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Clostridium difficile

References

Pathogenesis

Spectrum of Disease

Pathogenesis

Asymptomatic carrier

Ingestion

Diarrhea

Colitis

Pseudomembranous Colitis

Colonization

Colonic

Steps of Infection

Source: http://www.sickchirpse.com/wp-content/uploads/2013/01/C.-difficile.jpg

Bacteremia

Fulminant Colitis

Regulation

1) Surawicz CM, Brandt LJ, Binion DG, Ananthakrishnan AN, Curry SR, Gilligan PH, McFarland LV, Mellow M, Zuckerbraun BS. Guidelines for Diagnosis, Treatment, and Prevention of Clostridium difficile Infections. Am J Gastroenterol. Forthcoming 2013 Feb 26.

2) Fogliaa G, Shaha S, Luxemburgerb C, Pietrobona PJF. Clostridium difficile: Development of a novel candidate vaccine. Vaccine. 2012 June 19;30(29):4307-4309.

3) Rebeaud F, Bachmann MF. Immunization strategies for Clostridium difficile infections. Expert Review of Vaccines. 2012 April;11(4):469-479.

4) Soares GMS, Figueiredo LC, Faveri M, Cortelli SC, Duarte PM, Feres M. Mechanisms of action of systemic antibiotics used in periodontal treatment and mechanisms of bacterial resistance to these drugs. J Appl Oral Sci. 2012 May/June;20(3):295-309.

5) Nailor MD, Sobel JD. Antibiotics for Gram-Positive Bacterial Infection: Vancomycin, Teicoplanin, Quinupristin/Dalfopristin, Oxazolidinones, Daptomycin, Telavancin, and Ceftaroline. Medical Clinics of North America. 2011 July;95(4):723-742.

6) Ford M. Medical Microbiology. New York: Oxford University Press; 2010.

7) Peppler M. Immunological Memory & Vaccines [unpublished lecture notes]. MLSCI 242: Clinical Microbiology, University of Alberta; lecture given 2012 Sep 28.

8) Madan R, Petri WA. Immune responses to Clostridium difficile infection. Trends Mol Med [serial on the Internet]. 2012 Nov [cited 2013 Feb 21]; 18(11): 658-66. Available from: PubMed.

9) Aboudola S, Kotloff KL, Kyne L, Warny M, Kelly EC, Sougioultzis S, et al. Clostridium difficile vaccine and serum immunoglobulin G antibody response to toxin A. Infect Immun [serial on the Internet]. 2003 Mar [cited 2013 Feb 21]; 71(3): 1608-10. Available from: PubMed.

10) Kyne L, Warny M, Qamar A, Kelly CP. Association between antibody response to toxin A and protection against recurrent Clostridium difficile diarrhoea. The Lancet [serial on the Internet]. 2001 Jan 20 [cited 2013 Feb 21]; 357: 189-93. Available from: PubMed.

11) Peppler M. Microbe Cards. Canada: ASM Press; 2003.

12) Vaishnavi C. Clinical spectrum and pathogenesis of Clostridium difficile associated diseases. Indian J Res Med [serial on the Internet]. 2010 Apr [cited 2013 Mar 22]; 131:487-99. Available from: PubMed

13) Shen A. Clostridium difficile toxins: mediators of inflammation. J Innate Immun [serial on the Internet]. 2012 Feb [cited 2013 Mar 22]; 4(2): 149-158. Available from: PubMed

14) Boice JL. Reactive arthritis induced by Clostridium difficile. West J Med [serial on the Internet]. 1994 Feb [cited 2013 Mar 22]; 160(2): 171-2. Available from; PubMed

15) Voth DE, Ballard JD. Clostridium difficile toxins: mechanism of action and role in disease. Clin Microbiol Rev [serial on the Internet]. 2005 Apr [cited 2013 Mar 22]; 18(2): 247-263. Available from: PubMed

16) Incidence of and risk factors for community-associated Clostridium difficile infection: A nested case-control study. BMC Infectious Diseases [serial on the Internet]. (2011, Jan), [cited March 24, 2013]; 11(1): 194-200. Available from: Academic Search Complete.

17) Jennifer R. O, Stuart J, Dale N. G. Clostridium difficile Infection Caused by the Epidemic BI/NAP1/027 Strain. Gastroenterology [serial on the Internet]. (n.d.), [cited April 5, 2013]; 136(Intestinal Microbes in Health and Disease): 1913-1924. Available from: ScienceDirect.

18) CDC’s Frequently Asked Questions about Clostridium difficile for Healthcare Providers [Internet]. Atlanta, USA: Centers for Disease Control and Prevention; 2010 [updated 2012 Mar 6; cited 2013 Apr 6]. Available from: http://www.cdc.gov/hai/organisms/cdiff/cdiff_faqs_hcp.html

Reactive Arthritis

Mechanism

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3388264/

Toxins

Small Bowel

Extracolonic

Effects

Pathogenesis

Colonic Involvement

Toxin Regulation

Pathogenesis

Ingestion

http://cmr.asm.org/content/18/2/247/F1.expansion.html

  • vegetative cells
  • destroyed by stomach acid
  • spores
  • survive in the environment
  • germination stimulated by bile salts in small intestine
  • TcdA
  • enterotoxin
  • ABCD structure

  • TcdB
  • cytotoxin
  • ABCD structure

  • TcdC
  • negative regulator
  • decline in expression correlates to increased expression of TcdA and TcdB

  • TcdD
  • positive regulator
  • expressed in response to environmental conditions
  • increased in stationary phase

http://radiographics.rsna.org/content/20/2/399/F39.expansion.html

  • Asymptomatic carrier
  • contribute to disease transmission

  • Diarrhea
  • toxins present in feces
  • normal endoscopic and histological features

  • Colitis
  • most common clinical manifestation
  • malaise, diarrhea, abdominal pain, nausea, anorexia

  • Pseudomembranous Colitis
  • raised yellow plaques over colorectal mucosa

  • Fulminant Colitis
  • rapidly progressing
  • toxic megacolon, perforation, death

Colonization

  • normal intestinal flora disrupted
  • provides access to mucosal surface
  • adherence
  • Surface Layer Proteins (SLP's)
  • flagellin Fli C and Fli D
  • Cwp 66
  • Fbp68
  • s-layer protein
  • C diff Transferase (CDT)

Pathogenesis

Final points on C. diff

Extracolonic Involvement

Pathogenesis

Toxin Effects

Mechanism of TcdA and TcdB

  • inactivation of Rho causes
  • disaggregation of actin cytoskeleton
  • cell rounding
  • apoptosis

  • other toxic effects
  • chemokine and cytokine production
  • tight junction disruption
  • neutrophil infiltration
  • pseudomembrane formation
  • increased cell permeability causing diarrhea
  • Antibiotic treatment, hospitalisation and comorbidities are key risk factors
  • Can cause asymptomatic colonization to persistent diarrhea to severe colon disease
  • Toxins A and B are the main virulence factors
  • Diagnose by EIA for toxins in stool
  • Treat with antibiotics, fecal microbiota transplant or surgery
  • Prevent with antibiotic stewardship and hospital infection control practices (vaccines are in the works!)
  • Bacteremia
  • usually polymicrobial infection
  • positive blood culture

  • Reactive Arthritis
  • possibly due to antibody cross-reactivity with synovial tissue

  • Small Bowel
  • pseudomembranes on ileal mucosa
  • chances increased with surgical procedures like colectomy

http://www.nature.com/nrmicro/journal/v9/n7/full/nrmicro2592.html

  • ABCD structure
  • A= active
  • B= binding
  • C= cutting
  • D= delivery

  • Binding of B subunit
  • triggers clathrin-dependent receptor-mediated endocytosis

  • Acidification of endosome
  • induces conformational changes
  • pore formation by D domain
  • translocation, cleavage, and release of A domain into cytosol

  • Glucosylation
  • A domain glucosylates Rho GTPases
  • inactivates Rho causing pathogenic effects

http://cmr.asm.org/content/18/2/247.long

Our patient

  • 80-year-old woman recovering from bladder surgery

  • Took 7 days of oral cephalexin to treat UTI

  • After cephalexin: watery diarrhea, abdominal pain

  • No Salmonella, Shigella, Campylobacter or Yersinia in stool

  • EIA for Clostridium difficile toxin in stool was positive

Humoral Immunity

IgG anti-toxin A antibodies

Innate Immunity

A Double Edged Sword

Immunity

http://bitchspot.jadedragononline.com/wp-content/uploads/2012/07/double-edged-sword.jpg

  • Associated with protection against Clostridium difficile diarrhea
  • High levels of Ab are found in:
  • C. difficile symptom-free carriers,
  • Patients with a single episode of C. diff associated diarrhea (CDAD)

Innate Immunity

  • Role in prevention:
  • Includes normal gut flora, intact mucus barrier, healthy enteric epithelial cells

Immune Responses

  • However, patients with recurring CDAD had very low levels of Ab

Humoral Immunity

  • Can also cause damage:
  • Stimulation of the innate immune system to release cytokines and attract neutrophils and mast cells
  • Leads to inflammation, cell damage, tissue destruction

Single vs. Recurrent

CDAD

Levels of Protection

http://www.biospectrumasia.com/IMG/562/9562/antibody-r-d-alliances-is-the-way-forward-262x174.jpg

Practical Applications

Why do patients with recurrent CDAD have a lower

Antibody Titre?

  • Major risk factor for recurrent CDAD is the patient is unable to mount an immune response against Toxin A
  • Patients more likely to be older and sicker
  • More likely to receive a higher dose of antibiotics
  • Have a higher death rate

Control

Prevention - Barrier nursing

Prevention - Environmental removal

Environmental removal

Single-use disposable equipment :

  • example = rectal and oral thermometers

Barrier nursing

Prevention

Contact precautions :

  • Don and doff gowns and gloves
  • HAND-WASHING: most significant and cost-effective method
  • alcohol-based hand disinfectants have poor activity against spores.
  • Soap and water effective on both organism & its spores.

http://www.fitsugar.com/How-Wash-Hands-Prevent-Colds-27889115

Antibiotic stewardship

http://www.guvestindia.com/oral-rectal-underarm-thermometer.htm

Initial Infection and Immunization

Epidemiology

Non-disposable medical equipment :

  • dedicated to the patient's room
  • equipment thoroughly cleaned after use

Antibiotics

Cohorting :

  • placing a patient or patients with the exact same infection, into a separate room.

Incidence

Fecal microbiota transplant

http://www.cleanlink.com/productwatch/details/Activate-Bleach-Dilution-System--4074

  • Most common cause of nosocomial infectious diarrhea

  • Incidence and severity on the rise

Treatment

Disinfection of environmental surfaces :

  • Environmental Protective Agency (EPA)-registered disinfectant with C. difficile sporicidal label claim
  • 5000 p.p.m. chlorine-containing cleaning agents

These isolation precaution measures serve to minimize further spread of infection to staff or other patients.

Vaccines

http://nwitimes.com/app/gethealthy/?p=8385

Hypervirulent strains

Epidemiology

Surgery

An initial infection of C. diff can produce an antibody response to Toxin A, leading to reduced recurrence and lasting immunity to some degree.

This immune response is the basis for developing immunizations against C. diff to prevent its onset and recurrence.

More on this later..

Risk factors

C. difficile incidence figures for England and Wales. Figures from The Health Protection Agency.

Stephen T. C, John T. H, Sarah A. K, Alan C, Nigel P. M. Mini Review: The emergence of ‘hypervirulence’ in Clostridium difficile. International Journal Of Medical Microbiology [serial on the Internet]. (n.d.), [cited April 5, 2013]; 300(Nosocomial Infections): 387-395.

Treatment - Antibiotics

Treatment - Fecal microbiota (FMT)

Prevention - Antibiotic stewardship

http://healthwise-everythinghealth.blogspot.ca/2013/02/fecal-microbiota-transplants.html

http://4.bp.blogspot.com/-_qUGWI2V4Oo/TcQkyy6G2LI

/AAAAAAAAAEo/NyGGvsFek3I/s1600/Vaccines-full.jpg

Metronidazole Vancomycin Fidaxomicin

Patients with mild-to-moderate CDI

Similar efficacy as vancomycin

More efficacious in preventing recurrence

Newer drug (2011)

But... cost is significantly higher than that of vancomycin

Limit antibiotics used :

  • Ab's most strongly associated with CDAD
  • Unnecessary antimicrobial prescribing, both in outbreak and non-outbreak settings.

Antibiotic use is the biggest risk factor for CDI.

Patients with severe & severe complicated CDI

Bactericidal activity via inhibition of bacterial cell wall synthesis

  • Complexes with the D-alanyl-D-alanine peptide precursor units, inhibiting peptidoglycan polymerase and transpeptidation reactions

FMT usually reserved if antibiotics fail

Healthy donor stool transplanted to stomach, small intestine, or colon of patients with RCDI.

  • Has the highest rate of success (90%) compared with results of other therapies
  • Restores phylogenetic richness and colonization resistance
  • Appears safe, with no adverse effects or complications directly attributed to the procedure

Case 25

Group 3

Requires metabolic activation by strict anaerobe microorganisms

  • nitroreductases + metronidazole

active metabolites which inhibit nucleic acid synthesis

  • Reactive nitro group causes lethal

breaks in DNA strands of targeted microbe

Risk factors

Epidemic C. diff BI/NAP1/027

http://2012.igem.org/wiki/index.php?title=Team:NTU-Taida/Project/Safety&oldid=295963

http://www.weatherimagery.com/blog/grey-market-and-the-internet/dollar-sign/

But... potential for transmission of infectious agents is a concern

http://www.rowett.ac.uk/edu_web/DNA2.html

http://www.biologie.uni-hamburg.de/b-online/library/micro229/terry/229sp00/lectures/cells2.html

http://www.hse.gov.uk/workplacetransport/safetysigns/warning.htm

http://www.nlm.nih.gov/medlineplus/antibiotics.html

[Sarah Aziz, John Leung, Shannon Porter, Albert Zuehlke]

  • Antibiotic use! (especially fluoroquinolones and cephalosporins)

  • Hospitalisation

  • Co-morbidities

  • Immunosupression

  • Advanced age
  • Notorious for causing hospital outbreaks

  • Deletion in tcdC leads to increased toxin A and B production

  • Binary toxin

  • Fluoroquinolone-resistant

http://www.clearclinic.com/wp-content/uploads/2013/03/antibiotics-for-acne-best-acne-treatment.jpg

Clostridium difficile Infection Caused by the Epidemic BI/NAP1/027 Strain.

Jennifer R. O, Stuart J, Dale N. G. Clostridium difficile Infection Caused by the Epidemic BI/NAP1/027 Strain. Gastroenterology [serial on the Internet]. (n.d.), [cited April 5, 2013]; 136(Intestinal Microbes in Health and Disease): 1913-1924.

Treatment - Vaccines (toxins A & B)

Treatment - Surgery

Treatment - Vaccines (bacterial colonization)

Toxoid vaccine DNA vaccine

Salvage therapy

  • Mortality rates from 35% to 80% - key improvements if done earlier
  • CDI usually involves the entire colon

Involves identifying factors controlling tissue invasion by C. difficile

  • 30+ cell wall proteins identified
  • Oligosaccharides on cell surface potential targets but highly strain specific
  • Capsular polysaccharides identified but needs testing for vaccine protection against clinical manifestations.

Several vaccines are in trials

Surgery considered for the following CDI patients:

  • hypotension requiring vasopressor therapy
  • clinical signs of sepsis and organ dysfunction (renal and pulmonary)
  • mental status changes
  • white blood cell count 50,000 cells/μl, lactate 5mmol/l
  • failure to improve on medical therapy after 5 days

Safe and immunogenic in healthy volunteers

Synthetic gene encoding the receptor-binding domain of C. difficile toxin A has been produced and optimized for expression in human cells.

  • DCs take up DNA, express it and present to B and T cells. Can produce CTL memory on top of B cell and Th cell memory.

Future development: Multicomponent vaccines that prevent colonization and neutralize toxin activity = synergistic protection!!!

Loop ileostomy + intraoperative colon lavage + post-operative colonic vancomycin flushes

Sanofi Pasteur’s formalin-inactivated, highly purified preparation of toxoids A and B

  • Phase II studies: assessing vaccine on symptomatic CDI adults & confirming the immunogenicity and safety of the vaccine, evaluating optimal dose, formulation, and immunization schedule

Why? Reduce # of asymptomatic carriers by immunizing against colonization

Diagnostic Tests

Rapid tests

http://www.colorectal-surgeon.com/stomas.htm

http://www.chiroone.net/commonConditions/crohns.html

colon preservation in >90% of patients + improved survival compared to historical colectomy patients

http://www.influenza.org.nz/?t=919

Rapid tests

  • Enzyme immunoassay for toxins

  • Rapid antigen detection

  • PCR for toxin B gene

Diagnosis

Culture tests

Sample considerations

http://www.immunochemistry.com/products/elisa-solutions-for-immunoassay-development-in-vitro-diagnostic-assay-manufacturing.html

An even more rapid test?!

Culture tests

Sample considerations

ONLY test patients with active diarrhea (we don't treat or isolate carriers)

For toxin EIA: toxins degrade after 2 hours at room temperature which can cause false negatives

  • Toxigenic stool culture

  • Tissue culture cytotoxicity assay

http://depts.washington.edu/molmicdx/mdx/tests/cdiff.shtml

http://www.thestar.com/life/2012/12/13/cliff_the_beagle_can_detect_c_difficile_just_by_sniffing_air_around_infected_patients.html

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