Environmental shedding of toxigenic Clostridioides difficile by asymptomatic carriers: A prospective observational study

ObjectivesThe aim was to compare the burden of environmental shedding of toxigenic Clostridioides difficile among asymptomatic carriers, C. difficile-infected (CDI) patients and non-carriers in an inpatient non-epidemic setting.MethodsC. difficile carriage was determined by positive toxin-B PCR from rectal swabs of asymptomatic patients. Active CDI was defined as a positive two-step enzyme immunoassay/polymerase chain reaction (EIA/PCR) test in patients with more than three unformed stools/24 hr. C. difficile environmental contamination was assessed by obtaining specimens from ten sites in the patients' rooms. Toxigenic strains were identified by PCR. We created a contamination scale to define the overall level of room contamination that ranged from clean to heavy contamination.ResultsOne hundred and seventeen rooms were screened: 70 rooms inhabited by C. difficile carriers, 30 rooms by active CDI patients and 17 rooms by non C. difficile -carriers (control). In the carrier rooms 29 (41%) had more than residual contamination, from which 17 (24%) were heavily contaminated. In the CDI rooms 12 (40%) had more than residual contamination from which three (10%) were heavily contaminated, while in the control rooms, one room (6%) had more than residual contamination and none were heavily contaminated. In a multivariate analysis, the contamination score of rooms inhabited by carriers did not differ from rooms of CDI patients, yet both were significantly more contaminated than those of non-carriers odd ratio 12.23 and 11.16 (95% confidence interval 1.5–99.96 p 0.0195, and 1.19–104.49 p 0.035), respectively.DiscussionHere we show that the rooms of C. difficile carriers are as contaminated as those of patients with active CDI and significantly more than those of non-carriers.     

Clinical heterogeneity of patients with stool samples testing PCR+/Tox− from a two-step <Emphasis Type="Italic">Clostridium difficile</Emphasis> diagnostic algorithm

The clinical significance of indeterminate (PCR+/Tox?) results for patients tested with a two-step algorithm for Clostridium difficile infection (CDI) is uncertain. We aimed to evaluate the clinical presentation and 8-week outcomes of patients with indeterminate test results. Patients with stool samples testing positive by PCR and negative by toxin A/B immunoassay between February 1, 2017, and April 30, 2018, were assessed by antimicrobial stewardship program (ASP) clinicians and classified as colonized or infected. Retrospective chart review was performed to obtain outcomes occurring within 8 weeks of testing, including recurrent C. difficile diarrhea, subsequent treatment for CDI, follow-up C. difficile testing, all-cause mortality, and CDI-related complications. In total, 110 PCR+/Tox? patients were evaluated. ASP classified 54% of patients as infected and 46% as colonized. Patients assessed and classified as colonized did not have increased adverse outcomes by 8 weeks compared to those assessed as infected, despite not receiving treatment for CDI. We conclude that PCR+/Tox? patients are heterogeneous with respect to clinical presentation. Negative toxin A/B immunoassay in a two-step algorithm should not be interpreted in isolation to distinguish colonization from infection as many PCR+/Tox? results may be clinically significant for CDI.     

Toxigenic Clostridium difficile carriage in general practice: results of a laboratory-based cohort study

ObjectivesReported rates of community-acquired Clostridium difficile infections (CDIs) have been increasing. However, the true burden of the disease in general practice is unknown in France. Our objective was to determine the incidence of toxigenic C. difficile carriage and the percentage of stool samples prescribed by general practitioners (GPs) which contained free C. difficile toxins.MethodsDuring an 11-month period, all stool samples submitted for any enteric pathogen detection to 15 different private laboratories in Paris and the surrounding areas were tested for C. difficile, irrespective of the GPs' request. A clinical questionnaire was completed for each patient. Stool samples were screened using a rapid simultaneous glutamate dehydrogenase and toxins A/B detection test: any positive result (glutamate dehydrogenase or toxin) was further confirmed by the stool cytotoxicity assay (CTA) on MRC-5 cells and by toxigenic culture (TC) at a central laboratory. The C. difficile isolates were characterized by PCR ribotyping.ResultsA total of 2541 patients (1295 female, 1246 male) were included. The incidences of patients with a positive toxigenic culture and a positive CTA were 3.27% (95% CI 2.61%–4.03%) and 1.81% (95% CI 1.33%–2.41%), respectively. GPs requested C. difficile testing in only 12.93% of the stool samples, detecting 52.30% of all TC-positive patients. The 83 toxigenic C. difficile strains belonged to 36 different PCR ribotypes.ConclusionsToxigenic C. difficile carriage is frequent in general practice but remains under-recognized. It may affect young patients without previous antimicrobial therapy or hospitalization.     

Increased age and proton pump inhibitors are associated with severe Clostridium difficile infections in children

BackgroundClostridium difficile infection (CDI) is increasing in children. We aimed to compare the clinical characteristics between CDI and colonization and to identify the risk factors for severe diseases of CDI in children.MethodWe retrospectively reviewed 124 children (1–18 years old) from 2011 to 2018. CDI was defined as diarrhea (≥3 loose stool in the past 24 h) with confirmed toxigenic strain. Colonization was defined as presence of C. difficile without clinical symptoms. Severe diseases included ileus, acute kidney injury, gastrointestinal bleeding or mortality. Patients younger than 1 year old and coinfections with other enteric pathogens were excluded.ResultsAmong 124 patients with C. difficile identified, 49 of them fulfilled CDI definition and 75 had C. difficile colonization. Children with CDI were more likely to present with watery (74% vs. 1%, p < 0.01) and mucoid stool (25% vs. 7%, p < 0.01) and occult blood in stool (67% vs. 33%, p < 0.01) than children with colonization. In CDI cases, elevated age-adjusted creatinine (18% vs. 0%, p = 0.03) and hyponatremia (134 mEq/L vs. 137 mEq/L, p = 0.04) were found. Also, they had more complicated diseases (27% vs. 0%, p < 0.01). On multivariate analysis, age older than 4 years (adjusted odds ratio: 5.83; 95% confidence interval: 1.05–32.27) and proton pump inhibitor use (PPI) (adjusted odds ratio: 7.25; 95% confidence interval: 1.07–49.07) were the independent factors for severe diseases.ConclusionsWatery diarrhea, mucoid stool and occult blood in stool could differentiate CDI from colonization. Patients with increased age and previous PPI use were the independent risk factors for severe diseases in children.     

Universal screening for Clostridioides difficile in a tertiary hospital: risk factors for carriage and clinical disease

ObjectivesThe role of asymptomatic carriers in Clostridioides difficile infection (CDI) epidemiology is not fully understood. Our aim was to evaluate CD carriage prevalence on admission, associated risk factors, and the risk of developing CDI.MethodsA 10-week surveillance program for CD carriage of all medical patients admitted to the Sheba Medical Centre was implemented, utilizing an admission rectal swab PCR. Healthcare facility-onset CDI (HO-CDI) was recorded and divided into HO-CDI diagnosed in CD carriers and non-carriers.ResultsA total of 4601 admissions were recorded in 3803 patients; 2368 patients had technically analysable rectal swabs, of whom 81 (3.4%) were CD carriers. A multivariate logistic regression model showed that previous hospitalization, old age (>85 years) and low Norton scores were significant independent predictors of CD carriage. Carriers were more likely to receive antimicrobial therapy during hospitalization than non-carriers were. The incidence of HO-CDI in non-carriers was 4.6 cases per 10 000 patient-days; the incidence of HO-CDI in carriers was 76.7 cases per 10 000 patient-days (RR 16.6, 95% CI 4.0–69.1, p .002).ConclusionsIn a prospective study, the rate of CD carriage on admission in medical patients was 3.4%. CD carriers were older, frailer, and more likely to have been hospitalized recently. HO-CDI incidence was significantly higher among CD carriers than among non-carriers, with at least a third of CDI in screened patients developing in carriers. Targeted screening of high-risk groups for CD carriage should be further considered.     

Clostridium difficile and One Health

BackgroundFor over four decades, Clostridium difficile has been a significant enteric pathogen of humans. It is associated with the use of antimicrobials that generally disrupt the microbiota of the gastrointestinal tract. Previously, it was thought that C. difficile was primarily a hospital-acquired infection; however, with the emergence of community-associated cases, and whole-genome sequencing suggesting the majority of the hospital C. difficile infection (CDI) cases are genetically distinct from one another, there is compelling evidence that sources/reservoirs of C. difficile outside hospitals play a significant role in the transmission of CDI.ObjectivesTo review the ‘One Health’ aspects of CDI, focusing on how community sources/reservoirs might be acting as a conduit in the transfer of C. difficile between animals and humans. The importance of a One Health approach in managing CDI is discussed.SourcesA literature search was performed on PubMed and Web of Science for relevant papers published from 1 January 2000 to 10 July 2019.ContentWe present evidence that demonstrates transmission of C. difficile in hospitals from asymptomatic carriers to symptomatic CDI patients. The source of colonization is most probably community reservoirs, such as foods and the environment, where toxigenic C. difficile strains have frequently been isolated. With high-resolution genomic sequencing, the transmission of C. difficile between animals and humans can be demonstrated, despite a clear epidemiological link often being absent. The ways in which C. difficile from animals and humans can disseminate through foods and the environment are discussed, and an interconnected transmission pathway for C. difficile involving food animals, humans and the environment is presented.ImplicationsClostridium difficile is a well-established pathogen of both humans and animals that contaminates foods and the environment. To manage CDI, a One Health approach with the collaboration of clinicians, veterinarians, environmentalists and policy-makers is paramount.     

The roles of host and pathogen factors and the innate immune response in the pathogenesis of Clostridium difficile infection

Clostridium difficile (C. difficile) is the most common cause of nosocomial antibiotic-associated diarrhea and the etiologic agent of pseudomembranous colitis. The clinical manifestation of C. difficile infection (CDI) is highly variable, from asymptomatic carriage, to mild self-limiting diarrhea, to the more severe pseudomembranous colitis. Furthermore, in extreme cases, colonic inflammation and tissue damage can lead to toxic megacolon, a condition requiring surgical intervention.     

The role of <Emphasis Type="Italic">Clostridium difficile</Emphasis> in the paediatric and neonatal gut — a narrative review

Clostridium difficile is an important nosocomial pathogen in adults. Its significance in children is less well defined, but cases of C. difficile infection (CDI) appear to be increasingly prevalent in paediatric patients. This review aims to summarize reported Clostridium difficile carriage rates across children of different age groups, appraise the relationship between CDI and factors such as method of delivery, type of infant feed, antibiotic use, and co-morbidities, and review factors affecting the gut microbiome in children and the host immune response to C. difficile. Searches of PubMed and Google Scholar using the terms ‘Clostridium difficile neonates’ and ‘Clostridium difficile children’ were completed, and reference lists of retrieved publications screened for further papers. In total, 88 papers containing relevant data were included. There was large inter-study variation in reported C. difficile carriage rates. There was an association between CDI and recent antibiotic use, and co-morbidities such as immunosuppression and inflammatory bowel disease. C. difficile was also found in stools of children with diarrhoea attributed to other pathogens (e.g. rotavirus). The role of C. difficile in the paediatric gut remains unclear; is it an innocent bystander in diarrhoeal disease caused by other organisms, or a pathogen causing subclinical to severe symptoms? Further investigation of the development of serological and local host response to C. difficile carriage may shed new light on disease mechanisms. Work is underway on defining a framework for diagnosis and management of paediatric CDI.     

Successful treatment by fecal microbiota transplantation for Japanese patients with refractory Clostridium difficile infection: A prospective case series

We prospectively enrolled four Japanese patients with refractory Clostridium difficile infection (CDI) and were treated with a single fecal microbiota transplantation (FMT). The average age of the patients was 83.7 years. All patients had a successful clinical course for up to 3 months without any adverse events.     

Fecal Microbiota Transplantation and Successful Resolution of Multidrug-Resistant-Organism Colonization

We report a case in which fecal microbiota transplantation (FMT) utilized for relapsing Clostridium difficile colitis successfully eradicated colonization with several multidrug-resistant organisms (MDROs). FMT may have an additive benefit of reducing MDRO carriage and should be further investigated as a potential measure to eradicate additional potentially virulent organisms beyond C. difficile.     

How to: diagnose infection caused by Clostridium difficile

BackgroundClostridium difficile is recognized as the major agent responsible for nosocomial diarrhoea. In the context of recent increase in the incidence and severity of C. difficile infections (CDI), an accurate diagnosis is essential for optimal treatment and prevention, but continues to be challenging.AimsThe present article reviews each key step of CDI diagnosis including stool selection, methods and strategies used, and interpretation of the results.SourcesThe most recent guidelines for CDI diagnosis published by scientific societies were reviewed.ContentCDI diagnosis is based on clinical presentation and laboratory tests confirming the presence of toxigenic strain or toxins in stools. Stool selection is crucial and can be improved by implementing rejection criteria and a strict policy for appropriate testing. Multiple laboratory tests detecting different targets (free toxin or presence of a potentially toxigenic strain) are commercially available. However, none of these tests combine high sensitivity and specificity to diagnose CDI, low hands-on time and low cost. An optimized diagnosis can be achieved by implementing a two- or three-step algorithm. Algorithms currently recommended by the ESCMID comprise a screening test with high sensitivity followed by a more specific test to detect free toxins. Presence of free toxins in stools has been shown to better correlate with severe outcome whereas nucleic acid amplification tests may lead to an over-diagnosis by detecting asymptomatic carriers of a toxigenic strain.ImplicationTo date, no single test can accurately diagnose CDI. Guidelines from the ESCMID recommend a two- or three-step algorithm for optimal CDI detection.     

A Tetraspecific VHH-Based Neutralizing Antibody Modifies Disease Outcome in Three Animal Models of Clostridium difficile Infection

Clostridium difficile infection (CDI), a leading cause of nosocomial infection, is a serious disease in North America, Europe, and Asia. CDI varies greatly from asymptomatic carriage to life-threatening diarrhea, toxic megacolon, and toxemia. The incidence of community-acquired infection has increased due to the emergence of hypervirulent antibiotic-resistant strains. These new strains contribute to the frequent occurrence of disease relapse, complicating treatment, increasing hospital stays, and increasing morbidity and mortality among patients. Therefore, it is critical to develop new therapeutic approaches that bypass the development of antimicrobial resistance and avoid disruption of gut microflora. Here, we describe the construction of a single heteromultimeric VHH-based neutralizing agent (VNA) that targets the two primary virulence factors of Clostridium difficile, toxins A (TcdA) and B (TcdB). Designated VNA2-Tcd, this agent has subnanomolar toxin neutralization potencies for both C. difficile toxins in cell assays. When given systemically by parenteral administration, VNA2-Tcd protected against CDI in gnotobiotic piglets and mice and to a lesser extent in hamsters. Protection from CDI was also observed in gnotobiotic piglets treated by gene therapy with an adenovirus that promoted the expression of VNA2-Tcd.     

Relationship between remote cholecystectomy and incident Clostridioides difficile infection

ObjectivesCholecystectomy (CCY) is associated with increased faecal levels of secondary bile acids. Secondary bile acids confer resistance to Clostridioides difficile infection (CDI, formerly Clostridium difficile infection) in animal studies. This study tested the hypothesis that CCY confers protection against CDI by increasing gut levels of secondary bile acids.MethodsThis was a retrospective case–control study. Adults hospitalized between January 2010 and June 2017 at our institution were included. CDI cases were defined as a positive stool PCR followed by anti-CDI treatment and were matched 1:1:1 with two control groups (those who tested negative for CDI and those who were not tested for CDI) by sex, age group, body mass index (BMI), and exposure to antibiotics. CCY was defined as a history of CCY at least 6 months prior to the index C. difficile test or the index admission date in the untested controls. Conditional logistic regression modelling was used to estimate the relationship between remote CCY and risk for CDI.ResultsThe final study population was 7077 (2359 CDI cases, 2359 matched controls without CDI, and 2359 matched controls not tested for CDI). Rates of remote CCY did not differ among the three groups (14.4% vs. 15.5% vs. 14.2%) and this result was unchanged after adjusting for additional clinical factors (adjusted OR 0.90, 95% CI 0.76–1.06 comparing CDI cases vs. matched controls without CDI; adjusted OR 1.04, 95% CI 0.78–1.39 comparing CDI cases vs. matched controls not tested for CDI).ConclusionsThere was no association between remote CCY and risk for CDI.     

Genome engineering of Clostridium difficile using the CRISPR-Cas9 system

ObjectivesClostridium difficile is a notorious pathogenic species that can cause severe gastrointestinal infections in humans and animals. C. difficile infection (CDI) results in thousands of deaths worldwide every year. The elucidation of related mechanisms of CDI and exploration of potential therapeutic strategies are largely delayed due to the lack of efficient genetic engineering tools for C. difficile strains.MethodsPlasmids carrying the CRISPR-Cas9 system were constructed and transformed into C. difficile through conjugation. Mutants were identified using colony PCR with primers annealing to the regions flanking the target gene deletion/integration locus. Heat-survival assay was used to compare the sporulation frequency between the mutant with spo0A deletion and the wild type strain. The fluorescence in the mutant with the insertion of the green fluorescent protein (GFP) gene was inspected under a fluorescent microscope.ResultsAn efficient genome editing tool was developed for C. difficile based on the CRISPR-Cas9 system. With this tool, spo0A was deleted with a 100% mutation efficiency. Conversely, an anaerobic GFP gene was successfully inserted into the C. difficile chromosome (with a mutation efficiency of 80%).ConclusionsThe developed CRISPR-Cas9-based genome engineering tool will facilitate functional genomic studies in C. difficile as well as the elucidation of mechanisms related to host–bacteria interaction and pathogenesis of CDI. This will be highly beneficial for the development of innovative strategies for CDI diagnostics and therapies.     

Microbiologic factors affecting Clostridium difficile recurrence

BackgroundRecurrent Clostridium difficile infection (rCDI) places a huge economic and practical burden on healthcare facilities. Furthermore, rCDI may affect quality of life, leaving patients in an rCDI cycle and dependant on antibiotic therapy.AimsTo discuss the importance of microbiologic factors in the development of rCDI.SourcesLiterature was drawn from a search of PubMed from 2000 onwards with the search term ‘recurrent Clostridium difficile infection’ and further references cited within these articles.ContentMeta-analyses and systematic reviews have shown that CDI and rCDI risk factors are similar. Development of rCDI is attendant on many factors, including immune status or function, comorbidities and concomitant treatments. Studies suggest that poor bacterial diversity is correlated with clinical rCDI. Narrow-spectrum gut microflora-sparing antimicrobials (e.g. surotomycin, cadazolid, ridinilazole) are in development for CDI treatment, while microbiota therapeutics (faecal microbiota transplantation, nontoxigenic C. difficile, stool substitutes) are increasingly being explored. rCDI can only occur when viable C. difficile spores are present, either within the gut lumen after infection or when reacquired from the environment. C. difficile spore germination can be influenced by gut environmental factors resulting from dysbiosis, and spore outgrowth may be affected stage by some antimicrobials (e.g. fidaxomicin, ramoplanin, oritavancin).ImplicationsrCDI is a significant challenge for healthcare professionals, requiring a multifaceted approach; optimized infection control to minimize reinfection; C. difficile–targeted antibiotics to minimize dysbiosis; and gut microflora restoration to promote colonization resistance. These elements should be informed by our understanding of the microbiologic factors involved in both C. difficile itself and the gut microbiome.     

Host factors are more important in predicting recurrent Clostridium difficile infection than ribotype and use of antibiotics

ObjectiveA frequent complication of Clostridium difficile infection (CDI) is recurrent disease. The aim of this study was to determine whether early recurrence risk was higher after infection with ribotype 027 (outbreak strain) compared with infection with endemic strain types of C. difficile.MethodsConsecutive patients diagnosed with CDI between May 2013 and March 2014 were included (outbreak strain, and non-outbreak strains). Patients who developed recurrent CDI within 30 days after completion of CDI treatment, were compared with patients without a recurrence. Medical charts were reviewed for demographic and clinical characteristics. General practitioners were contacted to complete data about the occurrence of recurrent CDI, and the use of medication after hospital discharge.ResultsIn total, 135 patients were at risk for the development of recurrent CDI; 74 patients were infected by ribotype 027, and 61 patients by other ribotypes. Thirty-nine patients (29%) developed recurrent CDI within 30 days after completion of CDI treatment. In multivariable analysis, age ≥70 years (HR 3.05, 95% CI 1.54–6.03), and a duration of CDI treatment ≥11 days (HR 1.92, 95% CI 1.00–3.69) were clearly associated with recurrence; infection with ribotype 027 showed a HR of 1.72 (95% CI 0.88–3.33).ConclusionDuring this outbreak of C. difficile in a tertiary care centre, age and a prolonged duration of CDI therapy (which is most likely a marker of underlying disease severity) were the main risk factors for recurrent CDI. This points to host factors as more important predictors for recurrent CDI than strain type or antibiotic use.     

Spatial clustering and livestock exposure as risk factor for community-acquired Clostridium difficile infection

ObjectivesClostridium difficile infections (CDI) account for 1.5% of diarrhoeic episodes in patients attending a general practitioner in the Netherlands, but its sources are unknown. We searched for community clusters to recognize localized point sources of CDI.MethodsBetween October 2010 and February 2012, a community-based prospective nested case–control study was performed in three laboratories in the Netherlands with a study population of 2 810 830 people. Bernoulli spatial scan and space–time permutation models were used to detect spatial and/or temporal clusters of CDI. In addition, a multivariate conditional logistic regression model was constructed to test livestock exposure as a supposed risk factor in CDI patients without hospital admission within the previous 12 weeks (community-acquired (CA) CDI).ResultsIn laboratories A, B and C, 1.3%, 1.8% and 2.1% of patients with diarrhoea tested positive for CDI, respectively. The mean age of CA-CDI patients (n = 124) was 49 years (standard deviation, 22.6); 64.5% were female. No spatial or temporal clusters of CDI cases were detected compared to C. difficile–negative diarrhoeic controls. Except for one false-positive signal, no spatiotemporal interaction amongst CDI cases was found. Livestock exposure was not related to CA-CDI (odds ratio, 0.99; 95% confidence interval, 0.44–2.24). Ten percent of CA-CDIs was caused by PCR ribotype 078, spatially dispersed throughout the study area.ConclusionsThe absence of clusters of CDI cases in a community cohort of diarrhoeic patients suggests a lack of localized point sources of CDI in the living environment of these patients.     

How to: Surveillance of Clostridium difficile infections

BackgroundThe increasing incidence of Clostridium difficile infections (CDI) in healthcare settings in Europe since 2003 has affected both patients and healthcare systems. The implementation of effective CDI surveillance is key to enable monitoring of the occurrence and spread of C. difficile in healthcare and the timely detection of outbreaks.AimsThe aim of this review is to provide a summary of key components of effective CDI surveillance and to provide some practical recommendations. We also summarize the recent and current national CDI surveillance activities, to illustrate strengths and weaknesses of CDI surveillance in Europe.SourcesFor the definition of key components of CDI surveillance, we consulted the current European Society of Clinical Microbiology and Infectious Diseases (ESCMID) CDI-related guidance documents and the European Centre for Disease Prevention and Control (ECDC) protocol for CDI surveillance in acute care hospitals. To summarize the recent and current national CDI surveillance activities, we discussed international multicentre CDI surveillance studies performed in 2005–13. In 2017, we also performed a new survey of existing CDI surveillance systems in 33 European countries.ContentKey components for CDI surveillance are appropriate case definitions of CDI, standardized CDI diagnostics, agreement on CDI case origin definition, and the presentation of CDI rates with well-defined numerators and denominators. Incorporation of microbiological data is required to provide information on prevailing PCR ribotypes and antimicrobial susceptibility to first-line CDI treatment drugs. In 2017, 20 European countries had a national CDI surveillance system and 21 countries participated in ECDC-coordinated CDI surveillance. Since 2014, the number of centres with capacity for C. difficile typing has increased to 35 reference or central laboratories in 26 European countries.ImplicationsIncidence rates of CDI, obtained from a standardized CDI surveillance system, can be used as an important quality indicator of healthcare at hospital as well as country level.     

Emergence of Clostridium difficile infection in tuberculosis patients due to a highly rifampicin-resistant PCR ribotype 046 clone in Poland

Clostridium difficile infection (CDI) is a major cause of nosocomial diarrhea. CDI is known to develop after antibiotic administration, but anti-tuberculosis agents have rarely been implicated. We documented an outbreak caused by a highly rifampicin-resistant C. difficile strain of polymerase chain reaction (PCR) ribotype 046 in patients with active tuberculosis.     

Clostridium difficile as a cause of healthcare-associated diarrhoea among children in Auckland,New Zealand: clinical and molecular epidemiology

We aimed to determine the incidence of Clostridium difficile infection (CDI), the molecular epidemiology of circulating C. difficile strains and risk factors for CDI among hospitalised children in the Auckland region. A cross-sectional study was undertaken of hospitalised children <15 years of age in two hospitals investigated for healthcare-associated diarrhoea between November 2011 and June 2012. Stool specimens were analysed for the presence of C. difficile using a two-step testing algorithm including polymerase chain reaction (PCR). C. difficile was cultured and PCR ribotyping performed. Demographic data, illness characteristics and risk factors were compared between children with and without CDI. Non-duplicate stool specimens were collected from 320 children with a median age of 1.2 years (range 3 days to 15 years). Forty-six patients (14 %) tested met the definition for CDI. The overall incidence of CDI was 2.0 per 10,000 bed days. The percentage of positive tests among neonates was only 2.6 %. PCR ribotyping showed a range of strains, with ribotype 014 being the most common. Significant risk factors for CDI were treatment with proton pump inhibitors [risk ratio (RR) 1.74, 95 % confidence interval (CI) 1.09–5.59; p?=?0.002], presence of underlying malignancy (RR 2.71, 95 % CI 1.65–4.62; p?=?0.001), receiving chemotherapy (RR 2.70, 95 % CI 1.41–4.83; p?=?0.003) and exposure to antibiotics (RR 1.17, 95 % CI 0.99–1.17; p?=?0.03). C. difficile is an important cause of healthcare-associated diarrhoea in this paediatric population. The notion that neonatal populations will always have high rates of colonisation with C. difficile may not be correct. Several risk factors associated with CDI among adults were also found to be significant.