Tear anti-Chlamydia antibodies in males with chlamydial urethritis.

Conjunctival Chlamydia trachomatis was studied in 50 males with chlamydial urethritis. Conjunctival samples from 49 patients were negative for chlamydial antigen by enzyme immunoassay. However, tear anti-Chlamydia IgA antibodies were found in high titers (> or = 400) in 20 out of the 50 patients. Secretory IgA against Chlamydia in corresponding titers was found in 8 patients. The titers differed significantly from those of the 22 negative control subjects. IgG antibodies were not detected. Tear anti-Chlamydia IgA antibodies are not diagnostic for chlamydial conjunctivitis, but occur concomitantly in patients with chlamydial urethritis.

Avidity of IgG antibodies against mumps, parainfluenza 2 and Newcastle disease viruses after mumps infection.

Paired serum samples from 39 patients with recent mumps infection were assayed for IgG antibodies against mumps, parainfluenza 2 and Newcastle disease virus (NDV). A modified enzyme immunoassay was used, giving separate estimates of high avidity antibodies (EHAA) and total specific antibodies (ETSA). A marked cross-reaction was seen between mumps and parainfluenza 2 virus, with changes of ETSA between paired samples of about the same magnitude against both these viruses. The mean change of EHAA against mumps was, however, significantly greater than that against parainfluenza 2. There were 16 patients who had a change of ETSA greater against parainfluenza 2 than against mumps. When the EHAA responses were compared, there were only 8 such patients. The responses against NDV were negligible. Estimation of antibody avidity, even by the arbitrary method used, can distinguish between homotypic and cross-reactive heterotypic antibodies after mumps infection. The implications for expressing the results of enzyme immunoassay are discussed.

Solid-phase enzyme-immunoassay for the detection of herpes simplex virus antigens in clinical specimens.

An indirect solid-phase enzyme-immunoassay (EIA) for the detection of herpes simplex virus (HSV) antigens in clinical specimens was developed. Rabbits and guinea pigs were hyperimmunized with highly purified nucleocapsids of HSV type 1. Microtitre plates were coated with 0.25 microgram of guinea pig anti-herpes simplex type 1 immunoglobulins per well. Clinical specimens, diluted in phosphate buffered saline containing fetal calf serum and detergents, were sonicated and incubated in the test wells overnight at 37 degrees C. Rabbit anti-HSV immunoglobulins were added as a secondary antibody at a concentration of 3.2 micrograms per well, and peroxidase conjugated swine antibodies against rabbit immunoglobulins, diluted 1:1,000, were used as a fourth layer. Clinical specimens which were sent for virus isolation or for isolation of Chlamydia trachomatis were tested by the developed assay and 20 out of 27 isolation positive specimens were found positive by EIA. Five out of 67 specimens negative by isolation gave positive results by EIA. The specificity of the results was confirmed by a control test using wells coated with normal guinea pig immunoglobulins. The test detected antigens from both serotypes of HSV. Cross reactions with varicella-zoster- or with cytomegalovirus were not found, and antigens from uninfected cells did not result in false positive results.

Fatal influenza A myocarditis with isolation of virus from the myocardium.

A previously healthy 27-year-old woman developed an acute cardiac failure one week after onset of influenza-like respiratory infection, and died on her fourth day in hospital. Intravital differential diagnosis included myocardial infarction because of ECG changes and massive elevation of myocardial enzymes. Autopsy revealed severe myocarditis and intact coronary arteries. At microscopic examination the myocardium was heavily infiltrated with lymphocytes, and there was a marked myocytolysis. Influenza A virus was isolated from the myocardial tissue. An immunological mechanism of myocardial damage is suggested.

Time-resolved fluoroimmunoassay: a new test for rubella antibodies.

A new solid-phase immunoassay, time-resolved fluoroimmunoassay (TR-FIA), for rubella antibody was developed. The test used polystyrene beads coated with rubella antigen as the solid phase and a chelate of the rare earth metal europium as fluorescent label. A fast light pulse from a xenon flash lamp was used to excite the label, and after a 400-mus delay time the emission fluorescence was measured for 500-mus at 1-ms intervals during a total counting time of 1 s. Background fluorescence of short duration caused by fluorescent serum components and scattering could be eliminated by including the delay time. The TR-FIA was compared with hemagglutination inhibition, single radial hemolysis, and two types of radioimmunoassay (RIA) (a commercial RIA [GammaCoat] and a noncommercial RIA [T-RIA]) by using 60 serum specimens from patients with remote rubella infection. Overall agreement of TR-FIA with hemagglutination inhibition and GammaCoat was 96.7%, with single radial hemolysis 98.3%, and with T-RIA 100%. Linear regression coefficients varied from 0.83 to 0.94, the best being obtained with single radial hemolysis and T-RIA. TR-FIA was also found to be suitable for the diagnosis of acute infections, as significant increases of antibody level were detected in all 30 paired serum specimens tested from patients with an acute rubella infection. Sensitivity and specificity comparable to those of RIA and enzyme immunoassay were obtained with TR-FIA. Furthermore, the advantage that TR-FIA has over RIA is that it incorporates a nonisotopic and stable label; its advantage over EIA is that it is easier to standardize because no additional reaction with substrate is required.

An ELISA for the estimation of high-avidity and total specific IgG and IgM antibodies to rubella virus.

High-avidity and total specific IgG and IgM antibodies to rubella virus were measured by using a curve-fitting analysis for the dose-response curves of enzyme-linked immunosorbent assay (ELISA). The analysis of follow-up samples from 14 patients with primary rubella infection revealed that these two antibody categories had different kinetics during the time after rash. The high-avidity IgG antibodies increased more slowly than total specific IgG antibodies. In the IgM response, high-avidity antibodies reached their maximum slightly earlier than total specific antibodies. After reinfection with rubella, 14-30 days after the illness, convalescent patients had higher amounts of high-avidity IgG antibodies when compared to the patients who suffered from primary rubella infection. These findings are in agreement with the results obtained by measuring antibody affinity in experimental animals after immunization.

Persistence of humoral and cell-mediated immunity to rubella virus in cloistered nuns and in schoolteachers.

Humoral and cell-mediated immunity to rubella virus after naturally acquired infection were compared in 19 cloistered nuns (29-79 years of age), 18 female schoolteachers (21-61 years of age), and 21 female control subjects (20-30 years of age), who were all seropositive for rubella virus, by use of a hemagglutination-inhibition test, a passive hemagglutination test, a hemolysis-in-gel test, a radioimmunoassay, an enzyme-linked immunosorbent assay, and lymphocyte transformation tests. No significant differences were found among the groups by the radioimmunoassay, the hemolysis-in-gel test, and the enzyme-linked immunosorbent assay. The cloistered nuns had significantly lower cell-mediated immunity to rubella virus than did the teachers and the control subjects but nonetheless showed protective levels of antibody to rubella virus and significant lymphocyte transformation responses, which persisted until age 79 in the probable absence of reinfection.

A "reverse" solid-phase radio-immuno-assay for IgM-antibodies to hepatitis A virus.

A "reverse"solid-phase radio-immuno-assay for IgM antibodies to hepatitis A virus (HAV) was developed. Anti-human IgM immunoglobulins were bound on the wells of polyvinylchloride microtiter plates. Serum specimens were incubated in the anti-human IgM coated wells and bound IgM antibodies were then assayed for antigen specificity by subsequent incubations with HAV antigen and 125I-labelled human anti-HAV IgG. The test showed a high sensitivity and specificity for anti-HAV IgM antibodies. No false-positive reactions were observed either in the sera from patients with hepatobiliary disorders other than HAV infection or in the sera containing both rheumatoid factor and anti-HAV IgG antibodies. In acute HAV infections specific IgM antibodies were present already in the first specimens taken within a few days after the onset of jaundice. The persistence of the IgM antibodies was from 4 to 6 months. IgM antibody titers up to 1,000,000 were observed in the acute phase of HAV infection. In routine diagnostic work the titration of the sea was not necessary, since a reliable qualitative result was obtained by testing the sera in a single dilution of 1:100. A similar "reverse" immuno-assay principle may be adaptable for the diagnostic determination of IgM antibodies to different viral and microbial antigens.

Solid-phase enzyme immunoassay for determination of antibodies to cytomegalovirus.

A solid-phase enzyme immunoassay for the determination of immunoglobulin H (IgG) and IgM antibodies to cytomegalovirus is described. The enzyme immunoassay gave reliable and consistent results which were in concordance with those obtained by the complement fixation test and the indirect immunofluorescence test. Antibodies to herpes simplex and varicella-zoster viruses did not interfere in the enzyme immunoassay for cytomegalovirus IgM antibodies. In a few patients with IgM antibodies to Epstein-Barr virus, cytomegalovirus IgM antibodies were also detected. False-positive cytomegalovirus IgM antibody results were observed in sera containing both the rheumatoid factor and cytomegalovirus IgG antibodies. This rheumatoid factor interference was overcome by the absorption of sera with polymerized human gamma globulin. The absorption did not affect true cytomegalovirus IgM antibody titers. Also described is a simple enzyme immunoassay that makes possible a more sensitive detection of the rheumatoid factor than the latex agglutination test.

Prevalence of secretory otitis media in seven to eight year old school children.

One thousand two hundred and seven school children aged seven to eight years were examined in the spring of 1978 in order to determine the frequence of undiagnosed secretory otitis media (SOM). All children were without any subjective ear symptoms. Two and eight tenths percent of the children were found to have SOM. The relation between SOM and the appearance of allergy, the occurrence of earlier otitis media, the occurrence of repeated upper respiratory tract infections and the treatment of earlier middle ear infections are discussed.

[Ultrasound testing in the diagnosis and management of maxillary sinusitis in children (author's transl)]. / Die Verlaufskontrolle des Heilungsprozesses der kindlichen Kieferhöhlenentzündung mit der Ultraschalldiagnostik.

A- and B-mode ultrasound testing was used in conjunction with X-ray studies as screening tests for the detection of maxillary sinusitis in 280 children (aged 4-10 years) about to undergo adenoidectomy, and/or tonsillectomy and/or tympanostomy. X-rays were abnormal in 114 (41%) children, while sinus disease with the retention of discharge was diagnosed by ultrasound in 63 (23%) children. These latter children were then treated for ten days with amoxicillin and decongestant (Rinexin). At time of initial surgery, 63 maxillary sinuses (in 43 of the children) were punctured and irrigated. Another group (consisting of 27 sinuses in 20 children) was treated without antral puncture. All children were reexamined after 10, 20 and 30 days with ultrasound and radiography. After 10 days, there was no discharge found in 86% of the sinuses treated by antral puncture and in 85% of the control sinuses. After one month, all sinuses which had been irrigated were found to be free of disease, while three (11%) of the control group contained discharge. However, the healing process in all patients seemed to be slower, as evidenced by sequential sinus X-rays.--We believe that ultrasonic evaluation of the maxillary sinuses is a simple, rapid and reliable method for the diagnosis and followup of maxillary sinusitis in children. With this technique, it is possible to follow the disappearance of residual sinus discharge as well as diagnose cases which need active treatment.

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.

Persistent rubella-specific IgM-antibody in the cerebrospinal fluid of a child with congenital rubella.

Rubella-specific IgM-antibody was detected, using a solid-phase radioimmunoassay (RIA) method, in the CSF of a child with congenital rubella at ages 3 and 4 years. No rubella-specific Igm was found in the CSF of 20 other children with congenital rubella, and the ratios of rubella-specific IgG RIA antibody titres in serum and CSF were normal.

A new method for the identification of cerebrospinal fluid leakage.

Apart from the use of anamnestic data the most common techniques for the diagnosis of CSF leakage have included X-ray studies, and chemical analysis of glucose, protein, and electrolytes of the fluid obtained from the nose or ear, intrathecal staining, and radioactive cisternography. These studies, although useful, have not always succeeded in demonstrating the CSF leakage, especially when the leak is delayed, small, or contaminated. A new immunochemical technique for the identification of the CSF leakage is described. It is based on demonstration of an extra band of transferrin located in the beta 2-fraction of protein electrophoresis of CSF. This beta 2-transferrin is pathognomonic for liquor and could not be demonstrated in serum, nasal secretions, saliva, tears, or peri- and endolymph. After routine protein electrophoresis on cellulose acetate membranes, the transferrins are identified by application of anti-transferrin on beta-regions. Stained precipitates in both beta-regions demonstrate clearly the presence of CSF. Compared with other methods, the new technique offers many advantages. The amount of sample needed for the procedure is small, moderate contamination does not invalidate it, it makes it possible to localize the leak by differential suction, and it is absolutely safe for the patient.

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.

IgM-class rheumatoid factor interference in the solid-phase radioimmunoassay of rubella-specific IgM antibodies.

The interference of IgM-class rheumatoid factor (RF) in the solid-phase radioimmunoassay (RIA) of rubella virus IgM antibodies was studied. Acute rubella infections did not significantly activate RF. False-positive rubella antibody results were obtained, however, when patients with raised RF levels were tested. If a low rubella IgG antibody titre was present, a high level of RF was required to cause a false-positive IgM result; conversely, in sera with high IgG titres, only a low level of RF was required for interference. Although the false-positive IgM titres obtained were generally low, thet did show a positive correlation to both RF levels and rubella IgG titres. False-positive results were successfully avoided by removing the RF by absorption with heat-aggregated human gamma globulin. The absorption procedure did not affect true rubella IgM antibody titres.

Antibody responses in patients with rubella infection determined by passive hemagglutination, hemagglutination inhibition, complement fixation, and solid-phase radioimmunoassay tests.

Antibody responses in serial serum specimens collected from 31 patients with an acute rubella infection were determined by passive hemagglutination (PHA), hemagglutination inhibition (HI), complement fixation (CF), radioimmunoassay (RIA) immunoglobulin G (IgG), and RIA immunoglobulin M (IgM) tests to evaluate the effectiveness of these tests in diagnosing a recent infection. The HI, RIA IgG, and RIA IgM antibodies appeared almost simultaneously and reached the maximum level about 1 week after the onset of rash. Compared to these, the CF antibodies developed only slightly later, whereas the development of the PHA antibodies was much more delayed. The RIA IgM response was shown to be transient, lasting approximately 1.5 to 2.5 months postinfection. The results of this study indicate that demonstration of specific IgM antibodies is the best method for diagnosing a recent infection, one within 2 months after the onset of the illness. If an IgM test is not available, a combination of the HI and PHA tests is recommended.

Scanning electron microscopic appearance of viral-antigen-coated polystyrene balls.

A radioimmunoassay (RIA) was recently developed for the detection of antiviral IgG and IgM class-specific antibodies using antigen-coated polystyrene balls as the RIA solid-phase. In this communication the attachment and distribution of herpes simplex virus (HSV) capsid and envelope antigens and rubella viruses on the surface of the balls was examined by scanning electron microscopy (SEM). In SEM the surface of the untreated 'clear frosted' polystyrene balls appeared very uneven with innumerable pits and grooves. The viral particles were haphazardly distributed both in the grooves and on the exposed surface of the balls. The strength of adsorption of the viral antigens onto the balls seemed to be remarkably resistant to outside mechanical forces. HSV antigens frequently appeared in clusters, whereas rubella viruses were mostly found as single particles.