Norovirus and Sapovirus (Caliciviridae). Detection by real time PCR (Norwalk - like viruses and virus Sapporo virus) based method ISO 15216-2 standard.

Noroviruses are very contagious virus, implicated in 60 to 95% of gastroenteritis caused by viruses, and these, in turn, are the main cause (80%) of gastroenteritis.

Norwalk virus and other similar viruses belong to the same family (Caliciviridae). These viruses were called "Norwalk-like viruses", having been found for the first time in 1968 as a result of an outbreak of gastroenteritis in the city of Norwalk (Ohio, USA). Subsequently, other outbreaks were describing some phylogenetically very similar to the Norwalk virus, and other very similar to other viruses in this family, described in the city of Sapporo in Japan in 1977 and was called Sapporovirus. Currently, these producers gastroenteritis viruses are classified into 4 genera, two of them with species that affect humans (Norovirus and Sapovirus), and two others affect only animals (Lagovirus and Vesivirus). Norovirus two genogroups differ, and within each can differentiate phylogenetic groups (clusters).

These viruses are:

Norovirus (NLVs: Norwalk-like viruses)

I genogrupo

            • Norwalk virus (prototype: Norwalk / 68 / US)
            • Southampton virus
            • Desert Shield virus
            • Cruise ship virus
            • .......

genogrupo II

            • Snow Mountain agent (prototype: Snow Mountain / 76 / US)
            • Hawaii virus
            • mexico virus
            • Toronto virus
            • Lordsdale virus
            • Grimsby virus
            • Gwynedd virus
            • White River virus
            • .......

Sapovirus (SLVs: Sapporo-like viruses)

            • Sapporo virus
            • Manchester virus
            • Parkville virus
            • London virus
            • .......  

Caliciviridae infections  

Noroviruses are very contagious virus, the main cause of nonbacterial gastroenteritis in older children (30%) and adults (50%). Are the leading cause of acute gastroenteritis outbreaks in hospitals, homes for the elderly and child care. Also produce outbreaks of gastroenteritis in hotels, cruises.

After a short incubation period (12-48 hours) gastroenteritis characterized by predominance of vomiting and abdominal pain, along with nonbloody watery diarrhea it occurs. Headache and mild fever may exist. The process is self - limited and lasts between 1 and 3 days, except in young children, the elderly and hospitalized patients who can last 4 to 6 days. Viruses are eliminated with the feces for longer (up to 2 weeks or even longer). The concentration of virus in stool can be of March 10 to 10 6 particles per mL. Infectivity of affected is very high, because the minimum infective dose may be low (10-100 virions).

In affected you drink plenty of fluids to avoid dehydration, washing hands frequently with soap and water for at least 20 seconds, especially after using the bathroom and before preparing food. In restaurant workers should be avoided cooking, preparing or serving food. Contaminated should be disinfected with vomiting or diarrhea with products containing bleach surfaces. We recommend washing fruits, vegetables and cooking bivalve molluscs (mainly oysters careful) before consuming.

Caliciviridae transmission  

The reservoir is strictly human, and the main transmission mechanism is by direct contact or fecal-oral route so that infected individuals spread the virus to other people directly, or can contaminate food or beverage prepared for others. The virus can also contaminate surfaces and survive in them, contaminating food prepared. Aerogenically noted the way when considering small splashes that may be generated during vomiting, but is discussed. In many cases it is related to the consumption of oysters contaminated or polluted water ice made with water.

In September 2012 there was a huge outbreak of gastroenteritis in Germany in several schools and kindergartens in various federal states (Berlin, Brandenburg, Saxony, Saxony-Anhalt, and Thuringia, which affected more than 11,000 children and young people who had vomiting and diarrhea . the outbreak was due to ingestion of frozen raspberries, some consumed if no processing other with short and sugared warming.

The heating time to inactivate human Norovirus is not perfectly known, the inability to test heat treatment and infectivity, from being unable to grow the virus in the laboratory, but it is believed that heating at 60 for 30 minutes not completely inactivates . If you want to completely inactivate it would be necessary to reach 90 ° C and / or maintain longer with temperature above 70 ° C. Were performed experimental studies to determine the inactivation using similar animal virus (FCV: Feline Calicivirus; BCSEQ: Canine Calicivirus; MNV: Murine Norovirus), reaching the conclusion that 70 ° C for 1 minute reduced by 3 log the number of infectious virions; 70 for 15 seconds decreased 1 log; 70 for 5 minutes reduces 7.5 log, etc.

The virucidal disinfectant action against human Norovirus, is not exactly known for being unable to cultivate and test him. Evaluations using the FCV virus (Feline Calicivirus), have shown that it is very resistant to disinfectants marketed with virucidal activity, and phenolic disinfectants used 2 to 4 times the recommended to decontaminate surfaces concentration, can control outbreaks caused by Norovirus in restaurants. In raspberries and lettuce, peroxyacetic acid and hydrogen peroxide, when used at 4 times the recommended by the manufacturer for 10 minutes, concentration is effective. It has also been shown that water washing fruits or vegetables reduces the amount of virus in 2 log. The results of the evaluation of disinfectants suggest that these viruses are very resistant to commercial disinfectants.

Diagnosis of infections Caliciviridae

This virus can not be grown in cell cultures, so the only possible alternatives are:

        • Virus detection in stool samples by observing stool purified by electron microscopy. Low sensitivity (50%).
        • Viral antigen detection mediantes immunoenzymatic reaction using various types of formats (ELISA or immunochromatography). Generally low sensitivity (35%).
        • Detecting its nucleic acid (RNA). This test is considered more sensitive (98%).

Tests in IVAMI

        • Real - time PCR, following the guidelines of ISO 15216-2 noma, and verification by sequencing for identification in case of a positive result.  

Type and volume of samples / number of samples

        • Drinking water or other (5 liters).
        • Stool (10 mL og).
        • Vomiting (10 mL og).
        • Food (100 mL or 100 g; for red fruits sample suitable for washing at least 100 g).
        • Swabs impregnated in surfaces (swabs moistened and passed through suspicious surfaces).

Sample storage: RNA viruses because it should be stored frozen, refrigerated or at least the shortest time possible, avoiding repeated freezing if frozen.  

Transport: frozen or chilled, without breaking the cold chain.

Delivery of results

The samples begin to be processed when received, and the results are output in the shortest possible time allowing turnaround times. For testing Molecular Microbiology time it is 24 to 72 hours, as would be sequenced or not. If we have before it would communicate the results.

Cost of the test:

    • PCR positive test followed by sequencing to differentiate Norovirus and Sapovirus (only if positive PCR and request ID): consult .