Interferon-gamma blood test What is it and why do we use it? - April 2018 - TB Hub
←
→
Page content transcription
If your browser does not render page correctly, please read the page content below
Interferon-gamma blood test
What is it and why do we
use it?
April 2018
Interferon gamma (IFNγ) test • What is it? • How does it work? • How accurate is it? • Why do we use it? • When do we use it?
IFNγ test • Supplementary blood test used in addition to the tuberculin skin test • Approved for use in cattle under EU legislation and by the OIE (World Organisation for Animal Health) • It is the only blood test approved in the UK and the EU to supplement the skin test for TB in cattle
Why do diagnostic tests for bovine TB need to be
approved?
• Diagnostic tests need to be fully validated as per OIE
guidelines, so that we can interpret the results that they
generate
• Validation involves multiple steps to assess the
diagnostic performance characteristics of the test
• The test must also be shown to be repeatable and
standardised
• Tests for bovine TB need to be approved under EU legislation for trade purposes and to establish the official disease status of herds • IFNγ and IDEXX antibody tests are the only blood tests currently listed by the OIE as validated for TB in cattle
How does the IFNγ test work? The tuberculin skin test and IFNγ test both measure the host’s immune response to the TB bacterium (Mycobacterium bovis), rather than detect the bacterium itself
How does it work? • The IFNγ test is a comparative test like the skin test • It measures the animal’s immune response to avian and bovine tuberculins (same as used in the skin test) • Tuberculins are mixtures of proteins extracted from cultures of M. bovis bacteria grown in liquid media in the laboratory, and then killed by heat
How does it work?
Instead of injecting tuberculins into the skin, the animal’s
blood is stimulated with avian and bovine tuberculins in the
laboratory
White blood cell
Blood sample from an animal White blood cell releases
infected with TB the cytokine IFNγ
(chemical messenger)
Levels of IFNγ
measured
IFNγ
Stimulated by avian and bovine
tuberculin
How does it work? • Blood samples from TB-infected animals will release greater amounts of IFNγ in response to bovine tuberculin compared to avian tuberculin • Being a laboratory-based test, it is subject to strict quality controls, its readout is more objective and its protocol is easier to standardise compared to the skin test • Samples need to be transported to the lab quickly in temperature- controlled packaging systems as the white blood cells must be kept alive
How accurate is it?
No diagnostic test is perfect!
There is a always a trade off between
sensitivity and specificity
Find all infected animals Don’t remove uninfected
animalsHow accurate is it?
90%
Sensitivity
The probability that a test will correctly identify an infected
animal as positive
The higher the sensitivity of the test, the lower the
probability of incorrectly classifying an infected animal as
uninfected (a false negative result)How accurate is it?
The IFNγ test is 90% sensitive and will miss, on average,
10% of infected animals in a herd
20 truly infected animals – 2 missed by the IFNγ testHow does this compare with the skin test?
The IFNγ test is more sensitive than the skin test
The skin test is 80% sensitive at standard interpretation and
will miss, on average, 20% of infected animals
20 truly infected animals – 4 missed by the skin testHow accurate is it?
96.5% (minimum)
Specificity
The probability that a test will correctly identify an animal
that is free from infection as negative
The higher the specificity, the lower the probability of
incorrectly classifying an uninfected animal as infected (a
false positive result)How does it compare to the skin test? • With the IFNγ test you expect to get, on average, 3-4 false positives per 100 disease-free animals tested (false positive rate of 3.5%) • This means that the IFNγ test is less specific than the skin test • The skin test has a higher specificity of 99.98%, which means you only get one false positive per 5,000 disease-free animals tested (false positive rate of 0.02%)
So why use IFNγ if there is more chance of
false positives?
• Due to a higher probability of false positive results, the
IFNγ test is not used for testing of officially TB-free herds
• In most cases it is only applied to TB breakdown herds
with lesion and/or culture positive animals
• In these herds the risk of taking out low numbers of false
positives is outweighed by the need to identify and
remove all infected cattleSo why use IFNγ if there is more chance of
false positives?
• In 2017, 98,529 samples were IFNγ tested in England
• The overall proportion of test-positive results was 5.2%
(5094 animals slaughtered)
• The IFNγ test is used in a targeted, proportionate way in
TB breakdown herds to maximise the sensitivity of
testing
• The IFNγ test policy must balance costs and benefits in
the context of the strategy for achieving officially TB free
status for EnglandWhat are the benefits? The skin and IFNγ tests are used together to maximise the sensitivity of testing and increase the chances of finding all infected animals in the herd
What are the benefits? • The IFNγ test can identify cattle at an earlier stage of infection than the skin test (experimental cattle infection shows 3-4 weeks vs 7 weeks) • Studies in GB, NI & RoI consistently show that IFNγ- positive but skin test- negative cattle have a higher risk of becoming skin test reactors and develop visible lesions if not removed (compared with IFNγ and skin test- negative animals in the same herds)
What are the benefits? • Using the IFNγ test at the beginning of a new TB breakdown can shorten the duration of movement restrictions by increasing detection of infected animals • It can also reduce the likelihood of residual cattle infection in herds that regain Officially TB Free status after a breakdown • 58% of infected herds in the High Risk Area have had a breakdown in the last 3 years
What are the benefits?
448,971 animals from TB breakdown
herds IFNγ tested from 2006 to April
2017
Over 33,000 IFNγ-positive animals
identified that otherwise would have
been missedWhat about ‘no visible lesion’ (NVL) IFNγ and skin-
test positive animals?
If a skin test or
IFNγ positive
animal does not
have visible
lesions at
slaughter, it does
not mean that it
wasn’t infectedWhat about ‘no visible lesion’ (NVL) IFNγ and
skin-test positive animals?
• Post mortem meat inspection is a relatively
insensitive method of confirming TB infection
• Test positive (but NVL) animals may have been
in the early stages of infection when TB
lesions were too small to see with the naked eye
or not yet developed in the organs
• Small lesions can be missed at the
slaughterhouse
• Can get genuine false positives (3.5%), i.e. the
animal was not infected but tested positiveWhat about further IFNγ positive results after a
skin test?
• Residual infection, or the reintroduction of infection can
cause more test-positives downstream
• The skin & IFNγ tests identify slightly different
populations of infected individuals
• Overall in a confirmed infected group, while most infected
animals will be identified by both tests, there will always
be animals picked up by one test but not the other
• Infected individuals in a herd will not all be at the same
stage of infection. So while removing those that have
reacted to the skin or IFNγ test, there may still be others
that have only just been infectedWhen do we use the IFNγ test? Edge & Low Risk Areas • Mandatory for new TB breakdowns with lesion and/or culture positive animals • On 1st January 2018, Cheshire, Derbyshire, East Sussex, Oxfordshire and Warwickshire became fully incorporated into the Edge • Breakdown herds with lesion and/or culture positive animals in the former High Risk Area parts of those counties are now eligible for IFNγ testing
Why? The overall incidence of TB in the Edge Area is still increasing, therefore cattle measures need to be strengthened. IFNγ testing • Increases the overall sensitivity of testing in TB breakdown herds • Helps identify infected animals in the herd earlier • Potentially reduces the time under movement restrictions • ‘Mops up’ infection that the skin test leaves behind • Potentially helps reduce recurrence rates due to infected animals remaining in the herd and causing repeat breakdowns (or infecting other herds and badgers)
Increase in IFNγ use as a result of policy changes
since 2013
Annual number of supplementary IFNγ blood tests carried out in England, by risk areaWhen do we use the IFNγ test? High Risk Area (HRA) From April 2017, mandatory IFNγ testing in TB breakdown herds with lesion and/or culture positive animals that satisfy any of the following criteria: 1. New breakdowns in badger culling areas where at least two seasons of effective culls have been completed 2. Where there is clear evidence that repeated skin testing of the herd has failed to clear the infection (persistent/chronic breakdown herds) 3. Where APHA’s investigation concludes that the most likely transmission route for the affected herd is infected cattle (e.g. brought-in animals, contact with neighbouring cattle)
IFNγ test policy in badger culling areas
• Implemented from 1st April 2017
• New TB breakdown herds with lesion and/or culture positive
animals in eligible cull areas
Cull area Year culling started Year eligible for mandatory
IFNγ testing policy
Area-1 Gloucestershire
Area-2 Somerset
2013
1 April 2017
Area-3 Dorset 2015
Area-4 Cornwall
Area-5 Cornwall
Area-6 Devon
Area-7 Devon 2016 1 January 2018
Area-8 Dorset
Area-9 Gloucestershire
Area-10 HerefordshireWhy not wait four years in badger culling areas? • Areas that have undergone badger culling have a reduced risk of TB from badgers, and so the TB risk from cattle to cattle transmission becomes even more important • Residual cattle infection is an important cause of recurrent herd breakdowns in the HRA • Need to remove infection from cattle herds as quickly as possible to prevent ‘spill-back’ into the remaining badgers • Two badger culling seasons strikes a balance between allowing culling enough time to reduce the wildlife reservoir while not unnecessarily delaying the benefits of gamma testing
What about environmental contamination with
M. bovis?
• Sunlight, rain dilution, temperature and other
microorganisms (e.g. antibiotic producing fungi) all affect
the survival time of M. bovis in the environment
• IFNγ test is deployed 15 months after the onset of
badger culling. A review of published literature suggests
that M. bovis does not survive in soil beyond a few
months under different environmental conditions
• A study found no viable M. bovis bacteria in the bedding
of a sealed badger sett six months following vacation of
the sett (Gallagher 2000)*
• Viability of infection in badger carcases has been
examined, with no detectable M. bovis bacteria found
after six weeks following burial (Gallagher 2000)*
* J Gallagher, Clifton-Hadley RS (2000). Tuberculosis in badgers; a review of the disease and its significance for other animals. Res
Vet Sci. 2000 Dec;69(3):203-17.M. bovis survival times in soil
Conditions Length of Method Reference
survival
20-40ºC in up to 4 weeks Culture Duffield B, Young D (1985). Survival of Mycobacterium
Australia bovis in defined environmental
conditions. Vet. Microbiology,10: 193--197.
Autumn/winter in 28 days max. Culture Fine A, Bolin C, Gardiner J, Kaneene J (2011) A study of
Michigan, USA the persistence of Mycobacterium bovis in the
environment under natural weather conditions in
Winter/spring in 88 days max. Culture Michigan, USA. Veterinary Medicine International Volume
Michigan, USA 2011 (2011), Article ID 765430, 12 pages.
Spring/summer in 11 days max. Culture http://dx.doi.org/10.4061/2011/765430
Michigan, USA
4ºC in lab (moist up to 150 days Culture Barbier E, Rochelet M, Gal L, Boschiroli ML, Hartmann A.
dark) (2017) Impact of temperature and soil type on
Mycobacterium bovis survival in the environment. PLoS
4ºC in lab (moist up to 150 days qPCR One. 2017 Apr 27;12(4):e0176315
dark) https://doi.org/10.1371/journal.pone.0176315
22ºC in lab (moist up to 90 days Culture
dark)
22ºC in lab (moist up to 120 days qPCR
dark)IFNγ testing in badger culling areas –
early results
Badger cull area Total number Number of Proportion of
of samples positive IFN-γ samples with a
tested test results positive IFN-γ
test result (%)
Dorset 4069 257 6.3
Gloucestershire 464 41 8.8
Somerset 1557 108 6.9
All areas 6090 405 6.7
The overall proportion of samples with an IFN-γ positive test result was 6.7%
(data from first 9 months of the policy - April to December 2017 inclusive)Area-1 Gloucestershire
Proportion of
Submission Total Number of samples with a
and month number of positive IFN-γ positive IFN-γ test
(2017) Herd samples test results result (%)
November
1 43 2 4.7
September
October
2 188 14 7.4
November
November
3 183 24 13.1
December
4 50 1 2.0
8.8
464 41Area-2 Somerset
Proportion of
Submission Total Number of samples with a
and month number of positive IFN-γ positive IFN-γ
(2017) Herd samples test results test result (%)
September 1 50 4 8.0
November
November 2 682 50 7.3
November
November 3 243 19 7.8
November 4 140 7 5.0
October
November 5 153 10 6.5
September
October 6 134 12 9.0
November 7 48 3 6.3
December 8 43 1 2.3
December 9 64 2 3.1
1557 108 6.9Proportion of
Number of
Area-3 Submission and month
(2017)
Herd
Total number
of samples
positive IFN-γ
IFN-γ test
positive
test results
Dorset June
1 441 39
samples (%)
8.8
July
July
2 38 0 0.0
July
July
3 415 14 3.4
August
July
4 159 13 8.2
August
July
5 377 17 4.5
August
October (2nd round )
5 467 19 4.1
November (2nd round)
July
6 330 16 4.8
August
November (2nd round)
6 304 40 13.2
November (2nd round)
August
7 343 21 6.1
August
October (2nd round)
7 189 3 1.6
November (2nd round)
August 8 9 1 11.1
August
9 360 33 9.2
August
August
10 237 5 2.1
August
October
11 400 36 9.0
October
4069 257 6.3IFNγ testing in badger culling areas –
early results
• For the first 9 months’ worth of data, 6.7% of samples
from TB breakdown herds in the three badger culling
areas tested positive, in line with what Defra expected
• The percentage of IFNγ test positive samples in Area-3
Dorset has been lower than in Area-1 Glos. and Area-2
Somerset
• Some herds have yielded higher than average IFNγ %
positivity, whilst others have been below average
• ‘Outliers’ can occur as no ante-mortem test for TB in
cattle is perfectly sensitive or specific, and the herds that
undergo IFNγ testing are exposed to different levels of
M. bovis infectionWhen do we use the IFNγ test? Discretionary use by APHA • Severe (‘explosive’) TB breakdowns with high numbers of reactors that don’t automatically qualify for mandatory testing • To inform decisions about partial/complete herd slaughter (in extreme situations)
De-coupling of skin and IFNγ tests In England, IFNγ testing in TB breakdown herds is undertaken as soon as practicably possible for disease control reasons: • Increases the chances of finding infected animals. The IFNγ test is more sensitive than the skin test and identifies a different population of infected animals • IFNγ test can identify cattle at an earlier stage of infection than the skin test – infected animals are removed earlier (less time for spread of infection to other cattle and wildlife) • Potentially reduces the duration of the breakdown and the chances of residual infection remaining when movement restrictions are lifted
De-coupling of skin and IFNγ tests • To achieve this, the default position is to 'de-couple' the IFNγ test from the skin test and complete it as a stand- alone test as soon as practicably possible, giving the farmer a reasonable period of notice • By de-coupling, we are essentially removing infected animals that were missed by the previous skin test earlier, thus maximising the probability that the herd goes clear at its next skin test • On some occasions it may not be practical to decouple e.g. if the IFNγ test is to be undertaken very close to the next skin test • More information on decoupling on the TB Hub
• Animals under 6 months of age are excluded (their developing immune system interferes with the test) • TB breakdown herds eligible for IFNγ testing will initially undergo one round of blood testing • The IFNγ test is repeated up to three times as long as standard interpretation skin test reactors and/or animals with visible lesions (including slaughterhouse cases) continue to be found after application of the blood test i.e. if there is still evidence of residual infection in the herd which was not picked up by previous skin and IFNγ testing
Private IFNγ testing (England only) • Private vets can submit samples for IFNγ test at keeper’s cost with prior approval from APHA • Limited to specific scenarios outside of the government- funded testing programme where owners seek additional assurances as to the TB-free status of animals over and above statutory testing e.g. to supplement pre/post movement testing, screen animals joining high value herds, test animals following a negative routine or tracing skin test More information at: http://ahvla.defra.gov.uk/vet-gateway/ifng-testing/index.htm
Private IFNγ testing (England only)
Cattle not eligible for private testing:
• Test reactors & other cattle awaiting slaughter for TB
control purposes
• Cattle from TB breakdown herds undergoing
government-funded IFNγ testing
• Cattle from herds under restrictions for overdue skin
testing
If a positive result is received, the animal is (with few
exceptions) compulsorily slaughtered with compensation
paid, the herd will be placed under movement restrictions
and normal breakdown procedures followedPrivate IFNγ testing (England only)
Private IFNγ test cost (Oct 2017 to March 2018)
Cost per test (£) Single test 5+ tests 10+ tests
High specificity test 22.20 18.00 17.00
High sensitivity test 16.20 13.00 12.00Antibody (serological) tests for bovine TB • Blood tests that measure the antibody immune response of the animal to M. bovis (generally detect relatively later compared to IFNγ response) • Lower sensitivity than the IFNγ test and affected by the skin test - animals need to be skin tested before taking the blood sample, to ‘boost’ the serum antibody levels detected by such tests • Antibody tests are not currently approved for statutory TB testing in GB or the EU
Antibody (serological) tests for bovine TB • Data from GB show that antibody tests are less sensitive overall compared to the skin & IFNγ tests, but they can be useful for identifying small numbers of infected cattle that are skin and IFNγ test negative (~3-5% of cattle from fully confirmed TB breakdown herds in a Defra study)
Antibody (serological) tests for bovine TB • IDEXX ELISA test is OIE registered and used by APHA in exceptional cases • Requires prior informed consent from the keeper • Positive animals are slaughtered and compensation is paid • Used by APHA in exceptional cases as a targeted third- line test in difficult TB breakdown herds with lesion and/or culture positive animals, where skin & IFNγ tests are not clearing the herd of infection, and where new infection is not being introduced
Non-validated diagnostic tests for bovine TB
• Tests not officially approved for use in cattle due to
insufficient data on their performance characteristics
• Results may be unreliable and the test may not offer any
certainty about the presence or absence of infection
1. Private vet needs prior permission from APHA
2. Must follow Defra’s protocol for exceptional use of
non-validated diagnostic tests in persistent TB
breakdown herds
3. Owner/private vet decision to voluntarily slaughter or
retain test-positive animals, no compensation paid
4. Test-positive animals retained by the herd owner are
restricted to the holding for lifeTake-home messages • IFNγ test is more sensitive than the skin test. It can identify TB infected cattle that are earlier in the course of infection and those missed by the skin test • It is less specific than the skin test and so it is used in TB breakdown herds with lesion and/or culture positive animals. It cannot be used for surveillance testing or as a replacement for the skin test • IFNγ test positive animals do not necessarily show visible lesions at post mortem. This is because they are often in the early stages of infection when TB lesions are too small to be seen by the naked eye and sometimes lesions are missed at the slaughterhouse
Further information
• More information on bovine TB, including further
background on the IFNγ test is available on the TB hub
http://www.tbhub.co.uk/
• Biosecurity visits may be requested from the TB Advisory
Service
http://www.tbas.org.uk/You can also read
