iSpot Lyme Test - A Clinician's Review

iSpot Lyme is a type of ELISPOT assay used for borrelia diagnosis, which belongs to the group of interferon-gamma release assays. The test is manufactured by Pharmasan Labs. Similar testing are available in Germany (BCA-labs) and Australia (InfectoLab).

The EliSpot (Enzyme-linked Immunosorbent Spot Assay) measures the number of activated T-lymphocytes in cell cultures that release cytokines after being challenged by the relevant antigen. The proteins used are DopA, OspC, p100, VIsE1, which represent different life cycles of Borrelia burgdorferi

Image 1: T-Cells activate other white blood cells to attack antigens.

iSpot Lyme counts the number of anti-Borrelia effector T cells, each one being reported as a Spot Forming Unit (SFU). T cells are white blood cells that produce interferon-gamma, in a sample of blood. This gives an overall measurement of the host immune response against Borrelia, which can reveal the presence of infection with Borrelia species, the causative agent of Lyme disease. Because this does not rely on production of a reliable antibody response or recoverable pathogen, the technique can be used to detect low level infections, persistent Lyme disease or help determine Post-Treatment Lyme Disease Syndrome.

iSpot Lyme interpretation: Pharmasan Labs has validated testing to be positive for Lyme disease with SFUs >25.; with equivocal range between 17-24 SFUs. I use these provided ranges as guidelines due to observations made in my clinical usage and publications on other ELISPOT tests (QuantiFERON-TB Gold test (QFT-G)).

Possible limitations:

  • Fluctuation in IFN-γ responses (SFUs) with serial testing in individual patients. This may be due to normal changes in an individuals immune status, activity of the infective agent or due to changes in life stage of Borrelia infection.

  • Some decreases in IFN-γ response (SFUs) in individual persons might be attributed to antimicrobial treatments.

  • The magnitude of these fluctuations can be of sufficient size to cause test interpretations to change from negative to positive (conversion) or from positive to negative (reversion), especially when the IFN-γ responses are near cut points separating positive and negative results.

Practical suggestions:

  • Blood draws must be done Mon-Wed due to lab requirements.

  • Do not use smaller than 21 gauge needle for venipuncture due to increased chance of cell lysing.

  • Check for interruptions of lab service around holidays; check lab calendar.


  • iSpot Lyme SFUs can remain elevated in patients after a successful treatment for up to 12 months. SFUs gradually decrease; which may result in Post-Treatment Lyme Disease Syndrome.

  • Recent exposure will lead to increase in SFUs within 4-6 days.

  • Acute (active) exposure results in higher SFU counts (>50).

  • Persistent (chronic) exposure results in lower SFU counts (<50).

  • Hidden forms of Borrelia (e.g. cysts) do not appear to evoke SFU elevations.

  • Presence of a co-infection have been seen to suppress SFU levels and may result in false negatives.

  • SFUs will increase with successful antibiotic treatment. Frequently see increases 100 or more SFUs shortly (2-3 months) after initiation of an effective round of antibiotic treatment. Levels typically drop after 6 months but this may be longer is cases or poor cellular detoxification.

  • Reinfections often result in higher SFU counts (>50).

  • SFUs >14 strongly suggests past exposure or very weak immune response to active infection.

  • Borrelia species other than burgdorferi will elicit iSpot Lyme positives. I had a patient bitten in Europe in the 1970's test positive.

  • Low SFU levels on a patient clinically determined to have a tick-borne disease should be retested after treating for co-infection(s).

  • SFUs that remain elevated in Post-Treatment Lyme Disease Patients with resolving symptoms, should have their cellular detoxification pathways supported, treated with anti-inflammatory strategies and consider a biofilm reduction strategy.

  • Long-term SFU elevation often occurs in chronic inflammatory states resulting in decreased synthesis of monoamine neurotransmitters, sub-clinical hypothyroidism and alterations in HPA axis.

Map of Lyme borreliosis world-wide

Image 2: World map showing areas affected by Lyme boreliosis.

General Recommendations:

  • iSpot Lyme should be used as an aid in diagnosing Borreliosis. This tests may be used to identify persons likely to benefit from treatment or for surveillance or monitoring purposes.

  • Both the standard qualitative test interpretation and the quantitative assay measurements should be reviewed together for best clinical interpretation. This will permit more refined assessment of results and promote understanding of patient’s infective status.

  • iSpot Lyme should be run in conjunction with Western Blot testing for ideal base-line.

  • Patient exposure history must be taken into account when interpreting results.

  • Excellent test for diagnosis and monitoring Post-Treatment Lyme Disease Syndrome due to quantifiable assessment of circulating Borrelia proteins.

  • Best if avoid immunosuppressant medications for 2 weeks prior to testing.

  • Explain to patients that iSpot Lyme measures Borrelia protein activity. These proteins may be part of a bacteria that is alive or dead (debris material), the test does not differentiate.

  • A single test result is limiting and additional testing data points will improve clinical interpretation and utility of the iSpot Lyme test.

  • Inform patient that this is not a CDC approved test for the diagnosis of Lyme disease; current guideline is the 2-tiered algorithm with an enzyme immunoassay (EIA), followed by IgM/IgG Western immunoblots. If the iSpot Lyme is positive I note in the patient’s chart that testing shows elevated Borrelia protein activity suggesting recent infection with remaining and problematic debris material or an active case of Borreliosis.


  • Mazurek GH, Jereb J, Vernon A, LoBue P, Goldberg S, Castro K; IGRA Expert Committee; Centers for Disease Control and Prevention (CDC). Updated guidelines for using Interferon Gamma Release Assays to detect Mycobacterium tuberculosis infection - United States, 2010. MMWR Recomm Rep. 2010 Jun 25;59(RR-5):1-25.

  • Jin C, Roen DR, Lehmann PV, Kellermann GH. An Enhanced ELISPOT Assay for Sensitive Detection of Antigen-Specific T Cell Responses to Borrelia burgdorferi. Cells. 2013 Sep 13;2(3):607-20.

  • Nordberg M., Forsberg P., Nyman D., Skogman B.H., Nyberg C., Ernerudh J., Eliasson I., Ekerfelt C. Can ELISPOT be applied to a clinical setting as a diagnostic utility for Neuroborreliosis? Cells.2012;1:153–167.

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