METHODS: SfSAG2 and SfSAG3 genes were isolated from S. falcatula and expressed in Escherichia coli expression system. A total of 348 serum samples [volunteers from both islands (n = 100), non-Sarcocystis parasitic infections patients (n = 50) and healthy donors (n = 100)] were collected and tested with purified SfSAGs in Western blot and ELISA assays to measure the seroprevalence of human sarcocystosis.
RESULTS: None of the sera in this study reacted with rSfSAG2 by Western blot and ELISA. For rSfSAG3, relatively high prevalence of sarcocystosis was observed in Tioman Island (75.5%) than in Pangkor Island (34%) by Western blot. In ELISA, the different prevalence rate was observed between Tioman Island (43.8%) and Pangkor Island (37%). The prevalence rate in other parasitic infections (amoebiasis, cysticercosis, filariasis, malaria, toxocariasis and toxoplasmosis) was 30% by Western blot and 26% by ELISA. Only 8% (by Western blot) and 10% (by ELISA) of healthy donors showed reactivity towards rSfSAG3.
CONCLUSION: This is the first study reporting a seroprevalence of sarcocystosis in Pangkor and Tioman Islands, Malaysia. The combination of Western blot and ELISA is suitable to be used for serodiagnosis of sarcocystosis. With further evaluations, SfSAG3 can potentially be used to confirm infection, asymptomatic screening, surveillance and epidemiological studies.
METHODOLOGY/PRINCIPAL FINDINGS: Using in silico tools, we designed and expressed four novel P. knowlesi protein products to address the distinct lack of suitable serosurveillance tools: PkSERA3 antigens 1 and 2, PkSSP2/TRAP and PkTSERA2 antigen 1. Antibody prevalence to these antigens was determined by ELISA for three time-points post-treatment from a hospital-based clinical treatment trial in Sabah, East Malaysia (n = 97 individuals; 241 total samples for all time points). Higher responses were observed for the PkSERA3 antigen 2 (67%, 65/97) across all time-points (day 0: 36.9% 34/92; day 7: 63.8% 46/72; day 28: 58.4% 45/77) with significant differences between the clinical cases and controls (n = 55, mean plus 3 SD) (day 0 p<0.0001; day 7 p<0.0001; day 28 p<0.0001). Using boosted regression trees, we developed models to classify P. knowlesi exposure (cross-validated AUC 88.9%; IQR 86.1-91.3%) and identified the most predictive antibody responses.
CONCLUSIONS/SIGNIFICANCE: The PkSERA3 antigen 2 had the highest relative variable importance in all models. Further validation of these antigens is underway to determine the specificity of these tools in the context of multi-species infections at the population level.
METHODS: An indirect enzyme-linked immunosorbent assay (ELISA) was developed to evaluate the usefulness of USM.TOXO1 antigen for the detection of IgG antibodies against Toxoplasma gondii in human sera. Whereas the reactivity of the developed antigen against IgM antibody was evaluated by western blot and Dot enzyme immunoassay (dot-EIA) analysis.
RESULTS: The diagnostic performance of the new antigens in IgG ELISA was achieved at the maximum values of 85.43% and 81.25% for diagnostic sensitivity and specificity respectively. The USM.TOXO1 was also proven to be reactive with anti- T. gondii IgM antibody.
CONCLUSIONS: This finding makes the USM.TOXO1 antigen an attractive candidate for improving the toxoplasmosis serodiagnosis and demonstrates that multiepitope antigens could be a potential and promising diagnostic marker for the development of high sensitive and accurate assays.
SETTINGS AND DESIGN: Case-control study at Rheumatology Clinic of Universiti Sains Malaysia Hospital.
SUBJECTS AND METHODS: The sera of SLE patients and HCs were tested for the presence of anti-CLIC2 and anti-HMGB1 autoantibodies using human recombinant proteins and ELISA methodologies. Other serological parameters were evaluated according to routine procedures, and patients' demographic and clinical data were obtained.
STATISTICAL ANALYSIS: Mann-Whitney U-test, Chi-square test, Fisher's exact test, and receiver operating characteristic analysis.
RESULTS: Anti-CLIC2 autoantibody levels were significantly higher in SLE patients compared to HCs (P = 0.0035), whereas anti-HMGB1 autoantibody levels were not significantly elevated (P = 0.7702). Anti-CLIC2 and anti-HMGB1 autoantibody levels were not associated with ANA pattern, anti-dsDNA, and CRP. Interestingly, SLEDAI score (≥6) was associated with anti-CLIC2 (P = 0.0046) and with anti-HMGB1 (P = 0.0091) autoantibody levels.
CONCLUSION: Our findings support the potential of using anti-CLIC2 autoantibodies as a novel biomarker for SLE patients. Both anti-CLIC2 and anti-HMGB1 autoantibody levels demonstrated potential in monitoring SLE disease activity.