What is the timeline for administering the 1st and 2nd tuberculin skin test (TST) or interferon-gamma release assay (IGRA) in a two-step TB testing protocol for individuals with a high risk of exposure or history of TB exposure?

Two-Step TB Testing Timeline

For the two-step tuberculin skin test (TST) protocol, administer the first test on Day 0, read it at 48-72 hours (Day 2-3), then place the second test 1-3 weeks after reading the first test (approximately Day 9-24), and read it again at 48-72 hours. 1

Purpose of Two-Step Testing

The two-step TST protocol specifically detects the "booster phenomenon" in individuals with waned TB immunity, preventing misinterpretation of boosted reactions as new infections during subsequent serial testing. 1 This is critical because:

• Without two-step testing, a boosted reaction on later testing might be incorrectly classified as a new TB conversion, triggering unnecessary contact investigations and preventive therapy. 1
• The booster effect occurs when an initial TST "reminds" the immune system of prior TB exposure or BCG vaccination, causing a stronger reaction on the second test that reflects old—not new—infection. 1

Who Requires Two-Step Testing

Two-step testing should be performed on all newly employed healthcare workers who have an initial negative TST and no documented negative TST within the preceding 12 months. 1 This applies to:

• Healthcare workers in facilities conducting periodic TB screening 1
• Staff in correctional institutions and nursing homes 1
• Populations with high rates of prior TB exposure or BCG vaccination 1
• Older adults, who demonstrate the booster phenomenon more frequently 1

Detailed Timeline Protocol

First Test

• Day 0: Place the first TST using the Mantoux technique (0.1 mL of 5 tuberculin units intracutaneously on the forearm) 1
• Day 2-3 (48-72 hours): Read the first test by measuring only the induration (hardened area), not erythema 1

Second Test

• Day 9-24 (1-3 weeks after first reading): Place the second TST 1
• 48-72 hours later: Read the second test 1

Critical Timing Considerations

The interval between the first and second test must be 1-3 weeks. 1 Improper timing affects accuracy:

• Testing too soon (less than 1 week) can interfere with the immunological response and produce inaccurate results 2
• Testing beyond 3 weeks may miss the optimal window for detecting the booster phenomenon 1

Interpretation of Two-Step Results

• Both tests negative: True negative baseline established; individual can proceed with annual or periodic screening as indicated by risk classification 3
• First test negative, second test positive: This represents a boosted reaction from prior TB exposure or BCG vaccination, not a new infection 1
• First test positive: No second test needed; proceed with evaluation for latent TB infection or active disease 1

Common Pitfalls to Avoid

• Failure to implement two-step testing in appropriate populations leads to misclassification of boosted reactions as new infections, particularly problematic in healthcare settings with serial testing programs. 1
• Reading tests outside the 48-72 hour window reduces accuracy and should be avoided. 1
• Measuring erythema instead of induration leads to incorrect interpretation; only the palpable, raised, hardened area should be measured in millimeters. 1
• Performing the second test too soon (less than 1 week) interferes with immune response and yields unreliable results. 2

Alternative Considerations

For individuals unlikely to return for multiple visits, interferon-gamma release assays (IGRAs) may be considered as they require only a single blood draw without return visits for reading. 3 However, guidelines vary internationally on IGRA use, with some recommending TST followed by IGRA for confirmation in BCG-vaccinated individuals, while others suggest either test alone. 3