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The aim of this study was to evaluate the antibody responses to different VlsE protein IR(6) peptide variants and the synthetic C6 peptide in acute and convalescent (2-3 and 6 months) serum samples from Swedish patients with clinical erythema migrans (EM). Serum samples were prospectively collected from 148 patients with EM and compared to serum samples obtained from 200 healthy blood donors. The IgG responses to 3 IR(6) peptide variants originating from Borrelia burgdorferi (B. burgdorferi) sensu stricto, B. garinii, and B. afzelii were measured by enzyme-linked immunosorbent assays (ELISAs) and compared to a commercial C6 peptide ELISA. Seropositivity rate in the IR(6) or C6 peptide ELISAs ranged from 32% to 58% at presentation, 30-52% after 2-3 months, and 20-36% after 6 months. At presentation, positive antibodies in any of the 4 ELISAs were found in 66%. In 7/52 (13%), C6-negative EM cases, serological reaction was found to the B. burgdorferi sensu stricto-derived IR(6) peptide. In patients reporting previous LB compared to those without previous LB, significantly higher seropositivity rates were noted for all IR(6) peptides, but not for the C6 peptide. In the serology of EM in Europe, C6 ELISA does not seem to cover all cases. An ELISA using a mixture of B. burgdorferi sensu stricto IR(6) peptide and the C6 peptide could be of value in the serodiagnosis of LB in Europe. Further studies on combinations of variant IR(6) peptides and the C6 peptide in other manifestations of LB are needed to address this issue.