Emergence of Clostridium difficile-associated disease in Canada, the United States of America and Europe.

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1 Second concept, March 3th, Emergence of Clostridium difficile-associated disease in Canada, the United States of America and Europe. Background document prepared by dr. Ed. J. Kuijper and dr. Peet Tüll on behalf of the European Study Group for Clostridium difficile (ESGCD) and European Centre for Disease Prevention and Control (ECDC). dr. Ed J. Kuijper, Medical Microbiologist, Leiden University,Leiden, The Netherlands prof. Ian Poxton, Medical Microbiologist, University of Edinburgh, Edinburgh, UK dr. Jonathan Brazier Medical Microbiologist, Univ. Hospital of Wales, Cardiff, UK prof. Brian Duerden Medical Microbiologist, Department of Health, London, UK prof. Michel Delmée Medical Microbiologist, Univ. Cat. de Louvain, Bruxelles, Belgium dr. Petra Gastmeier Medical Epidemiologist, Hannover, Germany dr. Frédéric Barbut Medical Microbiologist, Hôpital-Saint-Antoine, Paris, France dr. Bruno Coignard Medical Epidemiologist, Saint-Maurice Cedex, France dr. Maja Rupnik Microbiologist, Maribor, Slovenia dr. H. Pituch Medical Microbiologist, Warsaw, Poland dr. Carl Suetens Medical Epidemiologist, Sc.Inst.of Public Health, Brussels, Belgium dr. Marco Baldari Senior Expert, ECDC, Stockholm, Sweden dr. Peet Tüll Senior Expert, ECDC, Stockholm, Sweden Advisors: dr Clifford McDonald, epidemiologist, CDC, Atlanta, Georgia, USA Ing. Tjallie van der Kooi, dr. Susan van den Hof and dr. Daan W. Notermans; Center for Infectious Disease Control, National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands. prof. dr. Elisabeth Nagy; Anaerobe Reference Laboratory of Hungary, Szeged, Hungary dr. Alaric Colville, Royal Devon and Exeter Hospital NHS Foundation Trust, Exeter, UK. 1

2 Content 1. Clostridium difficile-associated disease (CDAD); an increasing healthcare threat; pages Emergence of Clostridium difficile NAP1/027 in Canada and USA ; pages Emergence Clostridium difficile 027 in Europe (UK, The Netherlands, Belgium); pages Characteristics of Clostridium difficile PCR ribotype 027, PFGE type NAP1, toxinotype III; pages Implications for Europe; pages a. Clinical awareness of CDAD due to Clostridium difficile in all European member states. b. The variety of methods and strategies used for diagnosing CDAD underlines the need for guidelines. c. A proposal for CDAD case definitions d. Investigation and reporting of outbreaks on national and European level e. The incidence of CDAD in the member s states of EU is largely unknown. f. The role of the epidemiologists, medical microbiologists, infectious disease specialists and infection control practitioners in combatting CDAD. 6. References; pages

3 1. Clostridium difficile-associated disease (CDAD); an increasing healthcare threat The clinical spectrum of CDAD ranges from mild diarrhoea to severe life threatening pseudomembranous colitis. The disease is not always associated with previous antibiotic use. There is an increase of reports of community-acquired CDAD in individuals previously not recognized as predisposed. CDAD is also recognised increasingly in a variety of animal species. Clostridium difficile is an anaerobic bacterium, widely distributed in soil and intestinal tracts of animals. Its vegetative cells are capable of forming spores, which confer resistance to heating, drying and chemical agents including disinfectants. C. difficile was identified as the cause of pseudomembranous colitis and its milder form, C. difficile-associated diarrhoea, in the 1970s. The spectrum of disease ranges from asymptomatic carriage to a fulminant, relapsing, and potentially fatal colitis (8). Mortality of C. difficile-associated disease (CDAD) ranges from 6% to 30% when pseudomembranous colitis is shown to be present, and is substantial even in the absence of colitis (8). C. difficile also appears to be an important cause of enteric disease in a variety of animal species, including horses, dogs, cats, birds, rodents and especially in neonatal pigs (61). These results suggest that animals may serve as a reservoir for humans. The typical manifestations of CDAD are abdominal cramps, profuse diarrhoea consisting of mucoid, greenish, foul smelling and watery stools, low grade fever and leukocytosis (8). These can start a few days after antibiotic therapy is begun, or, in 20% of cases, up to 8 weeks after its discontinuation. Although colitis can occur throughout the colon, it is usually more severe in the distal colon and rectum. However, when patients develop colitis of the caecum and right side of the colon, they may have little or no diarrhoea. The clinical presentation then consists of fever, marked right sided abdominal pain, marked leukocytosis, and decreased intestinal motility. This presentation is not rare, but is especially serious because treatment is delayed due to the lack of diarrhoea. It seems to occur more frequently when anti-peristaltic agents or opiates have been administered. Traditionally CDAD has been considered as an antibiotic-associated nosocomial infection, but the role of antibiotics as predisposing factors for CDAD can be overestimated (13,17). Patients can develop CDAD after all kinds of conditions which have significant effects on the colonic flora, also outside hospitals. Community-acquired CDAD is increasingly recognised. Reports of the American Centers for Disease Control and Prevention (CDC) mention an increase of severe community-acquired CDAD in populations previously considered to be at low risk in Philadelphia and four surrounding counties (13). Of the patients who developed CDAD, 30% had no previous use of antibiotics. The minimum annual incidence of community-associated disease was estimated as 7.6 cases per 100,000 population or one case per 5,000 outpatient antimicrobial prescriptions. A recent report from the UK indicated that the incidence of C. difficile in patients diagnosed by their general practitioners had increased from fewer than one case per 100,000 in 1994 to 22 per 100,000 in 2004 (17). 3

4 The transmission of C. difficile can be patient-to-patient, via contaminated hands of healthcare workers or by environmental contamination. The role of symptom-free colonised healthcare workers and patients with C. difficile in the intestinal tract is unclear. There is a divergence of opinion on whether detergents or disinfectants should be used for routine hospital cleaning. Some evidence suggests that hypochlorite environmental cleaning may reduce CDAD. C. difficile can be cultured from the stool of 3% of healthy adults and up to 80% of healthy newborns and infants (8). The assumption that C. difficile is not pathogenic for neonates and children needs some reconsideration, since it is mainly based on anecdotal evidence. Stool carriage of C. difficile reaches 16 35% in hospital inpatients, with the percentage proportional to the duration of hospital stay and increasing with exposure to antibiotics. C. difficile persists in the stools of 10-40% of patients with CDAD, irrespective of the antibiotic used for treatment (42). The asymptomatic carrier state is probably the end result for most patients infected with C. difficile. Contaminated environmental surfaces, patients with CDAD and healthcare personnel hand carriage are considered to be potentially important vectors for C. difficile transmission in hospitals. Patients with CDAD are nursed in side rooms to minimize the spread of C. difficile. Diarrhoea can often be unexpected and explosive, and results in increased shedding of C. difficile spores. Consequently, spores have been found in far greater quantities in the environment around CDAD patients in comparison with those not infected with C. difficile (28, 42). C. difficile spores are highly resistant to many commonly used disinfectants and may persist for many months in hospital ward environments (28). It is not known if healthcare workers and patients with symptomfree colonization by C. difficile in the intestinal tract spread the bacterium. The frequency of C. difficile-positive healthcare personnel hand culture has been shown to correlate significantly with the intensity of environmental contamination. Clearly, good hand hygiene practice with water and soap (not alcohol hand gels) is essential in reducing hand carriage. The true significance of the environment as a potential reservoir for C. difficile and its role in subsequent patient infection remains unclear, primarily because it has been difficult to determine whether environmental contamination is a cause, or a consequence, of diarrhoea. Environmental contamination includes mainly floors, commodes, toilet floors and bed frames (69,72). The bed frame was found to be the most common site from which C. difficile was recovered, although the floor was the most contaminated site in terms of total numbers of colonies (69). There is a general lack of evidence about whether detergents or disinfectants should be used for routine cleaning in patient areas. Routine cleaning with detergent is often unsuccessful in removing C. difficile from the environment (72). There is evidence that contamination with C. difficile may still persist after environmental cleaning with hypochlorite. Kaatz et al. recovered C. difficile from 31% of ward environmental samples (26). Following disinfection with unbuffered hypochlorite (500 ppm available chlorine), surface contamination decreased to 21% of initial levels, and this coincided with the end of an outbreak of CDAD. Phosphate-buffered hypochlorite (1600 ppm available chlorine, ph 7.6) was found to be more effective at reducing environmental C. difficile levels (98% reduction in surface contamination). The results of another study found that unbuffered 1:10 hypochlorite solution was effective in decreasing patients' risk of developing CDAD (38). However, whether longterm environmental use of hypochlorite is sustainable given its corrosive nature is uncertain. The lack of sporicidal but environmentally friendly disinfectants is a problem. At ICAAC 2005, preliminary findings of a prospective study on the effect of hydrogen peroxide vapour (Bioquell, UK) demonstrated efficacy for the eradication of C. difficile in the environment in four intensive care wards (JC Boyce et al). 4

5 Pathogenic Clostridium difficile has a pathogenicity locus (PaLoc) with genes encoding enterotoxin A (TcdA) and cytotoxin B (TcdB). Genes for the binary toxin are located outside the PaLoc but the role of this toxin is unclear. C. difficile is divided into more than 150 PCR ribotypes and 24 toxinotypes. Host resistance to CDAD is associated with an acquired immune response to toxins A and B. Pathogenic C. difficile organisms release two potent toxins that ultimately mediate diarrhoea and colitis (8). These large exotoxins - toxin A, a 308-kd enterotoxin, and toxin B, a 269-kd cytotoxin, exhibit an overall homology of greater than 45% at the amino acid level. Most enteropathogenic strains produce both toxins simultaneously. It is suggested that toxins A (TcdA) and B (TcdB) work synergistically, based on the fact that a TcdB effect is dependent on tissue damage by TcdA. TcdA has been regarded to be the most important factor for causing diarrhoeal disease, but an increasing number of reports show disease caused by TcdA-negative strains, thereby implying a more important and TcdA-independent role for TcdB in pathogenesis. In recent years, epidemics have been described due to TcdAnegative strains (2, 32). Additionally, a recently discovered new binary toxin of C. difficile is currently being studied as a possible new virulence marker (15, 54). This binary toxin, an actin specific adenosine diphosphate ribosyltransferase, can be present in 4% of C. difficile strains (59). The binary toxin is encoded by the cdta gene (the enzymatic component) and the cdtb gene (the binding component) (54). Whether this toxin contributes to the pathogenicity of C. difficile is unknown; however, the C. difficile strain in which the binary toxin was first detected caused severe pseudomembranous colitis. In a very recent publication, binary toxin genes were present in nearly two-thirds of the C. difficile strains (41). C. difficile can be divided in more than 150 ribotypes and 24 toxinotypes (57, 68, Toxinotyping involves detection of polymorphisms in the toxin A and B and surrounding regulatory genes, an area of the genome known collectively as the pathogenicity locus or PaLoc (11). PCR ribotyping is based on differences in profiles generated by PCR amplification of the intergenic spacer regions between the 23S and 16S rrna genes (68). Diagnostic assays can be divided into test for the detection of C. difficile products, tests for the detection of C. difficile genes, and culture methods for isolation of a toxinproducing bacterium. Despite its low sensitivity and practical drawbacks, the cell cytototoxicity assay is still considered as the gold standard. There is no consensus on the optimal test strategy. Rapid diagnostic assays are available on a limited scale and need much improvement. Molecular tests are of special interest, since they also enable the detection of carriers of toxigenic and non-toxigenic strains. The diagnosis of CDAD requires the detection of C. difficile toxins or toxin producing C. difficile in diarrhoeal stool specimen. Diagnostic assays can be divided into test for the detection of C. difficile products (e.g glutamate dehydrogenase, volatile fatty acids, toxins), tests for the detection of C. difficile genes (16S rrna, toxin genes), and culture methods for isolation of a toxin-producing bacterium (16). A European survey of diagnostic methods for C. difficile revealed marked discrepancies between laboratories and between countries in the methods and strategies that are used for the diagnosis of C. difficile (6). Culture of toxigenic C. difficile takes at least 4 days before results are available and has, therefore, no rapid diagnostic value. As non-toxigenic strains exist, cultured C. difficile must be tested for toxin production. For typing, cultured isolates are necessary. Toxins of C. difficile can be detected either by virtue of their biological properties (cell cytotoxicity assay) or by immunological methods (latex agglutination, immunoassay). The cell cytotoxicity test remains the gold standard by which other tests are measured (8, 16, 20, 46, 63, 64) but suffers from some drawbacks. The laboratory requires a supply of cultured cell monolayers, the results are 5

6 known to vary according to the cell line, dilution factors, reagents used and storage conditions. Additionally, the turnaround time is very slow, typically 24 hours to demonstrate cytotoxicity and a further 24 hours to neutralise this cytotoxicity. Enzyme immunoassays are easier to perform and provide rapid results. Two types of immunoassay have been developed: conventional enzyme immunoassays and a membrane immunochromatography test. There are numerous publications comparing the performance of different kits for enzyme immunoassays, but no meta-analysis has been performed in an attempt to demonstrate the superiority of any particular test. The National C. difficile Standard Group in England recommend the use of EIAs that detect both toxin A and toxin B, because of an increasing awareness of toxin A-negative/toxin B-positive strains (2, 32, 46). A second generation of tests has been put on the market recently. The principal advantage of the membrane immunochromatography assay is speed, since results can usually be obtained within minutes. Another advantage is the simplicity of the assay, which does not require high technical skills. Recently, a new rapid immunoassay (Immunocard toxins A and B, Meridian) has been compared with an in-house real-time PCR in a prospective multicentre study with the cytotoxicity test as the gold standard (63). It was concluded that the new rapid immunoassay is a quick and easy-to-perform test for the diagnosis of CDAD, but that the test performances should be improved. The detection of C. difficile gene sequences in stool samples has focused on 16S rrna and toxin genes (9, 30). An important disadvantage of the 16S rrna approach is that non-toxigenic as well as toxigenic strains are detected. Therefore, more attention was given to the TcdB gene of C. difficile and a successful approach was published in 1993 (27, 31). Real-time PCR results can be obtained within a working day, which is shorter than for the cytotoxicity assay. The hands-on time is highly reduced compared to other methods as well, negating the use of post-pcr analysis. Other advantages of real-time PCR are the low risk of carryover contamination and that it will also detect asymptomatic carriers. The impact of CDAD on modern healthcare is significant. In terms of costs, this translates into 5-15,000 per case in England and $1.1 billion per year in the USA. Assuming a population for the European Union of 457 million, CDAD can be estimated to be potentially costing the Union 3,000 million per annum. It is expected to almost double over the next four decades. C. difficile associated disease (CDAD) is primarily a nosocomial disease which can be prevented by robust infection control practice. CDAD is currently the most frequently occurring nosocomial infection in many European hospitals, exceeding infections caused by methicillin resistant Staphylococcus aureus (MRSA). In the UK, for instance, in 2003 twice as many deaths were attributed to C. difficile as to MRSA. The organism is resistant to various antibiotics and capitalizes on the ensuing disruption of the normal intestinal flora to colonize and cause disease. Several factors have contributed to the worrying escalation in the incidence of CDAD. The elderly and immuno-compromised are particularly at risk (80% of cases occur in those over 65 years old). The proportion of the population in these high-risk groups is rising rapidly. Of concern has been the gradual development in Spain over the past few years of clones with increased resistance to metronidazole or vancomycin (49, 50). Very recently, metronidazole resistance has also been observed in Israel (10). The impact of CDAD in healthcare settings is considerable. Patients require isolation, revised supportive therapy for underlying disease and for CDAD, specific therapy to eliminate C. difficile, scrupulous hygiene in nursing, environmental decontamination, and (in outbreaks) cohort isolation and ward closure. Reports indicate that cases spend from 1 to 3 extra weeks in hospital. In terms of costs, this translates into 5-15,000 per case in England and $1.1 billion per year in the United States (35, 44, 56, 71). Taking the latest incidence figures for England (44,488 in 2004[DN: this is the figure from mandatory surveillance in England]), and allowing for a 3% annual inflation since 1996, the management of CDAD can be estimated to 6

7 be costing the NHS in England 340 million per annum. Assuming a population for the Europe Union of 457 million, CDAD can be estimated to be potentially costing the Union 3,000 million per annum. Moreover, as CDAD is predominantly a disease of the elderly (>80% of cases are over 65 years old), these costs are set to rise as the proportion of the European population over 65 years old increases. Thus, the latest figures from Eurostat (the statistical bureau for the European Union) estimate that the proportion of older people (aged 65 years and over) is expected to almost double over the next four decades, from 75.3 million in 2004 to million in Emergence of Clostridium difficile NAP1/ 027in Canada and the USA Since March 2003, increasing rates of CDAD have been reported in Canada and USA with a more severe course, higher mortality, and more complications. This increased virulence is assumed to be associated with higher amounts of toxin production by fluoroquinolone-resistant strains belonging to PCR ribotype 027, toxinotype III and PFGE NAP1. The rate and severity CDAD is increasing both in the USA and Canada. This increase may be associated with a new strain of C. difficile marked by increased virulence and/or resistance. Since March 2003, outbreaks of severe cases of CDAD have been reported in hospitals in Montreal and southern Quebec (51, 53). In 2004, institutions in the region of Quebec experienced a sharp rise in CDAD incidence involving more than 14,000 patients with CDAD (19, 45, 53). In January 2005, 30 hospitals in Quebec reported rates of nosocomial CDAD five-fold greater than their historical average. A new, more virulent variant (ribotype 027) strain was associated with this increase. This prompted the government of Quebec to reserve 13.7 million to combat CDAD in hospitals and nursing homes (18). A study conducted in 2004 at 12 hospitals in Quebec included a total of 1,703 patients with 1,719 episodes of nosocomial CDAD (37). The calculated incidence was 22.5 cases per 1,000 admissions with a 30-day attributable mortality rate of 6.9%. Both incidence and mortality increased with increasing age. Compared to matched controls, patients with CDAD were more likely to have received fluoroquinolones (odds ratio, 3.9; 95% CI ) or cephalosporins (OR, 3.8; 95% CI ). The most common strain, which was resistant to fluoroquinolones, was identified in 82.2% (129/157) of patients. In addition, 84.1% (132/157) of isolates had binary toxin genes as well as partial deletions of the tcdc gene (37). The CDC also reported a growing threat of CDAD in hospitals in the USA and found the strain associated with high morbidity and mortality during outbreaks in hospitals in 11 states (41). CDC also reported a steady increase from 2.7 per 10,000 hospital days in 1987 to 4.2 in 2001 and advised on standardization of definitions and notification of CDAD (5, 39). USA hospital discharges for which CDAD was listed as any diagnosis doubled between 1996 and 2003 (40). A study of isolates from the USA found a previously uncommon strain of C. difficile, resistant to fluoroquinolones and also manifesting genetic variation, was causing disease in geographically dispersed outbreaks (41). The USA study collected 187 C. difficile isolates from eight health care facilities in six states in which CDAD outbreaks had occurred between 2000 and At least half of the specimens from five of the eight facilities belonged to REA group B1 and PFGE type NAP1. B1/NAP1 isolates were positive for the CDT binary toxin, had a deletion in the tcdc locus and produced higher amounts of toxins A and B(41). Resistance to moxifloxacin and gatifloxacin was more common among B1/NAP1 C. difficile isolates than in other types (100% versus 42%, P<0.001). None of the historic B1/NAP1 isolates were resistant to moxifloxacin and gatifloxacin. Furthermore, reports from CDC mention an increase in severe community-acquired CDAD in populations previously 7

8 considered to be at low risk in Philadelphia and four surrounding counties (13). The minimum annual incidence of community-associated disease was estimated as 7.6 cases per 100,000 population or 1 case per 5,000 outpatient antimicrobial prescriptions. 3. Emergence Clostridium difficile PCR ribotype 027 in Europe (UK, Belgium, The Netherlands). Epidemics of CDAD due to the new, highly virulent strain of C. difficile PCR ribotype 027 have been recognized in 44 hospitals in England, eight hospitals in The Netherlands and six hospitals in Belgium. Retrospectively, PCR ribotype 027 was shown to have already caused outbreaks in 2002 in all three countries. The outbreaks are very difficult to control. Preliminary results from case-control studies indicate a correlation with the use of fluoroquinolones and cephalosporins. No information is available on community-acquired cases of type 027. Data on the incidence of type 027 in nursing homes are limited, but at least one outbreak has been detected. The Communicable Diseases Surveillance Centre (CDSC) for England and Wales had noticed that the number of CDAD reports had risen from 1,000 in 1990 to 15,000 in 2000 and 35,500 in2003, with PCR ribotype 027 being very rare. In the period February to June 2004, the PCR ribotype 027 strain was recognized in the UK in an outbreak involving 150 patients with 12 deaths at the Stoke Mandeville Hospital (3, 60). Shortly thereafter, the Royal Devon and Exeter Hospital submitted 18 representative isolates of C. difficile with a history of an outbreak that coincided with a change of antibiotic regimen for treatment of patients with community acquired pneumonia to moxifloxacin; almost all were type 027. Up to February 2006, 302 isolates of type 027 have been referred to the Anaerobe Reference Laboratory in Cardiff from 50 hospitals. Some were from clinically recognised outbreaks, others from the routine submission of if isolates as part of the mandatory surveillance of CDAD in England. In July 2005, the medical microbiological laboratory at the Leiden University Medical Center was requested to type C. difficile strains from an outbreak in a hospital in Harderwijk (33, 67). The incidence of CDAD in the hospital had increased from 4 per 10,000 patient admissions in 2004 to 83 per 10,000 in the months April to July Cultured isolates were subsequently identified as toxinotype III and PCR ribotype 027. Measures taken by the hospital included isolation of all patients with diarrhoea until twice tested C. difficile toxin negative, cohorting of all C. difficile-infected patients on a separate ward, banning all fluoroquinolone use and limiting the use of cephalosporins and clindamycin. In January 2006, the situation appeared to be under control as the number of positive patients per month had decreased. All nine patients with CDAD diagnosed from September 2005 until January 2006 were caused by non-027 ribotypes. However, a resurgence of CDAD due to type 027 was noticed in February 2006, when six new patients were diagnosed. A second epidemic occurred in another hospital 30 km from the first hospital and was probably related to the first outbreak through a transferred patient with CDAD (33, 67); 85 CDAD patients were identified to December 2005, 19 (22%) patients died and 16 (19%) relapses were observed. In response to the outbreaks in the Netherlands, the Center for Infectious Disease Control (CIb) at the National Institute for Public Health and the Environment (RIVM) in Bilthoven organized a meeting with experts in the fields of microbiology, infectious diseases, infection control, and epidemiology. The team agreed to bundle parts of existing national hospital guidelines relevant for infection control of CDAD, and to use national and international experience in drawing up specific CDAD guidelines for infection control and treatment, separately for hospitals and nursing homes. Furthermore, diagnostic facilities were increased and made accessible for all microbiology laboratories in The Netherlands. Relevant professionals were informed through several communication channels including the various scientific societies. Plans were made to register and monitor new outbreaks. Subsequently, three hospitals in the Western part of the country also reported an increase of the incidence of severe CDAD. A 8

9 nursing home in the same region was also found to harbour patients with CDAD due to PCR ribotype 027, with evidence of spread within the facility. A cluster of 12 patients with CDAD caused by PCR ribotype 027, toxinotype III was reported in July and August in a large teaching hospital in Amsterdam. One patient died due to the consequences of CDAD and two other patients developed severe complications (29). Another hospital in Amsterdam also reported an increase of severe cases of CDAD in July The patients were nursed at the Department of Geriatrics. Two hospitals in the centre of the Netherlands did not notice an increase in the incidence of patients with CDAD, but submitted strains to the reference laboratory for typing. Type 027 was found in 6 of 17 (35%) and 1 of 4 (25%) isolates tested, respectively (34). In September 2005, the PCR ribotype 027 strain was isolated from four patients with CDAD in Ieper, Belgium (25). The incidence had increased from 10/10,000 admissions to 33/10,000 admissions in September Subsequently, the same pattern was identified among strains from three outbreaks that had occurred in Brussels in involving 17, six and five patients, respectively. In Ostende (Belgium) another outbreak was found with four patients. More recently, 14 strains from a severe outbreak (still ongoing) in the Hospital of Libramont (south of Belgium) were identified. 4. Characteristics of Clostridium difficile PCR ribotype 027, PFGE type NAP1, toxinotype III. The epidemic strain isolated in Canada, USA, UK, Belgium and The Netherlands was characterised as toxinotype III, North American PFGE type 1, restriction-endonuclease analysis group type BI and PCR-ribotype 027. This strain carried the binary toxin gene cdtb and had an 18-bp deletion in tcdc. Strains of toxinotype III have been found sporadically and represent 2-3 % of isolates from large collections (21, 57, 58). Strains belonging to toxinotype III produce binary toxin in vitro, but the importance of binary toxin CDT as a virulence factor in C. difficile has not been established (54). The binary toxin, an actin specific adenosine diphosphateribosyltransferase, is encoded by the cdta gene (the enzymatic component) and the cdtb gene (the binding component) which are not located within the pathogenicity locus (15,54). Non-pathogenic strains containing cdta and cdtb genes but lacking the pathogenicity locus are also capable of producing binary toxin. The binary toxin is present in about 6% of all C. difficile isolates, irrespective of the toxinotype (15, 54, 59). The application of restriction analysis as a typing technique for C. difficile was published in 1987 and subsequently standardized in 1993 (12, 31). The REA patterns are visually compared to the restriction patterns of the reference REA types. At least 75 groups have been recognized by REA and the eight most common REA groups are B, G, L, E, S, N, AL and BD. REA group type BI, which was first identified in 1984, was uncommon (n=18) among isolates from the historic database ( ) of 6,000 isolates. PCR ribotype 027 was first assigned in 1988 from a culture collection of prof. M. Popoff (Paris) and originated from a 28-year-old woman with severe pseudomembranous colitis. Until March 2004, is was considered to be an unimportant and very rare PCR ribotype. The clonality of PCR 027 is currently a topic of research. PCR-ribotype 027 exhibits two PFGE patterns with 94% similarity (41); North American PFGE type 1a and 1b (NAP1a and NAP1b). As demonstrated for PCR ribotype 001, other typing techniques (DNA fingerprinting, REA, AP-PCR) may reveal additional subgroups. Preliminary data from Dale Gerding (Chicago, USA) indicate that REA is able to discriminate further at least 24 subtypes of BI within PCR ribotype 027. Currently, a large collection of strains from Canada, UK, USA and the Netherlands are being subjected to various typing techniques. 9

10 The importance of the 18-bp deletion in tcdc of the PCR ribotype 027, toxinotype III strains is also unknown. TcdC is considered to be a negative regulator of the production of toxins A and B, but it is not known whether this 18-bp deletion results in a nonfunctional product. A recent report, however, indicates that toxinotype III isolates produce toxins A and B in considerably greater quantities in vitro than toxinotype 0 isolates (70). On the other hand, deletions in tcdc are frequently present in toxigenic isolates. Of 32 toxigenic strains studied in 2002, eight belonged to toxinotypes 0, V and VI and contained deletions in tcdc of 18 bp or 39 bp without an association with clinical severity of disease (62). Little information is available on the sporulation characteristics and spread of NAP/027 into the environment in comparison with other types. At ICAAC 2005, one poster (LB : S. Underwood et al.) reported that sporulation levels of outbreak type 027 strains exceeded those of other strains. The highest sporulation levels were achieved when strains were exposed to non-chlorine-based cleaning agents. 5. Implications for Europe a. Clinical awareness of CDAD due to Clostridium difficile in all European Member States. CDAD has a broad clinical spectrum and is not always associated with previous antibiotic use. Although CDAD presents most frequently as a hospital acquired/ nosocomial infection, recent reports indicate an increase of community acquired CDAD in populations not considered at risk. Therefore, knowledge of CDAD is of importance for all health care workers. There exists a severe underestimation of CDAD in Europe, due to lack of awareness by physicians and lack of standardized diagnostic strategies (6). The spectrum of CDAD ranges from asymptomatic carriage to a fulminant, relapsing, and increasingly fatal colitis. The association of CDAD with antibiotic use may be overestimated, since it also occurs in patients without previous antibiotic treatment. Additionally, reports of CDC mention an increase of severe community-acquired CDAD in populations previously considered at low risk in Philadelphia and four surrounding counties (13). Physicians should consider the diagnosis of CDAD also in patients without traditional risk factors. b. The variety of methods and strategies used for diagnosing CDAD underlines the need for guidelines. Guidelines for CDAD diagnostic strategies should be formulated based on regional incidence rates of CDAD and local laboratory capacities. There is an urgent need for rapid diagnostic tests with a better performance than the currently available assays. A European surveillance study of diagnostic methods and testing protocols for C. difficile among 212 hospitals in eight countries in 2002, revealed marked differences between laboratories concerning the methods and the strategies that are used for diagnosing CDAD (6). In 58% of the cases, laboratories only undertook investigations for CDAD when specifically requested by the physician and only 55% of the laboratories were capable of culturing for C. difficile. These results are also in agreement with the observations of a 10

11 survey performed in the UK by the Healthcare Commission and Health Protection Agency among 118 acute NHS Trusts in This survey was conducted to improve knowledge on the reported incidence of CDAD, the approaches to prevent, manage and control outbreaks, and the views of professionals on what should be done for prevention. Secondly, the survey was undertaken to obtain information regarding diagnostic testing and processing of samples from suspected CDAD cases. In 2004, guidelines and recommendations were published by the National Clostridium difficile Standards Group and mandatory surveillance was introduced in 2004 in which were required to report infections in patients aged 65 years and over (4, 46). All laboratories were using a recommend test for diagnosing CDAD, but only 25% performed C. difficile culture. There was considerable variation in the use of culture and typing by different laboratories. As part of the mandatory surveillance, from January 2005 all laboratories were required to submit isolates of C. difficile for typing in a structured programme. During one week each year, in a rolling programme, a laboratory is asked to send toxin-positive stool samples (up to a maximum of 10) to the Regional Laboratory of the Health Protection Agency. Culture of the samples is performed at the Regiona Laboratory and the isolates of C. difficile are sent to the Anaerobe Reference Laboratory (Cardiff) for typing. In The Netherlands, a survey conducted among 12 laboratories also revealed marked discrepancies regarding the methods and the strategies that are used for diagnostic tests. Subsequently, in a 3-month pilot study using an optimal test algorithm at four university laboratories, a 50% increase in the number of diagnosed CDAD patients was found (manuscript submitted). This algorithm enabled the microbiological laboratories to test all faecal specimens of patients hospitalized more than 3 days who developed diarrhoea, irrespective of the physician s request. This algorithm has been introduced now in 15 laboratories. c. A proposal for CDAD case definitions (B. Coignard and F. Barbut). There is no consensus for a CDAD case definition. We therefore propose case definitions, which are based on past experiences and especially on those from the USA, Canada, UK and The Netherlands. These definitions focus on disease, and do not refer to a particular strain. Consensus on case definitions is the first step to be implemented, in order for European hospitals and countries to assess better and monitor the burden of CDAD and to compare themselves with each other. Case definition for CDAD - Criterion 1: - diarrhoeal stools 1 or toxic megacolon, AND - a positive assay for C. difficile toxin (either an immunoassay detecting toxin A or B, or a neutralised cell cytotoxicity assay), OR a positive stool culture with a toxigenic strain. 2 - Criterion 2: pseudomembranous colitis observed on lower gastrointestinal endoscopy. - Criterion 3: colonic histopathology characteristic of C. difficile infection (with or without diarrhoea), on a specimen obtained during endoscopy, colectomy or autopsy. 1 In a laboratory-based surveillance scheme: unformed stools, i.e., stools that take shape of their container; in a patient-based surveillance scheme: at least three liquid or unformed stools for at least 24 hours. 2 Toxigenic strains are defined as isolates capable of producing toxin A (TcdA) and/or toxin B (TcdB). The presence of binary toxin is excluded from this definition. 11

12 - Criterion 1 can be used in laboratory-based surveillance systems; criteria 1 to 3 can be used in patient-based surveillance systems. - Excluded from the above definitions: - Asymptomatic patients with a positive stool culture for C. difficile, - Asymptomatic patients with a positive assay for C. difficile toxin, - Neonates, - Diarrhoea associated with another cause, as diagnosed by the attending physician, - Recurrences Definition of CDAD recurrence (7, 24, 48, 52, 73) Two episodes of CDAD in the same patient are considered to be distinct events if they occur >2 months apart; an episode that occurs within 2 months of a prior episode (i.e., there is a return of symptoms less than 2 months after the end of the treatment) is considered to be a recurrence of the initial one. A recurrence can correspond either to a relapse with the same strain or to a re-infection with a different strain. 3 It is not possible in clinical practice to differentiate between these two mechanisms, and the term "recurrence" is used as a designation for both. 4 Definition of a severe case of CDAD A severe case of CDAD can be defined as a case-patient who: - is admitted to an ICU for CDAD (e.g., for shock requiring vasopressor therapy); - OR underwent surgery (colectomy) for megacolon, perforation or refractory colitis; - OR is readmitted for CDAD ; - OR died within 30 days after CDAD diagnosis, if the death can be associated with CDAD, i.e. : - CDAD is the primary (attributable) cause of death, - OR CDAD is a contributary cause of death. The severity of CDAD also can be graded (14): Severity of disease Criteria Mild Moderate Severe # stools / day Fever None 37,5 C 38,5 C 38,6 C Leukocytosis None x 10 6 /l 20 x 10 6 /l Abdominal None Severe abdominal pain Symptoms of peritonitis symptoms Complication None Lower gastro-intestinal haemorrhage (with stable blood pressures) The origin of CDAD can be defined as follows (22): - Lower gastro-intestinal haemorrhage (with unstable blood pressures) - Perforation of the colon - Sepsis due to colitis - Renal dysfunction 3 Even the definitions of relapse vs. reinfection are questionable. (ren) describes the simultaneous occurrence of multiple PCR ribotypes in 10-20% of faecal samples. It is therefore better, for surveillance purposes, to only use the term "recurrence". 4 ref. 14 uses an 8-week cut-off. Ref 53 used a 3-month cut-off. Ref 52 uses the term recurrence and a 2- month cut-off. 12

13 - Healthcare-associated, nosocomial: - CDAD in patients with onset of symptoms occurring at least 72 hours after admission, or within 4 weeks 5 after discharge. - Healthcare-associated, imported from another institution: - CDAD in hospitalized patients within 72 hours after admission or in outpatients, - AND a history of hospitalization or ambulatory care (dialysis, ambulatory surgery, ambulatory medical care, intravenous therapy) in the preceding 4 weeks. - Community-acquired : - CDAD in outpatients or in hospitalized patients within 72 hours after admission, - AND no history of hospitalization or ambulatory care in the preceding 4 weeks ; - Unknown: cases that cannot match the above definitions. Note: if a patient is diagnosed with CDAD and has a history of stay in another hospital with epidemic forms of C. difficile PCR ribotype 027, it is advised to request a stool culture for this patient in order to type the strain and identify this particular one. d. Investigation and reporting of outbreaks on national and European level. European countries first should develop early warning and response capability. In addition, and depending on the nature of the notifications they received, countries will implement laboratory-based or patient-based surveillance systems in specific, targeted populations. The above definitions can be used when implementing CDAD surveillance in specific populations: as in the UK, they can be restricted to patients over 65 years old, regardless of the presence or absence of any specific risk factors (e.g., prior antimicrobial therapy). However, implementing comprehensive, systematic surveillance systems at the national level in all European countries will require some time. In order to detect severe CDAD cases related to the PCR ribotype 027 strain of C. difficile, European countries first should develop early warning and response capability, and therefore encourage hospitals to notify to regional or national public health authorities: - Severe cases of CDAD, as defined above; - Outbreaks of CDAD, i.e., a significant increase in CDAD incidence over a defined time period in a facility, taking account of the background rate. This concept underlines the importance of making basic CDAD surveillance data available. - An outbreak can be defined as the occurrence of two or more related cases of CDAD over a defined period agreed locally, taking account of the background rate (46). - Only nosocomial cases, i.e., the ones reflecting cross transmission in the facility, should be included in this analysis (22, 47). In addition, and depending on the nature of the notifications they received, countries will implement laboratory-based or patient-based surveillance systems in specific, targeted populations (e.g., patients in hospitals, patients in nursing homes, etc.) 5 Ref 22 used an 8-week cut-off before the June 2005 surveillance report. Ref. 13 mentions a 3-month cut-off. 13

14 e. The incidence of CDAD in the Member States of EU is largely unknown. In order to gain insight in the prevalence of CDAD due to C. difficile 027 in 12 European Member States, the currently performed surveillance study of ESGCD should be completed as soon as possible. This study will also demonstrate the prevalence of the most frequently occurring PCR ribotypes and characterization of the strains for specific virulence markers and antimicrobial susceptibility patterns. A new surveillance study should be developed in which all European Member States participate and in which the incidence of CDAD will be determined in hospitals, long-term care facilities and in the community. Limited information on the incidence of CDAD is available from a European survey performed among 212 hospitals by the ESGCD in UK, France, Belgium, Denmark, Germany, Italy and Spain in 2002 (6). The incidence varied considerably, dependingt on the test strategies and tests applied. The incidence was 11 per 10,000 admissions. In contrast, data from studies in the USA showed that the incidence among hospitalized patients is much higher, ranging from 10 to 200 per 10,000 admissions (36). A second European surveillance study has been performed in 2005, but the financial budget to complete this study has not been found. In the USA, the number of hospital discharge diagnosis of CDAD during 2001, 2002 or 2003 exceeded the estimated annual number of methicillin-resistant S. aureus infections (40). The impact of CDAD on modern healthcare is significant and reports indicate that cases spend from 1 to 3 extra weeks in hospital. In terms of costs, this translates into 5-15,000 per case in England and US $1.1 billion per year in the USA. Reporting of cases in all EU Member States is on a voluntarily basis, except in England where mandatory reporting of cases was introduced in The first set of results from the mandatory surveillance scheme showed there were 44,488 cases of C. difficile reported in those over 65 years old in England during 2004 (4). Two thirds of the Trusts reported an increase of CDAD cases during the past 3 years and 25% have closed a ward during the past 12 months for CDAD care. It is very likely that these data underestimate the real incidence of CDAD, since the surveillance studies were restricted to patients over 65 years old and to nosocomial acquired CDAD. Information is lacking on the extent of CDAD in nursing or residential homes, in other health care facilities and in patients under the care of a general practitioner f. The role of the epidemiologists, medical microbiologists, infectious disease specialists and infection control practitioners in the combat against CDAD. Each EU Member State should institute a national working group for Clostridium difficile encompassing the National Institutes of Heath, epidemiologists and experts in prevention, diagnosing and treating of CDAD. The working group will collaborate intensively with national reference laboratories to be established. A European Centre will coordinate the activities of the national working groups and provide an overview of the incidence rates per member state on a regular basis. To reduce severe outcomes of CDAD, early diagnosis and initiation of specific antimicrobial treatment are important. Prevention of outbreaks of CDAD in hospitals can only be accomplished by early recognition, adequate isolation measures and prompt treatment. Based on the recent experiences with epidemics of C. difficile type 027, most microbiological laboratories are currently implementing a new rapid immunoassay test in routine diagnostics. Some microbiological laboratories have developed rapid molecular tests (real-time PCR) to 14

15 diagnose CDAD. It is expected that in the next few years microbiological laboratories will introduce other rapid diagnostic tests. Development of these new tests should lead to assays with a better performance than those currently available. The medical microbiology laboratories should have a central role for surveillance of CDAD in hospitals, other health care institutions and the community using well defined testing strategies and algorithms. The hospital hygiene/infection control department plays a central role in the prevention of nosocomial infections. They have also a major role in the prevention of CDAD by adequate isolation of affected patients, institution of precautions, and formulation of practice guidelines. The hospital hygiene team develops strategies for hand washing, environmental hygiene and outbreak control. Contamination with C. difficile spores has been demonstrated in 30-60% of sites in hospital wards. Appropriate and adequate cleaning of the hospital environment is an important part of the prevention programme for CDAD. Infection control is of special importance in the control of transmission in an outbreak situation. Patients with suspected or proven CDAD will be isolated or cohorted if isolation facilities are not sufficiently available. National Institutes have a central role in the monitoring of regional incidences of CDAD and the development of specific CDAD guidelines for infection control and treatment, separately for hospitals and long term health care institutions. The national institutes will coordinate outbreak management. Medical Microbiologists and Infectious Disease departments play a major role in the prevention of CDAD by reducing antibiotic prescriptions. Up to 50% of all antibiotic usage in hospitals is inappropriate. Hospital infections caused by antibiotic-resistant bacteria are associated with higher mortality, morbidity and prolonged hospital stay compared with infections caused by antibiotic-susceptible bacteria. A very recent analysis by the Cochrane Institute (2005, Oct 19;4:CD003543) showed that interventions to improve antibiotic prescribing to hospital inpatients are successful, and can significantly reduce antimicrobial resistance or hospital-acquired infections. They are also essential for the control of CDAD. Currently, PCR ribotyping has been considered as the gold standard for typing of C. difficile in Europe. Other typing methods have also been developed and applied, but standardization of these methods and exchange of data between laboratories has never been achieved. The results of PCR ribotyping can be stored as TIFF Files and for further analysis imported into the BioNumerics software (Applied Maths, Kortrijk, Belgium). The Anaerobe Reference Laboratory, University Hospital of Wales, Cardiff, UK holds isolates from all different PCR ribotypes in its database. This database will allow future epidemiological international investigations. It is possible to make this library available on-line through a NPHS server. In this way, other reference laboratories will be able to identify not just type 027 but other PCR ribotypes. Further characterization of the strains for the presence of virulence markers should be performed by the reference laboratories. Such virulence markers include genes for TcdA and TcdB, genes for the binary toxin, and deletions in a toxin regulator gene (TcdC). Finally, the reference laboratories should develop new typing techniques with better discriminative capacities than those currently available. Antibiotic susceptibility testing of C. difficile is not routinely performed at every microbiological laboratory. It is however of the utmost importance that surveillance of the antibiotic sensitivity of C. difficile be done, preferably by a single national centre. The antibiotic of choice for treatment of infections associated with C. difficile is metronidazole, followed by vancomycin as a second choice. Unfortunately, reports from some laboratories mention the occurrence of metronidazole resistance and vancomycin resistance in C. difficile, although the exact mechanism is unknown and confirmation of these findings by reference laboratories is urgently needed(10, 23, 49, 50). 15