[THE COMPARATIVE ANALYSIS OF EFFECTIVENESS OF QUICK TESTS IN DIAGNOSTIC OF INFLUENZA AND RESPIRATORY SYNCYTIAL VIRAL INFECTION IN CHILDREN].

The analysis was implemented concerning diagnostic parameters of commercial quick tests (immune chromatographic tests BinaxNOW Influenza A&B and BinaxNow RSV Alere, Scarborough Inc., USA) under detection of antigens of influenza virus A and respiratory syncytial virus in clinical materials. The polymerase chain reaction in real-time and isolation ofviruses in cell cultures. The analysis of naso-pharyngeal smears from 116 children demonstrated that sensitivity and specifcity of detection of influenza virus A using device mariPOC in comparison with polymerase chain reaction made up to 93.8% and 99.0% correspondingly at total concordance of results of both techniques as 98.3%. At diagnosing of respiratory syncytial virus using device mariPOC parameters made up to 77.3%, 98.9% and 862% as compared with polymerase chain reaction. The sensitivity, specificity and total concordance of results of immune chromatographic tests BinaxNOW in comparison ofpolymerase chain reaction made up to 86.7%, 100% and 96.2% correspondingly at detection of influenza virus A and 80.9%, 97.4% and 91.6% correspondingly at detection of respiratory syncytial virus. In comparison with isolation technique in cell cultures sensitivity of system mariPOC and immune chromatographic tests proved to be in 1.3-1.4 times higher at detection of influenza virus A and in 1.7-2 times higher in case of isolation of respiratory syncytial virus. There is no statistically significant differences between diagnostic parameters received for mariPOC and immune chromatographic tests at diagnosing influenza virus A and respiratory syncytial viral infection.

[The application of synthetic peptides to characterize the site-directed antiviral humoral immune response in patients with respiratory syncytial viral infection].

The article deals with the study of characteristics of epitope-specific humoral immune response to respiratory syncytial viral infection depending on nature of disease and patients' age. The couple serums from 226 children and adults with respiratory syncytial viral infection were analyzed. To detect in enzyme immunoassay the epitope-specific IgG the synthetic peptides were applied imitating the structure of functionally depended epitopes of F-protein of respiratory syncytial virus with amino acid sequences 221-232 (F = SP12), 479-491 (F-SP13) and G-protein with amino acid sequences 152-164 (G-H13), 184-198 (G-T15). The respiratory syncytial viral infection neutralizing antibodies were detected using the microneutralization reaction. The rate of seroconversions of epitope-specific IgG consisted 21-25% in children 3-18 years old under primary respiratory syncytial viral infection and increased up to 42-50% in children 3-18 years old and adults under recurrent episodes of diseases. In these groups, the seroconversions of respiratory syncytial virus neutralizing antibodies were observed in 42%, 65% and 58% of cases correspondingly. Independently of age of patients with diagnosed respiratory syncytial viral infection, the absence of conversions of neutralizing antibodies was statistically significant associated with the absence of response from epitope-specific IgG. The presence among B-cell epitopes of immune dominance of surface glycoproteins of respiratory syncytial virus in patients with recurrent (but not primary) respiratory syncytial viral infection is detected The hierarchy of epitope-specific immune response in case of complicated course of disease (response activity to epitopes: SP12 = G-T15 > G-H13) differed from the case of uncomplicated course of disease.

[The application of radial hemolysis technique in detection of antibodies to avian influenza virus A (H5N1) and pandemic virus A (H1N1) pdm09].

The article discusses the results of study that demonstrated the possibility of successful application of radial hemolysis reaction in analyzing the human inoculation immunity to new strains of influenza virus serotype A - A (H5N1) and A (H5N2). The radial hemolysis reaction provides accurate results on introduction of erythrocytes of horse or sheep into hemolytic system instead of erythrocytes of hens applied previously. The technique combines high sensitivity (in comparison with reactions of hemagglutination-inhibition and micro-neutralization, correlation coefficient 0.84-0.85) and total absence of inhibitors impact on the reaction results. During the investigation of immune response of patients who had pandemic virus A (H1N1) pdm09, radial hemolysis reaction demonstrated not only primary detection of antibodies to virus-agent (67%) during pandemic, but also elective heightened sensibility in the zone of low titer serums (1:20) in hemagglutination-inhibition reaction. These characteristics are very important in analysis of antibodies levels at early stages of disease. The radial hemolysis reaction continues to be a reliable instrument in evaluating qualitative and quantitative indicators of humoral immunity in ill patients and persons inoculated with new strains of human influenza virus.

[Development of competitive enzyme immunoassay for the detection of influenza A(H5) virus antibodies].

An indirect enzyme immunoassay (EIA) using the purified fraction of surface viral glycoproteins (GP) as an antigen for solid phase sensitization was not shown to be a specific method for the differential detection of influenza A(HS) (HS-Ab) virus antibodies (Abs) due to total conservative epitopes in the structure of GPs of influenza A(H5) and A(H1NI) viruses. The cross activity of some monoclonal Abs (MAbs) to influenza A(H5) and A(HIN1) viruses, which had been obtained at the Research Institute of Influenza, was proof of the presence of total immunodominant determinants in the structure of influenza H1 and H5 virus hemagglutinin (HA). In this connection, an EIA, which was based on the competition of influenza A(H5) H5-Ab virus HA-specific MAbs in the test sera for an association with influenza A(H5) virus, was proposed for the subtype-specific detection of H5 Ab. Comparison of the results of competitive EIA (cEIA), microneutralization (MN) test and HA inhibition test (HAIT) (using equine red blood cells) in the examination of sera obtained from 44 volunteers immunized with inactivated vaccine containing influenza A/Indonesia/5/2005 (H5N1) virus showed the high sensitivity and specificity of cEIA in detecting H5-specific Abs. The effectiveness of cEIA for the sera strictly positive for the content of H5 Abs was close to that of MN test and was 9-34% higher than HAIT (depending on those used in the analysis of H5 virus antigens). cEIA may be proposed to assess new influenza vaccines as an additional laboratory test. Since the infectious virus is not used during cEIA, it may be recommended for the serodiagnosis of influenza A(H5) at practical virological laboratories.

[Experimental investigation of Ingavirin antiviral activity against human parainfluenza virus].

The Ingavirin antiviral properties with respect to the parainfluenza virus, as an actual human respiratory tract pathogen, were investigated by two methods, i.e. immunoenzymatic analysis and microtetrazolium test. The results showed that along with the immediate antiviral activity Ingavirin had nonspecific cytoprotective properties. While affecting the virus proteins synthesis, Ingavirin lowered the virus cytopathogenic action. The drug significantly decreased the portion of the bronchial epithelium cells killed at the stage of acute infection.

[Determination of the diagnostic parameters of domestic immunoenzyme test systems versus foreign analogues in the detection of serum rubella virus antibodies].

Russian enzyme immunoassay (EIA) test systems (EATS) and foreign analogues (DSL and BCM-Diagnostics, USA) were compared in the detection of human serum rubella virus (RV) antibodies. Analysis of RV IgM levels ascertained a greater agreement of quality indices for the EATS "EKOlab-Krasnukha-IgM" than for the system "VectoRubella-IgM". As compared with the USA reference systems, the sensitivity, specificity, and total agreement of the data obtained by the EATS "EKOlab" were 100, 97.5, and 97.7%, respectively; those were 88, 84.4%, and 85.4% for "VectoRubella-IgM". In healthy individuals, strictly seropositive cases of IgM detection, revealed by the EATS "VectoRubella-IgM" and unconfirmed by the results obtained by the EATS "BCM-Diagnostics-IgM" (8%) are most likely to be regarded as false-positive due to the presence of serum rheumatoid factor (RF). The principle of indirect EIA used in the system "VectoRubella-IgM" makes it impossible to rule out the impact of RF on test results. The EATS "EKOlab-Krasnukha-IgM" and the systems made in the USA apply the principle of EIA with IgM involvement that, unlike indirect EIA, minimize to have nonspecific results. All three analyzed Russian EATS "EKOlab-Krasnukha-IgG", "Krasnukha-screening", and "VectoRubella-IgG" in the detection of RV IgG show rather high diagnostic parameters as compared with the systems made in the USA. The important additional advantage of the EATS "EKOlab-Krasnukha-IgG" over other Russian systems is that the kit has reference serum with the known content of RV IgG. This allows one to give results in absolute units and to standardize the calculations of some independent experiments. The performed study suggests that out of the Russian test systems, EATS "EKOlab-Krasnukha-IgM" and "EKOlab-Krasnukha-IgG" should be preferred.

[The age-related specificity of site-oriented immunity in respiratory-syncytial virus infection]. / Vozrastnye osobennosti saitnapravlennogo immuniteta pri respiratorno-sintsitial'noi virusnoi infektsii.

ELISA test systems were designed on the basis of synthetic peptides (SP) simulating the primary structure of functionally significant epitopes of the respiratory and cyncyntial virus (RCV) F-protein for the purpose of investigating the structure and age-related peculiarities of humoral immunity in respect to separate epitopes of RCV F-protein. One of them (221-232) simulates a part of RCV "virus-neutralizing domain" and another one (479-491) is highly important for the fusion mechanisms. New SP-based ELISA were used to examine pair sera in 159 patients with documented RCV infection including children, aged up to 3 years and 3 to 15, and adults. The activity of anti-RCV antibodies to SP was found to be significantly lower in children aged up to 3 years versus the older children and especially versus the adults. The virus neutralizing and, to a greater extent, fusion-inhibiting activities of antibodies were increasing with age, which collated with the results of detecting the antibodies to SP by immune-enzyme assay. The results testify to synchronism of formation of antibodies to different epitopes of the RCV F-protein. The shaping-up of antibodies with the above SP could denote the protective properties of humoral immunity, which justifies the use of the SP-based ELISA in its analysis, especially, in babies as well as in different-type immunodeficiency and immunopathology conditions.

[Development of immunoenzyme assay for subtype-specific detection of antibodies to influenza viruses A (H1N1) and A (H3N2)]. / Razrabotka immunofermentnykh test-sistem dlia podtipospetsificheskoi detektsii antitel k virusam grippa A (H1N1) i A (H3N2).

Conditions were developed for obtaining surface viral glycoprotein (GP) fraction intended for solid phase sensitization with the aim of constructing enzyme immunoassay test systems (EIATS) for detection of subtypical IgG and IgG to influenza A (H1N1) and A (H3N2) viruses. New variants of test systems were compared with the traditional methods for serological diagnosis of influenza. GP-based EIATS more often diagnosed influenza than EIATS based on purified whole-virion (WV) suspensions, hemagglutination inhibition and neutralization tests. Evaluation of conversions of subtypical IgG showed that the results coincided with the findings of neutralization and hemagglutination inhibition tests in 83-90% (EIATS-GP) and 50% (EIATS-WV). Cross-detection of antibodies to both virus subtypes in EIATS-GP and EIATS-WV was observed in 4 and 31% cases, respectively.

[Development of new approaches to standartization of enzyme immunoassay test systems for detection of antibodies to respiratory syncytial virus using electron microscopy and monoclonal antibodies]. / Razrabotka novykh podkhodov k standartizatsii immunofermentnykh test-sistem dlia detektsii antitel k respiratorno-sintsitial'nomu virusu s ispol'zovaniem élektronnoi mikroskopii i monoklonal'nykh antitel.

Respiratory syncytial virus (RSV), strain Long, was purified through 20-60% sucrose gradient. The virions from different sucrose density zones were tested by ELISA for reactivity with monoclonal antibodies (MAB) to F- (MAB 9C5) and N- (MAB 8B10) proteins of RSV. Comparative study of the same patterns of RSV by electron microscopy after negative staining showed a close relationship between the virion morphology and MAB binding in ELISA. MAB 9C5 were highly reactive with the surface domains of both mature RSV virions and "empty" virion envelopes without formed inner nucleocapsid structures. MAB 8B10 reacted well only with mature virions with completely assembled nucleocapsids. These MAB failed to reorganize the N-protein epitope of immature and destroyed virions, which indicated a conformation dependence of the 8B10 binding site. For practical purposes, MAB tests can be used to determine the RSV patterns, which can be used in ELISA for serologic diagnosis of RSV infection. Testing with these MAB demonstrate the stability of RSV to extreme exposures (lyophilization, storage, heating), which is important for creation of sensitive ELISA test systems and their standardization.

[Anti-RS viral IgE in respiratory syncytial viral infection in adult patients with a complicated course of bronchitis]. / Anti-RS-virusnyi IgE pri respiratorno-sintsitial'noi virusnoi infektsii u vzroslykh patsientov s oslozhnennym techeniem bronkhita.

In adult patients with different variants of the complicated course of bronchitis (prolonged, relapsing, chronic) respiratory syncytial (RS) virus infections were shown to occur very frequently (37-72%, depending on the form on bronchitis). In 220 cases of RS infection the content of anti-RS-virus IgE in the blood was analyzed. The reaction of specific IgE was more pronounced in RS infection observed in patients with acute of bronchitis, than in those with chronic forms. The occurrence of RS-virus infections and reinfections was shown to be directly related to the presence of the broncho-obstructive syndrome in patients. The direct correlation between high levels of serum anti-RS-virus IgE and the presence of bronchospasms in patients, revealed in this study, was indicative of the possible pathogenetic role of this immunoglobulin: the Pearson association factor (rA) was 0.32, p < 0.01. Depending on the specific features of the clinical course of bronchitis, differences in the dynamics and speed on immune response, in the duration of the preservation of anti-RS-virus IgE in the blood, as well as in the character of the reaction of this immunoglobulin to single and multiple RS-virus lesion, were observed.

[Characteristics of anti-respiratory syncytial humoral immunity during persistence of respiratory syncytial virus antigens in adult patients with chronic obstructive bronchitis]. / Osobennosti anti-respiratorno-sintsitial'nogo gumoral'nogo immuniteta pri persistentsii antigenov respiratorno-sintsitial'nogo virusa u vzroslykh bol'nykh khronicheskim obstruktivnym bronkhitom.

Specific humoral immune response has been studied in 8 patients with chronic obstructive bronchitis, in whom the persistence of RSV was detected, and in a group of patients without RSV persistence. Humoral immunity in patients with RSV persistence vs. those with acute and chronic bronchitis without viral persistence was characterized by the following features: a) earlier and more active production of IgG and, even more so, IgM and IgA; b) long persistence of high titers of anti-RSV IgG, IgM, and IgA-up to 17, 28, and 30 weeks, respectively; c) low anti-RSV neutralizing activity of serum antibodies.

[The use of halotherapy for the rehabilitation of patients with acute bronchitis and a protracted and recurrent course]. / Primenenie galoterapii dlia reabilitatsii bol'nykh ostrym bronkhitom s zatiazhnym i retsidiviruiushchim techeniem.

Halotherapy was used for rehabilitation in 25 patients with acute bronchitis of long-standing and recurrent types. The main therapeutic action was ensured by aerodispersed medium saturated with dry highly dispersed sodium chloride aerosol, the required mass concentration being maintained in the range of 1 to 5 mg/m3. Therapy efficacy was controlled through assessment of clinical, functional, immunological and microbiological findings. Metabolic activity values were taken into consideration as well. Positive dynamics of the function indices in the clinical picture resulted from elimination of pathogenic agents, control of slowly running inflammatory lesions and stimulation of some immune system factors. Favourable changes in metabolic activity were present: normalization of serotonin excretion, marked decrease of unbalance in lipid peroxidation-antioxidant system.

[The characteristics of the humoral response to respiratory syncytial viral infection in adult patients with different forms of bronchitis]. / Osobennosti gumoral'nogo otveta na respiratorno-sintsitial'nuiu virusnuiu infektsiiu u vzroslykh patsientov s razlichnymi formami bronkhita.

The levels of specific IgG and IgM were determined in 72 patients with verified respiratory syncytial (RS) virus infection: 11 patients with acute delayed bronchitis (ADB), 26 with recurring bronchitis (RB) 35 with chronic obstructive bronchitis (ChOB), by indirect enzyme immunoassay; RS virus was detected by direct immunofluorescent technique. The activity of the inflammatory process in the bronchi was shown to depend upon RS infection activity in cases of ChOB and RB. RS infection was observed to be significantly dependent on obstruction and bronchospasms. The specific humoral response was reduced and delayed in ChOB and RB as compared to acute bronchitis. The possibility of long-term persistence of RS virus antigen in chronic and recurring forms of bronchitis was demonstrated.

[Indices of the humoral response to respiratory syncytial virus infection in children with acute bronchitis]. / Pokazateli gumoral'nogo otveta na respiratorno-sintsitial'nuiu virusnuiu infektsiiu u detei s ostrymi bronkhitami.

Examinations of 30 children with acute bronchitis have revealed a respiratory syncytial (RS) viral infection in 18. A reduced response to RS virus was seen in patients with bronchitis complicated by obstruction, in contrast to that in cases without obstruction. Humoral response to this virus was reduced and delayed in younger children as against that in elder ones. Even high levels of specific IgG did not result in vitro neutralization of RS virus.