Evaluation of the host response to Clostridium difficile infection

Despite several interventional measures, Clostridium difficile infection (CDI) continues to be a major problem for healthcare services worldwide. Clinical classification of patients at initial disease presentation is very challenging which makes it complex to accurately predict who will respond favourably to the treatment or have adverse outcomes such as recurrence. This thesis is based upon work undertaken on a prospective CDI cohort, which was the preferred study design, as it allowed for careful assessment of both clinical and biological factors. In order to identify clinical risk predictors for poor CDI outcomes, such as mortality and recurrence, clinical and laboratory variables were analysed, and predictive models derived. Although some similarities were identified in the risk factors in our cohort when compared with previous published studies, overall, the potential for external replication was poor, indicating that many of the models had internal validity, but little external validity. We also attempted to assess clinical prediction rules, and applied to our dataset. Again, it was not possible to replicate the findings of the prediction rules. Most studies, including ours, are small with less than 500 evaluated patients, which may be the major factor in limiting their generalisability. Future studies need to focus on much larger cohorts. The genetic polymorphism rs4073/-251T>A in the pro-inflammatory IL-8 gene has previously been reported to predispose to CDI. We were unable to replicate these findings using both a discovery cohort (286 CDI cases versus 135 AAD controls; p=0.84) and a replication cohort (100 CDI cases versus 170 healthy controls; p=0.87), and no association was found upon meta-analysis with the original study data (OR, 1.72; 95% CI, 0.63-4.71). We also failed to replicate previous findings of a significant association between faecal IL-8 concentration and IL-8 rs4073 genotype in a sub-set of our CDI patients (p=0.28). These findings suggest that this polymorphism is unlikely to constitute a major risk factor for CDI. Faecal calprotectin and faecal lactoferrin have been used as biomarkers in inflammatory bowel disease. We analysed these biomarkers in CDI cases compared with a group of diarrhoea control inpatients. There was a significant difference between cases and controls (p 0.85), but there was no association with CDI clinical outcomes, including severity, recurrence, and length of stay, suggesting a limited applicability of both faecal biomarkers for disease stratification. An effective CDI vaccine would constitute an important breakthrough for tackling the disease, but progress in this area has been hampered in part due to the lack of reliable methods for quantitating toxin-specific immune-mediated responses. We have developed novel and enhanced assays to measure immune response to the major C. difficile toxin epitopes (tcdA, tcdB, cdtA and cdtB). Whilst lower anti-tcdA and anti-tcdB IgG titres correlated with severe disease at baseline (p<0.01 and p=0.04), lower anti-tcdB IgM titres were associated with recurrence (p=0.04) and decreased levels of anti-cdtB (the binding precursor of binary toxin) was linked with prolonged disease (p=0.01). Nonetheless, our overall findings did not confirm previous associations with disease recurrence, mortality or prolonged disease, which is probably related to the fact that we did not have access to longitudinal samples. The role of mannose binding lectin (MBL), a lectin protein whose deficiency has been linked with several acute infections, was investigated in CDI due to its immunomodulatory properties and association with inflammation and innate immunity. We demonstrated that MBL concentration, but not genotype, was a significant predictor of 90-day CDI recurrence at both <50 ng/ml (OR=3.18, P<0.001) and <100 ng/ml (OR=2.61, P<0.001). MBL seems to acts as an immunomodulator of CDI disease course, but not as a predisposing factor. In conclusion, the work in this thesis has focused on clinical and biological factors associated with differing clinical outcomes in patients with CDI. Further work is needed to define host factors that modulate disease severity, and how they interact with the bacterium, in order to better understand the pathogenesis of disease, allow for stratification of treatment and improve clinical outcomes.