IGRA / TUBERCULIN SKIN TEST.

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

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
operationl
characteristics
T.S.T IGRAS
Estimated sensitivity in
pt with active TB
75%-90% lower
immunocompromised
populations
75%-95% {inadequate
data 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
mycobacterium
tuberculosis exposure
YES YES
Benefits of treating
test positive based on
randomized controlled
trials
YES NO evidence
Boosting phenomenon YES NO

Performance and
operationl
characteristics
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

IGRA / TUBERCULIN SKIN TEST.

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