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Sensitivity and specificity of Clostridium difficile detection kits Kerrie Eastwood Clinical Scientist Leeds Teaching Hospitals NHS Trust.

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Presentation on theme: "Sensitivity and specificity of Clostridium difficile detection kits Kerrie Eastwood Clinical Scientist Leeds Teaching Hospitals NHS Trust."— Presentation transcript:

1 Sensitivity and specificity of Clostridium difficile detection kits Kerrie Eastwood Clinical Scientist Leeds Teaching Hospitals NHS Trust

2 Overview  Background on C. difficile  Purpose of study  Methods  Results  Which kit is best?  What’s next?  Acknowledgements

3 Background Anerobic spore-forming bacilli –survive in environment –Need to wash hands Nosocomial pathogen –Predisposing antibiotics Cephalosporins Clindamycin Fluoroquinolones Cross infection

4 C. difficile disease Symptoms –Mild to severe diarrhoea (over 10 episodes per day) –Pseudomembranous colitis –Megacolon –Relapse in 30% of patients

5 Diagnosis and treatment Laboratory diagnosis –Don’t just isolate organism –Detection of toxin Treatment –Stop predisposing antibiotics –Start oral metronidazole (or Vancomycin if severe or ribotype 027) –Infection control e.g. isolation/cohorting

6 Purpose of study No real comparison to date Evidence based on small studies –Debunked by manufacturers False positives?

7 Implications of false positive CDI diagnosis Inappropriate antibiotic cessation / modification Inappropriate treatment for CDI Unnecessary isolation Potentially harmful cohorting Inaccurate surveillance / infection control data Wasted resources Reimbursement / fines Medicolegal implications

8 Types of commercial toxin detection assay Enzyme immunoassay 96-well format manual Semi-automated Enzyme-linked Fluorescence assay Automated Lateral flow assay Rapid

9 Other commercial tests for C. difficile Glutamate dehydrogenase (GDH) –Cell surface associated enzyme –Found in many bacterial species –EIA assay specific for C. difficile GDH Real time PCR –Detection of toxin B gene –Doesn’t indicate toxin production –Alternative assays available to detect other toxin genes

10 Assays included in this evaluation

11 Gold standards Two gold standards used for comparison –Cytotoxin assay –Cytotoxigenic culture Cytotoxin assay performed on culture supernatants

12 Sample selection Collected 600 samples –Submitted for C. difficile testing –Diarrhoeal –Enough volume Picked daily (10 per day) Randomised and anonymised before testing PCR (n=554) and GDH (n=558) performed on freeze-thawed samples at later date

13 Sample processing Each sample –tested on every assay –Cultured on CCEYL agar in anaerobic cabinet –Cytotoxin –Cytotoxigenic culture –Isolates stored at -70°C –Isolates PCR-ribotype Discordant results for toxin detection assays –Majority rules –Repeated further 2 times (best of 3)

14 Results Cytotoxin positive = 108/596 (18%) Cytotoxigenic culture positive = 125/600 (21%)

15 Sensitivity and specificity CytotoxinCytotoxigenic culture Assay Sensitivity (%) Specificity (%) Sensitivity (%) Specificity (%) Cytotoxin ------- 86.499.2 Premier Toxin A + B 91.797.180.897.5 GA Clostridium difficile antigen 76.890.968.891.4 Ridascreen toxin A/B 66.795.160.095.6 Techlab Toxin A/B II 90.795.180.096.0 Remel ProSpecT 89.892.681.693.3 Vidas C. difficile Toxin A & B 89.896.780.097.3 Remel Xpect 77.898.868.899.4 Techlab Tox A/B Quik Chek 84.398.674.498.9 Premier Immunocard A + B 77.892.868.893.0 Techlab C. diff Chek-60 90.192.987.694.3 BD GeneOhm C. difficile

16 Sensitivity and specificity

17 Positive and negative predictive values Change depending on the prevalence of toxin positive C. difficile in faecal samples within the population 10% prevalence in hospital setting 2% prevalence in community setting

18 Positive and negative predictive values Vs CytotoxinVs Cytotoxigenic culture PPVNPVPPVNPV Prevalence: 2%10%2%10%2%10%2%10% Cytotoxin --------------- 67.792.099.798.5 Premier Toxin A + B39.578.099.899.139.578.099.697.9 GA Clostridium difficile antigen 14.848.699.597.314.047.099.396.3 Ridascreen toxin A/B21.760.199.396.321.760.199.295.6 Techlab Toxin A/B II30.170.199.898.929.069.099.697.7 Remel ProSpecT19.957.599.898.819.857.499.697.9 Vidas C. difficile Toxin A & B 35.975.399.898.837.376.499.697.8 Remel Xpect56.387.599.597.669.092.499.496.6 Techlab Tox A/B Quik Chek 54.686.899.798.359.188.799.597.2 Premier Immunocard A + B 18.154.799.597.416.852.499.396.4 Techlab C. diff Chek- 60 20.658.699.898.824.063.1 99.798.6 BD GeneOhm C. difficile 23.863.099.899.

19 Discordant results for toxin detection kits Assay % repeatability of discordant results Vs cytotoxin % repeatability of discordant results Vs cytotoxigenic culture Premier Toxin A + B78.383.9 GA Clostridium difficile Antigen78.376.9 Ridascreen toxin A/B68.363.5 Techlab Toxin A/B II51.648.3 Remel ProSpecT48.928.1 Vidas C. difficile Toxin A & B77.770.0 Remel Xpect93.395.0 Techlab Tox A/B Quik Chek87.587.9 Premier Immunocard A + B54.239.1 Note: These include samples where an equivocal or failure was reported

20 OD values for toxin detection EIA’s

21 Ribotypes 128 culture positive samples, of which 125 were cytotoxin positive There were 21 different ribotypes; most common ribotypes –106 (26.6%) –027 (18.8%) –002 (6.3%) No difference between assays for different ribotypes

22 Which kit is best? Depends on your population Cytotoxin gives best PPV for toxin detection assays –But is labour intensive and slow Lateral flow toxin detection assays have good PPV and are rapid –But have poorer NPV GDH gives best PPV overall –But is only detecting presence of C. difficile, not active disease PCR has highest NPV, good screening test –But only detecting presence of toxin gene Test results should be taken in context with the clinical presentation of the patient

23 Single tests? Advice from the Department of Health: The currently available kits for detection of C. difficile toxins have variable performance Currently available kits may miss about 1 in 5 to 1 in 10 cases of CDI and will falsely identify (1-2 out of 10) cases as positive when they are not The poor positive predictive values of toxin detection kits, especially in the context of widespread testing, and the possibility of missing true positives mean that there are limitations to using these as single tests for the laboratory diagnosis of CDI

24 What’s next? Algorithms –Two step –Three step –Which combination of tests? Requires further evaluation

25 Acknowledgements Prof. Mark Wilcox Patrick Else All the Enteric lab staff All the manufacturers/distributors Ann Prothero (Leeds Ethics) Keith Perry and Andre Charlett at HPA

26 Any questions? Useful references:  Comparison of nine commercially available Clostridium difficile toxin detection assays, a real-time PCR assay for C. difficile tcdB, and a glutamate dyhydrogenase assay to cytotoxin testing and cytotoxigenic culture methods. 2009. Eastwood K., Else P., Charlett A. and Wilcox M. Journal of Clinical Microbiology. 47: 3211- 3217  f CEP report on toxin detection methods. f  DOH advice on using single tests.

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