Rapid Point-of-Care Molecular Test for the Differentiation of Dengue and Chikungunya Viruses

 描述(申请人提供)：人类旅行、城市化和气候变化的增加导致登革热病毒(DENV)和基孔肯雅病毒(CHIKV)引起的发热疾病在地理上广泛传播。这两种感染在临床表现上有很大的重叠。此外，这两种疾病在许多国家也是共同流行的。这对临床医生构成了巨大的挑战，因为这两种疾病需要不同的临床管理。只有在血液样本中检测到病毒才能确认最近的感染，因为血清学检测的阳性结果可能表明以前感染过病毒。然而，随着抗体对感染的反应而产生，血液中的病毒载量会减少。因此，对这些病毒进行灵敏和特异的检测对于准确的诊断是必不可少的。通常，采用基于逆转录酶实时聚合酶链式反应(rtRT-PCR)检测病毒RNA的分子方法需要提取全血样本的核酸。在这个第一阶段的计划中，我们建议(1)开发一种用于直接检测血液样本中的DENV或CHIKV的多重分子检测方法，然后(2)将这种多重检测方法整合到收集-检测(C2T)系统中，以克服复杂样本处理的需要。C2T系统包括一种简单的血液采集和处理方法，不需要移液或样品处理，并结合装有干涸分析试剂的试剂盒，用于同时进行样品准备和模板放大。样品制备和装盒相当于目前使用的运行侧向流动装置的方法。该系统将允许通过RTRT-PCR从最少量的全血(即手指刺穿)中同时检测DENV和CHIKV，而不需要离心法或耗时的提取方法。关键是开发了一种基于rtRT-PCR的分析方法，该方法优化了直接从血液样本中检测病毒RNA，而不需要进一步提取样本。这些试剂与C2T系统一起，将与T-COR“8便携式热循环仪兼容。Tetracore在开发高灵敏度和特定的干燥、室温稳定的实时聚合酶链式反应分析方面拥有丰富的经验，包括之前与海军医学研究中心共同开发的DENV特异性分析。该方法在商业实验室的rtRT-PCR平台上使用临床样本进行了系统的评估和成功的测试。根据我们的初步数据和经验，我们有信心提出这一计划，开发一种用于DENV和CHIKV差异检测的多重rtRT-PCR检测方法，该方法可以与C2T试剂盒和T-COR 8便携式热循环仪结合使用，以创建一个在公共卫生实验室和低资源环境下同样有效的综合护理点系统。

 DESCRIPTION (provided by applicant): Increased human travel, urbanization and climate changes have resulted in a wide geographic spread of febrile illness caused by both Dengue Virus (DENV) and Chikungunya Virus (CHIKV). These two infections show a substantial overlap in clinical presentation. In addition, both are also co-prevalent in many countries. This poses a significant challenge to clinicians, as the two illnesses require different clinical management. A recent infection can only be confirmed by detection of the virus in a blood sample, as positive results in serological assays may instead be indicative of prior infection. However, as antibodies develop in response to the infection, the viral load in the blood decreases. Thus, sensitive and specific detection of these viruses is imperative for an accurate diagnosis. Typically, molecular methods that employ reverse transcriptase real-time polymerase chain reaction (rtRT-PCR) based detection of viral RNA require nucleic acid extraction of whole blood samples. In this Phase I plan, we propose to (1) develop a multiplexed molecular assay for the direct detection of either DENV or CHIKV in blood samples, and then to (2) incorporate this multiplexed assay into a Collect-to-Test (C2T) system in order to overcome the need for complex sample processing. The C2T system includes a simple blood collection and processing method that requires no pipetting or sample manipulation, combined with a cartridge containing the dried-down assay reagents for simultaneous sample preparation and template amplification. The sample preparation and cartridge loading are equivalent to methods currently used to run lateral flow devices. This system will allow for simultaneous detection of DENV and CHIKV by rtRT-PCR from a minimal volume of whole blood (i.e., a finger prick) without the need for centrifugation or time- consuming extraction methods. Critical to this is the development of a rtRT-PCR-based assay optimized for the detection of viral RNA directly from blood samples and requiring no further sample extraction. These reagents, together with the C2T system, will be compatible with the T- COR" 8 portable thermocycler. Tetracore has extensive experience in the development of highly sensitive and specific dried-down, room temperature-stable assays for real-time PCR, including a DENV-specific assay previously developed with Naval Medical Research Center. This assay was systematically evaluated and successfully tested using clinical samples on a commercial laboratory rtRT-PCR platform. Based on our preliminary data and experience we are confident to propose this plan to develop a multiplex rtRT-PCR assay for differential detection of DENV and CHIKV that can be combined for use with the C2T cartridge and the T-COR 8 portable thermocycler, to create an integrated point-of-care system that can be equally effectively in both public health labs and low resource settings.