Lyme Borreliosis Serology: Performance of Several Commonly Used Laboratory Diagnostic Tests and a Large Resource Panel of Well-Characterized Patient Samples

Abstract

The current recommendation for the laboratory confirmation of Lyme disease is serology-based diagnostics. Specifically, a standardized two-tiered testing (STTT) algorithm is applied that utilizes a first-tier immunofluorescence assay or enzyme immunoassay (EIA) that, if the result is positive or equivocal, is followed by second-tier immunoblotting. Despite the standardization and performance achievements, STTT is considered technically complex and subjective, as well as insensitive for early acute infection. These issues have prompted development of novel algorithms and testing platforms. In this study, we evaluated the performance of several commonly used assays for STTT. Several modified two-tiered testing (MTTT) algorithms, including a 2-EIA algorithm and modified criteria for second-tier IgG immunoblots, were also evaluated. All tests were performed on sera from a recently available, well-defined archive of positive-and negative-control patients. Our study demonstrates differences in the results between individual first-and second-tier tests, although the overall agreement of the different STTT algorithms used was strong. In addition, the MTTT algorithm utilizing 2-EIAs was found to be equivalent to all STTT algorithms tested, with agreement ranging from 94 to 97%. The 2-EIA MTTT algorithm slightly enhanced sensitivity in early disease compared to the STTT algorithms evaluated. Furthermore, these data add to the mounting evidence that a 2-EIA-based MTTT algorithm, where immunoblotting is replaced by the C6 EIA, performs as well or better than STTT. Serologic testing has been the mainstay of laboratory diagnostics for Lyme disease for over 20 years. To date in the United States, serologic tests are the only U.S. Food and Drug Administration (FDA) 510(k)-cleared diagnostic tests for Lyme disease (1). Standardization of serologic testing for Lyme disease occurred in 1994, when attendees of the Second National Conference on Serologic Diagnosis of Lyme Disease (Dearborn, MI) determined that a two-tiered algorithm, optimized for specificity and the highest sensitivity, was most appropriate for Lyme disease laboratory diagnosis (2). The standard two-tiered testing (STTT) algorithm utilizes a first-tier immunofluorescence assay (IFA) or enzyme immunoassay (EIA) that, if the result is positive or equivocal, is followed by a second-tier immunoblot (IgM and/or IgG). The interpretation criteria for a positive immunoblot requires that at least two of three or at least five of ten bands be considered positive for IgM and IgG reactivity, respectively. Further, it was recommended that patients only be tested by IgM immunoblotting if their duration of illness is 30 days or less. STTT and interpretation criteria continue to be recommended by the Centers for Disease Control and Prevention (CDC) for laboratory confirmation of Lyme disease (2).Several dozen individual first-and second-tier diagnostic tests have been FDA 510(k) cleared and are commercially available. Most first-tier tests use either Borrelia burgdorferi whole-cel...

Evaluation of Modified 2-Tiered Serodiagnostic Testing Algorithms for Early Lyme Disease

“…One of the MTTT protocols, a WCS EIA followed by a C6 EIA, has been evaluated by several independent groups with similar findings to those reported here [3,5,6,8,21]. Our study furnishes additional evidence that this algorithm provides similar or greater sensitivity in acute-or convalescent-phase EM, compared with any 2-tiered protocol involving WBs, while maintaining the specificity of algorithms involving WBs.…”

“…Therefore, a negative result does not rule out the diagnosis of early Lyme disease, if signs and symptoms have been present for ≤30 days [2]. Because first-tier assays typically become reactive earlier during seroconversion than WBs, the WB component substantially reduces the sensitivity of 2-tiered testing in patients with the earliest manifestation of LD, erythema migrans (EM) [3,5,8,10,11]. Although EM is the most common clinical manifestation of LD, only 10%-20% of EM lesions present in the form of a characteristic targetoid rash [12,13], creating diagnostic uncertainty and strong demand for a diagnostic test to support suspected LD.…”

“…The 2-EIA algorithm is more specific than either EIA alone, because the assays involved are orthogonal to some degree. Cross-reactive antibodies that affect the specificity of WCS EIAs often do not react with the C6 EIA, and vice versa [3,5,8].…”

“…Two-tiered testing protocols involving WBs are sensitive and specific in the later stages of LD [5,9,10], but they are insensitive in early infection [3,5,7,9,10] due to the slow development of the humoral immune response. Therefore, a negative result does not rule out the diagnosis of early Lyme disease, if signs and symptoms have been present for ≤30 days [2].…”

“…These assays are prepared from recombinant proteins or synthetic peptides that correspond to spirochetal antigens known to induce relatively early and robust humoral immune responses [7]. Because they contain far fewer antigens than WCS assays, these alternative first-tier assays are typically more specific [3,5,6,8], although none is completely specific. At present, these assays must still be used in the first tier of a 2-tiered testing protocol, with WBs used in the second tier.…”