[Impact of current approaches to laboratory screening of donated blood and its components on hepatitis B virus infection in patients with blood system diseases]. / Vliianie sovremennykh podkhodov k laboratornomu obsledovaniiu donorov krovi i ee komponentov na infitsirovannost' virusom gepatita V u bol'nykh s zabolevaniiami sistemy krovi.

AIM: To evaluate the detection rate of markers for hepatitis B virus (HBV) in the blood samples taken from patients with blood system diseases, by applying the current approaches to examining donated blood and its components for markers of viral infections. MATERIAL AND METHODS: The investigation included blood samples from patients with blood system diseases (n=364) and donors (n=5,011). The results of laboratory screening of donated blood samples (n=13,081) were retrospectively analyzed. Commercial kits of reagents were used for immunochemical assay and polymerase chain reaction. RESULTS: Patients with blood system diseases were recorded to have markers of active HBV infection in 12.6% of cases, anti-HBc in 31.3%, and anti-HBs in 37.6%. A retrospective analysis of the results of screening donated blood samples showed the presence of markers for active HBV infection in 0.28% of cases. A prospective examination of blood donors revealed markers of HBV infection in 4.83% of cases, including those of active forms in 0.54% and anti-HBc in 4.79%. The markers of active HBV infection in donors were only anti-HBc IgM in 0.42% of cases. The blood samples from donors with an anti-HBs titer of >200 mIU/ml contained anti-HBc IgM in 10.5%. CONCLUSION: In the last 5-7 years, the detection rate of markers of HBV infection in the blood samples of patients with blood system diseases have remained at a high level. Screening for decreed markers fails to identify people with inapparent infections among the donors. Even high anti-HBs concentrations in the donated blood may be a risk for HBV transmission by transfusion to a recipient.

[The screening of donor blood on antibodies to nuclear antigen of hepatitis B virus as a tool of increasing of safety of transfusion for patients with diseases of blood system.]

Despite application of decreed modes of laboratory analysis of components of donors' blood, the risk of infection of recipients with hepatitis B virus continues to be actual. The isolated identification of HBsAg provides no control of all categories of persons infected with hepatitis B virus. The analysis of presence of antibodies to nuclear antigen of hepatitis B virus that are the first out of antiviral ones and are preserved for life, is an expedient technique of screening testing of donor's blood that permits implementing an additional selection of donors. During March 2014 - March 2015, cohort of regular anti-hepatitis B virus negative donors of blood and its components. The testing of blood samples for anti-hepatitis B virus can be recommended as a routine test increasing viral safety of blood transfusions for patients with diseases of blood system.

[The PCR markers of viral infections under chronic gastritis in children].

The extended monitoring (up to 1 year 11 months) of PCR markers was implemented concerning viral infections: cytomegalovirus, Epstein-Barr virus, simple herpes virus type I and II, hepatitis B virus, hepatitis C virus and bacterial infection of Helicobacter pylori in bioassays (blood, biopsy material of mucous coat of stomach and inferior third of esophagus) from children with different types of chronic gastritis. In biological samples from patients with gastritis type A and type A + B DNA of hepatitis B virus (87% and 71% of patients correspondingly) and DNA of Epstein-Barr virus (63% and 67% of patients) were detected with high rate. Under gastritis type B and C these markers were detected significantly rarely (20-36%). Among patients with gastritis type A, B and A + B, the positive results on DNA of cytomegalovirus consisted 13-17%. In patients with gastritis type C DNA of cytomegalovirus was not detected. In any of analyzed samples no DNA of simple herpes virus type I and II was detected. The control of DNA of H. pylori demonstrated its presence in biological materials of 67% and 84% of patients with gastritis type B and A +B. This type of DNA was absent in patients with gastritis type A and C. Under gastritis type A, B and A+B, DNA of Epstein-Barr virus and DNA of hepatitis B virus detected more often in biological materials of mucous coat of stomach (71%-100%) and out of them simultaneously in blood in 33%-60% of examined patients and only in blood up to 29%. DNA of Epstein-Barr virus was detected in leukocytes of peripheral blood and DNA of hepatitis B virus both in plasma and leukocytes of peripheral blood. Under gastritis type C DNA of Epstein-Barr virus was always detected in leukocytes of peripheral blood (in 20% out of these patients simultaneously in biological material) and DNA of hepatitis B virus just as much in blood (plasma and/or leukocytes of peripheral blood) and biological materials. The lower concentrations (less than 700 copies/ml) DNA of hepatitis B virus in most samples were detected in absence of markers of hepatitis B virus. In patients with autoimmune gastritis and in absence of bacterial infection H. pylori (group I) or against its background (group III) PCR-markers of hepatitis B virus and Epstein-Barr virus were detected quite often. The evidence of persistence (in superior sections of digestive organs) of Epstein-Barr virus nad hepatitis B virus is detection of DNA of these viruses under their extended monitoring (up to 1 year 11 months) in biological samples from patients with autoimmune forms of gastritis type A and type A+B.

[Low concentrations of hepatitis C virus RNA in serologically mild infection.]

Occult HCV infection (OCI) provides significant interest recently. HCV RNA in this case can be detected not in plasma, but in blood cells and/or in liver tissue. In case of antibody genesis impairment anti-HCV detection may lead to negative or "uncertain" result. The aim of the study was to estimate infection type in blood donors and patients with hematological diseases by exploration of samples with uncertain anti-HCV detection results. Blood samples of 30 180 potential blood donors' and 4322 patients with hematological diseases were tested. Comparative analysis of wide pattern of HCV markers was performed. 33 blood donors and 42 patients were enrolled in follow-up examination. Uncertain results of Anti-HCV detection in donors' samples were in 0.18% of cases. Follow-up examination of 33 donors provided discordant results using immunochemiluminescence assay and ELISA. 15.2% donors' samples contained HCV RNA in low concentration. Follow-up observation of 42 patients with incomplete antiviral antibody pattern showed HCV RNA presence in 40.5% cases (21.4% high viremia and 19.0% low viremia). Samples with low RNA concentration contained low titers of anti-core antibodies. Samples with high titers of anti-core antibodies contained high HCV RNA level. Uncertain results of anti-HCV in 15.2% of potential blood donors' samples were confirmed by detection of HCV RNA in low concentration. It proved OCI presence in these individuals and called for testing for wide pattern of HCV markers in addition to routine screening. Patients with hematological diseases showed low level of HCV RNA along with low titers of antibodies against one or two viral antigens.

LABORATORY DIAGNOSTICS OF ACTIVE AND LATENT HHV 6-INFECTION IN PATIENTS WITH HEMATOLOGICAL MALIGNANCIES.

INTRODUCTION: Human herpes virus type 6 (HHV 6) can cause serious infectious complications in immunodeficient patients. It is also capable of integrating into the genome of the infected cell. Due to this, there can be a misdiagnosis between viral integration and active infection during laboratory diagnostics. Thus, determination of HHV 6 infection using proper laboratory tools is relevant. Also the data on viral interference of HHV 6 and other herpes viruses are very poor especially for patients with hematological malignancies. The aim of the study was to identify laboratory markers of HHV 6 and the form of infection in patients with hematological malignancies. MATERIALS AND METHODS: 98 patients with hematological malignancies positive for HHV 6 DNA during the infectious complication were enrolled in the study. Viral load in leukocytes and plasma of peripheral blood, antiviral M and G immunoglobulins and peripheral blood leukocytes count were evaluated. RESULTS: The majority of patients (66 out of 98, 67.3%) showed laboratory signs of latent HHV 6. Integrated HHV 6 was suspected in 2 patients due to high viral load (1.5x105 copies and 1.7x105 copies), but it was not confirmed subsequently. Additional testing of HCMV and EBV in patients with laboratory signs of active HHV 6 infection revealed the superiority of monoinfection over mixed infection (20 of 32, 62.5%). In cases of mixed infection, the most common co-infectant was HCMV observed in 9 out of 12 (75%) cases. Mild leukopenia accompanied HHV 6 active infection. CONCLUSION: Laboratory signs of latent HHV 6 tend to be prevalent in patients with hematological malignancies. In patients with laboratory markers of active HHV 6, the monoinfection demonstrated the superiority over mixed one. In cases of mixed infection, HCMV appeared to be the most commonly co-infectant. No cases of an integrated form of HHV 6 have been observed. The viral load of HHV 6 in leukocytes and blood plasma is almost 3 times lower in patients with a mixed infection than with a monoinfection. Active replication of HHV 6 was accompanied with mild leukopenia.

Hepatitis B and C viruses interference in coinfected multitransfused recipients.

Data on hepatitis B (HBV) and c (HCV) viruses interference in hematological patients are described. Patients with a hematological malignancy are at high risk of HBV and HCV infection as recipients of multiple transfusions. Results of the laboratory testing of 339 blood samples of patients treated at the National Research center for Hematology, Russian Federation, were studied. Among these patients, HBV/HCV coinfection markers were observed in 153 patients; HBV markers only, in 76 patients; HCV markers only, in 110 patients. The vast majority of coinfected patients had HBV DNA in blood (significantly more in HBsAg-negative patients: 100% vs. 82.8%, p = 0. 0005). HBsAg-negative coinfected patients had low HBV DNA levels (102-103ME/ml) and reduced (or completely absent) HCV RNA levels. The virus interference leads to a decrease in the viral nucleic acid concentrations. Thus, virus detection should include implementation of high sensitive molecular techniques (such as real-time PCR), and an enhanced set of serological HBV markers along with routine screening methods (HBsAg, anti-HCV).

A study of possible mechanisms of the RNA-polymerase involement in mutagenesis in phage T4.

Spontaneous and induced mutation frequencies of phage T4 have been measured in Escherichia coli strains containing altered RNA-polymerase. In the strain E. coli rif-r stl-r, with double RNA-polymerase mutation, spontaneous reversion rates were increased in different mutants of phage T4. The study of base analogues mutagenesis in rUV mutants of phage T4 has shown that the introduction of RNA-polymerase mutations did not increase reversion rates in a mutant of frame-shift type but enhanced the rate of mutagenesis in phage mutants with lesions of transition type.

Induced mutagenesis in bacteriophage T4 growing in strains of E. coli with altered RNA-polymerase.

Enhanced reversion frequencies of T4 r mutants in E. coli strains with altered RNA polymerase have been obtained. The results reported have confirmed previous data on the effect of RNA-polymerase on the process of mutagenesis [2]. No such effect has been found in the course of studies of the recombination process.

Physicochemical properties of Sendai virus RNA. II. Effect of ionic strength on thermostability of RNA.

The dependence of the melting point (Tm) and width of the melting range (deltaTm) on the ionic strength and pH of the medium was investigated for the double-stranded RNA formed through self-hybridization during the isolation of RNA from Sendai virus. It was shown that Tm is a linear function of the logarithm of the sodium ion concentration in the range of concentrations from 10(-1) to 10(-4) M, with a slope of 11.5 degrees toward the abscissa for each order of magnitude. The width of the melting range increased slightly with a decrease in the ionic strength. A change in the pH of the solutions from 5 to 8 had almost no effect on the melting point or the width of the melting range. The degree of purification of the preparations of RNA and the presence of EDTA in the solutions affected the form of the dependence of the mp on the logarithm of the sodium ion concentration very strongly, especially in the region of low ionic strengths.

Quantitative PCR as a method for monitoring retroviral infection on the gene level.

For monitoring retroviral infection on the gene level, we propose the use of quantitative PCR with two internal standards: one for a fragment of the viral genome and the other for the host cell marker gene. The standards (one for HIV and the other for a human DNA marker gene HLA-DQ alpha) were constructed by PCR-mediated joining of DNA fragments and were found to be effective in quantitative PCR despite rather different structures of amplified fragments in target and standard DNAs. The number of HIV DNA copies was found to be 2-500 per 1000 lymphocytes in blood from HIV-infected patients and up to 5000+ per 1000 cells in chronically infected cell lines. The degree of infection thus measured was found to change over the course of treatment.

Secondary structures of influenza and Sendai Virus RNAs.

The secondary structures of influenza and Sendai virus RNAs were investigated by thermal denaturation, circular dichroism and proflavine binding methods. In 0.1 M NaCl about 60% of the bases of both RNAs were involved in secondary structure. The melting temperatures (Tm) of both viral RNAs were linear functions of the logarithm of the sodium ion concentration in solution, but under all ionic conditions the melting temperatures of Sendai virus RNA were higher than those of influenza virus RNA. At all ionic strengths the melting range of Sendai virus RNA was less than influenza virus RNA, indicating that the helical regions in Sendai virus RNA were longer than those in influenza virus RNA. Although Sendai virus RNA had a higher thermal stability than influenza virus RNA, hyperchromicity and circular dichroism data showed that Sendai virus RNA had less G+C content (34%) within the double stranded regions than influenza virus RNA (48%). The binding isotherms of Sendai and influenza virus RNA-proflavine complexes were studied at different ionic strengths. The number of binding sites of proflavine with influenza virus RNA were significantly lower than those with Sendai virus RNA. These results demonstrate the essential difference between the secondary and tertiary structures of the RNAs under study.

Direct comparison of the subtilisin-like intracellular protease of Bacillus licheniformis with the homologous enzymes of Bacillus subtilis.

Intracellular serine proteases of Bacillus licheniformis and Bacillus subtilis are closely related.