Lyme Disease Workup

Updated: May 17, 2023
  • Author: John O Meyerhoff, MD; Chief Editor: Herbert S Diamond, MD  more...
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Approach Considerations

In endemic areas, patients with probable erythema migrans and a recent source of tick exposure should be started on treatment without blood tests. At this early stage (the first several weeks of illness), the clinical probability of Lyme disease is high and the sensitivity of serologic tests is low. If the lesion is indeed erythema migrans, improvement should occur within a few days after initiation of empiric antibiotics, along with resolution of any constitutional symptoms.

Alternatively, observing the rash over several days is safe. In most patients with erythema migrans, some expansion of the rash is expected over 2-3 days without antibiotics. This is a reasonable alternative to immediate empiric therapy.

In contrast, laboratory tests are important for establishing the diagnosis in the many patients with suspected Lyme disease who do not recall a tick bite and did not notice or do not have erythema migrans. However, much confusion can occur in the interpretation of the tests used for Lyme disease. [46]

The most widely used tests for Lyme disease are antibody detection tests, which can demonstrate that a patient has been exposed to Borrelia burgdorferi but cannot confirm infection. In the presence of typical clinical manifestations and laboratory results suggestive of current disease activity (eg, elevated synovial and spinal fluid cell counts), they support the clinical diagnosis.

The US Centers for Disease Control and Prevention (CDC) recommends a two-step testing procedure. The first step typically consists of a screening enzyme immunoassay (EIA) or enzyme-linked immunosorbent assay (ELISA); if results are positive or equivocal, a Western immunoblot test is performed to confirm the results. [3]

In 2019, the US Food & Drug Administration (FDA) approved the use of concurrent or sequential EIA testing for diagnosis of Lyme disease. The FDA approved the new indication on the basis of data from clinical studies showing that this alternative approach, referred to as a modified two-tier test, is as accurate as testing with EIA or IFA plus Western blot. [4]

Guidelines from the Infectious Diseases Society of America, American Academy of Neurology, and American College of Rheumatology (IDSA/AAN/ACR) suggest performing acute-phase serum antibody testing in patients with 1 or more skin lesions that are suggestive of erythema migrans but atypical for it. If the initial result is negative, repeat testing can be performed on a convalescent-phase serum sample collected at least 2–3 weeks afterward. [6]

Routine use of sequential serologic testing in individual patients with early Lyme disease should be discouraged. In addition, acute and convalescent-phase serologic testing has no role in Lyme disease. Because titers may remain elevated for extended periods (as can the positivity of Western blots), convalescent testing is not helpful.

Culturing B burgdorferi is impractical. Obtaining adequate samples requires an invasive procedure, such as biopsy or lumbar puncture, and the organism is difficult to grow.

Even if the tick that bit the patient is available, testing the tick for B burgdorferi is not recommended. The presence or absence of B burgdorferi in an Ixodes tick does not reliably predict the likelihood of Lyme disease. [6]

Biopsy of dermatologic lesions suggestive of borrelial lymphocytoma or acrodermatitis chronica atrophicans in patients without a clear history of other symptoms suggestive of Lyme disease may be helpful. Biopsy of other skin lesions should be restricted to research settings.

Most, but not all, patients with borrelial lymphocytoma are seropositive for antiborrelial antibodies. This is true for all early disseminated manifestations of Lyme disease. In addition, essentially all patients with acrodermatitis chronica atrophicans are seropositive for antiborrelial antibodies. Seriously question the diagnosis in seronegative patients.

In patients with clinical findings typical of Lyme disease, a complete blood cell count (CBC), erythrocyte sedimentation rate (ESR), and liver function tests generally are unnecessary. However, leukopenia or thrombocytopenia suggests co-infection with Ehrlichia or Babesia species. Elevation of at least one liver enzyme level is reported to occur in 40% of patients with Lyme disease. This finding also is common in ehrlichiosis.

On urinalysis, microscopic hematuria and mild proteinuria have been described. Urine antigen testing has not been studied sufficiently. Because it has not been proven reliable or accurate, it should not be used as a diagnostic tool.

Joint aspiration for diagnostic reasons is unnecessary if only Lyme arthritis is suspected. However, arthrocentesis may be appropriate to exclude other causes of effusions, such as septic arthritis or, in adults, gout and pseudogout. In Lyme arthritis, joint fluid may have 25,000-125,000 white blood cells (WBCs)/µL, often with a polymorphonuclear predominance.

A retrospective study of children in areas where Lyme disease is endemic who presented with knee monoarthritis found that the presence of a peripheral blood absolute neutrophil count of 10 × 103 cells/mm3 or higher and an ESR of 40 mm/hour or higher predicted septic arthritis; no child with values below those cutoffs had septic arthritis. These researchers suggested that those criteria could be used to identify children with knee monoarthritis who are at low risk for septic arthritis and might not require diagnostic arthrocentesis. [48]

A joint guideline from the Infectious Diseases Society of America (IDSA), the American Academy of Neurology (AAN), and the American College of Rheumatology (ACR) recommends testing for Lyme disease in patients with plausible exposure to high-risk ticks who present with meningitis, painful radiculoneuritis, mononeuropathy multiplex, acute cranial neuropathies, or evidence of spinal cord (or rarely brain) inflammation The guideline recommends against routine Lyme disease testing for patients with other neurologic syndromes or psychiatric illnesses. [6]

In patients with Lyme disease meningitis, cerebrospinal fluid (CSF) analysis often reveals a mild pleocytosis (< 1000 cells/µL) with lymphocyte predominance. CSF antibody is considered positive when the titer is higher than in serum. [49]

In children, the "Rule of 7's" can be used to identify those patients who are unlikely to have Lyme meningitis and can be managed in an outpatient setting while awaiting Lyme serology test results. [50] The Rule of 7’s classifies children as being at low risk when they meet the following three criteria:

  • < 7 days of headache
  • < 70% CSF mononuclear cells
  • Absence of seventh or other cranial nerve palsy

Electrocardiograms (ECGs) show fluctuating levels of atrioventricular block in patients with syncopal or near-syncopal symptoms secondary to Lyme carditis. In patients with possible exposure but without symptoms of myocardial ischemia, such changes should prompt further investigation for Lyme disease.

Imaging studies are almost never indicated in patients with Lyme disease who present with early syndromes. Patients with some clinical syndromes may require imaging studies to exclude other disorders, depending on the specifics of the case. For example, a patient with fever and severe back pain, with signs of radiculopathy, might require spine imaging.


Serologic Testing

Serologic testing for Lyme disease is complex. Rational ordering and interpretation of these test results requires some understanding of the basic underlying principles and performance characteristics of the tests. The test results do not rule in or rule out Lyme disease; however, the results make a clinical diagnosis of Lyme disease more (or less) likely.

The most frequently used test is the enzyme immunoassay (EIA) or enzyme-linked immunosorbent assay (ELISA). Much less often used for this purpose is the immunofluorescent assay (IFA). [3]

The principal limitation of these serologic tests has been the high frequency of both false-negative results and false-positive results. False-negative results occur during the acute phase of Lyme disease, when patients have not yet developed a sufficient antibody response to give a positive serologic test. Seroconversion can take as long as 6-8 weeks after a tick bite. The false-negative rate for ELISA is 32% in early disease.

A variety of diseases, including Rocky Mountain spotted fever, syphilis, systemic lupus erythematosus, and rheumatoid arthritis, can cause false-positive ELISA results. Also, a small percentage of the healthy population has positive test results with ELISA testing. For these reasons, confirmatory Western blot testing is recommended.

Patients with early Lyme disease who are treated with antibiotics may never develop positive titers. Of patients with early disseminated disease, 90% have a positive titer. Some patients with late disease are seronegative, but significant controversy exists regarding the frequency of late seronegativity. Most authorities suggest that this phenomenon is rare.

Two-tier testing

The US Centers for Disease Control and Prevention (CDC) recommends a two-step testing procedure. The first step typically consists of an EIA or ELISA. [3] The test for the first step may measure either a total Lyme titer or separate immunoglobulin G (IgG) and immunoglobulin M (IgM) titers.

If the results of the initial test are positive or equivocal, the second step is to confirm the results with a Western blot. If signs and symptoms have been present for 30 days or less, both IgM and IgG Western blot testing are performed; if signs and symptoms have been present for more than 30 days, only IgG Western blot testing is performed.

For IgM blots, the test is considered positive if any two of the following three bands are present, as they are the most commonly found in early disease:

  • 23 kd
  • 39 kd
  • 41 kd

For IgG blots, any five of the following bands are considered a positive test result:

  • 18 kd
  • 21 kd
  • 28 kd
  • 30 kd
  • 39 kd
  • 41 kd
  • 45 kd
  • 58 kd
  • 66 kd
  • 93 kd

A positive IgM titer is reliable only when measured 30 days or less from symptom onset. In patients with a high probability of having early Lyme disease, IgM testing is 96% specific and 93% predictive. In the absence of treatment, IgM titers usually peak 6-8 weeks after infection and disappear within 4-6 months, although levels sometimes remain elevated for several months or years.

IgG antibodies are typically detectable within 6-8 weeks after infection, peak within 4-6 months, and remain elevated indefinitely. In late-stage disease (>4-6 wk after infection), IgG results are more useful than IgM results.

Careful consideration of both IgG and IgM antibodies is essential because the IgG response may be negative in as many as 50% of patients (particularly those with early disease), whereas a persistence of IgM antibodies can lead to false-positive findings in patients infected for more than 1 month who subsequently receive effective treatment. Of note, serologic results can remain positive years after adequate treatment and cannot be used to distinguish active from inactive disease. Similarly, positive IgM titers with negative IgG more than 6-8 weeks after exposure in an untreated patient is thought to represent a false-positive test.

Two-step testing is not indicated for patients with erythema migrans, because the rash may develop before the antibodies. Nor is it recommended for patients who have not been in endemic areas, because of the high false-positive rates in that setting. In addition, inadequate antibiotic therapy for early Lyme disease may fail to control the infection yet still suppress the antibody response, potentially yielding a false-negative result.

Western blot testing should be performed only in conjunction with antibody titer testing, and only as followup of a recent positive or equivocal ELISA titer. Ordering a “Lyme titer with reflex testing” ensures that two-step testing is performed properly.

In the United States, patients with extracutaneous involvement in the absence of treatment almost universally have positive titers. [51] In Europe, negative serum titers have been reported in patients with neurologic Lyme disease that was confirmed by intrathecal antibody production.

The results of one study noted that differing sensitivity and specificity were found between various assays used to detect anti-Borrelia antibodies in patients suspected of having Lyme disease. False-positive results occurred in 7% of healthy controls in two of the eight ELISA assays tested. This variability makes it very difficult to compare results from different laboratories, both among different patients and in individual patients. [52]

C6 peptide testing

A newer serologic test that measures IgG to a peptide from the sixth invariant region (C6) of the variable major proteinlike sequence-expressed (VlsE) lipoprotein of B burgdorferi may be more sensitive in patients with erythema migrans. [51] However, because the recommendation in patients with erythema migrans is to treat without obtaining laboratory tests, [53] there is no clear reason to perform this assay in clinical practice. The C6 peptide test may be effective in differentiating southern tick-associated rash illness (STARI) from Lyme disease, as well as confirming infection in patients who may have been infected in Europe.


Polymerase Chain Reaction Testing

Polymerase chain reaction (PCR) testing is growing in uses and availability, but is not readily available to most clinicians in routine practice. PCR remains a research technique in part because laboratories performing PCR tests must be meticulous in technique to minimize the likelihood of false-positive results. In addition, no large clinical series have been reported that assess the performance of Lyme disease PCR in the nonresearch setting.

PCR can be used to detect B burgdorferi DNA in the blood, CSF, urine, or synovial fluid within weeks of infection. The result is positive in approximately 30% of patients with active Lyme disease.

A notable disadvantage of PCR testing is the likelihood of false-negative results because of a sparsity of spirochetes in infected tissues. Likewise, inexperience with the PCR technique can yield false-positive findings when care is not taken to prevent contamination and when incorrect primers are used in preparing the specimen.

Although most PCR results become negative within 2 weeks of antimicrobial therapy, results can remain positive for years after apparent cure. One of the most compelling uses of PCR may be in confirming persistent or recurrent disease, because a positive result is highly specific for exposure to B burgdorferi.

With the exception of synovial fluid, PCR testing is not recommended because of unacceptable low sensitivity, especially from the CSF (though it does have high specificity if the result is positive). CSF titers to B burgdorferi should not be used for diagnosis of Lyme meningitis but may have value in patients who have recurrent infection or for following serial markers in patients with persistent symptoms. CSF titers should be performed and interpreted at a reference laboratory.


Blood Studies

In patients with Lyme disease, the white blood cell count (WBC) can be normal or elevated. The erythrocyte sedimentation rate (ESR) is usually elevated. The serum aspartate transaminase (AST) may be elevated. On complement testing, C3 and C4 levels are generally normal or slightly elevated. Antinuclear antibody (ANA) and rheumatoid factor test results are negative.


Synovial Fluid Analysis

In patients with Lyme arthritis, synovial fluid is usually inflammatory, with cell counts ranging from 500-98,000/µL reported. In adult patients, the fluid should also be examined for crystals to rule out gout and pseudogout.

One study that included 63 patients with Lyme arthritis found that although the majority had positive polymerase chain reaction (PCR) results for B burgdorferi DNA in synovial fluid, none of the samples tested were positive for B burgdorferi messenger RNA (a marker of spirochetal viability), even when the specimen was obtained before initiation of antibiotic treatment. These results suggest that detection of B burgdorferi DNA in synovial fluid is not a reliable test for active joint infection in Lyme disease. [54]


Cerebrospinal Fluid Evaluation

A lumbar puncture should be performed if Lyme meningitis is in the differential diagnosis. Whether all patients with cranioneuropathy require lumbar puncture before treatment is controversial. Occasionally Lyme disease presents as pseudotumor cerebri, and in such cases an opening pressure is essential for diagnosis. In most patients with isolated Bell palsy and no associated signs of aseptic meningitis, most physicians do not perform a lumbar puncture.

For most other patients with cranioneuropathies and suspected Lyme disease, a lumbar puncture should be performed, particularly in patients who live in an endemic area and present during peak Lyme disease season. Computed tomography (CT) scan or magnetic resonance imaging (MRI) should be performed before the lumbar puncture if increased intracranial pressure or mass lesion is suspected.

Unlike most bacterial infections in the spinal fluid, Lyme disease produces a pleocytosis characterized by mononuclear cells. In addition, spinal fluid levels of IgM and IgG antibodies to B burgdorferi should be measured, and an index of cerebrospinal fluid (CSF) to serum antibody (immunoglobulin-to-albumin ratio) should be calculated. [55] This is particularly true in patients who have no other signs of Lyme disease.

Although CSF cultures are positive in fewer than 10% of Lyme disease patients with apparent meningitis, intrathecal antibodies and a lymphocytic pleocytosis (approximately 100 cells/µL) are present in more than 80%. Patients with meningitis typically have elevated protein concentrations (>50 mg/dL) but normal glucose levels (45-80 mg/dL). Oligoclonal bands specific for B burgdorferi may be present.

Ongoing controversy surrounds the diagnosis of neurologic Lyme disease. One of the most important concepts to understand is that a positive Lyme disease serology in CSF does not mean that the patient has neuroborreliosis. It could represent evidence of a previous infection or simply reflect potential leakage of serum antibodies across the blood-brain barrier. [56] IgG and IgM antibodies may persist in CSF long after adequate treatment and in the absence of evidence of active neurologic disease.

Intrathecal anti-Borrelia antibody production is typically seen within 3-6 weeks of infection. Anti-Borrelia antibody CSF-to-serum index has been reported to show a 97% specificity and 75% sensitivity for the diagnosis of neuroborreliosis. [57] A CSF-to-serum index greater than 1.0 suggests synthesis of antibody in the central nervous system (CNS).

It has been proposed that four of the following five criteria should be present in order to diagnose neuroborreliosis [58] :

  • No past history of neuroborreliosis

  • CSF anti-B burgdorferiantibodies

  • Positive anti-B burgdorferi antibody index

  • Favorable clinical outcome after proper antibiotic therapy

  • Absence of alternative diagnosis


Brain Imaging

Magnetic resonance imaging (MRI) shows abnormalities in approximately 15-20% of patients in the United States who have neurologic manifestations of Lyme disease. In European patients with CSF-confirmed Lyme disease, imaging findings have suggested that microvasculitis and macrovasculitis in the central nervous system may be responsible for neurologic sequelae and the MRI changes seen in patients with neuroborreliosis.

Punctate lesions of the periventricular white matter are common and resemble changes seen in demyelinating or inflammatory disorders. In an attempt to differentiate radiologic manifestations of neuroborreliosis and multiple sclerosis, one study proposed that occult brain tissue damage (seen by brain magnetization transfer and diffusion tensor magnetic resonance) are not common in neuroborreliosis, as opposed to multiple sclerosis. [59] Space-occupying lesions have also been reported as a rare manifestation.

Functional brain imaging, such as single-photon emission computed tomography (SPECT) scanning, may contribute to the diagnosis of chronic neurologic Lyme disease. In a study of SPECT scanning in 183 patients who met the clinical definition of chronic Lyme disease, 75% showed abnormalities in perfusion to various areas of the brain. By comparison, MRI demonstrated brain abnormalities in only 14% of study patients. In 70% of patients, the abnormalities resolved or improved over 1-2 years with antibiotic treatment. [60]



Because of the fastidious growth requirements for B burgdorferi, culture has not been a useful test in the past. In routine practice, borrelial cultures are often unavailable. [61]

In the skin, where findings are most likely to be positive, culturing is least likely to be clinically useful, except in cases of atypical rash. In other body fluids (eg, blood, synovial fluid, CSF), the yield is lower. Although one study from an endemic area reported positive blood culture results in 43.7% of untreated adult patients with erythema migrans, this required culturing specifically for Lyme disease. In addition, all but two of the 213 patients met CDC criteria for Lyme disease and warranted treatment, regardless of culture results. [8]



Approximately 60-80% of specimens isolated from the leading edge of a suspected erythema migrans lesion by means of saline-lavage needle aspiration or 2-mm punch biopsy reveal B burgdorferi. However, because the presence of a lesion along with a compatible history and clinical presentation are sufficient to initiate treatment, these skin biopsy procedures are seldom performed.


Histologic Findings

Histologic findings in erythema migrans are nonspecific, usually showing a perivascular cellular infiltrate consisting of lymphocytes, plasma cells, and histiocytes. Occasionally, mast cells and neutrophils are seen. Central biopsies may show eosinophilic infiltrates consistent with a local reaction to an arthropod bite. Spirochetes occasionally may be identified using silver or antibody-labeled stains, although usually, a paucity of spirochetes is found in the tissues of patients with Lyme disease.

Borrelial lymphocytoma

Histologic examination is recommended in patients with suspected borrelial lymphocytoma, when the location of the lesion or the clinical history is not clear enough to support a diagnosis. Borrelial lymphocytoma biopsy specimens show a dense dermal lymphocytic infiltrate with lymphoid follicles and pseudogerminal centers. Lymphocytes with both B- and T-cell markers, occasional macrophages, plasma cells, and eosinophils are seen.

Acrodermatitis chronica atrophicans

In acrodermatitis chronica atrophicans, biopsy specimens from early lesions show a lymphocytic dermal infiltrate, sometimes perivascular in location, with some vascular telangiectasia and lymphedema. Plasma cells also may be seen in the cellular infiltrate. Later lesions demonstrate epidermal thinning with loss of skin appendages. At this stage, plasma cells may be the only feature to distinguish acrodermatitis chronica atrophicans from morphea.

The fibrotic nodules show fibrosis of the deeper dermis and sometimes, hyalinization of collagen bundles. B burgdorferi occasionally can be cultivated from the lesions; in one patient, cultivation was successful more than 10 years after the lesion's first appearance.