Lyme disease is the fastest growing and most common tick-borne infectious disease in the United States and Europe. Borrelia burgdorferi sensu stricto is the pathogen that causes Lyme disease in the United States. Borrelia afzelii and Borrelia garinii are other closely related species that causes Lyme disease in Europe and Asia respectively. In the United States, Lyme disease hotspots are found on the East Coast and in mid-Atlantic states as well as in Minnesota and Wisconsin, with fewer cases reported on the Pacific Coast, Northern California and Oregon. Ticks are spreading to other states as well and bringing Borrelia along with them.

Lyme Disease Testing Available at Infectolab Americas

LYMESPOT

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Infectolab’s Lyme ELISpot/LymeSpot uses unique, cutting-edge ELISPOT assay technology to provide information about T-cell activity or the cellular immune response elicited against the Borrelia antigen. It has been reported that both the humoral and the cellular response develop in a Borrelial infection. The cellular response is usually elicited soon after exposure to the antigen, while the humoral response takes up to 2-4 weeks. In some cases, the humoral response either doesn’t develop or develops at a much later stage of the disease due to potential immune-evasion or immunesuppression by Borrelia; in other words, due to a curtailment of seroconversion. These are some of the challenges of traditional serology testing, which is why monitoring the cellular response is a good way to catch the Lyme disease early on and begin the treatment process. 

Fundamentals of LymeSpot Technology 

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The Lyme ELISpot assay works by isolating your immune cells and stimulating them with specific antigens purified from the US Borrelia strain; it uses a peptide library to sequence from multiple Borrelia strains in order to check for infections contracted inside and outside of the US. Analyzing with multiple sets of antigens autonomously will produce a more accurate test result. The test is designed to measure the number of antigen specific T-lymphocytes that release a cytokine IFN-γ (effector fighting T-cells) and IL-2 (central memory T-cells) based on the function that these cells have during an infection. Every individual spot count represents an activated T-cell. Two different colored fluorescent dyes are used to count the spots separately. 

The LymeSpot Advantage

The LymeSpot can be used for the detection of T-cell activity soon after exposure to Borrelia. It measures the activity of two different T-cell subsets: the effector fighting T-cell (releases IFN-γ) and the central memory T-cell (releases IL-2). A high number of IFN-γ spots indicates the presence of active fighting antigen specific effector T-cells, which shows an active Lyme infection. Once treatment begins, the level of IFN-γ producing T-cells should come down and the IL-2 producing central memory T-cell count should increase, marking the success of an effective treatment. The detection of the IL-2 producing T-cell is of great benefit when determining the success of the treatment and is something that no other marketable test provides. If both IFN-γ and IL-2 spots are found then this signifies that the infection is chronic controlled. 

The presence of both cellular and humoral responses to a Borrelia infection will give a patient’s physician greater confidence. 

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Reference Range​

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LymeSpot

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B. burgdorferi B31 antigen IFN-G

Negative:  ≤ 1.99 SI

Borderline/Equivocal: 2.00-3.00 SI

Positive: ≥ 4.00 SI

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B. burgdorferi B31 antigen IL-2

Negative:  ≤ 1.99 SI

Borderline/Equivocal: 2.00-3.00 SI

Positive: ≥ 4.00 SI

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B. burgdorferi OSP peptide IFN-G

Negative:  ≤ 1.99 SI

Borderline/Equivocal: 2.00-3.00 SI

Positive: ≥ 4.00 SI

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B. burgdorferi OSP peptide IL-2 

Negative:  ≤ 1.99 SI

Borderline/Equivocal: 2.00-3.00 SI

Positive: ≥ 4.00 SI

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Lyme ELISA IgG/IgM​

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Serology testing for Lyme disease identifies the presence of antibodies against Borrelia (IgG and IgM). IgM antibodies are the first antibody to appear during an early infection. It takes up to 2-4 weeks for IgM antibodies to be found in measurable amounts in the bloodstream, while IgG antibodies can be measured after 4-6 weeks. The presence of these antibodies against Borrelia indicates exposure to the disease but not the presence of the disease. A positive ELISA IgG/IgM will result in an automatic Western blot IgM/IgG in order to confirm the positive results. ​

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Fundamentals of ELISA​

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Enzyme-linked immunosorbent assay (ELISA) is based on principles of specific antibody and antigen interactions. Antibodies bind the target antigen, which are usually peptides or proteins. A detection is used to quantify the antibody present in a patient’s blood sample.

In more detail, an antigen is immobilized to a plate and then complexed with an antibody that is linked to an enzyme to facilitate detection of a substrate. The quantification of the bound product is done by optical densitometry of the substrate. ​

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Reference Range: ​

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Borrelia Burgdorferi ELISA

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B. burgdorferi IgM

Negative: ≤ 0.80 SI

Borderline/Equivocal: 0.81-1.09 SI

Positive: ≥ 1.10 SI

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B. burgdorferi IgG

Negative: ≤ 0.80 SI

Borderline/Equivocal: 0.81-1.09 SI

Positive: ≥ 1.10 SI

Lyme Western blot IgG/IgM

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The Western blot is a sensitive test that is usually used to confirm the results of an ELISA(IgM/IgG). The Lyme ELISA, used in conjugation with the Lyme Western blot, is a CDC-recommended test for Borrelial infection. Exposure to Lyme disease causes an immune response that results in the production of IgM and IgG antibodies. The IgM antibody response builds up quickly when compared to that of IgG. The antibodies usually stay in the blood much longer, even after treatment has stopped and the symptoms have disappeared. The Western blot is a qualitative assay that helps visualize the presence of antibodies.

Fundamentals of the Western Blot

Borrelia antigens are bound to a membrane and are usually recognized by antibodies present in a patient’s blood sample. The number of detected antigens will lead to a conclusion as to whether or not a patient has been exposed to the disease.

Generally, a mix of proteins will be separated by size and transferred on a membrane. This process is called blotting and lends its name to this testing method. And like in most immunological assays, an enzymatic reaction is used to visualize bound antibodies. The separation of proteins makes it possible to distinguish specific antigens that are recognized by the immune system. In most cases, the longer the progression of the disease, the more antigens there are that will be recognized by a patient’s immune system.

Reference Range:

Borrelia burgdorferi Western Blot

Blot IgG

Blot IgG p83/93

Blot IgG p66

Blot IgG p60

Blot IgG p58

Blot IgG p45

Blot IgG p41

Blot IgG p39

Blot IgG p34, OspB

Blot IgG p31, OspA 

Blot IgG p30

Blot IgG p28

Blot IgG p25, OspC

CDC indicates Positive result when 5 out of 10 bands react positively. This excludes Blot IgG p34, OspB and Blot IgG p31, OspA.

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Blot IgM

Blot IgM p41

Blot IgM p39

Blot IgM p25

CDC indicates Positive result when 2 of 3 bands react positively.

NK Cells (CD57+)

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CD57+ immune cells are measured with an established method of flow cytometry for cell characterization. They are a type of lymphocyte called Natural Killer (NK) cells. These NK cells contain a CD57 cell-surface marker correlated with clinical prognosis in chronic infections (Kared 2016). CD57+ NK cells measure at a lower frequency in patients suffering from chronic disease.

Normal CD57 results do not necessarily rule out Lyme disease, nor do abnormal results necessarily offer confirmation. When used in conjunction with other testing methods and symptomatology, CD57 results provide information that Health Care Practitioners can utilise in determining the stage of the illness or deciding on treatment for Lyme disease or other infections.

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