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.

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.

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.

[Results of a study of parvovirus B19 (Parvoviridae, Parvovirinae, Erythroparvovirus, Primate erythroparvovirus 1) prevalence and circulation activity in socially significant categories of the population].

Currently, along with the increasing need of medical organizations for blood preparations, algorithms for laboratory testing of blood donors are not available for all infections with hemo-contact mechanism of transmission. A representative example is infection caused by parvovirus В19. PURPOSE OF THE STUDY: The article presents the results of the original study, the purpose of which was to study the prevalence of antibodies to parvovirus B19 and the activity of the circulation of this virus in socially important categories of the population. MATERIAL AND METHODS: The materials of the study were blood samples from blood donors of Saint Petersburg, as well as parvovirus В19 sequences isolated from DNA-positive plasma samples. RESULTS AND DISCUSSION: According to the results of the laboratory examination, a high proportion of carriers of virus-specific IgG antibodies was found in studied group of donors, which confirms the previous infection of parvovirus B19 in them and illustrates the high prevalence of infection in this socially significant group. Based on the results of the blood preparations testing, the presence of parvovirus DNA В19 in a significant number of samples was determined by polymerase chain reaction method. This indicates an current parvovirus infection in the examined donors and points to a high epidemiological risk of the blood products obtained from them. Sequencing and phylogenetic analysis of a fragment of the VP1 gene demonstrated that the studied isolates belonged to А1 genotype and its subtype 1А2, which correlates with the genotypes of parvovirus В19 circulating in the European Union and Asia. In addition, two previously unknown В19 parvovirus isolates were isolated, the nucleotide sequences of which were deposited into the international GenBank database. CONCLUSION: Based on the results of the study, it is justified to include testing of blood samples for markers of В19 parvovirus infection in existing algorithms of laboratory examination of donors, which will ensure prevention of hemo-contact infection of blood recipients with parvovirus В19.

[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.

[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.

[TYPING OF LEPTOSPIRA SPP. STRAINS BASED ON 16S rRNA].

AIM: Comparative typing of Leptospira spp. strain collection based on analysis of 16S RNA fragment. MATERIALS AND METHODS: 2 pairs of primers were used for PCR, that jointly flank 1423b.p. sized fragment. Sequences of Leptospira spp. strain 16S rRNA, presented in the international database, were used for phylogenetic analysis. RESULTS: A high similarity, including interspecies, of the 16S fragment in Leptospira spp. strains was shown independently of the source, serovar and serogroup. Heterogeneity of the primary matrix, spontaneous mutations of hotspots and erroneous nucleotide couplings, characteristic for 16S sequence of pathogenic Leptospira spp. strains, are discussed. Molecular-genetic characteristic of certain reference Leptospira spp. strains by 16S sequence is obtained. CONCLUSION: Results of the studies give evidence on expedience of introduction into clinical practice of identification of Leptospira spp. by 16S sequence directly from the clinical material, that would allow to significantly reduce identification time, dismiss complex type-specific sera and other labor-intensive methods.

[MOLECULAR-BIOLOGICAL MARKERS OF HEPATITIS B IN PATIENTS WITH LIVER FIBROSIS/CIRRHOSIS IN UZBEKISTAN].

AIM: Evaluate prevalence of genetic variants of hepatitis B, viruses in population of various regions of Uzbekistan with hepatitis of various genesis and different severity levels of liver fibrosis and cirrhosis. MATERIALS AND METHODS: Blood plasma and liverbiopsy from 39 patients with different severity levels of liver fibrosis and cirrhosis served as study material. Genotyping based on direct sequencing of Pre-S1/Pre-S2/S HBV DNAregion was applied. RESULTS: Hepatitis B virus was detected in 32 samples ofthe 39 provided, frequency of occurrence - 82%, respectively. Phylogenetic analysis has shown, that only genotype D was detected among the examined,patients, hepatitis B virus subtype D1 predominated (84.38%) compared with D2 (3.12%) and.D3 (12.5%) subtypes. CONCLUSION: Large-scale sequencing of HBV in, Central Asia will allow to evaluate routes of transmission and time of evolutionary separation or virus isolates. Understanding the epidemiology of the infectious process is important for development of programs for prophylaxis and therapy of the infection.

[HBV COVALENTLY CLOSED CIRCULAR DNA AS A MARKER OF PREVALENCE OF OCCULT HEPATITIS B IN PATIENTS WITH HBV, HDVAND HCV INFECTION IN UZBEKISTAN].

AIM: Evaluate significance of covalently closed circular DNA of hepatitis B virus as a marker for detection of occult viral hepatitis B in Uzbekistan population with hepatitis of various genesis. MATERIALS AND METHODS: Blood plasma and liver biopsy from 39 patients with different severity levels of liver fibrosis and cirrhosis served as study material. HBV covalently closed circular DNA detection was carried out according to Pollicino T et al. (2004). RESULTS: Covalently closed circu- lar DNA of hepatitis B virus was detected in 82% of samples, including in 54.5% of patients with chronic viral hepatitis C (CVHC) and in 100% of patients with hepatitis of unknown etiology. Quantitative evaluation of content of covalently closed circular DNA of hepatitis B virus in liver tissue in patients with CVHB has shown an average of 2.5 copies of HBV genome as ccc DNA per cell, in patients with CVHB + D an average of 0.7 copies/cell, in patients with co-infection by HCV and HBV - 0.5 copies/cell, in patients with CVHC an average of 0.12 copies/cell, and in patients with cryptogenic hepatitis - 0.2 copies/cell. CONCLUSION: Detection of HBV DNA is a complex problem for effective laboratory diagnostics of hepatitis. Detection of HBV ccc DNA as a marker of occult hepatitis B in patients with CVHC and patients with hepatitis of unclear etiol- ogy is an. important factor for diagnostics, selection of adequate therapy, prognosis of disease outcome and prevention of development of severe liver diseases.

Expression of thrombospondin-1 gene mRNA and protein in the placenta in gestosis.

The expression of TSP-1 gene mRNA and TSP-1 protein in the placental tissue was studied during normal pregnancy and in gestosis. The formation of placental tissue in normal gestation was associated with expression of TSP-1 gene mRNA and of TSP-1 protein. Gestosis was associated with inflammatory reaction in the placenta characterized by increased counts of lymphocytes and macrophages in the villous stroma and involution degenerative changes in tissue. Disorders in placental villi maturation and branching in gestosis were paralleled by hyperexpression of TSP-1 gene mRNA by placental cells and hyperexpression of TSP-1 protein predominating in the stromal elements of terminal villi and near villous vessels.