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CLOSTRIDIUM DIFFICILE A Disease of the Antibiotic Era Resident Lecture 2010.

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Presentation on theme: "CLOSTRIDIUM DIFFICILE A Disease of the Antibiotic Era Resident Lecture 2010."— Presentation transcript:

1 CLOSTRIDIUM DIFFICILE A Disease of the Antibiotic Era Resident Lecture 2010

2 Objectives Learn the Microbiology of C. difficile Understand the Pathogenesis of Disease Introduction to the Epidemiology of Infection by this organism Overview of Advances in Diagnostic Testing

3 Wolf, P. L. et al. N Engl J Med 2005;353:2491 A 75-year-old man with peripheral vascular disease, chronic obstructive pulmonary disease, and diabetes mellitus presented with gangrene of the right fourth toe and underwent tarsal- metatarsal amputation to manage the progression of gangrene, followed by amputation of the right leg below the knee

4 C. difficile Strict anaerobe Gram variable rod Subterminal elongated spores Irregular, flat, rough colonies Fluoresce chartreuse




8 Isolation Media CCFA –2.5% egg yolk –Fructose –Neutral red –8-16 ugm/L cefoxitin –250-500 mgm/L cycloserine BA with lysed horse blood –Opaque grey-white colonies –Larger than when egg yolk is used –Fluoresce a green-yellow colour TCCFA –0.1% Na taurocholate –Enhances sporulation CDMN Selective Agar –Cysteine hydrochloride –Norfloxacin –Moxalactam –C. difficile grows better –Fecal Flora more inhibited –May eliminate the need for a selective broth Buchanen’s broth –CMC –Cefoxitin –Cycloserine

9 C. difficile Laboratory Identification GLC Acetic Acid Butyric Acid Isobutyric Acid Isovaleric Acid Isocaproic Acid

10 C. difficile Laboratory Identification Biochemicals C. difficile C. sporogenes Glucose +W+W+W+WV Mannitol WAWAWAWA- Milk- D (digested) Neya Lecithinase - -+-+-+-+ Lipase-+ Fructose AWAWAWAW -W-W-W-W

11 C. Difficile –Rapid ID RapID ANA –4 hour enzyme detection system –10 reaction cavities –18 tests Latex Agglutination

12 Toxins Produced by C. difficile Toxin A Toxin B Molecular Wt. 550,000360,000 Stability (-20 0 C. – 37 0 C.) 4 weeks Stability (56 0 C) <6 minutes Cytotoxicity (CHO cells) 3.8 X 10 6 cu/mgm. 5.3 X 10 9 cu/ mgm. Fluid accumulation in infant mice > 0.25 ugm. > 2.5 ugm. Death of infant mice None at 5 ugm. 50% at 1-5 ugm. Rabbit ileal loop Viscous hemorrhagic fluid response Variable fluid response

13 C. difficile Pathogenicity Locus Clinical Microbiology Reviews 18: 247-263


15 TcdA and TcdB Glucosyltransferases Inactivate Rho, Rac and Cdc42 Actin condensation Rounding of cells Membrane blebbing Apoptosis

16 Virulence I Bind to specific receptors on the colonic epithelium Transported into the cytoplasm Both toxins inactivate Rho proteins –GTP-binding proteins important in actin polymerization, cytoskeletal architecture and cell movement Disruption of the actin cytoskeleton Disruption of epithelial cell tight-junction proteins Defective pathogenicity locus may cause disease –22 Toxinotypes to date

17 Virluence II Production of inflammatory mediators –Macrophage inflammatory protein-2 (MIP-2) –Activate sensory nerves –Sensory nerves release pro-inflammatory neuropeptides Substance P and CGRP Stimulate inflammatory cells to release pro- inflammatory cytokines Neutrophil recruitment

18 Clinical Microbiology Reviews 18: 247-263

19 Binary Toxin Encoded by CDTa and CDTb 2 component ADP-ribosyltransferase activity >80% homology with C. perfringens iota toxin In 6.4% of isolates from the UK Due to genetic exchanges among several clostridial species Appears to enhance pathogenicity of NAP 1

20 C. Difficile Toxin Assay W. I. 38 cells Stool in MEM with antibiotics 12,800 g for 10 minutes 0.2 ml. on tissue culture C. difficile antitoxin



23 Epidemiology of C. difficile Normal fecal flora of newborns 2% of healthy adults 10-20% of elderly individuals colonized 20-40% of hospitalized patients colonized Nosocomial clusters Isolated from toilets, bedpans, floors Spores isolated for up to 6 months

24 Consequences of Ingestion of C. difficile Excretion Asymptomatic colonization of gut DiarrhoeaColitis Pseudomembranous colitis Death

25 Antibiotic Associated Colitis 3-5 loose stools per day No alternative cause Onset during or after antimicrobial administration –Erythromycin stimulates gut peristalsis Wide specturm

26 Pseudomembranous colitis Loose watery stools 8-11 days Prolonged if –Onset after antibiotics stopped –Antibiotics continued after onset

27 C. Difficile Infection CDI Diagnosis must be confirmed by laboratory tests Spectrum the same as AAD Not all AAD is CDI

28 Clinical Presentation Diarrhea within a few days of antibiotic therapy Faeces have a distinctive foul odour Abdominal pain +/- pyrexia Blood if pseudomembranous colitis Electrolyte disturbances Hyoalbuminaemia Paralytic ileus Toxic Megacolon/perforation/shock Increased WBC


30 PMC Microscopic Appearance

31 PMC Low Power Microscopic

32 Other Organisms Implicated in AAD Enterotoxigenic Clostridium perfringens –8% of cases in one study (Wilcox et al) Staphylococcus aureus –1/6 cases (Graves et al) –92% MRSA –9 bacteraemic –Fluroquinolones involved –Leukotoxins and Staphylococcal enterotoxin A

33 Antibiotic-related risk of C. difficile Infection High Risk Medium Risk Low Risk CephalosporinsMacrolidesAminoglycosides ClindamycinCo-trimoxazoleMetronidazole Amipicillin/amoxycillinTetracyclines Anti-pseudomonal penicillins FluoroquinolonesRifampicin Vancomycin

34 Antibiotic Risk Determinants Frequency of use Age Environmental contamination Association with an outbreak Combination of antimicrobials Duration of therapy –<3 days –Surgical prophylaxis


36 Microorganisms Which Inhibit the Growth of C. difficile Lactobacillus species Group D Streptococci Staphylococcus aureus Bacteroides species Bifidobacteria

37 Antineoplastic Agents Direct inflammatory effects of cytotoxic agents Neutropenia Agents implicated –Adriamycin –Cyclophosphamide –5-Fluorouricil –Bleomycin –Melphalan –Dichloromethotrexate

38 Immune Response 70% of people have antibody Patients who are colonized on admission are less likely to develop symptomatic infection Asymptomatic carriers had greater increases in serum IgG antitoxin A than those with diarrhoea Higher levels of serum IgM and IgG antitoxin A in those with one episode, vs. multiple episodes

39 Epidemiological Update Changes in antimicrobial drug use –Fluroquinolones Alcohol-based waterless, hand sanitizers New more virulent strains –Hyperproduction of toxins A and B Cut-backs in environmental hygeine

40 JID 2004:189 (1585-1589)

41 EID 12: 409-415.2006


43 Role of Aging Increased exposure to healthcare facilities Increased use of antimicrobials Use of histamine-2 receptor blockers or proton-pump inhibitors Decreased host defenses –Achlorhydria –Decreased immune responsiveness

44 N-CDAD in Canada: The CNISP Study 19 Health Care Facilities in 8 provinces Toxin assay or Toxin + culture Case: Acute onset of loose stools in a patient that was positive for C. difficile culture/toxin that persisted for at least 2 days, without an alternative explanation for diarrhea. Six continuous weeks/200 consecutive diarrhea specimens Jan. to April, 1997

45 CNISP Results 371 Potential Cases 269 met case definition of N-CDAD 13.0% of patients with diarrhea had N- CDAD 250 cases occurred >3 days after admission 19 patients readmitted for diarrhea within 1 month of discharge Can J Infect Dis 12: 82-88

46 CNISP Results II Incidence density was 66.3 cases/100,000 patient days and 5.9 cases/1000 patient admissions Mean # of N-CDAD per institution in 6 weeks was 13.7. Patients not treated with antibiotics were more likely to die than those that were treated (OR 2.69) 41 patients with CDAD died (15%) 4 died because of N-CDAD directly or indirectly Can J Infect Dis 12: 82-88

47 Copyright ©2004 CMA Media Inc. or its licensors Pepin, J. et al. CMAJ 2004;171:466-472 Fig. 1: Annual incidence (per 100 000 population) of Clostridium difficile-associated diarrhea (CDAD) in Sherbrooke, Que., 1991-2003

48 Copyright ©2004 CMA Media Inc. or its licensors Pepin, J. et al. CMAJ 2004;171:466-472 Table 1.

49 Copyright ©2004 CMA Media Inc. or its licensors Pepin, J. et al. CMAJ 2004;171:466-472 Fig. 2: Proportions of patients with CDAD by class of antibiotic received in the 2 months preceding the diagnosis of CDAD, 1991-2003

50 Loo, V. G. et al. N Engl J Med 2005;353:2442-2449 Age-Specific Incidence and Mortality Attributed to Clostridium difficile-Associated Diarrhea

51 Characteristics of the Highly Virulent Strain 1400 deaths in Quebec since 2003 PFGE: NAP 1 strain Toxinotype III Ribotype 027 Partial deletion of the tcdC gene Levels of Toxins A and B are 16 to 23 times higher than previous toxinotypes Produced Binary toxin Resistant to fluoroquinolones

52 CNISP Study 2004-5 Incidence 6 per 1000 hospital admissions NAP 1 strain preset in 8 provinces Mortality rate rose from 1.5% to 5.8% since 1997 NAP 1 strain 2.3 times more likely to have a serious outcome

53 Copyright ©2006 CMA Media Inc. or its licensors Eggertson, L. CMAJ 2006;174:607-a-608-a Fig. 1: Case-fatality rates among patients with Clostridium difficile-associated disease (CDAD) from Nov

54 Diagnosis of C. difficile Associated Diarrhea Tissue Culture Cytotoxin Assay the gold standard Toxigenic Culture –4-6 isolates to CMC –24 hr. filtrate for toxin detection Immunoassay for toxin detection Glutamate dehrydrogenase detection PCR

55 Controversies in Diagnosis Sensitivity of a single cytotoxin assay 79% Toxigenic culture may increase sensitivity to 96% Toxin positive, culture negative patients may progress to death Toxin A negative/B positive patients Repeat Testing: Is it necessary

56 Immunoassay Performance vs Cytotoxicity Assay TestSensitivitySpecificity Alexon-Trend Prospect II CD Toxin A 82-8590-100 Bartel Toxin A, Prima 8798 bioMerieux VIDAS Toxin A II 65-8599-100 Biosite Triage C. difficile Panel (antigen or toxin A) 68-10083-90 Meridian ImmunoCard Toxin A 65-9295-100 Meridian Premier Cytoclone CD Toxin A/B 77-8399-100 Meridian Toxin A 87-9497-99 Meridian Premier Toxins A and B 84-9597-00 TechLab/Wampole CD Tox A/B 80-9499-100

57 Immunoassay Performance vs Cytotoxic Culture AuthourTestSensitivitySpecificity Alcala et al Tox A/B (Remel) 49%95.8% Tox A/B (Quick Chek) 54.9%95.5% ImmunoCard A&B 66.7%95.1% Ticehurst et al Tox A/B II EIA 38%100% Fenner et al Tox A/B II EIA 50.7%99.4%

58 Commercial PCR Assays AuthourTestSensitivitySpecificity Stamper BD GeneOhm 83.6%98.2% Kvach 91.4%100% Novak-Weekly Xpert C. difficile assay 94.4%96.3% Swindells et al BD GeneOhm 100%97% Xpert C. difficile Assay100%97% Stamper ProGastro Cd assay 77.394.4

59 Testing Frequency Aichinger: Patients with >2 tests in 7 days –Repeat EIA: Increase yield by 1.9% –Repeat PCR: Increase yield by 1.7% Cardona: 8,256 tests from 3, 112 patients –49% of tests repeated Day % Positive 00.9 11.8 23.8 32.6 4-65.4 7-1010.6

60 Methods to Subtype C. difficile Restriction Endonuclease Analysis –Many subtypes –Outbreaks divided 50/50 between the outbreak strain and others –Requires expertise to read the gels –Time consuming

61 Methods to Subtype C. difficile Pulse Field Gel electrophoresis –Some strains autodigest and not typable using this method –Thiourea decreases the amount of autodigestion of DNA AP-PCR –Non-specific amplifications of segments of the bacterial chromosome by PCR using short primers under low-stringency conditions –Difficulties with reproducibility PCR-ribotyping –Specific primer complimentary to the 3’ end of the 16S rRNA gene and to the 5’ end of the 23 S rRNA gene –Variable-length intergenic spacer regions of up to 20 16-23 S rRNA gene pairs

62 Treatment of C. difficile Associated Colitis Discontinue preciptitating antibiotics Oral Vancomycin 125 mgm qds for 7-10 d Oral Metronidazole 400-500 mg tds for 7- 10d IV give both antibiotics together Recent reports of resistance to metronidazole

63 Treatment of C. difficile Associated Colitis Relapses common ¼ of symptom-free patients continue to excrete C. difficile in their faeces Relapses are often re-infections with different strains

64 Treatment of C. difficile Associated Colitis S. boulardii –Evidence mixed –Cases of fungaemia in immunocompromised patients –Virulence differs with the source Other agents –Lactobacillus acidophilus –Lactobacillus GG –Non-toxigenic C. difficile

65 Prevention of Infection Role of restrictive antibiotic policies Transmission via contaminated hands –Gloves –Antiseptic soaps –Alcohol-based hand rinse Transmission via the environment –Role of Na hypochlorite vs. Quaternary ammonium compounds

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