Circulating Bacteriophages for the Diagnosis of Sepsis

项目概要 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。 延迟诊断会导致大量不必要的抗生素处方和更糟糕的患者治疗结果。 一种用于改善脓毒症诊断的方法是循环游离 DNA (cfFNA)。在这里， 血清中存在的细菌 DNA 用于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 方法擅长识别一些脓毒症病原体，但 cfDNA 却表现不佳 区分细菌感染和细菌定植。在有大量背景信号的环境中 存在于肠道、皮肤或肺部的相关细菌中，现有方法常常将定植混淆为 感染。同样，将感染误解为定植也会损害敏感性。我们建议 这种缺乏分辨率的存在是因为 cfDNA 诊断只能识别物种水平的细菌。他们是 无法深入了解感染背后的细菌菌株动态。 为了解决这个问题，我们确定了一种新方法，使用以下方法来提高脓毒症中 cfDNA 的性能： 噬菌体——细菌产生的病毒。因为噬菌体对其特定的特性具有极高的特异性 宿主菌株，独特噬菌体的量化可以提供对细菌种群动态的深入了解 应变水平。我们的初步数据表明，噬菌体序列存在于从以下来源收集的 cfDNA 中： 患有败血症的人。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进行铜绿假单胞菌感染 单独的细菌序列。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 中的噬菌体，因为现有的 算法设计用于处理人类和细菌 DNA。 cfDNA 测序的另一个问题是速度。 cfDNA 有助于识别败血症病原体， 及时提供结果至关重要。为此，较新的纳米孔技术比更长的时间具有优势 消耗照明测序方法。然而，纳米孔测序对噬菌体 cfDNA 的实用性是 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 可以深入了解脓毒症的细菌发病机制。至 为了测试这一点，在本次拨款的 R21 部分中，我们将开发用于研究现有噬菌体的计算协议 脓毒症 cfDNA 数据集。然后，在这些研究的 R33 部分中，我们将开发快速测序方案 表征现有脓毒症生物样本库样本中的噬菌体 cfDNA 这些研究将共同产生调查作用所需的工具和概念框架。 脓毒症中的细菌菌株。此外，这些研究将为大型前瞻性人类队列研究奠定基础 测试噬菌体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.