Diagnosis of astrovirus gastroenteritis by antigen detection with monoclonal antibodies.

An enzyme-linked immunosorbent assay (ELISA), based on monoclonal antibodies to the astrovirus group antigen, was designed for the detection of astroviruses in stools of patients with gastroenteritis. Compared to immune electron microscopy used as the standard test, the sensitivity of the astrovirus ELISA was 91% (31/34) and the specificity was 96% (54/56). All five of the known astrovirus serotypes could be detected in 16 samples on which serotyping was done. In tests on 155 stools containing other enteric viruses, including adenoviruses, rotaviruses, caliciviruses, Hawaii virus, Snow Mountain virus, and Norwalk virus (30, 20, 70, 24, 4, and 7 samples, respectively), only 3 were positive in the astrovirus ELISA. The combined specificity for all astrovirus immune electron microscopy-negative samples was 98% (206/211). The results demonstrate that the new ELISA provides a sensitive and specific means for the diagnosis of astrovirus gastroenteritis.

Role of Norwalk virus in two foodborne outbreaks of gastroenteritis: definitive virus association.

Two separate food-associated outbreaks of gastroenteritis occurred among Erie County, New York residents in June 1986. In one outbreak, cases of illness were estimated to have occurred in 50% of the approximately 700 persons in 13 groups who ate at an out-of-county restaurant during a seven-day period, and, in the second outbreak, illness occurred in 26 (30%) of 87 persons who attended a graduation party held in a private home. Laboratory investigation included serology (blocking radioimmunoassay) to determine seroconversion to Norwalk virus and an enzyme immunoassay for detection of Norwalk virus antigen in stools, which the investigators have found to be more specific for Norwalk virus than serology. Seroconversion to Norwalk virus occurred in 11 (79%) of 14 restaurant-related cases and seven (100%) of seven graduation party cases. Seroconversion to Norwalk virus antigen was also found in four (40%) of 10 food handlers at the restaurant and in two (100%) of two food handlers at the graduation party. Antigen was detected in the stools of three (20%) of 15 restaurant-related cases and four (67%) of six graduation party cases. No stools for viral analyses were available for testing from food handlers. All seven of the patients with Norwalk virus-positive stools were also positive by seroconversion. Widespread availability of reagents for stool antigen detection would result in confirmation of more outbreaks due to Norwalk virus and in a more timely manner.

Antigenic relationships between human caliciviruses and Norwalk virus.

We investigated antigenic relationships between human calicivirus (HCV) strains and Norwalk virus by using immune electron microscopy (IEM) and radioimmunoassay (RIA). Three serologically distinct HCV strains, UK1, UK2, and Japan, were demonstrated by IEM, as was evidence for two additional strains, UK3 and UK4. Although HCV strains and Norwalk virus were distinct by IEM, 12 of 20 patients with gastroenteritis due to HCV UK4 and two of eight with gastroenteritis due to UK2 showed seroconversions to Norwalk virus by RIA. These naturally occurring antibody responses in humans, as detected by RIA, support the concept that Norwalk virus belongs to the family Caliciviridae. An RIA for HCV Japan antigen also detected HCV UK1, UK2, and UK4 and thus appears to identify a group-specific antigen for these viruses. An RIA for antibody to HCV Japan failed to identify seroconversions in 45 of 47 patients with gastroenteritis due to HCV UK. These results may reflect different reactivities of various immunologic tests for the identification of infections due to small gastroenteritis viruses.

Widespread outbreaks of clam- and oyster-associated gastroenteritis. Role of Norwalk virus.

Consumption of raw shellfish has long been known to be associated with individual cases and sporadic outbreaks of enteric illness. However, during 1982, outbreaks of gastroenteritis associated with eating raw shellfish reached epidemic proportions in New York State. Between May 1 and December 31, there were 103 well-documented outbreaks in which 1017 persons became ill: 813 cases were related to eating clams, and 204 to eating oysters. The most common symptoms were diarrhea, nausea, abdominal cramps, and vomiting. Incubation periods were generally 24 to 48 hours long, and the duration of illness was 24 to 48 hours. Bacteriologic analyses of stool and shellfish specimens did not reveal a causative agent. Norwalk virus was implicated as the predominant etiologic agent by clinical features of the illness and by seroconversion and the formation of IgM antibody to Norwalk virus in paired serum samples from persons in five (71 percent) of seven outbreaks in which testing was done. In addition, Norwalk virus was identified by radioimmunoassay in clam and oyster specimens from two of the outbreaks. Determining the source of the shellfish was not always possible, but northeastern coastal waters were implicated. The magnitude, persistence, and widespread nature of these outbreaks raise further questions about the safety of consuming raw shellfish.

Detection of Norwalk virus in stools by enzyme immunoassay.

The development of a solid-phase microtiter enzyme immunoassay (EIA) for detection of Norwalk virus antigen in stool samples is described. The EIA was compared with a previously developed radioimmunoassay (RIA) for detection of Norwalk virus antigen in stools obtained from 30 volunteers who received Norwalk virus. The EIA detected viral antigen in stools from 17 of the volunteers and the RIA detected viral antigen in 15. Seroconversion was a more sensitive indicator of infection in some patients. However, two samples from volunteers who were clinically ill but did not show seroconversion to Norwalk virus were positive for Norwalk virus antigen by both immunoassays. This indicates that antigen detection may be important for use in epidemiological studies. Neither of the immunoassays gave positive reactions for stools known to contain enteric adenovirus, rotavirus, or Hawaii virus, or in stools from patients with acute diarrhea of unknown cause. The stability of the EIA reagents and ease of use should provide a means for more extensive testing for Norwalk virus in outbreaks of gastroenteritis.

Comparative analysis of serum antibody responses to Pseudomonas aeruginosa exotoxin A by cystic fibrosis and intensive care unit patients.

Pulmonary infection with Pseudomonas aeruginosa is a major cause of morbidity and mortality in cystic fibrosis (CF) patients. P. aeruginosa toxin is one of several proposed virulence factors which may be responsible for chronic P. aeruginosa infections in these patients. With a highly specific, sensitive, and quantitative radioimmunoassay (RIA) and a cell culture assay, the humoral immune responses of CF patients in terms of total antitoxin, antitoxin immunoglobulins A and M, and neutralizing antitoxin were compared with those of P. aeruginosa-infected intensive care unit patients and controls. The P. aeruginosa-infected CF patients were divided into severe and moderate disease groups based on mortality observed over an 8-year period. The intensive care unit patients were divided by the site of infection and the controls were healthy children and uninfected CF patients. Antibodies to toxin were found in the sera of all subjects by radioimmunoassay. Neutralizing antibody was associated with current infection. Elevated titers of antitoxin immunoglobulin A were found only in subjects with pulmonary P. aeruginosa infections. No significant differences in any antibody class were observed between the severe and moderate disease groups. In addition, no differences were observed in the antitoxin immune response of chronically infected CF patients and intensive care unit patients with acute pulmonary infections.

Immunoglobulin M responses to the Norwalk virus of gastroenteritis.

Eighty-seven serum specimens from 20 human subjects experimentally inoculated one or more times with Norwalk virus were quantitatively examined for virus-specific immunoglobulin M (IgM). A sensitive and specific radioimmunoassay for anti-Norwalk virus blocking activity was applied to whole serum and to separate IgM and IgG fractions obtained by sucrose density gradient ultracentrifugation. The peak IgM response occurred at about 2 weeks after illness, but IgM was detectable at lower titers for up to 21 weeks after infection. The IgM response was seen in volunteers who became ill, whether or not prechallenge total serum antibody was present. On long-term (27 to 42 months) rechallenge, volunteers who were previously ill and had produced IgM antibody again developed illness, and a secondary IgM response greater than the first was detected. Inoculated volunteers who did not develop illness, as well as previously ill volunteers on short-term rechallenge (4 to 14 weeks), usually failed to generate an IgM response, whether or not an IgG response had occurred. In ill subjects, the rise in IgM and IgG occurred concomitantly. Virus-specific IgM is not necessarily indicative of primary infection with Norwalk agent inasmuch as reinfection produces an enhancement of the IgM response. Furthermore, Norwalk-specific IgM responses do not appear to be associated with subclinical illness.

Affinity chromatography purification of Pseudomonas aeruginosa exotoxin A on specifically linked NAD agarose.

The specific binding of P. aeruginosa exotoxin A to NAD was exploited for the rapid purification of the toxin. Affinity chromatography on a column of agarose-N6-(aminohexyl)carbamoylmethyl-NAD resulted in an enzymatically, biologically, and immunologically active purified toxin preparation. Other NAD-agarose resins were not efficient substrates for toxin purification.