A one-step solid phase immunoassay for simultaneous detection of serum IgG and IgM antibodies to Borrelia burgdorferi.

A one-step immunoassay for simultaneous detection of serum IgG and IgM antibodies to Borrelia burgdorferi has been developed. The assay is based on C1q, which binds to immune complexes containing IgG and/or IgM antibodies. Micro-beads pre-coated with antibodies to human C1q are mixed with human serum samples and fluorochrome-labelled B. burgdorferi flagellum antigen. In the presence of serum IgG and/or IgM antibodies to B. burgdorferi, fluorochrome-labelled antigen/antibody complexes are formed. These are then bound by serum C1q and are subsequently captured on the anti-C1q-coated beads. The sample is analysed on a flow cytometer and the presence of fluorescent beads is, thus, indicative of a positive test result. In the present study the sensitivity and specificity of the assay are compared to those of the indirect IDEIA B. burgdorferi IgG and the mu-chain capture IDEIA B. burgdorferi IgM ELISAs for separate determination of IgG and IgM. Detection using a flow cytometer can be performed without separation of the beads from the reaction mixture, which means that in practice, the method is carried out as a one-step assay and it is, thus, very suitable for automation. Other advantages of this kind of assay includes an antibody/antigen reaction which occurs in solution and the potential of using the method for the detection of antibodies against several antigens from the same or different infectious agents (multi-parameter screening).

Acute gastroenteritis in children attending day-care centres with special reference to rotavirus infections. I. Aetiology and epidemiologic aspects.

Acute gastroenteritis (GE) among 214 children (aged 6 months-7 years) attending day-care centres (DDCs) in the Copenhagen County was studied during a 12-month period. A total of 197 cases of GE was observed in 109 children (i.e. 51% of the participants). The aetiology was as follows: rotavirus (n = 48) (24%), pathogenic bacteria (n = 11) (6%), Giardia lamblia (n = 3) (2%), while the aetiology of 68% remains unknown. The pathogenic bacteria included Yersinia enterocolitica, thermophilic Campylobacter, Clostridium difficile (+/- toxin) and enteropathogenic E. coli. In 4% of the GE the infections were multiple and Cryptosporidium was seen in one of these cases. The rate of GE declined with age from 1.35 GE per child per year (age group 1.0- less than 2.0 years) to 0.36 (6.0- less than 8.0 years). Serum sampled at the start of the study period showed that the frequency of detectable rotavirus IgG increased with age from 48% in the 6 months- less than 1.0 year group to 96% in the 4.0- less than 7.0 year group. The highest rates of rotavirus GE occurred from January to April (i.e. the rotavirus season). Moreover, rotavirus GE was almost absent after the age of 4. Hence, the rates of rotavirus GE per rotavirus season per child were 0.80 (age group 6 months-less than 1.0 year), 0.32 (1.0-less than 2.0), 0.14 (2.0-less than 3.0), 0.16 (3.0-less than 4.0), 0.06 (4.0-less than 5.0) and 0.04 (5.0-less than 6.0). Only 2 out of the 48 rotavirus GE were reinfections.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute gastroenteritis in children attending day-care centres with special reference to rotavirus infections. II. Clinical manifestations.

In a prospective study of a cohort of 214 children (aged 6 months-7 years) attending day-care centres, a total of 197 episodes of acute gastroenteritis (GE) occurred in 109 children (i.e. 51% of the participants) during a 12-month observation period. Rotavirus, pathogenic bacteria and Giardia lamblia caused GE in 24%, 6% and 2% of the cases, respectively. The aetiology of the remaining 68% was not discovered. Generally, the symptoms of GE were light and only two episodes led to hospitalization. Thirty-two rotavirus infections were asymptomatic. Two rotavirus GE reinfections occurred. They showed less severe symptoms than the primary infections. The older children (greater than 1.5 years) with rotavirus GE had lighter symptoms than the younger ones (less than or equal to 1.5 years). Compared with children with non-rotavirus GE, those with rotavirus GE showed the following clinical features: (1) Age between 6 months and 2 years. (2) Occurrence of rotavirus GE almost exclusively during the rotavirus season, i.e. January to April (winter). (3) High frequency of vomiting, the onset of which often preceded that of diarrhoea. However, these signs did not form a safe basis for the clinical diagnosis of rotavirus GE. One or more upper respiratory manifestations (URM) were observed in 39% of the children with rotavirus GE and in 36% of those with non-rotavirus GE. The occurrence of URM was age-related being highest in children less than 2 years. Consequently, the existence of a rotavirus syndrome is questioned. It is argued that URM in children with rotavirus GE may be due to a co-infection of the upper respiratory tract by a different micro-organism.

Protective effect of preexisting rotavirus-specific immunoglobulin A against naturally acquired rotavirus infection in children.

The preexisting levels of rotavirus IgA and IgG were measured in 225 children aged 6 months to 7 years in November, ie, before the "rotavirus season" from January to April. During the following 6 months, all episodes of acute gastroenteritis (GE) were evaluated clinically according to a score system and feces was examined for rotavirus, pathogenic bacteria, and parasites. Furthermore, rotavirus GE (n = 45) as well as asymptomatic rotavirus infections (n = 29) were diagnosed serologically. The preexisting concentrations of rotavirus IgA and IgG measured by ELISA were similar in these two groups. However, preexisting rotavirus IgA in the group of children who developed rotavirus GE correlated with less severe symptoms. Thus vomiting was found in 24% and 63% of the children with detectable and undetectable rotavirus IgA, respectively (P less than 0.025). Moreover, according to the total symptom score of rotavirus GE, 52% of the children with detectable preexisting rotavirus IgA had mild symptoms compared with only 13% of those with undetectable concentrations (P less than 0.025). Rotavirus IgG did not have any protective effect. Age per se had a protective effect; older age (greater than 1.5 years) was related to mild symptoms. According to previous studies of local and intestinal antibody response to a rotavirus GE, it is suggested that rotavirus IgA in serum reflects the immunological status of the intestine concerning rotavirus. It is recommended that studies of rotavirus vaccines include rotavirus IgA response and its protective effect.

Detection by ELISA of immunoglobulin G subclass-specific antibody responses in rotavirus infections in children.

IgG subclass-specific antibody responses to a human subgroup 2 rotavirus were studied in 26 children by ELISA by use of monoclonal antibodies specific to the four human IgG subclasses. One hundred twenty-nine serum samples were obtained before, during, and after an episode of rotavirus-induced diarrhoea in these patients. When these sera were investigated, an increase in IgG1 and IgG3 subclass-specific antibodies was detected in all 26 patients. IgG3 antibodies reached a peak concentration 1 week after rotavirus was detected in faecal samples and then progressively declined over the following months, whereas the peak concentration of IgG1 subclass antibodies was found 2 months later and seemed to persist thereafter. IgG2 rotavirus-specific subclass antibodies were never found and IgG4 subclass antibodies were detected only in sera from seven of the 26 patients.

Antibody response in serum and intestine in children up to six months after a naturally acquired rotavirus gastroenteritis.

The aim of this study was to provide detailed information about the local and systemic antibody response and their relationship following a rotavirus gastroenteritis. Rotavirus-specific immunoglobulins were analyzed by enzyme-linked immunosorbent assay (ELISA). The study included 49 children referred to hospital with rotavirus gastroenteritis and 16 children with nonrotavirus gastroenteritis. The concentrations of rotavirus immunoglobulin A (IgA) in serum increased within the first 2 weeks and those of rotavirus IgG within the first month after the onset of diarrhea. Thereafter, they remained unchanged during the 6-month observation period. Rotavirus ScIg (i.e., antirotavirus immunoglobulin-containing secretory component) appeared in serum almost exclusively within 7-14 days after onset (i.e., 85% of the samples). After the first 2 weeks, rotavirus IgA could be detected in the majority of fecal samples, even up to 6 months after the disease. However, rotavirus ScIg was absent in the majority of fecal samples. The severity of illness correlated only with the increase of rotavirus IgG in serum. Conclusively, there is a longstanding immune response after a naturally acquired rotavirus gastroenteritis. Moreover, with the present methods, measurements of rotavirus IgA and IgG in serum can be safely used for serodiagnosis, even when samples are taken with 6-month interval. It is suggested that trials with rotavirus vaccines include measurements of rotavirus IgA and ScIg in serum and rotavirus IgA in feces.

Rotavirus antibodies in the mother and her breast-fed infant.

The transfer of rotavirus antibodies from 25 healthy mothers to their breast-fed infants was investigated during the period of lactation (mean, 3.9 months; range, 1-9 months). Furthermore, the destiny of these antibodies in the infants' gastrointestinal tract and serum was examined. Rotavirus-specific immunoglobulins were analyzed by the ELISA (enzyme-linked immunosorbent assay) technique. All the mothers had rotavirus IgA and IgG in serum. About 80% of the mothers had low concentrations of rotavirus ScIg (i.e., antirotavirus immunoglobulin containing secretory component) in serum at the beginning of the lactation period declining to about 45% at the end of the period. From a few days after delivery to about 2 weeks later, the concentrations of rotavirus IgA and ScIg in milk declined. Thereafter, they remained unchanged. There was a positive correlation among the concentrations of rotavirus IgA in serum and rotavirus IgA as well as ScIg in milk. Rotavirus IgG in the infants' serum correlated with that of the mothers. Few samples of the infants' duodenal fluid contained rotavirus IgA or ScIg. On the other hand, about 80% of the infants' fecal samples contained rotavirus ScIg and IgA. Rotavirus IgA and ScIg disappeared from the infants' feces after cessation of lactation. Hence, it may be concluded that infants receive rotavirus IgG through the placenta, and rotavirus ScIg and IgA in constant amounts via milk throughout the period of lactation. The small intestine is flushed with rotavirus ScIg and IgA at each breast-meal, and these antibodies survive proteolysis in the gut. A possible protectional effect of rotavirus ScIg or IgA requires frequent breast-meals, and the effect is limited to the period of lactation.

Intestinal and serum immune response to a naturally acquired rotavirus gastroenteritis in children.

Seventeen children (mean age: 2.0 years, range: 36 days-8 years) hospitalized with acute gastroenteritis were investigated. Thirteen children had a rotavirus infection while four did not. Rotavirus serum IgA as well as ScIg, i.e., antirotavirus immunoglobulin containing secretory component, increased rapidly after rotavirus infection. While rotavirus IgA persisted in serum for at least 6 months, rotavirus ScIg disappeared from serum in less than 4 months. Rotavirus IgG could be detected in serum during the early stage of the infection and was still high after 6 months. The patients with nonrotavirus acute gastroenteritis did not show any of the above-mentioned serological hallmarks of those with rotavirus infection. The amounts of rotavirus ScIg found in serum about 1 week after the infection correlated to the amounts of rotavirus ScIg in duodenal fluid. Six months after the infection, rotavirus IgA was found in the feces of the majority of the patients while rotavirus ScIg could be detected only in one patient. The amounts of rotavirus IgA in sera and intestinal secretions showed identical patterns in the acute phase of the disease as well as after recovery. The same applied to rotavirus ScIg. These findings could be useful in future evaluations of vaccines and immunity against rotavirus infections.

Nosocomial acute gastroenteritis in a paediatric department, with special reference to rotavirus infections.

Nosocomial acute gastroenteritis caused by rotavirus as well as by non-rotavirus gastroenteritis was registered during a 12-month period in the paediatric department of a district hospital. The number of patients in the two groups amounted to 27% (rotavirus) and 7% (non-rotavirus) of the total number of patients hospitalised with the corresponding type of acute gastroenteritis. The seasonal and age distributions for the two types of nosocomial acute gastroenteritis followed the pattern of the respective types of hospitalised community-acquired acute gastroenteritis. Nosocomial non-rotavirus gastroenteritis was found to occur scattered with regard to time and locality within the department. The same applied to one half of the cases with nosocomial rotavirus gastroenteritis, whereas the other half occurred during an epidemic outbreak in the general infant/toddler ward. Vomiting and diarrhoea were less significant in nosocomial rotavirus gastroenteritis as compared with community-acquired rotavirus gastroenteritis. As to the other manifestations no difference was found between the two groups. Nosocomial rotavirus gastroenteritis prolonged the stay in hospital with on the average 3.8 days. Guidelines are suggested for isolation of patients with acute gastroenteritis in order to reduce particularly the frequency of nosocomial rotavirus infections.

Clinical features in hospitalised children with acute gastroenteritis. Does the rotavirus syndrome exist?

In a prospective study of children referred to hospital, rotavirus was identified in 37% of 128 patients with acute gastroenteritis. As compared with patients with non-rotavirus gastroenteritis, those with rotavirus gastroenteritis showed the following clinical characteristics: Age between 5 months and 4 years. Occurrence of the rotavirus infection almost exclusively during the winter season. Severe vomiting. Absence of gross blood in the stools. However, these signs did not form a safe basis for the clinical diagnosis of rotavirus gastroenteritis. One or more signs of upper respiratory illness were observed in 36% of the patients with rotavirus gastroenteritis and in 35% of those with non-rotavirus gastroenteritis. Consequently, the existence of a rotavirus syndrome is questioned. It is argued that upper respiratory illnesses in patients with rotavirus gastroenteritis could be due to a separate infection occurring coincidentally.

Comparison of indirect double antibody and double antibody sandwich ELISA techniques with latex agglutination test for the diagnosis of human rotavirus infection.

94 faecal samples from infants and children suffering of acute gastroenteritis were investigated for rotavirus by indirect double antibody sandwich ELISA kit (WHO, Geneva), Rotavirus ELISA kit (DAKOPATTS A/S, Copenhagen) and Rotalex latex-agglutination kit (Orion Diagnostica, Helsinki). The ELISA techniques gave almost identical results and seemed to be of same sensitivity and specificity. Rotalex agglutination had an overall agreement of 88% with ELISA. It is concluded that strongly positive reactions found by Rotalex may be regarded as true positive reactions, whereas samples producing weakly positive and/or negative reactions should be retested in a more specific and sensitive assay, such as enzyme linked immunosorbent-assay (ELISA).

Detection by ELISA of IgA and IgM antibodies in secretion and IgM antibodies in serum in primary lower respiratory syncytial virus infection.

Twenty-six infants and children with primary lower RS virus infection, diagnosed by the detection of RS virus in nasopharyngeal secretion (NPS) by use of immunofluorescent antibody (FA) technique, were studied with respect to the presence of IgA and IgM antibodies. Samples of NPS and serum obtained during the first 3-4 months following the beginning of illness, were investigated. Employing a reverse ELISA technique, we found IgM antibodies in the acute, but not during the convalescent, phase of illness in NPS from 20 of the patients and in serum from 21 of the patients. The majority of the IgM antibody conversions observed occurred in NPS as well as in serum on days 5-8 following the illness. RS virus IgA antibodies, also detected by a reverse ELISA technique, were demonstrated in NPS in 22 of the patients, with antibody conversions being found in 19 of the patients on days 5-8 following the beginning of the illness. Two patients still had IgA antibodies in NPS approximately 3 months FSOI. By comparison, RS virus was detected in acute-phase NPS by double-antibody sandwich ELISA in 25 of the 26 patients investigated.

Production of antiserum to respiratory syncytial virus polypeptides: application in enzyme-linked immunosorbent assay.

By use of crossed immunoelectrophoresis techniques, respiratory syncytial (RS) virus-specific precipitates were produced between RS virus cellular antigen [solubilized in tris(hydroxymethyl)aminomethane-glycine buffer, pH 9] and antiserum raised in rabbits against semipurified RS virus. When these precipitates were employed as antigens for further immunizations in rabbits, antibodies (anti-RSV-precip.I) were produced which reacted with only one RS virus antigen when tested by the crossed immunoelectrophoresis technique. Precipitates obtained between RS virus cellular antigen (labeled with L-[35S]methionine) and anti-RSV-precip.I were examined by polyacrylamide gel electrophoresis, which showed that anti-RSV-precip.I precipitated RS virus polypeptides of molecular weights 28,000 to 84,000. Anti-RSV-precip.I was employed as capture antibodies in the enzyme-linked immunosorbent assay, in which RS virus cellular antigen was used as the second layer. Determination of human RS virus immunoglobulin G antibodies by this enzyme-linked immunosorbent assay technique showed a high degree of sensitivity, specificity, and reproducibility.

Optimized enzyme-linked immunosorbent assay for detection of human and bovine rotavirus in stools: Comparison with electron-microscopy, immunoelectro-osmophoresis, and fluorescent antibody techniques.

Detection of human and bovine rotavirus in stools is described using a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) with poly-styrene microtest plates as solid phase, immunoglobulin fraction of rabbit antiserum to rotavirus (human) as catching antibody, and the same reagent labelled with horseradish peroxidase as conjugate. The ELISA has been optimized with regard to simplicity, rapidity, sensitivity, and specificity. In a comparative study, stool specimens from 81 infants and children and 92 neonatal calves with diarrhoea were tested for rotavirus by ELISA, electron microscopy (EM), immunoelectro-osmophoresis (IEOP), and fluorescent antibody technique (FA). The relative sensitivity of the different assays for human and bovine rotavirus was: EM 68%, 76%; IEOP 80%, 76%; FA not determined, 85%; and ELISA 86%, 98%, respectively. Less than 1 ng of purified human rotavirus could be detected in ELISA, whereas 100 ng was the minimal amount detected by IEOP. It is concluded that the developed ELISA is a simple, rapid, reliable, and sensitive method for the diagnosis of human and bovine rotavirus infections.