Objectives Clostridium difficile Infections, So Many Tests, Which One to Choose?

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1 Objectives Clostridium difficile Infections, So Many Tests, Which One to Choose? March 9, 0 Raymond P. Podzorski, Ph.D., D(ABMM) Clinical Microbiologist ProHealth Care Laboratories raymond.podzorski@phci.org Background for Clostridium difficile Compare various tests for C. difficile toxins Discuss various toxin testing algorithms Review the FDA approved molecular toxin tests Acceptable specimens for toxin testing Disclosure Clostridium difficile Anaerobic, Gram positive, spore forming, rod Raymond P. Podzorski, Ph.D., D(ABMM) March 9, 0 Speaker for Roche Diagnostics Corporation Produces exotoxins, A enterotoxin, B cytotoxin -3% of healthy adult GI flora Up to 70% of children less than months GI flora Up to 50% of individuals with exposure to inpatient health care facilities may be asymptomatic carriers of C. difficile 3 4 Clostridium difficile Clostridium difficile Accounts for 5-5% of all antibiotic-associated diarrhea Accounts for 95-00% of antibiotic associated pseudomembranous colitis Fecal-oral transmission contaminated environment hands of healthcare personnel 5 Pathogenicity Locus Binary toxin tcdd tcdb tcde tcda tcdc cdta cdtb Spigaglia P and Mastrantonio P. J Clin Microbiol. 00 Sep;40(9): MacCannell DR, et al. J Clin Microbiol 006; 44:

2 Risk Factors - Antimicrobial exposure prior -3 months Acquisition of C. difficile Healthcare exposure in prior -3 months Advanced age Underlying illness Immunosuppression Tube feeds? Gastric acid suppression 7 Advanced age Underlying illness Pathogenesis of Antimicrobial therapy Disturbed colonic microflora C. Difficile exposure and colonization Acquisition of toxigenic C. difficile Toxin B & Toxin A production due to recent (re)acquisition of C. difficile Incubation period unknown: <7 days to several weeks? Antimicrobial exposure may or may not precede acquisition The two appear to be in proximity 8 Pathogenesis of Pathogenesis of. Ingestion of spores transmitted from other patients via the hands of healthcare personnel and environment 3. Altered lower intestine flora (due to antimicrobial use) allows proliferation of C. difficile in colon 4. Toxin B & A Production leads to colon damage +/- pseudomembrane. Germination into growing (vegetative) form Sunenshine et al. Cleve Clin J Med. 006;73: The Problem Hospital-acquired, hospitalonset: 65,000 cases, $.3 billion in excess costs, and 9,000 deaths annually Hospital-acquired, postdischarge (up to 4 weeks): 50, cases, $0.3 billion in excess costs, and 3,000 deaths annually Nursing home-onset: 63,000 cases, $. billion in excess costs, and 6,500 deaths annually Campbell et al. Infect Control Hosp Epidemiol. 009:30: Dubberke et al. Emerg Infect Dis. 008;4:03-8. Dubberke et al. Clin Infect Dis. 008;46: Elixhauser et al. HCUP Statistical Brief # Age-Adjusted Death Rate* for Enterocolitis Due to C. difficile, Rate R The Problem Male Female White Black Entire US population Year *Per 00,000 US standard population Heron et al. Natl Vital Stat Rep 009;57(4). Available at

3 Cytotoxin Neutralization Assay Microwell EIA Rapid Cartridge EIA GDH Based Combination Procedures Molecule Procedures Toxigenic Culture Peterson L R, Robicsek A Ann Intern Med 009;5: Swindells, et.al. 00. JCM, 48: Quinn, et.al. 00. JCM, 48: Noren, et.al. 00 JCM, 49: Novak-Weekley, et.al. 00. JCM, 48: ASM Guidance Algorithm # EIA Toxin + = Pos. GDH assay Positive Neg.= Neg. EIA Toxin Negative 3 Molecular Assay Neg.=Neg. Pos.=Pos. Karre et. al. 0, JCM, 49: August 4,

4 ASM Guidance Algorithm # ASM Guidance Algorithm #3 GDH Toxin A/B Both Neg. = Neg. Both Pos. = Pos. One Pos. One Neg. Molecular Assay Neg.= Neg. Pos.= Pos. August 4, 00 Molecular Assay August 4, Molecular Based Tests BD Cdiff Assay BD GeneOhm Cdiff Assay Gen-Probe Prodesse ProGastro Cd Cepheid Xpert C. difficile Meridian illumigene C. difficile ProGastro Cd ProGastro Cd Extracted nucleic acid Specimens Add buffer, Vortex & spin Clarified Stool Dilute Internal Control. Combine 0 µl clarified stool and 0 µl Internal Control Run Lysis, Add Beads Perform extraction and elute in 0 µl 0 µl of nucleic acid for analysis Mastermix Prepare controls Combine 5 µl of nucleic acid & 0 µl of Mastermix in reaction tube Insert each reaction tube in SmartCycler Program instrument and run assay 3 4 4

5 Xpert C. difficile illumigene C. difficile 5 6 Acceptable Specimens Repeat Testing Only diarrheal stools ( 3/day) should be submitted for testing (no asymptomatic patient stools) Only a single specimen should be tested Test should be used for diagnosis only and not test-of-cure One specimen per 7 days Children < year old? 3% Prev 0.8% Prev Peterson L R, Robicsek A Ann Intern Med 009;5: Repeat Testing Repeat Testing Conclusion:..little value of repeat testing for C. difficle by EIA or PCR. Aichinger et.al. J Clin Microbiol 008;46: Conclusion: Repeat PCR within 7 days appears rarely useful, except for patients with evidence of a new infection. Luo and Banaei, J Clin Microbiol, 00;48:

6 Guidance Conclusions Incidence of is increasing Many C. difficile toxin testing options Molecular assays perform very well If not solely molecular, use a multi-step algorithm Test only patients with diarrhea Repeat testing for toxin within 7 days of little value 3 3 6