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. The IgG antibody often persists long after symptoms have disappeared. The presence of antibody indicates exposure, not active disease. A positive or equivocal test must be confirmed by both IgG and IgM Western Blots.

Reference Range

Borrelia burgdoferi Antibody Serology IgG/IgM Negative <1.0

Equivocal ≥1.0 to <1. Positive ≥1.2

Clinical Significance

The IgG/IgM antibody serology indicates the presence of IgG and/or IgM antibodies to B. burgdorferi. The IgG antibody often persists long after the symptoms have disappeared while the IgM antibody commonly appears with initial infection. The presence of antibody indicates exposure, not active disease. This test is recommended at least four weeks after exposure. Patients with the diagnosis of 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.
2. Cross Reacting Antibodies:
   1. Sera from patients with other pathogenic spirochetal diseases such as syphilis, yaws, pinta, leptospirosis, and relapsing fever may give false positive results.
   2. Sera from patients with mononucleosis or lupus erythomatosis (LE) may also give false positive results.
   3. 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 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.

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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.

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The Immunetics® C6 B. 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.

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The Lyme mmunofluorescent assay (IFA) is designed to detect Borrelia burgdorferi specific antibodies, IgA, IgM and IgG in human serum. For diagnostic purposes, IFA 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. burgdorferiantibodies 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.

Special Instructions 

This test is not yet available for NY residents

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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.

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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.

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 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 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.

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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.

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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. In early or late stage of the disease when antibody levels are very low, western Blot 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.

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.  Western blot strip is a membrane strip with fixed B. burgdorferi antigens separated by size, 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. 

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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. 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 western blot be run along with the IgG western blot.

Reference Range

Negative <2 double started bands present on the blot

Clinical Significance

The Lyme Western Blot 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 Western Blot 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 Western Blot be run along with the IgG Western Blot.

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 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 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.

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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 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. 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.
2. Positive results for 31 and/or 34 kDa may be present after Lyme vaccination in uninfected persons.
3. A negative Lyme Western Blot IgG 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 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. 

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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.

Special Instructions
This test is not yet available for NY residents.

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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. 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. 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.

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

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.

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Principle

The B. burgdorferi IGXSpot is an Enzyme-Linked ImmunoSpot (ELISPOT) assay that detects human T-cells reactive to B. burgdorferispecific 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.

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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.

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Principle

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

1. Binding of anti-TBRF Borreliaspecific 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 substratewith bound antibodies on the strip.  A dark purple colored precipitate (band) develops on the antigen-antibody complexes.

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.

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The IgG ImmunoBlot is an immunoassay and qualitative test in which antibodies are visualized.  The IgG antibody can be present as early as 6 to 8 weeks after infection.

Reference Range

Negative <2 bands present on the blot

Clinical Significance

The TBRF ImmunoBlot IgG is a sensitive indicator of an exposure to B. burgdorferi.  The IgG antibody can be present as early as 6 to 8 weeks after infection.

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.

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IgM Reference Range

• Negative: Relapsing Fever Borrelia specific IgM antibodies not detected

IgG Reference Range

• Negative: Relapsing Fever Borrelia specific IgM antibodies not detected

Clinical Significance

The Relapsing Fever (TBRF) Western Blot is a sensitive indicator of an exposure to Relapsing Fever Borrelia. The TBRF Western Blot should be performed on patients with Lyme-like symptoms but negative on all Lyme tests.

Limitations

1. Patients with negative results should be re-tested using a fresh specimen after six weeks if clinical symptoms persist.
2. The results of this test must be interpreted based on patient’s clinical history and other laboratory results.

IGeneX Relapsing Fever Borrelia q-PCR detects the specific DNA of relapsing fever Borrelia group and speciates to B. miyamotoi in human specimens and ticks.

Reference Range

• Negative- Relapsing Fever Borrelia specific DNA not detected
• Negative- B. miyamotoi specific DNA not detected

Clinical Significance

The Relapsing Fever (RF) Borrelia group real-time PCR Assay is designed for qualitative detection of RF Borrelia group DNA in clinical samples. The RF group includes B. miyamotoi, B. hermsii, B. coriaceae, B. lonestari and B. anserine, etc. The assay detects RF Borrelia group genomic DNA and B. miyamotoi specific DNA.