Sensitivity and specificity of Clostridium difficile detection kits

Kerrie Eastwood Clinical 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

Well-type EIAs Automated immunoassay Vidas Tox A/B Membrane assays Remel Xpect Tox A/B Quik Chek Immunocard Stat Well-type EIAs PCR Premier Toxin A+B GA Clostridium difficile Antigen Ridascreen toxin A/B Toxin A/B II Remel ProSpecT C. diff Chek 60 GeneOhm C. difficile Toxin A + B Toxin A + B Toxin A + B Toxin A + B Toxin A + B Toxin A + B Toxin A + B Toxin A + B Toxin A + B GDH

tcd

B

Supplier

Meridian The Binding Site Biopharm Techlab Oxoid Vidas Oxoid Techlab Meridian Techlab 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%)

Assay

Cytotoxin Premier Toxin A + B GA Clostridium difficile antigen Ridascreen toxin A/B Techlab Toxin A/B II Remel ProSpecT Vidas

C. difficile

Toxin A & B Remel Xpect Techlab Tox A/B Quik Chek Premier Immunocard A + B Techlab C. diff Chek-60 BD GeneOhm C. difficile

Sensitivity and specificity

Cytotoxin Cytotoxigenic culture

Sensitivity (%) ------ 91.7

76.8

Specificity (%) ------ 97.1

90.9

Sensitivity (%) 86.4

80.8

68.8

Specificity (%) 99.2

97.5

91.4

66.7

90.7

89.8

89.8

77.8

84.3

77.8

90.1

92.2

95.1

95.1

92.6

96.7

98.8

98.6

92.8

92.9

94.0

60.0

80.0

81.6

80.0

68.8

74.4

68.8

87.6

88.5

95.6

96.0

93.3

97.3

99.4

98.9

93.0

94.3

95.4

Sensitivity and specificity

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

100 98 96 Ridascreen toxin A/B Remel Xpect Techlab Tox A/B Quik Chek cytotoxin Premier toxin A + B Vidas C. difficile A & B Techlab Tox A/B II GeneOhm Cdiff C. Diff Chek-60 94 92 Premier Immunocard A + B GA Clostridium difficile antigen Remel ProSpecT 90 60 65 70 75 80

Sensitivity (%)

85 90 95 100

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

PPV Vs Cytotoxin NPV Cytotoxin

Prevalence:

Premier Toxin A + B GA Clostridium difficile antigen Ridascreen toxin A/B Techlab Toxin A/B II Remel ProSpecT Vidas

C. difficile

Toxin A & B Remel Xpect Techlab Tox A/B Quik Chek Premier Immunocard A + B Techlab C. diff Chek 60 BD GeneOhm C. difficile 2% ------ 39.5

14.8

21.7

30.1

19.9

35.9

56.3

54.6

18.1

20.6

23.8

10% ------- 78.0

48.6

60.1

70.1

57.5

75.3

87.5

86.8

54.7

58.6

63.0

2% ------- 99.8

99.5

99.3

99.8

99.8

99.8

99.5

99.7

99.5

99.8

99.8

10% ------- 99.1

97.3

96.3

98.9

98.8

98.8

97.6

98.3

97.4

98.8

99.1

2% 67.7

39.5

14.0

21.7

29.0

19.8

37.3

69.0

59.1

16.8

Vs Cytotoxigenic culture PPV NPV 10% 92.0

78.0

47.0

60.1

69.0

57.4

76.4

92.4

88.7

52.4

2% 99.7

99.6

99.3

99.2

99.6

99.6

99.6

99.4

99.5

99.3

10% 98.5

97.9

96.3

95.6

97.7

97.9

97.8

96.6

97.2

96.4

24.0

28.1

63.1

68.0

99.7

99.7

98.6

98.7

Discordant results for toxin detection kits

Assay Premier Toxin A + B GA Clostridium difficile Antigen % repeatability of discordant results Vs cytotoxin 78.3

78.3

Ridascreen toxin A/B Techlab Toxin A/B II Remel ProSpecT Vidas

C. difficile

Toxin A & B Remel Xpect Techlab Tox A/B Quik Chek Premier Immunocard A + B 68.3

51.6

48.9

77.7

93.3

87.5

54.2

Note:

These include samples where an equivocal or failure was reported % repeatability of discordant results Vs cytotoxigenic culture 83.9

76.9

63.5

48.3

28.1

70.0

95.0

87.9

39.1

OD values for toxin detection EIA’s

3 2.5

2 1.5

1 0.5

0 0

Scattergram of OD values for Techlab ToxinA/B II assay

100 200 300

sample number

400 500 false neg correct false pos Cut-off value 600 4 3.5

3 2.5

2 1.5

1 0.5

0 0

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

100 200 300

Sample number

400 false neg correct false pos 500 Average cut-off value 600

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.p

df CEP report on toxin detection methods.

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