Clostridium Difficile Virulence Mechanism Study by Collecting Stool Samples From Clostridium Difficile Infection Patients (CDVM Study)
Conditions: Clostridium Difficile Infection, Clostridium Difficile
Sponsor: Chinese University of Hong Kong

 
 
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Title: Clostridium difficile virulence mechanism 
study (CDVM study) 
Date: 15 June 2018 
 
 
 
 
 
 
 

 
 
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Title: Clostridium difficile virulence mechanism study (CDVM study) 
Principal Investigator: Professor Sunny H Wong 
 
1. Research background and objectives 
Clostridium difficile is an anaerobic, gram-positive, sporulating bacterium that can give rise to a spectrum 
of gastrointestinal presentations, including diarrhoea, pseudomembraneous colitis and toxic megacolon. C 
difficile infection (CDI) is the most common cause of hospital-associated gastrointestinal infection. It 
accounted for approximately half a million infections and 29,000 deaths in the United States in 2011 
(Lessa et al., 2012), and it also represented a major healthcare burden in Canada and across Europe 
(Bauer et al., 2011). Meanwhile, the hyper-virulent ribotype 027 (BI/NAP1) strain has caused major 
outbreaks and deaths in North America and Europe. In Asia, the hyper-virulent ribotype 027 was only 
found sporadically; rather, the ribotype 002 was common in Asia. Our recent work showed that the 
ribotype 002 was the most common ribotype in Hong Kong (22.8%), and was associated with increased 
mortality compared to other ribotypes (47.6% versus 12.7%) (Wong et al., 2016). Despite its importance 
in this locality, there is a paucity of data regarding the virulence mechanism of this ribotype.  
This project aims (1) to investigate the virulence mechanisms of C. difficile ribotype 002, by analysing its 
growth, cytotoxicity, and metabolite production in vitro and in vivo. We also aim (2) to compare these 
virulence mechanisms with other common ribotypes in Asia, and (3) to correlate these with important 
clinical outcomes. The results of this study will have a major significance to the medical and scientific 
community. The mechanistic data will point to important molecular pathways that can be targeted for 
novel therapies; whereas the phenotypic data will inform us of its virulence and therefore will be useful 
for disease surveillance and public health interventions. The identified toxin(s) and metabolite(s) may be 
useful as prognostic biomarkers to stratify patients with different outcomes, as patients with a worse 
outcome may require more aggressive therapy. Given the prevalence and severity of this ribotype, our 

 
 
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results will be useful for gastroenterologists, microbiologists, infectious diseases physicians and public 
health specialists in the Asia Pacific region. 
 
2. Methodology 
Study subjects and sample collection 
Stool samples will be collected from the in-patients in the Prince of Wales Hospital. Patients newly 
diagnosed with Clostridium difficile infection will be invited to participate in this study. All participants 
will sign an informed consent form to be recruited into this study. Clinical data regarding patients’ age, 
gender, medical history, drug history and laboratory test results will be recorded. A fresh stool samples 
will be collected from the subjects using a 30-mL universal sample container with spoon cap. The stool 
samples will be delivered to laboratory and stoed at -80 degrees Celsius freezers. All research samples 
will be de-identified after collection and processing.  The study will be conducted in compliance with the 
Declaration of Helsinki guideline. 
 
Patients must meet the following criteria in order to be enrolled in the study:  
1. Patients with a confirmed diagnosis of CDI, as documented by diarrheal symptoms and positive stool 
test result for C. difficile toxin or toxigenic C. difficile, or colonoscopic findings of pseudomembranous 
colitis (PMC). 
2. Patients aged over or equal to 18 years old. 
3. Patients able and willing to provide informed consent. 
 

 
 
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Patients will be excluded if they meet the following criteria: 
1. Patients with concomitant infection by other microbes such as Salmonella, Campylobacter, Vibrio, 
Shigella, and Escherichia coli. 
2. Patients under 18 years old. 
3. Patients who cannot give consent. 
 
Laboratory and statistical methods 
The collected stool samples will be stored at -80C refrigerator. Only one tube of stool sample will be 
collected from each patient. 
Stool filtrate will be used to test for the presence of toxins, including toxin A and toxin B. There are some 
tests that can be used, including enzyme linked immunosorbent assay (ELISA) and cell cytotoxicity 
neutralization assay (CCNA). Commercial kits will be used for these assays. Stool DNA extraction can be 
done to obtain information about toxin A and toxin B through polymerase chain reaction (PCR).Strain 
typing will also be done to identify the ribotypes of the stool samples collected. This is to also determine 
which ribotype is the most common ribotype among others. 
The statistical software SPSS 13.0 (SPSS, Chicago, IL, USA) and the R Project for Statistical Computing 
will be used for all statistical analyses. For the microbiome data, the assembly and taxonomical 
assignment will be performed using the mothur software. 
 
 

 
 
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Sample size 
Assuming a power of 70% to detect a significant difference at 5% false positive rate, 227 samples will be 
required at an effect size (Cohen's d) of 0.195. 
 
 
3. Conclusion 
This proposed research aims to collect clinical stool samples to validate virulence mechanisms of 
different ribotypes of C. difficile being investigated in laboratory work. The study does not involve any 
invasive or harmful procedure. This important study will inform the characteristics of common ribotypes 
in Hong Kong to benefit the community.  
 
References 
Bauer MP, Notermans DW, van Benthem BH, Brazier JS, Wilcox MH, Rupnik M, Monnet DL, van 
Dissel JT, and Kuijper EJ (2011). Clostridium difficile infection in Europe: a hospitalbased survey. 
Lancet 377, 63-73. 
Lessa FC, Gould CV, and McDonald LC (2012). Current status of Clostridium difficile infection 
epidemiology. Clin Infect Dis 55 Suppl 2, S65-70.   
Wong SH, Ip M, Hawkey PM, Lo N, Hardy K, Manzoor S, Hui WW, Choi KW, Wong RY, Yung IM, 
Cheung CS, Lam KL, Kwong T, Wu WK, Ng SC, Wu JC, Sung JJ, Lee N (2016). High morbidity and 
mortality of Clostridium difficile infection and its associations with ribotype 002 in Hong Kong. The 
Journal of infection,73(2):115-22. doi: 10.1016/j.jinf.2016.05.010