Evaluation and comparison of two commercial enzyme-linked immunosorbent assay kits for detection of antigenically diverse human noroviruses in stool samples

Abstract

Two recently commercialized enzyme-linked immunosorbent assay kits, the SRSV(II)-AD (Denka Seiken Co. Ltd., Tokyo, Japan) and IDEIA NLV (DakoCytomation Ltd., Ely, United Kingdom) kits, that detect human norovirus (HuNV) antigens in stool samples were evaluated to assess whether they could be used instead of reverse transcription-PCR (RT-PCR) for routine diagnosis. The sensitivities and specificities of the two kits were tested with a panel of 103 stool samples containing HuNVs of 4 and 10 genetic subgroups within genogroups I and II (GI and GII), respectively, and 39 stool samples containing other enteric viruses. The Denka kit had a high sensitivity (>70% for 10 of the 14 subgroups) but a specificity of only 69%, and the Dako kit had a low sensitivity (<30% for 6 GII subgroups) but a high specificity of 100%. Statistical analysis suggests that HuNVs of four subgroups (subgroups GII/2, GII/5, GII/6, and GII/n) are likely to elude detection by the Dako kit. The two kits also demonstrated differences in reactivities. While the Dako kit discriminated between the GI and GII antigens of HuNVs, the Denka kit cross-reacted with samples containing all GI and GII subgroups of HuNVs. Moreover, the Denka kit also reacted with samples containing human sapovirus (HuSV). We demonstrate that the cross-reactivity of the Denka kit is not due to specific reactions with HuNV and HuSV antigens. These results indicate that neither the Denka kit nor the Dako kit has all the performance characteristics required to replace the RT-PCR methods used to detect HuNVs.

FIG. 1.

Comparison of OD values between Denka and Dako kits (A and B) and the GI and GII wells of the Denka kit (C and D) and the Dako kit (E and F), respectively. Points represent OD values in the GI and GII wells obtained from the same sample. The left (A, C, and E) and right (B, D, and F) groups of graphs provide OD values for samples with GI HuNVs and GII HuNVs, respectively. Dashed lines on horizontal and vertical axes indicate cutoff OD values. The dashed diagonal lines correspond to the locations where the OD values in the two wells are the same.

FIG. 2.

Box-and-whisker plots of homologous OD values, showing the 95% central ranges derived from an estimated normal distribution of OD values obtained with the Denka kit (A) and the Dako kit (B) before logarithmic transformation. The left and right panels indicate OD values for samples with GI and GII HuNVs, respectively. The bottom, middle, and top lines of each box correspond to the 25, 50, and 75% cumulative frequencies of the observed values, respectively. The upper and lower whiskers extending from the box correspond to 2.5 and 97.5% cumulative frequencies, respectively (either the whisker or the box line, or both, overlap each other for some subgroups). The plus signs and squares mark the mean OD value and the relative outliers, respectively. The dashed line indicates cutoff OD value. Non-HuNVs, negative reference samples.

FIG. 3.

Results of evaluation of the Denka kit with a panel of VLPs (see the text for details). ▴, OD values in GI wells; •, OD values in GII wells.

FIG. 4.

Reactivity of HuNV GI/3 antigen with the Denka kit after purification from stool samples by isopycnic CsCl density gradient centrifugation followed by dialysis. *, CsCl density; ▴, OD values in GI wells; •, OD vales in GII wells. Dashed lines indicate the cutoff values in GI wells (OD, 0.182) and GII wells (OD, 0.275).