Klinichescheskaya Laboratornaya Diagnostika最新文献

Simultaneous quantitative measurement of mRNA of the WT1, BAALC, EVI1, PRAME and HMGA2 genes in whole blood samples reflects the specific pathological proliferative activity in acute leukemia and their ratio is promising as a diagnostic marker. The transcriptome profile of acute leukemia cells is usually assessed using NGS or microarray techniques after a preliminary procedure for isolation of mononuclear cells. However, the results of using the multiplex PCR reaction for the simultaneous determination of all above mRNAs in whole blood samples have not been published so far. Determination of mRNA of WT1, BAALC, EVI1, PRAME and HMGA2 genes in venous blood level samples by multiplex RT-PCR. The study included 127 blood samples from patients who diagnosis of acute leukemia was subsequently confirmed. In the comparison group, 87 samples of patients without oncohematological diagnosis were selected, including 31 samples (K1) with a normal blood formula and 56 samples (K2) with a violation of the cellular composition - anemia, leukocytosis and thrombocytopenia. RNA isolation and reverse transcription were performed using the Ribozol-D and Reverta-L kits (TsNIIE, Russia). Determination of the mRNA expression level of the WT1, BAALC, EVI1, PRAME and HMGA2 genes by multiplex real-time PCR using a homemade multiplex PCR kit. The mRNA level was characterized by high interindividual variation and did not correlate with the rate of circulating leukocytes or blood blasts. Expression of WT1 mRNA was observed in whole blood only in one patient from the control group and in 112 (88%) patients with leukemia and was combined with a decrease in the level of HMGA2 mRNA expression and BAALC mRNA values. In contrast to the control groups, patients with leukemia had higher levels of BAALC mRNA in AML and ALL, increased PRAME mRNA in AML and APL, but lower levels of HMGA2 in APL.

The relationship between multiple sclerosis and the state of the human microbiome was studied, namely, the change in the representation of microbiota phylotypes, the proportion of coccal flora, the proportion of anaerobic, gram-negative, proteolytically active microflora, as well as the concentration of markers of bacterial plasmalogen and endotoxin in the blood. Microbiome studies were carried out by gas chromatography - mass spectrometry of microbial markers in the blood. A statistically significant increase in blood concentrations of the total level of microbial markers of bacterial plasmalogen and endotoxin was determined in multiple sclerosis, which may be associated with an increase in the permeability of the intestinal wall. In multiple sclerosis, the proportion of coccal, gram-negative, anaerobic microflora with a proteolytic type of metabolic activity increases. The correlations of the representation of microbiota phylotypes change due to the switching of the direct relationship Proteobacteria-Bacteroides to Proteobacteria-Firmicutes. In multiple sclerosis, Actinobacteria and Proteobacteria increase and Firmicutes decrease. Conclusion. The multiple sclerosis disease may be associated with pathological changes in the structure of the microbiome and the growth of endotoxemia, which may be one of the factors in the pathogenesis of the disease. New laboratory markers for diagnosing and predicting the course of MS have been proposed.

Despite of great number of investigations in the area of tinea pedis, question is opened: to what extent dermatophyte fungi are spread among modern population and does their occurrence interrelated with host age? Investigated group included 99 volunteers from 14 to 73 years old. Skin scales were collected from heel area of foot, and signs of heel skin trouble were expressed in points. In contrast to usual laboratory microscope magnification x900 we worked at x1750, what allowed to estimate not only fungal, but bacterial forms too. Average abundances of microbial morphotypes were expressed in points. Heel skin trouble increased in the process of aging (Pirsons` coefficient r=0.954). Bacilli occurred in all persons independently from age, but their abundance increased with aging (0.821). On the contrary cocci were more common and abundant in young person`s feet (-0.620). Occurrence of dermatophytes increased with age (0.891), at that relatively high values took place in young persons (10.5% with mycelium and 73.7% with spores) and in group without any heel skin trouble symptoms (7.7% and 76.9%), what allow to refer these fungi to normal habitats of foot skin.

One of the reasons for the emergence of highly resistant strains is associated with the ability of bacteria to form biofilms on various surfaces. The formation of a biofilm by pathogens leads to a decrease in the activity of the antibiotic, an increase in the time for the production of stress response genes by bacteria, and, as a result, an increase in antimicrobial tolerance. To investigate the effect of imipenem and cefepime on the activity of biofilm forms of K. pneumoniae bacteria isolated from the wounds of patients with chronic osteomyelitis. The object of the study is clinical strains of K. pneumoniae isolated from the wounds of patients with chronic osteomyelitis. In the control series, the level of biofilm formation of K. pneumoniae strains was assessed after 48 hours of cultivation on coverslips and 96-well polystyrene plates. In the second and third series, the biofilm form of K. pneumoniae bacteria was exposed to imipenem and cefepime, and after 24 hours the activity of biofilm formation was assessed according to previously developed criteria. The structure of the emerging biofilm on the surface of the coverslip in all series of the experiment was represented by single adherent cells and microcolonies of various sizes. Cultivation with antibiotics led to a decrease in the number of microcolonies ranging in size from 10 to 10,000 µm2 in the second and third series, however, significant differences from the control series were found only when exposed to cefepime. The intensity of film formation of K. pneumoniae in the control series by the tablet method was 0.350 (0.334; 0.368) units opt.pl. When cultivating biofilms together with antibacterial drugs, the biofilm-forming activity after 24 hours of the experiment was significantly lower than in the control group in all experimental series. K. pneumoniae bacteria isolated from patients with chronic osteomyelitis, when cultivated on polystyrene plates and on the surface of coverslips, actively form a biofilm, exhibiting highly adhesive properties. The studied antibiotics were shown to have a bacteriostatic effect on biofilm forms of K. pneumoniae bacteria. The bactericidal effect of imipenem and cefepime on biofilm forms was not revealed.

One of the most common reasons for the progressing of aseptic instability of implanted structures in patients with end-stage osteoarthrosis is a disorder of immunogenulatory processes of bone tissue remodeling along with chronic inflammatory response influenced by endoprosthesis wear components. This research features the specifics of systemic immune response in patients with inflammatory complications in late postoperative period after total replacements of large joints. The factor analysis enabled determining the most significant immunological mechanisms associated with the progressing of implant aseptic instability. Pathogenetically significant components involved in the formation of cellular and humoral immune responses in patients with signs of inflammatory activity in late postoperative period have been identified. Our findings can be used in designing diagnostic and prognostic criteria for systemic inflammatory response severity in preoperative monitoring of the condition of patients in need of large joint arthroplasties, and also in detecting the progress of implant aseptic instability.

Multiple myeloma (MM) is a malignant tumor occurring from plasma cells that produce an abnormal monoclonal immunoglobulin - a paraprotein. A distinctive feature of Bence-Jones myeloma is the excretion of monoclonal free light chains of immunoglobulins with 24h urine, and the absence of monoclonal intact immunoglobulins secretion. Comprehensive analysis of biochemical parameters in blood serum and 24h urine in patients with Bence-Jones multiple myeloma using electrophoretic and immunoturbidimetric methods to assess their sensitivity as biomarkers. 50 patients with a morphologically confirmed diagnosis of MM of the Bence-Jones immunochemical type were examined. 28 people without oncological diseases were examinedas a control. Detection of monoclonal secretion in blood serum and daily urine was performed by immunofixation electrophoresis on the Hydrasys 2 electrophoretic system (Sebia). The determination of free light chains of immunoglobulins (FLC) was performed by the immunoturbidimetric method (Binding Site) on an Advia 1800 analyzer (Siemens). Analysis of IgG, IgA, IgM, β2-microglobulin and C-reactive protein was performed on Cobas 6000 analyzer (Roche). The median excretion of Bence-Jones protein in 24h urine of MM patients was 0.49 g/24h (0.06-2.45 g/24h). In the blood serum, in 86% of cases, the presence of paraproteinemia, represented by κ and λ type light chains of immunogloublins was detected. At the same time, the frequency of detection of monoclonal secretion in blood serum in Bence-Jones type λ myeloma was 95.7%, which was statistically significantly higher than the frequency of detection of monoclonal secretion of type κ - 77.8%. In patients with identified paraproteinemia, Bence-Jones protein excretion in daily urine (median 0.82 g/day) was statistically significantly higher than in patients without a monoclonal component detected in blood serum (median 0.04 g/24h). The levels of FLC in blood serum obtained by immunoturbidimetry in Bence-Jones myeloma of the corresponding type were higher than the reference levels in 100% of cases. The median level of κ-FLC reached 4358 mg/l, λ-FLC - 2225 mg/l, which was statistically significantly higher than the control levels. The median concentrations of IgG, IgA and IgM in patients with Bence-Jones myeloma were statistically significantly lower than in the control group, while the medians of β2-microglobulin and C-reactive protein were significantly higher than in the control. Our investigation showed high diagnostic efficiency of electrophoretic and immunoturbidimetric analysis of monoclonal secretion in patients with Bence-Jones MM, while FLC analysis demonstrated maximum sensitivity. Bence-Jones MM revealed biochemical signs of secondary immunodeficiency and general inflammatory syndrome.

The study of the characteristics and dynamics of laboratory biomarkers in patients with cardiovascular diseases (CVD) with type 2 diabetes mellitus who underwent COVID-19-associated pneumonia is of great clinical importance for preventing the risk of adverse events. IN the study we used data from 65 patients in the present work. Patients were divided into 2 groups: group 1 included patients with CVD: arterial hypertension (AH) in combination with coronary artery disease (CAD) without DM2 (n=45), group 2 included patients with CVD and DM2 (n=20). Patients were examined at baseline in the infectious disease hospital and 3 months after discharge. During laboratory examination of blood biosamples we evaluated parameters of general blood test; biochemical and immunologicai parameters; elastic properties of the vascular wall. The analyzed leukocyte parameters and their index coefficients - increase in NLR ratio (neutrophils/lymphocytes) and decrease in LYM/CRP ratio (lymphocytes/CRP) were more significantly changed in DM2 group. Patients in both groups had a significant excess of baseline max CRP concentrations with decrease in parameters after 3 months, but with persistent excess values in group 2. Three months after discharge patients with DM2 had levels of hs-CRP, IL-1β and TNFa and NT-proBNP, that exceeded both the reference values and those in group 1, which reflected the presence of more pronounced vascular inflammatory potential for possible adverse events in this group of patients in post-COVID period. The method of multiple regression showed that DM2 is an independent risk factor for increased stiffness of the vascular wall. Thus, dynamic control of laboratory parameters has prognostic value in assessing the nature of the course of COVID-19 associated pneumonia in patients with CVD and DM2 developing an algorithm for personalized monitoring of patients in the post-COVID period with the aim of timely prevention of unwanted vascular complications.

The study compared the effectiveness of two different primer sets for detecting and evaluating the prevalence of the babA2 gene in 52 H. pylori clinical isolates from patients with chronic gastritis (n=32), duodenal ulcer (n=16) and stomach cancer (n=4) in St. Petersburg, Russia. The PCR was used for detection of the babA2 gene with 271 bp and 832 bp primer sets followed by sequencing of the PCR-amplicons. The largest proportion of babA2-positive strains - 90.4% (47/52) was detected using a 271 bp PCR primer set. Detection of the 832 bp PCR positive samples was observed only in 51.9% of cases (27/52). The largest proportion of babA2-positive strains - 90.4% (47/52) was detected using 271 bp PCR primer set; detection of 832 bp PCR product was observed only in 51.9% cases (27/52), however, there were no significant differences in the babA2 gene detection rates (p>0.05). Bioinformatic analysis revealed a homology of Sanger sequenced PCR products 271 bp and 832 bp of babA2 gene with regions of the babA2, babA1, and chimeric babA/B genes of H. pylori strains annotated in the NCBI database. Regardless of the primer set used, the presence of babA2 was not significantly associated with duodenal ulcer nor gastric cancer (p>0.05). The combination of the three babA2, cagA, and vacAs1 genes did not reveal any association between the presence of babA2 gene and cagA/vacAs1 genes in H. pylori strains (p>0.05). Thus, none of the two primer sets (271 bp and 832 bp) appears sufficiently informative for detecting the babA2 gene to assess virulence of H. pylori Russian strains.

Corynebacterium spp. are part of the human microbiome, but can cause the development of inflammatory diseases of various localization. Purpose - to evaluate the relationship between pathogenic properties and resistance to antimicrobial drugs (AMD) of Corynebacterium spp. from patients with inflammatory diseases of the respiratory tract. Strains of Corynebacterium spp. isolated from patients with inflammatory diseases of the respiratory tract (99 pcs.) and practically healthy individuals (33 pcs.). Isolates were identified by mass spectrometric method (MALDI-ToFMS), their adhesive and invasive activity on Hep-2 cells, cytopathic effect (CPE) in CHO-K1 cell culture, and resistance to antimicrobial drugs (AMD) were determined. Indicators of adhesion (3.65±0.679(CFU±m)x102/ml), invasion (1.72±0.230 (CFU±m)x102/ml), cytotoxicity (69.1±3.8% of dead CHO-K1 cells ) Corynebasterium spp. strains isolated from patients are higher (p≤0.05) than similar indicators in practically healthy people. 90.9% of isolates from patients had resistance to AMD, in most cases (57.6±4.9%) resistance to only one AMP was noted, less often to two (25.2±4.3%), three or more (8.08±2.7%). According to the results of correlation-regression analysis, pathogenic properties (adhesiveness, invasiveness, cytotoxicity) of Corynebacterium spp. strains isolated from patients are in close direct relationship with resistance to AMD. This indicates the importance of identifying strains of non-diphtheria corynebacteria resistant to AMDs, which, under the influence of developing resistance to AMDs, can increase their pathogenic potential, moving from commensalism to parasitism.

A method has been developed for HBV DNA finding in biological material at low viral load based on nested PCR with real-time detection of three viral targets. When developing the method, blood plasma samples were used from 128 CHB patients living in the regions of the Russian Federation and countries of Central Asia and 173 hemodialysis center patients living in the North-West Federal District. Analytical sensitivity was tested using the stepwise dilution method. HBV was detected by nested PCR. According to the method developed by us, at the first stage, the HBV DNA is amplified using at the first stage oligonucleotides complementary to the greatest similarity regions of the various HBV isolates genomes flanking the entire virus genome. At the second stage, when using the amplification product of the first stage as a template, PCR was performed using three pairs of oligonucleotides and the corresponding oligonucleotide fluorescently labeled probes to three virus genome regions (Core gene, S gene and X gene), as well as one pair of primers and the corresponding probe complementary to a human HPRT gene region. The method sensitivity for DNA extraction from plasma with a 100 μl volume was 10 IU/ml. Obtaining a threshold Ct cycle for only one fluorophore may indicate the presence of HBV DNA in the sample at a load of less than 10 IU/ml, HBV detection in this case is possible with a repeated PCR study of the corresponding sample with HBV DNA extraction from an increased plasma volume (200-1000 μl). The developed method makes it possible to identify the disease in various HBV subgenotypes and can be used to diagnose CHB in the population and risk groups, including those with the HBsAg-negative form of the disease.