Clostridium difficile Strain NAP-1 Is Not Associated With Severe Disease in a Nonepidemic Setting

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1 CLINICAL GASTROENTEROLOGY AND HEPATOLOGY 2009;7: Clostridium difficile Strain NAP-1 Is Not Associated With Severe Disease in a Nonepidemic Setting JEFFREY CLOUD,* LAURA NODDIN, AMANDA PRESSMAN, MARY HU, and CIARAN KELLY *Division of Gastroenterology and Hepatology, University of Virginia Medical Center, Charlottesville, Virginia; Division of Gastroenterology and Hepatology, Beth Israel Deaconess Medical Center, Boston, Massachusetts; Division of Gastroenterology and Hepatology, Brown University Medical School, Providence, Rhode Island See related article, Gradel KO et al, on page 495 in Gastroenterology. BACKGROUND & AIMS: Recent outbreaks of Clostridium difficile infection (CDI) in North America and in Europe with very high case-fatality rates have been associated with infection by North American Pulsed Field Type I (NAP-1) isolates. This study examined whether NAP-1 strains are associated with worse outcomes of CDI in a nonepidemic, nosocomial setting. METHODS: All cases of CDI that occurred over a 13-month period at a tertiary medical center were examined for risk factors associated with increased severity of CDI and other outcomes. Stool samples from each patient were cultured for C difficile and the resulting isolates were strain-typed by pulsedfield gel electrophoresis. RESULTS: Strain types were obtained from 236 of 272 CDI samples; the NAP-1 strain was identified in 59 (25%). In this inpatient cohort of patients with CDI, the incidence of in hospital death was 12.1% and of death caused by CDI was 4.0%. Of the patients with CDI, 22.1% met the combined outcome end point of severe CDI. In both univariate and multivariate analyses, patients infected with the NAP-1 strain did not have worse outcomes compared with those infected with non-nap-1 strains. Infection with the NAP-1 strain was correlated with admission from outside health care facilities regardless of whether symptoms of CDI began before or after admission to the study hospital. CONCLUSIONS: The NAP-1 strain of C difficile was found to cause 25% of cases of CDI in the hospital where the study was performed. However, compared with non-nap-1 strains, CDI was not associated with increased severity of disease in this nonepidemic setting. Over the last 10 years, the importance of Clostridium difficile infection (CDI) as a cause of nosocomial diarrhea has grown. In the United States, a 3-fold increase in the reported national rates of CDI was seen from 2000 to 2005 and a 4-fold increase in reported deaths related to CDI was seen between 1999 and ,2 In addition to endemic rates of CDI, a number of regional outbreaks of CDI have been reported, the largest of which has been in the Estrie region of Quebec. 3 In addition to a 3-fold increase in the incidence of CDI at involved hospitals in Quebec, there were also a higher number of cases complicated by intensive care unit (ICU) admission, toxic megacolon, colectomy, or death. The 30-day mortality of CDI patients increased from 4.7% in to 13.8% in A peripheral blood leukocyte count of greater than 20,000 cells/ L and an elevated serum creatinine ( 2.3 mg/dl or 200 mol/l) were found to be independent risk factors for complicated CDI. Strain typing was performed on isolates from involved Quebec hospitals, revealing that 129 of 157 isolates tested (82.2%) were of the North American Pulsed Field Type I (NAP-1) strain type. 4 This prompted researchers to search for reasons why this epidemic strain might be more virulent than other strains. The NAP-1 strain (also referred to as ribotype 027, toxinotype III, or restriction endonuclease analysis group BI depending on the typing method used) has since been found to carry certain virulence determinants that could contribute to an increase in disease severity. NAP-1 isolates from Quebec were found to produce 16 and 23 times as much toxin A and toxin B, respectively, in vitro as compared with historic non-nap-1 isolates (toxinotype 0) of C difficile. 5 This increase in toxin production is thought to be due to deletion mutations in the tcdc gene, a gene that encodes a putative negative regulator of toxin production. 6,7 The NAP-1 strain also produces a separate, third toxin called binary toxin, homologous to the iota toxin in Clostridium perfringens. 8 However, the role, if any, played by binary toxin in inducing severe CDI is currently unclear. 9,10 Despite the numerous reports of severe disease in outbreaks of CDI caused by the NAP-1 strain, evidence showing that individuals with the NAP-1 strain fare any differently from individuals infected with other strains of C difficile was lacking until recently. Investigators analyzing individual patient outcomes from the Quebec outbreak revealed that severe disease was 2.3 times more frequent (95% confidence interval, ) in patients infected by strains carrying both binary toxin genes and a partial tcdc deletion. 11 However, this effect was marginally short of statistical significance (P.054) after adjusting for patient age. Our institution is located in an area where NAP-1 isolates are commonly found and where NAP-1-associated CDI outbreaks have recently occurred. Despite the fact that we had not experienced an outbreak of CDI we suspected that at least some of our patients may be infected by the NAP-1 strain. The aims of this study were (1) to determine the relative frequency of NAP-1 versus non-nap-1 CDI in our institution; and (2) to compare disease Abbreviations used in this paper: BIDMC, Beth Israel Deaconess Medical Center; CDI, Clostridium difficile infection; CI, confidence interval; ICU, intensive care unit; NAP-1, North American Pulsed Field Type I; PCR, polymerase chain reaction; PFGE, pulsed field gel electrophoresis by the AGA Institute /09/$36.00 doi: /j.cgh

2 August 2009 SEVERE C DIFFICILE INFECTION AND NAP 1 STRAIN 869 severity outcomes in NAP-1 and non-nap-1 infected patients in an endemic (ie, nonepidemic) nosocomial CDI setting. Methods More detailed methods are provided in the Appendix. Briefly, we prospectively identified all inpatient cases of CDI at Beth Israel Deaconess Medical Center, Boston, from December 2004 to January We reviewed the electronic medical records for all subjects and recorded relevant demographic and clinical data. We also noted clinical outcomes of CDI including in hospital deaths, admission to the ICU, megacolon or colectomy performed for CDI, and all CDI recurrences that occurred within 60 days after completing treatment. Our primary outcomes of interest were in-hospital death and the combined severe CDI end point which was met if any 1 of the following was found: death caused by CDI or with CDI as a contributing factor, ICU admission caused by CDI or with CDI as a contributing factor, megacolon or colectomy for CDI. Secondary outcomes of interest were in-hospital death caused by CDI, inhospital death with CDI as a contributing cause, ICU admission (or lengthening of ICU stay) caused by CDI, and ICU admission (or lengthening of ICU stay) with CDI as a contributing cause. Other outcomes evaluated were toxic megacolon, colectomy necessitated by CDI, and failure of metronidazole. The physicians collecting data from the medical record had no knowledge of strain typing results when making determinations of clinical outcomes. The study was approved by the institutional review board at Beth Israel Deaconess Medical Center with a HIPAA waiver. All stool samples were cultured in an anaerobic chamber, C difficile isolates identified and typed as NAP-1 versus non-nap-1 by pulsed field gel electrophoresis (PFGE) as previously described. 12,13 Polymerase chain reaction (PCR) toxinotyping was performed to assign toxinotype III versus nontoxinotype III for a small number of cases not successfully typed by PFGE. 14 Also, in a subset of samples the results of PFGE were verified using PCR toxinotyping to confirm the validity of the PFGE technique. All laboratory studies were performed on coded specimens by investigators who were unaware of the baseline patient characteristics or clinical outcomes. All statistical analyses were performed using the SAS software system, version (SAS Institute Inc, Carey, NC). Statistical significance was defined at a level of.05, all P values were 2 sided. Results 276 stool samples positive for C difficile were collected over the 13-month study period. A total of 4 subjects were removed from the study cohort (2 asymptomatic, 2 outpatients). This resulted in a clinical cohort of 272 consecutive episodes of nosocomial CDI from 250 separate individuals. C difficile was successfully isolated from 257 of these samples (94.5%). The results of PFGE on 229 isolates (57 of which were NAP-1) were combined with the results of PCR toxinotyping on 7 further isolates (2 of which were toxinotype III), for a total of 59 NAP-1/toxinotype III isolates (25% of 236). During the period studied, the incidence of nosocomial CDI at our hospital was stable at 0.82, 0.95, and 0.84 cases per 1000 patient days in fiscal years 2004, 2005, and 2006, respectively. The percentage of CDI caused by NAP-1 isolates was similar between the first and second halves of the study period, 23.9% and 26.4%, respectively (P.65 for the difference). Clinical Characteristics of the Cohort (Table 1) The average age of individuals with CDI in our cohort was 67.2 years. Our cohort was evenly divided by gender. These individuals had received an average of 2.7 different antibiotics in the preceding 60 days, including a fluoroquinolone in 55% of subjects. Three quarters of our cohort had received either a Table 1. Description of the Clinical Cohort and Breakdown by NAP-1 Status Clinical cohort (272 episodes) NAP-1 (59 episodes) Non-NAP-1 (177 episodes) 2 P Age (y), mean (95% CI) 67.3 ( ) 73.0 ( ) 66.7 ( ).015 Gender 135 Females 26 Females 94 Females (50.4%) Males 33 (55.9%) Males 83 (46.9%) Males Charlson Score, mean (range) a 3.29 ( ) 3.2 ( ) 3.4 ( ).70 Number of antibiotics in prior 60 days, 2.7 ( ) 2.5 ( ) 2.7 ( ).35 mean (range) Recent fluoroquinolone exposure 55% 61.0% 52.0%.23 Recent cephalosporin exposure 37.5% 44.0% 37.3%.36 PPI or H2 blocker use in the 10 days 74.6% 67.8% 76.8%.17 prior to CDI symptoms Admitted from outside facilities b 94 (34.6%) 34 (57.6%) 50 (28.3%).0001 By location where CDI symptoms began At home 15 of 69 (21.7%) 54 of 69 (78.3%).0097 (home vs OSF) OSF 17 of 37 (45.9%) 20 of 37 (54.1%) At study hospital c 21 of 108 (19.4%) 87 of 108 (80.6%).0016 (OSF vs study hospital) H2, histamine-2; OSF, outside facility; PPI, proton pump inhibitor. a Index with a cumulative score based on a list of comorbidities. b Outside facilities include outside hospitals, rehabilitation facilities, and nursing homes. Individuals transferred from an outside emergency room where they presented from home were considered to have been admitted from home. c The study hospital subset only included patients who did not have diarrhea on admission to the hospital and whose CDI was diagnosed 3 or more days after admission.

3 870 CLOUD ET AL CLINICAL GASTROENTEROLOGY AND HEPATOLOGY Vol. 7, No. 8 Table 2. Outcomes by General Cohort and by NAP-1 Status General cohort (n 272) NAP-1 (n 59) Non-NAP-1 (n 177) P In-hospital death 33 (12.1%) 8 (13.6%) 22 (12.4%).82 In-hospital death from CDI 11 (4.0%) 3 (5.1%) 7 (4.0%).71 In-hospital death with CDI as contributor 23 (8.5%) 5 (8.5%) 16 (9.0%).90 ICU CDI 24 (8.8%) 7 (11.9%) 17 (9.6%).62 ICU CDI contributor 53 (19.5%) 12 (20.3%) 35 (19.8%).93 Megacolon 7 (2.6%) 2 (3.4%) 4 (2.3%).64 Colectomy 5 (1.8%) 1 (1.7%) 3 (1.7%) 1.0 Combined end point a 60 (22.1%) 14 (23.7%) 40 (22.6%).86 a A dichotomous end point that was met with any of the following: death caused by CDI or with CDI as a contributing cause, ICU admission caused by CDI or with CDI as a contributing cause, megacolon, or colectomy. proton pump inhibitor or a histamine receptor 2 antagonist within the 10 days before being diagnosed with CDI. Almost 94% of patients in our cohort were started initially on either oral or intravenous metronidazole for treatment of their CDI. Six percent of patients were initially started on a regimen that included oral vancomycin. In general, individuals infected with the NAP-1 strain tended to be slightly older (73.0 vs 66.7 years, P.01). They were evenly divided between male and female gender. There were no significant differences in Charlson Score, the number of antibiotics that they had received, or the proportion that had recently received fluoroquinolones or cephalosporins. However, there was a significantly higher proportion of individuals that were admitted to our hospital from outside facilities in the NAP-1 group compared with the non-nap-1 group. Clinical Outcomes (Table 2) Twelve percent of individuals with CDI in our inpatient cohort died during that hospital admission. Approximately a third of those individuals (4% of the general cohort) died as a direct result of CDI. In two thirds of those individuals who died (8.5% of the general cohort), CDI was at least a contributing cause. Of patients with CDI in our cohort 8.8% required admission to the ICU (or had their ICU stay lengthened) because of CDI and 19.5% required an ICU admission with CDI as at least a contributing cause. A total of 7 subjects (2.6%) developed megacolon and 5 individuals (1.8%) required a colectomy because of CDI. Of patients in our cohort 22.1% met our combined end point for severe CDI. However, none of the outcomes studied was significantly worse in the NAP-1 infected individuals compared with non-nap-1 infected individuals. The severity of disease was similar between the first and second halves of the study period for all 6 clinical outcomes of interest, 12.1% versus 12.2% for inpatient death (P.65 for the difference). A number of predictors including age, male gender, Charlson index score, fluoroquinolone exposure, and recent use of acidblocking agents were associated with 1 or more of the 6 clinical outcomes of interest on univariate analysis. However, only leukocyte count greater than 20,000 cells/ L at the time of CDI diagnosis and peak creatinine greater than 2.3 mg/dl were consistently associated with worse outcomes on multivariate analysis. This increased risk for severe disease was seen consistently for all 6 of the clinical outcomes of interest (see Table 3 and Figure 1), with leukocyte count 20,000 conferring a 3.7-fold (95% confidence interval [CI], 1.5-fold to 9.1-fold) increased odds of in-hospital death and creatinine 2.3 mg/dl conferring a 3.6-fold (range, 1.4-fold to 8.8-fold) increased odds of in-hospital death. Furthermore, a leukocyte count 20,000 conferred a 30-fold (range, 5-fold to 144-fold) increased odds of death attributed to CDI and creatinine 2.3 mg/dl conferred a 6.6-fold (range, 1.4-fold to 32-fold) increased odds of CDIattributed death. Male gender was associated with a higher risk of death with an odds ratio of 2.5 ( ). Fluoroquinolone exposure was associated with an increased risk of death with CDI as a contributing cause with an odds ratio of 3.1 (95% CI, ). In a similar analysis of those deaths not attributable to CDI (individuals with CDI who died in the hospital, but whose deaths were not thought to be contributed to by CDI), leukocyte count 20,000 and creatinine 2.3 mg/dl were not sig- Table 3. Multivariate Analysis: Independent Predictors of Outcomes WBC 20,000 cells/ L Serum creatinine 2.3 mg/dl Male gender Fluoroquinolone exposure NAP-1 status Death (all causes) 3.7 ( ) P ( ) P ( ) P ( ) P.37 Death from CDI 30.0 ( ) P (1.4 32) P ( ) P.75 Death with CDI as 6.46 (2.3 18) P (2.0 18) P ( ) P ( ) P 0.41 contributor ICU CDI 10.4 (3.5 31) P (4.0 36) P ( ) P.84 ICU CDI contributor 7.0 (3.1 16) P (2.7 15) P ( ) P.64 Combined end point a 5.5 (2.5 12) P (3.0 16) P ( ) P ( ) P.49 NOTE. Data are presented as odds ratio (95% confidence interval). WBC, white blood cell count. a A dichotomous end point that was met with any of the following: death caused by CDI or with CDI as a contributing cause, ICU admission caused by CDI or with CDI as a contributing cause, megacolon, or colectomy.

4 August 2009 SEVERE C DIFFICILE INFECTION AND NAP 1 STRAIN 871 count 20,000, and use of acid suppressive agents were not independently significant. Figure 1. Predictors of negative outcomes in CDI. WBC 20K denotes a peripheral white blood cell count of greater than 20,000 cells/ L on the date of CDI diagnosis. Creat 2.3 denotes a serum creatinine of greater than 2.3 mg/dl (203 mol/l). The outcomes reported are in hospital death caused by CDI, ICU admission caused or contributed to by CDI, and a combined end point (death, ICU admission, or colectomy). nificantly correlated with death, indicating that these predictors may be somewhat specific for CDI. Outcomes: NAP-1 Multivariate Analysis Although the individuals infected by the NAP-1 strain in general had slightly higher (but not statistically significant) rates of the 6 clinical outcomes of interest (Table 2), after adjusting for other covariates including age and admission from an outside facility, infection with the NAP-1 strain was not found to convey any increased risk (Table 3 and Figure 1). On the contrary, the odds ratios tended to show slight protection from more severe outcomes for individuals infected with NAP-1, although all P values were nonsignificant. Failure of Metronidazole Oral metronidazole was used as the initial treatment in 74% of episodes, and intravenous metronidazole was used as initial therapy in 19%. Initial treatment included oral vancomycin in 6.4% of episodes. These percentages did not differ significantly between the NAP-1 and non-nap-1 groups. Of the 251 individuals who were initially started on treatment using oral or intravenous metronidazole, 21.1% required change to or addition of vancomycin for treatment of their CDI. NAP-1 status was not correlated with failure of metronidazole on univariate or multivariate analysis (odds ratio, 0.69; P.38). The only independent predictor of failure of metronidazole in our cohort was having a prior history of CDI which had an odds ratio of 2.6 (95% CI, ). Predictors of NAP-1 Status On multivariate analysis admission from home versus an outside facility was the only independent predictor of NAP-1 status in our cohort. Age, gender, Charlson score, leukocyte Nosocomial Versus Community Acquired Infection with the NAP-1 strain was correlated with being admitted from an outside facility in our cohort (see Table 1). Only 21.7% of patients admitted from home with CDI were infected with the NAP-1 strain compared with 46% of those admitted from outside facilities including hospitals, rehabilitation facilities, and nursing homes. Only 19.4% of individuals whose CDI developed at our hospital (defined as not having diarrhea on admission, but being diagnosed with CDI after 3 or more days in the hospital) were infected with the NAP-1 strain. We also analyzed the subset of patients whose symptoms of CDI clearly developed within our hospital, restricting the set to patients whose CDI was diagnosed after hospital day number 5, in whom it was clearly recorded that they did not have diarrhea on admission nor within the first 72 hours after admission. We found that 0 of 31 patients with CDI who had originally been admitted from home were infected with the NAP-1 strain compared with 7 of 15 (46%) who were originally admitted from outside facilities, a difference that was highly significant (P.0001). Discussion This is the largest study reported to date comparing NAP-1 isolates to non-nap-1 isolates in a nonepidemic setting. A recent study by Morgan et al similarly examined the outcomes of 127 episodes of CDI in the East of England including 51 isolates of ribotype 027 (equivalent to NAP-1). 15 That study yielded the same finding as our own, that ribotype 027 (NAP-1) was not associated with worse clinical outcomes in endemic CDI. The findings of our study are in contrast to those reported by Hubert et al who found that, in the Quebec outbreak, there was an association between infection with NAP-1 strains (characterized by binary toxin production and an 18bp deletion in the tcdc gene) and more severe CDI. 11,15 However, since the association reported in the Hubert study lost statistical significance on multivariate analysis, residual confounding could partially explain this discrepancy in findings. It is not known why an increased severity of disease was not seen in episodes of CDI caused by the NAP-1 strain in our nonepidemic setting. Differences in substrains of NAP-1 or differences in unidentified virulence determinants between NAP-1 isolates collected in outbreak settings and NAP-1 isolates in our nonepidemic setting could explain the lack of increased severity of CDI from NAP-1 strains in this nonepidemic setting in contrast with the increased severity of CDI with NAP-1 strains in Quebec and elsewhere. 4,11 NAP-1/BI/027 strains can be divided further into different subtypes. Thus, it is possible that our NAP-1 isolates, collected in an endemic CDI setting, have a different propensity for causing severe disease than the NAP-1 isolates collected in the Quebec outbreak. If true, comparison of endemic and outbreak NAP-1 isolates may reveal additional important virulence determinants in addition to those already known. This hypothesis is supported by a recent study by Razaq et al, examining the mean time to death after inoculation of different strains of C difficile in the Syrian golden hamster model. 16 The investigators found some

5 872 CLOUD ET AL CLINICAL GASTROENTEROLOGY AND HEPATOLOGY Vol. 7, No. 8 variability between different restricted enzyme analysis type BI stains (equivalent to NAP-1/027) as well as significant differences in the time to death between the non-nap-1 strains. Toxinotype 0 strain K14 (a non-nap-1 strain) was similarly rapidly fatal in comparison with the several BI strains studied, but the BI strains and K14 were both significantly more rapidly fatal than toxinotype 0 strain 630 (a different non-nap-1 strain). Further testing of isolates in animal models may help identify the most virulent strains and hence those virulence factors that are most important in causing lethal CDI. Our study reaffirms the clinical significance of having a leukocyte count 20,000 cells/ L at CDI diagnosis and of having a peak creatinine 2.3 mg/dl in predicting severe CDI and CDI-related deaths. Both parameters were associated with over a 6-fold risk of ICU admission and death caused by CDI. These indicators may help to identify patients at high risk for complicated CDI who could benefit from oral vancomycin rather than metronidazole therapy. 17 The high proportion of NAP-1 isolates from our CDI patients admitted from outside care facilities gives insight into the local epidemiology of C difficile. Because our study design was not able to reliably assess for recent hospitalizations at other facilities we were unable to characterize each episode of CDI as nosocomial or community-acquired according to the same criteria used by prior studies. Nevertheless, our results indicate that in our cohort NAP-1 was not associated with admission from home, but rather was strongly associated with admission from other health care facilities in the Boston area. The reasons for these differences are unclear but may relate to differing antibiotic use and infection control practices as well as through selection of the patient population requiring transfer. In this study we found a continued strong association, even after adjustment for possible confounders, of NAP-1 status with admission to our hospital from outside facilities even for episodes of CDI that clearly developed after 4 or more days in our hospital. This finding suggests presymptomatic carriage of C difficile for longer than was previously hypothesized. 18 In this study we examined the prevalence of nosocomial NAP-1 CDI and the associations between NAP-1 and severe or fatal CDI. Although the NAP-1 strain was identified as causing a substantial proportion of CDI cases in our hospital, it was not associated with increased severity of disease in this nonepidemic setting. This lack of association occurred despite 22% of our patients meeting the combined end point for severe CDI and a 12% in-hospital mortality. Consistent with prior studies, a leukocyte count of 20,000 cells/ L and peak creatinine 2.3 mg/dl at the time of diagnosis of CDI were found to be independent predictors of CDI-related death and severe disease. Infection with the NAP-1 strain was not associated with prior exposure to fluoroquinolones or recent exposure to gastric acid antisecretory agents. NAP-1 infection was highly associated with admission to our hospital from outside health care facilities. This association of admission from outside facilities with infection with the NAP-1 strain persisted even for those individuals who clearly developed CDI within our hospital, suggesting presymptomatic carriage of the organism. Our study findings indicate that CDI outbreaks related to NAP-1 strains are likely to be multifactorial in their genesis resulting from a combination of host, environmental, and bacterial factors and are not simply related to a single hypervirulent strain of C difficile. Alternatively, as yet unrecognized, NAP-1 substrains may carry virulence determinants that predispose to severe CDI outbreaks but are not shared by other NAP-1 isolates in less severe endemic disease settings. Supplementary Data Note: to access the supplementary materials accompanying this article, visit the online version of Clinical Gastroenterology and Hepatology at References 1. McDonald LC, Owings M, Jernigan DB. Clostridium difficile infection in patients discharged from US short-stay hospitals, Emerg Infect Dis 2006;12: Redelings MD, Sorvillo F, Mascola L. Increase in Clostridium difficile-related mortality rates, United States, Emerg Infect Dis 2007;13: 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: Loo VG, Poirier L, Miller MA, et al. A predominantly clonal multiinstitutional outbreak of Clostridium difficile-associated diarrhea with high morbidity and mortality. N Engl J Med 2005;353: 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: MacCannell DR, Louie TJ, Gregson DB, et al. Molecular analysis of Clostridium difficile PCR ribotype 027 isolates from Eastern and Western Canada. J Clin Microbiol 2006;44: Noren T. Outbreak from a high-toxin intruder: Clostridium difficile. Lancet 2005;366: Goncalves C, Decre D, Barbut F, et al. Prevalence and characterization of a binary toxin (actin-specific ADP-ribosyltransferase) from Clostridium difficile. J Clin Microbiol 2004;42: Geric B, Carman RJ, Rupnik M, et al. Binary toxin-producing, large clostridial toxin-negative Clostridium difficile strains are enterotoxic but do not cause disease in hamsters. J Infect Dis 2006; 193: Geric B, Johnson S, Gerding DN, et al. Frequency of binary toxin genes among Clostridium difficile strains that do not produce large clostridial toxins. J Clin Microbiol 2003;41: Hubert B, Loo VG, Bourgault AM, et al. A portrait of the geographic dissemination of the Clostridium difficile North American pulsedfield type 1 strain and the epidemiology of C. difficile-associated disease in Quebec. Clin Infect Dis 2007;44: Klaassen CH, van Haren HA, Horrevorts AM. Molecular fingerprinting of Clostridium difficile isolates: pulsed-field gel electrophoresis versus amplified fragment length polymorphism. J Clin Microbiol 2002;40: Corkill JE, Graham R, Hart CA, et al. Pulsed-field gel electrophoresis of degradation-sensitive DNAs from Clostridium difficile PCR ribotype 1 strains. J Clin Microbiol 2000;38: Rupnik M, Avesani V, Janc M, et al. A novel toxinotyping scheme and correlation of toxinotypes with serogroups of Clostridium difficile isolates. J Clin Microbiol 1998;36: Morgan OW, Rodrigues B, Elston T, et al. Clinical severity of Clostridium difficile PCR ribotype 027: a case-case study. PLoS ONE 2008;3:e Razaq N, Sambol S, Nagaro K, et al. Infection of hamsters with historical and epidemic BI types of Clostridium difficile. J Infect Dis 2007;196: Zar FA, Bakkanagari SR, Moorthi KM, et al. A comparison of vancomycin and metronidazole for the treatment of Clostridium difficile-associated diarrhea, stratified by disease severity. Clin Infect Dis 2007;45:

6 August 2009 SEVERE C DIFFICILE INFECTION AND NAP 1 STRAIN Kyne L, Warny M, Qamar A, et al. Asymptomatic carriage of Clostridium difficile and serum levels of IgG antibody against toxin A. N Engl J Med 2000;342: Reprint requests Address requests for reprints to: Jeffrey Cloud, MD, MPH, Division of Gastroenterology and Hepatology, University of Virginia Medical Center, PO Box , Charlottesville, Virginia jwc5t@ virginia.edu; fax: (434) Conflicts of interest The authors disclose the following: Ciarán P. Kelly has acted as a scientific consultant for Acambis, Actelion, BioHelix, Genzyme, Merck, Replidyne, Salix, and ViroPharm, or has received research grant funding from Actelion, and Genzyme companies that were working toward developing new diagnostic tests or treatments for CDI. He has also served as an expert witness in medical legal cases related to CDI. The remaining authors disclose no conflicts. Funding This work was supported by grants from the National Institutes of Health (RO-1 AI to Ciarán P. Kelly; K30-HL04095 for the Scholars in Clinical Science Program at Harvard Medical School, in which Mary Y. Hu was enrolled. Jeffrey Cloud received grant support from Massachusetts Biologics Laboratories.

7 August 2009 SEVERE C DIFFICILE INFECTION AND NAP 1 STRAIN 873.e1 Appendix. A Detailed Description of the Methods Methods Case identification. We prospectively identified all inpatient cases of CDI at Beth Israel Deaconess Medical Center (BIDMC), a 585-bed academic tertiary care hospital in Boston, over a 13-month period from December 2004 to January CDI was defined as an episode of antibiotic-associated diarrhea where a stool specimen was positive for C difficile toxin A by immunoassay and where the physicians caring for the patient made a new diagnosis of CDI. Thus, asymptomatic patients with positive stool tests for C difficile were excluded. Positive stool samples were collected and frozen at 70 C for later culture. Clinical data. We reviewed the electronic medical records for all subjects and recorded the following data: age, gender, race, comorbidities (from which a Charlson comorbidity score was calculated), presence and duration of diarrhea, whether CDI symptoms began at home, prior history of CDI, admission from home or from an outside facility, admitting diagnosis, the number of days in hospital before the diagnosis of CDI, antibiotics received within the preceding 60 days, use of proton pump inhibitors or histamine-2 blockers within the 10 days prior to the onset of symptoms of CDI, leukocyte count at the time of diagnosis of CDI ( 24 hours), baseline creatinine prior to symptoms of CDI, creatinine levels during the episode of CDI including the peak creatinine while still symptomatic, antibiotics used to treat CDI (including dose and duration), failure of initial therapy using metronidazole (as defined below), length of diarrhea after starting CDI treatment, in-hospital deaths (and whether caused by CDI or contributed to by CDI), admission to the ICU (and whether caused by CDI or contributed to by CDI), development of megacolon by radiography, colectomy performed for CDI and/or toxic megacolon, discharge home versus discharge to a rehabilitation or skilled nursing facility, and all recurrences that occurred within 60 days after completing treatment as identified through our hospital laboratory or follow-up notes through our electronic medical record system. In the 2 cases that a patient with active C difficile colitis was discharged home on comfort care and as expected died within 14 days after discharge of anticipated causes, their death was counted as an in-hospital death. Outcomes. Our primary outcomes of interest were in-hospital death and the combined severe CDI endpoint as defined below. Secondary outcomes of interest were in-hospital death caused by CDI, in-hospital death with CDI as a contributing cause, ICU admission (or lengthening of ICU stay) caused by CDI, and ICU admission (or lengthening of ICU stay) with CDI as a contributing cause. We refer to these as the 6 clinical outcomes of interest. CDI was deemed to have been a contributing cause of an ICU admission or death if the disease process that directly caused the ICU admission or death was temporally related with active CDI symptoms and it was both physiologically plausible and likely that CDI contributed to that disease process. Other outcomes evaluated were toxic megacolon, colectomy necessitated by CDI, and failure of metronidazole. We created a dichotomous combined end point for severe CDI which was met if any of the following were found: death caused by CDI or with CDI as a contributing factor, ICU admission caused by CDI or with CDI as a contributing factor, or megacolon or colectomy for CDI. The inconsistent recording of duration of diarrhea in the medical record and the discharge of patients with continued diarrhea made our determination of duration of diarrhea unreliable. However, because electronic medication orders are highly accurate and universal within our system, we defined failure of metronidazole similarly to previous studies as receipt of at least 2 days of oral or intravenous metronidazole followed by the addition or substitution of oral vancomycin therapy for CDI. Recurrences were defined as a resumption of diarrhea believed secondary to CDI after discontinuation of antibiotic therapy for CDI for at least 48 hours. Because recurrences were identified only if a patient was still hospitalized at BIDMC, had a recurrence requiring or during a repeat hospitalization at BIDMC, had a repeat stool sample positive for C difficile submitted to the BIDMC laboratory, or had a recurrence noted in follow-up notes with outpatient physicians who contribute records to the BIDMC electronic medical record, we anticipated that our study design would underestimate recurrence rates. The physicians collecting data from the medical record had no knowledge of strain typing results when making determinations of clinical outcomes. The study was approved by the institutional review board at BIDMC with a HIPAA waiver. Microbiological analysis. All stool samples were cultured on taurocholate-cefoxitin cycloserine fructose agar (T-CCFA) in an anaerobic chamber at 37 C for 72 hours. Characteristic colonies were selected, subcultured onto anaerobic blood agar, and inoculated into trypticase soy broth. Agarose plugs were made from culture aliquots and PFGE was performed as previously described by Klaassen et al using a Bio-Rad CHEFDR II apparatus (Bio-Rad Laboratories, Hercules, CA) with the addition

8 873.e2 CLOUD ET AL CLINICAL GASTROENTEROLOGY AND HEPATOLOGY Vol. 7, No. 8 of 50 M thiourea. 12,13 Gels were stained using ethidium bromide, scanned under ultraviolet light, and analyzed using Bionumerics 4.0 (Applied Maths Inc, Austin, TX) to assign them as North American Pulsed Field Type I (NAP-1) versus non-nap-1. PCR toxinotyping was performed as described by Rupnik et al to assign toxinotype III versus nontoxinotype III on a small number of cases not successfully identified by PFGE. 14 Also, in a subset of samples the results of PFGE were verified using PCR toxinotyping to confirm the validity of the PFGE technique. All laboratory studies were performed on coded specimens by investigators who were unaware of the baseline patient characteristics or clinical outcomes. Statistical analysis. All statistical analyses were performed using the SAS software system, version Statistical significance was defined at a level of.05, all P values were 2 sided. Each clinically separate episode of CDI was considered independent, regardless of prior episodes of CDI in the same individual. The continuous variables of leukocyte count on day of CDI diagnosis and peak creatinine during the episode of CDI were categorized at a leukocyte count 20,000 cells/ L and peak creatinine 2.3 mg/dl (203 mol/l) on the basis of previous studies. 3 Missing values were excluded from analysis with the exception of the separate multivariate analysis of outcomes by NAP-1 status in which missing values of NAP-1 status were dummy-coded. The following covariates were analyzed for their association with each outcome: age, gender, Charlson score, admission from home versus an outside facility, whether the symptoms of CDI were present on admission, recent use of acid antisecretory agents, the number of different antibiotics used in the 60 days preceding the diagnosis of CDI, fluoroquinolone use, cephalosporin use, the number of days in hospital prior to the diagnosis of CDI, leukocyte count greater than 20,000 cells/ L, peak creatinine greater than 2.3 mg/dl (203 mol/l), and NAP-1 status. For associations between dichotomous variables, a 2 statistic was used, or Fisher s exact test when expected values were less than 5 in any cell. Determinations of statistical significance involving a dichotomous outcome and a continuous predictor were performed using simple logistic regression. To identify independent predictors for each outcome, multivariate logistic regression models were built in a stepwise fashion. Only covariates with a P value of.2 on univariate analysis were entered into each model, and only covariates with a P value of.05 were allowed to remain. In certain circumstances stated below, covariates were included in the model regardless of P value because of likely confounding determined a priori. Potential confounders of each outcome were then screened for by individually adding those excluded covariates with a P value of.2 on univariate analysis back into the model, looking for a change of more than 20% in the coefficient of significant predictors. Adjusted odds ratios and 95% confidence intervals were calculated from the final model. Since NAP-1 status did not have a P value of.2 for association with any of the clinical outcomes on univariate analysis, a separate set of models were created using an approach similar to that described above, but with NAP-1 status, age, and admission from home versus an outside facility forced into each model regardless of their association on univariate analysis. The odds ratios and P values from the association of NAP-1 status with each outcome were interpreted from this separate analysis.