Update on Clostridium difficile infections.

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General review

Update on Clostridium difficile infections Actualités sur les infections à Clostridium difficile A. Le Monnier a,∗,b , J.-R. Zahar c , F. Barbut d,e a

Unité de microbiologie clinique, groupe hospitalier Paris Saint-Joseph, 185, rue Raymond-Losserand, 75014 Paris, France b Université Paris-Sud, EA4043 écosystème microbien digestif et santé, 92296 Châtenay-Malabry cedex, France c Unité de prévention et de lutte contre les infections nosocomiales, université d’Angers, CHU d’Angers, 49100 Angers, France d Laboratoire Clostridium difficile associé au CNR des anaérobies et du botulisme, 75012 Paris, France e Groupe de recherche clinique no 2 EPIDIFF, université Pierre-et-Marie-Curie, 75012 Paris, France Received 27 February 2014; accepted 1st April 2014

Abstract Clostridium difficile infections (CDI) occur primarily in hospitalized patients with risk factors such as concomitant or recent use of antibiotics. CDI related additional costs are important for the global population and health-care facilities. CDI epidemiology has changed since 2003: they became more frequent boosted by large outbreaks, more severe, more resistant to antibiotic treatment, and spread to new groups of population without any risk factor. This is partly due to the emergence and worldwide dissemination of new and more virulent C. difficile strains such as the epidemic clone 027/NAP1/BI. The host immune response plays a central role in the pathogenesis of CDI and could also be involved in the occurrence of recurrent or severe forms. New guidelines including new molecular tests (NAAT) have recently clarified and simplified the diagnostic strategies for the microbiological diagnosis of CDI. The CDI incidence was proven to be related to the level of clinical suspicion and the frequency of microbiological screening for C. difficile. The current recommendations for the treatment of CDI mention oral metronidazole as the first line treatment for mild to moderate diarrhea. Oral vancomycin use should be restricted to severe cases. In the absence of consensus, the treatment of multiple recurrences remains a major concern. New and more targeted antibiotics and innovative therapeutic strategies (fecal transplantation, monoclonal antibodies, and vaccination) have emerged as new therapies for CDI. © 2014 Elsevier Masson SAS. All rights reserved. Keywords: Clostridium difficile; Post-antibiotic diarrhea; Colitis

Résumé Les infections digestives à Clostridium difficile (ICD) surviennent essentiellement chez des patients hospitalisés présentant des facteurs de risque notamment la prise récente ou concomitante d’antibiotiques. Les surcoûts des ICD sont importants pour la collectivité et les établissements de santé. Depuis 2003, l’épidémiologie des ICD s’est modifiée : elles sont devenues plus fréquentes amplifiées par des épidémies majeures, plus sévères, répondent moins bien au traitement antibiotique et touchent de nouvelles catégories de population sans facteur de risque. Ceci est en partie dû à l’émergence et la dissémination mondiale de nouveaux clones épidémiques plus virulents. Au regard des données récentes de la littérature, la réponse immune de l’hôte semble jouer un rôle central dans les processus physiopathologiques des ICD. Elle serait impliquée dans la survenue des formes récidivantes ou sévères. Concernant le diagnostic microbiologique des ICD, de nouvelles recommandations intégrant les techniques de biologie moléculaire ont permis de préciser et de simplifier les stratégies diagnostiques. Il a clairement été démontré que l’incidence pouvait varier en fonction du degré de suspicion clinique et de la fréquence des recherches microbiologiques. Concernant les recommandations actuelles sur le

∗

Corresponding author. E-mail address: [email protected] (A. Le Monnier).

http://dx.doi.org/10.1016/j.medmal.2014.04.002 0399-077X/© 2014 Elsevier Masson SAS. All rights reserved.

Please cite this article in press as: Le Monnier A, et al. Update on Clostridium difficile infections. Med Mal Infect (2014), http://dx.doi.org/10.1016/j.medmal.2014.04.002

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traitement des ICD, le métronidazole administré par voie orale reste la molécule de choix pour le traitement des formes simples ou peu sévères. La vancomycine per os est réservée aux formes sévères. En l’absence de consensus, le traitement des récidives multiples reste un problème majeur. Aujourd’hui, de nouvelles molécules antibiotiques plus ciblées sur C. difficile et de nouvelles stratégies thérapeutiques innovantes (transplantation de selles, anticorps monoclonaux, vaccination) apparaissent comme de nouvelles perspectives de traitement. © 2014 Elsevier Masson SAS. Tous droits réservés. Mots clés : Clostridium difficile ; Diarrhée post-antibiotique ; Colite

1. Introduction Clostridium difficile is an ubiquitous spore-forming Grampositive anaerobic bacillus widespread in the environment. It was first described in 1935 by Hall and O’Toole as part of the normal intestinal microbiota of healthy neonates, but it is rarely isolated in healthy adults. The pathogenic role of C. difficile was discovered in 1978 and it has since emerged as the leading cause of healthcareassociated diarrhea among adults in industrialized countries (15–25%); it causes more than 95% of pseudomembranous colitis (PMC) cases [1]. There has been a renewed interest for this bacterium since 2003 because of the increased incidence of infections worldwide, their severity, and poor outcome (complications and death) [2]. The re-emergence of C. difficile related infections (CDI) boosted clinical and basic research on the strain, as proved by the number of articles on C. difficile referenced in PubMed which increased 3-fold between 2000 and 2013 (Fig. 1). We present an overview of the pathophysiological processes including risk factors, clinical presentations, economic burden of the disease, and an update of epidemiological trends worldwide with a focus on Europe and France. It should provide a basis for the optimization of microbiological diagnosis, treatment, and prevention according to recent European and American guidelines.

The spores of the bacteria resist to gastric acidity and are transformed into vegetative cells, especially under the action of bile salts. Then, the infectious process of CDI can be broken down into two successive steps [4]. The first step is the colonization of the intestinal microbiota, an essential prerequisite for the second step of toxin A and B production, the major virulence factors responsible for the clinical symptoms. 2.3. Colonization process The first step for host colonization is the implantation and growth of C. difficile in the gut lumen favored by the disruption of the intestinal microbiota and of its barrier effect caused by antibiotics. Colonization is facilitated by C. difficile surface proteins. Several adhesins were identified in C. difficile such as the S-layer proteins (HMW and LMW proteins), Cwp66 (cell wall protein) Fbp68 (fibronectin binding protein), GroEL (heat shock protein), and the flagellar proteins FliC (flagellin) and FliD (flagellar cap). In addition, all strains of C. difficile produce hydrolytic enzymes such as hyaluronidase, chondroitin-4-sulphatase, and protease Cwp84 (clostridial wall protein of 84 kDa). This protease may promote the spread of infection by degrading the mucosal extracellular matrix proteins. The authors of a study of strain deleted for the cwp84 gene recently highlighted the role of this protease in the cleavage of the S-layer protein precursor.

2. Pathophysiology and risk factors for CDI

2.4. Major role of the toxins

2.1. Bacterial cycle

The second step is characterized by the production and release of toxins A (TcdA) and B (TcdB) responsible for clinical symptoms. Indeed, only toxigenic strains are pathogenic and cause infection [4]. TcdA (308 kDa) and TcdB (269 kDa) are high molecular weight toxins belonging to the family of large clostridial toxins (LCTs). They have a glucosyltransferase activity targeting small GTP-binding proteins or guanosine triphosphate (RhoA, Rac, Cdc42). Their glycosylation causes a major disruption of cell signaling pathways in enterocytes, with actin cytoskeleton modifications, disruption of tight junctions, and cytopathic effect [5]. They have a similar structure and mechanism of action, although their effects are not strictly similar. Moreover, toxins have a pro-inflammatory role. TcdA modulates the production of substance P (neurotransmitter present in the intestinal epithelium), increases the production of proinflammatory cytokines such as interleukin-Il 8, and stimulates macrophages in the basal lamina propria responsible for the production of tumor necrosis factor (TNF)-␣. Finally, both TcdA

C. difficile can be present in two forms: a pathogenic vegetative form, producing toxins and causing clinical symptoms; and spores, highly resistant to disinfectants and antibiotics that persist in the gut microbiota and may be shed in the environment. 2.2. Origin and transmission Contamination occurs by the oral fecal route. Spores are transmitted by the hands of health care workers or directly from the hospital environment of an infected patient. Transmission is correlated with the importance of environmental contamination cases [3]. Other factors, such as the resistance of spores to the action of conventional antiseptics and disinfectants, overcrowding of patients, and antibiotic pressure, promote this transmission. The spores can also be found in some foods such as meat products, suggesting a possible food-borne transmission of CDI.




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Fig. 1. Exponential literature and main dates of Clostridium difficile history attesting of a renewed interest for C. difficile. Caractère exponentiel de la littérature et principales dates de l’histoire de Clostridium difficile attestant de l’intérêt renouvelé pour C. difficile. (Last request on web of science on September 30, 2013, keyword “Clostridium difficile”; CD: Clostridium difficile; EAI: enzyme immunoassay; NAAT: nucleic acid amplification test; PMC: pseudomembranous colitis).

and TcdB induce apoptosis [5]. The production of toxins leads to inflammation and destruction of the intestinal epithelium. The influx of neutrophils in the intestinal epithelium leads to the characteristic pseudomembrane lesions visible at colonoscopy (volcano-like lesions) [6]. TcdA and TcdB are encoded respectively by genes tcdA (8133 bp) and tcdB (7098 bp) located on a pathogenicity locus of 19.6 kb (PaLoc). The toxigenic PaLoc element contains three additional genes: tcdR encodes an alternative sigma factor that acts as a positive regulator; tcdC encodes a negative regulator (anti-sigma factor), and tcdE encodes the protein TcdE, which is structurally similar to holing bacteriophage and could therefore be used for the secretion of toxins in the extracellular medium. A deletion in position 117 of the tcdC gene is observed in the epidemic clone 027/BI/NAP1, resulting in a non-functional TcdC protein, which is implicated in the down-regulation of toxin production. As a result, the strain is able to produce an excess of both toxins A and B in vitro, which may partly explain the hypervirulence of 027/BI/NAP1 strains [7]. However, this overproduction of toxins was challenged by the authors of a more recent study. Other possible mechanisms of increased virulence include higher sporulation rates, and polymorphisms in the binding domain of toxin B [7]. There is a wide variety of clones with various pathogenic and epidemic potential. The genetic polymorphism of the PaLoc is usually assessed by toxinotyping, a PCR-RFLP technique which can discriminate among 31 different toxinotypes. Most pathogenic strains secrete 2 toxins, however, some virulent strains produce a TcdA deleted in the C-terminal extremity (strain A− B+ ). A third toxin, the binary toxin (or CDT) is produced by 20% of C. difficile. This additional toxin which is produced by the

epidemic 027/BI/NAP1 strains could also be involved in virulence [8]. This binary toxin has an ADP-ribosyl transferasespecific activity. The authors of some clinical studies have suggested that these CDT-producing strains, including PCRribotype 027 strains, could be associated with more severe clinical presentations of CDI and higher mortality [9]. 3. Risk factors for CDI Several risk factors for CDI have been identified and sorted in three groups (Table 1): • environmental factors favoring exposure to C. difficile spores; • factors favoring colonization of the digestive microbiota; • host related factors [3,10,11]. First, patients have to be in contact with C. difficile spores. Thus, the risk increases with prolonged or repeated hospitalizations. Health care settings constitute important reservoir of C. difficile spores particularly in the direct environment of infected patients or asymptomatic carriers. High frequency of caregiving, overcrowding of patients, stay in an intensive care unit, important colonization pressure or environmental contamination are non exclusive additional factors favoring the transmission [12]. Furthermore, some factors may facilitate the implantation and growth of C. difficile in the digestive microbiota. Thus, antibiotics are the major risk for CDI. They disrupt the intestinal microbiota barrier, resulting in a decreased resistance to colonization by C. difficile [4]. It has now been acknowledged that all antibiotics – except for aminoglycosides administered parenterally – can cause CDI, since the first cases of PMC




Table 1 Major risk factors for Clostridium difficile infections classified in three groups. Principaux facteurs de risque répartis en 3 groupes. Factors favoring exposure

Factors favoring colonization

Host factors

High colonization rate Prolonged hospital stay ICU stay Roommate with an infected patient Being hospitalized in a room just after an infected patient

Antibiotic consumption (current or up to 6 months before) Chemotherapy Proton pump inhibitors Nasogastric tube and gastrointestinal surgery Anti-acids Enemas Laxatives

Age: > 65 years Comorbidity and their severity Immunodepression Host immune response (rate of neutralizing antibodies) Women > men Previous CDI episode

were reported after administration of clindamycin. The antibiotics most commonly associated with CDI include clindamycin, cephalosporins, amoxicillin + clavulanic acid [11], and in recent years, new fluoroquinolones (levofloxacin, moxifloxacin, gatifloxacin), whose spectrum of action is extended to anaerobic bacteria. The duration of antibiotic treatment seems correlated to an increased risk. Nevertheless, CDI can occur several weeks after antibiotic treatment because of the long term disruption of the intestinal flora [4]. Other factors could support the implantation of C. difficile by helping the bacterium to bypass the first host natural defenses. Some CDI have been reported after chemotherapy, including administration of methotrexate, doxorubicin, cyclophosphamide, and 5-fluorouracil. Globally, all the factors that cause a change in the intestinal microbiota or gut motility (laxatives, digestive barium enemas, antacids, antimotility drugs, all procedures concerning the gut including the use of naso-gastric tubes, and inflammatory bowel disease) can potentially enhance the risk of CDI [13]. Proton pump inhibitors and antacids are frequently associated as risk factors for CDI. Last but not least, host related factors are probably the leading risk factors for CDI. The incidence of CDI increases with age, with a significantly increased risk in patients more than 65 years of age. The reasons for this increased susceptibility to colonization and infection in elderly patients remain unclear. It could be due to a combination of several factors: more frequent and more severe underlying diseases, a decrease in the effectiveness of the immune response, a change in the resistance to colonization or repeated hospitalizations. Moreover, some co-morbidities, such as impaired immunity, gastrointestinal diseases, clearly predispose to CDI [3,11].

as mild fever, nausea, and malaise; but there is no significant deterioration of the general health status. Hyperleukocytosis and hypoalbuminemia are frequently observed. If endoscopy is performed (it is not necessary in this clinical presentation), the mucosa would appear normal or with erythema without ulceration or pseudomembranes.

4. Clinical presentations

• an optimal management of patients; • a homogenous categorization of patients in clinical studies; • an international comparison of epidemiological data.

The clinical presentations of C. difficile infections (CDI) range from mild self-limited diarrhea to severe life-threatening colitis [1]. 4.1. Post-antibiotic diarrhea It clinically presents as diarrhea (at least three unformed stools per day, without visible blood or mucus). It may be accompanied by lower abdominal pain and systemic symptoms such

4.2. Colitis and pseudomembranous colitis (PMC) Diffuse or patchy colitis, with or without pseudomembranes can be observed on colonoscopy. The characteristic PMC begins with profuse watery diarrhea (> 7 stools/day), usually bloodless, often accompanied by fever (> 65%), and abdominal pain (70%). Leukocytosis and a biological inflammatory syndrome (significant increased of C-reactive protein [CRP]) are common. The endoscopy shows a colonic mucosa covered with raised scattered or confluent yellowish plaques (pseudomembranes). The histological analysis of these pseudomembranes reveals a superficial necrosis of the mucosa, an exudate accumulation of leukocytes, tissue, debris, and mucus. 4.3. Complications and severe CDI presentations Major CDI complications include fulminant colitis, toxic megacolon, perforation, and septic shock syndrome which may be fatal. Diarrhea is the most common clinical presentation, but some severely ill patients may have little or no diarrhea due to a toxic megacolon or/and paralytic ileus. These complications require medical and surgical treatment [4]. The CDI classification made according to the severity criteria is essential for:

Variation in the rate of severe presentations is a good indicator to assess changes in the epidemiology of CDI over time. To date, there is no international consensus between American and European definitions of severe presentations [10,14]. Overall, the severity criteria include clinical signs (fever > 38.5 ◦ C, chills, hemodynamic instability, peritonitis, ileus, or toxic megacolon), biological parameters


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(WBC > 15 × 109 /L, increased serum lactate and creatinine, hypoalbuminemia < 25 g/L), and imaging criteria (dilation of the colon, thickening of the colonic wall, ascitis, and pseudomembranes observed during endoscopic examination) [10,14,15]. 4.4. Outcome: mortality and recurrences Mortality associated with mild diarrhea CDI ranges from 0.6 to 1.5% whereas mortality is higher with complications, ranging from 24 to 38%. According to the ECDIS study, one in 10 cases of CDI leads to or contributes to intensive care unit admission or death, or requires total or partial colectomy [16]. Variations in mortality rates reported in studies could be attributed to differences in the definitions (day 30 or day 90, attributable or overall cases, etc.) and/or populations observed, and/or variations in prevalent PCR ribotypes [16]. Recurrence is the major clinical issue for CDI. According to the most recent clinical studies, recurrences occur in up to 27% of patients in the month following the first episode [17,18]. Some authors reported that the recurrence rate had increased in recent years [19]. A patient with recurrence may enter a cycle of multiple recurrences causing exhaustion and protein loss enteropathy [18]. This cycle is a therapeutic challenge. Recurrences may be due to the intraluminal persistence of C. difficile spores (relapses) or to the acquisition of a new strain (reinfection). The authors of a recent clinical study reported that early recurrences (0–14 days post treatment) were relapses in 86.7%, and late recurrences were relapses in 76.7% of cases. Independent risk factors for recurrence include: age greater than 65 years, previous CDI recurrence, increased severity of underlying disease (Horn’s index), concomitant antibiotic therapy (other than those given for treating the first CDI episode), prolongation of hospital stay, low serum antibody response to toxin A during the initial episode, or being infected by the epidemic NAP1/027/BI epidemic strain [16,18,20]. 5. Predictive scoring system for CDI The wide spectrum of clinical presentations, ranging from asymptomatic carriage to fulminant, life-threatening colitis, is still not understood. All the risk factors described above do not fully explain why some people develop CDI while others remain asymptomatic or escape colonization, despite exposure to the same environmental conditions and risk factors [21]. The frequency of asymptomatic carriers of toxigenic or nontoxigenic strains increases with the duration of hospitalization and can reach 50% of hospitalized patients over four weeks. However, although asymptomatic carriage seems more common than infection, the data differs from one study to another and depends on the type of hospital care unit [3,22]. Moreover, it remains unclear why one in four patients presents with multiple recurrences whereas others present with a single episode of mild and self-limited diarrhea [21]. Several authors have proposed clinical scores to predict the initial episode of CDI, evolution to severe clinical presentations, or recurrences. However, these predictive scores for CDI and/or

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recurrences are mainly based on the analysis of clinical, laboratory data and patient history; they do not take into account the impact of the immune response [15,23]. Hu et al. first reported, in 2009, that the reliability of scores for the identification of patients at risk of recurrence was significantly increased by including the serum IgG anti-toxin A titer [24]. There is currently no test available for clinical practice. 6. Role of the host immune response The host immune response has become central in understanding the pathophysiology of CDI according to recent published data. The immune responses to C. difficile highlight the dramatic variations in disease presentation and outcome. There is little but controversial data on the effect of an excessive host inflammatory response. Toxins are able to initiate and propagate a pro-inflammatory cascade, likely responsible for host intestinal damage observed in severe CDI. These findings suggest dampening the host immune response to prevent a severe course of the infection [25]. However, some recent data from animal models suggests that dampening the immune response could lead to a higher virulence. More than a beneficial or harmful implication of the innate immunity, there must be a fine balance between pro and anti-inflammatory responses [6,25]. Moreover, Viscidi et al. found specific human antibodies directed against these toxins in approximately 60–70% of the global asymptomatic population [26]. Antibodies appear during early infancy and are maintained over time even if important inter-individual heterogeneity in antibodies titers is observed [26,27]. This proves that contact with the bacterium is probably early and frequent all life long. Fifty to 78% of patients presenting with CDI develop a systemic post-infection response, and serum IgA and IgG levels follow the same trend [27,28]. Since these first observations, authors of clinical studies have indicated that antitoxin responses in both serum and intestinal secretions could prevent CDI whereas an inadequate immune response or low titers in serum antibodies was correlated with the occurrence of a first CDI episode [29] or recurrent C. difficile diarrhea [20,28]. These authors suggest that tests evaluating the host immune response could be used to increase the performance of clinical scores including other previously identified risk factors [6,24]. However, the host immune response to C. difficile is still poorly understood. A better understanding of the host inflammation and immune mechanisms that modulate the course of disease and control host susceptibility to C. difficile could lead to novel host-targeted strategies. Moreover, there is now considerable interest in developing new options using passive (monoclonal antibodies) and active immunization for the prevention and treatment of CDI as well as multiple recurrences [30,31]. 7. Burden of the disease CDI cause significant additional costs for the global population and healthcare facilities. These costs are related to the management of infected patients including prevention and


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Table 2 Estimated attributable hospital costs and healthcare associated Clostridium difficile infections. Estimation des coûts hospitaliers et associés aux soins attribuables aux infections à C. difficile. Country

Year of study

Average additional cost per hospitalization

UK Ireland Germany Italy

1995 2000 2006 2009–2012

£ 4,107 £ 2,860 D 7,147 D 13,580

control measures for CDI, and to prolonged hospital stay, readmission, recurrence, and mortality [32]. Three to 30% of patients with healthcare-associated CDI die within 30 days [32]. However, many of these patients present with underlying diseases so that death cannot be directly attributable to CDI. There is some evidence that hospitalized patients with CDI are up to 3-fold more likely to die while in hospital or within 30 days, independently of their age, and underlying diseases. The authors of the ECDIS study reported that CDI caused or contributed to 40% of deaths that occurred within three months of CDI diagnosis, corresponding to 9% of all diagnosed patients with CDI [16]. There has been relatively little research on the impact of community-acquired CDI. The authors of an American retrospective study including 157 patients with community acquired CDI reported that 40% of these patients required hospitalization, 20% presented with a severe infection and 28% had recurrent CDI. In a French study, 28% of cases diagnosed in hospital were categorized as community acquired CDI in 2009 [8]. CDI have a considerable impact on the healthcare system. CDI significantly increase the cost of patient care in hospitals, primarily because they increase the duration of hospital stay. Most authors reported that nosocomial CDI extended the length of hospital stay from one to three weeks, as compared to noninfected controls matched according to risk factors (i.e. age and underlying diseases) [32,33]. CDI may also be prolonged intensive care unit (ICU) stay among patients admitted to these units. Moreover, additional costs for the institution’s budget include specific therapy for the infection (diagnosis, antibiotic treatment, ICU, and surgery for severe CDI), and infection control measures (isolation precautions, specific environmental disinfection). In case of uncontrolled outbreaks, wards may need to be closed or admission may need to be restricted. Various approaches have been used to estimate the extra cost induced by CDI (Table 2) [32–36]. Some recent epidemiological data from the US suggests that C. difficile might be responsible for as many as 333,000 initial cases of CDI per year, costing up to $3.2 billion per year. In 2006, an expert group including representatives of the ECDC and the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) estimated that CDI caused a direct cost of approximately three billion Euros per year in the EU [2]. These costs are related to healthcare associated CDI and do not include the additional cost of community-acquired CDI, and nursing home CDI, and the indirect costs such as disorganization of care units, loss of productivity, and social care.

8. Epidemiological data 8.1. Emergence of a hypervirulent C. difficile clone in North American healthcare facilities Outbreaks of severe CDI with increased morbidity and mortality were reported in North America in the early 2000s [37]. A 4-fold increase of CDI incidence was reported in Quebec province hospitals between 1998 and 2004. An estimated 15,000 to 20,000 patients die from CDI in the USA every year. This unexpected increase of CDI was partly due to the emergence and the rapid spread of a specific C. difficile clone belonging to PCR ribotype 027 (also characterized as toxinotype III, North America PFGE pulsotype 1 [NAP1] and restriction endonuclease analysis group BI) [38], in 2000. This epidemic strain was significantly associated with more complications (megacolon, septic shock syndrome, perforation), a higher death rate, and a poor clinical response to metronidazole [37]. The 027/BI/NAP1 C. difficile strain was extensively studied to better understand its increased virulence. The emergence and dissemination of this epidemic strain was correlated with its resistance to newer fluoroquinolones (moxifloxacin, gatifloxacin, levofloxacin), a trait that was not present in the rare 027/BI/NAP1 strains isolated before 2000. CDI cases due to the 027/BI/NAP1 strain were reported by hospitals in 40 USA states and in all Canadian Provinces between 2003 and 2008. In 2005, the 027/BI/NAP1 accounted for 36% of all strains collected as part of a multicenter clinical trial on the toxin-binding polymer, tolevamer. Two clinical trials focusing on fidaxomycin were performed between 2006 and 2009; the incidence of 027/BI/NAP1 strain was 38.1% and 45.9% in the American and Canadian populations, respectively [17,18]. 8.2. Epidemiology in Europe, France, and in other parts of the world A pan-European point prevalence study on healthcare infection was performed in 2012 under the auspices of the ECDC. CDI accounted for 48% of gastrointestinal infections which in turn accounted for 7.7% of all healthcare associated infections (http:// www.ecdc.europa.eu/en/publications/Publications/healthcareassociated-infections-antimicrobial-use-PPS.pdf). C. difficile ranked eighth among pathogens responsible for nosocomial infections. Various European countries have reported an increase of CDI, despite using different CDI surveillance systems [2]. A 2-month prospective study focusing on CDI was conducted in 38 hospitals in 14 European countries in 2005 [9]. The 027/BI/NAP1 epidemic strain was isolated in 6.2% of cases. A more comprehensive overview of CDI was obtained through a hospital-based survey performed in 34 European countries in 2008 [16]. The mean incidence of healthcare-associated CDI was 4.1 per 10,000 patient-days per hospital but with a great inter hospital variation (range 0.0–36.3). CDI was estimated to occur in approximately one out of 435 admissions per hospital. Sixty-five PCR-ribotypes were identified; 014/020 (16%), 001




(9%), and 078 (8%) were the most prevalent. The incidence of PCR-ribotype 027 was 5%. A 6-month prospective multicenter study was performed in France in 2009. The incidence of CDI was 2.28 or 1.15 cases per 10,000 patient-days in acute care (n = 1316 cases) or rehabilitation/long-term care (n = 295 cases), respectively. The five major PCR-ribotypes were 014/020/077 (18.7%), 078/126 (12.1%), 015 (8.5%), 002 (8%), and 005 (4.9%) [8]. There is more recent data indicating that some countries have successfully controlled the spread of the epidemic 027/BI/NAP1 strain and stopped the increased of CDI incidence. The notification of CDI cases has been mandatory since 2007 in England and the number of CDI reported every year has started to decrease, after years of steady increase. There was a 19% decrease in the incidence of 027/BI/NAP1 between 2007 and 2008 and a 29% decrease in the number of death certificates in which C. difficile was mentioned. There is little data on CDI incidence or microbiological characteristics of strains from Asia, Africa, and Middle East countries. Sporadic cases of CDI caused by the 027/BI/NAP1 strain were recently reported in hospitals in Japan, Korea, Hong Kong, and Australia. 8.3. Recent trends in C. difficile epidemiology Other C. difficile genotypes have also been reported as predominant or associated with outbreaks or severe cases. PCR ribotype 053 and 106 are predominant in Austria, and the UK, respectively. The prevalence of C. difficile ribotype 078 has increased from 3 to 13% in Europe. Strains of PCR ribotype 078 were recently associated with poor clinical outcome, higher neutrophil count, and greater 14-day mortality rate in the Netherlands. This strain infected younger people and was more frequently responsible for community-acquired CDI than PCR ribotype 027. PCR ribotype 078 was also isolated in pigs and the strains were shown to be genetically very similar to human isolates. Large outbreaks and severe PMC due to PCR ribotype 017 strains, producing toxin B but not toxin A, were reported in Asia. The profile of patients presenting with CDI also changed in the last decade. CDI not only occur in elderly patients with comorbidities and previous antibiotic treatment, but they can also affect populations previously considered at low risk (e.g. patients without previous exposure to antibiotics, young individuals, pregnant women) [39]. Furthermore, while CDI remains a major healthcare-related pathogen, there is also a better acknowledgment of community-acquired CDI (CA-CDI) in patients without any hospitalization in the previous 8 weeks (2, 10). The incidence of community-acquired CDI ranges from 6.9 to 46 cases per 100,000 person years in the USA, and is estimated at 20 to 30 per 100,000 individuals in the UK [39]. The origin of infection in patients with CA-CDI is still unknown. However, the isolation of C. difficile in livestock, meat from various animals, vegetables, and even water in the USA and Europe has raised concerns about a possible food-borne contamination [40]. Similar characteristics have been reported among strains isolated in animals or food and those isolated in humans, suggesting a possible relationship.

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Nevertheless, no documented outbreak has been related to food consumption yet, and there is no evidence of C. difficile as a food-borne pathogen. 9. Microbiological diagnosis of CDI 9.1. Indications for C. difficile testing A rapid diagnosis of CDI is crucial for the individual management of patients but also to implement rapid infection control measures to avoid cross contamination. It is also essential to get a reliable diagnosis to monitor CDI trends over the time within a hospital, or to compare CDI incidence in various healthcare facilities. The dramatic changes in the epidemiology of CDI has prompted companies to manufacture more sensitive and rapid methods such as nucleic acid amplification tests (NAATs) based on the detection of tcdA and/or tcdB genes, for the diagnosis of CDI. The results of a recent pan-European point prevalence study showed that CDI diagnosis was performed only upon the physician’s request in more than 52% of cases (Davies et al., ECCMID 2013, LB2968). However, the awareness of CDI as well as the testing frequency was highly variable across Europe and accounted for major differences in reported incidences among countries. For example, the authors of a Spanish study reported that two of every three CDI episodes were not diagnosed because C. difficile was not suspected and screened for in 47.6% of cases. C. difficile is a major healthcare related pathogen, consequently the European and American guidelines recommend implementing the “3-day rule” according to which any nosocomial diarrhea (defined as stool sample taken after day 3 of hospitalization) should be systematically tested for C. difficile, whatever the physician’s request (Cumitech 12A–Laboratory Diagnosis of Bacterial Diarrhea 1992, ASM Press). C. difficile should be also screened for in any patient presenting with acute diarrhea, having received antibiotics or been hospitalized in the previous two months. Some laboratories decided to screen every diarrheic stool (stool taking the shape of the container) for C. difficile because there as a better acknowledgment of CDI as a cause of community acquired infection in patients without any previous risk factors. 9.2. Definition of C. difficile infection A CDI case is defined as a patient with one or more of the following criteria [2,41]: • patients with diarrheic stools, or toxic megacolon, or ileus, and a positive laboratory assay for C. difficile TcdA and/or TcdB in stools, or a toxin producing C. difficile detected in stools by culture or other means; • patients with pseudomembranous colitis (diagnosed by lower gastrointestinal endoscopy, after colectomy, or autopsy). These definitions exclude diarrhea with an other etiology (diagnosed by the attending physicians), and asymptomatic




patients with toxin-producing C. difficile or an assay positive for TcdA and/or TcdB. 9.3. General recommendations for the diagnosis of CDI Some general and practical considerations should be recalled before going through the technical aspects of CDI diagnosis. First, only diarrheic stools should be tested for C. difficile to avoid identifying asymptomatic carriers of a toxigenic C. difficile strain who do not require any specific treatment. Second, a single stool specimen is sufficient to diagnose CDI and repeating testing within a 7-day period is not recommended. Although this is still quite commonly practiced in many laboratories, the diagnostic gain (defined by the frequency of results converted from negative to positive after repeating testing) is low, ranging between 1 and 2%. Moreover, repeating testing may lead to false positive results due to a weak specificity of methods used. Third, a “test-of-cure” is not recommended for the clinical management of CDI. The effectiveness of treatment should be only assessed according to clinical criteria (improvement or resolution of diarrhea) and not on the microbiological eradication of the bacterium from stools. Indeed, up to 56% of patients still had positive culture and/or expressed toxins in the one to four weeks following the end of treatment, despite the resolution of diarrhea [42]. Such follow-up examinations are likely to cause confusion and may lead to additional and unnecessary treatment. 9.4. Various methods, various targets, and various interpretations There is currently no “gold standard” for the diagnosis of CDI but there are two reference methods which actually detect two different targets. The stool cytotoxicity assay detects the presence of “free” C. difficile toxins in stools (primary toxin B but also toxin A) whereas the toxigenic culture detects C. difficile bacteria or spores that have a potential to produce toxins. Paradoxically, these methods are not frequently used in clinical practice because they lack standardization and they have a slow turn-around time. Nevertheless, they are still used to assess the performances of new diagnostic assays. A great number of methods are now commercially available for the diagnosis of CDI. These methods can be classified in three groups (Fig. 2): • methods detecting free toxins in stools: these methods include the stool cytotoxicity assay (CTA) on cell culture and the enzyme immunoassays (EIA) for toxins A and B. These methods are very specific, mainly because the asymptomatic carriage of free toxins in stools is infrequent (< 0.7%): a positive result usually confirms the diagnosis of CDI. However, the CTA requires specific skills and equipment in cell culture has a slow turn-around time and is quite time consuming. EIAs for toxins sometimes lack sensitivity (only 50 to 80%) and therefore cannot be used as standalone methods for the diagnosis of CDI;

• screening methods for C. difficile. These methods include culture on a selective medium and EIAs for the detection of glutamate dehydrogenase (GDH), a specific enzyme of C. difficile (including toxigenic and non toxigenic C. difficile strains). These methods are highly sensitive and have an excellent negative predictive value for the diagnosis of CDI. EIAs for GDH are rapid to perform and can be used as screening tests. A negative result for GDH can rule out the diagnosis of C. difficile. However, a positive result must be confirmed with a more specific method to identify patients carrying a toxigenic strain of C. difficile; • screening methods for toxigenic strains of C. difficile in stools. They include toxigenic culture which is a 2-step method: isolating the microorganism in stools followed by the in vitro detection of toxin secretion by the isolated strain. This technique is quite time-consuming and results are not available before 48 to 72 hours. Nucleic acid amplification tests (NAATs) for the diagnosis of CDI were developed to overcome this issue and became commercially available in 2009. Since then, a multitude of molecular methods based on real time PCR have been marketed. Most of these assays target a conserved region of tcdB (BD GeneOhm C. difficile, BD Max C. difficile [BD Diagnostics], ProGastro Cd [Prodesse]), or of both toxins tcdA and tcdB (RIDA® gene toxin A/B [rBiopharm]); whereas Xpert C. difficile (cepheid) is able to detect simultaneously tcdB, binary toxin gene, and a specific deletion in position 117 of tcdC as a surrogate and presumptive marker of the epidemic NAP1/BI/027 strains. The illumigene C. difficile assay (Meridian Bioscience) uses the loop mediated isothermal amplification reaction; the AmpliVue C. difficile (Quidell Molecular) uses a helicasedependent amplification. Both assays target a conserved region of the C. difficile tcdA gene. The mean sensitivity and specificity of NAATs were estimated at 90 and 96%, respectively [43] compared to the toxigenic culture. Furthermore, NAAT results are available within a working day, a shorter delay than for the cytotoxicity assay. Also, the hands-on time is markedly shorter than for other methods. However NAATs tend to be more expensive than toxigenic culture or EIA assays. The main issue with NAAT tests or toxigenic culture is the interpretation of the result. Indeed, a positive result means that a toxigenic strain is present, whether free toxins are present or not. Therefore it may be difficult to differentiate a truly infected patient from an asymptomatic carrier of a toxigenic strain. As a result, NAATs or toxigenic culture may overestimate the number of patients presenting with CDI. This issue can be prevented by an appropriate selection of stool samples and a positive result requires a clinical assessment. The significance of a positive-NAAT result (or a positive toxigenic culture) without free toxin is currently debated. Several authors have addressed this issue. On one hand, several authors reported that methods detecting the presence of a toxigenic strain in stools were more sensitive than methods detecting free toxins (EIAs or stool cytotoxicity assay). Gerding et al. reported that 11% of patients suspected of presenting with CDI who were stool-toxin negative and toxigenic culture positive were found




9

Fig. 2. Classification of methods used for the diagnosis of Clostridium difficile infections, according to their target. Classification des méthodes utilisées pour le diagnostic des infections à Clostridium difficile selon leur cible.

to have pseudomembranes on endoscopy [44]. The stool cytotoxicity assay was only positive in 27 (48%) cases in a cohort of 56 patients with rectosigmoidoscopy-proven PMC [45]. Frozen stools were only available for nine of these 29 CTA-negative patients, all of whom had a positive toxigenic culture, suggesting that toxigenic culture is more sensitive than direct detection of toxins in stools. On the other hand, the authors of a large study reported that patients with free toxins in stools had a significantly higher mortality at day 30 and a white blood cell count > 15,000/mm3 compared to patients with a positive toxigenic culture without any detectable free toxin [46], indicating that free toxin was correlated with a poor clinical outcome. The ESCMID guidelines (published in 2009), the Infectious Disease Society of America (IDSA), and the Society of Healthcare Epidemiology of America (SHEA) recommend to implement a 2-step algorithm for the diagnosis of CDI. The principle of the algorithm is to use a highly sensitive method (such as EIA for GDH or NAAT for toxins) as a screening method and subsequently to confirm any positive result with a more specific method (which usually detects free toxins in the stools) [10,47]. The most recent guidelines from the American Society for Microbiology (updated on September 21, 2010) mention that laboratories can use an NAAT to detect C. difficile toxin genes as a stand-alone diagnostic test or can use this assay to confirm GDH positive screening tests instead of using EIAs (http://www. asm.org/asm/images/pdf/Clinical/clostridiumdifficile9-21.pdf). A positive result means that CDI is likely and requires clinical assessment. 10. CDI treatment Exposure to antibiotics, antiperistaltic agents such as loperamide, and opiates should be avoided before the use of any specific medication. According to recent guidelines, the first

decision in case of mild to moderate infection is to stop the ongoing antibiotic treatment as soon as possible. The results of a meta-analysis of 12 observational studies and randomized control trials (RCTs) showed that concomitant use of antimicrobials for infections other than CDI was significantly associated with an increased risk of CDI recurrence. A retrospective review of 246 patients treated during the years 2004–2006 also confirmed an independent association of non-CDI antimicrobial use with recurrence, but only when non-CDI antimicrobials were given after CDI therapy was completed. In light of this consistent observational evidence, exposure to antibiotics other than those intended to treat CDI should be avoided unless absolutely indicated. Specific antibiotic treatment against C. difficile should be initiated except in case of self-limited diarrhea. Metronidazole (500 mg tid per os for 10 days) is considered as the reference treatment in case of mild to moderate CDI infection according to IDSA [10] and ESCMID guidelines issued in 2009 and recently updated at the end of 2013 [14,48] (Table 3). Vancomycin (125 mg qid per os for 10 days) is more appropriate for severe episodes (Table 3). Indeed, vancomycin is superior to metronidazole for patient presenting with severe CDI, according to the results of several controlled trials [15,49]. The authors of one study stratified 150 patients according to an ad hoc definition of CDI severity and then randomized to oral metronidazole or vancomycin [15]. Clinical cure was defined as a negative follow-up toxin assay and absence of diarrhea on day-6 of therapy. Ninety percent of patients treated with metronidazole and 98% treated with vancomycin were cured of mild CDI according to this definition, but cure rates were lower in the severe disease group treated with metronidazole (76%) compared with vancomycin (97%). Metronidazole (at the same dose and frequency) is suggested if oral therapy is not possible [14], combined with intracolonic




Table 3 Overview of therapeutic options according to clinical presentations of Clostridium difficile infections. Synthèse des différentes options thérapeutiques selon les présentations cliniques des infections à C. difficile. Clinical presentation of CDI

First-line treatment

Alternative therapeutics

Non severe first episode

Metronidazole 500 mg tid orally for 10 days

Severe first episode

Vancomycin 125 mg qid orally for 10 days or IV metronidazole 500 mg tid for 10 days plus intracolonic vancomycin 500 mg in 100 mL saline every 4 to12 hours and/or vancomycin 500 mg qid by nasogastric tube if oral therapy is impossible Metronidazole 500 mg tid orally for 10 days or vancomycin 125 mg qid orally for 10 days or fidaxomycin 200 mg bid 10 days Fidaxomycin 200 mg bid 10 days or vancomycin 125 mg qid orally for 10 days and pulse for 4 weeks or taper to 125 mg/2–3 days for 2–8 weeks

Vancomycin 125 mg qid orally for 10 days or fidaxomycin 200 mg bid 10 days Fidaxomycin 200 mg bid for 10 days

First recurrence

Second and later recurrences

vancomycin (500 mg in 100 mL of normal saline every 4–12 h) or vancomycin (500 mg qid by nasogastric tube) in case of severe CDI (Table 3). Therapeutic effectiveness is based on the resolution of clinical signs whatever the antibiotic used. “Test-of-cure” should be avoided. However, the most important issue is the high rate of recurrence highlighted in recent clinical trials despite adequate antibiotic therapy and the absence of resistant strains [19]. Indeed, an antibiotic treatment for an initial CDI is associated with 15 to 25% of recurrence. Moreover, the estimated effectiveness of antibiotic therapy after a first recurrence is only 60%. This suggests that therapeutic strategies may be improved especially to prevent relapse or to break the cycle of multiple recurrence. There is currently no consensus for the treatment of recurrence. Even if most recommendations suggested treating with the same agent initially used, stratified according to severity [10,14], several alternative strategies have been evaluated such as using tapered or pulsed vancomycin regimen over a 6-week period, feces infusion, probiotics, passive or active immunization (Table 3). Moreover, new antibiotics for CDI were recently labeled. All these new treatment options were reviewed in the recent ESMICD recommendations [48]. Fidaxomycin (trade name of dificlir) is a new class of narrow spectrum macrocyclic antibiotic recently introduced and could modify our therapeutic strategies. The authors of two published phase III trials reported that fidaxomycin was not inferior to vancomycin in the modified intention-to-treat and the per-protocol analysis for clinical response at the end of therapy [17,18]. The authors of the first study suggested that fidaxomycin was superior to vancomycin since there were fewer recurrences at 30 days after therapy [18]. More recently Cornely and al. suggested, for patients with cancer, that fidaxomycin treatment was superior to vancomycin, resulting in higher cure and sustained response rates, shorter time to resolution of diarrhea, and fewer recurrences [17]. Given the cost of fidaxomycin, and the limited clinical data, especially for severe CDI, it seemed important to define precisely what population could benefit from this antibiotic in first and second line therapy. Debast et al. reviewed the effectiveness of available treatments in the recent ESCMID Study Group

Fecal transplant

review on Clostridium difficile. Using fidaxomycin in case of multiple recurrences was moderately supported, with II level quality of evidence [48]. Fidaxomycin was approved for recurrence but also as the first-line treatment in France. Although each health care facility has to assess its indications, fidaxomycin is more often recommended as first line for hospitalized patients identified as at risk of recurrence and/or those with a known recent history of CDI. Other drugs (i.e. surotomycin, cadazolid, etc.) are currently being developed for the treatment of CDI. Infusion of feces from healthy donors was reported to be an effective treatment for recurrent CDI, in more than 300 patients [50]. Recently, in a small open-label, randomized, controlled trial, the authors compared three treatment regimens: infusion of donor feces preceded by a short vancomycin course and bowel lavage, a standard vancomycin regimen (500 mg/d, 14 days), and a standard vancomycin regimen with bowel lavage. Thirteen (81%) of the 16 patients in the infusion group were cured after the first infusion of donor feces. Donor feces cured 15 of the 16 patients (94%). The infection was cured in four of the 13 patients (31%) in the vancomycin-alone group and in three of the 13 patients (23%) in the group receiving vancomycin with bowel lavage. Donor-feces infusion was statistically superior to both vancomycin regimens (P < 0.01 for both comparisons after the first infusion and P < 0.001 for overall cure rate without relapse for recurrent C. difficile infection). [51]. There is a lot of evidence supporting an impaired immune response in the pathophysiological processes of CDI. Indeed, the authors of recent studies suggested a correlation between serum levels of antitoxin antibodies with protection against C. difficile infection [20,28,29]. This suggests the potential benefit of active or passive immunization. The authors of a recent randomized double-blind study suggest that the administration of fully human monoclonal antibodies favorably affected the natural history of CDI when added to metronidazole or vancomycin treatment. A single infusion of 2 monoclonal antibodies against C. difficile toxins A and B (CDA1 and CDB1) resulted in a 75%-reduction of the rate of recurrent CDI but with no impact on the resolution of current episodes [30]. An international phase-3 clinical trial is ongoing.




C. difficile toxoid vaccine was reported to induce high levels of antitoxin A antibodies with a neutralizing activity. The first assay in three patients with multiple recurrences resulted in a significant resolution of symptoms with no recurrence after a one year follow-up [31]. These first promising results should be confirmed by the results of the phase-2 clinical trial. An international phase-3 clinical trial is ongoing, to evaluate an injectable C. difficile toxoid vaccine for the primary prevention of CDI. Nevertheless, we must identify patients who will benefit from new strategies based on active immunization. Several authors have evaluated the use of probiotics to restore the intestinal microbiota. A systematic review and meta-analysis of 23 randomized controlled trials including 4213 patients yielded moderate quality evidence suggesting that probiotics are both safe and effective for the prevention of C. difficileassociated diarrhea [52]. 11. Prevention of CDI The surveillance and prevention of CDI remains, even today, an important public health concern due to the recent reemergence of CDI. Some systematic rules should be applied to avoid the spread of C. difficile. Prevention is based on contact isolation. The patient should be nursed preferably in a single bed room with dedicated equipment, gloves, and gowns. Isolation is the key measure to control C. difficile outbreaks. Hand hygiene is the second most important measure to reduce healthcare-associated C. difficile infection. Bacterial spores are not killed by alcohol, and none of antiseptic hand-wash or handrub preparations used can be reliably effective against C. difficile spores. Meticulous hand washing with soap and water is recommended for all staff after contact with body substances, or following any other potential hand contamination. The physical action of rubbing and rinsing is the only way to remove spores from hands. Health care workers should be encouraged to wear gloves when nursing patients with C. difficile infection. Wearing gowns and aprons should be encouraged to prevent contamination of occupational wear. The contamination occurs as a result of C. difficile infection, thus one of the most important measures is environmental cleaning and disinfection. Hypochlorite-based disinfectants are recommended for routine use. Hypochlorite use at a concentration of 1000 parts per million is associated with a significant reduction in the incidence of C. difficile infection. Hydrogen peroxide vapor recently proved to be effective for environmental C. difficile eradication [53]. 12. Conclusion CDI epidemiology has significantly changed over the last decade since the spread of new epidemic strains. Its incidence has increased worldwide, even if there are still great differences with higher incidences reported in countries where the frequency of testing is high. The concept of nosocomial infection should be revisited, because of the spread of CDI to the community in patients with no risk factor. Recurrence is currently the most important issue in the management of patient presenting with CDI, either for the diagnosis

11

or treatment. A better characterization of the host immune response is essential to develop a future vaccine and to develop relevant tools for an early identification of patients at risk for whom a prevention strategy based on vaccination could be proposed. The burden of CDI and their recurrence is huge including direct but also indirect extra-costs because CDI leads to a significant increase in morbidity, mortality, and length of hospital stay. The evolution of CDI epidemiology is worrying as the global population is becoming older, and new strains may emerge and spread. Disclosure of interest Alban Le Monnier was invited as auditor or participant for Astellas, Cepheid, and bioMérieux. Jean-Ralph Zahar reports no conflict of interest concerning this article. Frédéric Barbut was invited as auditor or participant, and consultant for various laboratories such as: Astellas, Sanofi Pasteur, Merck, Diasorin Cepheid, and bioMérieux. Authors’ contribution: all co-authors contributed equally to the drafting of this article. References [1] Bartlett JG. Narrative review: the new epidemic of Clostridium difficileassociated enteric disease. Ann Intern Med 2006;145(10):758–64. [2] Kuijper EJ, Coignard B, Tull P. Emergence of Clostridium difficileassociated disease in North America and Europe. Clin Microbiol Infect 2006;12 Suppl. 6:2–18. [3] Eyre DW, Cule ML, Wilson DJ, Griffiths D, Vaughan A, O’Connor L, et al. Diverse sources of C. difficile infection identified on whole-genome sequencing. N Engl J Med 2013;369(13):1195–205. [4] Rupnik M, Wilcox MH, Gerding DN. Clostridium difficile infection: new developments in epidemiology and pathogenesis. Nat Rev Microbiol 2009;7(7):526–36. [5] Voth DE, Ballard JD. Clostridium difficile toxins: mechanism of action and role in disease. Clin Microbiol Rev 2005;18(2):247–63. [6] Kelly CP, Kyne L. The host immune response to Clostridium difficile. J Med Microbiol 2011;60(Pt 8):1070–9. [7] Merrigan M, Venugopal A, Mallozzi M, Roxas B, Viswanathan VK, Johnson S, et al. Human hypervirulent Clostridium difficile strains exhibit increased sporulation as well as robust toxin production. J Bacteriol 2010;194:4904–11. [8] Eckert C, Coignard B, Hebert M, Tarnaud C, Tessier C, Lemire A, et al. Clinical and microbiological features of Clostridium difficile infections in France: the ICD-RAISIN 2009 national survey. Med Mal Infect 2013;43(2):67–74. [9] Barbut F, Mastrantonio P, Delmee M, Brazier J, Kuijper E, Poxton I. Prospective study of Clostridium difficile infections in Europe with phenotypic and genotypic characterisation of the isolates. Clin Microbiol Infect 2007;13(11):1048–57. [10] Cohen SH, Gerding DN, Johnson S, Kelly CP, Loo VG, McDonald LC, et al. Clinical practice guidelines for Clostridium difficile infection in adults: 2010 update by the society for healthcare epidemiology of America (SHEA) and the Infectious Diseases Society of America (IDSA). Infect Control Hosp Epidemiol 2010;31(5):431–55. [11] Bignardi GE. Risk factors for Clostridium difficile infection. J Hosp Infect 1998;40(1):1–15. [12] Dubberke ER, Reske KA, Olsen MA, McMullen KM, Mayfield JL, McDonald LC, et al. Evaluation of Clostridium difficile-associated disease



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An Update on Clostridium difficile Toxinotyping.

Clostridium difficile infection: A brief update on emerging therapies.

An update on antibody-based immunotherapies for Clostridium difficile infection.

Clostridium difficile infection: update on diagnosis, epidemiology, and treatment strategies.

Diagnosis of Clostridium difficile Infections in Children.

Faecal microbiota transplantation in Clostridium difficile infections.

Rational Therapy of Clostridium difficile Infections.

Clostridium difficile infections in Veterans Health Administration acute care facilities.

Clostridium difficile infection: responses, relapses and re-infections.

[Current treatment and epidemiology of Clostridium difficile infections].

Could fecal microbiota transplantation cure all Clostridium difficile infections?

Neutrophil-mediated inflammation in the pathogenesis of Clostridium difficile infections.

Using a Novel Lysin To Help Control Clostridium difficile Infections.

[Clostridium difficile infections in Internal Medicine Departments. Addendum].

Attributable inpatient costs of recurrent Clostridium difficile infections.

The prediction of complicated Clostridium difficile infections in children.

Extraintestinal Clostridium difficile infections: a single-center experience.

Assessing the burden of Clostridium difficile infections for hospitals.

Fecal microbiota transplantation in the treatment of Clostridium difficile infections.

Clostridium difficile infections before and during use of ultraviolet disinfection.

Predictors of first recurrence of Clostridium difficile infections in children.

Predictors of Clostridium difficile infections in hospitalized children.

Low vitamin D level and impact on severity and recurrence of Clostridium difficile infections.

Effects of fluoroquinolone restriction (from 2007 to 2012) on Clostridium difficile infections: interrupted time-series analysis.