Molecular epidemiology of Clostridium difficile for clinical practice

DOI: https://doi.org/10.4414/smw.2014.13995

Methodology

The search engine PubMed ( http://www.pubmed.com ) was used to identify publications. Reference lists of selected reports were searched to identify additional publications.

Publications were collected from 1980–2014. Studies published in English were identified and results of publications written in other languages were included if their abstracts were in English. Search terms included “Clostridium difficile”, “C. difficile”, “epidemiology”, “ribotype”, “strain”, and “North American pulsed field type”.

Global spread of different C. difficile-ribotypes

Clostridium difficile is the most frequent cause of healthcare associated diarrhoea resulting in significant morbidity and mortality among hospitalised patients [1]. In the United States, diagnoses of CDI doubled from 31/100,000 discharges in 1996 to 61/100,000 in 2003 – the overall rate during this period being several-fold higher in persons greater than 65 years of age [2]. Concurrently, an epidemic of CDI associated with a high case-fatality rate emerged in Canada in 2002 [3]. These developments were largely attributed to the emergence of a more virulent strain of C. difficile – namely PCR-ribotype 027 causing more severe and recurrent CDI [4, 5]. First reports of the further spread of this ribotype to the European continent derived from Great Britain in 2005 [6] and consecutively from the Netherlands [7]. Over time, numerous European countries – including Switzerland [8] – reported epidemics [9]. Evidence for the worldwide spread of this organism derives from reports of PCR-ribotype 027 from different Asian countries [10–12], Australia [13] and South America [14].

The successful establishment of PCR-ribotype 027 in healthcare settings has been attributed to its fluorochinolone-resistance, toxin-hyperproduction and the potential of this strain to produce binary toxin, possibly explaining more severe and recurrent cases of CDI. Fluorochinolone-resistance may have played a crucial part in the distribution of this strain as the increasing use of these antibiotic agents in highly susceptible patients precipitated the rapidly emerging international epidemic [15]. Overcrowding of hospitals and understaffing may have additionally contributed. To understand the evolution of PCR-ribotype 027, historic non-endemic 027 C. difficile strains were compared to recent epidemic strains by performance of comparative genome and phenotypic analysis. The genome of the epidemic PCR-ribotype 027 strain has five additional genetic regions compared with its historic counterpart (i.e., non-epidemic 027 strain) including a novel phage island, a two component regulatory system and transcriptional regulators possibly contributing to its successful emergence [16].

After its recognition as one of the principal drivers of the ongoing CDI-epidemic, ribotype 027 still accounts for the majority of all hospital-acquired CDI cases (62.7%) in Canada – this strain being predominant among patients with CDI, whereas asymptomatic patients were more likely to be colonised with other strain-types [17]. A comprehensive survey on the distribution of different C. difficile ribotypes was performed in 34 different European countries in 2008 [18]. Incidence of CDI was 4.1 per 10’000 patient-days, ranging from 0.0–36.3 and varying across hospitals. Interestingly, PCR-ribotype 027 only accounted for 5% of all strains, other ribotypes – most commonly 014/020, 001 and 078 being predominant in European hospitals [18]. These results differed from a previous report demonstrating a predominance of PCR-ribotypes 001 and 014 followed by 027 and 020 in 14 European countries [19]. PCR-ribotype 078 was the third most commonly identified strain correlating with reports on the emergence of this ribotype in the Netherlands [20]. Patients infected with PCR-ribotype 078 present with similar disease severity as patients infected with PCR-ribotype 027, but are younger and more commonly diagnosed with community-associated disease [20]. Table 1 summarises the global distribution of the two hyper-virulent PCR-ribotypes 027 and 078.

It is likely that other epidemic strains of C. difficile will emerge in the future [21] owing to the highly fluid genome of this pathogen, resulting in its adaptability to environmental changes. In a study on the molecular epidemiology of C. difficile over a 10–year course at a tertiary care hospital, years with high CDI-incidence were associated with large clusters of specific strains that changed yearly. The molecular epidemiology of CDI in this hospital was characterised by a wide diversity of C. difficile types and an ever-changing dominance of specific C. difficile types over time – leading to the conclusion that the molecular epidemiology of CDI is expected to continuously evolve [22].

The role of different ribotypes in community-associated disease

CDI is classified as community-associated in up to 41% of all cases [23]. In a population-based study, patients with community-acquired CDI were significantly younger, had lower co-morbidity scores, and lower rates of antibiotic exposure [23]. These findings have been supported by further studies reporting that otherwise healthy people without prior exposure to antibiotics [24, 25], peripartum women [26–28] and children [29] are increasingly at risk for CDI. Few studies have analysed the correlation of such trends with the circulation of more virulent C. difficile ribotypes in the community. A recently published article aimed to identify epidemiological and clinical characteristics of community-associated CDI and to explore potential sources of C. difficile acquisition in the community [30]. A total of 984 patients from eight US states diagnosed with community-associated CDI were included and North American pulsed-field gel electrophoresis (NAP) 1, corresponding to PCR-ribotype 027 was the most commonly (21.7%) isolated strain. Exposure to antibiotics during the last 12 weeks prior to diagnosis of CDI was not reported for 35.9% of all patients and only 18% had no outpatient exposure. Patients having CDI with no or low-level outpatient health care exposure were more likely to be exposed to infants younger than 1 year and to household members with active CDI compared with those having high-level outpatient health care exposure [30]. These findings were also supported by one European study suggesting that PCR-ribotype 027 is most commonly acquired in the community [31]. Potential reservoirs and vectors for community-associated CDI may include colonised asymptomatic infants [32]. A recent study reported an acquisition rate of 100% in infants followed during their first year of life from the age of 6 months [32]. Food borne transmission may be an additional explanation for increasing rates of community-associated CDI and affection of populations previously considered being at low risk. C. difficile has been recovered from retail meat and food-animals in Europe [33] – including Switzerland [34], the United States [35] and Canada [36].C. difficile PCR-ribotype 078 has been isolated from piglets with diarrhoea possibly suggesting ongoing transmission by introduction to the food chain, as isolates from humans and pigs were found to be highly genetically related [20]. A community component to ribotype 078 infections was also reported in a recent study deriving from Northern Ireland, where almost a 1:1 ratio of cattle to humans and a large pig population are present [37].

Associations between different ribotypes with disease severity and recurrence

Disease severity

The emergence of PCR-ribotype 027 not only resulted in an overall increase of CDI-incidence in many industrialised countries, but was also associated with an alarming increase in disease severity. High case-fatality rates were reported from Canada [3, 38, 39] and increasing colectomy- and death rates were reported from the United States [40]. These reports were mirrored by studies emerging from Europe also relating to higher case-fatality rates than previously experienced [15, 41]. In the UK, a six-fold increase in CDI-related mortality was observed from 1999–2006 [15]. In the Canada-wide CDI study performed in 2005 by the Canadian Nosocomial Infection Surveillance Program, 12.5% of the infections due to the NAP1 strain (corresponding to PCR-ribotype 027) resulted in a severe outcome, compared with only 5.9% of infections due to the other types. Patient’s age was strongly associated with severe outcome, and patients 60–90 years of age were approximately twice as likely to experience a severe outcome if the infection was due to NAP1, compared with infections due to other types [42]. In a recent study from the Netherlands, high mortality rates were reported for patients infected with ribotype 027 – however, underlying conditions were not accounted for [43]. In contrast, in a study performed at a teaching hospital in the United Kingdom assessing the relationship between strain type, clinical factors and outcome, strain type was not associated with any outcome measure [44]. This result is supported by the fact that the mortality rate among patients included in this study was typical of studies in which outbreaks of PCR-ribotype 027 have been reported [44]. More recent data deriving from the United States have also challenged the concept of disease severity being linked to strain-type. In a cross-sectional study including 310 independent cases of CDI, the association between ribotypes 027 and 078 and severe CDI was not significant after correcting for any of the other clinical covariates. After full adjustment, severe cases were predicted only by patients’ white blood cell count and albumin level. This result was supported by analysis of a validation data set containing 433 independent CDI cases [45] – an approach clearly underscoring the robustness of these findings. The authors concluded that presence of binary toxin or tcdC mutations may not be useful to guide patient care. No associations of ribotypes 027 and 078 with more complicated CDI were observed in the large European survey including 389 patients with CDI and characterisation of their respective strain-type [18]. However, PCR ribotypes 018 and 056 were significantly associated with complicated disease outcome after adjustment for possible confounders. This association has not been shown previously and the results should be interpreted with caution given the small number of cases with complicated CDI in the cohort [18]. A recent study from Austria confirms the lack of association between different ribotypes (including ribotype 027) and disease severity [46]. An outbreak with two different C. difficile PCR-ribotypes (017 und 027) occurred in the Netherlands providing a unique opportunity to asses ribotype-specific outcomes [47]. Thirty-day mortality rates were strikingly higher in patients infected with one of the two outbreak strains (23% for PCR-ribotype 017 and 26% for PCR-ribotype 027) compared to patients infected with other ribotypes (3%). This result is surprising as PCR-ribotype 017 does not harbour the gene encoding for toxin A and contains none of the proposed virulence markers typical for ribotype 027 [47]. One possible explanation provided by the authors may be that both ribotypes 017 and 027 are markers for underlying disease severity. Possibly, more severely ill patients are at higher risk for transmission of C. difficile in outbreak settings.

Increased disease severity associated with the emergence of ribotype 027 is thought to be related to binary toxin production and genetic mutations in a toxin regulator gene (tcdC) resulting in hyperproduction of toxins A and B [48, 49] – the primary virulence factor of C. difficile [50, 51]. A more recent study, however, suggests that PCR-ribotype 027 isolates do not produce more toxins in vitro than other strains but produce significantly more spores, which may in itself explain the successful spread of this ribotype in healthcare settings [52]. Similarly, functional status of tcdC based on nucleotide sequencing does not necessarily correlate with disease severity [53]. Another study found deletions in tcdC genes being common among C. difficile isolates but not associated with more severe disease [54].

The role of binary toxin in conferring virulence and contributing to disease severity remains conflicting. Higher mortality rates have been reported for patients infected with binary toxin-producing strains – however, it remains unclear if it is just a marker for more virulent strains or if it contributes directly to disease severity [55].

In conclusion, most of the evidence regarding increased virulence of specific ribotypes derives from settings in which such strains emerged rapidly, often resulting in outbreaks. In endemic settings, the association of disease severity has not been confirmed.

Recurrence

High rates of recurrence – defined as an episode of CDI occurring within 8 weeks after the onset of a previous episode, provided that CDI symptoms from the earlier episode resolved, with or without therapy are one of the hallmarks of CDI and approximately 20% of patients are affected [56]. Two phase 3 treatment trials with the same study design compared the safety and efficacy of fidaxomicin compared to vancomycin, and the combined study population from both trials was analysed with respect to treatment outcomes in relation to different C. difficile strain types [57]. Recurrence rates for patients infected with the B1 strain (corresponding to PCR ribotype 027) were higher than for patients infected with other strain types (27.4% versus 16.6%).

Suggested associations between different ribotypes with disease severity and recurrence are summarised in table 2.

Ribotypes and treatment of C. difficile infection-implications for clinical practice

Guidelines issued by both American (The Infectious Diseases Society of America (IDSA), the Society for Healthcare Epidemiology of America (SHEA)) [58] and European societies (European Society of Clinical Microbiology and Infectious Diseases [ESCMID]) [59, 60] do not make specific recommendations regarding treatment-adaption in accordance to different C. difficile ribotypes. All societies, however, recommend treatment with vancomycin instead of metronidazole for patients diagnosed with severe CDI. Given the conflicting data on the associations of different ribotypes with disease severity, it seems prudent to tailor treatment to clinical presentation rather than strain characteristics.

Regarding recurrent CDI, treatment with Fidaxomicin was associated with a significantly lower rate of recurrence of CDI associated with non–North American Pulsed Field type 1 strains. These findings, however, need to be confirmed [60, 61]. Treatment with monoclonal antibodies against C. difficile toxins A and B tended to show lower recurrence rates for patients infected with PCR ribotype 027 [62]. No specific recommendations – taking specific ribotypes into account – can be made at this point regarding further treatment options for CDI as fecal microbiota transplantation. The European Society (ESCMID) states that the value of the PCR ribotype as a prediction marker for disease severity may be limited, as the ribotype involved in an infection is commonly not known upon diagnosis. However, in an epidemic situation the PCR ribotype may be taken into account in deciding on the choice of empirical treatment regimen [60].

Conclusions

The molecular epidemiology of C. difficile is expected to continuously evolve. Ongoing surveillance of the distribution of different ribotypes and their independent associations with disease severity and outcome are crucial to drive future recommendations regarding prevention of CDI.

Community-associated CDI accounts for a high proportion of all CDI cases also affecting patients previously considered as being at low risk. Food borne transmission may be an important source for infection in this setting and requires further investigation.

Most of the evidence regarding increased virulence of specific ribotypes derives from settings in which such strains emerged rapidly, often resulting in outbreaks. In endemic settings, associations of disease severity with specific ribotypes are conflicting and require further confirmation.

Given the conflicting data on the associations of different ribotypes with disease severity, it seems prudent to tailor treatment to clinical presentation rather than strain characteristics. In epidemic settings, PCR ribotype may be taken into account for decisions on empirical treatment regimens.

References

  1 Kyne L, Hamel MB, Polavaram R, Kelly CP. Health care costs and mortality associated with nosocomial diarrhea due to Clostridium difficile. Clin Infect Dis. 2002;34:346–53.

  2 McDonald LC, Owings M, Jernigan DB. Clostridium difficile infection in patients discharged from US short-stay hospitals, 1996–2003. Emerg Infect Dis. 2006;12:409–15.

  3 Pepin J, Saheb N, Coulombe MA, et al. Emergence of fluoroquinolones as the predominant risk factor for Clostridium difficile-associated diarrhea: a cohort study during an epidemic in Quebec. Clin Infect Dis. 2005;41:1254–60.

  4 Loo VG, Poirier L, Miller MA, et al. A predominantly clonal multi-institutional outbreak of Clostridium difficile-associated diarrhea with high morbidity and mortality. N Engl J Med. 2005;353:2442–9.

  5 McDonald LC, Killgore GE, Thompson A, et al. An epidemic, toxin gene-variant strain of Clostridium difficile. N Engl J Med. 2005;353:2433–41.

  6 Smith A. Outbreak of Clostridium difficile infection in an English hospital linked to hypertoxin-producing strains in Canada and the US. Euro Surveill. 2005;10:E050630 2.

  7 Goorhuis A, Van der Kooi T, Vaessen N, et al. Spread and epidemiology of Clostridium difficile polymerase chain reaction ribotype 027/toxinotype III in The Netherlands. Clin Infect Dis. 2007;45:695–703.

  8 Fenner L, Frei R, Gregory M, Dangel M, Stranden A, Widmer AF. Epidemiology of Clostridium difficile-associated disease at University Hospital Basel including molecular characterisation of the isolates 2006–2007. Eur J Clin Microbiol Infect Dis. 2008;27:1201–7.

  9 Kuijper EJ, Coignard B, Brazier JS, et al. Update of Clostridium difficile-associated disease due to PCR ribotype 027 in Europe. Euro Surveill. 2007;12:E1–2.

10 Cheng VC, Yam WC, Chan JF, To KK, Ho PL, Yuen KY. Clostridium difficile ribotype 027 arrives in Hong Kong. Int J Antimicrob Agents. 2009;34:492–3.

11 Kato H, Ito Y, van den Berg RJ, Kuijper EJ, Arakawa Y. First isolation of Clostridium difficile 027 in Japan. Euro Surveill. 2007;12:E070111 3.

12 Kim H, Lee Y, Moon HW, Lim CS, Lee K, Chong Y. Emergence of Clostridium difficile ribotype 027 in Korea. Korean J Lab Med. 2011;31:191–6.

13 Riley TV, Thean S, Hool G, Golledge CL. First Australian isolation of epidemic Clostridium difficile PCR ribotype 027. Med J Aust. 2009;190:706–8.

14 Quesada-Gomez C, Rodriguez C, Gamboa-Coronado Mdel M, et al. Emergence of Clostridium difficile NAP1 in Latin America. J Clin Microbiol. 2010;48:669–70.

15 Clements AC, Magalhaes RJ, Tatem AJ, Paterson DL, Riley TV. Clostridium difficile PCR ribotype 027: assessing the risks of further worldwide spread. Lancet Infect Dis. 2010;10:395–404.

16 Stabler RA, He M, Dawson L, et al. Comparative genome and phenotypic analysis of Clostridium difficile 027 strains provides insight into the evolution of a hypervirulent bacterium. Genome Biol. 2009;10:R102.

17 Loo VG, Bourgault AM, Poirier L, et al. Host and pathogen factors for Clostridium difficile infection and colonization. N Engl J Med. 2011;365:1693–703.

18 Bauer MP, Notermans DW, van Benthem BH, et al. Clostridium difficile infection in Europe: a hospital-based survey. Lancet. 2011;377:63–73.

19 Barbut F, Mastrantonio P, Delmee M, et al. Prospective study of Clostridium difficile infections in Europe with phenotypic and genotypic characterisation of the isolates. Clin Microbiol Infect. 2007;13:1048–57.

20 Goorhuis A, Bakker D, Corver J, et al. Emergence of Clostridium difficile infection due to a new hypervirulent strain, polymerase chain reaction ribotype 078. Clin Infect Dis. 2008;47:1162–70.

21 Lessa FC, Gould CV, McDonald LC. Current status of Clostridium difficile infection epidemiology. Clin Infect Dis. 2012;55(Suppl 2):S65–70.

22 Belmares J, Johnson S, Parada JP, et al. Molecular epidemiology of Clostridium difficile over the course of 10 years in a tertiary care hospital. Clin Infect Dis. 2009;49:1141–7.

23 Khanna S, Pardi DS, Aronson SL, et al. The epidemiology of community-acquired Clostridium difficile infection: a population-based study. Am J Gastroenterol. 2012;107:89–95.

24 Severe Clostridium difficile -associated disease in populations previously at low risk--four states, 2005. MMWR Morb Mortal Wkly Rep. 2005;54:1201–5.

25 Surveillance for community-associated Clostridium difficile – Connecticut, 2006. MMWR Morb Mortal Wkly Rep. 2008;57:340–3.

26 Garey KW, Jiang ZD, Yadav Y, Mullins B, Wong K, Dupont HL. Peripartum Clostridium difficile infection: case series and review of the literature. Am J Obstet Gynecol. 2008;199:332–7.

27 Hecker MT, Riggs MM, Hoyen CK, Lancioni C, Donskey CJ. Recurrent infection with epidemic Clostridium difficile in a peripartum woman whose infant was asymptomatically colonized with the same strain. Clin Infect Dis. 2008;46:956–7.

28 Rouphael NG, O’Donnell JA, Bhatnagar J, et al. Clostridium difficile -associated diarrhea: an emerging threat to pregnant women. Am J Obstet Gynecol. 2008;198:635 e1–6.

29 Benson L, Song X, Campos J, Singh N. Changing epidemiology of Clostridium difficile -associated disease in children. Infect Control Hosp Epidemiol. 2007;28:1233–5.

30 Chitnis AS, Holzbauer SM, Belflower RM, et al. Epidemiology of community-associated Clostridium difficile infection, 2009 through 2011. JAMA Intern Med. 2013;173:1359–67.

31 Buffet-Bataillon S, Tattevin P, Senechal H, Cormier M, Vincent P. Clostridium difficile O27 colitis: hospital-onset but community-acquired. Eur J Clin Microbiol Infect Dis. 2012;31:2263–7.

32 Rousseau C, Poilane I, De Pontual L, Maherault AC, Le Monnier A, Collignon A. Clostridium difficile carriage in healthy infants in the community: a potential reservoir for pathogenic strains. Clin Infect Dis. 2012;55:1209–15.

33 Koene MG, Mevius D, Wagenaar JA, et al. Clostridium difficile in Dutch animals: their presence, characteristics and similarities with human isolates. Clin Microbiol Infect. 2012;18:778–84.

34 Hoffer E, Haechler H, Frei R, Stephan R. Low occurrence of Clostridium difficile in fecal samples of healthy calves and pigs at slaughter and in minced meat in Switzerland. J Food Prot. 2010;73:973–5.

35 Songer JG, Trinh HT, Killgore GE, Thompson AD, McDonald LC, Limbago BM. Clostridium difficile in retail meat products, USA, 2007. Emerg Infect Dis. 2009;15:819–21.

36 Rodriguez-Palacios A, Staempfli HR, Duffield T, Weese JS. Clostridium difficile in retail ground meat, Canada. Emerg Infect Dis. 2007;13:485–7.

37 Patterson L, Wilcox MH, Fawley WN, et al. Morbidity and mortality associated with Clostridium difficile ribotype 078: a case-case study. J Hosp Infect. 2012;82:125–8.

38 Pepin J, Valiquette L, Alary ME, et al. Clostridium difficile -associated diarrhea in a region of Quebec from 1991 to 2003: a changing pattern of disease severity. CMAJ. 2004;171:466–72.

39 Eggertson L. C. difficile may have killed 2000 in Quebec: study. CMAJ. 2005;173:1020–1.

40 Muto CA, Pokrywka M, Shutt K, et al. A large outbreak of Clostridium difficile -associated disease with an unexpected proportion of deaths and colectomies at a teaching hospital following increased fluoroquinolone use. Infect Control Hosp Epidemiol. 2005;26:273–80.

41 Coignard B, Barbut F, Blanckaert K, et al. Emergence of Clostridium difficile toxinotype III, PCR-ribotype 027–associated disease, France, 2006. Euro Surveill. 2006;11:E060914 1.

42 Miller M, Gravel D, Mulvey M, et al. Health care-associated Clostridium difficile infection in Canada: patient age and infecting strain type are highly predictive of severe outcome and mortality. Clin Infect Dis. 2010;50:194–201.

43 Hensgens MP, Goorhuis A, Dekkers OM, van Benthem BH, Kuijper EJ. All-cause and disease-specific mortality in hospitalized patients with Clostridium difficile infection: a multicenter cohort study. Clin Infect Dis. 2013;56:1108–16.

44 Wilson V, Cheek L, Satta G, et al. Predictors of death after Clostridium difficile infection: a report on 128 strain-typed cases from a teaching hospital in the United Kingdom. Clin Infect Dis. 2010;50:e77–81.

45 Walk ST, Micic D, Jain R, et al. Clostridium difficile ribotype does not predict severe infection. Clin Infect Dis. 2012;55:1661–8.

46 Wenisch JM, Schmid D, Kuo HW, et al. Hospital-acquired Clostridium difficile infection: determinants for severe disease. Eur J Clin Microbiol Infect Dis. 2012;31:1923–30.

47 Goorhuis A, Debast SB, Dutilh JC, et al. Type-specific risk factors and outcome in an outbreak with 2 different Clostridium difficile types simultaneously in 1 hospital. Clin Infect Dis. 2011;53:860–9.

48 Akerlund T, Persson I, Unemo M, et al. Increased sporulation rate of epidemic Clostridium difficile Type 027/NAP1. J Clin Microbiol. 2008;46:1530–3.

49 Warny M, Pepin J, Fang A, et al. Toxin production by an emerging strain of Clostridium difficile associated with outbreaks of severe disease in North America and Europe. Lancet. 2005;366:1079–84.

50 Barbut F, Decre D, Lalande V, et al. Clinical features of Clostridium difficile -associated diarrhoea due to binary toxin (actin-specific ADP-ribosyltransferase)-producing strains. J Med Microbiol. 2005;54:181–5.

51 Barbut F, Gariazzo B, Bonne L, et al. Clinical features of Clostridium difficile -associated infections and molecular characterization of strains: results of a retrospective study, 2000–2004. Infect Control Hosp Epidemiol. 2007;28:131–9.

52 Merrigan M, Venugopal A, Mallozzi M, et al. Human hypervirulent Clostridium difficile strains exhibit increased sporulation as well as robust toxin production. J Bacteriol. 2010;192:4904–11.

53 Goldenberg SD, French GL. Lack of association of tcdC type and binary toxin status with disease severity and outcome in toxigenic Clostridium difficile. J Infect. 2011;62:355–62.

54 Verdoorn BP, Orenstein R, Rosenblatt JE, et al. High prevalence of tcdC deletion-carrying Clostridium difficile and lack of association with disease severity. Diagn Microbiol Infect Dis. 2010;66:24–8.

55 Bacci S, Molbak K, Kjeldsen MK, Olsen KE. Binary toxin and death after Clostridium difficile infection. Emerg Infect Dis. 2011;17:976–82.

56 Aslam S, Hamill RJ, Musher DM. Treatment of Clostridium difficile-associated disease: old therapies and new strategies. Lancet Infect Dis. 2005;5:549–57.

57 Petrella LA, Sambol SP, Cheknis A, et al. Decreased cure and increased recurrence rates for Clostridium difficile infection caused by the epidemic C. difficile BI strain. Clin Infect Dis. 2012;55:351–7.

58 Cohen SH, Gerding DN, Johnson S, 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;31:431–55.

59 Bauer MP, Kuijper EJ, van Dissel JT. European Society of Clinical Microbiology and Infectious Diseases (ESCMID): treatment guidance document for Clostridium difficile infection (CDI). Clin Microbiol Infect. 2009;15:1067–79.

60 Debast SB, Bauer MP, Kuijper EJ, Committee. European Society of Clinical Microbiology and Infectious Diseases: update of the treatment guidance document for Clostridium difficile infection. Clin Microbiol Infect. 2014;20(Suppl 2):1–26.

61 Louie TJ, Miller MA, Mullane KM, et al. Fidaxomicin versus vancomycin for Clostridium difficile infection. N Engl J Med. 2011;364:422–31.

62 Lowy I, Molrine DC, Leav BA, et al. Treatment with monoclonal antibodies against Clostridium difficile toxins. N Engl J Med. 2010;362:197–205.

63 Lim PL, Ling ML, Lee HY, et al. Isolation of the first three cases of Clostridium difficile polymerase chain reaction ribotype 027 in Singapore. Singapore Med J. 2011;52:361–4.

64 Bishara J, Goldberg E, Madar-Shapiro L, Behor J, Samra Z. Molecular epidemiology of Clostridium difficile in a tertiary medical center in Israel: emergence of the polymerase chain reaction ribotype 027. Isr Med Assoc J. 2011;13:338–41.

65 Jalali M, Khorvash F, Warriner K, Weese JS. Clostridium difficile infection in an Iranian hospital. BMC Res Notes. 2012;5:159.

66 Jamal WY, Rotimi VO, Grubesic A, Rupnik M, Brazier JS, Duerden BI. Correlation of multidrug resistance, toxinotypes and PCR ribotypes in Clostridium difficile isolates from Kuwait. J Chemother. 2009;21:521–6.

67 Roberts S, Heffernan H, Al Anbuky N, et al. Molecular epidemiology and susceptibility profiles of Clostridium difficile in New Zealand, 2009. N Z Med J. 2011;124:45–51.