Vol 9, No 5-6 (2019)

There is no doubt that infectious agents and host undergo multilayered yet not fully understood interactions. This is primarily due to at least mechanisms resulting in chronic course of infectious process. Acute infection proceeds in parallel with primary immune response and its typical phases, each of which manifests as certain stage in clinical picture featured with disease onset and subsequent recovery. A whole process of immune response developing against infectious agent occurs in peripheral lymphoid organs and immune tissues. With regard to the role of immune system in infectious process, process, two main outstanding issues still remain unanswered: 1) what are the mechanisms of host death in the case of acute infectious process? 2) what is a “fault” of immune system in it? In its inferiority or in abruptly suppressed functions induced by infectious agent, when it “does not have time” to mount an immune response of sufficient power? So far, no answer is still found yet. The second question concerns mechanisms of converting to chronic course of infectious process. The obtained available in publications evidence about an intimately involved thymus as the central immune organ in infectious process of, the main function of which is to ensure developing central immune tolerance to self-antigens accomplished via T-cell positive and negative selection. It turned out that in case of some examined infections due to pathogens, which entered the thymus, such intimate events such as partial tolerance to pathogens and autoimmune reactivity are altered. Moreover, these processes are further aggravated by homeostatic proliferation, which is also induced by an infectious agent. In both cases, it accounts for decreased magnitude of immune response against a certain pathogen, burdened by emergence of autoimmune reactions.

The aim of the study was to identify an optimal research target for detection of Klebsiella michiganensis isolates, determine their genetic and phenotypic characteristics necessary for identification. Here, we examined 11 Klebsiella oxytoca strains, lacking (atypical, negative) a marker 5-aminosalicylate decarboxylase (detected by the chromogenic reaction by 5-aminosalicylic acid) unique for the genus Klebsiella bacteria. They were selected for genetic analysis subsequent to a phenotypic characterization of K. oxytoca clinical isolates, collected in within 2015–2018 period in medical institutions in St. Petersburg. Two K. oxytoca and two Raoultella ornithinolytica clinical strains displaying typical properties were used as a control. The presence of 5-aminosalicylate decarboxylase was detected by the chromogenic reaction with the “Klebsiella 5-ASK CHROME C” nutrient medium (Pasteur Institute, St. Petersburg). Substrate utilization as the sole carbon source was detected on a solid minimal synthetic medium added with 2 g/L substrate during incubation for 72 hours at 37°C. Biochemical bacteria features were studied by the microvolume method with the “Rapid-Entero” test system (Pasteur Institute, St. Petersburg). Genetic strain characterization was performed by estimating 16S rRNA, gyrA, rpoB by using a routine PCR with primer sequences described before. Two rpoB gene fragments with a total length 834 bp, 16S rRNA gene fragment — 387 bp, and gyrA gene fragment — 441 bp were amplified followed by their sequencing by Singer on an ABI 3130 automatic capillary sequencer (Applied Biosystems, USA) and subsequently determined similarity levels. Amplification pattern for pehX gene PCR fragments was performed by using a method described elsewhere with two primer pairs flanking fragment AD with a 513 bp length and 344 bp CD-long motifs. While examining 11 clinical bacterial strains identified earlier as Klebsiella oxytoca, lacking (atypical, negative) a 5-aminosalicylate decarboxylase (detected by the chromogenic reaction by 5-aminosalicylic acid) unique for the genus Klebsiella, molecular techniques identified 9 K. michiganensis strains and 1 strain highly homologous to Klebsiella kielensis based on the rpoB gene nucleotide sequence, confirming its high informative value. We used the methods for estimating a similarity level for sequenced fragments of 16S rRNA genes (fragment length 387 bp), gyrA gene (fragment length 441 bp), rpoB gene (rpoB-b and rpoB-e with a total fragments length 834 bp), and the analysis of marker amplicon patterns for pehX gene (AD, CD). It was shown that for the 4 K. oxytoca strains, 99–100% similarity to K. michiganensis was identified for all fragments in the sequenced genes. Moreover, similarity of all 9 strains detected with K. michiganensis was revealed only in the rpoB gene, hereby allowing to recommend it as the most informative approach. The pehX gene encoding polygalacturonase was verified by PCR in the majority of K. michiganensis strains, pointing that this approach is not rational for their identification (distinguish with K. oxytoca). The most informative for the phenotypic identification of K. michiganensis are were assays characterized by a common profile for the majority of strains: lack of 5-aminosalicylate decarboxylase, lack of utilized histamine, dulcitol, tricarballylic acid; positive for indole production, as well as D-melezitose and putrescine utilization.

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of paratuberculosis (John’s disease) mainly in large and small domestic and wild ruminants, and suspected causative agent in human Crohn’s disease. In Bulgaria, paratuberculosis is still poorly researched in both groups of ruminants. We present results of the first in-depth study of mouflon, grown free in one hunting reserve in the Western region of the country. The aim was to prove the presence of MAP in diagnostic materials from regularly hunted or dead mouflon suspected for paratuberculosis. Small intestine and mesenteric lymph nodes (MLN) from 12 hunted and 4 dead mouflon and 10 faecal samples (Fc) were studied in the period of 2009–2013. Typical for paratuberculosis pathomorphological lesions were observed in four mouflon (of 16 examined). The intestinal wall was thickened, strongly folded and soft, with severe hyperemia. The MLN were enlarged, soft, with marbled appearance. The affected section of the ileum showed hyperplasia of the mucous corion and submucosa with diffuse infiltration of epithelioid cells. Lymphadenopathy with atrophy of T and B lymphocytes areas was observed in the mesenteric lymph nodes. For bacteriological isolation of MAP, the tissue and faecal samples were decontaminated with NALC-NaOH, cultured in Middlebrook 7H9 Broth and on Herrold’s medium. The Ziehl–Neelsen stained smears and isolates were examined microscopically for acid-fast bacteria. Presence of MAP was observed in tissue samples of 4 (25%) mouflon and in 2 (20%) faecal samples. The same samples were confirmed by the IS900 PCR for the presence of specific for MAP fragments with a commercial amplification kit. The cases of paratuberculosis found at different times in the free-living mouflon in our study prove that the disease exists in Bulgaria and highlight the need for more serious control of the disease among wild and domestic ruminants.

Streptococcus pneumoniae are significant causative agents of severe and life-threatening acute pneumonia, meningitis, as well as otitis and sinusitis both in children and elderly. As many as 1.2 million pediatric lethal outcomes due to pneumonia and infections of the central nervous system (meningitis) caused by S. pneumoniae, are recorded worldwide annually, a large proportion of which occur in developing countries. Metal-dependent IgA1 proteases derived from pathogenic bacteria comprise an important group of bacterial enzymes cleaving human immunoglobulin A1 (IgA1) at the hinge region, thereby interfering with fully-executed host antibacterial immunity.

Objective. To study activity of IgA1proteinases and their class profile (Na₂-EDTA and PMSF-inhibited) in various pneumococcal serotypes isolated from nasopharyngeal carrier children.

Materials and methods. There were examined 585 children attending preschool facilities residing in Kazan (n = 331) and rural areas (n = 254). Microbiological, molecular genetics and immunochemical methods were used to identify, serotyping composition and protease activity of Streptococcus pneumoniae isolates. Data statistical processing was carried out by using software Graph Pad Prism version 5.0.

Results. Prevalence of S. pneumonie in pediatric carriers aged 1.5–3 years was 35.1%, 3–5 years — 23.4%, 5–7 years — 19.6%, and over 7 years — 21.9%. Vaccine serotypes 14, 19F, 23F as a part of current pneumococcal vaccines (Prevenar, Pneumavax-23) comprised as high as 55.8%. However, in 19% of cases were positive for non-vaccine S. pneumoniae strains. Non-typeable strains were detected in 5.8% isolates. IgA-proteinase activity was detected in cell lysates of 45 (86.5%) S. pneumoniae strains isolated from pediatric carriers. Cell lysates of S. pneumoniae strains showing no proteolytic properties, were assigned to serotypes 12F, Sg18. Thus, studies on development of alternative vaccines containing immunogenic proteins, adhesins or other virulence factors common to capsulated and non-typeable (encapsulated) pneumococcal strains hold promise. All the aforementioned accounts for a need for microbiological monitoring of S. pneumoniae carriage and search for new diagnostic approaches for etiological interpretation of S. pneumoniae-associated diseases.

Ixodes tick-borne borreliosis is a natural focal transmissive infectious multi-system disease featured with complex pathogenesis, which multiple aspects remain unclarified. The persistence stage during this infection associated with prolonged Borrelia presence in metastatic foci and repeated multiple dissemination is most complicated for clinical practice. It is assumed that the chronic process can be caused by an inadequate immune response associated with activated autoimmune mechanisms leading to emergence of permanent irreversible (degenerative and atrophic) changes in affected organs. Patients who experienced tick-borne borreliosis need dynamic observation for assessing disease prognosis providing a maintenance therapy. The purpose of the study was to evaluate an opportunity of using cellular immunity indices for predicting disease transition to a chronic course.

Materials and methods. The study was carried out at the Medical Association of the Far East Branch of the Russian Academy of Sciences. Case report form data, serum and blood plasma samples collected from 22 patients aged 29–83 years old were examined. Immunological examination data from patients with ixodes tick-borne borreliosis (12–18 months after the onset of acute period) were analyzed. Specific IgM and IgG against Borrelia burgdorferi were determined by using the OMNICS diagnostic test system (St. Petersburg). Lymphocyte immunophenotyping was performed on BD FACSCalibur flow cytometer (Becton Dickinson, USA) by using doublelabeled monoclonal antibodies (Beckman Coulter, France).

Results. A variability of activated peripheral blood lymphocytes was found in patients with tick-borne borreliosis evidencing about individual immune response. An imbalanced cellular immune response recorded in seronegative convalescents from tick-borne borreliosis, may be an indirect finding of ongoing Borrelia infection. Finding of specific serum IgG and IgM antibodies in convalescents at late stage coupled to impaired immune system is a warning sign presuming a risk to developing autoimmune reactions. Detection of specific IgM antibodies at late timepoint combined with increased immune cytotoxic potential may be referred to a marker of possible disease transition to chronic course.

A lead place among pathogens resulting in pediatric chronic bronchopulmonary diseases is held by S. pneumoniae and Haemophilus influenzae. Vaccination against pneumococcal and hemophilic infections is approved and recommended for patients with chronic pathologies, but no clear recommendations for combined use of Pneumo-23 and Act-HIB vaccines in children with congenital pulmonary malformations and bronchial asthma were proposed.

Materials and methods. There were enrolled 92 children aged 0–17 years old with chronic bronchopulmonary diseases; 55 healthy children, and 57 unvaccinated children with chronic broncho-pulmonary pathology were included into control group. Mono- and combination vaccination by Pneumo-23 and/or Act-HIB was performed in remission period. IgM and IgG level against H. influenzae antigens, H. influenzae type b, to S. pneumoniae (serotypes 3, 6B, 9N, 23F) vaccine-specific polysaccharide as well as polysaccharide complex antigens were measured by using ELISA developed by us. Statistical processing was carried out by methods of descriptive, parametric and nonparametric statistics by using Statistica 5.0 software.

Results. Vaccination with Pneumo 23 was accompanied by IgG production against serotypes 3, 6, 9N, 23F-derived polysaccharide. A markedly increased anti-serotype 3 and 23F antibody level was observed 6 months after vaccination. Moreover, a significant increase in anti-polysaccharide 23F and anti-vaccine-derived polysaccharide IgM levels was found 1 month after the onset. In addition, anti-serotype 6B and 9N IgM antibodies were maintained 18 months after vaccination at high level, whereas it was significantly elevated against serotypes 3, 23F. Assessing an effectiveness of vaccine prophylaxis against Hemophilus type b infection, it was shown that a significantly increased anti-vaccine-derived polysaccharide IgM level was found 1, 6, 18 months after Act-HIB vaccination. In addition, IgG to the anti-H. influenzae type b polysaccharide tended to rise 1 month after the vaccination.

Summary. Children with chronic bronchopulmonary diseases vaccinated by Pneumo-23 and Act-HIB demonstrated activated adaptive immunity manifested by increased vaccine-derived antigen-specific IgM and IgG antibodies.

Here we present the current data on the chronic gastritis prevalence in young people. An issue regarding involvement of Helicobacter pylori infection in formation of low bone density is discussed. Examining the features of bone tissue metabolism in infection-associated chronic gastritis to optimize diagnostic algorithm was shown to be of high significance. In the study there were enrolled 200 employees working at the EMERCOM of Russia, who suffered from the acid-dependent gastric diseases and risk factors for reducing bone mineral density. Depending on the bone mineral density parameters, examining functional activity of the gastric mucosa, verification of the H. pylori infection, examining parameters of mineral metabolism, vitamin D level, parathyroid hormone, bone tissue remodeling markers was performed. It was shown that atrophic changes in the gastric mucosa the played a lead role in developing low bone mineral density.

A total of 399 Neisseria gonorrhoeae clinical isolates collected in different regions of the Russian Federation in 20152017 were analyzed for tetracycline susceptibility and genetic markers of resistance. Drug susceptibility testing was performed by serial dilution method in agar and minimum inhibitory concentration (MIC) was measured according to the Russian “Guidelines for microbial susceptibility testing for antibacterial agents No. 4.2.1890-04”. Tetracycline resistance determinants were studied by using hydrogel microarray with immobilized oligonucleotide probes able to identify a series of chromosomal mutations and detect plasmid tetM gene. Different resistance determinants were found in 193 isolates (48.4%). Mutation in codon 57 in the rpsJ gene (41.2%) was most common that decreases tetracycline affinity to ribosome 30S subunit, mainly due to Val57Met substitution both as a point mutation as well as in combination with others. Mutations in the rpsJ gene were found in strains with the intermediate tetracycline susceptibility. Mutations in the porB gene (lower tetracycline influx) held the se cond place in prevalence pattern (23.1%); the Gly120Lys substitution usually led to emergence of tetracycline resistance either as a point mutation or in combination with other substitutions. Substitutions of Gly120 for other residues (Asp, Asn, and Thr) and Ala121 for Asp, Asn, and Gly had much less effect on resistance level. The –35 delA deletion in the promoter region of mtrR gene (increased expression of MtrC-MtrD-MtrE efflux pump) was observed in 11.3% strains. The tetM gene was found in 27 strains including 17 American and 10 Dutch type tetM determinants. Evolutionary tree was constructed for the tetM genes with the estimation of their homology with similar genes in genera Streptococcus, Enterococcus and Mycoplasma. Mutations in chromosomal genes resulted in increase of tetracycline MIC up to 2–4 mg/L; 4 mg/L MIC was observed in case of simultaneous presence of several mutations. Strains bearing tetM gene-containing plasmid showed extremely high resistance level: MIC ≥ 8 mg/L (64 mg/L for the two samples). Thus, long-lasting withdrawal of tetracycline use for treatment of gonococcal infections in Russia (since 2003) resulted in decreased percentage of resistant strains (including strains with intermediate susceptibility) from 75% down to 45.4%. However, currently tetracycline resistance in Russia remains elevated that is explained by the presence of different resistance determinants in the half of isolates under study.

Drug resistant tuberculosis (TB), especially multidrug (MDR) and extensively drug-resistant (XDR) TB, is still a serious problem in global TB control. Slovenia and North Macedonia are low-incidence countries with TB incidence rates of 5.4 and 10.4 in 2017, respectively. In both countries, the percentage of drug resistant TB is very low with sporadic cases of MDR-TB. However, global burden of drug-resistant TB continues to increase imposing huge impact on public health systems and strongly stimulating the detection of gene variants related with drug resistance in TB. Next-generation sequencing (NGS) can provide comprehensive analysis of gene variants linked to drug resistance in Mycobacterium tuberculosis. Therefore, the aim of our study was to examine the feasibility of a full-length gene analysis for the drug resistance related genes (inhA, katG, rpoB, embB) using Ion Torrent technology and to compare the NGS results with those obtained from conventional phenotypic drug susceptibility testing (DST) in TB isolates. Between 1996 and 2017, we retrospectively selected 56 TB strains from our National mycobacterial culture collection. Of those, 33 TB isolates from Slovenian patients were isolated from various clinical samples and subjected to phenotypic DST testing in Laboratory for Mycobacteria (University Clinic Golnik, Slovenia). The remaining 23 TB isolates were isolated from Macedonian patients and sent to our laboratory for assistance in phenotypic DST testing. TB strains included were either mono-, poly- or multidrug resistant. For control purposes, we also randomly selected five TB strains susceptible to first-line anti-TB drugs. High concordance between genetic (Ion Torrent technology) and standard phenotypic DST testing for isoniazid, rifampicin and ethambutol was observed, with percent of agreement of 77%, 93.4% and 93.3%, sensitivities of 68.2%, 100% and 100%, and specificities of 100%, 80% and 88.2%, respectively. In conclusion, the genotypic DST using Ion Torrent semiconductor NGS successfully predicted drug resistance with significant shortening of time needed to obtain the resistance profiles from several weeks to just a few days.

Currently, HIV infection is characterized by emergence of its severe and comorbid forms. Mid-1990 HIV epidemics was expanded due to injection drug users who brought viral hepatitis C to the cohort. Along with developing immunosuppression, opportunistic and AIDS-defining diseases, including tuberculosis emerged. Various combinations of coinfections (HIV infection+tuberculosis±viral hepatitis) affect clinical manifestations and clinical score, reduce the therapeutic efficacy and worsen disease prognosis.

Objective: to study an impact medical and social factors on course of TB-co-infection associated with immunosuppression related to HIV infection and viral hepatitis.

Materials and methods. Comorbid cases (HIV infection, tuberculosis and chronic hepatitis) dominated by verified TB-infection (n = 137) were analyzed.

Results. It was shown that socially maladapted young people with previous experience of intravenous drug and alcohol abuse dominated among subjects with co-infections, half of which were held in penal institutions. More-over, the mean CD4 lymphocyte level in generalized tuberculosis was significantly lower than in patients with significantly reaching 164±21.5 cells. In addition, lung-specific lesions were observed in 73.4% of patients with generalized tuberculosis that developed by initial targeting of the lymphoid system followed by affecting other organs, mainly the central nervous system, urinary system and abdominal organs. Introduction of antiretroviral drugs to anti-TB therapy reduced mortality rate by 8 times. Viral hepatitis was the most common concomitant disease found in co-infected patients, with dominating viral hepatitis C both as a mono-infection (86.8%) as well as in combination with viral hepatitis B (9.43%). In addition, co-morbid viral hepatitis resulted in progression of TB infection affected due to intra-thoracic lymph nodes being involved in tuberculosis process as well as development of opportunistic diseases due to a markedly decreased CD4 cell count. Analysis of potentially aggravating risk factors for developing hepatotoxicity (viral hepatitis, combined treatment with anti-TB and anti-retroviral drugs) did not reveal their any additional negative impact on hepatic functions. Thus, use of a combination therapy with anti-TB and anti-retroviral drugs in co-infected patients was shown to be safe and not accompanied by a high rate of hepatotoxic reactions.

Endogenous uveitis (EU) in children is a multifactorial sight-threatening disease that reduces patient’s quality of life. Proliferative syndrome (PS) coupled to developing adhesions, opacity of vitreous body, epiretinal and preretinal membranes is one of the most serious EU complications, with yet-unknown pathogenesis. Among the numerous trigger factors, a role for infections, particularly human herpes group-driven, is proposed. The goal of the study was to assess a potential role of Herpes simplex viruses type 1 (HSV-1), Herpes simplex viruses type 2 (HSV-2), Epstein–Barr virus (EBV) and Cytomegalovirus (CMV) played in the PS pathogenesis in children with endogenous uveitis. 112 patients aged 3–17 years (mean age 10 years) with (93 patients)/without PS (19 children) were examined. IgM and IgG antibodies (markers of chronic and active infection) against HSV-1/2, EBV and CMV were detected by ELISA. A significantly increased PS rate in infected vs. uninfected children was revealed solely for EBV infection (p = 0.03), but not for HSV-1/2 (p > 0.05) or CMV-positive patients (p > 0.05). However, PS emergence in EBV-negative patients also suggests that some factors might contribute to proliferation in intraocular inflammation. In addition, level of serum IL-8 and IL-6 were assessed by multiplex analysis in 28 children. It was found that IL-8 was detected in all patients, with great individual fluctuations (5.6–2743 pg/ml). Enhanced systemic IL-8 level tended to rise in patients with more prominent proliferation and serological markers of EBV reactivation. However, serum IL-6 was detected by about 2-fold less often reaching up to 55% cases (variation of individual indices 1.3–35.5 pg/ml). A correlation between PS severity, EBV infection activity and systemic IL-6 level was not observed. Further studies evaluating a role of EBV infection in PS pathogenesis pediatric endogenous uveitis are necessary, as it may underlie a rationale for including antiherpetic drugs into a combination therapy.

The sharp rising incidence of tick-borne encephalitis (TBE) in Komi Republic at the North-east of European Russia was recorded last decades. Tick-bite incidence also was grown. Rapid rise of TBE incidence growth and Ixodidae ticks depends on a number of factors, and the impact of climate change being one of them. Ixodes persulcatus ticks is considered as a main vector of TBE in Komi. Our objective is to estimate the influence of air temperature change on the tickbite incidence and Ixodes persulcatus population in Komi. Komi Republic is located near the Polar circle where the northern frontier of Ixodes persulcatus ticks situated and we expected the growth of tick’s population. The number of Komi inhabitants seeking medical care after tick bites in 1992–2014 was considered. Gridded monthly air temperature data with grid size 0.5 degree were recalculated to temperature referred to Komi administrative units. The time series of annual number of tick victims from 1992 till 2014 and model air temperature from 1948 till 2016 for all Komi administrative units were compiled. We analyzed the data on tick-bite incidence in Komi administrative units in relation to changes in local annual average air temperature within the study area. The linear dependence of the tick-bite incidence on air temperature was established when of the tick-bite incidence is represented in logarithm form. The tick population depends not only on temperature but humidity, landcover and hosts. Described areas of Komi belong to humid climate, where precipitations exceed evaporation. Most of the Komi territory is covered by taiga with underwood, grass and bush. Hosts of the first and the major levels are represented by birds and rodents. The dependence of tick-bite incidence and temperature looks like “Malthus’s law”, but the development of population depends on temperature not on time. The exponential growth in the nearest future will ceased and the population will proceed to stable phase. Ticks population in Komi Republic is moving to the North and the air temperature determines the dynamics of population.