Appropriate laboratory testing in Lyme disease
Release date: November 1, 2019
Expiration date: October 31, 2020
Estimated time of completion: 1 hour
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ABSTRACT
Testing for Lyme disease is challenging and if done incorrectly can lead to unnecessary treatment. To interpret serologic test results, first assess the patient’s pretest probability of infection based on the probability of exposure and clinical findings. Two-tiered testing remains the gold standard in diagnosing Lyme disease, although new guidelines may be published soon.
KEY POINTS
- Lyme disease, the most common tick-borne infection in North America, is a complex multisystem bacterial disease caused by Borrelia burgdorferi.
- Lyme disease preferably affects the skin, joints, and nervous system and presents with typical and atypical features. Certain clinical features are diagnostic. Its diagnosis is mainly clinical and epidemiologic and, when doubtful, is supported by serologic testing.
- Standard 2-tiered testing is the diagnostic testing method of choice—enzyme-linked immunoassay followed by Western blot. Interpretation of the bands depends on the duration of infection.
- When interpreting the test results, be aware of false-positives and the reasons for them.
Interpreting the IgM Western blot test: The ‘1-month rule’
If clinical symptoms and signs of Lyme disease have been present for more than 1 month, IgM reactivity alone should not be used to support the diagnosis, in view of the likelihood of a false-positive test result in this situation.18 This is called the “1-month rule” in the diagnosis of Lyme disease.13
In early localized infection, Western blot is only half as sensitive as ELISA testing. Since the overall sensitivity of a 2-step algorithm is equal to that of its least sensitive component, 2-tiered testing is not useful in early disease.13
Although currently considered the most specific test for confirmation of Lyme disease, Western blot has limitations. It is technically and interpretively complex and is thus not universally available.13 The blots are scored by visual examination, compromising the reproducibility of the test, although densitometric blot analysis techniques and automated scanning and scoring attempt to address some of these limitations.13 Like the ELISA, Western blot can have false-positive results in healthy individuals without tick exposure, as nonspecific IgM immunoblots develop faint bands. This is because of cross-reaction between B burgdorferi antigens and antigens from other microorganisms. Around 50% of healthy adults show low-level serum IgG reactivity against the FlaB antigen, leading to false-positive results as well. In cases in which the Western blot result is indeterminate, other etiologies must be considered.
,False-positive IgM Western blots are a significant problem. In a 5-year retrospective study done at 63 US Air Force healthcare facilities, 113 (53.3%) of 212 IgM Western blots were falsely positive.19 A false-positive test was defined as one that failed to meet seropositivity (a first-tier test omitted or negative, > 30 days of symptoms with negative IgG blot), lack of exposure including residing in areas without documented tick habitats, patients having atypical or no symptoms, and negative serology within 30 days of a positive test.
In a similar study done in a highly endemic area, 50 (27.5%) of 182 patients had a false-positive test.20 Physicians need to be careful when interpreting IgM Western blots. It is always important to consider locale, epidemiology, and symptoms when interpreting the test.
Limitations of serologic tests for Lyme disease
Currently available serologic tests have inherent limitations:
- Antibodies against B burgdorferi take at least 1 week to develop
- The background rate of seropositivity in endemic areas can be up to 4%, affecting the utility of a positive test result
- Serologic tests cannot be used as tests of cure because antibodies can persist for months to years even after appropriate antimicrobial therapy and cure of disease; thus, a positive serologic result could represent active infection or remote exposure21
- Antibodies can cross-react with related bacteria, including other borrelial or treponemal spirochetes
- False-positive serologic test results can also occur in association with other medical conditions such as polyclonal gammopathies and systemic lupus erythematosus.12
RECOMMENDATIONS FOR TESTING
Standard 2-tier testing
The CDC released recommendations for diagnosing Lyme disease after a second national conference of serologic diagnosis of Lyme disease in October 1994.18 The 2-tiered testing method, involving a sensitive ELISA followed by the Western blot to confirm positive and indeterminate ELISA results, was suggested as the gold standard for diagnosis (Figure 3). Of note, negative ELISA results do not require further testing.11
The sensitivity of 2-tiered testing depends on the stage of the disease. Unfortunately, this method has a wide range of sensitivity (17% to 78%) in stage 1 disease. In the same stage, the sensitivity increases from 14.1% in patients with a single erythema migrans lesion and early localized infection to 65.4% in those with multiple lesions. The algorithm has excellent sensitivity in late stage 3 infection (96% to 100%).5
A 2-step ELISA algorithm
A 2-step ELISA algorithm (without the Western blot) that includes the whole-cell sonicate assay followed by the VisE C6 peptide assay actually showed higher sensitivity and comparable specificity compared with 2-tiered testing in early localized disease (sensitivity 61%–74% vs 29%–48%, respectively; specificity 99.5% for both methods).22 This higher sensitivity was even more pronounced in early disseminated infection (sensitivity 100% vs 40%, respectively). By late infection, the sensitivities of both testing strategies reached 100%. Compared with the Western blot, the 2-step ELISA algorithm was simpler to execute in a reproducible fashion.5
The Infectious Diseases Society of America is revising its current guidelines, with an update expected late this year, which may shift the recommendation from 2-tiered testing to the 2-step ELISA algorithm.
Multiplex testing
To overcome the intrinsic problems of protein-based assays, a multiplexed, array-based assay for the diagnosis of tick-borne infections called Tick-Borne Disease Serochip (TBD-Serochip) was established using recombinant antigens that identify key immunodominant epitopes.8 More studies are needed to establish the validity and usefulness of these tests in clinical practice.
Who should not be tested?
The American College of Physicians6 recommends against testing in patients:
- Presenting with nonspecific symptoms (eg, headache, myalgia, fatigue, arthralgia) without objective signs of Lyme disease
- With low pretest probability of infection based on epidemiologic exposures and clinical features
- Living in Lyme-endemic areas with no history of tick exposure6
- Presenting less than 1 week after tick exposure5
- Seeking a test of cure for treated Lyme disease.
DIAGNOSIS IN SPECIAL SITUATIONS
Early Lyme disease
The classic erythema migrans lesion on physical examination of a patient with suspected Lyme disease is diagnostic and does not require laboratory confirmation.10
In ambiguous cases, 2-tiered testing of a serum sample during the acute presentation and again 4 to 6 weeks later can be useful. In patients who remain seronegative on paired serum samples despite symptoms lasting longer than 6 weeks and no antibiotic treatment in the interim, the diagnosis of Lyme disease is unlikely, and another diagnosis should be sought.3
Antimicrobial therapy may block the serologic response; hence, negative serologic testing in patients started on empiric antibiotics should not rule out Lyme disease.6
PCR or bacterial culture testing is not recommended in the evaluation of suspected early Lyme disease.
Central nervous system Lyme disease
Central nervous system Lyme disease is diagnosed by 2-tiered testing using peripheral blood samples because all patients with this infectious manifestation should have mounted an adequate IgG response in the blood.11
B cells migrate to and proliferate inside the central nervous system, leading to intrathecal production of anti-Borrelia antibodies. An index of cerebrospinal fluid to serum antibody greater than 1 is thus also indicative of neuroborreliosis.12 Thus, performing lumbar puncture to detect intrathecal production of antibodies may support the diagnosis of central nervous system Lyme disease; however, it is not necessary.11
Antibodies persist in the central nervous system for many years after appropriate antimicrobial treatment.
Lyme arthritis
Articular involvement in Lyme disease is characterized by a robust humoral response such that a negative IgG serologic test virtually rules out Lyme arthritis.23 PCR testing of synovial fluid for borrelial DNA has a sensitivity of 80% but may become falsely negative after 1 to 2 months of antibiotic treatment.24,25 In an algorithm suggested by Puius et al,23 PCR testing of synovial fluid should be done in patients who have minimal to no response after 2 months of appropriate oral antimicrobial therapy to determine whether intravenous antibiotics are merited.
Table 5 summarizes the tests of choice in different clinical stages of infection.
Acknowledgment: The authors would like to acknowledge Anita Modi, MD, and Ceena N. Jacob, MD, for reviewing the manuscript and providing valuable suggestions, and Belinda Yen-Lieberman, PhD, for contributing pictures of the Western blot test results.
