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

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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.