Interferon-gamma blood test What is it and why do we use it? - April 2018 - TB Hub

 
Interferon-gamma blood test What is it and why do we use it? - April 2018 - TB Hub
Interferon-gamma blood test
  What is it and why do we
            use it?

April 2018
Interferon-gamma blood test What is it and why do we use it? - April 2018 - TB Hub
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?
Interferon-gamma blood test What is it and why do we use it? - April 2018 - TB Hub
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
Interferon-gamma blood test What is it and why do we use it? - April 2018 - TB Hub
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
Interferon-gamma blood test What is it and why do we use it? - April 2018 - TB Hub
• 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
Interferon-gamma blood test What is it and why do we use it? - April 2018 - TB Hub
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
Interferon-gamma blood test What is it and why do we use it? - April 2018 - TB Hub
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
Interferon-gamma blood test What is it and why do we use it? - April 2018 - TB Hub
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
Interferon-gamma blood test What is it and why do we use it? - April 2018 - TB Hub
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
Interferon-gamma blood test What is it and why do we use it? - April 2018 - TB Hub
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
                                    animals
How 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 test
How 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 test
How 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 cattle
So 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 England
What 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 missed
What 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 infected
What 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 positive
What 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 infected
When 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 area
When 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 Herefordshire
Why 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           41
Area-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               6.9
Proportion 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             6.3
IFNγ 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 infection
When 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 followed
Private 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      12.00
Antibody (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 life
Take-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/
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