Clostridium difficile infections in a university hospital in Greece are mainly associated with PCR ribotypes 017 and 126

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1 RESEARCH ARTICLE Kachrimanidou et al., Journal of Medical Microbiology 2017;66: DOI /jmm Clostridium difficile infections in a university hospital in Greece are mainly associated with PCR ribotypes 017 and 126 Melina Kachrimanidou, 1 Olga Tsachouridou, 2, * Ioannis A. Ziogas, 2 Eirini Christaki, 2 Efthymia Protonotariou, 3 Symeon Metallidis, 2 Lemonia Skoura 3 and Ed Kuijper 4 Abstract Purpose. Data regarding the incidence and molecular epidemiology of Clostridium difficile infections (CDIs) in Greece are limited. Methodology. A retrospective study of all laboratory-confirmed CDI cases in a university hospital during a 9-month period. Stool samples from inpatients with diarrhoea were tested with a combined glutamate dehydrogenase (GDH) and toxin enzyme immunoassay (EIA) test, as part of a two-step algorithm for CDI testing. All GDH-positive samples were cultured and isolates were further tested for the presence of toxin genes and characterized by PCR ribotyping. Results. The incidence of CDI in our hospital was 25 per hospital admissions. Of 33 CDI cases, 72.7 % were hospitalacquired. Fourteen different PCR ribotypes were identified, of which 017 (21.2 %), 078/126 (15.1 %) and RT202 and RT106 (9 %) were the most prevalent. Most patients had a risk profile of recent antibiotic use, older age and comorbidities. Despite mild CDI clinical characteristics, six cases showed complications and led to 18.2 % mortality. Conclusion. The CDI incidence was comparable to that in other European countries. The hypervirulent PCR ribotype 027 was not found, whereas ribotypes 017 and 126 predominated. Most CDI cases were in patients who used antibiotics, emphasizing that antimicrobial stewardship should be considered as a cornerstone for the prevention of CDI. INTRODUCTION Clostridium difficile infections (CDIs) have emerged over the last few years as a major health problem, and are associated with prolonged hospitalization, morbidity and mortality [1, 2]. Despite this, clinical suspicion of CDI and therefore testing frequency remains low [3, 4]. CDI is defined as the acute onset of diarrhoea with a toxigenic C. difficile when other reasons for diarrhoea have been ruled out [5]. Exposure to antibiotics and contact with the spores of bacterium are the main risk factors for infection [6]. Comorbidities, use of proton-pump inhibitors (PPIs) and gastrointestinal tract operations are also considered to be predisposing factors [7, 8]. Data on CDI in Greece are limited [9]. Therefore, we designed a retrospective study to determine CDI incidence, combined with clinical and epidemiological data, and molecular characterization of C. difficile isolates. METHODS Patients and study design This is a retrospective study of all laboratory-confirmed CDI cases diagnosed at the Microbiology Department of AHEPA Hospital in Thessaloniki, Greece, a 600-bed tertiary hospital. The requirement for informed consent was waived, since the study was retrospective and patients records were anonymized and de-identified prior to analysis. During a 9- month period (June 2014 March 2015) patient admissions occurred. Based on clinical criteria, physicians requested the microbiological laboratory to test 144 diarrhoeal stool samples for CDI. Diarrhoea was defined as at least three episodes of unformed stools in 24 h (Bristol stool chart: types 5 7) [10]. Clinical and laboratory data Detailed demographic, clinical (vital signs, physical examination and number of stools) and laboratory [white blood Received 7 August 2017; Accepted 13 October 2017 Author affiliations: 1 First Department of Microbiology, Aristotle University of Thessaloniki, Medical School, Thessaloniki, Greece; 2 First Internal Medicine Department, Infectious Diseases Division, AHEPA Hospital, Medical School, Aristotle University of Thessaloniki, Thessaloniki, Greece; 3 Department of Microbiology, AHEPA University Hospital, Aristotle University of Thessaloniki, Thessaloniki, Greece; 4 Department of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, Leiden, Netherlands. *Correspondence: Olga Tsachouridou, olgat_med@hotmail.com Keywords: Clostridium difficile; diarrhoea; risk factors; epidemiology; antibiotics; toxin genes. Abbreviations: CA, community-acquired; CBA, Columbia blood agar; CCEY, cycloserine cefoxitin egg yolk; CDI, Clostridium difficile infection; ESR, erythrocyte sedimentation rate; GDH, glutamate dehydrogenase; HA, hospital-acquired; PPI, proton-pump inhibitor ã 2017 The Authors 1774

2 cell count, erythrocyte sedimentation rate (ESR), creatinine, serum albumin and imaging tests] data were recorded. Patients gender, age, date of admission, time of CDI onset, type of ward, duration of hospitalization, concurrent medication and co-morbidities were obtained from their medical records. The severity of CDI infection was also assessed and classified based on current global recommendations [11, 12]. Characterization of hospital-acquired (HA-CDI) and community-acquired (CA-CDI) infection and cases of recurrent infection was also performed for all cases per guidelines [6]. Isolates and growth media Stool specimens were primarily tested for both glutamate dehydrogenase (GDH) and toxins A/B of C. difficile by an enzyme immunoassay (EIA; C. diff quick check complete). All GDH-positive samples underwent culture onto modified Brazier s cycloserine cefoxitin egg yolk (CCEY) agar (CCEY agar base containing cycloserine cefoxitin supplement and 5 % defibrinated horse blood; Bioprepare, Greece), and the plates were incubated anaerobically at 37 C for up to 5 days. A single colony was subcultured onto a Columbia blood agar (CBA) plate and incubated for 48 h, after which colonies giving the characteristic odour and fluorescence under UV illumination were obtained. For long-term storage, isolates were suspended in nutrient broth containing 10 % glycerol and stored at 80 C [13]. DNA extraction Genomic DNA was prepared from C. difficile grown on blood agar incubated anaerobically for 48 h. A few colonies were emulsified in Tris/EDTA (TE) buffer (Sigma Aldrich Co. Ltd., Gillingham, United Kingdom) and heated at 100 C for 10 min. Debris was removed by centrifugation at r.p.m. for 2 min, and the supernatant was removed. DNA was stored at 20 C [13]. Detection of C. difficile toxin genes The presence of TcdA, tcdb, cdta and cdtb genes was investigated accordingly to previously described methods. The absence of the PaLoc was demonstrated using the primers lok1 and lok3, which amplify a 769-bp amplicon in strains without the PaLoc [13 16]. PCR ribotyping All PCR ribotyping of the cultured isolates described in the present study was performed at the Department of Medical Microbiology, National Reference Laboratory for Clostridium difficile, Leiden University Medical Center, Leiden, the Netherlands [17]. PCR ribotyping was performed by a standardized protocol for capillary gel-based PCR ribotyping and an international database with well-documented reference strains [18]. Statistical analysis The data were expressed as mean±standard deviation (SD) for continuous variables and as percentages for categorical data. The Kolmogorov Smirnov test was utilized for normality analysis of the parameters. Bivariate analyses were performed using the Student t-test, ANOVA model, the Chi-squared test and the Fisher exact test to analyse the relation between the outcome variables: CDI (healthcare vs community), severity (mild to moderate vs severe vs severe to complicated), and the quantitative and qualitative demographic and clinical variables, respectively. All of the tests were two-sided, and statistical significance was set at P<0.05. All analyses were carried out using the statistical package SPSS v (Statistical Package for the Social Sciences, SPSS, Inc., Chicago, Il, USA). RESULTS Clinical characteristics Over 9 months, stool samples from 144 patients with diarrhoea were sent to the microbiological laboratory for CDI testing. Of the 144 samples tested by GDH/ToxAB, 76.4 % were negative, 15.3 % were only positive for GDH and 8.3 % were positive for both GDH and ToxAB. Out of 144 stool samples suspected to be CDI-positive, 33 (22.9 %) contained C. difficile with 14 distinct PCR-ribotypes. Two GDH-positive samples remained negative for C. difficile culture. Of the 33 isolates, 4 were non-toxigenic. The patients from which C. difficile was isolated were aged 1 91 years, with a mean age of 62 years. The incidence of CDI was 25 per hospital admissions. Analysis of the patient demographics showed that most of them fitted the established risk profile, with almost two-thirds being older than 65 years, nearly all having significant comorbidities and all having received antibiotics in the 3 months prior to infection. Hospital-acquired vs community-acquired CDI distribution of C. difficile isolates in wards The incidence of HA-CDI (24 cases, 72.7 % of all CDI cases) in our hospital was higher than that for CA-CDI. With regard to other clinical data, half of the patients had been hospitalized recently, with a mean duration of current hospital stay of 26.5±28.7 days. The mean duration of hospitalization before CDI was diagnosed in patients with HA-CDI was 18.82±16.25 days. There was no difference between HA-CDI and CA-CDI in relation to the reason for hospitalization or past medical history (Table 1). Similarly, no significant difference was found between HA-CDI and CA-CDI concerning previous hospitalization in the last 6 months (62.5 vs 66.7 % respectively, P=1.000). Of the nine patients with CA-CDI, six patients (66.7 %) had experienced a previous hospitalization during the 3 months before the CDI was diagnosed. Variable rates of isolation from different wards were observed, with the highest rate of isolation being in internal medicine wards (18 cases, 54.5 %). The distribution of C. difficile isolates is shown in Fig

3 Table 1. Demographics/clinical characteristics of patients at baseline and previous/current antibiotic use stratified by CDI type Community-acquired CDI, onset of CDI outside a healthcare unit or within 48 h following admission to a healthcare facility without residence in/discharge from a healthcare facility within the previous 12 weeks; hospital-acquired CDI, onset of CDI at least 48 h after admission to a healthcare unit; GI, gastrointestinal disease; CDI, Clostridium difficile infection; heart disease, heart failure/coronary disease; blood disease, myelodysplastic syndrome/leukaemia/ lymphomas. Hospital-acquired (n=24, 72.7 %) Community-acquired (n=9, 27.3 %) P-value Age at diagnosis (years) mean±sd 63.24± ± Sex n (%) male/female 12 (50.0 %)/12 (50.0 %) 3 (33.3 %)/6 (66.7 %) Reason for hospitalization Fever n (%) 16 (66.6 %) 4 (44.4 %) Blood disease n (%) 5 (20.8 %) 3 (33.3 %) Pulmonary disease n (%) 11 (45.8 %) 1 (11.1 %) Heart disease n (%) 3 (12.5 %) 0 (0.0 %) GI disease n (%) 0 (0.0 %) 2 (22.2 %) Renal disease n (%) 3 (12.5 %) 1 (11.1 %) Past medical record Heart disease n (%) 8 (33.3 %) 5 (55.6 %) Pulmonary disease n (%) 3 (12.5 %) 1 (11.1 %) Renal disease n (%) 8 (33.3 %) 3 (33.3 %) Immunosuppression n (%) 6 (25.0 %) 1 (11.1 %) Blood disease n (%) 6 (25.0 %) 1 (11.1 %) GI disease n (%) 2 (8.3 %) 0 (0.0 %) Previous CDI n (%) 2 (8.3 %) 0 (0.0 %) Previous hospitalization n (%) 15 (62.5 %) 6 (66.7 %) PPI taken n (%) 15 (62.5 %) 6 (66.7 %) Previous antibiotic use n (%) 15 (60.0 %) 7 (77.8 %) Current antibiotic administration Penicillin n (%) 21 (87.5 %) 6 (66.7 %) Cephalosporins n (%) 9 (37.5 %) 4 (44.4 %) Penems n (%) 7 (29.1 %) 0 (0.0 %) Fluoroquinolones n (%) 5 (20.8 %) 3 (33.3 %) Previous antibiotic administration Cephalosporins n (%) 4 (16.0 %) 0 (0.0 %) Penems n (%) 0 (0.0 %) 1 (11.1 %) Fluoroquinolones n (%) 7 (28.0 %) 3 (33.3 %) Antibiotic use and CDIs All patients with CDIs were receiving antibiotic treatment. Analysis of patients medical records showed that in HA- CDI cases, penicillins (87.5 %), cephalosporins (37.5 %), carabapenems (29.1 %) and fluoroquinolones (20.8 %) were the most commonly administered antibiotics prior to CDI (Table 1). The respective consumption rates in CA-CDI cases were penicillins (66.7 %), cephalosporins (44.4 %) and fluoroquinolones (33.3 %). Previous studies have shown that CDIs can occur long after antibiotic exposure, and therefore we looked for a history of antibiotic use in the 3 months prior to diagnosis. The most commonly prescribed class of antibiotic was fluoroquinolones in outpatients and penicillin in hospitalized patients. There was no significant difference between healthcare- and community-acquired infection with respect to the use of separate antibiotics (currently or previously administered) (Table 1). Regarding PPI administration, in most healthcare- and community-acquired CDI cases the patients had been receiving PPIs during the onset of CDI or in the previous 3 months. There was no difference concerning current or previous PPI intake (62.5 vs 66.7 % respectively, P=1.000) and CDI onset (Table 1). Severity of CDI and mortality Most CDI cases (52.8 %) had a mild-to-moderate course of infection, whereas 21.2 % (n=7) experienced a complicated course of infection (Table 2). Six patients with CDI (18.2 %) died during hospitalization and mortality was attributable to CDI. They were all older than 70 years and their infection was HA, except for one case. All patients had significant comorbidities (four with heart disease, two with chronic renal disease, two with blood disease and one with pulmonary disease), although they were immunocompetent and none of them had a confirmed previous CDI or presented 1776

4 with abdominal pain or bloody stools at admission. The strains causing severe infections with a fatal outcome belonged to PCR ribotypes 017 (two patients), 220, 106 and 050, with one unknown ribotype. The patients with a complicated infection were aged (SD±26.92) and had chronic underlying diseases. Only one of these patients was immunocompromised. All of the patients had received antibiotics in the three months prior to admission, most commonly penicillin, carbapenems and fluoroquinolones. PCR ribotypes 017, 126, 202, 220, 106 and 050 were associated with the above complicated cases. There was a statistically significant correlation between severity status and fatal complication of CDI (death), as shown in Table 3. Patients with a complicated course had higher risk of in-hospital death, compared to patients with an uncomplicated course (mild moderate and severe 100 vs 0 %, 0 %; P<0001). No significant difference was found between HA and CA infection in relation to CDI symptoms, severity status and daily mean number of stools (Table 2). Remarkably, higher rates of severe CA-CDI compared to HA-CDI (33.3 vs 25.0 %) were recorded, and one fatal CA- CDI complicated case was observed. Interestingly, patients with mild or moderate CDI presented lower renal impairment during hospitalization monitoring, compared to those with severe and complicated infection (29.4 vs 77.8 %, 62.5 %; P=0.046). CDI treatment Most patients with CDIs (n=20, 60.6 %) received metronidazole 500 mg three times daily per os. Vancomycin 125 mg per os, four times daily, was administered to eight patients (24.2 %). Three patients received both regimens (9.1 %) and only two received fidaxomicin (6.06 %). The mean duration of therapy for all selected regimens was 9.4±5.0 days. Complicated or recurrent cases were equally distributed among patients who were treated with metronidazole or with vancomycin. Molecular characteristics Molecular detection of C. difficile toxin Twenty-nine out of the 33 C. difficile isolates were positive for both tcda and tcdb with PCR, although 7 isolates had a partial deletion in tcda (A-B+). The remaining four isolates were negative for both genes (A-B-, non-toxigenic C. difficile isolates). Despite the unclear clinical relevance of these non-toxigenic strains, we included them in the study as the stool specimens were A-/B-positive by enzyme immunoassay, and the patients responded well to C. difficile-specific therapy. Five of the A + B + C. difficile isolates produced a binary toxin (cdta/cdtb+), whilst the A - B + isolates were all binary toxinnegative. The five binary toxin isolates belonged to RT078/ 126, which were A+B+. In summary, four toxigenic profiles were revealed in this study: A+B+ CDT- (16/33, 48.5 %), A + B + CTD + (5/33, 15.2 %), A - B + CDT - (8/33, 24.2 %) and A-B-CDT- (4/33, 12.1 %). Typing PCR ribotyping was used for further characterization of the 33 clinical C. difficile isolates. Fourteen RTs were Fig. 1. Distribution of PCR ribotypes over time and by hospital wards, (a) monthly (b) according to hospital wards. CD, cardiology; HEM, haematology; ICU CD, coronary disease intensive care unit; ID, infectious disease unit; IM, internal medicine; ORL, otorhinolaryngology; PD, paediatric. 1777

5 Fig. 1. (cont.) recognized. The most prevalent RT in this study was 017 (21.2 %, 7/33). The next three most common RTs were 126 (12.1 %, 4/33), RT202 and RT106 (9 %, 3/33). The remaining RTs, 078, 012, 005, 024, 050, 070, 137 and 220, were detected sporadically and were represented by up to two isolates. The RT profiles of four isolates (4/ %), one of which was toxin A/B-negative, differed from each other and did not match any of the reference RTs available in this study (provided by the Leiden Reference laboratory). Table 2. Symptoms and severity of infection, stratified by CDI type WBC, white blood cell count; mild-to-moderate disease, diarrhoea plus any additional signs or symptom not meeting severe or complicated criteria; severe disease, serum albumin <3 g dl 1 plus WBC cells mm 3 or abdominal tenderness; severe and complicated disease, any of the following, attributable to CDI: admission to intensive care unit for CDI or hypotension with or without use of vasopressors being required or fever 38.5 C or ileus or significant abdominal distension or mental status changes or WBC cells mm 3 or <2000 cells mm 3 or serum lactate levels >2.2 mmol l 1 or end organ failure (mechanical ventilation, renal failure, etc.). Symptoms Hospital (n=24, 72.7 %) Community (n=9, 27.3 %) P-value Fever n (%) 17 (70.8 %) 4 (50.0 %) Abdominal pain n (%) 10 (41.6 %) 4 (44.4 %) Bloody stools n (%) 4 (16.6 %) 2 (22.2 %) Serum creatinine rise n (%) 12 (50.0 %) 5 (55.6 %) WBC increase n (%) 15 (62.5 %) 5 (55.6 %) Severity Mild moderate 12 (50.0 %) 5 (55.6 %) Severe 6 (25.0 %) 3 (33.3 %) Severe complicated 6 (25.0 %) 1 (11.1 %) Number of daily stools Mean±SD 5.2± ± Median (IQR) 4.0 (3.0) 5.0 (3.0)

6 Table 3. Relation of CDI and severity with complication of CDI type Percentage within death status. CDI All of the Greek isolates from RT 017 were only positive for tcdb with a partial deletion in tcda (A - B + CDT - ). Two patients >80 years old diagnosed with C. difficile healthcareassociated infection by RT 017 died. Both patients had severe comorbidity because of heart disease. All of the 126 RT isolates were found to be A + B + CTD +. Even though the presence of all three toxins (A + B + CTD + ) contributed to the more severe CDI course, only a 67-year-old patient in the cardiology ward presented a recurrent CDI. A single A + B + CTD + isolate typed as 078 was recovered from an 89-year-old haematology patient, who improved clinically after metronidazole treatment. Among the 33 cases, 4 were in respect of young children aged 2, 3 and 4 years old. The strains isolated from the three children indicated that their infections were acquired in the healthcare setting by ribotypes RT012 and RT024. DISCUSSION Death: no (n=27, 81.8 %) Death: yes (n=6, 18.2 %) P- value Healthcare n (%) 19 (79.1 %) 5 (83.3 %) Community n (%) 8 (29.6 %) 1 (16.7 %) Severity Mild moderate n (%) 17 (62.9 %) 0 (0.0 %) <0.001 Severe n (%) 9 (33.3 %) 0 (0.0 %) Severe complicated n (%) 1 (3.7 %) 6 (100.0 %) Over a 9-month period, we found a CDI incidence of 25 per hospital admissions in a Greek tertiary care hospital, which is similar to the rates reported in other European countries [19, 20]. However, a recently published pointprevalence survey of HA-CDI in Greek hospitals reported an overall incidence rate of CDI of 15.7 %, which is lower than the one found in our hospital (22.9 %) [9]. Of the patients diagnosed with CDI, 27.3 % of them acquired their infections in the community. The most frequently found PCR ribotypes were RT017 and RT126/078. Severe and complicated cases of CDI were observed in 48.5 % of patients and the overall mortality rate was 18.2 %. Remarkably, C. difficile toxin A-negative PCR ribotype 017 was the predominant type in our hospital. Historically, RT017 was initially reported in Asia, but it has now been reported worldwide, including in countries such as Poland, Korea, Japan, Bulgaria and Argentina [21], where it has been associated with severe disease [22 25]. Additionally, RT017 has been associated with a high level of resistance to clindamycin and erythromycin [21]. Studies of the outcome and clinical presentation of CDI caused by RT017 strains have discrepant results: some reported no difference from other ribotypes, while others reported more severe disease and worse outcome [26, 27]. The RT017, with its unique toxin profile and unusual global prevalence, has been overshadowed by the global outbreak of the ribotype 027 lineage. A recently published phylogeographic analysis of wholegenome sequencings from RT017 isolates of six continents showed a North American origin for ribotype 017, as has been found for the emerging epidemic RT027 lineage [28]. The authors also concluded that, despite having only one toxin, RT017 strains have evolved in parallel from at least two independent sources and can readily transmit between continents. The toxigenic and so-called hypervirulent PCR ribotype 078 is closely related to PCR ribotype 126 and differs by only one band by electropheris. C. difficile RT078 is one of the prevalent ribotypes in European hospitals, is generally characterized by the presence of all three toxins A + B + CTD + and is considered to be associated with complicated infection [26]. Similarly, ribotype 126 (RT126), which is phylogenetically close to RT078 [29], is also regarded as hypervirulent and has been implicated in cases of CDI. Epidemiological surveillance reports for C. difficile in Spain revealed that the RT078/126 complex is prevalent in human patients [30]. Interestingly, we found that RT126 was encountered more frequently in our hospital. According to Davies and colleagues, countries with low prevalence of ribotype 027 have higher overall ribotype diversity among C. difficile isolates, highlighting the diverse epidemiology of C. difficile across Europe. However, that study reported that ribotypes 078/126 and 017 were among the 10 most commonly isolated ribotypes from countries of southern Europe (Greece, Italy, Portugal and Spain) [31]. Previous studies from Europe have shown that the prevalence of cdta/cdtb in C. difficile human isolates varies from 1.6 to 20.8 %, which agrees with our data [32]. Infection with CDT-positive C. diffficile is associated with higher mortality and recurrence rates. This contrasts with our findings, as none of the CDT-positive patients had fatal outcomes, and only one patient experienced an episode of CDI recurrence [22]. In line with previous studies, most patients with CDI were of advanced age and had been exposed to antibiotics [9]. About three-quarters of cases were HA, in line with previous reports [19, 20, 27, 33]. However, since 2000 there have been several reports of increased incidence and severity of CDI with progressive increase of CA infection, a trend that was also evidenced in our hospital during the study period [34]. This is a finding that deserves more local and national attention. Of all the patients diagnosed with CDI, 27.3 % cases were acquired in the community. However, 63.6 % had experienced a hospitalization in the previous 3 months, suggesting that CA-CDIs could also be healthcare-associated. In the present study, all of the patients used antibiotics during the onset of CDI. The results are consistent with 1779

7 previous reports, since the prevalence of CDI is increasing globally because of high levels of antibiotic use, with thirdgeneration cephalosporins, fluoroquinolones and penicillin being the major predisposing factors for CDI [35]. Moreover, recent data from Greece confirmed the crucial role of current antibiotic treatment in the onset of CDI in patients with all-cause hospitalization [9]. The excessive use of fluoroquinolones in the Greek community is also illustrated in our data. The mortality rate (18.2 %) in our study was similar that in some other studies [36], while other authors have described even higher rates, related to the severity of CDI and the coexistence of comorbidities [37, 38]. Severe and complicated cases had a higher risk of death compared to mild and moderate cases [39, 40]. Recent reports indicate a remarkable increase in severe cases and deaths related to CDI [41 43]. Identifying patients who are at high risk for severe CDI early in the course of infection may improve outcomes. Our study has several limitations. Apart from its retrospective nature, another important limitation is the fact that the isolates originated solely from one hospital and this may not be representative of the national incidence rate or the general diversity of C. difficile. Furthermore, the relatively small sample size tested might have led to the under-representation of PCR ribotypes that can cause outbreaks in hospitals. To study the epidemiology of an emerging infection like CDI better, data on larger patient populations are needed. Nevertheless, this study is an important forward step towards improving the surveillance of CDI in Greece. Future prospective studies are needed to obtain more detailed information on CDI epidemiology in Greece. To conclude, the majority of C.difficile isolates in our hospital in Greece belonged to RT017, which is widely distributed in Europe but mainly found in Poland and Bulgaria. The emergence of community-acquired CDIs is alarming and probably due to excessive use of antibiotics, and it warrants continuous surveillance to prevent further spread of the toxigenic C. difficile isolates. Most CDI cases suffered from comorbidities and the mortality rates were high despite the absence of hypervirulent 027. This may have contributed to the low recurrence rate observed. However, to be able to draw firmer conclusions about the epidemiology of CDI in Greece, further large-scale multicentre sampling of C. difficile is necessary. Funding information The authors received no specific grant from any funding agency. Acknowledgements We would like to thank all staff members of the 1st Internal Medicine Department and Microbiology Department for their valuable cooperation. 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