Clostridium difficile disease.
A review of laboratory
investigations.
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Dr. Jon Brazier
Anaerobe Reference Laboratory
National Public Health Service for Wales
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Microbiology Cardiff
University Hospital of Wales, Cardiff.
Antibiotic Associated Diarrhoea
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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 = Enterotoxigenic C. perfringens (8-15%) and Staph. aureus = v.
rare cause (c.<0.1%)
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Idiopathic AAD = direct effect of antibiotic on gut eg.
erythromycin, and disturbance of balanced gut flora in
the absence of a known pathogen.
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.
Manchester Evening News: Winter 1991/2
C. difficile positive lab. reports for
England and Wales 1992-2002 (CDR 2nd
Oct. 2003)
30000
25000
20000
15000
10000
5000
2002
2001
2000
1999
1998
1997
1996
1995
1994
1993
1992
0
Figure 2 Age-specific rates of Clostridium
difficile reports: England & Wales, 2001
rate per 100,000 population
male
female
220
200
180
160
140
120
100
80
60
40
20
0
<1 y
1-4 y
5-9 y
10-14 y 15-44 y 45-64 y
age group
65 y+
Lab Diagnosis of CDD Two fundamental questions:
• When to test ?
• How to test ?
Testing Criteria: (NEQAS)
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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.
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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.
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.
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
National C. difficile Standards
Questionnaire 2002:
Lab diagnosis
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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.
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
17. 2 of the 3 patients died.
Hospitals with known toxin A neg/B pos
strains of C. difficile.
C. difficile - an “Alert Organism”
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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.
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.
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
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.
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
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,
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
EIA kits (contd)
• Sensitivity levels (range 68-98%)
• Specificity levels (range 75-100%)
• 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
TechLab EIA kit
“Dip-slide” Immuno-chromatography
kits:
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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)
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.
Vero Cell monolayer
Cytopathic effect of C. difficile on Vero Cells
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?
Survey by ESGCD of percentage of laboratories
testing for C. difficile by stool toxin assay by
European country (Clin.Micro.Infect. Oct.2003)
100
90
95.9
93.8
100
97.7
100
100
Spain
Sp
UK
GB
87
80
70
60
%
50
40
30
20
10
0
0
Belgium
Denmk.
France
B
Dk
F
Nether.
Germany
Italy
NL
G
I
Percentage of laboratories performing culture
for C. difficile by European country.
100
100
93.8
90
80
72.3
70
60
60
46.7
% 50
47.8
40
27.8
30
20
20
10
0
B
Belg.
Dk
Denmk.
F
NL
France
Neth.
G
I
Germany
Italy
Sp
Spain
GB
UK
C. diff in the papers.
C. perfringens AAD
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C.perfringens is normal flora in the gut 103-105/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 8 - 15% of AAD. Can be a crossinfection problem as with C. diff. CpEnt is very labile.
Lab diagnosis of C. perfringens
AAD
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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;51:891-894)
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 nonspecific reactions with faecal matter have been
reported.
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.
Summary: Current State of Play
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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. 2004 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 2004.
Training course to be held in ………………..