，皮肤或肺部的相关细菌，现有的方法往往混淆殖民作为 感染同样，敏感性也可能因将感染误解为定植而受到损害。我们提出 这种分辨率的缺乏是因为cfDNA诊断仅在物种水平上识别细菌。他们是 无法提供对感染的细菌菌株动力学的深入了解。 为了解决这个问题，我们已经确定了一种新的方法，用于改善cfDNA在脓毒症中的性能， 噬菌体-由细菌产生的病毒。因为噬菌体对它们特定的 宿主菌株，定量的独特的微生物可以提供洞察细菌种群动力学在 应变水平我们的初步数据显示，噬菌体序列存在于从大肠杆菌中收集的cfDNA中。 脓毒症患者。使用先前收集和测序的cfDNA数据，我们发现我们可以诊断 铜绿假单胞菌感染使用独特的噬菌体序列，不可能诊断与 细菌序列。也许可以将这种方法扩展到其他细菌病原体。 然而，首先，我们必须开发强大的计算管道来研究cfDNA中的DNA，如现有的计算管道。 算法被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有助于鉴定脓毒症病原体， 及时交付成果至关重要。为此，更新的纳米孔技术在更长的时间内具有优势 消耗Illumina测序方法。然而，噬菌体cfDNA的纳米孔测序的实用性是有限的。 未经测试我们必须证明纳米孔测序的准确性和及时性。 我们的假设是噬菌体cfDNA可以提供对脓毒症的细菌发病机制的洞察。到 为了测试这一点，在R21部分，我们将开发计算协议，用于研究现有的 败血症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， 在现有脓毒症生物储存库样品中表征噬菌体cfDNA 总之，这些研究将产生必要的工具和概念框架， 败血症中的细菌菌株。此外，这些研究将为大型前瞻性人类队列研究奠定基础 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.