Serological identification of Borrelia burgdorferi sensu stricto, B. garinii and B. afzelii in human sera: development, evaluation and optimization of novel peptide-based assay

AIM OF THE WORK
Current diagnostic tests for Lyme borreliosis (LB) assess antibody titres to one or more Borrelia outer surface protein. However, these tests do not determine which Borrelia species is infecting the patient. LB is caused by the Borrelia burgdorferi sensu lato (Bbsl) complex transmitted to humans by the bite of infected ticks. In North America, Borrelia burgdorferi sensu stricto (Bbss) is the dominant species; in Eurasia B. afzelii and B. garinii, are the two most prevalent pathogenic species. Here, we describe the development, optimization and evaluation of a multiplex peptide ELISA for serological identification of the three key Bbsl species.
RESEARCH METHODS
Systematic review of the literature was performed to identify outer surface proteins (Osp) of potential immune-diagnostic value. A total of 19 Osps were analysed in silico to identify B-cell epitopes with evidence of ortholog sequence divergence, and these were synthesised as oligopeptides to be used as capture antigens in ELISA. The IgM, IgG and simultaneous IgM + IgG antibody titres were determined by multiplex ELISA in 47 LB-negative and 39 LB-positive sera, where the Borrelia genospecies was inferred from 5S-23S rDNA intergenic spacer sequence in the associated biting tick. Additionally, the sensitivity and diagnostic accuracy of the multiplex assay was compared using passively bound peptide on standard ELISA plates (Greiner MicrolonTM600) and covalently coupled peptide on amnio-activated ELISA plates (NuncTM Immobilizer Amino Plates)
RESULTS
Twenty-one (21) peptide sequences across five Osps were selected for the multiplex ELISA. Sensitivity and correct species identification were 58.1% and 88.8% respectively for IgM responses, and 74.1% and 95.6% respectively for IgG, responses. Highest diagnostic power was observed when IgM + IgG antibody titre was simultaneously determined. This gave sensitivity and correct species identification of 93.5% and 96.5%, respectively. Overall, specificity of the anti-IgM, anti-IgG and anti-IgM + IgG multiplex peptide ELISA calculated on a total number of 47 LB-negative sera was 89.4%, 93.6 and 91.4%, respectively. Use of surface activated amino plates enhanced the performance of individual peptides and significantly increased (P <0.05) the discrimination power of the test.
CONCLUSION
The multiplex ELISA platform demonstrated accurate identification of Bbss, B. garinii and B. afzelii. Performance was enhanced with covalently coupled capture peptides, and this offers a prototype platform for an ELISA kit. As a research diagnostic this would offers the potential to retrospectively analyse biobank LB sera to investigate the relationship of infection genospecies to disease presentation and clinical manifestations, and we are currently evaluating this approach with neuroborreliosis sera.
As in vitro studies indicate differential antibiotic susceptibility between Borrelia species, the assay has further potential as a clinical diagnostic in guiding effective disease treatment and progress monitoring.