Bezlotoxumab Yielded Outcomes by Addressing Personalized Needs in Clostridioides Difficile Infection: The BEYOND Double-Blind Randomized Clinical Trial
Phase: PHASE2
Conditions: Clostridioides Difficile Infection, Stool Microbiome, Organ Dysfunction Syndrome, Clostridioides Difficile Infection Recurrence, Mortality
Sponsor: Hellenic Institute for the Study of Sepsis

BEZLOTOXUMAB YIELDED OUTCOMES BY ADDRESSING PERSONALIZED NEEDS IN CLOSTRIDIOIDES 
DIFFICILE INFECTION: THE BEYOND DOUBLE-BLIND RANDOMIZED CLINICAL TRIAL 
BEYOND Protocol 
Version 1.0, 5 October 2021 
 
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BEZLOTOXUMAB YIELDED OUTCOMES BY ADDRESSING PERSONALIZED 
NEEDS IN CLOSTRIDIOIDES DIFFICILE INFECTION: THE BEYOND DOUBLE-
BLIND RANDOMIZED CLINICAL TRIAL 
 
 
 
STUDY PROTOCOL 
 
 
Evangelos J. Giamarellos-Bourboulis, MD, PhD  
Professor of Internal Medicine  
National and Kapodistrian University of Athens, Medical School, Greece 
 
Chairman of the Board, Hellenic Institute for the Study of Sepsis 
 
 
 
 
EudraCT number: 2021-005473-10 
Protocol version: 1.0 
Protocol date: 5 October 2021 
 
 
Study Sponsor:  
Hellenic Institute for the Study of Sepsis 
88 Michalakopoulou Str., 115 25 Athens 
e-mail: insepsis@otenet.gr 
tel: +30 210 74 80 662 
 
 

BEZLOTOXUMAB YIELDED OUTCOMES BY ADDRESSING PERSONALIZED NEEDS IN 
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DISCLOSURE OF PRINCIPAL INVESTIGATOR 
 
Protocol Study Title: BEZLOTOXUMAB YIELDED OUTCOMES BY ADDRESSING 
PERSONALIZED NEEDS IN CLOSTRIDIOIDES DIFFICILE INFECTION: THE BEYOND 
DOUBLE-BLIND RANDOMIZED CLINICAL TRIAL 
 
 
 
The herein protocol became known to myself by the Study Sponsor. I understand that the 
protocol remains as yet unpublished; I certify that all disclosed information to myself for 
this protocol will remain strictly confidential.  
 
The Principal Investigator, 
 
 
 
Print Name 
 
 
Signature                                                                                  Date 
 
 
 

BEZLOTOXUMAB YIELDED OUTCOMES BY ADDRESSING PERSONALIZED NEEDS IN 
CLOSTRIDIOIDES DIFFICILE INFECTION: THE BEYOND PROJECT 
 
BEYOND Protocol 
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TABLE OF CONTENTS 
STUDY SYNOPSIS ......................................................................................................... 5 
ABBREVIATIONS ............................................................................................................ 8 
INTRODUCTION ............................................................................................................. 9 
AVAILABLE UNPUBLISHED DATA .............................................................................. 10 
AIM OF THE PROJECT ................................................................................................ 12 
STUDY DESIGN ............................................................................................................ 12 
Inclusion criteria ......................................................................................................... 13 
Exclusion criteria ........................................................................................................ 14 
Screening stage ......................................................................................................... 14 
Treatment stage ......................................................................................................... 15 
BLINDING PROCEDURES ........................................................................................... 21 
Circumstances for Urgent Unblinding ......................................................................... 21 
Reasons and Guidelines for Unblinding ..................................................................... 22 
Guidelines for Urgent Unblinding of Individual Participant Treatment Assignments for 
Medical Reasons ........................................................................................................ 22 
Guidelines for Early Non-Urgent Unblinding of Individual Participant Treatment 
Assignments for Medical/Safety Reasons .................................................................. 23 
STUDY ENDPOINTS .................................................................................................... 23 
Primary study endpoint ............................................................................................... 23 
Secondary study endpoints ........................................................................................ 24 
POWER CALCULATION ............................................................................................... 25 
STATISTICAL ANALYSIS ............................................................................................. 25 
ADVERSE EVENTS ...................................................................................................... 25 
QUALITY CONTROL AND ASSURANCE ..................................................................... 28 
ETHICAL CONSIDERATIONS ...................................................................................... 29 
DATA HANDLING AND RECORD KEEPING ................................................................ 29 
FINANCING AND INSURANCE .................................................................................... 30 
PUBLICATION POLICY ................................................................................................ 30 
PROTOCOL ADHERENCE AND AMENDMENTS ........................................................ 30 
REFERENCES .............................................................................................................. 31 

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APPENDIX I Final study report of the SPECIFY project ................................................ 33 
APPENDIX II The SOFA score ...................................................................................... 55 
APPENDIX IΙI The Charlson’s comorbidity index (CCI) ................................................. 56 
APPENDIX IV Study visits ............................................................................................. 58 
APPENDIX V List of study sites ..................................................................................... 59 
 
 
 
 

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STUDY SYNOPSIS 
Objective 
Previous data from our group have shown that integrated information 
from SNPs of the host DNA, IL-8 and the enrichment of the stool 
microbiome can indicate the patients with infection by Clostridioides 
difficile (CDI) at risk for unfavorable outcome. This integrated 
information is forming the BEYOND score. The aim of the BEYOND 
RCT is to investigate if adjunctive bezlotoxumab treatment to the 
current standard-of-care may decrease the likelihood of unfavorable 
outcome for patients who score positive by the BEYOND score. 
Study design 
Double-blind randomized clinical study powered by the primary 
endpoint for 44 patients. 
Inclusion criteria 
1. Age 18 years 
2. Both genders 
3. Written informed consent provided by the patient or by their legal 
representative in case of patients unable to consent 
4. In case of non-menopausal women, unwillingness to become 
pregnant during the study period. Women of child-bearing potential 
will be screened by a urine pregnancy test before inclusion in the 
study. 
5. Diarrhea defined as at least 3 episodes of unformed stool in the past 
24hours.  
6. Positive stool for C.difficile. This is defined as any stool sample 
positive for the presence of glutamate dehydrogenase (GDH) and for 
the presence of toxin A and/or B. 
7. Positive BEYOND score i.e. meeting any of the following: 
Gene score for susceptibility to CDI more than 53. The score is 
provided by the following equation: 
(Carriage of C allele of rs12148744 x 27) – (carriage of C allele of 
rs714024 x 27) - (carriage of C allele of rs721059 x 29) + (carriage of 
T allele of rs4311028 x 33) – (carriage of A allele of rs62183547 x 25) 

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+ (carriage of C allele of rs1128266 x 12) - (carriage of T allele of 
rs4279595 x 17) + (carriage of G allele of rs175006 x 11) + (carriage 
of T allele of rs3859214 x 17) + (carriage of G allele of rs7222870 x 
15) – (carriage of G allele of rs5086600 x 9) + (carriage of T allele of 
rs7240534 x 12) + (carriage of G allele of rs20911172 x 11) - (carriage 
of C allele of rs17680671 x 17) 
OR 
Score provided by the following equation more than 9= [Hemoglobin 
<9.5 g/dl x 10] + [serum urea >64.5 mg/dl x 14] + [serum interleukin-8 
>227 pg/ml x 19] – [carriage of G allele of rs2091172 x 17] 
OR 
More than 3log10 of gammaproteobacteria or Enterobacteriaceae or 
Enterobacteriales in the stool 
Exclusion criteria 
• Age below 18 years 
• Denial for written informed consent 
• Known allergy to bezlotoxumab  
• Pregnancy or lactation. Women of child-bearing potential will be 
screened by a urine pregnancy test before inclusion in the study 
Intervention 
Patients will be blindly assigned to adjunctive treatment with placebo or 
bezlotoxumab at a 1:1 ratio. All patients will receive standard-of-care 
treatment for CDI at the discretion of their attending physicians. 
Primary endpoint 
The superiority of bezlotoxumab over placebo to reduce the incidence 
of unfavorable outcome. Unfavorable outcome is defined as any of the 
following: progression into organ dysfunction; relapse of CDI; and 
death. The primary endpoint is tested on Day 40 from start of blind 
treatment. Organ dysfunction is defined as any increase of the baseline 
total SOFA score by at least 2 points. Need for colectomy or admission 
in the Intensive Care Unit counts as organ dysfunction. 
Secondary endpoints The superiority of bezlotoxumab over placebo when given for CDI of 
high likelihood for unfavorable outcome on the following: 

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• Incidence of organ failure and time to first organ failure 
• Time to relapse of CDI 
• Survival time 
• Cost of hospitalization 
• Validation of the BEYOND score 
Power of the study 
This is calculated for the primary endpoint. Power calculation is based 
on the hypothesis that the difference between bezlotoxumab and 
placebo will be at least 40%. It is anticipated that 60% of patients 
allocated to the placebo arm and 20% of patients allocated to the 
bezlotoxumab arm will achieve the primary endpoint of unfavorable 
outcome. To demonstrate this difference at the 10% level of 
significance with 80% power, 22 patients should be enrolled into each 
arm (44 patients in total). It is anticipated that one out of three screened 
patients with CDI will meet all inclusion criteria, so that a total of 132 
patients need to be screened. 
Duration 
2 years 
 
 
 

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ABBREVIATIONS 
AE: adverse event 
bid: two times daily 
CCI: Charlson’s comorbidity index 
CDI: Clostridioides difficile infection 
CI: confidence interval 
CRF: case report form 
eQTL: expression qualitative trait locus 
ICU: intensive care unit 
IL: interleukin 
IRB: institution review board  
GDH: glutamate dehydrogenase 
GWAS: genome-wide association study 
q6h: every 6 hours 
OR: odds ratio 
OTU: operational taxonomic unit 
PPV: positive predictive value 
SAE: serious adverse event 
SOFA: sequential organ failure assessment 
SNP: single nucleotide polymorphism 
RCT: randomized clinical trial 
RT-PCR: real time polymerase chain reaction 
TEAE: treatment-emergent adverse event 
tid: three times daily 
 
 

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INTRODUCTION 
 
Clostridioides difficile infection (CDI) is an emerging infection with increased point-
prevalence per year as reported both for the United States and for European countries (1, 
2). Current evidence suggests that CDI is a complex interaction between the host and the 
offending pathogen where physician intervention plays a pivotal role. Although originally 
conceived to be a hospital-acquired infection, it becomes more and more recognized that 
many cases are community-acquired. This underscores the significance of the host in the 
pathogenesis of CDI. CDI is traditionally considered to derive from the direct oral 
transmission from one host to the other in the hospital environment. However, a recent 
survey in three large hospitals in Wales analyzing the whole genome C.difficile isolated 
from 499 cases of diarrhea positive for glutamate dehydrogenase (GDH) revealed that the 
vast majority of isolates of C.difficile had different nucleotide sequences suggesting 
against the direct transmission from one patient to the other in the vast majority of cases 
(3). The complex interaction between the host and C.difficile ending to infection is 
modulated by environmental factors like antibiotic consumption, proton pump inhibitor 
intake and prolonged hospitalization (4). 
The main endpoint of all randomized clinical trials (RCTs) of new antimicrobials 
targeting C.difficile is sustained clinical response that is defined as the composite of the 
resolution of the infection at the end of treatment without any CDI relapse within the first 
40 days (5). This endpoint is mainly targeting to demonstrate if newly developed agents 
are superior over the comparator vancomycin in the minimization of relapse risk. However, 
recent data outscore the lack of substantial merit of this endpoint as many cases of CDI 
bear an intrinsic danger of unfavorable outcome without this being incorporated in the 
RCT endpoints. More precisely, a substantial rate of patients dies after the end of oral 
treatment between days 12 and 40 probably as a result of emerging organ dysfunction in 
the field of CDI colitis (6). Recent epidemiological data coming from the United States 
challenge even more the traditional way of our thinking of CDI. More precisely, in a 
prospective survey of 30,326 patients it was shown that the risk of unfavorable outcome 
and death was greater among first-time infected patients than in relapsed cases. This 
survey reports that mortality from the first CDI case may be as high as 28% (1). These 
cases of CDI associated with an unfavorable outcome leading to surgery and 

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hospitalization in an intensive care unit are associated with a tremendous cost mounting 
to $34,149 per case (7). Surprisingly when data of the two registration RCTs of fidaxomicin 
were combined reduced CDI mortality was shown over the comparator vancomycin 
treatment (6). This observation strengthens the belief that the substantial merit of new 
agents is not in preventing CDI relapse but in saving lives.  
 
The pivotal RCTs of fidaxomicin influenced the design of the MODIFY studies in a 
way that the inclusion criteria of these studies comprised not only cases of CDI infection 
of moderate severity but also patients with high chance of relapse. Half of the enrolled 
patient population was aged equal to or above 65 years and another quarter of enrolled 
patients had history of at least one episode of CDI. In these trials, patients with CDI 
receiving oral treatment with metronidazole, vancomycin or fidaxomicin were randomized 
to one single dose of adjunctive treatment with placebo or the monoclonal antibody 
targeting the toxin B of C.difficile bezlotoxumab, or the monoclonal antibody targeting the 
toxin A of C.difficile actoxumab or both antibodies. Results revealed that bezlotoxumab 
treatment reduced considerably the risk of relapse whereas the intake of actoxumab did 
not affect the clinical outcome of CDI. (8). However, the efficacy of bezlotoxumab 
treatment on other variables of unfavorable outcome was not analyzed. 
 
One main argument against new treatments targeting C.difficile is the substantial 
cost. This argument is based on the fact that there are no tools that can discriminate 
between patients at high risk for unfavorable outcome that are in need of new treatments 
from patients at low risk from unfavorable outcome that will benefit from standard-of-care 
vancomycin treatment. 
 
AVAILABLE UNPUBLISHED DATA 
SPECIFY (Scoring personalized needs in Clostridium difficile infections for 
fidaxomixin therapy) was a prospective study that was sponsored by the Hellenic Institute 
for the Study of Sepsis. The study was conducted during the period September 2015 to 
November 2016 in 11 departments of Internal Medicine in Greece (ClinicalTrials.gov 
NCT02573571) with the endpoint of the development of a score that could comprise 
clinical signs, biomarker and genetic elements and that could provide positive predictive 
value (PPV) equal to or more than 80% for the prediction of unfavorable outcome of CDI. 

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Unfavorable outcome was defined as any of the following: presentation or progression 
into severe CDI, relapse; and death. The SPECIFY study was conducted in two phases. 
In the first phase, a total of 153 patients with CDI and 150 comparators matched for their 
Charlson’s Comorbidity Index (CCI) were enrolled. All patients and comparators were of 
Caucasian origin. DNA was extracted from whole blood. Genome-wide association study 
(GWAS) was performed using one HumanCore-chip comprising 4,330,811 single 
nucleotide polymorphisms (SNPs). Following quality control of imputed data 134 
comparators and 134 CDI cases were analyzed. After Manhattan plotting, 17 single 
nucleotide polymorphisms (SNPs) were found to be strongly associated with CDI. These 
17 SNPs entered forward step-wise logistic regression analysis and 14 SNPs remained 
to be independently associated with CDI so as to construct a prediction score for the 
development of CDI when CCI was high. In the second phase of the SPECIFY study, 
these 14 identified SNPs were analyzed along with clinical and biomarker data to develop 
a score that could predict the risk of unfavorable outcome within the population of the 134 
patients who also participated in the first phase. Among these 134 patients, unfavorable 
outcome was found in 39 (29.1%). Using logistic regression analysis, a prediction score 
to early detect unfavorable outcome was developed. This score comprised only one of the 
14 SNPs, namely carriage of G alleles of rs2091172. This score is provided by the 
following equation: 
[Hemoglobin <9.5 g/dl x 10] + [serum urea >64.5 mg/dl x 14] +  
[serum interleukin-8 >227 pg/ml x 19] – [carriage of G allele of rs2091172 x 17] 
Any score more than 9 was associated with PPV 80% for the prediction of unfavorable 
outcome meeting successfully the study primary endpoint. At that cut-off, the prediction 
score has specificity 95.8% and negative predictive value 79.8% for the early detection of 
unfavorable outcome by CDI. Although the specificity of the score is fully satisfactory, the 
score has limited sensitivity mounting to 41.0% and mandating further improvement (full 
clinical study report of the SPECIFY study provided in APPENDIX I).    
 
In order to improve the sensitivity of the score, full microbiome analysis was done 
in the stool of patients. Unpublished results showed that the enrichment of the gut flora 
with gammaproteobacteria or Enterobacteriaceae or Enterobacteriales at an LDA score 
more than 3 log10 was associated with unfavorable outcome.  

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With this information, we introduce a score which is called BEYOND. In this score, 
patients with CDI is at increased likelihood for unfavorable outcome if they meet any of 
the following: 
• Gene score for susceptibility to CDI more than 53. The score is provided by the 
following equation: 
(Carriage of C allele of rs12148744 x 27) – (carriage of C allele of rs714024 x 27) - 
(carriage of C allele of rs721059 x 29) + (carriage of T allele of rs4311028 x 33) – 
(carriage of A allele of rs62183547 x 25) + (carriage of C allele of rs1128266 x 12) - 
(carriage of T allele of rs4279595 x 17) + (carriage of G allele of rs175006 x 11) + 
(carriage of T allele of rs3859214 x 17) + (carriage of G allele of rs7222870 x 15) – 
(carriage of G allele of rs5086600 x 9) + (carriage of T allele of rs7240534 x 12) + 
(carriage of G allele of rs20911172 x 11) - (carriage of C allele of rs17680671 x 17) 
• Score provided by the following equation more than 9. [Hemoglobin <9.5 g/dl x 10] + 
[serum urea >64.5 mg/dl x 14] + [serum interleukin-8 >227 pg/ml x 19] – [carriage of 
G allele of rs2091172 x 17] 
• More than 3log10 of gammaproteobacteria or Enterobacteriaceae or 
Enterobacteriales in the stool 
 
AIM OF THE PROJECT 
BEYOND (BEzlotoxumab Yielded Outcomes by addressing personalized Needs in 
Clostridioides Difficile infection) is aiming to investigate if adjunctive treatment with 
bezlotoxumab may benefit patients who are scoring positive for the BEYOND sore and 
prevent them from unfavorable outcome.  
 
STUDY DESIGN 
 
This is a prospective, proof-of-concept, double-blind, RCT to compare the efficacy 
of bezlotoxumab over placebo among patients with CDI and high-risk for unfavorable 
outcome. The study will be conducted according to the Helsinki declaration in Internal 
Medicine Departments (see APPENDIX V) participating at the network of the HSSG 

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(www.sepsis.gr). Patients will be enrolled after written informed consent provided by 
themselves or by their legal representatives in case of patients not able to consent. The 
study protocol will be approved by the National Organization for Medicines of Greece and 
by the National Ethics Committee of Greece. The study will be registered at 
www.clinicaltrials.gov before inclusion of the first patient. The study will be provided one 
EudraCT number and will be registered at the EudraCT public site of the EU 
(https://eudract.ema.europa.eu). 
The study shall be governed by: 
• The international standards for Good Clinical Principal (GCP) developed by the 
International Council for Harmonization of Technical Requirements for 
Pharmaceuticals for Human Use 
• Directive 2001/20/EC for Clinical trials 
• General Data Protection Regulation 679/2016 (EC) 
 
Inclusion criteria 
Enrolled patients should meet ALL the below inclusion criteria: 
1. Age 18 years 
2. Both genders 
3. Written informed consent provided by the patient or by their legal representative in 
case of patients unable to consent 
4. In case of non-menopausal women, unwillingness to become pregnant during the 
study period. Women of child-bearing potential will be screened by a urine 
pregnancy test before inclusion in the study. 
5. Diarrhea defined as at least 3 episodes of unformed stool in the past 24hours.  
6. Positive stool for C.difficile. This is defined as any stool sample positive for the 
presence of glutamate dehydrogenase (GDH) and for the presence of toxin A and/or 
B. 
7. Positive BEYOND score i.e. meeting any of the following: 
Gene score for susceptibility to CDI more than 53. The score is provided by the following 
equation: 

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(Carriage of C allele of rs12148744 x 27) – (carriage of C allele of rs714024 x 27) - 
(carriage of C allele of rs721059 x 29) + (carriage of T allele of rs4311028 x 33) – 
(carriage of A allele of rs62183547 x 25) + (carriage of C allele of rs1128266 x 12) - 
(carriage of T allele of rs4279595 x 17) + (carriage of G allele of rs175006 x 11) + 
(carriage of T allele of rs3859214 x 17) + (carriage of G allele of rs7222870 x 15) – 
(carriage of G allele of rs5086600 x 9) + (carriage of T allele of rs7240534 x 12) + 
(carriage of G allele of rs20911172 x 11) - (carriage of C allele of rs17680671 x 17) 
OR 
Score provided by the following equation more than 9= [Hemoglobin <9.5 g/dl x 10] + 
[serum urea >64.5 mg/dl x 14] + [serum interleukin-8 >227 pg/ml x 19] – [carriage of G 
allele of rs2091172 x 17] 
OR 
More than 3log10 of gammaproteobacteria or Enterobacteriaceae or Enterobacteriales 
in the stool 
 
Exclusion criteria 
Patients meeting ANY of the following criteria CANNOT be enrolled in the study: 
• Age below 18 years 
• Denial for written informed consent 
• Known allergy to bezlotoxumab  
• Pregnancy or lactation. Women of child-bearing potential will be screened by a urine 
pregnancy test before inclusion in the study 
 
The study comprises a screening stage and a treatment stage.  
Screening stage 
 
The screening stage starts from the time patients or their legal representatives sign 
the written informed consent form and it can be extended until up to 72 hours from the 
start of empirical treatment for CDI by the attending physicians. Study sites will be 
equipped with the quick C.DIFFICILE Complete test to run GDH and toxins A/B detection 
on the study site. If patients meet inclusion criteria 1 to 6 and they do not meet any 

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exclusion criteria, 5ml of blood is collected after venipuncture of one forearm vein under 
aseptic conditions; 3ml are collected into one EDTA-coated tube to be used for DNA 
isolation; and the remaining 2ml into one pyrogen-free tube to be used for centrifugation 
and serum collection. Then either 10ml of liquid stool or one rectal swab are collected. 
Samples are transported via courier at the central lab of the Laboratory of Immunology of 
Infections at the 4th Department of Internal Medicine of ATTIKON University hospital of 
Athens. The central lab provides service 24hours by 7 days. There, transported samples 
will be processed and the following will be done: 
• Isolation of patient DNA and RT-PCR for the presence of G alleles of rs2091172 
• Measurement of serum IL-8 by enzyme an immunosorbent assay 
• Measurement 
of 
the 
stool 
microbiome 
diversity 
by 
the 
RT-PCT 
OF 
gammaproteobacterial, Enterobacteriaceae and Enterobacteriales 
 
Lab results will be provided to the study site and the investigators may calculate the 
BEYOND score using the hemoglobin and urea biochemistry. If the BEYOND score is 
positive, the patient can be enrolled in the study.  
Patients who fail screening because they score negative for the BEYOND score 
will be visited/contacted after 40 days to capture information on progression into organ 
dysfunction; relapse of CDI; and/or death (see below section of Primary endpoint for full 
definition). 
All lab samples will be destroyed immediately after completion of procession. 
 
Treatment stage 
Once a patient is considered eligible for enrolment, they will be blindly allocated at 
1:1 ratio to adjunctive treatment with placebo or bezlotoxumab. The allocated sequence 
of the study drug (placebo or bezlotoxumab) will be generated by a biostatistician. The 
preparation of the study drug will be done in each study site by the un-blinded pharmacist 
investigators. The administration of the study drug will be done in each study site by the 
blinded investigators.  
Placebo or bezlotoxumab will be given as single intravenous infusions of one 
hour within 72 hours from the start of standard-of-care treatment. The dose of 

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bezlotoxumab will be 10mg per kg of body weight dissolved in normal saline 0.9% or 5% 
dextrose water up to maximum of 1000mg. Placebo-treated patients will receive an equal 
volume of normal saline 0.9% or 5% dextrose water. Standard-of-care treatment will be 
prescribed to all patients at the discretion of the attending physicians according to local 
guidelines or to their own decision. This standard-of-care treatment may be oral 
fidaxomicin 200 mg bid or oral vancomycin 125 mg q6h for 10 days or oral metronidazole 
500mg tid for 10 days or equivalent.  
 
Study visits 
The following study visits will be done: 
 
Visit 1 (baseline or day 1) 
The following procedures are done: 
• Recording of: a) demographics; b) vital signs including blood pressure, heart rate and 
oxygen saturation; c) laboratory findings including white blood cells, creatinine and 
liver biochemistry (if available); d) co-administered drugs and antimicrobials; e) 
underlying comorbidities, calculation of the Charlson’s Comorbidity Index (CCI) (10, 
see APPENDIX III) and predisposing illnesses; f) results of coloscopy if available; g) 
SOFA score (see APPENDIX II); h) any organ failure; i) need for colectomy or 
admission in the Intensive Care Unit; k) number of bowel movements; l) clinical cure 
or relapse; and m) survival or death. 
• Administration of the study drug (placebo/bezlotoxumab) 
 
Visit 2  
This visit is taking place on day 2. The following procedures are done: 
• Recording of: a) vital signs including blood pressure, heart rate and oxygen saturation; 
b) laboratory findings including white blood cells, creatinine and liver biochemistry (if 
available); c) co-administered drugs and antimicrobials; d) results of coloscopy if 
available; g) SOFA score (see APPENDIX II); h) any organ failure; i) need for 
colectomy or admission in the Intensive Care Unit; k) number of bowel movements; l) 
clinical cure or relapse; and m) survival or death. 

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• Recording of any treatment-emergent adverse event (TEAE) or serious TEAE  
 
Visit 3  
This visit is taking place on day 3. The following procedures are done: 
• Recording of: a) vital signs including blood pressure, heart rate and oxygen saturation; 
b) laboratory findings including white blood cells, creatinine and liver biochemistry (if 
available); c) co-administered drugs and antimicrobials; d) results of coloscopy if 
available; g) SOFA score (see APPENDIX II); h) any organ failure; i) need for 
colectomy or admission in the Intensive Care Unit; k) number of bowel movements; l) 
clinical cure or relapse; and m) survival or death. 
• Recording of any treatment-emergent adverse event (TEAE) or serious TEAE  
 
Visit 4  
This visit is taking place on day 4. The following procedures are done: 
• Recording of: a) vital signs including blood pressure, heart rate and oxygen saturation; 
b) laboratory findings including white blood cells, creatinine and liver biochemistry (if 
available); c) co-administered drugs and antimicrobials; d) results of coloscopy if 
available; g) SOFA score (see APPENDIX II); h) any organ failure; i) need for 
colectomy or admission in the Intensive Care Unit; k) number of bowel movements; l) 
clinical cure or relapse; and m) survival or death. 
• Recording of any treatment-emergent adverse event (TEAE) or serious TEAE  
 
Visit 5  
This visit is taking place on day 5. The following procedures are done: 
• Recording of: a) vital signs including blood pressure, heart rate and oxygen saturation; 
b) laboratory findings including white blood cells, creatinine and liver biochemistry (if 
available); c) co-administered drugs and antimicrobials; d) results of coloscopy if 
available; g) SOFA score (see APPENDIX II); h) any organ failure; i) need for 
colectomy or admission in the Intensive Care Unit; k) number of bowel movements; l) 
clinical cure or relapse; and m) survival or death. 
• Recording of any treatment-emergent adverse event (TEAE) or serious TEAE  

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Visit 6  
This visit is taking place on day 6. The following procedures are done: 
• Recording of: a) vital signs including blood pressure, heart rate and oxygen saturation; 
b) laboratory findings including white blood cells, creatinine and liver biochemistry (if 
available); c) co-administered drugs and antimicrobials; d) results of coloscopy if 
available; g) SOFA score (see APPENDIX II); h) any organ failure; i) need for 
colectomy or admission in the Intensive Care Unit; k) number of bowel movements; l) 
clinical cure or relapse; and m) survival or death. 
• Recording of any treatment-emergent adverse event (TEAE) or serious TEAE  
 
Visit 7  
This visit is taking place on day 7. The following procedures are done: 
• Recording of: a) vital signs including blood pressure, heart rate and oxygen saturation; 
b) laboratory findings including white blood cells, creatinine and liver biochemistry (if 
available); c) co-administered drugs and antimicrobials; d) results of coloscopy if 
available; g) SOFA score (see APPENDIX II); h) any organ failure; i) need for 
colectomy or admission in the Intensive Care Unit; k) number of bowel movements; l) 
clinical cure or relapse; and m) survival or death. 
• Recording of any treatment-emergent adverse event (TEAE) or serious TEAE  
 
Visit 8  
This visit is taking place on day 8. The following procedures are done: 
• Recording of: a) vital signs including blood pressure, heart rate and oxygen saturation; 
b) laboratory findings including white blood cells, creatinine and liver biochemistry (if 
available); c) co-administered drugs and antimicrobials; d) results of coloscopy if 
available; g) SOFA score (see APPENDIX II); h) any organ failure; i) need for 
colectomy or admission in the Intensive Care Unit; k) number of bowel movements; l) 
clinical cure or relapse; and m) survival or death. 
• Recording of any treatment-emergent adverse event (TEAE) or serious TEAE  
 

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Visit 9  
This visit is taking place on day 9. The following procedures are done: 
• Recording of: a) vital signs including blood pressure, heart rate and oxygen saturation; 
b) laboratory findings including white blood cells, creatinine and liver biochemistry (if 
available); c) co-administered drugs and antimicrobials; d) results of coloscopy if 
available; g) SOFA score (see APPENDIX II); h) any organ failure; i) need for 
colectomy or admission in the Intensive Care Unit; k) number of bowel movements; l) 
clinical cure or relapse; and m) survival or death. 
• Recording of any treatment-emergent adverse event (TEAE) or serious TEAE  
 
Visit 10  
This visit is taking place on day 10. The following procedures are done: 
• Recording of: a) vital signs including blood pressure, heart rate and oxygen saturation; 
b) laboratory findings including white blood cells, creatinine and liver biochemistry (if 
available); c) co-administered drugs and antimicrobials; d) results of coloscopy if 
available; g) SOFA score (see APPENDIX II); h) any organ failure; i) need for 
colectomy or admission in the Intensive Care Unit; k) number of bowel movements; l) 
clinical cure or relapse; and m) survival or death. 
• Recording of any treatment-emergent adverse event (TEAE) or serious TEAE  
 
Visit 11  
This visit is taking place on day 12. The following procedures are done: 
• Recording of: a) vital signs including blood pressure, heart rate and oxygen saturation; 
b) laboratory findings including white blood cells, creatinine and liver biochemistry (if 
available); c) co-administered drugs and antimicrobials; d) results of coloscopy if 
available; g) SOFA score (see APPENDIX II); h) any organ failure; i) need for 
colectomy or admission in the Intensive Care Unit; k) number of bowel movements; l) 
clinical cure or relapse; and m) survival or death. 
• Recording of any treatment-emergent adverse event (TEAE) or serious TEAE  
 
 

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Visit 12  
This visit is taking place on day 40. The following procedures are done: 
• Recording of: a) vital signs including blood pressure, heart rate and oxygen saturation; 
b) laboratory findings including white blood cells, creatinine and liver biochemistry (if 
available); c) co-administered drugs and antimicrobials; d) results of coloscopy if 
available; g) SOFA score (see APPENDIX II); h) any organ failure; i) need for 
colectomy or admission in the Intensive Care Unit; k) number of bowel movements; l) 
clinical cure or relapse; and m) survival or death. 
• Recording of any treatment-emergent adverse event (TEAE) or serious TEAE  
 
If a patient is in need colectomy or admission in the Intensive Care Unit or dies between 
visits 11 and 12, this information and the date of the event are recorded. 
 
In case of patient discharge prior to Day 40, information on final outcome is collected by 
phone calls. All information is recorded in one case report form (CRF).  
The cost of hospitalization of every patient comprising drugs, laboratory and 
radiology exams, accommodation and interventions will be provided at the end of day 40 
in Euros according to the itemized cost for each of the above given by the Greek state.  
 
 
Clinical cure is defined by the resolution of diarrhea (i.e. fewer than three unformed 
bowel movements per day for 2 days consecutively) for the duration of treatment and no 
further need for treatment.  
Relapse is defined as the return of more than three unformed bowel movements in 
24 hours with a positive stool toxin test necessitating retreatment (5, 11, 12).  
 
Study drugs 
Bezlotoxumab will be provided to each site in the form of vials. Bezlotoxumab 
needs to be stored at 2-8°C at the study site at a refrigerator with recording of temperature. 
In case recording indicates deviation of temperature below 0°C or above 10°C for more 
than a day, stored drugs need to be replaced. The placebo (N/S 0.9%) will be provided to 

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each site in the form of bottles and needs to be stored at room temperature with recording 
of temperature. The un-blinded site pharmacists are responsible for the recording of the 
storage conditions of the study drugs. They also need to prepare the test article according 
to instructions provided at Investigator’s Brochure (IB). The IB will be provided by the 
Sponsor. The bottles with active drug or placebo will be covered to conceal the identity of 
the test article. Covering materials will be provided by the Sponsor. At the exterior of each 
bottle after coverage, a label will be placed with the prefix “BEY-”, a letter and a 2-digit 
number. The prefix “BEY-” refers to the study code “BEYOND”. The letter refers to the 
study site. The 2-digit number refers to the serial number of the enrolled patient at the 
respective study site. For example, the code BEY-A-07 refers to study site A and patient 
number 07 at that study site. The un-blinded pharmacist will provide the covered bottles 
to the blinded nurse or blinded investigator who will administer the infusion. 
 
BLINDING PROCEDURES 
This study is designed to maintain blinding from participants, site investigators and 
their teams until completion of the study. At each center, there will be an unblinded 
pharmacist who will be in charge of randomizing and preparing the study drug for each 
participant according to the randomized intervention assignment. These pharmacists will 
not be involved in data acquisition, collection, adjudication of outcomes or adverse events, 
or any other study procedures. They will not disclose the treatment assignment to the 
study team members unless it is via a formal process of early unblinding as described 
below. 
Αn independent biostatistician will generate the assignment to blinding treatment. 
Under normal circumstances, all the treatment assignments of participants will remain 
blinded until the completion of the trial (completion of enrollment and follow-up or early 
termination of the trial). 
Circumstances for Urgent Unblinding 
• Urgent unblinding of an individual participant’s treatment assignment can be 
performed for emergency medical reasons to protect participant safety when, as 

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determined by the site Investigator or designee, knowing the participant’s treatment 
assignment would affect immediate medical management of the participant.  
• Non-urgent early unblinding of an individual participant’s treatment assignment for 
medical/safety reasons, when knowledge of treatment assignment would not affect 
immediate medical management but may affect other aspects of a participant’s 
medical care/safety.  
Reasons and Guidelines for Unblinding  
Conventionally, full unblinding of the investigator team takes place after all study data 
have been recorded and reviewed for all participants, endpoints have been reviewed (if 
applicable per protocol), and the protocol team has declared the study dataset to be 
complete and "locked".  
It is critical to the objectives of any blinded study that the objectivity of the Investigator 
team (including site Investigators), other site staff, and participants be maintained. Any 
unblinding prior to the conventional full unblinding date can result in bias and should 
therefore be avoided. This includes unblinding the treatment assignments of individual 
participants as they come off the study, since there is substantial potential for bias in the 
reporting of results for other participants.  
Given that such bias can compromise the integrity or objectivity of the trial, unplanned 
unblinding prior to the conventional full unblinding date should be undertaken only to 
protect participant safety or to fulfill safety reporting and other regulatory obligations. 
Guidelines for Urgent Unblinding of Individual Participant Treatment Assignments 
for Medical Reasons  
The need for emergency unblinding of individual participant treatment assignments is 
expected to be extremely rare. Emergency unblinding does not apply for participants who 
have died, because knowledge of treatment assignment will not affect immediate 
management in such cases.  
Requests for urgent unblinding should be made in writing to the center pharmacist 
such that he/she can provide the assignment information immediately. In cases of extreme 
emergency in which it is not possible for the unblinding request to be made in writing, the 
site investigator or designee may make the request orally but must provide a written 

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statement of the request within 24 hours, including the reason why the request could not 
initially be made in writing. In these cases, the center pharmacist can provide the 
information orally, and provide a written confirmation of the unblinded treatment within 24 
hours.  
The Site Investigator should alert the Sponsor of the urgent unblinding within 24 hours 
of the unblinding via email.  
 
Unblinded treatment assignments should be shared with as few individuals as 
possible on a need-to-know basis. Care should be taken to prevent additional unblinding 
to maintain study integrity. The site PI and Sponsor are responsible for preventing 
additional unblinding beyond those who need to know and for protecting information that 
may identify the participant. 
Guidelines for Early Non-Urgent Unblinding of Individual Participant Treatment 
Assignments for Medical/Safety Reasons  
Site PIs or designees may request a participant’s treatment assignment before a study 
is fully unblinded for reasons that are not urgent and do not require immediate 
(emergency) unblinding but may affect the participant’s medical care/safety.  
The site PI or designee will consult with the Sponsor via email or teleconference and 
then submit a written request for unblinding in writing (via email). Early unblinding for this 
reason should generally not occur until all primary outcome data have been submitted, all 
queries related to these data have been resolved, and any clinical endpoints have been 
reviewed by designated reviewers. In cases in which knowledge of a participant’s 
treatment assignment sooner may affect the participant’s medical care and/or would 
otherwise be in the participant’s best interest, this requirement can be waived.  
 
STUDY ENDPOINTS 
Primary study endpoint 
 
The primary study endpoint is the superiority of bezlotoxumab over placebo, when 
given for CDI of high likelihood for unfavorable outcome, to reduce the incidence of 
unfavorable outcome. Unfavorable outcome is a composite endpoint and comprises the 
incidence of at least of one of the following in each enrolled patient: progression into organ 

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dysfunction; relapse of CDI; and death. The primary endpoint is tested on Day 40 from 
start of blind treatment. Organ dysfunction is defined as any increase of the baseline total 
SOFA (sequential organ failure assessment) score before allocation into blind treatment 
by at least 2 points. This definition is provided according to the current consensus of the 
definition of organ dysfunction in critically ill patients (13, 14). The need for colectomy or 
admission in the Intensive Care Unit count as organ dysfunction. 
The study is aiming to demonstrate the superiority of adjunctive bezlotoxumab 
treatment on the composite endpoint of unfavorable outcome. Patients who do not meet 
any of the components of this composite endpoint on Day 40 will be considered to have 
succeeded treatment. 
 
Secondary study endpoints 
 
The comparisons of bezlotoxumab over placebo on the following four endpoints: 
• Incidence of organ dysfunction and time to first organ dysfunction. This is defined as 
the presentation of organ dysfunction in any of the enrolled patients of each group 
starting from the day of blind allocation until Day 40. For patients with more than two 
episodes of organ dysfunction, the first one is encountered in the endpoint analysis. 
Organ dysfunction is defined as any increase of the baseline total SOFA (sequential 
organ failure assessment) score before allocation into blind treatment by at least 2 
points.   
• Time to relapse of CDI; the relapse of CDI is under daily follow-up until Day 40. 
Relapse is defined as the return of more than three unformed bowel movements in 24 
hours with a positive stool toxin test necessitating retreatment. The day of CDI relapse 
from blind allocation will be captured for relapsed patients and compared between 
groups.  
• Survival until Day 40 from study enrolment 
• Overall cost of hospitalization; this is calculated as stated above starting from the day 
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• Validation of the BEYOND score; this is done by comparing the incidence of the 
primary endpoint between patients who have failed screening in the trial and patients 
who were enrolled in the trial and who were allocated to the placebo arm.  
 
POWER CALCULATION 
 
This is calculated for the primary endpoint using the Power and Sample Size 
Calculations software version 3.1.2. Power calculation is based on the hypothesis that the 
difference between bezlotoxumab and placebo will be at least 40% i.e. equal to the one 
reported in the MODIFY randomized clinical trials (8). It is anticipated that 60% of patients 
allocated to the placebo arm and 20% of patients allocated to the bezlotoxumab arm will 
achieve the primary endpoint of unfavorable outcome. To demonstrate this difference at 
the 10% level of significance with 80% power, 22 patients should be enrolled into each 
arm (44 patients in total). It is anticipated that one out of three screened patients with CDI 
will meet all inclusion criteria, so that a total of 132 patients need to be screened. 
 
STATISTICAL ANALYSIS 
 
Regarding the qualitative primary and secondary endpoints, groups of treatment 
will be compared by the Fisher exact test; odds ratio and 95% confidence intervals (CIs) 
will be calculated by the Maentel-Henzel’s statistics. Secondary endpoints of time to an 
event will be compared between groups Cox regression analysis. 
 
ADVERSE EVENTS 
Treatment-emergent Adverse events (TEAEs) and Serious TEAEs will be collected 
from baseline until the last patient’s evaluation. Investigators should monitor subjects for 
adverse events and are responsible for recording ALL adverse events and serious 
adverse events occurring to a patient during the trial. Mortality will not be reported as an 
SAE since this is the study secondary endpoint. 
A TEAE is any undesirable medical occurrence in a subject administered a 
pharmaceutical product and which does not necessarily have a causal relationship with 
this treatment. The time relationship is defined from the moment the AE occurs during 

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therapeutic treatment until 30 days or 5 half-lives after treatment discontinuation. The 
adverse event may be a sign, a symptom, or an abnormal laboratory finding. Reporting to 
Health Authorities and Ethics Committees will be done by investigator according to the 
local requirements.  
Serious TEAEs. Serious TEAEs must be reported by the investigator to the health 
authorities and ethics committee according to the local requirements within 24 hours. If 
an adverse event meets any of the following criteria, it is considered STEAE: 
• Life-threatening situation The subject was at risk of death at the time of the adverse 
event/experience. It does not refer to the hypothetical risk of death if the AE were more 
severe or were to progress. 
• Inpatient hospitalization or prolongation of existing hospitalization. 
• Persistent or significant disability/incapacity Any TEAE having an outcome that is 
associated with a substantial disruption of the ability to carry out normal life functions, 
including the ability to work. This is not intended to include transient interruption of daily 
activities. 
• Congenital anomaly/birth defects Any structural abnormality in subject’s offspring 
that occurs after intrauterine exposure to treatment. 
• Important medical events/experiences that may not result in death, be life-
threatening, or require hospitalization may be considered a serious adverse 
event/experience when, based upon appropriate medical judgment, they may 
jeopardize the subject and may require medical or surgical intervention to 
prevent one of the outcomes listed above, i.e., death, a life-threatening adverse 
event/experience, inpatient hospitalization or prolongation of existing hospitalization, a 
persistent or significant disability/incapacity, or a congenital anomaly/birth defect.  
Examples of such medical events/experiences include allergic bronchospasm 
requiring intensive treatment in an emergency room or at home, blood dyscrasias or 
convulsions that do not result in inpatient hospitalization, or the development of drug 
dependency or drug abuse. 
• Spontaneous and elective abortions experienced by study subject.  
 
A non-TEAE is any untoward medical occurrence in a patient or subject who is 
administered a pharmaceutical product, and which does not necessarily have a causal 

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relationship with this treatment. A non-serious adverse event is one that does not meet 
the definition of a serious adverse event given. Non-serious TEAEs must be reported by 
the investigator to the health authorities and ethics committee according to the local 
requirements at the end of the trial. 
 
Grading of severity  
The severity of the adverse events shall be graded as: 
• Mild the adverse event is transient and well tolerated by the patient  
• Moderate the adverse event causes discomfort and affects the usual activities of the 
patient.  
• Severe the adverse event affects the usual activities of the patient to an important 
degree and may cause disability or be life-threatening. 
 
Relationship to the drug 
The investigator will use the following definitions to assess the relationship of the 
adverse event to study drug: 
• Probably Related: The adverse event has a strong time relationship to the drug or 
relapses if re-induced, and another etiology is improbable or clearly less probable. 
• Possibly Related: The adverse event has a strong time relationship to the drug and 
an alternative aetiology is as probable or less probable. 
• Probably not Related: The adverse event has a slight or no time relationship to the 
drug and/or there is a more probable alternative aetiology.  
• Unrelated: The adverse event is due to an underlying or concomitant disease or to 
another pharmaceutical product and is not related to the drug (no time relationship and 
a much more probable alternative aetiology).  
If an investigator’s opinion of possibly related, probably not related or not related 
to study drug is given, an alternate etiology must be provided by the investigator. Please 
note that a severe adverse event/experience is not necessarily serious, as the term severe 
is a measure of intensity while a serious adverse event is determined based on the 

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aforementioned regulatory criteria. Individual un-blinding thought to be necessary for the 
management of an adverse event will be documented in the subject Case Report Form. 
All Investigators must report every adverse event and evaluate the severity and 
possible causality with the study drug according to aforementioned criteria. All adverse 
events/reactions are reported to Sponsor. The sponsor is responsible for evaluation of all 
serious TEAEs. All Serious Adverse Events/ Serious Adverse Reactions must be reported 
within 24 hours by completion of the SAE and faxing/emailing to Hellenic Institute for the 
Study of Sepsis. 
The Sponsor must evaluate whether an adverse event is expected or not. A SAE 
may qualify for expedited reporting to regulatory authorities if it is determined to be a 
suspected, unexpected serious adverse reaction (SUSAR). The Sponsor is responsible 
for submitting expedited safety reports to the appropriate regulatory agency for all 
confirmed SUSARs. In the case of a fatal or life-threatening SUSAR, the Sponsor will 
notify the appropriate regulatory agency as soon as possible but in no case later than 7 
calendar days after the Sponsor’s initial receipt of the information. For a non-life-
threatening SUSAR, the report will be submitted no later than 15 days after the Sponsor 
is made aware of the event. 
The Sponsor has the obligation to submit annually a drug safety updated report 
(DSUR) according to global experience to appropriate regulatory authorities. The 
electronic submission to Eudravigilance will be performed through the Organisation ID: 
HISS. 
The above pharmacovigilance procedures will be performed for the Sponsor 
(Hellenic Institute for the Study of Sepsis) by the Consultant Company SustChem 
Engineering S.A., 144 3rd Septemvriou str, 11251, Αthens, and the Qualified Person for 
Pharmacovigilance (QPPV) will be Ms Areti Voulomenou, MSc (contact details in 
Appendix V). 
QUALITY CONTROL AND ASSURANCE 
Quality control and assurance checks are performed by sponsor in order to allow 
periodic review of adequacy of the study activities and practices and allow for revising 
such practices as needed so the data and process are maintained, the study meets the 
protocol and procedural requirements, and is reproducible.  

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Before enrolling any subject in this study, sponsor personnel and the investigator have 
to review the protocol, the IB, the CRFs and instructions for their completion, the 
procedure for obtaining informed consent and the procedure for reporting AEs and SAEs. 
A qualified representative of the sponsor will monitor the conduct of the study by 
visiting the site and by contacting the site by telephone and e-mail. During these site visits, 
all source documents are reviewed, and information recorded in the CRFs is verified 
against them. 
Besides routine monitoring, quality assurance will be documented through 
independent auditing of the quality control activities and where applicable, by regulatory 
authorities through inspections. 
 
ETHICAL CONSIDERATIONS  
Prior to the initiation of this study, the study design will receive ethical, scientific, and 
where applicable, regulatory review. Investigators will conduct this study in accordance 
with the principles of the Declaration of Helsinki, GCP, and applicable regulatory 
requirements. 
Regarding Informed Consent Form obtaining procedures, before any procedure 
specified in the protocol is performed, a subject must: 
- 
Be informed of all pertinent aspects of the study and all elements of informed 
consent 
- 
Be given time to ask questions and to consider the decision to participate 
- 
Voluntarily agree to participate in the study 
- 
Sign and date the updated and approved by IEC/REB ICF version. 
DATA HANDLING AND RECORD KEEPING 
Each enrollment center will record patient data in Case Report Forms (CRFs). These 
forms were designed specifically for the trial according to the study protocol and will be 
completed for every study participant by a blinded member of the study team.  

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Source documents will be kept under secure file at individual enrollment centers. 
Delegated members of the Sponsor will have access to all the records from enrolled 
participants, for monitoring purposes. 
FINANCING AND INSURANCE 
The BEYOND trial is funded by the Hellenic Institute for the Study of Sepsis (HISS). 
PUBLICATION POLICY 
The design and primary results of the paper will be published regardless of the study 
results. HISS will not make final decisions regarding the contents of the publication(s). 
The results of the trial will also be posted in clinicaltrials.gov and any other public 
repositories. 
 
PROTOCOL ADHERENCE AND AMENDMENTS 
Investigators ascertain that they will apply due diligence to avoid protocol deviations. 
All significant protocol deviations will be recorded and reported in the clinical study report 
(CSR). Any change or addition to the protocol can only be made in a written protocol 
amendment that must be approved and signed by the sponsor, health authorities where 
required, and the IEC/REB. 
 
 
 
 
 

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8. Wilcox MH, Gerding DN, Poxton IR, Kelly C, Nathan R, Birch T, et al. Bezlotoxumab 
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9. Pakpour S, Bhanvadia A, Zhu R, Amarmani A, Gibbons SM, Gurry T, et al. Identifying 
predictive features of Clostridium difficile infection recurrence before, during, and after 
primary antibiotic treatment. Microbiome 2017; 5: 148 
10. Charslon ME, Pompei P, Ales KL, MacKenzie CR. A new method of classifying 
prognostic comorbidity in longitudinal studies: development and validation. J Chronic 
Dis 1987; 40: 373-383 
11. Louie TJ, Miller MA, Mullane KM, Weiss K, Lentnek A, Golan Y, et al. Fidaxomicin 
versus vancomycin for Clostridium difficile infection. N Engl J Med 2011; 364: 422-431 

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12. Cornely OA, Crook DW, Esposito R, Poirier A, Somero MS, Weiss K, et al. Fidaxomicin 
versus vancomycin for infection with Clostridium difficile in Europe, Canada, and the 
USA: a double-blind, non-inferiority, randomised controlled trial. Lancet Infect Dis 
2012; 12: 281-289 
13. Singer M, Deutschman CS, Seymour CW, Shankar-Hari M, Annane D, Bauer M, et al. 
The third international consensus definitions for sepsis and septic shock (Sepsis-3). 
JAMA 2016; 315: 801-810 
14. Giamarellos-Bourboulis EJ, Tsaganos T, Tsangaris I, Lada M, Routsi C, Sinapidis D, 
et al. Validation of the new sepsis-3 definitions: proposal for improvement in early risk 
identification. Clin Microbiol Infect 2017; 23: 104-109 
 
 

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APPENDIX I Final study report of the SPECIFY project 
 
 
 
 
 
 
 
 
SCORING PERSONALIZED NEEDS IN CLOSTRIDIUM DIFFICILE INFECTIONS FOR 
FIDAXOMIXIN THERAPY: 
THE SPECIFY PROJECT 
 
 
FINAL STUDY RESULTS REPORT 
 
 
 
Evangelos J. Giamarellos-Bourboulis, MD, PhD 
Principal Investigator 
Associate Professor of Medicine 
Chairman of the Board, Hellenic Institute for the Study of Sepsis 
 
 
 
 
 
 
 
 
ATHENS 2018 

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FIRST PART OF THE ANALYSIS 
GENETIC SUSCEPTIBILITY TO CLOSTRIDIUM DIFFICILE 
The first patient was enrolled on September 5 2015 and the last patient on November 11 
2016. During this study period, 153 patients and 150 comparators were enrolled from 11 
study sites. Comparators were patients hospitalized in the same wards during the same 
study period. The comparators were fully matched to patients regarding demographics 
and the Charlson’s Comorbidity Index (CCI) (Table 1). 
 
Table 1 Comparison between patients with Clostridium difficile infection (CDI) and 
comparators. 
 
CDI (n=153) 
Comparators (n=150) 
p-value 
Age (years, mean ± SD) 
76.2 ± 13.3 
66.7 ± 11.4 
<0.0001* 
Male gender (n, %) 
71 (46.6) 
80 (53.3) 
0.251** 
CCI (mean ± SD) 
5.79 ± 2.44 
6.03 ± 2.88 
0.443* 
Study site (n, %) 
 
 
 
Infections Unit, Evangelismos 
Hospital 
31  (20.3) 
30 (20.0) 
 
4th Department of Internal 
Medicine, ATTIKON Hospital 
29 (19.0) 
28 (18.7) 
 
1st Department of Internal 
Medicine, G.Gennimatas Hospital 
21 (13.7) 
20 (13.3) 
 
1st Department of Internal 
Medicine, Thriasio Hospital 
20 (13.1) 
20 (13.3) 
 
Infections Unit, Tzaneion Hospital 19 (12.4) 
19 (12.7) 
 
2nd Department of Internal 
Medicine, Thriasio Hospital 
17 (11.1) 
17 (11.3) 
 
3rd Department of Internal 
Medicine, Sotiria Hospital 
6 (3.9) 
6 (4.0)  
 

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1st Department of Internal 
Medicine, Laiko Hospital 
4 (2.6) 
4 (2.7) 
 
3rd Department of Internal 
Medicine, AHEPA Hospital 
3 (2.0) 
3 (2.0) 
 
2nd Department of Internal 
Medicine, Sismanogleion Hospital 
2 (1.3) 
2 (1.3) 
 
Department of Internal Medicine, 
Rion Hospital 
1 (0.7) 
1 (0.6) 
 
*Student’s t-test; **Fisher exact test 
 
DNA was extracted from all samples by the end of December 2016 and it was of 
appropriate quality. Then GWAS was performed with one HumanCore-chip. Running of 
the GWAS was completed by the end of April 2017. Bioanalysis started in May 2017 and 
the first round of results were provided end of July 2017. The quality control of imputed 
data is shown in Figure 1. Analysis was done for 4,330,811 SNPs in 134 remaining 
controls and 134 remaining cases. 
 
 

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Figure 1 Quality control of imputed data 
Abbreviations HWE: Hardy Weinberg equilibrium; MDS: multidimensional scaling; QC: 
quality control; SNP: single nucleotide polymorphism 
 
 
Logistic regression analysis taking into consideration age, gender and Charlons’ 
comorbidity index (CCI) did not disclose any SNP below the p level of 5x10-8 of 
significance. It was decided to focus on SNPs that according to the Manhattan plot were 
strongly associated with susceptibility for CDI (Figure 2). Among these four SNPs 
(rs4311028, rs1940913, rs474032 and rs2091172) were detected at a level of 
significance lower than 9x10-6 (Table 2). For the other SNPs, association with 
expression qualitative trait loci (eQTLs) was analyzed and another 13 SNPs were found 
to strongly influence the expression of several gene loci as shown in Table 3. The 

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frequencies of the total of the 17 gene SNPs were compared between controls and 
cases as shown in Tables 4 and 5. 
 
 
 
 
Figure 2 Manhattan plot indicating loci that show suggestive association with 
susceptibility to Clostridium difficile infection 

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Table 2 The 4 SNPs showing the strongest association with Clostridium difficile infection 
Chromosome 
SNP ID 
Position 
p-value* 
Known gene 
Gene function 
2 
rs4311028 
29706981 
9.35 x 10-7 
ALK 
Anaplastic lymphoma kinase (ALK) or ALK tyrosine kinase receptor 
or CD246; oncogenic kinase 
3 
rs474032 
8076409 
8.1 x 10-6 
LOC101927394 
uncharacterized 
7 
rs2091172 
27515580 
8.2 x 10-6 
 
HOX genes 
Homeobox; transcription factor 
18 
rs1940913 
13572220 
2.69 x 10-6 
 
LDLRAD4 
Low-density lipoprotein receptor class A domain-containing protein 4; 
negative regulator of TGF-beta signaling and thereby probably plays 
a role in cell proliferation, differentiation, apoptosis, motility, 
extracellular matrix production and immunosuppression 
 
*after logistic regression analysis 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Table 3 The 13 SNPs that are associated with an effect on gene expression loci 
 
 
 
 
GTEx-eQTLs 
BIOS-eQTLs 
 
 
Chromosome 
SNP ID 
Position 
p-value* 
Gene 
p-value 
Gene 
p-value 
Tissue 
Gene function 
2 
rs6735120 
55961771 
2.32 x 10-5 
 
 
SMEK2 
1.4 x 10-11 
Whole blood 
Protein Phosphatase 4 
Regulatory Subunit 3B/ 
Regulates the activity of 
PPP4C at centrosomal 
microtubule organizing centers 
2 
rs62183547 
172372438 
1.78 x 10-5 
 
 
 
CYBRD1 
2.0 x 10-9 
Whole blood 
Cytochrome B Reductase 1/ 
Plays a role in dietary iron 
absorption 
7 
rs1128226 
21941669 
7.03 x 10-5 
 
 
 
CDCA7L 
7.2 x 10-9 
Whole blood 
Cell Division Cycle Associated 
7 Like/ Transcriptional 
regulation as a repressor of 
monoamine oxidase A (MAOA)  
8 
rs4279595 
22907330 
9.45 x 10-5 
 
RP11-
875O11.3 
8.2 x 10-14 
RP11-
875O11.3 
5.4 x 10-94 
Whole blood 
TNF Receptor Superfamily 
Member 10b/ Promotes the 
activation of NF-kappa-B. 
Essential for ER stress-
induced apoptosis. 
14 
rs175006 
75445499 
4.38 x 10-5 
 
 
 
EIF2B2 
4.5 x 10-122 
Whole blood 
Eukaryotic Translation 
Initiation Factor 2B Subunit 
Beta/ Involved in protein 
synthesis  
15 
rs12148744 
41315357 
5.96 x 10-5 
 
 
 
CHP1 
1.6 x10-22 
Whole blood 
Calcineurin Like EF-Hand 
Protein 1/ Essential cofactor of 
NHE family members and 
mitogenic regulation of NHE1 
17 
rs3859214 
64460807 
6.24 x 10-5 
 
PRKCA 
9.4 x 10-10 
No 
 
PBMCs 
Protein Kinase C Alpha/ 
Tegulation of cell proliferation, 
apoptosis, differentiation, 
migration and adhesion, 
tumorigenesis, cardiac 
hypertrophy, angiogenesis, 
platelet function and 
inflammation, by directly 
phosphorylating targets such 
as RAF1, BCL2, CSPG4, 

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TNNT2/CTNT, or activating 
signaling cascade involving 
MAPK1/3 (ERK1/2) and 
RAP1GAP 
17 
rs7222870 
77652955 
7.65 x 10-5 
 
 
 
CTD-
2116F7.1 
1.7 x 10-17 
Whole blood 
Long Intergenic Non-Protein 
Coding RNA 2078 
18 
rs7240534 
51885897 
1.38 x 10-5 
POLI 
1.8 x 10-10 
POLI 
2.2 x 10-137 
Whole blood/ 
transverse colon 
Encodes for error-prone DNA 
polymerase involved in DNA 
repair 
18 
rs508660 
12391134 
6.75 x 10-5 
 
 
RP11-
861E21.2 
2.4 x 10-19 
Whole blood 
 
19 
rs17680671 
21576563 
5.44 x 10-5 
RP11-
678G14.2 
4.4 x 10-10 
ZNF429, 
RP11-
678G14.2 
2.9 x 10-77 
Whole blood 
Zinc Finger Protein 429/ 
Involved in transcriptional 
regulation 
20 
rs721059 
2524927 
3.34 x 10-5 
 
 
NOP56 
3.0 x 10-8 
Whole blood 
NOP56 Ribonucleoprotein/ 
Involved in early to middle 
stages of 60S ribosomal 
subunit biogenesis 
22 
rs714024 
47013535 
9.51 x 10-5 
 
GRAMD4 
2.5 x 10-6 
GRAMD4 
1.5 x 10-140 
Whole blood 
GRAM Domain Containing 4/ 
Mitochondrial effector of E2F1 
(MIM 189971)-induced 
apoptosis 
 
*after logistic regression analysis 
 
 
 
 
 

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Table 4 Comparison of the frequencies of the 4 SNPs that are associated with 
the strongest association between patients with Clostridium difficile infection 
(CDI) and controls. 
 
 
Controls (n=134) 
CDI (n=134) 
p-value* 
OR (95%CIs)** 
rs4311028 (n, %) 
 
 
 
 
CC 119 (88.8) 
86 (64.2) 
 
 
TC 15 (11.2) 
46 (34.3) 
<0.0001 
 
CC  
2 (1.5) 
 
 
Carriage of C allele 15 (11.2) 
48 (35.8) 
6.0 x 10-6 
4.43 (2.32-8.42) 
rs474032 (n, %) 
 
 
 
 
GG 58 (43.3) 
85 (63.4) 
 
 
AG 58 (43.3) 
43 (32.1) 
<0.0001 
 
AA 18 (13.4) 
6 (4.5) 
 
 
Carriage of A allele 76 (56.7) 
49 (36.6) 
0.001 
0.44 (0.69-0.71) 
rs2091172 (n, %) 
 
 
 
 
AA 54 (40.3) 
25 (18.7) 
 
 
CA 59 (44.0) 
70 (52.2) 
<0.0001 
 
CC 21 (15.7) 
39 (29.1) 
 
 
Carriage of C allele 80 (59.7) 
109 (81.3) 
1.5 x 10-4 
2.94 (1.68-5.12) 
rs1940913 (n, %) 
 
 
 
 
GG 35 (26.1) 
57 (42.5) 
 
 
CG 65 (48.5) 
64 (47.8) 
<0.0001 
 
CC 34 (25.4) 
13 (9.7) 
 
 
Carriage of C allele 99 (73.9) 
77 (57.5) 
0.007 
0.48 (0.29-0.80) 
 
 
 
 
 
 

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Table 5 Comparison of the frequencies of the 13 SNPs that are associated with 
eQTLs between analyzed patients with Clostridium difficile infection (CDI) and 
controls. 
 
 
Controls (n=134) 
CDI (n=134) 
p-value* 
OR (95%CIs)** 
rs6735120 (n, %) 
 
 
 
 
AA 117 (87.3) 
88 (65.7) 
 
 
GA 17 (12.7) 
43 (32.1) 
<0.0001 
 
GG 0 (0) 
3 (2.2) 
 
 
Carriage of G allele 17 (12.7) 
46 (34.3) 
4.4 x 10-5 
3.59 (1.93-6.69) 
rs62183547 (n, %) 
 
 
 
 
GG 79 (59.0) 
115 (85.8) 
 
 
AG 52 (38.8) 
18 (13.4) 
<0.0001 
 
AA 3 (2.2) 
1 (0.7) 
 
 
Carriage of A allele 55 (41.0) 
19 (14.2) 
1.0 x 10-6 
0.24 (0.13-0.43) 
rs1128226 (n, %) 
 
 
 
 
AA 65 (48.5) 
32 (23.9) 
 
 
CA 59 (44.0) 
79 (59.0) 
<0.0001 
 
CC 10 (7.5) 
23 (17.2) 
 
 
Carriage of C allele 69 (51.5) 
102 (76.1) 
4.2 x 10-5 
3.00 (1.78-5.06) 
rs4279595 (n, %) 
 
 
 
 
CC 52 (38.8) 
91 (67.9) 
 
 
AC 70 (52.2) 
39 (29.1) 
<0.0001 
 
AA 12 (9.0) 
4 (3.0) 
 
 
Carriage of A allele 82 (61.2) 
43 (32.1) 
3.0 x 10-6 
0.30 (0.18-0.49) 
rs175006 (n, %) 
 
 
 
 
CC 81 (60.4) 
50 (37.3) 
 
 
GC 45 (33.6) 
67 (50.0) 
<0.0001 
 
GG 8 (6.0) 
17 (12.7) 
 
 
Carriage of G allele 53 (39.6) 
84 (62.7) 
2.3 x 10-4 
2.56 (1.56-4.20) 

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rs12148744 (n, %) 
 
 
 
 
TT 119 (88.8) 
89 (66.4) 
 
 
TC 15 (11.2) 
44 (32.8) 
<0.0001 
 
CC 0 (0) 
1 (0.7) 
 
 
Carriage of T allele 15 (11.2) 
45 (33.6) 
1.6 x 10-5 
4.01 (2.10-7.65) 
 
rs3859214 (n, %) 
 
 
 
 
GG 48 (35.8) 
21 (15.7) 
 
 
GT 67 (50.0) 
76 (56.7) 
<0.0001 
 
TT 19 (14.2) 
37 (27.6) 
 
 
Carriage of T allele 86 (64.2) 
113 (84.3) 
2.5 x 10-4 
3.00 (1.67-5.39) 
rs7222870 (n, %) 
 
 
 
 
AA 87 (64.9) 
64 (47.8) 
 
 
GA 46 (34.3) 
62 (46.3) 
<0.0001 
 
GG 1 (0.7) 
8 (6.0) 
 
 
Carriage of G allele 47 (35.1) 
70 (52.2) 
0.007 
2.02 (1.24-3.31) 
rs7240534 (n, %) 
 
 
 
 
CC 44 (32.8) 
23 (17.2) 
 
 
TC 71 (53.0) 
66 (49.3) 
<0.0001 
 
TT 19 (14.2) 
45 (33.6) 
 
 
Carriage of T allele 90 (67.2) 
111 (82.8) 
0.005 
2.36 (1.32-4.19) 
rs508660 (n, %) 
 
 
 
 
AA 60 (44.8) 
90 (67.2) 
 
 
GA 54 (40.3) 
39 (29.1) 
<0.0001 
 
GG 20 (14.9) 
5 (3.7) 
 
 
Carriage of G allele 74 (55.2) 
44 (32.8) 
3.4 x 10-4 
 
rs17680671 (n, %) 
 
 
 
 
GG  35 (26.1) 
57 (42.5) 
 
 
CG 65 (48.5) 
64 (47.8) 
<0.0001 
 
CC 34 (25.4) 
13 (9.7) 
 
 

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Carriage of C allele 99 (73.9) 
77 (57.5) 
0.007 
0.47 (0.28-0.80) 
rs721059 (n, %) 
 
 
 
 
TT 43 (32.1) 
84 (62.7) 
 
 
TC 66 (49.3) 
46 (34.3) 
<0.0001 
 
CC 25 (18.7) 
4 (3.0) 
 
 
Carriage of C allele 91 (67.9) 
50 (37.3) 
8.22 x 10-7 
0.28 (0.17-0.47) 
rs714024 (n, %) 
 
 
 
 
TT 46 (34.3) 
81 (60.4) 
 
 
TC 69 (51.5) 
47 (35.1) 
<0.0001 
 
CC 19 (14.2) 
6 (4.5) 
 
 
Carriage of C allele 88 (65.7) 
53 (39.6) 
2.9 x 10-5 
0.34 (0.21-0.56) 
 
*by the Fisher exact test for 2 x 2 comparisons or by the Pearson’s chi-square test for 
2 x 3 comparisons  
**Mantel-Haenszel statistics 
Abbreviations CI: confidence intervals; OR: odds ratio 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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As a next step logistic regression analysis was done to identify which of the total of 
17 minor frequency SNP alleles (Tables 3 and 4) are independent predictors of CDI 
among a patient population of hospitalized patients with similar CCI. Results after 
nine steps are shown in Table 6; only nine of the 17 SNPs remained significant.  
 
 
 
 
 
Table 6 Final step of the stepwise logistic regression procedure with group 
(comparators vs CDI) as the dependent variable; odds rations less than 1 
indicate protective alleles.   
 
 
Β-SLOPE 
SE 
Wald 
p-value Odds ratio 
Lower 95%CI 
Upper 95%CI 
Carriage of at least one C 
allele of rs12148744 
2,721 
,737 
13,635 
,000 
15,192 
3,584 
64,385 
Carriage of at least one C 
allele of rs714024 
-2,715 
,635 
18,278 
,000 
,066 
,019 
,230 
Carriage of at least one C 
allele of rs721059 
-2,922 
,638 
20,954 
,000 
,054 
,015 
,188 
Carriage of at least one T 
allele of rs4311028 
3,280 
,755 
18,880 
,000 
26,584 
6,054 
116,741 
Carriage of at least one A 
allele of rs62183547 
-2,545 
,618 
16,981 
,000 
,078 
,023 
,263 
Carriage of at least one C 
allele of rs1128226 
1,239 
,498 
6,199 
,013 
3,452 
1,302 
9,155 
Carriage of at least one A 
allele of rs4279595 
-1,724 
,484 
12,680 
,000 
,178 
,069 
,461 
Carriage of at least one G 
allele of rs175006 
1,084 
,473 
5,247 
,022 
2,957 
1,169 
7,477 
Carriage of at least one T 
allele of rs3859214 
1,682 
,518 
10,531 
,001 
5,375 
1,947 
14,844 
Carriage of at least one G 
allele of rs70028870 
1,492 
,515 
8,388 
,004 
4,444 
1,620 
12,195 

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Carriage of at least one G 
allele of rs508660 
-,972 
,457 
4,520 
,033 
,378 
,154 
,927 
Carriage of at least one T 
allele  of rs7240534 
1,272 
,561 
5,151 
,023 
3,570 
1,190 
10,712 
Carriage of at least one G 
allele of rs2091172 
1,099 
,516 
4,542 
,033 
3,001 
1,092 
8,244 
Carriage of at least one C 
allele of rs17680671 
-1,675 
,547 
9,357 
,002 
,187 
,064 
,548 
 
 
 
Abbreviation CI: confidence interval 
 
 
Using the values of the B slope of the logistic regression analysis, we developed 
a gene score to differentiate between comparators and CDI. This is provided in the 
following equation: 
Gene score=  
(Carriage of C allele of rs12148744 x 27) – (carriage of C allele of rs714024 x 27) - 
(carriage of C allele of rs721059 x 29) + (carriage of T allele of rs4311028 x 33) – 
(carriage of A allele of rs62183547 x 25) + (carriage of C allele of rs1128266 x 12) - 
(carriage of T allele of rs4279595 x 17) + (carriage of G allele of rs175006 x 11) + 
(carriage of T allele of rs3859214 x 17) + (carriage of G allele of rs7222870 x 15) – 
(carriage of G allele of rs5086600 x 9) + (carriage of T allele of rs7240534 x 12) + 
(carriage of G allele of rs20911172 x 11) - (carriage of C allele of rs17680671 x 17) 
   
 
At the above equation, carriers of at least one the indicated alleles score 1 point and 
non-carriers score 0 points. 
 
 
The received operator characteristics (ROC) curve to differentiate between CDI and 
comparators is shown in Figure 3. 
 

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Figure 3 ROC curve of the gene score to discriminate between patients with 
CDI and comparators 
Area under the curve: 0.827 (0.780-0.897), p: 2.05 x 10-20 
 
 
 
 
 
 
Using a cut-off of sensitivity greater than 90%, we depicted through the co-ordinate 
points of the curve a cut-off greater than 53 of the gene score. The diagnostic 
characteristics of the gene score at that cut-off are provided in Table 7. It is concluded 
that this gene score at a state of elevated CCI, provides odd ratio 6.89 (3.53-13.42, p: 
1.39 x 10-8) for susceptibility to CDI. 
Table 7 Diagnostic performance of the gene score at a cut-off greater than 53 to 
differentiate between CDI and controls among a hospitalized patient population 
with similar CCI. 
 

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CDI 
Comparators 
Total 
Gene score >53 
57 
Sensitivity: 42.5% 
PPV: 81.4% 
13 
70 
Gene score ≤53 
77 
121 
Specificity: 90.3% 
NPV: 61.1% 
198 
Total 
134 
134 
268 
  
Abbreviations CDI: Clostridium difficile infection; NPV: negative predictive value; 
PPV: positive predictive value 
 
 
 
 
 
 
Special mentioning has to be done to the eQTL of rs7240537 that is associated with 
the expression of the PORI gene in the colon (Figure 4). This gene encodes for error-
prone DNA polymerase involved in DNA repair and it is associated with psoriasis and 
with the interaction of the microbiome with the host in psoriatic arthritis. 
 
 
 
 
 
 

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Figure 4 eQTL of rs7240534 associated with the POLI gene 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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SECOND PART OF THE ANALYSIS 
SUSCEPTIBILITY TO UNFAVORABLE OUTCOME OF CDI 
 
From the 134 analyzed CDI cases, 39 (29.1%; 95%CIs 22.1-37.3%) met the per 
protocol primary study endpoint of unfavorable outcome i.e. presenting or developing 
organ dysfunction and/or CDI relapse and/or death. From these 39 patients, 15 had an 
extreme CDI phenotype i.e. they had unfavorable outcome with ATLAS score equal to 
zero. The comparisons between the 95 patients who did not experience the primary 
endpoint and the 39 patients who met the primary endpoint are shown in Table 8. 
 
 
Table 8 Difference in baseline characteristics between patients with CDI without 
unfavorable outcome and patients with CDI and unfavorable outcome 
 
CDI without unfavorable 
outcome (n=95) 
CDI with unfavorable 
outcome (n=39) 
p-value 
Age (years, mean ± SD) 
76.3 ± 12.7 
76.1 ± 14.6 
0.951** 
Male gender (n, %) 
50 (52.6) 
21 (53.8) 
1.000*** 
Hemoglobin <9.45g/dl (n, %)* 
19 (20.0) 
17 (43.6) 
0.009*** 
Urea >64.5 mg/dl (n, %)* 
18 (18.9) 
17 (43.6) 
0.005*** 
Creatinine (mg/dl, mean ± SD) 
1.24 ± 1.28 
1.83 ± 2.02 
0.240** 
Hypoalbuminemia (n, %) 
7 (7.4) 
0 (0) 
0.106*** 
Intake of antimicrobials <3 months (n, %) 
68 (71.6) 
26 (66.7) 
0.678*** 
CCI (mean ± SD) 
5.55 ± 2.17 
6.38 ± 2.92 
0.117** 
Previous CDI 
7 (7.5) 
4 (10.3) 
0.731*** 
Extreme CDI phenotype (n, %) 
0 (0) 
15 (38.5) 
<0.0001*** 
White blood cells (/mm3, mean ± SD) 
12083.0 ± 8124.5 
12984.3 ± 9918.3 
0.586** 
C-reactive protein (mg/dl, median-range) 
40.0 (<3-331.0) 
39.8 (<3-279.0) 
0.471**** 
IL-6 (pg/ml, median-range) 
7.0 (7.0-2000) 
7.0 (7.0-133.1) 
0.779**** 
IL-8 (pg/ml, median-range) 
31.3 (<4-494.7) 
16.4 (<4-437.9) 
0.877**** 
Eotaxin (pg/ml, median, range) 
201.9 (<31-975.5) 
152.6 (<31-1753.3) 
0.899**** 
*selected for specificity >80% by the ROC curve of the indicated variable for 
unfavorable outcome 
**by the Student’s t-test 

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***by the 2-sided Fisher exact test 
****by the Mann-Whitney U test 
 
The eQTL of rs714024 is for the GRAMD4 gene that is a pro-apoptotic protein which 
binds to Nogo-B and negatively regulates immune responses initiated by nucleic 
acid–sensing Toll like receptors. Our findings correlate this eQTL with IL-8 and 
indicate significant differences of circulating IL-8 in relation to the genotype (Figure 
5). Following ROC curve analysis, a cut-off of 227 pg/ml is recognized based on 
which patients with IL-8 greater than 227 pg/ml have odds ratio 6.21 (95%CIs: 1.96-
19.64, p: 0.002) for unfavorable outcome. 
 
 
 
Figure 5 Association between eQTL of rs714024 and circulating levels of IL-8 
A) Circulating IL-8 on day 1 in relation to the genotype of rs714024; B) eQTL of 
rs714024 on TLR signaling. P values of significance are shown.  
 
 
 
 
 
 
Since the great majority of patients have positive gene score, it was cross-
tabulated which of the nine SNPs of the gene score either as carriers or in 
homozygosity is associated with unfavorable outcome. Significant results of the 
cross-tabulations are shown in Table 9. 

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Table 9 Difference in SNPs/genotypes between patients with CDI without 
unfavorable outcome and patients with CDI and unfavorable outcome 
 
CDI without unfavorable 
outcome (n=95) 
CDI with unfavorable 
outcome (n=39) 
p-value 
Carriage of G allele of rs2091172 (n, %) 
84 (84.4) 
25 (64.1) 
0.003 
GG genotype of rs6735120 (n, %) 
0 (0) 
3 (7.7) 
0.023 
 
Based on the findings of Tables 8 and 9 and of Figure 4, a logistic regression 
analysis was done to identify the variables that were independently associated with 
unfavorable outcome (Table 10). 
 
Table 10 Logistic regression analysis among the variables strongly associated 
with unfavorable outcome 
 
Variable 
β slope 
OR 
95% CIs 
p-value 
Hb<9.5 g/dl 
+1 
2.97 
1.17-7.46 
0.021 
Urea >64.5 mg/dl 
+1.44 
4.23 
1.64-10.92 
0.003 
IL-8 >227 pg/ml 
+1.87 
6.50 
1.79-23.68 
0.005 
G allele rs2091172 
-1.67 
0.19 
0.07-0.53 
0.002 
 
 
 
 
 
 
Based on the above Table, a prediction score is formatted from the following 
equation where the slope of the curves is used: 
 
Prediction score= (Presence of Hb <9.5 g/dl x 10) + (presence of urea >64.5 mg/dl x 
14) + (circulating IL-8 >227 pg/ml x 19) – (carriage of G allele of rs2091172 x 17). 
 

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In the above equation, the presence of each of the variable is scored as 1 and the 
absence of each of the variables is scores as 0. 
The ROC curve for prediction of unfavorable outcome with this score among patients 
is shown in Figure 6. 
 
 
Figure 6 ROC curve of the prediction score to early prognosticate unfavorable 
outcome among patients with CDI  
Area under the curve: 0.777 (0.686-0.869), p: 4.87 x 10-7 
 
 
 
 
 
Using a cut-off of 9, the positive predictive value of the score to predict 
unfavorable outcome is 80% (Table 11). The odds ratio for unfavorable outcome 
among patients with CDI and prediction score more than 9 is 15.82 (95%CIs: 4.83-
51.88, p: 5 x 10-6). 
 
 

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Table 11 Diagnostic performance of the prediction score at a cut-off greater 
than 9 for early prognosis of unfavorable outcome among patients with 
Clostridium difficile infection 
 
 
Unfavorable outcome 
Favorable outcome 
Total 
Prediction score >9 
16 
Sensitivity: 41.0% 
PPV: 80.0% 
4 
20 
Prediction score ≤9 
23 
91 
Specificity: 95.8% 
NPV: 79.8% 
114 
Total 
39 
95 
134 
  
Abbreviations NPV: negative predictive value; PPV: positive predictive value 
 
 
 
 

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APPENDIX II The SOFA score  
Variable 
0 points 
1 point 
2 points 
3 points 
4 points 
PaO2/FiO2 (mmHg) 
≥400 
<400 
<300 
<200 
<100 
Platelets (per mm3) 
≥150 
<150 
<100 
<50 
<20 
Hypotension 
MAP≥ 70 mmHg 
MAP< 70 mmHg 
Dobutamine 
whatever dose 
Adrenaline ≤0.1* or 
Noradrenaline≤ 0.1* 
Adrenaline>0.1* or 
Noradrenaline >0.1* 
Glasgow Coma Scale 
15 
13-14 
10-12 
6-9 
<6 
Bilirubin (mg/dl) 
<1.2 
1.2-1.9 
2.0-5.9 
6.0-11.9 
≥12 
Creatinine (mg/dl) or 
Urine output 
<1.2 
1.2-1.9 
2.0-3.4 
3.5-4.9 or  
<500 ml/day 
≥5.0 or 
<200 ml/day 
*μg/kg/min 
 
Each variable is scored between 0 and 4. The SOFA score is the sum of the score of each variable 
 

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APPENDIX IΙI The Charlson’s comorbidity index (CCI) 
CCI is defined after adding the score for age to the score for co-morbidities. 
The score for age is given below: 
Age group (years) 
Points 
0-49 
0 
50-59 
1 
60-69 
2 
70-79 
3 
80-89 
4 
90-99 
5 
 
The score for comorbidities is given below: 
Condition 
Points 
None 
0 
• Myocardial infarct 
(one or more definite or probable myocardial infarctions) 
1 
• Congestive heart failure 
(exertional or paroxysmal nocturnal dyspnea treated with digitalis, diuretics, or 
afterload reducing agents) 
 
• Peripheral vascular disease 
(intermittent claudication or  bypass for arterial insufficiency or gangrene or acute 
arterial insufficiency  or an untreated thoracic or abdominal aneurysm ≥6 cm) 
 
• Cerebrovascular disease (except hemiplegia) 
(history of a cerebrovascular accident with minor or no residua and  transient 
ischemic attacks) 
 
• Dementia (defined as chronic cognitive deficit) 
 
• Chronic pulmonary disease 
 
• Connective tissue disease 
(systemic lupus erythematous, polvmyositis. mixed  connective tissue disease, 
polymyalgia rheumatic and moderate to severe rheumatoid arthritis) 
 
• Peptic ulcer disease 
 
• Mild liver disease (cirrhosis without portal hypertension or chronic hepatitis ) 
 
• Diabetes (without complications) 
 

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• Diabetes with end organ damage 
(Retinopathy, neuropathy, nephropathy, previous hospitalizations for ketoacidosis, 
hyperosmolar coma, or juvenile onset or brittle diabetics) 
2 
• Hemiplegia 
 
• Moderate or severe renal disease 
(moderate defined as serum creatinine of 2-3 mg/dl, severe defined as need for 
dialysis/transplantation) 
 
• Solid tumor (non-metastatic) 
 (without  documented metastases, but initially treated in the last five  years) 
 
• Leukemia/lymphoma/multiple myeloma 
 
• Moderate or severe liver disease 
(Moderate: cirrhosis with  portal hypertension; severe: cirrhosis with portal 
hypertension and a history  of variceal bleeding 
3 
• Metastatic solid tumor 
6 
• AIDS (acquired immunodeficiency syndrome) (Definite or probable AIDS) 
 
 
 

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APPENDIX IV Study visits 
Visits days 
Screening 
 Days 1-10 
Day 12 
Day 40 
Informed consent 
x 
 
 
 
Exclusion criteria 
x 
 
 
 
Inclusion criteria 
x 
 
 
 
quick C.DIFFICILE Complete test  
x 
 
 
 
Blood sampling  
x 
 
 
 
Stool collection or rectal swab 
x 
 
 
 
BEYOND score calculation 
x 
 
 
 
Study drug administration 
 
x 
 
 
AE/SAE 
 
x 
x 
x 
Demographics 
 
x 
 
 
Comorbidities and predisposing illnesses 
 
x 
 
 
Vital signs 
 
x 
x 
x 
Laboratory findings including white blood cells, 
creatinine and liver biochemistry (if available) 
 
x 
x 
x 
Co-administered drugs and antimicrobials 
 
x 
x 
x 
Results of coloscopy (if done) 
 
x 
x 
x 
SOFA score 
 
x 
x 
x 
Organ dysfunction 
 
x 
x 
x 
Number of bowel movements 
 
x 
x 
x 
Need for colectomy 
 
x 
x 
x 
ICU admission 
 
x 
x 
x 
Clinical cure/relapse 
 
x 
x 
x 
Mortality 
 
x 
x 
x 

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APPENDIX V List of study sites 
• 4th Department of Internal Medicine, ATTIKON University General Hospital, (PI: 
Antonios Papadopoulos, Associate Professor of Internal Medicine and Infectious 
Diseases)  
• 2nd Department of Propaedeutic Internal Medicine, ATTIKON University General 
Hospital, (PI: Erifili Hatziaggelaki, Professor of Internal Medicine)  
• 3rd Department of Surgery, ATTIKON University General Hospital, (PI: Emmanouil 
Pikoulis, Professor of Surgery)  
• 3rd University Department of Internal Medicine, SOTIRIA General Hospital of Chest 
Diseases of Athens, (PI: Garyfallia Poulakou, Assistant Professor of Internal 
Medicine)  
• 1st University Department of Internal Medicine, LAIKO General Hospital of Athens, 
(PI: Michael Samarkos, Associate Professor of Internal Medicine and Infectious 
Diseases) 
• 1st Department of Internal Medicine, G. GENNIMATAS General Hospital of Athens, 
(PI: Georgios Adamis, Director of NHS)  
• 1st University Department of Propaedeutic Surgery, IPPOKRATEION General 
Hospital, (PI: Konstantinos Toutouzas, Associate Professor of Surgery)  
• 1st Department of Internal Medicine, THRIASIO General Hospital of Eleusis (PI: 
Styliani Symbardi, Director of NHS) 
• 2nd Department of Internal Medicine, THRIASIO General Hospital of Eleusis (PI: Zoi 
Alexiou, Director of NHS) 
• 2nd Department of Internal Medicine, TZANEIO General Hospital of Piraeus (PI: 
Chrysos Georgios, Director of NHS) 
• 1st Department of Internal Medicine, AHEPA University General Hospital of 
Thessaloniki (PI: Simeon Metallidis, Associate Professor of Internal Medicine and 
Infectious Diseases) 
• Department of Internal Medicine, University General Hospital of Patras PANAGIA I 
VOITHIA, (PI: Karolina Akinosoglou, Assistant Professor of Internal Medicine)  
• Department of Internal Medicine, University General Hospital of Larissa, (PI: 
George Dalekos, Professor of Internal Medicine) 

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• 2nd 
Department 
of 
Internal 
Medicine, 
University 
General 
Hospital 
of 
Alexandroupolis, (PI: Periklis Panagopoulos, Associate Professor of Internal 
Medicine) 
• 1st Department of Internal Medicine, University General Hospital of Ioannina, (PI: 
Charalampos Milionis, Professor of Internal Medicine) 
• 2nd Department of Surgery, G. GENNIMATAS General Hospital of Thessaloniki (PI: 
Ioannis Koutelidakis, Associate Professor of Surgery) 
 
 
Monitor Dr. Antigoni Kotsaki, MD, PhD  
e-mail: antigonebut@yahoo.com  tel: +30 694 66 37 164; +30 210 58 31 2562 
 
Pharmacovigilance supervisor Ms Areti Voulomenou, MEng, MSc  
e-mail: voulomenou@suschem.gr  tel +30 210 82 52 510