Volume 26 (2018): Issue 2 (April 2018)

Background To evaluate the effects of miR-99a on the migration and proliferation of glioma cells. Materials and Methods: Glioma cell line LN229 with stable up-regulation of miR-99a was constructed by transfection of hsa-miR-99a mimics, and cells with stable miR-99a knock-down were established by transfection of hsa-miR-99a inhibitor. The proliferation capacities of two groups were detected by the MTT assay, and their migration capacities were detected by the scratch assay. Results: LN229 cells with stable up-regulation and knock-down of miR-99a were successfully constructed. Up-regulating miR-99a inhibited the proliferation and migration of glioma cells, but knocking down this gene promoted their proliferation and migration. Conclusion: MiR-99a significantly affected the proliferation and migration of glioma cells, as a potentially eligible target for glioma therapy.

Both incidence and mortality of colorectal cancer (CRC) in Romania have shown a continuous increase during the last decades. Hereditary Non-Polyposic Colorectal Cancer (HNPCC), also known as Lynch syndrome, is mainly attributable to mismatch repair (MMR) genes MSH2, MSH6, and MLH1. Individuals carrying germ-line mutations of these genes present high lifetime risk of colorectal and other cancers, compared to non-carriers. Oncogenetics is developed worldwide nowadays, for identifying hereditary predisposition to cancer and offering appropriate clinical follow-up to patients and mutation carriers in Lynch families. Molecular oncogenetic diagnosis in Lynch syndrome is based on complete Sanger sequencing of entire MMR genes, which is time and resources consuming, therefore needing an appropriate and adapted optimization. Conventional sequencing requires a sufficient number of available samples to be processed simultaneously, which increases the waiting time for diagnostic results. Complete analysis for only one patient meets difficult technical problems due to the complex co-amplification of all gene regions of interest within the same conditions, therefore increasing the costs and reducing the cost-effectiveness of the test. Here we present an original and robust technical protocol for sequencing the entire MSH2, MSH6, and MLH1 coding sequence for one patient in a single PCR plate. Our optimized and verified system overcomes all technical problems and offers a quick, robust, and cost-effective possibility to personalize molecular oncogenetic diagnosis in Lynch syndrome.

Introduction: Breast cancer is the most common cancer in women worldwide, and Romania makes no exception from this trend. Genetic screening for Hereditary Breast and Ovarian Cancer began to be used on a larger scale after the introduction of Next Generation Sequencing. The aim of this study was to assess the association of deleterious mutations responsible for breast cancer with histopathological and immunohistochemical prognostic factors and to identify some genetic variants in the BRCA1 and BRCA2 genes. Method: 80 patients with breast cancer and negative genetic test or pathogenic variants on BRCA1/2, TP53, PALB2, CHEK2, ATM genes were included. All the cases had a prior histological diagnosis and complete immunohistochemical features. The genetic testing was conducted through a multigene panel. Results: 65% of patients had a deleterious mutation on BRCA genes. In 97.5% of cases the histology was invasive ductal carcinoma. Significant differences were identified between BRCA1 group and negative mutation group regarding estrogen receptor (ER) (p=0.0051), progesterone receptor (PR) (p=0.0004) and Ki67 (p=0.001). Seven breast cancer patients had BRCA1 c.3607C>T variant, which was statistically significantly associated with triple- negative breast cancer (p <0.0001). Of the 7 cases diagnosed with BRCA 2 mutations we identified the c.8755-1G>A variant in 3 cases and the c.9371A>T variant in 3 cases. Discussion and conclusion: Our study confirmed the association of BRCA1 mutations with negative ER, PR or triple negative breast cancer (TNBC). Description of BRCA1 c.3607C>T mutation for the first time in Romanian population and its association with TNBC will need further investigation.

Purpose: Our study focuses on elucidating if two common inflammatory biomarkers, easily performed in any laboratory - high-sensitivity C-reactive protein (hsCRP), as well as fibrinogen - could be used to assess the personal health risk of workers exposed to a complex occupational exposure to ultrafine particles (UFP) and a mixture of organic solvents. Methods: To assess the inflammatory response on the body, laboratory determinations were performed by testing the serum levels of hsCRP and fibrinogen, in exposed and unexposed groups. Results: There are no statistically significant differences for hsCRPs (p-0.25), medians were similar in groups. The mean values of fibrinogen in the three groups were: in the workers group (1st group): 346.2 mg/dl, in the office staff group (2nd group): 328.7 mg/dl, and in the control group (3rd group): 284.8 mg/dl, with significant differences between 1st group vs 3rd group and between 2nd group vs 3rd group (p-0.002). UFP levels differ between the groups, as follows: 1st group were exposed to the highest levels, ranging from 48349 to 3404000 part/cm3; 2nd group, ranging from 17371 to 40595 part/cm3; and 3rd group, ranging from 213 to 16255 part/cm3. Conclusions: Our study demonstrates that fibrinogen is a useful inflammatory biomarker for exposure to a mixture of UFP and organic solvents. On the other hand, hsCRP is not a useful inflammatory biomarker in occupational exposure to UFP and organic solvents. Further studies are needed to demonstrate the extent to which fibrinogen is more or less influenced by organic solvents or UFP alone.

This study investigated the antibiotic susceptibility patterns and genetic resistance markers of 35 C. difficile strains isolated from patients with C. difficile infection. Vancomycin, metronidazole, tigecycline, teicoplanin, rifampicin, moxifloxacin, cefotaxime, tetracycline, erythromycin, clindamycin, chloramphenicol, linezolid and imipenem MICs were determined for toxigenic strains belonging to PCR ribotypes (PR) 012 (2), 014 (4), 017 (3), 018 (2), 027 (17), 046 (2), 087 (3) and 115 (2). Results showed vancomycin, metronidazole, tigecycline and teicoplanin to be active against all isolates. High resistance rates were noticed against cefotaxime (n = 35), clindamycin (n = 33), imipenem (n = 31), moxifloxacin (n = 25), erythromycin (n = 25) and rifampicin (n = 22). Linezolid-resistance was found in three isolates (PR 017/2, PR 012/1), showing complex resistance (7-9 antibiotics). PR 012, 017, 018, 027 and 046 isolates (n = 26) were resistant to 5-9 antibiotics. Twelve resistance profiles (2-9 antibiotics) were detected. Rifampicin-moxifloxacin-cefotaxime-erythromycin-clindamycin-imipenem-resistance was predominant, being expressed by 18 strains (PR 027/17, PR 018/1). PCR results suggested tetracycline-resistance to be induced by the gene tetM. Three tetM-positive isolates (PRs 012, 046), were also tndX-positive, suggesting the presence of a Tn5397-like element. Only two MLSB-resistant strains (PR 012) had the ermB gene and chloramphenicol-resistance determinant catD was not detected, leaving room for further investigating resistance mechanisms. Multidrug resistance could be attributed to most analysed strains, underlining, once more, the impact of wide-spectrum antimicrobial over prescription, still a tendency in our country, on transmission of antimicrobial resistance and emergence of epidemic C. difficile strains generating outbreaks.

Introduction: The infection with Clostridium difficile has increased in incidence worldwide and it raises many problems with regard to therapy, resistance to treatment and especially recurrence. Recurrence is frequent in patients treated for Clostridium difficile infection, requiring vancomycin by mouth, with limited alternatives. The literature shows that one of the most efficient treatment methods in Clostridium difficile infection is the transplantation of gut microbiota, also known as fecal microbiota transplantation. Aim: We present our results following FMT performed in patients with recurrent Clostridium difficile infection, and propose a simple and effective protocol for fecal microbiota transplantation. Study design: The study was prospective. The phases of the FMT procedure: assessment of patient eligibility, patient’s consent, identification and screening of donors, discontinuation of antibiotics (vancomycin, metronidazole) 3 days prior to the procedure. Methods: Between 2013 and 2015, FMT was performed in 30 patients with recurrent Clostridium difficile infection, by direct infusion of extensively processed donor fecal matter via colonoscopy. We followed up the patients for 12 months. Results: Immediate post-transplantation outcome in what concerns stool frequency during the follow-up period (7 days) was encouraging in 93.33% of patients. The donors were healthy individuals (53% 1st degree relatives), previously screened for possible infections and infestations. This result was sustained at 6-month and 12-month follow-up. Post-transplantation recurrence occurred in 6.67% (2 patients), which responded well to treatment and did not require a new vancomycin course. Conclusions: Fecal microbiota transplantation via colonoscopy is effective, safe, easy to perform, it yields lasting results and is therefore a good option for recurrent or treatment-resistant Clostridium difficile infection.

The O26 verocytotoxin-producing Escherichia coli (VTEC)-associated outbreak of hemolytic uremic syndrome (HUS) cases in Romania during 2016 showed the need to improve the current methodology of non-O157 VTEC detection and surveillance. An in-house assay based on xTAG Luminex technology was optimized to identify seven of the most relevant diarrheagenic E.coli serogroups (O-specific wzx genes), two convenient VTEC virulence markers (eaeA and ehxA genes), and a species-specific control gene (uidA). Twenty-nine strains previously characterized in terms of serogroup and virulence genes were tested with the optimized protocol and the results were as expected. The ratio of sample signal to background varied from 66.7 (ehxA) to 7.6 (uidA) for positive samples, with a cut-off of 3. Sensitivity varied depending on the target to be amplified from approximately 102 genomic copies to approximately 104 genomic copies per reaction, respectively. The current approach seems an affordable alternative to commercially available assays that can be further exploited to improve existing autochthonous strategies to prevent future VTEC outbreaks.

Introduction: Infections due to carbapenemase-producing carbapenem-resistant Enterobacteriaceae (CPCRE) are an emerging global public health threat. The purpose of this study was to investigate phenotypic and genotypic features of CP-CRE strains isolated from hospitalized patients. Material and methods: Between 1st of January - 1st of July 2017, in the Department of Microbiology, “Dr. Constantin Opriş” County Emergency Hospital Baia Mare, Romania, 1110 strains of Enterobacteriaceae were isolated from bronchial secretions, urine, wounds and blood cultures. Bacterial identification and antimicrobial susceptibility tests were performed by conventional methods, Vitek 2 Compact and M.I.C.E. strips. We analysed all Enterobacteriaceae strains non-susceptible to carbapenems according to CLSI 2017 criteria. The modified Hodge test (MHT), the modified carbapenem inactivation method (mCIM) and the combination disks test (KPC, MBL, OXA-48 Confirm kit, Rosco Diagnostica) were used for phenotypic confirmation, whereas a multiplex PCR assay for genes blaKPC, blaNDM and blaOXA-48 was used for genetic confirmation. Results: 19 non-duplicate strains isolated from 16 patients were phenotypically identified as CP-CRE: Klebsiella pneumoniae (n=14), Escherichia coli (n=2), Providencia stuartii (n=2) and Serratia marcescens (n=1). Most strains were isolated from bronchial secretions (n=9). The carbapenem-hydrolizing enzymes were identified by the combination disks test as: KPC (n=9), OXA-48-like (n=5) and MBL (n=5). Molecular confirmation was performed in 18 phenotypically positive isolates with 100% concordant results with mCIM and combination disks test. Discrepant results were noticed with the MHT in case of 4 NDM-producers confirmed by PCR. All CP-CRE strains were resistant to all tested cephems. Three out of 9 K. pneumoniae strains tested against colistin were found resistant. Conclusions: The most common carbapenemase detected was KPC. Therapeutic options were limited in all positive cases. Rapid and reliable detection of CP-CRE is critical for preventing the spread of these pathogens

Aim: In vitro antibacterial activity of 6-substituted-3(2H)-pyridazinone-2-acetyl-2-(substituted/nonsubstitutedbenzal/ acetophenone) hydrazone derivatives were tested in common species causing hospital-acquired infections. Material and Method: Antimicrobial activities of the compounds were performed by determining minimum inhibitory concentration (MIC) value against four Gram-positive, five Gram-negative and four Candida species fungi. Modified serial microdilution method was carried out. Reference strains of American Type Culture Collection (ATCC) were used. Results: In general, eleven compounds exhibited considerable activity. Comparatively, compound 3 exhibited strong activity against Enterobacter hormaechei and 5, 11 were the most active against Acinetobacter baumannii at 31.25 μg/mL. Compounds 1,2,3,4,8 and 10 were found to be as active as positive control ampicillin trihidrate against Stenotrophomonas maltophilia. On the other hand, compounds 1,2,3,4,7,8,9,10 and 11 showed strong antifungal activitiy as much as fluconazole against Candida tropicalis. Compound 1 was mostly active against Candida albicans, Candida glabrata, Candida parapsilosis and Candida tropicalis. It was also revealed that the antifungal activity of compounds 1, 6, 7, 8 and 9 were higher than the others. Compound 1 and 8 exhibited the best activity against Candida glabrata and Candida parapsilosis respectively. Conclusions: All tested compounds showed better activity against Gram-negative bacteria and yeast than Gram-positive bacteria. These compounds may be considered as alternative antimicrobial agents in the treatment of multiple drug resistant Gram-negative, Gram-positive bacteria and fungal pathogens. Especially, we suggested that Compound 1 and 8 might be a promising candidate of new antifungal agents

Backgroud. Which factors determine venous thrombotic events in some antiphospholipid syndrome (APS) patients and arterial thrombosis or conditions related to pregnancy in others has not been established yet. Purpose. The aim of this study was to search the antiphospholipid antibodies (APLAs) correlates in regard to deep vein thrombosis (DVT) in patients with systemic lupus erythematosus (SLE) and APS. Methods. Twenty-nine patients fulfilling the criteria of both SLE and APS were included. Complete anamnesis and clinical examination was performed on inclusion. Also, for all patients, disease activity was assessed by the SLEDAI score. An extended APLAs profile, ten Abs, was searched. Results. The titers of IgG anticardiolipin (aCL), IgG anti-β2 glycoprotein I (aβ2GPI), IgG antiphosphatidylethanolamine (aPE), and also of IgG antiprothrombin (aPT) were significant higher in patients with DVT history. After analysis by ROC curve and univariate logistic regression, the strongest association was found for IgG aPE. Also, in multivariate analysis, SLEDAI score correlated with the DVT antecedents. Conclusions. IgG aPE might be involved in DVT pathogenic pathways in patients with SLE and APS as their titers remain significantly higher in patients with previous DVT. Lupus patients with DVT events represent a subgroup of patients with more severe underlying pathology.