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TB Nurse Case ManagementSan Antonio TexasSan Antonio, TexasNovember 3-5, 2010

Interferon Gamma Releasing Assays

Edward A. Graviss, PhD, MPHNovember 5, 2010

Interferon Gamma Release Assays

Edward A. Graviss, PhD, MPHeagraviss@tmhs.org

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Conflict of Interest Statement

• Cellestis, Ltd: Consultation reimbursement d l t h iand lecture honorariums

• Oxford Immunotec, Ltd: Research support, consultation reimbursement and lecture honorariums

Objectives

• Give an overview of LTBI and TST

• Discuss the theory of IGRAs and their• Discuss the theory of IGRAs and their development

• Summarize CDC guidelines regarding the use of Interferon Gamma Release Assays

• Compare IGRAs: QFT versus T-SPOT.TBCompare IGRAs: QFT versus T SPOT.TB

• Present two (2) IGRA case studies

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Latent TB Infection and the Tuberculin Skin Text

Natural History of TB Infection

4-6 Weeks Years-Decades

Elimination of Bacteria

Elimination of Bacteria

Infection

Aveolar Macrophage or Dendritic Cell

(Innate Response)

Onward Transmission

Elimination of Bacteria

Reactivation

Latent TB(LTBI)

Lifelong Containment

Inability to Control Bacteria

Initial Immune Control of Bacteria

T Cell(Adaptive Response)

Exposure

Macrophage

Granuloma

Macrophage

5% 5%

95%

90%

ID and Treatment

Active TBActive TB

Modified Oxford Immunotec , 2010

Dx and Treatment

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Tuberculin Skin Test (TST)

• Injection of tuberculin (5 TU / 0 1 ml) which contains0.1 ml), which contains hundreds of antigens for T-cells

• Requires two visits

• Requires skilled personnel for placement and interpretation p p(digits, induration)

Skin Testing Least Reliable in High-risk Groups

• Homeless, drug users less likely to , g yreturn for reading

• False-negative tests in immunocompromised patients

• False-positive tests in BCG-vaccinated foreign-born

• Health care worker - noncompliance

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IGRA Theory and Developmenty p

MTB Specific Antigens

• Sequencing the TB genome has revealed two antigens, ESAT-6(early secreted antigenic target 6

Clin Microb Rev, 2003

( y g gkD protein) and CFP10 (culture filtrate protein 10) found in the RD1 region and highly antigenic

• A highly specific peptide from TB7.7 (Rv2645, d-ribose, 5-phosphate isomerase) showing a response in 20-30% of TB ppatients. QFT- 3G only.

• RD1 antigens are not present in BCG ***

Science 1999;284:1520-23

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Principle of INF-gamma Assays (IGRAs)

• Effector memory T-cells from persons with LTBI d d d IFN i texpand and produce IFN-gamma in response to

ESAT-6 and CFP10

• IGRA tests quantitatively measure the IFN-gamma produced by M. tuberculosis–specific memory T-cells

Injection of antigens below epidermal layer causes

In-vitro and in-vivo diagnostic test

infiltration of antigen-specific lymphocytes and the elaboration of inflammatory cytokines

PBMCs from the peripheral blood are stimulated in vitro and production of INF-vitro and production of INF-gamma from sensitized T cells is measured by ELISA or cell countLancet 2000;356:1099-104

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Blood Tests for LTBI

• QuantiFERON TB-GOLD – (QFT-IT) FDA approved Interferon-gamma release assay (IGRA) (Cellestis Ltd, Carnegie Victoria Australia); ELISACarnegie, Victoria, Australia); ELISA-based, whole blood, indirect cytokine-based.

• T-SPOT TB – (T-SPOT) FDA approved (Oxford Immunotec Ltd, Abingdon, Oxon, UK) utilizes the same

Qmycobacterial antigens as the QFT, but employs a different methodology of measuring the interferon-gamma response (ELISPOT).

Historical Perspective - FDA and CDC IGRA Statements

2001: QFT (1st generation) – FDA approved

2003: CDC Guidelines – MMWR 2003:53 (RR-2); 15-18 Confirmation of QFT with TST prior to LTBI treatment

2005: QFT Gold (2nd generation) FDA approved2005: QFT –Gold (2nd generation) – FDA approved

2005: CDC Guidelines – MMWR 2005:54(RR-15); 49-55 QFT-G can be used in all circumstances where TST used

2007: QFT – Gold In-tube (3rd generation) – FDA approved

2008: T-SPOT.TB FDA approved

2008: CDC convenes expert panel (n = 27) to review scientific2008: CDC convenes expert panel (n = 27) to review scientific evidence, provide findings and opinions for guidelines

2010: CDC Guidelines – MMWR 2010:59(RR-5); 1-26

2010: T-SPOT.TB Xtend FDA approved

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Updated Guidelines Regarding th U f I t f Gthe Use of Interferon Gamma

Release Assays (IGRAs)

*Updated guidelines regarding the use of Interferon Gamma Release Assays (IGRAs)1

• TST or IGRAs should not be used for testing persons who have a low

*MMWR 2010; 59 - RR5: 1-26

for testing persons who have a low likelihood of MTB infection and of progression to TB disease if infected

• IGRAs may be used in place of tuberculin skin test (TST) in all situations, including contact investigation

• In certain situations IGRAs are the preferred test

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*Updated guidelines regarding the use of Interferon Gamma Release Assays (IGRAs)2

• Selection of the most suitable test should be based on the reasons and the context for testing and the test gavailability and cost

– IGRA is preferred for testing persons who seem unlikely to return for TST reading

– IGRA is preferred for testing BCG vaccinated

IGRA is preferred for testing persons with a low– IGRA is preferred for testing persons with a low likelihood of both MTB infection and of progression to TB disease if infected

*MMWR 2010; 59 - RR5: 1-26

*Updated guidelines regarding the use of Interferon Gamma Release Assays (IGRAs)3

• IGRA should be used in place of (and not in addition p (to) TST. Dual testing not recommended

• Both the qualitative and quantitative assay measurements should be reported, together with the criteria for test interpretation

• TST is preferred when testing children younger than 5 p g y gyears of age

*MMWR 2010; 59 - RR5: 1-26

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QFT-IT versus the T-SPOT.TB

Interpretation of IGRAs

QFT

• Nil well <= 0.8 IU/mL

• Mitogen well – Nil well > 0.5 IU/mL

• TB Antigen minus Nil well >= 0.35 IU/mL and >= 25% of Nil value

T-SPOT.TB

• Eight or more spots in the ESAT-6 or CFP10 well, compared to the negative control well

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Sample IGRA Results

QuantiFERON T-SPOT.TB

Nil Control

0.000

0.500

1.000

1.500

2.000

2.500

Op

tica

l D

ensi

ty

Control

ESAT-6Panel A

CFP 10Panel B

0 2 4 6 8 10

Concentration of standard (IU/mL) Positive Control

QFN-TB GOLD T-SPOT TB

FDA-approved FDA-approved

QFN-TB GOLD and T-SPOT TB Comparison

Whole blood assay Requires cell separation

High frequency of indeterminate results in immunocompromised

More data in children and HIV+ persons

Cost to laboratory is $42-$150 per test

Cost to laboratory is $45-$150 per test

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QFN-TB In-Tube T-SPOT.TBBlood samples incubated within 16 Blood samples processed with 8

Additional Key Issues:QFN-TB In-Tube and T-SPOT TB Comparison

phours of blood draw / tubes can be held for 3 days prior to processing.

p phours of blood draw (T-prep approval – 36 hours)

$3 / blood tube = set @ $9 USD

antigens in tube

Lithium heparin (use of Ficoll) or CPT tubes ($9 USD for 2 x 4mL)

Adequate mixing of blood and antigens

Hemocytometer for cell count or automated counterantigens automated counter

ELISA – based /

no CO2 incubation

ELISPOT – based /

CO2 incubation

Comparing the Sensitivity of ELISPOT vs ELISA Assays

• With ELISPOT – you can get a response with a handful of cells

• With ELISA - you need about 4,000 T cells to get a measurable response

Comparing the sensitivity of ELISPOT (left) vs. ELISA (right) using T cell clones.Data courtesy of David Lewinsohn, Oregon Health and Science University, Portland.

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Forest Plots of Studies Estimating Sensitivity of T-SPOT.TB and QFT-IT Detecting Active TB DiseaseT-SPOT.TB Sensitivity QFT-IT Sensitivity

Modified from Diel R et al. Chest 2010:137;952-968 ***The T-SPOT.TB is significantly more sensitive

than the QFT-GIT (P<0.001) False negative results: 125/1000 T-SPOT.TB vs 190/1000 QFT-GIT.

Forest Plots of Studies Estimating Sensitivity of QFT-IT Detecting Active TB Disease by Country

Developed Countries

Developing Countries

Modified from Diel R et al. Chest 2010:137;952-968

Developing Countries

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Forest Plots of Studies Estimating Specificity of T-SPOT.TB and QFT-IT Detecting Active TB Disease

QFT-IT SpecificityT-SPOT.TB Specificity

Modified from Pai M et al. Ann Intern Med. 2008; 3:177-84.

***Meta-analysis suggests the QFT-IT may be a little more specific than the T-SPOT.TB, but difference is not statistically significant. Sample size?

• Lack of published data –

IGRA variability – Conversions / Reversions

IGRA Variability and TST Boosting (PLoSone 2009; 4(12): e8517)

data available consists of 67 HCWs

• Although small sample size – variability does occur

• Variability occurs around assay cut points

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IGRA Variability and TST Boosting

• Growing evidence that the TST can boost subsequent IGRA results but the effect appears to be more apparent in those

(PLoSone 2009; 4(12): e8517)

Boosting effect of TST on IGRA Results

results, but the effect appears to be more apparent in those individuals who are already IGRA positive.

• Effect seems apparent after 3 days and potentially wanes after a short period of time (1 -3 months)

• Published studies used time points between 7 and 28 days for the second IGRA (post TST)

• In IGRA negative individuals (2-12% boost) following a TST. [**Caution sample size]

IGRA Case Studies

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Case Study A (1)

Patient history

• 28-year-old Asian female

• Moved to U.S. from Vietnam ≈ 10 years ago

• Plans to work in a TMC facility

• TST negative (1 yr ago); BCG vaccinated

• TST employment screen = 10 mm induration

• CXR normal

• No symptoms of TB disease• No symptoms of TB disease

• No known contact with a TB patient

Case Study A (2)

Questions

1. What are the patient’s risk factors for TB infection or disease?

2. What is the appropriate management for this patient?

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Case Study A (3)

Discussion of risk factors

• TST conversion from negative to positive (within a 2-year period)

• TST conversion increases risk for progressing from LTBI to TB disease

• Foreign-born status is less of a risk factor, i.e., she g , ,immigrated more than 5 years ago

Case Study A (4)

Discussion of management

• Patient’s TST conversion indicates failure to identify this person as high risk for recent exposure to TB

• Patient may have had extended travel to her country of origin or other high-prevalence parts of the world

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Case Study A (5)

Discussion of management

• Patient is a recent converter and, as such, is a candidate for treatment of LTBI with INH

• Candidate for IGRA testing

QuantiFERON Ordered – Positive (0.48 IU)

Case Study B (1)

Patient history

• 46-year-old Caucasian male physician46 year old Caucasian male physician

• U.S.- born; no recent travel

• Working in a large inter-city hospital

• TST negative (1 yr ago)

• TST 3 mm induration

• CXR normal

• No symptoms of TB disease

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Case Study B (2)

Questions

1. What are the patient’s risk factors for TB infection or disease?

2. What is the appropriate management for this patient?

Case Study B (3)

Discussion of risk factors

• Clinical health care provider (20 years)

- Entered IGRA study

• T-SPOT.TB positive (8 spots)

• QFT-IT negative (0 24 IU)QFT IT negative (0.24 IU)

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Case Study B (4)

Discussion of management

• Conversion ?

• Former patient subsequently identified as a TB case after discharge (6 weeks post-discharge)

• Treatment ?

6 month serial follow-upp

• T-SPOT.TB positive (12 spots)

• QFT-IT negative (0.31 IU)

Questions ?

TMHRIClinical

Lab

Justin Lew , BS Ngan Ha , BS

Sophie Im , BSMarsha Feske , BS, MPH