Enhanced cellular immunity following influenza vaccination has been undetectable in kidney transplant recipients so far. Protection from influenza is dependent on cellular and humoral immunity. Aim of the study was to investigate immune responses before and after vaccination with influenza A and B antigens in 65 kidney transplant recipients. A significant increase in proliferative responses was only observed towards influenza B (P < 0.0001) by lymphocyte transformation test. The enzyme-linked immunospot (ELISpot) assay was more sensitive and detected significant, 3- to 5-fold increases (P < 0.0001) in interferon-gamma secretion using influenza A and B antigens. Furthermore, influenza antibody titers increased significantly (P < 0.0001). At month 1 post-vaccination 85% of patients displayed specific cellular, and 95% or 92% humoral immunity against influenza A and B, respectively. Thus, applying the sensitive ELISpot assay, influenza-specific cellular immunity could be detected for the first time in kidney transplant recipients after vaccination.

Fifty volunteers, treated with an inactivated trivalent influenza vaccine containing A/Bangkok/1/79 (H3N2), A/Brazil/11/78 (H1N1) and B/Singapore/222/79 virus, were subdivided according to the estimated first exposure to influenza in their lifetime (priming) and the presence of antibodies against the vaccine components in the pre-vaccination sera. The isotypic antibody response (IgG, IgA, IgM) was determined by means of an antibody capture haemadsorption immunosorbent technique. For all three vaccine components, previously seropositive subjects produced antibodies of the IgG- and IgA-class more frequently than previously seronegative persons. Subjects primed to one of the influenza A subtypes showed more IgG and IgA responses in comparison with those unprimed (prime-effect). In contrast, IgM antibodies occurred in only 19 and 11% of primed, but in 59 and 54% of unprimed subjects, for A (H3N2) and A (H1N1), respectively. The incidence of IgM titre rises was not influenced by the prevaccination state. However, the mean magnitude of anti-A (H1N1)-IgM titre rises was greater in those previously seronegative. The concepts of primary and reinfection and of 'original antigenic sin' are discussed, and it is suggested that age and, if possible, serological state prior to antigen-exposure should be taken into account when studying isotypic antibody responses after influenza infection or vaccination.

Up to now, the complement fixation test (CFT) has been the basis for the serological diagnosis of influenza virus infection in routine laboratories. Generally, low CF titers (1:20 or 1:40) are difficult to interpret. This means that the differentiation between recent and remote influenza infections is not possible by CFTs on single sera. Nonetheless this is generally possible by the subtype- and immunoglobulin class-specific immunofluorescence test (IFT) reported in this paper. Sera from 76 patients with confirmed influenza infection were tested and we obtained the following results: only 27.6% contained antibodies of all immunoglobulin classes, 51% contained IgG and IgA antibodies (without IgM) and 3.9% responded only with the IgG isotype. The IFT-positive and CFT-negative were 5.2% and the IFT-negative and CFT-positive 4%. In 7.9% no antibody rises were detected by CFT or by IFT despite virus isolation. Results from IFT may permit the interpretation of low CF titers. In contrast to CFT, IFT makes possible the differentiation between vaccinated and unvaccinated persons because vaccinated persons regularly produce IgM antibodies against all strains of the vaccine.

The use of a radioimmunoassay and an enzyme immunoassay for early diagnosis of Q fever is described, both of which are based on the IgM antibody-capture principle. A commercially available phase II antigen and a labeled, purified anti-Q-IgG of human origin were employed. With these tests Q fever antibodies were detected earlier in the course of infection than with the complement fixation test.

Because it is not possible to distinguish clinically influenza from other respiratory infections, virological methods have to be used to establish the influenza etiology. Nasopharyngeal swabs from 202 children with respiratory symptoms were taken. Influenza A virus (H3N2) was isolated from 44 children, influenza A virus (H1N1) from 61 children and influenza B virus from 13 children. The maximal activity of the two influenza A virus subtypes was different. The following features permitted the classification of 3 groups; monophasic fever greater than or equal to 38.5 degrees C (81.35%), biphasic fever (14.41%), and pseudocroup (4.24%). 16.1% of the children with fever also had gastrointestinal symptoms. No relation between influenza type/subtype and type of manifestation could be established.

We report on results obtained with a direct immunofluorescence test for subtype-specific identification of influenza virus in detached cells of MDCK cultures after inoculation of 281 clinical specimens from patients with influenza-like disease. Influenza virus antibodies were produced in eggs from immunized hens and labelled with FITC. In 157 cases CPE was found in MDCK cells. A total of 57 cases of influenza A (H3N2), 86 cases of influenza A (H1N1), and 14 cases of influenza B were identified. In 33 cases of influenza A (H1N1) infection with massive CPE guinea pig but not chicken erythrocytes were agglutinated by the cell culture supernatants. The single step immunofluorescence test described proved easy to perform and results were obtained within 1 h after CPE was observed in contrast to the conventional HIT which is very time-consuming.

Up to now, the complement fixation test (CFT) has been the basis for the serological diagnosis of influenza virus infection in routine laboratories. Generally, low CF titers (1:20 or 1:40) are difficult to interpret. This means that the differentiation between recent and remote influenza infections is not possible by CFTs on single sera. Nonetheless this is generally possible by the subtype- and immunoglobulin class-specific immunofluorescence test (IFT) reported in this paper. Sera from 76 patients with confirmed influenza infection were tested and we obtained the following results: only 27.6% contained antibodies of all immunoglobulin classes, 51% contained IgG and IgA antibodies (without IgM) and 3.9% responded only with the IgG isotype. The IFT-positive and CFT-negative were 5.2% and the IFT-negative and CFT-positive 4%. In 7.9% no antibody rises were detected by CFT or by IFT despite virus isolation. Results from IFT may permit the interpretation of low CF titers. In contrast to CFT, IFT makes possible the differentiation between vaccinated and unvaccinated persons because vaccinated persons regularly produce IgM antibodies against all strains of the vaccine.

Among a population of 18,175 children below 7 years of age in medium sized towns and rural areas in south-western Germany 552 (3.03%) cases of croup were registered during a 12 months period in 1984-85 by their physicians. Distributions according to months, sex and age at the onset of the disease were the same as in other recent investigations: The level of the air-pollution measured (SO2, NOx, CO, CO2, ozone and dust) was low (highest monthly means in microgram/m3: SO2 88, NO2 73, NO 119, dust 41). There was no relevant influence of the degree of air pollution on croup-frequency. The rise of croup-frequency shortly after a period of several days of higher pollution was accompanied by an influenza epidemic as proved by virus isolations.

The suitability of serological surveys of roe deer (Capreolus capreolus) in determining the spread of tick-borne encephalitis virus (TBEV) was tested in a south German area with a low risk of TBEV infection to humans. Sera obtained from 192 hunted roe were screened by an haemagglutination-inhibition test (HAI) and in an ELISA developed in our laboratory. Those found positive were tested in a neutralization test (NT). Fifty (26.0%) sera reacted positive by ELISA and 43 (86.0%) of these were confirmed by HAI or NT. Forty-seven (24.5%) samples were positive by HAI, 44 (93.6%) of which were also positive in NT or ELISA. Only insignificant increase of the antibody prevalence with age (P = 0.17 for HAI antibodies) suggests that most infections occur at an early age in scattered natural foci. The antibody prevalence in females was lower than in males (OR = 0.63; P = 0.02 for HAI antibodies). In determining the distribution of seropositive roe we increased the sample size to 235 sera. No antibodies were detected in 56 (23.8%) sera collected in the eastern third of the county. The areas of high antibody prevalence in roe match those in which humans have been infected. We conclude that serosurveys of roe deer are useful in marking out areas in which humans face the risk of infection, provided that an adequate number of sera, preferably from males, is available.

Since virus isolation consumes a lot of work and time, and virus specific antibodies are not detectable before several days after the onset of illness we developed an enzyme immunoassay (ELISA) for the detection of influenza A and influenza B virus antigen in nasopharyngeal specimens (NPS). This test permits antigen detection within four hours. This ELISA was tested with 119 NPS from children, most of these between 1-12 years old. Virus isolation in MDCK-cells served as control. A total of 67 influenza A/H3N2-, 10 influenza A/H1N1, and 2 influenza B viruses were isolated from cell cultures. 68 (88.3%) of the NPS reacted positive in influenza A virus antigen ELISA, 2 in influenza B virus antigen ELISA, and 9 reacted falsely-negative. The failure to detect antigen could not be solely due to low antigen concentration in the NPS because in 5 materials high concentrations of infectious virus were shown in cell culture. The test allows the rapid diagnosis of influenza virus infections with high efficacy also for laboratories without the facility to perform tissue culture. For accelerating the diagnosis by isolation of viruses in cell cultures, ELISA is useful as cell culture confirmation test, because influenza virus antigen is detectable before a cytopathogenic effect appears.

We developed a direct enzyme immunoassay [EIA; Enzygnost Influenza A(Ag) and Enzygnost Influenza B(Ag)] for the direct detection of influenza A and B virus antigens in nasopharyngeal secretion specimens (NPS). The test is performed without sonification of specimens, and results are obtained within 4 h. A direct comparison between direct EIA and quantitation of virus shedding for influenza A and B virus antigen detection was carried out. A total of 210 NPS and 98 nasopharyngeal wash specimens (NPW) were investigated. We isolated influenza A viruses from 79 (37.6%) of 210 NPS; of these 79 cell-culture-positive NPS, 70 (88.6%) were also positive by direct EIA. Of 29 (13.8%) NPS from which influenza B virus was isolated, 24 (82.8%) NPS were positive by direct EIA. Virus shedding was determined quantitatively in 48 NPS from patients with influenza A and in 24 NPS from patients with influenza B. Only a crude correlation between optical density values and virus concentrations was observed. Detection of influenza virus antigens in NPS by direct EIA showed sensitivities of 89.7% for influenza A virus and 87.9% for influenza B virus and specificities of 99.3% for influenza A virus and 100% for influenza B virus. With direct EIA, all NPW were negative for influenza A virus, although virus was isolated from 21 (21.4%) NPW. Of 15 NPW from which influenza B virus was isolated, 7 showed positive results in direct EIA. In addition, direct EIA is suitable for detecting influenza A and B viruses in cell cultures before the appearance of any cytopathic effects and can be used as a cell culture confirmation test.

An indirect immunofluorescence assay (IFA) using a recently developed commercial kit for detecting antibodies against Coxiella burnetii (C.b.), the etiological agent of Q fever, has been evaluated using human field serum samples. The IFA was compared with an ELISA and a complement fixation test (CFT). The IFA was based on the corpuscular C.b. phase I and phase II antigens specific to anti-C.b. phase I and II antibodies, respectively. Fifty sera from persons with symptoms of Q fever were examined in this study. The IFA compared with the ELISA showed the sensitivities of 97.7% and 87.2% for IgG and 66.7% and 60.0% for IgM phase II and I antibodies, respectively and the specificities of 100% and 90.0% for IgG and 75.9% and 64.7% for IgM phase II and phase I antibodies, respectively. Due to a limited number of sera positive in the IgA antibody testing, the data presented should be considered with caution. It appears that the IFA strikes a very good balance between high specificity and sensitivity with phase II and phase I IgG antibodies and a less satisfactory one with IgM antibodies. The CFT failed in one of the above aspects showing a good specificity but a poor sensitivity, especially for phase I antibodies. The study demonstrated that the IFA is suitable for diagnosing Q fever and its therapeutic follow-up and is a good candidate for screening sera in large numbers. A certain limitation, especially in testing early stages of the chronic disease, could be a low fluorescence intensity of the IgA positive control in comparison with the IgA negative one.

Serology and nucleic acid amplification are the main diagnostic tools for the diagnosis of Mycoplasma pneumoniae infection. Since no reference standard is generally accepted, serologic assays for M. pneumoniae have not been evaluated on a broad scale. In this study, 12 commercially available serologic assays (for immunoglobulin G [IgG] and IgM) and the complement fixation test (CFT) were evaluated by using M. pneumoniae DNA detection by real-time PCR as the "gold standard." The assays tested were Platelia EIA (Bio-Rad), SeroMP EIA (Savyon), Serion classic EIA (Virion/Serion), Biotest EIA (Biotest), Ridascreen EIA (r-Biopharm), AniLabsystems EIA (Labsystems), Novum EIA (Novum Diagnostica), Diagnosys EIA (MP products), Genzyme/Virotech EIA, ImmunoWell EIA (Genbio), ImmunoCard EIA (Meridian), and SerodiaMycoII microparticle agglutination (Fujirebio). Serum samples (n = 46) from 27 PCR-positive patients with a known first day of disease and sera (n = 33) from PCR-negative controls were obtained from prospective studies of acute lower respiratory tract infections. Additionally, control sera (n = 63) from patients with acute viral or bacterial respiratory infections other than those caused by M. pneumoniae were tested. The results showed low specificities for both the Novum and the ImmunoCard IgM assays. The IgM assays with the best performances in terms of sensitivity and specificity were AniLabsystems (77% and 92%, respectively), SeroMP (71% and 88%, respectively), and CFT (65% and 97%, respectively). Good receiver operating characteristic areas under the curve were found for CFT (0.94), the Platelia assay (0.87), and the AniLabsystems assay (0.85). We conclude that there are few commercial serologic assays for the detection of M. pneumoniae infections with appropriate performances in terms of sensitivity and specificity and that PCR has become increasingly important for the diagnosis of M. pneumoniae infections in defined groups of patients.

This article describes the standardization and evaluation of an in-house specific IgG avidity ELISA for distinguishing recent primary from long-term human cytomegalovirus (HCMV) infection. The test was standardized with the commercial kit ETI-CYTOK G Plus (Sorin Biomedica, Italy) using 8 M urea in phosphate-buffered saline to dissociate low-avidity antibodies after the antigen-antibody interaction. The performance of the in-house assay was compared to that of the commercial automated VIDAS CMV IgG avidity test (bioM rieux, France). Forty-nine sera, 24 from patients with a recent primary HCMV infection and 25 from patients with a long-term HCMV infection and a sustained persistence of specific IgM antibodies, were tested. Similar results were obtained with the two avidity methods. All 24 sera from patients with recently acquired infection had avidity indices compatible with acute HCMV infection by the VIDAS method, whereas with the in-house method, one serum sample had an equivocal result. In the 25 sera from patients with long-term infection, identical results were obtained with the two methods, with only one serum sample having an incompatible value. These findings suggest that our in-house avidity test could be a potentially useful tool for the immunodiagnosis of HCMV infection.

AIMS--To compare the novel Serofast latex agglutination test (International Mycoplasma, Toulon-Cedex, France) with the complement fixation test and enzyme immunoassay (EIA) for diagnosing acute Mycoplasma pneumoniae infection. METHODS--Paired sera from 60 patients with respiratory infection who had tested positive for M pneumoniae by complement fixation test were analysed with Serofast and indirect EIA for specific IgG and IgM antibodies. RESULTS--Serofast was less sensitive than the two other tests. Only 30 (50%) out of 60 paired sera which showed a diagnostic seroconversion or had high positive, unchanged antibody titres by complement fixation test or EIA, or both, tested positive with Serofast. Positive test results with Serofast were associated with the presence of a complement fixation test titre of > or = 512 and high positive IgM antibody titres measurable by EIA; virtually all patients with a complement fixation test titre of < 256 or those responding primarily in the IgG class tested negative with Serofast. Based on analysis of sera taken at the acute phase of infection, 10 (17%) of the 60 patients tested positive by complement fixation test, 10 (17%) by EIA, and only four (7%) by Serofast. CONCLUSIONS--Serofast was less sensitive than complement fixation test and EIA and it cannot be recommended as a replacement for either test in routine diagnostic use. It might prove useful in laboratories where non-specific tests, such as the determination of cold agglutinins, are still used for the diagnosis of M pneumoniae infection. Testing paired sera is, however, a prerequisite for obtaining acceptable sensitivity by Serofast as well as other serological methods currently available.

An indirect immunofluorescent antibody test for early detection of IgM antibodies to Mycoplasma pneumoniae is described, using sera from which IgG antibodies have been removed. The results over a five-year period were studied and compared with complement fixation testing and isolation of the organism. During the epidemic season of November 1990 to April 1991, the immunofluorescent IgM test was used as a first-line test on specimens taken both early and late in the illness to provide a simple, inexpensive and clinically useful test, with 151 positive results being obtained from 886 sera tested.

The use of a gel filtration method allowed us to obtain pure IgM fractions to be tested for specific Toxoplasma serology. The good correlation found in our series between IgM-IFAT and LBCF-H100-TTE performed on pure IgM fractions suggests that LBCF-H100-TTE may be another useful serological test for detection of IgM antibodies although false negative results might occur when LBCF-H100-TTE is performed on IgM fractions of cord sera.

An enzyme-linked immunosorbent assay (ELISA) for the detection of IgG and IgM antibodies against Mycoplasma pneumoniae, performed with commercial antigen and reagents, is compared with the complement fixation test (CF) in a serological study of 209 human sera. Concordant results were usually obtained by CF test and by IgG ELISA in sera from patients with recent M pneumoniae infection. In contrast, when used for an immunological survey of a general population, approximately 27% of the sera negative in the CF test were positive for IgG by the ELISA, and sera with low CF titres were found to have a broad range of IgG titre by the ELISA. This may be due to the greater sensitivity of the ELISA technique and/or to different types of antibody measured by both tests. IgM was detected by ELISA in sera from all patients with recent M pneumoniae infection diagnosed on the basis of clinical findings and by CF assay. Occasionally false-positive IgM antibodies were due to rheumatoid factor (RF); this potential interference necessitates routine testing of IgM antibody positive sera for RF.

The Diffusion-In-Gel Enzyme Linked Immunosorbent Assay (DIG-ELISA) is a new and simple method for the quantitation of antibodies to diffuse from wells in gel in petri dishes and absorb to an antigen coated to the plastic surface prior to testing. The antigen-antibody reaction is visualized with horseradish-peroxidase conjugated class-specific anti-immunoglobulins. The DIG-ELISA permits detection of 0.1 microns/ml of specific antibodies. This method was used to determine the IgG, IgA and IgM antibody levels to Yersinia enterocolitica O:3 in sera from patients with antibody titres to this microorganism, as determined by direct agglutination and complement-fixation test. The DIG-ELISA IgG antibody values, in contrast to IgA and IgM, correlated well to titres obtained by the direct agglutination method. Low degrees of correlations were found for all three immunoglobulin classes when compared to the complement fixation test.

The sera of 30 patients with complement-fixing antibodies to Mycoplasma pneumoniae (titres larger than or equal to 32) were treated with staphylococcal protein A. This procedure effectively removed 90 to 95% of the IgG antibodies. The Mycoplasma-specific antibodies in the treated sera could then be measured by a complement fixation test. This provides a useful test for distinguishing serological responses to recent Mycoplasma pneumoniae infections from anamnestic responses due to past exposure to Mycoplasma pneumoniae.

One group of 51 cattle was vaccinated with B. abortus S19 (S19) and a further 51 cattle were vaccinated with B. abortus S45/20 (S45/20). Forty-eight cattle (24 from each group) and a control group of 12 cattle were subsequently challenged with B. abortus S544. The enzyme-linked immunosorbent assay (ELISA) was used to detect specific IgG and IgM antibodies in these groups. All cattle vaccinated with S19 had high levels of IgG and IgM, but the S45/20 vaccine produced detectable antibody in only a few cattle. In those cattle where the challenge induced infection, the mean levels of IgG and IgM were much higher than those of the uninfected cattle in the same groups. When the isolation of B. abortus was compared at slaughter with the serological results, the ELISA, when used to detect specific IgG, was more sensitive but less specific than the serum agglutination test, complement fixation test and indirect haemolysis test, and more sensitive and more specific than the Rose Bengal test.