Interferon Gamma Release Assays IGRAs

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Interferon Gamma Release Assays(IGRAs)

• John Jereb forJerry MazurekDivision of
  Tuberculosis EliminationCenters for Disease
  Control and PreventionApril 30, 2009

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Interferon Gamma Release Assays (IGRAs)

• Detect M. tuberculosis infection
• Do not differentiate latent infection from
  disease
  
• Several approved by FDA
• aid diagnosing infection with Mycobacterium
  tuberculosis
  

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Why Interferon gamma (IFN-?)?

• Component of cell mediated immune response
• Antigen specific secretion
• Measurable
• Associated with TST results
• Associated with TB exposure

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Types of IFN-? Release Assay

• Measure ? IFN-? concentration
• e.g. QuantiFERON-TB Gold In-Tube
• Whole Blood stimulated with TB antigens
• Measure IFN-? by ELISA
• Measure ? of cells releasing IFN-?
• e.g. T SPOT (ELISpot)
• PBMCs stimulated with TB antigens
• Count spots

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TB-specific antigens
Antigens specific for M. tuberculosis

ESAT-6 CFP-10
Shared mycobacterial antigens

Present in NTM BCG
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Antigen Specificity by Species
Andersen, et al. Lancet 2000356(9235)1099.
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QuantiFERON-TB Gold In-Tube (QFT-GIT)
Stage 1 Whole Blood Culture
Mtb
Nil
PHA
Centrifuge 5 minutes to separate plasma above
gel

Collect 1mL of blood in 3 tubes
Incubate at 37ºC for 16-24 hours.
Stage 2 Measure IFN-? Interpret
Mb
Nil
PHA
Collect 50 µL of plasma for ELISA
Measure IFN-? in Sandwich ELISA
Software calculates results and prints report
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QFT-GIT Interpretation
TB Response is the IFN-? concentration in plasma
from blood stimulated with a single cocktail
representing ESAT-6, CFP-10, and part of TB7.7,
minus the IFN-? concentration in plasma from
unstimulated blood.
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QFT-GIT Report

• Sample Info
• - Specimen ID
• - Collection date time
• Assay Info
• Test format QFT-G or QFT-GIT
• Run
• Result Info
• IFN-? in each plasma
• Interpretation as MTBI Likely, MTBI not
  likely, or Indeterminate

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T-Spot.TB

• Collect blood in CPT tube
• Recover, wash, count PBMCs
• Aliquot 250,000 PBMCs to 4 wells with anti-IFN-?
• Add saline, PHA, ESAT-6 or CFP-10 incubate
• Wash away cells
• Develop count spots where cells produced IFN-?

CFP-10
Saline
ESAT-6
PHA
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T-Spot.TB Interpretation
TB Response is the higher number of spots
resulting from stimulation of PBMCs with two
separate cocktails of peptides representing
ESAT-6 or CFP-10, minus the number of spots
resulting from incubation of PBMCs with saline.
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T-Spot TB Report Whats important?

• Sample Info
• - Specimen ID
• - Collection date time
• Assay Info
• Test format
• Run
• Result Info
• of spots in each well
• Interpretation as MTBI likely, MTBI not
  likely Borderline, or Indeterminate
  

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Evaluation of IGRAs

• Lack of gold standard for TB infection
• Sensitivity Compare to culture
• Sensitivity positives / culture () people
  tested
  
• Specificity In subjects at low risk for LTBI
• Specificity negative / low-risk people
  tested
  
• Agreement with TST
• Association of positive results with exposure
• Predicting TB disease

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IGRA Sensitivity

• 80 in subjects with untreated, culture TB
• Ranges from 56 to 100
• Similar to TST sensitivity
• Treatment ? IFN-? and ? TST
• Sensitivity of T-Spot may be better if
• HIV
• Renal failure

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IGRA Sensitivity

• Sensitivity in subjects with LTBI
• Extrapolated
• Immune differences LTBI ? TB
• Unable to accurately measure

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IGRA Specificity

• 99 in subjects at low risk for LTBI
• Ranges from 89 to 99.6
• Lower estimates where LTBI more likely
• 33 to 75 fewer IGRA than TST
• especially after BCG
• associated with NTM

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Agreement with TST

• Poor agreement may be a good thing
• Agreement varies widely
• Positive TST Negative IGRA discordance
• Associated with BCG, NTM, TB Prevalence
• Negative TST Positive IGRA discordance
• Less frequent, unpredictable

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IGRAs in Contact Investigations

• Recent exposure is associated with IGRA results
  more than TST results
  
• similar of exposed are IGRA TST
• fewer of unexposed are IGRA than TST

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IGRAs in Contact Investigations

• Ewer, et al (2003) T-Spot in 534 school contacts
  grouped in 4 categories from frequent close to
  possible exposure

OR for T-Spot 2.78 OR for TST 2.33
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Prediction of Subsequent TB

• Diehl et al (2008), studied 601 German contacts
• 11 QFT-GIT positive
• 40 TST positive
• 6 of 41 QFT-GIT w/o IPT developed TB
• 5 of 219 TST w/o IPT developed TB
• 1 of 6 who developed TB was TST negative

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Prediction of Subsequent TB

• Hill, et al (2008) studied 2,348 Gambian TB
  contacts
  
• 56 (14 of 25) secondary cases had been
  TST
  
• 52 (11 of 21) secondary cases had been
  ELISpot
  

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IGRA vs. TST

• in vitro
• TB specific antigens
• no boosting
• 1 patient visit
• results possible in 1 day
• unknown variability
• stimulate w/i 8 to 16 hrs
• Uncertain predictive value

• in vivo
• Less specific PPD
• boosting
• 2 patient visits
• results in 2 - 3 days
• inter-reader variability
• read in 48 - 72 hrs
• Increased TB risk if

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• Available evidence
• Expert opinion
• Guidelines

• Guidelines for using QFT-GIT and T-SPOT in the
  U.S. are being written

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Remaining Questions

• Sensitivity of IGRAs for LTBI?
• Risk of TB associated with a positive IGRA?
• Risk of TB when TST and IGRA are discordant?
• How stable are IGRA results?
• What is an IGRA conversion?
• Risk associated with IGRA conversion?
• Cost effectiveness of IGRAs?

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Conclusion

• IGRAs are useful aids for diagnosing Mtb
  infection
  
• Logistical advantages of IGRAs
• Specificity of IGRAs TST in some populations
• Sensitivity of IGRAs similar to TST?
• May replace or augment TST
• Unanswered questions
• TB incidence associated with IGRA results
• TB incidence associated with IGRA conversion

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Forecast of Guidelines

• TST or IGRAs (QFT-G QFT-GIT T-SPOT) may be used
  as aids in diagnosing infection with M.
  tuberculosis.
  

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• As with the TST, IGRAs generally should not be
  used for testing persons who have a low
  likelihood of M. tuberculosis infection except
  
• People who are, or will be at increased risk of
  progression to tuberculosis disease if infected
  (e.g. people taking or planning to take Tumor
  Necrosis Factor alpha Inhibitors)
• People whose activities increase their risk of
  exposure (e.g., health care workers)
  

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• As with the TST, IGRAs should not be used for
  excluding a diagnosis of TB disease.

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• IGRAs may be used in place of TST in most
  situations in which CDC recommends tuberculin
  skin testing as an aid in diagnosing M.
  tuberculosis infection with noted exceptions and
  preferences.

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Controversies

• Guidance for preferred test IGRA or TST?
• IGRA usage for unstudied populations
• Predictive values of IGRA results
• Unexpected or incongruent IGRA results
• Serial IGRA testing
• Strategies for using 1 type of test