Skip to content
SearchProgress
Display

Display

Theme
Density
Text size

Sign in

Add your earlier progress to your account?

Study progress is waiting to be saved

Lyme and tuberculosis testing

15 min

  • Read Lyme two-tier results, counting an IgM blot only in the first 30 days of illness
  • Interpret an IGRA with its controls and limits for active tuberculosis

Read the full reference

Try first

Try first

A patient has been ill for 3 months. The Lyme first-tier enzyme immunoassay (EIA) is positive. The IgM immunoblot shows the 23, 39 and 41 kDa bands. The IgG immunoblot shows 3 of the 10 scored bands. How does the result read?

The next section explains it.

Right. The next section explains why.

The next section explains it.

The next section explains it.

Get the idea

Lyme serology runs in two tiers

Borrelia burgdorferi is hard to find in blood, so Lyme testing reads the antibody response. Serology can stay negative for 4 to 6 weeks after infection.1

Standard two-tier testing starts with a sensitive EIA. A negative first tier ends the test. A positive or equivocal first tier reflexes to immunoblots, read by how long the patient has been ill.2

Illness durationBlots readPositive when
30 days or lessIgM and IgGIgM shows 2 of 3 bands (23, 39, 41 kDa), or IgG shows 5 of 10 bands
More than 30 daysIgG onlyIgG shows 5 of 10 bands

2

Modified two-tier testing replaces the blots with a second EIA that uses different antigens, in pairs cleared together by FDA. Positive or equivocal results on both tiers read positive.2 A positive result shows antibodies from recent or past infection. IgG lasts months to years after cure, so serology cannot follow treatment.2

An IGRA reads T-cell memory

An interferon-gamma release assay (IGRA) measures the interferon-gamma that T cells release when blood meets Mycobacterium tuberculosis antigens. Bacille Calmette-Guérin (BCG) vaccination does not affect it. It shows infection and cannot separate latent infection from active disease.3

The whole-blood ELISA format runs a Nil tube, two TB antigen tubes and a Mitogen tube. Nil is subtracted from each of the others.4

  1. Nil must be 8.0 IU/mL or less. Above that, the result is indeterminate.
  2. Positive when either TB tube minus Nil is at least 0.35 IU/mL and at least 25% of the Nil value.
  3. Negative when both TB responses fall short and Mitogen minus Nil is at least 0.50 IU/mL.
  4. Indeterminate when Mitogen minus Nil is below 0.50 IU/mL and neither TB response is positive.4

An indeterminate result means the test did not work, often from a failed mitogen response or a high Nil. It says nothing about infection. A repeat IGRA or a tuberculin skin test can resolve it. The laboratory reports the qualitative result together with the numbers.3

References
  1. Centers for Disease Control and Prevention. Clinical testing and diagnosis for Lyme disease. May 15, 2024. Accessed September 27, 2026. https://www.cdc.gov/lyme/hcp/diagnosis-testing/index.html
  2. Association of Public Health Laboratories. Suggested Reporting Language, Interpretation and Guidance for Lyme Disease Serologic Test Results. April 2024. Accessed September 27, 2026. https://www.cdc.gov/lyme/media/pdfs/2024/05/Suggested-Reporting-Language-Interpretation-and-Guidance-Regarding-Lyme-Disease-Serologic-Test-Results.pdf
  3. Centers for Disease Control and Prevention. Clinical testing guidance for tuberculosis: interferon gamma release assay. Updated May 9, 2024. Accessed September 27, 2026. https://www.cdc.gov/tb/hcp/testing-diagnosis/interferon-gamma-release-assay.html
  4. Qiagen. QuantiFERON-TB Gold Plus (QFT-Plus) Instructions for Use. HB-3303-011 R11. August 2025. Accessed September 27, 2026. https://www.qiagen.com/en-US/resources/download/kitHandbookAndProtocol/hb-3303-011-r11-qft-plus-elisa-0825-us

Watch one

A 58-year-old man who takes high-dose corticosteroids has an IGRA before a new immunosuppressive drug is started.

What is the qualitative result?

TestResultPreviousReference intervalFlag
Nil0.20 IU/mL
TB10.28 IU/mL
TB20.31 IU/mL
Mitogen0.40 IU/mL

Specimen: Not measured on whole blood. Whole blood, IGRA tubes incubated within the manufacturer's time limit

  1. Nil is 0.20 IU/mL, below the 8.0 IU/mL limit.

    A high Nil makes every other tube unreadable, so Nil is checked first.

  2. TB1 − Nil = 0.28 − 0.20 = 0.08 IU/mL. TB2 − Nil = 0.31 − 0.20 = 0.11 IU/mL. Neither reaches 0.35 IU/mL.

    A positive antigen response settles the result whatever the mitogen shows.

  3. Mitogen − Nil = 0.40 − 0.20 = 0.20 IU/mL, below 0.50 IU/mL.

    With no positive antigen response, the mitogen shows whether his T cells could respond at all.

  4. No antigen response is positive and the mitogen failed, so the result is indeterminate.

    A failed positive control means the negative antigen tubes prove nothing.

  5. Report indeterminate with the four values. A repeat IGRA or a skin test can resolve it.

    The reader needs to know the test gave no answer.

Indeterminate. Mitogen − Nil is 0.20 IU/mL, below 0.50 IU/mL, and neither TB response is positive. The corticosteroids may explain the weak mitogen response.

Your turn

Problem 1 of 3

A QuantiFERON-TB Gold Plus panel gives Nil 1.00, TB1 1.50, TB2 1.10, and Mitogen 1.20 IU/mL. Applying the QFT-Plus interpretation criteria, what is the qualitative result?

Correct. TB1 minus Nil is 0.50 IU/mL, which is at least 0.35 IU/mL and at least 25% of Nil (0.25 IU/mL), and Nil is 8.0 IU/mL or less. Mitogen enters the decision only when no antigen response is positive.

Incorrect. A positive antigen response with an acceptable Nil makes the panel positive. The mitogen response matters when neither antigen tube meets the criterion.

Incorrect. The low-mitogen indeterminate rule applies when no antigen response meets the positive criterion, and here TB1 does.

Hint
  1. Subtract Nil from each TB tube before anything else.
  2. A TB response has to meet two limits in the same tube. Check both for TB1.

Review IGRA results

Problem 2 of 3

A patient has been ill for 2 weeks. The first-tier EIA is equivocal. The IgM immunoblot shows the 23 and 41 kDa bands, and the IgG immunoblot shows 2 of 10 bands. How does the result read?

A positive or equivocal first tier reflexes to the second tier.

Within 30 days the IgM blot is read as well. It meets its own criterion.

Within 30 days an IgM blot with 2 of its 3 bands is positive, and the equivocal first tier counts toward a positive two-tier result.

The band criteria already allow for cross-reaction. The 41 kDa band counts toward the IgM criterion.

Hint
  1. Check whether an equivocal first tier goes on to the blots.
  2. At 2 weeks of illness, count which blots are read and what each needs.

Review Standard two-tier testing

Problem 3 of 3

An IGRA reads Nil 9.10, TB1 9.40, TB2 9.30 and Mitogen above 10.0 IU/mL. What is the qualitative result?

The Nil is above 8.0 IU/mL, so the TB tubes cannot be read. A negative report would suggest infection is unlikely when the test gave no result.

Called an indeterminate IGRA negative

An indeterminate interferon-gamma release assay (IGRA), usually from a failed mitogen control or a Nil above 8.0 IU/mL in the ELISA format, means the test did not work. Reporting it as negative suggests infection is unlikely when the test gave no result. A repeat IGRA or a tuberculin skin test can resolve it.

The TB values are read only after Nil is subtracted, and only when Nil is 8.0 IU/mL or less.

A Nil this high means background interferon-gamma swamps the antigen tubes. The test gave no answer, and a repeat IGRA or a skin test can resolve it.

Review IGRA results

Use it

  • Grace Adeyemi, 44, has had a swollen right knee for 10 weeks. She hikes in a Lyme-endemic area.
  • Her provider orders Lyme serology and, before a biologic drug for arthritis, an IGRA.
  • Lyme standard two-tier testing: first-tier EIA positive. IgM immunoblot shows the 23, 39 and 41 kDa bands. IgG immunoblot shows 7 of 10 bands.
TestResultPreviousReference intervalFlag
Nil0.60 IU/mL
TB10.80 IU/mL
TB21.10 IU/mL
Mitogen6.50 IU/mL

Specimen: Not measured on whole blood. Whole blood, IGRA tubes incubated within the manufacturer's time limit

Decision 1 of 2

Which blot supports the positive Lyme result?

After 30 days only the IgG blot is read. With 7 bands it meets the 5-of-10 criterion.

At 10 weeks the IgM blot is disregarded. The IgG blot carries the result.

Used a late isolated IgM blot to confirm Lyme disease

After 30 days of illness only the IgG blot counts, because false-positive IgM blots and prolonged IgM seropositivity are common. Reporting a positive IgM blot alone at that stage confirms an infection the algorithm does not support.

The IgM blot is not read after 30 days of illness, so it adds nothing here.

Review Standard two-tier testing

Decision 2 of 2

TB1 − Nil is 0.20 IU/mL, TB2 − Nil is 0.50 IU/mL and Mitogen − Nil is 5.90 IU/mL. How is the IGRA reported?

Either TB tube can make the result positive. TB2 − Nil meets both limits.

An IGRA cannot separate latent infection from active disease. Active disease needs its own clinical and microbiologic evaluation.

Indeterminate comes from a high Nil or a failed mitogen. Her Nil and mitogen are both acceptable.

TB2 − Nil is 0.50 IU/mL, at least 0.35 IU/mL and at least 25% of Nil (0.15 IU/mL). The IGRA shows infection, and evaluation for active disease follows.

Review IGRA results

The clue that settled the Lyme result is 10 weeks of illness, which leaves the IgG blot as the only one read. For the IGRA, a single TB tube meeting both limits made the result positive.

Keep

Sources checked