Treatment of Clostridium difficile-associated Diarrhea in the Era of Hypervirulence and Antibiotic Resistance

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1 Review Article Vol. 24 No. 3 Treatment of C. difficile in the era of hypervirulence:- Apisarnthanarak A & Mundy LM. 151 Treatment of Clostridium difficile-associated Diarrhea in the Era of Hypervirulence and Antibiotic Resistance Anucha Apisarnthanarak, M.D.* Linda M. Mundy, M.D.** ABSTRACT Severe morbidities and fatalities due to Clostridium difficile are usually rare. The emergence of hypervirulent strains containing binary toxin (toxinotype III, ribotype 027), mutation in tcdc variants, highlevel toxin A and B production in clinical isolates together with the evolution of a C. difficile strain with highlevel resistance to the newer quinolones (i.e. gatifloxacin and moxifloxacin) have contributed to the rise in C. difficile-associated diarrhea (CDAD) disease severity and mortality in recent years. Metronidazole or vancomycin is regarded as initial CDAD therapy, although several adjunctive strategies have been introduced to treat severe infection. Our current understanding of CDAD suggests that further characterization of virulence is imperative in order to effectively optimize CDAD treatment and prevention strategies. (J Infect Dis Antimicrob Agents 2007;24: ) INTRODUCTION Clostridium difficile was first discovered by Hall and O Toole in 1935 while examining the normal flora of newborn infants. 1 They described an obligate anaerobic, Gram-positive, spore-producing bacillus that grew slowly in culture and was difficult to isolate, giving the organism its descriptive name. 1 In 1974, Tedesco and colleagues were among the first researchers to establish a link between the use of antibiotics and colitis. 2 Four years later, Bartlett and colleagues identified C. difficile and its toxins as the cause of antibiotic-associated pseudomembranous colitis (PMC). 3 C. difficile is responsible for up to 20 percent of cases of antibiotic-associated diarrhea and almost all cases of PMC. 4-5 Penicillins, cephalosporins, and clindamycin are the most frequently associated antibiotics with C. difficile infection. Although most *Division of Infectious Diseases, Faculty of Medicine, Thammasart University Hospital, Pratumthani 12000, Thailand. **Saint Louis University School of Medicine, St. Louis, MO, USA. Received for publication: June 25, Reprint request: Anucha Apisarnthanarak, M.D., Division of Infectious Diseases, Faculty of Medicine, Thammasart University Hospital, Pratumthani 12000, Thailand. anapisarn@yahoo.com Keywords: Clostridium difficile, diarrhea, hypervirulence, emerging infectious diseases, treatment 151

2 152 J INFECT DIS ANTIMICROB AGENTS Sep.-Dec cases of C. difficile-associated diarrhea (CDAD) are associated with antibiotic use, CDAD without prior antibiotic exposure has been reported in patients with renal failure, diabetes mellitus, intestinal surgery, and those receiving antineoplastic chemotherapy. 6-8 Approximately 25 percent of patients with CDAD will experience relapse of the disease. 4-5,9-11 Although CDAD is a common problem, severe morbidity and fatality are rare. However, over the past 5 years a new hypervirulent, more drug-resistant strain has emerged in several countries resulting in large outbreaks and unusually high numbers of severe cases and deaths In this review article, we describe the current understanding of the epidemiology, pathogenesis, and treatment options for CDAD, along with an update on this hypervirulent strain. EPIDEMIOLOGY Approximately 3 million cases of CDAD and colitis are reported among hospitalized patients in the United States (US) each year, compared to an estimated 20,000 cases from the community These recent estimates reflect the importance of C. difficile as a major nosocomial pathogen and, furthermore, that the incidence of CDAD has doubled over the past 6 years in university-based US hospitals. 18 About 1-3 percent of healthy adults carry C. difficile as part of the normal intestinal flora. The colonization rate among healthy individuals with previous antibiotic exposure is 5-15 per cent. 9,19-20 Historically, many studies have consistently identified that 20 percent of hospitalized adults are colonized or acquire C. difficile during the inpatient stay, with subsequent diarrhea in one-third while the majority (two-thirds) comprise a potential asymptomatic reservoir. 4,9,21-22 Severe morbidity and fatality occur in approximately 3 percent of all cases. In contrast, a recent Canadian outbreak reported severe complications in 11 percent of patients infected with a hypervirulent C. difficile strain. 13,23 Transmission of C. difficile usually occurs through the oro-fecal route. Hardy C. difficile spores survive in the hospital environment for months. Previous studies showed a higher surface contamination rate in rooms of patients with CDAD, compared to asymptomatic carriers (49% versus 29%) Additional studies have also documented the presence of C. difficile and its spores on telephones, rectal thermometers, bath tubs, toilets, stethoscopes, and hands of hospital personnel. Patients in a double-bed room had a higher infection rate. 15,25,27-28 Routine use of disposable gloves, single use thermometers, hand washing with chlorhexidine, and environmental cleaning with a 10 percent hypochlorite solution have been shown to significantly reduce the rate of CDAD. 4,29-31 Exposure to antibiotics is the major risk factor for CDAD. 5,32 Administration of multiple antibiotics increases the likelihood of development of CDAD. However, CDAD has been reported even after a single prophylactic dose of antibiotic prior to surgery. Other risk factors include advanced age, recent gastrointestinal surgery, prolonged length of hospital stay, use of antineoplastic agents, enteral feeding, and use of laxatives. 5-6,24-25,33-34 Between 1989 and 1992, several large outbreaks of CDAD caused by clindamycin-resistant, ermbgene-positive strain of C. difficile (type J7 and J9) occurred in four US hospitals in four states, with an incidence ranging from 15.8 to 20 per 1,000 admissions. 35 Subsequently, clinical isolates of C. difficile with toxin B and without toxin A production (A-B + ) were reported from several countries between 1997 and The prevalence of CDAD caused by this toxin variant varied from 0.2 percent to 37 percent The epidemiology, risk factors, and outcomes for patients with CDAD were compared between in the era before and during the occurrence of strains with hypervirulence and antibiotic resistance (Table 1).

3 Vol. 24 No. 3 Treatment of C. difficile in the era of hypervirulence:- Apisarnthanarak A & Mundy LM. 153 Table 1. Epidemiological features of Clostridium difficile colitis before and after detection of hypervirulent C. difficile strains. Variable Historical era Hypervirulent era Geographic distribution Worldwide United States a Canada b United Kingdom Prevalence/incidence case per 1,000 admissions* 22.5 cases per 1,000 admissions* 8-12 cases per 100,000 person-year** Risk factors Age, antibiotic exposure, severity of illness, length of Receipt of fluoroquinolones and cephalosporins c, hospital and ICU stay, prior disease, CRF, chemotherapy, proton pump inhibitor, advanced age, severity of recent gastrointestinal surgery, enteral feeding, laxatives illness, length of hospital ICU stay, presence of and enemas binary toxin and 18-bp deletion in the tcdc gene Outcomes Recurrence: 5 percent to 50 percent Recurrence: 20 percent to 58 percent Mortality: percent Mortality: 1.2 to 22 percent d ICU: intensive care unit, CRF: chronic renal failure * In nosocomial setting, ** In community setting, a Including Georgia, Illinois, Main, New Jersey, Oregon, and Pennsylvania, b Including Quebec, British Columbia, and Ontario, c Including gatifloxacin, levofloxacin, moxifloxacin and first; second; third-generation cephalosporins, d Depends on age 153

4 154 J INFECT DIS ANTIMICROB AGENTS Sep.-Dec Although several sporadic cases of toxin variant (A- B + ) C. difficile diarrhea and PMC have been reported, to date there have been only two hospital outbreaks of CDAD caused by this strain Al-barrak and colleagues were the first to report a toxin variant (A- B + ) outbreak of CDAD in Canada in In this study, five patients had recurrent diarrhea (36%), three had severe colitis (20%), with the overall mortality of 66 percent. 40 Kuijper and colleagues described an outbreak in the Netherlands where 24 patients were diagnosed with CDAD caused by toxin-variant C. difficile. 41 Three patients (13%) had recurrent disease; there were seven cases of severe disease and one death (14%). The high mortality associated with this toxinproducing C. difficile strain may be attributable to an excess toxin B production. Toxin B is a more potent enterotoxin inducing ten-fold more damage in human colonic explants than toxin A Between 2000 and 2003, the spread of a hypervirulent, fluoroquinoloneresistant, binary toxin-producing strain of C. difficile (BI/NAP1) with a partial deletion of in the tcdc caused outbreaks in eight facilities in six US states. These epidemic strains reportedly produce 23 times more toxin B than wild type strains. 44 The same strain was also responsible for large outbreaks of CDAD in Quebec, Canada in 2003 where the incidence was 22.5 per 1,000 admissions and the mortality was 6.9 percent PATHOGENESIS Normal gut flora can inhibit growth of C. difficile in vitro and in vivo The pathogenesis of CDAD involves alteration of the endogenous intestinal flora, usually due to antibiotic use and exposure to pathogenic strains of C. difficile, most often during hospitalization. Approximately 25 percent of C. difficile stains are not pathogenic. 9,46 Development of disease depends on host defense and C. difficile virulence factors. Pathogenic strains of C. difficile produce two exotoxins, toxin A (enterotoxin) and toxin B (cytotoxin). Both bind to intraluminal colonic epithelial receptors. 19,24,43 Toxin A causes a cytokinemediated hypersecretion of fluids and a subsequent inflammatory hemorrhagic process. Toxin B causes cell death via alteration of the actin cytoskeleton. 4,19 Other virulence factors include adhesion factor (binding to colonic mucosal cells) and hyaluronidase (hydrolytic activity). In addition, a formation of spore allows C. difficile to survive in the environment for months. 4,47 Approximately, 6 percent of C. difficile isolates produce a binary toxin. Toxins A and B are encoded by the genes tcda and tcdb. Together with two regulatory genes and a porin gene (tcdc, tcdd, and tcde), they form the chromosomal pathogenicity locus (PaLoc). 14,48 Polymorphism or partial deletion of tcdc has been associated with 6-20-fold increase in toxin A and B production. During recent outbreaks in Canada and the US, more than 50 percent of the epidemic strains produced the binary toxin and had the partial deletion of the tcdc gene In a cross-sectional study of hospitalized patients with diarrhea, investigators developed a predictive model to identify C. difficile infection ,49 The final model suggested that the presence of at least 2 of 3 variables (fecal leukocytes, peripheral leukocytosis, and antecedent hospitalization) were associated with a 77 percent probability of positive C. difficile toxin assay. The clinical presentations range from mild diarrhea to severe colitis with pseudomembrane formation, ileus, perforation, or toxic megacolon. Nonimmunological host defense mechanisms include inhibition of C. difficile by normal flora, reduction of the number of spores and toxins through gastric acid, and elimination of the organism and toxins via intestinal peristalsis. The host immune response appears to play a major role in the development of CDAD, and several studies suggest that an inadequate immune response may result in severe or recurrent C. difficile

5 Vol. 24 No. 3 Treatment of C. difficile in the era of hypervirulence:- Apisarnthanarak A & Mundy LM. 155 colitis. 5,15,27,50 The presence of anti-toxin A IgG in adult sera has been associated with asymptomatic carriage and apparent neutralization of C. difficile toxins In addition, children with recurrent disease have been found to have lower levels of circulating IgG to toxin A. 53 Immunization to toxin A has been shown to prevent colitis in animal models and secretory IgA blocks the binding of toxin A to intestinal receptors. 4,54 RISK FACTORS AND OUTCOMES (Table 1) The major risk factor for CDAD is antibiotic exposure. Although any antibiotic can cause CDAD, penicillins, cephalosporins, and clindamycin are most frequently associated with CDAD. 4-5 Of interest, while fluoroquinolones have not been a risk factor in multivariate analyses, recent outbreaks of CDAD suggest an association with third-generation drugs in this class ,55 McDonald and colleagues characterized 187 C. difficile isolates from outbreaks occurring in eight health care facilities in six US states between 2000 and A molecular comparison with more than 6,000 isolates collected before 2001 revealed that the BI/NAP1, toxinotype III, binary-toxin-producing strain accounted for the majority (52%) of clinical isolates from the latter era. 13 All recent epidemic and historic (14 cases) BI/ NAP1 isolates had the 18-bp deletion in tcdc gene and resistance to gatifloxacin and moxifloxacin; most also had clindamycin and levofloxacin resistance. In contrast, none of the pre-2001 BI/NAP1 isolates showed resistance to third-generation fluoroquinolones. Patients infected with the BI/NAP1 strain tended to have more severe disease, pseudomembranous colitis, and higher peripheral leukocytosis. 13 In several facilities, the outbreak was preceded by a change in the hospital formulary from levofloxacin to gatifloxacin or moxifloxacin. It is hypothesized that the broad antianaerobic activity of the new fluoroquinolones selected for this outbreak. The same strain was responsible for 155 large outbreaks of CDAD among 1,703 patients in 12 Quebec hospitals in 2003 where the incidence of CDAD was 22.5 per 1,000 admissions with a 30-day mortality rate of 6.9 percent. 14 The majority of the isolates (84.1%) produced binary toxin, had the tcdc gene deletion and third-generation fluoroquinolone resistance (82.2%). In contrast, clindamycin use was not an identified risk factor for CDAD in this population. Pepin and colleagues studied 293 hospitalized patients with CDAD in Canada between 2003 and At least 50 percent of cases occurred in patients with more than 80 years of age, and the 30-day mortality was 21.8 percent. 55 Although cephalosporins, clindamycin, macrolides, and penicillins shared similar adjusted hazard ratios (ahrs, ) for causing CDAD, a use of fluoroquinolones (ahr, 3.44) was the most significant risk factor especially with longer duration. Recent data suggest that BI/NAP1 isolates of C. difficile produce significantly higher concentrations of toxin A and B, compared to non-bi/nap1 strains and may be responsible for more severe disease. Other risk factors for recent outbreaks may include the increasing hospitalized geriatric population with multiple co-morbidities and inadequate immune response, widespread use of alcohol-based hand-rubs, shortage of private rooms, and suboptimal response to metronidazole. TREATMENT OPTIONS One of the key steps in managing CDAD is to discontinue all current antimicrobial agents, if clinically plausible. Epidemiological studies in hospitalized patients indicate that the majority of people infected with C. difficile are asymptomatic. Treatment of these healthy carriers has no effect on toxin production. Patient with mild-to-moderate CDAD typically presents with diarrhea, abdominal pain, and peripheral leucocytosis. Oral therapy with vancomycin (125 mg

6 156 J INFECT DIS ANTIMICROB AGENTS Sep.-Dec every six hours) or metronidazole (250 mg every six hours) is recommended for patients with mild-tomoderate CDAD and usually results in clinical improvement within 3-5 days. 3-5 Intravenous metronidazole is reserved for patients who are unable to take oral medication because of ileus or abdominal surgery. Oral vancomycin is preferred to metronidazole among pregnant and breast-feeding women, individuals with known intolerance to metronidazole, or patients who fails to respond to metronidazole. Several comparative trials indicated that metronidazole and vancomycin were equally effective with the response rates of greater than 95 percent. 3-5 However, the excessive cost of vancomycin and its potential for selection of enteric vancomycin-resistant enterococci has led the Centers for Disease Control and Prevention (CDC) to recommend metronidazole as the treatment of choice for CDAD. A relapse is common and occurs in 25 percent of patients with CDAD. 3-5 Relapses are usually successfully treated with another course of metronidazole or vancomycin. Switching between these two antibiotics, and longer courses, have not reduced the frequency of relapses. However, during a large outbreak with a hypervirulent strain of C. difficile in Canada, an unusually high rate (47.2%) of recurrent CDAD was observed in patients who were initially treated with metronidazole. 56 Alternative oral treatment options for recurrent CDAD include pulsed doses of vancomycin, adjunctive cholestyramine or rifampin, and probiotics such as Saccharomyces boulardii. 4-5,9,19,25 Patients with fulminant CDAD may present with hypotension, ileus, hypoalbuminemia, toxic megacolon, or bowel perforation. 4-5,27 Among these cases, an abdominal X-ray may show thumbprinting of the colon, and on computed tomography colonic wall thickening will likely be evident. 4 In severe CDAD, treatment with standard oral or intravenous drugs has been limited by inadequate achievement of intracolonic drug concentrations and on-going toxin production. When oral anti-c. difficile drug is not a therapeutic option, the evidence is limited for selecting a biologically plausible alternative. Parenteral metronidazole drug excretion occurs mainly in the upper gastrointestinal tract with less than 14 percent recovered in feces Anecdotal reports suggest poor clinical responses of CDAD were noted in patients treated with intravenous metronidazole or infected with the strain with reduced metronidazole susceptibility ,60-63 Parenteral vancomycin has a limited penetration of the bowel as well, with stool concentrations of only 6.4 to 10 µg/ ml Available adjunctive CDAD treatment options for severe C. difficile colitis include intravenous immunoglobulin (IVIG), intracolonic vancomycin (ICV), and pancolectomy, while donor stool enemas and C. difficile vaccination have been successfully used in recurrent CDAD. Adjunctive treatment alternatives for severe CDAD colitis are summarized in Table 2. Several studies suggest a benefit of IVIG in severe or refractory CDAD In one retrospective study, two patients with persistent CDAD after treatment with metronidazole and/or vancomycin were given pooled human IVIG ( mg/kg). Both patients experienced prompt, dramatic resolution of diarrhea, abdominal distension and tenderness. Their sera subsequently were able to neutralize C. difficile toxin in cytotoxic assays. In a recent study, IVIG at mg/kg was used to treat 14 patients with severe, refractory, recurrent CDAD. 66 All patients tolerated IVIG without major side effects and nine (64%) had symptom resolution at a median of 10 days (range, 2-26); one patient had a partial response from two doses but died two months later after recurrent CDAD, and the other four died of noncdad-related comorbidities within three weeks of the IVIG treatment. 66 Beales reported a successful treatment of four elderly patients with recurrent CDAD after two doses of IVIG, three weeks apart. 67 A study by Leung

7 Vol. 24 No. 3 Treatment of C. difficile in the era of hypervirulence:- Apisarnthanarak A & Mundy LM. 157 Table 2. Adjunctive treatment alternatives for severe Clostridium difficile colitis. Option Indiation Dose Complications Pancolectomy No clinical response to standard therapy, multiple NA Short bowel syndrome, require ostomy care, malnutrition; organ failure, peritonitis, toxic megacolon, Despite surgery, the mortality has been reported to be radiographic evidence of fulminant colitis as high as 38 percent-48 percent Intracolonic vancomycin No clinical response to standard therapy, impaired 2-3 g/day with intervals of Colonic perforation, require monitoring of vancomycin oral intake, severe ileus resulting in cessation of 4-12 hours level in patients with renal insufficiency, potential diarrhea, clinical evidence of fulminant colitis development of VRE Intravenous immunoglobulin No clinical response to standard therapy, severe, mg/day Expensive refractory and recurrence CDAD CDAD: Clostridium difficile-associated diarrhea, NA: not applicable, VRE: vancomycin-resistant Enterococcus spp. 157

8 158 J INFECT DIS ANTIMICROB AGENTS Sep.-Dec and colleagues showed that IVIG was also effective in five pediatric patients with recurrent CDAD. 68 Together, these data suggest that IVIG may be an effective alternative for severe, refractory, or recurrent CDAD after failing conventional treatment. The administration of ICV as an adjunctive regimen, may occur via an 18-French Foley catheter or soft 6-French pigtail catheter as a 60-minute retention enema, lavage, or as a component of decompressive colonoscopy. 69 In a review of the archived literature for ICV treatment of CDAD, 20 of 24 patients (83%) had clinical and microbiological response to adjunctive ICV therapy As with most observational studies, the duration and dosing interval of ICV varied (2-3 g/day of ICV, with intervals of 4-12 hours), yet the observed findings suggested that ICV was safe and effective in the majority of ICV recipients. Lastly, pancolectomy is considered a treatment option for patients with multisystem organ failure, peritonitis, or radiographic evidence of fulminant colitis. The postoperative long-term sequelae include short gut syndrome, malnutrition, and ostomy care. And postoperative mortality is as high as 48 percent Donor stool via a nasogastric tube or enemas from healthy volunteers has been effective ways of treating recurrent CDAD Several studies indicated that an adequate antibody (IgG and IgA) response to C. difficile may prevent CDAD and that an inadequate response is associated with recurrent disease. Therefore, a development of a C. difficile vaccine seems prudent. Different vaccines have been tested in animals with various results In one study of 30 healthy volunteers, a highly immunogenic response was reported following the administration of an intravenous C. difficile toxoid vaccine. 79 Recently, a C. difficile toxoid vaccine showed promising results in three patients with refractory CDAD. 80 CONCLUSION The epidemiology of CDAD now encompasses hypervirulent and more fuoroquinolone-resistant strains, particularly among elderly patients. Recent outbreaks demonstrate the ability of C. difficile to adapt to an antibiotic-rich environment and for strains to emerge with toxin hyperproduction that are associated with higher morbidity and mortality than previously encountered. Any increased incidence of CDAD should be monitored while strict adherence to infection control measures as well as appropriate and judicious antibiotic therapy are employed. Recent outbreaks indicated that current management strategies have not prevented recurrent CDAD or reduced mortality especially in the elderly populations. The existing data suggests that adjunct use of ICV or IVIG should be vigorously studied and that the development of an effective C. difficile vaccine is warranted. Randomized, controlled trials with and without these adjunctive interventions will contribute to future streamlined treatment options of severe CDAD. Until such data are available, clinicians should carefully select the antimicrobial agent(s) for patients with CDAD and consider the potential benefits of adjunctive therapy. References 1. Hall IC, O Toole E. Intestinal flora in newborn infants with a description of a new pathogenic anerobe, Bacillus difficilis. Am J Dis Child 1935;49: Tedesco FJ, Barton RW, Alpers DH. Clindamycinassociated colitis: a prospective study. Ann Intern Med 1974;81: Bartlett JG, Chang TW, Gurwith M, Gorbach SL, Onderdonk AB. Antibiotic-associated Pseudomembranous colitis due to toxin-producing clostridia. N Engl J Med 1978;298: Kelly CP, Pothoulakis C, LaMont JT. Clostridium difficile colitis. N Engl J Med 1994;330:

9 Vol. 24 No. 3 Treatment of C. difficile in the era of hypervirulence:- Apisarnthanarak A & Mundy LM Bartlett JG. Clinical practice. Antibiotic-associated diarrhea. N Engl J Med 2002;346: Anand A, Glatt AE. Clostridium difficile infection associated with antineoplastic chemotherapy: a review. Clin Infect Dis 1993;17: Moskovitz M, Bartlett JG. Recurrent Pseudomembranous colitis unassociated with prior antibiotic therapy. Arch Intern Med 1981;141: Bartlett JG, Gorbach SL. Pseudomembranous enterocolitis (antibiotic-related colitis). Adv Intern Med 1977;22: Fekety R, Shah AB. Diagnosis and treatment of Clostridium difficile colitis. JAMA 1993;269: Gerding DN. Disease associated with Clostridium difficile infection. Ann Intern Med 1989;110: Bartlett JG. Treatment of Clostridium difficile colitis. Gastroenterology 1985;89: 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: McDonald LC, Killgore GE, Thompson A, et al. An epidemic, toxin gene-variant strain of Clostridium difficile. N Engl J Med 2005;353: 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: McFarland LV, Mulligan ME, Kwok RY, Stamm WE. Nosocomial acquisition of Clostridium difficile infection. N Engl J Med 1989;320: Johnson S, Clabots CR, Linn FV, Olson MM, Peterson LR, Gerding DN. Nosocomial Clostridium difficile colonisation and disease. Lancet 1990;336: Hirschhorn LR, Trnka Y, Onderdonk A, Lee ML, Platt R. Epidemiology of community-acquired Clostridium difficile-associated diarrhea. J Infect Dis 1994;169: Polk RE, Oinonen M, Pakyz A. Epidemic Clostridium difficile. N Engl J Med 2006;354: Bartlett JG. Management of Clostridium difficile infection and other antibiotic-associated diarrhoeas. Eur J Gastroenterol Hepatol 1996;8: Viscidi R, Willey S, Bartlett JG. Isolation rates and toxigenic potential of Clostridium difficile isolates from various patient populations. Gastroenterology 1981;81: Bartlett JG. Antibiotic-associated diarrhea. Clin Infect Dis 1992;15: Razavi B, Apisarnthanarak A, Mundy LM. Clostridium difficile: emergence of hypervirulence and fluoroquinolone resistance. Infection 2007;35: Pepin J, Routhier S, Gagnon S, Brazeau I. Management and outcomes of a first recurrence of Clostridium difficile-associated disease in Quebec, Canada. Clin Infect Dis 2006;42: Barbut F, Petit JC. Epidemiology of Clostridium difficile-associated infections. Clin Microbiol Infect 2001;7: Gerding DN, Johnson S, Peterson LR, Mulligan ME, Silva J Jr. Clostridium difficile-associated diarrhea and colitis. Infect Control Hosp Epidemiol 1995;16: Malamou-Ladas H, O Farrell S, Nash JQ, Tabaqchali S. Isolation of Clostridium difficile from patients and the environment of hospital wards. J Clin Pathol 1983;36: Kelly CP, LaMont JT. Clostridium difficile infection. Annu Rev Med 1998;49: Kim KH, Fekety R, Batts DH, et al. Isolation of Clostridium difficile from the environment and contacts of patients with antibiotic-associated colitis. J Infect Dis 1981;143: Johnson S, Gerding DN, Olson MM, et al. Prospective, controlled study of vinyl glove use to interrupt Clostridium difficile nosocomial transmission. Am J Med 1990;88: Brooks SE, Veal RO, Kramer M, Dore L, Schupf N, 159

10 160 J INFECT DIS ANTIMICROB AGENTS Sep.-Dec Adachi M. Reduction in the incidence of Clostridium difficile-associated diarrhea in an acute care hospital and a skilled nursing facility following replacement of electronic thermometers with single-use disposables. Infect Control Hosp Epidemiol 1992;13: Mayfield JL, Leet T, Miller J, Mundy LM. Environmental control to reduce transmission of Clostridium difficile. Clin Infect Dis 2000;31: Gerding DN, Olson MM, Peterson LR, et al. Clostridium difficile-associated diarrhea and colitis in adults. A prospective case-controlled epidemiologic study. Arch Intern Med 1986;146: Privitera G, Scarpellini P, Ortisi G, Nicastro G, Nicolin R, de Lalla F. Prospective study of Clostridium difficile intestinal colonization and disease following singledose antibiotic prophylaxis in surgery. Antimicrob Agents Chemother 1991;35: Brown E, Talbot GH, Axelrod P, Provencher M, Hoegg C. Risk factors for Clostridium difficile toxinassociated diarrhea. Infect Control Hosp Epidemiol 1990;11: Johnson S, Samore MH, Farrow KA, et al. Epidemics of diarrhea caused by a clindamycin-resistant strain of Clostridium difficile in four hospitals. N Engl J Med 1999;341: Johnson S, Sambol SP, Brazier JS, et al. International typing study of toxin A-negative, toxin B-positive Clostridium difficile variants. J Clin Microbiol 2003;41: Brazier JS, Mulligan ME, Delmee M, Tabaqchali S. Preliminary findings of the international typing study on Clostridium difficile. International Clostridium Difficile Study Group. Clin Infect Dis 1997;25 (Suppl 2):S199-S Brazier JS, Stubbs SL, Duerden BI. Prevalence of toxin A negative/b positive Clostridium difficile strains. J Hosp Infect 1999;42: Barbut F, Lalande V, Burghoffer B, Thien HV, Grimprel E, Petit JC. Prevalence and genetic characterization of toxin A variant strains of Clostridium difficile among adults and children with diarrhea in France. J Clin Microbiol 2002;40: al Barrak A, Embil J, Dyck B, et al. An outbreak of toxin A negative, toxin B positive Clostridium difficileassociated diarrhea in a Canadian tertiary-care hospital. Can Commun Dis Rep 1999;25: Kuijper EJ, de Weerdt J, Kato H, et al. Nosocomial outbreak of Clostridium difficile-associated diarrhoea due to a clindamycin-resistant enterotoxin A-negative strain. Eur J Clin Microbiol Infect Dis 2001;20: Savidge TC, Pan WH, Newman P, O brien M, Anton PM, Pothoulakis C. Clostridium difficile toxin B is an inflammatory enterotoxin in human intestine. Gastroenterology 2003;125: Riegler M, Sedivy R, Pothoulakis C, et al. Clostridium difficile toxin B is more potent than toxin A in damaging human colonic epithelium in vitro. J Clin Invest 1995;95: 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: Borriello SP, Barclay FE. An in-vitro model of colonisation resistance to Clostridium difficile infection. J Med Microbiol 1986;21: Borriello SP. The influence of the normal flora on Clostridium difficile colonisation of the gut. Ann Med 1990;22: Murray PR, Rosenthal KS, Kobayashi GS, Pfaller MA. Clostridium difficile. In: Murray PR, Rosenthal KS, Kobayashi GS, Pfaller MA, eds. Medical Microbiology. Mosby-Year Book, 1998: Spigaglia P, Mastrantonio P. Molecular analysis of the pathogenicity locus and polymorphism in the putative negative regulator of toxin production (TcdC) among Clostridium difficile clinical isolates. J Clin Microbiol 2002;40: Cooper GS, Lederman MM, Salata RA. A predictive

11 Vol. 24 No. 3 Treatment of C. difficile in the era of hypervirulence:- Apisarnthanarak A & Mundy LM. 161 model to identify Clostridium difficile toxin in hospitalized patients with diarrhea. Am J Gastroenterol 1996;91: Warny M, Vaerman JP, Avesani V, Delmee M. Human antibody response to Clostridium difficile toxin A in relation to clinical course of infection. Infect Immun 1994;62: Kyne L, Warny M, Qamar A, Kelly CP. Association between antibody response to toxin A and protection against recurrent Clostridium difficile diarrhoea. Lancet 2001;357: Kyne L, Warny M, Qamar A, Kelly CP. Asymptomatic carriage of Clostridium difficile and serum levels of IgG antibody against toxin A. N Engl J Med 2000;342: Leung DY, Kelly CP, Boguniewicz M, Pothoulakis C, LaMont JT, Flores A. Treatment with intravenously administered gamma globulin of chronic relapsing colitis induced by Clostridium difficile toxin. J Pediatr 1991;118: Kim PH, Iaconis JP, Rolfe RD. Immunization of adult hamsters against Clostridium difficile-associated ileocecitis and transfer of protection to infant hamsters. Infect Immun 1987;55: 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: Pepin J, Alary ME, Valiquette L, et al. Increasing risk of relapse after treatment of Clostridium difficile colitis in Quebec, Canada. Clin Infect Dis 2005;40: Musher DM, Aslam S, Logan N, et al. Relatively poor outcome after treatment of Clostridium difficile colitis with metronidazole. Clin Infect Dis 2005;40: Jang SS, Hansen LM, Breher JE, et al. Antimicrobial susceptibilities of equine isolates of Clostridium difficile and molecular characterization of metronidazole-resistant strains. Clin Infect Dis 1997;25 (Suppl 2):S266-S Bolton RP, Culshaw MA. Faecal metronidazole concentrations during oral and intravenous therapy for antibiotic associated colitis due to Clostridium difficile. Gut 1986;27: Barbut F, Decre D, Burghoffer B, et al. Antimicrobial susceptibilities and serogroups of clinical strains of Clostridium difficile isolated in France in 1991 and Antimicrob Agents Chemother 1999;43: Wong SS, Woo PC, Luk WK, Yuen KY. Susceptibility testing of Clostridium difficile against metronidazole and vancomycin by disk diffusion and Etest. Diagn Microbiol Infect Dis 1999;34: Pelaez T, Alcala L, Alonso R, Rodriguez-Creixems M, Garcia-Lechuz JM, Bouza E. Reassessment of Clostridium difficile susceptibility to metronidazole and vancomycin. Antimicrob Agents Chemother 2002;46: Browne RA, Fekety R Jr, Silva J Jr, Boyd DI, Work CO, Abrams GD. The protective effect of vancomycin on clindamycin-induced colitis in hamsters. Johns Hopkins Med J 1977;141: Geraci JE, Heilman FR, Nichols DR, Ross GT, Wellman WE. Some laboratory and clinical experiences with a new antibiotic, vancomycin. Proc Staff Meet Mayo Clin 1956;31: Wilcox MH. Descriptive study of intravenous immunoglobulin for the treatment of recurrent Clostridium difficile diarrhoea. J Antimicrob Chemother 2004;53: McPherson S, Rees CJ, Ellis R, Soo S, Panter SJ. Intravenous immunoglobulin for the treatment of severe, refractory, and recurrent Clostridium difficile diarrhea. Dis Colon Rectum 2006;49: Beales IL. Intravenous immunoglobulin for recurrent Clostridium difficile diarrhoea. Gut 2002;51: Bartlett JG. New drug for Clostridium difficile infection. Clin Infect Dis 2006;43: Apisarnthanarak A, Razavi B, Mundy LM. Adjunctive 161

12 162 J INFECT DIS ANTIMICROB AGENTS Sep.-Dec intracolonic vancomycin for severe Clostridium difficile colitis: case series and review of the literature. Clin Infect Dis 2002;35: Apisarnthanarak A, Khoury H, Reinus WR, Crippin JS, Mundy LM. Severe Clostridium difficile colitis: the role of intracolonic vancomycin? Am J Med 2002;112: Lipsett PA, Samantaray DK, Tam ML, Bartlett JG, Lillemoe KD. Pseudomembranous colitis: a surgical disease? Surgery 1994;116: Longo WE, Mazuski JE, Virgo KS, Lee P, Bahadursingh AN, Johnson FE. Outcome after colectomy for Clostridium difficile colitis. Dis Colon Rectum 2004;47: Aas J, Gessert CE, Bakken JS. Recurrent Clostridium difficile colitis: case series involving 18 patients treated with donor stool administered via a nasogastric tube. Clin Infect Dis 2003;36: Schwan A, Sjolin S, Trottestam U, Aronsson B. Relapsing Clostridium difficile enterocolitis cured by rectal infusion of normal faeces. Scand J Infect Dis 1984;16: Torres JF, Lyerly DM, Hill JE, Monath TP. Evaluation of formalin-inactivated Clostridium difficile vaccines administered by parenteral and mucosal routes of immunization in hamsters. Infect Immun 1995;63: Ryan ET, Butterton JR, Smith RN, Carroll PA, Crean TI, Calderwood SB. Protective immunity against Clostridium difficile toxin A induced by oral immunization with a live, attenuated Vibrio cholerae vector strain. Infect Immun 1997;65: Ward SJ, Douce G, Figueiredo D, Dougan G, Wren BW. Immunogenicity of a Salmonella typhimurium aroa arod vaccine expressing a nontoxic domain of Clostridium difficile toxin A. Infect Immun 1999;67: Pavliakova D, Moncrief JS, Lyerly DM, et al. Clostridium difficile recombinant toxin A repeating units as a carrier protein for conjugate vaccines: studies of pneumococcal type 14, Escherichia coli K1, and Shigella flexneri type 2a polysaccharides in mice. Infect Immun 2000;68: Aboudola S, Kotloff KL, Kyne L, et al. Clostridium difficile vaccine and serum immunoglobulin G antibody response to toxin A. Infect Immun 2003;71: Sougioultzis S, Kyne L, Drudy D, et al. Clostridium difficile toxoid vaccine in recurrent C. difficileassociated diarrhea. Gastroenterology 2005;128: