Clinical features and characteristics of Clostridium difficile PCR-ribotype 176 infection: results from a 1-year university hospital internal ward study

Background Clostridium difficile infection (CDI) is a major cause of antibiotic-associated diarrhoea. Given an increasing CDI incidence and global spread of epidemic ribotypes, a 1-year study was performed to analyse the molecular characteristics of C. difficile isolates and associated clinical outcomes from patients diagnosed with CDI in the Internal Medicine department at University Hospital Motol, Prague from February 2013 to February 2014. Results A total of 85 unformed stool samples were analysed and CDI was laboratory confirmed in 30 patients (6.8 CDI cases per 10,000 patient bed days and 50.6 CDI cases per 10,000 admissions). The CDI recurrence rate within 3 months of treatment discontinuation was 13.3% (4/30). Mortality within 3 months after first CDI episode was 26.7% (8/30), with CDI the cause of death in two cases. 51.9% of C. difficile isolates belonged to PCR-ribotype 176. MLVA of ribotype 176 isolates revealed two clonal complexes formed by 10/14 isolates. ATLAS scores and Horn’s index were higher in patients with ribotype 176 infections than with non-ribotype 176 infections. Conclusion This study highlights the clinical relevance of C. difficile PCR-ribotype 176 and its capacity to spread within a healthcare facility.


Background
Clostridium difficile infection (CDI) is a major cause of antibiotic-associated diarrhoea and a significant burden to healthcare services worldwide [1]. Results of a pan-European epidemiological study in 2008 indicated that the Czech Republic has a relatively low CDI incidence (1.1 per 10,000 patient bed-days and 7.0 per 10,000 hospital admissions) [2], although a recent epidemiological study suggested a CDI incidence rate of 4.4 and 6.2 cases per 10,000 patient bed-days in 2011-12 and 2012-13, respectively [3].
In response to the reported unfavourable global CDI epidemiological situation, including in Czech Republic, a 1-year study was initiated to monitor the incidence of CDI, clinical features and outcomes and to investigate the molecular characteristics of C. difficile isolates in patients with CDI hospitalised in the Internal Medicine department of University Hospital Motol, Prague, Czech Republic, from February 2013 to February 2014.

Annals of Clinical Microbiology and Antimicrobials
Microbiological testing
The tcdC gene was amplified with primers C1 and C2 [17] and the obtained sequence was compared to NCBI reference sequence NC_009089.1. Two deletions (position 117, which introduces a frame-shift mutation leading to protein truncation [17], and 330-347) in the tcdC gene were found in all 14 ribotype 176 isolates. One isolate, ribotype 078, revealed 39-bp deletion from nucleotides 341-379 in the tcdC gene. No deletion in other 12 isolates was found. The precise function of the tcdC gene is not yet clear [18].
The time intervals of hospitalisation of patients infected by C. difficile ribotype 176 did not overlap except for two patients. This finding suggests that the probable source of infection may have come from the hospital environment and, given the high incidence of this ribotype previously reported in the Czech Republic [4], it is possible that ribotype 176 is endemic in the country and this type has

Clinical and epidemiological data analysis
CDI was diagnosed in 30 patients (female n = 13, male n = 17; mean age 69.0 years). The overall CDI incidence in the Internal Medicine ward during the study period was calculated as 6.8 CDI cases per 10,000 patient beddays and 50.6 CDI cases per 10,000 admissions which indicated a higher CDI incidence compared with recently reported rates [3].
To assess the association between C. difficile ribotype and disease severity, the clinical outcomes of patients with ribotype 176 infections were compared to those with other ribotype infections ( Table 3). Analysis of ATLAS scores and the Horn's index found that 11/14 (78.6 %) patients with ribotype 176 infections had an ATLAS score of 6-9 or a Horn's index score of 3 or 4 compared with 6/13 (46.2 %) and 7/13 (53.9 %) of patients with nonribotype 176 infections. Furthermore, the mortality rate appeared to be higher in patients with ribotype 176 infections compared with non-ribotype 176 infections (35.7 versus 15.4 %). No significant ribotype-associated differences were noted in recurrence rates, ICU admission rates or prior antibiotic use (Table 3).
Clostridium difficile ribotype 027 strains are often thought to be associated with CDI outbreaks of increased disease severity [1,5], but the clinical severity associated with ribotype 176 infections has not yet been studied in detail with exception of clinical data on ten patients, of whom 50 % had severe form of CDI, reported by Obuch-Woszczatyński et al. [9]. Our finding of a trend towards increased Horn's index and ATLAS scores in patients with ribotype 176 infections compared with non-ribotype 176 infections provides some evidence to support the clinical importance of this ribotype. However, the small sample size of patients in this study indicates a need for further studies, incorporating a larger number of patients, to better understand the relative virulence of ribotype 176. The high incidence of epidemic C. difficile PCR-ribotype 176 in our study emphasises the importance of implementing continuous surveillance programmes for CDI at national and European level, including PCR ribotyping.