Human rotavirus antigen detection by enzyme-immunoassay with antisera against Nebraska calf diarrhoea virus.

A four-layer solid phase enzyme-immunoassay (EIA) with antisera against Nebraska calf diarrhoea virus (NCDV) as immunoreagents was developed to detect human rotavirus antigens from stool specimens of patients with acute rotavirus gastroenteritis. Polystyrene beads were used as the solid phase, guinea-pig and rabbit anti-NCDV immunoglobulin as the catching and secondary antibody, and peroxidase-conjugated swine anti-rabbit immunoglobulin as the indicator antibody. A comparison of the developed NCDV-EIA with an identical EIA, using antisera against human rotavirus (HRV-EIA) instead of NCDV antisera, was made with 216 stool specimens positive or negative for rotavirus. A complete agreement was obtained between the two methods provided that appropriate confirmatory tests were included. The developed NCDV-EIA was as sensitive and specific for rotavirus as the HRV-EIA, and it allowed the detection of both established rotavirus types 1 and 2 from stools with equal sensitivity. The difficulties in cultivating human rotavirus in vitro for immunisation and the relative ease of growing NCDV in widely-used continuous cell lines make NCDV a good alternative in the preparation of the highly specific and sensitive rotavirus antisera required in immunoassays, and facilitate the setting-up methods for the routine diagnosis of rotavirus gastroenteritis by EIA or RIA in diagnostic virus laboratories.

Comparison of enzyme-immunoassay and radioimmunoassay for detection of human rotaviruses and adenoviruses from stool specimens.

An enzyme-immunoassay (EIA) using polystyrene beads as the solid phase, guinea pig anti-rotavirus or anti-adenovirus immunoglobulin as primary antibody, rabbit anti-rotavirus or anti-adenovirus immunoglobulin as secondary antibody, and horseradish peroxidase-conjugated swine anti-rabbit immunoglobulin as indicator antibody, has been developed for the detection of human rotavirus and adenovirus antigens from stool specimens. A comparison of the developed EIA and radioimmunoassay (RIA) used previously in our laboratory was made with 250 stool specimens from children with acute gastroenteritis. Two specimens were found negative by both rotavirus and adenovirus EIAs but not by RIAs, but in each of these cases confirmatory EIA tests showed them to be false negatives. The confirmatory EIA tests were also necessary in several cases to prove the specificity of the binding or to eliminate non-specific reactions with specimens giving low positive reactions in EIA. The developed EIA was found to be as specific, sensitive and reliable as RIA in the routine diagnosis of rotavirus and adenovirus gastroenteritis provided that appropriate confirmatory tests were included.

Local IgA-class antibodies against respiratory viruses in middle ear and nasopharyngeal secretions of children with secretory otitis media.

The presence of antibodies against some important respiratory viruses in the middle ear and nasopharyngeal secretions of 52 children with secretory otitis media (SOM) was investigated in order to find out about the role of these viruses in the development of SOM. The method employed was a sensitive radioimmunoassay test. Antibodies to adeno, syncytial, and parainfluenza 3 viruses were detected in about 50%, 50% and 20% of the patients, respectively. In eight patients the middle ear secretion/nasopharyngeal secretion titer ratio of antibodies to one virus was selectively increased in comparison with the other viruses tested, indicating an active local production of specific antibodies in the middle ear. Further studies are required to determine the cause of such an active antibody synthesis and its possible role in the etiopathogenesis of SOM.

Solid-phase radioimmunoassay of IgA, IgG, and IgM antibodies to human rotavirus.

A solid-phase radioimmunoassay (RIA) has been developed for the detection of human rotavirus-specific IgA, IgG, and IgM antibodies. Nebraska calf diarrhea virus grown in LLC-MK2 cell cultures in the presence of trypsin was directly adsorbed onto polystyrene balls, and antibodies that attached to the virus-coated balls were detected by subsequent binding of 125I-labeled antibodies specific to human alpha, gamma or mu chains of human Iga, IgG, or IgM immunoglobulins. A total of 116 serum specimens from 58 adult patients were tested. Binding ratios between the positive and the negative serum varied between 5 and 15, occasionally being 20 or more in the IgA and IgG assays, but rarely exceeding 3 in the IgM assay. The RIA was found to be more sensitive in detecting antibodies to rotavirus than the complement fixation (CF) test, the RIA titers obtained being 50--100 times as high as the CF titers. The method described offers a possibility of evaluating the immune response to human rotavirus and of detecting recent infection.

Comparison of four-layer radioimmunoassay and electron microscopy for detection of human rotavirus.

A four-layer radioimmunoassay (RIA) using polystyrene beads as the solid phase, anti-rota guinea pig IgG as primary antibody, anti-rota rabbit IgG as secondary antibody, and 125I-labelled sheep anti-rabbit immunoglobulin as indicator antibody has been developed for the detection of human rotavirus in stool specimens. A comparison was made of the developed RIA, routine electron microscopy, and research electron microscopy of 147 unconcentrated stool specimens from patients with infantile gastroenteritis. In routine electron microscopy 17 (11.6%) false-positive or false-negative results were obtained when compared with research electron microscopy. Each specimen positive in research electron microscopy was positive in RIA, and six additional RIA positives were found from 58 electron microscopy negative specimens. A confirmatory test was necessary to find out marginally positive but nonspecific reactions in RIA. The developed radioimmunoassay is slightly more sensitive than research electron microscopy of unconcentrated stool specimens and considerably more sensitive and more specific than routine electron microscopy.

Quantitative aspects of rotavirus excretion in childhood diarrhoea.

The amount of rotavirus (antigen) in diarrhoeal stools was determined from serially diluted faecal specimens using a solid phase radioimmunoassay (RIA) method. The acute stage RIA titres for rotavirus antigen ranged from 1:200 to 1:200 000 with a mean titre of 1:2 936 in a group of 16 infants with age range 2 to 14 months and 1:4 703 in a group of 12 children aged 25 to 94 months. In the younger age-group the diarrhoea lasted longer (mean 7.8 days) than in the older children (mean 5.4 days) despite the fact that the quantity of rotavirus was similar. In most of the younger infants there was either no demonstrable rotavirus present at the time of cessation of diarrhoea, or the amount of virus was on the decrease. In contrast, among the older children there were cases with a high titre of rotavirus in the stools a few days after the diarrhoea had ceased. These children were considered as potential spreaders of rotavirus infection in the community. However, none of the long-term follow-up stool specimens, collected 28 to 47 days after the onset of diarrhoea, were positive for rotavirus RIA.

Type-specific detection of parainfluenza viruses by enzyme-immunoassay and radioimmunoassay in nasopharyngeal specimens of patients with acute respiratory disease.

A four-year solid phase enzyme-immunoassay (EIA) and radioimmunoassay (RIA) techniques were applied for the type-specific detection of parainfluenza type 1, 2 and 3 virus antigens in sonicated nasopharyngeal specimens of patients with acute respiratory disease. Guinea-pig antiviral immunoglobulins as the secondary antibodies, and horseradish peroxidase-labelled swine anti-rabbit immunoglobulins (EIA), or 125I-labelled sheep anti-rabbit IgG (RIA) as the indicator antibodies. A total of 174 nasopharyngeal specimens collected by mucus extractor were tested, and the results were compared with those obtained by a routinely used immunofluorescence (IF) technique. The same number of positive specimens were achieved by the EIA and the RIA and 3/4, 4/4, and 19/20 immunofluorescence (IF)-positive nasopharyngeal specimens were positive by the parainfluenza type 1, 2 and 3 immunoassays respectively. In addition, four parainfluenza type 1 and three parainfluenza type 3 virus-positive specimens were found by the immunoassays out of 146 parainfluenza IF-negative specimens. The type-specificities of the parainfluenza immunoassays were confirmed by showing that no cross-reactions occurred when purified immunizing antigens and the EIA- and RIA-positive clinical specimens were cross-tested. The results indicate that parainfluenza type-specific antigens can be detected directly in nasopharyngeal specimens by the immunoassays and the preliminary findings with a small number of positive specimens suggest that these assays have a diagnostic potential which is similar or slightly better than the IF techniques.

Detection of influenza A virus by radioimmunoassay and enzyme-immunoassay from nasopharyngeal specimens.

Four-layer (indirect) radioimmunoassay (RIA) and enzyme-immunoassay (EIA) techniques were developed for the detection of influenza A and B virus in the sonicated nasopharyngeal specimens from patients hospitalized for acute respiratory infection. Polystyrene beads (RIA) or polystyrene microtiter plates (EIA) were used as the solid-phase, guinea pig antivirus immunoglobulins as the catching antibodies, rabbit antivirus immunoglobulins as the secondary antibodies, and 125I-labeled sheep antirabbit (RIA) or horseradish peroxidase conjugated swine antirabbit (EIA) immunoglobulins as the detector antibodies. A comparison of the developed RIAs and EIAs with the immunofluorescence (IF) method was made with 41 influenza A IF-positive and 150 influenza A IF-negative specimens. Each of the 41 influenza A IF-positive specimens was positive by the influenza A RIA and negative by the influenza B RIA. Out of 150 influenza A IF-negative specimens 3 specimens were found with weakly positive results in influenza A and B RIAs, but in each of these 3 specimens the binding proved nonspecific by the corresponding confirmatory tests. Using the EIa technique and the same immunoreagents as in RIA, identical results were obtained in each selected specimen tested. The developed RIAs and EIAs proved to be as specific and sensitive as the IF technique, and they should be practical in the diagnosis of respiratory infections directly from nasopharyngeal specimens.

Detection of respiratory syncytial, parainfluenza type 2, and adenovirus antigens by radioimmunoassay and enzyme immunoassay on nasopharyngeal specimens from children with acute respiratory disease.

Four-layer antispecies radioimmunoassay (RIA) and enzyme immunoassay (EIA) procedures were developed for the detection of respiratory syncytial virus (RSV), parainfluenza type 2 virus, and adenovirus antigens in nasopharyngeal specimens from children hospitalized for acute respiratory disease. Polystyrene beads (RIA) or flat-bottomed polystyrene microtiter plates (EIA) were used as the solid phases, guinea pig anti-virus immunoglobulins were used as the captive antibodies, rabbit anti-virus immunoglobulins were used as the secondary antibodies, and 125I-labeled sheep anti-rabbit (RIA) or horseradish peroxidase-labeled swine anti-rabbit (EIA) immunoglobulins were used as the indicator antibodies. A comparison of the EIAs and RIAs with routinely used immunofluorescence (IF) techniques was made with 164 nasopharyngeal specimens collected from children with acute respiratory disease. Only 3 of 66 RSV IF-positive specimens were negative in RSV RIA, and of 83 RSV, parainfluenza type 2 virus, and adenovirus IF-negative specimens, 1 was positive in RSV RIA. Of 4 parainfluenza type 2 virus IF-positive and 11 adenovirus IF-positive specimens, each was positive in corresponding RIAs, and all 83 IF-negative specimens were negative in parainfluenza type 2 virus and adenovirus RIAs. The results of the RSV, parainfluenza type 2, and adenovirus EIAs confirmed the results of corresponding RIAs in each selected case tested. The RIAs and EIAs were found to be as specific and sensitive as IF techniques, and more practical in the rapid detection of respiratory viruses in nasopharyngeal secretions.

Rotavirus, adenovirus, and non-viral enteropathogens in diarrhoea.

The aetiology of rotavirus and adenovirus in acute gastroenteritis was studied in a prospective series that comprised 283 children admitted consecutively with diarrhoea during a 1-year period. Rotavirus was associated in 49% of the cases by solid-phase radioimmunoassay and electron microscopical examination of stool specimens, or by serology. Adenovirus was detected by radioimmunoassay in the stool specimens of 29 (11%) patients, including 8 cases of possible dual infection with rotavirus. Rotavirus infections showed a typical age distribution and seasonal clustering between January and June, whereas the adenovirus-associated cases did not form a distinctive subgroup. Enteropathogenic bacteria were found in 10% of cases, and were nearly as common in association with rotavirus infection as not. Respiratory symptoms accompanied diarrhoea in 34% of the patients with rotavirus and in 25% of those with neither rotavirus nor adenovirus. Therefore we could not confirm the existence of a 'rotavirus syndrome', nor could we confirm an association of respiratory symptoms with rotavirus infection. Use of antibiotics before the onset of diarrhoea was more common among those with non-viral diarrhoea (23%) than in the rotavirus group (13%). Rotavirus infections appeared to be common among cases of 'antibiotic-induced' diarrhoea.

Clinical evaluation of radioimmunoassay of nasopharyngeal secretions and serology for diagnosis of viral infections in children hospitalized for respiratory infections.

Viral diagnosis was performed using radioimmunoassay (RIA) for virus antigen in nasopharyngeal secretions (NPS) and complement-fixation (CF) tests of paired sera from specimens of 90 children hospitalized for acute respiratory infection. Major respiratory viruses sought for by both methods (adenoviruses, influenza A and B viruses, parainfluenza virus type 3, respiratory syncytial virus) were detected in 40 (44%) of the patients; 15% of the diagnoses were made by NPS-RIA alone. Serologic diagnosis of other viral infections was confirmed in six additional cases. In the different clinical entities a viral diagnosis was established as follows: pneumonia, 50%; upper or middle respiratory infection with no wheezing, 43%; acute laryngitis, 54%; and wheezing bronchitis, 29%. In each clinical entity the virus-positive and virus-negative patients had similar total leukocyte counts, mean C-reactive protein levels and mean erythrocyte sedimentation rates. There was no difference in the duration of hospitalization between the patients with positive and negative viral studies. It was not possible to divide the patients into clinical subgroups according to the presence or absence of detectable viral infection.

Rapid detection of rotavirus in stool by latex agglutination: comparison with radioimmunoassay and electron microscopy and clinical evaluation of the test.

A latex agglutination test (LX) using antisera prepared against Nebraska calf diarrhea virus (NCDV) is described for the detection of rotavirus in stool of children with acute gastroenteritis. The test was compared with electron microscopy (EM) and radioimmunoassay (RIA) with 100 stools positive or negative for rotavirus. Out of 53 stools positive in RIA or EM, 49 were positive in LX and 4 were negative. Two specimens negative in EM and RIA were falsely positive in LX. The method was also tested in two clinical series with 115 stools from 101 children. Altogether 67/115 stools were positive in RIA, and 62/115 in LX. Out of 7 stools with contradictory results, 6 were negative in LX but positive in RIA, and 1 was positive in LX but negative in RIA. The results indicate that the LX is suitable for rapid screening of rotavirus gastroenteritis in clinical practice.