Volume 23 (2015): Issue 1 (March 2015)

Backgound. Using influenza epidemiological and virological surveillance data, we aimed at investigating the profile of influenza viruses circulating in Romania during the season 2012-2013 and estimating the effectiveness (VE) of the seasonal vaccine. Methods. We tested all specimens collected from patients with influenza like illness (ILI) in the national surveillance system between week 40/2012 to week 20/2013. Influenza A/B positive specimens identified by molecular detection (RT-PCR) were further characterized. We used hemagglutination inhibition assay for antigenic characterization and chemiluminiscence assay for the antiviral susceptibility testing. Subsequently we conducted nucleotide sequencing of hemagglutinin and neuraminidase genes and phylogenetic tree analyses. We estimated influenza VE using the test negative case-control study design, as 1-odds ratio of vaccination among ILI cases positive for influenza and ILI negative controls. Results and Discussions. We tested 1087 specimens, and 537 cases were positive (56.2% influenza B, 40.6% A(H1N1)pdm09, 3.2% A(H3N2). Sixty-four influenza viruses were antigenically and/or genetically characterized. A(H1N1)pdm09 viruses were related to the vaccine strain A/ California/07/2009 and clustered with genetic group 6 similar to A/St. Petersburg/27/2011. Influenza B viruses belonged to clade 2 of type B/Yamagata lineage, related to B/Estonia/55669/2011 except one, B/Victoria lineage, representative strain B/Brisbane/60/2008. A(H3) viruses clustered with group 3C of the A/Victoria/208/2009 clade, similar to the vaccine strain A/Victoria/361/2011. All tested strains (57) demonstrated susceptibility to oseltamivir and zanamivir. The adjusted seasonal influenza vaccine effectiveness against influenza A(H1N1)pdm09 (N=119) was 76.9% (95% CI: -113.4, 98.5), suggesting a good protection, consistent with the good match between the vaccine and circulating strains.

Meningococcal infection requires a fast and accurate diagnostic method in order to correctly initiate the antibiotic therapy. The aim of our study was to assess the efficiency of Real Time PCR -Taq Man using sod C gene / N. meningitidis in comparison with the classical methods for the diagnosis of meningococcal infection - direct microscopy, cultivation, latex agglutination and blood culture. We have detected 24/44 (54.54%) patients with meningococcal infection. In both cases of patients with / without previous antibiotic therapy before admission, the AUC (area under curve) had the highest values for RT PCR in CSF and blood analysis. This sod C RT-PCR assay is a highly sensitive and specific method for detection of Neisseria meningitis and it would be useful to include this method like a multiplex in routine testing of patients with clinical meningococcal infection for other etiological agents also.

Objective: The aim of this research is to evaluate the value of the histological score based on a histological record compared to the histometry for monitoring cranial bone defect healing. Methods: We designed a case -control study with a control and a study group. For a number of 60 CD1 mice representing the study group, a bone defect in the cranial bone was surgically induced and grafted with bone grafts obtained by tissue engineering. Bone grafts were obtained using embryonic stem cells seeded on a scaffold obtained from the red deer antler, and osteogenic basal and complex medium was used as differentiation medium. For other 30 CD1 mice representing the control group, a bone defect in the cranial bone was induced and left to heal without grafts. The regeneration process was assessed after 2 and 4 months using the histological healing scoring system and histometry. Results: The healing score was statistically significantly correlated with the defect size obtained by means of histometry (p<0.001). The evaluation of the parameters comprised in the healing score shows that regeneration of the bone diastasis was the most advanced in the group sacrificed at 4 months after plasty, which employed embryonic stem cells, a complex osteogenic differentiation medium and deer antler as scaffold. Conclusion: histological method based on a histological score is a valuable quantification system of bone regeneration comparable to histometry. Clinical Relevance: This study proves that the presented histological score can help the clinician in the process of bone regeneration evaluation.

Primary open-angle glaucoma (POAG) represents the most common form of a heterogeneous group of glaucomatous optic neuropathies which are a worldwide cause of irreversible blindness. Immune dysregulation and the genetic background are considered important risk factors. The influence on susceptibility to POAG of single nucleotide polymorphisms (SNPs) of tumor necrosis factor-α (TNF-α) was intensively studied, mostly on Asian population. We investigated the possible association of two TNF-α SNPs (-308G/A and -857C/T) with susceptibility to POAG and its clinical characteristics. A case-control association study of aforementioned TNF-α SNPs was performed on 197 POAG patients (divided into two subgroups: high-tension and normal-tension glaucoma - HTG/ NTG) versus 208 ethnically matched controls. This is the first study performed on Romanian population. No significant differences were found in terms of allelic frequencies, genotype distribution of the studied SNPs, or their haplotypes between POAG and healthy control groups. In the subgroup analysis, TT genotype of TNF-α -857T-allele was found to be associated with higher values of central corneal thickness (CCT) in NTG subgroup (p-value 0.032). In order to confirm the association between -857C/T SNP of TNF-α and CCT in NTG subgroup of POAG patients, additional studies on different populations should be performed.

Background. Validating new sepsis biomarkers can contribute to early diagnosis and initiation of therapy. The aim of this study is to evaluate the sepsis predictive capacity of soluble urokinase plasminogen receptor (suPAR) and its role in evaluating the prognosis of bloodstream infections. Material and method. We conducted a prospective pilot study on 49 systemic inflammatory response syndrome (SIRS) patients admitted to the intensive care unit (ICU), that were divided, on the basis of bacteremia in group A (SIRS with bacteremia, n=14) and group B (SIRS without bacteremia, n=35). Hemoculture and blood samples were drawn on the first day to determine suPAR, C-reactive protein (CRP) and procalcitonin (PCT). We set to identify significant cut-off values in estimating bacteremia and mortality in septic patients. Results. In group A, suPAR values were 14.3 ng/mL (range 10-45.5 ng/mL) and in group B, 9.85 ng/mL (range 3.4-48 ng/mL) p=0.008. Area under the curve (AUC) for suPAR was 0.745 (95% CI: 0.600-0.859), for CRP 0.613 (95% CI: 0.522-0.799) and for PCT 0.718 (95% CI: 0.477-0.769). Cut-off value for suPAR in bacteremia prediction was 9.885 ng/mL, with 100% sensibility and 51.43% specificity. Mortality in group A was 85.7% (12/14) and in group B 74.3% (26/39), p>0.05. Area under the curve (AUC) for suPAR was 0.750 (95% CI: 0.455-0.936), for CRP 0.613 (95% CI: 0.413-0.913) and for PCT 0.618 (95% CI: 0.373-0.888). Cut-off value of suPAR in predicting mortality was 11.5 ng/mL, with 66.67% sensibility and 100% specificity. Conclusions. In our study suPAR had a predictive capacity for bacteremia and seems to be an independent factor for mortality prognosis in septic patients.

Malnutrition is a frequent and serious finding in surgical departments. Although its consequences include postoperative complications and higher costs, nutritional assessment is not part of the routine preoperative protocols. Nutritional assessment involves clinical and biological parameters and is vital in order to start treatment and improve outcome. Prealbumin is currently recognized as a faithful marker of malnutrition being introduced in practice guidelines. One of the most important aspects about prealbumin is the fact that its variations in time are more valuable than the absolute values. The aim of this study was to assess and compare the perioperative nutritional evolution of patients requiring thoracic surgery, with and without cancer, using prealbumin - preoperative and postoperative - as main marker. Thirty six patients from the Thoracic Surgery Department were assessed prior to surgery by body mass index, Subjective Global Assessment nutrition risk score and routine biochemical parameters. Prealbumin was assessed prior to surgery and 3 days after surgery. The age, length of postoperative stay and the presence was complications was noted. Patients with cancer (n=19) were significantly older than patients without cancer (p=0.007) and were more frequently, but not significantly, evaluated as malnourished through SGA (42.1% compared to 11.6%). Preoperative prealbumin and other parameters did not differ significantly between groups. However, there was a significant postoperative decrease in prealbumin only in patients with cancer. Therefore, prealbumin has been found to be valuable in assessing acute malnutrition in cancer patients, especially if variations are monitored in time, which could be useful in planning nutritional treatment

Introduction. In search for explanations of the clinical heterogeneity in patients with haemophilia (PwH) with the same mutation or degree of factor VIII deficiency, the coexistence of single or associated prothrombotic risk mutations has been widely evaluated. Objective. The evaluation of the frequency of prothrombotic risk mutations and polymorphisms in PwH in comparison with the general population. Method. The study was performed on 113 consecutive PwH consisting of PCR technology aiming to detect: factor V Leiden - G 1691A (FVL) and prothrombin (PT) - G 20210 A mutations, methylentetrahydrofolat - reductase (MTHFR) and plasminogen activator inhibitor type 1 (PAI-1) polymorphisms. Results. Within the whole study group, 52.21% patients have been identified with associated prothrombotic risk mutations or polymorphisms, 40.70% with one and 7.08% without any such alterations. The global frequency was characterized by the predominance of PAI-1 polymorphism present in 82.29% and MTHFR in 52.21% of patients. Heterozygous variants of PT G20210A, FV G1691A, MTHFR and PAI-1 were found in 7.96%, 9.73%, 39.82% and 53.98% cases, respectively. According to the disease severity, in 89 patients with severe hemophilia, the following frequencies of polymorphisms were found: for MTHFR 52.80%, for FV G1691A 5.61%, for PT G20210A 8.99% and for PAI-1 polymorphism 79.77%. Conclusions. The frequency of FV, PT and PAI-1 genes alterations in our group of hemophilia patients is higher than in the normal population. Nevertheless, considering their uneven distribution in different ethnic groups and geographical regions, more studies on a larger age- and sex-matched patient population are needed.

In this study, different aspects of anemia in chronic kidney disease have been observed, starting from the fact that the severity of anemia is associated with the degree of kidney dysfunction, the main cause being the erythropoietin deficiency, which is synthesized mostly by the kidneys. 58 persons were included in this study, 19 patients with non-dialysis-dependent chronic kidney disease, 18 patients with chronic kidney disease who received kidney transplantation and 21 apparently healthy persons. We evaluated the serum level of erythropoietin, serum creatinine, proteinuria, the glomerular filtration rate, the erythrocyte parameters and the correlations between them. The prevalence of anemia in patients with chronic kidney disease was of 51.35%. The hemoglobin concentration in patients with kidney transplantation (12.4 ± 2.7 g/dL) and in non-dialysis-dependent patients (11.7 ± 1.4 g/dL) is significantly different compared to the apparently healthy persons (14.6 ± 0.8 g/dL) (p<0.05). In the case of the non-dialysis-dependent patients who were not treated with erythropoiesis- stimulating agents we found positive associations between the glomerular filtration rate and the number of erythocytes (r = 0.71), hemoglobin (r = 0.65) and hematocrit (r = 0.73), as well as negative associations between creatinine and the number of erythrocytes (r = -0.72), hemoglobin (r = -0.86) and hematocrit (r = -0.88). In patients with kidney transplantation and anemia we observed positive correlations between erythropoietin and the number of erythrocytes (r = 0.69), between the glomerular filtration rate and the number of erythrocytes (r = 0.78) and erythropoietin (r = 0.97), as well as negative correlations between proteinuria and the number of erythrocytes (r= -0.89), hemoglobin (r= -0.72), hematocrit (r = -0.72), and erythropoietin (r = -0.67), and between creatinine and the number of erythrocytes (r = -0.75) and erythropoietin (r = -0.86).

Background: Women with polycystic ovary syndrome (PCOS) are at high risk for the development of diabetes mellitus, hypertension and coronary heart disease. Due to the inverse correlation between serum uric acid and insulin sensitivity, the measurement of uric acid may provide a marker of insulin resistance. Objective: To establish the relationship between uric acid and markers of insulin resistance in obese and overweight women with PCOS. Methods: Serum uric acid levels were measured in 38 PCOS obese and overweight patients and 30 controls matched for age and body mass index (BMI). Anthropometric variables, plasma glucose and insulin levels were measured. Insulin resistance was evaluated by homeostasis model assessment (HOMA-IR). Results: No statistically significant differences in uric acid levels between PCOS and non-PCOS women were found. Serum uric acid levels were positively correlated with BMI, waist circumference, insulin and HOMA. Following the use of stepwise linear regression analysis, BMI was the only parameter retained by the regression model, responsible for 42.1% of the variability of serum uric acid levels. Conclusions: In PCOS women obesity seems to be the main determinant of plasma uric acid levels. Insulin and HOMA are also involved to a lesser extent, but their role remains to be clarified by further studies.

A high-throughput liquid chromatography method with detection by tandem mass spectrometry (LC-MS/MS) was developed and validated for the quantification of apixaban in human plasma. The separation was performed on a Gemini-NX column under isocratic conditions using a 33:67 (v/v) mixture of acetonitrile and 1 mM ammonium formate in water at 40 ºC with a flow rate of 0.5 mL/min. The detection of apixaban was performed in multiple reaction monitoring mode (m/z 417.2 from m/z 460.2) with electrospray positive ionization. A single-step protein precipitation with methanol was used for plasma sample preparation. The method was validated with respect to selectivity, linearity (r > 0.994), intra-day and inter-day precision (CV < 14.4 %) and accuracy (bias < 9.5 %) over the range of 9.70 - 970.00 ng/mL plasma. The lower limit of quantification (LLOQ) was 9.70 ng/mL and the recovery was between 97.4 - 104.5 %. The method is fast, efficient, requires the processing of a small volume of plasma (50 μL), a short run-time (1 min) for chromatographic analysis, and a simple and rapid preparation of samples. It is very well suited for clinical therapeutic drug monitoring and pharmacokinetic studies.

A method of measuring in vivo nitric oxide (NO) levels is required to detect pathological conditions in which endogenous production is decreased or to identify agents able to release this biomolecule. Unfortunately, nitric oxide has a very short biological half-life and is very difficult to measure. Assay of the oxidative products’ of NO levels, nitrite (NO₂ -) and nitrate (NO₃ -), measured as total amount, after the reduction of nitrate to nitrite, determined after conversion in an azo dye, is usually the used method, named NOx test. The NOx test is frequently used as a NO biomarker in human studies and also in animal experiments. The aim of this work is to evaluate the suitability of the NOx test for the detection of an instant release of nitric oxide.

Rabbits were used as experimental animals, a validated HPLC-UV/VIS method was used for speciation of nitrite and nitrate. The following substances were administered: blank; “negative blank”: phenyl-N-tert-butylnitrone (PBN); “positive blank” (nitroglycerin); nitrite.

PBN administration significantly increased nitrate and decreased nitrite levels, nitrite administration excessively increased nitrate levels, while nitroglycerin (1 mg/kg) significantly increased both nitrate and nitrite levels.

Results show that NOx test cannot be considered accurate in acute nitric oxide status testing. Nitrite alone should be used as an in vivo released nitric oxide marker.