When is a Lyme disease test result considered positive or negative?

Lyme Disease Testing: Positive and Negative Criteria

Two-Tiered Testing Algorithm

Lyme disease testing is considered positive only when BOTH tiers are positive: a first-tier EIA/ELISA that is positive or equivocal, followed by a confirmatory Western immunoblot that meets strict band criteria. 1

First-Tier Testing (EIA/ELISA or IFA)

• Positive or equivocal result: Proceed to second-tier Western immunoblot 1, 2
• Negative result: Report as negative; no further testing needed unless disease duration is very short (consider repeat testing in 3-4 weeks) 1, 2
• This first tier measures overall IgM and IgG antibody response to B. burgdorferi antigens 1

Second-Tier Testing (Western Immunoblot)

For IgM Western Blot (only interpret if symptoms <30 days):

• Positive: ≥2 of 3 specific bands (21-24,39, and 41 kDa) 1, 3
• Negative: <2 bands present 1
• Critical pitfall: IgM testing beyond 4-8 weeks of symptoms is NOT clinically interpretable and should not be performed 3

For IgG Western Blot:

• Positive: ≥5 of 10 specific bands (18,21-24,28,30,39,41,45,58,66, and 93 kDa) 1, 3
• Negative: <5 bands present 1
• IgG testing alone is typically sufficient for symptoms >30 days, as most patients have detectable IgG response beyond this timeframe 1, 4

Critical Interpretation Rules

A single IgM band or fewer than 5 IgG bands does NOT constitute a positive result and should be reported as negative. 1, 3 This is a common source of misdiagnosis, as individual bands (particularly the 41 kDa band) cross-react with other bacterial flagellar proteins and were found in 43% of healthy controls in one study 1.

Timing-Based Testing Strategy

For symptoms <30 days (early disease):

• Perform first-tier EIA/ELISA 3
• If positive/equivocal, reflex to BOTH IgM and IgG Western blot 3
• Sensitivity is only 30-40% during early infection, improving to 61% in convalescent phase (3-4 weeks later) 2

For symptoms >30 days (disseminated disease):

• Perform first-tier EIA/ELISA 3
• If positive/equivocal, reflex to IgG Western blot ONLY 1, 3
• IgM is unnecessary at this stage and increases false-positive risk 1
• Sensitivity reaches 88-100% for disseminated manifestations 2

When Testing Should NOT Be Performed

Do not order serologic testing for patients with erythema migrans rash in endemic areas—diagnose clinically and treat immediately. 2, 3 Approximately 70-80% of Lyme disease patients present with erythema migrans, and early serologic testing has poor sensitivity (only 30-40%) during the first weeks of infection 1, 2.

Never order Western immunoblot without first performing EIA/ELISA, as this dramatically increases false-positive rates and violates the two-tiered testing principle 2, 3.

Geographic and Pretest Probability Considerations

Geographic exposure history is the most crucial factor determining whether to test. 2 Even highly specific tests produce false-positives when pretest probability is low:

• In low-incidence states without recent travel to endemic areas, positive predictive value is only 10% 2
• Only 0.7% of patients in non-endemic areas with arthritis, cranial neuropathies, or meningitis actually have Lyme disease 2
• Testing is recommended only when there is at least a 1 in 5 chance (20% pretest probability) that the patient has active Lyme disease 5

Special Circumstances

Chronic/Late Lyme Disease

• Patients with late/persistent infection typically demonstrate high IgG antibody titers, especially those with arthritis and acrodermatitis chronica atrophicans 4
• IgM antibodies are usually undetectable, found in only 10-40% of patients with late manifestations 4
• For chronic neuroborreliosis, intrathecal antibody production is the most important microbiological diagnostic criterion 4

Neuroborreliosis Testing

• Test for intrathecal antibody production in CSF 4
• Collect CSF and serum on the same day and dilute to match total protein or IgG concentration 1, 4
• CSF/serum IgG EIA optical density ratio >1.0 indicates active intrathecal antibody production 1, 4

Common Pitfalls to Avoid

Do not retest patients after treatment—antibodies persist for months to years after successful treatment and do not indicate active infection 2, 3. Neither positive serologic results nor history of previous Lyme disease ensures protective immunity, and reinfection can occur 4.

Avoid unvalidated tests: Urine antigen tests and CD57 tests lack validation and have been shown to be inaccurate 1, 2. Alternative laboratories using non-standard interpretation criteria have demonstrated false-positive rates as high as 58% 1.

PCR testing has limited clinical utility due to low sensitivity (particularly for blood and CSF samples) and potential for contamination 1, 4. Synovial fluid PCR is >75% sensitive for Lyme arthritis but CSF PCR yields positive results in only 38% of early neuroborreliosis cases 1.