Samples Of Biological Material Research Articles

A device for rapid calorimetric measurements on small biological tissue samples

AbstractA new calorimetric technique is described that allows high-throughput heat production rate measurements on small biological tissue samples. The technique is based on the widely used thermopile chip technology combined with an innovative method for precise transport and positioning of samples of different biological materials at the thermal power detector inside the calorimeter. The new transport and positioning technique is a combination of fluidic and mechanical transport, where the latter is realized by a magneto-motor drive. The transport facility ensures good diffusive oxygen penetration into the sample, which is essential for highly metabolically active materials. The proper functioning of the device is demonstrated by measuring the heat production of metabolically active brown adipose tissue, biopsied tegu lizard muscle, and live Drosophila larvae at different stages and temperatures.

Сytokines as promising diagnostic and prognostic biomarkers of microvascular complications of diabetes

Classical knowledge about the pathogenesis of diabetes and its complications in recent years has been supplemented by ideas about the role of chronic inflammation. It has been established that inflammatory reactions play a role in the beta cell dysfunction, the formation of insulin resistance and remodeling of the vascular wall. Cytokines, soluble low molecular weight proteins and peptides that perform informational and regulatory functions, play central role in the development of inflammation. A wide range of biological activity and involvement in many aspects of pathogenesis make it possible to consider cytokines as promising molecules for diagnosing and predicting the complications. In this review, we summarize data from studies that assessed the role of cytokines as diagnostic and prognostic markers for the development of microvascular diabetic complications. Current data indicate that angiogenic and pro-inflammatory cytokines (VEGF, TNF-α, IL-6, IL-8, IL-15, IL-17, MCP-1, IP-10, INF-γ, PEDF, etc.) are promising biomarkers for proliferative diabetic retinopathy, especially when their local production is assessed (in vitreous, aqueous humor and tears). The role of these molecules as biomarkers of non-proliferative diabetic retinopathy and diabetic macular edema needs further research. Serum proinflammatory and fibrogenic cytokines (primarily MCP-1, IL-6, TNF-α, YKL-40, TGF-β and bFGF) and cytokine receptors (sTNFR1, sTNFR2) are considered as promising diagnostic and prognostic markers of diabetic kidney disease. Urinary excretion of IL-6 and MCP-1 turned out to be a predictor of the progression of diabetic nephropathy. Multi-bead assay and mass spectrometry make it possible to study cytokine panels in small samples of biological material. Combined biomarkers, including several cytokines, may increase the reliability of the prognosis of diabetic complications.

The effect of the drug L-carnitine when administered intramuscularly on the blood parameters of rats

L-carnitine is a vitamin-like substance that plays an important role in the transport of fatty acids and their further use as an energy source. Most studies have identified beneficial effects of using L-carnitine for preventive and therapeutic purposes. The series of publications reviewed is devoted to the study of the results of the use of L-carnitine in metabolic disorders, diseases of various organs, organ systems, the central nervous system, as well as to elucidate the possible reasons for the decrease in the level of L-carnitine in the body. To date, L-carnitine is used in the Russian Federation only in the form of a feed additive; the purpose of our study was to study L-carnitine in the form of an injection solution. Toxicity studies were conducted on outbred rats in October 2021 in the vivarium of the St. Petersburg State University of Veterinary Medicine. The study involved females weighing 190-210 grams. When studying subchronic toxicity when administered intra-muscularly, L-carnitine was administered at 2 dose levels. 1/5 and 1/10 of the maximum tolerated dose in acute toxicity studies. In this regard, three groups of animals were formed. The first experimental group (n=10) was administered the drug intramuscularly at a dose of 0.08 mg/kg, the second group (n=10) at a dose of 0.04 mg/kg. The control group (n=10) was intramuscularly injected with a 0.09% sodium chloride solution at a dose of 0.08 mg/kg. The study was carried out over 42 days. Biological material samples were taken from 5 animals from each group the next day after the end of the drug administration (day 43), and 10 days after the last drug administration (day 53). The results of the study showed that dosages of 1/5 and 1/10 of the maximum tolerated do not cause external signs of toxicosis and death in rats. No significant changes were found in the biochemical parameters of the blood of animals in the experimental and control groups.

Epidemiological Situation on Cholera in the Russian Federation in 2023 and Forecast for 2024

The paper presents an analysis and assessment of risks of complication of epidemiological situation regarding cholera in the Russian Federation and a forecast for 2024. It briefly characterizes the dynamics of the epidemic process around the world during the last decade (2013–2022). The forecast of cholera importation into the Russian Federation, due to activity of the epidemic process in endemic countries, has been confirmed. In 2023, two cases of cholera importation from India were reported. It was established that the toxigenic strains isolated from patients belonged to the currently dominant in the world “post-Haitian” group, including in India and Bangladesh, with the genotype ctxB7 tcpACIRS rtxA4a VSP-IIΔ0495-0512. Furthermore, non-toxigenic Vibrio cholerae O1 strains were isolated from samples of biological material from persons arriving from India, containing preCTX prophage and distinct from the strain (ctxAB– tcpAEТ–) isolated from a patient with acute intestinal infection (Zaporozhye Region, Melitopol). Nontoxigenic NAG vibrios were isolated from 13 patients with acute intestinal infections, which is 1.9 times more than in 2022. Regardless of importations, a strain of V. cholerae O1 (ctxB1+tcpAEТ) of the “pre-Haitian” group was isolated from a water sample of a surface reservoir. 52 non-toxigenic V. сholerae O1 strains were found on the territory of 11 constituent entities of the Russian Federation, which is 18.9 % more than in 2022. In 2024, epidemiological risks of cholera importation from endemic countries to any administrative territory of the Russian Federation remain. The risks of epidemic manifestations of cholera are especially high in the territories of the constituent entities of the Russian Federation which are under ongoing emergency, due to migration processes, and the impact of other risk-forming factors that contribute to an increase in the degree of contact of the population with water bodies contaminated with cholera vibrios. If epidemic foci of cholera are to emerge in a neighboring country, there will be a high probability of spread of this infection in the stated constituent entities of the Russian Federation and in other territories of southern Russia. In the absence of epidemic manifestations of cholera, the possibility of occasional detection of toxigenic O1 serogroup strains in water samples due to unidentified introductions from cholera-affected areas cannot be ruled out.

Yeast fungi in the etiology of animal mycoses

760 samples of biological material from various regions of the Russian Federation were studied, 320 isolates of yeast fungi were isolated, which amounted to 42.10% of the total number of samples. Of the 760 samples, 29.0% belonged to cattle, 11.8% to small cattle, 13.0% to horses, 1.6% to camels, 7.2% to cats and 17.5% to dogs, 1.8% to raw milk and 0.8% to environmental objects (OVS). Most of the isolated isolates (240) of yeast fungi belonged to fungi of the genus Candida, which amounted to 75%. The species diversity of fungi of the genus Candida is represented by 15 species. The largest number of isolates belong to the species Candida albicans, Candida catenulata, Candida kefyr Candida krusei, Candida membranifaciens, Candida pararugosa and Candida tropicals. These species of fungi of the genus Candida are known as pathogens of fungal infections in humans and animals. The species diversity of species not belonging to the genus Candida is limited to 7 species in 5 genera – Geotrichum, Malassezia, Rhodotorula, Saccharomyces and Trichosporon. The fact of the development of resistance of some yeast species to the most commonly used antifungal drugs (fluconazole and voriconazole) has been established. It was revealed that 30% of the studied isolates of the genus Candida are resistant to fluconazole, 32% are dose–dependent, 38% are sensitive, 28% are resistant to voriconazole, 30% are dose–dependent, 42% are sensitive. Isolates not belonging to the genus Candida showed resistance to fluconazole 19%, 38% - dose–dependent, 43% - sensitive, voriconazole – 23% – resistant, 18% – dose–dependent and 59% - sensitive. The proportion of isolates of yeast fungi resistant to fluconazole and voriconazole isolated from animals is quite large, in some cases it is up to 50% (C. tropicalis). A high level of resistance was found in isolates of Candida albicans, Candida catenulata, Candida rugosa and Candida pararugosa, which can significantly complicate the treatment of infections caused by them and contribute to the worldwide problem of antibiotic resistance.

Effect of Paraquat toxicity on lung and kidney organs on histopathological image assessment

Background: Paraquat is one of the active ingredients of the gramoxone type herbicide, which has very high toxicity so that it can cause poisoning and death. Paraquat causes direct cell damage by producing free radicals, which can cause pulmonary fibrosis and kidney failure. This study aims to analyze the effects of paraquat toxicity on the lungs and kidneys on histopathological features. Methods: This research is an experimental research post-test only control group design. The research subjects used stored biological material in the form of 12 lung and kidney organ samples from white rats (Rattus norvegicus) that had been treated in previous research, which were divided into 2 groups, namely the negative control group (P1, given distilled water), the positive control group (P2; given paraquat 0.01 gr/kgBW [single dose intraperitoneal]). All samples of stored biological material were made for lung and kidney preparations. The lung condition was assessed using the American Thoracic Society criteria, and the kidneys were evaluated using the Mitchell criteria with the help of the ImageJ application. Data were analyzed using the Kruskal-Wallis test and the Mann-Whitney test. Results: The Kruskal-Wallis test showed a significant difference in lung damage scores between groups with a p-value of 0.01 (p <0.005). The results of the Kruskal-Wallis test of the kidneys showed substantial differences in glomerular and proximal tubular damage scores between groups with p values ​​of 0.004 and 0.009 (p <0.005). The results of the Mann-Whitney test also showed significant differences between groups P1 and P2 with a lung p value = 0.008 (p> 0.005) and kidney p = 0.009 (p > 0.005). Conclusion: In this study, it can be concluded that induction of paraquat 0.01 gr/kgBW causes changes in the histopathological appearance of the lungs and kidneys of white rats (Rattus norvegicus) in the form of lung and kidney damage as assessed by histopathological scores.

Prevalence and antimicrobial resistance of Enterobacteriaceae in the north of Kazakhstan.

An increasing number of drugs are used each year in the treatment of small pets (cats and dogs), including medicines (cephalosporins and fluoroquinolones) used in human therapy. The purpose of this study was to isolate and explore the antibiotic resistance of opportunistic Enterobacteriaceae (Escherichia coli, Klebsiella, Proteus, Ci trobacter, Enterobacter) from cats and dogs, and to isolate resistance genes in the microorganisms. In 2021, 808 samples of biological material from small domestic animals were collected in veterinary clinics in Kostanay. From these, 210 microorganisms were isolated and identified. A large majority of the strains sampled belonged to E. coli-149 (70.9%), Enterobacter-11 (5.2%), Klebsiella-28 (13.3%), Proteus-12 (5.7%) and 10 Citrobacter isolates (4.8%). In all isolates identified, antibiotic resistance/sensitivity was determined by disc-diffusion method to ampicillin, cefoxitin, gentamicin, levomycetin, tetracycline, ciprofloxacin, norfloxacin, ofloxacin, cefoperazone, cefpodoxime, streptomycin, kanamycin, doxycycline, gemifloxacin, nalidixic acid, furazolidone, furadonine, amoxicillin, and enrofloxacin. The study has demonstrated that the greatest number of Enterobacteriaceae were sensitive to the action of meropenem, which belongs to the group of beta-lactam antibiotics; resistance was demonstrated against tetracycline, doxycycline, ampicillin, amoxicillin, ofloxacin, and cefpodoxime. The most common genes encoding antimicrobial resistance were as follows: BlaTEM and OXA in 41 and 28 isolates, respectively, encoding resistance to beta-lactams; StrA and StrB in 45 and 48 isolates encoding aminoglycosides; and tetA and tetB in 43 and 28 isolates encoding tetracyclines. Obtained data demonstrate that uncontrolled and frequent use of beta-lactam and tetracycline antibacterials, in cats and dogs, results in the spread of genotypic resistance among micro-organisms of the family Enterobacteriaceae.

ЦИРКУЛЯЦИЯ ОРВИ У ДЕТЕЙ Г. БЕРДСКА НОВОСИБИРСКОЙ ОБЛАСТИ В СЕЗОН 2022-2023 Г.

Вackground. Acute respiratory diseases are the most common among all infectious diseases, both in the world and in the Russian Federation. Prevention of acute respiratory viral infections and timely detection of the viral etiology of these diseases is an urgent public health task. Purpose of the study. The aim of this work was to identify the viral etiology of acute respiratory diseases in children's age group patients who sought medical help with signs of upper respiratory tract infections at the Berdsk Central City Hospital. Patient Characterization and Research Methods. The target group of the study was patients aged 0-17 years in the acute stage of upper respiratory tract disease who presented with signs of infection. Identification of the genetic material of respiratory viruses was carried out by real-time PCR using the AmpliSens “ARVI - FL” diagnostic kits (FBIS Central Research Institute of Epidemiology of Rospotrebnadzor, Russia). Methods of statistical analysis. Microsoft Office Excel program was used. Results and conclusion. The result of the research revealed that 81.33% of samples of biological material received a positive result for at least one infection. Identified: RNA viruses SARS-CoV-2, influenza A (Influenza virus A), influenza B (Influenza virus B), respiratory syncytial virus (hRSv), metapneumovirus (hMPV), parainfluenza types 1, 2, 4 (hPiv), RNA coronaviruses species OC43, E229, NL63, HKUI (HCoV), RNA of rhinoviruses (hr v), DNA of adenoviruses of groups B, C and E (HAdV), DNA of bokavirus (HBoV), and in 12.59% of cases of the number of positive samples, the presence of coinfections is observed. The results of this work are aimed at verifying the etiological factor of ARVI and strengthening the prevention of the spread of upper respiratory tract infections in the population.

Possibilities of a biobank for serological monitoring of infectious morbidity

Currently, monitoring infectious morbidity, control and forecasting the epidemic process is a topical health problem.Aim. To analyze the capabilities of the biobank for serological monitoring of infectious diseases using the Epstein- Barr virus (EBV), which is associated with infectious mononucleosis, as well as somatic pathology, including cancer.Material and methods. Using the quantitative enzyme- linked immunosorbent assay for specific antibodies to various EBV proteins, we studied blood serum samples from healthy individuals (2010, 2016, 2019, 2020, 2021 and 2022) from the collection of the N. F. Gamaleya National Center of Epidemiology and Microbiology.Results. For the first time, the intensity of EBV epidemic process among apparently healthy adult residents of the capital was assessed using samples of biological material and a biobank information database. At the time of examination, 0,8% of individuals were not infected with EBV, and 9,4% suffered reactivation of a chronic infection. There were no significant changes in detection rate of markers over the years.Conclusion. Certified collections of blood sera can be effectively used to study population immunity, the prevalence of individual infections, population protection nationwide, etc.

Specific features of distribution and manifestation of cryptosporidiosis of calves on dairy complexes in Siberia

The results of the study of specific features of distribution and clinical manifestation of bovine cryptosporidiosis in association with pathogens of viral and bacterial nature are presented. From 2014 to 2023, 666 samples of biomaterial (tracheal mucosa, lungs, mediastinal and mesenteric lymph nodes, contents of rennet and intestine) collected from fallen and forcefully killed calves under 6 months of age with signs of enteritis and respiratory diseases were examined. 94 farms of six regions, two territories of the Siberian region and the Republic of Kazakhstan were surveyed. The findings confirm the widespread prevalence of Cryptosporidium in calves. Cryptosporidium parvum oocysts were most frequently detected and were present in 34 (5.1%) samples of biological material from calves in 22 (23.4%) of the surveyed farms. It was found that oocysts were rarely (22.7%) present in monovariant in animals, more often (77.3%) they were found in associations with viruses and bacteria during outbreaks of viral-bacterial infections. Young animals under 30 days of age are most susceptible to infection. Newborn calves are most susceptible to infection with oocysts, and those animals that have had the disease become lifelong sources of the pathogen for susceptible animals. Cryptosporidiosis is an independent disease and runs independently of the presence of the viruses and bacteria in the body and has no synergistic interaction with them, but the presence and multiplication in the body of animals of this pathogen contributes to the severity of the course of enteritis in calves and leads to difficulties in the treatment and preventive measures. The presence of Cryptosporidium in animals should be taken into account when carrying out complex anti-epizootic measures.

The rationale for the choice of preventive intraoperative anesthesia for uterine curettage in patients with individual intolerance to amide anesthetics

Background. Uterine curettage (UC) is widely used in global clinical practice owing to its great diagnostic and therapeutic value. At the same time, UC leads to a damage to the integrity of soft tissues, causing development of postoperative pain syndrome. Some patients have an individual intolerance to amide anesthetics, which limits their use in this cohort. Purpose: to improve the quality of providing anesthetic care in the early postoperative period after UC to women who have a history of individual intolerance to amide anesthetics through selection of a comprehensive method of preventive intraoperative anesthesia for this category of patients. Materials and methods. Ninety females took part in the study. Clinical, laboratory, and mathematical and statistical research methods were used to study the preventive analgesic effectiveness of the compared methods of anesthesia. Five samples of biological material, called the control points, were taken: before surgery, immediately after it, then 1, 3 and 6 hours after intervention. In this way, an idea was got about the initial level of the studied indicators and their changes during the postoperative stay in the hospital. Properties of various combinations of propofol, ketamine, fentanyl, and dexketoprofen were studied. Results. It was revealed that a combination of propofol (2 mg/kg), fentanyl (0.1 µg/kg), ketamine (0.1 mg/kg), as well as additional intravenous administration of dexketoprofen (50 mg) at the stage of premedication has the advantages in terms of preventive intraoperative analgesia. Effectiveness of the proposed method of anesthesia was proved by significantly better indicators of vital functions (heart rate, mean arterial pressure) and serum levels of some laboratory markers of stress (glucose, cortisol, insulin, cortisol-insulin index). Differences in the studied parameters were recorded both when comparing them between the groups at the same control time points and conside-ring the development and regression of the postoperative pain syndrome severity within the groups. Conclusions. The proposed method of anesthesia has a reliable advantage in terms of preventive reduction of the postoperative pain syndrome of UC in patients who cannot have application anesthesia due to individual intolerance to amide drugs. The specified method has a positive effect on vital functions and serum levels of some laboratory markers of stress, and the fastest recovery of patients is caused by a lower suppressive action of the anesthetic drugs used.

Awareness, Attitudes and Willingness to Donate Biological Samples to a Biobank: A Survey of a Representative Sample of Polish Citizens.

Biotechnology is developing at an ever-increasing pace, and the progressive computerization of health care and research is making it increasingly easy to share data. One of the fastest growing areas is biobanking. However, even with the best equipment and the best trained staff, a biobank will be useless without donors. For this reason, we have decided to gauge Polish citizens' awareness and attitudes towards biobanking and their willingness to donate biological samples. For this purpose the survey was conducted among a nationwide group of 1052 Poles aged 18 and over where the totals for gender, age and place of residence were selected according to their representation in the total population of adult Poles. The survey was conducted using the Computer Assisted Web Interview (CAWI) technique. Approximately two thirds of respondents N = 701 (66.6%) indicated that they had heard of scientific studies in which samples of biological material such as blood, saliva or urine are collected. More than half of respondents (N = 613, 58.3%) had a positive opinion regarding scientific research in which samples of biological material are taken. Only N = 220 (20.9%) of respondents had previously encountered the term biobanking. More than a half N = 687 (65.3%) of respondents would participate in a scientific study that biobanked biological material and health information. Almost half of the respondents (48.0%) would like specific consent to be used in biobanking. In our study we observed a negligible correlation between socio-demographic factors and a willingness to donate biological material to a biobank. Considering the results presented above, the level of knowledge and awareness of biobanks, and their role in scientific research and the health care system, among Polish citizens is low and requires education and information activities.

The role of culture-negative infection among infectious complications after total knee arthroplasty

Introduction Diagnosis of chronic periprosthetic joint infection (PJI) is difficult with the clinical signs of periprosthetic inflammation showing no growth of microorganism in the biomaterial. The frequency of culture-negative infection can reach 42.1 %. The objective of the study was to analyze outcomes of two-stage treatment of chronic PJI of the knee joint depending on the etiology of the infectious process.Material and methods A retrospective analysis of outcomes was produced for 103 patients: group I (n=30) showing no growth of microorganisms and group II (n = 73) demonstrating positive growth of pathogens. Knee PJI was diagnosed according to the 2018 ICM criteria. A favorable outcome suggested absence of recurrence for at least two years after reimplantation of endoprosthesis, arthrodesis, “life with a spacer” without signs of infection.Results Culture-negative infection was detected in 29.1 % (n = 30). Patients in the group were 1.5 times more likely to receive antibiotic therapy prior to admission and had average levels of CRP, ESR and articular leukocyte count being 1.5-2 times less than those in group II. Staphylococci (69.8 %) including MRSE (75 %) was the leading pathogen in group II. Recurrence of infection was 3.4 % in group I and 16.9 % in group II (p = 0.0928), the two-stage treatment was successful in 96.7 % and 74 %, respectively (p = 0.0064).Discussion Causes for the lack of growth of microorganisms in biological materials included previous antibiotic therapy, wound drainage, violations of the rules for sampling of biological material, absence of media for the growth of atypical microorganisms and the ability of microorganisms to form biofilms on implant surfaces. An emergency histological examination of the affected tissues was practical during surgery in doubtful situations for adequate surgical approach. The results of a meta-analysis (2023) showed that the replacement of an infected endoprosthesis was more effective for the treatment of a culture-negative infection compared to debridement and preservation of implant.Conclusion The culture-negative infection group in our series showed better success rate of a two-stage treatment of PJI using implant replacement and broad-spectrum empiric antibiotic therapy at a two-year follow-up period. The negative microbiological result in the group could be caused by antibacterial drugs administered prior to diagnosis of PJI.

The specifics of CpG islets methylation of some genes of peripheral blood T-lymphocytes in breast cancer patients before and after treatment

Introduction. The growth of primary breast tumor morbidity in the last ten years and increased number of patients with disseminated breast cancer in the Republic of Kazakhstan require the search for methods of early diagnosis of malignant tumors. Determination of breast cancer markers in epigenetic studies allows to use them as diagnostic signs of the presence of malignant tumor and as predictors of treatment effectiveness in patients with this pathology.Aim. To perform a search for therapeutic and prognostic breast cancer markers.Materials and methods. The study included samples of biological material (peripheral blood) of 50 deemed healthy individuals and 103 patients with locally advanced and disseminated breast cancer receiving special therapy. The following methods were used: blood collection, DNA extraction, creation of DNA methylation profiles, sequencing, statistical data analysis.Results. The results of search for epigenetic mutations in peripheral blood of patients with breast cancer showed their role as specific diagnostic, therapeutic and prognostic markers with specificity 0.91 % and sensitivity 0.94 %. The hypothesis on therapeutic significance of identified earlier diagnostic markers in patients with breast cancer, namely hypermethylation of CpG islands associated with genes JAM3, C17orf64, MSC, C7orf51 and CpG island associated with intragene part of chromosome 5 (chr5: 77,208,034–77,329,434) was tested and confirmed.Conclusion. A correlation between DNA methylation characteristics and disease progression during treatment was shown. The study results can be used in clinical practice: epigenetic markers, such as methylation in the CpG islets associated with the JAM3, C17orf64, MSC, C7orf51 genes, and in the CpG islet associated with the intragenic site of chromosome 5 (chr5: 77,208,034–77,329,434) can be used as prognostic markers and therapeutic predictors of breast cancer.

Poland - National regulation on processing data for scientific research purposes and biobanking activities

Biobanks raise great hopes for future research that can help understand many diseases and find ways to treat them. The purpose of the article is to study the legislative acts regulating the activities of biobanks in Poland and to formulate recommendations for improving the legal regulation of biobanking. For the purpose, tasks, object, and subject of research, both general scientific and special methods of scientific knowledge were used in the work. General scientific methods (dialectical; system analysis; formal-logical) are fundamental legal techniques, and means of research. To improve the current legislation of Poland, the following can be proposed. Develop a legal framework that regulates the legality of obtaining samples of human biological materials (both normal and pathologically altered) for submission to the biobank in Poland. Regulate at the legislative level the requirements regarding the organizational and legal form of the creation of a biobank, the legal basis of its functioning, the form of ownership, and financing. Thus, the creation of an effective system of biobanks in Poland with systematic collections of biological samples obtained from people with various diseases, their storage in appropriate conditions, the development of internal documentation and standard operating procedures, taking into account both the ethical and legal components, is of key importance for the formation and development of domestic science, development of innovative medicines, methods of diagnosis and monitoring of diseases.

#3822 POLYOMAVIRUS INFECTION MONITORING BY QUANTITATIVE PCR IN KIDNEY RECIPIENTS AS AN INSTRUMENT FOR PREVENTING POSTTRANSPLANT COMPLICATIONS

Abstract Background and Aims Polyomaviruses (PyV) are ubiquitous human viral pathogens. BKV and JCV representing this viral family are common causative agents of viral complications among kidney recipients. Viral load higher than 1 × 107 copies/ml in urine (viruria) or 1 × 104 copies/ml in serum (viremia) in posttransplantation period may lead to polyomavirus-associated nephropathy (PVAN), hemorrhagic cystitis (HC) or even kidney transplant failure. The aim of the study was to assess PyV reactivation frequency in patients during 12 months after renal transplantation (RT) and to identify molecular subtypes of BKV and JCV. Method We examined 3207 samples of biological material (serum and urine) of 763 adult (>18 years) patients who underwent renal transplantation (RT) at the State Institution “Minsk Scientific and Practical Center for Surgery, Transplantology and Hematology”, Healthcare institutions “Brest Regional Clinical Hospital”, “Vitebsk Regional Clinical Hospital Belarus”, “Mogilev Regional Clinical Hospital”. These patients were divided into 2 groups: group 1 included 394 patients examined only for BKV infection, group 2 – 356 recipients examined for both BKV and JCV infection. Serum and urine samples for regular monitoring were collected from patients before RT, every 2 weeks first 3 months, then at 6, 9, 12 months after RT. In the case of complication development samples from patients were collected later then 1-year monitoring period. PyV DNA was detected by real-time PCR. Viral DNAs from 17 BKV-positive and 11 JCV-positive patients were molecular typed by partial sequencing of VP1 genome region. Confidence intervals for the proportions were calculated using Wald's method. Results Results showed that BKV detection total frequency in the group 1 was 14.47% [11.32%; 18.3%], almost all patients developed viruria, only 2.54% [1.32%; 4.67%] had viremia. In the group 2 PyV DNA was detected in 46.07% [40.96%; 51.26%] of recipients: 19.10% [15.34%; 23.52%] had BKV infection, 19.94% [16.11%; 24.42%] – JCV, 7.02% [4.76%; 10.2%] – BKV+JCV mixed infection. Frequency of viremia was 6.74% [4.53%; 9.87%] in this group. Maximal BKV viral load levels reached 1.2 × 1012 copies/ml in urine and 5.9 × 107 copies/ml in serum. JCV loads were up to 3 × 109 copies/ml in urine and 1.2 × 108 copies/ml in serum. Then we analyzed frequency of PyV detection before RT and during the first year after RT among the 102 recipients. Results displayed on the Fig. 1 showed that the peak of PyV infection registration and the higher risk for patient had a place on the 1.5-2.5 months after RT. Quantitative monitoring of viral load in posttransplant period was the basis for the correction of the applied immunosuppressive therapy regimens in relation to the recipients with a high viral load (higher than 1 × 107 copies/ml in urine or 1 × 104 copies/ml in serum). The results of molecular typing showed that 17 BKV isolates belonged to subgroups Ib-2 and IVc-2 (12 and 5 isolates, respectively). Within subgroups Ib-2 isolates formed 3 clusters corresponding 3 separate genovariants. JCV isolates belonged to subtype 1A, 1B and 2A (7, 3 and 1 of isolates, respectively). The last one had 99% nucleotide sequence similarity with Greece and South Korea isolates. Conclusion Our data demonstrated an importance of PyV DNA monitoring of kidney recipients in the posttransplant period starting from the first days after RT to predict development of PyV complications as PVAN, HC or others by correcting the immunosuppressive therapy.

#3822 POLYOMAVIRUS INFECTION MONITORING BY QUANTITATIVE PCR IN KIDNEY RECIPIENTS AS AN INSTRUMENT FOR PREVENTING POSTTRANSPLANT COMPLICATIONS

Abstract Background and Aims Polyomaviruses (PyV) are ubiquitous human viral pathogens. BKV and JCV representing this viral family are common causative agents of viral complications among kidney recipients. Viral load higher than 1×107 copies/ml in urine (viruria) or 1×104 copies/ml in serum (viremia) in posttransplantation period may lead to polyomavirus-associated nephropathy (PVAN), hemorrhagic cystitis (HC) or even kidney transplant failure. The aim of the study was to assess PyV reactivation frequency in patients during 12 months after renal transplantation (RT) and to identify molecular subtypes of BKV and JCV. Method We examined 3207 samples of biological material (serum and urine) of 763 adult (>18 years) patients who underwent renal transplantation (RT) at the State Institution "Minsk Scientific and Practical Center for Surgery, Transplantology and Hematology", Healthcare institutions "Brest Regional Clinical Hospital", "Vitebsk Regional Clinical Hospital Belarus", "Mogilev Regional Clinical Hospital". These patients were divided into 2 groups: group 1 included 394 patients examined only for BKV infection, group 2 – 356 recipients examined for both BKV and JCV infection. Serum and urine samples for regular monitoring were collected from patients before RT, every 2 weeks first 3 months, then at 6, 9, 12 months after RT. In the case of complication development samples from patients were collected later then 1-year monitoring period. PyV DNA was detected by real-time PCR. Viral DNAs from 17 BKV-positive and 11 JCV-positive patients were molecular typed by partial sequencing of VP1 genome region. Confidence intervals for the proportions were calculated using Wald's method. Results Results showed that BKV detection total frequency in the group 1 was 14.47% [11.32%; 18.3%], almost all patients developed viruria, only 2.54% [1.32%; 4.67%] had viremia. In the group 2 PyV DNA was detected in 46.07% [40.96%; 51.26%] of recipients: 19.10% [15.34%; 23.52%] had BKV infection, 19.94% [16.11%; 24.42%] – JCV, 7.02% [4.76%; 10.2%] – BKV+JCV mixed infection. Frequency of viremia was 6.74% [4.53%; 9.87%] in this group. Maximal BKV viral load levels reached 1.2×1012 copies/ml in urine and 5.9×107 copies/ml in serum. JCV loads were up to 3×109 copies/ml in urine and 1.2×108 copies/ml in serum. Then we analyzed frequency of PyV detection before RT and during the first year after RT among the 102 recipients. Results displayed on the fig.1 showed that the peak of PyV infection registration and the higher risk for patient had a place on the 1.5-2.5 months after RT. Quantitative monitoring of viral load in posttransplant period was the basis for the correction of the applied immunosuppressive therapy regimens in relation to the recipients with a high viral load (higher than 1×107 copies/ml in urine or 1×104 copies/ml in serum). The results of molecular typing showed that 17 BKV isolates belonged to subgroups Ib-2 and IVc-2 (12 and 5 isolates, respectively). Within subgroups Ib-2 isolates formed 3 clusters corresponding 3 separate genovariants. JCV isolates belonged to subtype 1A, 1B and 2A (7, 3 and 1 of isolates, respectively). The last one had 99% nucleotide sequence similarity with Greece and South Korea isolates. Conclusion Our data demonstrated an importance of PyV DNA monitoring of kidney recipients in the posttransplant period starting from the first days after RT to predict development of PyV complications as PVAN, HC or others by correcting the immunosuppressive therapy.

Molecular Diagnostics of Oncological Disease: Prospects for the Development of a Reference Material for the HER2 gene Content

Cancer is the leading cause of death in the world. The development of oncopathology is closely related to various changes in the genetic material that occur in malignantly transformed cells. Medical decision-making requires a clear differentiation between normal and pathological indicators, which are, among other things, the results of application of quantitative methods in laboratory medicine. Studies of DNA isolated from a patient’s biological material, identification and measurement of the content of nucleotide sequences acting as oncopathology biomarkers allow to solve the problems of determining the genetic prerequisites for cancer, its early diagnosis, determining the treatment strategy, monitoring, and confirming the patient’s cure.The purpose of this research is to develop the main approaches to the design of DNA reference materials (RMs) for metrological support of molecular diagnostics of oncopathology through the example of the RM for the HER2 gene sequence content in the human genome, with the value of «the number of copies of the DNA sequence» which is metrologically traceable to the natural SI unit «one».In the course of the research, a technique for measuring the HER2 gene amplification (the number of copies of the gene sequence per genome) was developed based on the use of the digital PCR method (dPCR). Comparability of measurement results for the method developed by the authors, and the results obtained using a commercial kit by the MLPA method on samples of human biological material is shown.Five permanent cell lines obtained from the CUC «Vertebrate Cell Culture Collection» were characterized in relation to the copy number ratios of HER2 gene sequence and CEP17 and RPPH1 genes sequences. A cell line with the HER2 gene amplification was identified. The results obtained will be used to create the RM for the copy number ratio of the HER2 gene sequences and the RPPH1 and CEP17 gene sequences. Creation of matrix DNA RMs based on human cell cultures certified using dPCR will allow transferring the unit of copy numbers of the DNA sequence to calibrators included in medical devices, thereby ensuring the required reliability and comparability of measurement results in the laboratory diagnostics of oncopathology, as well as the possibility of calibrating routine methods of DNA diagnostics and intralaboratory quality control.

Biological and molecular genetic properties of <i>Trichophyton benhamiae</i>, a new pathogen of dermatomycoses in cats

The results of isolation of two strains OVB_T. b-19 and OVB_T. b-20 of a new microscopic fungus species Trichophyton benhamiae from the samples of biological material from cats with clinical signs of dermatomycosis are presented.This type of fungus was isolated from domestic cats for the first time in Russia. Molecular genetic studies, species identification and determination of the properties of the isolated cultures were carried out using approved methodological recommendations and determinants of pathogenic and opportunistic fungi. The keratinolytic and biochemical activity, cultural-morphological (phenotypic) and molecular-genetic properties of T. benhamiae were studied. Both strains of the fungus were characterized by a variety of phenotypic properties: they formed colonies on the nutrient media that differed in morphology and color of aerial and substrate mycelium. They revealed the similarity of micromorphology: the presence of a septate bamboo-like mycelium with characteristic branching of two-layer macroconidia and microconidia. The studied strains were characterized by similar biochemical properties (pronounced saccharolytic and urease activities) and keratinolytic activity. The identified keratinolytic activity of the T. benhamiae strains indicates their etiological role in the development of dermatomycoses in domestic cats. The phenotypic characteristics fully corresponded to the culture of the microscopic fungus T. benhamiae. Molecular genetic studies revealed that microscopic fungi isolated from cats belonged to the genus Trichophyton, species Benhamiae. Molecular genetic studies established that the sequences of OVB_T. b-19 and OVB_T. b-20 strains that had been obtained were identical. Both strains are listed in the GenBank database with individual numbers in the international NCBI database, ON479483 and ON479484.

Primer set for detecting enterococcal bacteriophages

Introduction. The development and use of therapeutic drugs based on bacterial viruses, or bacteriophages, is a promising direction in the fight against bacterial infections. The composition of phage preparations must be constantly updated, which requires the search for new viruses through the screening of biological material and samples from the environment.Purpose. Development of a method for the search and identification of virulent enterococcal bacteriophages based on the polymerase chain reaction (PCR).Materials and methods. The known diversity of enterococcal viruses was assessed by database searches of the National Center for Biotechnology Information (NCBI) and the International Committee on Taxonomy of Viruses (ICTV). Primers were selected using the NCBI PrimerBlast and Primer3 programs. Primers were tested on seven commercial phage cocktails and 46 biomaterial samples. The specificity of PCR was confirmed by determining the nucleotide sequences of PCR products.Results. The obligately virulent enterococcal bacteriophages described in the literature belong to five ICTV approved genera: Copernicusvirus, Efquatrovirus, Kochikohdavirus, Saphexavirus, and Schiekvirus. Representatives of the sixth genus, Phifelvirus, have a temperate life cycle. The PCR scheme developed by us is intended for specific amplification of fragments of the gene of the main capsid protein of the mentioned genera of bacteriophages. It was used to identify representatives of all five genera of virulent enterococcal bacteriophages in commercial phage cocktails. In samples of biological material, we identified representatives of the genera Efquatrovirus, Kochikohdavirus, Saphexavirus and Schiekvirus.Conclusion. The PCR scheme presented in this work makes it possible to detect all currently described obligately virulent bacteriophages infecting Enterococcus spp. in phagolysates and samples of biological material, and can also be used to determine the genera of viruses.