Rapid Low Cost Molecular Detection of Influenza Virus Infections

描述(申请人提供)：在发展中国家，急性呼吸道感染(ARI)每年导致约190万儿童死亡，其中20%在印度。幼童的流感与高危成人一样严重，而且经常没有被发现。在亚热带地区，流感是儿童住院的一个重要原因，其发病率超过了温带地区报告的发病率。在印度因急性呼吸道感染住院的儿童中，有15%发现了流感。在发展中国家了解病毒感染面临的挑战之一是，对诊断工具的要求超过了现有的实验室能力。这方面的一个例子是分子诊断学的应用，例如逆转录聚合酶链式反应(RTPCR)。虽然这些检测方法为病毒检测提供了优势，但它们的使用受到成本的限制。反转录环介导的等温扩增(LAMP)是一种新的、特异、高效、快速、低成本的RNA扩增技术。我们建议开发LAMP检测方法，用于印度流感病毒的研究。这将需要对人类和禽流感病毒进行检测，并确定其类型和亚型。在AIM中，将利用人类流感病毒和体外转录的人和禽流感目标基因的RNA建立LAMP检测。视觉检测系统和机器检测系统将进行比较。还将开发一种流感的LAMP定量检测方法。在目标2中，LAMP检测将与培养和RT-PCR检测流感病毒进行比较。将使用的样本来自德里独一无二的临床样本收集，包括预期从儿童病毒性ARI研究和全国流感病毒监测网络收集的样本。定量LAMP将被用来检验病毒载量与儿童疾病严重程度相关的假设。这项申请建议开发新的研究技术来解决流感的流行病学和发病机制。此外，它还涉及一个高度优先的流感研究领域，即开发敏感、特异和快速的流感临床诊断测试。虽然这项提案的重点是印度的流感，但其结果应该广泛适用，并有可能在全球范围内使用。叙述拟议的研究与公共卫生有关，因为流感病毒感染是发达国家和发展中国家儿童和成人患病的重要原因。改进的病毒检测方法可以改进流感诊断，并支持抗病毒药物和流感疫苗的研究。

DESCRIPTION (provided by applicant): Acute respiratory infections (ARI) result in ~ 1.9 million childhood deaths per year in developing countries, 20% of the deaths are in India. Influenza in young children is as severe as in high-risk adults and frequently goes unrecognized. In the subtropics, influenza is an important cause of hospitalization among children, with rates exceeding those reported for temperate regions. Influenza was found in 15% of the children hospitalized with ARI in India. Among the challenges to the understanding of viral infections in the developing world has been the requirement for diagnostic tools that exceed the available laboratory capacity. An example of this lies in the application of molecular diagnostics such as reverse transcription polymerase chain reaction (RTPCR). While these assays offer advantages for virus detection their use has been limited by cost. Reverse-transcription loop mediated isothermal amplification (LAMP) is a novel technique that can amplify RNA with high specificity, efficiency, rapidity and at a low cost. We propose to develop LAMP assays for use in the study of influenza virus in India. This will entail human and avian influenza virus detection and characterization as to type and subtype. In Aim 1 LAMP assays will be established using human influenza virus and in vitro transcribed RNAs from human and avian influenza target genes. Visual and machine detection systems will be compared. A quantitative LAMP assay for influenza will also be developed. In Aim 2 the LAMP assays will compared to culture and RTPCR for the detection of influenza virus. The samples to be used are from a unique collection of clinical samples available in Delhi, including prospectively collected samples from studies of viral ARI in children and a national influenza virus surveillance network. Quantitative LAMP will be used to test the hypothesis that viral loads correlate with disease severity in children. This application proposes to develop new research technology to address influenza epidemiology and pathogenesis. In addition, it addresses a high-priority influenza research area, the development of sensitive, specific, and rapid clinical diagnostic tests for influenza. Although the emphasis of this proposal is on influenza in India, the results should be broadly applicable, with the potential global use. NARRATIVE The proposed studies are relevant to public health because influenza virus infections are an important cause of disease among children and adults in both developed and developing countries. Improved virus detection methods can improve influenza diagnosis and support studies of antivirals and vaccines against influenza.