Technology development for point-of-care detection and antimicrobial susceptibility testing of Neisseria gonorrhoeae

项目摘要 淋病奈瑟菌（NG）中的抗菌素耐药性（AMR）是AMR 1威胁的最高级别， WHO.在过去的几十年里，NG已经对以前推荐用于治疗的所有抗菌药物产生了耐药性。 治疗淋病，留下头孢曲松加阿奇霉素的双重治疗作为目前唯一合适的 在全球大多数国家都是经验性一线治疗的选择。现在已经报道了NG菌株 对头孢曲松和阿奇霉素都有抗药性相反，尽管不再推荐使用环丙沙星 最近的研究表明，很大比例的GC感染可能是由CDC治疗NG， 可能用环丙沙星治疗随着AMR的不断发展，迫切需要 针对个体感染的个性化治疗方法，与目前的“全球性”相比， 统一的“经验方法”。然而，这就要求临床医生能够迅速了解耐药性或敏感性 足以决定处方。 为了减少NG中AMR的出现和传播，CDC定期发布STD治疗指南， 协助临床医生。这些指南是由国家疾病预防控制中心产生的敏感性数据提供信息的 淋球菌分离株监测项目。然而，GISP的影响正受到技术威胁， 进化确定AMR需要在复杂的环境中长时间（24-48小时）的微生物培养， 实验室设施。但是，随着核酸扩增检测（NAAT）的出现， 准确性已经取代了基于培养的NG感染诊断，导致有限的标本采集， 培养和丧失在大多数检测诊所进行NG培养的能力。因此， 广泛的NAAT无意中在AMR测试中造成了严重的空白。 我们建议开发一个完整的诊断解决方案，能够进行NG感染的识别（ID） 和表型抗菌药物敏感性测试（AST）。具体地，使用PCR检测NG-1来实现ID。 特异性DNA标记;而AST使用定量PCR进行，以测量核酸标记的差异。 与病原体的生理状态相关的酸性标记物（细菌DNA或RNA）在药物- 处理的样品和无药物对照。我们的联合ID-AST平台，利用创新的 NAAT和微流体技术的进步，有可能提供所有必要的NG诊断信息 针对POC的每一位疑似患者，量身定制个性化治疗;其实用设计也很好- 适合解决GISP在常规监测NG AMR方面面临的技术挑战。我们提出 以下目标：1）开发用于NG的集成ID和AST的简化诊断方案; 2） 开发实现集成ID-AST测定的液滴微流体盒; 3）系统集成， 仪器开发;和4）集成系统的分析和临床验证。以促进 技术翻译，提出了未来临床部署的产品开发计划。

PROJECT SUMMARY Antimicrobial resistance (AMR) in Neisseria gonorrhoeae (NG) is in the top tier of AMR l threats as defined by WHO. Over the past decades, NG has developed resistance to all antimicrobials previously recommended for treatment of gonorrhea, leaving dual therapy of ceftriaxone plus azithromycin as currently the only appropriate option for empirical first-line therapy in most countries world-wide. Now NG strains have been reported resistant to both ceftriaxone and azithromycin. Conversely, although ciprofloxacin is no longer recommended by the CDC for the treatment of NG, recent studies suggest that a large percentage of GC infections could be potentially treated with ciprofloxacin. With the continued evolution of AMR, there is an urgent need for personalized treatment approaches that target an individual infection, in contrast to the current “globally uniform” empiric approach. However, this requires clinicians to know drug resistance or susceptibility quickly enough to inform prescription decisions. To mitigate the emergence and spread of AMR in NG, CDC periodically publishes STD treatment guidelines to assist clinicians. These guidelines are informed by susceptibility data generated by the national CDC Gonococcal Isolate Surveillance Project (GISP). GISP’s impact, however, is being jeopardized by technological evolution. Determining AMR requires prolonged (24-48 hours) microbiological cultivation in sophisticated laboratory facilities. But the advent of nucleic acid amplification tests (NAAT) with enhanced speed and accuracy has supplanted culture-based diagnosis of NG infections, leading to limited specimen collection for culture and loss of capability to perform culture of NG in most testing clinics. Consequently, the success of widespread NAAT has inadvertently created a critical void in AMR testing. We propose to develop a complete diagnostic solution capable of performing identification (ID) of NG infection and phenotypic antimicrobial susceptibility testing (AST). Specifically, ID is achieved using PCR to detect NG- specific DNA markers; while AST is carried out using quantitative PCR to measure the difference in nucleic acid markers (bacterial DNA or RNA) which correlate with the physiologic state of pathogen between drug- treated samples and no-drug controls. Our combined ID-AST platform, which capitalizes on innovative advances in NAAT and microfluidics, has the potential to deliver all essential NG diagnostic information specific to each suspected patient at the POC to tailor personalized treatment; its practical design is also well- suited to resolve the technical challenges confronting GISP for routine surveillance of NG AMR. We propose the following aims: 1) to develop a streamlined diagnostic protocol for integrated ID and AST of NG; 2) to develop a droplet microfluidic cartridge implementing the integrated ID-AST assay; 3) system integration and instrument development; and 4) analytical and clinical validation of the integrated system. To facilitate technology translation, a Product Development Plan for future clinical deployment is proposed.