A simple, rapid and sensitive colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) method was established to detect norovirus genotype GII. The method employed a set of six specially designed primers that recognized eight distinct sequences of RNA-dependant RNA polymerase and capsid protein gene for amplification of nucleic acid under isothermal conditions at 65 degrees C for 60 minutes. The amplification process of RT-LAMP was monitored by the addition of HNB (Hydroxy naphthol blue) dye prior to amplification. A positive reaction was indicated by a color change from violet to sky blue and confirmed by agarose electrophoresis. The specificity of the RT-LAMP was validated by detecting several different diarrhea viruses including norovirus genotype GII. The sensitivity was determined by serial dilutions of RNA molecules from in vitro transcription of norovirus genotype GII in parallel with conventional RT-PCR detection. The assay was further evaluated with 93 clinical specimens of diarrhea patients. The results showed that the sensitivity of RT-LAMP was 1 000 copies/microL with a high specificity and the relative sensitivity was at the same level as that of conventional RT-PCR. Positive rate of RT-LAMP in analysis of clinical specimens was approximately the same as that of conventional RT-PCR as well. This colorimetric RT-LAMP assay was potential for rapid detection of norovirus genotype GII on spot due to the observation of visual result with high specificity and sensitivity, time-saving and cost benefit.
Caliciviridae Infections ; diagnosis ; virology ; Colorimetry ; methods ; Feces ; virology ; Genotype ; Humans ; Norovirus ; genetics ; isolation & purification ; Nucleic Acid Amplification Techniques ; methods

OBJECTIVEIn order to find out a more convenient, rapid and efficient way in detecting human Norovirus infections in specimens collected from hospitalized patients with acute non-bacterial diarrhea in Beijing.

METHODSTwo kits for enzyme immunoassay (EIA) were used to detect human Noroviruses in stool specimens collected from 69 infants and young children as well as 15 adults who were all diagnosed as acute non-bacterial diarrhea in 4 different hospitals. Reverse transcription-polymerase chain reaction(RT-PCR) was performed to evaluate the data from two kits in this study. Data were statistically analyzed by SPSS 11.5 software. chi2 test was used to test categorical variables.

RESULTSOut of 84 stool specimens collected from infants and young children or adults with acute non-bacterial diarrhea, 17 (20.2%) were Norovirus positive determined by EIA kit A and 31 (36.9%) were Norovirus positive determined by EIA kit B. chi2 test used to test categorical variables showed significant differences (P < 0.01), suggesting that the EIA kit B was superior to the EIA kit A. Among these 84 stool specimens, 20 were tested by RT-PCR simultaneously. Out of those 20 specimens, 11 (55.0%) were Norovirus positive as determined by RT-PCR, which was higher than that from 2 EIA kits.

RESULTSfrom 10 (50.0%) samples detected by EIA kit A were consistent with those detected by RT-PCR. Through chi2 test, the categorical variables showed significant differences with P < 0.05, suggesting that RT-PCR was superior to the EIA kit A. Results from 14 (70.0%) samples detected by EIA kit B were consistent with those detected by RT-PCR while chi2 test showed that the differences were not significant (P > 0.05), among categorical variables suggesting that EIA kit B was as sensitive as RT-PCR in detecting Norovirus. The were hospital acquired diarrhea outbreaks in these three hospitals since at least 2 Norovirus positive specimens were detected in each of the hospitals.

CONCLUSIONTo detect human Noroviruses infection, EIA seemed to be more convenient and time saving than RT-PCR which had been used worldwide. The EIA kit B in this study was comparable to RT-PCR for detecting Norovirus in stool specimens. Norovirus was a pathogen causing hospital acquired diarrhea outbreaks in these three hospitals.

Adult ; Caliciviridae Infections ; diagnosis ; Child ; Child, Preschool ; China ; Cross Infection ; diagnosis ; Diarrhea ; virology ; Feces ; virology ; Hospitals ; Humans ; Immunoenzyme Techniques ; Infant ; Norovirus ; genetics ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo study the clinical manifestations for norovirus gastroenteritis in infants and young children.

METHODSStool specimens were collected from infants and children with acute diarrhea who visited the affiliated Children's Hospital to Capital Institute of Pediatrics from January 2002 to December 2006. Enzyme-linked immunosorbent assay (ELISA) was used to detect human norovirus antigen in stool specimens and polyacrylamide gel electrophoresis (PAGE) was performed to detect rotavirus genome.

RESULTSOut of the 318 specimens under testing, 79 showed positive for norovirus antigen, with a positive rate of 24.8% (79/318). Among those positive specimens, 48(48/79, 60.8%) were detected in October to December, suggesting the seasonal preference of the virus. Most of the positive specimens (91.2%) were from those under 2 years of age. Rotavirus genome were detected from 16 out of 79 norovirus positive specimens (16/79, 20.3%), indicating those patients were co-infected by these two viruses. There was significant difference found in the severity of fever but not in the frequencies of diarrhea between rotavirus and norovirus co-infection group and noroviral infection group. Fourteen out of 79 norovirus positive patients were admitted to hospitals under the diagnosis other than gastroenteritis but started to develop symptoms of diarrhea between 1 to 11 days after hospitalization.

CONCLUSIONNorovirus seemed one of the most important pathogens for acute diarrhea among infants and young children and could cause nosocomial infectious gastroenteritis.

Caliciviridae Infections ; diagnosis ; virology ; Child ; Child, Preschool ; Electrophoresis, Polyacrylamide Gel ; Enzyme-Linked Immunosorbent Assay ; Female ; Gastroenteritis ; diagnosis ; virology ; Genome, Viral ; genetics ; Humans ; Infant ; Male ; Norovirus ; classification ; genetics ; pathogenicity

Because of limited viral replication and lack of cytopathic effect in cell culture, a new PCR-based rapid seroneutralization assay for detection of GII.4 norovirus neutralized antibodies was developed with serum samples from acute-phase patients, convalescent-phase patients and healthy controls. According to this study, neutralizing antibodies were detected in 100% of convalescent-phase sera, and in 2.5% of healthy controls sera. However, all of the acute-phase serum samples could not neutralize virus efficiently. Compared to the results from ELISA (96.2% at sensitivity and 80% at specificity), the present in vitro neutralization assay is more specific and more sensitive.
Antibodies, Neutralizing ; immunology ; Base Sequence ; Caliciviridae Infections ; diagnosis ; virology ; Case-Control Studies ; Cell Line ; DNA Primers ; Enzyme-Linked Immunosorbent Assay ; Gastroenteritis ; diagnosis ; virology ; Humans ; Norovirus ; immunology ; isolation & purification ; Polymerase Chain Reaction ; methods ; Sensitivity and Specificity

Rapid and accurate detection of norovirus is essential for the prevention and control of norovirus outbreaks. This study compared the effectiveness of a new immunochromatographic assay kit (SD BIOLINE Norovirus; Standard Diagnostics, Korea) and real-time reverse transcription-PCR (RT-PCR) for detecting norovirus in fecal specimens. Compared with real-time RT-PCR, the new assay had sensitivity, specificity, positive predictive value, and negative predictive value of 76.5% (52/68), 99.7% (342/343), 98.1% (52/53), and 95.5% (342/358), respectively. The sensitivity of the assay was 81.8% (18/22) for GII.3 and 75.7% (28/37) for GII.4. None of the 38 enteric virus-positive specimens (3 for astrovirus, 5 for enteric adenovirus, and 30 for rotavirus) tested positive in the cross-reactivity test performed by using this assay. The new immunochromatographic assay may be a useful screening tool for the rapid detection of norovirus in sporadic and outbreak cases; however, negative results may require confirmatory assays of greater sensitivity.
Acute Disease ; Adolescent ; Adult ; Aged ; Aged, 80 and over ; Caliciviridae Infections/*diagnosis ; Child ; Child, Preschool ; Feces/*virology ; Gastroenteritis/*diagnosis/virology ; Humans ; *Immunoassay ; Infant ; Middle Aged ; Norovirus/*genetics/isolation & purification ; RNA, Viral/analysis ; Reagent Kits, Diagnostic ; Reverse Transcriptase Polymerase Chain Reaction ; Sensitivity and Specificity

INTRODUCTIONNorovirus gastrointestinal disease (GID) outbreaks occur frequently in closed settings, with high attack rates. On October 16, 2008, a norovirus GID outbreak occurred at a Singapore military camp. This study describes the epidemiological investigations conducted to determine the cause of outbreak and the efficacy of the public health measures implemented.

METHODSEpidemiologic investigations included a case-control study of exposure to different food items and an environmental exposure survey. Stool samplings of patients and food handlers for common pathogens, and microbiologic testing of food and water samples were performed. Inspection of dining facilities and health screening of all food-handlers were also conducted.

RESULTSA total of 156 GID cases were reported on October 15-31, 2008. 24 (15.4%) personnel were positive for norovirus. The predominant symptoms were diarrhoea (76.3%) and abdominal pain (69.2%). There was no clinical correlation between any food item and the affected personnel. Testing of food and water samples, dining facility inspections and health screening of food handlers showed satisfactory results. The environmental exposure survey indicated possible transmission due to environmental contamination by vomitus in common areas. Comprehensive environmental decontamination was performed with hypochlorite solution, and personal hygiene measures were enforced. The outbreak lasted 17 days, with a decline in cases post intervention.

CONCLUSIONTimely notification and prompt response can curtail disease transmission. Swift implementation of public health measures, such as emphasis on personal hygiene, isolation of affected cases and comprehensive disinfection of the environment, effectively stopped norovirus transmission and may be adapted for future GID outbreaks.

Acute Disease ; Adolescent ; Caliciviridae Infections ; diagnosis ; epidemiology ; Case-Control Studies ; Communicable Disease Control ; methods ; Diarrhea ; virology ; Disease Outbreaks ; statistics & numerical data ; Feces ; virology ; Food Handling ; Gastroenteritis ; epidemiology ; virology ; Humans ; Male ; Military Facilities ; Norovirus ; isolation & purification ; Singapore ; epidemiology ; Water Microbiology ; Young Adult