What is the confirmatory test for a patient suspected of having Lyme disease with symptoms such as erythema migrans, fever, headache, or joint pain, and a history of exposure to ticks in endemic areas?

Confirmatory Test for Lyme Disease

The confirmatory test for Lyme disease is Western immunoblot (both IgM and IgG), performed only after a positive or equivocal first-tier enzyme immunoassay (EIA) or immunofluorescence assay (IFA), as part of the standard two-tiered testing algorithm. 1

Two-Tiered Testing Algorithm

The diagnostic approach follows a mandatory sequential process:

First-Tier Test

• Order EIA or IFA as the initial screening test, which detects overall antibody response (IgM and IgG) to Borrelia burgdorferi antigens 1
• Most U.S. laboratories use whole-cell sonicate preparation, though newer C6 peptide EIAs offer similar sensitivity with higher specificity 1
• If the first-tier test is negative, report as negative and stop—no confirmatory testing needed 1

Second-Tier Confirmatory Test

• Proceed to Western immunoblot only if the first-tier EIA/IFA is positive or equivocal 1
• Order both IgM and IgG Western blots if disease duration is <6-8 weeks 2, 3
• Order only IgG Western blot if disease duration is >6-8 weeks, as IgM loses clinical validity after this timeframe 2

Interpretation Criteria for Western Immunoblot

IgM Western Blot Positivity

• Requires ≥2 of 3 specific bands present (23,39,41 kDa) 2
• Valid only for disease duration <6-8 weeks 1, 2
• Must have preceding positive or equivocal EIA 2

IgG Western Blot Positivity

• Requires ≥5 of 10 specific bands present (18,21,28,30,39,41,45,58,66,93 kDa) 2
• This is the only valid confirmatory test for disease duration >6-8 weeks 1, 2

Test Performance Characteristics

The two-tiered approach achieves:

• Sensitivity of 70-100% for disseminated Lyme disease (neuritis, carditis, arthritis) 1, 3
• Specificity >95% across all disease stages 1, 3
• Sensitivity only 30-40% during early localized infection due to the antibody window period 1, 3

For early disease with erythema migrans, acute-phase sensitivity is only 40%, improving to 61% in convalescent-phase testing 3-4 weeks later 1, 3

Critical Pitfalls to Avoid

Never Order Western Blot as First-Line Test

• Ordering Western immunoblot without preceding EIA/IFA dramatically increases false-positive rates and violates the two-tiered algorithm 3
• The sequential approach is designed to increase pre-test probability with each step, maximizing positive predictive value 1

Avoid Invalid Testing Scenarios

• Do not order testing at the time of tick bite—antibodies are not yet detectable 2
• Never retest after treatment—antibodies persist for months to years after successful treatment and do not indicate active infection 2, 3
• Avoid urine antigen tests and CD57 tests—these lack validation and are not recommended by any guideline 2, 3

Consider Cross-Reactivity

• Following a positive screening assay, consider cross-reactivity with Treponema pallidum (syphilis), which can be clarified with a T. pallidum hemagglutination assay 1
• False-positive EIA results can occur with Epstein-Barr virus, cytomegalovirus, or autoimmune conditions, but these typically do not meet Western blot criteria 1

Special Clinical Scenarios

Erythema Migrans in Endemic Areas

• No laboratory testing required—diagnosis is clinical 1, 3
• Approximately 70-80% of Lyme disease patients present with erythema migrans 1, 3
• Testing during this early phase has poor sensitivity (30-40%) and may delay appropriate treatment 1, 3

Suspected Neuroborreliosis

• Order CSF analysis with cell count, differential, protein, and Lyme antibody index, along with concurrent serum sample for comparison 2
• CSF culture or PCR has only 10-30% sensitivity 1

Suspected Lyme Arthritis

• Consider synovial fluid or synovial biopsy for Lyme PCR, which has 50-70% sensitivity 1, 2

Alternative Testing Approaches

Recent evidence supports a two-EIA algorithm (whole-cell sonicate EIA followed by C6 peptide EIA) as an alternative to traditional two-tiered testing, with comparable sensitivity (87-100%) and specificity (99-100%) for disseminated disease 1. However, the traditional EIA followed by Western immunoblot remains the standard confirmatory approach endorsed by CDC and IDSA guidelines 1.