Vol 9, No 3-4 (2019)

Over the last years, an increasing attention in modern medical microbiology has been paid to examining Actinomycetaceae, Corynebacteriaceae, Mycobacteriaceae, Nocardiaceae, Gordoniaceae sps. Members of the Mycobacteriaceae family are increasingly examined in research and real-life practice, whereas bacteria belonging to families such as Nocardiaceae and Gordoniaceae remain poorly investigated despite novel methods emerging in practical microbiology that allow to more accurately identify microorganisms. According to the current classification, the genus Nocardia includes over 80 species, most of which rarely result in human disease development. Most often, members of the genus Nocardia cause lesions in bronchopulmonary system, which, however, may also cause development of pathological processes in other anatomical sites. Likewise, members of the genus Gordonia may also trigger infectious lesions in human, which were previously often incorrectly identified as other actinomycetes or mycobacteria. Owing to use of 16S rRNA sequencing, it substantially improved identification of these bacteria. Currently, an increasing number of microorganisms with potential clinical significance has been recorded. In addition, similar to nocardiosis, diverse primary and secondary immunodeficiencies play a primary role in gordonii-associated development of pathological processes. However, an additional risk factor may be represented by pathological conditions associated with ingestion of foreign bodies colonized by such microorganisms. Most often, members the genus Nocardia cause lesions in the bronchopulmonary system able, however, affect other anatomical areas. Half of all cases of pulmonary nocardiosis are accompanied by pathological processes of extrapulmonary localization, whereas as low as 20% of patients manifest with extrapulmonary form of the disease usually occurring when the pathogen spreads hematogenously or via other routes also highlighted by primary pulmonary lesion. Moreover, members of the genus Gordonia may result in similar infectious lesions. Currently, the number of aerobic actinomycetes of potential clinical significance is increasing that may be due to their role in diverse pathological processes of various etiologies, which have been more often reported in scientific publications. Few reports regarding infections caused by the genus Gordonia ara available which may be due to a paucity of microorganisms isolated from clinical material or false identification as mycobacteria or Nocardia. Similar to nocardiosis, diverse immunodeficiencies play a primary role in the development of pathological processes associated with Gordonia. However, an additional risk factor may be linked to pathological conditions associated with the ingestion of foreign bodies colonized by these microorganisms. Available publications allows to underline etiological significance of Gordonia in development of cholecystitis, granulomatous skin lesions, eyelid abscess of other soft tissues, granulomatous mastitis, brain abscess and meningitis, as well as external otitis, bronchitis, endocarditis and mediastinitis. In addition, all these microorganisms can cause bacteremia associated with use of a central venous catheter. Owing to emergence of new detection methods as well as elevated rate of immunocompromised patients, and subsequently increased amount of new cases caused by members of the Nocardiaceae and Gordoniaceae families, an interest they rise will grow progressively.

The review is devoted to the analysis of the available literature on the elimination of measles. The review focuses on the current measles epidemic situation in the African Region (AFR) and the implementation of the WHO strategic plan for the elimination of measles in AFR by 2020. Measles in the AFR is characterized by a severe course with a high risk of death due to malnutrition, vitamin A deficiency, concomitant bacterial and viral infections, and malaria. In 2015, 105,256 cases of measles were reported in the WHO African Region, most of them among children under 5 years old, 79% of whom were not vaccinated or had unknown vaccine status. Initially, the strategy for implementing the measles elimination program in AFRs was based on a combination of immunization campaigns for children under 14 years of age (coverage of more than 90%) and routine vaccination of at least 90% of children aged 9–15 months. It was recommended to repeat the campaign of mass immunization of children aged 9 months up to 4 years every 3–5 years. The use of this strategy has reduced the number of measles cases by 83–97% during the first year of additional immunization programs. The recommended age of routine measles vaccination in AFRs is 9 months — a strategy to reduce infant mortality, including that due to complications of measles. In 2016, measles vaccination was introduced into the national immunization schedule in all AFR countries, and 24 countries introduced revaccination. Currently, the measles elimination program in a number of AFR countries is based on two-dose immunization (MCV1 and MCV2). The measles prevention program in a number of AFR countries was disrupted due to the Ebola epidemic. There are some common problems in the realization of the program in AFR countries. All AFR countries are committed to the measles elimination program. The review provides information on strategies and successes in overcoming challenges to achieve the goals set for the WHO African Region in the implementation of the programme of measles elimination.

Human enterovirus infections comprise a group of infectious diseases caused by viruses of Enterovirus A-D species (genus Enterovirus, family Picornaviridae). Enterovirus infections can vary in clinical manifestations and severity, from asymptomatic infection to serious multisystem diseases. During evolution, enterovirus strains with increased neurovirulence or atypical pathogenicity may emerge exhibiting an epidemic potential. Recently, outbreaks of enterovirus infection with an increased rate of neurological manifestations, a significant percentage of severe cases and lethal outcomes have been observed worldwide, which were associated with enteroviruses EV-A71, EV-D68 etc. The World Health Organization has included EV-A71 and EV-D68 enterovirus infection together with some other dangerous viral diseases considered for inclusion in the List of Blueprint Priority Diseases. In connection with this, global enterovirus surveillance is important for controlling emergence and spread of epidemic enterovirus variants, prediction of establishing epidemic situation, timely conduction of preventive measures and vaccine development. A growing multi-field cooperation between Russia and Vietnam leads to increased two-way population migration, which actualizes scientific and practical collaboration in surveillance and control of infectious disease spread, including enterovirus infection. Currently, epidemiological surveillance of enterovirus infection in Vietnam is based on monitoring hand, foot and mouth disease (HFMD) rate, laboratory diagnostics of enterovirus infection and identification of enterovirus strains, mainly detected in severe patients. In 2001–2016, 34 non-polio virus types were identified in patients with enterovirus infection, largely represented by viruses EV-A71, CVA6, CVA10, and CVA16. Moreover, the peak incidence of enterovirus infection and related mortality rate were associated with the increased activity of EV-A71 virus. In Vietnam, EVA71 enterovirus of genotypes C1, C4, C5 and B5 circulated at different times. Over the last years, a new pandemic genotype virus CVA6 has been dominating as a causative agent of enterovirus infection in Vietnam as well as the majority of other countries. The data on phylogenetic relation between Vietnamese epidemic EV-A71 and CVA6 strains allowed to find that they underwent multiple betweencountry spreads, whereas their subsequent in-country dissemination resulted in 2011–2012 enterovirus outbreak and sustained high-level HFMD morbidity.

Chronic hepatitis B is a severe liver disease associated with persistent infection with hepatitis B virus. According to recent estimations, 250 million people in the world are chronically infected, including 3 million chronically infected people in Russia. Antiviral therapeutics (nucleos(t)ide analogues and PEGylated interferon) suppress viral transcription and replication, but do not eliminate the virus from infected cells. The key reason for HBV persistency is a stable form of the viral genome (covalently closed circular DNA, cccDNA) that exists as a minichromosome protected from novel cccDNA-targeting therapeutics. Novel therapeutic approaches aimed at elimination or inactivation of cccDNA are urgently needed. CRISPR/Cas9 systems induce double strand breaks in target sites of DNA sequences. Experiments with CRISPR/Cas9 demonstrated high antiviral activity and efficient cleavage of cccDNA, but a small part of cccDNA pool remains intact. One of the main reasons of incomplete cccDNA elimination might be the structural organization of cccDNA, which persists in a heterochromatinized, very compacted form and is not be accessible to CRISPR/Cas9 systems. Viral protein HBx unwinds cccDNA and regulates cccDNA epigenetically by recruiting transcription-remodeling factors. In this work, we analyzed effects of CRISPR/Cas9 in combination with an HBxencoding plasmid or plasmids encoding mutant forms of HBx (HBxMut, which does not interact with pro-apoptotic factors Bcl-2 и Bcl-xL, and HBxNesm is localized exclusively in the nucleus and does not generate reactive oxygen species and double strand breaks in the genome). We showed that HBx improves CRISPR/Cas9 efficiency, decreasing pregenomic RNA transcription level over 98%. Moreover, we analyzed optimal ratios of plasmids encoding CRISPR/ Cas9 and HBx proteins for better antiviral efficacy. Furthermore, we discovered that HBx proteins do not have an effect on proliferation and viability of the transfected cells. In conclusion, CRISPR/Cas9 with HBx proteins exhibit high antiviral effect.

Currently, live anthrax vaccine has been used for vaccine prophylaxis in Russia and neighbor countries for seve ral decades, but precise mechanism of post-vaccination protection mechanism remains unclear. Here, we provide data on examining serum antibody level against protective antigen (PA) and lethal factor (LF) in repeatedly vaccinated volun teers at early stage (5–8 days) and 1 month after the performing pre-scheduled annual revaccination. Amount of peripheral blood antigen-specific memory T cells after previous vaccinations was analyzed. It was showed that frequency of CD3+CD45RO+CD62L– memory effector T cells was increased in the majority of volunteers on day 5-8 day after performing pre-scheduled annual revaccination that peaked at day 7 by elevating it by 2-fold compared with the control group. Percentage of anthrax-specific central memory T cells did not increase at early stage after vaccination, whereas amount of activated CD3+CD45RO+CD62L+HLA-DR+ subset within this memory T cell population was increased. Likewise, percentage of activated CD3+CD45RO+CD62L–HLA-DR+ effector memory T cell subset was also increased. Moreover, serum anti-PA IgG were detected on day 5–8 day after pre-scheduled annual revaccination in half of volunteers, whereas anti-LF IgG were found only in a single volunteer. Rapidly elevated amount of serum anthrax-specific IgG antibodies evidences about sustained memory B cell response in peripheral blood samples in volunteers after pre-scheduled annual revaccination. However, percentage of CD19+CD27+ memory B cells was not significantly elevated at early stage after revaccination that tended to increase. Both helper and cytotoxic T cell subsets were activated on day 5–8 after revaccination revealed by upregulated expression of CD69 and/or CD25 markers, with the latter predominantly found on helper T cells, thereby accounting for their high proliferative activity, whereas the former — on cytotoxic T cell subsets. Detection of anti-PA IgG antibodies correlates with protection against anthrax, which was confirmed in animal models. Unfortunately, the level of serum anti-PA IgG antibodies rapidly declines after vaccination. Ability of memory B cells to rapidly trigger production of anthrax-specific antibodies in response to revaccination suggests that anti-anthrax immunity may be evaluated by measuring frequency of peripheral blood anthrax-specific memory B and T cells.

Effective eradication of Helicobacter pylori infection is an important means to reduce the risk of precancerous changes in the gastric mucosa and prevention of gastric cancer. A search for non-invasive diagnostic tools for Helicobacter pylori infection, evaluation of the effectiveness of eradication remains of high importance.

The aim of the study was to assess an informative value of detecting pepsinogen I and II as well as serum antibodies to Helicobacter pylori while assessing an efficacy of treated chronic Helicobacter gastritis and identifying preneoplastic changes in the stomach mucosa. There enrolled 113 male patients with chronic gastritis aged 41 to 76, average age- (56.7±0.7) years. Examination of patients was carried out at admission to the clinic, as well as at 2 and 12 months after administering a standard eradication therapy. It was found that Helicobacter pylori infection was detected in 101 (89.4%) patients. Groups of patients with effective eradication therapy were noted. A time-dependent level of antibodies to Helicobacter pylori, as well as measured concentration of pepsinogen I and II after the onset of eradication treatment were determined. Which were analyzed in connection with the results of histology examination of gastric mucosa biopsy specimens and expression of oncoproteins Ki-67, Bcl-2, c-erbB-2, p16 in the gastric mucosa depending on efficacy of eradication therapy. It is shown that effective eradication therapy was characterized by significantly decreased serum level of IgG antibodies to Helicobacter pylori 2 months after the onset of treatment. Moreover, a significantly decreased pepsinogen II and serum IgG antibodies to Helicobacter pylori during eradication therapy were accompanied by a significant decrease in Ki-67 expression in the gastric epithelium. Decreased concentration of pepsinogen II within the first year after Helicobacter pylori eradication therapy was due to a greater decrease in activity of inflammatory changes in the gastric mucosa than to dynamic changes in gastric atrophy and metaplasia. An inverse relation between the serum level of pepsinogen I and atrophy as well as intestinal metaplasia within the gastric mucosa were found.

Our study was aimed at investigating dynamic phenotype pattern of peripheral blood NK cells in patients with widespread purulent peritonitis (WPP) during postoperative period depending on disease outcome. A total of 48 patients aged 30–63 with acute surgical diseases and abdominal injuries complicated by WPP were examined. Blood sampling was performed before surgery (preoperative period) as well as on day 7, 14 and 21 during postoperative period. 40 apparently healthy age-matched subjects were included in control group. Peripheral blood NK cell phenotyping was performed by using flow cytometry with directly immunofluorescently tagged antibodies. Mean fluorescence intensity was measured to estimate expression levels of NK cell surface receptors was measured. It was found that in patients with a favorable WPP outcome during preoperative period the percentage of mature NK cells was decreased that was restored by the end of the postoperative period (21 days post-surgery) due to elevated mature, cytotoxic and cytokine-producing NK cell subsets. In addition, percentage of CD11b-positive NK cell subsets was increased upon favorable outcome by the end of postoperative period as well as frequency of CD57-positive NK cells relative to the preoperative period. However, frequency of mature NK cells with unfavorable WPP outcome vs. control vs. favorable outcome was decreased during preoperative and entire postoperative period. Moreover, amount of cytotoxic NK cells was elevated during examination period upon unfavorable WPP outcome. Further, percentage of mature CD11b-positive NK cells in this patient cohort was decreased during preoperative period and post-surgery. Percentage of CD57-positive NK cells was decreased during entire postoperative period in patients with unfavorable vs. favorable outcome vs. control group. At the same time, patients with unfavorable outcome of this infectious-inflammatory disease were shown to display upregulated expression of CD28 and CD57 markers on NK cells. such features identified in phenotype of peripheral blood NK cells in patients with unfavorable WPP outcome reflect abnormal mechanisms in NK cell maturation and migration, which, in turn, determines disturbance in events regulating acute inflammatory reaction in WPP. 

Vaccines for prophylactic immunization provide the most reliable and effective protection against the vast majority of infectious diseases. Tick-borne encephalitis (TBE) represents a high-priority medical issue at the territory of the Eurasian continent. Of great importance is assessing a role of distinct antibody titers especially low titers, observed quite often in vaccinated individuals, sometimes posing obstacles in determining a threshold of seropositivity as well as the level of specific protection against TBE virus. We aimed at obtaining data to assess antiviral activity of virus-specific antibodies with distinct titer levels based on the in vitro, ex vivo and in vivo experimental studies with a highly virulent Far-Eastern strain of tick-borne encephalitis virus. The in vitro, ex vivo and in vivo comprehensive experimental studies with a highly virulent Far-Eastern strain of tick-borne encephalitis virus (TBEV) were conducted and the dynamics of antiviral activity of virus-specific antibodies at variable titers (1:100–1:3200) was measured (timeframe ranged within 1–96 hours p.i.) to provide a rationale for evaluating the antiviral immune response. It was found that the in vitro experiments demonstrated that the IgG at 1:100 titer exerted a weak anti-TBEV neutralizing effect at all time-points examined. The IgG 1:400 titer caused a 2 log PFU/mL decline in TBEV Dal strain yield at 72 h post-infection, whereas at 1:3200 titer it completely suppressed TBEV replication throughout the observation period. The ex vivo experiments with blood serum obtained from vaccinated subjects demonstrating a range of TBEV antibody titers (sera from vaccinated individuals with varying anti-TBEV antibody titers) and in vivo (outinbred white mice) experiments revealed a delayed virus elimination for antibody titers at 1:100 and 1:200 as well as rapid virus elimination (1–2 days p.i.) for antibody titers greater than 1:400. Thus, antibody titer at 1:400 may be considered as the universal anti-TBEV protection threshold. In order to properly conclude regarding the revaccination schedule it is advised to start with testing blood serum for durability of anti-TBEV immune response. Subjects with TBEV antibody titers at 1:100 and 1:200 should be strongly recommended to undergo a mandatory revaccination. Such an approach is believed to be the most effective way toward enhancing efficacy of vaccine-mediated protection against TBE.

Objective. To compare epidemiological, clinical and laboratory characteristics of chronic hepatitis B (ChHB) associated with/without delta agent (ChHB+DV) study.

Materials and methods. The Kyrgyzstan State Reporting Form No. 12 covering 2010–2017 period was examined. For this, 133 and 130 case histories of ChHB and ChHB+DV patients, respectively, were analyzed. The data were statiastically processed by using Microsoft Office Excel software.

Results and discussion. Over the 2010–2017 period, prevalence of the “HBV Carrier” (60.4 ) was higher by 20-fold than that one for ChHB [3.8 , 95% CI (2.4–4.0)] and CVHD [3.4 , 95% CI (2.2–3.4)], as the vast majority of patients were not thoroughly examined after detecting HBsAg, and the HBV Carrier was empirically diagnosed at the primary health care units. As a result, routine case definitions for such conditions were revised and an improved system of epidemiological surveillance of viral hepatitis was developed, according to the 2016 WHO recommendations approved by the Ministry of Health of the Kyrgyz Republic (Order No. 524, dated of July 20, 2018). Asthenia was observed in ~60% of patients in both groups, whereas arthralgia — in ~5–10% of patients, more often in those comorbid with ChHB+DV, and myalgia — in as low as ~3% of cases. Impaired central nervous system functions manifested as headache and restless sleep were evenly recorded in about 10–15% of patients, without significant difference between groups. In contrast, dominating dyspeptic manifestations such as poor appetite (72±3.9% vs. 20.6±3.5%, p < 0,05), nausea (23.8±3.7% vs. 7.3±2.3%, p < 0,05), vomiting (12.3±2.6% vs. 3.3±1.5%, p < 0,05) and flatulence (27±3.9% and 13±2.9%, p < 0,05) were revealed in ChHB+DV patients. Pain in the right hypochondrium was noted in 52–56% of patients, insignificantly differed between patient groups. Incidence of yellowness of the sclera and skin layers as well as skin itching were recorded by 2–3 and 8 times, respectively, more frequently in ChHB+DV patients. A more profound cytolysis and signs of altered bilirubin metabolism were also more common in HBV patients comorbid with the delta agent. Thus, a more severe ChHB+DV course requires that all patients with primary HBsAg detection were mandatorily examined for anti-HDV antibodies to ensure early diagnostics and timely organization of the secondary and tertiary preventive measures in the Kyrgyzstan. 

Until now, psoriatic antigen as a specific antigen derived from some infectious agent potentially related to origin of psoriasis has not been identified, thereby strongly arguing against infectious theory of psoriasis. However, the lack of specific psoriasis-associated antigens may be theoretically accounted for by an idea that psoriatic antigen could be analogous to infectious prion proteins (PrPSc analogue). It might be identical to some epidermal protein in psoriasis-free subjects that might differ antigenically from related normal protein by resistance to digestive enzymes similarly to PrPSc. Since PrPSc cytopathogenic effect is associated with its location within cell structures of affected body tissue, by analogy with PrPSc we aimed at identifying a potential carrier of psoriatic antigen by examining peptic hydrolysates of psoriatic squamous elements. Psoriatic squamous elements and epidermal stratum corneum were collected from heel area of healthy subjects with a metal grater for further examination. Squamous elements were homogenized in isotonic saline followed by centrifugation to separate them from soluble components. Homogenates of squamous elements structures and their homogenate supernatants underwent peptic hydrolysis reaction. Antigens and their identity were analysed by Ouchterlony immunoprecipitation in agar with antiserum obtained after immunizing rabbits with homogenate of the psoriatic squamous element structures, before and after hydrolysis, using peptic hydrolysates of squamous element structures and relevant supernatants. It was found that two antigens were precipitated in peptic hydrolysates of homogenates of psoriatic squamous cell structures, underlying acidin-pepsin resistance of antigen carriers. However, compared to the remainder of antigens precipitated in examined substrates only one out of two antigens might be considered as a psoriatic antigen being some PrPSc analogue that was identical to one of supernatant antigens derived from epidermal stratum corneum homogenates collected from healthy subjects. Similar to PrPSc, it differed from antigenically related normal protein by acidin-pepsin resistance. Albeit confirming available reports revealing no psoriasis-associated pathogen-specific antigens, this, nonetheless, might not be sufficient for rejecting infectious theory of psoriasis, as psoriatic antigen exhibits some properties similar to those of PrPSc.

Aim of this study was to study the dynamics of the frequency of cytokinesis-blocked T-lymphocytes with micronuclei in peripheral blood and the frequency of buccal micronucleated epithelium cells for a period of half a year in patients with acute tick-borne encephalitis, depending on burden of active and inactive variants of glutathione-S-transferase genes (GSTM1 and GSTT1) in the patient's genotype. We carried out micronucleus assay in immunocompetent and non-immunocompetent cells in 54 patients with acute tick-borne encephalitis and 35 healthy persons (control) residing in the Tomsk and Tyumen regions. To analyze the frequency of cytokinesis-blocked micronucleated T-lymphocytes was used venous peripheral blood as material for phytohemagglutinin-stimulated cultures, and to study the frequency of buccal micronucleated cells, samples of the buccal mucous membrane epithelial cells were obtained. To carried out both techniques of micronucleus assay, cytological preparations were prepared, which were stained using the Giemsa or Felgen methods. The material for the study was obtained repeatedly during admission of patients to treatment, and also after 1 week, 1, 3 and 6 months.  Polymerase chain reaction was used to analyze the alleles of the GSTM1 and GSTT1 genes. As a result of this analysis was found a significant increase in the frequency of micronucleated cells in tick-borne encephalitis patients compared with the control group. In addition, the frequency of cytokinesis-blocked micronucleated T-lymphocytes was increased significantly higher than the one of micronucleated buccal cells. The most significant and prolonged increase in the frequency of micronucleated cells was associated with the mutant inactive variants of the genes GSTM1 (0/0) and GSTT1 (0/0). In the patients with burden the inactive forms of these genes, the cytogenetic instability of the cytokinesis-blocked blood T-lymphocytes could persist for up to six months. In case of buccal cells, the frequency of micronucleated cells was close to the one in the control group as early as 1-3 months after a course of treatment. Conclusion. It was found that the most increased and prolonged frequency of cytogenetically instable cells persisted in cytokinesis-blocked T-lymphocytes of peripheral blood of patients with tick-borne encephalitis who were carriers of the genotype with inactive variants of  both GSTM1 (0/0) and GSTT1 (0/0 ) glutathione-S-transferase genes.

Dynamics of serum proinflammatory cytokines such as interleukins (IL)-1, -2 and -6, tumor necrosis factor alpha (TNFα) was examined in patients of reproductive age suffering from acute inflammatory diseases of the pelvic organs. Our study was carried out with female patients at hospital admission prior to therapy applied with conventional methods of treatment and improved treatment by using vaginal low-frequency laser in a constant continuous magnetic field. The data obtained were compared with serum cytokine level from 20 healthy female volunteers of reproductive age consulted on better contraception methods. It was found that cytokine profile of in patients with acute inflammatory processes in pelvic organs was characterized by a high level of proinflammatory cytokines. It was shown that patients receiving conventional treatment contained decreased level of serum IL-1, IL-2, IL-6 and TNFα displaying slight dynamics, which did not reach it in control group. This may contribute to ongoing inflammatory process, despite the positive clinical dynamics. In turn, imbalance of immune responses leads to a persistently impaired fertility in women and need to perform subsequent comprehensive rehabilitation measures. Moreover, patients applied with intravaginal low-frequency laser radiation in a constant magnetic field were found to contain serum TNFα < 100 pg/ml observed in 59.7% of cases, IL-6 level was lower than 20 pg/ml (prevalent in control group) found in 54.2% of cases. Serum IL-2 level was decreased by 3.5-fold compared to baseline, whereas for IL-1β it was higher than 100 pg/ml in as few as 23.6% patients. Such temporal pattern of inflammatory markers with rapid significant decrease of serum proinflammatory cytokines in patients with preformed pathogenic factors can reduce probability of connective tissue formation and activate their own repair as well as regenerative events. The results obtained allow to wider use combined physical interventional factors for therapy of patients with acute inflammatory diseases of the pelvic organs.