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Lyme and tuberculosis testing

15 min

Skills you'll build2 skills
  • 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

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 is the result interpreted?

Keep going to see the reasoning.

Right. Keep going to follow the reasoning.

Keep going to see the reasoning.

Keep going to see the reasoning.

Get the idea

Use the explanation to check your first decision.

Two-tier Lyme serology

Borrelia burgdorferi is hard to find in blood, so Lyme testing measures 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 testing. A positive or equivocal first tier reflexes to immunoblots, interpreted 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 are interpreted as 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

Interferon-gamma release assays

An interferon-gamma release assay (IGRA) measures the interferon-gamma that T cells release when blood is incubated with 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 are below the positive cutoff 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 could not be interpreted, often because of a failed mitogen response or a high Nil. It gives no information about infection. A repeat IGRA or a tuberculin skin test can resolve it. The laboratory reports the qualitative result together with the numbers.3

Follow the bench demonstration

Sources for this step
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

Read each decision and its explanation.

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?

TestResultReference 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 uninterpretable, 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 makes the result positive regardless of the mitogen value.

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

    With no positive antigen response, the mitogen tube shows whether his T cells can respond.

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

    When the positive control fails, the negative antigen tubes cannot be interpreted.

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

    The provider needs to know the test gave no interpretable result.

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.

Practice this skill

Your turn

Problem 1 of 3 · Hints available

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. The mitogen result is used 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 · Hints available

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 is the result interpreted?

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 · On your own

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 cannot be interpreted. Reporting it as negative suggests infection is unlikely when the test gave no valid 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 masks any antigen response. The test gave no interpretable result, and a repeat IGRA or a skin test can resolve it.

Review IGRA results

Use it

Read the case evidence, then make each decision. The step is complete without it.

  • Grace Staines, 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.
TestResultReference 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 result is based on the IgG blot.

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 is not used 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

After 10 weeks of illness, only the IgG blot is read for the Lyme result. For the IGRA, one TB tube that meets both limits makes the result positive.

For reference

Sources checked