Antibiotics and hospital-acquired Clostridium difficile-associated diarrhoea: a systematic review

Claudia Thomas1,2,*, Mark Stevenson2 and Thomas V. Riley1,3

1 School of Biomedical and Chemical Sciences, The University of Western Australia, Queen Elizabeth II Medical Centre, Nedlands, Perth; 2 School of Population Health, The University of Western Australia, Clifton Street, Nedlands, Perth; 3 Division of Microbiology, The West Australian Centre for Pathology and Medical Research, Nedlands, Perth, Western Australia, 6009


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A systematic review of studies that investigated the association of antibiotics with hospital-acquired Clostridium difficile-associated diarrhoea (CDAD) was undertaken to summarize the strength of the evidence for this relationship. The results from the studies identified were considered after critically reviewing the design and conduct of each study. Although the majority of studies found an association with various antibiotics, antibiotic classes or components of antibiotic administration, most were limited in their ability to establish a causal relationship by the use of incorrect control groups, the presence of bias, inadequate control of confounding and small sample sizes. The limitations identified in this review prevented the pooling of results in a meta-analysis. Two studies of reasonable quality suggested an association between clindamycin, cephalosporins, penicillins and CDAD. Well-designed studies grounded in epidemiological principles are needed to identify true risk factors for CDAD and to provide reliable estimates of the strength of association.

Keywords: Clostridium difficile, systematic review


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Clostridium difficile-associated diarrhoea (CDAD) is the most commonly diagnosed infectious hospital-acquired diarrhoea in developed countries.1 Infection with C. difficile may produce a spectrum of outcomes that range from asymptomatic colonization to acute diarrhoea and pseudomembranous colitis, which can result in colonic perforation and death if left untreated.2 Although C. difficile was identified as the causative agent of CDAD during the late 1970s,3,4 it has only been since the late 1980s that it has received greater attention due to an increased incidence worldwide and outbreaks of CDAD in hospitals.59 Antibiotics are believed to be the most important risk factor for CDAD by reducing ‘colonization resistance’ of the bowel, allowing subsequent colonization and infection with C. difficile.10

Despite publication of numerous articles implicating almost all antibiotics with CDAD in hospitalized patients, it is still not clearly understood which antibiotics, or antibiotic classes, in particular are important and how these interact with other risk factors. Many narrative reviews of the subject have been published in recent years; however, only one systematic review that included a meta-analysis of the data has been published.11 It was noted in this review that most of the studies were small, indicating that the quality of CDAD epidemiological studies may be questionable. The pooling of data from observational studies of low internal validity can produce spurious results, particularly if bias and confounding are present.12 The aim of our study was to conduct a systematic review of epidemiological studies in order to determine the validity of reported associations of antibiotics with CDAD.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We identified all published epidemiological studies that investigated the association between antibiotics and hospital-acquired CDAD. Studies were included in the review if they met the following criteria: measurement of antibiotic use in hospitalized patients as an exposure, an outcome of laboratory-confirmed symptomatic CDAD and the inclusion of a comparison group without CDAD. Case reports and descriptive studies were excluded. Studies that used other CDAD cases as a comparison group, for example mild versus severe cases or single episodes versus recurrent CDAD, were also excluded. Non-English articles were excluded; however, their titles and abstracts were reviewed to assess eligibility based on the remaining criteria.

Computerized searches of MEDLINE (1966–2001) and EMBASE (1988–2001) were undertaken and the articles identified were downloaded to Endnote version 4.0. Search keywords used in the extraction were: Clostridium difficile; human; diarrhoea or colitis; antibiotic or antimicrobial; case control studies, cohort studies, prospective studies or retrospective studies. Literature cited in identified articles was examined for further studies. Assessment of the overall quality of the eligible studies was undertaken by critically reviewing each study in terms of study design, selection and information bias, confounding, precision and external validity.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Forty-eight articles, out of a total of 673 identified, fulfilled our eligibility criteria: 23 case–control, 22 cross-sectional and three cohort studies, all published between 1978 and 2001. One cohort study was designed to measure the incidence of C. difficile colonization and infection in tube-fed and non-tube-fed patients, but undertook a multivariate analysis of risk factors of cases and non-cases.13 All eligible studies measured symptomatic CDAD as an outcome, and defined CDAD cases based on the results of microbiology tests of stool samples and the presence of diarrhoeal symptoms. Eighty-five articles were excluded from the review because they were published in a language other than English. Of these, only four were eligible for inclusion based on the remaining criteria.1417

Study quality

The use of inappropriate control groups, the presence of bias, confounding, misclassification and lack of precision in the effect estimates were common problems identified in the study. Several studies used controls selected from populations that did not necessarily represent the source population from which the cases had come. Most commonly, patients that had been tested for C. difficile during their hospital admission and were negative were used.1830 One study used C. difficile carriers31 and another used patients with other nosocomial infections.32 Table 1 summarizes the results from the remaining 33 studies judged to have used appropriate comparison groups.


View this table:
[in this window]
[in a new window]
 
Table 1.  Summary of results from eligible studies
 
Diagnostic suspicion bias can lead to an overestimate of effect, and may have been present in studies that identified CDAD cases diagnosed through clinical management of patients, if the physician was aware of the relationship between antibiotics and CDAD. Of the 48 eligible studies, only 14 did not identify cases through the clinical management of patients.13,28,3142 The common feature of these 14 studies was the prospective identification of cases using objective diagnostic criteria; however, response rates were not reported in several studies.13,28,34,3942 Selection bias was also of concern in two studies that included antibiotic exposure in the case definition.26,43 Some studies were unclear in their method of control selection and therefore had potential for selection bias if the controls had not been selected randomly.19,4349 In particular, Thibault et al.44 used a ‘convenience sample’ of cases, and Nelson et al.45 used the first 33 cases in an outbreak of 195 cases in their case–control study.

Studies commonly failed to report sources of exposure information, raising the possibility of observer bias.24,3437,39,40,46,5052 Of the remaining studies, all obtained exposure information from medical records. The routine recording of antibiotic and medication exposures, procedures and diagnoses in medical records allows accurate information to be obtained; however, variation in the quality of the medical record may result in missing data, as acknowledged by Katz et al.22 Non-differential misclassification of exposures could not be excluded from most studies due to the method of data collection used. For example, the use of standardized data collection instruments, awareness of the purpose of the study by the abstractor, the use of single or multiple abstractors and whether the abstractors were blind to disease status, was poorly reported.

Misclassification of disease could not be excluded in many studies owing to the sensitivity and specificity of laboratory tests used to diagnose C. difficile infection. The most common test used was the direct detection of cytotoxin from faecal samples, which may have resulted in the inclusion of false-negatives among the controls, particularly in studies that used C. difficile-negative patients from laboratory records for comparison.1823,2527,29,30 Other studies included patients that were identified through the culture of C. difficile from stool samples, enhancing sensitivity but potentially including false-positives owing to the low specificity of culture.33,36,39,41,44,46 Others only included as cases those that had both positive cultures and cytotoxin results.34,37,43,53,54 Only three studies used culture of a toxigenic isolate of C. difficile as part of their case definition.13,24,38 This definition is considered to be the most appropriate for epidemiological studies.55 Others used enzyme immunoassay (EIA) tests to detect either toxin A40,47,49,5660 or toxins A and B28,31,50,61 from faecal samples. Several studies failed to use a definition of diarrhoeal symptoms, or ‘diarrhoea’ was stated with no clarification.19,20,24,25,32,37,4547,54,57,58,61,62 Of particular concern in some retrospective studies was the use of symptom definitions that required detail regarding the number of diarrhoeal episodes per day, which would be difficult to accurately obtain from notes in the medical record,21,27,29,48,49,51,52,56,59,63 although Katz et al.22 reported a 94% success rate. Because such information is not consistently recorded in medical records, some retrospective studies used the stool description, e.g. unformed, to enhance case ascertainment.30,64

The potential confounding effects of age, length of stay and severity of underlying illness, which may be associated with exposure to antibiotics and development of CDAD, were not adequately assessed in most studies. Age was most commonly considered either through restriction of the study population,18,24,28,29,37,50,58 matching27,40,43,44,47,49,56,60,65 or multivariate analysis.13,22,53,54,61,63,64 Some studies restricted the study population to those with a common underlying condition24,27,35,39,48,50,54,60 or an intensive care stay,20,31,64 or those who underwent particular procedures.32,33,41,47,51,52,61,62 This approach may have controlled for severity of underlying illness to some extent.

Many of the studies had small sample sizes that may have limited the precision of their estimation of effect (see Table 1). This applied in particular to cross-sectional studies and cohort studies, which commonly had small numbers of CDAD outcomes.

Evidence for the association of antibiotics with CDAD

Overall, 41 out of 48 studies found an increased risk of CDAD in hospitals associated with antibiotic exposure, but all had major weaknesses. For total antibiotics, exposure odds ratios (ORs) ranged from 2.8659 to 6.9236 in studies that used appropriate controls. A relative risk (RR) of 2.48 was recorded in one cohort study.33 For specific antibiotics, or antibiotic classes, ORs ranged from 2.1242 to 4254 for clindamycin, and from 3.8442 to 2656 for third-generation cephalosporins.

Only two studies were identified that had less serious threats to validity, and from which the reported results were considered the most reliable. McFarland et al.53 found an increased risk for CDAD after cephalosporin exposure for up to 1 week (RR 2.07, 95% CI 1.06–6.62) and penicillin exposure for between 1 and 2 weeks (RR 3.62, 95% CI 1.28–8.42) adjusted for age and severity of disease using Horn’s index. A moderately large cross-sectional study by Chang & Nelson63 provided precise estimates of effect for clindamycin (OR 4.22, 95% CI 2.11–8.45) and increased numbers of antibiotics (OR 1.49, 95% CI 1.23–1.81) adjusted for age, length of stay and proximity to patients with CDAD, but did not adjust for co-morbidities.

The ability to generalize study results to other populations was limited because of serious threats to internal validity in most of the studies. The association between CDAD and clindamycin, cephalosporins, penicillins and the number of antibiotics a patient received could be interpreted as representative of a more general association in other hospitalized patients.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Despite a large number of published studies supporting the association of various antibiotics with hospital-acquired CDAD, well-designed epidemiological studies are lacking. A few studies identified in this review were not designed to test causal hypotheses. For example, the studies by Hornbuckle et al.,27 Cooper et al.21 and Katz et al.22 were intended for use as clinical tools to predict the probability of a positive C. difficile test result in patients with hospital-acquired diarrhoea, and the study by Cheng et al.31 aimed to analyse differences in characteristics between patients with symptomatic CDAD and C. difficile carriers. However, the main objective of the majority of the studies was to estimate the risk of CDAD in hospitalized patients if exposed to various risk factors, notably antibiotics. The most serious threats to validity identified in this review were the use of incorrect control groups, lack of precision due to inadequate sample sizes and inadequate control of confounders. Diagnostic bias was a potential threat to many studies that identified cases through the clinical management of patients, but this is virtually unavoidable when laboratory databases are used for retrospective case identification. Information bias could not be excluded from many studies, mainly due to the provision of insufficient detail in the articles.

Almost half of the studies were described by their authors as ‘cohort’ or ‘prospective’ studies, although they were regarded in this review as cross-sectional studies because they did not meet the epidemiological classification of a cohort study.66 Cross-sectional studies are generally poor for testing causal hypotheses, owing to the potential for length-biased sampling (a person with a longer duration of illness will have a greater chance of being selected) and the use of measurements of current rather than past exposures, which may prevent the separation of cause and effect. Several cross-sectional studies identified in this review identified cases prospectively (incident cases), thus minimizing length-biased sampling, and collected risk factor information from the medical record rather than relying on self-reporting. However, the main limitation found with many of these studies was the low number of CDAD outcome events, which may have resulted in an imprecise estimate of effect.

The choice of controls in case–control studies of CDAD has been a contentious issue recently, with arguments for and against the use of symptomatic C. difficile-negative patients as controls.67,68 In order to accurately measure the risk of acquiring a disease if exposed to a particular factor, cases should be compared with controls selected from the source population, i.e. subjects who would have been included in the case group if they had developed the disease. Therefore, restricting the selection of controls to patients investigated for C. difficile is not representative of the source population from which the cases arose. This is likely to produce a result biased to the null either because exposures are likely to be similar for CDAD or other forms of nosocomial diarrhoea, or because of the classification of false-negatives as controls, depending on the sensitivity of the laboratory test used.

The different methods for laboratory diagnosis of CDAD used throughout the studies reviewed have implications for the misclassification of disease that can, as previously noted, produce a biased effect estimate. Direct detection of C. difficile cytotoxin (toxin B) from faecal specimens using mammalian tissue culture lines is considered the standard diagnostic test, and was the one used most commonly in the studies identified. However, this method lacks sensitivity due to inactivation of the cytotoxin in 20% of samples during storage and transport.69 Recent evidence indicates that C. difficile and its spores are affected relatively little by storage conditions compared with cytotoxin.70 In addition, ~60% of cytotoxin-negative faecal samples contain a cytotoxin-producing strain of C. difficile, suggesting a false-negative laboratory result.71,72 These methods are also time consuming, and EIAs that provide rapid results have been developed. Commercial EIAs for toxin A were used by several studies; however, this approach also requires culturing of C. difficile for adequate toxin detection.73 Furthermore, toxin AB+ variant C. difficile strains, found to account for 3% of isolates sent to a reference laboratory in the UK,74 would be missed using this method. Although toxin AB+ strains occur at a low prevalence, there is concern that these variant strains will spread through hospitals undetected where toxin A testing is used alone. To overcome this, EIAs that detect both toxins have been developed. These perform well against faecal cytotoxin tissue culture, but are still less sensitive than culture of toxigenic C. difficile.7577 Culture of C. difficile followed by detection of cytotoxin, either by tissue culture or immunoassay, has recently been recommended for confirmation of diagnosis in patients with cytotoxin-negative faecal samples.69,78 Therefore, in epidemiological studies of CDAD, culture of C. difficile followed by toxin demonstration is important in order to accurately identify cases.

In this study, we systematically reviewed the quality of published studies that investigated antibiotics as risk factors for hospital-acquired CDAD in order to decide whether a meta-analysis could be reliably conducted. A previous systematic review concluded that ‘the meta-analysis approach enabled the ranking of antibiotics in relation to the risk of C. difficile infection’.11 In our study, a critical review of studies that analysed antibiotics as risk factors for CDAD was undertaken in terms of study design, selection and information biases, control of confounding, precision of the effect estimate and external validity. Although our review was limited by including only published studies and excluding non-English articles, only two studies were identified that were considered to provide valid evidence for the role of antibiotics in hospital-acquired CDAD. Therefore, based on our findings we do not agree with Bignardi11 that a meta-analysis can be reliably conducted in order to assess the relationship between antibiotics and C. difficile.

Future studies designed to investigate the aetiology of hospital-acquired CDAD need to be aware of epidemiological principles. Investigators need to define a clear hypothesis when designing such studies so that appropriate controls are selected, an adequate sample size is used, bias minimized and confounding controlled. Adherence to these principles will ensure that risk factors are accurately identified and their association with CDAD validly and precisely estimated.


    Footnotes
 
* Correspondence address. School of Biomedical and Chemical Sciences, The University of Western Australia, Queen Elizabeth II Medical Centre, Nedlands, Perth, Western Australia, 6009. Tel: +618-9346-3440; Fax: +618-9382-8046; E-mail: c-thomas{at}cyllene.uwa.edu.au Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Riley, T. V. (1994). The epidemiology of Clostridium difficile-associated diarrhoea. Reviews in Medical Microbiology 5, 117–22.

2 . Kelly, C. & LaMont, J. (1998). Clostridium difficile infection. Annual Review of Medicine 49, 375–90.[CrossRef][ISI][Medline]

3 . Bartlett, J. G., Chang, T. W., Gurwith, M., Gorbach, S. L. & Onderdonk, A. B. (1978). Antibiotic-associated pseudomembranous colitis due to toxin-producing Clostridia. New England Journal of Medicine 298, 531–4.[Abstract]

4 . Larson, H. E., Price, A. B. & Honour, P. (1978). Clostridium difficile and the aetiology of pseudomembranous colitis. Lancet i, 1063–6.

5 . Cartmill, T. D. I., Panigrahi, H., Worsley, M. A., McCann, D. C., Nice, C. N. & Keith, E. (1994). Management and control of a large outbreak of diarrhoea due to Clostridium difficile. Journal of Hospital Infection 27, 1–15.[ISI][Medline]

6 . Frost, F., Craun, G. F. & Calderon, R. L. (1998). Increasing hospitalisation and death possibly due to Clostridium difficile diarrheal disease. Emerging Infectious Diseases 4, 619–25.[ISI][Medline]

7 . Wilcox, M. H. & Smyth, E. T. M. (1998). Incidence and impact of Clostridium difficile infection in the UK, 1993–1996. Journal of Hospital Infection 39, 181–7.[ISI][Medline]

8 . Djuretic, T., Wall, P. G. & Brazier, J. S. (1999). Clostridium difficile: an update on its epidemiology and role in hospital outbreaks in England and Wales. Journal of Hospital Infection 41, 213–8.[CrossRef][ISI][Medline]

9 . Johnson, S., Samore, M. H., Farrow, K. A., Killgore, G. E., Tenover, F. C., Lyras, D. et al. (1999). Epidemics of diarrhea caused by a clindamycin-resistant strain of Clostridium difficile in four hospitals. New England Journal of Medicine 341, 1645–51.[Abstract/Free Full Text]

10 . Johnson, S. & Gerding, D. N. (1998). Clostridium difficile-associated diarrhea. Clinical Infectious Diseases 26, 1027–34.[ISI][Medline]

11 . Bignardi, G. E. (1998). Risk factors for Clostridium difficile infection. Journal of Hospital Infection 40, 1–15.[ISI][Medline]

12 . Egger, M., Schneider, M. & Davey Smith, G. (1998). Spurious precision? Meta-analysis of observational studies. British Medical Journal 316, 140–4.[Free Full Text]

13 . Bliss, D. Z., Johnson, S., Savik, K., Clabots, C. R., Willard, K. & Gerding, D. N. (1998). Acquisition of Clostridium difficile and Clostridium difficile-associated diarrhoea in hospitalised patients receiving tube feeding. Annals of Internal Medicine 129, 1012–9.[Abstract/Free Full Text]

14 . Rabasa, M., Aguado, J. M., Lizasoain, M., Pedraza, M. A., Arribas, P., Lumbreras, C. et al. (1993). Yield of detection of Clostridium difficile toxin versus stool culture in the study of nosocomial diarrhea. Enfermedades Infecciosas y Microbiologia Clinica 11, 479–81.[Medline]

15 . Tacconelli, E., Leone, F., Mazzella, P. & Tumbarello, M. (1994). Clostridium difficile associated diarrhea: Two years of observation in a University Hospital. Igiene Moderna 102, 95–104.

16 . Ramos, A., Gazapo, T., Murillas, J., Portero, J. L., Valle, A. & Martin, F. (1998). Outbreak of nosocomial diarrhea by Clostridium difficile in a department of internal medicine. Enfermedades Infecciosas y Microbiologia Clinica 16, 66–9.[Medline]

17 . Selva O’Callaghan, A., Yuste, M., Armadans, L., Almirante Gragera, B., San Jose Laporte, A. & Vilardell Tarres, M. (2000). Factors for Clostridium difficile-associated diarrhoea in elderly patients. A case–control study. Medicina Clinica 115, 499–500.[ISI][Medline]

18 . Thompson, C. M., Jr, Gilligan, P. H., Fisher, M. C. & Long, S. S. (1983). Clostridium difficile cytotoxin in a pediatric population. American Journal of Diseases of Children 137, 271–4.[ISI][Medline]

19 . Zimmerman, R. K. (1991). Risk factors for Clostridium difficile cytotoxin-positive diarrhoea after control for horizontal transmission. Infection Control and Hospital Epidemiology 12, 96–100.[ISI][Medline]

20 . Jensen, G. L., Bross, J. E., Bourbeau, P. P., Naumovitz, D. W., Streater, M. & Gianferante, L. E. (1994). Risk factors for Clostridium difficile stool cytotoxin b among critically ill patients: role of sucralfate. Journal of Infectious Diseases 170, 227–30.[ISI][Medline]

21 . Cooper, G. S., Lederman, M. M. & Salata, R. A. (1996). A predictive model to identify Clostridium difficile toxin in hospitalized patients with diarrhea. American Journal of Gastroenterology 91, 80–4.

22 . Katz, D. A., Lynch, M. E. & Littenberg, B. (1996). Clinical prediction rules to optimize cytotoxin testing for Clostridium difficile in hospitalized patients with diarrhea. American Journal of Medicine 100, 487–95.[CrossRef][ISI][Medline]

23 . Watanakunakorn, P. W., Watanakunakorn, C. & Hazy, J. (1996). Risk factors associated with Clostridium difficile diarrhea in hospitalized adult patients: a case–control study—sucralfate ingestion is not a negative risk factor. Infection Control and Hospital Epidemiology 17, 232–5.[ISI][Medline]

24 . Burgner, D., Siarakas, S., Eagles, G., McCarthy, A., Bradbury, R. & Stevens, M. (1997). A prospective study of Clostridium difficile infection and colonization in pediatric oncology patients. Pediatric Infectious Disease Journal 16, 1131–4.[CrossRef][ISI][Medline]

25 . Lai, K. K., Melvin, Z. S., Menard, M. J., Kotilainen, H. R. & Baker, S. (1997). Clostridium difficile-associated diarrhea: epidemiology, risk factors, and infection control. Infection Control and Hospital Epidemiology 18, 628–32.[ISI][Medline]

26 . Climo, M. W., Israel, D. S., Wong, E. S., Williams, D., Coudron, P. & Markowitz, S. M. (1998). Hospital-wide restriction of clindamycin: effect on the incidence of Clostridium difficile-associated diarrhea and cost. Annals of Internal Medicine 128, 989–95.

27 . Hornbuckle, K., Chak, A., Lazarus, H. M., Cooper, G. S., Kutteh, L. A., Gucalp, R. et al. (1998). Determination and validation of a predictive model for Clostridium difficile diarrhea in hospitalized oncology patients. Annals of Oncology 9, 307–11.[Abstract]

28 . Guyot, A., Rawlins, M. D. & Barrett, S. P. (2000). Clarithromycin appears to be linked with Clostridium difficile-associated diarrhoea in the elderly. Journal of Antimicrobial Chemotherapy 46, 642–3.[Free Full Text]

29 . Shah, S., Lewis, A., Leopold, D., Dunstan, F. & Woodhouse, K. (2000). Gastric acid suppression does not promote clostridial diarrhoea in the elderly. QJM: Monthly Journal of the Association of Physicians 93, 175–81.[ISI][Medline]

30 . Mody, L. R., Smith, S. M. & Dever, L. L. (2001). Clostridium difficile-associated diarrhea in a VA medical center: clustering of cases, association with antibiotic usage, and impact on HIV-infected patients. Infection Control and Hospital Epidemiology 22, 42–5.[ISI][Medline]

31 . Cheng, S.-H., Lu, J.-J., Young, T.-G., Perng, C.-L. & Chi, W.-M. (1997). Clostridium difficile-associated diseases: comparison of symptomatic infection versus carriage on the basis of risk factors, toxin production and genotyping results. Clinical Infectious Diseases 25, 157–8.[ISI][Medline]

32 . Crabtree, T. D., Pelletier, S. J., Gleason, T. G., Pruett, T. L. & Sawyer, R. G. (1999). Clinical characteristics and antibiotic utilization in surgical patients with Clostridium difficile-associated diarrhea. American Surgeon 65, 507–11.[ISI][Medline]

33 . Keighley, M. R., Burdon, D. W., Alexander-Williams, J., Shinagawa, N., Arabi, Y., Thompson, H. et al. (1978). Diarrhoea and pseudomembranous colitis after gastrointestinal operations. A prospective study. Lancet ii, 1165–7.[CrossRef]

34 . Gerding, D. N., Olson, M. M., Peterson, L. R., Teasley, D. G., Gebhard, R. L., Schwartz, M. L. et al. (1986). Clostridium difficile-associated diarrhea and colitis in adults. A prospective case–controlled epidemiologic study. Archives of Internal Medicine 146, 95–100.[Abstract]

35 . Grube, B. J., Heimbach, D. M. & Marvin, J. A. (1987). Clostridium difficile diarrhea in critically ill burned patients. Archives of Surgery 122, 655–61.[Abstract]

36 . Johnson, S., Clabots, C. R., Linn, F. V., Olson, M. M., Peterson, L. R. & Gerding, D. N. (1990). Nosocomial Clostridium difficile colonisation and disease. Lancet 336, 97–100.[ISI][Medline]

37 . Cartmill, T. D., Shrimpton, S. B., Panigrahi, H., Khanna, V., Brown, R. & Poxton, I. R. (1992). Nosocomial diarrhoea due to a single strain of Clostridium difficile: a prolonged outbreak in elderly patients. Age and Ageing 21, 245–9.[Abstract]

38 . Talon, D., Bailly, P., Delmée, M., Thouverez, M., Mulin, B., Iehl-Robert, M. et al. (1995). Use of pulsed-field gel electrophoresis for investigation of an outbreak of Clostridium difficile infection among geriatric patients. European Journal of Clinical Microbiology and Infectious Diseases 14, 987–93.[ISI][Medline]

39 . Tumbarello, M., Tacconelli, E., Leone, F., Cauda, R. & Ortona, L. (1995). Clostridium difficile-associated diarrhoea in patients with human immunodeficiency virus infection: a case–control study. European Journal of Gastroenterology and Hepatology 7, 259–63.[ISI][Medline]

40 . Thamlikitkul, V., Danpakdi, K. & Chokloikaew, S. (1996). Incidence of diarrhea and Clostridium difficile toxin in stools from hospitalized patients receiving clindamycin, beta-lactams, or nonantibiotic medications. Journal of Clinical Gastroenterology 22, 161–3.[CrossRef][ISI][Medline]

41 . Kent, K. C., Rubin, M. S., Wroblewski, L., Hanff, P. A. & Silen, W. (1998). The impact of Clostridium difficile on a surgical service: a prospective study of 374 patients. Annals of Surgery 227, 296–301.[CrossRef][ISI][Medline]

42 . Schwaber, M. J., Simhon, A., Block, C., Roval, V., Ferderber, N. & Shapiro, M. (2000). Factors associated with nosocomial diarrhea and Clostridium difficile-associated disease on the adult wards of an urban tertiary care hospital. European Journal of Clinical Microbiology and Infectious Diseases 19, 9–15.[CrossRef][ISI]

43 . Pierce, P. F., Jr, Wilson, R., Silva, J., Jr, Garagusi, V. F., Rifkin, G. D., Fekety, R. et al. (1982). Antibiotic-associated pseudomembranous colitis: an epidemiologic investigation of a cluster of cases. Journal of Infectious Diseases 145, 269–74.[ISI][Medline]

44 . Thibault, A., Miller, M. M. & Gaese, C. (1991). Risk factors for the development of Clostridium difficile-associated diarrhea during a hospital outbreak. Infection Control and Hospital Epidemiology 12, 345–8.[ISI][Medline]

45 . Nelson, D. E., Auerbach, S. B., Baltch, A. L., Desjardin, E., Beck-Sague, C., Rheal, C. et al. (1994). Epidemic Clostridium difficile-associated diarrhea: role of second- and third-generation cephalosporins. Infection Control and Hospital Epidemiology 15, 88–94.[ISI][Medline]

46 . Pear, S. M., Williamson, T. H., Bettin, K. M., Gerding, D. N. & Galgiani, J. N. (1994). Decrease in nosocomial Clostridium difficile-associated diarrhea by restricting clindamycin use. Annals of Internal Medicine 120, 272–7.[Abstract/Free Full Text]

47 . Kreisel, D., Savel, T. G., Silver, A. L. & Cunningham, J. D. (1995). Surgical antibiotic prophylaxis and Clostridium difficile toxin positivity. Archives of Surgery 130, 989–93.[Abstract]

48 . Barbut, F., Meynard, J. L., Guiguet, M., Avesani, V., Bochet, M. V., Meyohas, M. C. et al. (1997). Clostridium difficile-associated diarrhea in HIV-infected patients: epidemiology and risk factors. Journal of Acquired Immune Deficiency Syndromes 16, 176–81.

49 . Yip, C., Loeb, M., Salama, S., Moss, L. & Olde, J. (2001). Quinolone use as a risk factor for nosocomial Clostridium difficile-associated diarrhea. Infection Control and Hospital Epidemiology 22, 572–5.[ISI][Medline]

50 . Ferroni, A., Merckx, J., Ancelle, T., Pron, B., Abachin, E., Barbut, F. et al. (1997). Nosocomial outbreak of Clostridium difficile diarrhea in a pediatric service. European Journal of Clinical Microbiology and Infectious Diseases 16, 928–33.[ISI][Medline]

51 . Bilgrami, S., Feingold, J. M., Dorsky, D., Edwards, R. L., Bona, R. D., Khan, A. M. et al. (1999). Incidence and outcome of Clostridium difficile infection following autologous peripheral blood stem cell transplantation. Bone Marrow Transplantation 23, 1039–42.[CrossRef][ISI][Medline]

52 . Chakrabarti, S., Lees, A., Jones, S. G. & Milligan, D. W. (2000). Clostridium difficile infection in allogeneic stem cell transplant recipients is associated with severe graft-versus-host disease and non-relapse mortality. Bone Marrow Transplantation 26, 871–6.[CrossRef][ISI][Medline]

53 . McFarland, L. V., Surawicz, C. M. & Stamm, W. E. (1990). Risk factors for Clostridium difficile carriage and C. difficile-associated diarrhea in a cohort of hospitalized patients. Journal of Infectious Diseases 162, 678–84.[ISI][Medline]

54 . Hutin, Y., Molina, J. M., Casin, I., Daix, V., Sednaoui, P., Welker, Y. et al. (1993). Risk factors for Clostridium difficile-associated diarrhoea in HIV-infected patients. AIDS 7, 1441–7.[ISI][Medline]

55 . Brazier, J. S. & Duerden, B. I. (1998). Guidelines for optimal surveillance of Clostridium difficile infection in hospitals. Communicable Disease and Public Health 1, 229–30.[Medline]

56 . Nath, S. K., Salama, S., Persaud, D., Thornley, J. H., Smith, I., Foster, G. et al. (1994). Drug risk factors associated with a sustained outbreak of Clostridium difficile diarrhea in a teaching hospital. Canadian Journal of Infectious Diseases 5, 270–5.

57 . Barthram, J. D., Dunstan, F. D. J., Hill, D., Hosein, I. K. & Pippen, C. A. R. (1995). Clostridium difficile: The association between antibiotic therapy and the incidence of infection in the elderly. Pharmaceutical Journal 255, 276–8.

58 . Impallomeni, M., Galletly, N. P., Wort, S. J., Starr, J. M. & Rogers, T. R. (1995). Increased risk of diarrhoea caused by Clostridium difficile in elderly patients receiving cefotaxime. British Medical Journal 311, 1345–6.[Free Full Text]

59 . Halim, H. A., Peterson, G. M., Friesen, W. T. & Ott, A. K. (1997). Case–controlled review of Clostridium difficile-associated diarrhoea in Southern Tasmania. Journal of Clinical Pharmacy and Therapeutics 22, 391–7.[CrossRef][ISI]

60 . MacGowan, A. P., Feeney, R., Brown, I., McCulloch, S. Y., Reeves, D. S. & Lovering, A. M. (1997). Health care resource utilization and antimicrobial use in elderly patients with community-acquired lower respiratory tract infection who develop Clostridium difficile-associated diarrhoea. Journal of Antimicrobial Chemotherapy 39, 537–41.[Abstract]

61 . Harbarth, S., Samore, M. H. & Carmeli, Y. (2001). Antibiotic prophylaxis and the risk of Clostridium difficile-associated diarrhoea. Journal of Hospital Infection 48, 93–7.[CrossRef][ISI][Medline]

62 . Gorecki, P., Schein, M., Rucinski, J. C. & Wise, L. (1999). Antibiotic administration in patients undergoing common surgical procedures in a community teaching hospital: the chaos continues. World Journal of Surgery 23, 429–32.[ISI][Medline]

63 . Chang, V. T. & Nelson, K. (2000). The role of physical proximity in nosocomial diarrhea. Clinical Infectious Diseases 31, 717–22.[CrossRef][ISI][Medline]

64 . Brown, E., Talbot, G. H., Axelrod, P., Provencher, M. & Hoegg, C. (1990). Risk factors for Clostridium difficile toxin-associated diarrhea. Infection Control and Hospital Epidemiology 11, 283–90.[ISI][Medline]

65 . Aziz, E. E., Ayis, S., Gould, F. K. & Rawlins, M. D. (2001). Risk factors for the development of Clostridium difficile toxin-associated diarrhoea: A pilot study. Pharmacoepidemiology and Drug Safety 10, 303–8.[CrossRef][ISI][Medline]

66 . Rothman, K. J. & Greenland, S. (1998). Cohort studies. In Modern Epidemiology, 2nd edn (Rothman, K. J. & Greenland, S., Eds), pp. 79–91. Lippincott-Raven, Philadelphia, PA, USA.

67 . Wilcox, M. (2001). Clarithromycin and risk of Clostridium difficile-associated diarrhoea. Journal of Antimicrobial Chemotherapy 47, 358–9.[Free Full Text]

68 . Guyot, A. & Barrett, S. P. (2001). What is an appropriate control group to identify risk factors for Clostridium difficile-associated diarrhoea? Journal of Antimicrobial Chemotherapy 48, 747–8.[Free Full Text]

69 . Brazier, J. S. (1993). Role of the laboratory in investigations of Clostridium difficile diarrhea. Clinical Infectious Diseases 16, S228–33.[ISI][Medline]

70 . Freeman, J. & Wilcox, M. H. (2003). The effects of storage conditions on viability of Clostridium difficile vegetative cells and spores and toxin activity in human faeces. Journal of Clinical Pathology 56, 126–8.[Abstract/Free Full Text]

71 . Lashner, B. A., Todorczuk, J., Sahm, D. F. & Hanauer, S. B. (1986). Clostridium difficile culture-positive toxin-negative diarrhea. American Journal of Gastroenterology 81, 940–3.[ISI][Medline]

72 . Riley, T. V., O’Neill, G. L., Bowman, R. A. & Golledge, C. L. (1994). Clostridium difficile-associated diarrhoea: epidemiological data from Western Australia. Epidemiology and Infection 113, 13–20.[ISI][Medline]

73 . Martirosian, G., van Belkum, A., Pituch, H., Obuch-Woszczatynski, P. & Meisel-Mikolajczyk, F. (2000). Are rapid immunoassays for in vivo detection of toxin A sufficient for diagnostic purposes of Clostridium difficile-associated diseases? Anaerobe 6, 15–9.[CrossRef][ISI]

74 . Brazier, J. S., Stubbs, S. L. & Duerden, B. I. (1999). Prevalence of toxin A negative/B positive Clostridium difficile strains. Journal of Hospital Infection 42, 248–9.[ISI][Medline]

75 . Doern, G. V., Coughlin, R. T. & Wu, L. (1992). Laboratory diagnosis of Clostridium difficile-associated gastrointestinal disease: comparison of a monoclonal antibody enzyme immunoassay for toxins A and B with a monoclonal antibody enzyme immunoassay for toxin A only and two cytotoxicity assays. Journal of Clinical Microbiology 30, 2042–6.[Abstract]

76 . Barbut, F., Kajzer, C., Planas, N. & Petit, J.-C. (1993). Comparison of three enzyme immunoassays, a cytotoxicity assay, and toxigenic culture for diagnosis of Clostridium difficile-associated diarrhoea. Journal of Clinical Microbiology 31, 963–7.[Abstract]

77 . Merz, C. S., Kramer, C., Forman, M., Gluck, L., Mills, K., Senft, K. et al. (1994). Comparison of four commercially available rapid enzyme immunoassays with cytotoxin assay for detection of Clostridium difficile toxin(s) from stool specimens. Journal of Clinical Microbiology 32, 1142–7.[Abstract]

78 . Delmée, M. (2001). Laboratory diagnosis of Clostridium difficile disease. Clinical Microbiology and Infection 7, 411–6.[CrossRef][ISI][Medline]