Optimized Growth Media to Detect Intracellular Bacteria Implicated in Chronic Dis

描述（由申请人提供）：CDC报告称，超过4000万美国人患有慢性疾病，其中大多数人报告患有多种慢性疾病。许多这些慢性疾病的病因尚不清楚，医生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许多慢性疾病的可能性，特别是那些涉及自身免疫反应的疾病，可能是由高度挑剔的免疫逃避病原体引起的。巴尔通体属细菌（立克次体目的一员）在医学病例系列文献中与人类和兽医学中的广泛疾病有关，包括多发性关节炎、皮肤血管炎、心内膜炎、心肌炎、鼻出血、肝盆腔炎、肉芽肿性炎症性疾病，以及最近的神经功能障碍和关节炎。在病人样本中检测细菌感染的传统方法已被证明在很大程度上对准确诊断巴尔通体病无效。北卡罗莱纳州立大学兽医学院的研究人员开发了一种高度敏感的巴尔通体检测平台，该平台结合了在新型液体培养基中进行血液预富集培养，然后使用属级引物和阳性结果序列id进行巴尔通体PCR检测。这种创新的巴尔通体检测平台的开发是为了促进在免疫能力强的患者中检测巴尔通体感染，这种高度挑剔的细菌感染水平如此之低，以至于测试点的采样误差导致直接PCR的假阴性率极高，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。这个分子诊断平台的基石是一种被称为巴尔通体α变形菌生长培养基（BAPGM）的专利预富集培养基。Galaxy Diagnostics拥有BAPGM的独家授权。初步数据支持BAPGM微生物学方法在人类医学中的有效性。这种新型的巴尔通体检测平台已经在慢性疾病和健康人类和动物的血液样本中检测到单一和多微生物巴尔通体感染。也许更重要的是，利用BAPGM-PCR检测平台的研究揭示了巴尔通体感染与严重慢性疾病之间的重要临床关联。我们的期望是，该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，并通过早期发现和预防使人衰弱的慢性病，大幅节省医疗支出。特别重要的是研究传播方式，以及确定特定慢性疾病（如关节炎、神经系统疾病、器官衰竭和其他难以诊断的慢性疾病）的病因或促成因素。由于巴尔通体可通过大量节肢动物媒介传播，包括跳蚤、虱子、沙蝇、蜱、叮蝇和其他可能的媒介，因此，由于各种巴尔通体向献血者传播的区域差异，确定适当的细菌进行筛查可能会变得复杂。由于慢性携带者和/或媒介的存在，不同地区巴尔通体种类的发病率应在未来得到解决，以便确定该属（及其每个物种）在美国的实际发病率。第一阶段的总体目标将是开发一种BAPGM无菌培养瓶配方，用于立即接种或微生物标本的运输。然后将标本运送到银河诊断公司，使用我们目前的培养加分子检测方案进行巴尔通体检测；目标1：开发并验证标准BAPGM平台培养基中的新配方，以满足BAPGM无菌培养瓶设计的要求；目标2将测试新配方能否实现合理的储存条件的可行性。这将通过检查之前在室温、4-8℃和-20℃储存后进行的生长敏感性测试来完成。第二阶段将专注于产品开发和监管里程碑，以开发包括BAPGM无菌培养瓶在内的测试试剂盒，该试剂盒将允许在全球销售和分销。Ii期研究还将解决BAPGM无菌培养瓶顶空气体组成的潜在变化，以最大限度地提高细菌的回收率和生长，这可能会导致测试平台中富集培养步骤所需时间的优化。

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.