InterferonGamma Testing in Tuberculosis

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Interferon-Gamma Testing in Tuberculosis

• Professor Peter Ormerod
• Royal Blackburn Hospital and Lancashire
  Postgraduate Medical School

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Tuberculin skin test
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Specific antigens

• ESAT 6
• CFP10
• Found in all M.tb complex organisms
• NOT present in BCG (RD1)
• NOT present in most non-TB
• mycobacteria (esp MAI, malmoense,
• xenopi)

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Specific CD4 response

• If individual has been TB exposed
• Memory CD4 cells should react to ESAT6
• and CFP 10
• These will then release interferon gamma
• This can then be measured
• Serum level
• Or by staining cells

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QuantiFERON-Gold In Tube
Stage One Blood collection and harvesting
Nil
TB
IFN-? stable refrigerated for at least 8 weeks.
Centrifuge tubes for 5 min
Incubation at 37ºC for 16-24 hours
3x1mL blood collection

• Option 1 Shipment of the blood collection tubes
  within 16 hours to a laboratory prior to
  incubation.

• Option 2 Shipment of the blood collection within
  3 days after incubation to the laboratory.

• Possibility to batch samples.

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QuantiFERON-Gold In Tube
Stage 2 Interferon-? ELISA
Incubate for 120min at room temperature
Add stop-solution and read absorbance
Software calculates and prints resuls
Add plasma and conjugate
Wash and add substrate

• Easy Standard-ELISA.

• User-friendly software supplied free-of-charge
  from Cellestis.

• No need for new equipment.

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T-spot TB

• Counting cells not supernatant

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Step 1 Preparation of cells

• Blood collected into Vacutainer CPT tube
• Tube centrifuged
• Lymphocyte band removed
• Cells washed counted
• Cells added to 96-well plate
• Antigens added to wells
• Incubate overnight

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Step 2 Forming spots

• Plate washed
• Add detection reagent for 60 minutes
• Plate washed
• Add substrate spots in 7 minutes
• Plate washed and dried

-ve
ve
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Step 3 Counting spots

• Count spots by eye using a magnifying glass or
  dissecting microscope
• Alternatively use an automated elispot reader
• Plate placed in reader and read automatically
• Computer stores all images, a tamperproof audit
  log, and calculates the number of spots in each
  well
• Camera and analysis settings set automatically

Above Automated T-SPOT plate reader
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Personal Declaration

• Have done no research for either
• company
• Have not received funding from either
• company
• Have declined invitations from both to be
• on their medical boards
• Hospital Lab use QFT because ELISA
• and can be batched

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IGRA Tests (1)

• IGRA testing in 2006 NICE TB Guidelines
• Contact and new entrant screening
• ON ECONOMIC GROUNDS
• PPV of positive IGRA and
• NPV of negative IGRA UNKNOWN

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New entrant screening

• NO strategy cost effective if LTBI rate less
• than 10
• At LTBI rates of 10-40 the 2-step
• tuberculin test followed by IGRA test on
• those tuberculin positive is cost effective
• IGRA testing alone is ONLY cost effective
• if LTBI rate 40 or greater

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IGRA Tests (2)

• Menzies et al. Meta-analysis New tests for the
  diagnosis of LTBI areas of uncertainty and
  recommendations for research. Ann Int Med
  2007146340-54
• New IGRAs show considerable promise and have
  excellent specificity. Additional studies are
  needed to better define their performance in high
  risk populations and in serial testing.
  Longitudinal studies are needed to define the
  predictive value of IGRAs

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IGRA Tests (3)

• Pai M, Zwerling A, Menzies D. Ann Int
• Med 2008 149 177-84
• Updated systematic review
• Excellent specificity of QFT (less data on
• commercial T-spot)
• TST high specificity in non-BCGd but low and
• variable in BCGd
• Sensitivity of TST and IGRAs not consistent
• across tests and populations
• ?? T-spot more sensitive than QFT/TST

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Data on PPV conflicting

• Diel et al 2008 AJRCCM
• German TB contacts of pulmonary TB
• Majority non-German born
• 14 QFT not taking treatment for LTBI
  developed TB in 2 years (5/42).
• TST 2.8 PPV

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PPV (2)

• Hill et al PLoS ONE 2008 i.e. 1379
• The Gambia
• High prevalence setting
• PPV 1.7 IGRA
• PPV 3.3 TST

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PPV (3)

• Kik SV et al (unpublished)
• KNCV (Holland)
• Immigrant contacts age 16yrs
• Sputum smear positive TB
• 1-2 year FU TST both IGRAs
• PPV TST 10mm 3.1 15mm 3.8
• PPV QFT 2.8
• PPV T-spot 3.3

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NPV

• The negative predictive value of a negative test
  is still not yet established in a either low or
  high prevalence settings

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? Role of IGRAs pre TNF

• Few cohort data
• Pratt A et al. Rheumatology 2007
• 101 RA patients 99 white
• Mean age 55 78.5 BCG
• 7 QFT ve 84-ve 11 indeterminate
• 6-30 months FU (mean 18.3)
• No TB (but)

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IGRA Testing pre TNF

• White TB rate at age 50 5/100000
• White LTBI rate at age 50 50/100000
• True positive rate therefore 1/2000
• Number of IGRA tests per true ve 2000
• QFT (at 35) 70000
• T-Spot (at 100) 200000

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IGRA testing pre TNF (2)

• In high risk groups (South Asian/Black-African
  born abroad)
• Say 200/100000 disease
• 2000/100000 LTBI
• 50 tests to detect 1 true positive
• 1750 QFT or 5000 T-spot
• But the NPV of a negative test is NOT
• YET KNOWN

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Conclusions (1)

• Justified economically in 2-step Testing
• as per NICE for TB contact and new
• entrant screening
• Rules out false positive reactions from
• BCG and NTMs
• Economic madness to use pre-TNF Rx in
• white ethnic group

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Conclusions (2)

• NOT licensed for active disease diagnosis
• or management
• Do NOT differentiate between disease
• and infection.
• False ve rate in active disease
• Of no proven ultility in monitoring
• treatment response
• Much more cohort data needed on both PPV
• and NPV

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2009

• NICE are going to review the data on
• IGRA testing from Aug 2005 onwards
• Probably wont be until Autumn 2009
• Conclusions early 2010
• TB algorithms may/may not be affected