Test Id : LNORO

Place vial in a sealed plastic bag and ship ambient. Specimens received at other temperatures will be rejected, and testing will be canceled.

Supplies: Culture and Sensitivity Stool Transport Vial (T058)

Container/Tube: Commercially available transport system specific for recovery of enteric pathogens from fecal specimens (15 mL of non-nutritive transport medium - either Cary-Blair, Para-Pak Culture and Sensitivity Media)

Specimen Volume: Representative portion of diarrheal fecal sample, 1 gram or 5 mL

Collection Instructions:

1. Collect fresh feces and place in preservative within 1 hour of collection.

2. Visibly formed feces are not consistent with Norovirus gastrointestinal disease and should not be submitted for testing.

Diagnosing gastrointestinal disease (diarrhea or vomiting) caused by norovirus genogroups 1 and 2

This test should not be used as a test-of-cure.

Noroviruses, previously known as Norwalk-like viruses, are highly contagious RNA viruses that cause acute gastroenteritis (diarrhea, vomiting). Although 6 genogroups of norovirus have been identified, only 3 genogroups (genogroup: G1, G2, and G4) cause disease in humans. Furthermore, the majority of outbreaks have been associated with G1 and G2, with G2 being most common.(1)

Noroviruses are transmitted through close, personal contact with an infected individual or via the fecal-oral route in which the virus becomes ingested in contaminated food or water. These viruses are extremely contagious, with fewer than 20 virions being able to cause disease.(1)

Once infected, the incubation period is typically short, between 24 and 72 hours. The onset of symptoms is abrupt, with vomiting and watery nonbloody diarrhea being common. Patients may also experience a low-grade fever, as well as headache and mild body aches.(1)

The diagnosis of norovirus infection can often be made on clinical grounds, and symptoms generally resolve in 24 to 48 hours. However, in certain patients, especially those who are immunocompromised or hospitalized, laboratory testing may be indicated for infection control purposes and to limit the use of antibiotics. Testing of diarrheal feces by real-time polymerase chain reaction allows for a rapid and sensitive means of detecting and differentiating norovirus G1 and G2 in clinical stool samples.

Negative

A positive result indicates that RNA from norovirus genogroups 1 and/or 2 was present in the clinical specimen.

A negative result suggests that RNA from norovirus genogroups 1 and 2 was absent in the clinical specimen.

A positive result suggests that norovirus is the cause of gastrointestinal disease (diarrhea or vomiting); however, in certain patients (eg, immunocompromised hosts), norovirus may be shed for weeks to months in the absence of symptoms.(2)

This test should not be used as a test-of-cure, due to the fact that norovirus nucleic acid may be detected in patients for weeks to months following the resolution of symptoms.(2)

A negative result suggests that norovirus is not the cause of gastrointestinal disease (diarrhea or vomiting); however, viral nucleic acid may be present at a level that is below the limit of detection for this test. The results should be interpreted in the context of the patient's clinical presentation and other laboratory findings.

Accuracy:

A total of 100 clinical stool specimens submitted to Mayo between 11/2015 and 03/2016 for testing by a commercial multiplex gastrointestinal panel (that includes norovirus) were aliquoted, blinded, and tested within 24 to 48 hours of receipt using the norovirus G1/G2 real-time polymerase chain reaction (PCR) assays. Specimens yielding discordant results between the multiplex panel and the real-time PCR were submitted to an outside reference lab and the Minnesota Department of Health (MDH) for norovirus molecular testing.

Table 1. Comparison of results following discordant analysis at an outside reference laboratory and MDH

Adjusted Sensitivity (95% confidence interval [CI])=96.9% (88.8, 99.8)

Adjusted Specificity (95% CI)=100% (88.2, 100)

Adjusted Agreement (95% CI)=98% (92.6, 99.9)

a. Samples showing discordant results between the norovirus real-time PCR and FilmArray were tested by a molecular method at Focus Diagnostics and MDH.

b. Samples that showed agreement between the norovirus real-time PCR and FilmArray were not tested further.

c. Four of these 63 samples were identified as G1 by the LDT; the remaining 59 were identified as norovirus G2.

d. One of these 2 samples was positive for norovirus at Focus Diagnostics and MDH, and upon repeat testing in triplicate by the LDT, was also positive. The second sample was positive for norovirus at MDH, but negative by Focus and the LDT upon repeat testing.

Testing of clinical stool samples yielded 59 specimens that were positive for norovirus G2 by the LDT; however, only 4 samples were determined to be positive for norovirus G1. In order to supplement the clinical data and increase the number of samples positive for norovirus G1, spiking studies were performed. Analyte-negative stool samples (n=26) were spiked with norovirus G1 RNA (ATCC) at 1 dilution above the defined limit of detection (LOD) (Table 2).

Table 2. Results of spiking studies for norovirus GI in stool specimens.

Analytical Sensitivity:

The limit of detection of both the G1 and G2 assays was determined to be 5 copies/mcL (5000 copies/mL) of stool.

Analytical Specificity:

A comprehensive specificity panel, consisting of bacteria (n=16), parasites (n=5), or viruses (n=5) known to cause gastrointestinal disease was tested by the norovirus G1/G2 assays. In addition, human DNA was tested. All members of the specificity panel were negative by both the norovirus G1 and G2 assays. In addition, a BLAST (basic local alignment search tool) analysis was performed on the primer and probe sequences and did not reveal significant cross-reactivity with organisms that may be present in stool samples.

1. National Center for Immunization and Respiratory Diseases, Division of Viral Diseases: Norovirus. Center for Disease Control and Prevention; 2013. Updated February 23, 2023. Accessed March 28, 2023. Available at www.cdc.gov/norovirus/index.html

2. Echenique IA, Stosor V, Gallon L, Kaufman D, Qi C, Zembower TR: Prolonged norovirus infection after pancreas transplantation: a case report and review of chronic norovirus. Transpl Infect Dis. 2016 Feb;18(1):98-104

This assay utilizes real-time, TaqMan-based polymerase chain reaction (PCR) technology to target the nonstructural polyprotein gene of norovirus G1 and G2. Nucleic acid is extracted from diarrheal stool samples in Cary-Blair media. Following extraction of viral RNA, reverse transcription is performed to convert norovirus genomic RNA to complementary DNA. Real-time amplification and detection are then performed. Two separate real-time PCR reactions are performed for each sample, one specific for norovirus G1 and the second targeting norovirus G2. The assay is able to accurately detect and differentiate these genogroups in clinical stool samples.(Unpublished Mayo method)

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This test was developed and its performance characteristics determined by Mayo Clinic in a manner consistent with CLIA requirements. It has not been cleared or approved by the US Food and Drug Administration.

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