Norovirus Rapid Test

Sample

faeces

Intended Use

Rapid-TEST Norovirus test is a chromatographic immunoassay for the qualitative detection, in separate bands, of Genogroup I (GI) and Genogroup II (GII) of Norovirus in human faeces. A positive signal in either test bands provides a good indication that we may be in presence of a Norovirus infection which should draw the attention of the clinician. The test is based on the immunological capture of coloured microbeads during its moving along a membrane on which specific monoclonal antibodies against GI and GII have been immobilized in two separate bands. Only for laboratory use.

Storage

Product may be stored at any temperature between +2 and +25°C.
The expiry date is printed on the tubes or aluminium packages.

Performance Characteristics

Repeatability
Ten replicates of each of the three concentrations established as PC ("positive control"), LPC ("low positive control") and NC ("negative control") with our internal standard were measured on the same day by the same person. A 100% repeatability was obtained with these three critical concentrations indicating a high intra-assay precision of the test.
Hook effect
The maximum amount of Norovirus that a person can eliminate during the acute phase of disease is about 1012 particles/gr of stool13, which is equivalent to 7.5*1010 particles/ml taking into account that Rapid-TEST Norovirus test uses a sample extraction of 75 mg of stool in 1 ml of buffer. Considering an approximate equivalence between VLPs and mass of 1 μg equal to 5.87*1010 VLPs/ml, 7.5*1010 particles/ml are about 1280 ng/ml of VLP. As the idea is to know the effect of a very high concentration of VLPs, well above the values that can be found among the population, a maximum concentration of 40000 ng/ml of both GI.1 and GII.4 was measured with the Norovirus test and, in both cases, positive results were obtained. This concentration of 40000 ng/ml is 13000-fold the detection limit of GI.1 and about 50000-fold the detection limit of GII.4. It is concluded that Hook Effect is not a problem for this test.
Interfering substances
The substances indicated in the below table at the concentration specified did not interfere with the results of the test when added to stool samples (positive and negative ones):

Precision

INTER-DAY PRECISION
Using a single lot of Norovirus test a sensitivity curve is measured through four days spaced in time. The results were very reproducible (the same sensitivity for both GI and GII through the four days of measurement).
INTER-OPERATOR PRECISION
Five operators measured in duplicate a sensitivity curve. Differences were observed but in no case exceeded 1 two-fold dilution.
INTER-LOT PRECISION
Using three different lots of Norovirus test a sensitivity curve was measured in duplicate. The analysis was performed by the same person on the same day. Only differences below to 1 two-fold dilution were observed, acceptable and tolerable for the assay.
The differences found in the different "Reproducibility" sections are acceptable for a qualitative immunochromatographic technique with its inherent variability.

Sensitivity

Analytical sensitivity
For the determination of the analytical sensitivity of Norovirus test, recombinant "virus like particles" (VLP) belonging to different Genotypes such as GI.1, GI.3, GI.6, GII.1, GII.2, GII.3, GII.4, GII.7 and GII.17 were used. The mean sensitivity values obtained were very different depending on the VLP assessed. The results obtained with GI.1 and GII.4 are included below as they are the most representative and common Genotypes within each Genogroup. For this reason all manufactured lots of Norovirus test are a mixture of GI.1 + GII.4.

Detection limit for GI.1 = 3 ng/ml
Detection limit for GII.4 = 0.75 ng/ml
It is important to note that the detection limit of the test was also analyzed using real stool samples. Values were consistent with those obtained from pure VLPs. These results demonstrate the robustness of the test.
Diagnostic sensitivity and specificity
The sensitivity and specificity of the Norovirus test was assessed by measuring a total of 96 negative samples, 23 positive GI samples and 100 positive GII samples, characterized by RT-PCR (reference technique). All samples analyzed were frozen.
The following results were obtained:
Total Sensitivity: 85 %
GI Sensitivity: 87 %
GII Sensitivity: 85 %
Specificity: 96 %
It can be seen that the specificity of the Norovirus test is very high, higher than 95%. With respect to sensitivity, it is very important to analyse the values with caution taking into account that the reference technique (RT-PCR) is ultra sensitive and sometimes it might detect as positive samples belonging to asymptomatic patients or patients who have overcome the disease. Anyway, a sensitivity values around 85% for both GI and GII are high ones for a rapid test.

General Description

Noroviruses are a kind of single-stranded, positivesense RNA viruses belonging to the family Caliciviridae (Sapoviruses also belong to this family, among others). They are highly contagious viruses whose main routes of transmission are: person-to-person contact and through contaminated food/water (in the U.S. is estimated that they account for 50% of gastrointestinal outbreaks by food poisoning). They often cause large outbreaks in closed communities in a variety of environments such as hospitals, nursing homes, schools, kindergarten, restaurants, cruises where, once the virus has been introduced, the infection spreads very quickly. Several studies show that Noroviruses are responsible for almost 50% of gastroenteritis outbreaks worldwide (considering all outbreaks of any aetiology). Human Noroviruses have been very difficult to study because of their high diversity and the lack of an efficient cell culture for "in vitro" replication as well as a suitable animal model. The cloning of genomes of Norovirus and expression of the viral capsid proteins in baculovirus and other expression systems, have allowed to obtain "virus-like particles" (VLP) which, once assembled, are able to recreate the real structure of the virus. Norovirus capsid is composed of a single major structural protein, the capsid protein (VP1) which can be divided into two main domains: the shell (S) and the protruding domains (P). The expression of VP1 in a eukaryotic system allows to obtain empty structural particles similar to the native present in the virus (VLP) that have been used as a substitute for native Norovirus for many years in any research process.
Classification of Noroviruses
Noroviruses are grouped into five Genogroups (GI to GV), of which GI and GII are involved in most acute cases of viral gastroenteritis in humans. Within each Genogroup viruses are classified into Genotypes (GI.1, GII.1,...). Over 25 different Genotypes have been described within Genogroups I and II. Of these, GII.4 is the most common Genotype representing around 60-80% of cases worldwide. Following are GII.2, GII.3 and GII.7 within GII and GI.1, GI.3 or GI.4 within GI although, in general, this Genogroup is rather less common than GII, representing less than 5%. Recent studies have shown that in recent years new variant strains of GII.4 have been identified, which arise as a result of genetic changes that, in some cases, affect only one amino acid in the sequence of the capsid protein but this is enough to make them different from previous GII.4 strains. This means that any type of immunity that an individual would have generated against GII.4 could be useless against these new strains. In short, a single Genotype is presented as a whole family, tremendously complex and variable over time.
Characteristics of Norovirus Infection
Although Noroviruses can be detected throughout the year, it has been clearly observed that there is a seasonal prevalence with peaks during autumn and winter. Norovirus infection has an incubation period of 24-48 hrs. and is characterized by nausea, vomiting, abdominal pain, fever,... Acute symptoms usually appear for 1-3 days, although safety hygiene actions must be maintained for a minimum of two weeks (the elimination of the virus in faeces may continue for days, even weeks); the patient may remain asymptomatic from third or fourth day after onset of symptoms. After overcoming the infection, immunity to Norovirus is usually incomplete and temporary (about 6 months) as well as specific for a particular Genotype. Given the high genetic variability of Noroviruses, individuals are likely to be infected several times throughout their lifetime. This may explain the high rates of infection that occur in all ages at an outbreak level. Recent studies suggest that susceptibility to Norovirus infection may be genetically determined.