1. No category

Public Health Wales Microbiology Cardiff
Wales Centre for Mycobacteria
Quality
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Public Health Wales Microbiology Cardiff
Wales Centre for Mycobacteria
User Manual
Document and Version: WCMQMS 035.5
Author: M Ruddy
Authorised by: P James
Date authorised: 8 May 2012
Publication/ Distribution:
NHS Wales (Intranet)
Public (Internet)
Review Date: 8 May 2014
Purpose and Summary of Document:
To provide information on the Wales Centre for Mycobacteria (WCM),
services offered by the WCM and guidance on requesting these services.
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Document amendments
Document amendments by completing the following table.
Date
Section
Page
Version
Description
No
No
No
15/12/09
2
2
Removal “collation/assistance”
insertion “Cellular interferon…”
Insert „in conjunction with
NPHS Microbiology Swansea‟
15/12/09
4
2
Replace Alan Paull with Paula
Brookes
15/12/09
4
2
Removed Paul Ellis and Debbie
Charles
15/12/09
6.2.2 (ii)
2
Insert reference to Mtb
complex being speciated by
phenotypic methods and NTMs
as necessary
15/12/09
6.2.5
2
Change NMRL London to NPHS
Swansea
Insert „£40‟
15/12/09
Appendix
2
Replaced HPA NMRL forms with
F
NPHS Wales forms
15/12/09
6.2.1
2
9am Tuesday changed to
midday tuesday
15/12/09
6.2.1
2
Addition of Negative results for
these specimens do not
exclude M. tb complex
infection
15/12/09
6.2.2 (i)
2
Molecular identification
changed to „identification by
Geneprobe‟
15/12/09
6.2.2 (ii)
2
„for these few isolates.‟ added
15/12/09
6.2.4
2
17regions (loci) changed to 15
15/12/09
2
15/12/09
2
15/12/09
Title page
2
15/12/09
3.0
2
all the M. tuberculosis complex
strains for molecular typing
identification and sensitivity
testing of Mycobacterium
tuberculosis complex isolates
Removal of “world TB day”
and change document number
(also in footer)
Removal “HAYS”
15/12/09
6.2.1
2
Removal of text: “the
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sensitivity …. Individual basis”
15/12/09
6.2.3
2
15/12/09
6.2.4
2
15/12/09
6.2.5
2
02/02/12
All
02/02/12
02/02/12
02/02/12
02/02/12
02/02/12
02/02/12
04/02/12
08/05/12
08/05/12
All
3
Removal “Dr Ian Campbell”
insertion “lead Consultant….”
Insertion of “extra MIRU loci
or..”
Insertion of “the original
ELIpsot assay..” “..an
enhanced version has been
developed…”
Delete section “until Oct
2007..kits to them”
Insert “WCM has introduced
QFT-G…”
Delete “to cover ….are
discarded/lost”
Delete “NMRL” insert “NPHS
Swansea”
Delete “£35” insert “£40”
Delete “introduction of…..
accommodate this”
Update to new format.
Change MOTT to NTM. Change
MRU to NMRL. Addition of
images.
Addition support to R&D
2
3
4
3
6.2.4
3
6.2.5
3
7
3
Additions to WCM staff
members
Details of change from 15 loci
to 24 loci.
Updates to CDC & NICE
guidance 2010/11
Additions to references
1 and 6
3
Addition of images
Metadata
4
Published to correct category.
No change to text.
Addition of test turnaround
times.
Format change to
QuantiFERON® form
6
8
5
Appendix
F
32
5
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Contents
1
INTRODUCTION ...................................................................... 5
2
SUMMARY OF SERVICES (JULY 2011) ..................................... 6
3
ADDRESS: ............................................................................... 6
4
WCM STAFF MEMBERS: ........................................................... 6
5
WCM CONTACT DETAILS ......................................................... 7
5.1 Contact Numbers: ................................................................. 7
5.2 Communications Algorithm: ................................................... 8
6
SERVICES AVAILABLE: ............................................................ 8
6.1 Turnaround times for Wales Centre for Mycobacteria ................. 8
6.2 Primary Cultures for mycobacteria: ......................................... 9
6.3 Reference services: .............................................................. 10
6.3.1
Direct molecular detection of MTBC on primary
specimens .............................................................................. 10
6.3.2
Identification of mycobacteria: .................................. 11
6.3.3
Sensitivity testing: ..................................................... 12
6.3.4
Typing of Mycobacterium tuberculosis isolates: ......... 13
6.3.5
Gamma- Interferon assays: ....................................... 17
7
SELECTED REFERENCES: ....................................................... 20
8
APPENDIX A ......................................................................... 23
9
APPENDIX B ......................................................................... 24
10 APPENDIX C.......................................................................... 25
11 APPENDIX D: NTM (NON TUBERCULOSIS MYCOBACTERIA) .. 26
12 APPENDIX E: MICROBIOLOGICALMYCOBACTERIAL ENQUIRIES:
............................................................................................. 28
13 APPENDIX F: WCM EXAMPLE FORMS..................................... 29
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Wales Centre for Mycobacteria (WCM)
Llandough Hospital ~1933 (courtesy Cardiff University)
1
Introduction
The WCM functions as the National Reference Laboratory for mycobacteria
for Wales and also as the regional mycobacterium reference unit for the
South West of England, along with providing a service to various other
users in England, the Channel Islands, Isle of Man, Ireland and veterinary
practices.
A mycobacterial reference laboratory has been located in Cardiff since at
least the early 1950s. From 1959 the laboratory was managed by the
Public Health Laboratory and was the UK reference unit until 1996 when it
became part of the PHLS network of regional reference laboratories. The
PHLS network was dissolved in April 2003 and since then the management
of the unit has transferred to the National Public Health Service Wales now
Public Health Wales. The other regional mycobacterial laboratories and the
UK National Mycobacterium Reference Laboratory (NMRL), London are
now managed by the Health Protection Agency in England, however we
continue to work closely within this network and provide services to the
South West of England as well as Wales.
There are three principal mechanisms for reporting on the work of the
laboratory:
through the Public Health Wales Microbiology Laboratory network as
part of Microbiology Cardiff
through the Public Health Wales Respiratory Disease Programme via
the Public Health Wales TB Programme Group and Public Health
Wales CDSC Wales
through established UK mycobacterial structures to the Department
of Health (e.g. via the HPA, with the HPA NMRL in London as the
lead UK mycobacterial reference centre and the HPA Centre for
Infection, Respiratory Division Colindale)
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Summary of Services (July 2011)
Processing of primary specimens for mycobacterial culture
Direct molecular detection of Mycobacterium tuberculosis complex
available on primary specimens.
Detection of mycobacteria from clinical specimens (direct
microscopic and molecular methods)
Isolation of mycobacteria (liquid and solid culture; plus culture
storage)
Identification of Mycobacteria sp. (both initial rapid molecular and
conventional phenotypic identification)
Primary sensitivity testing
Molecular strain typing and epidemiological surveillance of M.
tuberculosis (support to Public Health outbreak and control
management) using VNTR / MIRU technology
Cellular interferon gamma release assays (CIGRA) in conjunction
with Public Health Wales Microbiology Swansea
Clinical advice for case and outbreak investigation and management.
Technical laboratory advice.
Support to Research and Development into mycobacterial disease in
Wales.
3
Address:
Wales Centre for Mycobacteria
Microbiology Cardiff
Public Health Wales
Llandough Hospital,
Penlan Road,
Penarth
CF64 2XX
DX Address:
4
Telephone Number:
029 2071 6408
Secretary:
029 2071 5298
Wales Centre for Mycobacteria
Cardiff Public Health Wales (Llandough)
DX 6070400
Penarth 90 CF
WCM Staff Members:
Dr. Michael Ruddy
Consultant Microbiologist, medical lead for WCM
Paula Brookes
Technical Head of Bacteriology Department
Rhian Williams
Technical Lead WCM
Gwyneth Samuel
BMS 2
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Kay Parry
BMS 2
Dr. Sally Corden
Lead Clinical Scientist, Head of Wales Molecular
Centre UHW
Dr. Lewis White
Lead Clinical Scientist, mycology /
Mycobacteriology / Wales Molecular Centre UHW
Michael Perry
Clinical Scientist, bacteriology / Wales Molecular
Centre UHW
Rotational posts (shared with Food Water & Environmental Laboratory
Public Health Wales Cardiff):
David Tucker
BMS 1
Lynda El-Araby
BMS 1
Colin Mills
MLA
Plus rotational posts with Public Health Wales Microbiology Cardiff
5
WCM Contact Details
5.1
Contact Numbers:
Technical enquiries
TB laboratory (Llandough)
029 2071 6408
Molecular enquiries
Molecular Laboratory (UHW)
029 2074 4175
Clinical enquiries
Dr Michael Ruddy
029 2071 6408
Laboratory Access:
The WCM Llandough laboratory is staffed 08.45 to 17.00 Monday to
Friday.
Out of hours clinical advice and urgent AFB microscopy can be arranged (if
appropriate) via the relevant local NHS/Public Health Wales microbiology
laboratory. (For Cardiff contact University Hospital of Wales switchboard
02920 747747)
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Communications Algorithm:
Please see Appendix E
6
Services available:
6.1
Turnaround times for Wales Centre for
Mycobacteria
Test
AFB microscopy and culture
Geneprobe
culture
TB PCR
Target
Turnaround
time
4-6 weeks
test on positive 2 days
6 days
QuantiFERON®-TB Gold InTube test (Cellestis, Australia) 7 days
(QFT-G):
Comments
Positive microscopy
phoned 24hrs. Positive
culture results phoned.
Positive geneprobe test
for Mtb complex
phoned.
Once a week batch
testing (Wednesday).
Results available
Thursday afternoon
after 4pm.
Results will be available
by telephone after 7
days
For details of specimen containers, sample processing times and reporting
arrangements please refer to Public Health Wales Microbiology Cardiff User
Handbook (CDQMS 035) Appendices 1-3.
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Primary Cultures for mycobacteria:
We accept the following specimens for primary mycobacterial culture:
Sputum, Broncho-alveolar lavage, non-directed bronchial lavage
Tissue specimens
Pus
Gastric aspirate
Early morning urine
Blood/bone marrow (please request the appropriate blood culture
bottles before submitting)
Fluids (pleural, CSF, joint etc)
Microscopy is performed using both auramine and ZN stains. Culture is
performed using liquid culture media via a MGIT (Mycobacterial Growth
Indicator Tube) 960 system (Becton Dickinson) and backed up with solid
culture media (Lowenstein-Jensen media).
If we do not routinely perform your primary cultures, please discuss your
particular requirements for primary culture with the laboratory.
ZN stain
auramine stain
Mycobacterial Cultures
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6.3
Reference services:
6.3.1
Direct molecular detection of MTBC on primary specimens
Initial detection of Mycobacterium species (either MTBC or NTM) on
primary specimens is performed using the Seegene Anyplex MTB/NTM PCR
assay. Any samples positive for MTBC are then checked for common
rifampicin and isoniaizid resistance markers using the GenoType
MTBDRplus system (Hain Lifescience).
These systems are validated for use on smear positive respiratory
specimens. However, we accept the following specimens for Direct TB
PCR:
Sputum
Bronchio-alveolar lavage
Tracheal aspirates
Non-directed bronchial lavage
CSF
}
Minimum specimen
volume 500 L
The value of negative results from smear negative respiratory and CSF
specimens is limited. Negative results for these specimens do not exclude
M. tb complex infection.
Any other specimen types MUST be discussed with the laboratory and will
in general be referred to the Consultant Microbiologist covering the
service.
PLEASE NOTE THERE IS A CHARGE FOR THIS SERVICE
(Please refer to website or contact the WCM for latest charges).
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Identification of mycobacteria:
Isolates may be referred to us either from liquid culture systems (please
see appendix A for details of how to prepare a specimen for sending to us)
or on an L-J slope.
Molecular Identification:
i) M. tb complex & M. avium complex (Geneprobe):
Following culture or receipt of a referred mycobacterial isolate our
first line of identification is the application of a DNA probe
(Geneprobe). This allows identification of M.tb complex and M.avium
complex. The results are telephoned to the referring laboratory.
The target turnaround time for reporting identification by Geneprobe
is 2 working days from receipt of a culture.
ii) Non tuberculosis mycobacteria (NTM) (HAIN):
For the identification of non tuberculosis mycobacteria (NTM) the
GenoType Mycobacterium CM kit (Hain Lifescience) is used. This
improves the turnaround time to identification compared with
phenotypic methods. This kit permits the identification of the
following mycobacterial species: M. avium ssp., M. chelonae/M.
immunogenum,
M.
Abscessus/M.
immunogenum,
M.
Fortuitum/M. mageritense, M. gordonae, M. intracellulare, M.
scrofulaceum, M. interjectum, M. kansasii, M. Malmoense/M.
haemophilum/M. palustre, M. peregrinum/M. alvei/M.
septicum, M. marinum/M. ulcerans, M. tuberculosis and M.
xenopi. However not all isolates can be satisfactorily identified by
molecular methods and phenotypic systems will continue for these
few isolates.
Please note: using the GenoType Mycobacterium CM kit it is not
possible to differentiate species that are paired together
Automated geneprobe for ID of
Mycobacterial species
“HAIN GenoType”
HAIN GenoType Mycobacterium
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Phenotypic identification:
Phenotypic tests are set up on all positive cultures of M. tuberculosis
complex for speciation. Phenotypic tests are also set up on NTMs when
molecular results are unavailable or require confirmation. We currently
use solid media phenotypic methods to identify the mycobacteria.
6.3.3
Sensitivity testing:
Mycobacterium tuberculosis complex:
Sensitivity testing is carried out on all first isolates of Mycobacterium
tuberculosis and M.tb complex. (Routine repeat sensitivity testing is only
performed on subsequent isolates from samples > three months post the
original isolate).
1st line:
2nd line:
Isoniazid, Ethambutol, Rifampicin, Pyrazinamide
Clarithromycin, Ciprofloxacin, Streptomycin
Currently sensitivity testing is carried out on solid media (using the
“Absolute Concentration Method” measuring MIC on solid agar). To
improve turnaround times liquid culture drug sensitivity testing systems
are being validated, however solid media confirmation of resistance occurs
in all instances.
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If an isolate is found to be multi-drug resistant (i.e. 2 or more of 1st line
isolates testing resistant) the isolate will be referred to the HPA NMRL,
London for 3rd line testing (including capreomycin, amikacin, kanamycin,
moxifloxacin, ofloxacin, prothionamide)
Mycobacteria other than tuberculosis (NTM):
Drug sensitivity testing for NTM is not well standardised. The poor
correlation between in-vitro testing and in-vivo outcomes and the possible
colonisation/contamination from widespread environmental sources,
necessitate caution in general testing, particularly if isolated from only a
single sample. (See Appendix D).
Sensitivity testing will be routinely performed for:
M kansasii (if multiple respiratory isolates):
-Rifampin, Ethambutol, Ciprofloxacin, Clarithromycin
M marinum (any skin/soft tissue isolate):
-Rifampin, Ethambutol, Ciprofloxacin, Clarithromycin
MAIC (sterile sites eg blood/bone marrow):
-Clarithromycin
Rapid growing mycobacteria (M. fortuitum, M. chelonae, M. abscessus, M.
peregrinum) (for respiratory isolates if underlying lung damage eg cystic
fibrosis; for sterile sites eg blood/tissue if line associated or disseminated
disease):
-Clarithromycin, Ciproflxacin, Amikacin, Gentamicin, Tobramycin,
Kanamycin, Cefoxitin, Imipenem, Sulphonamide, Trimethoprim,
Piperacillin, Doxycycline, Linezolid, Augmentin
If sensitivity testing is required outside these parameters the case should
be discussed with the WCM Consultant Microbiologist.
Further advice on clinical management of NTM can be obtained from the
lead Consultant Chest Physician for mycobacterial disease, Llandough
Hospital and the Infectious Diseases unit at University Hospital Wales.
6.3.4
Typing of Mycobacterium tuberculosis isolates:
Since April 2005 all strains of Mycobacterium tuberculosis submitted to the
Wales Centre for Mycobacteria have been prospectively typed using the
UK wide standardised VNTR / MIRU method (Frothingham and MeekerO‟Connell, 1998 and Supply et al., 2001) agreed by the HPA DAME group.
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This molecular typing method initially involved analysis of 15 regions (loci)
of the TB genome using PCR amplification looking for repeat gene
sequences. The amplification products are further analysed using a
Beckman-Coulter sequencer. As two of the ETR loci overlap with the two
of 12 MIRU loci a final 17-digit number was reported as a 5-plus 12 digit
code incorporating the 5 digit “ETR” code followed by a 12 digit “VNTRMIRU” code. for each M. tuberculosis strain. The number generated
reflects the number of gene sequence repeats at each locus. Since April
2011 in line with UK HPA DAME group recommendations 9 extra loci have
been added. The 24 loci now reported (5 “ETR” + 10 original UK VNTRMIRU + 9 extra MIRU) help minimise false clustering and enable
international standardisation. Previous 15 loci reports can be compared to
the new 24 loci and if indicated further loci on previous isolates can be
assessed as all DNA is kept indefinitely.
When isolates of Mycobacterium sp. are submitted identification and
sensitivity testing is performed with submission of all the M. tuberculosis
complex strains for molecular typing. Our M. tuberculosis reports now
include confirmation of the strain identification, the results of sensitivity
testing and a 24-digit typing code.
As the WCM database of TB typing results builds up, BioNumerics software
will be used to build up a dendrogram showing the relationship of all TB
isolates. It is intended that these dendrograms will be routinely available
to Health Protection Teams in due course. On occasions the typing results
will throw up similar isolates, which will be highlighted to the relevant
microbiologists / Health Protection Teams for further investigation of any
epidemiological links. The strain type results will also be entered into a UK
wide database to further enhance TB control.
Investigation of possible outbreaks of TB:
It remains important, however, that if you suspect an outbreak of TB or a
cross-infection incident in your laboratory that you inform us which patient
strains are implicated so that we can prioritise the typing of these isolates
as an “outbreak / cross-infection” investigation.
The typing service will run alongside the routine identification and
sensitivity testing of Mycobacterium tuberculosis complex isolates.
Turnaround times of final reports for identification and sensitivities are not
affected by the addition of the typing service.
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VNTR (variable number tandem repeats)
– The Principle
TAAGGGCCA (X1)
Strain 1:
Strain 2:
2
TAAGGGCCA (X2)
4
3
1
3
ETR–A ETR-B
TAAGGGCCA (X3)
8
3
1
4
ETR-C
ETR-D
4
ETR-E
Digital Code for Strains generated by ETR – VNTR
Strain 1
1
3
2
2
3
Strain 2
3
3
2
3
3
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Typical MIRU VNTR results
4 3 0 7 A .B 1 1 _ 0 5 0 7 0 5 1 4 A 8
4 3 0 7 b .B 1 2 _ 0 5 0 7 0 5 1 5 M I
70 000
35000
60 000
4","5
MIRU
30000
26","2
MIRU
2","2
MIRU
50 000
39","3
MIRU
25000
27","3
MIRU
20","2
MIRU
40 000
20000
24","2
MIRU
30 000
15000
16","3
MIRU
20 000
10","4
MIRU
10000
40","3
MIRU
31","5
MIRU
10 000
23","10
MIRU
5000
0
100
200
300
400
500
600
700
Size (nt)
0
Dye Signal
Dye Signal
0
0
50
10 0
15 0
20 0
25 0
30 0
35 0
40 0
S ize (n t)
45 0
50 0
55 0
60 0
65 0
70 0
WCM 24 Loci TB Typing Report
A
B
C
D
31
2
10
16
20
23
24
26
27
39
40
Sample
42
19
21
23
24
31
36
40
41
4
55
63
47
01
71
90
52
56
1
8
5
2
1
5
2
4
3
2
6
2
2
3
1
3
2
10
6
3
2
3
6
4
1
2
4
2
2
3
5
2
6
4
2
5
1
5
3
3
4
3
4
2
4
2
3
3
-
8
3
4
2
2
3
5
2
5
4
2
5
1
7
3
3
3
4
4
2
4
2
3
2
5
-
4
8
4
4
4
5
2
4
2
2
6
2
2
3
1
3
2
11
2
3
2
3
2
7
1
5
4
2
2
3
5
2
6
3
2
5
1
7
3
3
3
4
4
2
4
2
3
3
-
-
6
3
2
3
3
2
2
3
3
2
5
1
5
3
2
4
4
3
3
4
4
3
3
-
3
7
4
2
2
3
5
2
5
4
2
5
1
5
3
3
3
3
4
2
4
2
3
3
6
3
8
3
2
4
3
3
2
3
3
2
6
1
5
3
2
3
2
4
2
4
4
3
3
4
3
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Gamma- Interferon assays:
Background:
Blood based commercial tests have been developed to aid the diagnosis of
infection with Mycobacterium tuberculosis (based on the release of
interferon-gamma from peripheral blood monocytes in patient blood
samples, by specifically testing the response to exposure to antigens
found in the Mycobacterium tuberculosis complex).
They have several distinct benefits compared to traditional tuberculin skin
testing (TST):
The antigens used (e.g. ESAT-6, CFP-10, TB7.7(p4)) are absent from the
vaccine-strain BCG and hence unlike TST these tests are not influenced by
prior BCG vaccination.
In addition to being absent from all BCG strains the antigens used are also
absent from most mycobacteria other than tuberculosis (NTM) (with the
exception of M. kansasii, M. marinum and M. szulgai) enabling the tests to
help distinguish positive TST reactions due to exposure to NTM.
Because interferon-gamma tests require single blood sampling, patients
do not need to return for a second visit for test reading as is required for
TST (where failure to return within 72 hours requires repeat testing).
Current systems:
Two main systems have been developed, one ELISA based measuring
interferon-gamma release in whole blood on exposure to the antigens
(QuantiFERON®-TB Gold In-Tube test, Cellestis, Australia) (QFT-G) the
other using the enzyme-linked immunospot principle involves counting of
individual activated specific T-cells (ELISpot, T-Spot.TB assay, Oxford
Immunotec, UK).
Both methods have been shown to be sensitive and specific. Whilst it
remains unclear whether one will prove more reliable in specific
circumstances, there is some suggestion that the ELISpot assay may have
greater sensitivity in young children and the immune-compromised.
Logistically the original ELISpot assay was more challenging as blood
needed to be processed within a few hours of sampling for optimal results,
requiring a special courier service to transport from centres in Wales to
the commercial laboratory in Oxford. An enhanced version (T-Spot.TB
Xtend, Oxford Imunotec, UK) has been developed which can be performed
at 24 hours allowing overnight transport (FDA approval September 2010,
usage studies ongoing). The QFT-G samples can be initially incubated in
the nearest local microbiology laboratory, prior to sending on to the
testing laboratory by regular transport.
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National recommendations for usage:
In the USA QFT-G had originally been recommended by the Center for
Disease Control and Prevention (2005) for use in all circumstances in
which TST is used, including as an aid for diagnosing both latent TB
infection and TB disease. In 2010 this updated guidelines were produced
(see reference).
Within the UK the National Institute for Health and Clinical Excellence
(2006) recommended that interferon-gamma testing should be considered
to diagnose latent TB in patients with positive results on Mantoux testing
(or in whom Mantoux testing may be less reliable, re the “Green Book”
2006) and for the six week follow up of close contacts of active TB with
initial negative TST.
Because the tests do not distinguish between latent infection and active
disease their role in the diagnosis of active disease is more contentious,
however NICE have stated they may have a utility in ruling out infection
when investigating for active disease with negative cultures results (in the
context of appropriate clinical/pathological/radiological investigations). In
March 2010 NICE released updated guidance (CG117 Tuberculosis, 23
March 2011).
The Department of Health commissioning “toolkit” for TB services (2007)
concluded that there was no evidence interferon-gamma tests are cost
effective in the diagnosis of active TB, however it did note their potential
value in diagnosis in difficult cases (e.g. children and the immune
compromised).
Interferon-gamma tests have also been suggested as tools to assist in TB
screening. NICE recommended they could be used instead of Mantoux for
screening employees new to the NHS with no evidence of prior BCG
vaccination. This has been included in the Department of Health
recommendations on new healthcare worker occupational health clearance
screens (2007). The potential utility of interferon-gamma testing for other
screening programmes (e.g. asylum seekers, prison health) is being
explored to assess if the cost benefit analysis compared to TST may be
favourable.
Additional screening with interferon-gamma testing has been suggested
for certain patient groups prior to planned immunosuppressive therapy
(e.g. anti TNF-α treatment).
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IFNg release: rationale
Andersen P et al, Lancet 2000
Current mechanisms for testing in Wales:
1) QuantiFERON®-TB Gold In-Tube test (Cellestis, Australia) (QFTG):
The Wales Centre for Mycobacteria (WCM) introduced interferon-gamma
testing via Public Health Wales Microbiology Swansea for laboratories in
Wales, using the QFT-G system since November 2009.
Stocks of kits (sampling tubes and instruction documentation) are kept at
the WCM. Appropriate users in Wales (nominated TB physicians/TB
nurses, consultant microbiologists, public health teams etc) requesting
interferon-gamma testing should contact their local microbiology
department or the WCM* for supplies of authorised kits which should then
be returned via the local microbiology laboratory to Public Health Wales
Microbiology Swansea as per the instructions kit (see attached sheets).
It would be advised that patient sampling is only performed Monday to
Wednesday (due to the distance involved the local laboratory would need
to do the initial overnight incubation then arrange transport to Public
Health Wales Microbiology Swansea to ensure samples arrive Monday to
Friday for further processing, avoiding the weekend).
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NB it is imperative that local teams discuss with their local
microbiology laboratory in advance of testing to ensure
appropriate transport and handling of samples.
It is anticipated the turn around of results will be within seven working
days. Unfortunately the £40 cost to the WCM needs to be re-charged to
the requestor pending any future business case.
*Wales Centre for Mycobacteria, Microbiology Cardiff, Llandough Hospital;
Tel: 029 20716408
Email: cardiff.cosurv@wales.nhs.uk or Michael.Ruddy@wales.nhs.uk
2) ELISpot T-Spot.TB assay and TB Xtend (Oxford Immunotec,
UK):
This service is currently not available through NHS laboratories in Public
Health Wales or the HPA but is accessible via the commercial company
Oxford Immunotec.
Enquiries should be made directly to:
Oxford Immunotec Limited, 91 Milton Park, Abingdon, Oxon, OX14 4RY.
Tel: 01235 442780
Fax: 01235 442781
Queries:
For any queries concerning interferon-gamma testing in TB management
(e.g. when and how to test) or for guidance with interpreting results
please contact the Wales Centre for Mycobacteria:
Wales Centre for Mycobacteria, Public Health Wales Microbiology Cardiff,
Llandough Hospital, Penlan Road, Penarth CF64 2XX
Tel: 02920 716408
Email: cardiff.cosurv@wales.nhs.uk or Michael.ruddy@wales.nhs.uk
7
Selected references:
Health Protection Agency (2006). Investigation of specimens for
Mycobacterium species. National Standard Method BSOP 40 Issue 5.
http://www.hpastandardmethods.org.uk/documents/bsop/pdf/bsop40dk.pdf.
Public Health Wales Microbiology Services Standard Operating Procedure
MSBSOP 040 Investigation of specimens for Mycobacterium species. NHS
Wales intranet
http://nww2.nphs.wales.nhs.uk:8080/QualityManagementDocs.nsf
Date Authorised: 8 May 2012
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For provisions for the transfer of infectious substances please refer to:
“The Carriage of Dangerous Goods and Use of Transportable Pressure
Equipment (Amendment) Regulations 2005” – ADR 2005. (Category A
infectious substance – Packing Instruction PI 620 applies to UN Nos. 2814
& 2900; Category B infectious substance – Packing Instruction PI 650
applies to UN No. 3373)
National Institute for Health and Clinical Excellence (2006). Tuberculosis:
Clinical diagnosis and management of tuberculosis, and measures for its
prevention and control. Clinical Guideline 33 developed by the National
Collaborating Centre for Chronic Conditions. www.nice.org.uk/CG033.
Replaced and updated by NICE update 2011
http://guidance.nice.org.uk/CG117
Control and prevention of tuberculosis in the United Kingdom: code of
practice 2000. Joint Tuberculosis Committee of the British Thoracic
Society.
Thorax. 2000 Nov;55(11):887-901.
Chemotherapy and management of tuberculosis in the United Kingdom:
recommendations 1998. Joint Tuberculosis Committee of the British
Thoracic Society.
Thorax. 1998 Jul;53(7):536-48.
Griffith DE et al. (2007) An Official ATS/IDSA Statement: Diagnosis,
Treatment, and Prevention of Nontuberculous Mycobacterial Diseases. Am
J Respir Crit Care Med. 175:367–416.
http://www.thoracic.org/sections/publications/statements/pages/mtpi/
nontuberculous-mycobacterial-diseases.html
Management of opportunist mycobacterial infections: Joint Tuberculosis
Committee Guidelines 1999. Subcommittee of the Joint Tuberculosis
Committee of the British Thoracic Society.
Thorax. 2000 Mar;55(3):210-218.
American Thoracic Society: Diagnosis and Treatment of Disease caused by
Non tuberculous mycobacteria. Am. J Respir. Crit Care Med 156: S1 S25
1997.
Barnard M, Albert H, Gerrit C, O‟Brien R, Bosman M. Rapid Molecular
Screening for Multidrug-Resistant Tuberculosis in a High-Volume Public
Health Laboratory in South Africa. Am. J Respir. Crit Care Med 177:787792; 2008
Date Authorised: 8 May 2012
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P. Supply, S. lesjean, E. Evgueni, K. Kremer, D. van Soolingen and C.
Locht. Automated high-throughput genotyping for study of global
epidemiology of Mycobacterium tuberculosis based on mycobacterial
interspersed repetitive units. Journal of Clinical Microbiology (2001), 39,
3563-3571.
R. Frothingham and W.A. Meeker-O‟Connell. Genetic diversity in the
Mycobacterium tuberculosis complex based on variable numbers of
tandem DNA repeats. Microbiology (1998), 144, 1189-1196.
Centers for Disease Control and Prevention. Updated Guidelines for Using
interferon Gamma Release Assays to Detect Mycobacterium tuberculosis
Infection – United States 2010. MMWR 2010;59(No.RR5 1-25)
Useful websites:
www.iechydcyhoedduscymru.wales.nhs.uk /www.publichealthwales.org
www.hpa.org.uk
http://www.cdc.gov/
http://www.tbalert.org/
http://www.theunion.org/
http://www.who.int/en/
http://www.hain-lifescience.de/en/
http://www.cellestis.com/IRM/content/aust/home.html
http://www.seegene.com/en
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Appendix A
Preferred procedure for sampling MGIT 960 tubes for
sending to WCM
Remove MGIT tube from MGIT 960 without disturbing the sediment
at the bottom of the tube.
Film small quantity of sediment to confirm presence of Acid Fast
bacilli
Using a sterile pipette, remove approximately 1 ml sediment from
the bottom of the tube and place in a small flat bottomed tube with
an „O‟ ring in the lid.
Ensure the top of the tube is secure and further secure with
parafilm.
Pack the vial according to the regulations governing transport of
infectious material.
In order to comply with the new regulations for transfer of mycobacteria
you are required to notify us of cultures being forwarded. Our preferred
option is that you contact us by E-mail using cardiff.cosurv@wales.nhs.uk
Please do not send the patients name, only the initials, date of birth and
your laboratory number. When the culture arrives we will then confirm
receipt by returning the message. If no parcel arrives within two working
days we will contact you.
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Appendix B
Packaging of Category III specimens for sending to WCM
Specimens – clinical samples and/or microbiological cultures must be
packed in a triple packaging system to 620 (602) standard as follows: The primary container must be watertight and leak-proof and may
be glass, metal or plastic. Screw caps must be re-enforced with
adhesive tape or parafilm. The container must be wrapped in
sufficient absorbent material to absorb the contents of the container
in the event of breakage or spillage.
The primary container must be placed into a secondary container
that is leak proof and watertight. Several wrapped primary
containers may be placed in the secondary container (not exceeding
50 ml or 50g). Sufficient absorbent material must be placed
between the specimens and the secondary container to prevent
rattling and also to absorb any spillage or leakage.
The secondary container must be placed in an outer package for
shipment. Include the request forms with the secondary container
and not inside the secondary container. Include the following details
on the outer package: o Name and address of Sender
o Name and address of receiver (see front of User Manual for
WCM address)
o Contents of Package
o Package orientation label/ THIS SIDE UP if liquids are being
sent
NB
Royal Mail “SafeBox” packaging system is 650 specification
only and should not be used to transport Hazard Group 3 biological
agents
In order to comply with the new regulations for transfer of mycobacteria
you are required to notify us of cultures being forwarded. Our preferred
option is that you contact us by E-mail using cardiff.cosurv@wales.nhs.uk
Please do not send the patients name, only the initials , date of birth and
your laboratory number. When the culture arrives we will then confirm
receipt by returning the message. If no parcel arrives within two working
days we will contact you.
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Appendix C
Prices for external services:
Please refer to the Public Health Wales website or contact the WCM for the
latest updates on prices.
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Appendix D: NTM (Non Tuberculosis Mycobacteria)
Mycobacterial sp.
Clinical syndromes
Pulmonary disease in
damaged lungs
M. avium
M. intracellulare
Lymphadenopathy in
children
Disseminated disease in
immunocompromised.
M. kansasii
M. marinum
M. ulcerans
Pulmonary Disease in
damaged lungs
Extra-pulmonary disease
Skin and soft tissue
disease. “Fish tank
granuloma”
Indolent ulceration on legs
Site of
Isolation
Respiratory
Lymph node or
aspirate
Sterile sites (e.g.
blood or bone
marrow)
Urine
Respiratory
Comment
Significant if isolated from repeated
specimens with appropriate clinical
history. Treat according to BTS guidance
2000*
Removal of node is best treatment
otherwise follow BTS guidance 2000*
Testing of clarithromycin susceptibility
may be relevant in patients who have
failed prior macrolide therapy.
Significance doubtful from urine
Significant if isolated from repeated
specimens with appropriate clinical
context. Treat according to BTS
guidelines 2000*
All isolates
All isolates
All isolates
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Mycobacterial sp.
Rapid growing
mycobacteria
M. chelonae
M. abscessus
M. fortuitum
M. peregrinum
Quality
Clinical syndromes
Pulmonary disease in
damaged lung e.g. CF
Line associated infection
Disseminated skin and
tissue infection
May disseminate in
immunocompromised
patients
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Site of
Isolation
Respiratory from
CF or other
damaged lung
Blood culture
isolate
Comment
Significance doubtful unless repeated
isolation from damaged lungs e.g. in CF
or bronchiectasis
Sterile sites
Doubtful significance unless isolated
repeatedly or from sterile site. Please
supply full clinical details if sensitivities
required.
Pulmonary infection
Respiratory
Significant if repeatedly isolated within
relevant clinical context. Treat according
to BTS guidance 2000*
All may disseminate in
immunocompromise
All isolates
Treat according to BTS guidance 2000*
M. gordonae
All isolates
Significance doubtful
Scotochromic
psychrophiles
All isolates
Environmental mycobacteria –
significance very doubtful.
M. xenopi
M. malmoense
M.
M.
M.
M.
M.
szulgai
scrofulaceum
celatum
simiae
genevense
References:
Joint Tuberculosis Committee Guidelines 1999: Management of opportunist mycobacterial infections Thorax 2000; 55:210-218.
American Thoracic Society: Diagnosis and Treatment of Disease caused by Nontuberculous mycobacteria. Am. J Respir. Crit Care Med 156:
S1 S25 1997 (see also 2007 update)
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Appendix E: MicrobiologicalMycobacterial Enquiries:
Communication Algorithm for Wales Centre for Mycobacteria (WCM)
Technical queries/ results enquiries
re:
Mycobacterial sampling
Microscopy
Culture
Identification
Drug sensitivity testing
Senior technical staff WCM, Llandough
Hospital:
Tel: 029 20716408
Email: cardiff.cosurv@wales.nhs.uk
Clinical enquiries or queries regarding:
epidemiological investigations
requests for IFN gamma testing
requests for PCR and molecular
resistance testing
1) WCM Consultant Medical Microbiologist (CMM)
(Dr Michael Ruddy)
Tel:
Llandough Hospital: 029 20716408
West Wales Hospital: 01267 237271
Email: Michael.ruddy@wales.nhs.uk
For urgent queries:
mobile: 07969 917709
Air call via Prince Philip Hospital: 01554 756567
2) if (1) above unavailable please contact Consultant
Medical Microbiologist on call at University Hospital
Wales (UHW):
Tel: 029 20744515
Senior clinical scientist Wales Molecular Centre,
University Hospital Wales: Tel: 029 20746581
Email: Lewis.white@nphs.wales.nhs.uk
Technical queries/results enquiries re:
M tb strain typing
PCR
(molecular resistance markers)
Additional expert advice on mycobacterial disease
is available from:
1) microbiological mycobacterial queries:
a. Dr Robin Howe CMM Microbiology
Cardiff; tel 029 20744515; email
Robin.Howe@wales.nhs.uk
b. Dr Mark Hastings CMM/Director Public
Health Wales Micro; tel 029 20744515;
email Mark.Hastings@wales.nhs.uk
2) Clinical queries:
a. Should be addressed in the first
instance to the local nominated TB
physician (contact via local hospital
switchboard)
b. The lead Consultant Chest Physician for
mycobacterial disease Llandough
Hospital.
3) Public health related queries:
Should be addressed in the first instance to
the local Health Protection Team (contact
via local authority/PHW directory)
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APPENDIX F: WCM EXAMPLE FORMS
1) WCM specimen referral form
2) Public Health Wales QuantiFERON-TB Gold Patient Data Sheet
and blood sampling instructions
3) UK MDRTB Services letter and Patient Data Sheet
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Wales Centre for Mycobacteria Microbiology Cardiff, Llandough Hospital Penlan
Road, Penarth Vale of Glamorgan, CF64 2X
DX: Public Health Wales Micro (Llandough)DX6070400
715134
Penarth 90 CF
Tell 02920 716408 Fax: 029 20
Source Laboratory
Reference Laboratory Nº
_________________________
Date Received by Ref. Lab. ________________________
Specimen Details
Source Lab Nº _________________________
Source Hospital _______________________________________
Specimen Date ________________________
Site of Lesion _________________________________________
Specimen Type ________________________
Microscopy + / - / not done / unknown
Please Indicate Test Required:
Culture

Direct PCR for TB complex
(please indicate below)
Identification


Sensitivity

Detection of Mycobacterium Tuberculosis
In non-processed smear-positive sputum

Typing

Detection of Mycobacterium Tuberculosis
In CSF (minimum 0.5ml needed)


If requesting the following please discuss with
our Consultant Microbiologist before submitting
the sample:
Smear negative and other samples
(please state site) _______________
Patient Details
Surname _______________________________
Forename (s) _____________________________________________
Male / Female / Unknown
Date of Birth/Age __________________________________________
Address ________________________________________________________ Post Code __________________________
Ethnic Origin
African
Caribbean
Indian
Pakistani




Previous TB
Bangladeshi

Indian Subcontinent Unspecified 
White

Mixed

yes / no / unknown.
Previous Anti-TB Therapy
Unknown 
Other (please state) __________________________
Country of Origin ____________________________
If yes, when and site of previous TB ___________________________________
yes / no / unknown.
Drugs used, if known ___________________
Is the culture related to a possible outbreak?
If yes, when ____________________________________________
Year Arrived in UK _______________________________________
Yes 
No 
Index Case (if known) ________________________
Place of contact:
________________________________________________________________________________
Why do you think this is an outbreak? ___________________________________________________________________
__________________________________________________________________________________________________
__________________________________________________________________________________________________
Do you agree to provide further information to the WCM if needed? Yes 
Is the culture a lab contaminant: Yes  No 
No 
Is the culture a bronchoscope contaminant Yes 
No 
If yes, please give details: ____________________________________________________________________________
Date Authorised: 8 May 2012
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Public Health Wales QuantiFERON®-TB Gold test
Patient Data Sheet
I, undersigned, authorise the WCM to carry out the Quantiferon®-TB test on this sample
Please print your name___________________________Department________________
Consultant/Registrar/TB.CNS
Signed_______________________________ Date_______________________________
Collection information
Time taken _______________ Date ______________
Laboratory information
Incubation
Time in____________ Date__________
Name_____________
Time out___________ Date__________
Name_____________
Sent to
___________ Date __________
Name _____________
Contact details:
Name of Consultant____________________________________________________
Department__________________________________________________________
Laboratory/Hospital____________________________________________________
____________________________________________________________________
Postal Address
____________________________________________________________________
____________________________________________________________________
____________________________________________________________________
Phone_____________________________DX address________________________
FOR WCM USE
Patient’s number_______________________
Date received_________________________ Time Received____________________
Date of test:
_____________
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If the answer requires specification, please specify. Please remember that
complete answers are essential for the correct interpretation of the test results
Baseline epidemiological data
Patient‟s Surname _________________First names____________________________
Address________________________________________________________________
________________________________Postcode code___________________________
DoB________________
Male / Female Occupation________________________
Was the patient born in the UK?
Yes
No
If No: Where? ________________When did the patient come to UK (year)?__________
Has patient lived in, or spent more than 2 months travelling in another country
Yes No Don’t know
History of BCG vaccination and TB skin tests
Has patient ever received the BCG vaccination?
Yes No
Don’t know
If “Yes”, please specify the age: Neonatal
School age (13-14)
Other
BCG scar: Yes No
Mantoux test done
Yes No Don’t know
Reading ________________ mm
Clinical data
Is patient taking any of the following medications?
None
Oral steroids
Cytotoxic drugs
Other immunosuppressive
drugs (please specify)__________________
Is patient immunocompromised?
Yes No
Don’t know
Is patient HIV positive?
Yes No
Don’t know
R
L
Does the patient have diabetes?
Yes No
Don’t know
Does the patient have any of the following:
Upper
(circle appropriate answer(s):
Fever
Night sweats
Loss of weight
Cough
Middle
Is patients CXR abnormal? If yes, please specify the location:
Lower
Cavities?
Yes No Don’t know
Consolidation? Yes No Don‟t know
Uni / Bilateral?
Other relevant clinical
data_____________________________________________________________
_______________________________________________________________________
_______________________________________________________________________
_______________________________________________________________________
_________________________________
History of TB disease, anti-TB treatment, and contacts with those with TB
Has the patient been diagnosed with TB before?
Yes
No Don’t know
If Yes: date (DD/MM/YYYY)____________________________
Previous TB treatment?
Yes
No Don’t know
Previous TB chemoprophylaxis?: Yes
No Don’t know
Has the patient had previous contact with TB? Yes
No
Don’t know
If Yes,
when? ___________
What was the nature of the contact?
Household
Work
Study/School
Prison
Other (please specify)________________________
Part of Outbreak/Contact screening?
Yes No
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GUIDELINES ON BLOOD COLLECTION, STORAGE AND
TRANSPORTATION
QuantiFERON®-TB Gold IT uses the following collection tubes:
1. Nil Control (Grey cap).
2. TB Antigen (Red cap).
3. Mitogen Control (Purple cap).
Blood
collection
tubes
should
be
kept
at
room
temperature
(DO
NOT
REFRIGERATE). Never use blood collection tubes after the expiry date (printed on
the tube label).
Antigens have been dried onto the inner wall of the blood collection tubes so it is
essential that the contents of the tubes be thoroughly mixed with the blood.
The following procedures should be followed for optimal results:
1. For each subject collect 1mL of blood by venepuncture directly into each of the
QuantiFERON®-TB Gold IT blood collection tubes (with red, grey, and purple caps).
As 1mL tubes draw blood relatively slowly, keep the tube on the needle for 2-3
seconds once the tube appears to have completed filling, to ensure that the
correct volume is drawn.
The black mark on the side of the tubes indicates the 1mL fill volume.
QuantiFERON®-TB Gold blood collection tubes have been validated for
volumes ranging from 0.8 to 1.2mL. If the level of blood in any tube is not close
to the indicator line, it is recommended to obtain another blood sample.
If a “butterfly needle” is being used to collect blood, a “purge” tube should be
used to ensure that the tubing is filled with blood prior to the QuantiFERON®TB Gold tubes being used.
2. Mix the tubes by turning the tube end-over-end 8 to 10 times or shaking the tube
for 5 seconds ensuring that the entire inner surface of the tube has been coated
with the blood. Thorough mixing is required to ensure complete mixing of the blood
with the tube’s contents.
3. Label tubes appropriately (Patient’s name, surname, DoB, Hospital No)
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4. After the blood collection and mixing, keep tubes in upright position in the rack at
room temperature. DO NOT REFRIGERATE OR FREEZE THE BLOOD SAMPLES.
After blood collection, bottles with blood can be:
-
either delivered to the WCM Cardiff or Public Health Wales Microbiology
Swansea on the same day (before 5 pm)
-
OR, where practical, incubated overnight in the incubator at 37 oC. There is no
need for CO2 supply in the incubator. The overall time of incubation at 37 oC
should not exceed 20 hours. After incubation at 37oC blood specimens should
be sent to Microbiology Swansea either with courier, or DX, or First Class
Royal Mail in appropriate biosafety containers. The overall time in transit
should not exceed 3 days.
Postal address:
FAO: Dr Michael Isaac
Microbiology Dept
PHW Microbiology Swansea
Singleton Hosp
Swansea SA2 8QA
DX:
Hays DX
DX 6070300 Swansea 90 SA
Date Authorised: 8 May 2012
Document and Version: WCMQMS 035.5
Page: 34 of 35
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Public Health Wales Microbiology Cardiff
Wales Centre for Mycobacteria
Quality
Status: Controlled for
Internet Publication
Wales Centre for Mycobacteria
Public Health Wales
Microbiology Cardiff
Dear Dr
Re: Case of multi-drug resistant tuberculosis (MDRTB)
We understand that you have recently had a patient diagnosed with multidrug resistant tuberculosis. You may be aware that there now exists a
national MDRTB service, whose aim is to collate information on all MDRTB
cases identified in the UK and, if required, provide advice and support in
the management of these patients. This initiative is endorsed by the
Department of Health and has financial support from Genus Pharma.
Therefore we are writing to you with two aims:(a) to invite you to provide information on the patient, their
management and their outcome to enable the collection of data on a
sufficiently large group of MDR TB cases to draw valid conclusions
and share good practice.
(b) to offer you ready access to expert advice on the management of
patients with MDR TB (see the attached list of experts and their
contact details ).
We would be grateful if you would complete and return the attached form
to the MDRTB service, via email, at MDRTBservice@ctc.nhs.uk
Yours sincerely
Dr Michael Ruddy
Reference Laboratory Microbiologist
Peter Davies, Coordinator
MDR TB Service
Date Authorised: 8 May 2012
Document and Version: WCMQMS 035.5
Page: 35 of 35
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