The Past, Present, and (Possible) Future of Serologic Testing for Lyme Disease


Lyme disease is the most commonly transmitted tick-borne infection in the United States. According to the Centers for Disease Control and Prevention (CDC), there were more than 30,000 confirmed and probable cases of Lyme disease in 2014. Additionally, nearly 300,000 presumed cases went unreported, and approximately 2.4 million specimens were submitted for Lyme disease testing.

Elitza Theel, Ph.D.
Elitza Theel, Ph.D.

To test for Lyme disease, the CDC, the National Institutes of Health, the Infectious Disease Society of America, and other health agencies endorsed the two-tiered testing algorithm (TTTA) in 1995. Today, this is still the most recommended testing. Elitza Theel, Ph.D., Director of the Infectious Diseases Serology Laboratory at Mayo Clinic in Rochester, wrote a review to discuss the evolution of serologic assays for Lyme disease and a summary of the performance characteristics for the recommended TTTA. Dr. Theel also analyzed a recently proposed alternative to the traditional TTTA and novel methodologies. The review was published in the Journal of Clinical Microbiology.

The accuracy of serologic testing for Lyme disease is extremely important for diagnosis and dependent on many factors, including the timing of specimen collection in relation to symptom onset and whether preemptive antibiotic treatment was initiated.

“Serologic assays detect antibodies specific to Borrelia burgdorferi, which can take two to three weeks to develop. So, if a patient sample is collected too soon following exposure, antibodies may not yet be detectable, leading to a falsely negative result,” explained Dr. Theel. “Along the same lines, for a small percentage of individuals who are started immediately on antibiotics for Lyme disease, their immune response may be blunted and likewise lead to negative serologic results.”

Prior to 1995, many laboratories used a variety of testing methods to detect B. burgdorferi specific antibodies and often applied their own unique interpretative criteria. This led to many conflicting and often inaccurate results. The current TTTA, which is based on an initial enzyme-linked immunosorbent assay (ELISA) screen followed by supplemental testing of positive results by an immunoblot, was developed out of a need to standardize and improve the accuracy of serologic testing for Lyme disease in U.S. laboratories.

Over the past 10 to 15 years, first- and second-tier serologic assays for detection of antibodies to Lyme disease have undergone significant modification, leading to improved clinical performance compared to their predecessor methods. However, they have their limitations. The lower specificity of some first tier screening assays necessitates supplemental testing by immunoblots, which can lack sensitivity depending on stage of disease.

In an effort to overcome these obstacles, a modified TTTA based on two sequential ELISAs, rather than supplemental testing by immunoblot, has been proposed by multiple groups.

“The current TTTA has fairly low sensitivity (42–73%) during the early stages of Lyme disease, and this is largely due to the lower sensitivity associated with the supplemental immunoblots. The advantage of the alternative TTTA is that immunoblots are no longer used and are in fact replaced with a second ELISA assay. The sensitivity of a TTTA based on serial ELISAs is significantly higher (53–100%) for early Lyme disease,” said Dr. Theel.

In addition to serology, Dr. Theel states that culture and molecular assays are also used to detect B. burgdorferi spirochetes or DNA. While the specificity of these assays is high, sensitivity remains an issue, and a negative result by either method cannot be used to exclude Lyme disease. The exception to this is the recently identified B. mayonii, for which PCR (polymerase chain reaction) is in fact the preferred diagnostic method. Additionally, there are numerous laboratories in the U.S. that “specialize” in Lyme disease diagnostics and offer a variety of alternative serologic and molecular assays. Importantly, however, these assays are rarely clinically validated and, if used, results should be interpreted with caution.

With so many testing methods available, how does one choose? According to Dr. Theel, “Diagnosis of Lyme disease should be based on clinical findings, exposure history, and laboratory testing.”

For example, for patients with a classic erythema migrans (EM) or “bull’s-eye” rash, laboratory testing is not indicated since the lesion itself is diagnostic for Lyme disease. For patients without an EM rash, but with proper exposure history and compatible clinical symptoms, serologic testing should be pursued. And at times, serial testing by serology may be needed to show seroconversion from an initially negative to a positive result.

“Lyme disease diagnostics have evolved significantly over the last 30 to 40 years and continue to improve. They are not perfect; we know this and are actively working on improving our current methods and algorithms,” said Dr. Theel.

To learn more about testing for Lyme disease, view Mayo Medical Laboratories’ comprehensive test offering


Kelley Schreiber

Kelley Schreiber is a Marketing Channel Manager at Mayo Medical Laboratories. She is the principle editor and writer of Insights and leads social media and direct marketing strategy. Kelley has worked at Mayo Clinic since 2013. Outside of work, you can find Kelley running, traveling, playing with her new kitten, and exploring new foods.


Your findings reflect the disease as if it were caught at an early stage, I’m told, just by my symptoms, to check it out and be tested…..
Should I have the TTTA and blood work? I have no rash. Chiefly arthritis, w/ fybro, thyroid, heat intolerance, poor sleep, weight gain, anxiety, hair loss, fatigue, brain fog, etc. No meds or supplements have helped. No dr has suggested Lyme….but I’ve asked them to think outside the box, because I’m just about had it!! Thank you.

Hi Karen, thank you for your comment/question. Unfortunately, we cannot diagnose conditions, provide second opinions, or make specific treatment recommendations through this correspondence. If you would like to seek help from Mayo Clinic, please call one of our appointment offices (Minnesota: 507-538-3270, Arizona: 800-446-2279, or Florida: 904-953-0853).

Can you back up serologic data with brain autopsies that look for persistent Borrelia?
We at the Paul H Duray Pathology Research Fellowship have done this, and we have found both Borrelia burgdorferi and Borrelia miyamotoi in the brains of seronegative Alzheimer’s patients. Doctors that look at negative serology tests too easily dismiss complicated patients as infection free. Also negative Lyme serology test results in a patient’s chart can often mean no antibiotic treatment will be authorized. The consequences are too high and the medical community does not accept responsibility for the serious sequelae in patients from delayed, denied or inadequate antibiotic treatments. If the medical community is going to accept flawed testing and deny treatment because of negative tests, then they should accept the possibility of being sued in court for patients that register for brain autopsies because they feel the Lyme disease tests failed to detect their brain infection and the doctor failed to address the possibility of seronegative neurological Lyme disease.

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