In this study, we evaluated the diagnostic accuracy, analytical sensitivity, and reactivity of the latest version of the IDEIA Norovirus assay. The assay showed excellent specificity (91.9%) and PPV (91.5%) but low sensitivity (57.6%) and NPV (59.0%). We demonstrated that the lack of sensitivity of the assay during the prospective study was primarily due to the low analytical sensitivity (or limit of detection) of the assay. The kit detects most of the NoV GI and GII genotypes circulating in humans (53
), although some uncommon genotypes could not be detected.
Laboratory diagnosis of NoV outbreaks relies on the detection of virus particles by EM, detection of viral RNA by RT-PCR, or detection of viral antigens by EIA (12
). A critical parameter for evaluating the performance of EIAs is the selection of the reference method. When using EM as the reference method we found that the sensitivity of the IDEIA Norovirus assay was 77.8% and the sensitivity of the rRT-PCR was 91.7%. The specificity of the rRT-PCR was only 50%, because 32 samples confirmed as NoV by sequence analysis tested negative by EM. Using conventional RT-PCR/bidirectional sequencing as the reference method, the sensitivity of the rRT-PCR increased to 100%, compared to 57.8% and 51.6% for the EIA and EM, respectively. Therefore, we chose an algorithm that combined conventional RT-PCR, rRT-PCR, and EM results as the reference method.
The low sensitivity of the IDEIA Norovirus assay in our study is similar or higher than previously reported results (5
). There are several reasons that could explain the low sensitivity of the EIA. First, collection of samples at >72 h after onset of symptoms is directly correlated with the lack of detecting NoV by the EIA (12
). In addition, storage of fecal samples for long periods of time before testing may also compromise the level of virus, as proteolytic degradation may occur. In our study, fecal samples were collected and tested within 72 h after onset of viral gastroenteritis symptoms (12
), while in other studies samples were tested by EIA after storage for 2 to 3 years (11
). Second, differences in inclusion criteria for the fecal samples between different studies may influence the outcome. For example, in our study, samples were only included when the clinical symptoms were consistent with a NoV outbreak. Other studies tested all samples that were submitted to a public health laboratory (12
) or a subset of samples reflecting the strain diversity, but not necessarily the prevalence of NoVs (5
). Finally, the antigenic diversity of the NoV strains may explain the discrepancy in sensitivity of the EIA kit found in different studies. The IDEIA Norovirus kit uses a cocktail of monoclonal and polyclonal antibodies generated against virus-like particles representing a selection of GI and GII genotypes, and certain genotypes may be missed (5
). This was clearly shown in a study where the sensitivity, but not the specificity, dropped significantly when samples from 2002, when the pandemic strain GII.4 Farmington Hills emerged, were tested (11
), or when samples were selected to reflect the known NoV genotypes at that time (5
The low sensitivity of the assay raises serious questions about the usefulness of this kit for routine screening for NoV. In our study, the sensitivity of the IDEIA Norovirus assay to diagnose a NoV outbreak compared to detecting NoV in a single fecal sample did not differ significantly (58.7% and 59.0%, respectively). However, the sensitivity of the assay to confirm a norovirus outbreak increased from 44.1% when three samples were tested to 76.9% when five samples were tested (19
). A previous study using statistical analysis demonstrated that when at least five fecal samples per outbreak are tested, finding two out of five samples positive was sufficient to diagnose an outbreak as NoV with at least 95% confidence, with a test sensitivity of 72% (14
). However, in our study, 71.6% of the outbreaks had less than five samples, which increased the risk of false-negative outbreaks.
With the great genetic and antigenic diversities of human NoV strains (3 genogroups [GI, GII, and GIV] and at least 25 genotypes) (53
) and the continuous emergence of new strains (43
), it is crucial that a diagnostic test detects the wide antigenic variety of contemporary NoV strains. The IDEIA Norovirus assay successfully detected strains from seven genotypes in fecal samples collected during the prospective arm of the study, but it failed to detect strains of another six genotypes (GI.7, GI.8, GI.14, GII.6, GII.9, and GII.14). However, in the retrospective study, 18 of the 21 genotypes were detected and only strains of GI.8, GII.10, and GII.16 were missed. The failure to detect specific NoV genotypes was also reported in previous studies (19
), but unlike our study, no contemporary strains were included in those earlier studies. For GII.4 strains, the majority of the samples with a CT
lower than 26.5 tested positive by the EIA kit, which suggests that although a few genotypes are not detected in the EIA kit (e.g., GI.8), the low sensitivity for samples collected during the prospective study is primarily caused by an insufficient amount of virus (analytical sensitivity).
Our study is the first to estimate the detection limit of a commercial norovirus EIA. The lowest number of GII.4 RNA copies that resulted in a positive signal by EIA was 4.20 × 108
of fecal sample, which equals a CT
value of 25.6 based on the standard curve. Fifty percent of the GII samples that tested negative by EIA had a CT
value higher than 26.5, suggesting that this is the approximate detection limit of the EIA. For GI.4, the lowest number of NoV RNA copies g−1
of fecal sample that resulted in a positive signal by EIA was 4.94 × 106
, which equals a CT
value of 27.9 based on the standard curve. During the first 48 h of a NoV outbreak, the viral load in fecal samples ranges from 107
), which suggests that samples that are collected later than 48 h after the onset of symptoms could be missed by the IDEIA Norovirus kit. However, additional studies are needed to evaluate the limit of detection of other genotypes.
In summary, the IDEIA Norovirus assay showed excellent specificity and PPV but low sensitivity and NPV both for outbreaks as well as for samples from sporadic cases. The low NPV indicates that EIA-negative results need to be confirmed by RT-PCR, but a positive result strongly correlates with the presence of NoV. The kit successfully detected 18 of the 21 genotypes evaluated and demonstrated that at least 107 virus particles g−1 of fecal sample are required for a positive signal. Taken together our data demonstrate that (i) the IDEIA Norovirus kit should only be used for samples collected during an outbreak; (ii) samples should be collected preferably during the first 48 h after onset of symptoms; and (iii) testing more than five samples per outbreak significantly increases the likelihood of a positive NoV diagnosis for the outbreak.
In conclusion, the IDEIA Norovirus kit may be useful for rapid screening of fecal samples collected during an outbreak of acute gastroenteritis. However, negative samples will have to be confirmed by a second technique, such as rRT-PCR.