1. Clostridium difficile disease. A review of laboratory investigations.
Dr. Jon Brazier
Anaerobe Reference Laboratory
National Public Health Service for Wales
Microbiology Cardiff
University Hospital of Wales, Cardiff.

2. Antibiotic Associated Diarrhoea
Disturbance of normal gut flora by antibiotics that remove “colonisation resistance barrier” allowing organisms such as C. difficile to proliferate. C.diff. is responsible for nearly 100% of PMC and up to 33% of AAD.
Other AAD associated infective agents = Entero- toxigenic C. perfringens (8-15%) and Staph. aureus = v. rare cause (c.<0.1%)
Idiopathic AAD = direct effect of antibiotic on gut eg. erythromycin, and disturbance of balanced gut flora in the absence of a known pathogen.

3. C. difficile disease:
C. difficile is the major identifiable cause of AAD and is responsible for significant levels of nosocomial morbidity and mortality.
Exact mortality rates are not recorded by OPCS but Wilcox et al. estimated a case of CDD costs c.£4,000 and is therefore a significant drain on health care resources of England and Wales. On 2002 figures = approx. £92m/yr.

8. Lab Diagnosis of CDD - Two fundamental questions:
When to test ?
How to test ?

9. Testing Criteria: (NEQAS)
In 1999 NEQAS issued a C. difficile survey of clinical diagnostic microbiology labs in the UK. Of 283 returns, 243 were included in the analysis.
92% of labs applied some degree of selection of specimens for C. difficile investigation.
63% using more than one criterion, 17% on clinicians request only, 12% on any in-patient with diarrhoea, and 3% on high risk patients with stated antibiotic therapy.
66% would not examine stools on patients under 2 yrs old.
Conclusion: Inconsistency in testing criteria leads to inconsistent data collection.

10. The “Three-day rule”
Current thinking is that for a patient who develops diarrhoea after being in hospital for three or more days it is a waste of resources to test for Salmonella, Shigella and Campylobacter.
More useful to test for C. difficile and C. perfringens.

11. How many labs test for the presence of both toxins A and B
How many labs test for the presence of both toxins A and B? (NEQAS 1999)
44% of labs test for toxin A only
23% tested for A and B
20% used cytotoxin assay
13% miscellaneous methods

12. National C. difficile Standards Questionnaire 2002: Lab diagnosis
Of 223 labs surveyed, 208 (93%) replied and 183 labs process faecal specimens for C. difficile.
179 (98%) of these perform toxin detection:
Of these, 21% cytotoxin assay,
23% EIA for toxin A only,
49% EIA for toxin A and B.

13. Toxin-variable (Aneg/B pos) C. difficile
Riegler (1995) reported that toxin B was 10 times more damaging to the human colon than toxin A.
Outbreaks with Aneg/Bpos strains have been reported in Canada, USA, Poland and Japan.
Clinical evidence of pathogenicity. Of 3 cases referred to ARU from Dublin, two patients had endoscopic proof of PMC, all three were stool toxin A negative but culture positive for C. difficile type of the 3 patients died.

14. Hospitals with known toxin A neg/B pos strains of C. difficile.

15. C. difficile - an “Alert Organism”
As of 1st April 2003, the HAISSG of DoH requires NHS Trusts to produce data on their incidence of C. difficile infections. In practice, 2004 will be the start date.
Surveillance includes the collection of isolates for epidemiology and antibiotic susceptibility monitoring.
HPA Regional labs are to call for toxin-positive stool samples from hospitals in their region on a rotational basis, isolate C. difficile and send them to ARL.
Approx. 1,000 per year are to be tested.

16. National C. difficile Standards Surveillance Policy
Test all patients >65yr with unformed stools
Test for toxins A and B by EIA for both toxins or neutralised cytotoxin assay.
Systematic and representative culturing of positive stools by HPA Regions to obtain isolates for referral to ARL to monitor antibiotic susceptibility and perform typing.

17. Methods of Laboratory Diagnosis of CDD
Detection of the organism in stools
Detection of C. difficile products in stools
Detection of toxin(s) in stools
Molecular methods eg. PCR for toxin genes

18. Laboratory methods for diagnosis of C. difficile disease
Isolation of C. difficile from stools
For: Easy, sensitive, (add-on data available)
Against: Not diagnostic of disease per se, low specificity
Detection of C. difficile by fluorescence microscopy - not generally applied.

19. Lab. diagnosis of CDD (detection of products of C. difficile)
GDH (glutamate dehydrogenase)
For: high sensitivity
Against: Low specificity
Method = Triage kit combines toxin A detection with GDH.
Old methods no longer used = GLC on stools

20. Lab. diagnosis of CDD (contd)
Toxin A or B detection in stools:
For: Diagnostic of disease, high specificity
Against: Labile toxins, some kits have low sensitivity, some detect toxin A only.
Methods = Cell cytotoxin assay, EIA assay, immuno-chromatography membrane kits,

21. EIA C. difficile Toxin kits
Advantages:
Rapid results (1-2 hours) microtitre-tray well format allows flexible batch sizes
Disadvantages: Cost per test is high with small numbers, lower sensitivity than tissue culture

22. EIA kits (contd) Sensitivity levels (range 68-98%)
Specificity levels (range %)
A+B kits available at no extra cost than A alone.
Automated EIA (Vidas) Low sensitivity 63-73%
NB. Some take 2 hours, others < 1 hour

23. TechLab EIA kit

24. “Dip-slide” Immuno-chromatography kits:
Immunocard Toxin A (Meridian)
Oxoid Toxin A (Oxoid) (centrifugation step)
Color PAC (Becton Dickinson)
Clearview C. difficile A (Unipath)
Triage (BioSite) - high negative predictive value
Rapyd Test - (false positive reactions, removed from the market)

25. Cell cytotoxin assay
Advantages: Very high sensitivity, (c.100% for Vero cells)
Disadvantages: Costly in MLSO time, maintaining cell line, lower specificity = neutralisation needed to prove CPE due to C. difficile.

26. Vero Cell monolayer

27. Cytopathic effect of C. difficile on Vero Cells

28. Criteria to consider in choosing a method for C
Criteria to consider in choosing a method for C. difficile testing- “Local Diagnostic Demand”
What is your throughput?
How often will you test? Would you batch test?
Do your clinicians expect a same-day result?
Do you have a virology department to supply you with cell-lines? Are your staff familiar with tissue culture techniques?

29. Survey by ESGCD of percentage of laboratories testing for C
Survey by ESGCD of percentage of laboratories testing for C. difficile by stool toxin assay by European country (Clin.Micro.Infect. Oct.2003)
Belgium Denmk. France Nether. Germany Italy Spain UK

30. Percentage of laboratories performing culture for C
Percentage of laboratories performing culture for C. difficile by European country.
Belg Denmk. France Neth Germany Italy Spain UK

31. C. diff in the papers.

32. C. perfringens AAD
C.perfringens is normal flora in the gut /gm but some wild strains (c.6%) produce an enterotoxin (CpEnt) - associated with food poisoning and antibiotic-associated diarrhoea. Cpe gene encodes for CpEnt which binds to gut epithelia altering cell permeability and causing cell death with loss of fluid = diarrhoea. No pseudomembrane formation as seen with C. diff. but symptoms are severe.
AAD due to CpEnt first reported in 1984, reported as causing between % of AAD. Can be a cross- infection problem as with C. diff. CpEnt is very labile.

33. Lab diagnosis of C. perfringens AAD
Currently only 1 EIA kit for CpEnt in UK (TechLab - BioConnections) is commercially available, this was used in a recent study on AAD in Leeds (Asha and Wilcox. J.Med.Micro. 2002;51: )
200 stools examined from pts. with symptoms of AAD; 16% +ve for C.diff toxins, 8% +ve for CpEnt, 2% +ve for both, weak reactions were doubtful.
An RPLA kit (Oxoid) for CpEnt is available but non- specific reactions with faecal matter have been reported.

34. C. difficile training day
For HPA staff involved in collection of isolates for surveillance, a one-day training course in isolation and identification methods for C. difficile is to be run on Jan. 15th 2004 at the Anaerobe Ref. Lab. in Cardiff.

35. Summary: Current State of Play
C. difficile is a bacterial nosocomial pathogen causing in the region of 28,000 recorded infections per year
C. perfringens causes approx. 10% of nosocomial AAD
C. difficile is costing NHS approx. over £1.5 million per week.
Lab diagnosis of CDD should include toxin A+B detection or neutralised CPE in Vero cells, for CpEnt use Tech Lab kit
As of Jan NHS Trusts are required to report their CD infection rates and representative samples for culture are to be performed by regional HPA labs for submission to ARL in Training course to be held in ………………..