Commercial Test Systems Research Articles

ТЕСТ-СИСТЕМА ДЛЯ ПРОВЕДЕНИЯ ПЦР В РЕЖИМЕ РЕАЛЬНОГО ВРЕМЕНИ ДЛЯ ОБНАРУЖЕНИЯ ДНК ТТV В БИОЛОГИЧЕСКОМ МАТЕРИАЛЕ

Background. The study of biological material for the presence of TTV DNA using the PCR method allows for a timely assessment of the functional state of the human liver and immune system. Objective. To develop components for real-time PCR for TTV DNA detection in biological material. Material and methods. The design and selection of optimal primers and probes (taking into account the size (length) of the amplicon, annealing temperature, nucleotide composition, distribution of nucleotides along the length of the primer, length of primers, the possibility of formation of hairpins and dimers by primers) were performed using the Primer-BLAST/Primer3, FastPCR programs. Since primers, even absolutely unique for certain DNA sequences, could anneal at nonspecific sites, not related to the gene analyzed, we checked the correspondence of the primers to the sequences of the target gene. For this purpose, we used the NCBI Primer BLAST online service and assessed the local pairwise alignment of each primer with all nucleotide sequences of the Refseq databases. Results. As the result of studies carried out on the selection of the optimal primer annealing temperature, primer concentrations, as well as the selection of the optimal nucleotide pair, the main parameters of the designed primers were determined. Conclusions. A kit for the detection and quantification of TTV DNA using the polymerase chain reaction method with hybridization-fluorescent detection in real time was created and became the basis for the development of a commercial test system.

Development of custom amplicon-based test systems for detecting KRAS, NRAS, and BRAF gene mutations in colorectal cancer using next-generation sequencing (NGS).

e15061 Background: Polymerase chain reaction (PCR)-based mutation diagnostics in colorectal cancer is widely available. However, PCR has limitations in identifying rare mutations and precise mutation coordinates. Targeted sequencing, though promising, often incurs high costs and longer research durations, demanding higher DNA sample quality. Our study demonstrates the benefits of using a customized amplicon test system for diagnosing somatic mutations in tumor tissue, including detecting rare mutations not captured by PCR, requiring less input biomaterial, and similar research costs. Methods: A targeted NGS assay system was developed using region-specific primers for KRAS (exons 2, 3, and 4), NRAS (exons 2, 3, and 4), and BRAF (exon 15). Library preparation involved a three-step PCR process: Target PCR incorporating Illumina overhang adapter sequences, Index PCR introducing multiplexing indices and sequencing adapters, and PCR cleanup using magnetic beads. Sequencing utilized MiSeq v2 reagents with paired 300‐bp reads, generating high-quality, full-length reads. Validation included re-testing known samples. Tumor cell percentages in FFPE specimens were determined, and DNA was extracted using the QIAamp DNA FFPE Tissue Kit. Data analysis utilized proprietary software. Results: Out of 1234 samples, 1133 were tested, with mutations detected in 685 samples (60.45%). KRAS mutations were found in 548 (48.36%), NRAS mutations in 40 (3.5%), and BRAF mutations in 88 (7.7%) samples. Double mutations were observed in 9 samples (0.79%). Data on rare and double mutations are presented in the table. Conclusions: The developed test system accurately identifies essential mutations in colorectal cancer, including rare and potentially clinically significant variants. The required DNA amount ranged from 10 to 30 ng in 5 μl, nearly ten times less than well-known commercial PCR test systems like COBAS (Roche) and therascreen (QIAGEN). The cost of the study is comparable to PCR with a minimum study duration of 4 days. [Table: see text]

Immune response and laboratory markers in the spectrum of severity of COVID-19

BACKGROUND: Serum antibodies to SARS-CoV-2, when measured early after disease onset, may add value to the diagnosis of COVID-19. AIM: To examine the levels of serum antibodies to SARS-CoV-2 and laboratory blood parameters in hospitalized patients with COVID-19 of varying severity. MATERIALS AND METHODS: In this retrospective cohort study, we examined laboratory markers of inflammation in patients with acute COVID-19 during the 1st week of hospitalization. The levels of serum antibodies to SARS-CoV-2 were studied using commercial test systems. RESULTS: In 47% of hospitalized patients with COVID-19, during the first week of hospital stay, IgM and IgG antibodies to SARS-CoV-2 were detected, both in the case of a positive and negative PCR test. An average positive cor- relation of detected IgM and IgG with antibodies to the receptor-binding site of the S-protein of the SARS-CoV-2 virus is shown. In total, IgM and IgG antibodies to SARS-CoV-2 were most often detected in patients with a favorable course of the disease. Laboratory parameters in patients with moderate and severe COVID-19 were characterized by asignificant increase in the level of serum C-reactive protein, an increase in the neutrophil-leukocyte ratio and fibrinogen level, in comparison with data from patients with a mild course of the disease. In mild cases of infection, a moderately negative correlation was revealed between the levels of antibodies to SARS-CoV-2 and NLR. CONCLUSIONS: Detection of antibodies to SARS-CoV-2 in the early stages of hospitalization may be a predictor of a favorable outcome of the disease and serve as an additional criterion for the diagnosis of COVID-19 along with PCR analysis.

HCV drug resistance mutations in HIV-infected patients

The aim of our work was to assess prevalence in the HCV drug resistance mutations in the NS3, NS5A, NS5B genes in HIV-infected patients. The material of the study was 157 blood plasma samples collected from HIV patients living in the Leningrad Region, with virological inefficiency of antiretroviral therapy. Samples were examined for the presence of anti-HCV antibodies and HCV RNA using commercial test systems in accordance with the manufacturer’s recommendations. In the case of detecting HCV RNA, amplification reaction was carried out using a set of primers co-flanking the NS3, NS5A, and NS5B genes. After sequencing the nucleotide sequences of the above genes, the virus subtype was determined. To assess HCV drug resistance mutations to direct-acting antiviral agents (DAAs), the Geno2pheno HCV resistance software (https:// hcv.geno2pheno.org) was used. The age of the patients varied from 18 to 65 and averaged 34.3±7.27 years. The number of males vs females prevailed comprising 71% and 28%, respectively. Antibodies against HCV were detected in 94.2% cases, whereas HCV RNA — in 98 (61.7%) HIV-infected persons. The distribution of HCV genotypes in this group was as follows: 1a — 7% (n = 7), 1b — 53% (n = 52), 2 — 2% (n = 2), 3a — 38% (n = 37). The results of determining the viral load varied from 5.3 × 103 to 2.3 × 108 IU/ml. The nucleotide sequence of all three regions NS3, NS5A, NS5B was determined in 73 samples. Analysis of drug resistance mutations in the NS3 region was performed for 82 HCV isolates of genotypes 1b (n = 46), 3a (n = 31), 1a (n = 4), 2 (n = 1). In total, the proportion of strains containing mutations in the NS3 region was 9.8%. The presence of significant amino acid substitutions in the NS5A region was tested for 83 HCV isolates of genotypes 1b (n = 47), 3a (n = 30), 1a (n = 6). The percentage of strains containing mutations in the NS5A region was 19.3%. Resistance mutations in the NS5B region were analysed for 87 HCV isolates of genotypes 1b (n = 48), 3a (n = 32), 2 (n = 2), 1a (n = 5). The proportion of strains containing mutations in the NS5B region was 11.5%. In the study group, mutations associated with hepatitis C virus resistance to DAAs in all regions were found in 16.6% (95% CI: 11.11–23.32%) cases (n = 26). In the NS3, NS5A and NS5B, there have been identified 6, 15 and 10 significant amino acid substitutions, respectively.

Association of polymorphic variants of cytokines genes, endothelial growth factor and matrix metalloproteinases with the development of uterine fibroids among russian women

One of the factors in the development of uterine fibroids is a genetic predisposition to its occurrence in some women, but the real molecular genetic mechanisms of this phenomenon remain unknown. Aim of the study was the distribution analysis of gene polymorphism of cytokines TNFα, IL-1β, IL-4, IL-6, IL-10, factors of angiogenesis (vascular endothelial growth factor, VEGF) and remodeling of extracellular matrix (metalloproteinases MMP2, MMP3, MMP9), which are associated with their levels. Material and methods. Genotyping was performed by real-time PCR using commercial test systems SYBR GreenI (Litech, Russia) and TaqMan (Syntol, Russia) in accordance with the instructions of the developer. Cytokine content in blood serum was determined by flow cytometry using microspheres coated with monoclonal antibodies to cytokines (Bio-Plex ProTM Human Cytokine 27-plex Assay), according to the instructions for Bio-Plex 200 (Bio-Rad Laboratories, USA).To evaluate the results obtained, in addition to the generally accepted methods of statistical processing for case – control studies, computational methods of bioinformatics were used for comparative analysis of the diagnostic value of various combined genetic traits. Results. It was shown that the maximum odds ratio value of uterine fibroids development are combined genetic traits that include representatives of all four regulatory factors: cytokines with pro-inflammatory activity, cytokines with anti-inflammatory activity, vascular endothelial growth factors and metalloproteinases (p = 0.002). Conclusions. The presented data reveal the real mechanisms of manifestation of the genetic predisposition of individual women to the uterine fibroids development, associated with the presence of polymorphism of certain genes in their genome, which provide features of the structure of cytokine networks with the predominance of certain activities in the regulation of tissue processes in the uterus. In addition to purely scientific interest, these results indicate the real possibility of their clinical application in the form of prognostic criteria with a certain level of prognostic significance.

Pathogenetic parallels and clinical relationships of HIV infection and Hodgkin’s lymphoma

Aim: to show the importance of the features of the development of Hodgkin’s lymphoma (HL) against the background of HIV infection, possible clinical relationships and consequences of simultaneous diseases in patients, as well as complex therapy. Material and methods. The article presents an analytical review of the problem and retrospective data on 63 patients with HIV[1]associated HL (HIV-HL) who were diagnosed with HL in St. Petersburg hospitals in the period 2000–2017. For the diagnosis of HIV-HL, regulated morphological, immunohistochemical, instrumental and laboratory research methods were used, the tumor stage was determined based on the Cotswolds-modified Ann Arbor classification. The diagnosis of HIV infection was confirmed by the detection of specific antibodies to HIV (and the p24 antigen) during serological and enzyme immunoassay of blood, by immune blotting. The number of copies of HIV RNA and the number of CD4 lymphocytes in the blood of patients were determined using commercial test systems approved for use in the territory of the Russian Federation. The analysis of overall survival was performed using the Kaplan–Meyer method. Statistical processing of the research results was performed using statistical programs MS Excel 2010, GraphPad Prism 8 (GraphPad Software, Inc., USA), SPSS version 22.Results. The study group was dominated by men (73%), the median age was 32 years, in 16 (25.4%) patients HIV and HL were detected simultaneously. The number of CD4 lymphocytes > 500 cl/μl at the HL debut was noted in 33.3% of patients, patients with severe immunosuppression prevailed (50–250 cl/μl in 20 (31.7%), 250–500 cl/μl in 11 (17.5%), less than 50 cl/μl in 11 (17.5%). The amount of HIV RNA>400 kop/ml was noted in 82.5%, HIV infection at stages 4B-5 was detected in 89% of cases, at the time of detection of HL ART was performed in 16 patients. EBV coinfection (77.8%), cytomegalovirus (60%), viral hepatitis (55.6%) with a predominance of viral hepatitis C, common opportunistic infections (tuberculosis, pneumocystis pneumonia, toxoplasmosis of the brain, common candidiasis), simultaneously occurring from 1 to 3 infections were observed in 77.8%. Stage IV HL was established in 54%, III — in 22%, II — in 24% of patients, the presence of B-symptoms was confirmed in 73% of cases. The predominant histological variant of HIV-HL was nodular sclerosis (58 patients), mixed[1]cell sclerosis in 4 patients, with lymphoid predominance in 1 case. Extranodal lesions were observed in 34 (54%), complications of the tumor process in 33 (37.5%) patients. 42 (66.7%) patients received antitumor treatment for HL: line 1 according to the ABVD scheme — 85.7% (80% achieved PET-negative complete remission (CR), according to the VEASORR esc or VEASORR schemes — 33.3%; line 2 — according to the ICE or DHAP schemes (n=10). An objective response was noted in 4 patients, PET-negative response in 2 of them, partial PET-positive regression in 2 patients. Progression was observed in 2 people. Autologous bone marrow transplantation was performed in 2 patients (in partial PET-positive regression); line 3 (n=3) — chemoimmunotherapy with bendamustine, gemcitabine (2 patients underwent autologous bone marrow transplantation). The cumulative life expectancy of patients for 1 year and 2 years was 44% and 37%, respectively, 1-year overall survival was 75%, 2-year — 60%. The factors negatively affecting survival and life expectancy were tumor progression and complications, ECOG≥2 (p=0.0001), candidiasis, pneumonia (p=0.001), viral hepatitis B and C (p=0.045), lack of antitumor treatment and ART (p=0.0001), age younger than 40 years, central nervous system damage, the presence of 1 or more concomitant infections (p=0.024). Conclusion. HIV-HL is one of the most common hematological malignancies, characterized by heterogeneity in its manifestations, polymorphism of pathogenetic and clinical features and relationships. During the dispensary supervision of PLHIV, special attention should be paid to the factors of an unfavorable prognosis of the disease, the timeliness of the appointment of ART and the assessment of the risks of developing lymphoproliferative diseases within the framework of the immune system restoration syndrome (IRIS) in order to increase their survival and quality of life. Further research is needed on the pathogenesis, early diagnosis and effective treatment of lymphomas associated with the human immunodeficiency virus

Tick-borne infections in the Leningrad oblast: incidence and seroprevalence

The study’s objective was to reveal trends in infections transmitted by ixodes ticks epidemic processes and the level of seroprevalence of the population to the tick-borne encephalitis virus, Borrelia burgdorferi sensu lato, Coxiella burnetii, Anaplasma phagocytophilum and Ehrlichia chaffeensis/ E. muris in the Leningrad Oblast.Materials and methods. A retrospective epidemiological analysis of the tick-bite incidence rate, the ixodic tick-borne borreliosis, tick-borne encephalitis, Q fever, human granulocytic anaplasmosis and human monocytic ehrlichiosis incidence rates, as well as vaccination against tick-borne encephalitis of the population of the Leningrad Oblast in 20112020. The official data of the tick-bite incidence rate, infections transmitted by ixodes ticks incidence rates and vaccination of the population was taken from the forms of federal statistical observation No. 2 and No. 5. Blood was sampled in 2019-2020 from randomly selected donors. Blood serum samples were examined by enzyme-linked immunosorbent assay for the presence of IgG antibodies to the pathogens of the studied infections using commercial test systems according to the manufacturer’s instructions.Results. In the Leningrad Oblast, in 2011-2020, there was a downward trend in the tick-bite incidence rate, as well as in the incidence rates of infections transmitted by ixodic ticks. The long-term average tick bite incidence rate was 369.0. The long-term average incidence rate of ixodic tick–borne borreliosis was 4.1; tick–borne encephalitis was 1.4; Q fever was 0.02. During the analyzed period, no cases of human granulocytic anaplasmosis and human monocytic ehrlichiosis were reported. The seroprevalence to tick-borne encephalitis virus was 1.7%; to B. burgdorferi s. l. – 3.7%; to C. burnetii – 1.8%; to A. phagocytophilum – 1.0%; to E. chaffeensis/ E.muris – 3.2%.Conclusion. The results obtained indicate the existence of active natural foci of infections transmitted by ixodes ticks, fairly frequent contact of the residents with these foci, and possible significant hypodiagnosis of these infections in the Leningrad Oblast.

DYNAMICS OF THE LEVELS OF ASYMMETRIC DIMETHYLARGININE AND PLASMINOGEN ACTIVATOR INHIBITOR TYPE 1 IN PATIENTS WITH ACUTE ST-SEGMENT ELEVATION MYOCARDIAL INFARCTION AND TYPE 2 DIABETES MELLITUS DEPENDING ON THE REPERFUSION STRATEGY

Introduction. Despite the major successes achieved in the treatment of acute coronary syndromes (ACS), acute myocardial infarction (AMI) remains the main cause of death among the working-age population of Ukraine. The means of treatment of interventional cardiology can actually reduce the mortality of patients with ACS, improve the course of the acute period of the disease and ensure less reduction in the functional capabilities of the heart in the future. Among the many pathogenetic mechanisms of vascular inflammation in coronary heart disease and type 2 diabetes, endothelial dysfunction is the determining factor.
 The aim of the study. To evaluate the levels of plasminogen activator inhibitor type 1, asymmetric dimethylarginine and endothelial nitric oxide synthase on the 10-14th day in patients depending on the presence or absence of concomitant diabetes type 2 and the type of reperfusion therapy.
 Materials and methods. 130 patients with acute myocardial infarction were examined, who were divided into 2 groups: 1 group consisted of patients with acute myocardial infarction with accompanying type 2 diabetes (n=73), 2 group – patients with acute myocardial infarction without type 2 diabetes (n =57). The quantitative content of the plasminogen activator inhibitor type 1 (PAI-1) was determined by the immunoenzymatic method using a commercial test system manufactured by Technoclone PAI-1 ELISA Kit (Austria), endothelial nitric oxide synthase (NOS) – Enzyme-Linked Immunosorbent Assay (ELISA) Kit For Nitric Oxide Synthase Endothelial, asymmetric dimethylarginine (ADMA) – Immunodiagnostik ADMA ELISA Kit (Austria).
 Results. Percutaneous coronary intervention (PCI) contributes to a more significant decrease in the content of the marker of endothelial dysfunction – ADMA and an increase in NOS on the 10-14th day of acute myocardial infarction in comparison with standard therapy. During PCI, the level of PAI-1 did not reliably change during treatment due to post-inflammatory and post-traumatic activation of platelets in the vascular wall.
 Conclusions. In patients with acute myocardial infarction with type 2 diabetes mellitus, percutaneous coronary intervention contributes to a significant decrease in the content of asymmetric dimethylarginine and an increase in NOS on the 10-14th day of acute myocardial infarction, but was not accompanied by a significant decrease in the level of PAI-1, which in general indicates positive effect of performed myocardial revascularization.

NATRIURETIC PEPTIDE: DIAGNOSTIC, PROGNOSTIC, AND CLINICAL RELEVANCE IN PATIENTS WITH TYPE 2 DIABETES MELLITUS, ARTERIAL HYPERTENSION AND OBESITY

Natriuretic peptides are one of the main biomarkers of chronic heart failure (CHF). The level of NT-proBNP makes it possible to evaluate the negative prognosis in patients with CHF, as well as to monitor the sensitivity of patients with left ventricular dysfunction to the appointment of treatment. The purpose of the study: to study the NT-proBNP biomarker in comorbid patients with hypertension, obesity, type 2 diabetes and determine the relationship with a number of hormonal and biochemical indicators. Materials and methods: all patients were divided into 4 groups depending on the presence of comorbid pathology in them: patients with hypertension – 1 group – 22 people; patients with hypertension combined with obesity – group 2 – 30 people; Hypertension in combination with type 2 diabetes – group 3 – 31 people; patients with hypertension, type 2 diabetes and obesity – group 4 – 28 people. Determination of the content of N-terminal brain natriuretic peptide (NT-proBNP) in the blood serum of patients was carried out by the immunoenzymatic method on the analyzer "Labline-90" (Austria) using a commercial test system manufactured by the company "Elabscience" (ELISA, China) according to the instructions, which was part of the set. Conclusions. It was established that in patients with hypertension, hypertension in combination with obesity, hypertension in combination with type 2 diabetes, as well as hypertension in combination with type 2 diabetes and obesity, there is a significant increase in NT-proBNP compared to the indicator of the control group (p<0.05). It has been proven that NT-proBNP has a direct correlation with creatinine (p=0.045), urea (p=0.038), triglyceride level (p=0.027) and cholesterol (p=0.031); direct correlation with the level of HbA1c (r=0.202; p=0.0038). The level of NT-proBNP has a significant predictive value for the risk of developing cardiovascular complications in patients with hypertension.

An open source platform for hearing research

The disparate nature and format of data obtained from existing commercial hearing test systems is a significant impediment to reproducibility of hearing research. Advancement of hearing diagnostic assays requires access to research-grade platforms that are easily customized while enabling broad collaboration across labs with access to varied populations. In this research, we present an open-source platform that combines the Tympan open-source audio processing device with the TabSINT open-source tablet-based hearing software The Tympan uses a Teensy 4.1 processor which leverages the Arduino Development environment, making it accessible to a wide variety of users to create, modify and configure the embedded software. The Tympan is also a powerful audio processing platform that has demonstrated good performance as a hearing aid in a study of 14 adult users with mild to moderate hearing loss. Separately, TabSINT has been used extensively across the Department of Defense for human studies of hearing that include a variety of speech-in-noise tests and questionnaires, with thousands of subjects tested over the past few years. In this project, the two platforms come together, with new hardware extensions, to create a powerful hearing research platform that is extensible and highly accessible, thanks to the low cost of the hardware.

Autoreactive processes in children in the initial period of multisystem inflammatory syndrome associated with SARS-CoV-2 and after rehabilitation

BACKGROUND: The problem of the pathogenesis of the multisystem inflammatory syndrome (MIS-C), associated with SARS-CoV-2, remains relevant, but is still far from being resolved. In this regard.
 AIM: The aim our research was to study and evaluate the levels of autoantibodies to native and denatured DNA in the blood serum of children who underwent MIS-C, associated with SARS-CoV-2, in comparison with the initial period of the pathological process.
 MATERIALS AND METHODS: 101 children aged 2 to 17 years old were examined, 77 of them was with a confirmed diagnosis of MIS-C associated with SARS-CoV-2. We have formed 3 groups: group 1 — children with MIS-C upon admission to the hospital; group 2 — children who underwent MIS-C after a course of rehabilitation; group 3 — comparison group. All children underwent determination of the concentrations of IgG-autoantibodies to native and denatured DNA in blood serum, which was performed by EIA ELISA using commercial test systems of Vector-Best LLC (Russia) according to the manufacturer’s instructions. Statistical processing of the obtained results was carried out using the Statistica v. 10.0 application package. Statistical processing used the Newman–Keuls multiple comparison test.
 RESULTS: It was noted that both in the initial period of MIS-C and after rehabilitation, an increased content of autoantibodies to nDNA and dDNA was determined compared to the control group. At the same time, there was a significant excess of the levels of indicators in the post-rehabilitation period compared with the initial period of MIS-C. During a clinical examination of children who underwent MIS-C, most of them noted the appearance of pain in the joints. The results obtained indicate the persistence of inflammatory reactions with the development of an autoreactive component. This requires both the search for risk markers for the development of MIS-C and further monitoring of children who have undergone MIS-C to prevent autoimmune complications, including arthritis, damage to the vascular endothelium with the possible formation of thrombotic complications.
 CONCLUSIONS: In the acute period of MIS-C and after rehabilitation, increased levels of the average content of autoantibodies to nDNA and dDNA were noted compared to the control. Individual values of autoantibodies to nDNA and dDNA exceeded normative levels of the indicator were found in a significantly larger number of children in the post-rehabilitation period after MIS-C, and this excess was more pronounced compared to the acute period of the process. When assessing the clinical condition of children after MIS-C, most of them showed pain in the joints during exercise, abdominal and neurological disorders, and decreased tolerance to physical activity, which indicates persistent inflammatory reactions.

The role of inflammatory markers and growth factors in progression of chronic kidney disease in patients with diabetes mellitus

Introduction: A Diabetes mellitus (DM) and chronic kidney disease (CKD) are two chronic non-communicable diseases that have exceeded epidemic thresholds in all countries of the world. The problem of early diagnosis, prevention and treatment of CKD continues to be relevant for modern medicine, and assessment of the severity of CKD and associated cardiovascular complications is of great practical importance for primary and secondary prevention. Objectives: The aim of the study was to assess the role of proinflammatory cytokines, chemokines and growth factors in progression of CKD in patients with DM. Patients and Methods: We screened 155 type 2 DM patients aged 65.00 (55.00-71.00) years. Control group included 21 healthy people with the same age. Renal function was assessed based on the levels of serum creatinine, cystatin C, estimated glomerular filtration rate (eGFR), which was calculated according to the CKD-EPI (chronic kidney disease epidemiology collaboration) equation, and albuminuria, which was assessed as albumin/creatinine ratio (A/C). The analysis of serum and urine biomarkers was carried out by enzyme immunoassay using commercial test systems. Results: Patients with DM had significantly higher levels of proinflammatory cytokines in comparison with the control group. The levels of interleukin-6 (IL-6), high-sensitivity C-reactive protein (hs-CRP), vascular endothelial growth factor A (VEGF-A), fibroblast growth factor-23 (FGF-23), regulated upon activation, normal T cell expressed and presumably secreted (RANTES), MIG, CRP, kidney injury molecule-1 (KIM-1) and homocysteine gradually increased with decreasing GFR. According to the results of univariate linear regression analysis, there were significant relationships between the levels of VEGF-A, FGF-23, RANTES, tumor necrosis factor alpha (TNF-alpha), MIG, CRP, hs-CRP, IL-6 and renal function. By multiple linear regression analysis adjusted for confounding factors, serum creatinine was significantly correlated with FGF-23 (β=0.40, P<0.001) and Il-6 (β=0.29, P<0.001). Conclusion: Our study demonstrated the important role of growth factors and proinflammatory cytokines in the development and progression of CKD in DM. However, despite these data, the pathogenesis of diabetic changes in the kidneys remains not fully understood and, in order to clarify the possibility of pathogenetic influences on the progression of diabetic nephropathy, it is necessary to study various aspects of its development, including genetic factors.

Electrochemical Biosensors for Express Analysis of the Integral Toxicity of Polymer Materials.

Biosensors based on an oxygen electrode, a mediator electrode, and a mediator microbial biofuel cell (MFC) using the bacteria Gluconobacter oxydans B-1280 were formed and tested to determine the integral toxicity. G. oxydans bacteria exhibited high sensitivity to the toxic effects of phenol, 2,4-dinitrophenol, salicylic and trichloroacetic acid, and a number of heavy metal ions. The system "G. oxydans bacteria-ferrocene-graphite-paste electrode" was superior in sensitivity to biosensors formed using an oxygen electrode and MFC, in particular regarding heavy metal ions (EC50 of Cr3+, Mn2+, and Cd2+ was 0.8 mg/dm3, 0.3 mg/dm3 and 1.6 mg/dm3, respectively). It was determined that the period of stable functioning of electrochemical systems during measurements was reduced by half (from 30 to 15 days) due to changes in the enzyme system of microbial cells when exposed to toxicants. Samples of the products made from polymeric materials were analyzed using developed biosensor systems and standard biotesting methods based on inhibiting the growth of duckweed Lemna minor, reducing the motility of bull sperm, and quenching the luminescence of the commercial test system "Ecolum". The developed bioelectrocatalytic systems were comparable in sensitivity to commercial biosensors, which made it possible to correlate the results and identify, by all methods, a highly toxic sample containing diphenylmethane-4,4'-diisocyanate according to GC-MS data.

Особливості ліпідного та вуглеводного обмінів у пацієнтів з гастроезофагеальною рефлюксною хворобою на тлі цукрового діабету 2-го типу залежно від наявності ожиріння

Особливості ліпідного та вуглеводного обмінів у пацієнтів з гастроезофагеальною рефлюксною хворобою на тлі цукрового діабету 2-го типу залежно від наявності ожиріння

Etiology of community-acquired pneumonia in children and adults based on 2015–2022 bacteriological and molecular genetic studies

The data on etiology of community-acquired pneumonia (CAP) remain contradictory, which is often assessed only according to bacteriological studies coming to a conclusion about the low frequency of major main pathogenStreptococcus pneumoniaеisolation. At the same time, there are very few observations of parallel examination of patients with CAP simultaneously by bacteriological and molecular genetic methods. There are no observations on the etiology of CAP in the long-term dynamics. The aim of the work is to analyze the etiology of CAP in children and adults based on 2015–2022 bacteriological and molecular genetic studies. The etiology of pneumonia was assessed using the data on posterior pharyngeal wall washes from 418 children and 483 adults hospitalized with a primary diagnosis of CAP at two large Perm medical organizations in 2015–2022. The samples were examined by the bacteriological method and polymerase chain reaction using commercial test systems with the amplifier “CFX96” (Bio-Rad, USA). The results showed thatS. pneumoniaе, despite implementing decreed vaccinations against pneumococcal infection, it remains the leading causative agent of CAP in both children and adults. The 2015–2022 frequency of pneumococcal discharge according to bacteriological studies was 9.7 per 100 examined, according to molecular genetic studies — 48.5 per 100 examined among children and adults in the study area. According to 2021–2022 vs 2015–2017 bacteriological studies, the frequency ofStaphylococcus aureusexcretion among children increased significantly, andS. pneumoniaеincreased among adults. According to the results of molecular genetic studies among children and adults, no significant changes in the etiology of CAP were found. In 2021–2022, SARS-CoV-2-positive vs SARS-CoV-2-negative patients with CAP infected had higher rate of isolatedS. aureus,KlebsiellapneumoniaеandHemophilus influenzae, i.e., those pathogens whose number increased in the long-term follow-up observations.

КЛИНИЧЕСКАЯ ЭФФЕКТИВНОСТЬ ПРИМЕНЕНИЯ ПУЛИРОВАННЫХ МЕЗЕНХИМАЛЬНЫХ СТВОЛОВЫХ КЛЕТОК ОБОНЯТЕЛЬНОЙ ВЫСТИЛКИ ДЛЯ ЛЕЧЕНИЯ ПАЦИЕНТОВ С ТЯЖЕЛЫМИ ВНЕБОЛЬНИЧНЫМИ ПНЕВМОНИЯМИ, ОСЛОЖНЕННЫМИ ОСТРЫМ РЕСПИРАТОРНЫМ ДИСТРЕСССИНДРОМОМ

Background. The difficulty in diagnosing COVID-19 is associated with the heterogeneity of clinical manifestations, and treatment is difficult because the course of the disease varies from asymptomatic to severe viral pneumonia, with a cytokine storm and development of acute respiratory distress syndrome (ARDS). Certain hopes in the treatment of COVID-19 and ARDS are currently pinned on the use of mesenchymal stem cells (MSCs), due to their ability to influence the immune system and activate the regeneration of damaged tissues. The aim of the study was to improve the clinical efficacy of existing methods of treating patients with severe community-acquired pneumonia complicated by acute respiratory distress syndrome using cell therapy with pooled MSCs. Material and methods. The subjects of the study were patients with COVID-19-associated pneumonia (U07.1 according to ICD-10), severe course, acute respiratory syndrome (U04) (study group (n=14) and comparison group (n=18)). Clinical and laboratory studies were carried out by routine methods using commercial test systems. Results. Good tolerability and safety of the method of treatment of patients with severe COVID-19 complicated by ARDS using pooled MSCs were shown. 57% of patients who received pooled MSCs did not need to be transferred to mechanical ventilation in the next 7 days. In the study group, 43% of patients survived (6 out of 14), which was significantly higher than in the control group (5.6%, 1 out of 18) (z=2.07; p=0.038). A correlation was found between lactate dehydrogenase (LDH) levels and the outcome of a severe form of coronavirus infection, and criteria were established for selecting patients for cell therapy based on the level of LDH increase by no more than 1.85 times. Administration of pooled MSCs at LDH level higher than 519.8 U/l was ineffective.

Склеростин-залежні механізми формування остеопорозу у хворих цукровим діабетом 2 типу та ожирінням

при обстеженні 103 пацієнтів на цукровий діабет 2 типу (ЦД), що у 83 випадкахперебігав на тлі збільшеної маси тіла або ожиріння, було встановлено вірогідне збільшеннявмісту склеростину у сироватці крові, яке мало пряму кореляційну залежність з індексоммаси тіла. Висловлено припущення, що зміни даного глікопротеїну, який входить до групиантагоністів кісткових морфогенетичних білків, відбуваються внаслідок багатьох процесів ворганізмі: збільшення синтезу ФНП-α адипоцитами, ендотелієм судин, клітинами нейроглії натлі пре- та менопаузи, що сприяє порушенню мінеральної щільності кісткової тканини, тобтоформуванню остеопорозу. Ендокринний остеопороз становить найбільшу групу вторинногоостеопорозу, провідною причиною якого є цукровий діабет 2 типу. В разі наявності ЦД 2 типу,який перебігає на тлі ожиріння, формуються умови до розвитку остеопоротичних станів, одниміз механізмів розвитку яких може виступати глікопротеїн склеростин. Метою дослідженнястало визначення структурно-функціонального стану кісткової тканини шляхом дослідженнявмісту склеростину у хворих на цукровий діабет 2 типу, що перебігає на тлі топографічнихособливостей розподілу жирової тканини. До роботи було залучено 103 хворих на ЦД 2 типу,який у 83 випадках перебігав в поєднанні з надлишковою вагою або ожирінням (основна група). Догрупи порівняння увійшло 20 хворих на ЦД 2 типу, що був діагностований у пацієнтів з незмінениміндексом маси тіла (ІМТ). Вік хворих коливався від 31 до 55 років та в середньому по групах складав43±4,6 років та 44,1±2,1 роки відповідно. В обох групах хворих переважали чоловіки 44 (53%) та11 (55%) осіб. Тривалість захворювання реєструвалась в межах від 1 до 13 років. Визначеннярозподілу жирової тканини в організмі досліджували за допомогою ваг для характеристикискладу тіла - OMRON BF511 (Японія). Ідентифікували вміст вісцеральної жирової тканини(ВЖТ) та підшкірної жирової тканини (ПЖТ). Активність склеростину в сироватці крові
 визначали імуноферментним методом з використанням комерційних тест-систем «Biomedi-ca» (США) згідно інструкції на імуноферментному аналізаторі «Labline-90» (Австрія). Обробку
 отриманих даних здійснювали методом варіаційної статистики за допомогою ліцензійногопрограмного забезпечення Stata 10 («StatSoft Inc.», США). Перевірка на нормальність розподілуданих виконувалася за допомогою критерію Шапіро-Уілка. Для параметрів з нормальнимрозподілом розраховували середню величину (M) ± стандартну помилку середньої (m). Отриманірезультати розглядалися як статистично значущі при p<0,05. В результаті було визначено, щозбільшення ІМТ супроводжувалось підвищенням показника склеростину. Тобто, виявлені зміниглікопротеїну, який входить до групи антагоністів кісткових морфогенетичних білків, дають змогу припустити порушення процесу диференціації остеобластів. Тобто, як ЦД 2 типу, такі кількісна зміна жирової тканини з її перерозподілом, негативно впливають на метаболізмкісткової тканини. Так, перебіг цукрового діабету супроводжується збільшенням активностіпрозапальних цитокінів, а саме, ФНП-α – складового цитокінового каскаду жирової тканини.Цей феномен також пов’язують із залученням до процесу судин (ендотеліальні клітинипродукують даний цитокін), нервової тканини (залучення клітин нейроглії) та метаболічнимизсувами в організмі. Окрім того F.Xu та співавт. в експерименті доведено, що остеокластогенезпригнічується при гіперглікемічних та гіперінсулінемічних станах. Тобто, у хворих на ЦД призбільшенні ІМТ складається негативний «тандем» через додаткове надходження з багатьохклітин ФНП-α та збільшення синтезу склеростину, що не тільки «запускає» процес порушеннямінеральної щільності кісткової тканини (МЩКТ), але й підтримує його впродовж всьогоперебігу захворювання. Також певна роль відводиться віковому аспекту обстежених хворихна ЦД (середній вік менопаузи у здорових жінок нашої країни становить 45 років), що такожсприяє пригніченню кісткового метаболізму. В такому разі збільшення активності склеростинув поєднанні з негативним фоном (ожиріння, період менопаузи) можна розглядати як один ізмеханізмів формування остеопоротичних змін. При цьому поєднуються механізми первинного тавторинного ОП, що посилює і прискорює процес порушення архітектоніки кістки. Тому можназробити висновки, що у хворих на цукровий діабет 2 типу у поєднанні з ожирінням виникаютьумови до зниження якості кісткової тканини через уповільнення кісткового метаболізму, щосприяє формуванню остеопоротичних станів.

Whole-genome sequencing provides insights into a novel species: Providencia hangzhouensis associated with urinary tract infections.

Providencia rettgeri is a clinically significant opportunistic pathogen that is involved in urinary tract infections. Due to the resolution limitations of identification, distinguishing P. rettgeri from closely related species is challenging by commercial biochemical test systems. Here, we first reported a novel species, Providencia hangzhouensis, which had been misidentified as P. rettgeri. Exhibiting ≤91.97% average nucleotide identity (ANI) and ≤46.10% in silico DNA-DNA hybridization values with all known Providencia species, P. hangzhouensis falls well beneath the established species-defining thresholds. We conducted a population genomics analysis of P. hangzhouensis isolates worldwide. Our study revealed that P. hangzhouensis has emerged in many countries and has formed several transmission clusters. We found that P. hangzhouensis shared the highest ANI values (91.54% and 91.97%) with P. rettgeri and P. huaxiensis, respectively. The pan-genome analysis revealed that these three species possessed a similar component of pan-genomes. Two genes associated with metabolism, folE2 and ccmM, were identified to be specific to P. hangzhouensis. Furthermore, we also observed that carbapenem-resistance genes frequently occur in P. hangzhouensis with the blaIMP-27 being the most prevalent (46.15%; 36/78). The emergence of P. hangzhouensis is often accompanied by extended-spectrum β-lactamase and carbapenem-resistance genes, and calls for tailored surveillance of this species as a clinically relevant species in the future. IMPORTANCE Our study has identified and characterized a novel species, Providencia hangzhouensis, which is associated with urinary tract infections and was previously misidentified as Providencia rettgeri. Through this study, we have identified specific genes unique to P. hangzhouensis, which could serve as marker genes for rapid PCR identification. Additionally, our findings suggest that the emergence of P. hangzhouensis is often accompanied by extended-spectrum β-lactamase and carbapenem-resistance genes, emphasizing the need for attention to clinical management and the importance of accurate species identification and proper drug use.

DEPENDENCE OF CARDIOTROPHIN-1 ON BIOCHEMICAL AND HORMONAL FACTORS IN PATIENTS WITH COMORBID PATHOLOGY: ARTERIAL HYPERTENSION, DIABETES MELLITUS TYPE 2 AND OBESITY

The prevalence of arterial hypertension, type 2 diabetes mellitus (type 2 DM), and obesity is constantly increasing worldwide. Today, a complex of factors, which primarily include chronic emotional stress, pandemics of infectious diseases, military conflicts, have led to a significant increase in chronic non-communicable diseases. The aim of the study. To determine the dependence of Cardiotrophin-1 on a number of biochemical and hormonal indicators in patients with arterial hypertension, type 2 DM, and obesity, representatives of the Ukrainian population. Materials and methods. The study included 211 patients aged 49 to 65 years, who were divided into groups depending on the pathology: patients with arterial hypertension – 49 persons, Group I; patients with arterial hypertension in combination with class 1 obesity – 54 persons, Group 2; patients with arterial hypertension in combination with type 2 DM – 57 persons, Group 3; patients with arterial hypertension, type 2 DM, class 1 obesity – 51 persons, Group 4; as well as 20 persons – the Control group. Determination of the content of Cardiotrophin-1, catestatin, leptin, cystatin C, neutrophil gelatinase-associated lipocalin (NGAL), N-terminal brain natriuretic peptide (NT-proBNP), 25-OH of common vitamin D (Vitamin D3), blood levels was carried out by enzyme immunoassay on Labline-90 analyzer (Austria), using commercial test systems manufactured by FineTest (ELISA, China), BT LAB (ELISA, China), DBC (ELISA, China), Elabscience (ELISA, Canada), Monobind Inc. (ELISA, USA). Statistical data analysis was performed, using the statistical software package Statistica, 12 (Stat Soft Inc, USA), Microsoft Office Excel 2013. Data are shown as mean (M) and standard deviations (δ). An error of less than 5% was considered to be significant (p<0.05). Stepwise regression analysis was used to determine the extent of exposure of the studied indicators to the CTF-1 level, with its effectiveness assessed by one-way analysis of variance (ANOVA). Results. Thus, the study indicates that comorbid patients with arterial hypertension, type 2 DM, and obesity are predicted to develop complications from the cardiovascular system and Cardiotrophin-1 is a marker of their development. In the examined patients as a whole, the level of Cardiotrophin-1 is closely related to the peptide-hormonal complex: catestatin, leptin, cystatin C, brain natriuretic peptide, and lipocalin associated with neutrophil gelatinase. Conclusions: The serum concentration of Cardiotrophin-1 in patients with arterial hypertension increases significantly in a progression with the rate of accession of comorbid pathology and its severity. The Cardiotrophin-1 level in patients with comorbid pathology is affected by catestatin, leptin, cystatin C, NT-pro BNP, as well as the level of triglycerides, HDL-C, creatinine, and BMI. Cardiotrophin-1 can be considered as a universal biomarker for the development and progression of cardiovascular disorders in patients with comorbid pathology of arterial hypertension, type 2 DM, and obesity.

Use of milk samples in epizootic surveillance of cattle diseases

Relevance. The analysis of the possibility of using milk as a non-invasive type of samples in the epizootological control of diseases of cattle is given. During pathogenesis, many etiologic agents cause breast lesions or are excreted together with milk, which makes milk an ideal sample for laboratory diagnostics of infectious diseases of cattle, since it is available in any quantity and its samples are easy to collect.Methods. Conventional methods of document analysis were used.Results. It is shown that milk samples can be used both at the individual and at the population level for early identification of infected herds, screening of infected herds and use to obtain evidence of the well-being of herds. The availability of commercial diagnostic test systems for detecting antibodies in milk to the causative agents of leukemia of cattle, viral diarrhea of cattle, Brucella abortus, Mycobacterium avium subspecies paratuberculosis, Fasciola hepatica, Q fever (Coxiella burnetii) makes available programs for the control and eradication of diseases in dairy herds and at the level of countries. The use of combined non-invasive milk samples makes it possible to combat slowly progressive and chronic cattle infections of dairy cattle (bovine leucosis, paratuberculosis, brucellosis), and exclude carrier animals from the production chain in real time, which indicates the feasibility of introducing this type of samples into veterinary practice in the Russian Federation.