Optimized Growth Media to Detect Intracellular Bacteria Implicated in Chronic Dis

描述(由申请人提供)：美国疾病控制与预防中心报告称，超过4000万美国人患有慢性病，其中大多数人报告说有多种慢性疾病的诊断。其中许多慢性疾病的病因还没有被很好地了解，医生们努力有效地诊断和治疗这些疾病。近年来，人们的注意力转向了这样一种可能性，即许多慢性疾病，特别是那些涉及自身免疫反应的慢性疾病，可能是由高度挑剔的、免疫逃避的病原体引起的。巴尔通体属细菌(属于立克次体菌目)在医学病例系列文献中被认为与人类和兽医中的一系列疾病有关，包括多发性关节炎、皮肤血管炎、心内膜炎、心肌炎、鼻出血、肝炎疱疹、肉芽肿性炎症性疾病，以及最近的神经功能障碍和关节炎。在患者样本中检测细菌感染的传统方法已被证明在很大程度上不能准确诊断巴顿氏菌病。北卡罗来纳州立大学兽医学院的研究人员开发了一种高灵敏度的巴氏杆菌检测平台，该平台结合了在新型液体介质中对血液进行预浓缩培养，然后使用属级引物和阳性结果的序列ID进行巴氏杆菌聚合酶链式反应(Bartonella PCR)检测。开发这一创新的巴氏杆菌检测平台是为了便于检测免疫能力强的患者中的巴氏杆菌感染，在这些患者中，这种高度挑剔的细菌的感染水平如此之低，以至于检测点的采样误差为直接PCR带来了极高的假阴性率，并且宿主免疫系统几乎没有机会产生针对这种细菌的抗体。这一分子诊断平台的基石是一种名为巴尔通体阿尔法变形杆菌生长介质(BAPGM)的专利预富集介质。Galaxy诊断公司拥有BAPGM的独家许可证。初步数据支持BAPGM微生物学方法在人类医学中的用处。这一新的巴通氏菌检测平台已经在从慢性病和健康的人和动物获得的血液样本中检测到单一和多个微生物巴通氏菌感染。也许更重要的是，使用BAPGM-PCR检测平台的研究揭示了巴尔通体感染和严重慢性病之间的重要临床联系。我们的期望是，这一测试平台的开发将导致慢性病诊断和治疗方面的医学突破，并将通过及早发现和预防令人衰弱的慢性病来广泛节省医疗支出。尤其重要的是关于传播方式的研究，以及确定特定慢性疾病的原因或促成因素，如关节炎、神经疾病、器官衰竭和其他难以诊断的慢性疾病。为巴尔通体属(Bartonella spp.巴尔通体是通过大量节肢动物媒介传播的，包括跳蚤、虱子、沙蝇、扁虱、叮咬苍蝇和可能的其他媒介，由于各种巴尔通体物种向献血者公众传播的区域差异，确定要筛选的适当细菌可能会变得复杂。由于慢性携带者和/或媒介的存在，未来应按地区处理不同巴尔通体物种的发病率，以便确定该属(及其每个物种)在美国的实际发生情况。第一阶段的总体目标将是开发一种BAPGM无菌培养瓶配方，用于立即接种或运输微生物标本。然后，标本将被运送到银河诊断公司进行巴氏杆菌检测；目标1将在标准的BAPGM平台培养基中开发和验证新配方，以满足拟议的BAPGM无菌培养瓶设计要求；目标2将测试新配方获得合理储存条件的可行性。这将通过检查之前在室温、4-8摄氏度和-20摄氏度下储存后执行的生长敏感性测试来实现。第二阶段将侧重于产品开发和开发试剂盒所需的监管里程碑，包括BAPGM无菌培养瓶，它将允许在全球范围内销售和分销。第二阶段的研究还将解决BAPGM无菌培养瓶盖-空间气体成分的潜在变化，以最大限度地促进细菌的恢复和生长，这可能会导致优化测试平台中的增菌培养步骤所需的时间。

DESCRIPTION (provided by applicant): The CDC reports that over 40 million Americans suffer from chronic disease, the majority of whom report multiple diagnoses of chronic disorders. The causes of many of these chronic diseases are not well understood and doctors struggle to effectively diagnose and treat these disorders. In recent years, attention has turned towards the possibility that many chronic disorders, especially those involving autoimmune response, may be caused by highly fastidious, immune-evasive pathogens. Bacteria of the genus Bartonella (a member of the Rickettsiales order) have been linked in the medical case series literature to a wide spectrum of diseases in human and veterinary medicine, including polyarthritis, cutaneous vasculitis, endocarditis, myocarditis, epistaxis, peliosis hepatis, granulomatous inflammatory disease, and, most recently, neurological dysfunction and arthritis. Traditional methods for detecting bacterial infections in patient samples have proven to be largely ineffective for the accurate diagnosis of Bartonellosis. Researchers at the North Carolina State University College of Veterinary Medicine have developed a highly sensitive Bartonella testing platform which combines the pre- enrichment culture of blood in a novel liquid media followed by a Bartonella PCR assay using genus level primers and sequence id of positive results. This innovative Bartonella testing platform was developed to facilitate the detection of Bartonella infections in immuno-competent patients where this highly fastidious bacteria infects at such low levels that sampling error at the point of testing introduces extremely high false negative rates for direct PCR and that the host immune system has little opportunity to develop antibodies against this bacteria. The cornerstone of this molecular diagnostic platform is a patented pre-enrichment medium called Bartonella Alpha Proteobacteria Growth Medium (BAPGM). Galaxy Diagnostics holds an exclusive license on BAPGM. Preliminary data support the usefulness of the BAPGM microbiological approach in human medicine. This novel Bartonella testing platform has resulted in the detection of single and polymicrobial Bartonella infections in blood samples obtained from both chronically-ill and healthy humans and animals. Perhaps more importantly research using the BAPGM-PCR testing platform have revealed important clinical associations between Bartonella infection and serious chronic disease. Our expectation is that the development of this testing platform will lead to medical breakthroughs in the diagnosis and treatment of chronic disease and will provide extensive savings in healthcare expenditures through early detection and prevention of debilitating chronic conditions. Of particular importance is research regarding modes of transmission and the identification as a cause or contributing factor in specific chronic disorders, like arthritis, neurological disease, organ failure and other hard- to-diagnose chronic conditions. As Bartonella spp. are transmitted by a large number of arthropod vectors, including fleas, lice, sand flies, ticks, biting flies and potentially others, determining the appropriate bacteria to screen for may be complicated by regional variations in the transmission of various Bartonella species to the blood donor public. Incidence of different Bartonella species by regions due to the existence of chronic carriers and/or vectors should be addressed in the future in order to establish the actual occurrence of this genus (and each of its species) in the US. The overall goal of Phase I will be to develop a BAPGM Axenic Culture Bottle formulation to be used for immediate inoculation or for transport of microbiological specimens. Specimens will be then transported to Galaxy Diagnostics for Bartonella testing using our current culture plus molecular testing protocol; Aim 1 develop and validate a new formulation in the standard BAPGM platform culture medium to fit the requirements of the BAPGM Axenic Culture Bottle design proposed; Aim 2 will test the feasibility that reasonable storage conditions on the new formulations can be achieved. This will be accomplished by examining growth sensitivity testing as performed previously after storage at room temperature, 4-8oC and -20oC. Phase II will focus on the product development and regulatory milestones required to develop test kits including the BAPGM Axenic Culture Bottle which will allow worldwide sale and distribution. Phase Ii research would also address the potential changes in the BAPGM Axenic Culture Bottle head-space gas composition required to maximize bacteria recovery and growth and that might lead to an optimization of the time required for the enrichment culture step in the testing platform.