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

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.

Next generation sequencing (NGS) based pathogen identification in bloodstream infection 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 and reducing the ratio of human:pathogen DNA < 10:1. This will enable us to develop sequencing based workflows for the rapid and accurate diagnosis of sepsis which will result in reduced patient morbidity and mortality.

Objectives:
(1) Further develop and optimise our current pathogen DNA enrichment strategy and to test two new enrichment strategies
(2) 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
(3) Run a clinical diagnostics evaluation study of an optimised NGS based diagnostic method

Objective 1:
Method 1 (current method): We will continue to develop the current method and determine the limit of detection of this method.
Method 2: We have devised a second novel approach to pathogen DNA enrichment involving the removal of human DNA using nucleic acid capture.
Method 3: This approach is designed to ensure detection of both intracellular/phagocytosed pathogens and cell free pathogen DNA while readily removing human DNA.

Objective 2:
Short read (Illumina MiSeq) sequencing technology will be compared to nanopore (MinION and PromethION) sequencing technology to determine which is best suited to infectious diseases diagnostics.

Objective 3:
The performance of the optimised sepsis diagnostic workflow will be tested on 50 biobanked blood samples from sepsis patients and controls. The arising NGS results will be compared to blood culture, PCR (Septifast) and the clinical gold standard diagnosis.

Industry, the NHS, society and academia will benefit from this research as follows:

Industry: Once we secure IP on our novel methods we plan to partner with industry to (1) enable better diagnostics for sepsis and other serious infections (2) enable narrow spectrum antibiotics development through better patient selection in clinical trials and (3) open up new potential markets and collaboration opportunities with the genomics industry. The translation of our research through industry will lead to significant economic and social impact including: (1) enhancing the research capacity of diagnostics, pharmaceutical and genomics businesses; (2) contributing toward wealth creation and economic prosperity by creating jobs and enhancing business revenue (3) attracting R&D investment from global business and (4) commercialisation and exploitation of scientific knowledge by creating new diagnostics products and possibly leading to a spin out company.

The NHS and society: We are working closely with the Norfolk and Norwich University Hospital Microbiology and Critical Care departments providing expertise and evidence to support the future implementation of sequencing based diagnostics methods. These methods have the potential to have a big impact on the NHS and society in general. By providing rapid and accurate diagnostics, clinicians will be able to make better informed treatment decisions faster. This will lead to a precision medicine approach to antibiotic treatment with improved antimicrobial stewardship and reduced patient morbidity and mortality. Sequencing based diagnostics will also improve infection control and epidemiology in hospitals and nationally. These benefits will enhance the health and well-being of the nation and lead to health policy changes at national and possibly international levels.

Academia: I am developing novel sample preparation based 'enabling technologies' that have wide potential diagnostic applications and can be used with any sequencing platform. Our methods can be used by academics involved in drug development and clinical trials, medical microbiology, veterinary and marine microbiology and cancer research (sequencing of exosomes and cell free DNA). Hence, we have the potential to make a large academic impact and enhance the knowledge economy by developing and utilising our new and innovative techniques and technologies.