The development of an M antibody capture ELISA for rubella IgM.

An M antibody capture enzyme-linked immunosorbent assay for rubella IgM was developed. The enzyme label was prepared from a monoclonal antibody raised against rubella haemagglutinin (Tedder et al., 1982). Paired sera from acute rubella infections and vaccines as well as sera from blood donors, antenatal patients and patients whose sera contained rheumatoid factor and patients with acute non-rubella infections were tested by this method.

Rapid diagnosis and management of parainfluenza I virus infection in common marmosets (Callithrix jacchus).

During 1983 a severe episode of respiratory infection occurred in a marmoset colony at these laboratories. Of 91 marmosets, 69 showed clinical signs of disease, one died and nine were so ill that euthanasia was necessary. Eight were examined post mortem and all showed consolidation of the lungs. Laboratory studies were carried out in an attempt to establish the cause of the outbreak and an interstitial pneumonia was found in seven animals which were examined histologically. Direct electron microscopy of nasal swabs and lung samples revealed the presence of a high titre of a paramyxovirus, and subsequent immunofluorescence studies established that the particular paramyxovirus involved was parainfluenza virus type I. Subsequent studies showed that surviving affected animals had seroconverted to parainfluenza I virus while animals that had not been implicated in the outbreak had not.

Rapid viral diagnosis of acute respiratory infections: comparison of enzyme-linked immunosorbent assay and the immunofluorescence technique for detection of viral antigens in nasopharyngeal secretions.

Nasopharyngeal secretions from adults and children were obtained in Stockholm, Sweden, for routine diagnosis of influenza A virus, influenza B virus, respiratory syncytial (RS) virus, parainfluenza type 3 virus, and adenovirus infections by demonstration of viral antigens directly in the specimens. The cells in nasopharyngeal secretions were pelleted by centrifugation for preparation of cell deposits for diagnosis by the immunofluorescence technique (IF) in London, England, and in Stockholm, whereas the supernatants were used to diagnose infection by the enzyme-linked immunosorbent assay (ELISA) in Stockholm. Titrations of the various purified viruses showed that ELISA could detect viral antigens in amounts corresponding to 1 to 10 ng of virus protein per test well. In a series of 73 specimens tested for influenza A, RS, and parainfluenza type 3 viruses by IF in London and by ELISA in Stockholm, 15 of 18 RS, 14 of 15 influenza A, and 2 of 2 parainfluenza type 3 viral infections were diagnosed by ELISA as compared with IF, giving sensitivities for RS and influenza A viral diagnosis of 83 and 93%, respectively, and a specificity of 100%. In another series of specimens from 35 patients tested for influenza B virus and adenovirus, five influenza B virus and four adenovirus infections were diagnosed by both methods; one additional influenza B infection was detected only by IF and another only by ELISA. Comparisons of diagnostic results between the two methods performed in Stockholm gave nonagreement of results for 37 of 1,593 tests (2.5%) for the five viruses. The conclusion reached was that the described ELISA, although a satisfactory test, had somewhat less sensitivity than did IF for the detection of respiratory viral infections. This could possibly be explained by unnecessary dilutions of specimens at the time of collection; transportation, processing, and storage of specimens were less complicated than for IF.

Public Health Laboratory Service IgM antibody capture enzyme linked immunosorbent assay for detecting rubella specific IgM.

A total of 468 sera were selected for the evaluation of the Public Health Laboratory Service's IgM antibody capture enzyme linked immunosorbent assay kit (MACELISA) for detecting rubella specific IgM. The results obtained were compared with those obtained by IgM antibody capture radioimmunoassay (MACRIA). Sera from patients with primary postnatal rubella, congenital rubella, remote rubella, infectious mononucleosis, and recent infection with other agents were included, in addition to sera taken after rubella immunisation and sera containing rheumatoid factor and rubella specific IgG antibody. The assay exhibited a similar ability and comparable specificity to MACRIA for detecting rubella specific IgM antibody. The Public Health Laboratory Service MACELISA can be recommended if, as for all assays that detect rubella specific IgM, all the available clinical and serological data are taken into account when the results are interpreted.

United Kingdom scheme for external quality assessment in virology. Part I. General method of operation.

Developments in the United Kingdom national external quality assessment scheme for virology are described. There are about 198 participants (170 in the UK) who are enrolled for examination of any or all of five categories of specimen (distribution types). These are detection of rubella antibody (128 UK participants), detection of hepatitis B surface antigen (130 UK participants), general virus serology (86 UK participants), virus identification (85 UK participants), and electron microscopy (56 UK participants). Specimens of a sixth category (rubella IgM antibody), not yet formally established, have also been distributed to 67 UK participants. Specimens in each distribution type are sent out once or twice a year, and, except for rubella IgM antibody, participants have been given a score of 2, 1, 0 or -1 marks for their reports on each specimen. Their cumulative scores and performance ratings are calculated retrospectively over a 12 month period for each distribution type separately and for combined distributions. The performance rating is defined by the number of standard errors by which the individual's cumulative score differs from the mean for all participants and carries a + or - sign depending on whether the cumulative score lies above or below the mean. Performance ratings have been found generally to be close to the mean in rubella serology and detection of hepatitis B surface antigen but are more variable in virus identification and electron microscopy. Ratings of less than -1.96 are considered to be significantly worse than average and to constitute poor performance.

United Kingdom scheme for external quality assessment in virology. Part II. Specimen distribution, performance assessment, and analyses of participants' methods in detection of rubella antibody, hepatitis B markers, general virus serology, virus identification, and electron microscopy.

Methods for the preparation and pre-distribution testing of specimens for external quality assessment in virology have been defined and criteria for allocation of scores for participants' reports on each category of specimen have been established. Specimens for detection of rubella antibody or markers of hepatitis B infection consist of human serum samples, which are distributed after detailed assessment of the expected results. In testing for rubella antibody or hepatitis B surface antigen (HBsAg) the scores given for reports of positive, equivocal, or negative depend on the specimen's content of antibody or HBsAg as established in the external quality assessment laboratory. For general virus serology two serum samples must be tested against a designated antigen by the complement fixation method; the score allocated for each participant's results depends on the ratio of the two titres he records, which is then compared with a target value derived from the results of a panel of participating laboratories. In virus identification and electron microscopy specimens are prepared from cultures or from clinical samples, and scores depend on the accuracy of identification. The pre-distribution tests necessary to establish the virus content and stability of these specimens have been defined, and media suitable for transporting specimens for virus culture, fluorescent antibody staining, or electron microscopy have been developed. A participant's overall success rate for each specimen is judged from the mean score (maximum 2) calculated from the scores of all participants examining the specimen. Mean scores were highest for detection of rubella antibody or HBsAg (from 1.67 to 1.96) and lowest for specimens containing certain small enteric viruses distributed for electron microscopy (0.82 to 1.12). Participants' reports on the methods used for each specimen have been analysed. Current changes and developments in methods have been recorded, and attempts have been made to relate the use of various techniques and test kits to successes or failures with various types of specimen.

Study of discrepancies in rubella haemagglutinin titrations and a reappraisal of diluents used in the rubella haemagglutination inhibition technique.

To elucidate inconsistencies in rubella haemagglutinin assays the components of the assay technique were examined. The results of carefully controlled assays of rubella haemagglutinin antigens from different sources in various plates and diluents with four species of indicator cells are reported. The quality and quantity of gelatin in the dextrose-gelatin-veronal buffered diluent commonly used in rubella haemagglutinin assays had a profound effect on the haemagglutination pattern and antigen titre. The veronal buffered saline used in the complement fixation test offered a valid alternative to the more complex diluents incorporating gelatin currently used in rubella haemagglutinin assays and haemagglutination inhibition tests.

Comparison of antibiotic susceptibility results obtained with Adatab and disc methods.

Strains of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, faecal streptococci, Proteus spp, and Klebsiella spp were distributed on two occasions to two groups of laboratories, one using a commercially produced break point method (Adatab, Mast Laboratories Ltd) and the other using a disc method for susceptibility testing. Minimum inhibitory concentrations of a range of antibiotics were determined for each of the strains in the Division of Microbiological Reagents and Quality Control and a correct result of sensitive or resistant was assigned where possible to each combination of strain and antibiotic. Laboratories were asked to determine the susceptibility of the strains to those antibiotics that they would test in routine practice. Results from each laboratory were compared with the correct results. The overall error rates obtained with the Adatab and disc methods, 8% and 8.2% respectively, were not significantly different. Fewer errors were made with trimethoprim, ticarcillin, and nitrofurantoin by laboratories using Adatabs than those using discs. Fewer errors were made with gentamicin by laboratories using discs than those using Adatabs. There was no significant difference between the two groups of laboratories in reproducibility of results on repeated testing of the same strains. Laboratories using Adatabs used a wide range of different break point concentrations. The Adatab method appeared to offer no overall advantages in terms of reduced error rates or increased reproducibility of results with the strains tested.

Detection of toxin production by Corynebacterium diphtheriae: results of a trial organised as part of the United Kingdom National External Microbiological Quality Assessment Scheme.

Four strains of Corynebacterium diphtheriae were sent to UK participants in the UK National External Microbiological Quality Assessment Scheme, who were asked to examine the strains for toxin production by in vitro methods. Laboratories achieved 162/176 (92%) and 160/175 (91%) correct results with two rapid toxin producers and 145/175 (82%) with a slow toxin producer. With a non-toxigenic strain 26/175 (15%) laboratories reported toxin production. Of the 173 laboratories reporting on all four strains, only 120 (69%) achieved the correct result for all. There was no significant association between the use of various methods and results, with the exception that laboratories using a full set of positive, weak positive, and negative controls made fewer errors than those not using controls. A number of unsatisfactory practices were revealed by the trial, however, and recommendations on preparation of inoculum, media, peptones, animal sera, and use of controls are made.

Comparison of results from two antibiotic susceptibility testing trials that formed part of the United Kingdom national external quality assessment scheme.

A susceptibility testing trial that formed part of the United Kingdom national external quality assessment scheme has been described previously. Results from this first trial showed an association between error rates and particular methods and practices. Changes in methods were recommended where appropriate. A second trial and survey of methods has shown reluctance to change methods and confirmed in most cases that high error rates were associated with the same methods and practices indicated by the first trial. Recommendations on disc content, method of methicillin testing, preparation of inoculum, use of controls and use of lysed blood for sulphonamide testing based on the results from these two trials are restated to encourage laboratories to review their methods critically. A statistical analysis of the results showed significant differences in performance among laboratories, and laboratories whose performance was significantly below the mean were identified. Poor performance was associated with the use of unsatisfactory methods. In view of the critical importance of susceptibility testing in patient care it is intended to use the results of susceptibility testing in the assessment of the performance of laboratories participating in the UK national external quality assessment scheme.

Viral diagnoses using the rapid immunofluorescence technique and epidemiological implications of acute respiratory infections among children in different European countries.

From November 1978 to October 1981, a total of 7716 specimens of nasopharyngeal secretions were examined by the rapid immunofluorescence technique to determine the frequency of infections caused by the respiratory syncytial virus (RSV), influenza virus A, and parainfluenza viruses 1 and 3. The tests were carried out in six different virus laboratories located in Newcastle upon Tyne (England), Copenhagen, Oslo, Stockholm, Turku (Finland), and Vienna; laboratories in Lisbon and Paris participated in the study for shorter periods. The specimens were collected from infants and children less than 6 years of age who had been admitted to hospital with an acute respiratory infection. Standardized techniques and quality controlled reagents were used. At least one of the above viruses was detected in 1927 (25%) of the specimens: RSV in 1475, influenza virus A in 123, parainfluenza virus 1 in 110, and parainfluenza virus 3 in 237 specimens. Respiratory syncytial virus dominated in all centres, but in some Scandinavian centres distinct outbreaks due to this virus occurred only once or twice during the 3 years' study period. Three outbreaks of RSV were observed in Newcastle, but here an unprecedented delay of the first winter's epidemic occurred. The delay was associated with prolonged school closures in the area, and with a very early outbreak of influenza. Parainfluenza virus 3, which was predominantly a summer virus in Newcastle, was most frequently encountered during the colder months of the year in the other centres.

Report of a joint DMRQC/Organon field trial to detect hepatitis A IgM by ELISA.

The results of a field trial of a joint DMRQC/Organon ELISA kit for the detection of hepatitis A IgM antibody are reported. The participating laboratories were asked to use the kit to test a panel of 360 specimens consisting of duplicate coded samples of 180 sera. The panel was also tested by MACRIA in the Virus Reference Laboratory, Colindale. The ELISA was shown to be specific and sensitive giving good discrimination between acute and late convalescent hepatitis A sera. It was proposed that the same cut-off control as is used in the RIA (equivalent to 10 RIA units) should be adopted for the ELISA also.

Enzyme-linked immunosorbent assay for the detection of human rotavirus in stools.

A simple method for the detection of human rotavirus in stools is described, using a double antibody sandwich enzyme-linked immunosorbent assay. Polysterene microtitre plates were used as solid phase. Four capture antibodies were tried, bovine, egg-derived, guinea pig and monoclonal antibody to rotavirus. Both bovine and egg-derived antirotavirus labelled with horseradish peroxidase were used as the detecting antibodies. The results obtained were compared with a commercially available ELISA, Rotazyme (Abbott Laboratories), and also with the direct detection of rotavirus by electron microscopy. Bovine antibody was found to be an unsuitable capture antibody due to non-specific false positive reactions.

Enzyme-linked immunosorbent assay (ELISA) for the detection of hepatitis Be antigen and antibody: report of a field trial.

A field trial of an enzyme-linked immunosorbent assay (ELISA) for the detection of the hepatitis Be markers is reported. It is simple to perform, is designed to be read by eye and does not require any expensive apparatus. When compared with a commercially available RIA kit for the detection of the same markers, ELISA was shown to be as sensitive as RIA for the detection of anti-HBe but slightly less sensitive for the detection of HBeAg. However if all specimens negative for both HBeAg and anti-HBe by ELISA are considered to be potentially infectious, the ELISA should prove to be as useful as RIA for determining the "e" status of HBsAg-positive patients and, therefore, provide a reliable indication of the risk of secondary spread of hepatitis B infection to contacts by needle stick accident, close personal contact or perinatal transmission.

An antibiotic susceptibility testing trial organised as part of the United Kingdom national external microbiological quality assessment scheme.

Organisms of known susceptibility to antimicrobial drugs were distributed for sensitivity testing to laboratories participating in the United Kingdom National External Microbiological Quality Assessment Scheme. The results obtained were correlated with the methods used. Laboratories differed in their standards of antimicrobial drug sensitivity testing. An association between error rates and particular methods and practices enabled recommendations to be made on disc content, method of methicillin testing, preparation of inoculum, use of controls and use of lysed blood for sulphonamide testing. Some media appeared significantly better than others but because of the many factors involved further information is being sought to clarify this.

Egg globulins in rapid virus diagnosis.

Egg-derived antibodies specific for a range of human viruses are now available commercially. These globulins are prepared by inoculating chickens with the relevant virus then harvesting antibodies from egg yolks. Reagents for influenza A and B, parainfluenza 1 and 3, adenovirus group antigen and rotavirus were tested. The successful use of these reagents on tissue culture and clinical material is described as well as their quality assessment. The advantages of this type of antibody are discussed.