Evaluation of a sequential enzyme immunoassay testing algorithm for Lyme disease demonstrates lack of test independence but high diagnostic specificity

To diagnose Lyme disease, a two-tier testing algorithm is used in which supplemental IgM and IgG immunoblots to detect antibody to Borrelia burgdorferi are reflexively performed if a first-tier assay, such as a whole-cell sonicate-based enzyme immunoassay (WCS EIA), is reactive. Recent data suggest that equal specificity is found by substituting the C6 peptide EIA for immunoblots. In this study using 3956 control sera, we demonstrated that although this two-tier testing algorithm does significantly improve diagnostic specificity compared with each of the EIAs individually, the WCS EIA and the C6 peptide EIA are not independent tests. Therefore, when the C6 peptide EIA is used as the second-tier test, it should be regarded as a supplemental rather than a confirmatory test. •To diagnose Lyme disease, two-tier testing is used with immunoblots (IB) as the second-tier assay.•The C6 peptide enzyme immunoassay (EIA) is a first-tier assay being considered to replace IB.•A whole-cell sonicate-based (WCS) EIA followed by the C6 EIA is more specific than a single EIA.•However, using 3956 control sera, we showed that WCS EIA and C6 EIA are not independent tests.•The C6 EIA in two-tier testing should be regarded as a supplemental rather than a confirmatory test.