Combatting Bacterial Resistance in Europe - Clostridium Difficile Infections (COMBACTE-CDI, Understanding the Burden of Disease
Conditions: Clostridium Difficile Infection
Sponsor: University of Leeds

 
 
 
 
FULL STUDY PROTOCOL for COMBACTE-CDI 
Protocol version number 1.6 17/04/18  
 
 
Author:  
 
Kerrie Davies 
Principal Clinical Scientist and Visiting Research Fellow 
Healthcare Associated Infections Research Group 
Leeds Institute of Biomedical and Clinical Sciences 
University of Leeds   
Sponsor:  
Innovative Medicines Initiative 2 (part of Horizon 2020).  
 
 
 
 
 
 
The information in this document must not be used or made public without the express permission 
of the European Coordinator for the study.  
 
 
 
 
 
 
 
 
 
 
 

 
 
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Study contributors 
 
COMBACTE-CDI  
COMBACTE-CDI partner 
Name of co-investigator 
Universitair Medisch Centrum Utrecht (UMCU) 
Prof Marc Bonten 
University of Leeds (UNIVLEEDS) 
Prof Mark Wilcox  
Mrs Kerrie Davies 
Academisch Ziekenhuis Leiden, also acting under 
the name Leids Universitair Medisch 
Centrum (LUMC) 
Prof Ed Kuijper 
National Laboratory of Health, Environment and 
Food (NLZOH) 
Prof Maja Rupnik 
Universitätsklinikum Köln (UKK) 
Prof Oliver Cornely 
Universiteit Antwerpen (UA) 
Prof Herman Goossens 
Istituto Nazionale Malattie Infettive Lazzaro 
Spallanzani, IRCCS (INMI) 
Dr Nicola Petrosillo 
Eberhard Karls Universitaet Tuebingen (EKUT) 
Prof Evelina Tacconelli 
Pfizer Limited (Pfizer) 
Dr Charles Knirsch 
GlaxoSmithKline Biologicals S.A. (GSK Bio) 
Johann Mols 
Nathalie Vos 
Jim Janimak 
BioMerieux (bMx) 
Mark Miller 
AstraZeneca AB/medImmune (AZ/MI) 
Andrew Nyborg 
Sanofi Pasteur (SP) 
Clarisse Demont 
Da Volterra (DAV) 
Annie Ducher 
Pierre-Alain Bandinelli 
 
 
European coordinators 
 
Name 
Function 
Prof Mark Wilcox 
Project lead 
Georgina Davis 
Research Manager 
Kerrie Davies 
Lead Scientist 
 
 
National coordinators 
 
National 
coordinator 
Country 
 
UK 
Kerrie Davies 
University of Leeds 

 
 
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Netherlands 
Dr Ed Kuijper 
Leiden University Medical Centre 
 
Ireland 
Dr Fidelma Fitzpatrick 
RCSI Education & Research Centre, Beaumont Hospital, Beaumont  
 
Spain 
Prof Emilio Bouza 
Catedrático-Jefe de Servicio, Microbiologica Clinica E. Infecciosas. Hospital General 
Universitario 'Gregorio Marañón ' 
 
France 
Dr Frederick Barbut 
Saint-Antoine Hospital, Paris 
 
Italy 
Dr Nicola Petrosillio 
National Institute for Infectious Diseases, Rome 
 
Sweden 
Dr Andreas Matussek 
Karolinska Institute, Stockholm 
 
Austria 
Prof Dr Franz Allerberger 
Austria Agency for Health and Food Safety 
 
Poland 
Dr Hanna Pituch 
Medical University of Warsaw 
 
Greece 
Prof Efthymia Petinaki 
University Hospital of Larissa 
 
Romania 
Dr Daniela Lemeni  
Cantacazino Institute, Bucharest 
 
Slovakia 
Dr Elena Novakova 
Jessenius Faculty of Medicine in Martin, Comenius University in Bratislava 
 
 
 

 
 
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Contents 
Study contributors .................................................................................................................................. 2 
COMBACTE-CDI ................................................................................................................................... 2 
European coordinators ....................................................................................................................... 2 
National coordinators ......................................................................................................................... 2 
1. Introduction ........................................................................................................................................ 7 
1.1 Study rationale .............................................................................................................................. 7 
1.2 Objectives and scope .................................................................................................................... 7 
1.2.1 Work-package 1 objectives .................................................................................................... 7 
1.2.2 Work-package 2 objectives .................................................................................................... 7 
1.3 Study design of COMBACTE-CDI (overview) ................................................................................. 8 
1.4 Ethical and regulatory approvals ................................................................................................ 10 
1.4.1 General overview ................................................................................................................. 10 
1.4.2 Description of studies within COMBACTE-CDI and their associated ethical considerations
 ...................................................................................................................................................... 11 
2. COMBACTE-CDI organisational structure ......................................................................................... 16 
2.1 Funding and Sponsorship ............................................................................................................ 16 
2.2 European coordinator ................................................................................................................. 16 
2.2.1 European Coordinator role .................................................................................................. 16 
2.3 National coordinating centres .................................................................................................... 16 
2.3.1 National Coordinator role .................................................................................................... 17 
2.4 Participating Sites........................................................................................................................ 17 
2.4.1 Participating Site recruitment .............................................................................................. 17 
2.4.2 Participating Site definition .................................................................................................. 17 
2.4.3 Participating Site packs ........................................................................................................ 18 
2.4.4 Materials required ............................................................................................................... 18 
3. Study methods .................................................................................................................................. 19 
3.1 Sample collection study (objective 1.1 and 1.2) ......................................................................... 19 
3.1.1 Study rationale ..................................................................................................................... 19 
3.1.2 Outcome measures .............................................................................................................. 20 
3.1.3 Sampling Criteria for sample collection study ..................................................................... 20 
3.1.4 Study methods ..................................................................................................................... 21 
3.1.5 Sample size ........................................................................................................................... 21 
3.1.7 Data analysis ........................................................................................................................ 22 
3.2 Case control study- objective 1.3 ................................................................................................ 22 
3.2.1 Study rationale ..................................................................................................................... 22 

 
 
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3.2.2 Outcome measures .............................................................................................................. 22 
3.2.3 Sampling criteria for case/control studies (objectives 1.3) ................................................. 23 
3.2.4 Study methods ..................................................................................................................... 23 
3.2.5 Sample size ........................................................................................................................... 23 
3.2.7 Data analysis ........................................................................................................................ 24 
3.3 Case control study- objective 1.4 (risk factors) ........................................................................... 24 
3.3.1 Study rationale ..................................................................................................................... 24 
3.3.2 Outcome measures .............................................................................................................. 24 
3.3.3 Sampling Criteria for sample collection study (objective 1.4) ............................................. 24 
3.3.4 Study methods ..................................................................................................................... 25 
3.3.5 Sample size ........................................................................................................................... 25 
3.3.6 Data collection ..................................................................................................................... 25 
3.3.7 Data analysis ........................................................................................................................ 25 
3.4 Transmission model (Objective 1.5) ........................................................................................... 26 
3.4.1 Study rationale ..................................................................................................................... 26 
3.4.2 Outcome measures .............................................................................................................. 26 
3.4.3 Sampling Criteria for sample collection study (objective 1.5) ............................................. 26 
3.4.4 Study methods ..................................................................................................................... 26 
3.4.5 Sample size ........................................................................................................................... 26 
3.4.6 Data collection ..................................................................................................................... 26 
3.4.7 Data analysis ........................................................................................................................ 26 
3.5 Surveillance study of current CDI detection, surveillance, treatment and attributable costs ... 27 
3.5.1 Study rationale ..................................................................................................................... 27 
3.5.2 Outcome measures .............................................................................................................. 28 
3.5.3 Sampling Criteria for surveillance studies (objectives 2.1-2.5) ............................................ 29 
3.5.4 Study methods ..................................................................................................................... 29 
3.5.5 Sample size ........................................................................................................................... 29 
3.5.6 Data collection ..................................................................................................................... 29 
3.5.7 Data analysis ........................................................................................................................ 29 
3.6 Measure the impact of CDI management on the healthcare system and the impact of current 
control measures (infection control/antibiotic stewardship). (Objective 2.6) ................................. 30 
3.6.1 Study rationale ..................................................................................................................... 30 
3.6.2 Outcome measures .............................................................................................................. 30 
3.6.3 Sampling Criteria for surveillance studies (objective 2.6) ................................................... 31 
3.6.4 Study methods ..................................................................................................................... 31 
3.6.5 Sample size ........................................................................................................................... 31 
3.6.6 Data collection ..................................................................................................................... 31 
3.6.7 Data Analysis ........................................................................................................................ 32 

 
 
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4.0 Data management .......................................................................................................................... 32 
5.0 Reporting......................................................................................................................................... 33 
6.0 References ...................................................................................................................................... 33 
Appendix 1 ............................................................................................................................................ 36 
Appendix 2 Participating site invitation letter ...................................................................................... 37 
 
 
 
 
 

 
 
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1. Introduction  
1.1 Study rationale 
COMBACTE-CDI addresses the specific challenge related to CDI as formulated in the Innovative 
Medicines Initiative (IMI) 2 grant call text,( http://www.imi.europa.eu/apply-funding/closed-
calls/imi2-call-9) and which the consortium was successfully awarded in 2017. The public-private 
partnership in COMBACTE-CDI will quantify the burden of CDI via a large, complex, multi-centre, 
multi-country study, describe current management practices and set-up a consolidated, European, 
CDI, clinical research platform. As indicated within the call, the need and opportunity for public-
private collaborative research is important to meet the clinical challenges provided by CDI in 
European healthcare systems. Therefore, different perspectives and a broad range of diverse 
evidence sources will be gathered and synthesised. A number of key stakeholders have been 
incorporated into this public-private partnership to ensure the success of the collaboration. In 
addition, mapping of and coordination with all stakeholders is integral in our proposal.  
 
COMBACTE-CDI fully addresses the specific scope as formulated. The better quantification of CDI 
burden, understanding of transmission and more complete description of control practices across 
Europe will provide a basis for the further development of public health interventions and practices.  
 
1.2 Objectives and scope  
This project, termed Combatting Bacterial Resistance in Europe – Clostridium difficile Infections 
(COMBACTE-CDI), aims to develop a detailed understanding of the epidemiology and clinical impact 
of CDI. More specifically, this project’s objectives are to:  
 
Align and understand the unmet public health needs relating to CDI;  
 
Identify and quantify the direct and long-term burden of CDI on healthcare systems;  
 
COMBACTE-CDI will synthesise all efforts made at European Union (EU) level on CDI, and crucially 
will align clinical/research consortia that have developed independently, one focussing on CDI and 
the other on AMR, and the CDI-related expertise of 7 EFPIA (industry) partners. This alignment 
between existing research consortia and EFPIA partners will optimize research and managerial 
efficiency. In addition, COMBACTE-CDI will perform a large, complex study to enable the delivery of 
data to answer multiple research questions on the epidemiology of CDI. 
 
1.2.1 Work-package 1 objectives 
The aim of this work-package is to deliver a more complete understanding of CDI epidemiology, risk 
factors and burden of disease across European countries, via a consistent, comprehensive data 
collection approach that builds on and extends existing information, and notably addresses 
knowledge gaps according to the whole healthcare economy within each country 
 
Objective 1.1  Collection and testing of residual diagnostic tissue to determine ‘missed’ cases 
Objective 1.2  Strain distribution (characterisation of strains) 
Objective 1.3  Collection of participant data regarding outcomes of CDI (case/control study)  
Objective 1.4  Collection of participant data regarding risk factors for CDI (Case/control study) 
Objective 1.5  Transmission model constructed from datasets collected in objectives 1.1-1.4. 
 
1.2.2 Work-package 2 objectives 
The aim of this work-package is to highlight current guidelines for CDI diagnosis and treatment, at 
both European and national levels, and measure compliance with these. We will also measure the 
impact of CDI on healthcare systems, and determine if better compliance with guidelines leads to a 

 
 
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lower burden in terms of reducing cases and costs. Objectives and deliverables for WP2 will be 
closely aligned with those for WP1, using a synergistic approach to study design (see Figure 1). The 
timing of the objectives has been planned so that data and analyses generated from WP1 will be 
available for analyses in the required objectives in WP2. In addition, much of the analysis in the cost-
effectiveness objective will be based on data generated from earlier objectives within WP2; the 
timing of these two objectives (O2.5 and 2.6) has therefore been planned accordingly. 
 
Objective 2.1  To highlight current guidelines for disease detection, notification and 
management/control 
Objective 2.2  To determine the heterogeneity in testing density and in following the existing 
guidance (including identification of missed diagnoses and empiric treatment 
approach practices) 
Objective 2.3  To determine the heterogeneity in current surveillance practices and ways to 
improve them. 
Objective 2.4  To describe current treatment pathways for patients with CDI infection and 
recurrences. 
Objective 2.5  To elucidate the overall costs of CDI, including management of multiples episodes 
and re-hospitalisation (healthcare perspectives incl. indirect financial impact on the 
healthcare system). 
Objective 2.6  To measure the impact of CDI management on the healthcare system and the 
impact of current control measures (infection control/antibiotic stewardship). 
 
1.3 Study design of COMBACTE-CDI (overview) 
To achieve our objectives, a large, complex study will be carried out (Figure 1), using proven 
methodology from a previous European wide (20 countries) CDI surveillance project (EUCLID 
network)1. In short, hospitals/laboratories of interest which carry out diagnostic testing of samples 
from both in-patients and community patients (including Long-Term Care Facilities patients) will be 
approached for inclusion. Hospitals/laboratories of interest will be selected to give respective 
coverage, based on population size, in the countries of interest and to represent North, South, East 
and West Europe.  
    
Two study days will be selected, and all diarrhoeal stool samples received on those days by the 
hospital/laboratory in the study will be sent (regardless of age of patient or test requested) to the 
European Coordinating Laboratory (Leeds), where samples will be tested for CDI using the 
recommended ‘optimal’ testing methodology (ESCMID guidelines) (O1.1). Relevant ethical approvals 
will be sought (please see ethics section 1.4).  Results will be compared to those from the submitting 
hospital/laboratory, including if the sample was even tested. Missed cases will be identified, 
enabling an estimation of the true burden of disease in both hospitals and the community.  
Isolates of C. difficile identified from the community and hospital samples will be sent for further 
analysis; including PCR-ribotyping, toxinotyping and whole genome sequencing to identify factors 
that may be driving clustering of strains (O1.2). Additional strains of animal and food origin collected 
from an existing network (NLZOH) will be added to the analysis for comparison between human and 
animal strains.  
 
A clinical report form (CRF) will be designed to capture data for risk factor analysis and outcome data 
on all positive (ECL defined) and a proportion of CDI negative samples; completed 6 months after 
sample collection data (O 1.3 and 1.4 ). Please see section 1.4 for relevant ethical considerations.   
 
All data collected from O1.1, 1.2, 1.3 and 1.4 will be brought into the analysis for transmission 
modelling in O1.5. 
 

 
 
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Hospitals/laboratories and their associated community practices will complete a surveillance 
questionnaire regarding: 
 
Testing policy and methodology; 
 
Surveillance practices; 
 
Guidelines available and compliance; 
 
The criteria for stratification of patients into ‘severe’ or ‘non-severe’ cases  
All these data will be uploaded (by the national coordinators) to a central website, thereby providing 
data for O2.1, 2.2, 2.3, 2.4  
 
All of the data from the previous objectives in WP1 and WP2 will be fed in to the cost effectiveness 
analysis in O2.5 and 2.6 
 
 
 
Figure 1. Schematic representation of study design (WP1 &WP2). A synergistic approach will enable 
completion of all the objectives from WP1 and WP2, with data created from a single, large complex 
study flowing into other objectives for further results analysis. Boxes in blue denote objectives in 
WP1, and those in orange denote for objectives in WP2. Due to the inter-related nature of the 
objectives within both WP1 and WP2, the flow of work has been planned to ensure maximum 
outputs within the timeframe of the grant. The directions of the arrows indicates data flow and, 
therefore, also the timing of activities;. i.e. Strains cannot be analysed (1.2) until after they have 
been collected (1.1). This ambitious comprehensive project design takes influence from several 

 
 
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existing, separate studies; the scope of producing all of these is data from one, well-designed and 
far-reaching study is novel and innovative. 
 
1.4 Ethical and regulatory approvals 
 
1.4.1 General overview 
The research involves residual diagnostic tissue (non-interventional study) and collection of clinical 
data from participants notes on outcomes and risk factors; therefore, the following ethical aspects 
have been considered: 
 
Research on humans (clinical studies); 
 
Observational, non‐interventional, research using data from the treatment of children; 
 
Use of human tissues (biological samples); 
 
Privacy/data collection (data collected from participant databases). 
 
Ethical standards and guidelines compatible with, and equivalent to, those of Horizon2020 will be 
rigorously applied, regardless of the country where the research is taking place. Every partner will 
agree to follow all relevant national and EU legislation, and associated guidelines, relating to the 
conduct of project work. We will ensure that as per H2020 guidelines we adhere to the prevailing EU 
law, being mindful of the fact that the exemptions under Article 89 of the GDPR 2018 may vary 
between countries. We have however tried to mitigate for this during the initial proposal, as we 
sought advice from several countries on their local laws, and this has helped to determine in which 
countries this research will be feasible.  Ethical approval will be sought from every country taking 
part in the study. All of the ethical implications of this project were reviewed by the ethical review 
board of IMI, as part of the grant application, to ensure that it met with all the relevant regulations 
(current and upcoming).  
 
1.4.1.1 Key EU legislation, standards, and guidelines for human research 
Key references that govern the conduct of human research are listed below. This list is not intended 
to be all‐inclusive. 
 
ICH GCP International Conference on Harmonisation of Technical Requirements for 
Registration of Pharmaceuticals for Human Use: Good Clinical Practice Guidelines; 
(2016) 
 
CIOMS: (1) International Guidelines for Ethical Review of Epidemiological Studies (1991); 
and (2) International Ethical Guidelines for Biomedical Research Involving Human 
Subjects (2002); 
 
WMA: Declaration of Helsinki (2008); 
 
UNESCO: Universal Declaration on Bioethics and Human Rights (2005); 
 
CoE: (1) Convention on Human Rights and Biomedicine (Convention of Oviedo), Articles 
15‐18 (1997); and (2) Additional Protocol on Biomedical Research (2005); 
 
EMA/219517/2012 publication ”Towards a robust global framework for conduct and 
oversight of clinical trials”. 
 
EU General Data Protection Regulation (GDPR) 2018 
 

 
 
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1.4.2 Description of studies within COMBACTE-CDI and their associated ethical 
considerations 
Work-package 1 
Objective 1.1 Collection and testing of residual diagnostic tissue to determine ‘missed’ cases 
A large study will be carried out, using proven project methodology from a previous European wide 
(20 countries) CDI surveillance project1. Hospitals/laboratories of interest which carry out diagnostic 
testing of samples from both in-patients and community patients (including Long-term Care facilities 
patients) will be approached for inclusion. The number of hospitals/laboratories will be selected to 
give proportional representation for each country included (with one study site/3 million 
population). In addition, the countries selected will proportionally represent each of the four zones 
of Europe (North, South, East and West, as defined by the UN Geoscheme for Europe), as stark 
differences between hospital CDI burden in these zones was shown in the previous study1.  
 
Two study days will be selected, and all diarrhoeal stool samples received on that day by the 
hospital/laboratory in the study will be sent (regardless of test requested) to the European 
Coordinating Laboratory (Leeds), where samples will be tested for CDI using the recommended 
‘optimal’ testing methodology (ESCMID guidelines). Results will be compared to those from the 
submitting hospital/laboratory, including if the sample was even tested. Missed cases will be 
identified, enabling an estimation of the true burden of disease in both hospitals and the 
community.  
 
All data held by the study team will be pseudonymised to a study number. Pseudonymisation of data 
in this way is in-line with the current EU Data Protection Directive and the upcoming General Data 
Protection Regulation (Spring 2018) specifically Article 89 (1), and all sites will abide by the principals 
of these legislative requirements regarding confidentiality and security of data. Only the original 
submitting laboratory will hold the code to link back to the patient. All samples will be sent for 
processing under the study number only. The link to the participant will be stored at the 
participating hospital/laboratory until after data verification (see objectives 1.3 and 1.4), and then 
destroyed. The anonymised research data (with no personal identifiers) will be securely stored for 10 
years. It is an widely accepted ethical principal to ensure that there is the maximum scientific output 
from the data collected, and future research may re-use the data that has been collected; this will 
only be permitted after this has been agreed by the data guardian for the project and full ethical 
approval for the new analysis has been granted after ethical/regulatory review. 
 
Informed consent is one of the cornerstones of the declaration of Helsinki. However, as detailed in 
the Recommendation Rec (2006)(4) of the Council of Europe Committee of Ministers to member 
states on research on biological material of human origin, Article 22 para 1 (i) ‘If contacting the 
person concerned is not possible with reasonable efforts, these biological materials should only be 
used in the research project subject to independent evaluation of the fulfilment of the following 
conditions; 
• 
The research addresses important scientific interest 
• 
The aims of the research could not reasonably be achieved using biological materials for 
which consent can be obtained and 
• 
There is no evidence that the person concerned has expressly opposed such research’. 
 
Informed consent is not required for the use of pseudoanonimised residual diagnostic material. In 
order to truly assess the burden of CDI we need to alleviate selection bias, which may be introduced 
by ‘selecting’ samples to include in the study. Understanding the true burden of CDI is one of the 
main objectives of the IMI2 grant award. It is also imperative for detection of C. difficile toxin that 
samples are tested within 3 days of receipt (from the participant) or the degradation of toxin may 

 
 
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lead to false negative results. It would be impossible therefore to contact each patient (after their 
sample has been identified) and ask for informed consent to use their sample within this time 
window, especially given that many of these samples will have come from patients in the 
community, who would need to be contacted by post. We feel therefore that it is justified not to 
obtain informed consent to use the residual diagnostic tissue (stool samples) for further CDI testing, 
subject to full ethical approval to do so (after ethical review). Articles 12(1) and 13(2) of the GDPR 
require ‘appropriate measures’ to inform participants of their inclusion in research and their related 
rights. In addition, under Article 9(1) ‘Processing of personal data revealing racial or ethnic origin, 
political opinions, religious or philosophical beliefs, or trade union membership, and the processing 
of genetic data, biometric data for the purpose of uniquely identifying a natural person, data 
concerning health or data concerning a natural person's sex life or sexual orientation shall be 
prohibited’. However, Article 9(2) states ‘Paragraph 1 shall not apply if’ one of a number of clauses 
are met. We believe that Article 9 para 2(j)) applies here; ‘processing is necessary for archiving 
purposes in the public interest, scientific or historical research purposes or statistical purposes in 
accordance with Article 89(1) based on Union or Member State law which shall be proportionate to 
the aim pursued, respect the essence of the right to data protection and provide for suitable and 
specific measures to safeguard the fundamental rights and the interests of the data subject.’ Under 
article 14, para 5(b) there is an exemption from providing information to potential participants 
regarding the research if this would require a disproportionate effort. Given that potential 
participants could be anyone within the catchment area of a particular hospital laboratory (both in-
patients and community patients) it would be exceedingly difficult to ensure that we could inform 
every one of the activity. We therefore believe, based on Article 9 para 2(j) and Article 14 para 5(b) 
the decision to waive consent and waive the requirement to take reasonable measures to inform 
participants of their inclusion, is compliant with the upcoming regulations. There is provision within 
the sub-contracts to ensure that the study sites comply with GDPR, and they should ensure they 
have appropriate measures in place to ensure data confidentiality and non-disclosure.   
 
Collection of human tissue 
We will not be prospectively collecting additional clinical samples. We will collect residual diagnostic 
stool samples that have been submitted to laboratories for microbiological testing. The following 
general principles will apply to all research conducted in COMBACTE‐CDI;  
• 
A human sample is defined as any human tissue or biological material, including any 
portion of an organ, any tissue, skin, bone, muscle, connective tissue, blood, 
cerebrospinal fluid, cells, or any derivative of such human biological material such as 
stem cells or cell lines; and any human biological product, including, but not limited to, 
hair, nail clippings, teeth, urine, faeces, breast milk, and sweat. Our study is limited to 
collecting residual diagnostic stool samples.  
• 
No additional samples will be collected. 
• 
In all cases, following initial planned analysis, remaining aliquots will be stored centrally 
at University of Leeds, UK, for the duration of COMBACTE-CDI to ensure samples can be 
re‐analysed as new data emerges to maximise benefit to society. At the end of the 
project, additional provisions will be made for storage or disposal as appropriate. This 
will be made clear in the ethical approval application. 
• 
All processing of clinical samples and experiments will be performed in laboratories 
equipped with class II safety cabinets. 
• 
When working with biological material, each partner must ensure that strict safety 
procedures are in place and in compliance with national and EU regulations on 
biosafety. 
 
Residual diagnostic faecal samples will be sent to the University of Leeds (European Coordinator) 
from each of the participating sites. Samples will be processed and stored within the Healthcare 

 
 
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Associated Infections Research Group Facilities at University of Leeds for the period of the 
COMBACTE-CDI project. A small portion of a selected number of samples will be sent to another 
COMBACTE-CDI partner (bioMerieux, France) for analysis by the SiMoA test, as this novel technology 
is not yet available elsewhere.  Once SiMoA testing is complete any residual material will be 
destroyed.  
 
Key EU legislation for human tissue use 
Key references that govern the conduct of research on human tissue samples are listed below. This 
list is not intended to be all‐inclusive. 
 
Directive 2004/23/EC on Setting Standards of Quality and Safety for the Donation, 
Procurement, Testing, Processing, Preservation, Storage, and Distribution of Human 
Tissues and Cells 
 
CoE: (1) Convention on Human Rights and Biomedicine (Convention of Oviedo), Articles 
21‐22 (1999) and (2) Recommendation Rec (2006) 4 of the Committee of Ministers to 
Member States on Research on Biological Materials of Human Origin (2006) 
 
WHO: Guideline for Obtaining Informed Consent for the Procurement and Use of Human 
Tissues, Cells, and Fluids in Research (2003) 
 
ITA: Infectious Substances and Diagnostic Specimens Shipping Guidelines (2005) 
 
ISBER: Best Practices for Repositories I: Collection, Storage and Retrieval of Human 
Biological Materials for Research (2005). 
 
EU General Data Protection Regulation (GDPR) 2018 
 
Objective 1.2 Strain distribution (characterisation of strains) 
C. difficile isolates collected from the study samples will be typed by various methods (PCR-
ribotyping, toxinotyping, whole genome sequencing), to determine strain relatedness and 
distribution. All of the residual diagnostic stool samples will be processed in the laboratory facilities 
at the Healthcare Associated Infection Research Group (HCAI) at the University of Leeds. The HCAI 
laboratory is also the Clostridium difficile reference laboratory for the UK and has >20 years of 
experience in handling stool specimens and bacterial isolates derived from the samples. The 
laboratory follows all relevant regulatory processes for the UK, and all staff are fully trained in the 
relevant health and safety procedures. All work is risk-assessed for hazards (microbiological, 
chemical, and procedural) before commencement. Samples are handled in a class II Microbiological 
safety cabinet to protect the researcher. Isolates of C. difficile will be sent to the National laboratory 
for health, environment and food (NLZOH), in Slovenia, a COMBACTE-CDI partner. Again the NLZOH 
has >20 years of experience in handling the organism C. difficile and the laboratory follows all 
relevant regulatory processes, and all staff are fully trained in the relevant health and safety 
procedures. All work is risk-assessed for hazards (microbiological, chemical, and procedural) before 
commencement. All laboratory work will be performed in the facilities mentioned above, which are 
containment level 2 facilities, who comply with all local and national laboratory health and safety 
regulations. Both laboratories follow strict waste disposal policies to ensure the correct disposal of 
microbiological material, to prevent accidental environmental contamination. 
 
No participant identifiable data is associated with the strains; each is identified by a sample code 
only. A dataset from Leeds will be provided to NLZOH which will give the origin (sample source site) 
of the sample from which the isolate came, so that comparisons can be made between isolates 
derived from different sample source sites. This dataset will not contain any personal or health data. 
Bacterial isolates derived from human biological materials do not require consent and/or IEC 
approval for use in research. 
 

 
 
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Objective 1.3 Collection of participant data regarding outcomes of CDI (case/control study) 
and Objective 1.4 Collection of participant data regarding risk factors for CDI (Case/control 
study) 
All CDI positive cases (as determined by the ECL), and a proportion of negative cases (at a ratio of 1 
case to 3 controls) will have clinical data collected (after 6 months) on patient outcomes (O1.3) and 
risk factors for disease (O1.4). Data will be compared to identify differences between countries and 
healthcare providers.  
 
Informed consent 
Informed consent will not be sought, as this is not required for collection of already recorded health 
data, that has been robustly pseudonymised at source. The researchers will never have access to 
personal identifiable information. CDI occurs in all ages, but the prevalence increases in the elderly, 
especially in those with co-morbidities and who are often frail. Recurrent visits to hospital are 
common in elderly patients who lack capacity, due to the complicated nature of their on-going care. 
This places them at increased risk of CDI and to exclude them from this project would be unethical. It 
is imperative that we understand the true burden of disease in these individuals, not just in hospital 
but within the community and the impact it has on their lives in term of morbidity and mortality. 
Whilst their inclusion in the testing of residual diagnostic tissue will allow us to estimate the burden 
of disease, only the case/control study will allow us to measure the impact it has on these 
individuals. The same arguments hold true for children, in which CDI may be an under-recognized 
condition. Understanding the true impact of CDI in these vulnerable groups will inform better 
diagnostic and treatment options for these groups in the future.  
 
  
 
Data protection 
The Data Protection Directive is the cornerstone of EU personal data protection. However, in January 
2012 the European Commission proposed a comprehensive reform of the data protection rules, to 
update and modernise the principles enshrined in the 1995 Data Protection Directive to guarantee 
privacy rights in the future. Central to these proposals is a new regulation (EU General Data 
Protection Regulation (GDPR) 2018), specifically Articles 12 (1), 13 (1) and 89 (1), which to set out a 
general EU framework for data protection and which includes special rules on the collection and use 
of personal data for medical research and public health purposes. Emerging legislation will be 
monitored to ensure the long‐term and on-going legitimacy of COMBACTE-CDI approaches to data 
privacy. 
 
The following general principles will apply to all research conducted in COMBACTE‐CDI.  
 
Data handling will comply with current national and EU legislation, including: 
• 
Personally Identifiable Information (PII) is adequately protected; 
• 
Anonymisation/de‐identification is conducted appropriately; 
• 
Ethical review is completed as required. 
 
Each COMBACTE‐CDI member will be responsible for the confidentiality and security of personal 
identifiable information and for complying with their own SOPs and any shared COMBACTE-CDI 
SOPs. 
 
In general terms, appropriate data protection principles will be observed, including: 
• 
Data are fairly and lawfully processed; 
• 
The amount of data collected is relevant and not excessive; 
• 
All reasonable efforts are taken to ensure data accuracy; 
• 
The data are used in accordance with the rights of the study participant; 

 
 
Protocol version 1.6 17/04/2018 
15 
• 
The data are stored securely; 
• 
Relevant international and national guidance will be consulted. 
 
All data held by the study team will be pseudonymised to a study number. Only the original 
submitting laboratory will hold the code to link back to the participant. All samples will be sent for 
processing under the study number only. After 6 months the original hospital will be asked to collect 
health data on a pseudonymised (study number only) clinical report form on all cases and a 
proportion of controls (CDI negative samples) identified by the central testing laboratory. Under 
GDPR pseudonimised data is classified as personal data. After collection of the data and 
verification/data querying has been completed (within one month of collection) the link will be 
destroyed at the local study sites, to ensure there cannot be any link to participants, rendering the 
data anonymous at this point. To add a further layer of protection all participating sites are also 
pseudonymised, to prevent link back to the site by consortium partners. The University of Leeds will 
hold the link back to the site code on a password protected spreadsheet on secure University of 
Leeds servers during the collection of the data; this will be destroyed after data collection and will 
not be archived.  
 
 
Key EU legislation for human data use 
Key references that govern the use of human data are listed below. This list is not intended to be all 
inclusive. 
 
Data Protection Directive 95/46/EC of the European Parliament and of the Council (1995) 
 
Convention for the Protection of Individuals with Regard to Automatic Processing of 
Personal Data (1985) 
 
Recommendation No. R (97) 5 on the Protection of Medical Data (1997) 
 
Declaration on Ethical Considerations Regarding Health Databases (2002) 
 
Data protection and privacy ethical guidelines of the Experts Working Group on data 
protection and privacy (2009) 
 
Article 29 Data Protection Working Party, Vademecum on Notification Requirements (2006) 
 
Article 29 Data Protection Working Party, Working Document on the processing of personal 
data relating to health in electronic health records (EHR) (2007)  
 
EU General Data Protection Regulation (GDPR) 2018) , specifically Articles 12 (1), 13 (1) and 
89 (1),   
 
Objective 1.5 Transmission model constructed from datasets collected in objectives 1.1-1.4.  
An anonymised  dataset, derived from the previous objectives, will inform the transmission 
modelling. The dataset will be anonymous at this point, as the link-back to the participants will have 
been destroyed. No participant identifiable data will be included in this dataset. All data will be held 
against a study number based on the sample number from objective 1.1. These data may include but 
may not be limited to demographic information, co‐morbidities, patient outcomes 
(mortality/morbidity), recurrence of CDI, linked clinical and laboratory tests and results. The 
principals of data protection discussed above in O1.3 and 1.4, are also applicable to this objective.  
 
Work-package 2 
Objectives 2.1-2.4 Collection of surveillance data from participating sites (hospital and 
community practices) 
Each participating centre and their associated community practices (sample source sites) will also be 
asked to complete a web-based questionnaire on guideline knowledge, compliance, testing policy 
and methodology, surveillance practices, and stratification of ‘severe’ patients. In addition, 10 

 
 
Protocol version 1.6 17/04/2018 
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nursing homes in each of the four regions of Europe will be asked to complete the surveillance form 
to address the knowledge gap with these facilities. Data will be compared to identify differences 
between countries and healthcare providers. Collection of surveillance data from participating sites 
and their associated community practices does not require approval from an ethical review board.  
 
Objectives 2.5 Evaluation of costs based on data collected from O2.1-2.4. 
The dataset used for this data analysis will be derived from objectives 2.1-2.4. This data was 
collected as part of surveillance and therefore does not require approval from an ethical review 
board.  
 
Objective 2.6 Construction of cost effectiveness model based on data in O2.5 and dataset 
generated from work-package 1.  
An anonymous  dataset, derived from the previous objectives in both work-package 1 and work-
package 2, will inform the cost-effectiveness model. No participant identifiable data will be included 
in this dataset. All data will be held against a study number based on the sample number from 
objective 1.1. These data may include but may not be limited to demographic information, co‐
morbidities, patient outcomes (mortality/morbidity), recurrence of CDI, linked clinical and laboratory 
tests and results. The principals of data protection discussed above in O1.3 and 1.4, are also 
applicable to this objective.  
 
2. COMBACTE-CDI organisational structure 
2.1 Funding and Sponsorship 
COMBACTE-CDI receives support from the Innovative Medicines Initiative Joint Undertaking under 
grant agreement no 115523 115620 115737 777362 resources of which are composed of financial 
contribution from the European Union Seventh Framework Programme FP7/2007-2013) and EFPIA 
companies in kind contributions. 
2.2 European coordinator  
The lead partner for COMBACTE-CDI is University of Utrecht; however work packages 1 and 2 are led 
by the University of Leeds and will be coordinated from the Healthcare Associated Infections 
Research Group in The University of Leeds. The European Coordinator for the main study that forms 
the basis for objectives 1.1-1.4 is the University of Leeds.   
2.2.1 European Coordinator role 
• 
Identify National coordinators and oversee study 
• 
Write standard operating procedures  
• 
Write materials required for submission for ethical approval  
• 
Design data capture sheets (manual and electronic) and databases 
• 
Collate data from all national coordinators 
• 
Data analysis and report writing 
• 
Control of budget 
2.3 National coordinating centres 
One national coordinating centre will be recruited per country.  Each national coordinating centre 
will correspond with the European coordinator directly. Each national coordinator will receive a 
budget to cover participating site recruitment, obtaining in-country ethical approval and 
coordinating all study activities within their country including distribution of study transport 

 
 
Protocol version 1.6 17/04/2018 
17 
packaging to participating sites. The national coordinator will also be responsible for the payments 
to participating hospitals for sample collection and follow up Clinical report forms and 
questionnaires. A full list of national coordinators can be found at the beginning of this document. 
2.3.1 National Coordinator role 
 
Obtain ethical approval for the study in their country  
 
Recruit participating sites in their own country to the study 
 
Distribution of study transport packaging to participating sites  
 
Coordination of sampling from all participating sites within their country. 
 
Distribute CRF packs to sites 
 
Coordinate the distribution of surveillance forms to participating sites (and nursing homes in 
selected countries) 
 
Report to the European coordinator  
2.4 Participating Sites  
A small fee will be paid to each participating site to cover labour costs incurred in sending the 
samples. An additional small fee will be payable for each CRF completed as part of the case/control 
study, and the completion of the surveillance form for their site and coordination of surveillance 
form completion (by all associated community practices) as part of work package 2.  
 
2.4.1 Participating Site recruitment 
The number of participating sites to recruit for each country is shown in table 1 in the appendices. 
Participating sites should be selected to cover as broad a geographical area of each country as 
possible, with all geographical regions within a country represented (dependent upon the number of 
participating sites to be included). In addition, the countries selected will proportionally represent 
each of the four zones of Europe (North, South, East and West, as defined by the UN Geoscheme for 
Europe), as stark differences between hospital CDI burden in these zones was shown in the previous 
study. The letter to invite the site to participate should be sent to the laboratory manager/lead 
Clinician of the Microbiology/Pathology department of the chosen site. A copy of the template 
Participating Site invitation letter can be found in Appendix 2.  
2.4.2 Participating Site definition 
A participating site is a hospital/laboratory facility that provides enteric testing and fulfils the criteria 
below.  
 
A sample source site is any location that sends a sample to the participating site to be tested on the 
sampling day (e.g. hospital, community doctor, nursing home, mental health facility).  
 
All participating sites must meet the following criteria: 
• 
Sites must carry out diagnostic testing of faecal samples from both in-patients and 
community patients  
• 
Sites must be able to collect residual diagnostic tissue of all faecal samples received by 
the laboratory on a specified date in May/June and October 2018, and export this to the 
UK the day after collection (transport provided) 
• 
Sites must be able to collect prospective patient data (outcome data) linked to the faecal 
samples provided during the sampling period (based on a provided Case Report Form) 
either directly or via the sample source site 
• 
Sites must be able to collect retrospective patient data (risk factor data) linked to the 
faecal samples providing during the sampling period (based on a provided Case Report 
Form) either directly or via the sample source site 

 
 
Protocol version 1.6 17/04/2018 
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• 
Sites must be able to collect data on attributable costs of CDI at their site (based on a 
provided Case Report Form) 
• 
Sites must be able to provide data in regards to their CDI testing policy and 
methodology, CDI surveillance practices, knowledge of national CDI guidelines and their 
local compliance, and CDI treatment (including according to patient level risk e.g. CDI 
severity) (based on a provided questionnaire). 
2.4.3 Participating Site packs 
Each participating site should be allocated a date, following the site sampling plan provided to each 
National Coordinator.  Each participating site should be sent a pack containing; the instruction leaflet 
to explain how to process the samples for transport to the European Coordinator, transport 
materials, the sampling date and copy of the data collection sheet (sample information only) at least 
a week before the date of sampling. Samples should be stored at 4-8°C until transport.  
2.4.4 Materials required 
2.4.4.1 Provided by European Coordinator 
 
National coordinator study packs (sample collection) 
o Site list and sampling plan 
o Template site invitation letter 
o National Coordinator instructions for sample collection 
 
Participating site study packs (sample collection) 
o Participating site cover letter following confirmation of participation 
o Participating site sampling and shipping instructions 
o Sample collection spreadsheet 
o CDI sample result spreadsheet 
o Template material transfer agreement 
 
Packaging materials for distribution to participating sites (to send samples to the European 
coordinator via DHL). 
 
National Coordinator study pack (case control study) 
 
Participating site study pack (case control study) 
 
National Coordinator study pack (surveillance study) 
 
Participating site study pack (surveillance study) 
 
Copy of UK ethical approval 
 
Study packs will be distributed to National Coordinators prior to the relevant sub-study (sample 
collection/case control/surveillance). 
3.2.2 Provided by National Coordinator 
 
Completed site list and sampling plan 
 
Copy of in-country ethical approval 
3.2.3 Provided by Participating site 
 
Completed and signed material transfer agreement 
 
Faecal samples and completed CDI sample result spreadsheet 
 
Clinical data on case control studies (CRF completion) 
 
Data for surveillance questionnaire 

 
 
Protocol version 1.6 17/04/2018 
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3. Study methods 
3.1 Sample collection study (objective 1.1 and 1.2) 
3.1.1 Study rationale 
Only two studies (in the UK & the Netherlands) have determined incidence rates, clinical 
characteristics and outcome of community-acquired CDI (CA-CDI), both reporting CDI positivity in 
1.5-2.1% of patients attending a general practitioner because of diarrhoea1,3. Given the number of 
patients that may never even be tested, this may well represent an underestimate of the true 
incidence of CA-CDI. The burden of CDI in LTCF and its relation with that in neighbouring hospitals is 
largely unknown. A systematic review identified only 8 studies for critical appraisal with none 
containing economic evaluations of interventions4. The estimated incidence of CDI in LTCF was 
0.41/10,000 residents per year. ECDC initiated surveillance of HAIs and antimicrobial use in 
European LTCF (HALT) project, including two point prevalence surveys5,6 in >750 LTCF in ~20 
European countries. This study reported HAI prevalence of 2.4-3.4%, of which 5.1% represented 
gastrointestinal infections; of which 18% were due to C. difficile. However, CDI was not surveyed 
systematically, and no information was collected on the diagnostic methodology. We aim to address 
this gap. 
 
PCR ribotyping is the method of choice for typing C. difficile strains. Currently, >800 PCR ribotypes, 
(designated 001, 002…) are known; the reference ribotype collection is held by the co-applicant’s 
(MHW) laboratory in Leeds, UK. Knowledge of strain distribution improves understanding of 
epidemiology (outbreaks), transmissions (within hospital, between reservoirs, e.g. animal to human) 
and potential differences in virulence. Indeed, some PCR ribotypes (e.g. ribotype 027, 078) appear 
more virulent, with emerging evidence that some are also more transmissible7.  
 
PCR ribotype distribution varies between European countries and over time, with some types 
remaining prevalent for decades (e.g. ribotypes 014, 002, 027, non-toxigenic 010)8-11 However, these 
studies were based on isolates from hospitalised patients collected within limited time intervals. 
Notably, comparable data on a European scale are lacking for CA-CDI and CDI in non-hospital 
settings. From studies at local or national level, it is known that different strains would be more 
likely associated with hospital (e.g. ribotypes 027, 176) and community (e.g. ribotypes 078, 244) 
infection, although there is also some overlap12. 
 
Strains collected from the main study (O1.1) will be compared, by PCR-ribotyping, along with basic 
clinical data. Comparisons will be made between strains identified from the community and those 
from hospital settings, including specific analysis of those within long-term care facilities. 
Comparison will be made between the strains isolated from cases and those who are colonised (no 
C. difficile toxin detected). Previous work within the consortium has shown that there is variable 
performance for current toxin assays for different PCR-ribotypes of C. difficile, but lack of a highly 
sensitive toxin detection method has so far hampered efforts to investigate this further13. Further 
analyses of those strains that were isolated from patients with toxin levels that were only detectable 
by the innovative highly sensitive SiMoA assay will help to elucidate a correlation between certain 
strains and low levels of toxin produced in-vivo.  
 
Recent data show that certain C. difficile strains may cause disease and yet only produce binary 
toxin, but not toxins A and B (A-B-CDT+ strains). These strains are likely to be missed by conventional 
microbiological testing which detects the main toxins A and/or B14. Other binary toxin producing 
strains (A+B+CDT+, A-B+CDT+) are associated with increased relapse rates. More data are needed to 
determine the prevalence of such strains and what their true potential is to cause CDI. In addition, 
therefore, strains will be toxinotyped; identification of the variation in toxin genes. Comparisons will 
be made between those strains seen within hospital and community settings, with emphasis on 

 
 
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long-term healthcare facilities. In addition, as with the PCR-ribotyping results, analyses will 
determine if there is any correlation between different toxinotypes and the healthcare setting from 
which they are isolated. Molecular variations in toxin genes and toxins could influence molecular 
diagnostic assays and vaccine approaches. The current European toxinotyping scheme is run by MR 
(NLZOH), who will be leading this task.  
 
Further analysis of strains will be completed by using highly discriminatory fingerprinting (whole 
genome sequencing), comparing within and between healthcare settings. Whole genome 
sequencing (WGS) studies have recently demonstrated the value of this approach for supplementing 
conventional surveillance based on ribotyping, and so determine the extent of true linkage between 
CDI cases and other patients. We will employ WGS to examine the true relatedness of strain types 
within and between CDI settings and identify factors that may be driving clustering of strains.  
Unpublished pilot data show that for some, but not all, common ribotypes there is evidence for 
widespread dissemination of very closely related strains. We will seek to validate these findings to 
deliver innovative information about potential reservoirs for infection and strain transmission. 
 
There is emerging evidence that transmission of C. difficile is more complex than originally assumed; 
hence, CDI case-case transmission, outside of the outbreak setting, is not the dominant route of 
spread of C. difficile. This has led to several on-going research projects by the participants (some 
with manuscripts submitted) to examine other possible reservoirs/routes of transmission of 
C. difficile. There is increasing recognition that the food chain may be an important vehicle for 
C. difficile dissemination, which we recognise. Additional strains (matched in size with strains from 
main study) from animal and food sources will be collected (contemporaneously) from an existing 
network, collated by NLZOH. These strains will also be analysed, as above, and then comparisons 
made between strains of human and animal origin.  
3.1.2 Outcome measures  
Primary outcome measure: Number of missed cases of CDI - Comparison of test result at 
participating site and test result at European Coordinating Laboratory (using optimised testing). 
Objective 1.1 
 
Secondary outcome measures:  
 
Number of missed cases of CDI - Comparison of test result at participating site and test 
result at European coordinating laboratory (using novel highly sensitive C. difficile assay for 
toxins A and B). 
 
Types of C. difficile, as measured by PCR-riboyping, toxinotyping and whole genome 
sequencing. Objective 1.2 
 
3.1.3 Sampling Criteria for sample collection study 
3.1.3.1 Inclusion criteria   
ALL unformed faecal samples (takes the shape of the container and/or stick will not remain vertical 
when inserted into the sample) from ALL patients (ALL ages) sent to the microbiology department of 
participating sites on their allotted testing day (irrespective of test requested).  
 
3.1.3.2 Exclusion criteria 
Only one sample per patient can be included 
 

 
 
Protocol version 1.6 17/04/2018 
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3.1.4 Study methods 
Two study days will be selected, and all diarrhoeal stool samples received on that day by the 
participating site in the study will be sent (regardless of test requested) to the European 
Coordinating Laboratory (Leeds), where samples will be tested for CDI using the recommended 
‘optimal’ testing methodology (GDH/Toxin (Techlab, US) testing on the DS2, as per ESCMID 
guidelines), reference methods (cell-cytotoxicity neutralisation assay and cytotoxigenic culture) and 
a novel, Single-molecule Array (SiMoA) immunoassay platform for C. difficile toxins A and B. Samples 
will also be tested for other potential gastrointestinal pathogens (using the bioFire Film array 
molecular method), to investigate either co-infections or alternative cause of diarrhoea (where C. 
difficile negative). Results will be compared to those from the submitting hospital/laboratory, 
including if the sample was even tested. The SiMoA test will be carried out at bioMerieux (France), a 
COMBACTE-CDI partner, as the novel technology is currently not available elsewhere. A small 
portion of selected samples will be sent to BioMerieux for this purpose. They will only be labelled 
with the study number.  
 
All data held by the study team will be pseudoanonymised to a study number. Only the original 
submitting laboratory will hold the code to link back to the patient. All samples will be sent for 
processing under the study number only.  
 
All of the residual diagnostic stool samples will be processed in the laboratory facilities at the 
Healthcare Associated Infection Research Group (HCAI) at the University of Leeds. The HCAI 
laboratory is also the Clostridium difficile reference laboratory for the UK and has >20 years of 
experience in handling stool specimens and bacterial isolates derived from the samples. The 
laboratory follows all relevant regulatory processes for the UK, and all staff are fully trained in the 
relevant health and safety procedures. All work is risk-assessed for hazards (microbiological, 
chemical, and procedural) before commencement. Samples are handled in a class II Microbiological 
safety cabinet to protect the researcher. 
 
C. difficile isolates collected from the study samples will be typed by various methods (PCR-
ribotyping, toxinotyping, whole genome sequencing), to determine strain relatedness and 
distribution. Isolates will be processed at both the Healthcare Associated Infection Research Group 
(HCAI) in Leeds and the National laboratory for health, environment and food (NLZOH), in Slovenia, a 
COMBACTE-CDI partner. Again the NLZOH has >20 years of experience in handling the organism C. 
difficile and the laboratory follows all relevant regulatory processes, and all staff are fully trained in 
the relevant health and safety procedures. All work is risk-assessed for hazards (microbiological, 
chemical, and procedural) before commencement. All laboratory work will be performed in the 
facilities mentioned above, which are containment level II facilities, who comply with all local and 
national laboratory health and safety regulations. Both laboratories follow strict waste disposal 
policies to ensure the correct disposal of microbiological material, to prevent accidental 
environmental contamination. 
 
In addition, NLZOH will collect contemporaneous isolates of C. difficile from environmental and food 
sources, within the same countries as the human samples. These will be typed using the 
methodologies described above.  
3.1.5 Sample size 
It is impossible to accurately predict the number of samples that will be collected, as this is based on 
what is collected on those selected days. However, given our previous experience of collecting 
samples from 482 hospitals across 20 European countries during the EUCLID study1, we are able to 
estimate that we would receive 1620 samples from the 120 hospitals included (see Table 1) on each 
sampling day, with an average positivity rate (for toxin positive samples) of 10%, which would give 
240 toxin positive samples. The culture positive rate will likely be ~15%; 252 strains would therefore 

 
 
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be collected for further typing per sampling day. The number of samples and the positivity rate for 
CDI will be examined after the first sample day collection, to decide if the next sampling day should 
be carried out on one (as planned) or two days. Contingency has been added to the budget to cover 
the additional costs of adding a testing day to increase sample numbers.  
 
We will aim to collect a similar number of contemporaneous isolates from the environmental and 
food samples.  
3.1.6 Data collection  
In addition to the laboratory data, data will be collected on the following variables; 
• 
Sample source sites (hospital/GP, nursing home) 
• 
CDI test result for that sample at the submitting laboratory 
3.1.7 Data analysis 
Results of the CDI testing at the ECL will be compared with the original result at the participating 
site, with ‘missed’ diagnoses recorded (either due to false negative result at participating site, or 
sample not tested at participating site). Results of ‘missed’ diagnoses will be compared between 
countries and sample source sites (hospital/GP/nursing home). In addition, the number of co-
infections and alternative GI pathogens (in CDI negative samples) will be compared between 
countries and sample source sites, to determine difference between in-patient and community 
populations.  
 
Types of C. difficile (by PCR-ribotyping, and toxinotyping) will be compared between countries, 
settings (community/hospital), cases/colonised participants and those detected by different 
commercial testing methodologies (optimised method vs SiMoA). Whole genome sequencing will be 
used to examine the true relatedness of strain types within and between settings 
(community/hospital). Comparison will be made between the types of C. difficile isolated from 
human, environmental and food sources. In addition, whole genome sequencing will be used to 
examine the true relatedness of strain types between sources.  
3.2 Case control study- objective 1.3 (outcomes) 
3.2.1 Study rationale 
Morbidity and all-cause mortality in relation to CDI within hospitals have been extensively studied8, 
15-17. However, CDI outcomes in community settings, including nursing/convalescent homes, are 
poorly understood. In addition, research studies have concentrated on one area of healthcare, i.e. 
either a hospital or a community setting, without examining the interplay between these two 
settings. It is plausible that a patient with hospital acquired CDI, who recovers and is discharged into 
the community, may suffer a recurrence that is diagnosed empirically (i.e. without testing) or via a 
different laboratory from the index diagnosis. Currently, such outcomes are not captured by either 
surveillance or research. Given that CDI cases occur within all parts of the healthcare system, it is 
imperative that data relating to recurrence, morbidity and mortality from the healthcare system as a 
whole are studied to understand the true disease burdens and the interplay between settings.  
3.2.2 Outcome measures   
Primary outcome measure: Crude mortality at 30 days and 6 months after the sample was taken will 
be compared between case and controls, those that received a correct diagnosis at the original 
hospital and those that were 'missed' (either due to lack of testing or inadequate testing) and 
between community and hospitalised patients. 
 
Secondary outcome measures: Patient length of stay, severity of infection (e.g. WCC, serum albumin, 
temperature), need for escalation of medical/surgical care due to gastrointestinal disease, post-
infectious sequelae. 

 
 
Protocol version 1.6 17/04/2018 
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3.2.3 Sampling criteria for case/control studies (objectives 1.3) 
3.2.3.1 Inclusion criteria   
 
The study will collect data on cases/controls identified during the sample collection study (section 
3.1 above) 
 
1. Any patient who had a sample sent to the European coordinating laboratory and was positive 
for CDI (GDH positive/toxin positive) at the European coordinating laboratory 
2. A matched (age/gender) patient from the same participating site as a patient in criteria 1, but 
that had a negative result for CDI at the European Coordinating Laboratory.  
 
3.2.3.2 Exclusion criteria   
None 
 
3.2.4 Study methods 
All CDI positive cases (as determined by the ECL), and a proportion of negative cases (at a ratio of 1 
case to 3 controls) will have clinical data collected (after 6 months) on participant outcomes. Data 
will be compared to identify differences between countries and healthcare providers. 
 
3.2.5 Sample size 
We estimate, based on previous experience (please see section on sample size in 3.1.4), that we 
would receive 480 C. difficile toxin positive samples over the two sampling days. These would form 
the cases for the case/control study. Controls (participants with C. difficile negative samples 
submitted during O1.1) would be recruited at a rate of 1:3, from the same participating centre. 
Where possible these will be age and gender matched. Given that we will only have a predefined 
group of participants to select from; it is possible that we are unable to completely match all 
controls, apart from at the geographical level.  
3.2.6 Data collection  
Data will be collected via a standardised CRF, with a full supporting SOP. The CRF will be completed 
by the clinical care team at the participating site that submitted the sample (during O1.1). The CRF 
will not contain any participant identifiable data; all data will be held against a pseudoanonymised 
study code, with the link back held by the clinical care team only. COMBACTE-CDI investigators will 
not access participant’s notes or access the link back. Once the CRF data has been collected and 
validated and verified the link-back will be destroyed by the local study site. 
 
Data will be collected on the following variables on outcomes of CDI; 
• 
Evidence of other enteric pathogens (+/- 7 days of the original sample)? 
• 
Symptoms at time of the sample 
• 
Alternative cause for the patient’s diarrhoea documented (e.g. laxatives, pathogen)? 
• 
Duration of diarrhoea/non-normal bowel habit? 
• 
Primary/recurrent CDI 
• 
Antibiotics at the time of the sample or the preceding 3 months? 
• 
Evidence of colitis? 
• 
Evidence of post-infectious diarrhoea 

 
 
Protocol version 1.6 17/04/2018 
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• 
CDI Severity markers (Temperature (nearest reading +/- 3 days of the sample), 
serum creatinine (nearest reading +/- 3 days of the sample), White cell count 
(nearest test +/- 3 days of the sample) 
• 
Treatment for CDI episode  
• 
Was treatment for CDI/diarrhoea escalated – medical/surgical? 
• 
Outcome 30 days and 6 months after the sample (discharged, in-patient, died) 
• 
Length of stay 
 
3.2.7 Data analysis 
Comparisons will be made between recurrence/Morbidity/Mortality (crude and attributable) rates in 
different healthcare settings and countries. Outcomes will also be compared for different strain 
types (as identified in O1.2).  
 
3.3 Case control study- objective 1.4 (risk factors) 
3.3.1 Study rationale 
Many studies have examined risk factors associated with CDI; there are, however, three major 
deficiencies of existing data; (1) most studies have been performed in the epidemic setting and so 
may be biased by a preponderance of cases caused by one or a few strain types; (2) most studies are 
in hospitalised patients; (3) most were biased to patients receiving antibiotics, identified antibiotic 
use as the most important risk factor, and so reduced the potential to identify other modifiable risk 
factors. Conversely, several studies have shown that exposure to prescribed antibiotics was not 
present in up to 50% of CA-CDIs2,3. CA-CDI, therefore, may be linked to other microbiota disturbing 
factors (other drugs, diet, etc.) currently not recognised as risk factors for CDI. Recent data have 
questioned whether PPIs are truly associated with CDI; earlier results may be confounded by choice 
of control patients19.  
 
A recent systematic review of risk factors associated with CDI recurrence and mortality noted that 
important variables were inconsistently reported, such as previous episodes and antibiotic use17. 
Also, there was substantial heterogeneity and methodological limitations, including sample sizes, 
definitions of outcomes and follow-up periods, thus precluding a meta-analysis. We have 
considerable experience of determining risk factors associated with CDI2,3,20. Notably, a recent 
University of Leeds study has determined CDI risk factors for cases determined according to a 
standardised diagnostic algorithm (data unpublished).  
 
3.3.2 Outcome measures   
Primary outcome measure: Episode of CDI 
 
3.3.3 Sampling Criteria for sample collection study (objective 1.4) 
3.3.3.1 Inclusion criteria   
The study will collect data on cases/controls identified during the sample collection study (section 
3.1 above) 
 
1. 
Any patient who had a sample sent to the European Coordinating Laboratory and was 
positive for CDI (GDH positive/toxin positive) at the European Coordinating Laboratory 
2. 
A matched (age/gender) patient from the same participating site as a patient in criteria 
1, but that had a negative result for CDI at the European Coordinating Laboratory.  

 
 
Protocol version 1.6 17/04/2018 
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3.3.3.2 Exclusion criteria   
None 
 
3.3.4 Study methods 
All CDI positive cases (as determined by the ECL), and a proportion of negative cases (at a ratio of 1 
case to 3 controls) will have clinical data collected (after 6 months) on possible risk factors for CDI. 
Data will be compared to identify differences between countries and healthcare providers. 
3.3.5 Sample size 
We estimate, based on previous experience (please see section on sample size in 3.1.4), that we 
would receive 480 C. difficile toxin positive samples over the two sampling days. These would form 
the cases for the case/control study. Controls (participants with C. difficile negative samples 
submitted during O1.1) would be recruited at a rate of 1:3, from the same participating centre. 
Where possible these will be age and gender matched. Given that we will only have a predefined 
group of participants to select from; it is possible that we are unable to completely match all 
controls, apart from at the geographical level. The same participants will be recruited for both 
case/controls studies (O1.3 and 1.4), using harmonised documentation (PIS, ICF and CRF).  
 
3.3.6 Data collection 
Data will be collected via a standardised CRF, with a full supporting SOP. The CRF will be completed 
by the clinical care team at the participating site that submitted the sample (during O1.1). The CRF 
will not contain any participant identifiable data; all data will be held against a pseudoanonymised 
study code, with the link back held by the clinical care team only. COMBACTE-CDI investigators will 
not access participant’s notes or access the link back. Once the CRF data has been collected and 
validated and verified the link-back will be destroyed by the local study site. 
 
Data will be collected on the following variables to examine risk factors for CDI; 
 
Prior treatment with cytotoxic drugs 
 
Prior treatment with PPIs 
 
Underlying diagnoses 
 
Co-morbidities 
 
Previous antibiotics (preceding 12 weeks) 
 
Previous hospitalisation (preceding 12 weeks) 
 
Admission history (if an in-patient) 
 
Residential status (if in the community), e.g. home/nursing home 
 
Previous CDI 
 
Kissing contact with an infant 
 
Known contact with CDI case 
 
Previous wards/movement within hospital 
 
3.3.7 Data analysis 
Odds ratios for different risk factors for CDI will be compared between settings 
(community/hospital) and countries. Risk factor comparison will also be made between different 
strain types (as identified in O1.2).  

 
 
Protocol version 1.6 17/04/2018 
26 
 
3.4 Transmission model (Objective 1.5) 
3.4.1 Study rationale 
Consideration of patient movement in hospitals, LTCFs and the community is crucial in 
understanding the transmission dynamics and control of C. difficile21. Data–driven estimates of CDI 
burden outside hospital settings are lacking (see 1.1). However, frequent interactions between 
hospital and LTCF populations mean their dynamics are inherently linked. Asymptomatic 
colonisation in LTCF is a potential reservoir for transmission22; reported rates vary widely (4-50%)23, 
likely due to differences in healthcare system structures, and variations in definitions and long-term 
care case-mix24. Overall, C. difficile transmission dynamics are poorly understood. Our molecular 
studies and statistical modelling17,29 have questioned the importance of symptomatic hospital 
patients, the traditionally assumed primary source of CDI acquisition 
3.4.2 Outcome measures   
Primary outcome measure: A C. difficile transmission model describing the relatedness of each part 
of each target country’s healthcare economy 
Secondary outcome: A modelling tool that enables country and setting-specific CDI burden 
estimation and evaluation of intervention (cost-) effectiveness. 
3.4.3 Sampling Criteria for sample collection study (objective 1.5) 
3.4.3.1 Inclusion criteria   
Data generated from the sample collection study and the case/controls studies described above.  
3.4.3.2 Exclusion criteria   
None.  
 
3.4.4 Study methods 
All CDI positive cases (as determined by the ECL), and a proportion of negative cases (at a ratio of 1 
case to 3 controls) will have clinical data collected (after 6 months) on possible risk factors for CDI. 
Data will be compared to identify differences between countries and healthcare providers. 
3.4.5 Sample size 
This is based on the collection of data from the previous objectives.  
 
3.4.6 Data collection 
An anonimised dataset of the combined data from objectives 1.1-1.4 (as above) will be included in 
the transmission modelling.  No additional data will be collected.  
 
3.4.7 Data analysis 
We will use novel Markov chain Monte Carlo algorithms, to quantify transmission from different 
settings across different countries, using the repeated point-prevalence data collected in O1.1, along 
with the analysis on strain distribution (O1.2), impact on patient outcomes (O1.3) and risk factors 
(O1.4). 
 
In addition, we aim to develop an innovative tool to enable country and setting-specific CDI burden 
estimation and evaluation of intervention (cost-)effectiveness. The model framework will be easily 
adaptable to different countries and settings. For example, so that settings of high antimicrobial use 
or CDI incidence can be simulated. The model could also provide a unique framework to perform 

 
 
Protocol version 1.6 17/04/2018 
27 
transmission dynamic model-based (cost-)effectiveness evaluations of interventions such as 
antimicrobial stewardship (e.g. targeting in- vs. outpatient prescribing) or admission screening 
 
3.5 Surveillance study of current CDI detection, surveillance, treatment and 
attributable costs 
3.5.1 Study rationale 
This part of COMBACTE-CDI (work-package 2) will highlight current guidelines for CDI diagnosis and 
treatment, at both European and national levels, and measure compliance with these. We will also 
measure the impact of CDI on healthcare systems, and determine if better compliance with 
guidelines leads to a lower burden in terms of reducing cases and costs. Objectives and deliverables 
for WP2 will be closely aligned with those for WP1, again using a synergistic approach to study 
design (see Figure 1). The timing of the objectives has been planned so that data and analyses 
generated from WP1 will be available for analyses in the required objectives in WP2. In addition, 
much of the analysis in the cost-effectiveness objective will be based on data generated from earlier 
objectives within WP2; the timing of these two objectives O2.5 and 2.6 has therefore been planned 
accordingly. 
 
Highlight current guidelines for disease detection, notification and management/control 
Objective 2.1 
Five international societies have recently issued/updated guidelines on diagnosis, prevention, 
treatment and control of CDI.20,34-37 This reflects the increasing burden of CDI worldwide, new 
knowledge, and because some issues remain contentious. Underdiagnosis, as reported by our 
EUCLID study1 is a main contentious area. Underdiagnosis can depend on suboptimal laboratory 
testing for CDI and/or lack of suspicion by some clinicians, which could be overcome by automatic 
testing for CDI on every liquid stool sample. Overdiagnosis of CDI is also a challenge; it is important 
to distinguish CDI infection from colonization in hospitalised patients with diarrhoea25.  
 
One of the pillars of control of CDI is communication through reporting, but notification represents 
another conundrum not addressed by guidelines. In many countries, both in community and 
healthcare settings, there is mandatory notification for infectious diarrhoea, but not for CDI. In a 
recent systematic survey of clinical practice guidelines, showing that recommendations are often not 
consistent with available evidence, there is no mention of notification strategies for CDI26. 
 
 
Determine the heterogeneity in testing density and in following the existing guidance on 
testing practices (objective 2.2) 
We will aim to fill the current knowledge gaps about hospital identification of missed diagnoses 
(either due to lack of clinical suspicion or inadequate laboratory diagnostics).  Existing programmes 
collecting European data on CDI testing heterogeneity are limited by either the number of hospitals 
surveyed per country or the number of countries13,27.  Also, current programmes focus on hospital 
acquired CDI; thus, data for the entire healthcare economy are lacking. More robust data 
comparisons could be made by expanding existing networks. 
 
Determine the heterogeneity in current surveillance practices and ways to improve them 
(objective 2.3) 
An evaluation of changes in local laboratory CDI diagnostics in the period 2011-14 concluded that an 
increased use of ‘optimal’ diagnostics for CDI surveillance should be further promoted27. Results of a 

 
 
Protocol version 1.6 17/04/2018 
28 
web-based electronic form sent to national coordinators showed that surveillance methods also 
varied considerably between countries27. This illustrates the need for a harmonised European 
protocol to allow consistent monitoring of the epidemiology of CDI at European level. To support 
this further, a new CDI diagnostic guidance document has been published in 201628. We aim to 
determine the heterogeneity in current surveillance practices within the study source sites recruited 
for the prospective study in O1.1, and ways to improve them (by compiling and comparing 
information on the current surveillance systems at both European and country levels, and to 
determine the best strategy for future surveillance. 
 
Describe current treatment pathways for patients with CDI infection and recurrences 
(objective 2.4) 
Current European treatment guidelines were published in 201429, but there are a paucity of data on 
national guidance and compliance with these guidelines. Guidelines often focus on in-patients and 
we will explore whether altered guidance is needed for different healthcare settings (see WP1). By 
building on networks used to establish variations in CDI diagnosis (EUCLID, LUCID, CLOSER), we will 
measure variability in treatment policies across Europe, focussing on the treatment/prevention of 
recurrent CDIs. By using the network of hospitals/laboratories established for the main study 
(O1.1.2), and their associated community practices, we will close the knowledge gaps within the 
community setting, with focus on long-term healthcare facilities. Comparisons will be made between 
healthcare locales/types and countries. Because information of outpatient costs of CDI are scarce, 
these new networks will also be used for further health-economic evaluations to analyse direct and 
indirect costs of patients with CDI outside hospitals (this data will feed in to O2.5 and 2.6). 
 
Current guidelines on CDI treatment take into account both initial and recurrent episodes. For initial 
CDI treatment, pathways are mainly based on stratification on the severity of the disease; for 
recurrence, on the frequency of CDI episodes. The main concerns/knowledge gaps of these 
guidelines are represented by 1) heterogeneous definitions of severity 1,30 increasing rates of 
resistance to the often 1st-line treatment option metronidazole 18,31,32 lack of data on the risk of 
poorer outcomes in selected populations with CDI e.g. inflammatory bowel disease33. Recently, 
Kanhafer et al30 demonstrated that the proportion of severe CDI cases varied widely (11-59%) 
depended on the definitions/risk factors used. This is important not only for stratifying patients for 
management strategies, including new treatments, but also for comparing data across studies. Some 
risk factors are also controversial; albumin levels could be the cause or effect of severe CDI30. Studies 
should be addressed to better identify the role of these factors. 
 
Elucidate the overall costs of CDI, including management of multiples episodes and re-
hospitalisation (healthcare perspectives incl. indirect financial impact on the healthcare 
system). (Objective 2.5) 
There are current knowledge gaps regarding the costs of managing CDI in various healthcare 
settings. While some data exist on European tertiary care hospitals34,35, the majority of CDI patients 
receive treatment in other hospital categories. Outside hospitals, little is known about costs in 
outpatients and nursing homes/LTCF. There are further knowledge gaps regarding costs of recurrent 
CDI. 
 
3.5.2 Outcome measures   
This section of COMBACTE-CDI is surveillance of existing guidelines, including local knowledge and 
compliance. There is no fixed outcome measure for this.  
 

 
 
Protocol version 1.6 17/04/2018 
29 
3.5.3 Sampling Criteria for surveillance studies (objectives 2.1-2.5) 
3.5.3.1 Inclusion criteria   
All participating sites (hospital based), and their associated community healthcare providers who 
submitted samples on the sampling days (sample source sites). In four selected countries 
(representing the four zones of Europe)  10 nursing homes that did not submit samples on the 
sampling days will also be asked to complete the surveillance questionnaire, as there are a paucity of 
data on CDI regarding these facilities.   
3.5.3.2 Exclusion criteria   
None  
 
3.5.4 Study methods 
All included sites (see definition in inclusion criteria 3.5.3.1) will be asked to fill in a surveillance 
questionnaire asking about knowledge and compliance with current guidelines on CDI diagnosis, 
notification and control, surveillance, treatment and will collect attributable costs. Data will be 
compared to identify differences between countries and healthcare providers. 
 
For cost data we will gather data on direct medical and non-medical costs of CDI in different 
healthcare settings to address the gaps and so elucidate the costs in all healthcare settings.  
Direct medical costs will include personnel (i.e. medical, nursing and technical service) as well as 
material costs (e.g. material and drug products). Direct non-medical costs will include for example, 
costs for carriage and expenditures of relatives 
3.5.5 Sample size 
This is based on the number of participating sites (n = 120) and the number of associated community 
healthcare providers that submit samples on the sampling days, plus up to 40 nursing homes.  
 
3.5.6 Data collection 
An online data capture system will be used to collect the data, built within the COMBACTE online 
network. This data will not contain any personal data, but details about individual sites knowledge 
and compliance with existing guidelines. Each site will have a unique anonymised study identifier for 
the online data capture system; investigators will therefore not be able to identify sites.  
 
The main dataset will then be accessed via the online portal by the investigators for each of the 
objectives. Rule bases will be added to ensure that each objective investigator can only access data 
relevant to their objective.  
 
3.5.7 Data analysis 
Comparisons of knowledge and compliance with guidelines for disease detection, notification and 
management/control will be made between countries and healthcare settings (community/hospital). 
Particular attention will be focussed on the dates of policies and the length of time it takes for 
European/national guidance to filter down to a local level. This may highlight a possible area for 
improvement in guideline communications. 
 
Comparisons between testing policies and methodologies will be made between countries and 
healthcare settings (community/hospital). In addition, data from 1.1 on ‘missed’ case rates in these 
settings will allow identification of ‘poor practice’.  
 

 
 
Protocol version 1.6 17/04/2018 
30 
Data will be compiled on current surveillance programmes for CDI and compliance with these, in 
different countries and settings. This will be used to determine a ‘best-practice model’ for 
surveillance and investigate mechanisms for increasing engagement with surveillance.  
 
Comparisons of knowledge and compliance with guidelines for treatment pathways will be made 
between countries and healthcare settings (community/hospital). In particular comparisons will be 
made of risk stratification of patients (via severity criteria) between different healthcare settings and 
countries. Again this will be used to determine a ‘best-practice model’ for risk stratification.  
 
We will aim to elucidate the overall costs in all healthcare settings. Data will be compared on the 
costs of CDI in different healthcare settings, and to address knowledge gaps regarding managing CDI 
outside hospitals, including in nursing homes. For the most comprehensive analyses on overall costs 
associated with CDI, a micro-costing approach will be combined with published data of the 
respective health systems for all health-economic evaluations. Additionally, by using data from 
Objective 1.3, indirect costs, i.e. productivity losses due to illness related disability and death before 
retirement age, in inpatient and outpatient settings will be addressed from societal perspectives to 
measure the full impact of CDI. The human capital and/or friction cost approaches will be used. All 
calculated costs will be expressed in an appropriate currency (e.g. EURO, €). Furthermore, 
discounting of all calculated costs will be performed by taking account of country and annual 
discount rates (normally 3% - 5% per year). Multivariate analyses will be performed to estimate 
relevant components of cost. Sensitivity analyses will be performed by using different annual 
discount rates.  
 
3.6 Measure the impact of CDI management on the healthcare system and 
the impact of current control measures (infection control/antibiotic 
stewardship). (Objective 2.6) 
 
3.6.1 Study rationale 
Although it appears self-evident that CDI can be prevented by infection control and antimicrobial 
stewardship, this has only been demonstrated in the setting of outbreaks by selected strains and 
only for complex intervention bundles36. It is currently unknown which measures are best at 
preventing CDI and which are most cost-effective. For CDI treatment, calculations have been 
modelled 37,38, but for preventive measures such data need to be generated. Using data from 
Objectives 1.5, 2.1, 2.2 & 2.4, we will determine the diagnosis and treatment costs for those cases 
managed vs. not managed according to current guidelines. Results of cost-effective measures will be 
presented/visualized by using threshold analyses. Potential cost-savings are likely by e.g. shortened 
overall length of stay, prevention of severe and multiple CDI, transfer to (expensive) intensive care 
unit (ICU), and morbidity/mortality due CDI. Calculation of overall costs (direct and indirect costs) 
will be based on methodology as stated in O2.5. 
 
3.6.2 Outcome measures   
This section of COMBACTE-CDI is surveillance of existing guidelines, including local knowledge and 
compliance. There is no fixed outcome measure for this.  
 

 
 
Protocol version 1.6 17/04/2018 
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3.6.3 Sampling Criteria for surveillance studies (objective 2.6) 
3.6.3.1 Inclusion criteria   
All data generated from samples and isolates collected on the sample days (O1.1 and 1.2), all 
participants from the case/controls studies (O1.3 and 1.4), all participating sites (hospital based), and 
their associated community healthcare providers who submitted samples on the sampling days 
(sample source sites). In four selected countries an additional 10 nursing homes that did not submit 
samples on the sampling days will also be asked to complete the surveillance questionnaire, as there 
are a paucity of CDI data regarding these facilities.   
3.6.3.2 Exclusion criteria   
None  
 
3.6.4 Study methods 
The investigators will a execute cost-benefit analyses of the different CDI management 
policies/pathways highlighted from Objectives 2.1, 2.2, 2.3 and 2.4, and identify the most cost-
effective. The most cost-effective policy is expected to differ between healthcare settings as 
identified in 2.5. Cross-country comparisons of prices for health services will be based on OECD data. 
Data from Objective 1.2 will enable us to calculate differential costs per C. difficile strain/ribotype, 
and to what extent cost-effectiveness of infection control/antimicrobial stewardship (AMS) 
measures is driven by local CDI epidemiology. So-called hypervirulent strains 027/078/244 may be 
pooled for such analysis. It is well known that CDI due to hypervirulent strains leads to high rates of 
morbidity and mortality. Excessive direct costs may be expected, especially if patients are treated on 
an Intensive Care Unit e.g. due to toxic megacolon or pseudomembranous colitis). Further indirect 
costs may arise in case of productivity losses as stated in O2.5. 
 
The investigators will calculate the value of infection-control measures including the identification of 
high-risk patients and, using data from Objective.1.5, we will analyse the economic value of 
preventing transmission. Using data from Objective 2.5, we will determine the potential reduction in 
CDI economic burden in individual healthcare settings by introducing or changing infection 
control/AMS measures. Therefore, expenditures for direct cost factors (e.g. personnel costs for 
Infectious Diseases specialists) will be compared with in- and outpatient cost-savings (e.g. due to 
prevention of CDI). Based on these results we will calculate the optimal approach, taking into 
account an institution’s economic burden. This could be helpful to implement standardised and 
effective infection control/AMS measures into clinical practice to improve patient outcomes and 
reduce overall costs associated with CDI across Europe in all healthcare settings. Furthermore, these 
results could be of interest for consideration of updated guidelines. Calculation of costs will be based 
on methodology as stated in O2.5. 
3.6.5 Sample size 
As this analysis requires data from the objectives O1.1-15 and O2.1-2.5, the sample size is 
dependent on the sample size of these previous objectives. It will include; 
 
The number of samples collected on the sampling days (O1.1) and 
 
The number of isolates of C. difficile (O1.2),  
 
The number of participants in the case/control studies (O1.3 and 1.4),  
 
The data generated in the transmission modelling (O1.5),  
 
The number of completed surveillance questionnaires (O2.1-2.5) as described above.   
 
3.6.6 Data collection 
 

 
 
Protocol version 1.6 17/04/2018 
32 
The anonymised datasets generated from work-package 1 (O1.1-1.5) will be combined by the 
University of Leeds (overall guardian of the data) and sent to University of Cologne (a COMBACTE-
CDI partner) for analysis as part of the cost-benefit modelling. This dataset will contain anonymised 
health information; it will not contain any personal identifiable data.  This will be combined with the 
surveillance data (O2.1-2.5).  
 
As described above, for the surveillance data, an online data capture system will be used to collect 
the data, built within the COMBACTE online network. This data will not contain any personal data, 
but details about individual sites knowledge and compliance with existing guidelines. Each sample 
source site will have a unique anonymised study identifier for the online data capture system; 
investigators will therefore not be able to identify the sample source sites.  
 
The main dataset will then be accessed via the online portal by the investigator for this objective. 
Rule bases will be added to ensure that each objective investigator can only access data relevant to 
their objective.  
 
3.6.7 Data Analysis 
A simulation of the economic impact of novel CDI treatment options will be made using data from 
Objectives 1.4 & 1.5. 
 
For the simulation of costs, a decision-analytic model (e.g. decision tree analysis, markov model) will 
be appropriate. Therefore, a separate analysis of direct/indirect costs factors as well as further 
methodology as stated above will be performed/used to determine the impact of treatment 
interventions as well as changes in diagnostic pathways. 
 
Building on data collected in WP1 (esp. the modelling in 1.5) and in O2.5, resource-utilization 
analyses will be performed for different CDI management approaches according to healthcare 
setting. 
 
Data from O1.2 will be used to calculate differential costs per C. difficile strain/ribotype, and to what 
extent cost-effectiveness of infection control/antimicrobial stewardship (AMS) measures are driven 
by local CDI epidemiology.  
 
The value of infection-control measures including the identification of high-risk patients will be 
calculated, and, using data from O1.5, the economic value of preventing transmission will be 
assessed. 
4.0 Data management 
 
All data generated during work package 1 will be under the guardianship of the University of Leeds. 
It will be held on secure, password protected encrypted University servers. No personal identifiable 
data will be held, only health data against a pseudoanonymised study number. The link back to the 
participant will only be held by the clinical care team, this link back will never be disclosed to the 
investigators or leave the clinical participating site. The link back (retained at the clinical site) is 
essential however to enable the clinical care team to fill in the follow up CRFs for the case/control 
study. Once the CRF data has been collected and validated and verified the link-back will be 
destroyed by the local study site. 
 
This anonymised dataset will also be transferred to a sub-contractor (still to be defined) for the 
transmission modelling. The sub-contractor will be within the EEA, and will be subject to a full 

 
 
Protocol version 1.6 17/04/2018 
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document review by the University of Leeds, to ensure they comply with all of the relevant 
regulatory requirements. Their responsibilities will be clearly defined within the sub-contract and 
data transfer agreement between the University of Leeds and the sub-contractor. Once finalised, the 
data required for, and generated by the transmission modelling will transfer back to the University 
of Leeds for storage on secure, password protected encrypted University servers. No data will be 
held locally by the sub-contractor after completion of the modelling.  
 
In work package 2, an online data capture system will be used to collect the surveillance data, built 
within the COMBACTE online network. The data will be held on secure, password protected servers 
at the University of Utrecht (a COMBACTE-CDI partner). This data will not contain any personal data, 
but details about individual sites knowledge and compliance with existing guidelines. Each sample 
source site will have a unique anonymised study identifier for the online data capture system; 
investigators will therefore not be able to identify sample source sites. The main dataset will then be 
accessed via the online portal by the investigators for each of the objectives. Rule bases will be 
added to ensure that each objective investigator can only access data relevant to their objective.  
 
The anonymised datasets generated from work package 1 (O1.1-1.5) will be combined by the 
University of Leeds (overall guardian of the data) and sent to University of Cologne for analysis as 
part of the cost-benefit modelling. This dataset will contain anonymised health information; it will 
not contain any personal identifiable data.  This will be combined with the surveillance data (O2.1-
2.5). These data will be held on the secure, password protected servers at the University of Cologne 
(a partner within COMBACTE-CDI). Data transfer agreements are in place between the University of 
Leeds and the University of Cologne, as part of the consortium agreement. As the University of 
Cologne is a partner within COMBACTE-CDI, this data will be archived locally, not transferred back to 
University of Leeds.   
 
5.0 Reporting 
This is strictly an observational (surveillance) study.  Results of additional testing will not be 
reported back to participating laboratories.  The study is therefore ‘non-interventional’ as the 
participant samples will be processed as per local policy at each hospital and those results acted 
upon; i.e. additional COMBACTE-CDI study results will NOT be available for patient management.  
 
Study reports and conclusions based on analysis of data at a national and European level will be 
prepared by the lead investigator for each objective.  
 
A full study report will be presented at the end of each objective. Results will be submitted for 
presentations at scientific meetings followed by preparation of manuscript and publication 
whenever applicable.  Authorship will be led by the lead investigator for each objective (see 
consortium agreement).  
 
6.0 References 
1. 
Davies et al. Underdiagnosis of Clostridium difficile across Europe: the European, 
multicentre, prospective, biannual, point-prevalence study of Clostridium difficile 
infection in hospitalised patients with diarrhoea (EUCLID). Lancet Infect Dis 
2014;14:1208-19. 
2. 
Wilcox et al. A case-control study of community-associated Clostridium difficile infection. 
J Antimicrob Chemother 2008;62:388-96. 

 
 
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3. 
Hensgens et al. Diarrhoea in general practice: when should a Clostridium difficile 
infection be considered? Results of a nested case-control study. Clin Microbiol Infect 
2014;20:O1067-74. 
4. 
van Dorp et al., for the European Clostridium difficile Infection Surveillance Network 
(ECDIS-Net) on behalf all participants. Standardised surveillance of Clostridium difficile 
infection in European acute care hospitals: a pilot study. Eurosurveillance 2013; Jul 
21;21(29). doi: 10.2807/1560-7917.ES.2016.21.29.30293.. 
5. 
European Centre for Disease Prevention and Control (ECDC). Point prevalence survey of 
healthcare-associated infections and antimicrobial use in European long-term care 
facilities, May–September 2010. Protocol. Stockholm. 2010. 
6. 
European Centre for Disease Prevention and Control (ECDC). Point prevalence survey of 
healthcare-associated infections and antimicrobial use in European long-term care 
facilities, April–May 2013. Protocol. Stockholm. 2013. 
7. 
Jessica SH Martin, David W Eyre, Warren N Fawley, David Griffiths, Kerrie Davies, 
Damian PC Mawer, Timothy EA Peto, Derrick W Crook, A Sarah Walker, Mark H Wilcox, 
Risk factors for and clinical consequences of Clostridium difficile transmission between 
cases: A retrospective cohort study based on whole-genome sequencing. In press. 
8. 
Bauer et al. Clostridium difficile infection in Europe: a hospital-based survey. Lancet 
2011;377:63-73. 
9. 
Barbut et al. Prospective study of Clostridium difficile infections in Europe with 
phenotypic and genotypic characterisation of the isolates. Clin Microbiol Infect 
2007;13:1048-57. 
10. 
Davies et al. Diversity of Clostridium difficile PCR ribotypes in Europe: results from the 
European, multicentre, prospective, biannual, point-prevalence study of Clostridium 
difficile infection in hospitalised patients with diarrhoea (EUCLID), 2012 and 2013. Euro 
Surveill 2016; Jul 21;21(29). doi: 10.2807/1560-7917.ES.2016.21.29.30294 
11. 
Fawley. Enhanced surveillance of community-associated Clostridium difficile infection. 
Eurosurveillance 2016; Jul 21;21(29). doi: 10.2807/1560-7917.ES.2016.21.29.30295 
12. 
Planche et al. Differences in outcome according to Clostridium difficile testing method: a 
prospective multicentre diagnostic validation study of C difficile infection. Lancet Infect 
Dis 2013;13:936-45. 
13. 
Eckert et al. Prevalence and pathogenicity of binary toxin-positive Clostridium difficile 
strains that do not produce toxins A and B. New Microbes New Infect 2015;3:12-7. 
14. 
Crobach et al. European Society of Clinical Microbiology and Infectious Diseases 
(ESCMID): data review and recommendations for diagnosing Clostridium difficile-
infection (CDI). Clin Microbiol Infect 2009;15:1053-66. 
15. 
Kwon et al. The morbidity, mortality, and costs associated with Clostridium difficile 
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difficile infection: a systematic review. PLoS One 2014;9:e98400. 
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van Dorp et al. Standardised surveillance of Clostridium difficile infection in European 
acute care hospitals: a pilot study, 2013. Euro Surveill 2016;21. 
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Novack et al. Acid suppression therapy does not predispose to Clostridium difficile 
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and non-epidemic Clostridium difficile strains among long-term care facility residents. 
Clin Infect Dis 2007;45:992-8. 

 
 
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Bowel Disease: The RECIDIVISM Study. Am J Gastroenterol 2016. 
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Best et al. Effectiveness of deep cleaning followed by hydrogen peroxide 
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Bartsch et al. Is fidaxomicin worth the cost? An economic analysis. Clin Infect Dis 
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Clostridium difficile infection: a decision analysis. Clin Infect Dis 2014;58:1507-14. 
 
 

 
 
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Appendix 1 
 
Country 
Recruitment of study sites which test both in-
patient and community (GP, long term 
healthcare facilities) stool samples 
 Collection of residual diagnostic tissue and 
export to the UK 
Collection of prospective patient data (outcome 
data) 
Collection of retrospective patient data (risk 
factor data) 
 Collection of attributable costs of CDI data 
Collection of data regarding testing policy and 
methodology, surveillance practices, national 
guidelines and compliance and patient testing 
and treatment stratification (severity markers) 
Region of Europea 
Population (average from two sourcesb) 
Study sites 1 per 3m population 
Slovakia 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Eastern 
5463801 
2 
Poland 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Eastern 
38421407 
13 
Romania 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Eastern 
20486235 
7 
UK 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Northern 
64770786 
22 
Ireland 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Northern 
4833233 
2 
Sweden 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Northern 
9511327 
3 
Spain 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Southern 
47314040 
16 
Italy 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Southern 
60904272 
20 
Greece 
Yes 
Yes 
Yes 
Maybe 
Yes 
Yes 
Southern 
10846356 
4 
France 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Western 
65617845 
22 
Netherlands 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Western 
16998349 
6 
Austria 
Yes 
Yes 
Yes 
Yes 
Yes 
Yes 
Western 
8397080 
3 
Table 1. Results of an initial feasibility assessment for conducting the study in O1.1 in Europe. 
a = UN geocheme for Europe, zones of Europe; b= bwww.geoba.se, and www.worldmeters.info. 
 
 
 
 

 
 
Protocol version 1.6 17/04/2018 
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Appendix 2 Participating site invitation letter 
 
 
 
 
 
Dear Sir/Madam, 
 
We are writing to invite you to be a participating site in the Combatting Bacterial Resistance in 
Europe – Clostridium difficile Infections (COMBACTE-CDI) project which aims to develop a detailed 
understanding of the epidemiology and clinical impact of CDI across Europe. 
 
This research project received support from the Innovative Medicines Initiative Joint Undertaking 
under grant agreement, resources of which are composed of financial contribution from the 
European Union Seventh Framework Programme (FP7/2007-2013) and EFPIA companies in kind 
contributions. 
 
We are the national coordinating laboratory for XXXXXXX and are recruiting sites to take part in this 
study.  
 
All sites must meet the following criteria: 
 
Sites must carry out diagnostic testing of faecal samples from both in-patients and 
community patients  
 
Sites must be able to collect residual diagnostic tissue of all faecal samples received by the 
laboratory on a specified date in May/June and October 2018, and export this to the UK the 
day after collection (transport provided) 
 
Sites must be able to collect prospective patient data (outcome data) linked to the faecal 
samples provided during the sampling period (based on a provided Case Report Form) either 
directly or via the sample source site 
 
Sites must be able to collect retrospective patient data (risk factor data) linked to the faecal 
samples providing during the sampling period (based on a provided Case Report Form) 
either directly or via the sample source site 
 
Sites must be able to provide data in regards to their CDI testing policy and methodology, 
CDI surveillance practices, knowledge of national CDI guidelines and their local compliance, 
and CDI treatment (including according to patient level risk e.g. CDI severity) (based on a 
provided questionnaire). 
 
If you agree to take part, you would be required to collect all unformed faecal samples (after the 
completion of all routine microbiology tests) received on two selected sampling days in the year 
(May/June and October) and send them to the European Coordinating Laboratory in Leeds, UK.   A 
one-off fee of €500 Euros is payable to yourselves, to cover the labour and consumables costs 
incurred in sending the samples to Leeds.   You will receive shipping packaging and shipping 
instructions to ship the samples to Leeds using DHL.  The cost of shipping is covered by the European 
Coordinating Laboratory. 
 
The European Coordinating Laboratory will test each sample included in the study for CDI using the 
recommended ‘optimal’ testing methodology (ESCMID guidelines). Results will be compared to 
those obtained from your submitting hospital/laboratory, including if the sample was even tested. 

 
 
Protocol version 1.6 17/04/2018 
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All C. difficile isolates collected from the study samples will then be typed by various methods 
including PCR-ribotyping, toxinotyping and whole genome sequencing. 
 
All CDI positive cases (as determined by the European Coordinating Laboratory), and a proportion of 
negative cases (at the ratio of 1 case to 3 controls) will have clinical data collected (6 months after 
each sampling date) on patient outcomes and risk factors for disease Data will be collected on a Case 
Report Form and sent to the European Coordinating Laboratory for analysis.  A fee of €200 Euros is 
payable to yourselves, to cover the labour costs incurred in completion of each CRF. 
 
Each participating site (and their associated community practices) will also be asked to complete a 
web-based questionnaire on guideline knowledge, compliance, testing policy and methodology, 
surveillance practices, and stratification of ‘severe’ patients. Data will be compared to identify 
differences between countries and healthcare providers.   A one-off fee of €100 Euros is payable to 
yourselves to cover the labour costs incurred in completion of the questionnaire and a further one-
off fee of €100 Euros is payable to yourselves to cover the administration in distributing the 
questionnaire to your associated community practices. 
 
If you wish to participate in this study please confirm your participation via email to XXXXXXXXX by 
the 16th February 2018.  You will then be provided with a study pack for the initial sampling and 
copy of the full study protocol and study manual.   You will also receive an email from our 
COMBACTE partner, LAB-NET combacte.lab-net@uantwerpen.be requesting you to complete a 
questionnaire so that you can be added to the COMBACTE LAB-NET system.  We ask that you 
complete this at your earliest opportunity. 
 
We hope that you understand the value of this project and agree to participate in this study.  
 
Yours sincerely 
 
 
 
Name 
 
National Coordinator of Country for COMBACTE-CDI 
 
Contact details