Clostridium difficile disease. A review of laboratory investigations.Dr. Jon BrazierAnaerobe Reference Laboratory National Public Health Service for Wales Microbiology CardiffUniversity Hospital of Wales, Cardiff.

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.

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)

Chart1

26.918417966128.9312915467

5.8727743525.1291586256

1.37184561251.8637816129

1.70696527181.440271059

3.92776946195.2435959014

18.867664343819.1044366244

182.5748972349207.235647506

male

female

age group

rate per 100,000 population

Figure 2 Age-specific rates of Clostridium difficile reports: England & Wales, 2001

T1

Table 1 Laboratory reports of Clostridium difficile bacteraemia

England & Wales, 2001

20002001

North East777698

Yorkshire & Humberside24232648

East Midlands16491394

Eastern27613248

London15071323

South East29132911

South West29413110

West Midlands14191811

North West28342320

Wales17441601

England & Wales2096821064

&L&8&D&R&8\\Animal\HCAI\Bacteraemia reports for CDR\E coli & proteeae\E coli, proteus, morganella & providencia 2001.xls

F1

Region-specific rates of Clostridium difficile bacteraemia laboratory reports

England & Wales, 2001

Region NameONS Population 2000

numberrate(lower 95% CIupper 95% CI)

North East69827.08(25.11-29.17)2577346

Yorkshire & Humberside264838.41(36.96-39.90)6893932

East Midlands139427.56(26.13-29.05)5057915

Eastern324877.19(74.56-79.89)4207925

London132324.80(23.48-26.17)5335361

South East291153.32(51.40-55.29)5459606

South West311042.17(40.70-43.68)7375065

West Midlands181122.32(21.30-23.37)8114848

North West232046.63(44.75-48.57)4975091

Wales160154.34(51.71-57.07)2946195

England & Wales1771833.47(32.98-33.96)52943284

* rates calculated using 2000 mid-year resident population estimates

&L&8&D&R&8\\Animal\HCAI\Bacteraemia reports for CDR\E coli & proteeae\E coli, proteus, morganella & providencia 2001.xls

F1

27.082122462425.11001429.167961

38.41059064736.96138839.902054

27.560763674426.13277229.046494

77.187687518274.55572579.88884

24.79682255823.47847626.169918

53.318865866951.39936455.291714

42.169119865440.69995143.677769

22.3171154921.30098923.369192

46.632312856244.75387648.569332

54.341277478251.71171657.069904

33.46600108932.97502133.96246

rate per 100,000 population

Figure 1 Region-specific rates of Clostridium difficile bacteraemia: England and Wales, 2001

T2

Table 2 Antibiotic susceptibilities for Clostridium difficile bacteraemia laboratory reports

England & Wales, 2000

sensitiveresistant (%)*no information (%)#

Clostridium difficile (n=21,064)

Vancomycin30(0%)20965(100%)310.000263342720968

Metronidazole10(0%)20967(100%)120.0000877809

* as a per cent of reports with susceptibility information

# as a per cent of total reports

Table 2 Antibiotic susceptibilities for Clostridium difficile bacteraemia laboratory reports

England & Wales, 2001

sensitiveresistant (%)*no information (%)#

Clostridium difficile (n=21,064)

Vancomycin00-20968(100%)00021064

Metronidazole10(0%)20967(100%)120.0000877809

&L&8&D&R&8\\Animal\HCAI\Bacteraemia reports for CDR\E coli & proteeae\E coli, proteus, morganella & providencia 2001.xls

T3

Antibiotic susceptibilty data for Clostridium difficile bacteraemia, England and Wales: 2001

ANTIBIOTICREGIONresistant(%)sensitiveno informationtotal

vancomycinNorth East0(0%)0698698

Yorkshire & Humberside0(0%)026482648

East Midlands0(0%)013941394

Eastern0(0%)032483248

London0(0%)013231323

South East0(0%)029112911

South West0(0%)031103110

West Midlands0(0%)018111811

North West0(0%)023202320

Wales0(0%)016011601

Total0(0%)02106421064

resistant(%)sensitiveno informationtotal

metronidazoleNorth East0(0%)0698698

Yorkshire & Humberside0(0%)026482648

East Midlands0(0%)013941394

Eastern0(0%)032483248

London0(0%)013231323

South East0(0%)129102911

South West0(0%)031103110

West Midlands0(0%)018111811

North West0(0%)023202320

Wales(0%)016011601

Total0(0%)12106321064

Please could you add another axis showing

the percentage resistance?

&L\\Animal\HCAI\Bacteraemia reports for CDR\Pseudomonads and related\figures.xls

T3

000

000

000

000

000

000

000

000

000

000

resistant

sensitive

no information

Number of reports

Figure 4 Ampicillin/amoxycillin susceptibility data for Escherichia coli bacteraemia laboratory reports, England and Wales: 2001

F2

000

000

000

000

000

000

000

000

000

000

resistant

sensitive

no information

Number of reports

Figure 6 Ceftazidime/cefotaxime susceptibility data for Escherichia coli bacteraemia laboratory reports, England and Wales: 2001

111

resistant

sensitive

no information

Number of reports

Figure 5 Cefuroxime susceptibility data for Escherichia coli bacteraemia laboratory reports, England and Wales: 2001

000

000

000

#REF!

#REF!

#REF!

Number of reports

Figure7 Ciprofloxacin susceptibility data for Escherichia coli bacteraemia laboratory reports, England and Wales: 2001

000

000

000

000

000

000

000

000

000

#REF!

#REF!

#REF!

Number of reports

Figure 8 Gentamicin susceptibility data for Escherichia coli bacteraemia laboratory reports, England and Wales: 2001

Age-specific Clostridium difficile bacteraemia reporting rate

England & Wales, 2001

ONS Population 2000Rate per 100,000Rate per 100,000

AgegroupmalefemalemissingTotalmalefemalemalefemale

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

How to test ?

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.

The Three-day ruleCurrent 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 only23% tested for A and B20% used cytotoxin assay13% miscellaneous methods

National C. difficile Standards Questionnaire 2002:Lab diagnosisOf 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 OrganismAs 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 StandardsSurveillance PolicyTest all patients >65yr with unformed stoolsTest 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 CDDDetection of the organism in stoolsDetection of C. difficile products in stools Detection of toxin(s) in stoolsMolecular methods eg. PCR for toxin genes

Laboratory methods for diagnosis of C. difficile diseaseIsolation of C. difficile from stoolsFor: Easy, sensitive, (add-on data available)Against: Not diagnostic of disease per se, low specificityDetection of C. difficile by fluorescence microscopy - not generally applied.

Lab. diagnosis of CDD (detection of products of C. difficile)GDH (glutamate dehydrogenase)For: high sensitivityAgainst: Low specificityMethod = 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 specificityAgainst: 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 kitsAdvantages:Rapid results (1-2 hours) microtitre-tray well format allows flexible batch sizesDisadvantages: 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:Immunocard Toxin A (Meridian)Oxoid Toxin A (Oxoid) (centrifugation step)Color PAC (Becton Dickinson)Clearview C. difficile A (Unipath)Triage (BioSite) - high negative predictive valueRapyd Test - (false positive reactions, removed from the market)

Cell cytotoxin assayAdvantages: 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 DemandWhat 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)Belgium Denmk. France Nether. Germany Italy Spain UK

Nb of labs

52

67

18

23

6

20

16

10

2

Nb of labs

Nb of labs

CRITERIA REQU

6.5

9.1

13

23.4

40.3

45.5

57.1

%

culture

93.8

100

72.3

60

46.7

47.8

27.8

20

Culture

% culture

cult+tox

93.893.8

10033.3

72.395.9

60100

46.797.7

47.891.3

27.8100

20100

Culture

Toxins

%

tox

93.8

0

95.9

100

97.7

87

100

100

Toxins

%

cytotox-EIA

26.773.3

DkDk

19.180.9

4060

4.795.3

1090

5.694.4

4060

cytotoxicity

EIA

EIA classique rapid

206.746.7

000

12.82.161.7

30030

20.953.516.3

351045

55.65.622.2

153510

EIA A

EIA A+B

EIA rapid

%

specific criteria

18.8

66.7

41.7

40

44.4

13

22.2

95

specific criteria

%

jars chambres

13.386.7

1000

8.891.2

16.783.3

38.161.9

18.281.8

1000

7525

Chambers

Jars

strategies

06.381.312.5

027.156.316.7

0405010

2.345.543.29.1

8.747.839.14.3

072.227.80

0801010

66.7033.30

Not adapted

Standard

Optimal

Other

incid toxin

0.8523.0391.93

0.4021.2340.738

1.3783.0612

0.161.090.329

0.4873.871.24

0.4412.171.082

25%ile

75%ile

mediane

Incidence for 1000 patients

incid cult

0.51.381.02

24.322.78

1.245.482.3

0.7352.761.54

25%ile

75%ile

mediane

Incidence for 1000 patients

Feuil1

FGSpIDkGBBNLSl

Nb of labs5267182362016102

OtherAgeAll the stoolsSpecific departmentsLiquid stoolsATBNosocomial

6.59.11323.440.345.557.1

BDkFNLGISpGB

Culture93.810072.36046.747.827.820

Toxins93.833.395.910097.791.3100100

BDkFNLGISpGB

Toxins93.8095.910097.787100100

BDkFNLGISpGB

cytotoxicity26.719.1404.7105.640

EIA73.380.96095.39094.460

BDkFNLGISpGB

EIA A20012.83020.93555.615

EIA A+B6.702.1053.5105.635

EIA rapid46.7061.73016.34522.210

BDkFNLGISpGB

specific criteria18.866.741.74044.41322.295

BDkFNLGISpGB

Chambers13.31008.816.738.118.210075

Jars86.7091.283.361.981.8025

BFNLGISpGBDk

Not adapted0002.38.70066.7

Standard6.327.14045.547.872.2800

Optimal81.356.35043.239.127.81033.3

Other12.516.7109.14.30100

BFGISptotal

25%ile0.8520.4021.3780.160.4870.441

75%ile3.0391.2343.0611.093.872.17

mediane1.930.73820.3291.241.082

FGBtotal

25%ile0.521.240.735

75%ile1.384.325.482.76

mediane1.022.782.31.54

Feuil2

Feuil3

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

Nb of labs

52

67

18

23

6

20

16

10

2

Nb of labs

Nb of labs

CRITERIA REQU

6.5

9.1

13

23.4

40.3

45.5

57.1

%

culture

93.8

100

72.3

60

46.7

47.8

27.8

20

Culture

%

cult+tox

93.893.8

10033.3

72.395.9

60100

46.797.7

47.891.3

27.8100

20100

Culture

Toxins

%

tox

93.8

0

95.9

100

97.7

87

100

100

Toxins

%

cytotox-EIA

26.773.3

DkDk

19.180.9

4060

4.795.3

1090

5.694.4

4060

cytotoxicity

EIA

%

EIA classique rapid

206.746.7

000

12.82.161.7

30030

20.953.516.3

351045

55.65.622.2

153510

EIA A

EIA A+B

EIA rapid

%

specific criteria

18.8

66.7

41.7

40

44.4

13

22.2

95

specific criteria

%

jars chambres

13.386.7

1000

8.891.2

16.783.3

38.161.9

18.281.8

1000

7525

Chambers

Jars

strategies

06.381.312.5

027.156.316.7

0405010

2.345.543.29.1

8.747.839.14.3

072.227.80

0801010

66.7033.30

Not adapted

Standard

Optimal

Other

incid toxin

0.8523.0391.93

0.4021.2340.738

1.3783.0612

0.161.090.329

0.4873.871.24

0.4412.171.082

25%ile

75%ile

mediane

Incidence for 1000 patients

incid cult

0.51.381.02

24.322.78

1.245.482.3

0.7352.761.54

25%ile

75%ile

mediane

Incidence for 1000 patients

Feuil1

FGSpIDkGBBNLSl

Nb of labs5267182362016102

OtherAgeAll the stoolsSpecific departmentsLiquid stoolsATBNosocomial

6.59.11323.440.345.557.1

BDkFNLGISpGB

Culture93.810072.36046.747.827.820

Toxins93.833.395.910097.791.3100100

BDkFNLGISpGB

Toxins93.8095.910097.787100100

BDkFNLGISpGB

cytotoxicity26.719.1404.7105.640

EIA73.380.96095.39094.460

BDkFNLGISpGB

EIA A20012.83020.93555.615

EIA A+B6.702.1053.5105.635

EIA rapid46.7061.73016.34522.210

BDkFNLGISpGB

specific criteria18.866.741.74044.41322.295

BDkFNLGISpGB

Chambers13.31008.816.738.118.210075

Jars86.7091.283.361.981.8025

BFNLGISpGBDk

Not adapted0002.38.70066.7

Standard6.327.14045.547.872.2800

Optimal81.356.35043.239.127.81033.3

Other12.516.7109.14.30100

BFGISptotal

25%ile0.8520.4021.3780.160.4870.441

75%ile3.0391.2343.0611.093.872.17

mediane1.930.73820.3291.241.082

FGBtotal

25%ile0.521.240.735

75%ile1.384.325.482.76

mediane1.022.782.31.54

Feuil2

Feuil3

C. diff in the papers.

C. perfringens AADC.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 cross-infection problem as with C. diff. CpEnt is very labile.

Lab diagnosis of C. perfringens AADCurrently 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 non-specific reactions with faecal matter have been reported.

C. difficile training dayFor 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 PlayC. difficile is a bacterial nosocomial pathogen causing in the region of 28,000 recorded infections per yearC. 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 kitAs 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 ..