IGRA / Tuberculin Skin Test

Dr.Haifa Naser

Interferon-gamma release assays (IGRAs) are diagnostic tools for

latent tuberculosis infection (LTBI).

They are surrogate markers of Mycobacterium tuberculosis

infection and indicate a cellular immune response to M.

tuberculosis.

IGRAs

IGRAs cannot distinguish between latent infection and active

tuberculosis (TB) disease, and should not be used as a sole method

for diagnosis of active TB, which is a microbiological diagnosis.

A positive IGRA result may not necessarily indicate active TB;

however, a negative IGRA result rules out the possibility of both

active and latent tuberculosis.

Because IGRAs are not affected by Bacille Calmette-Guérin

(BCG) vaccination status, IGRAs are useful for evaluation of

LTBI in BCG-vaccinated individuals, particularly in settings

where BCG vaccination is administered after infancy or multiple

(booster) BCG vaccinations are given.

In contrast, the specificity of tuberculin skin test (TST) varies

depending on timing of BCG and whether repeated (booster)

vaccinations are given.

QuantiFERON

also known as QFT, is the

registered trademark of the test

for tuberculosis infection or

latent tuberculosis.

QFT is an interferon-γ release

assay (IGRA) used in

tuberculosis diagnosis.

The QFT-GIT assay is an ELISA-based, whole-blood test that uses

peptides from three TB antigens (ESAT-6, CFP-10, and TB7.7) in an

in-tube format.

The result is reported as quantification of IFN-gamma in international

units (IU) per mL. An individual is considered positive for M.

tuberculosis infection if the IFN-gamma response to TB antigens is

above the test cut-off (after subtracting the background IFN-gamma

response in the negative control).

QuantiFERON-TB Gold Test .

T-SPOT-TB Test .

Commercially Available IGRAs Tests

Comparison

of commercially available IGRAs

QuantiFERON –TB Gold

Collect 1ml of blood into

each of three tube: nil

control ESAT-6 /CFP-

10/TB7.7 .

Incubate for 16-24 h at 37

c co2 not required .

T SPOT-TB

Collect 4-8 ml blood in lymphcyte

separation tube . Certrifuge for 30 min.

Collect PBMC layer and add to 10 ml

sterile culture media .

Wash PBMC twice by resuspension and

centrifugation 7 min .

Stain and count viable cells in culture

media .

Add cells and anti gens ESAT-6/CFP-10

to coated microtiter plate.

Incubate for 16-20 h at 37 c co2

incubator required .

Comparison of commercially available IGRAs

QuantiFERON-TB Gold

Harvest plasma [ centrifuge tube 15 minutes optional .can store samples for up to 8 weeks at 4 c.

Add plasma and conjugate to ELISA plate incubate for 120 min at room temperature.

Wash microtiter plate and add substratefor 30 minutes.

Add stop reagent and reed optical densities.

Software calculates and print results.

T SPOT-TB

Wash off cells and add conjugate to

ELISPOT plate . Incubte for 60 min at

room temperature .

Wash microtiter plate and add

preciptating substrate for 7 minutes .

Wash plate dry for 3 h and count spots

visually or using an ELISPOT plate read.

Software with reader calculates results .

QuantiFERON-TB (QFT).

QuantiFERON-TB Gold In-Tube (QFT-GIT), the third

generation test, has replaced QuantiFERON-TB (QFT)

QuantiFERON-Gold, which are no longer marketed.According

to the U.S. Centers for Disease Control,[1] in 2001, the

QuantiFERON-TB test (QFT) was approved by the Food and

Drug Administration (FDA) as an aid for detecting latent

Mycobacterium tuberculosis infection.

This test is an in vitro diagnostic aid that measures a

component of cell-mediated immune reactivity to M.

tuberculosis.

The test is based on the quantification of interferon-gamma

(IFN-γ) released from sensitized lymphocytes in whole blood

incubated overnight with purified protein derivative (PPD)

from M tuberculosis and control antigens.

Advantages of QuantiFERON –TB Gold

Requires a single patient visit to draw a blood sample.

Results can be available within 24 hours.

Does not boost responses measured by subsequent tests, which can happen with tuberculin skin tests (TST).

Is not subject to reader bias that can occur with TST.

Is not affected by prior BCG (bacille Calmette-Guérin)

vaccination.

Disadvantages and limitation of QuontiFERON-TB Gold

Blood samples must be processed within 12 hours after collection

while white blood cells are still viable.

There is limited data on the use of QFT-G in children younger than

17 years of age, among persons recently exposed to M. tuberculosis,

and in immunocompromised persons (e.g., impaired immune

function caused by HIV infection or acquired immunodeficiency

syndrome [AIDS], current treatment with immunosuppressive

drugs, selected hematological disorders, specific malignancies,

diabetes, silicosis, and chronic renal failure).

Errors in collecting or transporting blood specimens or in

running and interpreting the assay can decrease the accuracy of

QFT-G.

Limited data on the use of QFT-G to determine who is at risk for

developing TB disease.

False positive results can occur with Mycobacterium szulgai,

Mycobacterium kansasii, and Mycobacterium marinum.

Tuberculin skin tests (TST) are administered to detect the presence

of Mycobacterium tuberculosis, the bacterium that causes

tuberculosis (TB).

The terms Mantoux, TB skin test, tuberculin skin test, and PPDs are often used interchangeably. Mantoux refers to the technique for administering the test.

Tuberculin Skin Test

Tuberculin (also called purified protein derivative or PPD) is the

solution used to administer the test.

The preferred term for the test is tuberculin skin test, or TST.

Tuberculin Skin Test

Mantoux Tuberculin Skin Test

Administration of Tuberculin skin test

The TST is performed by injecting 0.1

ml of tuberculin purified protein

derivative (PPD) into the inner surface

of the forearm.

The injection should be made with a

tuberculin syringe, with the needle

bevel facing upward.

The TST is an intradermal injection.

When placed correctly, the injection

should produce a pale elevation of the

skin (a wheal) 6 to 10 mm in diameter.

Reading of Tuberculin skin test

The skin test reaction should be read

between 48 and 72 hours after

administration.

A patient who does not return within 72

hours will need to be rescheduled for

another skin test.

The reaction should be measured in

millimeters of the induration (palpable,

raised, hardened area or swelling).

The reader should not measure erythema

(redness).

The diameter of the indurated area

should be measured across the forearm

(perpendicular to the long axis).

Skin test interpretation depends on two factors:

Measurement in millimeters of the induration.

Person’s risk of being infected with TB and of

progression to disease if infected.

How Are TST Reactions Interpreted?

Some persons may react to the TST even though they

are not infected with M. tuberculosis. The causes of

these false-positive reactions may include, but are not

limited to, the following:

Infection with nontuberculosis mycobacteria

Previous BCG vaccination

Incorrect method of TST administration

Incorrect interpretation of reaction

Incorrect bottle of antigen used

What Are False-Positive Reactions?

Some persons may not react to the TST even though they are infected with M. tuberculosis.

The reasons for these false-negative reactions may include, but are not limited to, the following:

Cutaneous anergy (anergy is the inability to react to skin tests because of a weakened immune system)

Recent TB infection (within 8-10 weeks of exposure)

Very old TB infection (many years)

What Are False-Negative Reactions?

Very young age (less than 6 months old).

Recent live-virus vaccination (e.g., measles and smallpox).

Overwhelming TB disease.

Some viral illnesses (e.g., measles and chicken pox).

Incorrect method of TST administration.

Incorrect interpretation of reaction.

Performance and

operationlcharacteristics

T.S.T IGRAS

Estimated sensitivity in pt with active TB

75%-90% lower immunocompromised populations

75%-95% {inadequatedata in immunocompromised populations}

Estimated specificity in healthy individuals with no known TBdisease orexposure

70%-95%lower in BCG vaccinated

90%-100% maintained in BCG vaccinated

Comparison of tuberculin skin test and IGRAs

Cross- reactivity with BCG

YES Less likely

Cross-reactivity with NTM

YES Less likely

Association between test-+ve and subsequent risk of active TB during follow-up

Moderate to strong positive association

Insufficient evidence

Correlation with mycobacteriumtuberculosis exposure

YES YES

Benefits of treating test positive based on randomized controlled trials

YES NO evidence

Boosting phenomenon YES NO

Performance and

operationlcharacteristics

T.S.T IGRAS

Potential for

conversions and

reversions

YES Insufficient evidence

Adverse reactions Rare Rare

Material costs LOW Moderate to high

Patient visits TWO ONE

Laboratory

infrastructure required

NO YES

Time to obtain a result 2 to 3 days 1 to 2 days

Thank You