A Multiplexed Serologic Test for Diagnosis of Lyme Disease for Point-of-Care Use

Abstract

Single multiplexed assays could replace the standard 2-tiered (STT) algorithm recommended for the laboratory diagnosis of Lyme disease if they perform with a specificity and a sensitivity superior or equal to those of the STT algorithm. We used human serum rigorously characterized to be sera from patients with acute- and convalescent-phase early Lyme disease, Lyme arthritis, and posttreatment Lyme disease syndrome, as well as the necessary controls (n = 241 samples), to select the best of 12 Borrelia burgdorferi proteins to improve our microfluidic assay (mChip-Ld). We then evaluated its serodiagnostic performance in comparison to that of a first-tier enzyme immunoassay and the STT algorithm. We observed that more antigens became positive as Lyme disease progressed from early to late stages. We selected three antigens (3Ag) to include in the mChip-Ld: VlsE and a proprietary synthetic 33-mer peptide (PepVF) to capture sensitivity in all disease stages and OspC for early Lyme disease. With the specificity set at 95%, the sensitivity of the mChip-Ld with 3Ag ranged from 80% (95% confidence interval [CI], 56% to 94%) and 85% (95% CI, 74% to 96%) for two panels of serum from patients with early Lyme disease and was 100% (95% CI, 83% to 100%) for serum from patients with Lyme arthritis; the STT algorithm detected early Lyme disease in the same two panels of serum from patients with early Lyme disease with a sensitivity of 48.5% and 75% and Lyme arthritis in serum from patients with Lyme arthritis with a sensitivity of 100%, and the specificity was 97.5% to 100%. The mChip-Ld platform outperformed the STT algorithm according to sensitivity. These results open the door for the development of a single, rapid, multiplexed diagnostic test for point-of-care use that can be designed to identify the Lyme disease stage.

Serum Epitope Repertoire Analysis Enables Early Detection of Lyme Disease with Improved Sensitivity in an Expandable Multiplex Format

“…Lyme disease (13-15, 41, 42). For example, a point-of-care microfluidic assay (mChip-Ld) using three Lyme antigens showed a 29% and 5% improvement in sensitivity in two separate early Lyme cohorts (13). Another multi-antigen test developed to improve IgM plus IgG serology demonstrated sensitivities in acute and convalescent early Lyme samples of 70% and 84%, respectively, equivalent to sensitivities of SERA (41).…”

“…These problems prompted national efforts to assemble specimen biobanks and develop new diagnostic tools to improve early disease detection (8,12). As a result, several novel assays have been developed and exhibit on November 25, 2020 by guest http://jcm.asm.org/ Downloaded from improved performance (13,14). Meanwhile, recognition of the high burden of non-Lyme tickborne infections, either alone or in combinations, including babesiosis, anaplasmosis, and rickettsioses (6), has spurred development of multiplex tests, for example using solid phase peptide arrays (15).…”

Quantitative multiplexed strategies for human Lyme disease serological testing

“…47,48 Alternatively, positive diagnosis can be made using a weighted score combining the measured titers of each target based on a multivariate regression model. 45,[49][50][51] Other multiplexed assays do not distinguish between targets, but instead directly measure a combined signal to predict infection status. 44,52 Cut-off and/or threshold establishment, whether it is for detection of individual targets or for scoring a positive sample, is another important decision in assay design.…”

Lyme Disease Biosensors: A Potential Solution to a Diagnostic Dilemma

“…In an effort to improve the mChip-Ld test, Arumugam et al (2019) tested 12 B. burgdorferi proteins to assess their corresponding antibody levels in early and late Lyme disease [ 33 ]. Of the tested proteins, three were selected for inclusion in the improved assay: VlsE, PepVF, and OspC.…”

“…Likewise, the MTT approach demonstrates only slightly improved sensitivity, at approximately 50%, for patients with early Lyme disease [ 18 ]. Some of the biosensors presented here ( Table 1 ) report sensitivities for early Lyme that exceed the STT/MTT approaches and have the potential to develop into a much-needed alternative [ 32 , 33 , 42 ]. However, some biosensors also demonstrate reduced specificity in comparison to the two-tiered approaches.…”