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. Author manuscript; available in PMC: 2016 Jun 1.
Published in final edited form as: Infect Dis Clin North Am. 2015 Jun;29(2):295–307. doi: 10.1016/j.idc.2015.02.005

Laboratory Diagnosis of Lyme Disease - Advances and Challenges

Adriana R Marques 1
PMCID: PMC4441761  NIHMSID: NIHMS663549  PMID: 25999225

Synopsis

Lyme disease is the most common tick-borne illness in the United States and Europe. Culture for B. burgdorferi is not routinely available. PCR can be helpful in synovial fluid of patients with Lyme arthritis. The majority of laboratory tests performed for the diagnosis of Lyme disease are based on detection of the antibody responses against B. burgdorferi in serum. The sensitivity of antibody-based tests increases with the duration of the infection, and patients who present very early in their illness are more likely to have a negative result. Patients with erythema migrans should receive treatment based on the clinical diagnosis. The current Centers for Disease Control and Prevention recommendations for serodiagnosis of Lyme disease is a 2-tiered algorithm, an initial enzyme immunoassay (EIA) followed by separate IgM and IgG Western blots if the first EIA test result is positive or borderline. The IgM result is only relevant for patients with illness duration of less than a month. While the 2-tier algorithm works well for later stages of the infection, it has low sensitivity during early infection. A major advance has been the discovery of VlsE and its C6 peptide as markers of antibody response in Lyme disease. Specificity is extremely important in Lyme disease testing, as the majority of tests are being performed in situations with low likelihood of the disease, a situation where a positive result is more likely to be a false positive. Current assays do not distinguish between active and inactive infection, and patients may continue to be seropositive for years. There is a need to simplify the testing algorithm for Lyme disease, improving sensitivity in early disease while still maintaining high specificity and providing information about the stage of infection. The development of a point of care assay and biomarkers for active infection would be major advances for the field.

Keywords: Lyme disease, Borrelia burgdorferi, Laboratory diagnosis, Serology

Introduction

Lyme disease, or Lyme borreliosis, is a multisystem illness caused by the spirochete Borrelia burgdorferi and it is the most common tick-borne illness in the United States and Europe. Newly revised estimates from the Centers for Disease Control and Prevention (CDC) suggest that there are likely to be around 300,000 new cases of Lyme disease per year in the United States1. B. burgdorferi is transmitted by the bite of infected ticks of the Ixodes ricinus complex. In the United States, most cases of Lyme disease are due to the blacklegged tick (Ixodes scapularis), occurring at the mid-Atlantic, northeast, and upper Midwest regions.

B. burgdorferi is a gram-negative bacteria, and has the elongated and spiral shape of the spirochetes2. It varies from 10 to 30 μm in length and 0.2 to 0.5 μm in width. It has a linear chromosome and a variable number of circular and linear plasmids3. The B. burgdorferi sensu lato group includes at least 20 genospecies4. Three genospecies are most commonly associated with human infections: B. burgdorferi sensu stricto, which causes disease in North America and Europe, B. afzelii and B. garinii, which occur in Europe and Asia5. Additional genospecies have been demonstrated to at least occasionally cause human disease in Europe (e.g., B. spielmanii and B. valaisiana). 5 There is some variation in the clinical presentation depending on the infecting genospecies, with B. burgdorferi sensu stricto predominating in arthritis, B. garinii in neurologic disease and B. afzelii in chronic skin manifestations6. Even within the same genospecies, there is variation in presentation and dissemination capability7,8.

For clinical purposes, Lyme disease is divided into early localized, early disseminated and late stages. Lyme disease usually begins with the characteristic skin lesion, erythema migrans (EM), at the site of the tick bite9–11. After several days or weeks, the spirochete may disseminate and patients can develop neurologic, cardiac and rheumatologic involvement12–15. The infection is characterized by low number of bacteria, which can persist in collagen rich tissues. While antibiotic therapy will accelerate resolution of the disease, manifestations can spontaneously regress without antibiotic therapy. The resolution of disease is mediated by immune responses, which control the infection. However, without antibiotic therapy, it can recur and/or new manifestations can appear9,16,17.

The available laboratory methods for the diagnosis of Lyme disease fall into two categories: direct methods to detect B. burgdorferi, and indirect methods that detect the immune response against it, mainly the detection of antibodies against B. burgdorferi. It is important to recognize that laboratory tests should be ordered and interpreted in the context of the clinical evaluation and the likelihood that the patient has Lyme disease. This chapter reviews the laboratory diagnostics for Lyme disease (with focus on the United States), and discusses current recommendations and new developments in the field.

Direct Methods for Detection of B. burgdorferi

Laboratory tests for direct detection of B. burgdorferi are hampered by very low numbers of spirochetes in the majority of clinical samples. The lack of sensitive, relatively easy, fast, direct tests for the presence of B. burgdorferi is one of the main challenges in the laboratory diagnosis of Lyme disease. While direct tests for B. burgdorferi can sometimes be helpful, none are required for the diagnosis of the disease. The main direct test modalities used are culture and PCR. Histopathology has limited utility, being used mostly to exclude other diseases, and in the evaluation of suspected cases of borrelial lymphocytoma and acrodermatitis chronica atrophicans18,19. Detection of B. burgdorferi is difficult and time-consuming due to the extreme scarcity of organisms20–23. Warthin-Starry and modified Dieterle silver stains, focus-floating microscopy, as well as direct and indirect immunofluorescence assays with anti-borrelial antibodies have been used, but can be difficult to interpret and require special expertise and careful use of controls24–26. At present, no antigen assays are recommended for the diagnosis of Lyme disease. A research test for detection of OspA has been used in cerebrospinal fluid27. An assay to detect antigens in urine has been shown to be unreliable28.

Culture

Culture is not a routinely available diagnostic method for the diagnosis of Lyme disease in clinical practice, due to its relatively low sensitivity, long incubation and the requirement of special media and expertise. However, the ability to isolate and culture B. burgdorferi is essential in Lyme disease research, and culture remains the gold standard to confirm the diagnosis. Methods that would improve sensitivity and simplify the procedure, so it could be adopted more extensively, are needed.

B. burgdorferi has a limited metabolic capacity and requires a complex growth media for cultivation. Media used for culturing B. burgdorferi include variations of the Barbour-Stoenner-Kelly (BSK) medium29 and the modified Kelly–Pettenkofer (MKP) medium30. Cultures are examined using dark-field microscopy or fluorescent microscopy after staining aliquots with acridine orange, but sensitivity is improved by testing aliquots with PCR methods31. B. burgdorferi replicates slowly and cultures are kept for 8 to 12 weeks before being considered negative31.

The probability of culturing B. burgdorferi depends on the specimen, the stage of the disease, and the expertise of the laboratory. It may also depend on the genotype32. Antibiotic therapy with agents effective against B. burgdorferi (even a single dose) will significantly impact the recovery rate33,34.

Culture of skin biopsies from EM has a sensitivity of 40 to 60%30,34–41. In the US, where disease is caused by B. burgdorferi sensu stricto, positive cultures are associated with shorter duration of the disease and smaller lesions35,42. Positive skin biopsy cultures in central Europe (where most of the isolates were B. afzelii) were associated with larger lesions (up to about 15 cm of diameter) and increased duration (up to 30 days)43. These findings are likely related to the different Borrelia species and the host immune response that eventually controls the infection. B. afzelii causes slow-growing EM lesions with few systemic symptoms, while B. burgdorferi sensu stricto is associated with more rapidly expanding skin lesions and more systemic symptoms11. Culture is moderately successful in skin biopsies of acrodermatitis chronica atrophicans lesions34.

Culture of 9-ml plasma samples from untreated patients with early and early disseminated infection has a sensitivity of around 40%, which can be increased to 75% by frequently testing culture aliquots with a sensitive PCR. Blood cultures are more likely to be positive in patients with multiple EM31,36. B. burgdorferi is seldom cultured from the blood of Lyme disease patients with later manifestations of the disease44,45. Culture of cerebrospinal fluid is rarely positive41,46–48. B. burgdorferi has not been reliably cultivated from synovial fluid49.

There are serious concerns50 regarding a new serum culture assay that claims a high positivity rate51 and further validation is needed. Results from another culture assay reported as having high positivity rates in patients with chronic disease52 could not be replicated53,54.

PCR

In general, sensitivity of PCR assays for detection of B. burgdorferi DNA directly in skin or blood samples appears similar to culture, but there is more variation due to methodology, gene targets and primers sets used34–38,42,45. When optimal culture methods are employed, PCR seems to be less sensitive, particularly for plasma samples, which may relate to the smaller sample volume tested in PCR assays36. A new assay using broad-range PCR and electrospray ionization mass spectrometry appears promising55. At this point, the main use of PCR assays is for evaluating synovial fluid samples in patients with Lyme arthritis, where B. burgdorferi DNA can be detected in up to 70 to 85% of patients42,56,57. A positive PCR may not necessarily mean an infection is active.42 Sensitivity of PCR in cerebrospinal fluid samples of patients with early neuroborreliosis is low (10–30%) and even lower in late disease58.

Indirect methods

Indirect methods detect the immune response of the host against the causative organism. The majority of laboratory tests performed for Lyme disease are based on detection of the antibody responses against B. burgdorferi in serum. Antibody-based assays are the only type of diagnostic testing for Lyme disease approved by the US Food and Drug Administration.

A major problem in laboratory diagnostics of Lyme disease is the appropriate use of tests. About 3.4 million Lyme serologic tests are done in the United States every year59, vastly above the estimated number of 300,000 cases of the disease. It is likely that tests are being used in situations where they are not recommended, including ruling out Lyme disease in populations with a low probability of having the disease. The predictive value of a test is determined by its sensitivity, specificity and the prevalence of the disease in the population to be tested. Consequently, in a patient with low probability of disease, a negative test rules out the disease while a positive test is more likely to be a false positive.

To improve the specificity of serologic testing for Lyme disease, a 2-tier approach (Figure 1) was recommended in 1995 by the CDC60. The first step uses a sensitive enzyme immunoassay (EIA) or rarely, an indirect immunofluorescence assay (IFA). If the test is negative, there is no further testing. If the test is borderline or positive, the sample is retested using separate IgM and IgG Western blots (WB, also referred to as immunoblots in the literature) as the second step. The WB is interpreted using standardized criteria, requiring at least two of three signature bands for a positive IgM WB, and 5 of 10 signature bands for a positive IgG WB. The IgM WB results are used only for disease of less than 4 weeks of duration. These recommendations apply to infection acquired in the US, as other species within the B. burgdorferi sensu lato complex can cause disease in Europe and Asia.

Figure 1. Current CDC Recommendations on Serologic Diagnosis of Lyme Disease: 2-Tier Algorithm.

Figure 1

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aBoth IgG and IgM WB results will be reported, but an IgM WB positive result is only significant for patients who have been ill for less than a month. ELISA: Enzyme-linked immunosorbent assay, IFA: Immunofluorescence Antibody Assay) IgM: immunoglobulin M, IgG: immunoglobulin G, WB: Western Blot. Adapted from 60

The use of specialty laboratories offering non-validated Lyme diagnostic tests, including unique interpretation of western blot results, is discouraged. They offer no documented advantage in terms of sensitivity while there is a large decrease in specificity61. The use of antibody assays in synovial fluid is not recommended62. There is little published information about use of western blot in the cerebrospinal fluid for the diagnosis of neuroborreliosis.

The current 2-tier algorithm works relatively well when used as recommended, but there are many areas for improvement. Problems include the low sensitivity during early infection, subjective interpretation of bands, and confusion by health care providers and patients regarding how to interpret results.

Most assays are based on whole cell sonicate (WCS) derived from cultured B. burgdorferi. WCS-based assays can have a significant number of false-positive results because of the presence of cross-reactive antigens63. Also, proteins expressed in culture can differ from in vivo expressed antigens. An example is the VlsE lipoprotein, which causes a rapid and strong humoral response during infection, while there is minimal VlsE expression in cultured B. burgdorferi. Adding VlsE to both 1st and 2nd tier tests has improved their performance64. Tests using the C6 peptide (a 26-amino acid peptide derived from invariant region 6 of VlsE) have comparable sensitivity to WCS-based EIAs, with significantly improved specificity, most markedly in patients with other diseases64–72. The C6 ELISA can also be used in patients who acquire the infection in Europe, as it is able to detect antibody responses elicited by other B. burgdorferi sensu lato species, and can be used as a stand-alone diagnostic strategy when such cases are evaluated in the US70,73. A variety of other recombinant and synthetic antigens have been evaluated for use in serodiagnosis of Lyme disease, including antigens combining portions of different proteins. Conserved regions of OspC, an antigen recognized early during the course of infection by B. burgdorferi, have been explored to develop diagnostic peptides, used as single or as part of multi-peptide assays66,68,74–77.

The sensitivity of antibody-based tests increases with the duration of the infection, and there will be a lag from initial infection until the time when there are sufficient levels of antibodies to be detected. Patients who present very early in their illness are more likely to have a negative result. Less than 50% of the patients with EM are positive at presentation, and these patients should receive treatment based on the clinical diagnosis. Serological tests are most helpful in patients with clinical findings indicating later stages of Lyme disease.

Figure 2 demonstrates how the duration of illness substantially affects the result of antibody-based tests. In a large study comparing the C6 ELISA with a WCS ELISA and the two-tier algorithm69, patients with single EM lesions were less likely to be seropositive than patients with multiple EM; and patients in the convalescent phase were more likely to be positive than patients in the acute phase. The vast majority of patients with Lyme arthritis or late neuroborreliosis were positive (Figure 2A). In another study78, less then 50% of patients with a single EM were positive by WCS ELISA or C6 ELISA and only 14% were positive by 2-tier testing when the patients were tested within the first week of illness, but the sensitivity of the tests increased with each weekly time point thereafter (Figure 2B).

Figure 2. Serological Results, Clinical Presentation and Duration of Illness.

Figure 2

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Panel A shows rates of seropositivity for the C6 ELISA, WCS ELISA and 2-tier algorithm in relationship to disease presentation and time of sample (acute and convalescent) while panel B compares rates of seropositivity in relationship with duration of disease in patients with a single erythema migrans. EM: erythema migrans, LNB: Lyme neuroborreliosis, WCS: whole cell sonicate. Adapted from 69,78.

As shown in many studies64,69,71,72,78, the additional IgM WB step decreases sensitivity in early disease, the only situation where its use is indicated. Positive IgM results for Borrelia can occur in more than 40% of parvovirus B19 infections79, has been observed in patients with human granulocytic anaplasmosis80, Epstein-Barr virus infections and patients with autoimmune diseases. Additionally, false positive IgM WBs are common in commercial laboratories81, and there is misinterpretation of “positive” IgM results in those patients with symptoms > 4 weeks. Therefore, there is a need to change the testing algorithm for early Lyme disease, avoiding the use of the IgM WB. Possible strategies include the use of the WCS ELISA followed by the C6 ELISA71,82, the addition of the VlsE band64, and the use of multi-peptide assays75,76.

Future developments that are needed include point of care tests. These tests would be particularly useful in evaluating patients with stage 2 manifestations of Lyme disease like facial palsy or carditis. Currently, if these patients do not have other manifestations of Lyme disease or a very suggestive history, the diagnosis may depend on serological tests results resulting in a delay in appropriate therapy.

Current assays do not distinguish between active and inactive infection, and patients may continue to be seropositive for years, including an IgM response, even after adequate antibiotic treatment83,84. Hopefully, with further studies utilizing new, promising immunoassay techniques, a combination of multiple antigens can be developed that will help in early diagnosis, inform on the stage and disease manifestations, and on the presence of active versus past infection85–87.

Intrathecal antibody production

The concomitant analysis of serum and cerebrospinal fluid is used to demonstrate selective production of anti-B. burgdorferi antibodies in the central nervous system. Measuring the antibody concentration only in the cerebrospinal fluid can be misleading, as a positive result may be due to passive transfer of antibodies from the serum. Evidence of intrathecal antibody production is considered a gold standard for the diagnosis of Lyme neuroborreliosis in Europe, where the vast majority of the studies originate and where B. garinii is the species most often associated with neurologic disease. There are many difficulties in the interpretation of results from these studies, due to the lack of a gold standard, the use of different case definitions, different assays and interpretative criteria, retrospective evaluation, and little comparison among assays and among laboratories. Overall, the sensitivity of intrathecal antibody production in acute Lyme neuroborreliosis is around 50%41,46,88–95. Intrathecal antibody can persist after therapy96,97. While there are few studies, positive intrathecal antibody production seems to be found less frequently in neuroborreliosis patients in the US14,27,93.

CXCL13

CXCL13 is a B lymphocyte chemoattractant chemokine that is increased in the cerebrospinal fluid of patients with acute Lyme neuroborreliosis and may be helpful in certain clinical settings, but its diagnostic value remains to be established46,98,99. At this point, this test is not routinely available to the clinician.

Other Tests

The clinical usefulness of cell proliferation assays, ELISPOT assays, cytokine measurements, complement split products and lymphocyte transformation tests have not been established, and these tests should not be used for the diagnosis of Lyme disease. Natural killer cell measurements (CD57) are not helpful100.

Xenodiagnosis, using the natural tick vector (Ixodes scapularis) to detect evidence of infection in Lyme disease, is an experimental test, and its clinical applications will depend on the results of future studies. Although xenodiagnosis is unlikely to be used in routine practice, it can offer researchers a tool to develop new tests for the disease.

Conclusion

Over the past years, major advances in laboratory testing for Lyme disease have occurred, but there is need for further progress. Improvements of several aspects of the currently recommended testing algorithm are needed. These include making the algorithm simpler, possibly as a single test or procedure, with objective, quantitative data, that is more sensitive in early disease and independent of disease duration. The use of the current IgM Western blot should be avoided, as it decreases the sensitivity in the clinical situations where it is recommended (early Lyme disease), while having lower specificity than tests for IgG antibody generally. There is a need to improve direct methods for detection of B. burgdorferi, and to develop accurate, sensitive and rapid diagnostic tests for early Lyme disease, preferably point-of-care tests. No current test can be used to follow the response to antibiotic therapy; the development of biomarkers for active infection would be a major advance for the field.

Key Points.

  • It is difficult to demonstrate B. burgdorferi by direct techniques (culture and polymerase chain reaction [PCR]). The spirochete is more easily found in the skin and plasma samples of patients with early disease (erythema migrans), and in the synovial fluid of patients with Lyme arthritis (using PCR).

  • The sensitivity of antibody-based tests increases with the duration of the infection. Less than 50% of the patients with erythema migrans are positive at presentation. These patients should receive treatment based on the clinical diagnosis.

  • Serological tests are most helpful in patients with clinical findings indicating later stages of Lyme disease.

  • Many tests for Lyme disease are being performed in patients with low likelihood to have the disease, a situation where a positive result is more likely to be a false positive.

  • The current assays do not distinguish between active and past infection, and patients may continue to be seropositive for years.

  • The use of nonvalidated Lyme diagnostic tests is not recommended.

Acknowledgments

This research was supported by the Intramural Research Program of the NIH, National Institute of Allergy and Infectious Diseases.

Footnotes

Disclaimer:

The findings and conclusions in this article are those of the author and do not necessarily represent the official views of the National Institute of Allergy and Infectious Diseases.

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