<p><strong>BACKGROUND: </strong>Variability in 2-tier Lyme disease test results according to the specific first-tier enzyme immunoassay (EIA) in children has not been examined rigorously. In this study, we compared paired results of clinical 2-tier Lyme disease tests to those of the C6 peptide EIA followed by supplemental immunoblotting (C6 2-tier test).</p>

<p><strong>METHODS: </strong>We performed a prospective cohort study of children aged ≥1 to ≤21 years who were undergoing evaluation for Lyme disease in the emergency department at 1 of 6 centers located in regions in which Lyme disease is endemic. The clinical first-tier test and a C6 EIA were performed on the same serum sample with supplemental immunoblotting if the first-tier test result was either positive or equivocal. We compared the results of the paired clinical and C6 2-tier Lyme disease test results using the McNemar test.</p>

<p><strong>RESULTS: </strong>Of the 1714 children enrolled, we collected a research serum sample from 1584 (92.4%). The clinical 2-tier EIA result was positive in 316 (19.9%) children, and the C6 2-tier test result was positive or equivocal in 295 (18.6%) children. The clinical and C6 2-tier test results disagreed more often than they would have by chance alone (P = .002). Of the 39 children with either a positive clinical or C6 2-tier test result alone, 2 children had an erythema migrans (EM) lesion, and 29 had symptoms compatible with early disseminated Lyme disease.</p>

<p><strong>CONCLUSIONS: </strong>Two-tier Lyme disease test results differed for a substantial number of children on the basis of the specific first-tier test used. In children for whom there is a high clinical suspicion for Lyme disease and who have an initially negative test result, clinicians should consider retesting for Lyme disease.</p>