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

人们普遍认识到，快速诊断对于(1)对抗抗菌素耐药性（AMR）至关重要，允许更早和更精确地使用窄谱抗生素靶向病原体，以及(2)改善对脓毒症等危及生命的感染的管理。目前的方法——血液培养和基于聚合酶链反应的分子检测——不适合这种情况。血培养方法周期长，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/或耐药性标记。诊断微生物学的范式转变是迫切需要的。基于下一代测序（NGS）的诊断有可能实现这一步骤的改变，可能像PCR一样迅速，像培养一样全面。然而，在血液感染诊断中，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（没有任何显著的病原体DNA损失），并将人类：病原体DNA的比例从10:9:1降低到< 10:1。我们的概念验证数据表明，我们的方法与MinION纳米孔测序技术相结合，可以在8小时内成功地识别脓毒症患者血液样本中的病原体及其耐药基因。有了这种方法，如果它能被引入临床，患者只需要接受一剂经验性广谱抗生素，就可以为病原体/患者量身定制治疗——这是一种真正的“精准医学”抗生素治疗方法。对抗菌药物管理（英国公共卫生）的“开始聪明-然后集中”方法的这一显著改进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。它还将减少因感染而接受不适当抗生素治疗的患者人数，从而偶然降低发病率和死亡率。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，成本和测序结果的全面性方面最合适-运行临床诊断评估，测试50个表型良好的，来自败血症患者和对照组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，并将确保英国和NHS在基于基因组学的分层和精准医学方面处于全球领先地位。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，例如卫生保健相关肺炎和复杂的尿路感染。全面的基于测序的诊断不仅将使窄谱抗生素的应用更加广泛，而且将促进窄谱抗生素的临床开发。最后，他们将鉴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，以及我和我的合作者的专业知识，使我处于独特的地位，可以完成这个前沿的、雄心勃勃的、高影响力的转化研究项目。

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.