Method for hepatitis B virus DNA detecting in biological material at low viral load based on nested PCR with detection on three viral targets in real-time mode.

A method has been developed for HBV DNA finding in biological material at low viral load based on nested PCR with real-time detection of three viral targets. When developing the method, blood plasma samples were used from 128 CHB patients living in the regions of the Russian Federation and countries of Central Asia and 173 hemodialysis center patients living in the North-West Federal District. Analytical sensitivity was tested using the stepwise dilution method. HBV was detected by nested PCR. According to the method developed by us, at the first stage, the HBV DNA is amplified using at the first stage oligonucleotides complementary to the greatest similarity regions of the various HBV isolates genomes flanking the entire virus genome. At the second stage, when using the amplification product of the first stage as a template, PCR was performed using three pairs of oligonucleotides and the corresponding oligonucleotide fluorescently labeled probes to three virus genome regions (Core gene, S gene and X gene), as well as one pair of primers and the corresponding probe complementary to a human HPRT gene region. The method sensitivity for DNA extraction from plasma with a 100 µl volume was 10 IU/ml. Obtaining a threshold Ct cycle for only one fluorophore may indicate the presence of HBV DNA in the sample at a load of less than 10 IU/ml, HBV detection in this case is possible with a repeated PCR study of the corresponding sample with HBV DNA extraction from an increased plasma volume (200-1000 µl). The developed method makes it possible to identify the disease in various HBV subgenotypes and can be used to diagnose CHB in the population and risk groups, including those with the HBsAg-negative form of the disease.

[Genetic diversity of the human immunodeficiency virus (HIV-1) in the Kaliningrad region].

INTRODUCTION: As is currently known, the epidemic process in the Kaliningrad Region was mainly associated with the spread of the recombinant form of HIV-1 (CRF03_AB); however, regular HIV importations from other countries and continents has created favorable conditions for emergence and spread of various recombinant forms of the virus.The most complete information on the diversity of recombinant forms in the region is also necessary to understand the structure of drug resistance (DR). The aim of the study was to explore the HIV-1 genetic diversity in the Kaliningrad Region. MATERIALS AND METHODS: We studied 162 blood plasma samples obtained from patients from the Kaliningrad Region, both with confirmed virological failure of antiretroviral therapy (ART) and with newly diagnosed HIV infection. For reverse transcription and amplification of HIV genome fragments, diagnostic «AmpliSense HIVResist-Seq¼. RESULTS AND DISCUSSION: The various recombinants between subtypes A and B (74%) were predominant in study group: recombinant was between CRF03_AB and subtype A (33.95%) and CRF03_AB-like (13.58%) were the most common. Among the "pure" subtypes of the virus, subtype A6 (16.67%). The circulation of subtypes B (3.70%) and G (1.23%) was also noted.Ninety-six patients (59.26%) were identified with at least one mutation associated with antiretroviral (ARV) drug resistance. CONCLUSION: The observed diversity of subtypes and recombinant forms of the virus implies that the new recombinants are actively emerging in the studied region, both between existing recombinant forms and "pure" subtypes, as well as between "pure" subtypes.

[Prevalence of viral hepatitis B markers among blood donors in the Republic of Guinea].

INTRODUCTION: The problem of transfusion safety in relation to parenteral viral hepatitis still remains relevant. Viral hepatitis B (HB) remains the most common viral infection transmitted through transfusion procedures. One of the natural phases of chronic hepatitis B (CHB) is occult hepatitis B infection (OBI), characterized by an undetectable HBsAg (regardless of the other serological markers content) in the presence of hepatitis B virus (HBV) DNA in the liver tissue and an extremely low, up to undetectable, level of viral load in the blood. In the Republic of Guinea, as in most countries on the continent, the prevention of HBV transmission through transfusion is still based on HBsAg serological testing of donors only. In this connection, OBI remains as a potential threat to blood transfusion safety. Detection of HBV DNA is a reliable preventive measure against transmission of the virus from donors with HBsAg-negative HBV infection, especially in highly endemic regions. In this regard, the study was conducted to substantiate recommendations for improving blood safety against the background of significant HBV prevalence in the Republic of Guinea.The aim of the work was the evaluation of serological and molecular markers of HBV infection in blood donors in the Republic of Guinea. MATERIAL AND METHODS: We examined 250 blood samples obtained from donors living in Conakry, Republic of Guinea. Samples were tested for the presence of serological (surface antigen, HBsAg; antibodies (ABs) to surface (anti-HBs IgG) and core (anti-HBc IgG) antigens) and molecular (DNA) markers of HBV infection. RESULTS AND DISCUSSION: The overall detection rate of hepatitis B markers was 83.2%; HBsAg was detected in 16.4% of all individuals. The high incidence of HBsAg in men (19.55%) compared to women (8.45%) was shown, the relative risk of HBV infection with the formation of HBsAg-positive chronic hepatitis B in males was also significantly higher. The prevalence of the HBV DNA in the study group was 30.4%, the OBI cases accounted for 15.6%. The prevalence of this form of the disease was shown in donors aged 30-49 years (24.78%), in the group of people younger than 30 years, the incidence was lower (8.73%), and at the age of over 50 years, OBI was not detected. Based on the phylogenetic analysis of 76 virus isolates, it was shown that genotype E prevails in the examined group (85.53%).Cases of pathogen DNA detection occurred in HBsAg-negative blood donors in the presence of anti-HBs IgG (n = 4), as well as in the simultaneous presence of ABs anti-HBs IgG and anti-HBc IgG (n = 7). The viral load exceeded 200 IU/ml in OBI samples. Escape mutations were detected by sequencing in each OBI sample, contributing to the virus escaping from diagnostic based on screening for HBsAg. CONCLUSION: Assessment of the prevalence viral hepatitis B markers in blood donors, determination of genotypes and clinically significant mutations of virus variants are necessary to ensure safe medical manipulations, control and prevention of the spread of this infectious agent.

Algorithm for laboratory diagnostics of parvoviral infection in risk groups.

Parvovirus infection (PVI) is widespread, characterized by airborne, bloodborne and vertical transmission routes. Parvovirus B19 (PVB19) exhibits tropism to erythropoietic cells. According to the increased likelihood principle of PVB19 infection and the severity of the consequences, immunocompromised individuals, especially those with hematological manifestations of diseases, are in increased risk group. Based on the own research results and analysis of the published data, we have proposed specific algorithms for PVI laboratory testing in individual risk groups, taking into account the peculiarities of the development and infection manifestation in each group: in HIV-infected patients, in oncohematological patients with to whom allogeneic hematopoietic stem cell transplantation (allo-HSCT) have been prescribed (blood and bone marrow recipients), as well as in patients with chronic anemia of parasitic etiology. For each group, the main clinical or laboratory marker, treatment procedure, or patient physiological parameters have been determined, based on which it was recommended to test for PVI. For HIV-infected patients, the main criterion for PVI testing is persistent anemia. For oncohematological patients, the basis for PVI testing is allo-HSCT procedure, which is planned or performed for this particular patient. For malaria patients, the patient's age was considered as major criterion, since in malaria and PVI coinfected young children can lead to a fatal outcome. The proposed PVI diagnostics algorithms usein risk groups can help to predict the severe course of underlying disease associated with PVB19 infection, and timely correct the therapy used.

Peptidoglycan of Bacterial Cell Wall Affects Competitive Properties of Microorganisms.

We studied the effect of bacterial wall peptidoglycan of 7 bacterial species on the competitive properties of human-associated microorganisms. Addition of peptidoglycan to the culture medium did not change the growth characteristics of the test cultures; however, an increase in the antagonism and hydrophobicity of Bifidobacterium sp. and Enterococcus sp. was observed, while the effect on enterobacteria was predominantly indifferent or inhibitory. The effect did not depend much on the source of peptidoglycan and was equally manifested on both indigenous and probiotic strains. The observed new property of peptidoglycan indicates its participation in the formation and functioning of microbiota. The obtained data on the regulation of the properties of microorganisms provide new possibilities for the correction and maintenance of host homeostasis through host-associated microbiota.

Method for detecting hepatitis B virus in blood plasma at low viral load using real-time PCR.

A method for detecting HBV DNA in peripheral blood at low viral load using real-time PCR was developed and its significance in identifying HBsAg-negative viral hepatitis B was evaluated. When developing the method, blood plasma samples and liver tissue biopsy material were used from 128 patients living in St. Petersburg, in various regions of the Russian Federation, as well as in the Central Asia countries. We also used blood plasma samples from 96 pregnant women and 37 hemodialysis center patients living in Northwestern Federal District, 199 foreign citizens undergoing medical examination to obtain work permits at the Directorate for Migration in the Northwestern Federal District, 397 conditionally healthy people living in the Socialist Republic of Vietnam. HBV was detected by nested PCR. Analytical sensitivity was tested using the stepwise dilution method. According to the method developed by us, at the first stage, the HBV DNA is amplified using at the first stage oligonucleotides flanking the genome region 2932-3182 ... 1-1846 nt., and at the second stage two oligonucleotides pairs to the genome virus regions (gene S and gene X) and corresponding oligonucleotide fluorescently labeled probes complementary to the amplified fragments regions carrying fluorophores at the 5'-end, and non-fluorescent quenchers at the 3'-end. The channel corresponding to the FAM fluorophore detects the HBV DNA S-region amplification product, and the channel corresponding to the ROX fluorophore detects the HBV DNA X-region amplification product. The method sensitivity for DNA extraction from plasma with a 100 µl volume was 10 IU/ml. Obtaining a threshold cycle Ct for only one FAM or ROX fluorophore may indicate the HBV DNA presence in a sample at a load of less than 10 IU / ml, HBV detection in this case is possible with a repeated PCR study of the corresponding sample with HBV DNA extraction from an increased plasma volume (200-1000 µl). The developed method makes it possible to identify various HBV genovariants, both characteristic and rare in the Russian Federation, circulating in other world regions. The method can be used to detect HBV in risk groups, in the population, as well as in screening blood donors in order to ensure the blood transfusions safety.

Serological diagnosis and prevalence of HIV-1 infection in Russian metropolitan areas.

BACKGROUND: HIV infection is a major health problem in Russia. We aimed to assess HIV prevalence in different population groups and to compare the characteristics of 4th generation immunoassays from Abbott, Bio-Rad, Vector-Best, Diagnostic Systems, and Medical Biological Unit. METHODS: The study included 4452 individuals from the general population (GP), 391 subjects at high risk of HIV infection (HR) and 699 with potentially interfering conditions. HIV positivity was confirmed by immunoblot and by HIV RNA, seroconversion and virus diversity panels were also used. HIV avidity was employed to assess recent infections. RESULTS: The prevalence in GP was 0.40%, higher in males (0.62%) and in people aged < 40 years (0.58%). Patients attending dermo-venereal centers and drug users had a high prevalence (34.1 and 58.8%). Recent infections were diagnosed in 20% of GP and in 4.2% of HR. Assay sensitivity was 100% except for one false negative (99,54%, MBU). Specificity was 99.58-99.89% overall, but as low as 93.26% on HR (Vector-Best). Small differences on early seroconversion were recorded. Only the Abbott assay detected all samples on the viral diversity panel. CONCLUSION: HIV infection rate in the high-risk groups suggests that awareness and screening campaigns should be enhanced. Fourth generation assays are adequate but performance differences must be considered.

Optimization of the algorithm diagnosis chronic hepatitis B markers in patients with newly diagnosed HIV infection.

The possibility of modifying the algorithms for chronic viral hepatitis B laboratory diagnosis in individuals with newly diagnosed HIV infection is analyzed. Plasma samples were used from 196 patients residing in the Northwestern Federal District. Serological HBV markers were found in 79.6% of cases. However, HBsAg was detected in 5.6% of patients. Anti-HBcore IgG antibodies are found in 62.24% of cases, anti-HBe IgG antibodies in 27.55%, anti-HBs IgG antibodies in 52.55% of cases. Using a commercial kit with a 100 IU / ml sensitivity, HBV DNA was detected in 4.6% of patients, that is, 81.8% of HBsAg-positive individuals. Using the method developed by us, HBV DNA was found in 18.36% of HIV-infected individuals, including 12.75% of cases was HBsAg-negative (latent) disease form. In the examined group, HBV of genotype D prevailed (91.7%), genotype A was detected in 8.3% of cases. The distribution of subgenotypes is presented in the following ratios: D2 - 55.6%, D1 - 22.2%, D3 - 13.9%, A2 - 8.3%. Mutations were detected in the reverse transcriptase (RT) region in 91.6% of patients, in the SHB region in 83.3%, in the Core and Precore regions in 72.2% and in 27.7% of patients, respectively. Three HBV isolates (8.3%) were identified with drug resistance mutations to lamivudine, entericavir, telbivudine and tenofovir, which are amino acid substitutions in the HBV polymerase gene at positions L180M, T184A, M204V. Vaccine escape mutations were detected in 61.1% of patients. In all samples with drug resistance mutations, escape-mutants were simultaneously present. When analyzing the basal nucleus promoter, Precore and Core regions, 22.2% of patients with the double mutation A1762T / G1764A, 25% with the mutation G1896A were identified. In one person, all three substitutions were found. In the Core region, 77.7% of patients showed mutations in one of the hot spots (codons 87, 97, 112, and 130 substitution), which can play a role in immunomodulation in CHB. Analysis of the HBV genetic structure, mutations detection early in the virus in patients with HBV can help predict the clinical course and disease progression, and ART complications. To reduce the HIV HBV co-infection burden and to appointer anti-HBV therapy, it is necessary to introduce detection the occult HBV to modify the algorithm for CHB laboratory diagnosis.

Plasma cytokine profile in synucleinophaties with dementia.

Immune response may play a pivotal role in the pathogenesis of the common synucleinopathy as Parkinson's disease (PD) and could be mediated with the accumulation of neurotoxic alpha-synuclein. There is limited evidence for immune response in another synucleinopathy as dementia with Lewy bodies (DLB). Recent data suggest that immune response may contribute to cognitive impairment. We aimed to estimate plasma cytokine profile in patients with synucleinopathies with dementia (PD dementia (PDD), DLB). Plasma cytokine levels (interferon-gamma (IFN-gamma), interleukin (IL)-4 (IL-4), IL-6, IL-10, tumor necrosis factor alpha (TNF-alpha), monocyte chemoattractant protein-1 (MCP-1)). were estimated in 16 patients with DLB, 19 patients with PDD, 28 patients with PD without dementia (PD) and 19 individuals without neurological disorders (controls) using Luminex array system. Cognitive status was assessed with the Mini-Mental State Examination (MMSE). TNF-alpha and IL-6 plasma levels were elevated in patients with synucleinopathies with dementia (DLB, PDD) compared to controls and IL-10 plasma level was increased in PDD compared to controls (p < 0.05). IFN-gamma levels were decreased in PD and PDD patients compared to controls (p < 0.001, p = 0.026, respectively) and in PD patients than in DLB patients (p = 0.032). Patients with PD, PDD, and DLB were characterized by increased plasma levels of MCP-1 compared to controls (p < 0.001). At the same time, no differences in TNF-alpha, IL-10, IL-6 plasma levels in PD patients compared to controls were found. Our study demonstrated more pronounced immune response in synucleinopathies associated with dementia compared to PD without demetia.

[The prevalence clinically significant virus mutations among patients with chronic viral hepatitis B.]

The prevalence of clinically significant virus mutations in patients with chronic viral hepatitis B from the Kyrgyz Republic was analyzed. Blood plasma samples of 64 patients with verified chronic viral hepatitis B obtained from Kyrgyzstan indigenous people were used in the work. Asymmetric PCR was carried out with extended oligonucleotides and the first reaction amplification product was further used in a new PCR with one of the nested pairs overlapping primers that flanked the entire HBV genome together, followed by sequencing. Based on the phylogenetic analysis of 64 HBV isolates obtained from patients from the Kyrgyz Republic, it was shown that only the genotype D virus was present in the examined group, the HBV subgenotype D1 (68.75%) prevailed compared with the HBV subgenotype D2 (18.75%) and subgenotype D3 (12.5%). For all subgenotypes, several independent infection sources are obvious, subclusters that include isolates from Kyrgyzstan, Kazakhstan and Uzbekistan are distinguished, as well as subclusters that include isolates only from Kyrgyzstan, which are less similar to isolates previously deposited in the international database, which probably indicates an independent HBV homologous evolution in the region. Clinically significant mutations were identified in 26.5% of patients. Including 12.5% with escape mutations that prevent the virus detection and / or allow the virus to replicate despite the vaccine (122K, 128V, 133I, 134N). Another 12.5% of the isolates are characterized by mutations that are independently associated with the liver cirrhosis and hepatocellular carcinoma development, including 21, 24, 27 nucleotides deletions in the Pre-S2 region and the S11F mutation in the PreCore region. In one case, unusual 236S and 250P mutations were found in the positions described as drug resistance sites of the P region associated with the resistance development to adefovir, tenofovir, and entecavir. The hepatitis B virus genetic structure analysis, early virus mutations detection in patients with chronic hepatitis B virus can help to choose the right vaccination strategy, antiviral and immunosuppressive therapy, as well as predict the clinical course and disease progression.

Plasma Cytokines Profile in Patients with Parkinson's Disease Associated with Mutations in GBA Gene.

Plasma cytokine concentration in patients with Parkinson's disease and mutation in GBA gene, in patients with sporadic Parkinson's disease, and in healthy volunteers were measured by ELISA and multiplex analysis. In patients with Parkinson's disease and mutation in GBA gene, elevated plasma concentrations of IL-1ß and TNFα were revealed by ELISA in comparison with both controls and patients with sporadic form of Parkinson's disease. Multiplex analysis revealed enhanced secretion of IL-1ß, IL-2, IFNγ and reduced plasma levels of monocyte chemoattractant protein-1 (MCP-1) in patients with Parkinson's disease and mutation in GBA gene (in comparison with other groups) and increased plasma levels of IL-13 (only in comparison with the healthy volunteers). Our results support the hypothesis that the concentrations of inflammatory mediators are increased in patients with Parkinson's disease and mutation in GBA gene.

[Hepatitis B virus identification in a blood plasma at a low viral load.]

To analyze the method for detecting HBV DNA in peripheral blood at low viral load and evaluate its significance in identifying HBsAg-negative viral hepatitis B. In this work, samples of blood and liver tissue biopsy material were used from 128 patients living in the Russian Federation and the Republic of Uzbekistan without CHB and with CHB confirmed detection of circle covalently closed HBV DNA in hepatocytes. Plasma viral load was measured using the «AmpliSens® HBV-Monitor-FL¼ kit. HBV at low viral load was detected by nested PCR. Analytical sensitivity was checked by step dilution. According to our method, at the first stage, an asymmetric PCR is carried out using extended oligonucleotide primers with different melting points, complementary to the hepatitis B different genotypes genomes greatest similarity region. To increase the sensitivity, a second PCR is performed using the first reaction amplification product and internal primers. The sensitivity of the method for DNA extraction from 100 µl of plasma was 5 IU / ml, specificity 100%. Since, in spite of the HBV genotypes characteristic geographical distribution, the detection of "alien" genovariants for certain territories is becoming more frequent, we tested the method in geographically remote but active international relations with the Russian Federation regions with a high frequency of hepatotropic viruses. The developed method for detecting HBV DNA in blood plasma at low viral load based on PCR technology allows the various HBV gene variants identification and genotyping, both characteristic and rare in the Russian Federation, circulating in other world regions. The method can be used to detect HBV in risk groups, in a population, as well as when screening blood donors in order to ensure the blood transfusions safety.

[Identification and molecular-genetic characteristics of the hepatitis B virus among HIV-infected patients in Arkhangelsk.]

AIM: To estimate the prevalence and characterize the hepatitis B virus among HIV-infected patients with virological failure of antiretroviral therapy in Arkhangelsk. MATERIAL AND METHODS: HBV markers determinations (HBsAg, anti-HBs IgG, antiHBcor IgG, DNA HBV) were performed in isolates from blood plasma samples 64 HIV-infected patients with virological failure of antiretroviral therapy (viral load >50 IU / ml after 6 months of antiretroviral therapy or an increase in viral load after primary suppression of viral replication). For the detection of the hepatitis B virus, nucleic acids were isolated using the commercial kit «AmplePrime Ribo-prep¼. The virus presence analysis was performing by the polymerase chain reaction (PCR) method with hybridization-fluorescence detection in "real time" using the commercial set of «AmpliSens® HBV-FL¼. In the future, we used the method developed by the Saint-Petersburg Pasteur Institute, which allows detecting HBV in biological material with a low viral load. RESULTS: HBsAg-negative (occult) HBV was detect in 28 (43.8%) HIVinfected patients. Only HBV genotype D was detected, and the HBV subgenotype D1 prevailed (39.3%) compared with the HBV subgenotype D2 (32.1%) and D3 (28.6%). Serological markers in 42.8% of patients with HBV DNA were founding. Two HBV isolates with drug resistance mutations in the polymerase gene, leaded to amino acid substitutions (L180M, M204V) associated with the resistance development to lamivudine, entecavir, telbivudine and tenofovir were identifying. CONCLUSION: The occult (HBsAg-negative) HBV high prevalence among HIV-infected patients suggests the need to use molecular-biological diagnostic methods to identify HBV, as well as to analyze the HBV drug resistance mutation before starting antiretroviral therapy for HIV.

[The quantitative determination method of covalently closed circular DNA HBV in puncture biopsy specimens of the liver.]

To analyze the method HBV covalent-closed circular DNA quantitative determination in liver puncture biopsies and evaluate its significance in identifying HBsAg-negative viral hepatitis B. In this work, samples of liver tissue biopsy material were used from 128 patients living in St. Petersburg, in various regions of the Russian Federation, as well as in the Republic of Uzbekistan. For quantitative analysis of HBV covalently closed circular DNA in a biopsy material a method was developed based on real-time PCR using TaqMan probes for the target fragment and for the endogenous reference gene, based on the detecting ccc HBV DNA method of Pollicino T. et al. When quantifying ccc DNA HBV in liver tissue of 18 moderately HBV activity with HBV DNA PCR positive results patients and 16 inactive HBsAg carriers, the ccc DNA HBV content was significantly different between groups (p<0.034) and in terms 1 copy of the ß-globin gene among moderate activity HBV patients amounted to 1.71±1.32 copies/cell, and for inactive HBsAg carriers 0.15±0.14 copies/cell. In the group of patients with severe liver fibrosis and cirrhosis, the amount of ccc DNA HBV in liver tissue in patients with HBV averaged 2.5±0.4 copies/cell, in patients with HBV + D on average 0.7±0.25 copies/cell, in patients with HCV + HBV co-infection 0.45±0.07 copies/cell, in patients with a preliminary diagnosis of chronic hepatitis C hepatitis, on average 0.12±0.04 copies/cell, in patients with cryptogenic hepatitis 0.2± 0.05 copies/cell. A significant difference was shown between the group of patients with chronic hepatitis B with marked fibrosis and cirrhosis of the liver with other patients groups, except for the group of 18 moderate activity chronic hepatitis B patients. The values of Student's t-test when compared with other groups were respectively: for patients with a HCV preliminary diagnosis t=5,92 p<0,05 f = 19, patients with cryptogenic hepatitis t=5,71 p<0,05 f = 18, with «inactive HBsAg carriage¼ t=5,55 p<0,05 f = 29, with HCV + HBV co-infection t=5,05 p<0,05 f = 15 and HBV + D co-infection t=3,82 p<0,05 f = 17. The covalently closed circular DNA HBV quantitative assessment method in liver puncture biopsies allows identifying HBsAgnegative chronic viral hepatitis B forms and also reflects the virus replication activity, which, in turn, makes it possible to assume further disease progression and evaluate the antiviral therapy effectiveness.

Nanocrystalline SiC film thermistors for cryogenic applications.

We developed a heat-sensitive material based on nanocrystalline SiC films obtained by direct deposition of carbon and silicon ions onto sapphire substrates. These SiC films can be used for resistance thermometers operating in the 2 K-300 K temperature range. Having high heat sensitivity, they are relatively low sensitive to the magnetic field. The designs of the sensors are presented together with a discussion of their thermometric characteristics and sensitivity to magnetic fields.

Complex Analysis of Diffusion Transport and Microstructure of an Intervertebral Disk.

We studied the relationship between diffusion transport and morphological and microstructural organization of extracellular matrix of human intervertebral disk. Specimens of the lumbar intervertebral disks without abnormalities were studied ex vivo by diffusion-weighed magnetic resonance imaging, histological and immunohistochemical methods, and electron microscopy. Distribution of the diffusion coefficient in various compartments of the intervertebral disk was studied. Significant correlations between diffusion coefficient and cell density in the nucleus pulposus, posterior aspects of annulus fibrosus, and endplate at the level of the posterior annulus fibrosus were detected for each disk. In disks with nucleus pulposus diffusion coefficient below 15×10-4 mm2/sec, collagens X and XI were detected apart from aggrecan and collagens I and II. The results supplement the concept on the relationship between the microstructure and cell composition of various compartments of the intervertebral disk and parameters of nutrient transport.

[The possibilities for diagnostics of prescription of death coming based on the changes in the lumbar intervertebral disks (the comparison of the morphological, immunohistochemical and topographical findings)]. / Vozmozhnosti diagnostiki davnosti nastupleniia smerti po izmeneniiam v poiasnichnykh mezhpozvonkovykh diskakh (sopostavlenie morfologicheskikh, immunogistokhimicheskikh i tomograficheskikh rezul'tatov).

The objective of the present study was the comprehensive analysis of the postmortem changes in the lumbar intervertebral disks within different periods after death. A total of seven vertebromotor segments were distinguished in the lumbosacral region of the vertebral column based on the examination of 7 corpses. All these segments were divided into three groups in accordance with the prescription of death coming as follows: up to 12 hours (group 1), between 12 and 24 hours (group 2), and between 24 and 36 hours (group 3) after death. The models of the segments thus obtained were subjected to the study by means of diffusion weighted MRI. The removed intervertebral disks were used for morphological and immunohistochemical investigations. The comparison of the diffusion coefficients (DI) revealed the significant difference between the intervertebral disks assigned to groups 1 and 2 (p<0.01). The number of the cells in the pulpal core, the vertebral end plate, and the fibrous ring in all the above groups of the intervertebral disks was significantly reduced (p<0.01). The analysis of the correlation dependence between cell density and diffusion coefficients has demonstrated the well apparent relationship between these characteristics of the intervertebral disks comprising groups 1 and 2. It is concluded that diffusion weighted MRI in the combination with the calculation of diffusion coefficients for the intervertebral disks provides a tool for diagnostics of prescription of death coming as confirmed by the results of the morphometric studies and immunohistochemical analysis.

[ZIKA FEVER: CLINICAL, EPIDEMIOLOGICAL, LABORATORY, AND DIAGNOSTIC ASPECTS].

The authors overview data on the prevalence of Zika fever with reference to biological properties of the causative agent, epidemiological process, pathogenesis, and clinical symptoms of the disease. Special attention is given to the identification of the virus in pregnant women, microcephaly in the babies born by Zika-infected women, algorithm of laboratory diagnostics, and measures needed to prevent and control mosquitoes that spread viruses.

[THE IDENTIFICATION OF STENOTPHOMONAS MALTOPHILIA USING THE TECHNIQUES OF DIRECT SEQUENATION 16S P RNA AND MALDI-TOF MASS-SPECTROMETRY].

The in-hospital infections are one of the most serious problems of medicine, especially if patients have a background immunosuppression of various genesis conditioned by both disease itself and corresponding therapy. The detection of presence of infection and identification of agent and detection of its resistance are needed for choosing adequate therapy. At that, high heterogeneity of strains and multiple resistance of nosocomial infections to antibiotics and antimicrobial pharmaceuticals and standardization of antibacterial prevention and number of other causes becomes an obstacle for both determination of medicinal sensitivity of bacterium and for identification of pathogen itself in patient. One of the most complicated in antibacterial therapy pathogens causing pyo-septic diseases, are bacteria Stenotrophomonas spp., the only significant species out of them - Stenotrophomonas maltophilia has primary multiple antibiotic resistance. The significance of early identification of S.maltophilia is obvious. The application of MALDI-ToF mass-spectrometry requires shortage of of time of species identification of primary bacterial culture up to 1-2 hours including sampling preparation and analysis of obtained specters. The sequencing of 16S rRNA requires shortage of of total time od species identification of pathogen from clinical sample (blood) up to 1-12 hours, including sampling preparation ans comparison with successions presented in international data base. The given technique permits to exclude out of analysis prolonged period of obtaining a pure hemoculture of agent. The application of sequencing of 16S rRNA and MALDI-ToF mass-spectrometry as an alternative high-precision techniques shorten time of identification of bacteria, including detection of agent directly in blood of patient. Hence, occurs optimization of complex treatment and shortage of time of selection of adequate therapy that is especially important in case of oncological patients because sensitivity of cultural methods can be diminished due to preventive antibiotics' therapy.