[Resistance to antiviral drugs in human viruses from the subfamily Betaherpesvirinae].

The review provides information on the mechanisms of the emergence of resistance to antiviral drugs in human viruses from the subfamily Betaherpesvirinae. Data on the principles of action of antiviral drugs and their characteristics are given. The occurrence rates of viral resistance in various groups of patients is described and information about the possible consequences of the emergence of resistance to antiviral drugs is given. Information is provided regarding the virus genes in which mutations occur that lead to viral resistance, and a list of such mutations that have described so far is given. The significance of the study of mutations leading to the resistance of the virus to antiviral drugs for medical practice is discussed.

[Mutations in the UL97 gene of cytomegalovirus (Herpesvirales: Herpesviridae: Cytomegalovirus: Human betaherpesvirus 5) associated with ganciclovir resistance in recipients of allogeneic hematopoietic stem cells].

INTRODUCTION: Infection caused by cytomegalovirus (CMV) is a serious problem for patients with weakened immunity, including patients with hematopoietic depression. The cases of complications associated with cytomegalovirus require antiviral therapy. However, during the natural mutation process, especially with prolonged use of drugs in suboptimal doses, CMV strains resistant to the action of antiviral drugs (such as ganciclovir, valganciclovir) may occur. Hypothetically, the emergence of resistance in the virus may cause a more aggressive course of infection, the ineffectiveness of antiviral therapy and, as a result, an increase in the number of deaths. In this regard, timely detection of mutations that can potentially lead to the resistance of the virus to antiviral drugs during hematopoietic stem cell transplantation (HSCT), as well as during organ and tissue transplantation, may be important when making a therapeutic decision. We describe three clinical cases for which the dynamics of the appearance of a mutant strain of CMV by the UL97 gene, which correlates with the viral load and clinical picture, is analyzed.The aim of the study was to determine the timing of the occurrence of mutations in CMV phosphotransferase UL97 gene associated with resistance to antiviral drugs in patients with hemoblastoses after allogeneic hematopoietic stem cell (allo-HSCs) transplantation. MATERIAL AND METHODS: The study included 48 samples of CMV DNA isolated from the peripheral blood of three allo-HSCs recipients with CMV infection who were treated in the clinics of the FSBI «National Medical Research Center for Hematology¼ of the Ministry of Health of Russia with oncohematological diseases during 2015-2017. Patients received conditional codes (PR, PD, and FS). Mutations associated with antiviral therapy (AVT) resistance were identified in all patients. Sanger sequencing was used for mutation detection. The obtained DNA sequences were analyzed using Nucleotide BLAST and Genome compiler software. Mutations were searched in MRA mutation resistance analyzer software. The nucleotide sequences were compared with the UL97 reference sequence of the Merlin CMV strain using this software environment. RESULTS AND DISCUSSION: For all patients in whom the virus strains containing C592G (PR), C607F (PD) and C603W (FS) mutations were detected, the timing of the mutation occurrence was determined at days 187, 124 and 1184, respectively. The emergence of mutations with a high resistance factor was shown to be accompanied by an increase in viral load (VL), the appearance of a clinical picture characteristic of CMV infection and a lack of an adequate response to therapy with ganciclovir and its derivatives. CONCLUSION: Using these results, it is proposed to develop the test system based on random polymerase chain reaction (rPCR) to detect mutations in the most frequently encountered codons: M460I/V, C592G, A591V, A594T/V, L595F/S, C603W. Given that the data on the prevalence of these mutations were obtained from foreign sources, it is advisable to conduct similar studies on the frequency of mutations in the UL97 gene among the population of the Russian Federation in order to improve the quality and accuracy of test systems.

[Herpesviruses in patients after renal transplantation].

AIM: To estimate graft function after kidney transplantation during active herpesviruses or superinfection Materials and methods. The study included 32 patients (men 21, women 11) with end-stage chronic kidney disease. The median age was 43 years. Cytomegalovirus (CMV), EpsteinBarr virus (EBV) and human herpes virus 6 (HHV-6) DNAs were screened by RT-PCR in the donor's transplant biopsy, and recipients peripheral blood and urine after kidney transplantation (KT) on 0, 1, 2, 4, 6, 12 months. Antiviral antibodies (IgM and IgG) were also screened by Enzyme-linked immunoassay analysis (ELISA) along with PCR. The 500 or less copies of viral DNA per 105 nuclear cells or 1 ml of urine was considered as low, more than 1000 copies high. RESULTS: On the first month after KT CMV DNA was detected in 50% of pts., EBV DNA in 40% and HHV-6 DNA in 33%. During first year after KT two or three viruses simultaneously were found in 12 recipients: CMV, EBV, and HHV-6 were detected in 5 recipients; CMV and EBV in 4 patients; CMV and HHV-6 in 2 pts; EBV and HHV-6 in 1 pt. Graft dysfunction was observed in 9 patients with a high concentration of viral DNA of one, two or three viruses simultaneously. An upraise of the concentration of virus DNA (CMV, EBV and HHV 6) was detected primarily in the urine, while in the blood its concentration was less than 500 cop or undetectable. Renal dysfunction was not observed on the background of low concentrations of viral DNA in urine and blood. However, with an increase of DNA concentration, an impaired graft function in 8 of 12 patients appeared. Low viral DNA level proved to be a background for another virus activation or bacterial/fungal superinfection. CONCLUSION: Graft dysfunction occurs at high viral DNA levels detection during mono-or superinfection. Low viral load can serve as a background for another virus activation and/or bacterial/fungal superinfection.

[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.

[Hepatosplenic T-cell lymphoma: The problems of diagnosis and treatment]. / Gepatolienal'naia T-kletochnaia limfoma: problemy diagnostiki i lecheniia.

In the past decade, a notable advance has been made in the understanding of the pathogenesis of NK/T-cell lymphomas; however, their diagnosis remains difficult because of their rarity and clinical and morphological variabilities. The paper generalizes the ten-year experience of the Hematology Research Center, Ministry of Health of Russia, in diagnosing and treating hepatosplenic T-cell lymphoma (HSTL), considers the problems of differential diagnosis with other hematological diseases occurring with similar clinical and laboratory symptoms, and lays down current approaches to the diagnosis and treatment of this condition. A clinician's view of the problem of diagnosis and treatment of this disease is given. HSTL is shown to be a heterogeneous group of diseases differing in a T-cell receptor chain gene rearrangement, the clinical course of the disease, and overall survival (OS). According to our data, 3-year OS was 12%; the median survival was 26 months. Two-year OS for γδ and αß HSTL was equal to 25 and 70%, respectively. The difference in OS for the variants of HSTL failed to reach statistical significance (because the sample might be insufficient).

[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.

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).

[The laboratory diagnostic of herpes viral infections under nosocomial pneumonia in oncologic hematologic patients].

The study was organized to discover diagnostically valuable clinical material for detection of etiologic agent of pneumonia in oncological hematological patients, rate of association of nosocomial pneumonia with herpes viruses and evaluation of viral load in patients with depressed immunity. In oncological hematological patients, half of nosocomial pneumonia cases is associated with herpes virus. In every third patient DNA of Epstein-Barr virus and DNA of type I and II are detected. The most informative material in this case is broncho-alveolar lavage fluid and the most convenient diagnostic technique is polymerase chain reaction in real-time. The low viral load in broncho-alveolar lavage fluid is specfic for Epstein-Barr virus, cytomegalovirus and human herpes virus type VI. The concentration of DNA ofsimple herpes virus type I and type II is located in both high and low values. The paradox phenomena is established concerning more benevolent course of nosocomial pneumonia associated with simple herpes virus type I and II in patients with higher viral load in broncho-alveolar lavage fluid. The further research in this direction is needed.

[The alternative view on diagnostic of hyperlipoproteinemia, cholesterol lipoproteins of low density and effect of statins: a lecture].

The effect of statins occur in several stages: 1) inhibition in hepatocytes of synthesis of functionally specific pool of spirit cholesterol, polar mono-layer of lipoproteins of very low density; 2) activation of hydrolysis of triglycerides in lipoproteins of very low density, formation of apoE/B-100-ligand and absorption of lipoproteins of very low density by insulin-depended cells; 3) decreasing of content of and spirit cholesterol-lipoproteins of very low density in blood plasma; 4) activation of hydrolysis of triglycerides in lipoproteins of low density, formation of apoB-100-ligand and absorption of lipoproteins of low density by insulin-independent cells; 5) decreasing of level of and increasing of content of lipoproteins of high density. During first weeks of effect of statins occurs decreasing of concentration of triglycerides and unesterified spirit cholesterol-lipoproteins of very low density in blood plasma. Then, slower and more durational decreasing of level of spirit cholesterol-lipoproteins of low density occurs. The value of spirit cholesterol-lipoproteins of low density is primarily determined by content of palmitic saturated fatty acid in food, its endogenous synthesis from glucose and concentration of palmitic triglycerides and lipoproteins of very low density of the same name in blood plasma. The effect of preparations is biologically valid and corresponds to alternative hypolipidemic preparations. All these preparations have an effect following a common algorithm: they activate, using different mechanisms, receptor absorption of lipoproteins of very low density or lipoproteins of low density by cells. The level of spirit cholesterol-lipoproteins of low density in full measure depends on content of triglycerides in blood. The concentration of spirit cholesterol in blood plasma has a reliable diagnostic significance only under physiological content of triglycerides. The main criterion of diagnostic and control of hypolipidemic therapy biologically is content of triglycerides. The comprehension of differences in effect of hypolipidemic preparations within framework of common algorithm permits rationally combine them under treatment of both primary inheritable phenotypes of glucolipoproteins and secondary symptomatic types of glucolipoproteins under obligatory observation of strict dietary treatment.

[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.

[Viral mucosal lesions of the gastrointestinal tract organs in patients with lymphoma].

Infectious complications are one of the main causes of the lower efficiency of chemotherapy in hematologic oncology. The common infectious pathogens are herpes group viruses. The manifestations of herpesvirus infection or reactivation may be extremely diverse; just the same, digestive tract injury is rarely associated with herpesvirus infection in clinical practice. Viral mucosal injury of the intestine and pharynx is described in 2 patients with lymphomas during agranulocytosis. Virus-specific DNA was absent in blood; however, it was detected at high titers (the number of copies of 10(3) 10(5) genome-equivalent/mI) in feces and mucosal biopsy specimens. Addition of antiviral therapy could rapidly abolish infectious complications in both cases. Virological examination of material from the injury focus makes it possible to reveal a pathogenic virus even though the latter is undetectable in blood.

[Prolonged bone marrow aplasia in patients with acute leukemia after chemotherapy].

AIM: To analyze the causes of prolonged hematopoietic tissue aplasias in patients with acute leukemias (AL) after chemotherapy courses. MATERIALS AND METHODS: Data on 7 patients with acute myeloid leukemia, followed up at the Hematology Departments, Hematology Research Center, Russian Academy of Medical Sciences, over the period 2003 to 2007, who had developed deep bone marrow aplasia (BMA) inadequate to cytostatic drug exposure during chemotherapy, were analyzed. The authors compared in all the patients the values of peripheral blood and bone marrow (BM) puncture specimens and the results of blood tests using the polymerase chain reaction at different AL development stages with the results of an immunohistochemical study using the markers of viruses of hepatitis C and B, a herpes group (EBV, CMV, HSV-1, HSV-2) and parvovirus B19. RESULTS: The marker of hepatitis C was detected in 6 of the 7 patients with prolonged BMA; 3 of these 6 patients showed a simultaneous infection with hepatitis B. Six of the 7 patients were found to have concomitant BM lesion with various herpes group viruses. Two patients had a resistant form of AL. CONCLUSION: Hepatitis C virus infection in patients and the resistant form of the disease were the principal causes of the development of BMA inadequate to cytostatic drug exposure. Affliction of abundant bone marrow cells with herpes group viruses was not a direct cause, but might substantially aggravate BMA.

[Laboratory diagnosis of herpesvirus infections in patients with acute leukemias].

The paper presents the results of monitoring the markers of herpes simplex viruses types 1 and 2, cytomegalovirus, Epstein-Barr virus, and human herpesvirus type 6 in the blood and bone marrow of patients with acute leukemias during induction multidrug therapy. Whether it is expedient to diagnose herpesvirus markers in patients with acute leukemias in the period of remission induction is discussed.