Metagenomic Analysis of Arthroplasty Failure

描述(由申请人提供)：假体关节感染(PJI)的发病率正在增加，并对诊断和治疗提出了挑战。目前被归类为PJI的病例中，有四分之一到五分之一使用经典技术是微生物阴性的，即使在检测到微生物的情况下，我们最近的数据也表明，存在着使用现有策略没有检测到的生物亚群。此外，区分感染和无菌植入物失败可能是有问题的。{除了机械原因，如种植体错位，所谓的“无菌”种植体失败的原因没有明确界定，可能是由于未被识别的微生物感染，这些微生物是PJI的非传统原因。与PJI相关的微生物在种植体表面的生物膜中被发现。我们已经开发出一种使用涡流和超声波对种植体表面进行采样的方法，并一直表明，从种植体表面脱落的含有生物膜的材料提供了理想的微生物检测标本，比假体周围组织和滑液更好。然而，仍然有许多文化阴性的PJI病例。为了解决这个问题，在R01 AR056647的支持下，我们开发了一组针对属/组水平的PCR分析，目标是已知的导致PJI的最常见细菌。当对移位的含有生物膜的材料进行检测时，PCR板的敏感性为77%，而培养的敏感性为73%；两者的特异性均为98%。{基于这些发现，我们正在评估/开发一种术中使用的快速聚合酶链式反应方法。}尽管在我们之前的资助期间开发的方法的敏感度有所提高，但仍有PJI病例检测呈阴性。{在我们的续订R01申请中，我们将重点确定培养和平板PCR阴性PJI的病原学。我们还将描述与PJI相关的相同物种和混合物种生物膜的亚群，我们的新初步数据表明存在这些亚群，并可能为患者管理提供信息，确保理想的结果。最后，我们将确定我们的聚合酶链式反应小组没有针对的生物是否可以解释目前被归类为与设备故障无关的非感染性人工关节置换失败的原因。}我们建议使用自我们最初的R01项目期以来出现并成熟的深度测序技术。后基因组学和后转录组学方法将被用来分析从骨科植入物表面移出的含有生物膜的材料中的宿主和微生物核酸。利用深度测序，我们将分析微生物DNA和总表达的RNA，后者分离为微生物和人的RNA。我们将测试这样一种假设，即当应用于从骨科植入物表面移出的含有生物膜的材料时，新的深度测序方法将以比培养和面板PCR更高的临床灵敏度来检测PJI。我们还将定义以前未检测到的多微生物PJI的流行率，并评估同一类型细菌的亚群，我们的初步数据表明，这些细菌将存在，并可能影响治疗以及告知发病机制。我们将分析从移植关节置换中取出的材料中人类基因的表达，以检验存在PJI特异性人类基因组表达特征的假设。{无菌失败的原因在许多情况下没有完全确定；有几条证据表明，非由设备相关的失败引起的病例可能是由细菌引起的。为了解决这个问题，我们将使用元基因组学和元基因组学方法来研究无菌失败，分别分析颗粒磨损碎片导致的失败，植入物错位/结构装置相关的失败，以及更严格地归类为“无菌”失败。}除了定义PJI和无菌失败的病因外，我们的 结果将为开发直接针对微生物和/或与其相关的宿主反应的PJI新的诊断分析提供信息，并将为PJI的预防(例如疫苗接种)和治疗(即药物开发)提供目标。此外，我们将确定一些因“无菌”关节置换失败而接受髋关节或膝关节翻修术的患者是否有以前未被发现的感染。

DESCRIPTION (provided by applicant): Prosthetic joint infection (PJI) is increasing in incidence and poses diagnostic and therapeutic challenges. One quarter to one fifth of cases currently classified as PJI are microbiologically-negative using classic techniques and even in cases in which microbes are detected, our recent data shows that there are subpopulations of organisms present which are undetected using existing strategies. Further, differentiating infection from aseptic implant failure can be problematic. {Outside of mechanical reasons such as implant malposition, the cause of so-called "aseptic" implant failure is not well-defined and may be due by unrecognized infection with organisms that are nontraditional causes of PJI.} Microorganisms associated with PJI are found in biofilms on the surface of the implant. We have developed a method that uses vortexing and sonication to sample the implant surface and have consistently shown that biofilm-containing material dislodged from the implant surface provides the ideal specimen for microbial detection, better than periprosthetic tissues and synovial fluid. Nevertheless, there remain many cases of culture-negative PJI. To address this issue, with support of R01 AR056647, we developed a panel of PCR assays targeting, at the genus-/group-level, the most common bacteria known to cause PJI. The sensitivity of the PCR panel was 77% whereas that of culture was 73% when performed on dislodged biofilm-containing material; specificities were 98% for both. {Based on these findings, we are evaluating/developing a rapid PCR panel strategy for intraoperative use.} Despite the improved sensitivity of the approach developed in our prior funding period, there remain PJI cases that test negative. {In our renewal R01 application, we will focus on determining the etiology of culture- and panel PCR- negative PJI. We will also delineate subpopulations of same- and mixed-species biofilms associated with PJI, which our new preliminary data indicate are present and which may inform patient management, ensuring an ideal outcome. Finally, we will determine whether organisms not targeted by our PCR panel might account for what are currently classified as non-infectious arthroplasty failures unrelated to device failure.} We propose to use deep sequencing technology which has emerged and matured since our original R01 project period. Metagenomic and metatranscriptomic approaches will be used to analyze host and microbial nucleic acids in biofilm-containing materials dislodged from the surfaces of orthopedic implants. Using deep sequencing, we will analyze microbial DNA and total expressed RNA, the latter separated into microbial and human RNA. We will test the hypothesis that the new deep sequencing approaches will detect PJI with improved clinical sensitivity compared to culture and panel PCR when applied to biofilm-containing materials dislodged from surfaces of orthopedic implants. We will also define the prevalence of previously undetected polymicrobial PJI, and assess for subpopulations of same-type bacteria, which our preliminary data suggest will be present, and which can impact treatment as well as inform pathogenesis. We will analyze human gene expression in material dislodged from explanted arthroplasties to test the hypothesis that there are PJI-specific human genomic expression signatures. {The cause of aseptic failure is in many cases incompletely defined; several lines of evidence suggest that cases not caused by device-related failure may be caused by bacteria. To address this, we will use metagenomic and metatranscriptomic approaches to study aseptic failure, separately analyzing failure due to particulate wear debris, implant malposition/structural device-related failure and more strictly classified "aseptic" failure.} Beyond definition of the etiologies of PJI and aseptic failure, our results will inform development of new diagnostic assays for PJI targeting microbes directly and/or their associated host response and will provide targets for prevention (e.g., vaccination) and therapy (i.e., drug development) of PJI. In addition, we will determine whether some patients undergoing revision hip or knee arthroplasty for "aseptic" arthroplasty failure have previously unrecognized infection.