Title: Interferon Gamma Release Assays IGRAs
1Interferon Gamma Release Assays(IGRAs)
- John Jereb forJerry MazurekDivision of
Tuberculosis EliminationCenters for Disease
Control and PreventionApril 30, 2009
2Interferon 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
3Why Interferon gamma (IFN-?)?
- Component of cell mediated immune response
- Antigen specific secretion
- Measurable
- Associated with TST results
- Associated with TB exposure
4Types 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
5TB-specific antigens
Antigens specific for M. tuberculosis
ESAT-6 CFP-10
Shared mycobacterial antigens
Present in NTM BCG
6Antigen Specificity by Species
Andersen, et al. Lancet 2000356(9235)1099.
7QuantiFERON-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
8QFT-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.
9QFT-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
10T-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
11T-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.
12T-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
13Evaluation 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
14IGRA 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
15IGRA Sensitivity
- Sensitivity in subjects with LTBI
- Extrapolated
- Immune differences LTBI ? TB
- Unable to accurately measure
16IGRA 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
17Agreement 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
18IGRAs 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
19IGRAs 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
20Prediction 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
21Prediction 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
22IGRA 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
23- Available evidence
- Expert opinion
- Guidelines
- Guidelines for using QFT-GIT and T-SPOT in the
U.S. are being written
24Remaining 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?
-
25Conclusion
- 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
26Forecast of Guidelines
- TST or IGRAs (QFT-G QFT-GIT T-SPOT) may be used
as aids in diagnosing infection with M.
tuberculosis.
27- 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)
28- As with the TST, IGRAs should not be used for
excluding a diagnosis of TB disease.
29- 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.
30Controversies
- 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