Determination of rubella virus-specific cell-mediated immunity using IFN gamma-ELISpot.

Immunity to rubella virus (RV) is conventionally determined by measuring specific immunoglobulin G (IgG). However, several individuals may be considered immune despite undetectable antibody levels. In the present study RV-specific interferon-gamma (IFN gamma)-ELISpot and rubella-IgG-ELISA were compared in 75 young adults aged between 20 and 30 years. In a subgroup, not only rubella-like particles (RLP), but also HPV77 rubella vaccine derived antigen was used in IFN gamma-ELISpot. The results from both, ELISA and ELISpot were independent of previous encounter to RV (vaccination, exanthematous disease, or childhood infection). There was no difference between RLP and RV vaccine antigen in IFN gamma-ELISpot response, and there was no correlation between IFN gamma-ELISpot and RV-specific IgG levels. IFN gamma-producing cells were found in 78.7% of all tested persons, and 83.8% of them were positive in ELISA. In almost all individuals seronegative for RV antibody, IFN gamma-producing cells were detected. Considering both humoral and cell-mediated immune responses, a positive RV immune reaction was seen in 98.6%. The results indicate that the IFN gamma-ELISpot can provide valuable additional information in seronegative individuals.

[Quantitative laboratory methods for the diagnosis of cytomegalovirus infection in premature infants].

31 prematures with signs of the cytomegalovirus infection (CMV) were examined. The blood and urine samples were tested for direct viral markers, i.e. for infectious CMV by the rapid culture method (RCM) and for viral DNA by quantitative PCR. Besides, the parameters of the specific immune response were studied in the babies. CMV was detected by RCM and/or PCR in 25 of the 31 examined babies during their 1st life week. The highest content of CMV within the investigated samples, i.e. 100 antigen-containing cells per 2.5 x 10(5) culture cells and above 2000 copies/ml of viral DNA was detected in 8 (32%) children. The quantity of viral DNA did not exceed 1000 copies/ml and one to three of stained cells was detected by PCR in 13 (42%) children. A study of anti-CMV in sera revealed high-titer of AT IgG in all 30 children. High avidity of anti-CMV-IgG was demonstrated to correlate with a low viral load and a low CMV infection activity in the newborns. According to the results, at least 3 laboratory diagnosis tools should be used in the diagnosis, they are PCR, RCM and determination of the anti-CMV avidity.

[Detection of direct markers of cytomegalovirus. Research of the spectrum and avidity of antiviral antibodies in infants and preschool children].

Thirty-three children aged 1 month to 3 years were examined within the case study. spELISA, immunoblot (IB), shell vial method (SVM) and PCR, were used for the detection of anti-CMV IgM and IgG, in the diagnosis of cytomegalovirus (CMV). Clinical signs of CMV infection (CMVI) were registered in 20 children (group 1); no CMVI specific signs were detected in the remaining 13 children (group 2). Class M antibodies were identified in 50% of group-1 sera. Around 80% of children in the group had anti-CMV-IgG. AI < 0.6 was in 3 (20%) of 15 examinees. Direct CMV markers (DNA and infection activity) were detected in 13 (65%) of 20 children. Sera of 13 children with non-specific symptomatology (group 2) had no anti-CNV-IgM, while IgG were found in 54% examinees in the group. The infectious active virus was not detected in a single baby. The used laboratory tools enhance the efficiency of CMVI diagnosis and denote a disease variation.

[Dynamic of immune response in primary cytomegalovirus infection and after reactivation of cytomegalovirus in patients with organ allotransplantation]. / Dinamika immunogo otveta pri pervichnoi tsitomegalovirusnoi infektsii i pri reaktivatsii tsitomegalovirusa u bol'nykh posle allotransplantatsii organov.

A total of 44 serum specimens from 7 patients with kidneys or liver transplanted from donors who had antibodies (Ab) to human cytomegalovirus (CMV) were studied. In 4 recipients anti-CMV Ab were found before transplantation and in 3 others they were not detected. It was shown by EIA that IgM and IgG anti-CMV appeared in the sera of primarily infected patients after 1-2 weeks and their titers were 5-10 times lower than in patients with reactivated CMV infection. Immunoblotting of Ab to individual CMV proteins showed a narrower spectrum of Ab during the initial period of primary CMV infection in comparison with the same period of reactivation and delayed production of AB to conformation-dependent determinants. Hence, analysis of anti-CMV Ab during the first 4-6 weeks after organ transplantation by EIA and immunoblotting differentiates primary CMV infection from its reactivation by Ab titers and spectrum. These parameters vary in some patients.

High incidence of parvovirus B19 DNA in synovial tissue of patients with undifferentiated mono- and oligoarthritis.

A common problem in rheumatological practice is inflammatory joint disease that cannot be classified. The prognosis of such undifferentiated arthritides is uncertain. The synovial tissue of 41 consecutive patients with various forms of arthritis was tested for the presence of viral DNA in a diagnosis-unaware fashion, using the polymerase chain reaction (PCR). Of all tested viruses, cytomegalovirus and parvovirus B19 were positive (each in 10 patients, two double-positives), whereas herpes simplex virus was positive in two patients. Rubella virus RNA was detected in three specimens. When the positivity for viral material was analysed in terms of distribution among the various diagnostic groups, it became evident that five out of 10 parvovirus B 19-positive patients belonged to the undifferentiated arthritis group, whereas cytomegalovirus-positive patients were spread among all diagnostic groups. This indicates the possibility of a new diagnostic category of undifferentiated mono- and oligoarthritis, which can be identified by the presence of parvovirus B19 DNA in synovial tissue.

Persistent fetal rubella vaccine virus infection following inadvertent vaccination during early pregnancy.

Inadvertent immunisation of seronegative women with RA27/3 rubella virus live-attenuated vaccine several weeks before and after conception is described. Whereas in 5 cases the vaccine virus was not transmitted vertically, in 1 case vaccination led to the development of persistent fetal infection with prolonged virus shedding for more than 8 months. Sequence analysis carried out on isolates from amniotic fluid, from cord blood leukocytes as well as from infantile urine confirmed an infection by the vaccine strain. At birth, the newborn infant exhibited none of the symptoms compatible with the congenital rubella syndrome and signs indicative for development of late onset disease are not apparent. This observation constitutes the first unequivocal documented case of rubella vaccine virus related to persistent fetal infection.

Papillomavirus-induced genital warts in a girl--management by surgery and immunomodulating therapy.

A 4-year-old girl with condylomata acuminata of the vulva and papular warts of the surrounding skin is presented. The lesions were removed by surgery. Histologic investigation showed koilocytosis of the squamous epithelium and in-situ hybridization revealed human papilloma virus type 6 infection. There were no signs of sexual abuse or sexual transmission of the virus. After ablation, an interferon-containing ointment was applied. In order to prevent recurrence, a low-molecular-weight immunomodulating leucocyte fraction was given for more than 1 year, during which time no relapse was observed.

Predictive value of serological tests in rubella virus infection during pregnancy.

In an attempt to define diagnostic criteria that may help to distinguish the congenital rubella syndrome (CRS) from subclinical intrauterine rubella virus (RV) infection, maternal and fetal serum samples were analyzed using (1) enzyme immunoassay employing RV synthetic peptides as antigen, (2) IgG avidity assay, and (3) immunoblot under nonreducing conditions, in addition to hemagglutination inhibition and commercial enzyme immunoassays. Infants born with CRS and their mothers were shown to reveal low or undetectable levels of E2-specific antibodies and deficient IgG recognizing the major neutralizing antibody-inducing epitope on the E1 protein (SP15). Antibody responses were normal in mothers with presumed RV reinfection as well as in asymptomatic infants born after maternal primary rubella. The results indicate that the maturation of specific humoral immune responses is obviously less efficient when intrauterine RV infection results in CRS. The detection of high avidity IgG, conformational E2-specific as well as SP15-reactive antibodies may serve as a potential predictor for a benign outcome of intrauterine RV infections.

The role of rubella-immunoblot and rubella-peptide-EIA for the diagnosis of the congenital rubella syndrome during the prenatal and newborn periods.

Rubella infection during the first trimester of pregnancy can cause the congenital rubella syndrome (CRS). Patients with CRS were shown to have a decreased humoral and cellular immunity. It is not known whether asymptomatic newborns who had experienced intrauterine infection with rubella virus (RV) differ in their antibody response from newborns with CRS. In this study we compared both groups for a difference which might be a useful diagnostic criterion for CRS during the prenatal and newborn periods. We used the nonreducing Rubella-Immunoblot and the Rubella-IgG-Peptide-Enzyme Immunoassay (EIA) to determine the antibodies directed to rubella proteins E1, E2 and C. The results showed that only newborns with CRS who had experienced RV infection during the first 12 weeks of gestation showed significantly reduced levels of antibodies directed to both the linear RV E1 epitope (SP 15) and the topographic RV E2 epitope. Asymptomatic newborns infected mostly later than week 10 of gestation showed normal levels of antibodies. These data suggest that the lack of antibody response in CRS is linked to the immaturity of the fetal immune system during the first trimester of gestation. Rubella-IgG-Peptide-EIA and Rubella-Immunoblot should be used additionally for CRS diagnosis in the prenatal/newborn periods. These results may have an impact on the early treatment of late-onset symptoms of CRS patients.

Evaluation of recombinant rubella-like particles in a commercial immunoassay for the detection of anti-rubella IgG.

BACKGROUND: We have investigated the performance of the novel rubella serology assay, Cobas Core Rubella IgG EIA recomb, which uses rubella-like particles (RLPs) expressed in transfected BHK-21 cells as the antigen. STUDY DESIGN: Evaluation of the assay included comparison with the hemagglutination inhibition (HAI) assay and another enzyme immunoassay (EIA) using native rubella virus (RV) as antigen, i.e. the Abbott IMx Rubella IgG. The assay was calibrated against the WHO 1000 IU/ml reference serum and showed good correlation with the HAI test in the analysis of 404 serum samples. However, quantitative differences in IgG values measured in the Cobas Core and the Abbott IMx assays were noted. RESULTS: Values obtained for patient sera as well as CDC and WHO standards were generally more than twice as high in the Abbott IMx assay as in the Cobas Core test. CONCLUSIONS: For sera whose IgG levels in the immunoassay and HAI test were discordant, immunoblotting proved valuable as a confirmatory method and indicated that a significant number of HAI-negative samples were correctly interpreted as positive by the immunoassay.

[Brush cytology and HPV detection in chronic laryngitis and papillomatosis]. / Abstrichzytologie und HPV-Nachweis bei chronischer Laryngitis und Papillomatose.

BACKGROUND: We investigated the utility of cytologic studies (including follow-up studies) in our specialty in determining the prevalence of human papillomavirus, specifically in the larynx. We utilized Papanicolaou's method of exfoliative cytology and in situ hybridization of the culture (biotin-marked DNA probes). PATIENTS: 486 patients with chronic hyperplastic laryngitis and 74 patients with papilloma were enrolled in the study. RESULTS: In 198 typings in 132 patients (59 patients with papilloma, 41 with chronic hyperplastic laryngitis, 17 with cancer, and 15 healthy), we found distributions of the individual types that were independent of the diagnosis. Particularly often, we encountered human papillomavirus types 18 and 31 in the presence of dysplasia, human papillomavirus type 16 in the presence of cancer, and type 11 in general. CONCLUSIONS: Cytologic examination and human papillomavirus typing of the culture permit us to define a third risk category in addition to the known risk groups smokers and dysplasia patients. Besides this, cytologic follow-up studies can provide further insight into the behavior of preneoplastic epithelial changes.

[Preparation of monoclonal antibodies to super early human cytomegalovirus proteins and their use for detecting infected cells]. / Poluchenie monoklonal'nykh antitel k sverkhrannim belkam tsitomegalovirusa cheloveka i ikh primenenie dlia vyiavleniia infitsirovannykh kletok.

Twenty-six mouse hybridomas producing monoclonal antibodies (MAB) to human cytomegalovirus (CMV) proteins have been obtained. MAB produced by three hybridomas were studied in detail. MAB were active in indirect immunofluorescence and solid-phase enzyme immunoassay, being directed to the super-early viral protein p72. Use of the resultant MAB for analysis of CMV-infected cells demonstrated that by specificity and sensitivity of viral antigen detection they were not inferior to anti-CMV antibodies manufactured by Ortho, USA. Screening of 258 patients with suspected CMV infection showed that these MAB may be used for immunofluorescent detection of CMV antigens in the material isolated from patients and infected subjects.

Evaluation of Cobas Core Rubella IgG EIA recomb, a new enzyme immunoassay based on recombinant rubella-like particles.

Cobas Core Rubella IgG EIA recomb (Roche), a new, commercially available rubella enzyme immunoassay using recombinant rubella-like particles, was compared with a hemagglutination inhibition assay (Rubenosticon; Organon Teknika) and two whole-virus enzyme immunoassays (IMx [Abbott] and Platelia [Sanofi Diagnostics Pasteur]). Compared with those of these reference assays, the relative sensitivities of Cobas Core Rubella IgG recomb were 100, 94, and 95.9%, with specificities amounting to 80.8, 98, and 98.2%, respectively.

[Antibodies to retroviral proteins in progressive scleroderma]. / Antikörper gegen retrovirale Proteine bei progressiver Sklerodermie.

In 8 out of 29 patients with scleroderma we found antibodies to HIV retroviral proteins in the Western blot analysis. The sera each reacted only to one or two of the p 18, p 24, p 55, and p 65 bands, and the reactions were relatively weak. There were no evident clinical correlations with the reactivity of certain bands and signs of direct HIV infection in our patients. Apart from three cases with positive CMV reactivity (IgM) there was no cross-reactivity to HTLV I or EBV (IgM) or to topoisomerase (Scl 70) or other autoantibodies to various nuclear antigens related to scleroderma. It is not clear whether retroviruses are involved into the pathogenesis of scleroderma or whether these antibodies are due to molecular mimicry.

Antibodies to retrovirus proteins in scleroderma.

In 8 out of 29 patients with scleroderma we found antibodies to HIV retroviral proteins in the Western blot analysis. The sera reacted only to one or two of the following bands: p 18, p 24, p 55, p 65 in relatively weak grades. There were no evident clinical correlations with the reactivity of certain bands, nor signs of direct HIV infection in our patients. Apart from 3 cases with positive CMV reactivity (IgM), there was no cross-reactivity to HTLV I or EBV (IgM) and to topoisomerase (Scl 70) or other autoantibodies to various nuclear antigens related to scleroderma. It is not clear whether retroviruses are involved in the pathogenesis of scleroderma or whether these antibodies are due to molecular mimicry.

[Serological diagnosis of cytomegalovirus-induced interstitial pneumonia in patients following bone marrow transplantation]. / Serologische Diagnostik zytomegalievirus-bedingter interstitieller Pneumonien bei Patienten nach Knochenmarktransplantation.

For diagnosis of the interstitial pneumonia conditioned by cytomegalovirus (CMV) an indirect immunofluorescence test was used to measure IgM and IgG antibodies to CMV in 369 serum specimens from 41 patients after bone marrow transplantation (BMT). An interstitial pneumonia conditioned by CMV was diagnosed in 5 patients either serological (4 patients) or by detection of cytomegaloviral infection typical inclusion bodies in lung cells (1 patient). The detection of antibodies to CMV for diagnosis of interstitial pneumonia conditioned by CMV is problematical in the phase immediately after BMT because bone marrow recipients are severely immunosuppressed by radiation and cytotoxic drugs received before transplantation. New method for detection of CMV-specific antigens are necessary.

[Detection of IgG antibodies against rubella virus using the "Rubella-IgG-EIA SSW" test kit]. / Nachweis von IgG-Antikörpern gegen Röteln-Virus mit dem Testsatz "Rubella-IgG-EIA SSW".

A new commercial ELISA test kit "Rubella-IgG-EIA SSW" is described for the determination of IgG antibodies to rubella virus. A panel of 99 sera was tested by "Rubella-IgG-EIA SSW" and by hemagglutination-inhibition (HI test). The results of enzyme-immunoassay (EIA) and of HI test correlate well; the coefficient of correlation is 0.95, the specificity is 90.9% and the sensitivity is 100%. A coefficient of correlation was found out of 0.92 between EIA and HI test in the evaluation of the quantitative procedure. The test kit can be used for determination of immune status to rubella virus and for quantitative detection of rubella-IgG antibodies.