Diagnostic Tests for Lyme Disease

Lyme disease (Lyme borreliosis) is the most common vector-borne disease in the United States according to the CDC.

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ABOUT GENUS BORRELIA - LYME DISEASE

Borreliosis is a worldwide infectious disease caused by spiral-shaped bacteria known as Borreliae, carried by ticks and louse. Although about 20 classifications of Borrelia exist, the species of Borreliae known to cause disease in humans are split into the following two groups:

  1. B. burgdorferi sensu lato, which causes Lyme disease
  2. Relapsing Fever Borrelia, which causes Tick-Borne Relapsing Fever (TBRF)


It’s important to note that each of these broad groups includes several Borrelia sub-species and strains, and researchers continue to discover more. It is not uncommon for patients to receive false negatives simply because the test they took was not designed to detect the species of Borrelia causing their infection.

THE IMPORTANCE OF DIAGNOSTIC TESTS FOR BORRELIA

Called the “great imitator,” Lyme disease can present a variety of symptoms that mimic a wide range of illnesses, including chronic fatigue syndrome, fibromyalgia, ALS, Alzheimer’s disease, depression, insomnia, and autoimmune disorders such as RA and Multiple Sclerosis (MS). In addition, many tick-borne infections go misdiagnosed for months because these nonspecific symptoms mirror other illnesses. Thus, laboratory testing is frequently required to confirm a diagnosis.

CDC-RECOMMENDED TWO-TIERED BORRELIA TESTING

Lyme disease is typically diagnosed by a two-tiered testing (TTT) approach involving an enzyme-linked immunosorbent assay (ELISA) followed by a Western blot test. However, the sensitivity of these commercially available tests is poor, meaning they can miss active infections. Experts advise against this testing technique due to the ambiguity of its results.

IGeneX has developed several Borrelia burgdorferi tests that provide higher sensitivity to detect and speciate the B. burgdorferi group. When used in conjunction with clinical symptoms and patient history, the tests listed below can better assist physicians in accurately diagnosing patients. Keep reading to learn more about Lyme borreliosis, including symptoms, prevalence, and, if necessary, which IGeneX test to order.

WHAT TESTS ARE AVAILABLE FOR LYME DISEASE?

IGeneX offers ImmunoBlots, Serologies, Western Blots, PCRs (Polymerase Chain Reaction), Culture Enhanced PCR (cePCR), IgXSpot, Broad Coverage Assays, and LSA tests to confirm the diagnosis of Lyme Disease.

Lyme IgG/IgM Antibody Serology

The IgG/IgM Antibody Serology test is an ELISA (enzyme linked immunoassay), which indicates the presence of both IgG and IgM antibodies to B. burgdorferi. IgM antibodies are present shortly after infection takes place. IgG antibodies often persist long after symptoms have disappeared. The presence of either IgG/IgM antibodies indicates exposure to Lyme-causing Borrelia, not the active disease. A positive or equivocal test must be confirmed by both IgG and IgM ImmunoBlots Blots.

Reference Range
Borrelia burgdoferi Antibody Serology IgG/IgM

Negative <1.0

Equivocal 1.0 to <1.2

Positive 1.2

Clinical Significance
This test is recommended at least four weeks after exposure. Patients diagnosed with Lyme disease based on clinical history have positive IgG/IgM serology results within one year of the tick bite, approximately 70% of the time. The percentage of patients with a positive serology is reduced in subsequent years.

All samples with positive or equivocal results should be tested with B. burgdorferi Western Blots.

Limitations

  1. This test should only be performed in conjunction with Western Blots/ ImmunoBlots.
  2. Cross Reacting Antibodies:
    • Sera from patients with other pathogenic spirochetal diseases such as syphilis, yaws, pinta, leptospirosis, and relapsing fever may give false-positive results.
    • Sera from patients with mononucleosis or lupus erythomatosis (LE) may also give false-positive results.
    • In cases where false-positive results occur, clinical epidemiological and laboratory workups should be carried out. Active syphilis and Lyme disease can be differentiated by the use of VDRL or RPR tests. In active syphilis, the VDRL and RPR are positive, whereas in Lyme disease they are not.
  3. Antibiotic therapy given early in the disease may prevent the development of an antibody response. Negative results early in the disease have a low predictive value. Retesting may be warranted if symptoms consistent with Lyme disease persist.
  4. The evaluation must include a review of all test results, the clinical history presented by the patient, the patient’s exposure to endemic regions for Lyme disease, epidemiological data, and potential exposure to other spirochetal diseases.
  5. The use of this assay has not been evaluated for individuals who have received a Lyme disease vaccine.


Stage of Disease

Screen test

Results Interpretation >
View Lyme disease serology tests >

Lyme IgM Antibody Serology

The IgG/IgM Antibody Serology test is an ELISA (enzyme linked immunoassay) format, and it detects the presence of IgM antibodies to B. burgdorferi after exposure to an infected tick. Because IgM antibodies appear early in response to infection, this test may be positive two to six weeks after exposure. But since the level of IgM rapidly declines over time, testing for IgM antibodies too late can cause a missed infection.

A positive or equivocal IgM antibody test must be confirmed by an IgM Western Blot or Lyme ImmunoBlot IgM. NOTE: The sensitivity concerns mentioned for the IgG/IgM assay also affect this assay.

Reference Range
Borrelia burgdorferi Antibody Serology IgM

Negative <0.8

Equivocal 0.8 to <1.2

Positive 1.2

Clinical Significance
The Lyme IgM antibody ELISA is a serological test for the detection of IgM antibodies to B. burgdorferi after possible exposure to an infected tick. IgM antibodies appear early in response to infection; therefore, this test may be positive between 2 to 6 weeks after exposure. Though the level of IgM declines over time, the IgM response may persist in patients with prolonged illness, and a new IgM response may appear later in persistent or recurrent disease or from re-infection. Therefore, this test is recommended approximately 2 weeks after suspected exposure to Lyme.

All samples with positive or equivocal results should be tested with B. burgdorferi Western Blots or Lyme ImmunoBlots.

Limitations

  1. This test should only be performed in conjunction with Western Blots/ ImmunoBlots.
  2. Cross Reacting Antibodies:
    • Sera from patients with other pathogenic spirochetal diseases such as syphilis, yaws, pinta, leptospirosis, and relapsing fever may give false positive results.
    • Sera from patients with mononucleosis or lupus erythomatosis (LE) may also give false positive results.
    • In cases where false positive results occur, clinical epidemiological and laboratory workups should be carried out. Active syphilis and Lyme disease can be differentiated by the use of VDRL or RPR tests. In active syphilis, the VDRL and RPR are positive, and in Lyme disease they are not.
  3. Antibiotic therapy given early in the disease may prevent the development of an antibody response. Negative results early in the disease have a low predictive value. Retesting may be warranted if symptoms consistent with Lyme disease persist.
  4. The evaluation must include all test results, the clinical history presented by the patient, the patient’s exposure to endemic regions for Lyme disease, epidemiological data, and any potential exposure to other spirochetal diseases.
  5. Positive or equivocal first-tier test results should not be reported until second-tier testing of the specimen is performed using a method that is more specific, such as Western Blot/ ImmunoBlot.
  6. The use of this assay has not been evaluated for individuals who have received a Lyme disease vaccine.


Stage of Disease

Screen test

Results Interpretation >
View Lyme disease serology tests >

Lyme Screen Immunoassay (LSA) - NEW!

Note: The LSA replaces the Lyme IFA and should be used as a screening test in conjunction with the Lyme ImmunoBlots. The sensitivity and specificity of the LSA is superior to the IFA, but is not meant to be a standalone test. The LSA has also replaced the IFA in test panels.

The Lyme Screen Immunoassay is a qualitative test designed to detect IgM or IgG antibodies to Lyme Borreliae group-specific antigens in human serum. The combined sensitivity of the Lyme Screen Immunoassay is 85%, and the combined specificity is 94%.

Principle
The test detects IgM or IgG antibodies to the Lyme Borreliae group, and should be used in conjunction with ImmunoBlots, as well as patient clinical symptoms and history.

Results
Positive: Lyme Borreliae antigens detected
Negative: Lyme Borreliae antigens not detected

Clinical Significance
A positive test suggests exposure to the Lyme Borreliae group and should be used in conjunction with ImmunoBlots, as well as the patient’s clinical symptoms and history.

Limitations

  1. This test should be used only for screening purposes.
  2. A negative LSA does not exclude the possibility of infection with B. burgdorferi.
  3. IGeneX interpretation is based on internal validation studies.
  4. The results of this test must be interpreted in relation to patient’s clinical history, epidemiological data, stage of disease, clinical symptoms or other laboratory results.


Special Instructions

This test is not yet available for NY residents.

Stage of Disease
Screen test

View LSA tests >

Lyme Broad Coverage Ab Assay

Principle
The Lyme Broad Coverage Antibody (BCA) Assay is a qualitative test designed to detect IgM/IgG antibodies to Lyme Borreliae group-specific antigens in human serum.

Clinical Significance
A positive test suggests exposure to the Lyme Borreliae group and should be used in conjunction with the patient’s clinical symptoms and history. The BCA Assay is a simple and cost-effective test that gives straightforward positive or negative results.

Performance Characteristics
The sensitivity of the Lyme BCA Assay is 90%, and the specificity is 97%.

Limitations

  1. For specific protein or band information, an ImmunoBlot IgM or IgG test should be ordered to provide more information and possible speciation of the Borrelia.
  2. A negative Lyme ImmunoBlot IgG does not exclude the possibility of infection with B. burgdorferi.
  3. IGeneX interpretation is based on internal validation studies.
  4. The results of this test must be interpreted in relation to patient’s clinical history, epidemiological data, stage of disease, clinical symptoms or other laboratory results.


Stage of Disease

Early disease / Re-activation later

Results Interpretation >
View BCA tests >

Lyme ImmunoBlot

The Lyme ImmunoBlot is a qualitative immunoassay in which antibodies specific to the B. burgdorferi antigens on a membrane strip are visualized. It is a qualitative test and is more sensitive and specific than the ELISA, IFA, and traditional Western Blot tests for lyme. When antibody levels are very low in the early or late stage of the disease, ImmunoBlot can be positive, whereas ELISA and IFA tests can be negative.

This test must be used if the Lyme IgG/IgM antibody or Lyme IgG/IgA/IgM IFA is positive or equivocal.

Principle

The Lyme ImmunoBlot assay is based upon an antigen-antibody complex formation in the following steps:

  1. Binding of anti-Borrelia-specific antibodies in human serum to the ImmunoBlot strip. The ImmunoBlot strip is a membrane strip with fixed B. burgdorferi recombinant antigens on it.
  2. Binding of enzyme labeled anti-human IgG or IgM antibodies to the human anti Borrelia antibodies bound to fixed B. burgdorferi antigens on the membrane.
  3. Reaction with BCIP/NBT, a chromogenic substrate with bound antibodies on the strip. A dark purple colored precipitate (band) develops on the antigen-antibody complexes.


Stage of Disease

Early disease / Re-activation later

Results Interpretation >
View Lyme disease ImmunoBlot tests >

Lyme ImmunoBlot IgM
The Lyme ImmunoBlot IgM is a very sensitive indicator of exposure to B. burgdorferi. It may be positive as early as two weeks after a tick bite and will usually remain positive for six to eight weeks after initial exposure; in some patients, it can remain positive for even longer. Re-exposure will also cause this test to be positive for a brief period of time.

For the Lyme ImmunoBlot testing to be complete, the IgM ImmunoBlot should be run along with the IgG ImmunoBlot.

Reference Range
Negative <2 starred bands present on the blot

Clinical Significance
This test is recommended to be performed at least two weeks after possible exposure. The Lyme ImmunoBlot test must be performed on any sample with positive or equivocal results for Lyme IgG/IgM antibody serology or Lyme IgG/IgA/IgM IFA. It should, ideally, be run alongside the IgG ImmunoBlot.

Limitations

  1. Patients with other spirochetal disease and/or who test positive for rheumatoid factor or Epstein Barr virus may have cross-reacting antibodies and may have positive results for 31, 41, and/or 83 kDa antigens.
  2. Positive results for 31 kDa antigens may be present after Lyme vaccination in uninfected persons.
  3. A negative ImmunoBlot does not exclude the possibility of infection with B. burgdorferi.
  4. IGeneX interpretation is based on internal validation studies.
  5. The results of this test must be interpreted in relation to the patient’s clinical history, epidemiological data, stages of the disease, clinical symptoms, or other laboratory results.


The presence of bands 23-25, 31, 34, 39 or 83-93kDa as indeterminate or presence of only one of these bands in a negative report may indicate clinical significance. Therefore, we recommend testing with another method and/or retesting in 4-6 weeks.

Stage of Disease
Early disease / Re-activation later

Results Interpretation >
View Lyme disease ImmunoBlot tests >

Lyme ImmunoBlot IgG
The IgG ImmunoBlot is an immunoassay and qualitative test in which antibodies are visualized. The IgG antibody is typically present a few months following initial infection.

Reference Range
Negative <2 starred bands present on the blot

Clinical Significance
The Lyme ImmunoBlot IgG is a sensitive indicator of an exposure to B. burgdorferi. IgG antibody is typically present a few months following the initial infection. Lyme ImmunoBlot IgG/IgM must be performed on any sample with positive or equivocal results for Lyme IgG/IgM antibody serology or Lyme IgG/IgA/IgM IFA.

Limitations

  1. Positive results for 31 and/or 34 kDa may be present after Lyme vaccination in uninfected persons.
  2. A negative Lyme ImmunoBlot IgG does not exclude the possibility of infection with B. burgdorferi.
  3. IGeneX interpretation is based on internal validation studies.
  4. The results of this test must be interpreted in relation to patient’s clinical history, epidemiological data, stage of disease, clinical symptoms or other laboratory results.


The presence of an indeterminate number of double-starred bands in a negative report may indicate clinical significance. Therefore, we recommend testing with another method and/or retesting in 4-6 weeks.

Stage of Disease
Later disease

Results Interpretation >
View Lyme disease ImmunoBlot tests >

Lyme Western Blot

The Lyme Western Blot is a qualitative immunoassay in which antibodies specific to the B. burgdorferi antigens on a membrane strip are visualized. It is a qualitative test and is generally more sensitive and specific than the ELISA and IFA tests. When antibody levels are very low in the early or late stage of the disease, the Western Blot can be positive, whereas ELISA and IFA tests can be negative. If the Lyme IgG/IgM antibody serology or Lyme IgG/IgA/IgM IFA is positive or equivocal, this test must be used.

Principle

The Lyme Western Blot assay is based upon an antigen-antibody complex formation in the following steps:

  1. Binding of anti-Borrelia specific antibodies in human serum to the Western Blot strip. The Western Blot strip is a membrane strip with fixed B. burgdorferi antigens separated by size.
  2. Binding of enzyme labeled anti-human IgG or IgM antibodies to the human anti-Borrelia antibodies bound to fixed B. burgdorferi antigens on the membrane.
  3. Reaction with BCIP/NBT, a chromogenic substrate with bound antibodies on the strip. A dark purple colored precipitate (band) develops on the antigen-antibody complexes.


Stage of Disease

Early disease / Re-activation later

Results Interpretation >
View Lyme disease Western blot tests >

Lyme Western Blot IgM
The Lyme Western Blot IgM is a very sensitive indicator of exposure to B. burgdorferi. It may be positive as early as 1 week after a tick bite, will usually remain positive for six to eight weeks after initial exposure, and in some patients will remain positive for much longer. Re-exposure will also cause this test to be positive for a brief period of time. For the testing to be complete, it is preferable that the IgM Western Blot be run along with the IgG Western Blot.

Reference Range
Negative: <2 double starred bands present on the blot

Limitations

  1. Patients with other spirochetal diseases and/or who test positive for rheumatoid factor or Epstein Barr virus may have cross-reacting antibodies and may have a positive result for 31, 41, and/or 83 kDa antigens
  2. Positive results for 31 and/or 34 kDa antigens may be present after Lyme vaccination in uninfected persons.
  3. A negative Western Blot does not exclude the possibility of infection with B. burgdorferi.
  4. IGeneX interpretation is based on internal validation studies.
  5. The results of this test must be interpreted in relation to patient’s clinical history, epidemiological data, stages of disease, clinical symptoms or other laboratory results.


Presence of bands 23-25, 31, 34, 39 and 83-93 kDa as indeterminate, or presence of only one of these bands in a negative report, may indicate clinical significance. Therefore, we recommend testing with another method and/or retesting in 4-6 weeks.

Stage of Disease
Early disease / Re-activation later

Results Interpretation >
View Panel & Test Price List >
View Lyme disease Western blot tests >

Lyme Western Blot IgG
The IgG Western Blot is an immunoassay and qualitative test in which antibodies are visualized. The IgG antibody is typically present a few months following initial infection.

Clinical Significance
The Lyme Western Blot IgG is a sensitive indicator of exposure to B. burgdorferi. IgG antibody is typically present a few months following the initial infection. Lyme Western Blot IgG must be performed on any sample with a positive or equivocal result for Lyme IgG/IgM antibody serology or Lyme IgG/IgA/IgM IFA.

Limitations

  1. Patients with other spirochetal disease and/or who test positive for rheumatoid factor or Epstein Barr virus may have cross-reacting antibodies and may have a positive result for proteins 31, 41.
    Positive results for 31 and/or 34 kDa may be present after Lyme vaccination in uninfected persons.
  2. A negative Lyme Western Blot IgG does not exclude the possibility of infection with B. burgdorferi.
  3. IGeneX interpretation is based on internal validation studies.
  4. The results of this test must be interpreted in relation to patient’s clinical history, epidemiological data, stages of disease, clinical symptoms or other laboratory results.


Presence of an indeterminate number of double starred bands in a negative report may indicate clinical significance. Therefore, we recommend testing with another method and/or retesting in 4-6 weeks.

Stage of Disease
Later disease

Results Interpretation >
View Panel & Test Price List >
View Lyme disease Western blot tests >

Lyme IgM and IgG 31kDa Epitope Test

The Lyme IgG or IgM 31kDa Epitope test is a qualitative immunoblot assay that determines whether the 31kDa band present on a Lyme Western Blot IgG or IgM is due to B. burgdorferi specific antibodies.

Reference Range
Negative – No visible bands present

Clinical Significance
It is known that Western blot tests, especially IgM, can give false positive results with some viral and bacterial infections. This test determines whether the band present at position 31 kDa on the IGeneX Lyme Western Blot IgM is specific for B. burgdorferi. Serum from the patient is tested against a Western Blot strip with fixed Borrelia burgdorferi-specific recombinant antigen fragments.

Limitations

  1. This test is only performed on samples previously tested by Lyme Western Blot IgM at IGeneX and have a band at 31kDa position on the blot.
  2. Positive results for the 31kDa band may be present after vaccination in uninfected persons.
  3. IGeneX interpretation is based on internal validation studies.
  4. The results of this test must be interpreted in relation to patient’s clinical history, epidemiological data, stages of disease, clinical symptoms or other laboratory results.


Special Instructions

This test is not yet available for NY residents.

Stage of Disease
Screen test

Results Interpretation >
View Lyme disease Epitope tests >

Lyme Multiplex PCR

The Lyme Multiplex PCR-based diagnostic test for Borrelia burgdorferi is performed directly on a clinical specimen. The combination of the following three steps imparts very high specificity and sensitivity:

  • Hybridization/Selection
  • Amplification of the specific DNA (genomic and plasmid)
  • Detection of B. burgdorferi-specific amplified DNA fragments by dot-blot assay using B. burgdorferi-specific probes.

Reference Range
Negative- Genomic: B. burgdorferi DNA not detected

Negative- Plasmid: B. burgdorferi DNA not detected

Clinical Significance
The Lyme Multiplex PCR test is a 3-step amplified nucleic acid assay that detects B. burgdorferi-specific DNA sequences. The gene fragments are first selected with specific probes. Then, DNA is amplified in two independent PCR assays using different primers from the Osp A gene and flagellin gene. Lastly, the amplified products are detected by hybridizations to specific probes in a Southern Dot-Blot Assay. This test detects DNA from B. burgdorferi, B. afzelii, B. andersonii, B. garinii, and B. mayoni (based on sequence information). The sample is considered positive if either the genomic or plasmid result is positive.

Limitations

  1. Results should be interpreted in conjunction with other laboratory and clinical findings.
  2. Test results can only help the physician in confirming clinical diagnosis.


Stage of Disease

Any stage of disease (Early to late / Chronic stage)

Results Interpretation >
View Lyme disease PCR tests >

Lyme Dot-blot Assay (LDA)

The Lyme Dot-blot Assay (LDA) is a qualitative immunoassay for detecting Borrelia specific antigens in urine using anti-B. burgdorferi antibodies.

Reference Range
Negative: No B. burgdorferi antigens detected

Limitations

  1. Negative tests do not exclude diagnosis of Lyme disease
  2. Cross-reactions with Leptospira may occur.
  3. False positive may occur for patients with a Urinary Tract Infection (UTI), currently being treated for a UTI, or who have undergone treatment for a UTI within the last two weeks.
  4. Test results should be interpreted in conjunction with other laboratory and clinical findings.


Special Instructions

This test is not yet available for NY residents. Test 805 is included in panel 875.

Stage of Disease
Any stage of disease (Early to late / Chronic stage)

Results Interpretation >
View Lyme disease LDA tests >

Lyme IgXSpot

The Lyme IgXSpot is an Enzyme-Linked ImmunoSpot assay that detects human T cells reactive to B. burgdorferi-specific antigens in vitro. It is well documented that both humoral and cellular immune responses develop in Borrelia infection. The cellular immune response develops much earlier than the humoral response in most patients infected with B. burgdorferi. In some patients, sero-conversion from cellular to humoral response does not occur or occurs much later in disease; and in some patients with chronic form of the disease, the humoral response is poor. Thus, the Lyme IgXSpot test is recommended for detection of very early and/or late B. burgdorferi infection and in seronegative patients’ whole blood samples.

Download Lyme IgXSpot Data Sheet >

Principle
IgXSpot is an Enzyme-Linked ImmunoSpot (ELISPOT) assay that detects human T-cells reactive to B. burgdorferi-specific antigens in vitro. ELISPOT is a widely used method for detecting and monitoring cellular immune responses to specific antigens. The IgXSpot assay allows visualization of the secretory product(s) of individual activated or responding cells to B. burgdorferi-specific antigens. Each spot that develops in the assay represents a single reactive cell.

Advantage
The IgXSpot detects specific T-cell responses soon after infection, when antibodies to the organisms are not detectable, or late in the disease when the levels of antibodies are minimal. When combined with Lyme ImmunoBlot tests, IgXSpot provides information on the full spectrum of a patient’s immune response to infection and stage of the disease. It is, therefore, especially useful for seronegative patients.

Reference Range
>2 colonies positive.

Clinical Significance
Assessment of the function and frequency of Borrelia-specific T cells is crucial for evaluating the cellular immune response to and diagnosis of Borrelia infection. Due to the clonal expansion (proliferation) of antigen-specific T cells in vivo during an immune response— increased frequencies of Borrelia antigen-specific effector/memory T cells in peripheral blood suggest prior infection/exposure. In addition, because of the apparent prevalence of either humoral or cellular immunity in infected individuals, the combination of the B. burgdorferi IgXSpot with Lyme serology assay would further increase the sensitivity of Lyme disease diagnosis.

Limitations
For diagnostic purposes, the IgXSpot test results should be used in conjunction with clinical symptoms and other evidence available to the diagnosing physician.

Stage of Disease
Early disease / Later re-activation

Results Interpretation >
View Lyme disease IgXSpot tests >

Lyme cePCR - New!

Culture testing is widely considered to be the “gold standard” for diagnosis of Lyme disease. For many years, Lyme disease cultures were too expensive and tedious to be practical for laboratory use. Until now. After many years of research and development, IGeneX is pleased to introduce cePCR (Culture-Enhanced PCR) for Lyme disease.

Download the cePCR datasheet >

Principle
In culturing, a clinical sample from the body (e.g. blood) is incubated in media.
During this incubation period, micro-organisms in the sample grow and multiply. The sample is then tested by PCR to identify the pathogens.

Advantages of Culture-Enhanced PCR
• Provides higher sensitivity than standard PCR testing.
• The only 100% specific method for identification of Lyme disease.
• Obtaining cultures before antibiotic use improves the chances of identifying the offending microorganism, which improves patient care.

Stage of Disease
Any stage

View Lyme disease cePCR tests >

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The first step in getting tested with IGeneX is to order a collection kit. Choose between a Blood, Urine, or Miscellaneous kit. Doctors can order unlimited quantities of kits at no charge. Patients are required to deposit $20, which is applied to the testing fees.