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BackgroundAcute rheumatic fever (ARF) is a serious sequela of Group A Streptococcus (GAS) infection associated with significant global mortality. Pathogenesis remains poorly understood, with the current prevailing hypothesis based on molecular mimicry and the notion that antibodies generated in response to GAS infection cross-react with cardiac proteins such as myosin. Contemporary investigations of the broader autoantibody response in ARF are needed to both inform pathogenesis models and identify new biomarkers for the disease.MethodsThis study has utilised a multi-platform approach to profile circulating autoantibodies in ARF. Sera from patients with ARF, matched healthy controls and patients with uncomplicated GAS pharyngitis were initially analysed for autoreactivity using high content protein arrays (Protoarray, 9000 autoantigens), and further explored using a second protein array platform (HuProt Array, 16,000 autoantigens) and 2-D gel electrophoresis of heart tissue combined with mass spectrometry. Selected autoantigens were orthogonally validated using conventional immunoassays with sera from an ARF case-control study (n=79 cases and n=89 matched healthy controls) and a related study of GAS pharyngitis (n=39) conducted in New Zealand.ResultsGlobal analysis of the protein array data showed an increase in total autoantigen reactivity in ARF patients compared with controls, as well as marked heterogeneity in the autoantibody profiles between ARF patients. Autoantigens previously implicated in ARF pathogenesis, such as myosin and collagens were detected, as were novel candidates. Disease pathway analysis revealed several autoantigens within pathways linked to arthritic and myocardial disease. Orthogonal validation of three novel autoantigens (PTPN2, DMD and ANXA6) showed significant elevation of serum antibodies in ARF (p < 0.05), and further highlighted heterogeneity with patients reactive to different combinations of the three antigens.ConclusionsThe broad yet heterogenous elevation of autoantibodies observed suggests epitope spreading, and an expansion of the autoantibody repertoire, likely plays a key role in ARF pathogenesis and disease progression. Multiple autoantigens may be needed as diagnostic biomarkers to capture this heterogeneity.

Acute rheumatic fever (ARF) is a serious post-infectious immune sequelae of Group A streptococcus (GAS). Pathogenesis remains poorly understood, including the events associated with collagen autoantibody generation. GAS express streptococcal collagen-like proteins (Scl) that contain a collagenous domain resembling human collagen. Here, the relationship between antibody reactivity to GAS Scl proteins and human collagen in ARF was investigated. Serum IgG specific for a representative Scl protein (Scl1.1) together with collagen-I and collagen-IV mimetic peptides were quantified in ARF patients (n = 36) and healthy matched controls (n = 36). Reactivity to Scl1.1 was significantly elevated in ARF compared to controls (P < 0.0001) and this was mapped to the collagen-like region of the protein, rather than the N-terminal non-collagenous region. Reactivity to collagen-1 and collagen-IV peptides was also significantly elevated in ARF cases (P < 0.001). However, there was no correlation between Scl1.1 and collagen peptide antibody binding, and hierarchical clustering of ARF cases by IgG reactivity showed two distinct clusters, with Scl1.1 antigens in one and collagen peptides in the other, demonstrating that collagen autoantibodies are not immunologically related to those targeting Scl1.1. Thus, anti-collagen antibodies in ARF appear to be generated as part of the autoreactivity process, independent of any mimicry with GAS collagen-like proteins.

Streptococcal serology is a cornerstone in the diagnosis of acute rheumatic fever (ARF), a postinfectious sequela associated with group A Streptococcus infection. Current tests that measure anti-streptolysin O (ASO) and anti-DNaseB (ADB) titers require parallel processing, with their predictive value limited by the low 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 titers for a third antigen, SpnA. 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 more than 350 subjects, including 97 ARF patients, were used to validate the assay and explore immunokinetics. Operating parameters demonstrate that the triplex assay produces accurate and reproducible antibody titers 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

Streptococcal serology is a cornerstone in the diagnosis of acute rheumatic fever (ARF), a postinfectious sequela associated with group A Streptococcus infection. Current tests that measure anti-streptolysin O (ASO) and anti-DNaseB (ADB) titers require parallel processing, with their predictive value limited by the low 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 titers for a third antigen, SpnA. 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 more than 350 subjects, including 97 ARF patients, were used to validate the assay and explore immunokinetics. Operating parameters demonstrate that the triplex assay produces accurate and reproducible antibody titers 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

Objectives Streptococcal serology provides evidence of prior Group A Streptococcus (GAS) exposure, crucial to the diagnosis of acute rheumatic fever (ARF) and post-streptococcal glomerulonephritis. However, current tests, which measure anti-streptolysin-O and anti-DNaseB antibodies, are limited by false positives in GAS endemic settings, and incompatible methodology requiring the two tests to be run in parallel. The objective was to improve streptococcal serology by combining the novel GAS antigen, SpnA, with streptolysin-O and DNaseB in a contemporary, bead-based immunoassay. Methods Recombinant streptolysin-O, DNAseB and SpnA were conjugated to polystyrene beads with unique fluorescence positions so antibody binding to all three antigens could be detected simultaneously by cytometric bead array. Multiplex assays were run on sera collected in three groups: ARF; ethnically matched healthy children; and healthy adults. Results The ability of the antigens to detect a previous GAS exposure in ARF was assessed using the 80th centile of the healthy children group as cut-off (upper limit of normal). SpnA had the highest sensitivity at 88%, compared with 75% for streptolysin-O and 56% for DNaseB. Conclusions SpnA has favorable immunokinetics for streptococcal serology, and can be combined with anti-streptolysin-O and anti-DNaseB in a multiplex format to improve efficiency and accuracy.