Outbreak of norovirus illness associated with a swimming pool.

On 3 February 2004, the Vermont Department of Health received reports of acute gastroenteritis in persons who had recently visited a swimming facility. A retrospective cohort study was conducted among persons attending the facility between 30 January and 2 February. Fifty-three of 189 (28%) persons interviewed developed vomiting or diarrhoea within 72 h after visiting the facility. Five specimens tested positive for norovirus and three specimen sequences were identical. Entering the smaller of the two pools at the facility was significantly associated with illness (RR 5.67, 95% CI 1.5-22.0, P=0.012). The investigation identified several maintenance system failures: chlorine equipment failure, poorly trained operators, inadequate maintenance checks, failure to alert management, and insufficient record keeping. This study demonstrates the vulnerability of recreational water to norovirus contamination, even in the absence of any obvious vomiting or faecal accident. Our findings also suggest that norovirus is not as resistant to chlorine as previously reported in experimental studies. Appropriate regulations and enforcement, with adequate staff training, are necessary to ensure recreational water safety.

Molecular analysis of noroviruses involved in acute gastroenteritis outbreaks in military units in Israel, 1999-2004.

The study presented here was conducted to determine the genetic properties of noroviruses (NoVs) identified between 1999 and 2004 in army recruits with acute gastroenteritis. Partial sequence analysis of the RNA-dependent RNA polymerase gene revealed the presence of two major sub-genogroups, all of which were related to genogroup II of NoV. Serological analysis using recombinant antigens confirmed this observation. Local strains associated with a 1999 outbreak were closely related to GII-6 strains, while those identified later were very closely related to GII-4 strains. GII-4 strains were also associated with an outbreak in civilian nursing homes in Israel in 2002 and samples from this outbreak were included in this study for comparison. This is the first report describing the molecular properties of NoV strains associated with diarrhea-related morbidity in Israel.

Use of stool collection kits delivered to patients can improve confirmation of etiology in foodborne disease outbreaks.

BACKGROUND: In 68% of foodborne disease outbreaks, no etiologic pathogen is identified. In two-thirds of outbreaks with no identified etiology, no stool specimens are submitted for testing. METHODS: From April 2001 to March 2003, we pilot-tested use of prepackaged, self-contained stool specimen collection kits in 3 states, delivered to and from patients by courier or mail, to improve rates of specimen collection in the outbreak setting. Specimens were tested for bacterial and viral pathogens at health department laboratories, and results were correlated with epidemiological investigation data. RESULTS: Specimens were returned by > or =1 person in 52 (96%) of 54 outbreaks in which kits were deployed; in total, 263 (76%) of 347 persons who received kits returned specimens. Resolution of symptoms was the most commonly cited reason for nonsubmission of kits. An etiology was confirmed in 37 (71%) of 52 outbreaks with specimens returned; 28 (76%) were attributable to norovirus, and 9 (24%) were attributed to bacterial pathogens. Stool kits were well received and cost an average of approximately 43 dollars per specimen returned. CONCLUSIONS: In two-thirds of foodborne disease outbreaks in which delivered stool collection kits were successfully deployed, an etiologic organism was identified. Delivery of kits to and from patients to improve rates of stool collection in outbreaks in which specimens might otherwise not be submitted could substantially reduce the number of outbreaks with an unknown etiology.

Norwalk virus gastroenteritis among Israeli soldiers: lack of evidence for flyborne transmission.

BACKGROUND: Paired sera collected from subjects before and after a fly-control intervention trial conducted in the Israel Defense Force (IDF) were tested for seroconversion to Norwalk virus (NV) to examine the role of NV as a cause of diarrhea in this population and to ascertain whether flies might also be implicated in transmission. MATERIALS AND METHODS: An enzyme-linked immunosorbent assay (ELISA), using recombinant NV capsid proteins (rNV) as antigen was employed to determine the seroconversion rate in a sample of 444 subjects. RESULTS: During 11-week field training cycles, 18% of IDF soldiers who were tested had an NV infection defined as a > or = 4-fold rise in antibody, yielding a cumulative incidence of nearly one infection (0.95) per soldier per year. The rate of seroconversion was nearly twice as high among soldiers who recalled having diarrhea as among those who did not, but the rates did not differ significantly between soldiers in the fly intervention areas and those in the control areas. CONCLUSION: NV is a common cause of enteric infections and diarrhea among Israeli soldiers who serve under field conditions, but unlike infections with Shigella and enterotoxigenic Escherichia coli, transmission of NV cannot be interrupted with an aggressive program of fly-control.

Multistate outbreak of Norwalk-like virus gastroenteritis associated with a common caterer.

In February 2000, an outbreak of gastroenteritis occurred among employees of a car dealership in New York. The same meal was also supplied to 52 dealerships nationwide, and 13 states reported illness at dealerships where the banquet was served. A retrospective cohort study was conducted to identify risk factors associated with the illness. Stool samples were collected to detect Norwalk-like virus, and sera were drawn and tested for immunoglobulin A antibodies to the outbreak strain. By univariate analysis, illness was significantly associated with consumption of any of four salads served at the banquet (relative risk = 3.8, 95% confidence interval: 2.5, 5.6). Norwalk-like virus was detected by reverse transcription-polymerase chain reaction assay in 32 of 59 stool samples from eight states. Nucleotide sequences of a 213-base pair fragment from 16 stool specimens collected from cases in eight states were identical, confirming a common source outbreak. Two of 15 workers at caterer A had elevated immunoglobulin A titers to an antigenically related Norwalk-like virus strain. This study highlights the value of molecular techniques to complement classic epidemiologic methods in outbreak investigations and underscores the critical role of food handlers in the spread of foodborne disease associated with Norwalk-like virus.

Characterization of capsid genes, expressed in the baculovirus system, of three new genetically distinct strains of "Norwalk-like viruses".

"Norwalk-like viruses" (NLVs), members of a newly defined genus of the family Caliciviridae, are the most common agents of outbreaks of gastroenteritis in the United States. Two features of NLVs have hindered the development of simple methods for detection and determination of serotype: their genetic diversity and their inability to grow in cell culture. To assess the immune responses of patients involved in outbreaks of gastroenteritis resulting from infection with NLVs, we previously used recombinant-expressed capsid antigens representing four different genetic clusters, but this panel proved insufficient for detection of an immune response in many patients. To extend and further refine this panel, we expressed in baculovirus the capsid genes of three additional genetically distinct viruses, Burwash Landing virus (BLV), White River virus (WRV), and Florida virus. All three expressed proteins assembled into virus-like particles (VLPs) that contained a full-length 64-kDa protein, but both the BLV and WRV VLPs also contained a 58-kDa protein that resulted from deletion of 39 amino acids at the amino terminus. The purified VLPs were used to measure the immune responses in 403 patients involved in 37 outbreaks of acute gastroenteritis. A majority of patients demonstrated a fourfold rise in the titer of immunoglobulin G to the antigen homologous to the outbreak strain, but most seroconverted in response to other genetically distinct antigens as well, suggesting no clear pattern of type-specific immune response. Further study of the antigenicity of the NLVs by use of VLPs should allow us to design new detection systems with either broader reactivity or better specificity and to define the optimum panel of antigens required for routine screening of patient sera.

"Norwalk-like viruses" as a cause of foodborne disease outbreaks.

While outbreaks of foodborne disease remain an important public health concern, their aetiology is not identified in a majority of instances. In targeted studies, the application of newly developed molecular assays has demonstrated that a large proportion of these outbreaks may be caused by the "Norwalk-like viruses" (NLV), a genus of genetically related viruses belonging to the family Caliciviridae. NLV outbreaks associated with consumption of faecally contaminated oysters are frequently reported and can best be controlled by preventing contamination of oyster-harvesting waters. Infectious foodhandlers are another frequent source of contamination, and such transmission can be minimised by exclusion of ill foodhandlers and the maintenance of strict personal hygiene. Molecular assays have greatly refined the epidemiological investigation of foodborne NLV outbreaks, allowing the linking of outbreaks in different locations and permitting the identification of the virus in the implicated vehicle. The development of simpler and more sensitive assays and their use on a broader scale will assist in defining the true burden of foodborne NLV outbreaks and improve strategies for their prevention and control.

Molecular epidemiology of human astroviruses.

Human astroviruses (HAstVs) are associated with 5-9 percent of cases of gastroenteritis in young children. Seven serotypes (HAstV-1 to -7), which correlate with genotypes, have been defined by using immune typing methods. We have used partial nucleotide sequence information from the capsid protein gene for molecular typing of 29 unique human astrovirus strains obtained from prospective studies of children with gastroenteritis in Egypt and Malawi. HAstV-1 was the most commonly detected strain, consistent with previous studies, but a surprising variety of strains were identified in both collections. An eighth astrovirus type, HAstV-8, has been defined on the basis of the complete capsid protein gene sequence and was detected in both collections analysed in this study. Although HAstV-8 and HAstV-4 strains segregate into well resolved clades by analysis of sequences from the region encoding protein P2 (VP32), the pair-wise distances between these types are less than those between strains of the other serotypes. In contrast, analysis of sequences from the region encoding protein P3 unambiguously resolve HAstV-4 and HAstV-8 strains, consistent with their classification as distinct serotypes. Overall, strains representing six of the eight serotypes were detected in two collections of samples from prospective studies of gastroenteritis in young children indicating that multiple astrovirus types are frequently co-circulating within communities.

Gastroenteritis viruses: an overview.

Acute gastroenteritis is among the most common illnesses of humankind, and its associated morbidity and mortality are greatest among those at the extremes of age, children and the elderly. In developing countries, gastroenteritis is a common cause of death in children < 5 years that can be linked to a wide variety of pathogens. In developed countries, while deaths from diarrhoea are less common, much illness leads to hospitalization or doctor visits. Much of the gastroenteritis in children is caused by viruses belonging to four distinct families--rotaviruses, caliciviruses, astroviruses and adenoviruses. Other viruses, such as the toroviruses, picobirnaviruses, picornavirus (the Aichi virus), and enterovirus 22, may play a role as well. Viral gastroenteritis occurs with two epidemiologic patterns, diarrhoea that is endemic in children and outbreaks that affect people of all ages. Viral diarrhoea in children is caused by group A rotaviruses, enteric adenoviruses, astroviruses and the caliciviruses; the illness affects all children worldwide in the first few years of life regardless of their level of hygiene, quality of water, food or sanitation, or type of behaviour. For all but perhaps the caliciviruses, these infections provide immunity from severe disease upon reinfection. Epidemic viral diarrhoea is caused primarily by the Norwalk-like virus genus of the caliciviruses. These viruses affect people of all ages, are often transmitted by faecally contaminated food or water, and are therefore subject to control by public health measures. The tremendous antigenic diversity of caliciviruses and short-lived immunity to infection permit repeated episodes throughout life. In the past decade, the molecular characterization of many of these gastroenteritis viruses has led to advances both in our understanding of the pathogens themselves and in development of a new generation of diagnostics. Application of these more sensitive methods to detect and characterize individual agents is just beginning, but has already opened up new avenues to reassess their disease burden, examine their molecular epidemiology, and consider new directions for their prevention and control through vaccination, improvements in food and water quality and sanitary practices.

A prospective case-control study of the role of astrovirus in acute diarrhea among hospitalized young children.

This study examines the importance of astroviruses as a cause of acute diarrhea in hospitalized children <10 years old during a 5-year period. Stools were screened by electron microscopy and were tested for astrovirus, rotavirus, and enteric adenovirus by EIA. During the study, 14.6% of hospitalized children had diarrhea. Astroviruses were second only to rotaviruses as etiologic agents of both community-acquired and nosocomial diarrhea. Community-acquired astrovirus infection occurred in 6.8% of patients, and nosocomial disease occurred in 16.2%. Most cases occurred from March through June, and astrovirus type 1 was the most common. The symptoms of astrovirus-infected children were similar to those of children with rotavirus infection. However, astrovirus-infected children had a lower median age, less dehydration, and lower symptom severity scores and were less likely to have been admitted for gastroenteritis than were children with rotavirus. Astrovirus, for which only rehydration therapy is required, should be considered as another common diarrheal pathogen in children <2 years old.

Molecular epidemiology of human astrovirus diarrhea among children from a periurban community of Mexico City.

Human astroviruses (HAstVs) were detected in 23 stool samples from 365 diarrhea episodes among 214 children (<18 months old) prospectively monitored for diarrhea in Mexico City. Stool samples were tested by EIA and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. EIA was less sensitive (74%) and equally specific, compared with RT-PCR analysis using type-common primers for HAstV detection. Of 31 HAstV isolates, EIA typed 18 (69%) of 26 EIA-positive samples, and RT-PCR analysis typed 26 (84%) of 31 RT-PCR-positive samples. Phylogenetic analysis of the 3' end of the capsid region (363 nucleotides) confirmed the type assignment by EIA and RT-PCR analysis and determined the type for 5 previously untyped samples. Six HAstV antigenic types cocirculated in the community: HAstV-2 (42%), HAstV-4 (23%), HAstV-3 (13%), HAstV-1 (10%), HAstV-5 (6%), and HAstV-7 (6%). RT-PCR and sequence analysis provided more detailed epidemiology of HAstV in the community than did antigenic detection methods.

Characterization of "Norwalk-like viruses" and astroviruses by liquid hybridization assay.

"Norwalk-like viruses" (NLVs) and human astroviruses are causative agents of gastroenteritis in all age-groups. The typing of these agents is generally done by nucleotide sequencing, blot hybridization, or enzyme immunoassay. These techniques are expensive, time-consuming, and sometimes require scarce reagents, which limits the typing of NLVs and astroviruses to a few reference laboratories. This report describes a liquid hybridization assay that uses broadly reactive probes whose sequences are based on data from specimens in collections available at CDC and GenBank. Two astrovirus genogroup-specific probes were designed and tested successfully on 26 wild strains from all serotypes. Fourteen GII and 16 GI representative NLV strains were typed without cross-hybridization by using P1B- and P2A-specific probes, described previously, and new P2B- and P1A-specific probes. Analysis of the specificity of the probes, the effect of the mismatches during hybridization, and the sensitivity of hybridization assay demonstrates this method to be a rapid and simple technique for molecular typing of NLVs and preliminary characterization of astroviruses.

"Norwalk-like viruses". Public health consequences and outbreak management.

"Norwalk-like viruses" (NLVs) cause outbreaks of gastroenteritis and are spread frequently through contaminated food or water. Molecular diagnostics now enables detecting viruses in clinical and environmental specimens, linking of NLV strains causing outbreaks in multiple geographic locations, and tracing them to their sources in contaminated food or water. This report reviews recent advances in NLV detection and provides guidelines and recommendations for investigating NLV-related outbreaks, including specimen collection and disease prevention and control. This report also updates information provided in CDC's previously published, Viral Agents of Gastroenteritis: Public Health Importance and Outbreak Management (MMWR 1990;39 [No. RR-5]: 1-24). These CDC recommendations are intended for public health professionals who investigate outbreaks of acute gastroenteritis but could be useful in academic and research settings as well.

Cloning of human picobirnavirus genomic segments and development of an RT-PCR detection assay.

Nearly full-length genomic segments 2 and a partial-length genomic segment 1 of human picobirnavirus were cloned and sequenced. The clones were derived from viruses obtained from human immunodeficiency virus (HIV)-infected patients in Atlanta, Georgia (strains 3-GA-91 and 4-GA-91) and a nonHIV-infected person from China (strain 1-CHN-97). The picobirnavirus genomic segments lacked sequence similarities with other viral sequences in GenBank and EMBL. Comparison of genomic segment 1 from a human and a rabbit picobirnavirus identified a region of 127 nucleotides with 54.7% identity. The genomic segments 2 of the 4-GA-91 and 1-CHN-97 strains had 41.4% nucleic acid identity and 30.0% amino acid similarity and contained amino acid motifs typical of RNA-dependent RNA polymerase genes. Reverse transcription-PCR detection assays were developed with primers targeted to the genomic segments 2 of strains 4-GA-91 or 1-CHN-97. Picobirnaviruses related to the China strain were the predominant viruses detected in stool samples from people in four countries on three continents. Picobirnaviruses were detected in samples from two outbreaks of gastroenteritis in long-term elder care facilities but were not determined to be the primary pathogen. Our findings support the view that picobirnaviruses constitute a distinct family of viruses.

RT-PCR identification and typing of astroviruses and Norwalk-like viruses in hospitalized patients with gastroenteritis: evidence of nosocomial infections.

BACKGROUND: Astroviruses (HAstVs) and 'Norwalk-like viruses' (NLV) are frequent causes of gastroenteritis worldwide, though no data on the strains in circulation or their prevalence is available for France. OBJECTIVES: We applied molecular methods to detect HAstVs and NLVs by reverse transcription-polymerase chain reaction (RT-PCR) in fecal samples collected during a 2-year period from children and adults hospitalized with gastroenteritis. STUDY DESIGN: All samples negative for rotavirus and adenovirus by latex agglutination which contained small (25-40 nm) viral particles observed by electron microscopy (EM) were examined by RT-PCR. RT-PCR products were sequenced to characterize the HAstV and NLV strains present. RESULTS: A total of 75 samples were analyzed by RT-PCR, of which 15 were positive for HAstV and 24 for NLV. Several distinct strains of serotype 1 HAstV, the predominant serotype, circulated during the period. Nineteen of the 24 NLVs were of the G2 genogroup including Mexico-like (n=10), Bristol-like (n=8), and Hawaii-like viruses (n=1); two were genogroup 1. Overall, seven (47%) of the 15 HAstV infections and nine (37.5%) of the 24 NLV infections appeared to be nosocomially acquired based on the date of admission in hospital and the date of illness. CONCLUSION: This study provides additional evidence of the importance of nosocomial infections caused by NLV and HAstV.

Astrovirus diarrhea in Egyptian children.

This study describes the epidemiology of astrovirus diarrhea among a population-based cohort of 397 children aged <3 years residing in rural Egypt from 1995 to 1998. The age-specific incidence rates of astrovirus diarrheal episodes per person-year were 0.38 for infants aged <6 months, 0.40 for those aged 6-11 months, 0.16 for those aged 12-23 months, and 0.05 for those aged 24-35 months. The overall incidence rate of astrovirus diarrhea was the same as that of rotavirus diarrhea, 0.19 episodes per person-year. Astrovirus infection was pathogenic and associated with severe dehydration in 17% of the cases. The most frequent serotype was HAstV-1, and, in order of decreasing frequency, HAstV-5, HAstV-8 and HAstV-3, HAstV-6, HAstV-4, and HAstV-2. In determining whether astrovirus diarrhea was associated with a reduced incidence of subsequent disease, there was evidence to suggest HAstV-1 homotypic immunity but not heterotypic immunity. Because we observed 38% of the incidence of astrovirus diarrhea to occur in infants aged <6 months, a candidate astrovirus vaccine would have to confer immunity very early in life.

The epidemiology of enteric caliciviruses from humans: a reassessment using new diagnostics.

In the United States, acute gastroenteritis is one of the most commonly noted illnesses on hospital discharge records and death certificates, yet few of these cases have an etiologic diagnosis. The application of new molecular diagnostic methods has shown caliciviruses (previously referred to as the Norwalk family of viruses or small round structured viruses) to be the most common cause of acute gastroenteritis (AGE) outbreaks in the United States, and they may emerge as a common cause of sporadic cases of AGE among both children and adults. Novel molecular methods have permitted outbreak strains to be traced back to their common source and have led to the first identification of virus in implicated vehicles of infection-water, shellfish, and foods contaminated both at their source and by food handlers. The broad application of these methods to routine diagnosis in hospitals and public health laboratories is advancing our appreciation of the full burden of calicivirus-associated diarrhea, and it is opening new avenues for its prevention and control.

Detection of norwalk-like virus in shellfish implicated in illness.

In the 1990s, Norwalk-like viruses (NLVs) were identified in patient specimens as the primary pathogen associated with shellfish-borne gastroenteritis in the United States. Identification of these viruses from implicated shellfish has been difficult due to inefficient recovery of viruses, natural polymerase chain reaction (PCR) inhibitors in shellfish, and low virus contamination. Recent improvements to the method of detecting NLVs in shellfish include enhanced processing of virus and shellfish samples, application of nested PCR and nucleotide sequencing, and increased knowledge of NLV genetic diversity. Using a newly developed and sensitive method, an NLV G2 strain was identified in 2 oyster samples implicated in a 1998 California outbreak involving 171 cases. NLV capsid primers demonstrated a greater specificity of PCR detection than did polymerase primers. The 175-base viral capsid nucleotide sequences derived from oysters were 100% identical to those derived from a patient stool sample. This finding supports the epidemiologic associations indicating that contaminated shellfish serve as the vehicle for NLV transmission.