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How Accurate are Lyme Disease Tests?


How Accurate are Lyme Disease Tests

Lyme disease testing is difficult, and if not performed accurately, it can result in unneeded therapy. To interpret serologic test results, consider the patient's pretest probability of infection, which is based on exposure probability and clinical signs. Although new guidelines may be issued shortly, two-tiered testing remains the gold standard for identifying Lyme disease.


Key points

Lyme disease is a complex, multisystemic, bacterial disease caused by Borrelia burgdorferi, the most common tick-borne infection in North America.

Lyme disease is mostly found in the skin, joints, and nervous system, with normal and atypical symptoms. Certain clinical symptoms can be used to make a diagnosis. Its diagnosis is primarily clinical and epidemiologic, with serologic tests supporting it when necessary.

The preferred diagnostic testing method is a two-tiered approach, with an enzyme-linked immunoassay followed by a western blot. The bands are interpreted differently depending on how long the infection has been present.

Be aware of false-positive results and their causes while evaluating test findings.


How does one get Lyme disease?

During an outbreak of arthritis in children in the town of Lyme, Connecticut, in the 1970s, the whole spectrum of sickness was first recognized, and the condition was named Lyme disease.

The epidemiology and etiology of Lyme disease, as well as the benefits and drawbacks of current diagnostic tools and algorithms, are discussed.

Annually, more than 30,000 cases are reported in the United States. According to the US Centers for Disease Control and Prevention (CDC), the number of cases in 2017 was about 42,000, indicating a 16% increase from the previous year (CDC).

B. burgdorferi, a highly arthritogenic spirochete, is spread by Ixodes scapularis (the black-legged deer tick) and Ixodes pacificus (the Western black-legged tick). It causes the infection. Although the infection can occur at any time of year, it is most common from May to late September, when outdoor recreational activities are more common in tick-infested areas. The usual tick habitat is deciduous woods with a good layer of rotting plants to provide enough humidity. People can, however, get Lyme disease from their backyard.


IgM comes first, then IgG

The etiology of Lyme disease and the various stages of the infection should guide laboratory testing.

Only 5% of infected ticks that bite individuals pass their spirochetes to the human host, according to estimates.

The patient's innate immune system develops a response once infected, resulting in the typical erythema migrans (rash at the bite site). About 85% of infected people get a rash, which can develop anytime between 3 and 30 days after infection, but it most usually occurs after 7 days. Hence, a rash that appears within minutes of tick contact is not erythema migrans and does not suggest infection, but rather an early reaction to tick salivary antigens.

In the first 7 days following exposure, antibody levels remain below the detection limits of currently available serologic testing. Immunoglobulin M (IgM) antibody titers peak between 8 and 14 days following a tick encounter. However, if the patient is treated early with adequate antibiotic medication, IgM antibodies may never develop.

If the infection is not treated, the spirochete can spread from the bite site to other tissues through the bloodstream.

At this point, both cell- and antibody-mediated immunity kick in to kill the spirochetes. In 1-2 weeks, the IgM antibody response begins, followed by a significant IgG response in 2-4 weeks.

The IgM test for Lyme disease is less specific than the IgG test because it can cross-react with antigens other than those associated with B. burgdorferi.

Even after successful antibiotic treatment and eradication of the disease, once a patient is exposed to the spirochete and mounts an antibody-mediated response, the antibody profile may linger for months or years.

Untreated B. burgdorferi infections can persist despite the immune system's strong defenses. It can reduce the production of particular immunogenic surface-exposed proteins, modify its antigenic characteristics through recombination, and bind to extracellular matrix proteins in the patient's extracellular matrix to aid further dissemination.

The HLA-DR4 allele, which has been linked to antibiotic-refractory Lyme-related arthritis, is one example of host-genetic variables that play a role in the pathogenesis of Lyme disease.


The Lyme stages and testing

Lyme disease progresses through stages that are broadly characterized as early and late infections with a wide range of symptoms.

Infection in its early stages

Early disease is divided into two stages:

  • Localized infection or stage I: This is characterized by a single erythema migrans lesion and local lymphadenopathy.
  • Disseminated infection or stage II: This is characterized by multiple erythema migrans lesions far from the bite site, facial nerve palsy, radiculoneuritis, meningitis, carditis, or migratory arthritis or arthralgia.

Erythema migrans, cranial nerve palsy, high-grade or progressive conduction block, and recurrent migrating polyarthritis are highly specific physical presentations of Lyme disease. Arthralgia, myalgia, neck stiffness, palpitations, and myocarditis are some of the less specific symptoms and indicators of Lyme disease. At least 85% of individuals with the early illness have erythema migrans lesions.

Moreover, 60% of infected people may acquire disseminated infections if therapy is not initiated early in the disease. Lyme arthritis and neuroborreliosis are symptoms of an untreated, progressive infection.

Because of increasing awareness of the disease and starting treatment early, noncutaneous symptoms are less common now than in the past.

Infection that occurs later

Oligoarthritis (affecting any joint but most commonly the knee) and neuroborreliosis are symptoms of a late (stage III) infection. Even after adequate antimicrobial medication is completed, clinical signs and symptoms of Lyme disease can take months to resolve. This should not be understood as an ongoing, permanent infection but as an action mediated by the patient's immune system.


The gold standard for Lyme testing

The use of a laboratory test is determined by the patient's pretest probability of infection, epidemiologic risk of exposure, and clinical Lyme disease symptoms. Patients with a high pretest probability (for example, those who have had a tick bite followed by the typical erythema migrans rash) do not need to be tested and can begin antibiotic medication right away.

Serologic testing

Early Lyme illness is easily curable and has no long-term consequences.

Direct methods identify the spirochete by culture or polymerase chain reaction (PCR), whereas indirect methods detect antibodies. Lyme disease tests identifies two types of antibodies, i.e., IgM and IgG. Because direct testing for Lyme disease lacks sensitivity, serologic tests remain the gold standard. A typical two-tier testing technique using an enzyme-linked immunosorbent assay (ELISA) followed by a western blot for confirmation is now suggested.

The sensitivity of direct culture to diagnose Lyme disease is limited for various reasonsB. burgdorferi is difficult to develop in culture as it needs specific media, low temperatures, and extensive incubation periods. To begin with, there are only a few spirochetes in human tissues and body fluids, and bacterial levels decrease as infection duration and spread increase. These factors reduce the likelihood of discovering this bacterium.

In some cases, PCR may be beneficial.

Molecular assays should only be used in conjunction with serologic testing and are not part of the usual evaluation.

These tests have high specificity, but their sensitivity is inconsistent.

PCR testing may be beneficial in the following cases:

  • Before antibody responses arise in the early stages of infection
  • When serologic tests are unreliable because antibodies linger for several years after an infection in many patients and reinfection occurs
  • Because of the high baseline population seropositivity for anti-Borrelia antibodies induced by subclinical infection, serologic testing in endemic locations has a significant false-positive rate.

Plasmid-rich “blebs” are shed in higher concentrations than chromosomal DNA during active infection. Therefore, PCR assays that target plasmid-borne genes encoding outer surface proteins A and C (OspA and OspC) and variable major protein-like sequence expressed (VisE) are more sensitive than those that detect chromosomal 16s ribosomal RNA (rRNA) genes.

However, these plasmid-containing genes can be found in body tissues and fluids long after the infection has been cleared, and their presence does not always indicate the presence of disease. The presence of 16s rRNA genes on chromosomes is a stronger indicator of real organism viability.

The sensitivity of PCR for Borrelia DNA varies by sample type. The sensitivity and specificity of a skin biopsy sample collected from the leading edge of an erythema migrans lesion are 69% and 100%, respectively. The sensitivity of PCR of synovial fluid in individuals with Lyme arthritis is up to 80%. However, PCR of the cerebral fluid of individuals with neurologic symptoms of Lyme disease has a sensitivity of just 19%. Spirochetes are generally confined to tissues, and only a few are found in these body fluids. Therefore, PCR of other clinical samples, such as blood and urine, is not suggested.

The disadvantage of PCR is that a positive result does not always indicate a current infection because the DNA of the dead microorganism can survive for months after therapy is completed.


Indirect methods

Enzyme-linked immunosorbent assay

An ELISA is a type of immunoassay that uses enzymes.

Anti-Borrelia antibodies are detected using ELISA tests. Many laboratories still use whole-cell extracts of B. burgdorferi in early-generation ELISAs. The Wampole B. burgdorferi IgG/M EIA II assay and the Vidas Lyme screen are two examples. Recombinant proteins are used in newer ELISAs.

Flagellin (Fla), outer surface protein C (OspC), and VisE, particularly the invariable region 6, are two major targets for ELISA antibodies (IR6). The VisE-IR6 region is most preserved in B. burgdorferi.

Early-generation assays had an 89% sensitivity and a 72% specificity.

However, antibodies in the patient's serum may react with unrelated bacterial antigens, resulting in false-positive results. When assay results are unclear or positive, whole-cell sonicate assays are not suggested as an independent test and must be verified by western blot testing.

Because the number of antigens expressed at this stage is restricted, older and newer ELISAs are less sensitive during early infection.

All patients who are seronegative at the time of first testing and are suspected of having early Lyme disease should undergo follow-up testing to check for seroconversion.


Western blot analysis

IgM and IgG antibodies against specific B. burgdorferi antigens are detected by western blot (immunoblot) testing. It is considered positive if it identifies at least two of the three possible specific IgM bands in the first 4 weeks of disease or at least 5 of the 10 possible specific IgG bands after that time.

The “1-month rule” to interpret the IgM western blot test

Because of the risk of a false-positive test result, IgM reactivity alone should not be used to support the diagnosis if clinical symptoms and indications of Lyme disease have been present for more than 1 month.

In the diagnosis of Lyme disease, this is called the “1-month rule.”

Western blot testing is only half as sensitive as ELISA testing in early localized infection. Two-tiered testing is not beneficial in early disease because the overall sensitivity of a two-step algorithm is equal to that of its least sensitive component.

Western blot has limitations; however, it is now considered the most specific test for confirming Lyme disease. Because it is technically and interpretively difficult, it is not widely available. Although densitometric blot analysis techniques and automated scanning and scoring seek to alleviate some of these shortcomings, visual evaluation of the blots compromises the test's repeatability. Western blot, like ELISA, can produce false-positive results in healthy people who have not been exposed to ticks because nonspecific IgM immunoblots produce faint bands. This is because antibodies from B. burgdorferi react with antigens from other bacteria. About half of healthy people have low-level serum IgG reactivity to the FlaB antigen, which might lead to false-positive results. Other etiologies must be investigated when the western blot result is uncertain.

Serologic tests that are currently available have several drawbacks

  • It takes at least a week for antibodies against B. burgdorferi to develop.
  • In endemic locations, the background incidence of seropositivity might be as high as 4%, reducing the value of a positive test result.
  • Because antibodies can persist for months to years following effective antimicrobial therapy and illness cure, serologic tests cannot be used as cure tests. Consequently, a positive serologic result could indicate an active infection or remote exposure.
  • Antibodies can react with bacteria that are related to Borrelia, such as other Borrelia or Treponema spirochetes.
  • Other medical disorders, such as polyclonal gammopathies and systemic lupus erythematosus, might cause false-positive serologic test results.


Testing recommendations

Two-tier testing is standard

This gold standard for diagnosis was suggested to be a two-tiered testing procedure that included a sensitive ELISA followed by a western blot to confirm positive and inconclusive ELISA results. Negative ELISA results, however, do not necessitate additional testing.

An ELISA algorithm with two steps

In early localized disease, a two-step ELISA algorithm (without the western blot) that included the whole-cell sonicate assay followed by the VisE C6 peptide assay had higher sensitivity and comparable specificity than two-tiered testing.

In early disseminated infections, the increased sensitivity was significantly more pronounced (sensitivity 100% vs 40%, respectively). Both testing methodologies' sensitivity had reached 100% by late infection. The two-step ELISA technique proved easier to conduct in a repeatable manner than the western blot.

The Infectious Diseases Society of America is updating its existing standards, with an update coming later in 2022 that may shift the recommendation away from two-tiered testing and toward the two-step ELISA technique.


  • Bratton RL, Whiteside JW, Hovan MJ, Engle RL, Edwards FD. Diagnosis and treatment of Lyme disease. Mayo Clin Proc. 2008;83:566-571.
  • Schriefer ME. Lyme Disease Diagnosis: Serology. Clin Lab Med. 2015;35:797-814.