The results of testing for cross-reactivity by MAC-ELISA by virus infection and days postonset are shown in Table . By use of a P/N cutoff of 3.0, of the 36 WN virus IgM-positive serum samples tested (average P/N, 10.27; standard deviation [SD], 3.17; range, 4.04 to 14.48), 6 (17%) reacted with SLE virus antigen (average P/N, 3.88; SD, 0.66; range, 3.0 to 4.95) and 19 (53%) reacted with JE virus antigen (average P/N, 4.35; SD, 1.25; range, 3.16 to 7.73). Of the 32 SLE virus IgM-positive serum samples (average P/N, 5.47; SD, 1.3; range, 3.06 to 7.73), 20 (63%) reacted with WN virus antigen (average P/N, 5.17; SD, 2.14; range, 3.04 to 11.36) and 7 (22%) reacted with JE virus antigen (average P/N, 4.06; SD, 1.65; range, 3.04 to 7.63). Of the 35 JE virus IgM-positive serum samples (average P/N, 5.28; SD, 1.65; range, 3.05 to 9.16), 1 (3%) reacted with SLE virus antigen and 11 (31%) reacted with WN virus antigen (average P/N, 4.4; SD, 0.85; range, 3.19 to 5.6). Table provides P/Ns for a set of WN virus SD IgM-positive CSF specimens tested against WN and SLE virus antigens. For the 28 WN-positive CSF specimens (average P/N, 17.44; SD, 9.22; range, 6.14 to 34.48), 14 (50%) reacted with SLE virus antigen (average P/N, 4.79; SD, 1.47; range, 3.19 to 7.84). All WN virus IgM-positive serum and CSF specimens had P/Ns significantly higher with the WN virus antigen than with the two heterologous flaviviral antigens. The P/Ns produced with SLE virus IgM-positive serum samples with SLE virus antigen were more comparable to the P/Ns observed with the other two flaviviral antigens. Six of these specimens had greater reactivities with WN virus antigen. Thirty-four of the 35 JE virus IgM-positive serum samples yielded higher P/Ns with JE virus antigen than with the heterologous virus antigens. One of these specimens had a greater reactivity with WN virus. The timing of serum collection postonset did not correlate directly to either the IgM cross-reactivity or the magnitude of the P/N in the first 40 days of infection.
P/N for confirmed flavivirus-positive human serum specimens tested against three related flaviviruses
P/N for confirmed WN virus-positive human CSF specimens tested against a related flavivirus
The virus cross-reactivity data, including the reactivities of a subset of each of these serum specimens with antigen from members of another flavivirus serogroup, the DEN serogroup (which includes important human pathogens but not members of the JE virus serocomplex) are depicted in Fig. . The WN virus specificity of a set of CSF specimens from WN virus-infected humans was observed to be similar to the WN virus-infected human serum specimens depicted in Fig. (Fig. ). As observed in previous studies, the MAC-ELISA cross-reactivities of these immune serum specimens from patients with WN, SLE, and JE virus infections with a polyvalent DEN virus antigen were low (Fig. ).
Line scatter graphs illustrating the extent of cross-reaction between four flaviviral antigens and WN, SLE, and JE virus IgM-positive human serum specimens.
Line scatter graphs illustrating the extent of cross-reaction between WN virus and SLE virus antigens in WN virus IgM-positive CSF.
Computed bootstrap estimates and associated 95% confidence intervals (CIs) for the ratio of the mean P/N for homologous antigen to the mean P/N for heterologous antigen (11
) for WN virus IgM-positive sera were 4.55 (95% CIs = 3.83, 5.32) for SLE virus antigen and 3.22 (95% CIs = 2.66, 3.84) for JE virus antigen. The same ratio for WN virus-infected CSF specimens for SLE virus antigen was even more significant at 5.13 (95% CI = 3.89, 6.67). The ratio of the mean P/N for homologous antigen to the mean P/N for heterologous antigen for SLE virus IgM-positive serum samples were lower: 1.35 (95% CI = 1.04, 1.59) for WN virus antigen and 2.34 (95% CI = 1.84, 2.77) for JE virus antigen. Lastly, JE virus IgM-positive serum samples gave ratios of 1.92 (95% CI = 1.6, 2.3) for WN virus antigen and 3.34 (95% CI = 2.83, 3.88) for SLE virus antigen.
The empirical ROC curves for the three heterologous antigens are plotted in Fig. . WN virus had the highest AUC (AUC = 0.99; 95% CI = 0.98, 1.0), followed by JE virus (AUC = 0.94; 95% CI = 0.90, 0.98) and SLE virus (AUC = 0.87; 95% CI = 0.80, 0.94). Positivity was determined by fixing the sensitivity at 95 and 99% to maximize specificity. The resulting estimates of specificity and associated 95% bootstrap confidence intervals were computed by using the methods of Platt et al. (14
). The resulting specificities were lowest with SLE virus and highest with WN virus. Use of this assay to detect WN virus infections appears to be highly specific, particularly compared to use of this assay to detect SLE virus (Table ).
Empirical ROC curves by use of P/Ns for WN, SLE, and JE viruses.
Observed specificities at fixed sensitivities using P/N for WN virus, SLE virus, and JE virus determined by MAC-ELISA