Discriminating viral and bacterial meningitis infections with iDDS probes

用iDDS探针鉴别病毒性和细菌性脑膜炎感染机密PI：Shafer，大卫A.，博士 项目摘要 快速准确的分子诊断分析使医生能够做出明智的治疗决定， 特别是当疾病状态可能是由未经这种测试而无法确定的可变原因引起时。 脑膜炎可能是由微生物感染引起的，如病毒、革兰氏阴性（GN）和革兰氏阴性（GN）。 阳性（GP）细菌、真菌或寄生虫。最常见的是，脑膜炎是由非脊髓灰质炎病毒感染引起的。 肠道病毒（EV）和双埃柯病毒（PV），其通常在10天内自行消退。相反，细菌 脑膜炎的死亡率很高，如果不治疗，死亡率接近100%。患者表现相似 因此，及时诊断病原体对患者护理至关重要。 在确诊之前，患者通常采用“经验性”抗生素治疗方案。 核酸扩增检测（NAAT）可将诊断周转时间缩短数天， 标准的培养方法，允许缩短住院时间，适当使用抗生素， 对患者和医疗保健系统造成经济负担。目前，只有两种FDA批准的NAAT用于 诊断病毒性和/或细菌性脑膜炎。不幸的是，这些测试只被批准用于 制造商的全自动系统，其成本往往是令人望而却步。此外，这些测定法检测 高度可变的病毒基因组。总的来说，这些限制突出了对 不依赖仪器的检测，可区分病毒性和细菌性脑膜炎，并进行错误检查 在病毒基因组中的冗余位点的特性和确证性检测。 GeneTAG技术公司开发了错误检查DNA检测开关（iDDS）探针， 使用荧光标记的探针和稍微错配的淬灭剂标记的反探针。在没有 预期的目标，反探针与探针杂交，淬灭它们的荧光并防止关闭， 目标探测最近，我们开发了检测EV和PV的方法，每种方法使用两种iDDS探针。 目标，提供同时2色信号和自动确认阳性或阴性测试结果。 我们之前开发了用于GN和GP检测的iDDS探针。最终，我们有兴趣开发一种 FDA批准的基于iDDS探针的脑膜炎检测试剂盒，采用Sample-ReadyTM冻干板形式， 我们的合作者BioGX Inc.开发了平行的类似测试，其中所有试剂、引物和探针均 包括在内，并且测试将仅需要添加样品和分子级水。目标1研究将侧重于 优化双iDDS探针EV和PV测定，以在与商业EV的多重形式中表现良好， PV菌株和患者脑脊液，将多重检测转换为Sample-ReadyTM格式， 使用原始样品测试Sample-ReadyTM检测试剂盒。目标2将采用类似的战略， 准备Sample-ReadyTM GN/GP细菌测定。提供高保真、经济高效的脑膜炎检测 应该对脑膜炎诊断产生积极影响，并为公共卫生提供直接利益。

Discriminating viral and bacterial meningitis infections with iDDS probes Confidential PI: Shafer, David A., PhD PROJECT SUMMARY Rapid and accurate molecular diagnostics assays empower physicians to make informed treatment decisions, especially when a disease state may result from variable causes that are indeterminable without such testing. Meningitis may be caused by infection with microorganisms such as viruses, gram-negative (GN) and gram- positive (GP) bacteria, fungi, or parasites. Most frequently, meningitis results from infection with non-polio enteroviruses (EV) and parechoviruses (PV), which typically self-resolve within 10 days. In contrast, bacterial meningitis has a high mortality rate that approaches 100% if not treated. Patients present with similar symptoms regardless of origin; thus, timely diagnosis of the causative agent is paramount for patient care. Patients are commonly treated with an `empiric' regimen of antibiotics until a definitive diagnosis can be made. Nucleic acid amplification tests (NAATs) reduce diagnosis turnaround times by up to several days compared to standard culturing methods, allowing shortened hospital stays, appropriate use of antibiotics, and a reduced financial burden to patients and the health care system. Currently, there are only two FDA-approved NAATs for diagnosing viral and/or bacterial meningitis. Unfortunately, these tests are only approved for use on the manufacturer's fully automated systems, the cost of which is often prohibitive. Further, these assays detect the highly variable viral genomes at a single target site. Collectively, these limitations highlight the need for an instrument-independent assay that can discriminate between viral and bacterial meningitis, with error-checking properties and confirmatory detection at redundant sites in the viral genomes. GeneTAG Technology, Inc. has developed error-checking DNA Detection Switch (iDDS) probes, which employ a fluorescent labeled probe and a slightly mismatched quencher-labeled antiprobe. In the absence of the intended target, the antiprobes hybridize to the probes, quenching their fluorescence and preventing off- target detection. Recently, we developed assays for detecting EV and PV employing two iDDS probes for each target, providing simultaneous 2-color signaling and automatic confirmation of positive or negative test results. We previously developed iDDS probes for GN and GP detection. Ultimately, we are interested developing an FDA-approved iDDS probe-based meningitis assay in a Sample-ReadyTM, lyophilized plate format that will parallel similar tests developed by our collaborator, BioGX Inc., where all reagents, primers, and probes are included, and the test would only require adding sample and molecular grade water. Aim 1 studies will focus on optimizing the dual-iDDS probe EV and PV assays to perform well in multiplex format with commercial EV and PV strains and patient cerebrospinal fluids, converting the multiplex test to Sample-ReadyTM format, and re- testing the Sample-ReadyTM assay with the original samples. A similar strategy will be employed in Aim 2 to prepare a Sample-ReadyTM GN/GP bacteria assay. Delivering a high fidelity, cost-effective meningitis assay should positively impact meningitis diagnostics and provide a direct benefit to public health.