Validation of Multiplex Serology for human hepatitis viruses B and C, human T-lymphotropic virus 1 and Toxoplasma gondii

Abstract

Multiplex Serology is a high-throughput technology developed to simultaneously measure specific serum antibodies against multiple pathogens in one reaction vessel. Serological assays for hepatitis B (HBV) and C (HCV) viruses, human T-lymphotropic virus 1 (HTLV-1) and the protozoan parasite Toxoplasma gondii (T. gondii) were developed and validated against established reference assays. For each pathogen, between 3 and 5 specific antigens were recombinantly expressed as GST-tag fusion proteins in Escherichia coli and tested in Monoplex Serology, i.e. assays restricted to the antigens from one particular pathogen. For each of the four pathogen-specific Monoplex assays, overall seropositivity was defined using two pathogen-specific antigens. In the case of HBV Monoplex Serology, the detection of past natural HBV infection was validated based on two independent reference panels resulting in sensitivities of 92.3% and 93.0%, and specificities of 100% in both panels. Validation of HCV and HTLV-1 Monoplex Serology resulted in sensitivities of 98.0% and 95.0%, and specificities of 96.2% and 100.0%, respectively. The Monoplex Serology assay for T. gondii was validated with a sensitivity of 91.2% and specificity of 92.0%. The developed Monoplex Serology assays largely retained their characteristics when they were included in a multiplex panel (i.e. Multiplex Serology), containing additional antigens from a broad range of other pathogens. Thus HBV, HCV, HTLV-1 and T. gondii Monoplex Serology assays can efficiently be incorporated into Multiplex Serology panels tailored for application in seroepidemiological studies.

Fig 1. Comparison of quantitative antibody measurements (MFI) with reference serostatus in pathogen-specific Monoplex Serology for HBV (Ia, Ib), HCV (II), HTLV-1 (III) and T. gondii (IV).

Ia, Ib, II-IV indicate corresponding reference panels. red lines: optimized cut-offs for single antigen performance; cut-offs were determined by optimizing specificity and sensitivity analogous to Receiver Operator Characteristics analysis. No cut-offs were determined for antigens that showed a reduced capacity to distinguish between reference assay positives and negatives. MFI: Median Fluorescence Intensities.

Fig 2. Statistical performance of pathogen-specific assays (overall seropositivity to HBV, HCV (AND/OR), HTLV-1, T. gondii) in monoplex (blue) and multiplex (orange) format.

To enhance visualization, Cohen’s kappa statistics are illustrated as percentages. Sensitivity, specificity and Cohen’s kappa statistics are illustrated by boxes while the corresponding 95% CI is indicated by horizontal lines. Performance of Monoplex and Multiplex Serology was directly compared on the corresponding reference panel using ICCs. The comparison yielded moderate to excellent reliability between monoplex and multiplex format: ICC_{HBV RPIa}: 0.94 (95% CI 0.92–0.95), ICC_{HBV RPIb}: 0.97 (95% CI 0.96–0.98), ICC_HCV: 0.97 (95% CI 0.96–0.98), ICC_HTLV-1: 0.94 (95% CI 0.92–0.95), ICC_T.gondii: 0.69 (95% CI 0.61–0.76). kappa: Cohen’s kappa. ICC: Intraclass correlation coefficient. CI: confidence interval. RP: reference panel.