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，通常是传染性的过程，可导致显著的发病率和死亡率。及时，适当的治疗是至关重要的，但确定感染病因可能是困难和耗时的。包括细菌和病毒在内的各种各样的病原体都可能参与其中。培养是细菌性脑膜脑炎（ME）的诊断标准，病毒核酸PCR是“无菌”病因的诊断标准。然而，目前的方法要么1)太慢，不具有临床相关性；2)技术复杂；3)目标数量有限；4)没有得到FDA的批准。爱达荷技术公司（ITI）开发了一种名为“FilmArray”的基于pcr的诊断系统，该系统快速、易于使用，可以同时检测大量感染因子。一种可以检测15种呼吸道病原体的FilmArray眼袋被FDA批准为CLIA“中等复杂性”测试。我们建议将FilmArray技术应用于ME诊断问题。SA1：开发FilmArray脑膜脑炎（FAME）检测组：我们将构建一个FilmArray检测袋，将现有检测组的PCR检测与五种新检测相结合，直接从脑脊液（CSF）中检测以下引起me的生物体：肠杆菌科、大肠杆菌、肠球菌、流感嗜血杆菌、单核增生乳杆菌、肺炎支原体、脑膜炎奈瑟菌、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、化脓性链球菌、病毒链球菌、腺病毒、肠病毒、Parechovirus、HSV1、HSV2、VZV、EBV、CMV、HHV-6。SA2：优化脑脊液中病毒和细菌的核酸提取：脑脊液是在FilmArray上处理过的较不复杂的样品基质之一（与鼻拭子、血培养和粪便相比）。然而，直接从脑脊液中检测细菌和病毒可能需要最高的灵敏度。我们将优化囊袋内部的核酸纯化步骤，并研究在囊袋SA3之前浓缩细菌和病毒的简单步骤：对300份脑脊液样本进行FAME测试：我们的合作者将对疑似ME的儿童和成人患者的脑脊液样本进行测试。我们将这些结果与他们在样品上进行的标准分析进行比较，并辅以SA1中描述的比较分析结果。该提案的成功结果将是能够检测和识别常见ME病原体的FilmArray袋。它将为向FDA提交510(k)所需的分析和临床试验做好准备。这将是未来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.