Interferon Gamma Release Assays (IGRAs) for the Diagnosis of TB:

Can We Replace the TST?

Helene M. Calvet, MDHealth Officer and TB Controller

Long Beach Department of Health and Human Services

Overview

• Diagnosis of active and latent TB

• In-Vitro Interferon Gamma Release Assays (IGRAs)

• Sensitivity and specificity of IGRAs

• Implementation data from San Francisco and Long Beach

Definitions• “Positive PPD”: a tuberculin skin test (TST)

that is indurated:– >5 mm: HIV+, recent contact of TB case, CXR

c/w old TB, organ transplant or other immunosuppression

– >10 mm: everybody else (in California)• Latent TB Infection (LTBI): TB infection

without evidence of clinically active disease (+PPD, but no symptoms); CXR usually normal, or may be abnormal, but sputa negative

• TB Disease: active tuberculous infection of any organ

Diagnosis of TB

• TB disease

• Latent TB infection (LTBI)

Toolbox for Diagnosis of TB Disease

History

Cultures

Sputa

CXR

Physical Exam

PCR

Pathology

Response to therapy

TST

IGRAs

Gold Standard =

Culture

Toolbox for Diagnosis of Latent TB

TST

IGRAsCXR

History

No Gold Standard!

Comparison of Toolboxes

TB Disease Toolbox LTBI Toolbox

Abundant tools Paucity of tools

Gold standard No gold standard

No need to rely on one test May need to rely on one test

The TST…A Very Old, and Not-So-Perfect Test • Discovered by Koch in 1890, skin test agents

standardized in 1976• False negatives:

– Anergy (immunocompromise or malnutrition)– Recent TB infection– Very young age (< 6 months)– Overwhelming TB disease– Recent live virus vaccination– Poor placement or reading of TST

• False positives:– BCG or nontuberculous mycobacterial infections– Inaccurate reading of TST

• Requires two visits…substantial proportion do not come for reading

Tuberculin Skin TestingMantoux Method

48 to 72 hours5 TU of PPD

Interpretation depends on person’s risk factors

BCG and TST (1)• General teaching is that reactivity from BCG wanes

after a few years and is unlikely to persist > 10 years, but may be boosted by PPD.

• Study done in Switzerland* suggests that false positives due to BCG may be much more common than we thought:– 40% of 5000 HCW had positive TST– Prior BCG strongest risk factor for positive TST among

those less than age 40 with TSTs <18 mm (was not as strong a risk factor for those > 40 years old and those with TSTs > 20 mm)

*CID 2005; 40:211 – 217.

BCG and TST (2)

• Review of studies that compared TST responses to BCG during and after infancy

• Vaccination during infancy estimated to cause false-positive TST in 6.3% overall, but only 1% of those tested more than 10 years after vaccination

• Vaccination at 2 years of age or older estimated to cause false-positive TST in 40% of persons overall, 20% of those tested 10 years or more after vaccination

Farhat M et al, Int J Tuberc Lung Dis 2006; 10: 1192-204

In-Vitro Interferon Gamma Release Assays (IGRAs) for TB

• QuantiFERONQuantiFERON®® TB Gold (QFT-G): FDA approved

• QuantiFERONQuantiFERON® In-Tube (QFT-IT): ® In-Tube (QFT-IT): submitted for FDA approvalsubmitted for FDA approval

• T-Spot TB : submitted for FDA approval

QuantiFERON® (QFT) History

• First generation (QuantiFERON ® – TB) test FDA-approved in 2001

• 2003 CDC guidelines advise use of test in selected groups only

• Second generation test (QuantiFERON® - TB Gold) FDA-approved March 2005

• CDC guidelines December 2005* allow use of QuantiFERON® - TB Gold (QFT-G) in any situation in which a TST would be used; however, points out lack of data in many groups (pediatrics, immunocompromised, etc.)

*MMWR 2005, 54 (RR-15): 49-55

In Vivo and In Vitro Diagnostic Tests

Antigenpresenting

cell

MemoryT-cell

Presentation ofmycobacterial antigens

IFN-

IFN-

IL-8, etc.

IL-8, etc.

TNF-

TNF-

Andersen P, et al. Lancet 2000;356:1099

Why Measure Interferon-?

• TB infection induces T-cell response (CMI)• IFN- is the ‘classic’ CMI cytokine• Produced in vitro in response to specific antigen• Secreted in measurable and stable amounts• Absent from normal circulation• Extensive literature showing importance of IFN- in

TB infection

QuantiFERON®-TB Gold Principle

• Based on the quantitative measurement of IFN- secreted from stimulated T cells in human whole blood

– T-cells reactive to M. tuberculosis - specific antigens are only present in those infected

• A two stage process:

– Incubation of whole blood with TB-specific and control antigens (mitogen) and nil control

– Detection of IFN- using a rapid, single-step, sandwich ELISA

Species specificity of ESAT-6 and CFP-10Species specificity of ESAT-6 and CFP-10Environmental strains

Antigens

ESAT CFP

M 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 - -

Tuberculosis complex

Antigens

ESAT CFP

M tuberculosis + +

M africanum + +

M bovis + +

BCG substrain    

gothenburg - -

moreau - -

tice - -

tokyo - -

danish - -

glaxo - -

montreal - -

pasteur - -

Whole Blood IFN- AssayQuantiFERON-TB Test

ESAT-6 CFP 10MitogenControl

TMBTMB

COLORCOLOR

Stage 1 Whole Blood CultureStage 1 Whole Blood Culture

Stage 2 IFN-gamma ELISAStage 2 IFN-gamma ELISA

NilControl

Incubate Incubate →→ INF- INF- from sensitized T-from sensitized T-

cellscells

Draw blood Draw blood + heparin+ heparin

Aliquot blood Aliquot blood & add antigen& add antigen

Harvest plasma Harvest plasma from above settled from above settled

cellscells

Measure [ IFN-Measure [ IFN-] in ] in ‘Sandwich’ ELISA‘Sandwich’ ELISA

ComputerizedComputerizedinterpretationinterpretation

Cellestis

QuantiFERON®-TB Gold Test MethodAdvantages and Disadvantages

• Advantages:– Only one visit required– Objective and reproducible; not operator-dependent

– No cross reactivity with BCG, little cross-reactivity with non-tuberculous mycobacteria

– Controls for low or no immune response

– No chance of ulceration due to brisk skin test reaction

• Disadvantages:– Blood must be received in lab within 12 hours– Labor intensive for the lab– Not much data for some patient groups

T-Spot.TB: “Six easy Steps”

Nil Control

Positive Control

Infection

Infection

Oxford Immunotec

IGRA Possible Results

• Positive: indicates TB infection, does not differentiate between active disease and latent infection

• Negative: no TB infection

• Indeterminate: not sure

What Does an Indeterminate Mean?• Indeterminate can occur as a result of low mitogen

response (due to patient immunocompromise, poor specimen handling or storage, lab error, etc.) or high nil response (due to patient illness, recent vaccinations, etc.)

• Estimated rate of indeterminates for QGT-G: approximately 1-2% among HCW, about 5-10% among patients

• Upon retesting, approximately ½ of the indeterminates come out with a definitive result (unpublished data)

• Retesting indeterminates once, and if indeterminate again, stop

• Indeterminate rate much higher among children

Estimating Sensitivity and Specificity of IGRAs

• Sensitivity in people with culture + TBSen = # positives / # tested

• Specificity in people at low risk for TB infection

Spec = # negative / # tested

QFT-G Sensitivity EstimatesReference Population + IFN-

(n) + TST (n)

Mori; 2004Untreated Cult+TB; Japan 89% (118) 66% (76)

Kang; 2005 Pulmonary TB; Korea 81% (54) 78% (54)

CDC; Unpub. Untreated Cult+TB; US 81% (41) 81% (41)

Ravn; 2005 Active TB; Denmark 85% (48) Not done

Lee, 2006 Active TB, Korea 70% (61) 67% (58)

Menzies* 2007 Meta-analysis (9 studies) 80% (393) 74% (394)

*Menzies, D. et al, Annals of Int Med 2007;146 (5): 340-354

TB Suspects: QFT-G Performance

• Among 242 suspects, 23 of 37 had culture-confirmed tuberculosis and +QFT-G results

• QFT-G sensitivity: only 64% (95% CI, 48% - 78%), but negative predictive value 89%

• Sensitivity of the TST was 88% in this review• Very poor performance in extrapulmonary TB (14%

sensitivity), but numbers were low• Conclusion: lowish sensitivity in TB suspects means

it’s probably not all that useful for that purpose

Dewan et al, Clin Infect Dis 2007, 44:69-73.

Why Poor Performance in Active TB?

• CMI response likely diminished in active TB, particularly in those with more advanced disease, malnutrition and older age

• If their CMI was working well, they wouldn’t have TB disease; so TB disease likely not a great surrogate for LTBI

• Pooled sensitivity from 10 studies: 75% (71-78%)• Lower cutoff may be needed in those who are

highly suspect to have TB

Pai and Menzies, Clin Infect Dis 2007; 44: 74 - 77

QFT-G Specificity Estimates

Reference Population + IFN- (n)

+ TST (n)

Mori; 2004 Nursing Students; Japan 2% (213) 65% (113)

Kang; 2005 Med Students; Korea 4% (99) 51% (99)

CDC; Unpub.

Navy recruits; US .2% (532) .9% (532)

Menzies* 2007

Meta-analysis (9 studies) 3% (711)+BCG: 44% (516)

No BCG: 2% (156)

*Menzies, D. et al, Annals of Int Med 2007;146 (5): 340-354

T-Spot TB Sensitivity Estimates

Reference Population + IFN- (n)

+ TST (n)

Lalvani; 2001

Untreated Cult+TB; UK 96% (45) 69% (32)

Pathan, 2001 Active TB, UK 92% (33) N/A

Meier, 2005 Active TB, Germany 97% (70) N/A

Lee, 2006 Active TB; Korea 95% (83) 67% (58)

Menzies* 2007

Meta-analysis (11 studies) 88% (557) 74% (394)

*Menzies, D. et al, Annals of Int Med 2007;146 (5): 340-354

T-Spot TB Specificity Estimates

Reference Population+ IFN-

(n)Spec.

Lalvani; 2001Low risk BCG-vaccinated subjects, UK 0% (26) 100%

Pathan, 2001Low risk BCG-vaccinated subjects, UK 0% (32) 100%

Lalvani, 2001Low risk BCG-vaccinated subjects, India 0% (40) 100%

Menzies* 2007 Meta-analysis (4 studies) 8% (229)+ BCG: 44% (516)

No BCG: 2% (156)

*Menzies, D. et al, Annals of Int Med 2007;146 (5): 340-354

T-SPOT.TB vs. QFT-TB Gold!

Comparison of T-Spot.TB and QFT-TB Gold

• Site - Italy• Design - Prospective study of 393 consecutively

enrolled patients with LTBI or suspected TB• Agreement with the TST and the two IGRAs was

similar, but T-Spot TB more likely to yield positive results in close contacts

• Indeterminate results were more common with QFT-TB Gold than T-Spot.TB (11% vs 3%).

• Indeterminate results were more likely in young children (< 5 yrs) and immunosuppressive treatment

Ferrara G, et al. Lancet. 2006;367:1328-1334

Comparison of Test Results Among Contacts

TST QFT-G T-SPOT.TB

All contacts 62 (54%) 25 (22%) 39 (34%)

<0.0001 <0.0001

Non-BCG vac. 36 (43%) 21 (25%) 29 (35%) contacts

0.0001 0.0923

Ferrara G, et al. Lancet. 2006;367:1328-1334

Comparison of T-Spot.TB and QFT-TB Gold

Test results Active TB (%) Low risk for TB

No. of subjects 87 131

TST + (≥ 10 mm) 58 (66.7) 28 (21.4)

QFT-G positive 61 (70.1) 11 (8.4)

T-SPOT.TB positive 83 (95.4) 20 (15.3)

Lee JY, et al. ERJ. 2006

Three Ways to Interpret This Data

• QFT-G is less sensitive than TST and T-Spot TB

OR

• QFT-G is a lot more specific than TST and T-Spot TB

OR

• A little bit of both

Diagnosis of TB: The Truth*?

Truth

Sensitivity Specificity

TSTTST

QFT-G

QFT-G?T-Spot TB

Active

Latent

T-Spot TB?

QFT-GTST T-Spot TB

* My opinion only, based on impression of available data

QuantiFERON Testing: The San Francisco Experience

• Implemented QuantiFERON-TB (QFT-1) screening in 4 community clinic sites (2 homeless, 1 methadone, 1 immigrant) and TB clinic in November 2003

• Switched to QuantiFERON-TB Gold (QFT-G) in March 2005

• Over 6100 samples run between March 2005 and February 2006

QFT-G Test Results by Age Category March 2005 – February 2006

0%

20%

40%

60%

80%

100%

<5 5 to 14 15 to24

25 to44

45 to64

65 to84

Perc

en

t

Indeterminate

Negative

Positive (%) (3) (1) (6) (7) (12) (31)

TB Infection Prevalence by Testand Clinic Type

HomelessTB

ClinicMethadone Immigrant

TST

(2001-2003)26% >50% 10% 37%

QFT-1 (11/04-2/05)

17 %

n=1848

48 %

n=292

18 %

n=346

37 %

n=344

QFT-Gold (3/05-2/06)

Decline in positive rate from TST

6 %

n=3594

77%

26 %

N=693

48%

4 %

n=546

60%

12 %

n=626

66%

QFT Implementation – LB Experience

• Lab was experienced with the technology after doing a contact investigation study in a dialysis center using QFT in 2003– Over 120 patients and staff tested twice– QFT-Gold found to be more closely associated with

contact to case, less affected by albumin, good reproducibility

• Waited for FDA approval of QFT-Gold and completion of lab reconstruction before implementing

• Performed validation study Nov. 2005 started running patient samples in Jan. 2006

QFT in the Lab

First Experience

• From January through June 2006, 193 samples run on patients in TB clinic

• Some testing also done in Occupational Health

• Of 193 samples in TB, 137 (71% foreign-born, 130 (67%) with history of BCG

• Many samples done as “confirmatory” testing for positive TST

Results in First 6 Months• 108 (56%) negative, 57 (30%) positive and 25

(13%) indeterminate• Excluding indeterminates, 65% negative and 35%

positive• Of those with known prior TSTs (n = 100,

excluding indeterminates)– 33 (33%) positive QFT-G– 67 (67%) negative QFT-G

• Average size of prior TST by QFT-G result category– QFT-G positive: median 18 mm, (range 10 -30)– QFT-G negative: median 14 mm (range 10 – 30)

QFT-G in Occupational Health (OH)

• Police recruit class, May ’06: – 80 healthy young folks

– 30% positive, 14% indeterminate

– Much higher positivity rate than historical, but no prior tests

• Firefighter screening, September ‘06– 384 healthy, low risk firefighters with prior negative

TSTs

– 19% positive, 5% indeterminates, 76% negative

• What the heck was going on?

Tube B or Not Tube B…?• QFT-G users had been reporting unusual results

for several months• High nil values noted in many of the positive

samples in LB firefighters in September• October 3 letter from Cellestis notifying users of

increased numbers of indeterminates and false positives (usually with high nil values) associated with certain lots of Becton Dickinson Na heparin tubes; recommended switching to lithium heparin tubes

High Nil Value

• High nil value does not always lead to an indeterminate result – If reactivity to ESAT-6 or CFP-10 is > 50% higher than

nil, computer calls it positive

• Not sure cause for this outcome: blood drawing technique, inadequate mixing, lab phenomenon, contaminated tubes etc.

• SF also had experience with this, and change of blood drawing venue took care of it

• Planned to repeat Fireman testing using both Na heparin and lithium heparin tubes

Repeat Testing on Firefighters

• 93 firefighters retested January 2007 with both lithium and sodium heparin tubes

• 1/92 (1%) positive on both (subsequently found to have hx of prior +TST), 86/92 (93%) negative on both, 5/92 (5%) discordant

• Among 5 discordant, all positive on lithium heparin but negative on sodium heparin; repeat of assay gave same result on sodium heparin, but varying results (+, -, indeterminate) on lithium heparin = likely gray zone, or borderline, values

• So, is lithium heparin really better or not?

QFT Reproducibility

Reproducibility good, but conversions and reversions possible

Actual breakpoints of conversion & reversion not well defined (aside from change from positive to negative or back) Since normal variations not known, thresholds not

established (as for TST) Conversion and reversion more likely with results near

gray zone, or for results discordant with the TST. What is the natural history of T-cell response to

TB infection??

Facilitating Interpretation

Might it be better to report continuous variables (actual IFN readings) instead of dichotomous variables (positive/negative)?

Could assist clinician in interpreting the results Might give more data in serial tests to help determine

conversion versus normal variation

Recent recurrent problems with false positives (associated with high nil values) have led the state to suggest that labs should call all specimens with high nil value indeterminate, regardless of the ESAT-6 and CFP-10 values

QFT-G: My Concerns about Sensitivity• There definitely are false positive skin tests, but are

there really that many??• The test relies on effector lymphocytes, which have

recently encountered antigen in vivo, to produce interferon within hours

• Memory cells, which have encountered antigen in the distant past, would require several days in the presence of antigen to produce interferon

• Could we be missing distant, latent infection that is not immunologically active? (I think we may be)

….and/or…• Could this be telling us who has infection that is

requiring more of an immunological response, and thus possibly at increased risk of progression??

The IGRA Research Agenda

• Group of experts met March 2006• Comprehensive Research Agenda Generated• 58 key questions in 7 general areas

Biological issues Test performance in high-risk populations Test reproducibility & serial testing Responses during treatment Epidemiological & field applications Health systems & economic research

Pai, M et al, Lancet Infect Dis 2007; 7: 428-38

Key Questions (1)

• What are appropriate cut-off points for different groups?

• What is the normal variation in T-cell responses?• What is the risk of active disease in those with

positive and negative IGRA results? How does this relate to risk associated with positive TST?

• Is there a cut-off point that will predict incipient active TB?

• How should conversions and reversions be defined (incrementally), and how often do they occur?

Pai, M et al, Lancet Infect Dis 2007; 7: 428-38

Key Questions (2)

What is the biological basis for discordance between IGRAs and TST?

What is the role of IGRAs in monitoring response to therapy of active or latent TB?

Can IGRAs be used to revise estimates of worldwide TB infection and the lifetime risk of TB disease?

How useful is the combined approach of using TST to screen and IGRA to confirm?

Pai, M et al, Lancet Infect Dis 2007; 7: 428-38

Things to Think About in Implementing QFT-G in Occupational Health

• Realize the limitations and benefits of the test!– Lowish sensitivity for LTBI, but probably highest specificity,

especially in BCG-vaccinated population

• Cost of tests and turn-around time• Educating everybody involved about this test• Specimen collection, handling and transport• Obtaining appropriate waivers for use in HCW screening• Changing all documentation that has TST on it (screening

cards, charts, forms, etc.) to include TST results• What to do with indeterminates• Mode of implementation: stepwise, or across –the –board?• Relationship with your lab; tracking indeterminates,

identifying problems

IGRAs: Summary• QFT-G:

– Like the TST, not a perfect test!– Very specific (usually), but somewhat lacking in

sensitivity– Revised cut-offs and quantitative reporting may be

helpful– 3rd generation submitted for FDA-approval; will be

easier for clinicians (does not need to be to lab in 12 hours), easier for lab

• T-Spot TB:– Superior sensitivity to QFT-G, but …– Possibly less specific than QFT-G– Much more difficult from lab standpoint– More expensive– Not yet commercially available

IGRAs: Summary (2)

• IGRAs represent an exciting new opportunity to learn more about a very old disease

• IGRAs are not perfect tests, so clinicians need to be aware of their limitations

• Can we replace the TST? – Not yet!

Questions?