Rapid, automated, detection of viral and bacterial pathogens causing meningitis

描述(由申请人提供)：脑膜炎和脑炎是中枢神经系统的炎症性过程，通常是传染性的，可导致严重的发病率和死亡率。及时、适当的治疗是至关重要的，但确定感染病原体可能是困难和耗时的。可能涉及广泛的病原体，包括细菌和病毒。细菌培养是诊断细菌性脑膜脑炎的标准，病毒核酸的聚合酶链式反应是诊断无菌病因的标准。然而，目前的方法要么是1)太慢而不能用于临床；2)技术复杂；3)靶标数量有限；4)FDA没有批准。爱达荷州技术公司(ITI)开发了一种基于聚合酶链式反应(PCR)的“袋装实验室”诊断系统，名为“FilmArray”，该系统快速、易用，可以同时检测大量感染性病原体。FDA批准了一种可检测15种呼吸道病原体的FilmArray邮袋，作为CLIA“中等复杂性”测试。我们提出将FilmArray技术应用于ME诊断问题。SA1：开发薄膜阵列脑膜炎(FAME)小组：我们将构建一个薄膜阵列脑炎(FAME)小组：我们将构建一个薄膜阵列小组，将现有小组的PCR分析与五种新的测试相结合，直接从脑脊液(CSF)中检测下列引起脑脊液脑炎的微生物：肠杆菌科、大肠杆菌、肠球菌、流感、单核细胞增多症、肺炎支原体、脑膜炎奈瑟氏菌、铜绿假单胞菌、葡萄球菌、金黄色葡萄球菌、链球菌、无乳链球菌、肺炎链球菌、化脓性葡萄球菌、病毒链球菌、腺病毒、肠道病毒、细小病毒、HSV1、HSV2、VZV、EBV、CMV、HV-6。SA2：优化脑脊液中病毒和细菌的核酸提取：脑脊液是FilmArray上处理过的较不复杂的样本基质之一(与鼻拭子、血液培养和粪便相比)。然而，直接从脑脊液中检测细菌和病毒可能需要尽可能高的灵敏度。我们将优化袋子内部的核酸纯化步骤，并研究在袋子SA3之前浓缩细菌和病毒的简单步骤：在300个脑脊液样本上测试FAME小组：我们的合作者将测试疑似脑脊液的儿科和成人患者的脑脊液样本。我们将把这些结果与他们对样品进行的标准分析进行比较，并补充SA1中描述的比较器分析结果。这一提议的一个成功结果将是一种能够检测和识别常见ME病原体的Film阵列袋。它将为向FDA提交510(K)计划所需的分析和临床试验做好准备。这将是今后提交的第二阶段报告的主题。

DESCRIPTION (provided by applicant): Meningitis and encephalitis are inflammatory, often infectious, processes of the central nervous system that can result in significant morbidity and mortality. Prompt, appropriate therapy is crucial, but determining the infectious etiology can be difficult and time-consuming. A wide-range of pathogens can be involved including both bacteria and viruses. Culture is the standard for diagnosis of bacterial Meningoencephalitis (ME), PCR of viral nucleic acid is the standard for "aseptic" causes. However the current methods are either 1) too slow to be clinically relevant; 2) technically complex; 3) limited in the number of targets r 4) not cleared by the FDA. Idaho Technology (ITI) has developed a "lab in a pouch" PCR-based diagnostic system termed "FilmArray" that is rapid, easy to use, and can test for large panels of infectious agents simultaneously. A FilmArray pouch that can detect 15 respiratory pathogens was cleared by the FDA as a CLIA "moderate complexity" test. We propose to apply the FilmArray technology to the problem of ME diagnosis. SA1: Develop a FilmArray Meningoencephalitis (FAME) panel: We will construct a FilmArray pouch that combines PCR assays from existing panels with five new assays to detect the following ME-causing organisms directly from cerebrospinal fluid (CSF): Enterobacteriaceae, E. coli, Enterococcus species, H. influenza, L. monocytogenes, Mycoplasma pneumoniae, N. meningitidis, P. aeruginosa, Staphylococci species, S. aureus, Streptococcus species, S. agalactiae, S. pneumoniae, S. pyogenes, viridians streptococci, Adenovirus, Enterovirus, Parechovirus, HSV1, HSV2, VZV, EBV, CMV, HHV-6. SA2: Optimize nucleic acid extraction for virus and bacteria from CSF: CSF is one of the less complex sample matrices that have been processed on the FilmArray (when compared to nasal swabs, blood culture and stool). However, detecting bacteria, and virus, directly from CSF, may require the highest possible sensitivity. We will optimize the nucleic acid purification steps internal to the pouch as well as investigate simple steps to concentrate bacteria and viruses before the pouch SA3: Test the FAME panel on 300 CSF samples: Our collaborators will test CSF samples from pediatric and adult patients with suspected ME. We will compare these results to their standard assays performed on the samples, supplemented with the results of the comparator assays described in SA1. A successful outcome of this proposal will be a FilmArray pouch capable of detecting and identifying the common ME pathogens. It will be ready for the analytical and clinical trials necessary for a 510(k) submission to the FDA. This would be the subject of a future phase II submission.