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

描述(由申请人提供)：淋球菌(GC)感染的改进诊断代表着一个严重的未得到满足的医疗需求。疾控中心估计，每年约有70万人感染GC，其中约一半报告了这些病例。GC感染的临床范围包括尿道炎和宫颈炎症、盆腔炎(PID)和播散性疾病。感染还可能导致不孕症、宫外孕和低出生体重。大约10%的受感染男性和50%的受感染女性没有症状，这加速了疾病的传播。耐药的GC菌株是一个主要的、长期存在的问题；尤其令人担忧的是最近有报道称，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POC诊断用于筛查。虽然在美国有四种商业上可用的GC分析方法，但它们受到几个限制。关键的是，它们不能在PoC执行。商业测试速度很慢，需要在设备和技术专业知识上进行大量投资。此外，目前的检测容易受到目标基因组突变的影响，这些突变将有效地使病原体看不见(最近看到的是欧洲主要的沙眼衣原体菌株)，无法进行菌株分型(使可靠的流行病学研究和接触者追踪变得不可能)，并且无法确定耐药性曲线。NetBio正在提议开发一种廉价的基于微流控技术的GC诊断方法，该诊断方法将在POC实时提供灵敏和特异的GC检测。开发这种有效的POC诊断将使及时、适当的治疗成为可能，从而减少与这些感染直接相关的急性和慢性发病率。这一第一阶段应用的具体目标是：1)对40株GC菌株进行全基因组测序，使现有的关于GC基因组结构的公共知识增加两倍以上；2)基于这些遗传信息设计一种多重扩增分析，允许同时扩增总共12个GC基因座(包括MLST和抗生素耐药基因座)；以及3)针对两种领先的商业GC诊断方法进行面对面的诊断测试。在SBIR第二阶段，我们将把多重扩增分析整合到一个完全集成的系统中，在45分钟内完成DNA的纯化、扩增和电泳分离。基于这项工作的第一个商业产品将基于12个基因座的扩增来诊断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博士和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，最终为GC获得快速的医疗诊断。 公共卫生相关性：淋病奈瑟菌(GC)每年感染美国约70万人。其中许多感染是无症状的，没有被发现，这会导致传播增加，并导致盆腔炎和不孕不育的后遗症。目前的GC基于核酸的诊断不能在护理点(POC)执行，需要广泛的技术专业知识、昂贵的设备和数天才能产生结果。NetBio正在开发一种易于使用、成本效益高的基于核酸的诊断方法，它可以在POC的一小时内检测到GC。

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. PUBLIC HEALTH RELEVANCE: Neisseria gonorrhoeae (GC) infects approximately 700,000 people in the US annually. Many of these infections are asymptomatic and go undetected, which leads to increased transmission and the sequelae of pelvic inflammatory disease and infertility. Current GC nucleic acid-based diagnostics cannot be performed at the point-of-care (POC), requiring extensive technical expertise, expensive equipment, and days to generate results. NetBio is developing an easy to use, cost effective nucleic acid-based diagnostic that detects GC within one hour at the POC.