Inflammation and Infective Endocarditis
Conditions: Infective Endocarditis (IE)
Sponsor: Barts & The London NHS Trust

 
 
 
 
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Joint Research Management Office (JRMO) Research Protocol for Research Studies 
 
 
 
 
Full Title  
Inflammation and infective endocarditis  
 
 
Short Title 
Inflammation and infective endocarditis 
Sponsor 
 
§ 
Queen Mary University of London (Queen Mary) 
 
Contact person: 
 
Dr Mays Jawad 
Research Management Office (JRMO) 
Barts Health NHS Trust & Queen Mary University of London 
Dept W, 81 Mile End Rd, London E1 4UJ. 
 
Phone: 020 7882 7275/6574 
 
Email: research.governance@qmul.ac.uk  
 
IRAS Number 
353031 
Integrated Research Application System 
 
Sponsor (EDGE) Number 
Pending  
REC Reference 
 
Research Ethics Committee 
 
Chief Investigator (CI) 
Dr Daniel Harding 
Clinical Lecturer in Cardiology 
07503192714 
d.harding@qmul.ac.uk 
 
List of sites 
St Bartholomew's Hospital 
West Smithfield 
London 
EC1A 7BE 
d.harding@qmul.ac.uk 
 
 
List of laboratories 
Biochemical Pharmacology 
 
Professor Federica Marelli-Berg 
f.m.marelli-berg@qmul.ac.uk 
 
Charterhouse Square  
 
London, EC1M 6BQ 
 
List of technical departments 
 
 
List of central facilities 
 

 
 
 
 
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Contents 
 
1. 
Contents 
 
 
 
2 
2. 
Glossary 
 
 
 
3 
3. 
Signature page 
 
 
4 
4. 
Summary and synopsis 
 
5 
5. 
Introduction 
 
 
 
6 
6. 
Study objectives 
 
 
6 
7. 
Study population 
 
 
7 
8. 
Study design  
 
 
7 
9. 
Study procedures 
 
 
7 
10. 
Statistical considerations 
 
8 
11. 
Ethics  
 
 
 
8 
12. 
Annual Safety Reporting 
 
9 
13. 
Public involvement 
 
 
9 
14. 
Data handling and record keeping 
9 
15. 
Laboratories   
 
 
10 
16. 
Safety reporting 
 
 
11 
17. 
Monitoring and auditing 
 
11 
18. 
Study committees 
 
 
11 
19. 
Finance and funding  
 
12 
20. 
Insurance and indemnity 
 
12 
21. 
Dissemination of research findings 12 
22. 
References 
 
 
 
12 
 
 
 
 
 
 
 
 
 

 
 
 
 
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2. Glossary 
 

 
 
 
 
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3. Signature page 
 
 
 
CI Agreement 
 
The study, as detailed within this Research Protocol, will be conducted in accordance with the 
principles of Good Clinical Practice (GCP), the UK Policy Framework for Health and Social Care 
Research, and the Declaration of Helsinki and any other applicable regulations. I agree to take 
responsibility for the statistical analysis and oversight of this study. 
 
 
CI Name: 
________________ 
 
Signature: 
_________________ 
 
Date: 
_______ 
 

 
 
 
 
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4. Summary and synopsis 
 
Short title 
Inflammation and infective endocarditis 
Methodology 
Research 
Objectives / aims 
1. Define the peripheral (blood) and local (valve 
tissue) 
immune 
landscape 
in 
infective 
endocarditis (IE).  
2. Confirm that cMET+ T-cells are present in patients 
with infective endocarditis (peripheral and local) 
and interrogate their functional role by flow 
cytometry and RNA sequencing. 
Number of 
participants 
100 
Inclusion and 
exclusion criteria 
Inclusion: 
• 
The principal inclusion criteria is a clinical 
diagnosis of IE made according to the Duke 
Criteria. Patients aged 18-85 are eligible.  
 
Exclusion: 
 
• 
We will exclude patients with an active solid 
organ or haematological malignancy as these 
conditions can influence immune cell frequency 
and phenotype.  
Statistical 
methodology and 
analysis (if 
applicable) 
With the support of our local NIHR bioinformatics and 
bio-repository facility (http://www.whri.qmul.ac.uk/core-
facilities/nihr-bioinformatics-and-bio-repository), Multiple 
parameters will be analysed using multiple group 
comparisons (ANOVAs) to increase power. 
Study duration 
18 months 
 

 
 
 
 
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5. Introduction 
 
Background 
 
Infective endocarditis (IE) is challenging to diagnose and manage. Its frequency is increasing as 
we perform more interventions on patients with pre- existing valve disease2,3. Current strategies 
are relatively historical; patients require positive blood cultures for a definitive diagnosis and are 
typically managed with intravenous antibiotics and cardiac surgery4. While the immunological 
complications of IE are well recognised (e.g. glomerulonephritis, rheumatoid factor positivity), 
there has been very little research to explore the immune system’s role in disease onset and 
progression5. Our laboratory has discovered and characterised a group of circulating immune 
cells (cMET+ T-cells) that appear to have a direct role in the progression of another inflammatory 
heart disease known as myocarditis6. Pilot data (also from our laboratory, n=3 patients) suggests 
that these cells are detectable in explanted valve tissue from patients undergoing endocarditis 
surgery. We believe that the presence of these cells in IE implies that there may be a significant 
contribution to disease progression from the immune system. It is possible that the exposure of 
cardiac proteins by damage caused by a bacterial infection primes the immune system against 
self, and that these auto- reactive T-cells then go on to attack and destroy native valve tissue.  
 
Rationale 
 
We would like to investigate whether circulating and valvular cMET+ T-cells are detectable across 
the wide spectrum of patients with IE and further explore their role in this condition. We are planning 
to functionally characterise both circulating immune cells and those that are detectable in explanted 
valve tissue using a combination of flow cytometry and spatial RNA sequencing. We hypothesise 
that cMET+ T-cells may have utility in the diagnosis of IE and that in patients with a significantly 
elevated frequency of cMET+ T-cells, we might be able to offer novel (and highly specific) targeted 
immunological therapy that could improve patient outcomes. To address this hypothesis, we will 
undertake an observational cohort study in patients with IE to analyse the immune cell status in the 
peripheral blood and valve tissue (obtained at surgical explant) 
Risks / benefits 
 
We do not envisage any risks to this project as we will only be taking blood samples via 
venepuncture. Only a slight discomfort may be experienced by the patient at the injection site and 
slight bruising. To minimise bruising, the patient will be advised to apply pressure to the 
venepuncture site using a cotton swab that will be provided to the patient once the blood has been 
taken. The patient will also be advised to sit for 10 minutes to ensure that they are feeling well after 
the blood is taken. 
 
The potential benefit of this projects outweighs the potential risks. The immediate outcomes for this 
project is to gain a detailed understanding of how the immune system could affect the development 
and progression of infective endocarditis. This could help to inform our current understanding of 
why infective endocarditis occurs and help us to develop new treatments.  
 
 
6. Study objectives 
 
1. We will report a comprehensive analysis of immune cell frequency and function in the 
peripheral blood and valve tissue from patients with IE. This has never before been performed.  

 
 
 
 
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2. We will confirm whether cMET+ T-cells are present in patients with IE (blood and valve), and 
determine if they may have a diagnostic and therapeutic potential.  
 
3. We will compare immune phenotype during the acute disease phase in IE with 
convalescence/recovery. 
 
7. Study population 
 
All patients with IE who are managed within the Barts Heart Centre.  
 
Inclusion criteria 
 
• 
The principal inclusion criteria is a clinical diagnosis of IE made according to the Duke 
Criteria.  
• 
Patients aged 18-85 are eligible.  
• 
Fluency in English (otherwise interpreters will be used) 
• 
Informed, written consent 
 
Exclusion criteria 
 
• 
Unwilling or unable to give consent 
• 
Participants who are unable to understand written English 
• 
Patients with an active solid organ or haematological malignancy 
 
8. Study design 
 
This is a prospective cohort study.  
 
Dr Daniel Harding and allocated research nurse (Innocent Bvekerwa) will identify and recruit IE 
patients whilst inpatients at the Barts Heart Centre (~300 patients/year in this service). Peripheral 
blood will be obtained at the time of recruitment (DH and IB to perform phlebotomy), valve tissue 
will be obtained as/when these patients undergo cardiac surgery. Samples will be stored at 4 
degrees prior to transfer to QMUL for processing by the scientific team (DH and post-doctoral 
scientists). Samples will be processed and either stored at -80 for subsequent analysis (blood), 
fixed in formalin and embedded in paraffin (valve tissue for spatial analysis) or processed and 
analysed immediately (valve tissue for flow cytometry). 3. Relevant (anonymised) clinical data will 
be extracted at the point of recruitment (demographic, blood test results, imaging findings) and 
recorded for downstream correlation analysis. 4. Patients will be followed remotely, and outcome 
data (death, further surgery, recurrent disease) will be recorded. When the patients attend for their 
outpatient clinic (in those that survive), they will be offered a further blood test. We will use this 
sample to perform repeat immune cell analysis in the convalescence phase of their illness. 5. Flow 
cytometry analysis of immune cells in blood and valve tissue will be performed locally (Biochemical 
Pharmacology, basement laboratory), whereas spatial RNA analysis will be commissioned from a 
third party provider (Azenta life sciences). 
 
 
9. Study procedures 
 
• 
Laboratory assessments (Please see the laboratories section 12) 

 
 
 
 
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• 
Participant withdrawal (including data collection, retention or replacement for withdrawn 
participants: 
 
Participants will have the option to submit a written request to be withdrawn from the study at any 
stage. Participants will also have the option to withdraw consent from further participation in which 
circumstance no further data will be collected for inclusion. In the unfortunate event that a 
participant loses capacity, information gathered will continue to be used but no further data will be 
recorded. The end of the study will be defined by achieving the proposed patient numbers per 
patient group (outlined above).  
10. Statistical considerations 
 
The CI declares responsibility for the statistics and statistical oversight of the study (see CI 
Signature Agreement section 3). 
 
 
Sample size and power calculations 
 
The total sample size for this project is 100 patients with IE. We will aim to recruit to 5 IE subgroups 
(n=20 per subgroup), to reflect the heterogeneous nature of this condition: native valve, prosthetic 
valve, cardiac-device related, uncorrected GUCH and recurrent IE. Power calculations were 
performed using G*Power (University of Kiel, Germany) with the support of the QMUL 
Bioinformatics department. Sample size estimates were based on a Wilcoxon-Mann-Whitney test 
using the following parameters: α = 0.05, β = 0.2, predicted effect size 0.87. The predicted effect 
sized used for this power calculation was based on a previously published study comparing the % 
of peripheral cMET+ T-cells in acute myocarditis and DCM6. Using the above calculation, we 
determined that a minimum sample size of 10 patients per subgroup would yield statistical 
significance. To avoid under-powering, we chose to double this target to 20 patients per subgroup. 
 
Method of analysis  
 
1) Flow cytometry of immune cell. Whole blood flow cytometry is a well-established method. 
Dr Harding’s laboratory has access to a flow cytometry core facility, which has machines 
that can identify up 12 phenotypic markers on any given cell. Moreover, we, have 
extensive experience in immune cell phenotyping using this method. The key advantages 
are: 
• 
It is the gold-standard for immune cell phenotyping 
• 
A minimal amount of blood is needed (max 200μls; 50μls per patient per antibody 
panel -see table 3 below) 
• 
Antibodies are commercially available and have been extensively validated by 
ourselves and others. 
 
All samples will be processed an analysed within 1 hour of sample collection to minimise 
degradation of proteins on the surface of the immune cells.  
 
 
11. Ethics 
 
Application for the current study is pending following submission (IRAS ID 353031).  

 
 
 
 
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Informed consent will be obtained and recorded as described as above. Allowances for special 
groups are detailed above.  
The CI will ensure that this study is conducted in full conformity with the current revision of the 
Declaration of Helsinki (last amended October 2013) on research involving human subjects. 
  
The CI will ensure that the study is conducted in accordance with all applicable regulatory 
requirements which are based on ICH Guidelines for GCP, Trust and Research Office policies and 
procedures and any subsequent amendments.   
 
The study can only start after approval from a REC, HRA and confirmation of local capacity and 
capability at each of the participating centres.  
 
Members of the study team do not have any conflicts of interest to declare. 
 
Incidental Findings 
Should any incidental findings of clinical significance arise during the course of the study, the 
participants GP and clinical care team will be informed. Consent to contact the GP will be sought 
during the enrolment process.  
 
12. Annual Safety Reporting 
 
The CI will send an Annual Progress Report to the REC and the sponsor using the HRA template 
on the anniversary of the REC “favourable opinion”. 
 
 
13. Public involvement 
 
There are no UK groups specifically dedicated to raising funds or awareness for infective 
endocarditis. We have therefore been unable to involve any specific advocacy groups related to 
this condition in the study design. We do however intend to utilise current public engagement 
schemes run at the William Harvey Research institute and would also be keen to take part in any 
events organised by the Barts Charity to help disseminate our findings. Dr Harding has previously 
been involved in the writing and dissemination of myocarditis patient information resources for 
Cardiomyopathy UK. We would be very enthusiastic as a team to develop these sort of resources 
for infective endocarditis and plan to approach Cardiomyopathy UK to facilitate this.We plan to 
disseminate aspects of our research via newsletters, web-based information, and open forum 
patient information days. The proposed research will be incorporated into our current public 
engagement schemes. 
 
 
14. Data handling and record keeping 
 
Data management 

 
 
 
 
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All data generated will be stored within an Excel spreadsheet combining clinical and lab data. The 
spreadsheet will be password protected stored with a secure hard drive on a protected computer. 
Access to the password and data will be on a strict need-to-know basis. A full audit trail will be in 
place. 
 
Source Data 
 
Source data for this study will comprise of a diagnosis of infective endocarditis, which will be 
obtained from the adult cardiology team, led by Dr Simon Woldman at the Barts Heart Centre. 
Source data will also be obtained from the laboratory procedures of blood samples taken from 
patients at the time points mentioned above. Investigators who are directly involved with the 
study will have access to the source data.  
 
 
Confidentiality 
 
Dr Daniel Harding, the PI, will ensure only the clinical team has access to patient identifiable 
information, such as name, date of birth and hospital number. Any data that needs to be sent to 
the research team at the laboratory at Queen Mary (CI Dr Daniel Harding), a pseudo-anonymised 
database will be created where each participant will be given a unique study number, ensuring 
the research team at Queen Mary will not have access to patient identifiable data. 
 
Record retention and archiving 
 
The study data and documentation will be archived in accordance with the relevant regulatory 
requirements and site SOPs.   
 
A unique alphanumeric patient identifier will be allocated to each participant at the start of the 
study. Data will be stored using the unique participant identifiers on a secure database. Data will 
not be stored on personal computers. Confidential documents will be stored in a locked cupboard 
located in a secure room. Clinical notes will be handled according to trust protocol. The Data 
Protection Act and the NHS confidentiality code of practice will be adhered to throughout this 
study. Data may be shared with members of the clinical team to allow ongoing clinical care. 
 
GCP guidelines require that the investigator or the institution maintains all Case Report Forms 
and all source documents that support the data collected from each participant plus all trial 
documentation. Measures will be taken to prevent accidental or premature destruction of these 
documents. Essential documents must be retained for 5 years. It is the responsibility of the 
sponsor to inform the investigator when these documents no longer need to be retained. If the 
responsible investigator retires or leaves the institution responsibility for the documentation must 
be transferred to a person who will accept their custody 
 
 
15. Laboratories  
 
Central and local laboratories  
 
All laboratory work will be carried out within the Centre for Biochemical Pharmacology, William 
Harvey Research Institute, Charterhouse Square, London EC1M 6BQ 
 

 
 
 
 
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Sample preparation and collection 
 
Blood samples to assess immune cell phenotypes will be collected from patients who have read 
the information sheet and signed the consent form. A maximum volume of 15mls will be taken 
from the patient at any one time and collected into lavender EDTA blood tubes.  
 
Laboratory procedures 
 
The majority of the blood samples will undergo phenotypic analyses using the flow cytometry 
method. Briefly, 50 microlitres of blood will be labelled with commercially-available antibodies that 
are conjugated to flurochromes that can be detected by lasers on the flow cytometer. Specialised 
software will enable the analyses of the assessment of phenotypic markers that are expressed on 
immune cells. The samples will also be isolated for specific immune cell including B and T-cells, 
which will be stored in a secure -80oC freezer. Plasma will also be collected from these samples 
to assess circulating factors, to be assessed by commercially-available kits. 
 
Human valve samples will be formalin fixed and paraffin embedded before being shipped to a 
third party provider (Azenta life sciences) for spatial RNA sequencing at a later date.  
 
Sample storage and transfer 
 
Blood and valve samples will be taken by Dr Harding (or a dedicated member of the research 
team) and will be transferred to the laboratory using secure sample transfer boxes. All blood and 
heart valve tissue samples will be processed within one hour of collection. Blood samples will be 
stored within a secure -80C freezer at the Heart Centre, where the Centre for Biochemical 
Pharmacology is based. All samples will be fully anonymized. 
 
 
16. Safety reporting 
 
The study involves taking blood samples from the participant. One of the main risks of blood tests 
are discomfort and bruising at the site where the needle goes in. To minimise bruising, the 
participant will be encouraged to put pressure at the puncture site immediately after the sample 
has been taken. 
 
17. Monitoring and auditing 
 
The Sponsor or delegate retains the right to audit any study, study site or central facility. In 
addition, any part of the study may be audited by the funders where applicable. 
 
On-site monitoring will be performed as per the study monitoring plan. Monitoring will include 
source data verification. 
Monitoring of the centre may be undertaken by a trained study monitor or monitors, to verify the 
study documentation. The sponsor reserves the right to audit trial sites, subject to giving a 
suitable period of notice (minimum period of notice 10 working days). 
 
 
18. Study committees 
A study management group will be in place where we will hold monthly meetings. The study 
management group will comprise of the investigators directly involved in this study: 

 
 
 
 
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Dr Daniel Harding (CI) 
Professor Simon Woldman, Consultant Cardiologist 
Professor Federica Marelli-Berg, British Heart Foundation Chair of Cardiovascular Immunology 
(QMUL) 
 
 
19. Finance and funding 
 
This study is in the process of peer review and a funding application has been made to the Barts 
Charity (ID 27360).  
 
20. Insurance and indemnity 
 
The insurance that Queen Mary has in place provides cover for the design and management of 
the study as well as "No Fault Compensation" for participants, which provides an indemnity to 
participants for negligent and non-negligent harm. 
 
21. Dissemination of research findings 
 
In addition to publishing our key findings in peer-reviewed articles, we plan to disseminate 
aspects of our research via newsletters, web based information, and open forum patient 
information days. The proposed research will be incorporated into our current public engagement 
schemes 
 
22. References 
 
1.  
Harding D, Prendergast B, Kutarski A, Rouzet F, Bernard BC. Advanced imaging improves the 
diagnosis of infective endocarditis. F1000Research 2018 7:674. 2018;7:674. 
doi:10.12688/f1000research.13791.1 
2. 
Cresti A, Chiavarelli M, Scalese M, et al. Epidemiological and mortality trends in infective 
endocarditis, a 17-year population-based prospective study. Cardiovasc Diagn Ther. 
2017;7(1):27-35. doi:10.21037/CDT.2016.08.09 
3. 
Harding D, Cahill TJ, Redwood SR, Prendergast BD. Infective endocarditis complicating 
transcatheter aortic valve implantation. Heart. 2020;106(7). doi:10.1136/heartjnl-2019-315338 
4. 
Holland TL, Baddour LM, Bayer AS, Hoen B, Miro JM, Fowler VG. Infective endocarditis. Nat Rev 
Dis Primers. 2016;2(1):16059. doi:10.1038/nrdp.2016.59 
5. 
Garnier JL, Touraine JL, Colon S. Immunology of infective endocarditis. Eur Heart J. 
1984;5(suppl_C):3-9. doi:10.1093/EURHEARTJ/5.SUPPL_C.3 
6. 
Fanti S, Stephenson E, Rocha-Vieira E, et al. Circulating c-Met-Expressing Memory T Cells 
Define Cardiac Autoimmunity. Circulation. 2022;146(25):1930-1945. 
doi:10.1161/CIRCULATIONAHA.121.055610 

 
 
 
 
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7. 
Investigating infective endocarditis: teaching hospitals to choose wisely. British Journal of 
Cardiology. Published online 2023. doi:10.5837/BJC.2023.032 
  
 
 
 
This protocol is based on JRMO Protocol template for Research Studies; 
v4.0 07.04.2022