Vol 9, No 2 (2019)

REVIEW ARTICLES

Congenitally impaired pattern-recognition receptors in pathogenesis of pediatric invasive and recurrent pneumococcal infection

Tereshchenko S.Y., Smolnikova M.V.

Abstract

Here we review currently available data showing that innate immune signs predisposing to recurrent and invasive pneumococcal infections were identified in children. Streptococcus pneumoniae (pneumococcus) belongs to Grampositive bacteria being the major cause of morbidity and mortality in infants, especially in developing countries and in communities with low socioeconomic status. Due to the lack of anti-pneumococcal vaccination, the significant proportion of pneumococcus carriers develop non-invasive (pneumonia, otitis media, sinusitis) and severe invasive (bacteremia/septicemia, meningitis) pneumococcal infection. A great deal of diverse factors related to pneumococcus biological features (virulence factors) as well individualized host-specific immunity are implicated in efficient bacterial penetration across the mucous membranes. The TLR signaling system plays a crucial role in the human nonspecific defense upon the first encounter with the pathogen. Various TLRs comprise the first pattern recognition receptor fami ly ever described which sense ligands derived from the outer bacterial wall. The complement system is the ancient innate immunity component mainly involved in intravascular elimination of bacterial agents. In addition, the complement proteins serve as a bridge between innate and adaptive immunity, ensuring optimal conditions for B- and T-cell maturation and differentiation. Because pneumococcus secretes the IgA protease, a local protective effects related to IgA antibodies might not be so prominent. Therefore, B-cell immunodeficiency and impaired complement system hold a lead place among congenital causes resulting in severe and recurrent pneumococcal infections in children. Thus, based on available data, we concluded that impaired B-cell function, the complement components deficiency as well as receptor-recognition receptors (TLR-2, -9, -4, MYD88 adapter protein, TLR cascade enzymes: IRAK4, NEMO, NOD-like receptors: NOD2, NLRP3; C-type lectins: MBL, Dextin-2, and, possibly, ficoline) play the most important role among congenital immunodeficiencies predisposing to invasive and recurrent pneumococcal infections play the most important role among congenital immunodeficiencies predisposing to invasive and recurrent pneumococcal infections, and should be used as a rationale for immunological surveillance and organizing immunogenetics screening in these patients. 

 

Russian Journal of Infection and Immunity. 2019;9(2):229-238
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Dendritic cells in the pathogenesis of viral hepatitis C

Chernykh E.R., Oleynik E.A., Leplina O.Y., Starostina N.M., Ostanin A.A.

Abstract

Infection with hepatitis C virus (HCV) is a public health problem; it establishes a chronic course in most (up to 85%) infected patients and increases the risk for developing liver cirrhosis, hepatocellular carcinoma, and severe extrahepatic manifestations. The mechanisms of HCV persistence are largely related to the inefficient antiviral response of the host immune system. The effective clearance of the virus requires early activation of innate immune system together with the induction of a strong multiepitopic adoptive T cell response and long-term antiviral memory. Dendritic cells (DCs), which represent a heterogeneous population of antigen-presenting cells, contribute to the production of type I interferon, activate natural killer cells and induce adoptive immune response thus playing a major role in antiviral defense. In this case, DCs dysfunction in HCV-infection is considered to be the one of the mechanism that allows the virus to escape from the immune surveillance. The present review  includes current data focusing on the role of DCs in the anti-HCV immune response and highlights a number of key issues related to the phenotypic and functional changes of various DC subpopulations in HCV-infection, the mechanisms of DC impairments and the prospects for treatment of chronic hepatitis C based on the use of ex vivo generated DCs.
Russian Journal of Infection and Immunity. 2019;9(2):239-252
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Mechanisms of interacting Helicobacter pylori with gastric mucosal epithelium. II. A reaction of gastric epithelium on Helicobacter pylori colonization and persistence

Pozdeev O.K., Pozdeeva A.O., Valeeva Y.V., Gulyaev P.E., Savinova A.N.

Abstract

Gastric and duodenal recurrent inflammatory diseases have a high prevalence, but the role played by microbes in its development remained unclear. However, the data published in 1983 by Marshall and Warren about isolating Helicobacter pylori from the stomach mucosa of the patient with gastritis and proposing relevant cultivation methods was the turning point in investigating etiology of the upper digestive tract inflammatory disorders. Moreover, it was shown that the majority of H. pylori spp. are found within the gastric lumen upon colonization, whereas around 20% of them are attached to the epithelial cells in the stomach. In addition, effects of interacting H. pylori with gastric epithelium and activation of some defense mechanisms due to bacterial colonization and spreading were analyzed. It was found that along with triggering pro-inflammatory response induced by proteins VacA as well as phosphorylated/unphosphorylated CagA, wherein the latter is able to induce a set of protective reactions H. pylori disrupts intercellular contacts, affects epithelial cell polarity and proliferation, and activates SHP-2 phosphatase resulting in emerging diverse types of cellular responses. The activation mechanisms for the mitogen-activated protein kinase (MAPK) pathway were discussed. The ability of H. pylori to regulate apoptosis, particularly via its suppression, by expressing ERK kinase and protein MCL1 facilitating bacterial survival in the gastric mucosa as well as beneficial effects related to bacterial circulation on gastric epithelial cell survival elicited by anti-apoptotic factors were also examined. Of note, persistence of H. pylori are mainly determined by activating transcriptional factors including NF-κB, NFAT, SRF, T-cell lymphoid enhancing factor (TCF/LEF), regulating activity of MCL1 protein, in turn, being one of the main anti-apoptotic factors, as well as induced production of the migration inhibitory factor (MIF). The role of VacA cytotoxin in triggering epithelial cell apoptosis via caspase-mediated pathways was also considered. Infection with H. pylori is accompanied by release of proinflammatory cytokine cocktail detected both in vitro and in vivo. In particular, bacterial urease activating transcriptional factor NF-κB was shown to play a crucial role in inducing cytokine production. Moreover, such signaling pathways may be activated after H. pylori is attached to the cognate receptor in the gastric epithelial surface by interacting with CD74 and MHC class II molecules. Finally, a role for various CD4+ T cell subsets, particularly type 17 T helper cells (Th17) in inducing immune response against H. pylori antigens in gastric mucosa was revealed were also discussed. 

Russian Journal of Infection and Immunity. 2019;9(2):253-261
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ORIGINAL ARTICLES

Features of humoral answer in experimental animal tularemia with different sensitivity to infection

Gorbatov A.A., Titareva G.M., Kombarova T.I., Shaikhutdinova R.Z., Kravchenko T.V., Mironova R.I., Bakhteeva I.V., Aronova N.V., Pavlovich N.V., Mokrievich A.N., Firstova A.A.

Abstract

Tularemia is an anthropozoonotic infection caused by Francisella tularensis. In clinical and sanitary-epidemiological practice, traditional diagnostics methods in tularemia are based on serological assays for detecting specific antibodies, allowing to diagnose it and estimate durability of patients’ immunity after vaccination. Previously, it was shown that specific serum antibodies in patients recovered after tularemia, unlike to those vaccinated with the live tularemia vaccine F. tularensis 15 NIIEG, can interact with specific epitopes on lipopolysaccharides isolated from strains of various subspecies — F. tularensis (Ft) and F. novicida (LPS Fn), while LPS Fn-specific immunoglobulins are lacked in the blood of vaccinated individuals. A set of experiments on identifying antibodies with similar specificity in laboratory animals of various species — mice, guinea pigs and rats with differed sensitivity to tularemia, administered with live tularemia vaccine strain as well as virulent F. tularensis strains to simulate vaccine-mediated and infectious processes, respectively was conducted. A methodical approach has been developed that allows to analyze humoral response in modelled infectious process in animals highly sensitive to tularemia such as BALB/c mice and guinea pigs that consisted of preliminary immunization with live tularemia vaccine followed by infection with virulent F. tularensis strains. It was shown that induction of specific anti-LPS Ft antibodies occured in these animal species, both after vaccination and infection with virulent strains. It was noted that, unlike guinea pigs and rats, mice both during vaccination and infection were characterized by significantly lower titers of LPS Ft-specific antibodies. However, no specific interaction between mouse serum and LPS Fn might be detected. Moreover, two types of immunoglobulins with different antigen specificities to the LPS Ft and LPS Fn epitopes were detected by dot-blot analysis in guinea pigs immunized with live tularemia vaccine, followed by infection with a virulent strain. In addition, antibodies to LPS Fn were also detected in the serum of rats infected with virulent, but not vaccine-based, F. tularensis strains. Thus, previous experimental data on the production of immunoglobulins with different antigenic specificity were confirmed in an experimental tularemia modelled in rats and guinea pigs that demonstrated a diagnostic significance and feasibility of using LPS Fn to confirm tularemia infection in humans. 

Russian Journal of Infection and Immunity. 2019;9(2):262-272
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Comparative efficacy and safety of preventive treatment with cytovir-3 and arbidol in children during seasonal outbreak of respiratory viral infection (an open-label randomized clinical study)

Smirnov V.S., Saveliev S.A., Petlenko S.V., Redlich G., Erofeeva M.K., Lyovina A.V., Zavialova N.I.

Abstract

Aim: To compare the safety and prophylactic efficacy of Cytovir-3 and Arbidol in an open-label randomized clinical trial in children at seasonal risk of infectious respiratory disease.

Methods: This study was performed in the period preceding a seasonal outbreak of acute respiratory viral infections (ARVI) and influenza. The study enrolled apparently healthy children aged 6 to 18 years without contra-indications to either Cytovir-3 or Arbidol. Cytovir-3 was given as 1 capsule per day, each day in a fasted state for 12 days. Arbidol was taken as a 100 mg capsule twice a week for three weeks. Following dosing, subjects were followed-up for a further three weeks. Overall health and presence of symptoms of either ARVI or influenza were monitored daily in all subjects. The level of sIgA in saliva was baselined before treatment and measured at the end of the follow-up period.

Results: Most subjects did not exhibit any change in overall health status during the dosing and follow-up periods. The first two cases of illness in subjects receiving Cytovir-3 were diagnosed at the end of the third week after completion of dosing. In those subjects receiving Arbidol the first cases of illness were reported at the end of the second week following the end of prophylactic dosing. All patients displayed a significant increase in sIgA level at the end of the dosing period, but this was more pronounced in the Cytovir-3 group. 

Conclusion: The results of this comparative randomized clinical trial confirmed the safety and tolerability of both medications, and that the prophylactic efficacy of Cytovir-3 is equal to that of Arbidol.

Russian Journal of Infection and Immunity. 2019;9(2):273-278
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Hepatitis B virus and site-specific nucleases: effects of genetic modifications in CRISPR/Cas9 on antiviral activity

Kostyusheva A.P., Brezgin S.A., Zarifyan D.N., Chistyakov D.S., Gegechkory V.I., Bayurova E.O., Volchkova E.A., Kostyushev D.S., Chulanov V.P.

Abstract

Chronic hepatitis B is a severe liver disease caused by persistent infection of hepatitis B virus in human hepatocytes. Chronic hepatitis B is one of the most common diseases in the world. According to recent estimations, more than 250 million people are chronically infected and more than 1 million of people die annually due to consequences of chronic hepatitis B: liver cirrhosis and hepatocellular carcinoma. The key factor of hepatitis B virus persistency is a special form of viral genome called circular covalently closed DNA. Current therapeutics suppress viral replication but have no effect on circular covalently closed DNA as it exists in the nuclei of hepatocytes as a minichromosome and is not accessible for therapeutics. Commonly, viral reactivation occurs after cessation of treatment. Therefore, duration of antiviral treatment is supposed to be indefinitely long. One of the most promising approaches to target circular covalently closed DNA is the technology of site-specific nucleases CRISPR/Cas9 from Streptococcus pyogenes. A short guide RNA recruits an SpCas9 protein to the viral genome and induces generation of DNA double strand breaks. However, there are several limitations of CRISPR/Cas9 hampering translation of this technology into the clinic. First, efficacy of CRISPR/Cas9 needs to be improved. Second, CRISPR/Cas9-mediated off-target mutagenesis represents a menacing problem which has to be addressed. To overcome these limitations, several approaches have been devised to improve CRISPR/Cas9 activity (modification of guide RNAs) and reduce off-target mutagenesis (a Cas9 protein with enhanced specificity, eSpCas9). In this study, we compared antiviral activity of a classic SpCas9 with an eSpCas9 system as well as analyzed effects of gRNAs modification on anti-HBV effects. Here, we demonstrated that SpCas9 has the highest antiviral potency, reducing transcription and replication of HBV over 90%. Hepatitis B virus covalently closed circular DNA declined over 90% post CRISPR/Cas9 transfection. Although it was previously shown that modified guide RNAs increase nucleolytic activity of CRISPR/Cas9, our results indicated that this modification impairs antiviral activity of CRISPR/Cas9. To conclude, CRISPR/Cas9 effectively suppress viral replication and transcription per se. Described modifications do not potentiate antiviral activity of CRISPR/Cas9 system and should not be used for development of future therapeutics. The best strategy to improve CRISPR/Cas9 efficacy is to design new highly effective guide RNAs. 

Russian Journal of Infection and Immunity. 2019;9(2):279-287
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A role of impaired neonatal T cell activation upon late CMV detection

Kravchenko L.V.

Abstract

Objective: examine signs of impaired neonatal T cell activation upon late cytomegalovirus (CMV) DNA detection in the blood and urine.

Materials and methods. 147 neonates with non-specific clinical symptoms were enrolled to the study. Peripheral blood lymphocyte subsets were immunophenotyped by using anti-human antibody panel to stain for surface markers CD3, CD4, CD8, CD20, CD3+CD28-, CD3+CD28+, CD3CD28+, CD20+CD40+, CD28, CD40 (IMMUNOTECH, France) for further analysis on a laser flow cytometer Beckman Coulter Epics XL II. CMV infection in 123 neonates was verified by PCR, whereas it was negative in 24 cases. Further, 24 children aged 1.5–3 months negative for CMV DNA during the first month of life were later shown to contain it in the blood and urine samples as well as elevated anti-CMV IgG antibodies allowing to verify CMV infection.

Results. It was found that upon late CMV DNA detection, amount of CD3+CD28+ T cells in peripheral blood was directly related to count of total CD3+ T cells regardless of CD28 expression. Moreover, the peak percentage of CD3+CD28+ (70–80%) T cells ranged within 85–90%. Whereas children upon early CMV detection the maximum percentage of CD3+CD28+ T cells was associated with lowered CD3+ T cell count (45–55%), it was also paralleled with high CD28 expression (higher than 3%). Importantly, T cells co-expressing CD28+ activation marker necessary for co-stimulation represent one of significant parameters used in diagnostics. Down-modulated CD28 expression abrogating costimulatory signaling and resulting in T cell anergy contributes to developing immunological failure in neonatal CMV infection. The data obtained confirmed importance of cell-cell immune and non-immune interactions for executing anti-CMV immune response within the first months of life. Finally, it is proposed to predict developing CMV infection depending on percentage of CD3+ and CD28+CD3+ T cell subsets in neonates manifested with non-specific clinical signs upon late CMV DNA detection. 

Russian Journal of Infection and Immunity. 2019;9(2):288-294
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Chemokine receptor expression on peripheral blood T-helper cells in Helicobacter pylori-associated diseases: chronic gastroduodenitis and peptic ulcer disease

Talayev V.Y., Talaeyva M.V., Voronina E.V., Zaichenko I.Y., Neumoina N.V., Perfilova K.M., Babaykina O.N.

Abstract

Helicobacter pylori represents a pathogen causing chronic infection in around a half of the global human population, which manifestations vary from asymptomatic infection to developing gastritis and peptic ulcer. The data accumulated suggest that overt clinical types of this infection are associated with lost immunoregulation and increased pro-inflammatory cell-mediated immune response triggered by H. pylori-specific T helper cells. Here, we examined the degree of peripheral blood CD4+ T cell maturity and related expression of chemokine receptors involved in migration to gastrointestinal tract (CCR9 and CCR6), as well as Тand B-cell zones of lymphoid organs (CCR7 and CXCR5). It was shown that overt H. pylori-infection was coupled to changes in expression pattern of chemokine receptors on T helper cells. In particular, percentage of mature CD4+CD45RO+ T cells bearing CCR9 and immature CD4+CD45RO Т cells expressing CXCR5 was increased in peripheral blood of patients with chronic gastroduodenitis. However, increased amount of activated mature CD4+CD45RO+ICOS+ T cells was observed in patients with chronic gastroduodenitis comorbid with peptic ulcer that was also associated with elevated amount of mature CCR6+ T helpers (mainly CD4+CD45RO+CCR7 CCR6+ cells) and follicular T helper cells as well as emerging minor CD4+CD45RO+CXCR5+CCR6+.T cell subset, not affecting CD4+CCR9+ Т cells. Thus, the data obtained evidence that tissue-specific T-helper cell migration is controlled separately in of H. pylori-associated diseases. 

Russian Journal of Infection and Immunity. 2019;9(2):295-303
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Altered peripheral blood Th17 and follicular T-helper subsets in patients with pulmonary tuberculosis

Kudryavtsev I.V., Serebriakova M.K., Starshinova A.A., Zinchenko Y.S., Basantsova N.Y., Belyaeva E.N., Pavlova M.V., Yablonskiy P.K.

Abstract

Tuberculosis (TB) is one of the most common infections worldwide. Eradication of an intracellular pathogen M. tuberculosis requires to induce a Th1 response by activating IFNγ-producing tissue macrophages. Along with Th1 cells, various subsets of Th17 and follicular T-helper cells (Tfh) able to secrete a broad range of cytokines, including IFNγ, can also be involved in eliminating bacterial pathogens. It justified analyzing in this study changes in percentage of various peripheral blood Th subsets, including Th1, Th2, Th17 and Tfh cells, in TB patients. For this, major CD3+CD4+ T cell subsets were assessed by using multicolor flow cytometry in TB patients (n = 40) and healthy volunteers (n = 30). It was found that in TB patients vs. control group percentage of peripheral blood CD45RACCR7+ central memory (CM) Th was decreased also affecting frequency of some functional T cell subsets, e.g. either lowering Th2 cells (9.11% (6.95; 13.77) vs. 7.21% (5.64; 9.84), p = 0.012) or elevating CCR6+ Th17 subsets (35.92% (27.72; 41.06) vs. 40.39% (35.41; 47.79; p = 0.016), respectively, but not influencing Th1 and Tfh subsets frequencies. Moreover, percentage of total CCR6+ CM Th cells in TB patients vs. control was decreased in CCR4CXCR3+ Th17.1 cell subset (42.87% (33.64; 49.45) vs. 52.26% (46.45; 56.95), p < 0.001), whereas standard CCR4+CXCR3 Th17 and CCR6+ DP Th17 subsets were elevated (p = 0.005 and p = 0.002, respectively). In addition, altered Tfh subset composition associated with the increased (p = 0.021) percentage of CXCR3–CCR6 Tfh2 cells, but decreased CXCR3+CCR6 Tfh1 cells (p = 0.036) was observed. Finally, frequency of peripheral blood Th subsets noted above was also analyzed within effector memory (CD45RACCR7) cells. It was found that in TB patients vs. volunteers frequency of Th17.1 cells was also significantly lower (p = 0.006) in CCR6+ EM Th (54.43% (41.19; 91.92) vs. 61.76% (54.01; 65.63), whereas percentage of double-positive Th17 was significantly increased (20.83% (15.12; 30.87) and 12.93 % (9.80; 19.01), respectively, p < 0.001). Thus, it suggests that during M. tuberculosis infection percentage of IFNγ-producing Th17 and Tfh cells was reduced compared to control group also affecting both central memory Th cells patrolling peripheral lymphoid organs as well as effector memory Th cells able to exit to site of infection. 

Russian Journal of Infection and Immunity. 2019;9(2):304-314
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An association between parameters of Th1 and Th2 cell-related functional activity and HLA gene polymorphism in individuals after anti-plague vaccination

Kudryavtseva O.M., Bugorkova S.A., Shchukovskaya T.N., Mikshis N.I., Goncharova A.Y., Klyueva S.N., Shcherbakova S.A.

Abstract

In theRussian Federation, Y. pestis NIIEG strain-based live attenuated vaccine is used for immunization against plaque on epidemiological indications, displaying high efficiency. However, individual fluctuations in adaptive immunity after vaccination necessitate conducting a search for genes underlying variability of developing immune response. Of note, HLA (Human Leukocyte Antigen) gene polymorphism plays an important role in this process. In our study, we identified HLA class II haplotypes for HLA-DRB1, HLA-DQA1, and HLA-DQB1 in 120 residents of theterritoryofPre-Caspiannatural plague focus, who were immunized against plague. In addition, level of TNFα production correlated with detecting allelic groups HLA-DRB1*04 (р = 0.05) and DRB1*12 (р = 0.01). The data obtained show that HLA class II gene polymorphism can affect the level of cytokine secretion in response to plague immunization. Examining the genes regulating immune factor production will allow to get better insight into the mechanisms underlying immune response variations after vaccination as well as contribute to predicting immunogenicity and efficiency of developing vaccine preparations. 

Russian Journal of Infection and Immunity. 2019;9(2):315-324
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Measles vaccination of children born to HIV-infected mothers (clinical and immunological aspects)

Snegova N.F., Kostinov M.P., Pakhomov D.V., Ilina N.I.

Abstract

Vaccination of children born to HIV-infected mothers in accordance with the national Calendar of preventive vaccinations represents an important issue. Upon that, measles incidence rate especially among unvaccinated children has been substantially elevated. Use of live measles vaccine in both HIV-infected children and mothers has some features primarily due to HIV-associated immunocompromised state that was poorly investigated. Here we monitored 212 children aged 12–36 months born to HIV-infected women, 184 HIV-negative children born to HIVinfected women who were included into the group with the diagnosis “Perinatal HIV contact” (R75) after verification. In addition, 28 children were included into the group” HIV infection “(B23), whereas 42 children — into the control group. Live measles vaccine was administered subcutaneously at a dose of 0.5 ml. All children were followed up for 36 weeks. HIV status, measles antibodies level by measured by ELISA and RPG methods, as well as total immunoglobulin A, M, G classes and CD3+, CD4+, CD8+, CD19+ lymphocyte counts were examined. No post-vaccination complications or severe vaccine-induced reactions were reported in HIV-infected patients group. A satisfactory course of the vaccine process in all children born to HIV-infected mothers was observed that did not significantly differ from that one in control group. Children with perinatal HIV contact are able to respond adequately to vaccination with live measles vaccine. The number of children with conditionally protective antibody levels, still remaining seronegative among children with perinatal HIV contact (R75) was comparable to that one in healthy control group. At the same time, children with perinatal HIV contact were shown to display protective antibody level after administering live measles vaccine only in 36.4% of cases, whereas the remainder (63.6%) demonstrated a conditionally protective antibody level or lack it (compared to 76% of children with perinatal HIV contact). Children with perinatal HIV infection who did not respond to the first administered live measles vaccine produced low amount of antibodies after re-vaccination. Seroconversion was observed in up to 68% of cases, whereas protective antibody level was found in up to 33.6% of child ren. On the contrary, children from the perinatal HIV contact group were shown to have protective antibody level in 69.6% of cases upon seroconversion rate reaching 91.3% that did not differ them from those in control group. In the post-vaccination period, suppressive modality of immune reaction tended to increase indirectly evidenced by increased percentage of CD8+ T cell subset. 

Russian Journal of Infection and Immunity. 2019;9(2):325-336
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Assessing natural herd immunity to tick-borne encephalitis in Republic Sakha (Yakutia)

Dragomeretskaya A.G., Ignatyeva M.E., Trotsenko O.E., Mzhelskaya T.V., Budatsirenova L.V., Grigoryeva V.I., Romanova A.P.

Abstract

Tick-borne encephalitis (TBE) is a natural focal viral neuroinfection vastly spread in the mid-latitude climate zone of theEurasia. Lethal cases and disability related to TBE determine its high epidemiological significance as well as underlie undiminished attention to prevent it in endemic territories. At the same time, TBE epidemiologic features significantly differ in various geographic areas. Importantly, Republic Sakha (Yakutia) is not among TBE endemic areas in theRussian Federation. However, in the last decade an increased incidence of ixodic tick bites was registered in the Republic Sakha. A pattern of pathogen-specific population immunity is one of the most valuable criteria for assessing magnitude of epidemic process in TBE foci. Hence, our study was aimed at assessing natural herd immunity TBEV in Republic Sakha associated with elevated incidence of tick bite visits. Here, we analyzed the data regarding the rate of tick attacks in the period of 2001–2007 in Republic Sakha. Residents and individuals unvaccinated against TBEV in the city ofYakutskas well as various administrative regions were examined to record the peak incidence of tick attacks. It was found that a range of the administrative regions recording with registered of ixodic tick attacks and elevated incidence of tick bites was expanded. Moreover, the top incidence of tick bites was annually recorded in the south regions of the republic Aldanian, Neryungri, Olyekminsk, Leninsk, Khangalassky districts, city ofYakutskand its suburbs. Furthermore, TBEV-specific immunoglobulins were detected in 5.7±0.68% of the residents examined suggesting about contacts with the pathogen. In addition, the peak count of seropositive people (8.9±1.85%) was identified in Namsky district located in the central part of the republic. Overall, detection rate of TBEV-specific antibodies among general adult population (6.9±0.95%) was significantly higher compared to those observed in children (3.9±0.89%, р < 0.05). no significant differences in urban (5.8±1.12%) vs. rural population (5.63±0.84%) was found. 

Russian Journal of Infection and Immunity. 2019;9(2):337-346
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Experience of investigation the microflora and ejaculate proteins with different ultrasound picture of prostate

Godovalov A.P., Danielyan T.Y., Karpunina T.I., Vavilov N.V.

Abstract

At present, there are practically no studies deciphering the mechanisms of the possible influence of opportunistic pathogens on the morphological changes in the prostate gland. Many authors doubt that bacteria are the cause of chronic prostatitis. In clinical practice with prostate diseases preference is given to ultrasound examination as a reliable and time-consuming diagnostic test. At the same time, many authors note that for the diagnosis of inflammatory diseases of reproductive organs, the study of relevant secrets is an actual and promising scientific direction.

The aim of the study was the investigation of qualitative and quantitative composition of microflora, a number of ejaculate proteins against the background of a different echoscopic picture of prostate.

Materials and methods. The study included 18 men was in infertile couples for more than 3 years with a previously diagnosed chronic prostatitis (N41.1, observation group), and 28 healthy volunteers (comparison group). All participants in the study performed transrectal ultrasound. In the ejaculate, the concentrations of immunoglobulins of the main classes, total protein and albumin, as well as the level of oxidative modification of proteins were determined. Microbiological examination of the ejaculate was performed according to generally accepted methods. For statistical evaluation of the data obtained, an unpaired version of Student's t-test was used. Differences were considered statistically significant at p<0.05.

Results. Microflora of the ejaculate in observation group was represented mainly by gram-positive cocci. In 39% of cases, the growth of Gram-negative microflora was established, from which Escherichia coli was isolated in 71% of the samples. In most samples, microorganisms were in the association, which is more typical for diffuse changes in prostate tissue. In cases with fibrosis, a noticeable narrowing of the spectrum of the released microflora was recorded: from Gram-negative bacteria only E. coli was isolated, and Gram-positive microorganisms were absent altogether. It was shown that the development of fibrosis in the prostate gland tissue is accompanied by an increase in the concentration of total protein and albumin in the ejaculate. When the process goes to the calcification stage, the levels of these indicators decrease. It was found that in all cases, with the exception of severe fibrosis, the concentration of IgG increases and IgA decreases. The development of fibrosis is accompanied by a decrease in the level of IgG. In the present study, in determining the level of oxidative modification of proteins, a decrease in the concentration of such molecules was found in patients of observation group.

Discussion. In general, chronic inflammation in prostate of men of reproductive age in more than 60% of cases was characterized by fibrosis of the organ tissue and/or the formation of calcifications. In such situation, conditions are created for the persistence of opportunistic pathogenic microflora in fibro-modified tissue beyond the reach of immune system factors. In addition, there are phenomena of secondary immunodeficiency at the local level, manifested in a decrease in the number of leukocytes and their radical-producing function. That is why we consider the detection of conditionally pathogenic microorganisms as an essential fact of the pathogenesis of prostatitis, especially in the case of fibrosis and the formation of calcifications. Thus, the qualitative and quantitative study of microflora, the presence and degree of modification of the main protein components of the ejaculate, to a greater or lesser extent reacting to infectious inflammation, is primarily an asymptomatic one, broadens the concept of the pathogenesis of chronic prostatitis. As an addition to the ultrasound, its results allow us to clarify the duration of the inflammatory process, and to significantly decipher the echographic changes recorded at different stages of the development of the disease.

Russian Journal of Infection and Immunity. 2019;9(2):347-353
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The problem of pertussis in some regions of the world

Basov A.A., Tsvirkun O.V., Gerasimova A.G., Zekoreeva A.K.

Abstract

Pertussis infection remains a high-priority issue both for Russian health care and abroad. A rise of pertussis incidence in various human age groups instigates a search for new ways to fight this infection and improve methods for its laboratory diagnostics. By taking into consideration a short-term effect induced by acellular and whole-cell vaccines, a feasibility of introducing the second or even the third pertussis revaccination is vigorously debated. Objective of the study was to analyze the experience and effectiveness of acellular pertussis vaccines in countries, which use the second and third pertussis revaccination in the National Immunization Schedule in order to have an insight into adjusting strategy and tactics of pertussis immunization In Russia. Analyzing pertussis prevalence demonstrated that despite a wide immunization coverage pertussis incidence in the last years (2008–2015) was increased in a large number of countries in the European region, as well as inAustralia,CanadaandUSA. However, the reasons for elevated pertussis incidence have not been clarified yet. On one hand, it may be accounted for by low vaccination coverage in adolescents and adults; weakened immune protection after vaccination; genetic changes in Bordetella pertussis; shortened durability of protective immunity in children vaccinated with acellular vs. whole-cell vaccine; improved monitoring and morbidity reporting, as well as improved laboratory diagnostics due to shifting from serological and bacteriological to molecular genetic assays. In an attempt to solve this issue, researchers from several countries collaborate to discuss and develop a strategy to reduce pertussis incidence. ForRussia, the most important is to empower and/or improve existing infant immunization strategy in order to provide wide coverage with the four dose pertussis vaccine for decreasing the risk of pertussis morbidity and mortality. It is worth noting the “cocoon” strategy given the high risk of pertussis infection in children of the first months of life. We believe that forRussiait is worth investigating an opportunity of using children 2–3 months of life an acellular vaccine as the first vaccination, which is expected to increase the coverage of this cohort and allow to increase proportion of children who might complete vaccination by 5 months of age. At the same time, more reasonable might be to preserve a number of age groups for pertussis vaccination in the current National Immunization Schedule, as expanding age limits for vaccination might put a risk at increasing pertussis morbidity in older individuals, which could be hard to diagnose. 

Russian Journal of Infection and Immunity. 2019;9(2):354-362
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SHORT COMMUNICATIONS

Bioregulation of amplitude-phase biological activity of Candida albicans by women reproductive tract microsymbionts

Nikolenko M.V., Baryshnikova N.V., Bakhlykova E.A., Zykova A.V., Romanchenko K.V.

Abstract

In this study, we propose a chronobiological method for examining inter-microbial interactions in bacterial and fungal associations in female reproductive tract. Fungal and bacterial species were isolated in 45 women of reproductive aged 19–35, with regular menstrual cycle, applying no hormonal contraceptives, without previous gynecological surgery, abortions, miscarriages with vaginal eubiosis and dysbiosis in history. Sexually transmitted diseases (HIV infection, syphilis, gonorrhea, trichomoniasis, chlamydiosis) were excluded in all subjects. Proliferation rate, morphogenesis and phospholipase activity were examined within the 48-hour period every 4 hours, in winter time, Moon phase IV. The data obtained were assessed by using Student’s t-test, Wilcoxon test, and least squares method. All subjects were divided into the groups: group 1 — women with vaginal eubiosis, group 2 — women with vaginal dysbiosis. It was shown that in all subjects experimental parameters of C. albicans cultures showed a diurnal dynamics characterized in healthy women by circadian rhythms with a single peak of activity. However, in women with vaginal dysbiosis C. albicans was characterized by significant ultradian (around 12 hours long) rhythms with two peaks of biological activity. Concurrence and consistency in manifested physiological functions related to clinical isolates was coupled to temporal pattern of distributed biological resources in fungi depending on course of infectious process. It was found that in vaginal eubiosis exometabolites released by dominant associated microbiota did not significantly change microbiota-related amplitude-phase parameters. The data obtained evidenced that temporal pattern of parameters related to C. albicans from healthy individuals was stable and independent on bacterial metabolites. In contrast, dominant microsymbiont in vaginal dysbiosis inhibited fungi-related rhythms, which might be important in establishing lactobacillus-associated biotope colonization resistance. Effects of metabolites released by the associated microbiota typical to dysbiosis was revealed by increased mesor, amplitude, preserved biorhythm spectral pattern in examined properties as well as amplitude-phase characteristics indicating at enhanced or sustained C. albicans adaptive potential. Therefore, the amplitude-phase parameter of C. albicans physiological activity served as a marker of opposite (enhanced/weakened) effect of microsymbiont survival described in “microbial dominant-associate” pairs. 

Russian Journal of Infection and Immunity. 2019;9(2):363-368
pages 363-368 views

Inflammation induced by different plasmid types of russian Yersinia pseudotuberculosis strains

Somova L.M., Shubin F.N., Drobot E.I., Lyapun I.N., Plekhova N.G.

Abstract

In the 2000s, a scientific interest to the Far Eastern scarlet-like fever (FESLF) mainly recorded In Russia and Japanwas remarkably increased. Such clinical and epidemic manifestation of human pseudotuberculosis is related to a certain bacterial clonal lineage characterized by a specific plasmid profile (pVM82, pYV48), sequence type (2ST) as well as the yadA gene allele (1st allele). In our study we examined features of inflammatory changes characterizing plasmidassociated pathogenicity of the FESLF pathogen. In addition, organ histopathology in experimental animals infected intraperitoneally with Y. pseudotuberculosis strains of the four plasmid types 48+:82+; 48+:82-; 48-:82+; 48-:82-; and 48-:82- was investigated. It was found that the mortality rate in animals infected with Y. pseudotuberculosis H-5015 strain (82+:48+) bearing two plasmids with a molecular weight of 82 and 48 MDa was 40%. A picture of diffuse pneumonia with moderate inflammatory infiltration in pulmonary tissue and changes in lymphoid organs characterizing immunodeficiency we observed as early as 3 days postinfection (p.i.). On the contrary, animals infected with Y. pseudotuberculosis H-5015 strain (82+:48–) bearing a single plasmid 82 MDa pVM, mortality rate was 30%. A vascular reaction in the lungs and liver as well as deteriorated vascular destructive changes were revealed starting from day 3 and on day 5–7 days p.i., respectively, which was paralleled with perivascular infiltration mainly by mononuclear cells and focal pneumonia as well as a reaction of bronchialassociated lymphoid tissue and minimal organ destructive changes. On day 7 p.i., signs of granulomatous inflammation in the liver in a form of small mononuclear cell clusters and perivascular compact infiltrates were found. At all time points, lymphoid organ hyperplasia was noted. In case the infection caused by H-5013 strain (48+), the mortality rate was 80%. Inflammatory changes with dominant mononuclear cells were detected as early as 1 day p.i. associated with a picture of large focal bronchopneumonia, more pronounced in the non-survivor animals, also demonstrating signs of severe immunosuppression in the lymphoid organs. Starting from day 3 p.i., lymphoid hypoplasia in the spleen and lymph nodes was detected in all infected animals paralleled with pathogen-associated tissue damage in various organs. Animals infected with the plasmid-free H-5013 strain 48-) resulted in 25% mortality rate. Moreover, starting from day 3 p.i., a slight mononuclear inflammatory infiltration in the lungs and liver, a moderate giant cell reaction in the splenic pulp, and loose perinodal inflammatory infiltration in the lymph nodes were observed. Thus, while modeling infection caused by different plasmid types of Y. pseudotuberculosis, the data on differences in histopathology of changes in diverse organs regarding dynamics and generalization of the inflammatory response, as well as the severity of pathogen-associated damage in the lymphoid tissue were obtained. In case Y. pseudotuberculosis strains contained pVM82 plasmid with or without virulence plasmid pYV vs. single pYV-positive strains, an area of the inflammatory response as well as severity of immunosuppression manifested by splenic and lymph node delymphatization were decreased. It allowed to suggest that pVM82 plasmid found In Russia-originating Y. pseudotuberculosis strains might be implicated in limiting intensity of inflammatory response, bacterial dissemination and severity of lymphoid organ damage. 

Russian Journal of Infection and Immunity. 2019;9(2):369-374
pages 369-374 views

Characteristics of acute diarrhea adult cases with samples positive for Norwalk virus

Kozhukhova E.A., Gorbova I.V.

Abstract

Remaining unmanageable,Norwalkvirus infection is clearly tended to be recorded at higher rate, including adult patients. In many cases, clinical picture of adult acute diarrhea in patients positive for Norwalk virus in clinical samples vs. pathogenetically-caused norovirus infection differs, thereby justifying comparison of clinical and laboratory data. A cohort retrospective study with 146 hospitalized adult patients suffering from acute moderate diarrhea positive for fecalNorwalkvirus was performed. Along with standard laboratory tests (culturing, serologic for detecting 4-fold increase in titer between paired serum samples, ELISA), detection of diarrhea-linked agents included PCR kit Amplisens® AII-bacto-screen-FL Lab (Interlabservice). The data obtained demonstrated that in adult patients with acute diarrhea 54.1% of cases were positive forNorwalkvirus as well as for other enteric pathogens, including bacteria found in 36.3% of cases. Moreover, clinical samples of patients with acute diarrhea hospitalized at least on day 4 vs. day 3 after the onset were significantly more often (by 1.5-fold) positive for association between Norwalk virus and other acute enteric infection agents mainly due to astrovirus (р = 0.03; PCR data) and Shigella spp. (culture-based and serologic methods; р = 0.03). In addition,Norwalkvirus was associated with rotavirus, but not other enteric pathogens, at 2.1-fold higher rate in clinical samples from patients treated vs. untreated with antimicrobials before hospitalization. Finally, clinical samples positive for Norwalk virus from patients with vs. without developed colitis syndrome were at higher risk of developing virus-bacterial enteric infection detecting Salmonella spp. at 7.6and 3-fold higher rate verified by culture-based and PCR assay, respectively. Importantly, patients with vs. without hemorrhagic colitis Salmonella spp. verified primarily a culture-based method was detected by 11-fold more frequently (p = 0.01). 

Russian Journal of Infection and Immunity. 2019;9(2):375-380
pages 375-380 views

A mathematical model for developing hepatitis E virus infection in human population

Kontarov N.A., Yuminova N.V., Alatortseva G.I., Lukhverchik L.N., Nurmatov Z.S., Pogarskyia I.V.

Abstract

Hepatitis E is an acute viral infectious disease transmitted by fecal-oral route mainly through fecally contaminated drinking water, with cyclic outbreaks and frequent development of acute hepatic encephalopathy in pregnant women. Hepatitis E epidemic outbreaks occur in Central Asia, Africa and Latin America, whereasChina,India,Turkmenistan,Kazakhstan,Tajikistan,Uzbekistan,Kyrgyzstan,Bolivia,Mexico, andTaiwanrepresent endemic geographic regions. Hepatitis E in the structure of acute viral hepatitis morbidity during outbreaks ranges from 64.7% to 80%, whereas sporadic morbidity may be up to 10 to 18.8%. In contrast, percentage of hepatitis E in acute viral hepatitis varies from 0.5% to 12.6% in European countries and some territories of theRussian Federation. The latent active virus circulation was confirmed in various regions of theRussian Federation. All introduced cases were related to recent traveling to the regions with high incidence of hepatitis E, which course clinically did not differ from standard hepatitis E infection, but no cases of infection were recorded after exposure. Lack of contact transmission in this case was associated with low virus survival in environment. Patients with any clinical form including anicteric serve as a source of infection. An increased risk of hepatitis E infection is typical for livestock workers dealing with pigs, employe es of meat processing plants engaged in primary meat carcass processing and working at slaughterhouse. According to the World Health Organization, 20 million cases of hepatitis E virus infection are recorded annually, among which 3 million cases account for acute hepatitis E and related 70 000 lethal outcomes. Chronic liver disorders comprising up to 70% followed by death of pregnant women (40%) as well as acute liver and kidney failure reaching as low as 4% result in lethal outcome in hepatitis E patients. Creating a mathematical model for development of hepatitis E infection could allow to predict changes in its morbidity rate at controlled area. Here, for the first time we propose a mathematical model for developing hepatitis E in human population based on disease course, which may potentially predict an incidence rate for the most dangerous icteric hepatitis E as well as assess amount of individuals susceptible to it at morbidity rise in the geographic region. 

Russian Journal of Infection and Immunity. 2019;9(2):381-384
pages 381-384 views

METHODS

Coherent fluctuation nephelometry in clinical microbiology

Gur’ev A.S., Shalatova O.Y., Rusanova E.V., Vasilenko I.V., Volkov A.Y.

Abstract

In this article data concerning coherent fluctuation nephelometry (CFN) use in clinical microbiology is presented. CFN-analyzer allows to solve two important problems – fast urine screening for bacteriuria within 2-4 hours and antibiotic susceptibility testing within 3-6 hours. Altogether more than 650 urine samples were tested, and the effectivity of CFN-analyzer for preliminary selection of samples for further analysis was shown. Method allows to detect negative samples, reducing the number of urine analyses by 70-80%. Simultaneous analysis of growth curves and concentration of microorganisms shows high sensitivity and specificity (95.2% и 96.9%). Also more than 250 antibiotic susceptibility tests were performed using CFN-analyzer to show its effectiveness for determination of resistant properties of both pure cultures and urine microflora without isolation of bacteria. The agreement with traditional methods was from 84% to 88%. The use of CFN-analyzer with express methods of identification of microorganisms (chromogenic nutrient broths or mass-spectrometry) allows to make full urine analysis within 1-2 days. In the future CFN-analyzer gives an opportunity to screen different human biological liquids, and finds an application for other microbiological tasks, including standardization and speeding-up in sanitary bacteriology.

Russian Journal of Infection and Immunity. 2019;9(2):385-392
pages 385-392 views

A microchip developed for detecting antibodies against plaque-derived antigens

Utkin D.V., Kireev M.N., Guseva N.P., Kaplun G.A., Kuklev V.E., Osina N.A.

Abstract

Currently available Russia-made preparations intended for serological plague diagnostics are usually aimed at detecting antibodies to single bacterial antigens in the blood serum. To improve reliability of the data obtained, it is rational to use test systems to simultaneously quantify antibodies to several immunodominant Y. pestis antigens. An opportunity of using biochip technology for quantifying specific antibodies to Yersinia pestis antigens was investigated. To do this, 5 commercially available sera, 35 blood sera obtained from individuals vaccinated with live plague vaccine collected 1, 4, 5, 18 months after immunization, as well as 5 sera obtained from healthy donors were analyzed. The objective of this work was to develop a biological microchip (immunochip) for detecting antibodies specific to Y. pestis-derived antigens. In particular, amino-modified slides were sensitized by immunodominant Yersinia pestis-derived antigens: capsule antigen F1, lipopolysaccharide (LPS), main somatic antigen (MSA), fibrinolysin, and pestin PP. Diagnostic specificity and sensitivity of the immunochip were assessed by using the approved homoand heterologous immune-biological preparations and experimental animal sera. It was found that the immunochip demonstrated a 100% diagnostic efficiency. An opportunity of applying this immunochip to determine specific antibody profile in individuals vaccinated with live plague vaccine was estimated. A commercially available ELISA-AB-F1 of Yersinia pestis kit was used for comparison that allowed to detect antibodies to Y. pestis F1 antigen in 77.1% of vaccinated individuals within the examined time period covering between 1 to 18 months post-vaccination, at titer 1:160–1:2560. In contrast, using the immunochip resulted in detecting F1 antigen-specific antibodies in 91.4% of samples post-vaccination at titer 1:320–1:2560. Moreover, such immunochip additionally allowed to detect antibodies specific to the remainder of Y. pestis-derived LPS, MSA, pestin PP in 54.3%, 20%, 42% of vaccinated individuals, respectively. The percentage of positive seroconversion in individuals vaccinated with live plague vaccine was 77.1% based on the ELISA data, 91.4% — to the F1 antigen according to the immunochip data, and 94.3% — by analyzing an extended antigen panel. Combining multiple antigenic markers in our immunochip allowed to identify greater seroconversion among vaccinated people compared to a standard ELISA. Thus, the data obtained suggest that the proposed immunochip technology might be promising in assessing developing humoral immunity.
Russian Journal of Infection and Immunity. 2019;9(2):393-398
pages 393-398 views

Choosing a proper approach for isolating bacteriophages specific to Flavobacterium genus bacteria

Semanin A.G., Sadrtdinova G.R.

Abstract

The article presents results of research related to the selection of an effective method for isolating bacteriophages active against bacteria of the genus Flavobacterium. The number of bacterial strains used in the work — 3. All strains cultures were obtained from the museum of the department of microbiology, virology, epizootiology and veterinary-sanitary examination of federal state budgetary educational institution of higher education Ulyanovsk «State Agricultural University named after PA Stolypin» and have tinctorial and biochemical properties characteristic for this genus (and species). The studies consisted of 4 series of experiments. The first series of experiments was connected with the approbation of a technique for isolating a bacteriophage without using an influencing factor. As a result, the desired bacteriophages were not isolated. The second series of experiments consisted in testing the isolation method using an inducing factorultraviolet irradiation. The distance to the objects of irradiation is0.3 m. Exposure time: 1 min, 3 min, 5 min, 10 min, 15 min. The third series of experiments was also associated with the use of inducing factor X-ray irradiation. In the course of the work, three operating modes were determined, each of which differed in the duration of the exposure, the periodicity of the exposure, and the dose received. As a result of studies related to the use of inducing factors, the desired bacteriophages were not identified. The last series of experiments was to allocate bacteriophages from the objects of the natural places сommemorative (possible) existence of the «owner» (the studied microorganism) and viruses (bacteriophage desired). Three phage isolates were isolated from environmental objects: FL-j-1, FL-pec-2, FL-aq-3. Bacteriophage FL-j-1 is active against a strain of F. johnsoniae VKM B-1426 bacteriophage FL-pec-2 is active against strain F. pectinovorum VKM B-1171 bacteriophage FL-aq-3 strain active against F. aquatile VKPM B-8534. Further studies may be related to study properties of the isolated phages: study of the specificity in respect of this kind of sensitivity study, the study and higher lytic activity, selection of the most promising strains phages further selection of selected phages, as well as studying the possibility of bacteriophages from use in the method of the reaction increase titer phage for the purpose of indicating the microorganism being studied. 

Russian Journal of Infection and Immunity. 2019;9(2):399-403
pages 399-403 views

The possibility of identifying individual strains of glanders and melioidosis pathogens with a combination of immunoblotting and DNA amplification methods

Vetchinin S.S., Shchit I.Y., Shevyakov A.G., Biketov S.F.

Abstract

Causative agents of melioidosis and glanders are among the most dangerous bacterial pathogens for human. Moreover, Burkholderia pseudomallei and Burkholderia mallei are considered to be potential bioterrorism agents. In connection with this, timely diagnostics of such bacteria is of high importance. In our study, we made an attempt to develop an approach for detecting pathogenic Burkholderia spp. by combining species-specific amplification and strain-specific dot blotting assay with monoclonal antibodies. The following pathogenic Burkholderia strains were used in experiments: B. mallei (C-4, C-5, t-12, B-120, P-1, Мuksuwar-11, Z-12,Zagreb, Ivanovich, 5534), and B. pseudomallei (100, 102, 115, 116, 132, 135, 301, 51274, 60913, 61503). Real-Time PCR (RT-PCR) and dot blotting with monoclonal antibodies against surface Burkholderia epitopes were used to detect such pathogens. RT-PCR was carried out by using primers designed to recognize DNA fragments in B. mallei IS407A-fliP and the gene Orf12 from B. pseudomallei. For this, DNA was isolated from bacterial cells suspended at 1 × 104 microbial cells/ml. accumulation of the end reaction products was visualized by staining with dye SYBR Green I. Specificity of amplification reaction was determined by measuring melting temperature (Tm) for end products followed by running gel electrophoresis. It was demonstrated that all ten strains of either B. mallei or B. pseudomallei examined in the study were detected by using primers against IS407A-fliP DNA fragment and the gene Orf12, respectively. It was demonstrated that all ten strains of either B. mallei or B. pseudomallei examined in the study were detected by using primers against IS407A-fliP DNA fragment and the gene Orf12, respectively. Importantly, no signals specific to heterologous microbial DNA (isolated from bacterial cell suspension at concentration of 1 × 107 microbial cells/ml) were detected by using RT-PCR. Thus, RT-PCR provides an opportunity for assessing an inter-species diversity among pathogenic Burkholderia species. A genus-specificity was observed by using monoclonal antibodies 3D3 which bind to both Burkholderia strains, whereas antibodies 2D11 exhibited no selective binding to strain Р1 B. mallei and strain 100 B. pseudomallei, thereby displaying a strain-specific interaction. Thus, it allowed to conclude that combining a species-specific DNA amplification particularly RT-PCR together with immune-based assay such as dot blotting by using a panel of monoclonal antibodies seems to be a promising approach for assessing intra-species diversity among pathogenic Burkholderia. 

Russian Journal of Infection and Immunity. 2019;9(2):404-408
pages 404-408 views

Russia-made Mueller–Hinton agar: compliance with contemporary requirements

Domotenko L.V., Kosilova I.S., Shepelin A.P.

Abstract

At present, a rise of antimicrobial resistance requires that susceptibility of infectious agents to antimicrobial agents could be accurately evaluated as related errors may lead to selecting improper therapeutics provoking spread of drug resistance. Pathogen sensitivity to antimicrobial agents is commonly determined by a disc diffusion method. A quality of nutrient medium used in assays plays a crucial role influencing final results. In Russia, it turned out that regulatory documents such as the nationwide guidelines and clinical recommendations outlining methodology for antimicrobial susceptibility testing underlay availability in domestic market few nutrient media, including Mueller–Hinton Agar, AGV medium etc. exhibiting sometimes unsatisfactory quality. To harmonize such methodology with international requirements, theStateResearchCenterfor Applied Microbiology and Biotechnology has developed a technology and promoted manufacture of Russia-made Mueller–Hinton agar satisfying requirements of EUCAST documents, clinical guidelines, and ISO/TS 16782:2016. The main objective of this study was to compare quality of new agar product with five similar foreign media while examining 11 test strains by disc diffusion method. As a result, some of nutrient media available to the Russian market turned out to be off-standard: not all of them satisfy to the EUCAST requirements and clinical guidelines since diameter distribution for growth inhibition recommended by EUCAST for quality control does not fit into permissible range. Moreover, susceptibility of P. aeruginosa ATCC 27853 to aminoglycosides, fluoroquinolones, Meropenem, as well as S. aureus ATSS 25923 and E. faecalis ATCC 29212 to tigecycline was assessed with certain mistakes. The data obtained by us were analyzed in accordance to the new document ISO/TS 16782:2016 “Clinical laboratory testing — criterion for acceptable lots of dehydrated Mueller–Hinton agar and broth for antimicrobial susceptibility testing”, not approved yet In Russia. To determine potential reason for deviation of data from reference range, we measured concentration of bivalent metals in all nutrient media examined by atomic emission spectrometry with inductively coupled plasma. We determined new patterns affecting reliability of results on microbial antibiotic susceptibility. A need to check intralaboratory quality control of nutrient media was emphasized. 

 

Russian Journal of Infection and Immunity. 2019;9(2):409-416
pages 409-416 views

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