Rapid, on-chip, multiplexed detection of sepsis-causing organisms from blood samples

Lead Research Organisation: University of Hull
Department Name: Clinical Biosciences Institute

Abstract

This proposal brings together BioGene's expertise in development of rapid PCR, instrument design & manufacture and
software development, with the University of Hull's proven track-record in microfluidic chip design & optimisation, and
fabrication of devices with real world interfaces, e.g. scene of crime or point of care. Together the group will build a rapid
and portable, highly-accurate, system capable of extracting pathogen DNA from a blood sample, to determine the presence
of multiple bacterial species with relevant sub-type specificity, e.g. coagulase status for Staphylococci. All reagents for DNA
extraction, PCR amplification and separation will be preloaded in the device. The operation and stability of this instrument
are based on published work from the Hull team.
BioGene have a proven optical system capable of separating more than 10 dyes, and when this technology is coupled with
the ability to electrophoretically separate PCR products down to 2 base pair resolution, using Hull's microfluidic device, the
new instrument will be able to discriminate 1000s of distinct PCR fragments. The detection system is based on optical
deconvolution of the fluorescence signal generated by the multitude of differentially-labelled products. This method enables
the detection of many more targets than is currently possible with conventional sequencers. In this proof of concept study
we intend to characterise at least 10 specific factors, i.e. 8 organisms and two species-specific factors; it is anticipated
however that a considerably greater level of multiplexing will be achieved.
Blood is widely recognised as a "difficult medium" for pathogen nucleic acid amplification, both due to the presence of a
wide range of PCR inhibitors and the vast excess of human genomic DNA present. The blood sample will first be treated to
lyse red blood cells and facilitate collection of bacterial cells, initially by filtration, followed by recovery of the concentrated
cells to provide the starting material for the DNA analysis. The aim is to produce a platform suitable for handling microlitre
volumes as this would allow detection of sepsis in neonates and children where blood collection is via capillary tubes. An
additional benefit of using smaller volumes of blood is a reduction in complexity and cost of the overall unit.
The focus of the PCR system will be to maximise sensitivity and speed. Optimal PCR primer pairs for each target will be
identified after extensive in silico modeling before being tested in a real-time PCR format and for compatibility in a multiplex
mix. A selection of the most common organisms will be chosen from a list of sepsis-cuasing organisms in Hull & East
Yorkshire patients during 2010. Other key factors that will be addressed to deliver the system are: an effective sample
interface and automation of DNA extraction; incorporation of the ultra-rapid PCR unit and optical system onto a microfluidic
platform; and bespoke control software that includes data storage and user-friendly operator display.
Each part of the system will be tested extensively during design of the assays themselves as well as construction of the
proof of concept unit using blood samples from donors spiked with genome equivalents of the targets in order to test
functionality. Testing of the fully-functional device will be completed on blood drawn from patients: i) newly-presenting at
Accident & Emergency with classic symptoms of sepsis (n=50) or ii) on the Intensive Care Unit (n=15). The pathology
reports, obtained for clinical assessment, will give a "gold-standard" for comparison. Dr W Townend (Emergency Medicine),
Dr S Bennett (Cardiothoracic Anaesthesia and Intensive Care) and Dr R Meigh (Microbiologist), all Consultants at Hull &
East Yorkshire NHS Trust, will act as a clinical advisory team. Appropriate certification, eg. CE marking and IVD
certification (98/79/EC), will be applied for once the clinical trials have been completed.

Planned Impact

Biology - Impact on fundamental and applied research
The main output from the project is the generation of an optimized, rapid PCR machine capable of simultaneously
analyzing multiple genes (>10) in a single automated test, directly from a biological sample. The amount of data, which at
this point cannot be fully scoped, generated from any sample is highly likely to have significant benefit in the relevant area
of biology. The equipment will allow new science to be undertaken; analysis of gene transcription activity will become as
routine as measurements of protein concentration. It is anticipated that this will become generic technology that can be modified for numerous potential applications investigating physiological, pathological or pharmaceutical responses in cells,
tissues, organs and even whole organisms. The equipment will be extremely useful for pharmaceutical companies in their
drug-discovery programmes as changes in gene expression elicited by drug treatment; both intended and unwanted sideeffects,
can be identified. Any increase in efficiency of drug development means faster delivery of new drugs, less wastage
and cost savings.
Health
As highlighted in the main proposal the main aim of the new device is to rapidly identify bacterial species in patients with
sepsis. The benefit of this knowledge will allow clinicians to commence accurate and personalized treatment many hours
earlier than is possible with conventional testing methods; this will prevent disease progression and thereby considerably
reduce patient morbidity and mortality. There will be a concomitant reduction in NHS costs because there will be less need
for patients to occupy the highly-expensive intensive care beds (high staff costs and expensive equipment) further there will
be a reduction in the need for therapeutic drugs as only those indicated need be given. Even if patients are started on a
cocktail of broad acting prophylactic drugs, once the information on the organism typing is obtained (within an hour of
sampling) the regimen can subsequently be altered to that which is most effective. The idea of understanding a disease
profile and/or the effects of treatment better are equally applicable in many other acute and chronic diseases. Personalized
medicine is a major strategy of NIHR and the MRC; the current device will offer a flexible and powerful tool to help make
this a reality.
Security
The security services have a pro-active role in anti-terrorist operations; the ability to identify individuals accurately and
rapidly is a key issue. The current PCR instrument could be modified to give a genetic fingerprint of an individual, and even
if the identity of the person was unknown, their movements could be tracked if the fingerprint was detected. The
combination of sensitivity and speed of analysis means that the police and other security services can undertake this
testing whilst people are detained. Therefore the device is likely to have substantial benefits when utilized by the security
agencies.
Economic
The UK is a world-leading centre for biomedical research and healthcare innovation. The current sepsis device is a new
platform technology with the sepsis unit as the initial iteration. As others adopt and adapt the platform there will be a need
for a growth in manufacturing, chip device fabrication and functionalisation, sales, consumable suppliers and after-sales
support. These needs will generate opportunities for existing and new diagnostic device manufacturers to create further
highly skilled jobs in the UK to meet these requirements and support further innovation of the platform technology. The
uses of PCR described above and in the main document are not restricted to the UK thus, in conjunction with the breadth
of the device's potential applications, there will be significant export market opportunities in North America and Western
Europe, as well as the rapidly expanding markets of China and India.

Publications

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John Greenman (Author) (2013) International Innovation

 
Description The proposed unit for rapid-detection of sepsis-causing organisms has been built and the prototype tested on lab samples. The planned testing on patient samples was due to take place at the end of 2017, outside of the grant funded period, however the company then found the device could not be manufactured reliably or economically thus further development is on-going.
Exploitation Route It is still hoped that the clinical study work can be undertaken through the collaboration in 2018. Ethics and regulatory issues are in place. Unfortunately because of issues with the collaborative partner the University decided that it was not possible to continue to progress any clinical work.
Sectors Healthcare,Manufacturing, including Industrial Biotechology

 
Description Cafe Scientifique presentation (Beverley) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Personalised medicine and the miniature lab, was the title. It was a general discussion.
Year(s) Of Engagement Activity 2015
URL http://www.cafescientifique.org/index.php/find-your-local-cafe/uk/details/1/18/uk-beverley
 
Description Pint of Science Event - Microfluidic chips, can they customise treatment for cancer? 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact A night devoted to the leaps and bounds being made in the field of cancer research. Discover new and innovative ways of finding cancerous tumours as well as the potential of customised cancer treatment. How microfluidic devices (or chips) can be used to study pieces of tissue from the human body and the information could be used to inform clinical decisions.
Year(s) Of Engagement Activity 2017
URL http://www.bbc.co.uk/programmes/p053mwb1
 
Description School Visit (Beverley) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Overview of microfluidic approach - highlighting animal replacement opportunities.
Year(s) Of Engagement Activity 2017