Modified Two-Tiered Serodiagnostic Testing Algorithms for Acute Phase or Early Disseminated Lyme Disease

Lyme disease is the most common vector-borne illness, with more than 476,000 cases annually in the United States and can be challenging to diagnose.¹ The presence of erythema migrans is considered diagnostic in patients who live in or have recently traveled to an endemic area. However, serologic testing is recommended if the cause of the skin lesion is unknown and antibiotics have not been administered. Serologic testing is also recommended for early disseminated and late-stage Lyme disease.²

Traditional serologic two-tiered testing for Lyme disease involves initially obtaining an enzyme immunoassay (EIA) or immunofluorescence assay, followed by confirmatory immunoglobulin M (IgM) and IgG Western blot tests if the initial test result is positive or equivocal.³ The U.S. Food and Drug Administration approved modified two-tiered testing in 2019 by replacing the confirmatory Western blot test with another enzyme immunoassay.⁴ Since then, the Centers for Disease Control and Prevention has recommended modified two-tiered testing as the method of choice, especially in the acute phase or early disseminated Lyme disease, when slow humoral response may lead to false negatives.⁴

A multicenter prospective clinical trial of three separate modified two-tiered testing protocols was conducted to determine sensitivity and specificity for early Lyme disease detection (30 days or less of symptoms). Fifty-five patients with erythema migrans (considered a true positive) were examined to assess sensitivity. Specificity was determined using 50 patients with other illnesses and 1,227 healthy patients.³ This type of diagnostic case-control design has the potential to inflate specificity compared with evaluation in patients who have clinically suspected Lyme disease. All testing methods were compared with the conventional two-tiered testing protocol of whole-cell sonicate EIA and follow-up IgM and IgG Western blot tests if needed. Sensitivity for patients with acute erythema migrans was higher in all three modified two-tiered testing groups (35% to 54%) compared with the control group (25%); however, this reached statistical significance for only one protocol (variable major protein-like sequence, expressed [VlsE] chemiluminescence immunoassay [CLIA] with C6 EIA; P = .03). The three modified two-tiered testing protocols were similarly specific when compared with each other. The specificity was not statistically significant when compared with the control group.³

The positive and negative likelihood ratios for sensitivity of 54% and specificity of 99% are 54 and 0.46, respectively. Therefore, a negative test provides only weak evidence against the diagnosis. For a patient with a 50% pretest probability of having Lyme disease, a positive VlsE CLIA with C6 EIA test result increases the likelihood to 98%, whereas a negative result reduces it to only 32%.

Conventional Lyme disease testing protocols involving Western blots are expensive, require subjective interpretation of data, and have demonstrated poor sensitivity in patients with early symptoms. Many laboratories send out Western blot assays to other laboratories for testing, which further delays results.⁵ Modified two-tiered tests are more cost-effective, are less objective, and can be completed in-house by most laboratories.⁶