Elena Costa

Extended-spectrum beta-lactamase-positive Escherichia coli causing complicated upper urinary tract infection: Urologist should act in time

Objective: Recently, many articles reported increased incidence of urinary tract infection (UTI) due to Extended-Spectrum Beta-Lactamase (ESBL)-producing E. coli. No data are available to date regarding patients presenting with complicated upper ESBL-positive E. coli UTI and sepsis. We report the clinical presentation, management, and outcomes in seven cases. Materials and Methods: This prospective study was carried out between January 2008 and September 2011. Follow-ups varied in patients according to their disease presentation and clinical outcomes. All strains were cultured and identified by the Clinical Microbiology Laboratory and were recovered from blood and urine cultures. In-vitro presence of ESBL was confirmed with Clinical and Laboratory Standard Institute double disc method. Results: In the study period, 49 patients needed hospitalization for upper UTI. Overall, in 25 patients (51%), cultures were negative. In the remaining, seven patients (14.3%) presented positive blood and urine-culture for ESBL + E. coli. Of these, four were female and three were male. Their median age was 73 years (range 66-84). The median hospital stay of these patients was 23 days (range 13 to 45 days). Conclusions: The current situation of multiple bacterial antibiotic resistance has become a worrisome issue in UTI. Multi-drug-resistant E. coli can be readily encountered in hospital settings during daily clinical practice, and urologist should act timely. The management of such infections is extremely important for the future, with particular reference to prevention of new antibiotic resistance patterns.

Identification and characterization of DM1 patients by a new diagnostic certified assay: neuromuscular and cardiac assessments.

The expansion of the specific trinucleotide sequence, [CTG], is the molecular pathological mechanism responsible for the clinical manifestations of DM1. Many studies have described different molecular genetic techniques to detect DM1, but as yet there is no data on the analytical performances of techniques used so far in this disease. We therefore developed and validated a molecular method, “Myotonic Dystrophy SB kit,” to better characterize our DM1 population. 113 patients were examined: 20 DM1-positive, 11 DM1/DM2-negative, and13 DM1-negative/DM2-positive, who had a previous molecular diagnosis, while 69 were new cases. This assay correctly identified 113/113 patients, and all were confirmed by different homemade assays. Comparative analysis revealed that the sensitivity and the specificity of the new kit were very high (>99%). Same results were obtained using several extraction procedures and different concentrations of DNA. The distribution of pathologic alleles showed a prevalence of the “classical” form, while of the 96 nonexpanded alleles 19 different allelic types were observed. Cardiac and neuromuscular parameters were used to clinically characterize our patients and support the new genetic analysis. Our findings suggest that this assay appears to be a very robust and reliable molecular test, showing high reproducibility and giving an unambiguous interpretation of results.

Development and Validation of a New Molecular Diagnostic Assay for Detection of Myotonic Dystrophy Type 2.

BACKGROUND Myotonic dystrophy (DM) is the most common adult form of muscular dystrophy, characterized by autosomal dominant progressive myopathy, myotonia, and multiorgan involvement. Myotonic dystrophy type 2 (DM2) is caused by a [CCTG] expansion in the ZNF9/CNBP gene. The aim of this work was the validation of the new molecular diagnostic test Myotonic Dystrophy type 2 kit-FL. RESULTS A cohort of 126 individuals was analyzed. The results show that 126/126 patients were correctly identified using the new molecular assay. In particular, 74 were DM2 positive, 39 were DM2/DM1 negative and 13 DM2 negative/DM1 positive. Approximately 9.5% (7/74) of the DM2-positive samples had a single sizeable expansion and 85% (63/74) showed multiple bands or smears. Comparative fluorescence in situ hybridization (FISH) analyses, on muscle biopsies, revealed that the sensitivity and specificity were very high (>99%). Equivalent analytical performances were obtained using different DNA extraction methods. Among affected individuals 87.5% (28/32) had electrical myotonia, 69% (22/32) proximal weakness, 41% (13/32) cataracts, and about 37.5% (12/32) cardiac conduction defects. FISH analysis and clinical data were used to support the genetic analysis.

Emergence of Carbapenem-Resistant Klebsiella pneumoniae: Progressive Spread and Four-Year Period of Observation in a Cardiac Surgery Division

Frequent use of carbapenems has contributed to the increase to K. pneumoniae strains resistant to this class of antibiotics (CRKP), causing a problem in the clinical treatment of patients. This investigation reports the epidemiology, genetic diversity, and clinical implication of the resistance to drugs mediated by CRKP in our hospital. A total of 280 K. pneumoniae strains were collected; in particular 98/280 (35%) were CRKP. Sequencing analysis of CRKP isolated strains showed that 9/98 of MBL-producing strains carried the bla VIM-1 gene and 89/98 of the isolates were positive for bla KPC-2. Antimicrobial susceptibility tests revealed a complete resistance to third-generation cephalosporins and a moderate resistance to tigecycline, gentamicin, and fluoroquinolones with percentages of resistance of 61%, 64%, and 98%, respectively. A resistance of 31% was shown towards trimethoprim-sulfamethoxazole. Colistin was the most active agent against CRKP with 99% of susceptibility. Clonality was evaluated by PFGE and MLST: MLST showed the same clonal type, ST258, while PFGE analysis indicated the presence of a major clone, namely, pulsotype A. This finding indicates that the prevalent resistant isolates were genetically related, suggesting that the spread of these genes could be due to clonal dissemination as well as to genetic exchange between different clones.

Adenine phosphoribosyltransferase (APRT) deficiency: identification of a novel nonsense mutation

BackgroundAdenine phosphoribosyltransferase deficiency (APRTD) is an under estimated genetic form of kidney stones and/or kidney failure, characterized by intratubular precipitation of 2,8-dihydroxyadenine crystals (2,8-DHA). Currently, five pathologic allelic variants have been identified as responsible of the complete inactivation of APRT protein.Case presentationIn this study, we report a novel nonsense mutation of the APRT gene from a 47- year old Italian patient. The mutation, localized in the exon 5, leads to the replacement of a cytosine with a thymine (g.2098C > T), introducing a stop codon at amino acid position 147 (p.Gln147X).This early termination was deleterious for the enzyme structural and functional integrity, as demonstrated by the structure analysis and the activity assay of the mutant APRT protein.ConclusionThese data revealed that the p.Gln147X mutation in APRT gene might be a new cause of APRT disease.

Tibialis anterior muscle needle biopsy and sensitive biomolecular methods: a useful tool in myotonic dystrophy type 1.

Myotonic dystrophy type 1 (DM1) is a neuromuscular disorder caused by a CTG repeat expansion in 3’UTR of DMPK gene. This mutation causes accumulation of toxic RNA in nuclear foci leading to splicing misregulation of specific genes. In view of future clinical trials with antisense oligonucleotides in DM1 patients, it is important to set up sensitive and minimally-invasive tools to monitor the efficacy of treatments on skeletal muscle. A tibialis anterior (TA) muscle sample of about 60 mg was obtained from 5 DM1 patients and 5 healthy subjects through a needle biopsy. A fragment of about 40 mg was used for histological examination and a fragment of about 20 mg was used for biomolecular analysis. The TA fragments obtained with the minimally-invasive needle biopsy technique is enough to perform all the histopathological and biomolecular evaluations useful to monitor a clinical trial on DM1 patients.

Asymmetric Dimethylarginine: Relationship with Circulating Biomarkers of Inflammation and Cardiovascular Disease Risk in Uncomplicated Obese Women

In recent years, the link between obesity, inflammation and atherosclerosis has attracted increasing interest. Recently, besides the classical inflammatory markers, the competitive nitric oxide synthase antagonist asymmetric dimethylarginine (ADMA) has been shown to be involved in the pathogenesis of atherosclerosis and cardiovascular diseases. Since obese people present a condition of chronic low-grade inflammation and endothelial dysfunction, in the present study we quantified ADMA levels in uncomplicated obese women (with no clinical, cardiac or metabolic complications) and normal-weight control subjects. We investigated the relationship of ADMA with some anthropometric measurements, abdominal visceral and subcutaneous adipose tissue accumulation, and biochemical and proinflammatory factors of the subjects [interleukin-6 (IL-6), soluble IL-6 receptor (sIL-6R), IL6-R/IL-6 ratio, tumor necrosis factor alpha (TNFα), homocysteine (Hey) and plasminogen activator inhibitor-1 (PAI-1)]. ADMA and all the other pro-inflammatory parameters resulted higher in obese patients than in healthy subjects. ADMA significantly correlated with Hey, PAI-1, TNFα and with sIL-6R/IL-6 ratio but not with other anthropometric and biochemical parameters. In a stepwise regression analysis ADMA correlated most closely with Hey and TNFα. In conclusion, in our obese uncomplicated patients TNFα and Hey emerged as strong predictors of ADMA which might be a potential mediator of the effects of different risk factors affecting the cardiovascular system.

Anti-TNF-Mediated Modulation of Prohepcidin Improves Iron Availability in Inflammatory Bowel Disease, in an IL-6-Mediated Fashion

Background. Anaemia is common in inflammatory bowel disease (IBD), frequently resulting from a combination of iron deficiency and of anaemia of chronic disease (ACD). ACD is characterized by macrophage iron retention induced by proinflammatory cytokines. Hepcidin is the master inducer of iron accumulation during ACD, and its production is mainly regulated by IL-6 and the novel erythroid hormone erythroferrone (ERFE). This study evaluates whether anti-TNF monoclonal antibodies therapy modurates hepcidin production and the levels of its main regulators, leading to a restoration of iron homeostasis. Methods. Sera were collected from 21 IBD patients, before each anti-TNF administration, for the first 6 weeks of therapy. Prohepcidin, erythropoietin, erythroferrone, C reactive protein, interleukin-6, iron markers, and haemoglobin levels were measured and clinical activity indexes were evaluated. Results. Serum prohepcidin, IL-6, CRP, and ferritin were significantly reduced after 6-week treatment; an increase in serum iron and total transferrin was observed. No changes in the EPO-ERFE axis were found. Remarkably, haemoglobin was significantly increased. Conclusions. Anti-TNF therapy improves iron metabolism and, subsequently, anaemia in IBD. This effect appears to be related to the modulation of the cytokine network and specifically IL-6 leading to a relevant decrease of hepcidin, a master regulator of ACD.

Blood Volume Determination Through New Generation 130/0,4 Hydroxyethyl-Starch: A Propaedeutic, In-Vitro Study

Background: The importance of knowing the blood volume, in critically ill patients, collides with the difficulty to have its direct measure through a safe and economic method. Hydroxyethyl starch (HES) was introduced by Tschaikowsky as a useful marker for the dilution method, calculating the HES concentration (HESC) in a solution by inducing an in-vitro hydrolysis of starch molecules into glucose monomers and dosing the consequent increase of the solution glucose level (Δ GLUCOSE). Objective: This study develops a simple and cheap laboratory technique which uses a new generation 6% 130/0,4 Hydroxyethyl starch as a possible “dilution marker” for the measurement of patient’s blood volume maintaining Tschaikowsky’s study protocol. The aim is to refocus attention on an interesting method that could lead the way to a number of possibilities in critical area. Method: We designed a two-phase in-vitro experiment. Firstly, we found out the suitable treatment duration to ensure a complete hydrolysis of starch molecules. Secondly, we aimed to the achievement of a univocal constant of proportionality (K) between Δ GLUCOSE and HES concentration. HESC will be expressed as HESV/PV (μl/mL) where HESV represents the HES volume and PV the plasma volume. Plasma volumes were calculated as BV*(1-Ht). Results: K was planned by means of a linear regression analysis between HESV/PV and Δ GLUCOSE on 133 validated samples collected from 30 healthy volunteers. The obtained hematocrit values ranged between 39.9 and 48 (mean ± CI 95%=42,62 ± 2,93). This corresponded to HESC ranging from 0,033 to 0,038 HES (mL)/PV (mL) (mean ± CI 95%=0,035 ± 0,002). While hydrolysis times increase, glucose values tended to augment until they reached stable plateau. During the second phase we handled a total of 720 specimens. Hematocrit of collected samples ranged from 33,9 to 49 (mean ± CI 95%=41,3 ± 1,21). HESC ranged between 0,015 and 0,089 mL HES/mL PV (mean ± CI 95%=0,037 ± 0,003). The regression analysis showed that HESC equals 0,592 times Δ GLUCOSE (R2=0,947). Conclusion: This study might be the first step in reintroducing starches into the clinical management of critical patients, not just as therapeutic agents for volume resuscitation but even as useful markers in the diagnosis of hemodynamic derangements, improving fluid and blood therapy strategies.

Biomolecular diagnosis of myotonic dystrophy type 2: a challenging approach

Myotonic dystrophy type 1 (DM1) and type 2 (DM2) are the most common adult form of muscular dystrophy, characterized by autosomal dominant progressive myopathy, myotonia, and multiorgan involvement. The onset and symptoms of the myotonic dystrophies are diverse, complicating their diagnoses and limiting a comprehensive approach to their clinical care. Diagnostic delay in DM2 is due not only to the heterogeneous phenotype and the aspecific onset but also to the unfamiliarity with the disorder by most clinicians. Moreover, the DM2 diagnostic odyssey is complicated by the difficulties to develop an accurate, robust, and cost-effective method for a routine molecular assay. The aim of this review is to underline by challenging approach the diagnostic limits and pitfalls that could results in failure to recognize the presence of DM2 disease. Understanding and preventing delays in DM2 diagnosis may facilitate family planning, improve symptom management in the short term, and facilitate more specific treatment in the long term.