What is the recommended laboratory testing approach for a patient suspected of having Lyme disease, particularly in relation to enzyme-linked immunosorbent assay (ELISA) and Western blot tests, and how should results be interpreted in the context of patient demographics and clinical presentation?

Laboratory Testing for Lyme Disease

Standard Two-Tiered Testing Algorithm

The recommended laboratory approach for suspected Lyme disease is a two-tiered serologic testing algorithm: first-tier enzyme immunoassay (EIA) or immunofluorescence assay (IFA), followed by reflex Western immunoblot (both IgM and IgG) only if the first-tier test is positive or equivocal. 1, 2, 3

First-Tier Testing

• Order an EIA (or IFA) that measures total antibody response (IgM and IgG) to Borrelia burgdorferi antigens 1
• Most U.S. laboratories use whole-cell sonicate EIA, which provides high sensitivity but suboptimal specificity due to cross-reactive antigens 1
• Newer FDA-cleared EIAs using VlsE or C6 peptide antigens offer higher specificity with similar sensitivity 1
• Never proceed directly to Western blot without performing the first-tier test—this dramatically increases false-positive rates 3

Second-Tier Testing (Reflex Only)

• Perform Western immunoblot only when first-tier EIA/IFA is positive or equivocal 1, 2
• For disease duration <6-8 weeks: interpret both IgM and IgG Western blots 2, 3
• For disease duration >6-8 weeks: interpret only IgG Western blot, as IgM loses clinical validity 2, 3
• IgG positivity requires ≥5 of 10 specific bands present 2
• IgM positivity requires ≥2 of 3 specific bands present (plus positive/equivocal EIA) 2

Test Performance Characteristics

Sensitivity by Disease Stage

• Early localized disease (erythema migrans, acute phase): 40% sensitivity 1, 3
• Early localized disease (convalescent phase, 3-4 weeks later): 61% sensitivity 1, 3
• Disseminated disease (neuritis, carditis, arthritis): 88-100% sensitivity 1, 2, 3
• Noncutaneous manifestations: 96% sensitivity 1

Specificity

• Two-tiered testing achieves >95% specificity for disseminated Lyme disease 1, 3
• Specificity in healthy controls from endemic areas: 98-99% 1
• Specificity in healthy controls from non-endemic areas: 99.8-100% 1

Critical Clinical Context for Test Interpretation

When Testing Is Appropriate

• Patients with erythema migrans in endemic areas should be diagnosed clinically without laboratory testing, as 70-80% of Lyme disease patients present with this rash and serologic testing has poor sensitivity (30-40%) during early infection 3
• Order testing for acute neurologic manifestations: meningitis, painful radiculoneuritis, mononeuropathy multiplex, acute cranial neuropathies, or spinal cord inflammation with epidemiologically plausible tick exposure 2
• Order testing for acute myocarditis/pericarditis of unknown cause in endemic areas, particularly with dyspnea, palpitations, chest pain, syncope, or conduction abnormalities 2

When Testing Is Inappropriate

• Never order testing at the time of a tick bite, as antibodies are unlikely to be detectable and results would be misleading 2
• Do not test patients without recent travel to endemic areas—in low-incidence states, positive predictive value drops to only 10%, and only 0.7% of patients with arthritis/neuropathy actually have Lyme disease 2, 3
• Routine testing is not recommended for typical ALS, relapsing-remitting MS, Parkinson's disease, dementia, new-onset seizures, psychiatric illness, or developmental/behavioral disorders 2

Geographic and Epidemiologic Considerations

Geographic exposure history is the most crucial factor determining whether to test—even highly specific tests produce false-positives when pretest probability is low 3

• Document tick exposure history and geographic location meticulously 4
• In non-endemic areas without tick exposure, positive predictive value is only 10% 2, 3
• Identify accompanying features: fever with sudden onset, chills, headache, myalgia, arthralgia, and lymphadenopathy 4

Special Testing Scenarios

Neuroborreliosis

• Order CSF analysis with cell count, differential, protein, and Lyme antibody index 2
• Obtain concurrent serum sample for comparison 2

Lyme Arthritis

• Consider synovial fluid or synovial biopsy for Lyme PCR 2

Cross-Reactive Conditions

• Rule out syphilis by performing T. pallidum hemagglutination assay to exclude cross-reactivity 4
• Be aware of potential cross-reactivity with Epstein-Barr virus, cytomegalovirus, and other conditions 1

Tests to Avoid

Clinicians should only use Lyme disease tests that have been clinically validated and cleared by the FDA 1

• Never order urine antigen tests or CD57 tests—these lack validation and have been shown to be inaccurate 1, 3
• Avoid alternative laboratories using unvalidated interpretation criteria, which have demonstrated false-positive rates as high as 58% in healthy controls 1
• Never retest after treatment—antibodies persist for months to years after successful treatment and do not indicate active infection 2, 3

Emerging Alternative Algorithms

Two-Tiered EIA Approach

• A novel approach using whole-cell sonicate EIA followed by reflex C6 EIA (both FDA-cleared) has shown improved sensitivity for early Lyme disease (61% vs. 48%) while maintaining equivalent specificity (99.5%) 1
• This approach eliminates the subjectivity of Western immunoblot and is easier to automate 1
• Further research is needed before widespread adoption, but this may become a valid alternative in the future 1

Common Pitfalls

• The antibody window period means negative serology does not exclude Lyme disease in the first 2-4 weeks of infection 4
• Do not misinterpret persistent antibodies as active infection—antibodies remain detectable for months to years after successful treatment 4
• Screen-negative samples do not require confirmatory Western blot testing 5
• If questions arise regarding testing protocols or interpretation, consult an infectious disease specialist 1