iSpot Lyme: A New Approach to Lyme Disease Testing

It looks like spring has finally arrived! Along with warmer weather, this means the return of ticks to certain regions and new cases of Lyme disease. The Centers for Disease control (CDC) reported approximately 32,500 new cases of Lyme disease in 20111. However, it is estimated that the actual number could be up to 10-fold higher2 due to the potential for misdiagnosis and underreporting.

Lyme disease is caused by the bacterium Borrelia burgdorferi. Lyme disease is a vector-borne disease transmitted by the Ixodes (blacklegged) tick, more commonly known as the deer tick (Figure 1).

Figure 1. Ixodes tick

Figure 1. Ixodes tick

Symptoms may include:

  • Observed tick bite
  • Bullseye rash
  • Flu-like symptoms
  • Lymphadenopathy
  • Joint pain
  • Neurological symptoms
  • Heart palpitations
  • Severe fatigue

How is Lyme Disease Diagnosed?

The current CDC guidelines for diagnosing Lyme disease involve a two-tiered approach that consists of antibody-based Enzyme Linked Immunosorbent Assay (ELISA) and Western Blot (WB) testing. These tests are serological assays that detect antibodies to B. burgdorferi. The low sensitivity of the two-tier tests (about 30% in early Lyme disease and 50% in late Lyme disease) and the significant seronegativity of Lyme patients (as many as 30% to 50% of cases) suggests that more sensitive cellular immune-based laboratory tests should also be developed3. A comprehensive evaluation for Lyme requires testing for both a humoral (WB) and an immune cell-mediated (ELISPOT) response to B. burgdorferi.

What is iSpot LymeTM?

Enzyme-linked immunosorbent spot (ELISPOT) is an effective method for assessment of T cell immunity by measuring stimulated antigen-specific T cells4,5. The ELISPOT method utilized in iSpot LymeTM is a highly sensitive technique for detecting immune cells that secrete signature proteins (such as a given cytokine). It is the most sensitive technology that accurately detects, measures, and performs functional analysis of these immune cells. iSpot Lyme has a sensitivity of 84% and specificity of 94% for the detection of B. burgdorferi; making it an excellent complement to the current two-tiered antibody methods.

How does this Test Detect Lyme Disease?

The iSpot Lyme detects a cellular immune response against Lyme antigens, which appears earlier in the disease process (2 weeks) than the antibody response detected by the traditional WB test (4-6 weeks)6. More importantly, iSpot Lyme can even detect antigen-specific T cell responses in seronegative patients7.

iSpot Lyme test results are produced by measuring interferon-gamma (IFN- γ) secreted by T cells in response to stimulation by the B. burgdorferi antigens DbpA, OspC, p100, and VisE-1 (Figure 2). This test measures frequency of antigen-specific T cells by measuring those that produce INF- γ in response to Lyme antigens.

Figure 2: When peripheral blood mononuclear cells (PBMCs) from a B. burgdorferi-infected patient are exposed to B. burgdorferi protein antigens (A), B. burgdorferi-specific T cells are activated and secrete small proteins called cytokines (B). T cells that are not specific for B. burgdorferi do not become activated.  iSpot Lyme™ measures the cytokine IFN- secreted by the patient’s T cells. Cytokine proteins (IFN-gamma) are captured near the cells that secreted them and are then detected using a color reagent (C).

Figure 2: When peripheral blood mononuclear cells (PBMCs) from a B. burgdorferi-infected patient are exposed to B. burgdorferi protein antigens (A), B. burgdorferi-specific T cells are activated and secrete small proteins called cytokines (B). T cells that are not specific for B. burgdorferi do not become activated. iSpot Lyme™ measures the cytokine IFN-gamma secreted by the patient’s T cells. Cytokine proteins (IFN-gamma) are captured near the cells that secreted them and are then detected using a color reagent (C).

The iSpot Lyme test counts B. burgdorferi-sensitized T cells by capturing IFN-γ secreted by these T cells. More specifically, when IFN- γ is released, a “spot” of insoluble precipitate is formed at the site of the reaction. Evaluating the number of spot forming units (SFUs) provides a measurement of B.burgdorferi-sensitive effector/memory T cells in the peripheral blood. The SFU count correlates to a patient’s T cell reaction to B. burgdorferi.

In conclusion, the diagnosis and treatment of Lyme disease is controversial even as Lyme disease rates continue to increase. A more comprehensive approach to diagnosis can lead to faster treatment and better clinical outcomes. The very high sensitivity and specificity of the new cellular-based iSpot Lyme test combined with the traditional antibody-based western blot analysis improves Lyme disease diagnosis. This can benefit many people who are currently suffering from misdiagnosed or undiagnosed Lyme disease.

References
  1. http://www.cdc.gov/lyme/stats/chartstables/casesbyyear.html
  2. Yound JD. Underreporting of Lyme disease. N Engl J Med. 1998. 338(22):1629-1629.
  3. Lehmann PV, Zhang W. Unique Strengths of ELISPOT for T Cell Diagnostics. In: Alexander Kalyuzhny Ed. Handbook of ELISPOT: Methods and Protocols, Methods in Molecular Biology, vol. 792. 2nd Ed. New York, NY: Spriner Science+Business Media, LLC; 2012: 3-23.
  4. T-Spot.TB 96 [Package Insert]. Oxfordshire, UK: Oxford Immunotec Limited; 2009.
  5. Tary-Lehmann M, Hamm CD, Lehmann PV. Validating reference samples for comparison in a regulated ELISPOT assay. In: Uma Orabhakar and Marian Kelley Eds. Validation of Cell-Based Assays in the GLP Setting: A Practical Guide. 1st Ed. West Sussex, England: John Wiley & Sons. Ltd; 2008: 127-146.
  6. Ekerfelt C, Forsberg P, Svenvik M et al. Asymptomatic Borrelia-seropositive individuals display the same incidence of Borrelia-specific interferon-gamma (IFN-g)-secreting cells in blood as patients with clinical Borrelia infection. Clin Exp Immunol. 1999. 115: 498-502.
  7. Dattwyler RJ, Volkman DJ, Luft BJ, et al. Seronegatice Lyme Disease- Dissociation of Specific T- and B-Lymphocyte Responses to Borrelia burgdorferi. New England Journal of Med. 1988. 319(22): 1441-1446.

For more detailed information please see our White Paper: iSpot Lyme: A New Generation of Lyme Disease Testing

Guest authors: Chenggang Jin, MD, PhD, holds a PhD in immunology and has 10 years of experience in medical research at Princeton University, the University of Iowa Hospitals and Clinics, and Beth Israel Deaconess Medical Center/Harvard Medical School.  Dr. Jin also holds certification from the Educational Commission for Foreign Medical Graduates in the field of medicine.  His specialized training includes many years of technical and research experience in cellular immunology and molecular biology.  He is currently the director of laboratory immunology at Pharmasan Labs and led the creation of a new clinical laboratory assay, iSpot-Lyme, for Lyme disease.

Deanna Fall is currently a scientific writer and research analyst for NeuroScience, Inc. and holds a bachelor’s degree in biology from Ferris State University in Big Rapids, Michigan.  Deanna joined NeuroScience in 2009 with experience in pharmaceutical research and development in the field of neurotoxicology.  She is currently pursuing a master’s degree in health informatics at the University of Minnesota.

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