Development and Evaluation of a New Triplex Immunoassay That Detects Group A Streptococcus Antibodies for the Diagnosis of Rheumatic Fever

Abstract

Objectives: Streptococcal serology is a cornerstone in the diagnosis of acute rheumatic fever (ARF), a post-infectious sequalae associated with Group A Streptococcus infection. Current tests that measure anti-streptolysin-O (ASO) and anti-DNaseB (ADB) titres require parallel processing, with predictive value limited by the slow rate of decay in antibody response. Accordingly, our objective was to develop and assess the diagnostic potential of a triplex bead-based assay, which simultaneously quantifies ASO and ADB together with titres for a third antigen, SpnA. Methods: Our previous cytometric bead assay was transferred to the clinically appropriate Luminex platform by coupling streptolysin-O, DNaseB and SpnA to spectrally unique magnetic beads. Sera from over 350 subjects, including 97 ARF patients, were used to validate the assay and explore immunokinetics. Results: Operating parameters demonstrate the triplex assay produces accurate and reproducible antibody titres which, for ASO and ADB, are highly correlative with existing assay methodology. When ARF patients were stratified by time (days following hospital admission) there was no difference in ASO and ADB between PConclusions: Anti-SpnA immunokinetics support very recent Group A Streptococcus infection, and may assist in diagnostic classification of ARF. Further, bead-based assays enable streptococcal serology to be performed efficiently in a high-throughput manner.