Tuberculosis

Let’s tackle a truly unique challenge in infectious disease diagnostics: Tuberculosis. This is a perfect topic for an immunology review because our main diagnostic methods don’t look for antibodies or the organism itself. Instead, they look for the footprint of a cell-mediated immune response

The culprit, Mycobacterium tuberculosis, is an intracellular pathogen. It gets inside our macrophages and lives there. A healthy immune system can’t always eliminate it, but it can contain it by building a wall around the infected cells. This structure is called a granuloma. Inside this biological prison, the bacteria can lie dormant for decades. This leads to the single most important concept you must understand for TB diagnostics:

• Latent TB Infection (LTBI): The person is infected, the immune system has the bacteria walled off, and they are not sick and not infectious. The bacteria are alive but dormant—a sleeping spy awaiting orders. The majority of infected people are in this state
• Active TB Disease: The immune system’s containment fails, the granuloma breaks down, and the bacteria begin to multiply. The person is sick and highly infectious, typically with symptoms like a persistent cough, fever, night sweats, and weight loss

Our primary immunologic tests are designed to answer one question: “Has this person’s immune system ever been exposed to and mounted a response against M. tuberculosis?” Critically, these tests cannot distinguish between latent infection and active disease. Their job is to identify the roughly 10% of people with LTBI who are at risk of progressing to active disease so they can be treated

Classic Method: Tuberculin Skin Test (TST) or PPD Test

This test has been the cornerstone of TB screening for over a century. It’s a classic, in-vivo (in the body) demonstration of a Type IV, or Delayed-Type, Hypersensitivity (DTH) reaction

• The Principle: We are intentionally provoking a localized memory T-cell response in the skin
• The Procedure
  1. A small, standardized dose of Purified Protein Derivative (PPD) is injected intradermally (just under the top layer of skin) in the forearm. PPD is a crude cocktail of antigens derived from a culture of M. tuberculosis
  2. If the patient has been previously infected (has LTBI or active TB), they will have memory T-helper cells (CD4+) that are sensitized to these TB antigens
  3. Over the next 48-72 hours, these memory T-cells will migrate to the injection site. They recognize the PPD antigens, become activated, and release a flood of cytokines (like interferon-gamma)
  4. These cytokines recruit a large number of macrophages and other inflammatory cells to the site, causing localized swelling and hardening
• Reading and Interpretation
  • A trained healthcare worker must “read” the test 48-72 hours later. They are not looking at the redness (erythema), but are palpating for the diameter of the hard, raised bump, which is called induration
  • The interpretation of a “positive” result depends on the size of the induration and the patient’s risk factors. This is a key concept: the cut-off balances sensitivity and specificity
    • ≥ 5 mm: Positive in the highest-risk individuals (e.g., HIV-positive, recent close contacts of someone with active TB)
    • ≥ 10 mm: Positive in medium-risk individuals (e.g., healthcare workers, people from high-prevalence countries)
    • ≥ 15 mm: Positive for anyone, regardless of risk factors
• Limitations of the PPD
  • Subjectivity: Reading the induration can be subjective and requires training
  • Logistics: The patient must return for a second visit to have the test read
  • False Positives (The Biggest Problem): The PPD antigens are not specific to M. tuberculosis. A patient will have a positive test if they have been vaccinated with the BCG (Bacille Calmette-Guérin) vaccine, which is common in many countries outside the U.S. They will also cross-react with some other environmental non-tuberculous mycobacteria

Modern Method: Interferon-Gamma Release Assays (IGRAs)

IGRAs were developed to overcome the major limitations of the PPD, especially the BCG cross-reactivity problem. This is an in-vitro (in a test tube) blood test that measures the same T-cell response, but with much greater precision and specificity

• The Principle: We directly challenge a patient’s T-cells in a blood sample with highly specific TB antigens and measure the amount of interferon-gamma (IFN-γ) they release in response
• The Procedure
  1. A fresh whole blood sample is collected from the patient
  2. The blood is aliquoted into tubes containing different reagents. The key tubes contain synthetic peptide antigens, such as ESAT-6 and CFP-10. These antigens are incredibly specific; they are encoded by a region of the M. tuberculosis genome that is deleted from the BCG vaccine strain and is absent from most other mycobacteria. This is the key to the test’s specificity
  3. If the patient’s blood contains memory T-cells sensitized to TB, these cells will recognize the ESAT-6 and CFP-10 antigens and, in response, will produce and secrete the cytokine interferon-gamma
  4. The lab then measures the amount of IFN-γ produced
• The Two Main IGRA Platforms
  • QuantiFERON-TB Gold Plus (QFT-Plus): This is an ELISA-based method. After incubation, the plasma is harvested and the amount of IFN-γ is measured using a standard sandwich ELISA. The test includes multiple tubes:
    • Nil Control: The patient’s blood with no added antigen. This is the baseline
    • TB Antigen Tubes (TB1 & TB2): Contain the ESAT-6/CFP-10 antigens to stimulate both CD4+ and CD8+ T-cells
    • Mitogen Control: Contains a potent, non-specific T-cell activator. This is the positive control for the patient’s immune system. If the mitogen tube is negative, it means the patient is anergic or their T-cells aren’t working properly, and the test result is “indeterminate.”
  • T-SPOT.TB: This is an ELISpot method. After incubation with the antigens, the patient’s cells are washed away, and a reagent is added that creates a “spot” everywhere a single T-cell was sitting and secreting IFN-γ. The result is determined by counting the number of spots
• Advantages of IGRAs over the PPD
  • Specificity: Not affected by prior BCG vaccination
  • Objectivity: The result is a quantitative, numerical value from the lab, not a subjective measurement of a bump
  • Logistics: Requires only a single patient visit to draw blood

Final Word: Clinical Context is Everything

Remember, a positive result from either a PPD or an IGRA does not diagnose active TB disease. It simply identifies an individual with a cellular immune response to M. tuberculosis. To diagnose active TB, a patient with a positive immunologic test must also have clinical symptoms, a suggestive chest X-ray, and—the gold standard—a positive sputum smear (for acid-fast bacilli) and culture performed by the microbiology lab