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

MICR 420

Emerging and Re-EmergingInfectious Diseases

Lecture 4:C. difficile

Dr. Nancy McQueen & Dr. Edith Porter

Overview

Morphology Growth and metabolic characteristics Virulence factors including toxins Diseases Diagnosis

Endoscopy Culturing Cytotoxicity Assays for toxins

Therapy Threats

Clostridium difficile

Clostridium difficile

Cycloserine-cefoxitin-egg yolk-fructose agar

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

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

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

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

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

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

http://www.cfpc.ca/cfp/2004/Nov/_images/Fig0376_104_C.jpg

http://www.gihealth.com/images/imgNormalColon.gif

Colonic pseudomembranous plaques

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

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 test Turn-around time Sensitivity Advantages Disadvantages

Endoscopy 2 hours 51% Diagnostic of pseudomembranous colitis

Low sensitivity

Anaerobic culture 72 hours 89%-100% Results useful for molecular typing

Does not distinguish toxin-producing strains

Tissue toxicity assay 48 hours 94%-100% Detects A-B+ strainsGold standard

False positives. Results vary with experience of technologist

Common antigen 15-45 minutes 58%-92% Detects A-B+ strainsEasy to use

Does not distinguish toxin producing strains. Cross reacts with other anaerobes.

Enzyme-linked immunosorbent assay (ELISA)- toxin A

2 hours 80%-95% Easy to use Does not detect A-B+ strains

ELISA – toxin A +B 2 hours 80%-95% Detects A-B+ strains Increased sensitivity for low level toxin production

Immunochro-motographic toxin A

<1 hour 6-%-85% Simple to useRapid

Does not detect A-B+ strains

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)

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

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.

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)

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

Additional Resources

ASM Microbe library Madigan & Matinko, Brock Biology of

Microorganism, 11th edition. www.cdc.org