Interferon Gamma Release Assays (IGRAs)
John Jereb for Jerry Mazurek Division of Tuberculosis Elimination Centers for Disease Control and Prevention April 30, 2009Interferon Gamma Release Assays (IGRAs)
Detect M. tuberculosis infectionDo not differentiate latent infection from diseaseSeveral approved by FDA“aid diagnosing infection with Mycobacterium tuberculosis”Why Interferon gamma (IFN-)? Component of cell mediated immune response Antigen specific secretion Measurable Associated with TST results Associated with TB exposure
Types of IFN- Release Assay Measure IFN- concentratione.g. QuantiFERON®-TB Gold In-TubeWhole Blood stimulated with TB antigensMeasure IFN- by ELISAMeasure # of cells releasing IFN-e.g. T SPOT™ (ELISpot)PBMCs stimulated with TB antigensCount spots
TB-specific antigens
Antigens specific for M. tuberculosis ESAT-6 & CFP-10Shared mycobacterial antigens Present in NTM & BCG
ESAT
CFPM. abcessus
-
-
M. avium
-
-
M. branderi
-
-
M. celatum
-
-
M. chelonae
-
-
M. fortuitum
-
-
M. gordonii
-
-
M. intracellulare
-
-
M. kansasii
+
+
M. malmoense
-
-
M. marinum
+
+
M. oenavense
-
-
M. scrofulaceum
-
-
M. smegmatis
-
-
M. szulgai
+
+
M. terrae
-
-
M. vaccae
-
-
M. xenopi
-
-
ESAT
CFP
M. tuberculosis
+
+
M. africanum
+
+
M. bovis
+
+
BCG substrain
Gothenburg
-
-
Moreau
-
-
Tice
-
-
Tokyo
-
-
Danish
-
-
Glaxo
-
-
Montreal
-
-
Pasteur
-
-
Andersen, et al. Lancet 2000;356(9235):1099.
Antigen Specificity by Species
Centrifuge 5 minutes to separate plasma above gel
Collect 1mL of blood in 3 tubes
Stage 1 Whole Blood Culture
NilMtb
TMB
COLOR
Stage 2: Measure [IFN-] & Interpret Measure [ IFN- ] in ‘Sandwich’ ELISA Software calculates results and prints report
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Nil
MbPHA
QFT-GIT Interpretation
InterpretationTB Response
Nil
Mitogen – Nil Positive
> 0.35 IU/ml and > 25% of Nil
< 8.0
Any
Negative
< 0.35 IU/ml or < 25% of Nil
< 8.0
> 0.5
Indeterminate
< 0.35 IU/ml or < 25% of Nil
< 8.0
< 0.5
Any
> 8.0
Any
TB Response is the IFN-γ concentration in plasma from blood stimulated with a single cocktail representing ESAT-6, CFP-10, and part of TB7.7, minus the IFN-γ concentration in plasma from unstimulated blood.
QFT-GIT Report
Sample Info:- Specimen ID- Collection date & timeAssay Info:Test format: QFT-G or QFT-GITRun #Result Info:[IFN-] in each plasmaInterpretation as “MTBI Likely”, “MTBI not likely”, or “Indeterminate”
T-Spot.TB
Collect blood in CPT tubeRecover, wash, & count PBMCsAliquot 250,000 PBMCs to 4 wells with anti-IFN-Add saline, PHA, ESAT-6 or CFP-10 & incubateWash away cellsDevelop & count spots where cells produced IFN- INF- Antibody Sensitized T cellINF- Captured Detection Antibody
Chromogen Spot
Saline
ESAT-6
CFP-10
PHA
T-Spot.TB Interpretation
InterpretationTB Response
Nil
Mitogen
Positive
> 8 spots
< 10 spots
any
Negative
< 4 spots
< 10 spots
> 20 spots
Borderline
5, 6, or 7 spots
< 10 spots
> 20 spots
Indeterminate
< 8 spots
< 10 spots
< 20 spots
any
> 10 spots
any
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.
T-Spot TB Report: What’s important? Sample Info:- Specimen ID- Collection date & timeAssay Info:Test format:Run #Result Info:# of spots in each wellInterpretation as “MTBI likely”, “MTBI not likely” “Borderline”, or “Indeterminate”
Evaluation of IGRAs
Lack of “gold standard” for TB infectionSensitivity – Compare to cultureSensitivity = # positives / # culture (+) people testedSpecificity – In subjects at low risk for LTBISpecificity = # negative / # low-risk people testedAgreement with TSTAssociation of positive results with exposurePredicting TB diseaseIGRA Sensitivity
80% in subjects with untreated, culture + TBRanges from 56 to 100%Similar to TST sensitivityTreatment ↓ IFN- and ↑ TSTSensitivity of T-Spot may be better if—HIV+Renal failureIGRA Sensitivity
Sensitivity in subjects with LTBI ExtrapolatedImmune differences: LTBI TB Unable to accurately measureIGRA 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 NTMAgreement 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, & unpredictableIGRAs 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 +IGRAs in Contact Investigations
Ewer, et al (2003): T-Spot in 534 school contacts grouped in 4 categories from frequent close to possible exposure20 of 20 + (100%)
43 of 81 + (53%)
18 of 47 + (38%)
66 of 387 + (17%)
OR for T-Spot = 2.78 OR for TST = 2.33
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 negativePrediction 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 +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 valuein 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 +
Available evidence + Expert opinion = Guidelines
Guidelines for using QFT-GIT and T-SPOT in the U.S. are being writtenRemaining 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?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 conversionForecast of Guidelines
TST or IGRAs (QFT-G; QFT-GIT; T-SPOT) may be used as aids in diagnosing infection with M. tuberculosis.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)