Optimizing use of multi-antibody assays for Lyme disease diagnosis: A bioinformatic approach

Abstract

Multiple different recombinant and peptide antigens are now available for serodiagnosis of Lyme disease (LD), but optimizing test utilization remains challenging. Since 1995 the Centers for Disease Control and Prevention (CDC) has recommended a 2-tiered serologic approach consisting of a first-tier whole-cell enzyme immunoassay (EIA) for polyvalent antibodies to Borrelia burgdorferi followed by confirmation of positive or equivocal results by IgG and IgM immunoblots [standard 2-tiered (STT) approach]. Newer modified 2-tiered (MTT) approaches employ a second-tier EIA to detect antibodies to B. burgdorferi rather than immunoblotting. We applied modern bioinformatic techniques to a large public database of recombinant and peptide antigen-based immunoassays to improve testing strategy. A retrospective CDC collection of 280 LD samples and 559 controls had been tested using the STT approach as well as kinetic-EIAs for VlsE1-IgG, C6-IgG, VlsE1-IgM, and pepC10-IgM antibodies. When used individually, the cutoff for each kinetic-EIA was set to generate 99% specificity. Utilizing logistic-likelihood regression analysis and receiver operating characteristic (ROC) techniques we determined that VlsE1-IgG, C6-IgG, and pepC10-IgM antibodies each contributed significant diagnostic information; a single-tier diagnostic score (DS) was generated for each sample using a weighted linear combination of antibody levels to these 3 antigens. DS performance was then compared to the STT and to MTT models employing different combinations of kinetic-EIAs. After setting the DS cutoff to match STT specificity (99%), the DS was 22.5% more sensitive than the STT for early-acute-phase disease (95% CI: 11.8% to 32.2%), 16.0% more sensitive for early-convalescent-phase disease (95% CI: 7.2% to 24.7%), and equivalent for detection of disseminated infection. The DS was also significantly more sensitive for early-acute-phase LD than MTT models whose specificity met or exceeded 99%. Prospective validation of this single-tier diagnostic score for Lyme disease will require larger studies using a broader range of potential cross-reacting conditions.

Immunoblot Criteria for Diagnosis of Lyme Disease: A Comparison of CDC Criteria to Alternative Interpretive Approaches

“…CDC serologic approaches to LD diagnosis currently utilize a two-tiered method: serum from patients with clinically suspected LD are first tested by EIA, IFA, or chemiluminescent assay, followed by a second-tier EIA or immunoblot assay to confirm positive or equivocal first-tier results [1]. First-tier EIAs and chemiluminescent assays for polyvalent IgG/IgM antibodies to B. burgdorferi may yield false-positive rates between 2% and 5% when using recombinant proteins [2,3] and between 10% and 15% when using whole-cell lysate [4,5]. Because of concerns about false-positive first-tier test results, particularly in low-risk settings, the CDC recommends a confirmatory second-tier test [1].…”

“…Some MTT approaches, however, do not routinely identify which B. burgdorferi antigens are responsible for a positive result. When potentially cross-reacting medical conditions are suspected, such as syphilis, tick-borne coinfections (e.g., human granulocytic anaplasmosis), and some viral illnesses (e.g., Epstein-Barr virus), greater diagnostic information about the immune response to B. burgdorferi may be afforded by IgG immunoblots [4,10].…”

Gene set predictor for post-treatment Lyme disease

“…In addition, there is a recently introduced modified TTT (MTTT) and a test for antibodies reactive to the VlsE1 antigen. 3 The TTT test has a sensitivity of 17%–43% during the early stage of infection. 2 In the absence of a laboratory diagnostic tool, the diagnosis of early LD is reliant on clinical demonstration of the erythema migrans (EM) skin lesion that occasionally does not present or is not observed.…”