Metagenomic Analysis of Arthroplasty Failure

描述（由申请人提供）：人工关节感染（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，我们最近的数据显示，存在使用现有策略未检测到的生物体亚群。此外，区分感染与无菌植入失败可能是有问题的。{除了植入物错位等机械原因外，所谓的“无菌”植入物失效的原因尚未明确，可能是由于未识别的微生物感染，这些微生物是PJI的非传统原因。} 与PJI相关的微生物存在于植入物表面的生物膜中。我们开发了一种使用涡旋和超声处理对植入物表面进行取样的方法，并一致表明从植入物表面脱落的含生物膜材料为微生物检测提供了理想的标本，优于假体周围组织和滑液。尽管如此，仍有许多文化阴性的PJI病例。为了解决这一问题，在R 01 AR 056647的支持下，我们开发了一组PCR检测试剂盒，在属/组水平上靶向已知引起PJI的最常见细菌。PCR面板的灵敏度为77%，而文化是73%时，进行的脱落的生物膜含材料，特异性为98%。{基于这些发现，我们正在评估/开发用于术中使用的快速PCR面板策略。}尽管我们在上一个资助期开发的方法的敏感性有所提高，但仍有PJI病例检测为阴性。 {In在我们的R 01更新申请中，我们将集中于确定培养和PCR阴性PJI的病因。我们还将描述与PJI相关的相同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，这些亚群可以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PCR面板未靶向的微生物是否可能解释目前分类为与器械失效无关的非感染性关节置换术失效。 我们建议使用深度测序技术，该技术自我们最初的R 01项目期间以来已经出现并成熟。宏基因组学和元转录组学方法将用于分析从骨科植入物表面脱落的含生物膜材料中的宿主和微生物核酸。使用深度测序，我们将分析微生物DNA和总表达RNA，后者分为微生物和人类RNA。我们将检验以下假设：当应用于从骨科植入物表面脱落的含生物膜材料时，与培养和面板PCR相比，新的深度测序方法将以更高的临床灵敏度检测PJI。我们还将确定以前未检测到的多微生物PJI的患病率，并评估相同类型细菌的亚群，我们的初步数据表明将存在这些亚群，这些亚群可以影响治疗并告知发病机制。我们将分析从关节成形术取出的材料中的人类基因表达，以检验存在PJI特异性人类基因组表达特征的假设。{The在许多情况下，无菌性失效的原因未完全确定;几条证据表明，并非由器械相关失效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和元转录组方法来研究无菌性失效，分别分析由于颗粒磨损碎屑、植入物错位/结构性器械相关失效以及更严格分类的“无菌”失效而导致的失效。 除了PJI和无菌性失败的病因定义外， 结果将为直接靶向微生物和/或其相关宿主反应的PJI的新诊断测定的开发提供信息并将提供预防的靶点（例如，疫苗接种）和治疗（即，药物开发）。此外，我们将确定一些因“无菌性”关节置换术失败而接受髋关节或膝关节翻修术的患者是否存在先前未识别的感染。

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.