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Lyme Testing

Lyme Testing

Borreliosis is caused by two groups of Borrelia, B. burgdorferi group and the Tick-Borne Relapsing Fever (TBRF) Borrelia group. Until recently, it was believed that B. burgdorferi group was the only group that caused Lyme-like symptoms. However, we now know that TBRF Borrelia also causes Lyme-like symptoms. Therefore, IGeneX developed two sets of Immunoblots, Lyme ImmunoBlot IgM and IgG and TBRF ImmunoBlot IgM and IgG.

Many patients with Lyme-like symptoms are misdiagnosed because: (1) The current serological tests cannot detect antibodies to all the different strains of B. burgdorferi (2) For TBRF Borrelia, only two serological tests, an IFA test for B. hermsii and a serological test that detects only B. miyamotoi GlpQ protein, are available. In addition, with the increase in tick populations, Borreliosis is on the rise. Thus, there is an urgent need for better diagnostic tests for Borrelisosi.

Because symptoms of Lyme and Tick-Borne Relapsing Fever are often similar, a more comprehensive battery of tests is critical for proper diagnosis.

“IGeneX’s new Lyme ImmunoBlot and the TBRF ImmunoBlot tests are the first, most inclusive and specific serologic tests for Lyme disease and TBRF,” said Dr. Jyotsna Shah, President and CEO of IGeneX. “These tests, when combined with Lyme IgX spot and PCR, cover the full spectrum of the disease.”

Lyme ImmunoBlots IgM and IgG

Although IGeneX Lyme Western blot prepared from Borrelia burgdorferi strains B31 and 297 is one of the most sensitive tests, it does not detect antibodies to all the B. burgdorferi sensu lato antigens. To develop all-inclusive Western blots would be very expensive and impractical. Therefore, IGeneX has developed a Lyme ImmunoBlot that is very specific and inclusive of most species of B. burgdorferi sensu lato for clinical use with the following advantages over a Western blot:

TBRF ImmunoBlots IgM and IgG

The TBRF ImmunoBlot is designed to detect antibodies to specific antigens of TBRF Borrelia in human serum. It detects antibodies to B. hermsii, B. miyamotoi, B. turicatae and B. coriaceae. Based on in-house studies these blots detect antibodies to North American, European and Australian strains of TBRF Borrelia in patient serum samples. The specificity of the TBRF ImmunoBlot is 94% and 98% for IgM and IgG respectively.

Lyme IGX Spot

The Lyme 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 Lyme IGX Spot test:

  1. Detects specific T-cell responses soon after B. burgdorferi infection, when antibodies to the organisms are not detectable or late in the disease, when the levels of antibodies are very low.
  2. When combined with Lyme ImmunoBlot tests, information on the full spectrum of patient’s immune response to infection and stage of disease is obtained.
  3. Is especially useful for seronegative patients.

 

Borreliosis is a world-wide infectious disease caused by spiral-shaped bacteria of the Genus Borrelia, which is carried by ticks and louse. The two groups of Borrelia known to cause disease in humans are:

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). As a result, laboratory testing is often essential to confirming diagnosis of this disease. Generally, Lyme disease is diagnosed by a two-tiered testing approach involving an enzyme-linked immunosorbent assay (ELISA) followed by a Western blot test. Unfortunately, the sensitivity of these commercially available tests is poor. IGeneX has developed several B. burgdorferi tests that provide higher sensitivity to detect and speciate B. burgdorferi. When used in conjunction with clinical symptoms and patient history, these tests can better assist physicians in accurately diagnosing patients.

TBRF is a complex and progressive systemic illness that also can be difficult to diagnose due to its similarity to other diseases, including Lyme disease. As a result, laboratory testing is also typically required for accurate diagnosis. TBRF Borrelia can be seen in a Giemsa-stained blood smear only very early on in the disease. IGeneX, however, offers a variety of TBRF Borrelia tests that can aid in the clinical diagnosis of patients at multiple stages of the disease.

Lyme IgM Antibody Serology

The Lyme IgM antibody assay is another serologic test in ELISA 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. The level of IgM rapidly declines over time. A positive or equivocal IgM antibody test must be confirmed by an IgM Western Blot. 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 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. 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. This test is recommended approximately 2 weeks after suspected exposure.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.
  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.
  6. The use of this assay has not been evaluated for individuals who have received a Lyme disease vaccine.

C6 Peptide

The Immunetics C6B. burgdorferi (Lyme) ELISA Kit is intended for use in the presumptive detection of IgG and IgM antibodies to B. burgdorferi in human serum. The assay should be used only on samples from patients with clinical history, signs or symptoms consistent with B. burgdorferi infection, including individuals who have received the licensed recombinant OspA Lyme disease vaccine (Lymerix). Positive or equivocal results should be supplemented by testing with a standardized Western Blot (second step) method. Negative results should not be used to exclude Lyme disease.

Reference Range

0.91-1.09 Equivocal result

1.10 Positive result

Clinical Significance

The Immunetics C6 B.burgdorferi (Lyme) ELISA kit is intended for use on patients with clinical history, signs or symptoms consistent with B. burgorferi infection, including individuals who have received the licensed recombinant OspA Lyme Disease vaccine (Lymerix). This test detects IgG and IgM antibodies to B.burgdorferi in human serum. A synthetic peptide (C6 peptide) in the serum samples is bound by an immobilized antigen and detected by a HRP goat anti-human IgG/IgM conjugate. A blue-green product is produced where the antibodies have been bound to the antigens. The optical absorbance of each sample is measured at 450nm. A Lyme index value of 1.10 is a positive result. All samples with positive or equivocal results should be tested with B. burgdorferi Western Blots.

Limitations

  1. Negative result does not exclude the possibility of B. burgdorferi infection.
  2. Patients who have received antibiotic treatment and those who are in the early stages of Lyme disease may not exhibit detectable antibody titers.
  3. In the event that the initial test result is negative, patients with clinical history, signs or symptoms suggestive of Lyme disease should be re-tested in 2-4 weeks.
  4. A positive result is not a definitive evidence of infection with B. burgdorferi. It is possible that other disease conditions may produce artifactual reactivity in the assay. All samples with equivocal or positive results should be tested on a standardized B. burgdorferi Western Blots.
  5. The C6 B. burgdorferi (Lyme) ELISA kit has been tested on serum samples from individuals vaccinated with a licensed OspA vaccine (Lymerix). The performance of the test has not been determined on serum samples from recipients of other Lyme vaccines.
  6. This assay should only be used on patients with clinical symptoms of Lyme disease or suspected exposure to B. burgdorferi and should not be used to screen general populations.
  7. Lipemic, hemolyzed, bilirubinemic or turbid samples might produce artifactual results. Fresh samples should be collected for re-testing.
  8. Serum obtained from patients with diseases other than Lyme disease such as syphilis, periodontal disease, rheumatoid arthritis, systemic lupus erythematosis and other autoimmune diseases may result in false positive results.

Lyme IFA

The Lyme Immunofluorescent assay (IFA) is designed to detect Borrelia burgdorferi specific antibodies, IgA, IgM and IgG in human serum. For diagnostic purposesIFA test results should be used in conjunction with other data available to the physician.

Principal

The Lyme IFA assay is a two-stage sandwich assay, based upon an antigen-antibody complex formation in the following steps:

  1. Binding of anti-Borrelia specific antibodies in human serum to fixed B. burgdorferi on a slide.
  2. Binding of fluorescent-labeled anti-human IgG/IgM antibodies to the human anti B. burgdorferi antibodies bound to fixed B. burgdorferi on the slide.

Reference Range

Borrelia burgdorferi Antibodies IgG/ IgM/IgA Negative <1:40

Equivocal 1:40

Positive 1:80

Clinical Significance

The Lyme Immunofluorescense Antibody (IFA) assay detects IgA, IgG and IgM antibodies against B. burgdorferi. Seroconversion usually occurs 2-3 weeks after infection and may remain elevated in the case of persistent disease.

Limitations

  1. This test should only be performed in conjunction with Western Blots.
  2. Cross reactions occurs with other Borrelia species and spirochetes.

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. In early or late stage of the disease when antibody levels are very low, ImmunoBlot can be positive whereas ELISA and IFA tests can be negative. This test must be used if the Lyme IgG/IgM antibody serology or Lyme IgG/IgA/IgM IFA is positive or equivocal.

Download Data Sheet >

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. 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.

Lyme ImmunoBlot IgM

The Lyme ImmunoBlot IgM is a very sensitive indicator of exposure to B. burgdorferi. It may be positive as early as 1 week after a tick bite. This test will usually remain positive for six to eight weeks after initial exposure and in some patients will remain positive for a very long time. 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 ImmunoBlot be run along with the IgG ImmunoBlot.

Reference Range

Negative <2 stared bands present on the blot

Clinical Significance

The Lyme ImmunoBlot IgM is a sensitive indicator of exposure to B. burgdorferi. It may be positive as early as one week after a tick bite. This test will usually remain positive for six to eight weeks after initial exposure. Re-exposure will also cause this test to be positive for a brief period of time. In some patients this test can be positive for a very long time. Lyme ImmunoBlot IgM must be performed on any sample with positive or equivocal result for Lyme IgG/IgM antibody serology or Lyme IgG/IgA/IgM IFA. For the testing to be complete, it is preferable that the IgM ImmunoBlot be run along with 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 a positive result 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 patient’s clinical history, epidemiological data, stages of disease, clinical symptoms or other laboratory results.

Presence of any of the following bands: 23-25, 31, 34, 39 and 83-93 kDa as indeterminate or if only one of these bands is present in a negative report, it may have clinical significance. Therefore, we recommend testing with another method and/or retesting in 4-6 weeks.

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 stared 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 Western Blot IgG must be performed on any sample with positive or equivocal result 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.

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.

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 antibody or not.

Reference Range

Negative – No visible bands present

Clinical Significance

It is known that Western blots, 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 B. burgdorferi specific recombinant antigen fragments.

Limitations

  1. This test is only performed on samples that are 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.

Lyme Multiplex PCR

The Lyme Multiplex PCR-based diagnostic test for Borrelia burgdorferi, 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. burgdorferiB. afzelii, B. andersoniiB. garinii and B. mayoni (based on sequence information). The sample is considered positive if either the genomic or plasmid result is positive.

Limitations

  1. This test should only be performed in conjunction with Southern Dot-Blot.
  2. Results should be interpreted in conjunction with other laboratory and clinical findings.
  3. Test results can only help the physician in confirming clinical diagnosis.

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 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 patient?s whole blood samples.

Download Data Sheet >

Principle

The B. burgdorferi 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

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 very low. When combined with Lyme ImmunoBlot tests, provides information on the full spectrum of patient’s immune response to infection and stage of disease. Especially useful for seronegative patients.

Reference Range

Reference Range: >2 colonies positive.

Clinical Significance

It is well documented that both humoral and cellular immune responses develop to Borrelia infection. Assessment of both the function and the 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, the presence of increased frequencies of Borrelia antigen-specific effector/memory T cells in peripheral blood suggests prior infection/exposure to Borrelia. Because of the apparent prevalence of either humoral or cellular immunity in infected individuals, 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.

Tick-Borne Relapsing Fever (TBRF) ImmunoBlot

The Tick-Borne Relapsing Fever (TBRF) ImmunoBlot is a qualitative immunoassay in which antibodies specific to the TBRF Borrelia antigens on a membrane strip are visualized. It is a qualitative test and is more sensitive and specific than the IFA and traditional Western Blot tests. In early or late stage of the disease when antibody levels are very low, ImmunoBlot can be positive whereas IFA tests can be negative.

Download Data Sheet >

Principle

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

  1. Binding of anti-TBRF Borrelia specific antibodies in human serum to the ImmunoBlot strip. ImmunoBlot strip is a membrane strip with fixed TBRF Borrelia recombinant antigens on it.
  2. Binding of enzyme labeled anti-humanIgG or IgM antibodies to the human anti TBRF Borrelia antibodies bound to fixed TBRF Borrelia 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.

Tick-Borne Relapsing Fever (TBRF) ImmunoBlot IgM

The TBRF ImmunoBlot IgM is a very sensitive indicator of exposure to TBRF Borrelia. It may be positive as early as 2 weeks after a tick bite. This test will usually remain positive for six to eight weeks after initial exposure and in some patients will remain positive for a very long time. 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 ImmunoBlot be run along with the IgG ImmunoBlot.

Reference Range

Negative <2 bands present on the blot

Clinical Significance

The TBRF ImmunoBlot IgM is a sensitive indicator of exposure to TBRF Borrelia. It may be positive as early as 2 weeks after a tick bite. This test will usually remain positive for six to eight weeks after initial exposure. Re-exposure will also cause this test to be positive for a brief period of time. In some patients this test can be positive for a very long time. For the testing to be complete, it is preferable that the IgM ImmunoBlot be run along with the IgG ImmunoBlot.

Limitations

TBRF ImmunoBlot can be false negative early in the disease.

  1. A negative TBRF ImmunoBlot does not exclude the possibility of infection with TBRF Borrelia.
  2. IGeneX interpretation is based on internal validation studies.
  3. 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.
  4. Indeterminate result may have clinical significance. Therefore, we recommend testing with another method and/or retesting in 4-6 weeks.