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

项目总结 淋球菌对抗生素的耐药性是L对抗生素耐药性威胁的顶级威胁，其定义如下 谁。在过去的几十年里，NG已经对以前推荐的所有抗菌素产生了抗药性 治疗淋病，头孢曲松和阿奇霉素是目前唯一合适的双重疗法 世界上大多数国家的经验性一线治疗的选择。目前已有NG菌株的报道 对头孢曲松和阿奇霉素均耐药。相反，尽管环丙沙星不再被推荐使用 由疾病预防控制中心用于治疗NG，最近的研究表明，很大比例的GC感染可能是 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，而不是目前的“全球” 统一的“经验主义”方法。然而，这需要临床医生迅速了解耐药性或敏感性。 足以为处方决定提供信息。 为了减少AMR在NG中的出现和传播，CDC定期发布STD治疗指南，以 协助临床医生。这些指南是由国家疾控中心生成的易感性数据提供信息的 淋球菌分离监测项目(GISP)。然而，GISP的影响正受到技术上的威胁 进化论。测定AMR需要在复杂的环境中长时间(24-48小时)进行微生物培养 实验室设施。但核酸扩增测试(NAAT)的出现提高了速度和速度 准确性已经取代了基于培养的NG感染诊断，导致收集的样本有限 在大多数检测诊所，培养和丧失进行NG培养的能力。因此，成功的 广泛存在的NAAT无意中在AMR测试中造成了一个关键的空白。 我们建议开发一个完整的诊断解决方案，能够对NG感染进行识别(ID) 表型药敏试验(AST)。具体地说，ID是通过使用PCR来检测NG- 特异的DNA标记；而AST则用定量聚合酶链式反应来测量核差异 与病原体的生理状态相关的酸标记(细菌DNA或RNA) 处理过的样本和非药物对照。我们的联合ID-AST平台，利用创新的 NAAT和微流控技术的进展，有可能提供所有必要的NG诊断信息 在POC为每个疑似患者量身定做个性化治疗；其实用设计也很好- 适用于解决GISP面临的常规监测NG AMR的技术挑战。我们建议 以下目标：1)开发一种简化的NG ID和AST综合诊断方案；2)为 开发一种实现集成ID-AST分析的液滴微流控色谱柱；3)系统集成和 仪器开发；以及4)集成系统的分析和临床验证。为了方便 技术转换，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.