Introduction

Infection with toxin-producing strains of Clostridium difficile is a common cause of diarrhoea and varies from mild to severe cases of diarrhoea. Cases are frequently antibiotic-associated and occur mostly in hospitals. Pseudomembranous colitis in already impaired patients e.g. with an underlying condition is a serious manifestation of C. difficile infection (CDI) and can result in death.

Reports from North America, Europe and Japan have drawn attention to a recently discovered strain of C. difficile that is characterised as PCR ribotype 027, toxinotype III (CD027) [1-4]. This strain has an increased pathogenic capacity, possibly a higher infectious potential and a particular resistance profile. The increased pathogenicity is thought to be associated with an enhanced production of toxin A and toxin B caused by mutations in a regulatory gene, but the fact that this strain in addition produces a binary toxin CDT may also contribute to increased pathogenicity. This strain has caused severe outbreaks of CDI in hospital environments, but has also been described as the cause of outbreaks and sporadic cases outside hospitals [2-4].

The aims of the present report are to summarise national hospital data with a discharge diagnosis of CDI and to describe the first outbreak of CD027 in Denmark.

Methods

Because of the international emergence of CD027 and the subsequent recommendations from ECDC [5], we obtained hospital discharge data on CDI in Denmark from 1997 to 2007 and conducted a retrospective characterisation of C. difficile isolates from November 2006 to March 2007. In addition, Statens Serum Institut (SSI) asked Danish departments of clinical microbiology to continuously report C. difficile findings and to forward isolates for typing on suspicion of an outbreak or severe disease.

The hospital discharge data were obtained from the statistics of the Danish National Board of Health (http://sundhedsdata.sst.dk). Specifically, we obtained the annual aggregated number of discharges with the ICD10 diagnosis code DA04.7 (“enterocolitis caused by C. difficile”, i.e. enterocolitis independent of PCR ribotype) as well as the annual number of all discharges from somatic hospitals, i.e. hospitals treating only somatic and not psychiatric diseases. Data about consumption of fluoroquinolones and cephalosporins were obtained from The Danish Integrated Antimicrobial Resistance Monitoring and Research Programme (DANMAP) [6].

Isolates of C. difficile were characterised by PCR ribotyping, toxin gene profiles, and deletion studies undertaken by the National Reference Laboratory for Enteropathogenic Bacteria at SSI.
Stool samples were cultured on cycloserine cefoxitin fructose agar (CCFA) (SSI Diagnostica, Hillerød, Denmark) in an atmosphere composed of 86% N2, 7% H2 and 7% CO2 at 37°C for 48 hours. Colonies with typical morphology and distinctive odour were identified. The colonies were analysed by 5-plex PCR directed towards tcdA, tcdB, cdtA, cdtB and 16S rDNA and by sequencing of the 5’-end of tcdC in order to search for premature stop codons and internal deletions [7]. PCR ribotyping was performed according to Bidet et al. [8].

Results

Hospital discharges of CDI in Denmark
The aggregated number of discharges of enterocolitis caused by C. difficile increased from 86 (eight per 100,000 discharges) in 1997 to 282 (23 per 100,000 discharges) in 2007. In the same period, the consumption of fluoroquinolones and cephalosporins used in primary healthcare and hospitals taken together, increased from 384 to 1,162 kg and from 626 to 2,285 kg active component per annum, respectively (see Figure 1) [6].

Figure 1. Annual number of hospital discharges with enterocolitis caused by Clostridium difficile (ICD10 diagnosis code DA04.7) and annual consumption of fluoroquinolones and cephalosporins for human use, Denmark, 1997-2007
 

Detection of CD027 in Denmark
In the retrospective survey, isolates obtained between November 2006 and March 2007 were characterised; eight CD027 cases were found (Figure 2). The isolates came from eight hospitalised patients from the Region of Southern Denmark (the former Ribe County). Seven of the patients had been admitted to the same small hospital A, while the last case was a patient in another hospital in the same local area. Prompted by this cluster, active surveillance for CD027 was established in the area, and an additional 22 isolates of C. difficile were received between June and August 2007, of which five were CD027 (see Figure 2).

Figure 2. Number of patients with Clostridium difficile infection caused by CD027, Denmark, October 2006-August 2007 (n=13)
 

Thus, a total of 13 patients with CD027 were identified. Mean age was 79 years (age range 64 to 96 years), and 10 cases were women. The patients were admitted to hospital in the period October 2006 to July 2007. Nine of the patients were admitted to the same medical ward at the small hospital A, which consisted of only the one ward and a surgical day clinic. Most of these patients had overlapping periods of admission. The CD027-positive stool sample from one of these patients was requested by the general practitioner 13 days after the patient’s discharge from hospital. The other eight were obtained during admission. The remaining four patients were admitted to three different medical departments at the larger hospital B. Two of these patients had overlapping periods of admission at the same ward. One of these four patients was moved to another medical ward at another small hospital C (see Figure 3).

Figure 3. Distribution of cases of CD027 in the three hospitals, Denmark, October 2006-August 2007

 
The isolates were all PCR ribotype 027, carried the binary toxin gene, had an 18 bp deletion in the regulatory gene tcdC, and a 1 bp deletion at position 117 of tcdC. They were all resistant to fluoroquinolones (including moxifloxacin), but susceptible to erythromycin and clindamycin. Interestingly, at the same time and in the same geographical area, but unrelated to the outbreak  another isolate was found that also carried the binary toxin gene, had the 18 bp and the 1 bp deletion at position 117 in the regulatory gene tcdC, but was not PCR ribotype 027. In contrast to the CD027 strains it was sensitive to moxifloxacin.

As this cluster of 13 cases was detected in a setting with ample possibilities of transmission and at the time represented the only detection of CD027 in Denmark, it is reasonable to assume that an outbreak with CD027 occurred during this period. Multilocus variable-number tandem-repeat analysis (MLVA) or restriction endonuclease analysis (REA) [9,10] will be performed in order to elucidate the connection between the isolates.

All of the 13 patients were treated with broadspectrum antibiotics prior to positive culture of CD027. Eleven patients received cephalosporins and nine fluoroquinolones; seven received both cephalosporins and fluoroquinolones, either simultaneously or consecutively. Thirty days after confirmation of diagnosis, three of the 13 patients had died. It is unknown if the deaths were directly attributable to C. difficile.

Discussion

It is not known with certainty why the number of patients discharged after an episode of enterocolitis caused by C. difficile is increasing. However, it is certain that the patients with a discharge diagnosis of ICD10 code DA04.7 only comprise a modest fraction of the true number of cases. In 2007, 1,342 culture-confirmed cases of C. difficile infections were reported to the national surveillance system in Denmark (25 per 100,000 population). Surveillance was established in 2007. Data before this is therefore not available. Although increased diagnostic activity and awareness may play a role, it is also likely that changes in the strains’ pathogenicity are important contributing factors to the emergence of CDI. This includes the appearance of CD027 and possibly other hypervirulent strains. Several factors may be of importance to understand the emergence of C. difficile and in particular of CD027. The CD027 strain is resistant to the newer fluoroquinolones, including moxifloxacin, and it has been suggested that this may be the main reason for its wide dissemination [2,3]. This hypothesis is supported by the almost parallel increase in CDI discharge diagnoses and the consumption of fluoroquinolones as illustrated in Figure 1. However it should be emphasised that resistance to moxifloxacin and several other fluoroquinolones is also seen in other C. difficile PCR ribotypes [11,12]. Furthermore, increased use of other broadspectrum antibiotics including cephalosporins may also be related to the emergence of C. difficile since the same almost parallel increase is observed in CDI discharge diagnoses and consumption of cephalosporins (Figure 1).
 
However, these possible relations should be interpreted with caution. Other circumstances may also be of considerable importance, such as the increasing challenges in the area of hospital hygiene. For example, increased virulence of C. difficile resulting in pronounced diarrhoeal symptoms may have promoted spread and cross-infection within healthcare institutions, possibly because of dissemination of spores by incontinent patients [3]. The emergence of C. difficile and CD027 in particular is likely to be a result of environmental as well as person-to-person transmission in healthcare facilities rather than solely a result of increased antibiotic pressure. Finally, demographic changes such as an age distribution with an increasing proportion of elderly people and changes in the patterns of hospitalisation towards increased “turn-over” of patients may also contribute.

The recognition of the outbreak of CD027 in this particular geographical area of Denmark may not be an isolated observation. The initial cluster was detected in a convenience sample of stool specimens from diarrhoeal patients as part of a project including molecular characterisation of C. difficile isolates. Hence, it is conceivable that the cases discovered only represent the tip of the iceberg. On a voluntary basis, strains from all different geographical areas of Denmark are now being submitted for surveillance to the National Reference Laboratory for Enteropathogenic Bacteria to identify CD027.

Although we cannot conclude a cause-and-effect relation between the increase in fluoroquinolone and cephalosporin consumption and the increase in CDI discharge diagnoses, we consider it important to present these data to stimulate additional research. Studies are needed to determine the burden of disease associated with CD027 and other hypervirulent C. difficile strains, while integrated public health and microbiological surveillance should be established to determine trends, detect clusters in healthcare institutions, and facilitate more focused infection control. To prevent spread, it is essential to focus on hospital hygiene and promote prudent antibiotic policies, including the limitation of unnecessary use of broadspectrum antibiotics, including fluoroquinolones and cephalosporins.

Acknowledgements
We wish to thank J. Nevermann Jensen for extensive technical assistance.

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