Clostridium Difficile (C-Diff) testing - West Hertfordshire Hospitals ...

Trust Offices
Watford General Hospital
Vicarage Road
Watford
Hertfordshire
WD18 0HB
Tel: 01923 436209
RE: Freedom of Information Request
Thank you for your request under the Freedom of Information Act. I have pleasure in providing
the information requested below:
Does the Trust currently provide in-house
laboratory testing for Clostridium difficile on
stool samples
Is testing for Clostridium difficile available
seven days a week in the Trust
As at 21 April 2010 please provide the exact
method of testing for Clostridium difficile used
in the laboratory. If a commercial kit is used
please state the name and manufacturer e.g.
Meridian Premier Toxins A/B Enzyme
Immunoassay.
Please provide the Standard Operating
Procedure used in the laboratory for testing
of Clostridium difficile.
How many samples were submitted to the
laboratory for testing of Clostridium difficile in
2008 (1 January 2008 – 31 December 2008)
and how many of these tested positive
How many samples were submitted to the
laboratory for testing of Clostridium difficile in
2009 (1 January 2009 to 31 December 2009),
and how many of these tested positive
Yes, in house testing is provided
Yes
Premier Toxin A+B (Launch Diagnostics)
See attached
Number of samples tested positive 215
Number of samples submitted to the laboratory
2,751
Number of samples tested positive 152
Number of samples submitted to the laboratory
3,667
I hope that this information is helpful. The Trust operates an internal complaints procedure in
relation to FOI and if you are not satisfied with this response, in the first instance you should
contact Gail Samuel – Executive Assistant to the Chief Executive on 01923 436209. In the event
that you remain dissatisfied, you have the right to complain to the Information Commissioner for
a determination on whether or not the Trust has complied with the requirements of the FOIA.
The Information Commissioner’s contact details are:
Wycliffe House
Water Lane
Wilmslow
Cheshire
SK9 5AF

West Hertfordshire NHS Hospitals Trust
Microbiology Department Page 1 of 13
Title: Clostridium difficile Toxin Detection by
E.I.A
Date of Issue June 2008
PROCEDURES
TITLE
Document No. (Q-PULSE)
Clostridium difficile Toxin Detection by E.I.A
MI/SOP/040
EDITION No. 2
HOSPITAL SITE
LABORATORY DISCIPLINE
HHGH & WGH
Microbiology
DATE OF ISSUE June 2008
REVIEW DATE June 2010
AUTHOR
REVIEWED BY
AUTHORISED BY
Kathy Weber
Bill Champion/Rachel Hilson
Dr Prema Seetul-Singh
COPY 1
LOCATION OF COPIES
COSHH REFERENCE
UNCONTROLLED IF:
1. Q-Pulse
2. HHGH Microbiology Laboratory
3. WGH Microbiology Laboratory
Body Fluids/Waste, TECHLAB C.DIFFICILE TOX
A/B, Hycolin, hazard class 2 pathogens
Not printed on blue paper
Document review history
Review date Reviewed by Signature
June 2008
Rachel Hilson
MI/SOP/040 Clostridium difficile Toxin Detection by E.I.A
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West Hertfordshire NHS Hospitals Trust
Microbiology Department Page 2 of 13
Title: Clostridium difficile Toxin Detection by
E.I.A
Date of Issue June 2008
1. HEALTH & SAFETY
1. Gloves must be worn when handling potentially infectious stool samples and
whilst performing the test procedure.
2. The kit should be handled as though capable of transmitting infection.
Please refer to the following documents:
The laboratory Health and Safety policy
Relevant COSHH data sheets
Risk assessment for the Procedure
2. PRINCIPLE OF ASSAY
Background
Toxin producing Clostridium difficile is a major cause of antibiotic associated diarrhoea
and is an important nosocomial pathogen. It causes a wide spectrum of symptoms,
from asymptomatic carriage to severe diarrhoea and life-threatening
pseudomembraneous colitis.
Laboratory diagnosis is based on detection of this organism and/or either of its two
major toxins, toxins A and B. Toxin A is an enterotoxin, causing increase intestinal
permeability and subsequent fluid accumulation and diarrhoea. Toxin B is cytotoxic for
most mammalian cell lines in vitro. It has been hypothesized that the two toxins have
an additive or synergistic effect in vivo.
Not all strains of Clostridium difficile produce toxins and those, which do, can produce
toxin over a wide range of concentrations.
Diagnosis of Clostridium difficile associated diarrhoea (CDAD) has generally been
achieved by isolation of Clostridium difficile from the stool specimen and detection of
toxin B in the specimen using cell culture with neutralisation of the toxin by specific
antiserum.
Although the assay is very sensitive and has a high correlation to patients with severe
disease, it requires cell culture capability and up to 72 hours of incubation to complete.
This therefore has certain limitations in terms of patient intervention. Also the cytotoxin
assay is not standardised, is subjective in interpretation and detects the actions of toxin
B rather than toxin A.
MI/SOP/040 Clostridium difficile Toxin Detection by E.I.A
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West Hertfordshire NHS Hospitals Trust
Microbiology Department Page 3 of 13
Title: Clostridium difficile Toxin Detection by
E.I.A
Date of Issue June 2008
Principle
Clostridium difficile toxin detection at WHHT is performed by Enzyme Immuno Assay
(EIA) using the Premier Clostridium Difficile Toxins A + B kit supplied by Meridian
Diagnostics (1) .
The test utilises mouse monoclonal anti toxin A antibody and polyclonal goat antitoxin
B antibody absorbed to microtitre plates. An enzyme conjugated antitoxin A + B
polyclonal antibody and diluted patient stool samples are incubated in wells of a
microtitre plate. If either toxin is present in the patient sample, an enzyme-antibody
complex is formed which binds to the solid phase forming a sandwich. After washing to
remove unbound conjugate the substrate tetramethylbenzidine (TMB) is added and
incubated. A coloured product develops in the presence of any bound enzyme.
3. RESPONSIBILITY
Test set-up – Trained MLA or BMS staff
Interpretation – BMS staff or Trainee BMS staff under supervision
4. SAMPLE REQUIREMENTS
Stool specimens must be collected into clean, airtight containers with no preservatives.
Prior to testing, specimens should be stored at 2-8°C for a maximum of 72 hours.
If testing cannot be performed within this time specimens should be frozen immediately
upon receipt and stored at –80C until tested.
Do not use stools that have dried out.
All stool samples must be mixed thoroughly, regardless of consistency, to ensure a
representative sample prior to specimen preparation.
5. EQUIPMENT/REAGENTS/CALIBRATORS
12 x 75 mm plastic disposable test tubes
Wooden applicator sticks
Vortex mixer
Incubator 37 C +-2 C
Timer
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West Hertfordshire NHS Hospitals Trust
Microbiology Department Page 4 of 13
Title: Clostridium difficile Toxin Detection by
E.I.A
Date of Issue June 2008
Premier Clostridium Difficile Toxins A + B kit supplied by Meridian Diagnostics
Allow all reagents to reach room temperature before use.
Antibody coated Microwells.
(COSHH – Biological)
Coated with mouse monoclonal antitoxin A antibody + polyclonal goat anti toxin B
antibody.
Positive Control
(COSHH – Biological)
Inactivated toxin A + B in buffered protein solution with a preservative.
Sample Diluent / Negative control
Protein solution with a preservative.
(COSHH – Biological)
20X Wash Buffer
(COSHH – Irritant)
Concentrated wash buffer with a preservative.
Prepare 1 x wash buffer as required from 20 x stock concentration provided i.e. 5ml
concentrated wash buffer + 95ml distilled H 2 0. Place in a clean wash bottle. The
working wash buffer should be stored at room temperature for up to three months.
Label clearly with the date of preparation and date of expiry.
Enzyme Conjugate
(COSHH – Harmful)
Polyclonal goat anti-toxin A _ anti-toxin B antibodies conjugated to horseradish
peroxidase in buffered protein solution containing a preservative.
Substrate
(COSHH – Harmful)
Buffered solution containing urea peroxide and tetramethylbenzidine.
Stop Solution
1M phosphoric acid.
(COSHH – Irritant)
Caution : Avoid contact with skin.
6. PROCEDURE/CALIBRATION
Criteria for C.difficile Toxin Testing
1. Only diarrhoeal samples that are runny, like water and conform to the Kings
stool chart (KSC) J, K or L
2. And are from patients > 65 years old (in-patients, out patients or GP’s)
3. If specifically requested and conform to the KSC as J, K or L
The following samples will not be tested:
1. Samples that do not conform to the Kings stool chart J, K, and L.
MI/SOP/040 Clostridium difficile Toxin Detection by E.I.A
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West Hertfordshire NHS Hospitals Trust
Microbiology Department Page 5 of 13
Title: Clostridium difficile Toxin Detection by
E.I.A
Date of Issue June 2008
2. Samples that are from patients under 2 years old.
3. Samples from patients who have tested positive within the previous 28 days.
NB Refer to appendix B: Criteria for testing C.difficile
Specimen Preparation
1. Add 200µl of sample diluent to a 12 x 75 mm plastic disposable test tube.
2. Mix stool as thoroughly as possible prior to pipetting.
3. Using a transfer pipette, sample stool to 50µl calibration point (first mark on
pipette) and dispense into the sample diluent. Rinse pipette out in the sample
diluent several times to expel the entire stool sample. Seal tube with Para film
or a cap and vortex for 15 seconds.
Test Procedure
1) Print a WQS worksheet. Using ARR (refer to PA/SOP/IT/101 ARR Microbiology
Medical Abstract Reporting) check for any samples that have been positive within
the last 28 days.
2) Break off the required number of microwells (one well for each specimen plus one
positive and one negative control well per batch). Place the microwells in the
microwell strip holder. Record microwell positions of controls and specimens.
Unused strips must be resealed in the pouch immediately.
3) Using a transfer pipette add 100µl of diluted stool (second calibrated mark on
pipette) to the appropriate well. Place pipette tip halfway into well and allow sample
to run slowly down the side of the well.
4) Add two free falling drops of Positive control and for the negative control add 200µl
sample diluent to a tube, using a transfer pipette add 100µl (second calibrated mark
on pipette) to the appropriate well.
5) Add one free falling drop of enzyme conjugate (50µl) to all wells.
6) Seal wells with a strip of plate sealer and mix by firmly shaking or swirling the plate
(or use a plate shaker) for 30 seconds.
7) Incubate for 50 minutes at 37°C (± 2°C).
8) Carefully remove the plate sealer and wash the wells as follows:
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West Hertfordshire NHS Hospitals Trust
Microbiology Department Page 6 of 13
Title: Clostridium difficile Toxin Detection by
E.I.A
Date of Issue June 2008
a) Firmly tap the contents into a waste receptacle.
b) Tap the inverted plate on a clean pad of absorbent paper.
c) Fill all wells with 1 x wash buffer directing the stream of buffer to the sides of the
wells to avoid foaming.
d) Repeat this cycle four times. Ensure that the last tap onto absorbent paper
removes as much excess wash buffer as possible, but do not allow the wells to
completely dry out at any time.
e) Wipe the underside of the wells with lint free tissue.
9) Add two drops of substrate to each well.
10) Gently swirl the micro-titre plate (or use a plate shaker) for 30 seconds to mix the
substrates.
11) Incubate for 10 minutes at room temperature.
12) Add two drops of Stop Solution to all wells and again shake the plate for 30
seconds. Allow colour to develop for two minutes before reading. Leave for 15
minutes before reporting negatives.
13) Read the plate either
(a) Visually
(b) Spectrophotometrically using an EIA microwell reader at 450nm
(Reference filler 630nm) within 30 minutes of adding the stop solution.
Visual reading
NEGATIVE control - colourless to faint (barely visible) yellow
POSITIVE control - definite yellow colour
Spectrophotometric reading Using dual wavelength (450/630nm).
NEGATIVE control – OD values should be less than 0.100 but greater than 0.000.
POSITIVE control – OD values should be less than 2.000 but greater than 0.100.
MI/SOP/040 Clostridium difficile Toxin Detection by E.I.A
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West Hertfordshire NHS Hospitals Trust
Microbiology Department Page 7 of 13
Title: Clostridium difficile Toxin Detection by
E.I.A
Date of Issue June 2008
7.QUALITY CONTROL AND ASSESSMENT
The Positive and Negative controls must be used with each batch of specimens to
provide quality assurance of reagents and the results recorded on the work list.
8. LIMITATIONS
Premier Toxins A + B detects the presence of C.difficile toxin A + B in stools.
Failure to detect toxin A or toxin B in stool from patients with suspected
C.difficile associated disease may not preclude actual disease, but may be due
to such factors as improper collection, handling and storage of specimens.
The level of Toxin A or B in the stool has not been shown to correlate with either
the presence or severity of disease. Results should therefore be interpreted
together with other laboratory tests and clinical information.
Specimens should ideally be stored at 2-8°C for less than 24 hours. Prolonged
storage will cause deterioration of Toxin A in the sample, especially so in liquid
stools.
Most stools will remain reactive for up to 3 days if stored at 2-8°C.
9. REPORTING RESULTS
Recording Results
As well as recording the results on the form, also fill in the following information on the
WQS worksheet:
Results
Positive and negative control results
Initials of staff performing and validating the test
Batch number and expiry date of kit and buffer
This should then be photocopied twice and stored in the CDT file on the faeces bench.
Copies given to:
1) ICN
2) Consultant
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West Hertfordshire NHS Hospitals Trust
Microbiology Department Page 8 of 13
Title: Clostridium difficile Toxin Detection by
E.I.A
Date of Issue June 2008
Negative results are reported on the computer without referral to medical staff using
the Pathnet code: CDT N
Positive results are reported on the computer without referral to medical staff using
the Pathnet code: CDT P
Copies of positive forms are given to:
1) ICN
2) Debbie Dover
Centre For Infections
Once reported, place the request form on the COSERV clip. Refer to current
guidelines on notification and microbiological surveillance. (Refer to MI/RP/001 on
CDSC and COSURV reporting).
Infection Control Staff
Inform the infection control team of all positive results.
Weekly Sheet
All Positives must be entered onto the weekly C. difficile data Table MI/F/011.
At weekends record if all the tests were negative, to enable quick collation of the data
for Trust offices, micro consultants and ICN’s.
10. REFERENCES
1. http://www.mdeur.com/products/616096.htm
2. Advisory Committee on Dangerous Pathogens 2004 Approved List of Biological
Agents. http://www.hse.gov.uk/pubns/misc208.pdf. p. 1-17.
3. Public Health Laboratory Service Standing Advisory Committee on Laboratory
Safety. Safety Precautions: Notes for Guidance. 4th ed. London: Public Health
Laboratory Service (PHLS); 1993.
4. Control of Substances Hazardous to Health Regulations 2002. General COSHH
Approved Code of Practice and Guidance, L5. Suffolk: HSE Books; 2002.
5. Health and Safety Executive. 5 steps to risk assessment: a step-by-step guide to
a safer and Healthier workplace, IND (G) 163 (REVL). Suffolk: HSE Books;
2002.
6. Health and Safety Executive. A guide to risk assessment requirements: common
provisions in health and safety law, IND (G) 218 (L). Suffolk: HSE Books; 2002.
7. Health Services Advisory Committee. Safety in Health Service laboratories. Safe
working and the Prevention of infection in clinical laboratories and similar
facilities. 2nd ed. Suffolk: HSE Books;
MI/SOP/040 Clostridium difficile Toxin Detection by E.I.A
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West Hertfordshire NHS Hospitals Trust
Microbiology Department Page 9 of 13
Title: Clostridium difficile Toxin Detection by
E.I.A
Date of Issue June 2008
Related Documents
MI/LP/013 CDT Testing Notes
MI/LP/016 CDT Test Procedure
MI/F/011 Clostridium difficile Data Table
MI/SOP/040 Clostridium difficile Toxin Detection by E.I.A
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West Hertfordshire NHS Hospitals Trust
Microbiology Department Page 10 of 13
Title: Clostridium difficile Toxin Detection by
E.I.A
Date of Issue June 2008
APPENDIX A: MI/F/011
CLOSTRIDIUM DIFFICILE DATA
Week:
Name Sex Date of
birth
Lab
Number
Hospital
number
NHS
Number
Ward /
Location
Hosp
or
GP
Date of
specimen
Date of
test
65 Date entered
on CoSurv
Date entered
on MESS
reporting
These sheets need to be filled in weekly and returned to Debbie Dover. Please write legibly and do not use green ink.
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West Hertfordshire NHS Hospitals Trust
Microbiology Department Page 11 of 13
Title: Clostridium difficile Toxin Detection by
E.I.A
Date of Issue June 2008
Appendix B Criteria for Testing C.difficile
PATIENT
CDT
requested
Diarrhoeal
sample
(Kings
stool
chart J, K,
L)
Age 265 COE Test Computer code
Inpatient/outpatient/GP / X X X X
Inpatient/outpatient/GP X / X X X X X
CDT R *(not
tested because..)
Inpatient/outpatient/GP X X
Inpatient/outpatient/GP X X X
X
CDT N/ CDT P
CDT NT
Inpatient/outpatient/GP X X X X X X
Inpatient/outpatient/GP X X X X X
Inpatient/outpatient/GP X X CDT N/ CDT P
Inpatient/outpatient/GP X X X X CDT NT
Inpatient/outpatient/GP X X X CDT N/ CDT P
Inpatient/outpatient/GP X X X X X X
Patients who have
tested positive within
the previous 28 days
X CDT PR
* CDT R free text in: The performance of this assay has not been evaluated in a paediatric population + asymptomatic carriers
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West Hertfordshire NHS Hospitals Trust
Microbiology Department Page 12 of 13
Title: Clostridium difficile Toxin Detection by
E.I.A
Date of Issue June 2008
Authorised users
I have read the above procedure and been trained to use it. I undertake to follow it in all
details
NAME Post Signature Date
MI/SOP/040 Clostridium difficile Toxin Detection by E.I.A
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West Hertfordshire NHS Hospitals Trust
Microbiology Department Page 13 of 13
Title: Clostridium difficile Toxin Detection by
E.I.A
Date of Issue June 2008
PROCEDURE AMENDMENT SHEET – FOR MINOR CHANGES ONLY
Number Date Page
No.
1
Amendment
Authorised
by:
2
3
4
5
6
7
8
The amendment must be authorised by the author, where possible.
The amendment must be underlined and an asterisk written in the margin alongside the
change – DO NOT USE LIQUID PAPER
No more than 8 amendments will be made before the procedure is revised.
MAJOR CHANGES MUST RESULT IN AN IMMEDIATE PROCEDURE REVISION
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Yours sincerely
Jan Filochowski
Chief Executive