1. MICR 420 Emerging and Re-Emerging Infectious Diseases Lecture 4: C. difficile Dr. Nancy McQueen & Dr. Edith Porter

2. Overview Morphology Growth and metabolic characteristics Virulence factors including toxins Diseases Diagnosis Endoscopy Culturing Cytotoxicity Assays for toxins Therapy Threats Clostridium difficile

3. Cycloserine-cefoxitin-egg yolk- fructose agar

4. C. difficile:Morphology and Metabolic Characteristics Large Gram-positive anaerobic rods Spore producer Saccharolytic and proteolytic GLC Negative for lipase Negative for lecithinase Smells like a horse stable Ubiquitous in nature May be found in the vagina, urethra, or stool of healthy individuals

5. C. difficile: Virulence Characteristiccs Capsule Flagella Produces two exotoxins Enterotoxin A (TcdA) Cytotoxin B (TcdB) Both toxins act as glycosyltransferases Modify and inactivate Rho, Rac, and Cdc42 proteins  Fluid accumulation  Inflammatory response  Cell death  Pseudomembranous plaque formation

6. Mechanisms of action of TcdA and TcdB Voth, D. and Ballard, J. (2005)Clostridium difficile toxins: Mechanism of Action and Role in Disease (2005). Clinical Microbiology Reviews. 18 (2); 247-263

7. C. difficile: Diseases Nosocomial antibiotic-associated infection (80% of cases) Community acquired infection (beginning in 2005 – now 20% of cases) Asymptomatic carriage Watery diarrhea Colitis Pseudomembranous colitis Paralytic ileus and toxic megacolon death

8. Sunenshine, R and McDonald, L. (2006) Clostridium difficile- associated disease: New challenges from an established pathogen. Cleveland Clinic Journal of Medicine. 73(2): 187-197

9. http://www.health-res.com/EX/07-28-04/37FF1.jpeg

10. http://www.cfpc.ca/cfp/2004/Nov/ _images/Fig0376_104_C.jpg http://www.gihealth.com/imag es/imgNormalColon.gif Colonic pseudomembranous plaques

11. Immunochemical stain showing presence of C. difficile in pseudomembrane Sunenshine, R and McDonald, L. (2006) Clostridium difficile- associated disease: New challenges from an established pathogen. Cleveland Clinic Journal of Medicine. 73(2): 187- 197

12. C. difficile: Diagnosis Sunenshine, R and McDonald, L. (2006) Clostridium difficile- associated disease: New challenges from an established pathogen. Cleveland Clinic Journal of Medicine. 73(2): 187-197 More on this later Advantages and disadvantages of diagnostic testing methods for C. difficile Diagnostic testTurn-around timeSensitivityAdvantagesDisadvantages Endoscopy2 hours51%Diagnostic of pseudomembranous colitis Low sensitivity Anaerobic culture72 hours89%-100%Results useful for molecular typing Does not distinguish toxin-producing strains Tissue toxicity assay48 hours94%-100%Detects A-B+ strains Gold standard False positives. Results vary with experience of technologist Common antigen15-45 minutes58%-92%Detects A-B+ strains Easy to use Does not distinguish toxin producing strains. Cross reacts with other anaerobes. Enzyme-linked immunosorbent assay (ELISA)- toxin A 2 hours80%-95%Easy to useDoes not detect A-B+ strains ELISA – toxin A +B2 hours80%-95%Detects A-B+ strainsIncreased sensitivity for low level toxin production Immunochro- motographic toxin A <1 hour6-%-85%Simple to use Rapid Does not detect A-B+ strains

13. C. difficile:Therapy Stop inciting antibiotic Fluid and electrolyte replacement Metronidazole for mild disease Vancomycin for moderate disease Surgical consult and intraluminal vancomycin for severe disease (paralytic ileus, toxic megacolon, dehydration, or sepsis)

14. C. difficile:Threats Hospitalizations with a discharge diagnosis of C. difficile associated disease was 31 per 100,000 in 1996 and increased to 61 per 100,000 in 2003. From McDonald LC, et al. Emerg Infect Dis. 2006;12(3):409-15

15. C. difficile:Threats In 2000 a new strain of C. difficile (North American Pulse Field-type 1 – NAP1) was reported in hospitalized patients. This strain produces 16 times more toxin A and 23 times more toxin B than other strains From Warny M, et al. Lancet. 2005;366:1079-1084.

16. C. difficile:Threats MMWR. April 4, 2008. Surveillance for Community-Associated Clostridium difficile- Connecticut, 2006. Documents the presence of occasionally severe CDAD among healthy patients living in the community with no established risk factors for infection. Rodriguez-Palacios, A, et al. Clostridiunm difficile in retail ground meat, Canada Emerg, Infec Dis 2007:13:485-487. The NAP-1 strain of C. difficile has been detected in retail ground beef. It is not known how much C. difficile in food one would have to ingest to become sick. There are NO documented cases of people getting CDI from eating food that contains C. difficile. *Therefore, at this time, CDI is not thought to be a foodborne illness. C. difficile may be found in healthy companion animals such as horses, dogs, and cats. *There are no documented animal to human cases of CDI. *http://www.cdc.gov/ncidod/dhqp/id_Cdiff_in_meat.html (April 22, 2009)

17. Take Home Message C.difficile is ubiquitous and can be found as normal flora C. difficile causes antibiotic associated pseudomembraneous colitis C. difficile disease is caused by toxins Major public threat from new strains of C. difficile that produce substantially more toxin

18. Additional Resources ASM Microbe library Madigan & Matinko, Brock Biology of Microorganism, 11 th edition. www.cdc.org