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

Jedhan U. Galula; Gwong-Jen J. Chang; Shih-Te Chuang; Day-Yu Chao

doi:10.1128/JCM.02469-15

DOI: 10.1128/JCM.02469-15

Article Figures & Data

Figures

Tables
Additional Files

Open in new tab
Download powerpoint
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).
Open in new tab
Download powerpoint
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.
Open in new tab
Download powerpoint
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.
Open in new tab
Download powerpoint
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.
Open in new tab
Download powerpoint
FIG 5
Diagnostic algorithm for confirmation of acute JEV or WNV infection in human serum using multiantigen VLP- and NS1-MAC-ELISAs.

TABLE 1

Test accuracy of MAC-ELISA using VLP and/or NS1 antigens^a

Control panel by disease panel	Antigen	ELISA result	True status		AUC (95% CI)	% sensitivity (95% CI)	% specificity (95% CI)
Control panel by disease panel	Antigen	ELISA result	Disease	No disease	AUC (95% CI)	% sensitivity (95% CI)	% specificity (95% CI)
JEV
Other groups^b	JEV VLP	Positive	16	5	1.00 (1.00–1.00)	100.00 (79.41–100.00)	92.54 (83.44–97.53)
		Negative	0	62
	JEV NS1	Positive	16	0	1.00 (1.00–1.00)	100.00 (79.41–100.00)	100.00 (94.64–100.00)
		Negative	0	67
	JEV VLP and NS1	Positive	16	0	NA^c	100.00 (79.41–100.00)	100.00 (94.64–100.00)
		Negative	0	67
WNV and other groups	JEV VLP	Positive	16	23	0.99 (0.99–1.00)	100.00 (79.41–100.00)	82.44 (74.83–88.53)
		Negative	0	108
	JEV NS1	Positive	16	41	0.99 (0.96–1.01)	100.00 (79.41–100.00)	68.70 (60.02–76.52)
		Negative	0	90
	JEV VLP and NS1	Positive	16	13	NA	100.00 (79.41–100.00)	90.08 (83.63–94.61)
		Negative	0	118
WNV
Other groups	WNV VLP	Positive	64	0	1.00 (1.00–1.00)	100.00 (94.40–100.00)	100.00 (94.64–100.00)
		Negative	0	67
	WNV NS1	Positive	64	2	0.99 (0.98–1.01)	100.00 (94.40–100.00)	97.01 (89.63–99.63)
		Negative	0	65
	WNV VLP and NS1	Positive	64	0	NA	100.00 (94.40–100.00)	100.00 (94.64–100.00)
		Negative	0	67
JEV and other groups	WNV VLP	Positive	64	8	0.99 (0.98–1.00)^d	100.00 (94.40–100.00)	90.36 (81.89–95.75)
		Negative	0	75
	WNV NS1	Positive	64	16	0.89 (0.83–0.94)	100.00 (94.40–100.00)	80.72 (70.59–88.56)
		Negative	0	67
	WNV VLP and NS1	Positive	64	8	NA	100.00 (94.40–100.00)	90.36 (81.89–95.75)
		Negative	0	75

↵a Accuracy was defined as the ability to distinguish the disease panel (JEV or WNV serum panels) from the control panel using the positive-cutoff criterion (P/N ≥ 3.0) as the evidence of infection.
↵b Other groups include DENV, YFV, Zika, HTN, CHIKV, and negative serum panels.
↵c NA, not available.
↵d P < 0.05.

Additional Files

Figures
Tables

Supplemental material

Supplemental file 1 -
Table S1 (Summary of VLP- and NS1-MAC-ELISA results on 147 human sera tested with JEV and WNV antigens)

PDF, 479K