[Line immunoassay for detection of IgG antibodies to hepatitis E virus (Hepeviridae, Orthohepevirus, Orthohepevirus A)].

INTRODUCTION: The diagnostic efficacy of methods for hepatitis E serodiagnostic varies over a wide range; therefore, the combined use of tests of various formats is recommended. The aim of the research was to develop a test system for the detection of IgG antibodies to hepatitis E virus (HEV) in human serum by linear immunoassay (LIA). MATERIAL AND METHODS: Serum samples from patients with hepatitis and healthy individuals were tested using commercial enzyme-linked immunosorbent assay systems for the presence of IgG antibodies to viral agents causing hepatitis and other infections associated with liver pathology. Recombinant antigens ORF2 and ORF3 of HEV genotypes 1 and 3 were used. The "RecomLine HEV IgG/IgM" reagent kit (Mikrogen GmbH, Germany) was used as a comparison test system. RESULTS: The first Russian diagnostic kit "Blot-HEV", designed to detect IgG antibodies to individual HEV proteins in human serum using LIA, was developed. The antigenic base is represented by strips of a nitrocellulose membrane with immobilized recombinant antigens ORF2 (aa 406-660) and ORF3 (aa 1-113) of HEV genotypes 1 and 3, and control antigens in the form of discrete lines. The conjugate was mouse monoclonal antibodies to human class G immunoglobulins labeled with horseradish peroxidase. The chromogen solution contained the 3,3',5,5'-tetramethylbenzidine. A visual and digital recording of results was provided. The analytical sensitivity of the test kit was 0.625 IU/ml for ORF2 antigens and 2.5 IU/ml for ORF3 antigens. The absence of the influence of endogenous interfering substances on the results of the analysis and the absence of cross-reactions with antibodies to hepatitis pathogens of the other etiologies had been shown. The sensitivity of the test system compared to the "RecomLine HEV IgG/IgM" kit was 92%, specificity 97%. Shelf life in condition of storage was determined to be 12 months. CONCLUSIONS: The developed test can be used to confirm the results of ELISA in laboratory diagnosis of hepatitis E.

[Seroprevalence of hepatitis E virus (Hepeviridae: Orthohepevirus: Orthohepevirus A) among pregnant women in the highly endemic region of Kyrgyzstan].

INTRODUCTION: Hepatitis E (HE) is an important public health problem worldwide and is especially significant for pregnant women, among whom the associated mortality rate reaches 25%. The distribution of HE serological markers in this cohort in the endemic regions of Central Asia is poorly understood. The aim of the study was to assess the seroprevalence of HEV among pregnant women in the region of Kyrgyzstan where an increased incidence of HE is reported. MATERIAL AND METHODS: Blood serum of pregnant women, obtained from medical institutions in Bishkek, city of Osh and Osh region in the period from September 2016 to October 2019, and the control group of clinically healthy women were tested using the test systems "DS-IFA-ANTI-HEV-G" and "DS-IFA-ANTI-HEV-M" (NPO "Diagnostic Systems", Russia). RESULTS: IgG antibodies to HEV were detected in 87 (5.9%) of 1472 examined pregnant women, IgM antibodies -in 64 (4.8%) of 1378, while 5 (0.34%) samples were simultaneously positive for IgG and IgM antibodies. The rates of detection of antibodies to HEV in women of three age categories from 17 to 36 years old in the studied and control groups were similar. The analysis of the seasonal dynamics of HEV seroprevalence in pregnant women in the period from February to September 2019 showed a tendency towards an increase in the values of the positivity coefficient of specific IgM antibodies by the beginning of the autumn. Antibodies to HEV were detected with highest frequency in women from Osh region. CONCLUSIONS: A high risk of HEV infection for pregnant women in the surveyed region had been shown.

[The estimation of the hepatitis E proportion in the etiological structure of acute viral hepatitis in certain regions of of Kyrgyzstan.]

Despite the fact that the Kyrgyz Republic (KR) belongs to the highly endemic regions of the world for hepatitis E, the true extent of the spread of this infection in the country remains poorly understood. It was estimated the prevalence of serological markers of hepatitis E virus (HEV) infection among patients with acute viral hepatitis (AVH) from the regions of the Kyrgyz Republic with a high level of seroprevalence previously established by us. Blood sera samples of hepatitis patients who were admitted to hospitals of Kyrgyzstan in the period 2018-2019 were examined by the enzyme immunoassay method using the kits «DS-ELISA-Anti-HEVIgG¼ and «DS-ELISA-ANTI-HEV-IgM¼ (RPC Diagnostic Systems, Russia). IgG and IgM antibodies to HEV were detected in 103 of 344 studied samples (29.9%). Most often, seropositive specimens were detected among people of age groups under 20 and over 40 years old. Hepatitis with the fecal-oral mode of transmission was dominated in the structure of AVH: the specific gravity of hepatitis E was 47.9%, hepatitis A - 35.32%. Markers of mixed infections with other hepatitis viruses have been detected in 40.4% IgM-positive individuals. Thus, high prevalence of serological markers of HEV infection in the territory of Kyrgyzstan during the interepidemic period had been shown. The necessity of including the determination of serological markers of hepatitis E into the algorithm for the comprehensive diagnosis of AVH in patients of all age groups with liver pathology had been confirmed.

[The analysis of sero-prevalence of virus of hepatitis E among patients with HIV-infection and syphilis.]

The infection with hepatitis E virus is one of causes of liver diseases in patients with secondary immunodeficiency, including HIVinfected ones. The study was carried out concerning analysis of rate of detection of serological markers of infection with hepatitis E virus in samples of blood serums of HIV-infected patients and other group of patients with expressed immuno-suppression - patients with syphilis. The sero-prevalence of hepatitis E virus on IgM-antibodies among HIV-infected patients in Moscow made up to 21.7% and 2.9% correspondingly. In the examined group from the Far-East region the highest sero-prevalence of hepatitis E virus on specific IgE-antibodies (73%) is established in the group of patients infected with HIV subtype B. The most frequently IgG and IgM antibodies to hepatitis E virus were detected in HIV-infected patients at the stage of disease 4B. The examined group of patients with syphilis the sero-prevalence of hepatitis E virus made up to 14.7% that significantly surpasses the given indicator in the group of healthy persons (1.7%). The increasing of the level of sero-postivity is demonstrated in the groups of patients with latent syphilis and on the second stage of disease. Therefore, the highest values of sero-prevalence of hepatitis E virus is observed in the groups of HIV-infected patients and patients with syphilis at late stages of disease. In the Russian Federation, the identification of antibodies to hepatitis E virus in HIV-infected patients depends of the region of residence.

[Model of chronic salmonellosis: parameters of infection and immune response in inbred mice genetically variable in susceptibility to salmonellosis].

AIM: Study parameters of chronic infection and immune response in I/St and A/Sn line mice in the model of per oral infection of mice with Salmonella enterica serovar Typhimurium. MATERIALS AND METHODS: Studies were carried out in I/StSnEgYCit (I/St), A/JsnYCit (A/Sn) inbred line mice as well as their back crossing hybrids [I/StrxF1(I/StxA/Sn)]BC. Mice were infected per os by S. enterica serovar Typhimurium strain IE147 at a dose of 2 x 10(5) PFU per mice. The number of salmonellae was determined at days 3, 5 and 7, weeks 3 and 4 after the infection in various organs, the number of antibody producers--by cell EIA. Pathomorphologic changes in mice spleens were studied histologically by using hematoxylin and eosin staining. In offspring of back crossing [I/St x F1(I/St x A/Sn)]BCl segregation genetic analysis of sensitivity to salmonella infection trait and mapping of loci taking part in salmonella infection were carried out. RESULTS: The course of chronic salmonellosis in susceptible I/St line was characterized by the presence of more pronounced pathomorphologic changes in spleen and significantly higher microbial load in organs (approximately by 1000 times) when compared with A/Sn mice. Interlinear differences in susceptibility to infection correlated with differences in the type of early local and systemic immune response. In I/St mice a higher level of salmonella specific IgG2a-, IgG1- and IgA forming cells in spleen compared with A/Sn mice was detected which correlates with a pronounced splenomegaly and high concentration of salmonellae. On the contrary A/Sn mice demonstrated a higher level of salmonella specific IgA forming cells in Peyer patches that probably leads to protection of A/Sn line during per oral infection. Genetic analysis of susceptibility to salmonellosis trait inheritance showed the presence of its coupling with D9Mit89 locus of chromosome 9 on which previously Tbs2 locus was mapped that plays a role in the control of tuberculosis infection. CONCLUSION: There is a probability of the presence of general mechanisms of genetic control of tuberculosis and salmonella infections in A/Sn and I/St mice.

[The role of the type-three secretion system of the gram-negative bacteria in regulation of chronic infections].

The role of the type-three secretion system of the gram-negative bacteria in regulation of chronic infections is discussed. Recent research showed that most of severe chronic somatic diseases are derived from chronic infection induced in the first place by infectious agents. The role of the T3SS of different species in transition from an acute infection to persistence is reviewed. Clinical and bacteriological research showed that microorganisms are persistent in the form resistant to antibiotics. That is why one of the promising targets for the development of antibacterial new-generation treatment is T3SS that conducts transport of bacteria pathogenicity factors into eukaryotic cell. The presence of this structure is necessary for the development of an acute infectious process and chronization of an infection is essential for its functioning.

[Methods of identification and assessment of safety of genetically modified microorganisms in manufacture food production].

Methods of identification of genetically modified microorganisms (GMM), used in manufacture food on control probes are presented. Results of microbiological and molecular and genetic analyses of food products and their components important in microbiological and genetic expert examination of GMM in foods are considered. Examination of biosafety of GMM are indicated.

[Development of pulmonary chlamydia infection in inbred mice lines differentiated by genetically determinated sensitivity to tuberculosis infection].

Mice of I/St strain develop severe lung inflammation and die shortly following infection with virulent mycobacteria. The susceptibility does not depend on the Nramp1 gene, as I/St mice carry its resistant allele, but is controlled by little interacting QTL mapped to chromosomes 3, 9, 17. To find out whether the tuberculosis-susceptible I/St mice are susceptible to other intracellular bacteria taxonomically distant pathogen of Chlamydia pneumoniae was studied. Comparison of I/St and TB-resistant A/Sn mice (both Nramp1r) demonstrated that the former were more susceptible to chlamydia, displaying a significantly shortened survival time following challenge (I/St, 9.2 +/- 1.2 days; A/Sn, 22.0 +/- 0 days (p < 0.001)). To estimate the degree of chlamydial multiplication in the lungs, we suggested a quantitative real-time polymerase chain reaction (PCR)-based method which allows enumeration of the parasite's genome equivalents in infected tissue from 1 to 16 days after challenge. The interstrain difference of chlamydia burden in lungs was observed only after 24 hours after infection. Multiplication of chlamydia in the lungs was controlled efficiently after day 4 of infection. The numbers of genome equivalents dropped slightly by day 8 both in I/St and A/Sn mice. Lung pathology develops more rapidly in I/St compared to A/Sn mice following infection with chlamydia despite their similar ability to control bacterial multiplication. Lung tissue of susceptible I/St mice was markedly infiltrated with macrophages (p < 0.01), which differed significantly from the lungs of resistant A/Sn mice. In agreement with higher macrophage content in the lungs, significantly more macrophage-derived proinflammatory cytokines TNF-? and IL-6 were detected in lung tissue homogenates obtained from I/St mice (p < 0.05). Because the prominent difference in survival time did not correlate with permanent difference in bacterial multiplication, we suggested that both infections trigger fatal pathological processes whose dynamics depends strongly upon the host genetics.

[Persistence of Burkholderia cenocepacia bacteria in vivo in dependence of their ability to form biofilms].

AIM: To select the most susceptible line of mice which allows to conduct comparative studies of infectious process caused by different strains of B. cepacia in order to explore correlation between ability to form biofilms and persistence of bacteria in organs of infected animals. MATERIALS AND METHODS: Strain B. cenocepacia 370, which is a clinical isolate, and its mutants with modified ability to form biofilms were used. Conditional microbiologic methods and biological models of intraperitoneal and intranasal inoculation of mice belonging to 4 lines: BALB/c, BLACK, I/St, and A/Sn derived in Central Institute of Tuberculosis were employed. Criteria of persistence was duration of isolation of different strains of bacteria from lungs and spleen of inoculated animals as well as number of CFU. RESULTS: The most susceptible line of mice which enables to conduct comparative studies of infectious process caused by Burkholderia species was determined. It was shown that even after intraperitoneal inoculation the agent was better preserved in lungs than in spleen that corresponds to natural localization of this infection. At any time of observation the number of cells of mutant strain, which is a superproducer of biofilms, isolated from organs of inoculated mice was 2 - 10 times higher than number of isolated cells of mutant, which do not produce biofilms. CONCLUSION: Correlation of more prolonged persistence of B. cenocepacia in organs of inoculated animals in vivo with ability of the agent to form biofilms determined in vitro is experimentally established. The susceptible line of mice which allows to conduct comparative studies of dynamics of infectious process caused by various strains of Burkholderia species was revealed. It was shown that irrespective from method of inoculation B. cepacia are able to continuously persist in organism of susceptible animals with lungs as a predominant localization.

[Requirements to a medical and biologic assessment and the hygienic control of the food production received from recombinant-DNA microorganisms].

In work the characteristic of the created in the Russian Federation system of an estimation of safety of the foodstuff received from/or with use of genetically modified microorganisms (GMM) is given, at their admission to realization and the hygienic control of given production over a revolution. It is shown, that strategy of a safety at a stage of registration GMM, the established order and accepted control measures of the foodstuff received from/or with use GMM, in Russia their large-scale commercial use, and the normative-legal and methodical base based on the federal legislation on state regulation in the field of genetically engineering activity, about quality and effectively outstrip safety of foodstuff about protection of the rights of consumers, is harmonized with approaches of the international organizations.

Mycobacterium tuberculosis-susceptible I/St mice develop severe disease following infection with taxonomically distant bacteria, Salmonella enterica and Chlamydia pneumoniae.

Mice of I/St strain develop severe lung inflammation and die shortly following infection with virulent mycobacteria. To find out whether tuberculosis (TB)-susceptible I/St mice are susceptible to other intracellular bacteria, we investigated two different taxonomically distant pathogens, Chlamydia pneumoniae and Salmonella enterica serovar Typhimurium. Comparison of I/St and TB-resistant A/Sn mice (both Nramp1(r)) demonstrated that the former are more susceptible to both salmonella and chlamydia, displaying a significantly shortened survival time following challenge. Lung pathology develops more rapidly in I/St compared to A/Sn mice following infection with chlamydia, despite their similar ability to control bacterial multiplication. Following infection with salmonella, substantial ( approximately 3 log) but very short (second day post-infection) interstrain differences in bacterial loads were observed, accompanied by higher levels of interleukin (IL)-6 and tumour necrosis factor (TNF)-alpha in the peritoneal cavities of I/St mice. I/St macrophages were more permissive for salmonella growth during the first 24 h following infection in vitro. Because the prominent differences in survival time did not correlate with permanent differences in bacterial multiplication, we suggest that both infections trigger fatal pathological processes whose dynamics depend strongly upon the host genetics.

[Prevalence of antibodies to hepatitis E virus among residents of different climate and geographic zones of Russia Federation]. / Chastota vstrechaemosti antitel k virusu gepatita E u naseleniia razlichnykh klimatogeograficheskikh zon Rossiiskoi Federatsii.

The detection frequency of antibodies to Hepatitis E Virus (HEV) was studied in residents of the South and of the Middle European Part of the Russia Federation as well as of Siberia. Antibodies to HEV were most often found both in patients with hepatic pathologies and in subjects with diseases unrelated with a primary hepatic lesion, in particular, in patients with skin and venereal diseases and with HEV. A higher concentration of antibodies to HEV was noted also in blood donors, medical personnel and isolated communities, like prisons or psychiatric clinics. A correlation was established between the rate antibodies to HEV are registered and such risk factor as contacting with blood or a gross violation of the hygienic rules.

[Propagation of hepatitis E virus in an endemic and nonendemic regions]. / Osobennosti rasprostraneniia virusa gepatita E v éndemichnom i neéndemichnom regionakh.

The role of hepatitis E (HE) in sporadic morbidity at an endemic territory (Southern Uzbekistan) and the incidence of anti-HEV in different populations of a nonendemic region (Russia) were evaluated. Antibodies to HEV were detected in 22.1% of patients with acute HE, including mixed infections (+ HA or HB), in the Dekhkanabad district of Kashkadarya region in 1993. The estimated incidence of acute HE was 51.7 per 100,000 population. Analysis of monthly incidence of acute HE demonstrated a seasonal pattern of the morbidity: more than 80% of total recorded cases occurred in August-September. These data indicate the presence of group HEV infections and an important role of this infection in the structure of acute viral hepatitis at the endemic territory. Anti-HEV were found in some population groups at a nonendemic territory: in free-of-charge blood donors in Surgut (4%), in patients with HIV infection (1.6%), and in medical workers in Moscow (1.1%).

[Comparative evaluation of the efficacy of Borrelia indication in ixodes ticks (Ixodidae) using dark field microscopy and polymerase chain reaction (PCR)]. / Sravnitel'naia otsenka rezul'tativnosti indikatsii borrelii v iksodovykh kleshchakh (Ixodidae) metodami temnopol'noi mikroskopii i polimeraznoi tsepnoi reaktsii (PTsR).

Indication of Borrelia (B. burgdorferi sensu lato) in 205 adult unfed I. persulcatus ticks from a natural focus was carried out simultaneously by methods of PCR and dark-field microscopy of vital preparations. PCR method revealed Borrelia prevalence in considerable number of ticks, in which Borrelia were not found by microscopy of 250 microscopic fields in a preparation from each individual tick. At the same time, PCR method didn't give positive results for approximately 8% of ticks, which contained rather high concentration of Borrelia (more than 10 per 100 microscopic fields). In general, PCR method doesn't have advantages in comparison with a microscopy of vital preparations for study the Borrelia prevalence in ticks.

[Laboratory diagnosis of toxoplasmosis using polymerase chain reaction]. / Razrabotka laboratornoi diagnostiki toksoplazmoza s momoshch'iu polimeraznoi tsepnoi reaktsii.

Polymorphism of clinical manifestations in Toxoplasma infection and variegated disease patterns virtually rule out the diagnosis based solely on clinical symptoms, which makes modern laboratory tests particularly important. Amplification test system based on the polymerase chain reaction has been developed for the diagnosis of Toxoplasma infection. Computer analysis of nucleotide sequence of Toxoplasma gondii surface antigen gene P30 was analyzed, which helped choose and synthesize specific oligonucleotide primers. A method for biological material processing was selected, allowing sufficient DNA output. Optimal conditions for amplification reaction, ensuring absolute specificity and high (10-100 cells/sample) sensitivity, were determined.

[Antigenic specificity of recombinant polypeptides, containing fragments of hepatitis E virus proteins]. / Antigennaia spetsifichnost' rekombinantnykh polipeptidov, soderzhashchikh fragmenty belkov virusa gepatita E.

Antigenic specificity of recombinant polypeptides HE40 and HE60 containing fragments of gene ORF2 and ORF3 protein products of hepatitis E, strain Burma, produced in E. coli cells, is analyzed. Blood sera from patients with acute hepatitis from an endemic region in Uzbekistan were tested for IgG to recombinant antigens by solid-phase enzyme immunoassay with a protein fragment coded by PRF3 gene, a synthetic peptide previously characterized in a commercial test system, as the positive control. 93% sera reacting with recombinant polypeptide HE60 and 32% reacting with HE40 reacted with the synthetic peptide. No antibodies to the studied polypeptides were detected in the sera of Moscow patients with hepatitis A, B, or C confirmed by laboratory findings. Antigenic specificity of recombinant polypeptide HE60 was confirmed by competitive enzyme immunoassay with the same peptide as the competitive antigen. Test system based on recombinant polypeptides HE40 and HE60 was used for deciphering the etiological structure of acute viral hepatitis which occurred in a hepatitis E endemic region of Uzbekistan in 1993.

[Preparation of recombinant polypeptides containing antigenic determinants of hepatitis E virus]. / Poluchenie rekombinantnykh polipeptidov, soderzhashchikh antigennye determinanty virusa gepatita E.

DNA fragments complementary to hepatitis E Burma strain ORF2 and ORF3 obtained by oligonucleotide synthesis were cloned in expressing bacterial system. Recombinant polypeptides isolated from E. coli producer strains, immobilized on solid phase (polystyrene plates and nitrocellulose membranes), are studied in enzyme immunoassay to detect their ability to react with sera of patients with acute viral hepatitis from an Uzbekistan region endemic for hepatitis E. Two polypeptides reacting with the greatest number of sera, containing hepatitis E virus ORF2 and ORF3 gene fragments, were selected for further study of antigenic specificity.

[Genomic polymorphism in Mycobacterium tuberculosis strains]. / Issledovanie genomnogo polimorfizma shtammov Mycobacterium tuberculosis.

Different genomic fingerprinting techniques (universal probes, such as rRNA genes, phage M13 DNA, IS 6110 probe) have been used to investigate the genomic polymorphism of Mycobacterium tuberculosis strains isolated in different geographical regions of Russia and in some CIS countries. As shown with the use of these techniques and a specially developed PCR-mediated system for genetic typing, M.tuberculosis strains are genotypically heterogeneous in regions with a sporadic level of tuberculosis morbidity and genotypically homogeneous in regions with elevated morbidity and mortality levels. The evaluation of the effectiveness of the genetic typing of M.tuberculosis with the use of different genomic fingerprinting techniques has made it possible to propose the optimum 3-stage scheme for the differentiation of M.tuberculosis strains: (1) the typing of all isolated strains of the PCR-mediated test system; (2) the typing of several selected M.tuberculosis strains with the use of 1S 6110 probe (2-3 strains of each detected PCR-RFLP [correction of PLRF] genotypes); (3) the typing of M.tuberculosis strains, containing 1 copy of 1S 6110 or not containing such sequence, with the use of probes (phage M13 DNA) detecting hypervariable sequences in M.tuberculosis genomes.

[The detection of mycobacteria in children and adolescents by using the polymerase chain reaction]. / Vyiavlenie mikobakterii s pomoshch'iu polimerazno-tsepnoi reaktsii u detei i podrostkov.

A polymerase chain reaction (PCR) was used for determination of mycobacterial DNA in clinical samples from children and adolescents. The results were positive in 23 and 53% of cases, respectively, while standard microbiological methods failed to show presence of any bacteria in the samples. Case histories contained information on high incidence of positive Mantoux test. Microbiologically the diagnosis was confirmed only in 7 adolescents. In children mycobacteria were not found.

[Identification of tuberculosis pathogens in clinical material using the polymerase chain reaction]. / Identifikatsiia vozbuditelia tuberkuleza v klinicheskom materiale s pomoshch'iu metoda polimeraznoi tsepnoi reaktsii.

The authors examined the possibility of detecting M. tuberculosis cells in various types of diagnostic material (sputum, blood, bone marrow, bronchoalveolar lavage fluid) from tuberculosis patients using polymerase chain reaction (PCR). The developed PCR-based test systems helped detect M. tuberculosis in 48 (90.6%) out of 53 tuberculosis patients, in contrast to much slower microbiological methods which permitted detection of Mycobacteria in only 21 (39.6%) patients. High specificity and virtually no false-positive results of PCR were demonstrated in testing diagnostic material from patients with chronic nonspecific pulmonary diseases and from children with lympholeukemia and anemia.