Sensitivity and specificity of Clostridium difficile detection kits

Kerrie EastwoodClinical Scientist

Leeds Teaching Hospitals NHS Trust

Overview

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

Background

• Anerobic spore-forming bacilli– survive in environment– Need to wash hands

• Nosocomial pathogen– Predisposing antibiotics

• Cephalosporins• Clindamycin• Fluoroquinolones

• Cross infection

C. difficile disease

• Symptoms – Mild to severe

diarrhoea (over 10 episodes per day)

– Pseudomembranous colitis

– Megacolon– Relapse in 30% of

patients

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

Purpose of study

• No real comparison to date

• Evidence based on small studies– Debunked by manufacturers

• False positives?

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

Types of commercial toxin detection assay

Enzyme immunoassay

• 96-well format

• manual

• Semi-automated

Enzyme-linked Fluorescence assay

• Automated

Lateral flow assay

• Rapid

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

Assays included in this evaluation

Table 1. C. difficile detection assays included in this evaluation

Type Assay Target Supplier

Well-type EIAs Premier Toxin A+B Toxin A + B Meridian GA Clostridium

difficile Antigen Toxin A + B The Binding Site

Ridascreen toxin A/B Toxin A + B Biopharm Toxin A/B II Toxin A + B Techlab Remel ProSpecT Toxin A + B Oxoid Automated immunoassay

Vidas Tox A/B Toxin A + B Vidas

Membrane assays Remel Xpect Toxin A + B Oxoid Tox A/B Quik Chek Toxin A + B Techlab Immunocard Stat Toxin A + B Meridian Well-type EIAs C. diff Chek 60 GDH Techlab PCR GeneOhm C. difficile tcdB BD

Gold standards

• Two gold standards used for comparison

– Cytotoxin assay

– Cytotoxigenic culture • Cytotoxin assay

performed on culture supernatants

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

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)

Results

• Cytotoxin positive = 108/596 (18%)

• Cytotoxigenic culture positive = 125/600 (21%)

Sensitivity and specificityCytotoxin Cytotoxigenic culture

AssaySensitivity

(%)Specificity

(%)Sensitivity

(%)Specificity

(%)

Cytotoxin ------- ------- 86.4 99.2

Premier Toxin A + B 91.7 97.1 80.8 97.5

GA Clostridium difficile antigen

76.8 90.9 68.8 91.4

Ridascreen toxin A/B 66.7 95.1 60.0 95.6

Techlab Toxin A/B II 90.7 95.1 80.0 96.0

Remel ProSpecT 89.8 92.6 81.6 93.3

Vidas C. difficile Toxin A & B

89.8 96.7 80.0 97.3

Remel Xpect 77.8 98.8 68.8 99.4

Techlab Tox A/B Quik Chek

84.3 98.6 74.4 98.9

Premier Immunocard A + B

77.8 92.8 68.8 93.0

Techlab C. diff Chek-60

90.1 92.9 87.6 94.3

BD GeneOhm C. difficile

92.2 94.0 88.5 95.4

Sensitivity and specificity

Scattergram showing sensitivity Vs specificity for C. difficile detection assays in comparison with cytotoxigenic culture

Premier toxin A + B

GA Clostridium difficile antigen

Ridascreen toxin A/BTechlab Tox A/B II

Remel ProSpecT

Remel Xpect Techlab Tox A/B Quik Chek

Premier Immunocard A + B

cytotoxin

Vidas C. difficile A & B

C. Diff Chek-60GeneOhm Cdiff

90

92

94

96

98

100

60 65 70 75 80 85 90 95 100

Sensitivity (%)

Sp

ecif

icit

y (%

)

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

Positive and negative predictive values

Vs Cytotoxin Vs Cytotoxigenic culture

PPV NPV PPV NPV

Prevalence: 2% 10% 2% 10% 2% 10% 2% 10%

Cytotoxin ------- -------- -------- -------- 67.7 92.0 99.7 98.5

Premier Toxin A + B 39.5 78.0 99.8 99.1 39.5 78.0 99.6 97.9

GA Clostridium difficile antigen

14.8 48.6 99.5 97.3 14.0 47.0 99.3 96.3

Ridascreen toxin A/B 21.7 60.1 99.3 96.3 21.7 60.1 99.2 95.6

Techlab Toxin A/B II 30.1 70.1 99.8 98.9 29.0 69.0 99.6 97.7

Remel ProSpecT 19.9 57.5 99.8 98.8 19.8 57.4 99.6 97.9

Vidas C. difficile Toxin A & B

35.9 75.3 99.8 98.8 37.3 76.4 99.6 97.8

Remel Xpect 56.3 87.5 99.5 97.6 69.0 92.4 99.4 96.6

Techlab Tox A/B Quik Chek

54.6 86.8 99.7 98.3 59.1 88.7 99.5 97.2

Premier Immunocard A + B

18.1 54.7 99.5 97.4 16.8 52.4 99.3 96.4

Techlab C. diff Chek-60 20.6 58.6 99.8 98.8 24.0 63.1 99.7 98.6

BD GeneOhm C. difficile 23.8 63.0 99.8 99.1 28.1 68.0 99.7 98.7

Discordant results for toxin detection kits

Assay% repeatability of discordant

results Vs cytotoxin% repeatability of discordant

results Vs cytotoxigenic culture

Premier Toxin A + B 78.3 83.9

GA Clostridium difficile Antigen 78.3 76.9

Ridascreen toxin A/B 68.3 63.5

Techlab Toxin A/B II 51.6 48.3

Remel ProSpecT 48.9 28.1

Vidas C. difficile Toxin A & B 77.7 70.0

Remel Xpect 93.3 95.0

Techlab Tox A/B Quik Chek 87.5 87.9

Premier Immunocard A + B 54.2 39.1

Note: These include samples where an equivocal or failure was reported

OD values for toxin detection EIA’s

Scattergram of OD values for the Ridascreen toxin A/B assay

0

0.5

1

1.5

2

2.5

3

3.5

4

0 100 200 300 400 500 600

Sample number

OD

va

lue

false neg

correct

false pos

Average cut-off value

Scattergram of OD values for Techlab ToxinA/B II assay

0

0.5

1

1.5

2

2.5

3

0 100 200 300 400 500 600

sample number

OD

va

lue

false neg

correct

false pos

Cut-off value

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

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

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

What’s next?

• Algorithms – Two step– Three step– Which combination of tests?

• Requires further evaluation

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

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

http://www.pasa.nhs.uk/pasa/Doc.aspx?Path=%5bMN%5d%5bSP%5d/NHSprocurement/CEP/CEP08054.pdf

CEP report on toxin detection methods.

http://www.hpa.org.uk/hpr/archives/2009/news1209.htm#cdtdks DOH advice on using single tests.

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

http://www.pasa.nhs.uk/pasa/Doc.aspx?Path=%5bMN%5d%5bSP%5d/NHSprocurement/CEP/CEP08054.pdf

CEP report on toxin detection methods.

http://www.hpa.org.uk/hpr/archives/2009/news1209.htm#cdtdks DOH advice on using single tests.