IMMUNOLOGICAL TECHNIQUES

LAB DIAGNOSIS OF TUBERCULOSIS {TB}

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 body’s immune responses to MTB antigens.

• Can detect both :• Active TB• Latent TB

• Can detect both :• Pulmonary TB • Extra-pulmonary TB.• Genitourinary• Lymph nodes• Skin• …

Tuberculosis

Diagnosis

Latent TB

Infection

TST & IGRAs

Indirect tests

Active TB

Disease

Microscopy & culture

Conventional

Molecular

Advanced

Direct tests

Overview of body’s immune response

MANTOUX sk in test or tubercul in

1. Tuberculin Skin Test “TST”

Tuberculin skin test…TST

• 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• Can’t 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 mm2. Person’s 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 received Bacille Calmette–

Guérin (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 T-spot 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 can’t 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

37°C 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 γ 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.