Low-Cost Instrument-free Point-of-Care Diagnostic for Neisseria gonorrhoeae

摘要 淋病奈瑟菌(GC)是细菌性传播疾病的第二大常见原因。 (STD)，估计世界流行率为7800万。每年约有820,000例病例发生在 美国，但超过一半的人没有报告，利率继续上升，从2010年到2010年的10.5%的增长表明 2014年。GC是导致盆腔炎的主要原因，可导致输卵管因素不孕、异位 怀孕和慢性盆腔疼痛。GC还有助于HIV-1感染的传播。当我们依赖于 抗生素治疗GC感染，经验性疗法加剧了本已成问题的GC耐药问题 在美国和世界上。阻止GC感染传播的主要障碍是缺乏接入点 关怀(POC)诊断，可提供监控和早期检测，降低感染率和 后遗症。目前的GC诊断依赖于商业核酸扩增测试(NAAT)，这些测试 价格昂贵，需要设备和训练有素的操作员，需要一天到几天的时间才能取得效果，并可能导致损失 对患者进行随访。因此，当前的NAAT不适用于PoC。我们的团队由迪恩博士组成 以及性传播疾病和POC开发专家Gaydos，以及拥有专有技术的初创公司Diessate POC诊断技术，有初步数据表明，我们：1)可以快速提取GC核 宫颈拭子中的酸在没有仪器或在原型样品制备中受过训练的操作员的情况下 模块；2)具有无需仪器的多路检测模块，用于GC核的比色检测 酸；3)检测10种微生物/化验；4)已验证的检测GC菌株和 检测与其他性病病原体没有交叉反应的人类RNA/DNA的检测；以及5)表明 我们的系统可以可靠地检测临床宫颈拭子中的GC，这些拭子是商业NAAT已知的阳性。 我们将扩大我们的初步研究，目标如下。目标1：使用不断扩大的 参考和临床GC基因组序列，我们将改进我们的检测，根据需要更换失败的检测， 并确保我们的检测方法检测到不同的GC临床菌株，而不与STD病原体或 常见的阴道/宫颈物种；目标2：优化核酸提取的样品制备化学 从尿路、阴道和宫颈拭子，这些拭子类型的检测设计和比色化学， 没有干扰物质的假阴性或阳性结果；目标3：我们将评估灵敏度 和特异性，以及我们完全集成的系统(结合在一起)的正面和负面预测价值 样品制备模块和检测模块)与商用NAAT进行比较。到阶段结束时 我们将准备好制造和使用我们的快速(&lt；35分钟)、廉价、用户友好、无仪器、 在美国进行临床试验的灵敏和特异的GC POC测试，以获得FDA的监管批准。我们的整体 我们的目标是部署我们的GC POC诊断程序，用于医生办公室、城市和农村的大小诊所、青少年、 家庭和性病诊所、急诊室和医院诊所、其他检测地点以及周围资源有限的环境 整个世界。

Abstract Neisseria gonorrhoeae (GC) is the second most common cause of bacterial sexually transmitted diseases (STD) with an estimated world prevalence of 78 million. Approximately 820,000 cases occur annually in the U.S. but over half are unreported, and rates continue to rise as indicated by a 10.5% increase from 2010 to 2014. GC is a major cause of pelvic inflammatory disease (PID) that can lead to tubal factor infertility, ectopic pregnancy and chronic pelvic pain. GC also facilitates the transmission of HIV-1 infection. While we rely on antibiotics to treat GC infections, empiric therapy has fueled the already problematic issue of drug-resistant GC in the U.S. and the world. The main obstacle to stemming the spread of GC infections is the lack of a point-of- care (POC) diagnostic that could provide surveillance and early detection, reducing infection rates and sequelae. Current GC diagnostics rely on commercial nucleic acid amplification tests (NAATs) that are expensive, require equipment and highly trained operators, take a day to days for results, and can result in loss to follow up of patients. Thus, current NAATs are not suitable at the POC. Our team comprised of Drs. Dean and Gaydos, experts on STDs and POC development, and Diassess, a startup company with proprietary technology for POC diagnostics, have preliminary data showing that we: 1) can rapidly extract GC nucleic acids from endocervical swabs with no instruments or trained operator in a prototype Sample Preparation Module; 2) have an instrument-free multiplexed Detection Module for colorimetric detection of GC nucleic acids; 3) have a limit of detection of 10 organisms/assay; 4) have validated assays to detect GC strains and assays to detect human RNA/DNA with no cross reactivity with other STD pathogens; and 5) have shown that our system can detect GC reliably in clinical endocervical swabs that are know positive by a commercial NAAT. We will expand on our preliminary studies with the following aims. Aim 1: Using the expanding aggregate of reference and clinical GC genome sequences, we will refine our assays, replacing failed assays as needed, and ensure that our assays detect diverse GC clinical strains without cross-reactivity with STD pathogens or common vaginal/cervical species; Aim 2: Optimize sample preparation chemistry for nucleic acid extraction from urethral, vaginal and endocervical swabs, assay design and colorimetric chemistry for these swab types, without false negative or positive results from interfering substances; Aim 3: We will evaluate the sensitivity and specificity, and positive and negative predictive values of our fully-integrated system (the combined Sample Preparation Module with Detection Module) compared to commercial NAATs. By the end of Phase II, we will be poised to manufacture and use our rapid (<35 min), inexpensive, user-friendly, instrument-free, sensitive and specific GC POC test for clinical trials in the U.S. to obtain FDA regulatory clearance. Our overall goal is to deploy our GC POC diagnostic for use in doctor’s offices, small to large city and rural clinics, teen, family and STD clinics, ERs and hospital clinics, other testing sites, and resource-constrained settings around the world.