Low-Cost Instrument-free Point-of-care Platform for Multiplexed Chlamydia Diagnostics

摘要 沙眼衣原体(CT)是细菌性传播疾病(STD)的最常见原因，与 2014年，全球每年约有1.08亿例病例，美国约有144万例。大多数女性和男性感染 无症状，便于无节制的传播，可导致盆腔炎，不孕不育， 慢性盆腔疼痛和危及生命的宫外孕。CT也是侵袭性鳞状细胞癌的危险因素 宫颈癌是HIV-1感染的一个复杂因素。病人通常在没有接受治疗的情况下接受治疗 明确的诊断，这可能导致不适当的抗生素使用，并可能产生耐药性。主 阻止CT感染的障碍是缺乏护理点(POC)诊断，以增加早期发现 减少感染率和后遗症。目前的CT诊断依赖于商业化的核酸扩增试验 (NAAT)灵敏度和特异度各不相同，同一样本普遍缺乏一致的结果 打字。NAAT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结果，以及 可能导致失访和延误治疗。因此，当前的NAAT不适合PoC诊断。 我们的团队包括德博拉·迪恩博士，一位CT性病和POC发展方面的专家，以及一家初创公司DiVisate 这家拥有POC诊断专有技术的公司在第一阶段展示了我们：1)可以提取CT核 在原型样品制备模块中不使用仪器，5分钟内从宫颈拭子中提取酸液；2) 拥有免仪器的多路检测模块，可对Ct核酸进行30分钟的比色检测；3) 已经验证了检测所有CT参考菌株的方法，区分了淋巴肉芽肿(LGV)和 以及4)检测结果与标准相符。 200例残留宫颈标本的NAAT检测结果。在第二阶段，我们将推进第一阶段的成果。 目的1：利用不断扩大的参考和临床CT基因组序列集合，提纯我们的引物， 根据需要更换不合格的引物，并确保我们精制的Ct引物扩增分析检测到Ct 参考和多样化的CT临床菌株，与性传播病原体和 常见的阴道/宫颈物种；目标2：优化样品制备化学，扩增分析设计 阴道、尿路和宫颈拭子以及干扰物质的比色化学；目标3： 评估我们完全集成的系统(组合样品准备模块)的灵敏度和特异度 带有检测模块)与商业NAAT进行比较。到第二阶段结束时，我们将做好准备 制造和使用我们的快速(35分钟)、廉价、用户友好、无需仪器、灵敏和特定的CT 在美国进行临床试验的POC测试，以通过510(K)途径获得FDA的监管批准。我们的整体 目标是将我们的CT POC诊断部署在医生办公室、城市和农村的小型诊所和大型诊所、青少年和 世界各地的性病诊所、急诊室、其他检测地点和资源有限的环境。

Abstract Chlamydia trachomatis (Ct) is the most common cause of bacterial sexually transmitted diseases (STD) with ~108 million annual cases worldwide and ~1.44 million U.S. cases in 2014. Most female and male infections are asymptomatic, facilitating unchecked transmission that can result in pelvic inflammatory disease, infertility, chronic pelvic pain, and life-threatening ectopic pregnancy. Ct is also a risk factor for invasive squamous-cell carcinoma of the cervix and a complicating factor in HIV-1 infection. Patients are often treated without a definitive diagnosis, which can lead to inappropriate antibiotic use and possibly drug resistance. The main obstacle to stemming Ct infections is the lack of a point-of-care (POC) diagnostic to increase early detection to reduce infection rates and sequelae. Current Ct diagnostics rely on commercial nucleic acid amplification tests (NAATs) that vary in sensitivity and specificity with a general lack of concordant results for the same sample type. NAATs are expensive, require equipment and highly trained operators, take a day to days for results, and can result in loss to follow up and delay in treatment. Thus, current NAATs are not suitable POC diagnostics. Our team of Dr. Deborah Dean, an expert on Ct STDs and POC development, and Diassess, a startup company with proprietary technology for POC diagnostics, showed in Phase I that we: 1) can extract Ct nucleic acids from endocervical swabs in 5 min with no instruments in a prototype Sample Preparation Module; 2) have an instrument-free multiplexed Detection Module for 30 min colorimetric detection of Ct nucleic acids; 3) have validated assays to detect all Ct reference strains, differentiate lymphogranuloma venereum (LGV) from non-LGV strains and detect human DNA; and 4) have demonstrated assay results consistent with standard NAAT results on 200 remnant endocervical patient samples. In Phase II, we will advance on Phase I results. Aim 1: Using the expanding aggregate of reference and clinical Ct genome sequences, refine our primers, replacing failed primers as needed and ensure that our refined Ct primer amplification assays detect Ct reference and diverse Ct clinical strains without cross-reactivity with sexually transmitted pathogens and common vaginal/cervical species; Aim 2: Optimize sample preparation chemistry, amplification assay design and colorimetric chemistry for vaginal, urethral and endocervical swabs, and interfering substances; Aim 3: Evaluate sensitivity and specificity 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 Ct POC test for clinical trials in the U.S. to obtain FDA regulatory clearance via the 510(k) pathway. Our overall goal is to deploy our Ct POC diagnostic for use in doctor's offices, small to large city and rural clinics, teen and STD clinics, emergency rooms, other testing sites and resource-constrained settings around the world.