Effect of <i>Yersinia pestis</i> on the Soil Nematodes <i>Panagrolaimus</i> sp. from the Gorno-Altai High-Mountain Focus of Plague

The aim of the work was to study interaction of Yersinia pestis with soil nematodes isolated on the territory of the Gorno-Altai high-mountain plague focus. Materials and methods. We used the fluorescent Y. pestis strain KM2083, a derivative of the natural strain of the 4.ANT phylogenetic line, the antique biovar of the main subspecies, and a nematode culture isolated in the same area of the Gorno-Altai plague focus. The taxonomy of nematodes was determined by the region of the 18S rRNA gene; phylogenetic analysis was performed using the Maximum Likelihood method based on the Tamura-Nei model in the Mega 7.0 software. The interaction of the Y. pestis KM2083 strain and the nematodes was studied during cultivation on a solid NGM agar medium. Nematodes were observed using microscopes Stemi-2000C (Carl Zeiss, Germany) and Axio Imager Z2 (Carl Zeiss, Germany). Results and discussion. It has been established that the nematodes from the Gorno-Altai high-mountain plague focus used in the work belong to the genus Panagrolaimus. Cultivation of nematodes on the lawn of the Y. pestis strain of the main subspecies of antique biovar, the 4.ANT phylogenetic line for 24 hours did not lead to a reduction in the lifespan of nematodes compared to the control sample, which indicates the absence of toxicity of the used strain towards Panagrolaimus nematodes. On the cuticle of nematodes, the formation of a biofilm in the genital area and tail has been noted, and accumulations of fluorescent cells of the plague pathogen observed in the digestive tract. The data obtained can indicate the ability of nematodes to carry the plague pathogen in the soil biocoenosis

Neurofibromatosis: analysis of clinical cases and new diagnostic criteria

Neurofibromatoses are a group of genetic disorders with predisposing for central and peripheral nervous system tumor development. The group includes three entities: neurofibromatosis type I, neurofibromatosis type II and schwannomatosis, which are characterized by gradual phenotype development and have a partially overlapping spectrum of manifestations, which complicates diagnosis establishing, especially at the stage of clinical onset. At the same time, the emergence of new pathogenetic therapy and the high risk of transmission to descendants actualize the necessity of early diagnosis. DNA tests allow us to reliably confirm the presumed diagnosis. This article presents a review of neurofibromatoses, their clinical features and courses, modern diagnostic criteria and indications for DNA tests

Development of an Integrated System for Molecular-Genetic Identification of <i>Yersinia pestis</i> Strains

The paper describes a developed comprehensive system for molecular-genetic identification of Yersinia pestis strains according to their appurtenance to certain subspecies, biovars, phylo-geographic populations, using realtime PCR (RT-PCR), allele-specific RT-PCR, and multiplex PCR with hybridization fluorescent registration of results on a solid substrate. Application of this system makes it possible to establish the appurtenance of Y. pestis strains to the following phylogenetic branches: 0.ANT1, 0.ANT2, 0.ANT3, 0.ANT5, 3.ANT, 4.ANT of antique biovar of the main subspecies; 2.MED0, 2.MED1, 2.MED2, 2.MED3, 2.MED4 of medieval biovar of the main subspecies; 1.IN1, 1.IN2, 1.IN3 of intermedium biovar of the main subspecies; 1.ORI1, 1.ORI2, 1.ORI3 of oriental biovar of the main subspecies; 0.PE3 (angolica subspecies), 0.PE7 (tibetica subspecies) and 0.PE10 (qinghaica subspecies). The first stage of the studies within the frames of the developed system is indication of plague agent using registered diagnostic drugs. The second stage is the determination of belonging to individual subspecies through RT-PCR or by the method of multiplex PCR system with hybridization-fluorescent registration of results on a solid substrate, which also allows for establishing to which biovars of the main subspecies and the main phylogenetic lines of the ancient biovar the strains belong. The third stage is the identification of strain appurtenance to phylogenetic branches by the AS-RT-PCR method. The designed complex system for molecular-genetic identification of Y. pestis strains can be applied at the regional and federal levels of the laboratory network of the Russian Federation for diagnostics of infectious diseases. Its use will considerably facilitate and increase the efficiency of intraspecific differentiation of Y. pestis strains within the framework of the epidemiological investigation of outbreaks or importation of strains of plague pathogen into the territory of the Russian Federation or during the certification of strains in collection activities

Long-Term Persistence of <i>Yersinia pestis</i> in Association with Acanthamoeba castellanii in Experiment

The aim of the study was to test the feasibility of long-term survival and preservation of the properties of Yersinia pestis in association with soil amoeba Acanthamoeba castellanii. Materials and methods. Y. pestis strains and acanthamoeba isolated in the common area of the Gorno-Altai high-mountain plague focus were used for the study. The systematic affiliation of protozoa was determined through analyzing the 18S rRNA gene fragment sequencing data, followed by alignment with amoeba sequences from the NCBI GenBank database. A fluorescent Y. pestis strain was obtained by electroporation using the pTurboGFP-B plasmid. Co-cultivation was carried out in saline buffer in the absence of nutrients for the cells of plague pathogen. The influence of co-culturing with protozoa on Y. pestis properties was determined using microbiological, biological, and molecular-genetic methods. Results and discussion. The cell viability preservation for 22 months of the experiment in Y. pestis strain belonging to the main subspecies of the antique biovar, the 4.ANT phylogenetic line in co-culture with amoeba cells in the absence of additional nutrients has been established. Co-cultivation with amoebae did not lead to a change in the cultural, morphological, genetic and virulent properties of the plague pathogen strain. The data obtained confirm the possibility of using Acanthamoeba castellanii by the plague microbe to persist in soil biocenoses and open up the prospect of studying the mechanisms of plague pathogen surviving during extended inter-epizootic periods

Specific Appurtenance, Numbers, and Dynamics of Interaction of Acanthamoeba from Soils of Gorno-Altai High-Mountain Plague Focus with Yersinia pestis Strains

Objective of the study is to analyze the appurtenance, numbers, and dynamics of interaction of acanthamoeba from soils of GornoAltai plague focus with Yersinia pestis 367 strain, isolated in 2016 in enzootic territory of this focus. Materials and methods. Utilized were soil amoeba from Gorno-Altai high-mountain focus and the strain Y. pestis 367 of the main subspecies of antique biovar, isolated there in 2016. Determination of systematic relation of the isolated amoeba was carried out using PCR with genus specific primers and sequencing of the obtained PCR fragments followed by identification of nucleotide sequences against GenBank database. Localization of Y. pestis cells in acanthamoeba was performed using fluorescent antibody technique by means of Axio Imager Z2 (Carl Zeiss, Germany). Results and conclusions. For the first time ever established has been the presence of Acanthamoeba castellanii in soils of burrows of Marmota altaica in the numbers of up to 300000 cells/gr in Gorno-Altai high-mountain focus. Investigated has been the dynamics of interaction of these microorganisms. Preservation of the agent in vacuoles of endoplasmatic reticulum within 14 days has been revealed. It is an indicative of the possibility of Y. pestis persistence in amoeba of Acanthamoeba subspecies in soil biocoenosis of Gorno-Altai high-mountain plague focus

Нейрофиброматоз: анализ клинических случаев и новые диагностические критерии

Neurofibromatoses are a group of genetic disorders with predisposing for central and peripheral nervous system tumor development. The group includes three entities: neurofibromatosis type I, neurofibromatosis type II and schwannomatosis, which are characterized by gradual phenotype development and have a partially overlapping spectrum of manifestations, which complicates diagnosis establishing, especially at the stage of clinical onset. At the same time, the emergence of new pathogenetic therapy and the high risk of transmission to descendants actualize the necessity of early diagnosis. DNA tests allow us to reliably confirm the presumed diagnosis. This article presents a review of neurofibromatoses, their clinical features and courses, modern diagnostic criteria and indications for DNA tests.Нейрофиброматозы – группа наследственных заболеваний, которые характеризуются развитием опухолей центральной и периферической нервной системы. Включают 3 нозологии: нейрофиброматоз I типа, нейрофиброматоз II типа и шванноматоз. Эти заболевания отличаются динамическим развитием и имеют схожие клинические проявления, что осложняет постановку клинического диагноза, особенно на этапе клинического дебюта. В то же время появление новых методов патогенетической терапии и высокий риск передачи заболевания потомству обусловливают необходимость ранней диагностики. Одним из методов подтверждения предполагаемого диагноза является молекулярно-генетическое тестирование. В данной статье приведен анализ данных литературы, посвященных особенностям клинического течения нейрофиброматозов, актуальные диагностические критерии, а также показания к проведению молекулярно-генетической диагностики

Клинико-эпидемиологическая характеристика гепатитов В и дельта в Республике Дагестан

Aim: The analysis of the incidence of hepatitis B in the Republic of Dagestan (RD) and clinical and epidemiological characteristics of HBV/HDV coinfection in the region.Materials and Methods. The dynamics of the hepatitis B incidence rates and the coverage of vaccination against this infection in the RD in 2008-2022 were analyzed based on the data from of the statistical forms of Rospotrebnadzor. The clinical and epidemiological characteristics of delta hepatitis were analyzed in 371 patients under dispensary observation at the Republican Center for Infectious Diseases named after S.-А.М. Magomedov.Results. Over the past 10 years, the incidence of CHB in the RD has increased more than 4.5 times, from 1.4 per 100 thousand population in 2008 to 6.7 per 100 thousand population in 2022. A decrease in the rates of hepatitis B child immunization in the RD is observed since 2009. Hepatitis B vaccination coverage rates in adult population fell sharply after 2010, both in the RD and in the Russian Federation  on average. The frequency of HDV co-infection in persons infected with HBV in the RD is 13.8%, but reaches 15% in some regions of the republic, indicating the moderate level   of endemicity. Patients with HBV/HDV coinfection are predominantly males aged 25–45 years with advanced fibrosis or cirrhosis. All cases of HDV infection in the RD are caused by viral genotype 1.Conclusions. The obtained results testify to the significance of the problem of hepatitis B and delta in the RD. The number of identified patients and, accordingly, the rate of co-infection, apparently, will increase with the expansion of screening for markers of HDV infection, when patients who were registered as HBsAg carriers will be examined according to the patient routing guidelines. The late diagnosis of delta hepatitis in RD and the limited possibilities of antiviral therapy are another significant issues.Цель: анализ заболеваемости гепатитом В в Республике Дагестан и клинико-эпидемиологическая характеристика ко-инфекции HBV/HDV в регионе.Материалы и методы: проведен анализ динамики заболеваемости гепатитом В и охвата вакцинацией против этой инфекции в Республике Дагестан в 2008–2022 гг. по материалам форм статистического учета Роспотребнадзора. Проанализированы клинико-эпидемиологические  характеристики  гепатита   дельта  у 371 пациента, находящихся на диспансерном наблюдении в Республиканском центре инфекционных болезней им. С.-А.М. Магомедова.Результаты: за последние 14 лет заболеваемость хроническим гепатитом В в Республике Дагестан увеличилась более чем в 4,5 раза, с 1,4 на 100 тыс. населения в 2008 г. до 6,7 на 100 тыс. населения в 2022 г. Результаты анализа охвата вакцинацией против гепатита В свидетельствуют о снижении уровня иммунизации детского населения в Республике Дагестан после 2009 г. Показатели охвата вакцинацией против гепатита В взрослого населения резко упали после 2010 г., как в Республике Дагестан, так и в Российской Федерации в среднем. Частота ко-инфекции HDV у лиц, инфицированных HBV, в Республике Дагестан составляет 13,8%, но достигает 15% в отдельных районах республики, что позволяет отнести ее к регионам с умеренной эндемичностью. Среди пациентов с ко-инфекцией HBV/HDV преобладают мужчины в возрасте 25–45 лет, с продвинутыми стадиями фиброза или циррозом. Во всех случаях HDV-инфекция была вызвана 1 генотипом вируса.Заключение:  полученные  результаты  свидетельствуют о значимости проблемы гепатитов В и дельта в Республике Дагестан. Количество выявленных больных и, соответственно, показатель частоты ко-инфекции, повидимому, будут увеличиваться при расширении скрининга на маркеры HDV-инфекции, когда пациенты, находившиеся на диспансерном учете как носители HВsAg, будут обследованы по алгоритму согласно приказу о маршрутизации. Не меньшей проблемой является поздняя диагностика гепатита дельта в Республике Дагестан и ограниченные возможности противовирусной терапии

Phylogeny and Historical-Geographical Analysis of <i>Yersinia pestis</i> Strains from Vietnam

Objective of the work was to identify molecular-genetic peculiarities, to conduct whole genome sequencing and phylogenetic analysis of Yersinia pestis strains isolated inVietnam between 1962 and 1989.Materials and methods. We have studied the properties of 50 Y. pestis strains, carried out whole genome sequencing of 18 and fragment sequencing of 32 strains from Vietnam. Phylogenetic analysis was performed on the basis of whole genome SNPanalysis by 1391 identified SNPs. Whole genome SNP-analysis and search for marker SNPs were conducted applying Wombac 2.0 software package. Phylogenetic diagram construction was done using Maximum Likelihood algorithm.Results and discussion. Several phylogenetic branches and Y. pestis populations coinciding with geographical and historical dissemination of the strains have been distinguished. The major part of the strains from Vietnam falls under the branch designated by us as 1.ORI2v. Two strains form a separate branch together with the strain from India belonging to 1.ORI2 line, one more strain, 55-801 Saigon, is set among the strains of 1.ORI1 line. Based on the data obtained and evidence from the literature sources it can be assumed that introduction of plague into Vietnam occurred through several waves: Nha Trang city in 1898, by sea; north provinces of the country – 1908. The second wave of Y. pestis dissemination across the territory of Vietnam began in 1960s with the emergence of the strains from the natural plague focus in Yunnan province, China

Analysis of the structural and functional state of human cord blood hematopoietic progenitor cells after cryopreservation with DMSO and antioxidants and transfer to conditions close to physiological

У роботі проведено порівняльний аналіз впливу кріопротекторних сумішей, що містять різні концентрації проникаючого кріопротектора ДМСО та антиоксидантів, при кріоконсервуванні гемопоетичних прогеніторних клітин кордової крові людини та після перенесення їх до умов, наближених до фізіологічних. Показано, що додавання до кріозахисного середовища аскорбінової кислоти, N-ацетил-L-цистеїна або глутатіона сприяє підвищенню показників збереженості та життєздатності гемопоетичних прогеніторних клітин після перенесення їх до умов, що моделюють фізіологічні. Найбільший цитопротекторний ефект спостерігається в пробах, кріоконсервованих із 7,5 і 10% ДМСО з додаванням глутатіону в концентрації 1 та 3 мМ, де зберігається до 80% гемопоетичних клітин в життєздатному стані, порівняно з двома іншими антиоксидантами в оптимальних концентраціях, які забезпечують життєздатність клітин до 70%; В работе проведен сравнительный анализ влияния криопротекторных смесей, содержащих различные концентрации непроникающего криопротектора ДМСО и антиоксидантов, при криоконсервировании гемопоэтических прогениторных клеток кордовой крови и после переноса их в условия, приближенные к физиологическим. Показано, что добавление к криозащитной среде аскорбиновой кислоты, N-ацетил-L-цистеина или глутатиона способствует повышению показателей сохранности и жизнеспособности гемопоэтических прогениторных клеток после переноса их в условия, моделирующие физиологические. Наибольший цитопротекторный эффект наблюдается в пробах, криоконсервированных с 7,5 и 10% ДМСО с добавлением глутатиона в концентрации 1 и 3 мМ, где сохраняется до 80% гемопоэтических клеток в жизнеспособном состоянии, по сравнению с двумя другими антиоксидантами в оптимальных концентрациях, которые обеспечивают жизнеспособность только до 70% клеток; The use of cord blood (CB) hematopoietic progenitor cells (HPCs) for the past 15 years has been firmly establishing in practical medicine as an effective treatment for diseases of various origins. In this regard, it remains relevant for the development of protocols for cells storage at low temperature. Most of them are based on the use of penetrating cryoprotectant DMSO in effective concentrations. However, they do not take into account that during cryopreservation HPCs are subjected to a significant stress, resulting in the decrease of the antioxidant system work, disruption of the membrane structure and the glutathione release from the organelles, which leads to an increase in the level of ROS in cells after transferring them to the bloodstream and the development of lipid peroxidation. In addition, there are suggestions that in the initial period after warming, the level of lipid peroxidation is limited by the structural antioxidants of the glutathione peroxidase system. In this regard, to increase the efficiency of cryopreservation of cord blood preparations, an important task is not only to evaluate them immediately after thawing, but also to determine the delayed survival of cells after transport them to conditions close to physiological. The addition of antioxidants to the cryoprotective medium can improve the preservation rate and viability of HPCs both immediately after cryopreservation and after transfer to conditions that are close to physiological. Based on this, the aim of the work was to analysis of the structural and functional state of human cord blood hematopoietic progenitor cells after cryopreservation with cryoprotectant DMSO and antioxidants and transfer to conditions close to physiological The efficacy of ascorbic acid, N-acetyl-L-cysteine and glutathione application in combination with DMSO in cryopreservation of HPCs has been evaluated. It has been shown that in the process of cryopreservation with DMSO and after transferring to conditions that are close to physiological, the preservation and viability of HPCs decreases on 20% in the samples, cryopreserved with 7.5-10% DMSO. One of the reasons for this may be the accumulation of reactive oxygen species (ROS) in cells during freezing. The addition of antioxidants to the cryoprotective medium can significantly increase the stability of the HPCs against the effects of cryopreservation factors and improve the preservation and viability indices. A comparative analysis of antioxidants revealed that ascorbic acid at concentrations of 0.1 and 0.15 mM and N-acetyl-L-cysteine (10 and 15 mM) in combination with 7.5% and 10% DMSO increased the number of preserved viable HPCs up to 70% in comparison to the samples without adding the antioxidants. Addition of glutathione at a concentration of 1 and 3 mM to cryoprotective medium with 7.5% and 10% DMSO was able to maintain a viable state of up to 80% of the HPCs. This may be due to the fact that glutathione reduced the number of cells with excess content of ROS after transferring the cells to conditions that are close to physiological