Improving the management of sepsis through rapid pathogen and antibiotic resistance detection in blood

通过快速检测血液中的病原体和抗生素耐药性来改善脓毒症的治疗

• 批准号: MR/N013956/1
• 负责人: Justin O'Grady
• 金额: $ 25.94万
• 依托单位: University of East Anglia
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2016
• 资助国家: 英国
• 起止时间: 2016 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=MR%2FN013956%2F1
• 关键词: Improving; management; sepsis; through; rapid

It is widely recognised that rapid diagnostics are crucial (1) in the fight against antimicrobial resistance (AMR), allowing earlier and more precise targeting of pathogens with narrow-spectrum antibiotics, and (2) for improving the management of life threatening infections such as sepsis. Current methods - blood culture and PCR based molecular tests - are not fit-for-purpose in this context. Blood culture methods have long turn-around times and offer poor clinical sensitivity; PCR based methods are not sufficiently comprehensive, detecting only selected pathogens and/or resistance markers. A paradigm shift in diagnostic microbiology is urgently required.Next generation sequencing (NGS) based diagnosis has the potential to deliver this step change, being potentially as swift as PCR and as comprehensive as culture. However, sequencing-based pathogen identification in bloodstream infection diagnosis is very challenging owing to the vast amount of human DNA present compared with pathogen DNA (the ratio can be as high as 10^9:1). Therefore, pathogen DNA enrichment is crucial and we are developing novel strategies to achieve this, removing the vast majority of the human DNA from blood (without any significant loss of pathogen DNA) and reducing the ratio of human:pathogen DNA from 10^9:1 to < 10:1. We have proof-of-concept data to demonstrate that our approach, combined with MinION nanopore sequencing technology, can be used successfully to identify pathogens and their resistance genes in blood samples from patients with sepsis within 8h.With this approach, if it can be introduced to the clinic, patients need receive only one dose of empirical broad-spectrum antibiotics before treatment can be tailored for the pathogen/patient - a true 'precision medicine' approach to antibiotic treatment. This dramatic improvement to the 'Start Smart - then Focus' approach to antimicrobial stewardship (Public Health England) will lead to a reduction in the use of broad-spectrum antibiotics, mitigating selection pressure for antibiotic resistance. It will also reduce the number of patients who receive inappropriate antibiotics for their infections, with contingent decreases in morbidity and mortality.We propose to: - Further develop and optimise our current pathogen DNA enrichment strategy and to test two new enrichment strategies- Test a number of NGS technologies/platforms to determine the most suitable in terms of analysis time, flexibility, complexity of bioinformatics analysis, cost and comprehensiveness of sequencing results- Run a clinical diagnostics evaluation, testing 50 well-phenotyped, biobanked human blood samples from sepsis patients and controls to validate the performance of the optimised NGS based method. This project, combining our novel pathogen DNA enrichment strategies with NGS, represents the cutting edge of clinical microbiology and genomics, and will ensure the UK and the NHS are among the global leaders in genomics-based stratified and precision medicine.The pathogen DNA enrichment and NGS workflows will be applicable to diagnostic samples from other life-threatening infections e.g. healthcare-associated pneumonia and complicated urinary tract infections. Comprehensive sequencing-based diagnostics will enable not only the wider use, but also the clinical development of narrow spectrum antibiotics. Lastly, they will identify bacterial strains and their variants, providing information that can be used for infection control and for both local and national epidemiology purposes. The preliminary work that I have performed, along with my expertise and that of my collaborators, make me uniquely positioned to deliver this cutting edge, ambitious, high impact translational research project.

人们普遍认为，快速诊断是至关重要的（1）在对抗抗生素耐药性（AMR），允许更早和更精确地靶向病原体与窄谱抗生素，和（2）改善危及生命的感染，如败血症的管理。目前的方法-血培养和基于PCR的分子测试-不适合在这种情况下的目的。血培养方法的周转时间长，临床敏感性差;基于PCR的方法不够全面，只能检测选定的病原体和/或耐药标志物。基于下一代测序（NGS）的诊断技术有可能实现这一步的改变，它可能像PCR一样快速，像培养一样全面。然而，血流感染诊断中基于测序的病原体鉴定是非常具有挑战性的，因为与病原体DNA相比存在大量的人类DNA（比例可高达10^9：1）。因此，病原体DNA富集是至关重要的，我们正在开发新的策略来实现这一目标，从血液中去除绝大多数人类DNA（没有任何病原体DNA的显著损失），并将人类：病原体DNA的比例从10^9：1降低到< 10：1。我们有概念验证数据证明，我们的方法结合MinION纳米孔测序技术，可以成功地用于在8小时内从脓毒症患者的血液样本中鉴定病原体及其耐药基因。患者只需要接受一剂经验性广谱抗生素，然后就可以针对病原体/患者进行治疗-一种真正的“精确医学”抗生素治疗方法。这种对抗菌剂管理的“聪明开始-然后集中”方法的巨大改进（英国公共卫生）将导致广谱抗生素的使用减少，减轻抗生素耐药性的选择压力。我们建议：- 进一步开发和优化我们目前的病原体DNA富集策略，并测试两种新的富集策略-测试一些NGS技术/平台，以确定在分析时间、灵活性、生物信息学分析的复杂性方面最合适的技术/平台，测序结果的成本和全面性-运行临床诊断评估，测试来自败血症患者和对照的50个良好表型的生物库人类血液样本，以验证优化的基于NGS的方法的性能。该项目将我们的新型病原体DNA富集策略与NGS相结合，代表了临床微生物学和基因组学的前沿，将确保英国和NHS成为基于基因组学的分层和精准医学的全球领导者。病原体DNA富集和NGS工作流程将适用于其他危及生命的感染的诊断样本，例如医疗相关肺炎和复杂尿路感染。基于测序的全面诊断不仅将使窄谱抗生素的应用更广泛，而且还将使其临床开发成为可能。最后，他们将确定细菌菌株及其变种，提供可用于感染控制以及地方和国家流行病学目的的信息。我所做的初步工作，加上我和我的合作者的专业知识，沿着，使我处于独特的地位，能够提供这个尖端的，雄心勃勃的，高影响力的转化研究项目。

项目成果

Rapid inference of antibiotic resistance and susceptibility by genomic neighbour typing

• DOI: 10.1038/s41564-019-0656-6
• 发表时间: 2020-02-10
• 期刊: NATURE MICROBIOLOGY
• 影响因子: 28.3
• 作者: Brinda, Karel;Callendrello, Alanna;Hanage, William P.
• 通讯作者: Hanage, William P.

The variability and reproducibility of whole genome sequencing technology for detecting resistance to anti-tuberculous drugs.

• DOI: 10.1186/s13073-016-0385-x
• 发表时间: 2016-12-22
• 期刊: Genome medicine
• 影响因子: 12.3
• 作者: Phelan J;O'Sullivan DM;Machado D;Ramos J;Whale AS;O'Grady J;Dheda K;Campino S;McNerney R;Viveiros M;Huggett JF;Clark TG
• 通讯作者: Clark TG

Improving the diagnosis and management of serious infection using rapid point-of-care metagenomic sequencing

使用快速护理点宏基因组测序改善严重感染的诊断和管理

• 发表时间: 2017
• 期刊: INTERNATIONAL JOURNAL OF ANTIMICROBIAL AGENTS
• 影响因子: 10.8
• 作者: O'Grady Justin

Application of highly portable MinION nanopore sequencing technology for the monitoring of nosocomial tuberculosis infection.

• DOI: 10.1016/j.ijmyco.2016.10.035
• 发表时间: 2016-12-01
• 期刊: International journal of mycobacteriology
• 影响因子: 1.2
• 作者: Bates, Matthew;Polepole, Pascal;O'Grady, Justin
• 通讯作者: O'Grady, Justin

MinION Analysis and Reference Consortium: Phase 2 data release and analysis of R9.0 chemistry.

• DOI: 10.12688/f1000research.11354.1
• 发表时间: 2017
• 期刊: F1000Research
• 作者: Jain M;Tyson JR;Loose M;Ip CLC;Eccles DA;O'Grady J;Malla S;Leggett RM;Wallerman O;Jansen HJ;Zalunin V;Birney E;Brown BL;Snutch TP;Olsen HE;MinION Analysis and Reference Consortium
• 通讯作者: MinION Analysis and Reference Consortium