Lyme diagnosis and testing can be very confusing. This confusion has contributed to false positive diagnoses and unnecessary treatment.
As in other infections like HIV, testing for Lyme disease involves looking for antibodies produced by the body’s immune system in response to infection. This is called serologic testing because the antibodies are found in blood serum.
Testing positive for antibodies is called seropositive and testing negative for antibodies is called seronegative.
One known problem is that we can produce antibodies for years or decades after a Lyme infection has been eradicated. Therefore, a seropositive test on its own is not necessarily indicative of active infection.
In addition, it can can take a few weeks for detectable antibodies to build up in the body. Pseudoscience advocates frequently mislead about Lyme antibody testing by failing to differentiate testing performance in early infection from testing performance in late infection.
What the experts say
According to the CDC:
- Patients who have had Lyme disease for longer than 4-6 weeks, especially those with later stages of illness involving the brain or the joints, will almost always test positive.
- A patient who has been ill for months or years and has a negative test almost certainly does not have Lyme disease as the cause of their symptoms.
- Serologic testing is generally not useful or recommended for patients with single EM rashes. For this manifestation, a clinical diagnosis (alone) is recommended.
Experts around the world agree with the CDC. A 2018 French review of 16 Lyme diagnostic guidelines from 7 countries “revealed a global consensus regarding diagnosis at each stage of the infection.” The only outlier was the pseudoscience group German Borreliosis Society (Deutsche Borreliose-Gesellschaft, DBG), a German counterpart to the pseudoscience group ILADS.
Lyme testing: Accurate when used appropriately
A 2016 systematic review  that included 8 studies of CDC-recommended two tier test performance in “Late Lyme” showed Lyme antibody testing to be 99.4% sensitive and 99.3% specific** in North America.
In other words, out of 100 people with Late Lyme disease, 99-100 of them will test positive. Out of 100 people who may believe they have Late Lyme disease but do not, 99-100 of them will test negative.
Misinformation can convince patients to ignore or misinterpret negative tests to justify a false “Chronic Lyme” diagnosis.
Avoid unnecessary and unscientific testing
Don’t test for Lyme disease as a cause of musculoskeletal symptoms without an exposure history and appropriate exam findings. 
Experts also warn against unvalidated testing [5, 9]. Quackwatch  and Science-Based Medicine  both provide accessible explanations about the differences between validated and unvalidated tests.
How Lyme antibody testing works
Antibody testing for Lyme disease requires two different tests to establish a positive result. If either the first tier test or the second tier test is negative, the test result is negative overall.
But in the event of a negative result, Dr. Adriana Marques of the NIH states:
For patients with signs or symptoms consistent with Lyme disease for less than or equal to 30 days, the provider may treat the patient and follow up with testing of convalescent-phase serum.
The first tier of the “two-tiered testing” system is an Enzyme Immunoassay (EIA aka ELISA).
The second tier of the well-established Standard Two-Tiered Testing (STTT) involves one or two Western Blot tests, which can be complicated to understand. The Western Blot is also called an immunoblot or a line blot.
According to the CDC:
1) if the patient has had symptoms for less than or equal to 30 days, an IgM Western Blot is performed;
2) if the patient has had symptoms for more than 30 days, the IgG Western Blot is performed.
The IgM should not be used if the patient has been ill for more than 30 days.
In 2019, the CDC added a second test system to its recommended tests, the Modified Two-Tiered Test (MTTT). In the MTTT, the second tier is a different ELISA that is FDA-cleared specifically for the MTTT.
By using the MTTT, testing is simpler, costs may be reduced, and early Lyme disease is more likely to be detected. A drawback of the MTTT is that potentially useful information is no longer determined about which types of antibodies are being produced by the body.
More about Western Blot testing in North America
The IgG Western Blot test measures 10 different types of antibodies, which act as a fingerprint to identify Borrelia burgdorferi, the bacteria that cause Lyme disease. For Lyme disease, a positive IgG Western Blot test displays at least 5 of 10 measured antibody “bands.”
The scored IgG bands are 18 kDa, 24 kDa (OspC)†, 28 kDa, 30 kDa, 39 kDa (BmpA), 41 kDa flagellin (Fla), 45 kDa, 58 kDa (not GroEL), 66 kDa, and 93 kDa.
The IgM Western Blot test measures 3 different types of antibodies. If at least two of the three IgM bands are positive, the IgM Western Blot is considered positive.
The scored IgM bands are 24 kDa (OspC)†, 39 kDa (BmpA), and 41 kDa (Fla).
Beware IgM false positives
The IgM Western Blot is notorious for producing false positive results, which is why it is only used in very limited circumstances, i.e. during the first 30 days of illness before detectable IgG antibodies would be produced in the event of a Lyme infection.
† According to the CDC, “Depending upon the assay, OspC could be indicated by a band of 21, 22, 23, 24 or 25 kDA.”
Important: Don’t misinterpret a negative test as positive
Many people without Lyme disease will test positive for some bands. Therefore, the CDC cautions:
It is not correct to interpret a test result that has only some bands that are positive as being “mildly” or “somewhat” positive for Lyme disease.
For example, in one study, 43% of healthy people and 75% of syphilis patients tested positive for band 41. In a study of US veterans in New York, 76% of those without Lyme disease tested positive for band 41.
Even without a Borrelia burgdorferi infection, many of us produce antibodies that will react on a Lyme test. Notably, harmless bacteria found naturally in our mouths can cause us to test positive for band 41.
A positive Lyme antibody test requires both tiers to be positive, as many without Lyme infections can test positive on single tests. For example, one study found up to 40% of patients with Lupus and other rheumatic diseases test positive on the first tier ELISA test. The second tier test is necessary to stop a false positive diagnosis.
The American Society for Microbiology recommends against ordering the Western blot without a positive ELISA screening:
The Lyme immunoblot test is designed only as a confirmatory test, so it is important not to test screen-negative samples.
LymeScience recommends against:
- Tests from any of the following labs: IgeneX, DNA Connexions, Galaxy Diagnostics, Medical Diagnostic Laboratories (MDL), Milford Molecular Diagnostics Laboratory, Advanced Lab, Fry Laboratories, Ceres Nanosciences (Nanotrap), Global Lyme Diagnostics, Pharmasan Labs (iSpot Lyme), Coppe Laboratories (myLymeTest), ArminLabs, BCA-Lab (also known as InfectoLab), Australian Biologics, Melisa Labs, Moleculera Labs (Cunningham Panel), R.E.D. Labs, Immunosciences Lab, Aperiomics, Te?ted Oy (Tezted Limited, TICKPLEX), Lyme Diagnostics Ltd. (DualDur cell technology), ProGene (DX Genie), Ionica Sciences (IonLyme), T Lab Inc., Veramarx
- Any lab on Quackwatch’s list of “Laboratories Doing Nonstandard Laboratory Tests“
- CD57 testing
- Diagnosis with microscopy, e.g. Live Blood Cell Analysis.
- Diagnosis based on magical beliefs, for example applied kinesiology a.k.a Autonomic Response Testing (ART)
- Urine tests
- Electrodermal testing
- Lymphocyte Transformation Test (LTT)
- ELISpot tests
- MELISA testing
- Phelix Phage tests
- Failing to follow testing strategies recommended by the CDC or local infectious diseases organizations
- Failing to perform and acknowledge both tests of the established two-tiered test
- Clinical diagnosis without a science-based rationale
- Diagnosis via a questionnaire of non-specific symptoms
- Diagnosis from an unscientific practitioner, including those who market themselves using the following terminology: Lyme literate (especially those affiliated with ILADS), integrative, functional, alternative, complementary, Traditional Chinese Medicine, holistic, natural, Biological, Ayurvedic, chiropractic, naprapathic, homeopathic, and naturopathic
- Any testing not recommended by the CDC
See also the following LymeScience pages:
- How chronic Lyme recruits followers
- Lyme misdiagnosis illustrated
- Red flags of chronic Lyme quackery
- CDC scientist: Why bands 31 and 34 aren’t used to test for Lyme disease
- When do symptoms mean Lyme disease?
Other misconceptions about Lyme disease diagnosis and testing are discussed below.
Table excerpted and reformatted* from the longer 2013 paper (ref 2 below, which is worth reading):
There is substantial evidence supporting the accuracy of FDA-cleared tests once antibodies build up in the body (4-6 weeks post-infection).
The CDC concurs:
You may have heard that the blood test for Lyme disease is correctly positive only 65% of the time or less. This is misleading information. As with serologic tests for other infectious diseases, the accuracy of the test depends upon the stage of disease. During the first few weeks of infection, such as when a patient has an erythema migrans rash, the test is expected to be negative.
Several weeks after infection, currently available ELISA, EIA and IFA tests and two-tier testing have very good sensitivity.
It is possible for someone who was infected with Lyme disease to test negative because:
- Some people who receive antibiotics (e.g., doxycycline) early in disease (within the first few weeks after tick bite) may not develop antibodies or may only develop them at levels too low to be detected by the test.
- Antibodies against Lyme disease bacteria usually take a few weeks to develop, so tests performed before this time may be negative even if the person is infected. In this case, if the person is retested a few weeks later, they should have a positive test if they have Lyme disease. It is not until 4 to 6 weeks have passed that the test is likely to be positive. This does not mean that the test is bad, only that it needs to be used correctly. 
* LymeScience reformatted the table and added punctuation, emphasis, a note, and changed the word “evidence” to “science”. Excerpted for educational and commentary purposes.
** 99.4% sensitive (95% confidence interval: 95.7–99.9) and 99.3% (95% confidence interval: 98.5-99.7%) specific.
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Updated November 5, 2020