MOLECULAR DIAGNOSIS OF ORTHOPAEDIC BACTERIAL INFECTIONS

描述（改编自申请人摘要）：本研究计划 解决了骨科社区对快速、准确 和廉价的方法来确定是否整形外科标本， 例如滑液和其它整形外科组织，例如骨和骨 骨髓，被感染了。 标准实验室培养和革兰氏染色试验 检测关节中的细菌感染， 由于假阴性测试的频率相对较高， 结果 申请人正在开发，测试和改进一种分子 使用聚合酶链反应（PCR）的生物技术， 快速、准确地确定微生物DNA的存在， 整形外科的标本 该方法涉及一种新的处理方式， 骨科样本，以实现细菌裂解和DNA的释放， 通过PCR对DNA进行直接分子检测。 细菌特异性PCR 使用引物扩增高度保守的通用16 S 细菌基因组特有的核糖体RNA（rRNA）基因。 初探 正在进行测试这种诊断技术的有效性， 检测滑液中的感染。 最初，提议的过程是 用无菌滑液进行测试， 各种骨科相关细菌物种的浓度。 其次，申请人提出将该方法扩展到测试滑膜， 接受全膝关节置换术患者的液体标本 (TKA)手术，谁可能已经获得细菌感染， 受影响的关节 还提出了旨在使用PCR的策略， 用于确定感染原种类的诊断。 一 将对申请人的PCR进行统计比较 检测方法与标准诊断模式。 放置 由于假阴性，将翻修植入物植入受污染的关节 术前微生物学数据将导致感染的翻修TKA， 需要额外的手术，在医疗费用方面， 患者不适。 调查人员认为， 应用高灵敏度的PCR诊断技术， 准确鉴定受感染的标本可以大大有助于 建立手术和/或抗生素治疗方案。 照经上所 我推测，这项拟议中的研究可以提供一种有价值的方法， 对于骨科临床医生和基础研究人员， 正试图明确地确定存在和水平， 临床骨科标本中的感染因子。

DESCRIPTION (Adapted from the Applicant's Abstract): This research proposal addresses the need within the orthopaedic community for a rapid, accurate and inexpensive method for determining whether or not orthopaedic specimens, such as synovial fluid, and other orthopaedic tissues, such as bone and bone marrow, are infected. Standard laboratory culturing and Gram stain testing to detect bacterial infections in articular joints have been particularly disappointing due to the relatively high frequency of false negative test results. The applicants are developing, testing and refining a molecular biological technique using the polymerase chain reaction (PCR) to provide rapid and accurate determination of the presence of microorganismal DNA in an orthopaedic specimen. The methodology involves a novel way of processing orthopaedic samples to achieve bacterial lysis and release of DNA, allowing the direct molecular detection of that DNA by PCR. Bacteria-specific PCR primers are being used that amplify the highly conserved, universal 16S ribosomal RNA (rRNA) gene unique to bacterial genomes. A preliminary study is being undertaken to test the efficacy of this diagnostic technology for detecting infection in synovial fluid. Initially, the proposed process is being tested with sterile synovial fluid inoculated with known concentrations of various orthopaedically relevant bacterial species. Secondly, the applicants propose to extend this methodology to test synovial fluid specimens from patients who have undergone total knee replacement (TKA) surgery, and who may have acquired bacterial infections in the affected joints. Also proposed are strategies designed to use PCR diagnostics for the determination of the species of the infectious agent. A statistical comparison will be performing comparing the applicants' PCR detection methodology with standard diagnostic modalities. Placement of a revision implant into a contaminated joint because of false negative preoperative microbiological data will result in an infected revision TKA, requiring additional surgery at great expense, in terms of medical costs and patient discomfort. It is suggested by the investigators that, with the application of highly sensitive PCR diagnostic technology, rapid and accurate identification of infected specimens could greatly assist in the establishment of surgical and/or antibiotic treatment regimens. Thus, it is speculated that this proposed research could provide a valuable methodology for the orthopaedic clinician, as well as the basic researcher, when either is attempting to determine unambiguously the presence and level of infectious agents in a clinical orthopaedic specimen.