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IGRAs for Detection ofM. tuberculosis Infection

W ld TB D 2011World TB Day, 2011

John Jereb, MDAdapted from presentations by Jerry Mazurek

Division of Tuberculosis EliminationNational Center for HIV, Viral Hepatitis, STD, and TB Prevention

Disclaimers

• The views presented here are those of the speaker and not CDC.

• Off-label usage of the products under g pdiscussion may be broached during questions and answers, and will be verbally indicated as such.

Overview

• Need for testing• Interferon Gamma Release Assays (IGRAs)• FDA approved IGRAsFDA approved IGRAs• Recommendations• Controversies

Need for Testing

• 2008 Global Statistics– 9.4 million new cases

• increasing

• 2008 US Statistics– 12,898 new cases

• declining

– 1.8 million deaths• 5 thousand deaths / day

– 2 billion infected• 1/3 of world population

– ~290 deaths

– 12 million infected• 4 % of US population

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• Close contacts of persons with TB disease• Persons from areas with high incidence of TB • Persons who visit areas with a high prevalence of TB,

especially if visits are frequent or prolonged• Residents and employees of high-risk congregate

Focus #1: Persons with Increased Prevalence of Infection with M. tuberculosis

Residents and employees of high risk congregate settings

• Health care workers who serve high-risk clients• Populations defined locally as having an increased

incidence of infection or disease due to TB• Infants, children, and adolescents exposed to adults

in high-risk categories

Focus #2: Persons at Increased Risk for Disease from M. tuberculosis if Infected

• Persons with HIV infection*• Infants and children aged <5 years*• Persons receiving immunosuppressive therapy*• Persons recently infected with M. tuberculosis (within 2 yrs)• Persons with history of untreated/inadequately treated TB• Persons with silicosis, diabetes mellitus, chronic renal failure,

leukemia, lymphoma, or cancer of the head, neck, or lung• Persons with gastrectomy or jejunoileal bypass• Persons who weight less than 90% of ideal body weight• Populations defined locally as having an increased incidence of

infection or disease due to M. tuberculosis.

* Indicates groups at increased risk of a poor outcome such as meningitis, disseminated disease, or death due to M. tuberculosis

TST vs IGRA

Andersen et al. Lancet 2000;356:1099

Interferon Gamma (IFN-γ )Release Assays

• Indirect tests for M. tuberculosis infection• Do not differentiate latent infection from

disease• Three approved by FDA

– “as an aid in the diagnosis of infection with Mycobacterium tuberculosis”

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Types of IGRA

• Measure Δ IFN-γ concentration– Measure IFN-γ concentration by ELISA– Whole blood stimulated with & w/o antigen– e g QuantiFERON®-TB Golde.g. QuantiFERON TB Gold

• Measure Δ # of cells releasing IFN-γ– Count cells by ELISpot– PBMCs* stimulated with & w/o antigens– e.g. T-Spot™.TB

*peripheral blood mononuclear cells

FDA Approved IGRAs

• QuantiFERON®-TB Gold (QFT-G)– FDA approved May 2005

• QuantiFERON®-TB Gold In-Tube (QFT-GIT)FDA d O t 2007– FDA approved Oct 2007

• T-Spot®.TB (T-Spot)– FDA approved July 2008

Antigens

Antigens specific to M. tuberculosis, e.g. ESAT-6 & CFP-10

M. tuberculosisantigens shared with NTM, & BCG

Ganguly et al, 2008: 88, 510-517

ESAT-6 CFP-10M. abcessus - -M. avium - -M. branderi - -M. celatum - -M. chelonae - -M. fortuitum - -M. gordonii - -M intracellulare - -

ESAT-6 CFP-10

M. tuberculosis + +M. africanum + +M. bovis + +BCG substrain

Gothenburg - -Moreau - -

Antigen Specificity by Species

M. intracellulare - -M. kansasii + +M. malmoense - -M. marinum + +M. oenavense - -M. scrofulaceum - -M. smegmatis - -M. szulgai + +M. terrae - -M. vaccae - -M. xenopi - -

Tice - -Tokyo - -Danish - -Glaxo - -Montreal - -Pasteur - -

Andersen, et al. Lancet 2000;356(9235):1099.

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Stage 1 Whole Blood CultureStage 1 Whole Blood Culture

QuantiFERON®-TB Gold (QFT-G)

Incubate Incubate overnight: overnight: INFINF--γγfrom sensitized from sensitized lymphslymphs

Draw blood Draw blood iinto heparinnto heparin

Make 1 ml aliquots & Make 1 ml aliquots & add antigenadd antigen

ESAT-6CFP-10

MitogenControl

NilControl

Antigens

Stage 2: Measure [IFNStage 2: Measure [IFN-- γγ] & Interpret] & Interpret

pp

Harvest plasma from Harvest plasma from above settled cellsabove settled cells

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

Software calculates Software calculates results and prints reportresults and prints report

QFT-G InterpretationInterpretation TB Response* Nil Mitogen – Nil

Positive > 0.35 IU6/mland > 50% of Nil Any Any

Negative < 0 35 IU/ml < 0 7 > 0 5Negative < 0.35 IU/ml < 0.7 > 0.5

Indeterminate < 0.35 IU/ml < 0.7 < 0.5

< 50% of Nil > 0.7 Any

TB Response* is the higher IFN-γ concentration resulting from stimulation by ESAT-6 or CFP-10 minus the IFN-γ concentration in plasma incubated with saline.

QuantiFERON®-TB Gold In-Tube (QFT-GIT)

Centrifuge 5 minutes to separate plasma above gel

Collect 1mL of bl d i 3 t b

Incubate at 37ºC f 16 24 h

Stage 1 Whole Blood CultureStage 1 Whole Blood Culture

Nil

Mtb

PHA

separate plasma above gelblood in 3 tubes for 16-24 hours.

Stage 2: Measure [IFN-γγ] & Interpret] & Interpret

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

Software calculates results and prints report

Collect plasma for ELISA

Nil

Mb

PHA

(QFT-GIT adds TB7.7 to ESAT-6 and CFP-10)

QFT-GIT Interpretation

Interpretation TB Response Nil c Mitogen – Nil d

Positive > 0.35 IU6/mland > 25% of Nil < 8.0 Any

< 0 35 IU6/ lNegative < 0.35 IU6/mlor < 25% of Nil < 8.0 > 0.5

Indeterminate

< 0.35 IU6/mlor < 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.

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T-Spot.TB• Collect blood• Recover, wash, & count

PBMCs• Aliquot 250,000 PBMCs to 4

wells with anti-IFN-γγ• Add di l ESAT 6

INF-γγ Antibody

Sensitized T cell

• Add media alone, ESAT-6, CFP-10 or PHA, & incubate

• Wash away cells• Develop & count spots where

cells produced IFN-γγ

INF-γγ Captured

Detection Antibody

Chromogen Spot

Media ESAT-6 CFP-10 Mitogen

T-Spot.TB InterpretationInterpretation TB Response Nil Mitogen

Positive > 8 spots < 10 spots any

Borderline 5, 6, or 7 spots < 10 spots any

Negative < 4 spots < 10 spots > 20 spots

Indeterminate < 5 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.

IGRA Test Interpretation• Based on IFN-g response to TB antigens relative to

nil value• Interpretation criteria differ for different IGRAs• Unlike TST not risk stratified (i e there are notUnlike TST, not risk stratified (i.e., there are not

multiple cutoffs for different risk groups)• Still somewhat complicated

Differences in Currently Available IGRAsFormat QFT-G QFT-GIT T-Spot

Sample Tested

Whole Blood, by 12 hrs

Whole Blood, by 16 hrs; refrigerate plasma ≤ 28 days

PBMCs from whole blood by 30 hrs(Use T-Cell Xtend if > 8 hrs)

TB Antigen ESAT-6 & CFP-10in separate wells

ESAT-6, CFP-10, & TB 7.7 combined

ESAT-6 & CFP-10in separate wellsin separate wells TB 7.7 combined

in blood draw tubein separate wells

Measured Concentration of IFN-γ

Concentration of IFN-γ

Number spots (=cells) producing IFN-γ

Results Positive, negative, indeterminate

Positive, negative, indeterminate

Positive, negative, indeterminate, borderline

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IGRA vs. TST• In vitro• Specific antigens• No boosting• 1 patient visit

• in vivo• Less specific PPD• Boosting• 2 patient visits• 1 patient visit

• Results possible in 1 day• Stimulate within hours• Specialized lab test

• 2 patient visits• Results in 2 - 3 days• Read in 48 - 72 hrs• Point-of-care test

Evaluation of IGRAs• Problems:

– Lack of “gold standard” for TB infection– Inconsistent methods and test interpretation

• Sensitivity – Compare to cultureSe s t ty Co pa e o cu u e– Sensitivity = # positives / # culture (+) people tested

• Specificity – In subjects at low risk for LTBI– Specificity = # negative / # low-risk people tested

• Agreement with TST• Association with exposure• Forecast subsequent TB disease

IGRA Sensitivity

• 80% in subjects with untreated, culture + TB– Similar for different IGRAs– Ranges in studies from 57 to 100%Ranges in studies from 57 to 100%– Similar to TST sensitivity

• 95% if either +IGRA or +TST

IGRA Sensitivity

• Sensitivity in subjects with LTBI – Extrapolated– Unable to accurately measureUnable to accurately measure

• Longitudinal studies are needed

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IGRA Specificity

• 99% in subjects at low risk for LTBI– Ranges from 89 to 99.6%– Similar to TSTSimilar to TST

• 15 mm cutoff

– But appears more specific than TST• 10 mm cutoff

•after BCG vaccination•with NTM disease

Agreement with TST

• Poor agreement may be a good thing?• Agreement varies widely• Positive TST & Negative IGRA discordancePositive TST & Negative IGRA discordance

– Associated with •BCG, NTM: suggests IGRAs more specific?

•TB Prevalence: suggests TST more sensitive?

• Negative TST & Positive IGRA discordance– Infrequent, and still unexplained

Association with exposure• Recent exposure is better associated with IGRA

results than with TST results– Fewer unexposed are IGRA+ than TST +Fewer unexposed are IGRA+ than TST +– Similar number positive if exposed

• Long-term forecasting of TB disease?

Available evidence+ Expert opinion

= Guidelines

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Recommendations (1)• TST or IGRAs (QFT-G; QFT-GIT; T-Spot) should be used

as aids to diagnose infection with M. tuberculosis.– using FDA approved test formats, and in compliance with CCLIA standards.– reporting both the qualitative interpretation and quantitative measurements.– arranging for IGRA testing prior to blood collection.

• As with the TST IGRAs should not be used for testing• As with the TST, IGRAs should not be used for testing persons with low risk of infection and low risk of disease due to M. tuberculosis (with exception for those likely to be at increased risk in the future).

Recommendations (2)• IGRAs may be used in place of (and generally not in

addition to) TST in all situations in which CDC recommends tuberculin skin testing (with noted preferences and special considerations).– Despite the indication of a preference, use of the alternative test (FDA-

approved IGRA or TST) is acceptable medical and public health practice.

• Test selection should be based on the reasons for testing, test availability, and overall cost effectiveness of testing.

Recommendations (3)• An IGRA is preferred for testing persons from

groups that historically have poor rates of return for TST reading.

• An IGRA is preferred for testing persons who have received BCG (as a vaccine or for cancer therapy)received BCG (as a vaccine or for cancer therapy).

• TST is preferred for testing children younger than 5 years old.

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Recommendations (4)• IGRAs or TST (without preference) to test recent

contacts of persons with infectious tuberculosis with special considerations for follow-up testing.– Negative results prior to 8 wks typically should be confirmed by repeating

the test 8–10 weeks after the end of exposure – Repeating the same test minimizes misclassification due to test

discordancediscordance

• IGRAs may be used in place of TST (without preference) for periodic screening that addresses occupational exposure (with special considerations regarding conversions and reversions because criteria for interpreting changes in IGRA results that identify new infection remain uncertain).

Recommendations (5)• Sequential TST & IGRA may be useful if the initial test

is negative and:– the risk of infection, the risk of progression , and the risk of a

poor outcome are high (such as when persons with HIV infection, or children < 5 years old are at increased risk for M. tuberculosis infection), or

– there is clinical suspicion for active tuberculosis (such as in persons with symptoms, signs, and/or radiographic evidence suggestive of active tuberculosis) and confirmation of M. tuberculosis infection is desired.

Recommendations (6)• Sequential TST & IGRA may be useful if the initial test

is positive and:– additional evidence of infection is required to encourage

compliance (such as in foreign-born healthcare workers who believe their positive TST is due to BCG); or

– in healthy persons who have a low risk of both infection and progression.

Recommendations (7)• Repeating an IGRA or administering a TST may be

useful when the initial IGRA result is indeterminate, borderline, or invalid, and a reason for testing persists.

• Each institution and TB control program shouldEach institution and TB control program should evaluate the availability, overall cost effectiveness, and benefits of IGRAs in prioritizing IGRA use in their own setting.

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Recommendations (8)• A diagnosis of M. tuberculosis infection, and decisions

about medical or public health management should include epidemiological, historical, and other clinical information when using IGRA or TST results.

• Persons with a positive TST or IGRA result should be• Persons with a positive TST or IGRA result should be evaluated for – likelihood of M. tuberculosis infection– risks of progression to tuberculosis disease if infected– symptoms and signs of tuberculosis disease.

• With these risks, symptoms, or signs, additional evaluation is indicated.

Recommendations (9)

• A diagnosis of LTBI requires that tuberculosis disease be excluded by medical evaluation.

• In persons with symptoms, signs, or radiographic evidence of TB disease and in those at high riskevidence of TB disease, and in those at high risk of progression to TB disease if infected, a positive result with either an IGRA or TST may be taken as evidence of M. tuberculosis infection. – However, negative IGRA or TST results are not sufficient to

exclude infection in these persons.

Recommendations (10)• In healthy persons who have a low likelihood both

of M. tuberculosis infection and of progression to TB disease if infected, a single positive IGRA or TST result should not be taken as reliable evidence of M. tuberculosis infection.– Reevaluate to confirm lack of risk and consider repeat

testing on a case-by-case basis; ortesting on a case-by-case basis; or – Alternatively, assume, without additional testing, that the

initial result is falsely positive.

Recommendations (11)

• In persons with discordant test results (one positive and the other negative) decisions about medical or public health management requires individualized judgment in

iassessing:– the quality & magnitude of each result,– the probability of infection,– the risk of disease if infected, and– the risk of a poor outcome if disease occurs.

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Need For Additional Research• Further studies should be focused on determining

the value and limitations of IGRAs in situations critical to TB control. – Are IGRAs better at predicting subsequent tuberculosis p g q

disease than TST? – Do IGRAs perform differently in children as compared to

adults?– Discordance: why do simultaneously performed TST,

QFT-GIT, QFT-G, and T-Spot results differ?

Controversies

• Pediatrics• Boosting after tuberculin skin test• Sequential testing and stability of resultsSequential testing and stability of results• Longitudinal (predictive, prognositic) value

– Can IGRAs forecast who will get TB?– Can they forecast who will not get TB?

Forecast Study Design

Selected population Enrolled subjects

Follow-up period

Test for infection

Positive result

TB disease

Disease free

Negative result

TB disease

Disease free

Six Studies: Direct Comparison of IGRA with TST for Prognosis of TB Disease

Author; IGRA Place Popu-

lation Number* Mean Follow-up

IGRA Performance Comment

HillELISpot Gambia Household

contacts 1736 24 mo. Non-inferior; HIV predicts TB TB after neg.

HiguchiQFT-G Japan School

contacts 206 12 mo. Non-inferior † Little or no transmissionQFT-G contacts transmission

LeungT-Spot.TB

Hong Kong

Silicosis patients 241 29 mo. Superior TB after neg.

and high ratesBakirELISpot Turkey Pediatric

contacts 908 16 mo. Non-inferior † Explored combined tests

HarstadQFT-GIT Norway Asylum

seekers 820 23–32 mo. Non-inferior †

DielQFT-GIT Germany Household

contacts 953 42 mo. Superior † All cases in children found

* Recalculated for comparability † Greater efficiency with IGRA

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References on Prognosis (p.1)• Higuchi K, Harada N, Mori T, Sekiya Y. Use of QuantiFERON-TB Gold to investigate tuberculosis

contacts in a high school. Respirology 2007; 12:88–92.• Diel R, Loddenkemper R, Meywald-Walter K, et al. Predictive value of a whole blood IFN-gamma

assay for the development of active tuberculosis disease after recent infection with Mycobacterium tuberculosis. Am J Respir Crit Care Med 2008; 177:1164–1170.

• Higuchi K, Kondo S, Wada M, Hayashi S, Ootsuka G, Sakamoto N, Harada N. Contact investigation in a primary school using a whole blood interferon-gamma assay. J Infect 2009; 58:352-357. Epub2009 Apr 1.

• Aichelburg MC, Rieger A, Breitenecker F, Pfistershammer K, Tittes J, Eltz S, Aichelburg AC, Stinglg , g , , , , , g , gG, Makristathis A, Kohrgruber N. Detection and prediction of active tuberculosis disease by a whole-blood interferon-g release assay in HIV-1–infected individuals. Clin Infect Dis 2009;48:954-962.

• Hill PC, Jackson-Sillah DJ, Fox A, Brookes RH, de Jong BC, Lugos MD, Adetifa IM, Donkor SA, Aiken AM, Howie SR, Corrah T, McAdam KP, Adegbola RA. Incidence of tuberculosis and the predictive value of ELISPOT and Mantoux tests in Gambian case contacts. PLoS ONE. 2008;3:e1379.

• Bakir M, Millington KA, Soysal A, Deeks JJ, Efee S, Aslan Y, Dosanjh DP, Lalvani A. Prognostic value of a T-cell-based, interferon-gamma biomarker in children with tuberculosis contact. Ann Intern Med 2008;149:777-787.

Reference on Prognosis (p.2)• Harstad I, Winje BA, Heldal E, Oftung F, Jacobsen GW. Predictive values of QuantiFERON®-TB

Gold testing in screening for tuberculosis disease in asylum seekers. Int J Tuberc Lund Dis2010;14;1209-11.

• Yoshiyama T, Harada N, Higuchi K, Sekiya Y, Uchimura K. Use of the QuantiFERON-TB Gold test for screening tuberculosis contacts and predicting active disease. Int J Tuberc Lung Dis2010;14:819-27.

• Qumseya BJ, Ananthakrishnan AN, Skaros S, Bonner M, Issa M, Zadvornova Y, Naik A, Perera L, Binion DG. QuantiFERON TB Gold testing for tuberculosis screening in an inflammatory bowel disease cohort in the United States. Inflamm Bowel Dis 2010 May 10. [Epub ahead of print]

• Kik SV, Franken WP, Mensen M, Cobelens FG, Kamphorst M, Arend SM, Erkens C, Gebhard A, Borgdorff MW, Verver S. Predictive value for progression to tuberculosis by IGRA and TST in immigrant contacts. Eur Respir J 2010;35:1346-53.

• Leung CC, Yam WC, Yew WW, Ho PL, Tam CM, Law WS, Au KF, Tsui PW. T-Spot.TB outperforms tuberculin skin test in predicting tuberculosis disease. Am J Respir Crit Care Med. 2010 May 27. [Epub ahead of print]

Reference on Prognosis (p.3)• Diel R, Loddenkemper R, Niemann S, Meywald-Walter K, Nienhaus A. Negative and positive

predictive value of a whole-blood IGRA for developing active TB – an update. Am J Respir Crit Care Med 2010 Aug 27. [Epub ahead of print]

• Jonnalagadda S, Lohman Payne B, Brown E, Wamalwa D, Maleche Obimbo E, Majiwa M, Farquhar C, Otieno P, Mbori-Ngacha D, John-Stewart G. Latent tuberculosis detection by interferon γ release assay during pregnancy predicts active tuberculosis and mortality in Human Immunodeficiency Virus Type 1-Infected Women and Their Children.. JID 2010;202:1826-1835

Acknowledgements• Expert Consultants: (met in Aug 2008)

• TB Controllers in U.S.• Professional Organizations: (ATS, IDSA, AAP)

• Authors of guidelines: (Gerald Mazurek, John Jereb, Andy Vernon Phil LoBue Stefan Goldberg Ken Castro)Andy Vernon, Phil LoBue, Stefan Goldberg, Ken Castro)

• CDC Consultants: (Sean Toney, Suraj Sable, Kashef Ijaz, Patrick Moonan, Phil Talboy, Kathi Kellar, Bonnie Plikaytis, Carla Jeffries, Val Robinson, Bob Pratt)

• DTBE Diagnostics Team: (Brandon Campbell, Kit Whitworth, Stella Chuke, Elizabeth Henry, Joe Caba, Laura Daniels)