Tuberculosis

Tuberculosis (TB) serodiagnosis aims to identify individuals infected with Mycobacterium tuberculosis, the bacterium responsible for TB. The two primary methods for detecting TB infection are the Tuberculin Skin Test (TST), also known as the Purified Protein Derivative (PPD) test, and Interferon-Gamma Release Assays (IGRAs). This review will cover both, highlighting their principles, procedures, interpretations, advantages, and disadvantages

Understanding Tuberculosis Infection

• Causative Agent: Mycobacterium tuberculosis, an acid-fast bacterium
• Transmission: Airborne droplets from person to person
• Types of Infection
  • Latent TB Infection (LTBI): The bacteria are present in the body but are inactive. Individuals with LTBI do not have symptoms, are not contagious, and cannot spread TB to others. However, LTBI can progress to active TB disease
  • Active TB Disease: The bacteria are actively multiplying in the body, causing symptoms such as cough, fever, weight loss, and night sweats. Individuals with active TB disease are contagious and can spread TB to others

Tuberculin Skin Test (TST) / Purified Protein Derivative (PPD) Test

• Principle: The TST is based on the principle of delayed-type hypersensitivity. When tuberculin (a purified protein derivative of M. tuberculosis) is injected into the skin of a person who has been infected with TB bacteria, their immune system recognizes the tuberculin and mounts a local immune response, resulting in induration (a firm, raised area) at the injection site
• Procedure
  1. Injection: A small amount (0.1 mL) of PPD tuberculin is injected intradermally into the forearm, creating a wheal (a small, raised blister-like area)
  2. Reading: The test is read 48-72 hours after injection. The diameter of the induration (not the redness) is measured in millimeters
  3. Interpretation: The interpretation of the TST depends on the size of the induration and the person’s risk factors for TB

TST Interpretation Criteria

• ≥5 mm Induration is Considered Positive in
  • HIV-infected persons
  • Recent contacts of persons with active TB disease
  • Persons with fibrotic changes on chest radiograph consistent with prior TB
  • Organ transplant recipients
  • Persons who are immunosuppressed for other reasons (e.g., taking TNF-alpha antagonists)
• ≥10 mm Induration is Considered Positive in
  • Recent immigrants (≤ 5 years) from high-prevalence countries
  • Injection drug users
  • Residents and employees of high-risk congregate settings (e.g., prisons, nursing homes, homeless shelters)
  • Mycobacteriology laboratory personnel
  • Persons with certain medical conditions (e.g., diabetes, end-stage renal disease, silicosis, leukemia, lymphoma, head or neck cancer)
  • Children < 4 years old
• ≥15 mm Induration is Considered Positive in
  • Persons with no known risk factors for TB

Factors Affecting TST Results

• False Positives
  • Prior BCG vaccination (Bacille Calmette-Guérin vaccine)
  • Infection with nontuberculous mycobacteria (NTM)
  • Incorrect administration or interpretation of the test
• False Negatives
  • Anergy (inability to mount an immune response due to immunosuppression)
  • Recent TB infection (before the immune system has had time to respond)
  • Very old TB infection (waning immune response)
  • Incorrect administration or interpretation of the test

Advantages of TST

• Inexpensive
• Easy to perform (requires minimal training)
• Widely available

Disadvantages of TST

• Requires two visits (one for administration, one for reading)
• Subjective interpretation
• Affected by BCG vaccination and NTM infection
• Can give false-positive and false-negative results

Interferon-Gamma Release Assays (IGRAs)

• Principle: IGRAs are blood tests that measure the release of interferon-gamma (IFN-γ) by T cells in response to stimulation by M. tuberculosis-specific antigens. These antigens (ESAT-6 and CFP-10) are present in M. tuberculosis but are not present in BCG vaccine strains or most NTM
• Types of IGRAs
  • QuantiFERON-TB Gold In-Tube (QFT-GIT): A whole-blood assay in which blood is collected directly into tubes containing the M. tuberculosis antigens, a negative control, and a positive control. The tubes are incubated, and then the plasma is tested for IFN-γ using an ELISA
  • T-SPOT. TB: A cell-based assay in which peripheral blood mononuclear cells (PBMCs) are isolated and incubated with the M. tuberculosis antigens, a negative control, and a positive control. The cells are then washed, and IFN-γ-producing cells are detected using an enzyme-linked immunospot (ELISPOT) assay

Procedure (QFT-GIT)

1. Blood Collection: Collect blood directly into the QFT-GIT tubes (Nil, TB Antigen, Mitogen)
2. Incubation: Incubate the tubes at 37°C for 16-24 hours
3. Plasma Collection: Centrifuge the tubes and collect the plasma
4. ELISA: Perform an ELISA to measure the concentration of IFN-γ in each plasma sample
5. Interpretation: Calculate the IFN-γ response to the TB antigens by subtracting the Nil value from the TB Antigen value. The result is interpreted based on the manufacturer’s guidelines

QFT-GIT Interpretation Criteria

• Positive: TB Antigen value minus Nil value ≥ 0.35 IU/mL
• Negative: TB Antigen value minus Nil value < 0.35 IU/mL
• Indeterminate: Control values do not meet the manufacturer’s criteria

Advantages of IGRAs

• Requires only one patient visit
• Objective interpretation (no subjective reading)
• Not affected by BCG vaccination
• More specific than TST (less likely to give false-positive results due to NTM infection)

Disadvantages of IGRAs

• More expensive than TST
• Requires specialized equipment and training
• Blood sample must be processed within a certain timeframe
• May be less sensitive than TST in some populations (e.g., young children, immunocompromised individuals)

Comparison of TST and IGRAs

Feature	TST	IGRA
Principle	Delayed-type hypersensitivity to tuberculin	IFN-γ release in response to M. tuberculosis-specific antigens
Procedure	Intradermal injection of PPD, reading induration after 48-72 hours	Blood test, incubation with antigens, measurement of IFN-γ
Number of Visits	Two	One
Interpretation	Subjective (measurement of induration)	Objective (measurement of IFN-γ)
Effect of BCG Vaccination	Can cause false-positive results	Not affected by BCG vaccination
Effect of NTM Infection	Can cause false-positive results	More specific (less likely to be affected by NTM infection)
Cost	Less expensive	More expensive
Availability	Widely available	Requires specialized equipment and training

Recommendations for TB Testing

• IGRAs are generally preferred over TST in the following situations
  • Persons who have received BCG vaccination
  • Persons who are unlikely to return for TST reading
  • Persons who have difficulty with TST reading (e.g., young children)
• TST may be preferred in situations where IGRAs are not available or are too expensive
• Both TST and IGRAs have limitations, and clinical judgment should be used when interpreting test results
• Neither TST nor IGRAs can distinguish between latent TB infection and active TB disease. Additional tests (e.g., chest radiograph, sputum culture) are needed to diagnose active TB disease

Key Terms

• Tuberculosis (TB): An infectious disease caused by Mycobacterium tuberculosis
• Latent TB Infection (LTBI): A condition in which TB bacteria are present in the body but are inactive
• Active TB Disease: A condition in which TB bacteria are actively multiplying in the body, causing symptoms
• Tuberculin Skin Test (TST): A test to detect TB infection based on delayed-type hypersensitivity to tuberculin
• Purified Protein Derivative (PPD): A protein extract of M. tuberculosis used in the TST
• Induration: A firm, raised area at the site of a TST injection, indicating a positive reaction
• Interferon-Gamma Release Assay (IGRA): A blood test to detect TB infection based on the release of interferon-gamma by T cells in response to M. tuberculosis-specific antigens
• BCG Vaccination: A vaccine against tuberculosis
• Nontuberculous Mycobacteria (NTM): Mycobacteria other than M. tuberculosis that can cause infection