Qualitative Polymerase Chain Reaction Research Articles

Hematological, clinical, cytogenetic and molecular profiles of confirmed chronic myeloid leukemia patients at presentation at a tertiary care teaching hospital in Addis Ababa, Ethiopia: a cross-sectional study

BackgroundIn low-income countries there is insufficient evidence on hematological, clinical, cytogenetic and molecular profiles among new CML patients. Therefore, we performed this study among newly confirmed CML patients at Tikur Anbesa Specialized Hospital (TASH), Ethiopia.ObjectiveTo determine the hematological, clinical, cytogenetic and molecular profiles of confirmed CML patients at tertiary care teaching hospital in Addis Ababa, Ethiopia.MethodsA facility-based cross-sectional study was conducted to evaluate hematological, clinical, cytogenetic and molecular profiles of confirmed CML patients at TASH from August 2021 to December 2022. A structured questionnaire was used to collect the patients’ sociodemographic information, medical history and physical examination, and blood samples were also collected for hematological, cytogenetic and molecular tests. Descriptive statistics were used to analyze the sociodemographic, hematological, clinical, cytogenetic and molecular profiles of the study participants.ResultsA total of 251 confirmed new CML patients were recruited for the study. The majority of patients were male (151 [60.2%]; chronic (CP) CML, 213 [84.7%]; and had a median age of 36 years. The median (IQR) WBC, RBC, HGB and PLT counts were 217.7 (155.62–307.4) x103/µL, 3.2 (2.72–3.6) x106/µL, 9.3 (8.2–11) g/dl and 324 (211–499) x 103/µL, respectively. All patients had leukocytosis, and 92.8%, 95.6% and 99.2% of the patients developed anemia, hyperleukocytosis and neutrophilia, respectively. Fatigue, abdominal pain, splenomegaly and weight loss were the common signs and symptoms observed among CML patients. Approximately 86.1% of the study participants were Philadelphia chromosome positive (Ph+) according to fluorescence in situ hybridization (FISH). P210, the major breakpoint protein, transcript was detected by both qualitative polymerase chain reaction (PCR) and quantitative real time polymerase chain reaction (PCR).ConclusionDuring presentation, most CML patients presented with hyperleukocytosis, neutrophilia and anemia at TASH, Addis Ababa. Fatigue, abdominal pain, splenomegaly and weight loss were the most common signs and symptoms observed in the CML patients. Most CML patients were diagnosed by FISH, and p120 was detected in all CML patients diagnosed by PCR. The majority of CML patients arrive at referral center with advanced signs and symptoms, so better to decentralize the service to peripheral health facilities.

Development and validation of a multiplex real-time PCR method for screening genetically modified plants

AbstractThe diversity of genetically modified plants used for food and feed is increasing worldwide. For the detection and control of these products, efficient and reliable analytical tools are a prerequisite. This can be done by screening for specific DNA-elements and constructs characteristic of transgenic plants. In the past, numerous methods have already been published. However, several genetically modified plants are not covered by common screening methods. Here, a new qualitative triplex real-time polymerase chain reaction (PCR) method is presented, detecting two transgene flanking sequences and the transition between the Cassava Vein Mosaic Virus Promotor (P-CsVMV) and the phosphinothricin-N-acetyltransferase (pat) gene. These sequences are present in several transgenic plants and therefore, the described triplex method can be used as a screening tool to guide further analysis and increase the efficiency of the analysis strategy for GMO detection. The method is characterized by high specificity, sensitivity and robustness and is provided as a ring-trial validated method in the Official Collection of Methods according to the German Food and Feed Act.

Expression of JAK2 V617F Mutation in BCR - ABL Negative Myeloproliferative Neoplasms

OBJECTIVE: To detect the presence of JAK2V617F mutation in BCR - ABL negative myeloproliferative neoplasms and to stratify high risk patients for targeted therapy.PLACE AND DURATION OF STUDY: Department of Haematology, Armed forces institute of Pathology (AFIP), Rawalpindi, from Dec 2023 to Oct 2023.STUDY DESIGN: Descriptive cross sectional studyMETHODOLOGY: A total of 40 consecutive patients of BCR - ABL 1 negative myeloproliferative disorders were included in the study after being diagnosed according to WHO defined haematological criteria for each disorder and performing bone marrow examination under aseptic measures. Informed consent and ethical approval were obtained from each individual. All patients had their blood samples examined for the G-T point mutation (V617F) in the JAK2 gene located on chromosome 9, utilizing allele-specific real-time qualitative polymerase chain reaction (RT-qPCR). Results were analyzed by using SPSS version 22.RESULTS: Out of total 40 patients included in our study 21(52.5%) had Polycythemia Vera (PV), 15 (37.5%) had Primary myelofibrosis (PMF) and 4 (10%) had Essential thrombocythemia (ET). JAK2 mutation was found in 20 (95.2%) patients of PV, 9 (60%) patients of PMF and 2 (50%) patients of ET with a significant p-value of ˂ 0.001.CONCLUSIONS: JAK2 V617F is a useful clinical marker for establishing diagnosis and separating high risk patients for specific targeted therapy.

Endogenous hsa-circ_0007113 binds hsa-miR-515-5p to regulate senescence in human embryonic lung fibroblasts.

Cellular senescence can lead to many diseases. However, the roles and regulation of circular RNAs (circRNAs) in senescence are poorly understood. To investigate the altered expression pattern and mechanism of circRNA during cellular senescence and find potential targets to prevent senescence. The Arraystar Human circRNA Array and bioinformatics were used to profile the differentially expressed circRNAs in human embryonic lung fibroblasts (IMR-90) between young cells and senescent cells and quantification in the clinical materials. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed. The miRNA targets were predicted using TargetScan and miRanda. A total of 113 differentially expressed circRNAs were identified, including 109 upregulated and 4 downregulated circRNAs (fold change >2 and p-value <0.05). Real-time qualitative polymerase chain reaction (qPCR) showed that the expression levels of 4 circRNA were significantly increased in senescent cells, and that of hsa_circ_0007113 was significantly decreased, consistent with the microarray. siRNA against hsa_circ_0007113 increased p21 and p53 expression levels and β-gal staining. The hsa_circ_0007113 has a binding site for miR-515-5p, which is involved in regulating the p53/p21 signaling pathway. The expression level of hsa_circ_0007113 was also decreased in aged people. The study showed an altered circRNA expression pattern in cellular senescence, which might play important roles in senescence-related physiological processes. These findings provide a new direction for studying the molecular mechanism underlying senescence and a new possibility for the treatment of senescence by modulating circRNAs.

Modified Danzhi Xiaoyao Powder (MDXP) improves the corneal damage in dry eye disease (DED) mice through phagocytosis

Ethnopharmacological relevanceModified Danzhi Xiaoyao Powder (MDXP) is a traditional Chinese medicine formula remedy for treating Dry Eye Disease (DED). It showed the function of dispersing stagnated liver Qi for relieving Qi stagnation and clearing heat, which can be effective in treating conditions such as Dry Eye Disease (DED) and irregular menstruation due to liver depression and fire transformation. Aim of the studyThis study investigated the mechanism of the effect of MDXP in mice with DED. Materials and methodsA DED model was induced in mice using chronic painful stimulation (tail clamping) in combination with Benzalkonium Chloride Solution drops administered in a dry box for 28 days. After modeling, the MDXP groups were given Chinese medicine with different dosages by gavage for 14 days. The following parameters were recorded in each group: body mass, anal temperature, tear secretion, tear film rupture time, and corneal fluorescein staining. Behavioral changes were evaluated by elevating cross-maze and open-field experiments. The levels of inflammatory factors serum tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β), fcγR-mediated phagocytosis pathway cell division control protein 42 homolog (CDC42), actin-related protein 2/3 complex subunit 2 (ARPC2), and actin-related protein 3 (ACTR3) were measured by using Enzyme-linked immunoassay (ELISA), immunohistochemical staining, and real-time fluorescent qualitative polymerase chain reaction (RT-qPCR). ResultsMDXP increased body mass and lowered body temperature, prolonged tear film break-up time, promoted tear secretion, repaired corneal damage, decreased horizontal and vertical scores, elevated percentage of open arm times and boom opening time percentage, and reduced the expression levels of inflammatory factors of TNF-α, IL-1β and pathway-related proteins CDC42, ARPC2, and ACTR3 in mice. MDXP also reduced the expression levels of inflammatory factors of TNF-α and IL-1β in human corneal endothelial cells (HCECs), mouse mononuclear macrophage cells (RAW264.7), and human myeloid leukemia mononuclear cells (THP-1). ConclusionsMDXP can relieve tension and anxiety, inhibit apoptosis, reduce phagocytosis, reduce the expression of pro-inflammatory factors, repair corneal damage, and improve the symptoms in DED mice. The mechanism of action may be through the fcγR-mediated phagocytosis pathway.

Subgingival Plaque as a Potential Reservoir for SARS-CoV-2 Virus in Patients Recovered from COVID-19 Infection: A Cross-sectional Study

Background Subgingival plaque biofilm is a potential harboring site for bacteria and viruses, serving as a source of entry into the bloodstream. The presence of Severe Acute Respiratory Syndrome Coronavirus 2 RNA (SARS-CoV-2 RNA) in subgingival plaque could be a possible source for cross infection due to aerosol contamination during dental procedures. Objectives The objective of this study was to investigate the presence of SARS-CoV-2 RNA in the subgingival plaque biofilm of patients who recovered from COVID-19 infection. Materials and Methods Ten subjects visiting the Outpatient clinic with a previous history of COVID-19 infection were recruited for this cross-sectional study. Subgingival plaque sample was collected from all four quadrants using a sterile curette. Nasopharyngeal and oropharyngeal swabs were taken from all the subjects. Reverse transcriptase real-time qualitative polymerase chain reaction (RT-PCR) was used to determine the presence of SARS-CoV-2 RNA using BAG SARS-CoV-2 RT-PCR kit targeting E gene, RdRp gene, and internal control. Results Ten participants (seven females and three males) with a previous history of COVID-19 infection were included in the study. Six participants previously presented with mild COVID-19 infection (uncomplicated upper respiratory tract infection, mild fever, cough, sore throat, nasal congestion, malaise, headache, without evidence of breathlessness or hypoxia), and four participants previously presented with moderate COVID-19 infection (dyspnea and or hypoxia, fever, cough, SpO2 90%–94% on room air, respiratory rate more or equal to 24 per minute) according to clinical management protocol for COVID-19, Government of India. The nasopharyngeal, oropharyngeal, and subgingival plaque samples tested negative for SARS-CoV-2 RNA. Conclusion The absence of the SARS-CoV-2 virus in the subgingival plaque sample of patients recovered from COVID-19 infection suggests that dental plaque could not be a potential reservoir of the virus. However, clinicians should take at most precautions while treating the patients recovered from COVID-19 infection and should anticipate a change in subgingival environment leading to alteration in periodontal disease progression.

Disease Response Assessment Modalities in Chronic Myeloid Leukemia: Past, Present, and Future

AbstractChronic myeloid leukemia (CML) is a myeloproliferative neoplasm caused by the BCR::ABL1 fusion gene, which results from a reciprocal translocation between chromosome 9 and 22 t(9;22)(q34;q11). The use of tyrosine kinase inhibitor (TKI) against the chimeric BCR::ABL1 fusion protein has led to a paradigm shift in CML patient outcomes. Despite generational advancements in TKI, a fraction of patients harbor residual disease or exhibit resistance to TKI. The importance of disease monitoring and detection of resistance mechanisms has gained prominence with increasing knowledge about disease evolution. In the past, cytogenetic techniques such as karyotyping and fluorescence in situ hybridization were widely utilized for monitoring disease and prognostication. These techniques had various challenges related to limited sensitivity in minimal residual disease (MRD) monitoring; however, their importance still holds in the detection of additional chromosomal aberrations and in cases with cryptic insertions, variants, and masked Philadelphia chromosome. Molecular genetics has evolved significantly from the past to the present times for MRD monitoring in CML patients. Qualitative reverse transcription polymerase chain reaction (RQ-PCR) can be performed at diagnosis to detect the BCR::ABL1 transcript, while quantitative RQ-PCR is the most widely used and well-standardized MRD monitoring method. The DNA-based assays demonstrated high sensitivity and specificity, with many efforts directed toward making the laborious step of BCR::ABL1 breakpoint characterization less tedious to increase the utility of DNA-based MRD approach in the future. Flow cytometric–based approaches for the detection of the BCR::ABL1 fusion protein have been under trial with a scope of becoming a more robust and convenient methodology for monitoring in the future. Upcoming techniques such as digital PCR and ultra-deep sequencing next-generation sequencing (UDS-NGS) have shown promising results in residual disease monitoring and detection of resistance mutations. Novel MRD monitoring systems that are independent of BCR::ABL1 fusion such as the detection of CD26+ leukemic stem cells and microRNA mutations are the future of residual disease monitoring, which can go up to the level of a single cell. In this review, we tried to discuss the evolution of most of the above-mentioned techniques encompassing the pros, cons, utility, and challenges for MRD monitoring and detection of TKI resistance mutations.

Identification of Escherichia coli Sub Type Enterotoxigenic (ETEC) from Food Samples Using Pcr (Polymerase Chain Reaction) Technique

Escherichia coli is a normal flora of the digestive tract of humans and animals and is important in the digestion of food. This bacteria lives in feces, can cause health problems, such as diarrhea and vomiting. This research was conducted for consideration in the prevention of diarrhea, processing, marketing of food. This study aims to determine the presence of enterotoxigenic Escherichia coli sub-type (ETEC) from food samples. This research is an explorative descriptive study, as many as 5 samples from food, food samples taken are food consumed every day and some include traditional food in Mataram City. The samples were cultured on BHI-Broth media, followed by inoculation on Eosin Methylene Blue (EMB) medium and continued with the Gram stain test. Colonies that are metallic green in colour and show the characteristics of Escherichia coli bacteria on Gram staining are then carried out for purification of the bacteria on Nutrient Agar (NA) medium, if the classification of bacteria shows the characteristics of Escherichia coli bacteria on Gram staining then proceed with the Indole/sugar-test. The results showed that of the 5 food samples isolated, only 1 sample showed positive E.coli bacteria. Then proceed to the PCR (Polymerase Chain Reaction) stage using the qualitative PCR method to determine the presence of Enterotoxigenic Escherichia coli sub-type (ETEC) bacteria in food samples. , with a target gene of 237 bp.

Baseline Cytomegalovirus Viremia at Cryptococcal Meningitis Diagnosis Is Associated With Long-term Increased Incident TB Disease and Mortality in a Prospective Cohort of Ugandan Adults With HIV.

Adults with HIV-associated cryptococcal meningitis have overlapping burdens of cytomegalovirus (CMV) and tuberculosis (TB) coinfections. CMV infection/reactivation is strongly associated with CMV-specific memory T-cell activation and upregulation of type 1 interferons, which may lead to increased risk of TB disease and poor outcomes. We conducted a cohort study of 2-week survivors of cryptococcal meningitis during 2010-2021 to determine TB incidence and all-cause mortality over time stratified by baseline CMV status. We followed 497 Ugandans with HIV-associated cryptococcal meningitis for a median (interquartile range) of 4.6 (2.6-53.9) months. Overall, 42% (210/497) developed incident TB disease or died. One-fifth (98/497, 19.7%) developed incident TB disease, and 29% (142/497) of participants died during follow-up. Of 259 participants with CMV viral load measured at baseline, 37% (96/259) had concurrent CMV viremia (defined as anyone with detectable CMV DNA in plasma/serum by qualitative polymerase chain reaction [PCR] detection). Of 59 with measured CMV immunoglobulin G (IgG), 100% had positive CMV IgG antibody serology (≥10 enzyme-linked immunosorbent assay units/mL). CMV viremia was positively associated with higher HIV viral load (196 667 vs 73 295 copies/mL; P = .002) and higher cerebrospinal fluid fungal burden (68 500 vs 14 000 cfu/mL; P = .002) compared with those without. Participants with high-level CMV viremia (defined as CMV viral load ≥1000 IU/mL) had twice the risk of incident TB (subdistribution adjusted hazard ratio [aHR], 2.18; 95% CI, 1.11-4.27) and death (aHR, 1.99; 95% CI, 1.14-3.49) compared with participants with no or low-level CMV viremia. There was no association between the CMV IgG index and the incidence of TB/death (P = .75). CMV viremia >1000 IU/mL at meningitis diagnosis was associated with increased incident TB disease and mortality during long-term follow-up. Future studies to determine the causal relationship and potential for therapeutic intervention are warranted.

CORRELATION OF ECHOCARDIOGRAPHIC INDICATORS AND CLINICAL-LABORATORY PARAMETERS TAKING INTO ACCOUNT THE AGT GENE (RS4762) IN PATIENTS WITH ARTERIAL HYPERTENSION

The аim of the research – to assess the relationship between echocardiographic indices and clinical- laboratory parameters, taking into account genetic markers AGT (rs4762) in patients with essential arterial hypertension (EAH).&#x0D; &#x0D; Material and methods. The case-control study involved 100 patients with EAH stage II, 1-3 degrees of blood pressure (BP) increase, high and very high cardiovascular risk. Among the patients there were 21 % men, 79 % women. The middle age of the patients was 59.86±6.22y.o. The control group consisted of 60 practically healthy individuals, relevant in age (49.13±6.28y.o.) and gender distribution (63 % – women, 37 % – men). To study polymorphism of AGT (rs4762) and GNB3 (rs5443) genes a qualitative polymerase chain reaction (PCR) was performed in real time. The lipidogram indices were investigated in the blood plasma using diagnostic kits «Accent 200» firm (Poland). All patients were tested for levels of fasting glucose, ionized calcium, parathyroid hormone (PTH), 25-hydroxyvitamin D. The patients were performed echocardiography to determine the left ventricle hypertrophy. Results. The structural parameters of the myocardium, which determine the size of the LVH and the type of its geometric model, in patients with essential arterial hypertension (EAH) are influenced by the following indicators with a moderate strength of connection: concentric hypertrophy (CH LV) develops at a low level of high-density lipoprotein cholesterol (LPHD) (r= –0.59; р=0.008), ionized blood Ca2+ (r= –0.50; р=0.03) and increased PTH content (r=0.40; р=0.05) and high EchoCG indicators: increase in the size of the left atrium (LA), posterior wall thickness (PWT), interventricular septum in diastole (IVSd), left ventricle mass (LVM), left ventricular mass index (LVMI) and the relative left ventricular wall thickness (RWT) (r=0.47-0.65; p≤0.04-0.002). Conclusions. Structural and functional changes of the myocardium are correlated with a number of clinical, anthropometric, metabolic indicators and depend on polymorphic variants of the AGT gene (rs4762).

Reliability of nasopharyngeal PCR for the detection of otopathogens in children with uncomplicated acute otitis media compared to culture

Otopathogens in acute otitis media (AOM) have implications for care because the likelihood of resolution without antibiotics and optimal antibiotic agent varies by microorganism. We aimed to determine the sensitivity, specificity, positive predictive value, and negative predictive value of nasopharyngeal (NP) qualitative polymerase chain reaction (PCR) for common bacterial otopathogens in children with AOM compared to NP culture. NP flocked swabs collected from enrolled children aged 6 to 35 months with uncomplicated AOM in Denver, CO were tested by culture and multiplex PCR. The sensitivity and negative predictive value of PCR using culture as a reference were high (H. influenzae 93.3%, 98.0%; S. pneumoniae 94.2%, 95.1%; M. catarrhalis 92.3%, 86.4%); whereas the specificity and positive predictive value were lower and varied by organism (54.2%–84.1%, 55.1%–69.2%, respectively). PCR detected 1.5 times more organisms than culture. NP PCR has a high predictive value for excluding otopathogens compared to culture and warrants exploration as a diagnostic tool.

Colonic cytomegalovirus DNA detection by polymerase chain reaction does not influence outcomes in inflammatory bowel disease and immunosuppressed cohorts.

Cytomegalovirus (CMV) colitis is associated with negative outcomes in inflammatory bowel disease (IBD) and immunosuppressed cohorts and therefore requires timely recognition for appropriate management. We aimed to evaluate the diagnostic tools for CMV colitis and their associations with clinical outcomes. A retrospective cohort study of patients in a metropolitan health service with colonic samples analysed for CMV between 2012 and 2022, stratified into IBD and non-IBD groups, was performed. The main outcome measures were the prevalence of positive and negative results for each CMV test, as well as need for colectomy, use of antiviral and hospital length of stay. Five hundred eighty-two biopsies from 418 patients were included; the median age was 36 years (interquartile range, 24-52 years) and 223 (53.3%) were men. Four hundred sixty-one (79.2%) biopsies were from patients with IBD and 121 (20.8%) were from those without IBD. There were similar proportions of positive CMV histology (IBD 5.9% and non-IBD 7.4%) and tissue CMV polymerase chain reaction (PCR) in the two groups (IBD 5.6% and non-IBD 5.0%), but within each group, results were discordant. Positive CMV histology was significantly associated with need for colectomy in the IBD group, while positive tissue CMV PCR was not. Positive CMV histology, and tissue and serum CMV PCR were all significantly associated with antiviral use. Positive serum CMV PCR was significantly associated with colectomy. Histopathology remains the most predictive tool in assessing CMV colitis, while qualitative tissue CMV PCR was found to have limited utility. Quantitative serum CMV PCR may be useful but requires further evaluation.

Molecular changes in skeletal muscle in chronic kidney disease: A systematic review

AbstractBackgroundLoss of skeletal muscle mass is prevalent among patients affected by chronic kidney disease (CKD). It is associated with significant morbidity and mortality. The underlying molecular pathogenesis has yet to be fully understood. The aim of this systematic review is to summarize the current evidence on molecular changes in the skeletal muscle of humans and rodents with CKD and to assess the strength of such evidence.MethodsThe PubMed and EMBASE databases were searched using three main themes: messenger ribonucleic acid/protein/microRNA expression, skeletal muscle and CKD. This study was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta‐Analyses (PRISMA) standards.ResultsA total of 98 studies were included in the systematic review, comprising 26 prospective human clinical studies, four human and rodent studies, and 68 rodent‐only studies (32 mouse and 36 rat models respectively). The sample sizes of human studies were largely small (40% of studies had ≤20 participants). Qualitative polymerase chain reaction (qPCR) was the most commonly used method for gene expression and none of the studies fulfilled the Minimum Information for Publication of qPCR Experiments criteria for quality assessment. Majority of the studies investigated only a few genes or a specific signalling pathway. FBXO32, TRIM63, MSTN, IL6, TNF and IGF1 were the most investigated genes. The identified differentially expressed genes and proteins belonged to eight major pathways, including apoptosis, autophagy, inflammation, insulin/insulin‐like growth factor 1 signalling, lipid metabolism, mitochondrial function, muscle cell growth and differentiation, and protein degradation, similar to other chronic disease states.ConclusionsThe current evidence regarding molecular alterations in the skeletal muscle in CKD is largely derived from small and heterogenous studies. Markedly similar modifications in the major biological pathways between CKD and other chronic diseases supports shared deleterious molecular mechanisms producing muscle atrophy, irrespective of the underlying specific disease.

PCR-screening of genetically modified organisms in food and feed products sold in Kuwait's market

Derivatives of biotech or genetically modified (GM) crops are widely used for food, feed, or environmental release. In the present study, 31 samples of food and feed products of corn, soybean, and rice, randomly selected from the local market of Kuwait, were screened for the presence of potential genetically modified organisms (GMOs). Conventional (qualitative) polymerase chain reaction (PCR) test was used to amplify the sequences of the GMO regulatory elements, the cauliflower mosaic virus 35S promoter (p35S), and the Agrobacterium nopaline synthase terminator (Tnos). Obtained results demonstrated the presence of GMO sequences in 6 out of 21, 1 out of 5, and 2 out of 5 products derived from corn, soybean, and rice, respectively. The study also reports the detection of GMOs in corn-derived animal feed products for the first time. To date, Kuwait has no routine certified system to regulate the import of biotech crops. This study highlights the need to establish legislation on GMOs and to develop a reliable regulation and labeling system for imported biotech crops and their derivatives in Kuwait.

Topical Dihydroartemisinin Improves Wound Healing in Diabetic Mice.

Impaired skin wound healing is a common complication of diabetes mellitus. Angiogenesis is a critical step in wound healing because it allows oxygen and nutrients to reach the injured area, thereby promoting wound cell proliferation, re-epithelialisation, and collagen regeneration. However, the neovascularisation ability of patients with diabetes often decreases. Therefore, finding ways to improve diabetic angiogenesis is important for treating diabetic wounds that do not heal. To the best of our knowledge, it is unclear whether dihydroartemisinin (DHA) affects diabetic wounds. This study sought to determine how topical DHA affects the healing of diabetic wounds and how it is related to markers of angiogenesis. We topically applied DHA to full-thickness cutaneous lesions in a streptozotocin (STZ)-induced diabetic mouse model. Under a fluorescence microscope, the pathological morphology of the wound skin was observed, together with the positive expression of platelet endothelial cell adhesion molecule-1 (CD31) and vascular endothelial growth factor (VEGF). Western blotting was used to determine the CD31 and VEGF protein expression levels. The mRNA expression was determined using qualitative real-time polymerase chain reaction (qRT-PCR). We found that DHA can improve the expression of CD31 and VEGF, and accelerate wound healing in diabetic mice. We believe that DHA promotes angiogenesis, which is associated with increased VEGF signalling in vivo. Therefore, DHA can effectively accelerate the process of diabetic wound healing by promoting angiogenesis, implying that DHA may be used as a topical drug for the treatment of diabetic wounds.

POLYMORPHIC VARIANTS OF AGT (RS4762) AND GNB3 (RS5443) GENES AS RISK FACTORS FOR SEVERE ARTERIAL HYPERTENSION

Objective: The aim of our study was to evaluate the role of AGT (rs4762) and GNB3 (rs5443) gene polymorphisms as risk factors for severe hypertension. Design and method: The case-control study involved 100 patients with EAH stage II, 1-3 degrees of blood pressure (BP), high and very high cardiovascular risk. Among the patients there were 21% (21) men, 79% (79) women. The mean age of patients was 59.86±6.22 y.o. The control group consisted of 60 almost healthy individuals with relevant age (49.13±6.28y.o.) and gender distribution (63% - women, 37% - men). The AGT (rs4762) gene polymorphism was studied by a qualitative polymerase chain reaction (PCR) in real time. Results: Severity of EAH does not depend on polymorphic variants of AGT (rs4762) and GNB3 (rs5443) genes. The distribution showed no statistically significant differences in the aforementioned distribution. Individuals with the first degree of hypertension met more often with the CC genotype of the GNB3 gene (rs5443) by 22.23% than among patients with the T allele (x2 = 3,66; p = 0,055) in patients with EAH. Conclusions: Epidemiological analysis did not confirm the polymorphic variants of the AGT (rs4762) and GNB3 (rs5443) genes as predictors of the severe course of EAH according to the degrees of BP elevation.

RISK CONFERRED BY POLYMORPHIC VARIANTS OF VITAMIN D RECEPTOR (VDR) AND ANGIOTENSIN II TYPE 1 RECEPTOR (AGTR1) GENES FOR ESSENTIAL HYPERTENSION

Objective: The aim of this study was to establish the role of 1166A&gt;C polymorphism of the AGTR1 gene (rs5186) and A/G polymorphism of the VDR gene (rs2228570) in risk prediction of essential hypertension (EH). Design and method: The study included patients with EH and hypertensive-mediated organs damage (2nd stage), moderate, high/very high cardiovascular risk. 100 subjects were involved in the case-control study. There were 70.84% females, 29.16% males among them, the mean age was 57.86±7.81 yo. Age- and gender-matched controls (n = 60) whose blood pressure measurements were in normal range and without any apparent diseases were randomly selected to compare with the patient data. In order to detect AGTR1 (rs5186) and VDR (rs2228570) gene polymorphism the qualitative real-time polymerase chain reaction was done. AGTR1 gene genotyping was performed for 72 patients and 48 healthy individuals and VDR gene – for 100 patients and 60 healthy subjects. Results: The distribution of genotypes and alleles AGTR1 (rs5186) and VDR (rs2228570) in the study and control groups did not differ significantly (p&gt;0.05). C-allele of AGTR1 gene (rs5186) increases the risk of EAH more than 2 times [OR–2.31; 95% CI OR:1.19–4.47; p = 0.011], as well as AC- and the combination of AC- + CC-genotypes [OR–2.09; 95% CI OR:1.03–4.25; p = 0.038 and OR–2.30; 95% CI OR:1.14–4.64; p = 0.017]. The epidemiological analysis showed that polymorphic variants VDR (rs2228570) genes are not the risk factors of EH in the observed. Although, the combination of wild alleles of both genes in the homozygous state (AAAGTR1/AAVDR) makes a protective effect [OR = 0,42; OR 95%CI:0,18-1,0; ⇙2 = 3,74; p = 0,05], the combination of minor alleles (C-allele AGTR1 /AAVDR+C-allele AGTR1/AGVDR) increases the risk of EH more than threefold [OR = 3,36; OR 95%CI:1,24-9,09; ⇙2 = 5,88; p = 0,015]. Conclusions: Genotypes and alleles of VDR (rs2228570) gene are not associated with the risk of developing EH in the examined. C-allele of AGTR1 gene (rs5186) increases the risk of hypertension more than 2 times [OR–2.31; p = 0.011]. Combination of minor C-allele of AGTR1 gene and A-allele of VDR gene (C-allele AGTR1 /AAVDR+C-allele AGTR1/AGVDR) escalates the risk of EH more than 3 times [OR = 3,36; p = 0,015].

POLYMORPHIC VARIANTS OF AGT (RS4762) AND GNB3 (RS5443) GENES AS PREDICTORS OF ESSENTIAL ARTERIAL HYPERTENSION DEVELOPMENT

Objective: To evaluate the role of the polymorphism of AGT genes (rs4762) and GNB3 genes (rs5443) in the development of the essential arterial hypertension separately and by combining their polymorphic variants. Design and method: The case-control study involved 100 patients with EAH stage II, 1-3 degrees of blood pressure (BP), high and very high cardiovascular risk. Among the patients there were 21% (21) men, 79% (79) women. The mean age of patients was 59.86±6.22y.o. The control group consisted of 60 almost healthy individuals, relevant in age (49.13±6.28y.o.) and gender distribution (63% - women, 37% - men). To study of polymorphism of AGT (rs4762) and GNB3 (rs5443) genes was performed a qualitative polymerase chain reaction (PCR) in real time. Results: The mutated T-allele of the AGT gene (rs4762) occurs in 15.97% of hypertensive residents of Northern Bukovyna, that is more frequent than in healthy individuals by 9.72% (p = 0.023); homozygous mutations of the AGT gene were not found in the control group at all. Whereas, the relative frequency of polymorphic variants of the GNB3 gene (rs5443, 825C&gt;T) did not differ significantly between groups. The wild C-allele statistically significantly prevails over the mutated T-allele in both groups according to the AGT (rs4762) and GNB3 (rs5443) genes (p&lt;0.001). Binary logistic regression confirmed an increase in the risk of hypertension inheritance according to dominant and additive models in minor T-allele carriers of the AGT gene (rs4762) almost 3 times higher than in C-allele homozygotes (p = 0.04 and p = 0.03). Inheritance of EAH is not associated with polymorphic variants of the GNB3 gene (rs5443). Conclusions: The T-allele of the AGT gene (rs4762) increases the risk of hypertension almost 3 times, whereas, polymorphic variants of the GNB3 gene (rs5443) are not predictors of EAH in the observed.

PREVELANCE OF CARBAPENEMASE PRODUCING ORGANISMS AND MOLECULAR RAPID DETECTION OF DIFFERENT GENES FROM VARIOUS CLINICAL ISOLATES IN PATIENTS ADMITTED AT TERTIARY CARE HOSPITALS OF VADODARA

Introduction- Carbapenemase-producing organisms (CPO) have been identied as an urgent healthcare threat. The spread of carbapenemase-producing Enterobacterales (CPE) is a global health problem of great concern. Rapid detection of carbapenemase-producing organisms is clinically desirable for hospital infection control and antibiotic stewardship. Recently, an on-demand polymerase chain reaction (PCR) assay, namely, the Xpert CarbaR assay, that requires less than an hour of turnaround time, had been developed for CPO detection in clinical samples to identify and guide infection control programs to contain the spread of CPO within a hospital. Objective- Carba-R assay is a qualitative multiplex real-time PCR method that qualitatively detects and differentiates ve common carbapenemase genes blaKPC, blaNDM, blaVIM, blaOXA-48, and blaIMP) directly from clinical samples or puried colonies within approximately 1 hour. This benets hospitals and patients by facilitating timely Infection Prevention &amp; Control measures, thereby reducing risk of exposure, transmission and bed-days lost. Materials &amp; Methods- In this study, the Xpert Carba-R assay was evaluated for detection of the ve carbapenemase genes (blaKPC, blaNDM, blaIMP, blaOXA-48, and blaVIM) in total of 40 non duplicate various clinical samples of admitted patients in tertiary care hospitals of Vadodara. Result- We performed Carba-R on 40 isolates: 18 blood samples, 06 urine, 05 rectal swabs, 03 Endotracheal secretion, 03 Pus, 02 wound discharge, 01 Bronchoalveolar lavage uid, 01 sputum, and 01 ERCP stent isolates. 36/40 (90%) isolates had one or more carbapenemase genes. They were as follows: 19/40 (47.5%) both OXA48 and NDM , 12/40 (30%) NDM and 05/40(12.5%) OXA-48. There were 04/40 (10 %) isolates which were Carbapenem resistant on disc diffusion &amp; VITEK but none of the resistant genes were detected possibly due to other resistant mechanism like efux pump and porin channels. Klebsiella pneumoniae was the most common isolate with CR, 34/40 (85%). The most frequent genes encountered in Klebsiella pneumoniae were both OXA48 and NDM, 19/34 (55.88%), NDM 08/34 (23.53%) followed by OXA 48, 05/34 (14.71%) and Out of 34 Klebsiella isolates, 02 isolates failed to detect any of these ve carbapenemase genes. Conclusion- Xpert Carba-R assay provides good reliable results for detection and differentiation of ve carbapenemase genes in clinical isolates. Compared to bacterial culture followed by PCR identication of resistance genes from colonies, the Carba-R assay reduced turnaround time from 48 to 72 hours to less than 1 hour. Carbapenemase genes were detected by the Carba-R assay in Klebsiella pneumoniae (34/40), Escherichia coli (5/40), Acinetobacter spp (1/40). The Carba-R assay detected 19 both OXA48 and NDM (47.5%), 12 blaNDM (30% and 05 blaOXA-48 (12.5%) genes. Laboratory detection of these genes may help improve patient outcomes by tailoring therapy. This study was conducted for understanding the molecular epidemiology of Carbapenemase producing Enterobacteriaceae in a tertiary care hospital. The combined use of the Xpert Carba-R assay and culture produces rapid and reliable results for the active surveillance of CPO in patients

Investigation of Blood Plasma Viral Nucleocapsid Antigen as a Marker of Active Severe Acute Respiratory Syndrome Coronavirus 2 Omicron Variant Infection.

Nasopharyngeal qualitative reverse-transcription polymerase chain reaction (RT-PCR) is the gold standard for diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, but it is not practical or sufficient in every clinical scenario due to its inability to distinguish active from resolved infection. Alternative or adjunct testing may be needed to guide isolation precautions and treatment in patients admitted to the hospital. We performed a single-center, retrospective analysis of residual clinical specimens and medical record data to examine blood plasma nucleocapsid antigen as a candidate biomarker of active SARS-CoV-2. Adult patients admitted to the hospital or presenting to the emergency department with SARS-CoV-2 ribonucleic acid (RNA) detected by RT-PCR from a nasopharyngeal swab specimen were included. Both nasopharyngeal swab and a paired whole blood sample were required to be available for analysis. Fifty-four patients were included. Eight patients had positive nasopharyngeal swab virus cultures, 7 of whom (87.5%) had concurrent antigenemia. Nineteen (79.2%) of 24 patients with detectable subgenomic RNA and 20 (80.0%) of 25 patients with N2 RT-PCR cycle threshold ≤ 33 had antigenemia. Most individuals with active SARS-CoV-2 infection are likely to have concurrent antigenemia, but there may be some individuals with active infection in whom antigenemia is not detectable. The potential for high sensitivity and convenience of a blood test prompts interest in further investigation as a screening tool to reduce reliance on nasopharyngeal swab sampling and as an adjunct diagnostic test to aid in clinical decision making during the period after acute coronavirus disease 2019.