Agents Of Viral Gastroenteritis Research Articles

Detection of Viral Agents of Gastroenteritis: Norovirus , Sapovirus and Astrovirus

Norovirus, Sapovirus and Astrovirus are causative agents of viral gastroenteritis affecting all age groups, but most frequently the young, the elderly and persons in semi-closed communities such as hospitals, nursing homes, military bases and cruise ships. The sensitive and rapid detection of causative agents of viral gastroenteritis is key to the effective implementation of infection control systems. Traditional detection methods such as electron microscopy and antigen detection assays lack sensitivity. The detection of gastrointestinal viruses by molecular methods has resulted in increased levels of detection, and enables the epidemiological investigation of viral strains. The significant diversity of gastrointestinal viruses, in particular Norovirus and Sapovirus, are compounded by increasing reports of virus recombination, and pose an ongoing challenge to the development of sensitive and specific molecular detection assays.

Real-time reverse transcription PCR detection of norovirus, sapovirus and astrovirus as causative agents of acute viral gastroenteritis

The design and development of highly sensitive real-time reverse transcription PCR assays for the detection of norovirus genogroups I, II and IV, sapovirus genogroups I, II and IV, and human astrovirus from stool samples is described. Examination of 140 stool samples from paediatric patients exhibiting symptoms of diarrhoea and/or vomiting resulted in increased detection levels as compared to examination by electron microscopy. Real-time PCR resulted in a 200% increase in the rate of detection of norovirus as compared to electron microscopy. Only genogroup II noroviruses were detected in the stool specimens and when examined using partial-genotyping primers all were identified as clustering with the genogroup II/4 (Bristol/Lordsdale) cluster. Sapovirus was not detected in any of the stool specimens by electron microscopy while 11% (15/140) of specimens were sapovirus positive by real-time RT-PCR, accounting for 36% of calicivirus diarrhoea. Real-time RT-PCR resulted in a tenfold increase in the rate of detection of astrovirus when compared to detection by electron microscopy with both type 1 and type 4 human astroviruses being detected in circulation. The results highlight the importance of the introduction of molecular methods for the routine screening of stool samples for causative agents of viral gastroenteritis.

Prevalence of sapovirus infection among infants and children with acute gastroenteritis in Dhaka City, Bangladesh during 2004–2005

Sapovirus, a member of the family Caliciviridae is one of the major causative agents of viral gastroenteritis affecting all age group. Sapovirus was detected in 25 of 917 stool specimens from infants and children with acute gastroenteritis in a Children Hospital in Dhaka City, Bangladesh during 2004-2005. All fecal specimens were examined for sapovirus by reverse transcription-polymerase chain reaction. Molecular analysis of sapovirus was carried out by sequencing methods. Sapovirus detected in this study was clustered into only one distinct genogroup I. Sapovirus GI/1 was predominant, followed by GI/2 and accounted for 92% (23 of 25) and 8% (2 of 25), respectively. The results clearly indicated that sapovirus infections were observed most commonly in the autumn to winter seasons (September to January) in Dhaka City. The common clinical symptoms of sapovirus infected patients were dehydration (88%), vomiting (76%), and abdominal pain (60%). This is the first report of sapovirus in Bangladesh.

Prevalence of human astrovirus genotypes associated with acute gastroenteritis among children in Belém, Brazil

Worldwide human astroviruses (HAstV) have increasingly been recognized as causative agents of viral gastroenteritis, mainly in infants and young children. The aim of this study was to assess the epidemiology and genotype diversity of HAstVs detected in children who participated in a trial in Belém, Brazil with the rhesus human reassortant rotavirus vaccine tetravalent (RRV-TV). From April/1990 to August/1992, 624 diarrheic stool samples were tested by enzyme immunoassay (EIA) for HAstV, with a positive rate of 4.0%. Reverse transcription-polymerase chain reaction (RT-PCR) was done in 129 samples (25 positive and 104 with twice the optical density (OD) value of negative control by EIA) being 33 positive. The overall positivity yielded by both methods was 5.4% (34/624). Genotyping of the 33 positive samples was done by type-specific RT-PCR and confirmed by sequence analysis. Phylogenetic analysis was performed using a 348-bp fragment of the ORF2 region of the capsid gene. HAstV-1 was the most prevalent, accounting for 45.5% of the isolates, followed by HAstV-2 (27.3%), HAstV-3 (12.1%), HAstV-4 (12.1%), and HAstV-6 (3.0%). The monthly distribution showed that HAstV-1 was predominant in the first year of study (May/1990 to May/1991) with highest prevalence in January/1991. HAstV-2 was predominant from July to November/1991 and HAstV-4 from September to October/1990. At 24 months of age, 30.6% of children had been infected by HAstV. The clinical symptoms registered during HAstV associated-diarrhea were usually mild. These data highlight the circulation of the different HAstV genotypes in Belém during the study period.

Sapovirus detection by quantitative real-time RT-PCR in clinical stool specimens

Sapovirus (SV) is one of the major causative agents of viral gastroenteritis affecting all age groups worldwide. A new method for the quantitative detection of SV from clinical stool specimens by real-time reverse transcription-polymerase chain reaction (RT-PCR) based on TaqMan ® MGB technology was described. Primers and probe were designed to target the RNA-dependent RNA polymerase/capsid genes junction. Performance of the newly developed assay was validated against a panel of 244 clinical stool specimens collected for patients with gastroenteritis. SV was detected in eight (3.3%) specimens. Phylogenetic analysis of the positive isolates suggested that the assay could detect at least SV genogroups I, II and IV. In addition, the assay had an increased detection rate compared with a widely used conventional RT-PCR assay. Quantitative analysis showed that the assay could detect as low as 10 copies of viral cDNA per reaction. No cross-reactivity with norovirus and rotavirus was observed. In conclusion, the assay is a sensitive and specific method for the detection of SV from clinical stool specimens.

Viral Gastroenteritis in Mongolian Infants

Viral Gastroenteritis in Mongolian Infants

One-year survey of astrovirus infection in children with gastroenteritis in a large hospital in Hungary: occurrence and genetic analysis of astroviruses.

Human astroviruses (HAstV) are the causative agents of viral gastroenteritis mainly in children worldwide. This study investigated the epidemiology and genotype diversity of HAstVs detected in children admitted to hospital with gastroenteritis in Hungary. Stool samples were collected from children with diarrhea at the Municipal "Szent László" Hospital, Budapest, Hungary, between January 2002 and December 2002. Of 2,758 samples, 607 were negative for both rotaviruses and bacterial pathogens and were tested for astroviruses using a reverse transcriptase-polymerase chain reaction (RT-PCR) targeting the open reading frame (ORF2), capsid region. Astrovirus was detected in 10 samples (1.6%) by RT-PCR. Astrovirus infection was more frequent among children 49 to 60 months of age. Genotyping of positive samples was performed by type-specific RT-PCR and confirmed by sequence analysis. Phylogenetic analysis was performed using a 203 nucleotide consensus length of the 3'-end of the capsid gene. Type-specific RT-PCR and sequence analysis detected genotypes 1 (50%), 4 (30%), 3 (10%), and 8 (10%) among the children admitted to hospital. Genotype 1 was the predominant genotype, but genotypes 3, 4, and 8 were also present indicating the importance of emerging genotype 8 infections. Two distinct genotype 4 variants were observed during this study. Sequence analysis confirmed type-specific RT-PCR results in the capsid region. This is the first comprehensive report on the occurrence of HAstV infections in Central/Eastern Europe.

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Background: With rotavirus and Norwalk-like viruses, astroviruses are now recognized as important etiologic agents of viral gastroenteritis in all age groups. However, astrovirus is neither routinely screened for in stool samples, nor in environmental samples, and data on the health impact of waterborne astrovirus are lacking. Objectives: To assess the potential impact of astrovirus in drinking water on the incidence of acute digestive conditions (ADC) among a panel of volunteers. Study design: The Epidemiology and MIcrobial Risk Assessment (E.MI.R.A.) study combined a daily epidemiological follow-up of digestive morbidity among a panel of 544 volunteers supplied by French public water systems, and a microbiological surveillance of drinking water. Cases of digestive morbidity were collected through weekly telephone calls. The bacterial, virological and parasitic quality of tap water was assessed monthly. Additional samples were collected if the incidence of ADC increased. The relationship between incidence of ADC during a 7-day period centered about the water sampling day and astrovirus RNA prevalence in drinking water was modeled by regression techniques, taking into account several confounders. Results: 12% (8/68) of the analyzed water samples were positive for astrovirus, and presence of astrovirus RNA was associated with a significant increased risk of ADC: RR=1.51 (95% CI=[1.17–1.94], P value=0.002). Conclusions: This result suggests a role for waterborne astrovirus in the endemic level of digestive morbidity in the general population. Perhaps astrovirus is a candidate test target for viral surveillance of drinking water.

Are waterborne astrovirus implicated in acute digestive morbidity (E.MI.R.A. study)?

With rotavirus and Norwalk-like viruses, astroviruses are now recognized as important etiologic agents of viral gastroenteritis in all age groups. However, astrovirus is neither routinely screened for in stool samples, nor in environmental samples, and data on the health impact of waterborne astrovirus are lacking. To assess the potential impact of astrovirus in drinking water on the incidence of acute digestive conditions (ADC) among a panel of volunteers. The Epidemiology and MIcrobial Risk Assessment (E.MI.R.A.) study combined a daily epidemiological follow-up of digestive morbidity among a panel of 544 volunteers supplied by French public water systems, and a microbiological surveillance of drinking water. Cases of digestive morbidity were collected through weekly telephone calls. The bacterial, virological and parasitic quality of tap water was assessed monthly. Additional samples were collected if the incidence of ADC increased. The relationship between incidence of ADC during a 7-day period centered about the water sampling day and astrovirus RNA prevalence in drinking water was modeled by regression techniques, taking into account several confounders. 12% (8/68) of the analyzed water samples were positive for astrovirus, and presence of astrovirus RNA was associated with a significant increased risk of ADC: RR=1.51 (95% CI=[1.17-1.94], P value=0.002). This result suggests a role for waterborne astrovirus in the endemic level of digestive morbidity in the general population. Perhaps astrovirus is a candidate test target for viral surveillance of drinking water.

Experimental infection of Macaca nemestrina with a Toronto Norwalk-like virus of epidemic viral gastroenteritis.

Norwalk virus (NV) and Norwalk-like viruses (NLVs) are common etiologic agents of viral gastroenteritis. Viral gastroenteritis is a common disease that is highly transmissible, spreading rapidly through families, institutions, and communities. Because methods for in vitro cultivation of Norwalk etiologic agents are not available, information regarding this syndrome has come largely from studies in human volunteers. Sequential passaging of an NLV through an immunoincompetent newborn pigtail macaque (Macaca nemestrina) may allow for the adaptation of a human NLV to a primate host, thus providing an animal model for investigating this disease. A fecal filtrate of human origin containing NLV, Toronto virus P2-A, was obtained from a patient during an epidemic of viral gastroenteritis. The filtrate was administered via nasogastric tube to three newborn pigtailed macaques. Clinical illness, which was characterized by diarrhea, dehydration, and vomiting, occurred in three monkeys. Reverse transcription-polymerase chain reaction (RT-PCR) and oligonucleotide probe analysis of RNA extracted from the stool samples following infection revealed viral RNA in all inoculated monkeys. Infection was also transmitted experimentally by feeding two additional newborn macaques a fecal filtrate prepared from the three previously infected animals. Detection of viral RNA in the stools of animals that received the fecal filtrate indicates that viral replication occurred in association with clinical illness. The susceptibility of Macaca nemestrina to infection with a Norwalk-like agent will facilitate the study of the mechanisms of the pathogenesis of NLV. This system may also have the potential to serve as a vaccine test model for human epidemic viral gastroenteritis.

Determination of a universal nucleic acid extraction procedure for PCR detection of gastroenteritis viruses in faecal specimens

Four nucleic acid extraction protocols were examined for their suitability for extraction of the ssRNA, dsRNA and dsDNA genomes of gastroenteritis viruses, for PCR detection. Protocol (A), employed specimen lysis with guanidinium thiocyanate, extraction with phenol–chloroform–isoamyl alcohol and nucleic acid purification by size-fractionated silica particles. Protocol (B), utilised specimen lysis with guanidinium thiocyanate and nucleic acid purification by silica, followed by phenol–chloroform–isoamyl alcohol extraction. Protocol (C), employed specimen lysis with guanidinium thiocyanate and nucleic acid purification by RNAID glass powder. Protocol (D), employed specimen lysis with sodium dodecyl sulphate, proteinase K digestion and extraction with phenol–chloroform–isoamyl alcohol. Of the four protocols, (B) appeared to be a suitable candidate ‘universal’ nucleic acid extraction procedure for PCR detection of different viral agents of gastroenteritis in a single nucleic acid extract of a faecal specimen, irrespective of genome composition. Omission of the phenol–chloroform extraction step did not affect negatively the ability of protocol (B) to allow PCR detection of gastroenteritis viruses in faecal specimens. PCR detection of NLVs, astroviruses, rotaviruses and adenoviruses, in single nucleic acid extracts of faecal specimens obtained from the field, confirmed the universality of the modified protocol (B). We propose the modified protocol (B) as a ‘universal’ nucleic acid extraction procedure, for monoplex PCR detection of gastroenteritis viruses in single nucleic acid extracts of faecal specimens and for development of multiplex PCR for their simultaneous detection.

Detection of sporadic cases of Norwalk-like virus (NLV) and astrovirus infection in a single Irish hospital from 1996 to 1998.

'Norwalk-like viruses' (NLV) and astroviruses are recognised as the most important etiologic agents of viral gastroenteritis, excluding rotaviruses. However, neither of these two groups of viruses is routinely screened for in Irish hospital laboratories. The objective of this study was to examine faeces collected from patients with non-bacterial, non-rotaviral gastroenteritis and examine if NLVs and astroviruses could be identified as the causative agents of the illness. Faecal specimens were collected from a single Irish hospital from February 1996 to June 1998. Three hundred and sixty samples were tested for the presence of NLVs using newly designed inosine-containing degenerate primers. Two hundred and three faecal specimens from paediatric patients were screened for the presence of astroviruses. the results of the screening study were that 29 (8%) specimens were found to be positive for NLV by reverse transcription polymerase chain reaction (RT-PCR) and 15 (7%) specimens from paediatric patients were found to be positive for astroviruses. Genotyping of the NLV-positive samples determined that four of the isolates were from genotype I (G1) and 25 were G2. The G2 positive specimens were further subtyped by oligonucleotide probing and the majority (n = 21) were found to be subtype P2-B, with four isolates being typed as P2-A. No P1-B isolates were found. This is the first report of detection of sporadic cases of NLV and astrovirus in Ireland. The results obtained highlight the need for continued surveillance of these viruses and the development of rapid detection systems for use in clinical laboratories.

Perinatal risk factors for infant hospitalization with viral gastroenteritis.

A tetravalent vaccine against rotavirus, the most commonly identified etiologic agent of viral gastroenteritis (GE), has recently been licensed for use in the United States. To evaluate whether specific groups of infants might be at sufficiently high risk to warrant a focused rotavirus vaccine policy, we investigated perinatal risk factors for hospitalization with viral GE and rotavirus in the first year of life. Population-based, case-control study. Washington State linked birth certificate and hospital discharge abstracts from 1987 through 1995. Infants, 1 through 11 months of age, hospitalized for viral GE (N = 1606) were patients in this study. Control subjects were 8084 nonhospitalized infants, frequency-matched to patients on year of birth. Maternal and infant characteristics associated with infant hospitalization for viral GE. We found a significant association between birth weight and the risk for hospitalization. Very low birth weight infants (<1500 g) were at the highest risk (odds ratio [OR] 2.6; 95% confidence interval [CI]: 1.6,4.1);, low birth weight infants (1500-2499 g), at intermediate risk (OR 1.6; 95% CI: 1.3,2.1); and large infants (>4000 g), at reduced risk (OR 0.8; 95% CI: 0.6,0.9). Other characteristics associated with GE hospitalization were male gender (OR 1.4; 95% CI: 1.3,1.6); maternal smoking (OR 1.2; 95% CI: 1.1,1. 4); unmarried mother (OR 1.2; 95% CI: 1.1,1.4); Medicaid insurance (OR 1.4; 95% CI: 1.3,1.7); and maternal age <20 years (OR 1.2; 95% CI: 1.0,1.5). Infants born October through December were at decreased risk for hospitalization (OR 0.8; 95% CI: 0.7,0.9), as were infants born to Asian mothers (OR 0.5; 95% CI: 0.3,0.7), and infants born to mothers >34 years of age (OR 0.7; 95% CI: 0.6,0.9). Using these factors, the area under a receiver operating characteristic curve was 0.63. Therefore, to achieve a sensitivity of 90% in identifying high-risk infants, specificity would fall to 10%. Subanalyses of children admitted for viral GE during the peak of the Northwest rotavirus season (January to March) and children with confirmed rotavirus infection demonstrated similar risk factors and receiver operating characteristic curves. We conclude that a focused rotavirus vaccination policy using readily identifiable potential high-risk groups would be unlikely to prevent most infant hospitalizations associated with rotavirus infection. However, the safety of rotavirus vaccine in low birth weight and premature infants must be established, because these children appear to be at greater risk for hospitalization with viral GE and rotavirus.

Six-year retrospective surveillance of gastroenteritis viruses identified at ten electron microscopy centers in the United States and Canada

To identify the prevalence, seasonality and demographic characteristics of patients with viral gastroenteritis, we reviewed 6 years of retrospective data on viral agents of gastroenteritis screened by electron microscopy at 10 centers in the United States and Canada. From 52,691 individual electron microscopic observations, a virus was detected in 16% of specimens, and the yearly positive detection rate among centers ranged from 8 to 34%. Rotavirus was the agent most commonly observed (26 to 83%), followed by adenoviruses (8 to 27%, respiratory and enteric combined), and small round viruses (SRVs) (0 to 40%) which were second most common at two of the centers. Rotavirus and astrovirus detections occurred more often in the winter but seasonal trends in detection were not apparent for the other viruses. Of all astroviruses detected 64% were found in infants (less than 1 year); unlike the other agents studied SRVs were detected in a large percentage of infants (48%) and older children and adults (20%). Among hospitalized patients a majority of all astroviruses, caliciviruses and SRVs were detected 7 days or more after admission in contrast to both rotaviruses and adenoviruses which were more likely to be detected earlier. The data suggest that SRVs are common agents of gastroenteritis and may be important causes of nosocomial infections. Because of the relative insensitivity of direct electron microscopy as a screening method for SRVs, astroviruses and caliciviruses, these data probably underestimate the true prevalence of disease caused by these agents.

Development of an enzyme immunoassay for the Hawaii agent of viral gastroenteritis

The Hawaii agent is a Norwalk-like virus of acute gastroenteritis in humans which is antigenically distinct from the prototype Norwalk agent. We established a solid phase sandwich type microtiter enzyme immunoassay (EIA) for Hawaii antigen employing sera and stools from experimentally challenged volunteers as reagents. This assay detected the Hawaii agent in stools from 3 of 8 volunteers who were ill after oral challenge with the Hawaii agent, including one specimen which was positive to a dilution of 1 320 . Virus shedding occurred on days 3 to 7 after challenge. The Hawaii-positive stools did not react in the EIAs for Norwalk and Snow Mountain agent (SMA), nor did Norwalk or SMA-positive stools react in the Hawaii EIA. Human rotavirus, enteric adenovirus, feline calicivirus, and several enteroviruses also did not react in the Hawaii EIA. A blocking EIA to detect serum antibody to the Hawaii agent was also developed employing a diarrheal stool containing Hawaii as a source of antigen. Serum antibody rises were detected in 15 of 16 individuals with experimentally induced illness after challenge and in 3 of 5 individuals who remained well after challenge. The EIA for the Hawaii agent should permit epidemiologic studies of the Hawaii agent to be carried out as well as allow further characterization of the Hawaii virion.

Antigenic Characterization of Cell-Cultivated Astrovirus Serotypes and Development of Astrovirus-Specific Monoclonal Antibodies

Cultivation of human astroviruses in human embryonic kidney or LLCMK2 cell cultures was corroborated for four of the five serotypes originally reported (types 1, 2, 4, and 5). By using type-specific rabbit antisera and immunofluorescence of virus-infected cells, we readily distinguished between serotypes of astrovirus; however, these serotypes showed a high degree of cross-reactivity by enzyme-linked immunoassay, a result indicating the presence of a group antigen. We prepared monoclonal antibodies to astrovirus type 2 antigen and selected them on the basis of group antigen reactivity. The antibodies were reactive with the four astrovirus serotypes that we could cultivate, as well as with the Marin County strain of astrovirus. A previously reported cell-cultivated astrovirus type 3 also reacted with the monoclonal antibodies. These monoclonal antibodies, and the finding of group reactivity among the human astroviruses, should facilitate studies on the importance of these viruses as agents of viral gastroenteritis.

Viral Hepatitis and Gastroenteritis Transmitted by Shellfish and Water

The epidemiology and clinical presentation of water-borne viral diseases, including shellfish-associated viral illnesses, are discussed in this article. Hepatitis A virus, Non A-Non B hepatitis and the agents of viral gastroenteritis, Norwalk agent, Snow Mountain agent, rotavirus, the small round viruses, caliciviruses, and astroviruses are included. The technical problems associated with evaluating the viral contamination of water or shellfish are noted.

Radioimmunoassay for detection of the Snow Mountain Agent of viral gastroenteritis.

The Snow Mountain Agent (SMA) is a Norwalk-like viral agent of acute gastroenteritis that has been detected only by immune electron microscopy (IEM). We established a solid phase microtiter radioimmunoassay (RIA) for SMA antigen employing pre-and post-challenge sera from volunteer studies as capture antibodies. SMA was detected in 12 of 67 stool samples from volunteers who were ill after oral challenge with SMA. All samples in which virus particles were detected by IEM were positive by RIA, but the RIA was 10-80 times more sensitive than IEM. To detect serum antibody to SMA, a blocking test was developed, employing diarrheal stool containing SMA as a standard source of antigen. Serum antibody rises were detected in eight out of nine volunteers with experimentally induced illness following challenge with SMA, as well as in three out of three naturally occurring cases. A preliminary sero-epidemiologic survey suggested that infection with SMA was common in the population surveyed. This RIA should permit large scale seroepidemiologic studies of SMA to be carried out, and should also facilitate characterization of this agent.

Immunoassays for the diagnosis of viral enteric pathogens

The accurate diagnosis of infectious diseases is important for the optimal management of infected patients as well as for the prevention of disease transmission to susceptible individuals. Because viral gastroenteritis constitutes an important cause of morbidity in children living in developed countries and of mortality in children living in developing countries, there has been a great deal of interest in the development of effective methods for the diagnosis and study of this disease. While there are a number of assay systems that are capable of accurate detection of the agents of viral gastroenteritis in the intestinal contents of infected individuals, solid phase enzyme immunoassays have been used widely for this purpose. The widespread utilization of enzyme immunoassays is based on the advantages inherent in the use of enzymatic markers. These advantages include the stability of enzyme-immunoglobulin conjugates, the high degree of sensitivity inherent in the magnifying nature of enzyme-substrate interactions, and the low cost of assays performed in simple reaction formats. Enzyme immunoassays have thus been developed and widely used for the detection and epidemiologic study of rotaviruses, adenoviruses, coxsackie viruses, hepatitis A virus, and other agents that replicate in the human gastrointestinal tract. In addition, latex agglutination assays and nucleic acid hybridization techniques have been applied to the rapid detection of enteric viruses. It is likely that the application of a number of assay systems will promote accurate identification of a wide range of viral pathogens under different clinical, epidemiologic, and laboratory situations.

Public Health and Water Quality Significance of Viral Diseases Transmitted by Drinking Water and Recreational Water

A review of the available information from case and outbreak reports and the results of prospective and retrospective studies revealed that the potential for the spread of viral diseases by the use of fecally contaminated drinking and recreational water has only been realized for infectious hepatitis and viral gastroenteritis. These two, however, are the most serious and prevalent, respectively, of the water-related diseases. A model is described for predicting the beach specific, swimming-associated rates and annual number of cases of gastroenteritis. The inputs are the swimming-associated, gastroenteritis rate-bathing water enterococcus density regression equation developed from the USEPA prospective epidemiologic-microbiologic studies and the distribution of enterococcus density estimates and annual number of swimmers at the beach in question. In general, the gastroenteritis rates predicted from the enterococcus model were less than those from the corresponding E. coli model. Detectable rates at the 75 percentile level and rates approaching or exceeding 5/1000 swimmers at the 90 percentile level were predicted for 7 and 14, respectively, of the 87 sampling station-beach associations. All but one of the fourteen stations were potentially impacted by known municipal wastewater discharges and two of the associated beaches were posted as unsafe for swimming. The inapplicability of the model to beaches impacted with small, immediate sources of fecal wastes or stormwater run-off is noted. The limitations in the use of findings from the analysis of outbreak reports in the evaluation and establishment of water quality criteria and, hence, the need for prospective, drinking water and shellfish epidemiological studies is discussed. The need for such studies also derives from the results of the bathing beach study in that they suggest that cases of gastroenteritis are occurring with acceptable drinking and shellfish growing waters. The need for tissue culture enumerative methods for the viral gastroenteritis agents and methodology for determining the biological decay coefficients for these agents and their indicators is noted.