Volume 22 (2014): Issue 2 (June 2014)

Intellectual disability (ID) is a common disorder, with major consequences for individual, family and society. Due to clinical and genetic heterogeneity of ID, in about 50% of cases an etiologic diagnosis cannot be established. The aim of this study was to evaluate the ability of a combination of MLPA kits to establish the diagnosis in 369 patients with syndromic ID and normal or uncertain routine karyotype results. All patients were assessed for chromosome imbalance using SALSA MLPA P064 or P096 kits, if the phenotype was suggestive of a microdeletion syndrome (subgroup A - 186 patients), or subtelomeric P036 and P070 kits, if the phenotype was not suggestive of a microdeletion syndrome or if the result of the standard karyotype was uncertain (subgroup B - 183 patients). Abnormal results detected by these kits were further characterized using appropriate follow-up MLPA kits (Telomere Follow-up set, P029-A1, P250-B2, ME028-B1). In subgroup A we identified 25 patients with microdeletions (13.4%). Using subtelomere screening and follow-up kits in subgroup B we detected cryptic rearrangements in 7.5% cases and identified the origin of the unknown material noticed in the standard karyotype in 10 out of 11 patients. Summarizing data from the two groups, the combined use of MLPA kits led to the diagnosis in 10.6% (38/358) patients with normal karyotype. Using follow-up MLPA kits allowed us both to confirm abnormalities and to determine their size, which facilitated the interpretation of the clinical significance of these rearrangements. For laboratories that do not have yet access to microarray technology, using several MLPA kits represents an effective strategy for establishing the diagnosis in ID patients.

The aim of this work was to study for the first time in Romania Insertion/Deletion (I/D) polymorphism of the Vascular Endothelial Growth Factor (VEGF) gene in a group of patients with established type 2 diabetes mellitus (DM) and diabetic peripheral neuropathy (DPN) compared with a control group.

This was a case-control study consisting of a group of 84 patients with type 2 DM and DPN, diagnosed by clinical neurological examination and electrophysiological nerve conduction studies and a control group of 90 healthy volunteers. For deoxyribonucleic acid (DNA) isolation, a DNA purification kit from Zymo Research was used. In vitro amplification of DNA sequences was achieved by polymerase chain reaction (PCR). Selective in vitro amplification of a DNA fragment of known sequence is based on the principle of extension of a primer (“primer and PCR amplicon”). DNA fragments were separated by gel electrophoresis. For proper viewing and interpreting of agarose gels Vilber Lourmat system was used. D allele frequency of VEGF was significantly higher in patients with diabetic peripheral neuropathy (53.57%) compared with controls (25%), p=0.0001.

There is a positive association between I/D polymorphism of VEGF gene and the presence of diabetic peripheral polyneuropathy. Our study suggests that D allele of VEGF gene is a risk factor for the occurrence of DPN.

The aim of this study was to evaluate the vitamin B₁₂ status in a non-vegetarian sample of the adult urban population from Romania. The studied sample included 80 non vegetarian subjects aged between 19 and 74.6 years who did not use vitamin B₁₂ supplements or vitamin B₁₂ fortified food. The serum vitamin B₁₂ assay was performed using electrochemiluminescence immunoassay on a Roche Elecsys 2010 analyzer. Among all subjects, 93.75% (95% CI 86.2-97.3%) had serum vitamin B₁₂ concentrations in the reference range of the employed method (191-663 pg/ml). In these subjects, we found no statistically significant correlations between serum vitamin B₁₂ concentration and age or between serum vitamin B₁₂ concentration and hemoglobin level. The serum vitamin B₁₂ levels did not differ significantly (p>0.05-Student’s t-test) between women and men. Subnormal serum levels of vitamin B₁₂ were observed in 5% (95%CI 1.96-12.16%) of the investigated subjects, associated with irritable bowel syndrome and with long-term therapy with proton pomp inhibitors or birth control pills. In the present study, we have identified a large number of subjects with marginal depletion of vitamin B₁₂ in the population aged below 50 years. These results suggest the need of a screening for vitamin B₁₂ status in people from all age groups, the importance of the identification of responsible causes for the high prevalence of vitamin B₁₂ marginal status, as well as the fact that monitoring the vitamin B₁₂ status is especially important in subjects undergoing long-term treatment with certain drugs

Wilson’s disease is an inherited autosomal recessive disorder of copper balance leading to accumulation of copper mainly in liver and brain result from absent or reduced function of copper-transporting P-type ATPase. Copper is an essential trace element but in Wilson’s disease it accumulate to the point of toxicity. D-penicillamine is a classic drug for treatment of Wilson’s disease. Its major effect is to promote the urinary copper excretion. The use of D-penicillamine in the therapy of Wilson’s disease is known to be complicated by the development of various glomerular diseases. In this report we describe the development of nephrotic syndrome after 2 years treatment with D-penicillamine in a 31-year-old male undergoing treatment for Wilson’s disease, with a prompt regression at the discontinuation of the drug. We present this case to draw attention to the rare complication as nephrotic syndrome in patients with Wilson’s disease under D-penicillamine treatment and possible underlying causes. It is strongly necessary the therapy and clinical condition of patients with Wilson’s disease to be monitoring regularly - we recommended monthly.

Pseudothrombocytopenia is an in vitro sampling problem which may mislead the diagnosis towards the more critical condition of thrombocytopenia. The phenomenon occurs when the anticoagulant used while testing the blood sample causes clumping of platelets which mimics low platelet count without any clinical signs. This may determine unnecessary, expensive and invasive investigations and even treatment. In this article we report two cases of pseudothrombocytopenia diagnosed in pediatric patients.

Patients with acute leukemia develop abnormalities of haemostasis, leading not only to bleeding, but also to thrombotic complications. The pathogenesis of these complications is complex and multifactorial. Because platelets and platelet derived microparticles are key players in haemostasis and thrombosis, we presumed their roles in the prediction of bleeding and thrombotic complications. Our study groups included 24 patients with acute leukemia and 16 healthy volunteers. Platelet aggregation evaluation was performed by impedance whole blood aggregometry and the ennumeration of platelet derived microparticles was done by means of flow cytometry. Eight patints developed hemorrhagic complications associated with reduced platelet aggregation response at hospital admission. Major thrombotic events occurred in 5 patients, being preceded by increased platelet aggregation in 3 cases and high level of platelet derived microparticles in 2 cases. Our findings reveal that whole blood platelet aggregometry could be a valuable tool especially in the detection of platelet hyperreactivity and in the prediction of thrombotic events. A high level of platelet derived microparticles could also predict thrombosis. These hypotheses need further evaluation and confirmation on larger number of patients.

Aim: Our objective in conducting this study was to estimate the presence of lymphatic, blood vessel and neural invasion on Hematoxylin & Eosin (H&E) staining and also, the microvessel density detected by immunohistochemistry (MVD), in gastric adenocarcinoma, as well as their relationship with the clinical, pathological and biological characteristics of the tumors. Materials and Methods: To assess the vascular and neural invasion in our study, we included 367 patients diagnosed with gastric cancer. For the immunohistochemical study of MVD, from all cases with gastric carcinoma, we selected 28 patients, 12 patients with gastric biopsy and 16 patients with total gastric resection, which established the TNM stage. All the gastric biopsies and surgical samples were prepared using the paraffin-embedding method and H&E staining and using anti-CD31 and anti-CD34 antibodies for the assessment of intratumoral MVD. Results: The positive blood vessel invasion was associated in a significant way with advanced stages (p <0.01) and high grade carcinomas (p<0.01), while lymphatic invasion was very significant associated only with advanced stage tumors (p < 0.001). Regarding peri- and intraneural invasion, there was a significant matching with the female gender (p < 0.05), advanced stages of disease (p < 0.001), the diffuse type of gastric carcinoma (p <0.05), and with poorly differentiated tumors (p<0.05). There was a close relationship between CD34 MVD and the diffuse type of gastric carcinomas, according to Lauren’s classification (p<0.05), and poorly differentiated tumors (p<0.05). The CD34 MVD values mean was significant correlated with TNM stage, especially III and IV stages (p<0.01). Conclusions: Blood vessel invasion, neural invasion and CD34 MVD play a significant role in tumors biological behavior and they can be used as important prognostic parameters which describes the aggressiveness of gastric carcinomas.

Erythema migrans (EM) is the most common and recognized clinical manifestation of early Lyme Borreliosis (LB) in Europe. Purpose: to evaluate clinically and serologically all the patients with EM and to correlate the serology with clinical approach. Material and method: A prospective analyses of all patients that came with EM in our University Hospital of Infectious Diseases from Cluj-Napoca Romania between 1st of April to 31st of August 2011. We registered data regarding: age, gender, interval between tick bite and EM occurrence, interval between EM appearance and hospital presentation, serology (IgM and IgG for Borrelia burgdorferi sl) at presentation and one year after, antibiotic treatment, duration of antibiotic treatment and clinical outcome. Results: 44 patients with EM, 2 had Multiple EM, 23 male (52 %), average age 41.3 years (min 3 years, max 84 years, median 43.5 years). Serology at presentation was positive in 15 patients for IgM and in 9 patients for IgG with ELISA and for 13 for IgM and 16 for IgG with WB. At the one year follow up positive results for IgM were found in 14 patients with ELISA and in 11 patients with WB. IgG was positive in 7 patients with ELISA and in 5 with WB. The average time interval between the tick bite and EM was not significant different for patients with negative or positive IgM or IgG. The interval between EM occurrence and treatment initiation had a strong significance for IgG. After one year no statistical significance was found for positive IgM for any of the analyzed factors while for IgG positivity we found a significant importance for age and duration of incubation. No influence of antibiotic or duration of treatment was found on serology of our patients. Conclusions: Our data brings additional evidence that the serological profile is unpredictable.

Lyme borreliosis (LB) is the most frequent infection transmitted by ticks from the Ixodes genus, both in the United States of America (USA), and in Europe. The objectives of this study were the monitoring of the incidence and clinical manifestations of the disease in Mures County, Romania. Material and method. The study was conducted over a period of two years, January 1st 2010 -December 31st 2011, on a number of 120 consecutive patients. The definite or probable diagnosis of LB was established on the Centers for Disease Control and Prevention (CDC, Atlanta, USA) criteria, and on the European Union Concerted Action on Lyme borreliosis (EUCALB). ELISA and Western-Blot techniques were used for identification of IgM and IgG antiBb from the serum and CSF. Results. There were 44 cases in 2010 and 76 cases in 2011. According to the case definition, 106 cases were confirmed as LB and 14 were probable. LB was more frequently diagnosed in children, young adults and adults, compared to the over 60 years old age group. The incidence of disease was higher in women patients, 68 (56.66%) compared to men - 52 patients (43.33%), with a higher proportion in people coming from an urban setting, 78 patients (65%), compared to people coming from rural settings 42 patients (35%). Clinical manifestations were acute, like erythema migrans (EM) in 64.16% of the patients, neurological in 22.5% of the patients, joint related in 1.66% of the patients and cardiac in 0.83% of the cases. Conclusions. In Mures County we have observed an increased incidence of this disease in 2011 vs. 2010. The predominant clinical manifestations were the acute dermatological and neurological ones.

The clinical relevance of Staphylococcus aureus strains with heterointermediate susceptibility to vancomycin (hVISA) is still controversial, however they could be responsible for treatment failures in patients treated with vancomycin. The lack of standardization and the complexity of testing methods are the main challenge in indentifying such strains. The aim of our study was to evaluate the frequency of hVISA strains in Targu-Mures Clinical Emergency Hospital. One hundred twenty-two, non-duplicate, methicillin-resistant S. aureus (MRSA) isolates susceptible to vancomycin using standard E-test (MIC≤2 mg/L) were screened for heteroresistance with Glycopeptide Resistance Detection test (E-test GRD). Population analysis profile-area under the curve (PAP/AUC) method was used for confirmation. Twenty-four strains (19.5%) were found positive with the screening method. Two of them (1.63%) were confirmed having hVISA phenotype and no strains with intermediate vancomycin susceptibility (VISA) were detected. In conclusion, the rate of MRSA strains with reduced vancomycin susceptibility was low. However, their monitoring may be useful, taking into consideration the wide usage of glycopeptides in the treatment of serious MRSA infections.

Listeria monocytogenes has a ubiquitous distribution in nature and could contaminate food of animal origin, causing severe infections in humans. Till present, little is known about the antibiotic resistance profiles of these strains in Romania. The aim of this study was to determine the antibiotic susceptibility patterns of 37 L. monocytogenes strains isolated from animal derived foods and from clinical samples. Food samples were collected from meat and dairy products, between 2009 and 2013. Clinical samples were collected from patients with septicemia, meningitis/meningo-encephalitis, abortion cases and newborns, hospitalized during April 2010 - April 2013 in three medical institutions from Bucharest: Babes Hospital, Elias Hospital, National Institute of Infectious Diseases (INBI) Matei Bals. All tested isolates exhibited resistance to cephalosporins and nalidixic acid; one strain isolated from boiled shell snails was resistant to trimethoprim/sulfamethoxazole. The resistance to the first choice antibiotic ampicillin in L. monocytogenes strains isolated from severe infections is underlining the need of in vitro antibiotic susceptibility testing of each clinical isolate to establish the efficacy of different antibiotics, as well as of extended epidemiological studies to highlight the resistance profiles of L. monocytogenes strains circulating in our country.

Background. Patients infected with human immunodeficiency virus (HIV), especially at advanced stages of HIV infection and low CD4+ T-lymphocytes levels, were reported to be less frequently co-infected with Helicobacter pylori than general population, according to literature data. Purpose: to study Helicobacter pylori infection in HIV-positive hosts with digestive complaints. Methods: retrospective, analytical, case-control study (November 2011 - December 2013), upon two groups of patients with gastro-intestinal symptoms tested for Helicobacter pylori stool antigen at the Infectious Diseases Laboratory, Clinical County Hospital Mureş. Group A included 44 HIV-positive patients, group B: 58 HIV-negative subjects. We first compared groups A and B regarding the frequency of Helicobacter pylori infection. Group A was afterwards divided into two sub-groups, according to the status of Helicobacter pylori infection: group A1: 5 Helicobacter pylori-positive subjects, group A2: 39 Helicobacter pylori-negative ones. We collected information regarding CD4+ T-lymphocytes level, HIV-RNA plasma viral load, previous antibiotic and antiretroviral therapy, co-morbidities, comparing A1 and A2 subgroups. Data were processed using GraphPad Prism 5 programme. Results. The frequency of Helicobacter pylori infection was 11.36% among HIV-positive patients and 13.79% in HIV-negative ones, without statistically significant difference. We found no statistically significant differences between subgroups A1 and A2 regarding CD4+ T-lymphocytes level, HIV-RNA plasma viral load, antibiotic / antiretroviral therapy. Conclusions. Though Helicobacter pylori infection may represent one of the causes of gastro-intestinal symptoms in HIV-positive patients, its frequency did not differ to that registered in the general population, in our study.

Prostate cancer is, after lung cancer, the most common malignant disease diagnosed in the male population. The introduction into the practice used during the 80’s and 90’s of the determination of serum Prostate-Specific Antigen (PSA) levels, as a component of screening for prostate cancer, was a turning point in the medical practice. Due to this enzyme produced exclusively by the prostate gland, the prostate cancer detection rate (in curative, intracapsular stages) improved significantly. Serum PSA is a better predictive factor for prostate cancer (PC) than digital rectal examination or transrectal prostatic ultrasound.