CHARACTERIZATION OF PATIENTS IN THE POST-ACUTE PHASE OF COVID-19 WHO PRESENTED THEMSELVES TO AN EMERGENCY DEPARTMENT.

Background: Patients that recovered from COVID-19 may remain with symptoms which can persist for an uncertain period of time. Aim: The purpose of this study was to investigate the reasons why patients who passed the acute phase of COVID-19 presented themselves to the Emergency Department. Patients and Methods: We selected 87 patients admitted to the Emergency Department of the Bucharest University Emergency Hospital between 01.01.2021-31.05.2021. Patients had pulmonary fibrosis (11.49%), pleural effusion (16.09%) or a history of hypertension (73.56%), type 2 diabetes (42.53%), stroke (24.14%), malignant diseases (10.34%). Results: Association between neutrophil levels and acute stroke and between fibrinogen levels and alveolar condensation were identified. The percentage of deaths was significantly higher in the subgroup of subjects that had maxim 11 days of hospitalization (p=0.004); we observed a trend of association between the age of more than 51 years old and admission in the Emergency Unit at less than a month after the SARS Cov2 infection, the positive result at the RT-PCR test or a lung damage of over 30% (p<0.05). Conclusion: A significant percentage of patients that were admitted to the Emergency Unit post COVID-19 had chronic pathologies and their characteristics were associated with neutrophilia, high fibrinogen levels or length of hospitalization.

Impact of gender and dialysis modality on early mortality risk in diabetic ESRD patients: data from a large single center cohort.

BACKGROUND: Diabetes mellitus (DM)-associated chronic kidney disease (diabetic renal disease) became the predominant etiology of the end-stage renal disease (ESRD) in the western countries and shows the same trend in the developing countries. Early mortality (EM) after the dialysis initiation, defined as death of all causes within the first 3 months after initiation of renal replacement therapy (RRT), is of concern especially for the high-risk renal patients including diabetics. The goal of the present study was to identify demographic and clinical risk factors associated with EM in a retrospective cohort of Romanian DM patients initiated on dialysis. METHODS: A retrospective case-control study was designed. Clinical recordings from all patients initiated on hemodialysis (HD) or peritoneal dialysis between January 1996 and December 2005 in the Dialysis Center of NIDNMD Paulescu, Bucharest, were collected and analyzed. Patients were classified accordingly in two groups: those who displayed EM formed the "cases" group, while the others were included in the "controls" group. Both univariate (subgroup analysis) and multivariate analyses (logistic regression, Cox regression) were used to analyze the impact of risk factors on EM outcome. RESULTS: Data from 788 patients were included in the analysis. The factors significantly associated with EM in the univariate analysis were female gender, late initiation (LI) of dialysis, old age and HD used as the first/only method. Applying the multivariate analysis, only the use of HD (OR = 4.20, p < 0.0001) and the LI of dialysis (p < 0.0001; 95 % CI 1.36-2.30) were associated with EM, while female gender showed only a trend to a higher EM (OR = 1.29, p = 0.052). CONCLUSIONS: Hemodialysis used as a first/single method for RRT and the LI of dialysis were independent predictors of EM in our ESRD diabetic patients. A possible explanation for the first factor could be our specific center procedure, which allows only the HD as rescue therapy method for the most severe cases, managed in the intensive care unit.

Rapid detection of human torque teno viruses using high-resolution melting analysis.

Torque teno viruses (TTVs) are recently discovered DNA viruses, with heterogeneous genomes, highly prevalent in populations worldwide. The species that infect humans are Torque teno virus (TTV), Torque teno midi virus (TTMDV) and Torque teno mini virus (TTMV). High-resolution melting analysis (HRMA) is a sensitive and effective method for genotyping and mutation scanning. Up to now, HRMA has not been utilized for detection of TTVs. The aim of this study was to asses if HRMA is suitable for detecting TTVs variants. DNA was extracted from the blood and saliva of 13 healthy subjects for method optimization. Additionally, saliva samples from 100 healthy individuals were collected for estimating the TTVs' prevalence. Viral DNA was amplified by heminested polymerase chain reaction (PCR). Second round amplicons were used for the HRMA. The samples were analyzed using two fluorescent dyes, SYBR (®) Green I and EvaGreen®. The prevalence values for TTV, TTMDV and TTMV were 71.0, 31.0 and 54.0%, respectively. The three major melting curve patterns corresponding to TTV, TTMDV and TTMV on HRMA can be easily distinguished regardless of kit used. Our results showed that HRMA is a rapid and efficient method of detecting human TTVs.

Analysis of several BRCA1 and BRCA2 mutations in a hospital-based series of unselected breast cancer cases.

BACKGROUND: The distribution of BRCA mutations varies significantly between populations. The spectrum of BRCA1 and BRCA2 mutations in breast cancers in the Romanian population is incompletely known. The aim of the present study is to investigate the presence of nine BRCA mutations in patients with breast cancer identified in a surgical clinic from Bucharest. METHODS: Unrelated women diagnosed with breast cancer from Coltea Hospital (n=114) and healthy controls (n = 150) were selected for this study. Seven mutations in BRCA1 (185delAG, 5382insC, 943ins10, E1250X, 1294del40, E1373X, R1443X) and two in BRCA2 (IVS16-2A4G and 6174delT) were tested using PCR based protocols. In addition, the presence of BRCA1 185delAG, BRCA1 5382insC, BRCA2 6174delT mutations were tested with a post amplification mutation detection system, based on the ELISA method. RESULTS: Two patients with sporadic breast cancer (2%) and one patient with family history of the disease (7.14%) have the BRCA1 5382insC mutation. No other mutation was detected in patient and control groups. The mutations were not present in the control lot. CONCLUSIONS: Our results indicate that BRCA1 5382insC is a common mutation in Romanian women with breast cancer (3 114).

Rating the environmental and genetic risk factors for colorectal cancer.

Colorectal cancer (CRC) is a disease preventable in up to 50% of the patients by lifestyle modifications. The preventive strategy for the decrease in the incidence and mortality of CRC is based on understanding the relations between the environmental and genetic factors. The most important identified risk factors for CRC are aging, personal and familial history of CRC or adenomas, hereditary colon cancer syndromes, dietary patterns, and inflammatory bowel disease. The purpose of this review is to update data referring to environmental and genetic documented factors and CRC risk. Using data from the Medline database, we analyzed reports on CRC risk published between 2000 and 2010. We realized a classification taking into consideration the relative risk (RR) reported for each analyzed factor (RR ranged between 1 and 6.87). The highest RR were represented by the patients with distal advanced cancer (RR = 6.7) and those with high dysplasia adenomas (RR = 6.87). In the future, evaluation and optimisation of screening options will stay at the base of new prevention strategies that will be implemented based on the influence of risk factors identified in each population.

Investigation of P213S SELL gene polymorphism in type 2 diabetes mellitus and related end stage renal disease. A case-control study.

SELL (L-selectin) is a candidate gene for several complex diseases including diabetes mellitus and renal failure. Our aim was to investigate the involvement of P213S SELL gene polymorphism (rs2229569) in type 2 diabetes mellitus (T2DM) and related end stage renal disease (ESRD). Type 2 diabetes mellitus patients without ESRD (n=250) or with ESRD (n=90), ESRD patients without diabetes (n=119) and sex and age matched healthy subjects (n=459) were analyzed in this study. DNA samples from all these subjects were genotyped for the P213S polymorphism by PCR-RFLP technique. Statistical analysis indicated that SELL P213S genotypes and alleles were similar distributed in the patients and control groups (ORSS=0.37, CI 95%: 0.131>0.372>1.06, p=0.05, Yate's correction p=0.09, for T2DM patients without ESRD, ORSS=2.04, CI 95%: 0.365>2.047>1.465, p=0.4, Yate's correction p=0.67, for T2DM patients with ESRD and ORSS=1, CI95%: 0.198>1>5.057, p=1, Yate's correction p=0.67, for non-diabetic with ESRD patients). Also, no significant differences were noticed when we compared the ESRD subjects with diabetes vs. non-diabetic ones (OR=1.798, CI 95%: 0.392>1.798>8.245, p=0.44, Yate's correction p=0.7). No statistically significant results were found in order to sustain the hypothesis of association between SELL gene P213S polymorphism, type 2 diabetes mellitus and end stage renal disease.

Detection of possible restriction sites for type II restriction enzymes in DNA sequences.

In order to make a step forward in the knowledge of the mechanism operating in complex polygenic disorders such as diabetes and obesity, this paper proposes a new algorithm (PRSD -possible restriction site detection) and its implementation in Applied Genetics software. This software can be used for in silico detection of potential (hidden) recognition sites for endonucleases and for nucleotide repeats identification. The recognition sites for endonucleases may result from hidden sequences through deletion or insertion of a specific number of nucleotides. Tests were conducted on DNA sequences downloaded from NCBI servers using specific recognition sites for common type II restriction enzymes introduced in the software database (n = 126). Each possible recognition site indicated by the PRSD algorithm implemented in Applied Genetics was checked and confirmed by NEBcutter V2.0 and Webcutter 2.0 software. In the sequence NG_008724.1 (which includes 63632 nucleotides) we found a high number of potential restriction sites for ECO R1 that may be produced by deletion (n = 43 sites) or insertion (n = 591 sites) of one nucleotide. The second module of Applied Genetics has been designed to find simple repeats sizes with a real future in understanding the role of SNPs (Single Nucleotide Polymorphisms) in the pathogenesis of the complex metabolic disorders. We have tested the presence of simple repetitive sequences in five DNA sequence. The software indicated exact position of each repeats detected in the tested sequences. Future development of Applied Genetics can provide an alternative for powerful tools used to search for restriction sites or repetitive sequences or to improve genotyping methods.

Polymorphism of catalase gene promoter in Romanian patients with diabetic kidney disease and type 1 diabetes.

Hyperglycaemia leads to ROS (Reactive oxygen species) generation, affecting the cells that cannot decrease glucose uptake such as: glomerular epithelial cells, mesangial cells and proximal tubule cells. ROS excess seems to activate important pathogenic pathways of development of diabetic nephropathy. The decrease of CAT activity, one of the most important antioxidant enzymes, following to some genetic defects, may be a risk factor for diabetic nephropathy. The purpose of this study is to investigate the association of 21A/T (rs7943316) polymorphism of CAT gene with advanced diabetic nephropathy in patients with type 1 diabetes in Romania. There have been studied 238 patients with T1D (type 1 diabetes), divided into the group with diabetic nephropathy (DN) (106 patients) and the group without renal affectation (132 patients). The genotyping has been made by using PCR-RFLP technique. The analysis of association has been made by using DeFinetti programme. The value considered significant has been p < 0.05. There has been a deviation from Hardy-Weinberg equilibrium in the group with diabetic nephropathy (p = 0.019), the equilibrium being preserved by the control group (p = 0.771). T allele does not confer a risk for advanced diabetic nephropathy (ORT = 0.757, 95% C.I. = 0.405-1.414; P = 0.381), the result being statistically insignificant even taking into consideration the risk allele A (ORA = 0.793, 95% C.I. = 0.465-1.350; P = 0.392). The results remain concordant too after applying the Cochran -Armitage test. Our data do not suggest an effect of 21A/T (rs7943316) polymorphism in the susceptibility for diabetic nephropathy in Romanian patients with type 1 diabetes. Further studies are necessary in order to demonstrate or exclude the role of CAT gene in diabetic nephropathy in patients with type 1 diabetes.

MTRR polymorphism and the risk for colorectal and breast cancer in Romanian patients--a preliminary study.

BACKGROUND: The risk of colorectal cancer (CRC) and breast cancer (BC) is influenced by polymorphisms located in the genes encoding enzymes of the folate pathway. The aim of this study was to evaluate if A66G MTRR (rs1801394) polymorphism is involved in predisposition for colorectal and breast carcinogenesis in Romanian patients. MATERIALS AND METHODS: In the present case-control study, 300 individuals divide in four groups: sporadic CRC patients (n = 120), control CRC (n = 60), BC patients (n = 60) and control BC (n = 60), were genotyped by PCR-RFLP method. RESULTS: Frequency of genotype AA was 11.7% in CRC control and 5% respectively in BC control. For cancer groups the frequency of genotype AA was 9.2% in CRC and 0% in BC. CONCLUSIONS: Study results do not demonstrate an association between A66G MTRR polymorphism and CRC or BC in Romanian patients.

Spinal muscular atrophy disease: a literature review for therapeutic strategies.

Currently, there is no cure for the treatment of spinal muscular atrophy (SMA). Based on the available clinical and molecular findings, different therapeutic strategies were tested in vitro and in vivo and clinical trials are ongoing. The main therapeutic direction is focused on the enhancement of SMN expression by increasing the full-length (fl) SMN2 transcript levels, preventing the SMN exon 7 from skipping or from protein stabilizing. In addition, the action of neurotrophic, neuroprotective or anabolic agents is tested and stem cell and gene therapy approaches are in a promising development.

Association of +35A/C (intron3/exon3) polymorphism in SOD1-gene with diabetic nephropathy in type 1 diabetes.

Diabetic nephropathy is a major complication of type 1 diabetes whose pathogenesis is insufficiently known, but oxidative stress and genetic susceptibility seem to be involved. The purpose of this study is to assess the possible association of +35A/C (rs2234694) polymorphism in SOD1-gene with advanced stages of diabetic nephropathy in patients with type 1 diabetes in Romania. There have been enrolled 238 unrelated patients, having type 1 diabetes, divided into group A (106 patients) with diabetic nephropathy - macroalbuminuria or ESRD (End Stage Renal Disease) and group B (132 patients) without diabetic nephropathy. The genomic DNA was extracted from the peripheral venous blood and the genotyping of +35A/C (rs2234694) polymorphism has been made using the PCR-RFLP technique. The statistical analysis has been made using De Finetti's program. There has not been a significant deviation from the Hardy-Weinberg equilibrium for any group (p=0.229 and p=0.894, respectively). The data analysis revealed that the presence of a C-allele confers a significant risk (p=0.008) for the advanced diabetes nephropathy (OR=4.940, 95% C.I.=1.341-18.198), and the CA-genotype (p=0.015) confers a little lower risk (OR=4.491, 95% C.I.=1.203-16.766). This study shows the association of a mutant C-allele of rs2234694 polymorphism in SOD1-gene with the advanced stages of diabetic nephropathy in patients with type 1 diabetes in Romania, suggesting the involvement of the defense against oxidative stress, as an important link in the pathogeny of diabetic nephropathy.

Lack of association between ACE ID polymorphism and colorectal cancer in Romanian patients.

BACKGROUND: The insertion/deletion polymorphism of the angiotensin I-converting enzyme (ACE) gene has recently been linked to the pathogenesis of human cancers. The goal of this study was to analyze the possible association between ACE gene I/D polymorphism and colorectal cancer in Romanian patients. METHODS: Blood samples were obtained, after informed consent, from individuals with colorectal cancer (n=108, M:W = 64:44), and healthy persons (n=150, M:W = 84:66). Genomic DNA was extracted from peripheral blood leucocytes using commercial kits and the insertion (I) / deletion (D) polymorphism was assessed by PCR. Statistical analysis was done using the chi2 test. We determined the odds ratio using the genotype II as risk factor. A p value < 0.05 was considered statistically significant. RESULTS: The distribution of ACE II: ID: DD genotypes was 23.1%: 46.3%: 30.6% in patients and respectively 20%: 48.7%: 31.3% in controls. The distribution of genotype (chi2 0.37, p = 0.54) and alleles (chi2 0.19, p = 0.65) did not differ significantly between cancer patients and control. CONCLUSIONS: Study results do not demonstrate an association between ACE ID polymorphism and colorectal cancer in our patients.

Molecular analysis of mutations for the adenomatous polyposis coli (APC) gene in Romanian patients with colorectal cancer.

Mutations in adenomatous polyposis coli (APC) gene have not been previously characterized among Romanian patients with colorectal cancer (CRC). We initiate this study to detect the mutations in APC gene in blood and tumor samples collected from 16 patients (10 men and 6 women) and blood samples from 21 first and second degree relatives of the patients. For this the presence of mutations in exons 6, 7, 12, 13, 14 as well as in regions B, L and W of exon 15 was investigated using PCR multiplex. In the same time, we have searched for 5 bp deletions at codon 1061 of APC gene by PAGE and SSCP methods. These methods allowed us to evidence identification of the presence of mutations in samples from 7 individuals. In one patient, was detected a deletion of exon 13th of APC gene both in DNA extracted from blood and tumor samples. Multiple deletions (e.g. in exon 6, 12, and in 15L and 15W regions) in DNA extracted from the tumor sample were detected, but not in DNA probe obtained from blood cells. We can speculate that these mutations are an example of genomic instability accompanying the malignancy. Till now, no mutation affecting 1061 codon of APC gene was identified in the patients investigated in our study.

[When and what will we do with the descendants of colorectal cancer patients? Case study and research project presentation]. / Când si ce vom face cu descendentii pacientilor cu cancer colo-rectal? Studiu de caz si prezentarea unui proiect de cercetare.

The polygenic etiology of familial colorectal cancer and other digestive tract cancers has been acknowledged and therefore a study subject by means of various techniques such comparative genomic hybridization, serial genetic analysis (APC, CGH) or DNA arrays. Our paper is the first presentation of a CNCSIS research project named: "Analiza unor factori moleculari implicati in stabilirea riscului statistic de îmbolnavire la descendentii probanzilor cu cancer rectocolonic" and also presents the case of a 12 members family in which 3 had already been diagnosed with colonic or rectal cancer. The APC gene methylation profile was studied in order to establish both the gene implication in cancer development within the family and the risk of colorectal cancer for the healthy family members. The paper shows the present means of interaction between the surgeon and the familial colorectal cancer cases and the research project advocates for the necessity of a genetic counseling network to which such cases should be referred.

Screening for microdeletions in human Y chromosome--AZF candidate genes and male infertility.

About 30% of couple infertilities are of male origin, some of them caused by genetic abnormalities of the Y chromosome. Deletions in AZF region can cause severe spermatogenic defects ranging from non-obstructive azoospermia to oligospermia. The intracytoplasmatic sperm injection technique (ICSI) is rapidly becoming a versatile procedure for human assisted reproduction in case of male infertility. The use of ICSI allows Y chromosome defects to be passed from father. The goal of our study is to evaluate the frequency of microdeletions in the long arm of Y chromosome, within the AZF regions, in these cases of infertilities, using molecular genetics techniques. Thirty infertile men with azoospermia or oligozoospermia, determined by spermogram, were studied after exclusion of patients with endocrine or obstructive causes of infertility. Peripheral blood DNA was extracted from each patient, then amplified by multiplex PCR with STS genomic markers from the Y chromosome AZF zones. Each case was checked by multiplex PCR through coamplification with the SRY marker. Three men with microdeletions of the long arm of the Y chromosome were diagnosed among the 30 patients, corresponding to a proportion of 10%. The relatively high proportion of microdeletions found in our population suggest the need for strict patient selection to avoid unnecessary screening for long arm Y chromosome microdeletions. The molecular diagnostics was performed according to the current European Academy of Andrology laboratory guidelines for molecular diagnosis of Y chromosomal microdeletions.

Gastrointestinal histoplasmosis in HIV infection: two cases of colonic pseudocancer and review of the literature.

Primary gastrointestinal infection is an uncommon manifestation of histoplasmosis. It is almost always associated with disseminated disease and/or an immunocompromised host. The ileum and cecum are the most common sites involved. We report two cases of primary gastrointestinal histoplasmosis in HIV-seropositive men who presented with annular constricting right colon lesions.