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

Lead Research Organisation: University of East Anglia
Department Name: Norwich Medical School

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

10 25 50
 
Description CRITICAL ASSESSMENT OF THE USE OF NGS FOR THE MONITORING OF AMR ACROSS DIFFERENT ECOLOGICAL COMPARTMENTS
Geographic Reach Europe 
Policy Influence Type Participation in a advisory committee
URL https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6039958/
 
Description The role and implementation of next generation sequencing technologies in the coordinated action plan against antimicrobial resistance
Geographic Reach Europe 
Policy Influence Type Participation in a advisory committee
URL https://ec.europa.eu/jrc/en/publication/role-and-implementation-next-generation-sequencing-technolog...
 
Description Development of Key Technologies for Real-Time Diagnosis, Surveillance and Intervention of Resistant-Bacterial Infections Based on Nanopore Sequencing
Amount £730,000 (GBP)
Funding ID 104991 
Organisation Innovate UK 
Sector Public
Country United Kingdom
Start 05/2019 
End 04/2022
 
Description Grifols collaboration on detection of pathogens in blood
Amount £150,000 (GBP)
Organisation Grifols 
Sector Private
Country Global
Start 03/2017 
End 09/2017
 
Description MRC iCASE programme
Amount £400,000 (GBP)
Funding ID MR/R015937/1 
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 10/2018 
End 03/2022
 
Description Prostate Cancer UK grant
Amount £208,610 (GBP)
Organisation Prostate Cancer UK 
Sector Charity/Non Profit
Country United Kingdom
Start 11/2016 
End 10/2018
 
Title Method for depleting host nucleic acid from urine for the rapid metagenomic diagnosis of urinary tract infections 
Description The method is based on a combination of differential centrifugation and differential lysis to remove human DNA from infected urine before applying rapid MinION metagenomic sequencing to identify the pathogen and associated antimicrobial resistance genes. 
Type Of Material Technology assay or reagent 
Year Produced 2017 
Provided To Others? Yes  
Impact Successful application of metagenomics for acute infectious diseases diagnostics requires short turnaround time. The time required to complete a metagenomic sequencing based test is related to sample preparation and depth of sequencing required. The proportion of host compared to pathogen nucleic acid in a clinical sample can be very high and increases to time and cost of metagenomic pathogen detection. We demonstrated a sample-to-result turnaround time for the diagnosis of pathogens and associated antimicrobial resistance in urine of 4 hours, including host depletion, library preparation and sequencing. Our research is being used by Oxford Nanopore Technologies as evidence to secure UK Government funding to trial MinION technology for pathogen and AMR diagnosis. 
 
Description Applying clinical metagenomics for the diagnosis of pneumonia 
Organisation University of Pittsburgh
Department Department of Medicine
Country United States 
Sector Academic/University 
PI Contribution I transferred out methods for performing clinical metagenomics on respiratory samples to my collaborator at Pittsburgh Medical School (Georgios Kitsios). He is now applying these methods to patient samples in Pittsburgh and achieving excellent results.
Collaborator Contribution Georgios Kitsios is now applying our respiratory metagenomics methods in Pittsburgh, currently in a research context, to diagnose hospital acquired and ventillator associated pneumonia. We have submitted an conference abstract together on his study and a journal publication will follow later this year.
Impact Metagenomic DNA sequencing of respiratory microbial communities for detection of etiologic pathogens of pneumonia in mechanically-ventilated adult patients - American Thoracic Society abstract
Start Year 2018
 
Description Bioinformatic tool development for the rapid analysis of clinical metagenomics data 
Organisation Harvard University
Department Harvard T.H. Chan School of Public Health
Country United States 
Sector Academic/University 
PI Contribution We transferred out respiratory clinical metagenomics data to Harvard (William Hanage) for bioinformatic tool development and analysis. Nobody else in the world had nanopore respiratory metagenomics data.
Collaborator Contribution Bill Hanage and colleagues developed a bioinformatic tool to rapidly genotype respiratory pathogens. He tested this on bacterial isolated, which worked well. Then he heard we had respiratory metagenomes and he applied his tools on our data. It was then possible to predict antimicrobial resistance/susceptability directly from metagenomics data within 5 mins.
Impact Lineage calling can identify antibiotic resistant clones within minutes - BioXriv preprint currently under review in Nature Biotechnology.
Start Year 2018
 
Description Grifols collaboration 
Organisation Grifols
Country Global 
Sector Private 
PI Contribution A collaboration on the metagenomic sequencing based detection of pathogens in blood. We provide the knowhow and the background intellectual property.
Collaborator Contribution Grifols will provide the pathogens and fund some of the research.
Impact No outputs to date as the collaboration has just begun.
Start Year 2017
 
Description Mayo Clinic 
Organisation Mayo Clinic
Country United States 
Sector Charity/Non Profit 
PI Contribution We have provided our human DNA depletion methods to Professor Robin Patel at the Mayo Clinic (under NDA) for use in clinical metagenomics with a particular focus on prosthetic joint infection. I will give a Grand Round at the Mayo Clinic in May 2018 and will provide some training on the method while there.
Collaborator Contribution Prof Patel will test clinical samples using our methods with an eye on future clinical implementation and publication.
Impact No outputs yet. The collaboration is multidisciplinary including clinicians, microbiologists biomedical scientists and bioinformaticians.
Start Year 2017
 
Description Oxford Nanopore Technologies collaboration 
Organisation Oxford Nanopore Technologies
Country United Kingdom 
Sector Private 
PI Contribution We are working closely with ONT to develop clinical metagenomics applications in diseases such as UTIs, pneumonia and meningitis.
Collaborator Contribution ONT have co-funded a PhD student in my lab, are an iCASE partner and have provided financial and in-kind contributions to a number of my research projects. They have hosted one PhD student and will host another next year at ONT for 3 months where they have been trained in bioinformatics and library preparation techniques.
Impact 1. ASHTON, P. M., NAIR, S., DALLMAN, T., RUBINO, S., RABSCH, W., MWAIGWISYA, S., WAIN, J. & O'GRADY, J. 2015. MinION nanopore sequencing identifies the position and structure of a bacterial antibiotic resistance island. Nat Biotechnol, 33, 296-300. 2. BRINDA, K., CALLENDRELLO, A., COWLEY, L., CHARALAMPOUS, T., LEE, R. S., MACFADDEN, D. R., KUCHEROV, G., O'GRADY, J., BAYM, M. & HANAGE, W. P. 2018. Lineage calling can identify antibiotic resistant clones within minutes. bioRxiv. 3. CHARALAMPOUS, T., RICHARDSON, H., KAY, G. L., BALDAN, R., JEANES, C., RAE, D., GRUNDY, S., TURNER, D. J., WAIN, J., LEGGETT, R. M., LIVERMORE, D. M. & O'GRADY, J. 2018. Rapid Diagnosis of Lower Respiratory Infection using Nanopore-based Clinical Metagenomics. bioRxiv, 387548. 4. IP, C. L. C., LOOSE, M., TYSON, J. R., DE CESARE, M., BROWN, B. L., JAIN, M., LEGGETT, R. M., ECCLES, D. A., ZALUNIN, V., URBAN, J. M., PIAZZA, P., BOWDEN, R. J., PATEN, B., MWAIGWISYA, S., BATTY, E. M., SIMPSON, J. T., SNUTCH, T. P., BIRNEY, E., BUCK, D., GOODWIN, S., JANSEN, H. J., O'GRADY, J., OLSEN, H. E., MIN, I. O. N. A. & REFERENCE, C. 2015. MinION Analysis and Reference Consortium: Phase 1 data release and analysis. F1000Res, 4, 1075. 5. JAIN, M., KOREN, S., MIGA, K. H., QUICK, J., RAND, A. C., SASANI, T. A., TYSON, J. R., BEGGS, A. D., DILTHEY, A. T., FIDDES, I. T., MALLA, S., MARRIOTT, H., NIETO, T., O'GRADY, J., OLSEN, H. E., PEDERSEN, B. S., RHIE, A., RICHARDSON, H., QUINLAN, A. R., SNUTCH, T. P., TEE, L., PATEN, B., PHILLIPPY, A. M., SIMPSON, J. T., LOMAN, N. J. & LOOSE, M. 2018. Nanopore sequencing and assembly of a human genome with ultra-long reads. Nat Biotechnol, 36, 338-345. 6. JAIN, M., TYSON, J. R., LOOSE, M., IP, C. L. C., ECCLES, D. A., O'GRADY, J., MALLA, S., LEGGETT, R. M., WALLERMAN, O., JANSEN, H. J., ZALUNIN, V., BIRNEY, E., BROWN, B. L., SNUTCH, T. P., OLSEN, H. E., MIN, I. O. N. A. & REFERENCE, C. 2017. MinION Analysis and Reference Consortium: Phase 2 data release and analysis of R9.0 chemistry. F1000Res, 6, 760. 7. SCHMIDT, K., MWAIGWISYA, S., CROSSMAN, L. C., DOUMITH, M., MUNROE, D., PIRES, C., KHAN, A. M., WOODFORD, N., SAUNDERS, N. J., WAIN, J., O'GRADY, J. & LIVERMORE, D. M. 2017. Identification of bacterial pathogens and antimicrobial resistance directly from clinical urines by nanopore-based metagenomic sequencing. J Antimicrob Chemother, 72, 104-114.
Start Year 2014
 
Description Simcere Diagnostics 
Organisation Simcere Diagnostics
PI Contribution We are in the late stages of negotiating a collaboration agreement and possible patent licence with Simcere Diagnostics. We plan to co-develop a 'laboratory developed test' (LDT) for nosocomial pneumonia to be offered as a service in hospitals in China. The agreement will likely involve funding for my laboratory for a Post-doc plus consumables and a potential licence agreement for the human DNA depletion patent developed during this MRC Innovation Award. We have intellectual property and knowhow in the metagenomics infection diagnostics space that Simcere want access to. This IP and knowhow has been developed through funding from MRC (human DNA depletion patent), Rosetrees Trust (human DNA depletion patent) and NIHR (INHALE trial on nosocomial pneumonia diagnosis).
Collaborator Contribution I was introduced to Simcere (a large Chinese pharmaceutical company with a diagnostics division that invest in Oxford Nanopore Technologies, ONT) by the CEO of ONT at the Nanopore Community Meeting in New York in Dec 2017. Simcere were very interested in my presentation on metagenomics based diagnosis of nosocomial pneumonia and were interested in collaboration. I visited their headquarters in Nanjing in late January 2018 and return in late March 2018 to complete the collaboration agreement. Simcere have an extensive network of customers/collaborators in hospitals throughout China, expertise in nanopore sequencing based metagenomics and a track record in LDT development. They will provide the expertise to drive our diagnostics tests from the research lab to the bedside, through the difficult regulatory landscape, and can access the huge healthcare market in China.
Impact No outputs from this project yet. The collaboration is multidisciplinary, including industry, academia, genomics, bioinformatics, rapid diagnostics, clinical microbiology, intensive care medicine, sample preparation, molecular biology.
Start Year 2017
 
Description Simcere collaboration 
Organisation Simcere Diagnostics
PI Contribution We have transferred methods to Simcere and will continue to work with them as part of a new grant (Innovate UK UK-China AMR grant) to develop these methods for clinical implementation.
Collaborator Contribution Simcere are further developing our methods to clinical standards for future implementation.
Impact Follow on funding - an Innovate UK grant
Start Year 2018
 
Title METHOD FOR NUCLEIC ACID DEPLETION 
Description Provided is a method for depleting host nucleic acid in a biological sample, said sample having been previously obtained from an animal host, said method comprising the steps of (a) adding a cytolysin, or an active variant thereof, to said sample; and (b) carrying-out a process to physically deplete nucleic acid released from host cells within said sample or otherwise render such nucleic acid unidentifiable. 
IP Reference WO2018109454 
Protection Patent application published
Year Protection Granted 2018
Licensed Yes
Impact No notable impact to date, this will develop over the coming years
 
Title Rapid metagenomics based diagnosis of nosocomial pneumonia 
Description We have developed a rapid (6hr) metagenomics sequencing method for diagnosis of nosocomial pneumonia. This method is currently being evaluated in comparison with PCR tests in the INHALE trial (NIHR, £2.5M). 
Type Diagnostic Tool - Non-Imaging
Current Stage Of Development Early clinical assessment
Year Development Stage Completed 2018
Development Status Under active development/distribution
Clinical Trial? Yes
UKCRN/ISCTN Identifier NIHR funded trial: RP-PG-0514-20018 INHALE: Potential of Molecular Diagnostics for Hospital-Acquired and Ventilator-Associated Pneumonia in UK Critical Care
Impact This method has generated a lot of interest and is being used by a lot researchers around the world. The ones that I am aware of, as I am directly collaborating with them, are: Pittsburgh Medical School, University of Bergen, PHE, University of Oxford, University of Cambridge, Harvard and one large Chinese multinational company. 
URL https://www.biorxiv.org/content/10.1101/387548v1
 
Description Cambridge PHE talk 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact I was invited to give a talk at Public Health England, Addenbrookes on my metagenomics sequencing based infection diagnostics work. Clinicians and scientists attended and there was lively discussion afterward. PHE Addenbrookes are interested in applying the MinION in research and potentially for certain services in the future.
Year(s) Of Engagement Activity 2017
 
Description Press release on rapid MinION sequencing based diagnosis of urinary tract infection 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact We reported our work on rapid UTI diagnosis using MinION metagenomic sequencing at an international conference in the US. This was accompanied by a press release by UEA and the conference (ICAAC 2015) and was reported widely, including an article on the BBC News website.
Year(s) Of Engagement Activity 2015,2016
URL http://www.bbc.co.uk/news/health-34295481
 
Description Royal Norfolk Show - AMR research tent and Superbugs panel 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Demonstrating MinION naopore sequencing to school kids and the general public and explaining how we use it for the detection on pathogens and antimicrobial resistance. Also on a discussion panel re superbugs and how to control them. Both activities led to multiple questions on the nanopore technology and how we can use this British technology to control superbugs and fight AMR.
Year(s) Of Engagement Activity 2018
URL https://royalnorfolkshow.rnaa.org.uk/discovery-zone/
 
Description SAW Trust school visit 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Schools
Results and Impact I, together with post-docs from my lab an artist and a writer, ran a SAW (Science, art and writing) day for year 6 students at my children's primary school. The focus was on antibiotic resistance and the role rapid diagnostics can play in the fight against AMR. A description of the science and example poems and art created by the students formed part of a book, SAW Antibiotics, published by the SAW trust. Professor Dame Sally Davies is quoted in the book stating: "I thoroughly enjoyed this stunning book. Educating children in such an interactive way is so important. I am passionate about addressing AMR, as are the researchers, and I hope this can help inspire future generations to engage in the efforts to save modern medicine". SAW now runs in a number of countries and the book is sold globally.
Year(s) Of Engagement Activity 2016,2017
URL http://www.sawtrust.org/buy-the-books/saw-antibiotics/
 
Description Science Museum Lates - Superbugs 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Interactive presentation on the rapid diagnosis of infection and antimicrobial resistance using metagenomics performed multiple times over the evening. 50-100 people were present for each presentation. Several people asked questions afterwards about the technology and the importance of diagnostics in the fight against AMR.
Year(s) Of Engagement Activity 2018
URL https://www.sciencemuseum.org.uk/see-and-do/lates