LAB DIAGNOSIS OF

TUBERCULOSIS {TB}
I M M U N O LO G I C A L T E C H N I Q U E S

Immunologic lab diagnosis of TB

1. Tuberculin skin test.
2. Interferon Gamma Releasing Assay (IGRA).
3. ALS assay.
4. Full blood count.
5. Role of regulatory T cells in diagnosis of MTB.

Immunologic lab diagnosis of TB

Based on measurement of bodys immune
responses to MTB antigens.
Can detect both :
Active TB
Latent TB

Can detect both :

Pulmonary TB
Extra-pulmonary TB.

Genitourinary
Lymph nodes
Skin

Tuberculosi
s

Diagnosis

Latent TB

Active TB

Infection

Disease

TST &
IGRAs

Indirect
tests

Microscopy
& culture
Conventional

Direct
tests

Molecular
Advanced

Overview of bodys immune response

1. Tuberculin Skin Test

TST
M A N T O U X s k i n t e s t o r t u b e rc u l i n

Tuberculin skin testTST

Routine diagnostic method for TB
Based on delayed type hypersensitivity rx.
PPD purified protein derivative antigen
Dose of tuberculin is 5TU = 0.0001 mg PPD
Test for exposure to MTB
Cant differentiate between active disease
or latent infection

1. 0.1 ml of PPD is injected intradermal

2. Examine after 48-72 hr from injection

3. Interpret result by measuring induration

Interpretation of TST
Skin test interpretations depends on 2
factors:
1. Measurement of induration in mm
2. Persons risk of being infected with TB

Two step TST

Drawbacks of TST

Gamma Interferon Release Assays

IGRAs
Measure how the immune system reacts to
MTB.
IGRAs are the preferred method of TB
infection testing for the following:
People who have receivedBacille Calmette
Gurin (BCG).
People who have a difficult time returning for a
second appointment to look for a reaction to
the TST.

There is no problem with repeated IGRAs.

Gamma Interferon Release Assays

IGRAs
2 FDA approved & commercially available tests:
1. Enzyme-linked immunospot assay (ELISpot or Tspot TB test)
2. Enzyme-linked immunosorbent assay (ELISA)
(QuantiFERON-TB Gold In-Tube assay).

Gamma Interferon Release Assays

IGRAs
Both have high sensitivity and high specificity
But still cant differentiate between active & latent
TB
use of antigens encoded by Regions of Difference 1
(RD1) in the MTB genome, which is absent in BCG
vaccination or NTB.
Among the nine antigens encoded by RD1:
Early Secreted Antigenic Target 6kDa (ESAT-6) and
Culture Filtrate Protein 10kDa (CFP-10)
are used as a stimulatory antigens.

ADVANTAGES OF IGRA
Requires a single patient visit to conduct the test
Results can be available within 24 hours
Dose not boost responses measured by
subsequent tests
Prior BCG vaccination dose not cause a false
positive IGRA test results

DISADVANTAGES OF IGRA
Blood samples must be processed within 8-30 hr
after collection while blood cell still viable
Errors in collecting or transporting blood
specimens or in running and interpretation the
assay can decrease accuracy of the test
Limited data on the use of the IGRA to predict
who will progress to TB disease in the future
Tests may be expensive

Comparison

Immunological responses
to MTB

HIV/TB COINFECTION
Rare lethal combination
Urgent issue in global health
Leading cause of mortality among HIV patients.
HIV weakens immune system leading to false
negative results
HIV virus can weaken the immune system, LTBI
can be activated resulting in pulmonary or
extrapulmonary TB.

ALS ASSAY

ALS ASSAY
Antibodies from Lymphocyte
Secretion or Antibody in Lymphocyte
Supernatant or ALS Assay is an
immunological assay to detect active
diseases like tuberculosis, cholera,
typhoid etc.

ALS ASSAY
Procedure:
PBMCs (peripheral blood mononuclear cell) were
separated from blood by differential
centrifugation
PBMCs were suspended in 24-well tissue culture
plates culture medium.
Different dilutions of PBMCs were incubated at
37C with 5% CO2.
Culture supernatants were collected at 24, 48, 72,
and 96 h after incubation and the supernatants
were test against PPD by ELISA.
The ELISA titer indicates the positive or negative
result.

ALS ASSAY

Advantages:
High Sensitivity >93 %.
Early detection of active TB.
This method does not require a specimen taken
from the site of disease; it also may be useful in
diagnosis of childhood TB.
Secreted antibody may be preserved for long
time for further analysis

FULL BLOOD COUNT

FULL BLOOD COUNT

Full blood count is never diagnostic but normocytic
anemia and lymphopenia are common.
Neutrophilia is rarely found [iron deficiency anemia may
develop with isoniazid treatment].
Urea and electrolytes are usually normal, although
hypocalcaemia and hypernatremia are possible in
tuberculous meningoencephalitis due to SIADH (syndrome
of inappropriate antidiuretic hormone secretion).
In advanced disease: hypoalbuminemia,
hyperproteinemia, and hyperglobulinemia may be
present.
Erythrocyte sedimentation rate is usually raised.

ROLE OF T REG CELLS IN

DIAGNOSIS

ROLE OF T REG CELLS IN DIAGNOSIS

Immunosuppressive regulatory T-cells (T-Regs)
and CD4+ T-lymphocytes in general are
important in the host immune response to LTBI.
T-Regs down regulate the immune system to
prevent excessive immune responses which may
eventually lead to autoimmune disease and
immunopathology.
Activated T-Regs as they limit host immunity
they can inhibit pathogen clearance hence
facilitating pathogen multiplication and
dissemination.

ROLE OF T REG CELLS IN DIAGNOSIS

Treg cells were able to suppress IFN and IL-10
production in TB patients. This mechanism is
thought to contribute to the pathogenesis of
human TB .
Treg cell expansion is believed to predispose or
be a marker of the progression of latent TB to
active disease.
Method: Cryo-preserved peripheral blood
mononuclear cells (PBMCs) were used to
determine the number and phenotypic markers of
T-Regs using multi-color flow cytometry.