A Rapid Point-of-care Diagnostic for Neisseria gonorrhoeae STDs

描述（由申请方提供）：淋病奈瑟菌（GC）感染的诊断改善代表了一项关键的未满足医疗需求。CDC估计，每年约有70万人感染GC，其中约有一半报告了这些病例。GC感染的临床谱包括尿道炎和宫颈炎、盆腔炎和播散性疾病。感染还可导致不孕、宫外孕和低出生体重。大约10%的男性感染者和50%的女性感染者没有症状，加速了疾病的传播。耐药GC菌株是一个长期存在的主要问题;特别令人担忧的是最近报道的菌株对头孢曲松的敏感性降低或耐药。降低GC发病率的一个关键障碍是缺乏廉价的、基于核酸（NA）的床旁（POC）诊断筛查。 尽管在美国有四种市售的GC测定法，但它们受到若干限制。关键是，它们不能在POC中执行。商业试验进展缓慢，需要在设备和技术专长方面进行大量投资。此外，目前的测试容易受到目标基因组突变的影响，这将有效地使病原体不可见（如最近看到的一种主要的欧洲沙眼衣原体菌株），无法进行菌株分型（使得可靠的流行病学研究和接触者追踪成为可能），并且无法确定耐药性。 NetBio正在提议开发一种廉价的基于微流体的GC诊断方法，该方法将在POC实时对GC进行灵敏和特异性检测。开发这种有效的POC诊断将允许及时、适当的治疗，这将减少与这些感染直接相关的急性和慢性发病率。该I期申请的具体目的是：1）对40种GC菌株进行全基因组测序，使关于GC基因组结构的现有公共知识增加两倍以上; 2）基于该遗传信息设计多重扩增测定，其允许总共12个GC基因座（包括MLST和抗生素抗性基因座）同时扩增;和3）针对两种领先的商业GC诊断进行我们的诊断的头对头测试。在SBIR第二阶段，我们将把多重扩增检测纳入一个完全集成的系统，在45分钟内进行DNA纯化，扩增和电泳分离。基于这项工作的第一个商业产品将根据12个基因座的扩增诊断GC，后续产品也将对这些基因座进行测序。 鉴于Tim Read博士的基因组和测序专业知识，Dean和Shafer博士的STD和菌株分型专业知识，以及Selden博士的微流体和分子生物学专业知识，该应用程序提供了一个独特的合作机会，最终获得GC的快速即时诊断。

DESCRIPTION (provided by applicant): Improved diagnosis of Neisseria gonorrhoeae (GC) infections represents a critical unmet medical need. The CDC estimates that approximately 700,000 people are infected by GC annually, with approximately half of these cases reported. The clinical spectrum of GC infection includes urethritis and cervicitis, pelvic inflammatory disease (PID), and disseminated disease. Infection can also lead to sterility, ectopic pregnancy, and low birth weight. Approximately 10% of infected males and 50% of infected females are asymptomatic, hastening the spread of the disease. Drug resistant GC strains are a major, longstanding problem; particularly worrisome are recent reports of strains expressing decreased susceptibility or resistance to ceftriaxone. A critical obstacle to reducing the incidence of GC is the lack of an inexpensive, nucleic acid (NA)-based point-of-care (POC) diagnostic for screening. Though there are four commercially available GC assays in the US, they suffer from several limitations. Critically, they cannot be performed at the POC. Commercial tests are slow, and require extensive investment in equipment and technical expertise. Furthermore, current tests are vulnerable to mutations in the target genome that would effectively render the pathogens invisible (as was recently seen a major European Chlamydia trachomatis strain), incapable of strain typing (making reliable epidemiology studies and contact tracing impossible), and incapable of determining drug resistance profiles. NetBio is proposing to develop an inexpensive microfluidics-based diagnostic for GC that will provide sensitive and specific detection of GC to be made at the POC in real-time. Development of such an effective POC diagnostic would allow timely, appropriate treatment that would reduce the acute and chronic morbidity that is directly associated with these infections. The Specific Aims of this Phase I application are to: 1) Perform whole genome sequencing on 40 GC strains, more than tripling the available public knowledge about GC genomic structure; 2) Design a multiplexed amplification assay based on this genetic information that allows a total of 12 GC loci (including MLST and antibiotic resistance loci) to be amplified simultaneously; and 3) Perform a head-to-head test of our diagnostic against two of the leading commercial GC diagnostics. In the SBIR Phase II, we will incorporate the multiplexed amplification assay into a fully integrated system that performs DNA purification, amplification, and electrophoretic separation within 45 minutes. The first commercial product based on this work will diagnose GC based on the amplification of 12 loci, and a follow-on product will also sequence these loci. Given the genomic and sequencing expertise of Dr. Tim Read, the STD and strain-typing expertise of Drs. Dean and Shafer, and the microfluidic and molecular biology expertise of Dr. Selden, the application provides a unique collaborative opportunity to finally obtain a rapid point-of-care diagnostic for GC.