What is a two-step tuberculin skin test (TST) for detecting latent tuberculosis infection (LTBI) in individuals with a history of BCG vaccination or previous TB exposure?

What is a Two-Step Tuberculin Skin Test (TST)?

A two-step TST is a baseline testing procedure designed to detect the "booster phenomenon" in individuals with waned tuberculosis immunity, preventing the misclassification of boosted reactions as new TB infections during subsequent serial testing programs. 1

Purpose and Mechanism

The two-step procedure addresses a critical immunological phenomenon where individuals previously infected with M. tuberculosis may have diminished tuberculin reactivity that can be restored ("boosted") by an initial TST. 1 Without this two-step approach, a boosted reaction on subsequent routine testing might be incorrectly interpreted as a new infection (conversion), triggering unnecessary contact investigations and preventive therapy. 1

The booster phenomenon occurs because remote TB infection or BCG vaccination can cause tuberculin sensitivity that wanes over time, but the initial TST can stimulate an anamnestic immune response that increases reactivity on subsequent testing. 2

When Two-Step Testing Should Be Used

Recommended Populations

• All newly employed healthcare workers with an initial negative TST and no documented negative TST within the preceding 12 months 1
• Individuals entering serial tuberculin testing programs (healthcare facilities, correctional institutions, nursing homes) 1
• Populations with high rates of prior TB exposure or BCG vaccination where establishing accurate baseline status is critical 1

When Two-Step Testing Should NOT Be Used

The two-step procedure typically should not be used for contact investigations of active TB cases. 2 In contact tracing, a person whose second test is positive after an initial negative result should be classified as recently infected, not as demonstrating a booster phenomenon. 2

Procedure and Timing

• First TST is administered using standard Mantoux technique (0.1 mL of 5 tuberculin units intradermally) 1
• If the first test is negative, a second TST is administered 1-3 weeks later 1
• The timing window of 1-3 weeks is critical—performing the second test too soon can interfere with immunological response and lead to inaccurate results 1
• Both tests must be read by trained personnel at 48-72 hours after injection, measuring only induration (not erythema) 1

Interpretation

• If the first test is positive (≥5 mm, ≥10 mm, or ≥15 mm depending on risk category), the person is classified as infected and no second test is needed 1
• If the first test is negative but the second test is positive, this represents a boosted reaction, and the person should be considered previously infected 1
• If both tests are negative, the person is classified as uninfected, and this establishes the baseline for future serial testing 1

High-Risk Populations for Booster Phenomenon

The booster effect is particularly common in:

• Older adults (though it can occur at any age) 1
• Individuals previously vaccinated with BCG 1, 3
• Foreign-born persons from high TB prevalence countries 2, 1
• Persons exposed to nontuberculous mycobacteria 1

The boosting effect can occur years or even decades after initial infection or BCG vaccination. 1

Critical Pitfalls to Avoid

• Failure to implement two-step testing in appropriate populations leads to misclassification of boosted reactions as new infections during subsequent surveillance 1
• Improper timing between first and second test (must be 1-3 weeks) affects accuracy of detecting the booster phenomenon 1
• Using two-step testing in contact investigations can delay identification of true new infections 2
• Measuring erythema instead of induration or reading outside the 48-72 hour window reduces accuracy 1

Special Considerations for BCG-Vaccinated Individuals

In BCG-vaccinated persons entering serial testing programs, the two-step baseline procedure is particularly important because these individuals are more likely to demonstrate boosting. 3 However, a positive TST in BCG-vaccinated contacts of infectious TB cases should be interpreted as evidence of recent M. tuberculosis infection, not BCG effect, and these individuals should be evaluated for TB disease and offered treatment for latent TB infection. 2

Alternative Approaches

For individuals unlikely to return for TST reading or in BCG-vaccinated populations, interferon-gamma release assays (IGRAs) may be preferred as they require only a single visit and are not affected by BCG vaccination. 2, 3 However, research shows that TST can boost subsequent IGRA results when performed 2-4 weeks later, with conversion rates of approximately 17% in TST-positive individuals. 4