Establishment of an Algorithm Using prM/E- and NS1-Specific IgM Antibody-Capture Enzyme-Linked Immunosorbent Assays in Diagnosis of Japanese Encephalitis Virus and West Nile Virus Infections in Humans

Abstract

The front-line assay for the presumptive serodiagnosis of acute Japanese encephalitis virus (JEV) and West Nile virus (WNV) infections is the premembrane/envelope (prM/E)-specific IgM antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA). Due to antibody cross-reactivity, MAC-ELISA-positive samples may be confirmed with a time-consuming plaque reduction neutralization test (PRNT). In the present study, we applied a previously developed anti-nonstructural protein 1 (NS1)-specific MAC-ELISA (NS1-MAC-ELISA) on archived acute-phase serum specimens from patients with confirmed JEV and WNV infections and compared the results with prM/E containing virus-like particle-specific MAC-ELISA (VLP-MAC-ELISA). Paired-receiver operating characteristic (ROC) curve analyses revealed no statistical differences in the overall assay performances of the VLP- and NS1-MAC-ELISAs. The two methods had high sensitivities of 100% but slightly lower specificities that ranged between 80% and 100%. When the NS1-MAC-ELISA was used to confirm positive results in the VLP-MAC-ELISA, the specificity of serodiagnosis, especially for JEV infection, was increased to 90% when applied in areas where JEV cocirculates with WNV, or to 100% when applied in areas that were endemic for JEV. The results also showed that using multiple antigens could resolve the cross-reactivity in the assays. Significantly higher positive-to-negative (P/N) values were consistently obtained with the homologous antigens than those with the heterologous antigens. JEV or WNV was reliably identified as the currently infecting flavivirus by a higher ratio of JEV-to-WNV P/N values or vice versa. In summary of the above-described results, the diagnostic algorithm combining the use of multiantigen VLP- and NS1-MAC-ELISAs was developed and can be practically applied to obtain a more specific and reliable result for the serodiagnosis of JEV and WNV infections without the need for PRNT. The developed algorithm should provide great utility in diagnostic and surveillance activities in which test accuracy is of utmost importance for effective disease intervention.

FIG 1

Comparison of NS1-MAC-ELISAs with and without serum preabsorption with VLP antigens. (A) P/N values of anti-NS1 IgM on JEV-infected human serum with (+) or without (−) preabsorption with JEV VLPs before JEV NS1-MAC-ELISA. (B) P/N values of anti-NS1 IgM on WNV-infected human serum with (+) or without (−) preabsorption with WNV VLPs before WNV NS1-MAC-ELISA. (C) Detection of VLP antigens by anti-JEV mouse hyperimmune ascitic fluid (MHIAF) and prM/E antibody-VLP antigen immune complexes by anti-human IgM on the Ag-ELISA plate used in the preabsorption of the JEV-infected serum (JEV +ve) and normal human serum (NHS). The dotted lines indicate the P/N cutoff value of ≥3.0 for the positive detection of serum IgM. All data were obtained from the results from two independent experiments, and the error bars represent the standard deviations. Statistical significance is indicated with two asterisks (P < 0.01).

FIG 2

Fitted ROC curves of VLP- and NS1-MAC-ELISAs on JEV-infected (A) and WNV-infected (B) human sera. Assay performances between JEV VLP- and NS1-MAC-ELISAs on the target JEV-infected serum panel (A) or between WNV VLP- and NS1-MAC-ELISAs on the target WNV-infected serum panel (B) against the control panel, including DENV, YFV, Zika, HTN, CHIKV, and negative panels, were compared.

FIG 3

Bland-Altman analyses between VLP- and NS1-MAC-ELISAs on JEV- and WNV-infected human sera. (A and B) Bland-Altman plots showing the differences in the average log-transformed P/N values of VLP- and NS1-MAC-ELISAs on JEV-infected (A) and WNV-infected (B) human serum specimens. The solid horizontal lines indicate the mean bias of the systematic difference between the two methods. The dashed horizontal lines indicate the 95% limits of agreement as the mean bias ±1.96 its standard deviation (SD). The dotted lines indicate the line of equality or zero difference between the two methods. The dashed and dotted horizontal lines indicate the 95% CI of the mean bias.

FIG 4

P/N values of VLP- and NS1-MAC-ELISAs and the ratios of JEV-to-WNV P/N value (JEV/WNV IgM ratio) and WNV-to-JEV P/N value (WNV/JEV IgM ratio). (A and B) P/N values (upper panel) and JEV/WNV IgM ratios (lower panel) of VLP- (A) and NS1-MAC-ELISAs (B) using JEV and WNV antigens on JEV-infected human sera. (C and D) P/N values (upper panel) and WNV/JEV IgM ratios (lower panel) of VLP-MAC-ELISA (C) and NS1-MAC-ELISA (D) using JEV and WNV antigens on WNV-infected human sera. The open circles indicate the P/N values obtained by using JEV antigens, and the closed circles indicate the P/N values obtained by using WNV antigens. The JEV/WNV and WNV/JEV IgM ratios are indicated with open and closed diamonds, respectively. All data points were obtained from the results from two independent experiments in duplicates. The dotted lines denote the cutoff values of ≥3.0 for P/N and >1.0 for JEV/WNV and WNV/JEV IgM ratios.

FIG 5

Diagnostic algorithm for confirmation of acute JEV or WNV infection in human serum using multiantigen VLP- and NS1-MAC-ELISAs.