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.

Aerotolerant Clostridium tertium brain abscess following a lawn dart injury.

A young girl developed an intracranial abscess and necrotizing cellulitis following penetrating injury from a lawn dart. Initial identification of a gram-positive rod growing aerobically from clinical specimens was as a Bacillus organism, but the observation that the isolate grew poorly in subcultures for susceptibility testing but quite well under standard anaerobic culture techniques led to the identification of the organism as an aerotolerant Clostridium tertium. Early management of penetrating head trauma should include cranial imaging studies to detect fractures and intracranial pathology. Clinical microbiologists and clinicians should be aware of the phenomenon of aerotolerance in anaerobic bacteria to avoid errors in choice of antibiotic therapy.

Comparison of the reactivities of baculovirus-expressed recombinant Norwalk virus capsid antigen with those of the native Norwalk virus antigen in serologic assays and some epidemiologic observations.

Since the discovery of the Norwalk virus (NV) by immune electron microscopy (IEM) in 1972, serologic studies with this virus have relied on particle-positive fecal material from infected volunteers as the source of antigen because it has not been possible to propagate this virus in cell culture. However, the recent cloning of the NV (strain 8FIIa) genome and expression of the capsid protein in a baculovirus system to form "virus-like particles" has provided a consistent source of antigen (designated rNV). The purpose of the present study was to compare the antigenicities of these rNV particles with those of native NV antigen derived from human fecal material by using well-characterized sera obtained from earlier studies. In IEM studies, the rNV antigen reacted with NV-specific antibodies in a manner similar to that observed previously when particle-positive fecal material was used as antigen. In addition, a direct enzyme-linked immunosorbent assay, in which the rNV antigen was used as antigen, proved efficient and specific for the detection of serologic responses to NV compared with the previously established techniques of IEM and blocking antibody immunoassays in which particle-positive fecal material was used as the antigen. The availability of an unlimited source of antigen will enable serologic studies that will greatly increase our understanding of the epidemiology of NV and its role in human enteric illness.

Molecular characterization of Hawaii virus and other Norwalk-like viruses: evidence for genetic polymorphism among human caliciviruses.

Hawaii virus (HV), from a 1971 family outbreak of gastroenteritis, is serotypically distinct from Norwalk virus (NV), recently identified as a human calicivirus by molecular analysis. About 2600 consecutive nucleotides of the HV genome (including those encoding the viral capsid protein) and part of the polymerase region of three other viruses (MDV1, MDV6 and SV7) were sequenced. Comparison of the amino acid sequence of the capsid protein of HV with NV and other human caliciviruses (Toronto virus [TV24], Desert Shield virus [DSV395], and Southampton virus [SHV]) demonstrated the existence of two major genetic groups (genogroups) typified by HV and NV. HV had 76% identity with TV24 and 48% identity with NV, DSV395, or SHV. In addition, comparison of part of the polymerase protein of HV with other human caliciviruses also showed that there were these two genogroups. The large genetic diversity between the capsid sequence of HV and NV is consistent with their serotypic distinctiveness.

Hospitalizations involving gastroenteritis in the United States, 1985: the special burden of the disease among the elderly.

While diarrheal disease is a well-recognized problem in children, its impact in the elderly has not been adequately assessed. Among the 4.06 million hospitalizations in 1985 in the McDonnell-Douglas Health Information System database, 98,185 hospitalizations, including 1,130 deaths, had gastroenteritis recorded as a discharge diagnosis. The authors analyzed the 87,181 hospitalizations and 514 deaths for which gastroenteritis was one of the top three diagnoses. Gastroenteritis was among the top three diagnoses in 9% of all hospitalizations of children 1-4 years of age, compared with 1.5% of hospitalizations throughout adulthood (greater than or equal to 20 years). Only 0.05% of hospitalizations involving gastroenteritis were fatal for children younger than 5 years, compared with 3% in individuals 80 years or older. While children aged less than 5 years and adults aged 60 years or more each comprised one-fourth of hospitalizations involving gastroenteritis, the older group represented 85% of diarrheal deaths. Age was the most important risk factor for death subsequent to a hospitalization involving gastroenteritis (odds ratio = 52.6, 95% confidence interval 37.0-76.9 for age greater than or equal to 70 years vs. less than 5 years). Gastroenteritis is a large, underemphasized public health problem among the elderly, among whom its case-fatality ratio is higher than in children.

Laboratory-based surveillance for rotavirus United States, January 1989-May 1991.

Geographic and temporal trends of rotavirus detections in the United States for the period January 1989-May 1991 were determined by analyzing data reported monthly by 47 virology laboratories participating in the North American Rotavirus Surveillance System. Reports included complete information on the number of specimens tested, the number of test results positive for rotavirus, and the method used to detect rotavirus. Consistent trends in regional and geographic area were identified, with distinctly different peaks of rotavirus activity in the western and eastern states. Each year in the western states, rotavirus activity began in November and peaked in December-January, whereas in the eastern states activity began in January and peaked in February-March. These differences do not correlate with obvious trends in strain variation of rotavirus and remain unexplained. Unexpected reporting of summer rotavirus activity by some laboratories in 1989 was traced to the use of a single diagnostic kit and to two questionable laboratory practices: having more than six medical technologists perform the test and failure to use controls with the test. Laboratory-based surveillance of rotavirus activity has proven to be useful in identifying and correcting problems in laboratory methods for detecting rotavirus and will be a sensitive means for monitoring coverage of the rotavirus vaccine now being developed.

Molecular characterization and expression of the capsid protein of a Norwalk-like virus recovered from a Desert Shield troop with gastroenteritis.

Norwalk virus (NV) infection was recently found to be associated with gastroenteritis in U.S. military troops stationed in Saudi Arabia during the 1990 Desert Shield Operation. We identified a Norwalk-like virus in the stools of two military personnel with gastroenteritis by ELISA and IEM. By RT-PCR and sequence analysis, the nucleotide sequence of part of the polymerase region of each of these two "Desert Shield" strains (DSV275 and DSV395) was found to be 73% identical to the corresponding region of NV. In addition, one of the strains (DSV395), which underwent sequence analysis of approximately 2900 consecutive bases, had a genomic organization characteristic of the Caliciviridae. Comparison of the DSV395 amino acid sequence of the capsid region with that of three other viruses in the Norwalk group (Norwalk, Southampton, and Toronto viruses) showed amino acid identity of 47-68%. Consensus sequence analysis of these capsid proteins identified two regions of conserved amino acids that flanked an area of variable amino acids. In addition, the proteins corresponding to the capsid regions of DSV395 and NV were expressed in an in vitro translation system. Immunoprecipitation studies using the expressed capsid proteins and paired DSV395 or NV infection sera indicated the presence of shared antigenic sites between the capsid proteins of DSV395 and NV. However, hyperimmune sera specific for the self-assembled recombinant NV capsid protein did not react with DSV stool antigen in an ELISA, suggesting that there may also be unique antigenic sites not shared between DSV395 and NV.

Diarrheal deaths in the United States, 1979 through 1987. A special problem for the elderly.

OBJECTIVE: --Diarrhea is an important cause of death among young children in both developing and developed countries, but little is known about diarrheal death among adults. In this study, we examined trends in diarrheal deaths among all age groups in the United States. DESIGN/SETTING/PARTICIPANTS: --We reviewed national mortality data complied by the National Center for Health Statistics, Hyattsville, Md, which consists of information from all death certificates filed in the United States for the period 1979 through 1987. A death for which diarrhea was listed as an immediate or underlying cause was considered a "diarrheal death" and included in the analysis. RESULTS: --We found that 28,538 persons died of diarrhea cited as either an immediate or the underlying cause of death during the 9-year period. A majority of diarrheal deaths occurred among the elderly (older than 74 years of age, 51%), followed by adults 55 to 74 years of age (27%), and young children (younger than 5 years of age, 11%). For the elderly, adjusted risk factors for dying of diarrhea included being white, female, and residing in a long-term care facility. Only the elderly and young children had clear, distinct winter peaks of diarrheal deaths, suggesting that the diarrhea may, in part, be infectious in origin. CONCLUSION: --For the elderly, more directed studies of those at risk, such as nursing home residents, are needed to determine if oral rehydration therapy, vaccines, or other preventive measures might benefit this population.

Protection conferred by neonatal rotavirus infection against subsequent rotavirus diarrhea.

A cohort of newborns in New Delhi who were nosocomially infected with rotavirus during their first days of life were followed twice weekly for 14-23 months to determine whether neonatal infection protected them against subsequent episodes of rotavirus diarrhea. Infection occurred in 60% by the fourth day of life, was asymptomatic, and was caused predominantly by an unusual rotavirus strain (G9 P11) not previously identified in humans. The 148 children with neonatal rotavirus infection had 46% fewer attacks of rotavirus diarrhea in the follow-up period than the 56 infants without nosocomial infection (0.23 vs. 0.42 episodes/child-year, P < .05). This protection was concentrated among infants in their first year of life and was not associated with a significant decrease in disease severity. Consideration of this strain as a vaccine candidate will require further assessment of its natural protection under field conditions.

Identification of minireovirus as a Norwalk-like virus in pediatric patients with gastroenteritis.

In 1977, 30- to 32-nm virus-like particles, named minireovirus because of their unique morphologic appearance, were detected by electron microscopy in the stools of infants and young children with gastroenteritis. Sequence analysis of approximately 2,800 consecutive bases derived from overlapping PCR clones of a recent minireovirus clinical isolate showed 52% nucleotide sequence identity with the Norwalk virus sequence and, in addition, demonstrated that the genomic organizations of these two viruses were similar. Our data show that minireovirus is a Norwalk-like virus and should now also be included in the Caliciviridae family.

Estimates of morbidity and mortality rates for diarrheal diseases in American children.

Although the importance of diarrhea as a prime cause of morbidity and death in developing countries is well recognized, the disease burden in the United States has never been thoroughly examined. We have prepared national estimates of the annual number of cases of diarrhea in children less than 5 years of age and of the outcome, measured in terms of visits to a physician, hospitalizations, and deaths. The annual number of diarrheal episodes was estimated by reviewing longitudinal studies of childhood diarrhea conducted in the United States and extrapolating these data to the nation. Estimates of physician visits, hospitalizations, and deaths were prepared from a variety of national data sources. We estimate that 16.5 million children less than 5 years of age have between 21 and 37 million episodes of diarrhea annually. Of these, 2.1 to 3.7 million episodes lead to a physician visit, a total of 220,000 patients are hospitalized, and 325 to 425 children die. The major cost of diarrhea lies in the high numbers and cost of hospitalizations, because approximately 10.6% of hospitalizations in this age group are for diarrhea. Diarrheal deaths occur in relatively small numbers, are more common in the South and among black persons, are potentially avoidable, and could represent as much as 10% of the preventable postneonatal infant death in the United States. These estimates underscore the extensive burden of diarrheal illness in children in the United States and suggest that interventions to prevent disease or decrease its severity could be cost-effective.

Expression and self-assembly of recombinant capsid protein from the antigenically distinct Hawaii human calicivirus.

The Norwalk and Hawaii viruses are antigenically distinct members of the family Caliciviridae and are considered to be important etiologic agents of epidemic gastroenteritis, with most studies focusing on the role of Norwalk virus. To further investigate the importance of Hawaii virus, Hawaii virus-like particles (VLPs) were produced by expression of its capsid protein in the baculovirus system and these VLPs were used as the antigen in an enzyme-linked immunosorbent assay that was efficient in the detection of a serologic response to Hawaii virus. The ready availability of Hawaii VLPs should enable larger-scale epidemiological studies to further elucidate the importance of this agent.

Detection of Norwalk virus or Norwalk-like virus infections in Finnish infants and young children.

Norwalk virus (NV) and Norwalk-like viruses are important causes of epidemic nonbacterial gastroenteritis in older children and adults. Serologic responses to NV of 154 Finnish infants and young children participating in a rotavirus vaccine study were examined by ELISA with a recently available baculovirus-expressed recombinant NV capsid protein. In 4 serially collected sera (at the median ages of 3, 4, 14, and 23 months), 49% of children had at least one NV infection over the approximately 2-year study period. Children with low NV-specific IgG titers (< 1:50) at the median age of 4 or 14 months were significantly more likely to acquire an NV infection by the median age of 14 or 23 months, respectively, than children who had higher NV IgG titers (> 1:50) (P < .05). Thus, NV or Norwalk-like virus infections are more common in infants and young children than previously believed, and antibody to NV may be protective against such infections.

An outbreak of shigellosis aboard a cruise ship caused by a multiple-antibiotic-resistant strain of Shigella flexneri.

From October 23 to October 27, 1989, an outbreak of gastroenteritis occurred aboard a cruise ship in the Caribbean. The 818 passengers and 518 crew members were surveyed for gastrointestinal symptoms; 72 (14%) of 512 passengers and 12 (3%) of 388 crew members who answered the survey reported having a diarrheal illness. Multiple-antibiotic-resistant Shigella flexneri 4a was isolated from 19 ill passengers and two ill crew members. Thirteen people were hospitalized, and prolonged duration of illness was associated with taking an antibiotic to which the isolated strain of Shigella was resistant. A case-control study of food items implicated German potato salad as the vehicle of transmission. It was prepared and probably infected by a food handler from a country where multiple-antibiotic-resistant Shigella is common. Spread may have been facilitated by the limited availability of toilet facilities for the galley crew. This outbreak demonstrates how antibiotic-resistant strains can be introduced into the United States, where they can pose treatment problems. The continuing problem of foodborne gastrointestinal disease in settings such as cruise ships underscores the need for basic hygienic control for food handlers and food preparation areas. In addition, the availability of adequate working conditions for crew members, including appropriately furnished toilet facilities, may be important issues that must be addressed in order to decrease the frequency of diarrhea outbreaks aboard cruise ships.

Recommendations for collection of laboratory specimens associated with outbreaks of gastroenteritis.

Recent discoveries have implicated a number of "new" (i.e., previously unrecognized) infectious agents as important causes of outbreaks of gastroenteritis. Unfortunately, the ability to detect these agents in an outbreak can be limited by two factors: 1) the lack of appropriate assays-many of which are still in developmental stages and are not readily available to clinical laboratories, and 2) inadequately or improperly collected specimens. At CDC, many newly developed assays are being used for research and for outbreak investigations. The information in this report is especially intended for public health agencies that collaborate with CDC in investigating outbreaks of gastroenteritis. The report provides an update on guidelines and recommendations for the proper collection of specimens to be sent to CDC, gives general background information concerning some recently discovered pathogens, lists some of the tests available at CDC, and provides a list of CDC contacts. The guidelines and the general information provided on causes of outbreaks of gastroenteritis can be also used by public health workers for investigations when specific testing is available and appropriate.

Viral agents of gastroenteritis. Public health importance and outbreak management.

Each year, infectious gastroenteritis causes greater than 210,000 children in the United States to be hospitalized and 4-10 million children to die worldwide. Since the mid-1970s, knowledge has increased dramatically concerning the viral agents that are responsible for much of this public health burden. Rotavirus, the most common cause of diarrhea among children, infects virtually every child in the United States by the age of 4 years and causes potentially lethal dehydration in 0.75% of children less than 2 years of age. Other recently identified pathogens include the enteric adenoviruses, calicivirus, astrovirus, and the Norwalk family of agents. Conclusive diagnosis of these viruses requires electron microscopic examination of stool specimens, a laboratory technique that is available only at a few large centers, including CDC. Stool samples from an outbreak that are submitted to CDC for detection of viral pathology should be collected in bulk from 10 ill persons during their first 48 hours of illness, while feces are still liquid, and should be stored at 4 C (not frozen). Acute- and convalescent-phase serum samples should be collected from the same persons, plus from an equal number of controls, during the first week of illness and 3 weeks thereafter. Control measures for outbreaks of viral gastroenteritis should focus on the removal of an ongoing common source of infection (e.g., an ill food handler or the contamination of a water supply) and on the interruption of person-to-person transmission that can perpetuate an outbreak in a population after the common source has been removed. Because improvements in environmental hygiene may not be accompanied by reductions of endemic diarrhea caused by viruses, immunization may play an important role in future control; vaccine trials for rotavirus are in progress. In anticipation of vaccine development and use, CDC recently began national surveillance for the viral agents of gastroenteritis. Health-care facilities involved in the detection of rotavirus or the other viral agents of diarrhea can participate.

Sequence diversity of small, round-structured viruses in the Norwalk virus group.

We have determined the nucleotide sequences of a highly conserved region of the RNA-dependent RNA polymerase of the prototype Snow Mountain agent (SMA) and of four other small, round-structured viruses (antigenically Norwalk virus [NV]-like or SMA-like) following reverse transcription-PCR amplification of viral RNA obtained from human stools. The stool samples were either from volunteers administered SMA or from sporadic cases and outbreaks of gastroenteritis that occurred in Japan and the United Kingdom between 1984 and 1992. The GLPSG and YGDD RNA polymerase motifs were in the proper locations in the sequences of the five SRSVs, but each sequence was distinct from the 8FIIa prototype NV sequence and from each other. Analysis of the sequences and reactivities in a new NV antigen enzyme-linked immunosorbent assay showed that the five viruses could be divided into two groups (serogroups) with NV and SMA, respectively, being the prototypes. The sequences of the capsid region and a nonstructural region (2C) were determined from one strain from each group. One virus (SRSV-KY-89/89/J), isolated in Japan and antigenically similar to the prototype NV (isolated 21 years earlier in Ohio), showed a remarkable level of sequence similarity to NV. KY-89 and the 8FIIa NV showed 87.2% nucleotide similarity over 2,516 continuous nucleotides amounting to 96 to 98.9% amino acid similarity in three distinct domains in two open reading frames. Between the prototype SMA and NV, the polymerase region showed 63% nucleotide and 59% amino acid similarity, respectively. Two other antigenically SMA-like isolates (SRSV-925/92/UK and SRSV-OTH-25/89/J), from the United Kingdom and Japan, showed 80% nucleotide and 88 to 92% amino acid similarity in the polymerase region to the prototype SMA isolated 16 and 13 years earlier in the United States. The capsid region of the antigenically SMA-like OTH-25 virus showed 53% nucleotide and 65% amino acid similarity to the prototype NV capsid region. Domains of sequence diversity and conversation were identified within the capsid protein of these two distinct prototype serotypes of virus. These results indicate that NV-like and SMA-like agents are still circulating, and sequence comparisons will be useful to identify and classify distinct viruses in the NV group.

Characterization of a variant strain of Norwalk virus from a food-borne outbreak of gastroenteritis on a cruise ship in Hawaii.

A gastroenteritis outbreak affecting at least 217 (41%) of 527 passengers on a cruise ship was caused by a variant strain of Norwalk virus (NV) that is related to but distinct from the prototype NV strain. Consumption of fresh-cut fruit served at two buffets was significantly associated with illness (P < or = 0.01), and a significant dose-response relationship was evident between illness and the number of various fresh-cut fruit items eaten. Seven (58%) of 12 paired serum specimens from ill persons demonstrated at least fourfold rises in antibody response to recombinant NV capsid antigen. A 32-nm small round-structured virus was visualized by electron microscopy in 4 (29%) of 14 fecal specimens, but none of the 8 specimens that were examined by an enzyme immunoassay for NV antigen demonstrated antigen. Four (40%) of 10 fecal specimens were positive by reverse transcriptase-PCR by using primer pairs selected from the polymerase region of NV. In a 145-bp region, the PCR product shared only 72% nucleotide sequence identity with the reference NV strain and 77% nucleotide sequence identity with Southampton virus but shared 95% nucleotide sequence identity with UK2 virus, a United Kingdom reference virus strain. In addition, the outbreak virus was serotyped as UK2 virus by solid-phase immune electron microscopy. The genetic and antigenic divergence of the outbreak strain from the reference NV strain highlights the need for more broadly reactive diagnostic assays and for improved understanding of the relatedness of the NV group of agents.

A comparison of peripheral blood coculture versus 18- or 24-month serology in the diagnosis of human immunodeficiency virus infection in the offspring of infected mothers. Women and Infants Transmission Study.

The Women and Infants Transmission Study (WITS) has established virologic definitions of human immunodeficiency virus (HIV)-infected and uninfected children that have been widely used but never formally compared with serologic definitions of infection. Data from the offspring of HIV-infected women in the WITS with frequent HIV cultures during the first year of life and with HIV serology at 18 and/or 24 months of age were analyzed. Seventy-seven infants were HIV-infected and 430 uninfected by both virologic and serologic criteria. Thirteen were virologically infected (> or = 2 positive cultures) but either seronegative or serologically indeterminate. All but 1 of these had clinical HIV disease at the time of analysis. In this pediatric cohort, children defined as infected by virologic criteria often (13/90) had negative or indeterminate serology despite symptoms of HIV disease. Results suggest that serology at 18-24 months has high specificity but poor sensitivity. It should not be considered the reference standard in identifying HIV infection in perinatally exposed children.

Characterization of Toronto virus capsid protein expressed in baculovirus.

Toronto virus (TV), previously called "minireovirus", a human calicivirus classified as genogroup 2 and phylogenetic type P2-A, was originally described in association with diarrhea in children. The second open reading frame, encoding the capsid protein of TV24, was expressed in a baculovirus recombinant. The recombinant baculovirus produced a protein (rTV) with an apparent molecular mass of 58 kDa that self-assembled into virus-like particles approximately 30 nm in diameter with a density of 1.29 g/ml. Antigenic and immunogenic characteristics of these particles were determined by protein immunoblot, immunoprecipitation, and enzyme immunoassay. Seroconversion to the rTV protein was detected in 6 of 8 (75%) patients from a recent outbreak of gastroenteritis associated with a virus of similar phylogenetic type. These results confirm and extend the previous reports of the expression of the Norwalk and Mexico virus capsid proteins.