In this study, the performance of multiple STTT and MTTT strategies was evaluated using a well-defined study population of patients with early and late disseminated LB and multiple control groups. Furthermore, the contribution of solitary IgM results in the screening EIA to the final strategy result, and the impact of antibiotic treatment on seroreversion were evaluated.

The sensitivity of STTT with recomLine immunoblot as a second-tier assay was slightly, though not significantly, lower compared to the Euroline immunoblot, while the opposite was observed for specificity. One previous study that compared recomLine and Euroline immunoblots, reported equal sensitivity and specificity for IgM and IgG separately [19]. Unfortunately, the combined IgM/IgG results were not reported, which complicated comparison to the present study’s observations. In our study, MTTT strategies showed slightly higher sensitivities, with specificities comparable to those of STTT strategies. However, in contrast to previous MTTT evaluations in both early localized and disseminated LB patients in various epidemiological settings, the observed differences among STTT and MTTT strategies were not statistically significant in our study [4, 10, 20]. This may be due to the relatively small study population.

The specificities observed here were comparable to those reported previously [3], and reflect the background seroprevalence of 5.3% in the endemic region where the hospital is located, which is comparable to the 4.4% seroprevalence observed in the general population of the Netherlands [21]. Participants who reported treatment for LB in the past were grouped separately and, therefore, the seropositivity observed in the uHI group is likely the result of a previous (asymptomatic) Borrelia infection. Cross-reactivity seems less probable, as IgG was the dominant isotype in this group, whereas IgM positivity dominated the CR group, consistent with previous findings [9, 10].

The role of IgM testing for LB diagnosis has been debated for some time [9, 22]. In the present study, solitary IgM results were predominantly observed in the control groups, and disregarding them could possibly improve specificity for all strategies with minor loss of sensitivity. Due to the small sample size in the present study, this observation was not statistically significant, and should be confirmed in a larger cohort. The observed trend, however, underscores the recommendation that IgM testing should only be considered when clinical symptoms are suggestive for active LB with symptom duration until six to ten weeks [23, 24].

In our study, isotype-specific seropositivity in post-treatment sera seemed dependent on the disease stage in which antibiotics were administered. The solitary IgM responses observed among healthy individuals that were treated for early localized manifestations up to 6.2 years prior to inclusion suggest that seroconversion was abrogated before the isotype switch from IgM to IgG had taken place. For the disseminated manifestations LNB and LA, combined IgM/IgG seropositivity was predominantly observed among active patients, indicating that the isotype switch preceded initiation of antibiotic treatment. After antibiotic treatment, a significant decrease in seropositivity was observed among LNB patients, which was attributable to decreased IgM and IgG levels, consistent with previous findings [25]. Among treated LA patients, no seroreversion was observed; however, decreased IgM antibody levels resulted in increased solitary IgG seropositivity. As pre- and post-treatment sera were only available for some of the LB patients, associations with antibiotic treatment were made on group level. In future studies, pairwise comparisons should determine whether the observations in this study also apply to patient level.

This study was conducted on a well-defined population of patients diagnosed with early and late disseminated LB patients and various control groups. The LNB patients in our study were diagnosed based on clinical symptoms with pleocytosis and/or intrathecal B. burgdorferi s.l.-specific antibody production. For the diagnosis of LNB patients, the detection of intrathecal B. burgdorferi s.l.-specific antibody production is preferred over serology, because up to 20% of the LNB patients with intrathecal antibody synthesis had not seroconverted (yet) at the time of diagnosis [26, 27]. The LA patients in this study were diagnosed based on clinical symptoms and positive Borrelia PCR on synovial fluid, confirmed by serology. This selection bias could be considered a limitation that is difficult to resolve as serology is part of the diagnosis, and as shown here antibodies persist at least a year post-treatment. Furthermore, exclusion of EM patients in this study likely has resulted in an overestimation of the sensitivity. EM is, however, a clinical diagnosis, for which laboratory testing should not delay treatment – even with improved sensitivity of MTTT [10].

Most MTTT evaluations tested the off-market C6 Lyme ELISA as second-tier assay [4, 10, 20]. As reflected by the high DORs in this study, all test strategies that included this assay demonstrated the best performance in terms of sensitivity and specificity. As the C6 Lyme ELISA assay was discontinued, we also evaluated the Liaison as a second-tier EIA, and this MTTT strategy also outperformed commercially available STTT strategies. One could argue, however, that the antigens used in the Liaison might also be part of the screening assay, which could introduce bias. This could partially be overcome using a peptide-based and/or multiplex second-tier assay, such as the recently introduced Zeus VlsE1/pepC10 IgM/IgG ELISA (Zeus ELISA) or the BioPlex 2200 Lyme IgG and Lyme IgM assays.

The Zeus ELISA has previously been evaluated as a single-tier or first-tier assay, and performed comparably to the C6 Lyme ELISA in both single-tier and STTT strategies [9, 28]. In North America, the FDA-approved MTTT strategy uses Zeus ELISA as first-tier assay with Zeus’ whole cell sonicate (WCS)-based IgM/IgG ELISA as second-tier assay. This strategy outperformed STTT and MTTT strategies with second-tier C6 Lyme ELISA in terms of sensitivity, especially in early LB, without loss of specificity [29, 30]. However, these WCS ELISAs use inactivated B. burgdorferi B31 antigen, and might therefore be suboptimal for detection of the heterogenic B. burgdorferi s.l. population in Europe. The Bioplex, on the other hand, was also evaluated on European patient and control populations, and demonstrated comparable sensitivity, but inferior specificity to the C6 Lyme ELISA in a STTT strategy [31]. Also, the Bioplex requires specific equipment and expertise and is therefore less suitable for use in routine clinical settings. Hence, future assay developments should focus on accessible, low-cost assays for improved performance of TTT or to replace TTT altogether.

Both STTT and MTTT strategies do not discriminate active from past disease, and offer no measure for therapeutic success or reinfection. Here, dynamics in isotype-specific immune responses among different LB manifestations were demonstrated that should be explored further to ameliorate these shortcomings of TTT. Moreover, the output of TTT is currently categorical (i.e., positive or negative) and provides no detailed information about the type and magnitude of the antigen-specific immune responses. Preliminary data from our research group have shown promising results regarding antigen-specific immune responses. We are in the process of confirming these findings in a larger cohort of LB patients, including paired sera from active and treated patients diagnosed with EM, LA and LNB. Also, the relationship between antibody levels and predictive values has already been demonstrated for some assays [32, 33], and could be explored further to improve LB diagnostics.

In conclusion, this study showed that for disseminated LB, MTTT strategies demonstrated a slightly higher sensitivity, although not statistically significant, while maintaining similar specificity compared to STTT strategies. Since solitary IgM reactions were predominantly observed among healthy individuals, the use of IgM assays for the diagnosis of LB should be carefully considered based on disease duration and manifestation. Lastly, although antibiotic treatment does not always result in seroreversion, decline of isotype-specific antibody levels was observed, and could possibly be used to assess disease status for different Lyme manifestations.