Vol 10, No 1 (2020)

Unveiling origin of infectious diseases with unknown etiology is one of the major issues in contemporary medicine, since a laboratory-confirmed diagnosis may, unfortunately, be obtained solely in very few cases. Because the majority of the most common mid-latitude infections display a typical overt clinical picture, this problem has not been paid a proper attention until recently. Recent rise in incidence rate of infectious diseases lacking typical clinical signs observed lately makes it extremely important to consider the problem more closely. It is believed that such trend is due to a whole body of reasons, including impaired sanitary control, increased both internal and external migration flows, refusal of vaccination in case of long-lasting epidemic wellbeing, emergence of atypical bacterial strains of bacteria resulting from irrational antibiotic therapy etc. Viruses constitute the largest group of organisms on our planet accounting for them as the most common causative agent of infectious diseases of unknown etiology. Some estimates obtained by mathematical modeling propose that at least 320,000 types of viruses capable of infecting mammals may exist, most of which have not been described yet. Hence, monitoring circulation of the known viral pathogens, tracing down their spreading and changes in genome nucleotide sequence as well as revealing new types of viruses become important aspects of epidemiological surveillance necessary for timely response to emerging threats, prediction and early detection of outbreaks both in humans and animals. This review summarizes traditional molecular genetics methods for detection of viral pathogens, such as PCR, real-time PCR, Sanger sequencing with pre-cloning, and methods based on the second and third generation sequencing. Therefore, a more detailed overview was provided to diverse methods based on using such technologies because viral infectious agents investigated with high-throughput sequencing (or NGS — Next Generation Sequencing) has been increasingly appreciated as feasible for diagnostics, disease control, molecular epidemiology and infection control. Finally, a special attention was also paid to the approaches used to enrich the viral genetic material in samples containing low amount of pathogen nucleic acids.

According to the World Health Organization, over 10 million new tuberculosis cases are reported annually worldwide. According to the 2017 Federal State Statistics Service Report, incidence rate for active TB infection in the Russian Federation was 109.8 cases per 100,000 population, of which 41.3% accounted for chronic disease form. Regardless of climatic conditions, high prevalence of TB infection, is not only due to high Mycobacterium tuberculosis viability, but also its ability for long persistence in human body and reactivation after an unlimited period of dormancy. The outcome of infection is largely determined by host immunoreactivity and its ability to develop protective immune response. In addition, status of immune system also underlies tuberculosis course after the onset: either as a localized form, or as a form with extensive damage to the lungs and even other organs observed in generalized infection. In recent decades, a great attention was paid to examining mechanisms of adaptive cell immunity played in pathogenesis of TB infection. No doubt, adaptive immunity is a powerful defense system providing a targeted specific immune response, but now it is becoming clear that it represents solely an effector arm of innate immunity. Innate immunity is a phylogenetically more ancient, inherited system largely aimed at ensuring rapid pathogen elimination and preventing development of infection at early stages when adaptive immunity ongoing antigen-specific maturation. Mechanisms of innate immunity mediated by cells, diverse receptors, molecules and their complexes, found on various cells. Activation of innate immunity begins with recognition of conserved molecular groups present in various pathogens called pathogen-associated molecular patterns (PAMPs), which are sensed by pathogen recognition receptors (PRRs). Here, we review current data on the role of innate receptors in recognizing M. tuberculosis-derived PAMPs, production of immunoregulatory cytokines and activation of signaling pathways playing a crucial role in the regulation of necroptosis, apoptosis and autophagy of infected macrophages. Significance of innate mucosal factors in implementing immune response to M. tuberculosis is discussed. In particular, Toll-like receptors, scavenger-receptors, mannose receptor, DC-SIGN etc. were described to participate in development of M. tuberculosis immunity. The data on single nucleotide polymorphic variants for innate genes are shown, which predispose to developing tuberculosis and affecting its course.

The pathogenesis of poststreptococcal glomerulonephritis (PSGN), a major complication of acute infections caused by group A streptococci (GAS) remains unclear. Several theories, based on the role of certain streptococcal virulence factors, as well as immunological mimicry between GAS and renal tissue, have been proposed. Earlier, we reported that many virulent clinical GAS isolates showing confirmed nephritogenic activity were capable of nonimmune Fc binding of monomeric or aggregated IgG. Moreover, a rabbit model of PSGN allowed to obtain findings regarding a crucial role of streptococcal IgG Fc binding proteins belonging to the M family surface proteins, in the onset of PSGN. Rabbits injected with inactivated IgGFcBP-positive streptococci, acquired changes in the renal tissue with deposited IgG and complement C3, as well as signs of immune inflammation characteristic for human PSGN. Also, it was shown that the induction of experimental glomerulonephritis could be inhibited after normal IgG or its purified Fc fragments were inoculated at early stages of the process. The data obtained in rabbits injected with group A streptococcal type M60 also showed pathogenic functions of the IgA Fc-binding proteins of GAS. The aim of the study was to examine inhibiting activity of the purified rabbit IgG Fc fragments on the manifestations of glomerulonephritis induced by S. pyogenes strains capable of binding diverse forms of immunoglobulins such native IgG, immune complexes, and IgA.

Materials and methods. GAS strains of emml, emml2 and emm60 genotypes were used to induce PSGN or IgA-nephropa-thy in rabbits. Fc fragments derived from rabbit IgG were obtained by enzymatic digestion and purified by affinity chromatography on a protein G-sepharose FF column. Immunomorphological changes of renal tissue were estimated by morphometric analysis.

Results. In the present study, using the rabbit model, we revealed pathological changes of different intensity and localization in the renal tissue. For streptococci of the emm1 and emm12 genotypes, PSGN was characterized by deposition of IgG or IgG-anti-IgG immune complexes within the basal glomerular membrane. Morphological changes were evaluated as a membranous-proliferative glomerulonephritis. Meanwhile, IgA-glomerulonephritis is characterized by deposition of IgA in mesangial cells of glomeruli, leading to the mesangial-proliferative glomerulonephritis or IgA-nephropathy. Intravenously administered purified Fc fragments derived from normal rabbit IgG varied in effects on pathological processes: (i) IgG Fc fragments of fully inhibited development of the PSGN induced by IgG Fc binding strain of emml genotype, (ii) IgG Fc fragments of partially reverted changes caused by the emm12 genotype strain, which was binding only to immune complexes, and (iii) had no effects on pathological changes caused by the emm60 genotype GAS strain, which was binding only IgA.

Conclusion. The data obtained point and emergence of differences in mechanisms of renal lesions development at glomerulonephritis, depending on the emm genotype of GAS strain. In addition, it also confirmed GAS-derived involvement for various IgFc-receptor proteins in the pathology. Further studies on potential prophylactic or curative effects of IgG Fc fragments in PSGN should therefore be of interest. The findings might suggest a new therapeutic approach for non-suppurative poststreptococcal diseases.

The causative agent of Zika fever (ZIKV) belongs to the genus Flavivirus of the family Flaviviridae. The flavivirus genus consists of more than 70 members. Based on virion structural organization and amino acid composition of proteins, this virus resembles other flaviviruses such as dengue (DENV), yellow fever and West Nile (WNV) posing a threat to human health. ZIKV is an arbovirus and may be transmitted by diverse mosquito species of the genus Aedes. It is believed that the main carriers are also able to transmit dengue virus, yellow fever virus as well as other flavivirus infections. In 1947, ZIKV was isolated for the first time from blood samples obtained from rhesus macaques inhabiting the Zika Forest (Uganda). Long time this virus was not considered as a dangerous to human pathogen, as Zika fever mostly occurs asymptomatically. However, analysis of Zika fever course in pregnant women unveiled a link between this disease and severe congenital disorders of the nervous system, including microcephaly, that allowed to deal with it as a dangerous infection thereafter. Rapid ZIKV spread outlined a number of problems faced by medical doctors, among which the main issue was the lack of assays for its virus-specific diagnostics. ZIKV displays a marked antigenic similarity with other flaviviruses. The majority of dengue-specific monoclonal antibodies binds to Zika virus. It is expected given the high degree of amino acid sequence similarity found for flavivirus polyprotein. Several antigens bearing ZIKV E surface protein fragments were constructed to assess an opportunity for conducting differential diagnostics for distinct flaviviruses based on detection of virus-specific antibodies. Vector plasmid pET32 was selected for producing recombinant antigens in E. coli cells. After creating constructs encoding the ZEa187 and ZEa40 proteins, the chimeric proteins were produced in amount necessary for performing ELISA with blood serum samples. Protein samples were prepared by isolating them from bacterial biomass via lysis followed by chromatographic purification. Blood sera obtained from human subjects recovered after Zika, Dengue and West Nile fevers were used to examined immunochemical properties of chimeric proteins. Human sera containing no antibodies against flavivirus types were used as a negative control. It was found that serum IgM class antibodies derived from patients with flavivirus infections demonstrated a high level of cross-reactivity by interacting with ZEa187 and ZEa40. Upon that, despite increment of mean specific interaction signal observed for such proteins and IgG of ZIKV sera, a marked cross-reactivity with the IgG of WNV and DENV sera was found. Thus, with some certainty it may be concluded that in immunochemical assays use of natural amino acid sequence specific to Zika virus surface protein as antigenic material does not allow to achieve high specificity for antibody detection.

Since April 2016 after global cessation of using trivalent oral poliovirus vaccine (tOPV) and switch to bivalent OPV consisting of polioviruses types 1 and 3 (the “switch”), any isolation of type 2 poliovirus has been regarded as an event of extreme importance requiring investigation, risk assessment and decision making. In 2016, 2 cases of isolated vaccine-derived poliovirus type 2 from healthy children was registered in Russia. Our study was aimed at on the assessing a risk of further spread of vaccine-derived poliovirus type 2 and provide measures for preventing its further spread based on epidemiological investigation and genetic characteristics of the isolated viruses. The cases were revealed within the surveillance program for poliomyelitis and acute flaccid paralysis syndrome conducted in the Russian Federation. The laboratory investigation was carried out in accordance with the algorithm adopted in the Russian Federation and recommended by the WHO standards: virus isolation on RD, L20B and Hep2C cell cultures, identification in the neutralization reaction, intratyping differentiation by using RT-PCR in real-time mode, sequencing of the poliovirus genome fragments encoding the VP1 protein. A risk assessment for spread of vaccine-derived poliovirus type 2 was performed in accordance with the WHO recommendations. There was uncovered a genetic relationship between virus strains isolated in September and December from unvaccinated Moscow resident boy (1 year old) who arrived from the Chechen Republic and from unvaccinated girl resident of the Chechen Republic (1 year old) with impaired humoral and cellular immunity. The virus strains were found to bear 10 and 13 genomic nucleotide substitutions, respectively, at the site encoding the VP1 protein compared with the Sabin type 2 vaccine strain that allowed to classify them as vaccine-derived polioviruses. In particular, both virus strains were shown to originate from the type 2 strain presented in the tOPV used shortly before the “switch”. Epidemiological investigation revealed family ties and probable contact between both children in the same premises. A series of organizational and vaccination measures was undertaken, as well as polio surveillance was strengthened in the region. No new type 2 polioviruses of vaccine origin were detected in the territory of the Chechen Republic during 18-month monitoring follow-up. The risk assessment of spread for vaccine-derived poliovirus type 2 in a region, Russian Federation as well as cross-boundary spread identified it as “low,” requiring no use of type 2 monovalent OPV. Such experience for countermeasures may be taken into account to oppose the risks before and after the global certification for poliomyelitis eradication.

Meningococcal, pneumococcal, streptococcal A and Haemophilus influenzae infections are manifested in different clinical forms, ranging from bacterial carriage to generalized life-threatening conditions. However, a connection between bacterial carriage and disease development has not been fully explored. A PCR assay was performed with adenoid biopsy samples collected from 112 children after planned adenotomy to detect Neisseria meningitidis, Streptococcus pneumoniae, Streptococcus pyogenes, H. influenzae carriage. A DNA specific to at least one of the four studied microbial species was found in 104 samples (92.86%) so that: meningococcal DNA was detected in one sample (0.9%), pneumococcal — in 98 (87.5%), H. influenzae — in 19 (16.96%), and streptococcal A — in 42 (37.5%) samples. However, none of these species was found in 8 children (7.14%). A sole S. pneumoniae was detected in 54 samples (48.2%), whereas S. pyogenes — in 5 samples (4.5%). Moreover, two bacterial species were simultaneously as follows: N. meningitidis and S. pneumoniae — in 1 sample (0.9%), S. pneumoniae and H. influenzae — in 7 samples (6.3%); H. influenzae and S. pyogenes — in 1 sample (0.9%); S. pneumoniae and S. pyogenes — in 25 samples (22.3%). A triple combination consisting of S. pneumoniae, H. influenzae and S. pyogenes bacteria were detected together in 11 patients (9.8%). Meningococcal serogrouping revealed no connection with any of the 6 most common global serogroups responsible for epidemic incidence rise (A, B, C, W-135, X, Y). A clear tendency for prevalence of S. pyogenes DNA in adenoid pediatric biopsies in children diagnosed with “Adenoids and tonsils hypertrophy” vs. “Adenoids hypertrophy” was observed. It is noteworthy, a high relative prevalence of pneumococcal carriage (87.5%), found by us was of special importance. Pediatric carriers serving as a reservoir for virulent pneumococcal species pose a threat both for themselves and surrounding people. Thus, PCR-based data of adenoid biopsies may be a promising approach for future studies, as a potential to identify live viable but nonculturable bacteria in clinical specimens will contribute to a more accurate assessment of carriage rate of meningococci, pneumococci, H. influenzae and group A streptococci.

Within a framework of the state measles elimination program, in April, 2018 a level of measles herd immunity was assessed in 1899 Moscow hospital medical workers aged from 19 to 69 years and older. All subjects enrolled in the study were vaccinated against measles or recovered after measles infection. Serum samples were collected from subjects and examined by ELISA for measles IgG antibodies with the Vector-Best IgG-measle test system (Russia). It was found that 278 (14.6%) and 1621 (85.4%) subjects were seronegative (< 0.18 IU/mL) and seropositive (> 0.18 IU/ml), respectively. Age-related group distribution of 1855 serum samples revealed that percentage of seronegative subjects was in: aged 19—23 years was -38.5%; 24-28 and 29-33 years - 22.2%; 34-38 years - 27.5%; 39-43 years - 25.8%; 44-48 years - 16.8%; 49-53 and 54-58 years — 8.6% and 8.3%, respectively; 59-63 years old — 4.9%; 64-68 and over 69 years old — 0%. Moreover, mean level of measles IgG antibodies increased proportionally to age of medical workers from 0.58 IU/ml (19-23 years) to 2.94-2.72 IU/ml (64-68 and over 69 years). The data obtained indicate that a cohort of measles susceptible subjects (from 38.5% to 16.8%), respectively, is identified among young and middle age (from 19 to 48 years) individuals. It is assumed that two-dose measles vaccination in childhood does not contribute to the long-term preservation of protective levels of measles antibodies, thereby justifying a need to administer a three-dose measles vaccine.

Introduction. External genital endometriosis is an inflammatory estrogen-dependent disease characterized by implantation and proliferation of endometrial tissue outside the uterus, accompanied by increased production of proinflammatory cytokines, prostaglandins, components of the complement, hydrolytic enzymes, increased angiogenesis and anomalies of ectopic endometrium. According to implantation theory, external genital endometriosis develops from viable endometrial cells transferred retrogradely through the fallopian tubes to the abdominal cavity during menstruation, while a disturbed local immunity is an important factor in its pathogenesis. Genital pathogens may be involved in the formation of the immune environment of the peritoneal cavity in women with endometriosis.

Purpose. To study the peculiarities of local immunity in women with external genital endometriosis and genital infection pathogens.

Materials and methods. 159 women with external genital endometriosis were examined. The total number of leukocytes, the absolute and relative number of viable cells, counts of neutrophils, macrophages and their functional activity, the level of IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IFNα, IFNγ, TNFα in the peritoneal fluid were evaluated. The study of local immunity was performed in women with endometriosis, stage 1—2 and 3—4, depending on detected genital pathogens. Chlamydia trachomatis, Ureaplasma spp., Mycoplasma genitalium, HSV1,2/CMV, high carcinogenic risk HPV were analyzed by using PCR in samples collected from the endometrium, peritoneal fluid, and endometrioid heterotopies. Statistical processing was performed by using the IBM SPSS Statistics Version 22.2 statistical analysis software package.

Results. In the presence of HPV and Ureaplasma spp. in women with endometriosis, stage 1—2, the decreased functional activity of peritoneal neutrophils and macrophages was found. At 3—4 stage, a correlation analysis revealed that detected HPV and Ureaplasma spp. obtained increased both pro-inflammatory and anti-inflammatory cytokines in the peritoneum. However, the higher activity of Th-2 cells in the peritoneal fluid secreting IL-4 and IL-10 and suppressing cellular immunity, was observed in HPV-positive samples. In addition, HPV also correlated with a decreased IL-2 and IL-4 levels.

Conclusions. The most prominent changes in the immunological parameters from peritoneal fluid samples were observed in case of detected genital infection pathogens, particularly HPV. Thus, immune disturbances emerged upon bacterial and viral pathogen detection may contribute to the implantation of endometrial cells in the pelvic organs and disease progression.

Medical community has noted that the prevalence of allergic diseases by steadily tends to rise resulting from using synthetic drugs ecological catastrophe. In this regard, more and more attention is increasingly being paid to natural therapeutic approaches, one of which is based on applying hirudotherapy. There were used 40 pubescent outbred female rats and their 200 pups born during experiment. In particular, female rats underwent leeching in the subscapular region 2 weeks before and 2 weeks after mating by applying medical leeches weighing 400 ± 10 mg. Next, relevant progeny was examined at day 1, 15, 30, 45, and 60 after birth, whereas female rats– after weaning. Experiment was designed to assess diverse parameters in accordance with the age-related rat development: newborn pups – 1-5 days old, day 6-21 – suckling period, day 22-50 – onset of puberty, day 60 onward – puberty. After measuring body morphometric parameters, female rats were sacrificed by decapitation under ether narcosis on day 60, whereas rat pups – at various time points. followed by autopsy and collecting blood sample into a sterile centrifuge tube by using 2% Spofa crystalline heparin (8:1) to be further examined by standard assays: morphometry of rat body, spleen and thymus; measuring total leukocyte count; CBC with differential; RBC; hemoglobin level; color index. Statistical data processing was performed by calculating arithmetic mean, error of arithmetic mean, standard deviation by using SPSS version 21.0 and Microsoft Office Excel 2010 software. Significance of differences was estimated by using Student's t test, and set at p≤0.05. Our study demonstrated medical leech saliva components applied during hirudolotherapy exerted an immune-augmenting effect of on morphometric body, thymus and spleen parameters. Moreover, starting from day 1, total leukocyte count, RBC, and hemoglobin level were increased in the experimental group. Moreover, we convincingly demonstrated a stimulatory effect of medical leech saliva components on histogenetic reactions at various ontogenetic stages in both female rats and their progeny. In the former, such morphogenetic effect was maintained for more than 70 days after the last leeching: on week 2 after mating and 30 days after delivery. Changes in morphological and hematological evidence about enhanced morphogenetic function of immune system in response to saliva-derived biologically active substances released by medicinal leeches.

Tick-borne bacterial and viral infections are widespread in middle latitudes of the Northern hemisphere. Natural foci of such infections coincide with geographic areas inhabited by ixodid ticks. Ixodid tick-borne borreliosis is a pressing issue for some territories of Russia, especially for the North-Western Federal District and St. Petersburg megalopolis as well as adjacent areas of the Leningrad District, where people may become infected after tick bite in recreational zones in suburban park areas. Currently, very few publications regarding prevalence of tick-borne pathogens in St. Petersburg area are available. In our study, questing ticks flagged in park zone (northern coast of Finnish Gulf, Kurortny District) were examined with PCR for carriage of pathogenic B. burgdorferi sensu lato complex. In addition, samples positive for Borrelia DNA signal were further genotyped with species-specific primers against rpoBgene fragment. It was found that Ixodes persulcatus dominated in this area. Prevalence of Borrelia burgdorferi s.l. complex comprised 9.33%. Genospecies B. afzelii and less frequently B. garinii were detected. A mixt-infection with two Borel-lia species was detected in one sample. Interestingly, all Borrelia-infected ticks belonged to I. persulcatus suggesting a closer association for certain species in «pathogen-vector» system. Our findings are essential in investigating distribution of ixodid borreliosis foci in St. Petersburg and suburbs, obtaining new data regarding epidemiology, diagnostics, treatment and prevention of this infection. It is noteworthy than prevalence of pathogenic Borrelia spp. vs. tick-borne encephalitis virus in vectors was higher thereby accounting for its higher morbidity. Comparing our data with those published elsewhere by European researchers allows to note that prevalence of pathogenic Borrelia spp. in ticks varies broadly in diverse geographic regions. It is necessary to take into consideration that prevalence of Borrelia markers achieves ~10% in ticks given frequent attendance of park areas near St. Petersburg that point at risk of developing bor-reliosis in recreational zones.

At present, the level of Helicobacter pylori infection is determined by geographic area, gender and age of the examined individuals, and can reach up to 95% of the total population. Environmental adaptation of H. pylori is exhibited in its ability to adhere to the gastric mucosal epithelium and modulated expression of its own virulent factors. Current concepts implicate that H. pylori can survive inside epithelial cells, evading host immune response. Cytokines are produced by immune cells and act to regulate its major stages. A cytokine cascade launched after Helicobacter pylori infection triggers immune reactions, progression of chronic inflammatory and destructive processes in the gastric mucosa. The role of cytokines in precancerous diseases of the stomach is ambiguous because, on the one hand, they activate immune response aimed at eliminating the pathogen, whereas on the other hand, they do contribute to the disease progression. The aim of our study was to examine profile of some cytokines and features of cytokine regulation in H. pylori-infected middle-aged males with chronic gastritis (CG) as well as chronic atrophic gastritis (CAG). In patients with CG with H. pylori, CAG and CAG with H. pylori, an increase in the cytokine IL-2 was observed that might contribute to augmented damaging effect of cytotoxic lymphocytes, as well as implementation of antitumor effect. CAG with H. pylori was featured with IL-8 hyperproduction, which resulted in increased absolute numbers of band neutrophils in peripheral blood and their decreased phagocytic activity evidencing about altered host defense mechanisms. There was increased amount of IFNy involved in recognition of malignantly transformed cells and upregulated expression of the major histocompatibility complex molecules on antigen-presenting cells. In patients with CG with H. pylori and CAG with H. pylori, production of IL-4 was increased, which might serve as a contributing factor to the chronicity of H. pylori-associated diseases. Overproduction of type 1 and type 2 cytokines indicates about activated Th1 and Th2 type immune reactions in H. pylori-associat-ed CG. A potent pro-inflammatory cytokine cascade triggers inflammatory changes in gastric mucosa with developing neutrophil infiltration and lymphocyte activation. Damage and death of epithelial cells upon inflammation form erosive and ulcerative defects, or changes manifested as gastric mucosal atrophy, metaplasia and neoplasia. The data obtained may be used as additional diagnostic criteria in early diagnostics of precancerous stomach diseases.