Detection and differentiation of cytomegalovirus antibodies by radioimmunoassay.

A sensitive radioimmunological method was adapted for the assay of anticytomegalovirus IgG and IgM antibodies. Binding of immunoglobulins G with cytomegalovirus (CMV) antigens was greatly reduced when an antigen prepared by sonication of the infected cells in glycine buffer was used in place of the antigen obtained by freezing and thawing of the cells. The application of labelled serum against the Fc fragment of IgG made it possible to identify selectively the antibodies bound with the virus antigen by antigenic determinants.

Characterization of rotaviral RNA isolated from children with gastroenteritis in Poland.

Rotaviruses were detected in 37% (331/895) of stool specimens collected from children with gastroenteritis attending three hospitals in Warsaw between January 1981 and March 1994. An analysis of rotavirus electrophoretypes circulating in Warsaw and Cracow, Poland, over 6 years showed that all 176 electrophoretypes encountered were characteristic of group A rotaviruses. Based on variations in the migration pattern of RNA genome segments when passed through polyacrylamide gels, 10 different electrophoretypes were identified: 86% were long patterns and 14% were short patterns. Mixed infections were observed in two cases.

Complement and circulating immune complexes in children with mumps meningitis.

Haemolytic activity of complement (CH50) and occurrence of circulating immune, complexes (CIC) were studied in serum samples from children with mumps meningitis taken at regular intervals. The decrease of CH50 was observed in 46 out of 77 samples (60%) in the Ist examination, between 1-6th day from the appearance of symptoms, in 43/67 samples (64%) in the IInd examination, between 12--16th day of disease, and in 7/22 samples (32%) in the IIIrd examination, between 12--14th month of observation since the appearance of symptoms. Typical changes of complement level during the disease were of such profile that the most pronounced decrease was in the Ist and IInd examination with the normalization in the IIIrd examination. The presence of CIC was found in 21%, 15% and 14% of samples in the Ist, IInd, and IIIrd examination respectively. Serum sample form the child on 5th day of disease, with high concentration of CIC (482 micrograms/ml), was subjected to sepharose 6B gel filtration on the calibrated column. The molecular weight of thus analyzed immune complexes was as follows: 2.5 X 10(6) and 5.0 X 10(5) daltons. CIC contained anti-mumps antibodies, determined by Elisa, in IgG, IgA and IgM classes.

Radioimmunoassay of measles virus antibodies in SSPE.

A sensitive radioimmunological assay (RIA) was introduced for detection of measles virus IgG and IgM antibodies. The hyperimmune response to measles virus could be demonstrated more precisely by RIA than by haemagglutination inhibition (HI). The ratio between RIA and HI antibody titres was decidedly higher in sera and cerebrospinal fluids (CSF) of patients with subacute sclerosing panencephalitis (SSPE) than in those of other groups tested.

[Subacute sclerosing panencephalitis (SSPE) in Poland in 1996-1999. Phase VII of epidemiologic studies]. / Podostre stwardniajace zapalenie mózgu (SSPE) w Polsce w latach 1996-1999. VII etap badan epidemiologicznych.

In the seventh phase of epidemiological studies of SSPE data were gathered on patients who developed the disease in 1996-1999. In this time period the diagnosis was confirmed in only 10 cases (4 in 1996, 4 in 1997, 0 in 1998 and 2 in 1999). This is a significant reduction of incidence in relation to the preceding stages, in particular to the years 1993-1995 in which 49 new cases were still reported. This is not ruling out the possibility that at the beginning of the years 2,000 cases would be diagnosed in which the first symptoms developed several or more months earlier.

[Clinical aspects of herpes simplex meningoencephalitis]. / Klinika opryszczkowego zapalenia mózgu i opon mózgowo-rdzeniowych.

On the basis of the observed 10 cases the authors discuss the diagnostic, clinical and therapeutic problems of herpes encephalomeningitis. The diagnosis in early phase of the disease but already after appearance of psychic and neurological symptoms and signs is based on clinical criteria and also on the result of examination of the cerebrospinal fluid and rapid serological method ELISA. In view of the already available possibilities of virostatic treatment early diagnosis is of utmost importance, for beginning treatment (preferably with Vidarabine) before the development of extensive necrotic brain lesions.

[Evaluation of changes in the epidemiological situation of SSPE in Poland in the years 1977-1991 (in the course of 15 years after the introduction of mandatory antimeasles vaccination)]. / Ocena zmian w sytuacji epidemiologicznej SSPE w Polsce w latach 1977-1991 (tj. w okresie 15 lat po wprowadzeniu obowiazkowych szczepien przeciwodrowych).

The authors present the results of an epidemiological study of SSPE in Poland conducted since 15 years. Since the years covered by the study (1977-1991) included the first period after the introduction of obligatory vaccinations against measles in our country, the most important aim of this analysis was to find out the possible influence of the effect of these vaccinations on SSPE incidence. A comparison of the data obtained in successive stages of the study shows that a slow gradual decrease of the annual incidence of SSPE has been taking place in our country (from at least 1.6 per million before 1983 to 0.69 in 1991). The effect of the vaccinations is visible also in a significant increase of the mean age of SSPE onset which is a result of ever more widespread vaccination of the youngest children. These observations indicate unequivocally that the aetiological factor in SSPE is a wild strain of measles virus and not that used for vaccinations.

[Changes in the level of humoral response for measles virus in patients with subacute sclerotic panencephalitis during 1984-1989]. / Zmiany w poziomie odpowiedzi humoralnej dla wirusa odry u chorych na podostre stwardniejace zapalenie mózgu w latach 1984-1989.

Immunological response to measles virus in serum and cerebro-spinal fluid of 338 patients with subacute sclerotic panencephalitis (SSPE) during the years 1984-1989, was investigated. It was found that mean titer and distribution of results of antibody determination by the hemagglutination inhibition (OZHA) method in patients sera are visibly differing during consecutive years of investigation. Analysis of these parameters in determination by the immunoenzymatic methods (ELISA) has shown their higher stability. Also results of determinations of IgG concentration in sera of patients with SSPE did not demonstrate significant differences between consecutive years of investigation. Systematic decrease of mutual relation between antibodies to measles virus detected by ELISA and OZHA was found. Analysis of local synthesis of antibodies to measles virus in the central nervous system has revealed that it most strongly expressed by hemagglutinin.

[Interpretation of the results of identification of measles virus antibodies in the serum and cerebrospinal fluid from patients with suspected subacute sclerosing panencephalitis (SSPE)]. / Interpretacja wyników oznaczen przeciwcial dla wirusa odry w surowicy krwi i plynie mózgowo-rdzeniowym od chorych z podejrzeniem podostrego stwardniejacego zapalenia mózgu (SSPE).

Clinical diagnosis of subacute sclerosing panencephalitis+ (SSPE) requires laboratory confirmation relying on an evaluation of immune response in serum and in cerebrospinal fluid (CSF) to measle virus. In our study a comparison of antibody level for this virus by ELISA and haemagglutination inhibition test was made in materials derived from 1396 patients with SSPE, sclerosis multiplex, acute measles++ neuroinfections and other diseases of central nervous system (CNS). Statistical analysis permitted to settle criteria for differentiation of SSPE from remaining diseases of CNS and usefulness for diagnosis of particular methods as well as analysis of results obtained with single sample of serum or CNS. An advantage of ELISA for diagnostic purposes and CSF samples were confirmed.

[Influence of patient age and year of developing the disease on immunologic response to measles virus in subacute sclerosing panencephalitis (SSPE)]. / Wplyw wieku chorych i roku zachorowania na odpowiedz immunologiczna dla wirusa odry u osób z podostrym stwardniejacym zapaleniem mózgu (SSPE).

Analyses were concerning 338 SSPE patients investigated in the years 1984-1989 from a group in which 305 persons were known regarding year of illness development and their age at the time of sample taking. The analysis has shown statistically significant decrease of antibodies against measles virus with increase of age of tested patients. At the same time, it was found that in persons who developed disease in 1983-1985 and in 1988, increase of mean value of antibodies against measles virus was evident during first 2-3 years from beginning of the SSPE. Comparison of mean values of antibodies against measles virus in age groups of SSPE patients and normal population, revealed existence of relation between these groups where source of antibodies constituted of a wild measles virus. Our analyses indicate influence of measles wild virus circulating in the population on immunological response to this virus in sera of patients with SSPE.

[Evaluation of the usefulness of the ELISA method for determining the level of enterovirus antibodies in serum and cerebrospinal fluid]. / Ocena przydatnosci metody ELISA do oznaczania poziomu przeciwcial dla enterowirusów w surowicy i plynie mózgowo-rdzeniowym.

The usefulness of the methods was compared: complement fixation test (CFT), neutralization test (NT) and ELISA IgG and IgM against enteroviruses for the evaluation of specific immune reaction in sera and cerebrospinal fluid (CSF) samples of patients with confirmed enterovirus infections. The criteria were established for the assessment of ELISA results in rapid diagnosis of enterovirus neuroinfections. The criteria accepted by the producer lowered the sensitivity of the method and the possibility of recognition of local synthesis of antibodies in the CNS. The use of serum negative in CFT and negative CSF as reference for the determination made possible using of that kit for rapid diagnosis of neuroinfections. The modified ELISA IgG test makes possible determination of antibodies in CSF and serum, and accepting the generally recognized criteria for local production of antibodies in the CNS the ELISA test makes possible rapid diagnosis of neuroinfections which is not possible by other methods.

[Electrophoretic analysis of RNA from rotavirus strains isolated from children in the years 1986-1990]. / Elektroforetyczna analiza RNA szczepów rotawirusów izolowanych od dzieci w latach 1986-1990.

Double stranded RNA of rotaviruses is composed of 11 segments which in standard and field strains may differ in electrophoretic mobility in polyacrylamide gel electrophoresis. This study was aimed at determination of electrophoretic differentiation of rotavirus strains found in samples of feces of children treated because of diarrhoea in two hospitals in Warsaw and in Kraków region in the years 1986-1990. Out of 85 positive samples from Warsaw and 90 from Krakow region, for investigation 44 and 36 strains, respectively were included. Totally for 71 strains well stained 11 segments of RNA were obtained which permitted for determination of their electrophoretic-types. Majority of strains, 60 (84%) were characterized by a long electrophoretic-type, whereas 11 (16%) had a short electrophoretic-type. Considering differences in migration speed of 1-4 and 7-9 segments, within long and short types, eight subtypes denominated as a-h were present. Subtypes a, c and d constituted 73% of all tested strains. These studies indicate that in Poland occur at least eight electrophoretic subtypes of rotaviruses and that this feature is partly correlated with belonging of strains to subgroups and antigenic serotypes.

[Use of the western blot method for detecting antibodies against proteins of various rotavirus strains]. / Zastosowanie metody western blot do wykrywania przeciwcial dla bialek róznych szczepów rotawirusów.

The aim of this work was to adapt the Western blot method to analyse the humoral response to proteins of rotavirus strains having various antigens. 10 serum samples from animals immunized with SA11, 18 serum samples from hospitalized children with rotavirus infections and 17 serum samples from healthy adults were examined for rotavirus antibodies using Western blot. Antibodies against all structural rotavirus proteins were detected. The examined sera reacted differently with rotavirus proteins depending on the strain used. The highest IgG reactivity was observed for Wa and SA11, while the lowest was noted for DS-1. At the same time a different level of cross-reactivity of human sera with specific proteins of antigenically varying rotaviruses was observed. The sera taken from adults showed a lower reactivity with proteins of antigens used compared with sera from children. The exception was VP6 of the Ito strain for which the reactivity of adult sera was statistically higher. Essential differences in the reactivity of children's and adult sera were found mainly in the case of VP2 and VP4 belonging to the SA11 strain and VP4 and VP7 of the Ito strain. From these investigations we can conclude that the Western blot method may be useful in assessing immune response caused by rotaviruses. The results of reactivity of some human sera with proteins of strains used indicates that the persons had previously been infected by antigenically varied rotaviruses.

[Use of peroxidase-labeled antigen of tick-borne encephalitis virus for analysis of specific immunoglobulin response and the evaluation of its diagnostic usefulness for detecting the etiological factor of human neurologic infections]. / Uzycie znakowanego peroksydaza antygenu wirusa kleszczowego zaplaenia mózgu do analizy swoistej odpowiedzi w klasach immunoglobulin i ocena diagnostycznej przydatnosci tej metody do ustalania etiologicznego czynnika neuroinfekcji czlowieka.

Peroxidase labelled virus of tick-borne encephalitis (k.z.m.) was used for humoral immune response analysis in humans. Dot-ELISA methods elaborated in a version of "catching antibodies" (cAb-ELISA) was compared in respect to its diagnostic usefulness to indirect immunoenzymatic method (i-ELISA) and to haemagglutination inhibition test (OZHA). High sensitivity and diagnostic value of both methods were confirmed. As compared to i-ELISA, cAb-ELISA was characterised by higher specificity of IgM antibody determination resulting from a lack of influence of rheumatoid factor and specific IgG on above mentioned determinations of antiviral IgM in cAb-ELISA. Moreover, parallel analysis by cAb-ELISA technique of serum and cerebro-spinal fluid permits to evaluate in a simple manner a synthesis of specific viral antibodies present in central nervous system.

[Preparation of Staphylococcus aureus antigens for evaluation of their immunological reactivity with the human sera by the western blot method]. / Przygotowanie antygenów Staphylococcus aureus do oceny odpowiedzi immunologicznej w surowicy metoda western-blott.

Extracellular antigens as well as cell wall extracts of 4 S. aureus strains isolated from different kinds of infection were analysed by Western-Blott technique. Materials obtained in two systems of bacteria cultivation (with and without aeration) were compared. Four systems of PAGE (native conditions, with 8.0 M urea, with SDS and SDS after previous reduction of the material with 2-mercaptoethanol) were compared in order to get the best differentiation of proteins and antigens. Immunological reactivity of the antigens mixture with two human sera: highly positive (with three S. aureus antigens in ELISA) from patient with staphylococcal sepsis and negative (from blood donor) were analysed. The best results were obtained after reduction of the cell wall extracted material in SDS-PAGE. The different protein patterns depending on the strain and the method of bacteria cultivation were observed. The standardisation of Western-Blott technique was performed, including titration of the sera to get the best differentiation of the antigens. The difference in immunological reactivity of the positive and negative sera with staphylococcal antigens mixture showed rather quantitative than qualitative character.

[Humoral response to Staphylococcus aureus antigens evaluated by the western blotting method]. / Humoralna odpowiedz na antygeny Staphylococcus aureus oceniana metoda western-blott.

Cellular antigens extracted from the cells of four Staphylococcus aureus strains from different kinds of infections (sepsis, osteomyelitis, furunculosis) were analysed by the western blotting technique. Antibiotic sensitivity pattern of the strains was compared. One isolate was found to be MRSA strain. Sera samples from patients of whom strains were isolated and four sera from blood donors (as a control) were used in the investigation. IgG levels for purified staphylococcal antigens (lipase, alpha-toxin and teichoic acid) were estimated. Interaction between extracted bacterial antigens and serum antibodies of IgG class were analysed in homologous and heterologous systems. The most strong immunological reaction of the investigated sera with staphylococcal antigens was observed in the case of homologous system. Serum from sepsis patient was found to be the most reactive serum with all staphylococcal antigens mixtures.

[Use of western blotting methods in diagnosis of viral infection causing tick-borne encephalitis]. / Zastosowanie metody western blott w diagnostyce zakazen wirusem kleszczowego zapalenia mózgu.

A Western blot method for investigation of antigens and of immunological response to tick-borne encephalitis was prepared. An analysis was performed testing cross reactions of proteins of prepared antigen with sera for four Polish strains of tick-borne encephalitis and four other flaviviruses. Usefulness for diagnosis of infections in humans was determined by comparison of Western blot method with ELISA immunoenzymatic test. It was found that elaborated method permits equally as ELISA for evaluation of immune response in immunoglobulin classes. By evaluation of response for individual virus components, Western blot method enables at the same time verifications of nonspecific determinations which result from reaction of antibodies present in tested diagnostic materials with nonviral antigens.

[Use of western-blotting technique and computer analysis for comparing cellular antigens of coagulase-negative staphylococci]. / Zastosowanie techniki western-blotting i analizy komputerowej do porównania antygenów komórkowych szczepów gronkowców koagulazo-ujemnych.

Western-Blotting technique and computer programme have been used to analyse cellular antigenic patterns of coagulase-negative staphylococci mainly of S. epidermidis and S. saprophyticus species. It was shown that antigenic pattern among strains of one species was similar. Intra strains differences are mainly quantitative. The qualitative differences concern not quantitatively dominant antigens. The main antigens of S. epidermidis and S. saprophyticus strains were of 28 and 44 kD molecular weight respectively. The antigens of molecular weight around 44, 48, 89 and 105 kD were detected in antigenic extract of strains from different species. Only one of the quantitatively dominant antigen of m. w. 48 kD, was localised in all analysed materials. The method presented here was used for the first time to analyse staphylococcal antigens. Such analysis could be performed not only for bacterial antigens.

[Usefulness of immunoenzyme diagnostic kits and latex tests for isolation of rotaviruses]. / Ocena przydatnosci immunoenzymatycznych zestawów diagnostycznych i testów lateksowych do wykrywania rotawirusów.

The aim of this study was to compare characteristic parameters of 4 diagnostic kits available in Poland-immunoenzymatic Rotazyme II and Enzygnost kits and latex kits Rotalex and Slidex. Studies were performed on 67 samples of feces of children treated because of diarrhea. The sensitivity, specificity, frequency of positive tests, false positives and false negatives and the accuracy of the tests under evaluation were determined. The results obtained were further verified using a reference test electrophoresis of RNA of rotavirus. The highest sensitivity was found for Enzygnost, Rotazyme II and Rotalex, 97%, 92% and 90%, respectively, and the lowest for Slidex- 79%, while the specificity was higher for latex kits than for immunoenzymatic kits. The accuracy of the results was highest for Rotalex kit (92%), next for Enzygnost kit (88%), Rotazyme (87%), and Slidex (84%). The significant correlation between OD value readings in spectrophotometer in Rotazyme II kit and the results of visual reading in latex test was found. All tested kits were found to be useful for diagnostic purposes. Rotalex kit due to the high accuracy of the results obtained, methodological simplicity, short time of testing and relatively low price could be a based test in hospital laboratories.

[Use of RT-PCR and nested PCR for diagnosing enterovirus infections]. / Zastosowanie metody RT-PCR i nested-PCR do wykrywania zakazen enterowirusami.

An application of reverse transcription (RT-PCR) and nested-PCR for the detection of human enteroviruses isolated in Poland is described. A specific 297-bp fragment was amplified by the method from all studied enteroviruses strains. Amplification system and the nested primers allowed to detect RNA isolated from 10(1) TCID50/ml which corresponded to one molecule of viral RNA in tested volume. However, applicability of the method for routine diagnosis of enterovirus neuro-infection should be reassessed in extensive clinical studies.