Norovirus infection laboratory findings
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Editor-In-Chief: C. Michael Gibson, M.S., M.D. [1]
Overview
In the last 10 years, diagnosis of norovirus as cause of outbreaks of acute gastroenteritis has improved with the increasing use of the reverse transcriptase polymerase chain reaction (RT-PCR). Currently, state public health laboratories of 47 states have the capability to test for noroviruses by (realtime) RT-PCR. RT-PCR detects the norovirus RNA and can be used to test stool and emesis samples, as well as environmental swabs in special studies. Identification of the virus can be best made from stool specimens taken within 48 to 72 hours after onset of symptoms, although good results can be obtained by using RT-PCR on samples taken as long as 5 days after symptom onset. Virus can sometimes be found in stool samples taken as late as 2 weeks after recovery.
Sequencing of norovirus strains found in clinical and environmental samples has greatly helped in conducting epidemiologic investigations by linking cases to each other and to a common source and by differentiating outbreaks that were mistakenly connected. Sequences can be entered into CaliciNet, a recently developed sequence database on the basis of the PulseNet model. In the next years CaliciNet will be further implemented to be able to help to determine links (e.g., norovirus contaminated foods) between outbreaks across the U.S.
Older methods for diagnosis include direct and immune electron microscopy of fecal specimens, and detection of a fourfold increase of specific antibodies in acute- and convalescent-phase blood samples. Several commercially available enzyme-linked immunosorbent assays for detection of virus in stools have been developed but await evaluation further evaluation regarding sensitivity and specificity.
Other Diagnostic Studies
Real Time PCR Assays (RT-PCR)
Real time reverse transcriptase polymerase chain reaction (RT-qPCR) assays are the preferred laboratory method for detecting norovirus. These assays are very sensitive and can detect as few as 10 to 100 norovirus copies per reaction. They use different primers to differentiate genogroup I and genogroup II norovirus. RT-qPCR assays are also quantitative and can provide estimates of viral load. The assays may be used to detect norovirus in stool, vomitus, foods, water, and environmental specimens.
Conventional RT-PCR Assays for Genotyping
Conventional RT-PCR followed by sequence analysis of the RT-PCR products is used for norovirus genotyping. Typically, a partial region of the capsid gene, such as region D, is used by laboratories participating in CaliciNet, a national laboratory surveillance network for norovirus outbreaks coordinated by CDC.
Enzyme Immunoassay
Rapid commercial assays, such as enzyme immunoassays (EIAs), that detect norovirus antigen have recently been developed. However, these kits have poor sensitivity (50%) and are not recommended for diagnosing norovirus infection in sporadic cases of gastroenteritis. The RIDASCREEN Norovirus 3rd Generation EIA was recently cleared by Food and Drug Administration for preliminary identification of norovirus when testing multiple specimens during outbreaks. However, samples that test negative should be confirmed by a second technique, such as RT-qPCR. Thus, EIA kits should not replace molecular methods during outbreak investigations.
Specimen Collection
Clinical Specimens
- Stool: Whole stool is the preferred clinical specimen for laboratory diagnosis of norovirus. Ideally, specimens should be collected during the acute phase of illness (within 48 to 72 hours after symptoms start) while stools are still liquid or semisolid. Virus is excreted in the greatest amount during this time. Norovirus can sometimes be detected in stool specimens that are collected later in the illness or after the symptoms have resolved (up to 7 to 10 days after onset). Whole stool specimens should be kept refrigerated at 39°F (4°C) if testing is done within 2 to 3 weeks. If the specimens are shipped to a laboratory for testing, each sample should be sealed in a separate bag, and kept on frozen refrigerant packs in an insulated, waterproof polystyrene container. If testing will be done more than 3 weeks after the specimens are collected, they should be frozen at -4°F (-20°C) or -94°F (-70°C). When the specimens are stored in this way, norovirus can be detected after at least 5 years.
- Vomitus: Vomitus can be collected to supplement stool specimens during an investigation. These specimens should be collected, stored, and shipped in the same way as stool specimens.
- Serum: Serum specimens are not recommended for routine laboratory diagnosis of norovirus. If feasible and warranted for special studies, acute- and convalescent-phase serum specimens may be collected and tested for a greater than fourfold rise in IgG titer to noroviruses. Acute-phase serum specimens should be collected during the first 5 days after the symptoms start. Convalescent-phase specimens should be collected during the third to fourth week after the symptoms start.
Food, Water, and Environmental Specimens
- Food and Water Specimens: In principle, norovirus can be detected in water, food, and environmental specimens. However, the virus first needs to be concentrated or extracted or both from the specimen. Validated methods for these techniques are available only for water (at CDC) and shellfish [at the Gulf Coast Seafood Laboratory, Food and Drug Administration (FDA)]. If food or water is the suspected cause of a norovirus outbreak, samples should be collected as soon as possible after people were exposed. Food specimens should be stored frozen at -4°F (-20°C). Water can be tested for norovirus by processing large volumes (up to 100L) through specially designed filters. Water samples should be stored refrigerated or chilled on ice at 39°F (4°C).
- Environmental Specimens: Norovirus RNA has been detected in swabs of environmental surfaces collected in specific outbreak settings. However, obtaining virus from swabs is highly variable. Results should be interpreted with caution and in the context of the available epidemiologic evidence.