Evaluation of Modified Two-Tiered Testing Algorithms for Lyme Disease Laboratory Diagnosis Using Well-Characterized Serum Samples

Abstract

Standard two-tiered testing (STTT) is the recommended algorithm for laboratory diagnosis of Lyme disease (LD). Several limitations are associated with STTT that include low sensitivity in the early stages of disease, as well as technical complexity and subjectivity associated with second-tier immunoblotting; therefore, modified two-tiered testing (MTTT) algorithms that utilize two sequential first-tier tests and eliminate immunoblotting have been evaluated. Recently, a novel MTTT that uses a VlsE chemiluminescence immunoassay followed by a C6 enzyme immunoassay has been proposed. The purpose of this study was to evaluate the performance of the VlsE/C6 MTTT using well-characterized serum samples. Serum samples from the CDC Lyme Serum Repository were tested using three MTTTs, VlsE/C6, whole-cell sonicate (WCS)/C6, and WCS/VlsE, and three STTTs (immunoblotting preceded by three different first-tier assays: VlsE, C6, and WCS). Significant differences were not observed between the results of the MTTTs assessed; however, the VlsE/C6 MTTT resulted in the highest specificity (100%) when other diseases were tested and the lowest sensitivity (75%) for LD samples. Significant differences were present between the results for various MTTTs and STTTs evaluated. Specifically, all MTTTs resulted in higher sensitivities than the STTTs for all LD groups combined and were significantly more accurate (i.e., higher proportion of correct classifications) for this group, with the exception of the WCS/ViraStripe STTT. Additionally, when other diseases were tested, only the results of the VlsE/C6 MTTT differed significantly from those of the WCS/ViraStripe STTT, with the VlsE/C6 MTTT resulting in a 6.2% higher accuracy. Overall, the VlsE/C6 MTTT offers an additional laboratory testing algorithm for LD with equivalent or enhanced performance compared to that of the other MTTTs and STTTs evaluated in this study.

Revisiting the Lyme Disease Serodiagnostic Algorithm: the Momentum Gathers

“…As shown here and in previous studies (4,16,(26)(27)(28)(29), MTT algorithms will be positive in only about 50% of acute-phase samples from patients with EM, while the STT algorithm will be positive in about 40% (Table 1). Therefore, patients with EM should receive treatment based on the clinical diagnosis (1).…”

“…This brings us to the current study (4), in which the authors compared three MTT algorithms with three STT algorithms using the same assays as the initial tier test, followed by WB. The samples used in the study were from the CDC Lyme Serum Repository, and they have been described and analyzed in other studies (28,29).…”

Evaluation of a Novel Microarray Immunoblot Assay for Detection of IgM- and IgG-Class Antibodies to Borrelia burgdorferi

“…Additionally, we show that performance of the ViraChip MIB assays on an open-system, automated ELISA analyzer is associated with a significant time savings, particularly for highthroughput laboratories, and decreases the risk of ergonomic injury due to repetitive motions associated with manual specimen pipetting. Finally, it deserves mention that there is increasing data to support amendment of the current STTT algorithm for LD and, specifically, to replace the supplemental blot testing with a second-tier B. burgdorferi-specific ELISA (14,27,28). This modified two-tiered testing (MTTT) algorithm would obviate many of the aforementioned limitations associated with blot testing, significantly improving both the sensitivity and accuracy of LD testing overall.…”

A fluorescent plasmonic biochip assay for multiplex screening of diagnostic serum antibody targets in human Lyme disease

“…The STTT has been a useful diagnostic tool since its standardization by the Centers for Disease Control (CDC) in 1995 [13], but critical limitations have become increasingly apparent [16,17]. These include low sensitivity and low specificity for early disease [18,19], inability to monitor treatment progress or diagnose re-infection [20], inconsistencies across tests [21][22][23], and subjective interpretation of Western blot results [18,24]. Experts agree that new strategies for diagnosing LD are necessary to address these concerns, pointing to modified two-tier algorithms using only ELISAs [23,[25][26][27], as well as novel assays in various stages of development [28][29][30].…”