Phylogenetic characterization of Central/Southern European lineage 2 West Nile virus: analysis of human outbreaks in Italy and Greece, 2013-2014.

In recent years, West Nile virus (WNV) lineage 2 has been spreading and causing disease outbreaks in humans and animals in Europe. In order to characterize viral diversity, we performed full-length genome sequencing of WNV lineage 2 from human samples collected during outbreaks in Italy and Greece in 2013 and 2014. Phylogenetic analysis showed that these WNV lineage 2 genomes belonged to a monophyletic clade derived from a single introduction into Europe of the prototype Hungarian strain. Correlation of phylogenetic data with geospatial information showed geographical clustering of WNV genome sequences both in Italy and in Greece, indicating that the virus had evolved and diverged during its dispersal in Europe, leading to the emergence of novel genotypes, as it adapted to local ecological niches. These genotypes carried divergent conserved amino acid substitutions, which might have been relevant for viral adaptation, as suggested by selection pressure analysis and in silico and experimental modelling of sequence changes. In conclusion, the results of this study provide further information on WNV lineage 2 transmission dynamics in Europe, and emphasize the need for WNV surveillance activities to monitor viral evolution and diversity.

Whole genome sequencing and phylogenetic analysis of West Nile virus lineage 1 and lineage 2 from human cases of infection, Italy, August 2013.

A human outbreak of West Nile virus (WNV) infection caused by WNV lineage 2 is ongoing in northern Italy. Analysis of six WNV genome sequences obtained from clinical specimens demonstrated similarities with strains circulating in central Europe and Greece and the presence of unique amino acid changes that identify a new viral strain. In addition, WNV lineage 1 Livenza, responsible for a large outbreak in north-eastern Italy in 2012, was fully sequenced from a blood donor during this 2013 outbreak.

Use of grafts from anti-HBc-positive donors in liver transplantation: a 5-year, single-center experience.

INTRODUCTION: Liver transplantation (OLT) is the treatment of choice for advanced hepatic disease. The growing gap between waiting list patients and the number of donations has led to acceptance of less than optimal donors. The aim of this study was to evaluate the 5-year experience with anti hepatitis B core antigen (HBc)-positive liver donors. PATIENTS AND METHODS: All recipients of anti-HBc-positive grafts from January 2005 to December 2010 were evaluated annually after OLT for liver disease etiology, Model for End-Stage Liver Disease (MELD) score, and the presence of hepatocellular carcinoma (HCC) liver biopsy histology and serology for hepatitis B virus (HBsAg, anti-HBs, HBV-DNA), hepatitis C virus, and hepatitis D virus as well as antiviral prophylaxis to prevent de novo HBV. RESULTS: Among the 249 OLT performed from January 2005 to December 2010, (9.3%) cases used grafts from anti-HBc-positive donors. Etiologics of liver disease among the recipients were HBV (n = 13; 32.5%), HCV (n = 13; 32.5%) or other causes (n = 14; 35%). In 20 of the 40 patients (50%), HCC was found in the explanted organ. Of 40 recipients of anti-HBc-positive grafts 11 died, and 7 (17.5%) required retransplantation. Various regimens were employed as post-transplantation antiviral prophylaxis: (l) Immune globulin (25.8%); (2) Oral antiviral drugs (9.7%); and (3) combined prophylaxis (51.6%) or no treatment (12.9%). No difference was observed in patient or graft survival in relation to the etiology of liver disease, the MELD score, or the presence of HCC at the time of OLT, except graft survival was significantly reduced among recipient who underwent transplantation for non-HBV or non-HCV liver diseases compared with those engrafted due to viral hepatitis (P = .0062). No difference was observed in histologic features (grading and staging) compared with the antiviral prophylactic therapy; the 2 patients (5%) who developed de novo HBV had not received prophylaxis after OLT. CONCLUSIONS: Matching anti-HBc-positive grafts to recipients without HBV infection before OLT, may be especially safe.

Clinical and virological findings in the ongoing outbreak of West Nile virus Livenza strain in northern Italy, July to September 2012.

In July-September 2012, one month earlier than in previous years, 13 confirmed human cases of West Nile virus infection were diagnosed in northern Italy, including five with neuroinvasive disease, three with West Nile fever, and five West Nile virus (WNV)-positive blood donors. In nine cases, the presence of the WNV lineage 1a Livenza strain, characterised in 2011, was ascertained. Symptomatic patients had prolonged viruria with high viral load.

Novel West Nile virus lineage 1a full genome sequences from human cases of infection in north-eastern Italy, 2011.

During 2008-2009, several human cases of WNV disease caused by an endemic lineage 1a strain were reported in areas surrounding the Po river in north-eastern Italy. Since 2010, cases have been recorded in nearby northern areas, where, in 2011, both lineage 1a and 2 were detected. We describe here two new WNV complete genome sequences from human cases of WNV infection occurring in 2011 in the Veneto Region. Phylogenetic analysis showed that both genome sequences belonged to lineage 1a and were related to WNV strains of the Western Mediterranean subtype. The novel WNV genomes had high nucleotide and amino acid sequence divergence from each other and from the WNV strain circulating in Italy in 2008-2009. The presence of different WNV strains in a relatively small geographical area is a novel finding with unpredictable impact on human disease that requires further investigation.

New endemic West Nile virus lineage 1a in northern Italy, July 2012.

We report here the first blood donation positive for West Nile virus (WNV) by nucleic acid amplification testing collected in north-eastern Italy in July 2012.Partial sequencing of the WNV RNA demonstrated identity with a WNV lineage 1a genome identified in the same area in 2011 and divergence from the strain responsible for the outbreak in northern Italy in 2008­09. These data indicate that WNV activity in northern Italy is occurring earlier than expected and that different WNV strains are circulating.

Human cases of West Nile Virus infection in north-eastern Italy, 15 June to 15 November 2010.

In 2010, for the third consecutive year, human cases of West Nile virus (WNV) infection, including three confirmed cases of neuroinvasive disease and three confirmed cases of West Nile fever, were identified in north-eastern Italy. While in 2008 and 2009 all human cases of WNV disease were recorded in the south of the Veneto region, cases of WNV disease in 2010 additionally occurred in two relatively small northern areas of Veneto, located outside those with WNV circulation in the previous years. WNV IgG antibody prevalence in blood donors resident in Veneto was estimated as ranging from 3.2 per 1,000 in areas not affected by cases of WNV disease to 33.3 per 1,000 in a highly affected area of the Rovigo province. No further autochthonous human cases of WNV disease were notified in Italy in 2010. The recurrence of human cases of WNV infection for the third consecutive year strongly suggests WNV has become endemic in north-eastern Italy.

Novel method for simultaneous aqueous in situ derivatization of THC and THC-COOH in human urine samples: validation and application to real samples.

The present work describes the validation of a novel aqueous in situ derivatization procedure with trimethyloxonium tetrafluoroborate (TMO) as methylating agent for the simultaneous, quantitative analysis of Δ(9)-tetrahydrocannabinol (THC) and 11-nor-Δ(9)-tetrahydrocannabinol carboxylic acid (THC-COOH) in human urine. The derivatizing agent is directly added to the urine sample and the methyl-derivatives are then recovered by liquid-liquid extraction procedure. Gas chromatography-mass spectrometry was used to detect the derivatives in selected ion monitoring mode. The limits of detection were 0.7 ng/mL for THC and 0.5 ng/mL for THC-COOH, whereas the limits of quantification were 1.9 and 0.9 ng/mL, respectively. The method has been applied to 60 real samples both positive and negative to immunochemical screening test resulting to be very useful and reliable in routine analysis of THC-COOH in human urine for toxicological and forensic purposes.

Prevalence of IgM and IgG antibodies to West Nile virus among blood donors in an affected area of north-eastern Italy, summer 2009.

Following reports of West Nile neuroinvasive disease in the north-eastern area of Italy in 2009, all blood donations dating from the period between 1 August and 31 October 2009 in the Rovigo province of the Veneto region were routinely checked to exclude those with a positive nucleic acid test for West Nile virus (WNV). Only one of 5,726 blood donations was positive (17.5 per 100,000 donations; 95% confidence interval (CI): 0.4­97.3). In addition, a selection of 2,507 blood donations collected during the period from 20 July to 15 November 2009 were screened by ELISA for IgG and IgM antibodies against WNV. A positive result was received for 94 of them. The positive sera were further evaluated using immunofluorescence and plaque reduction neutralisation test (PRNT), in which only 17 sera were confirmed positive. This corresponds to a prevalence of 6.8 per 1,000 sera (95% CI: 4.0­10.9). In a case-control study that matched each of the 17 PRNT-positive sera with four negative sera with the same date of donation and same donation centre, we did not find a significant association with age and sex of the donor; donors who worked mainly outdoors were significantly more at risk to have a positive PRNT for WNV.

Genome sequence analysis of the first human West Nile virus isolated in Italy in 2009.

In 2009, six new human cases of West Nile neuroinvasive disease (WNND) were identified in Veneto region, following the six cases already reported in 2008. A human West Nile virus (WNV) isolate was obtained for the first time from an asymptomatic blood donor. Whole genome sequence of the human WNV isolate showed close phylogenetic relatedness to the Italy-1998-WNV strain and to other WNV strains recently isolated in Europe, with the new acquisition of the NS3-Thr249Pro mutation, a trait associated with avian virulence, increased virus transmission, and the occurrence of outbreaks in humans.

Detection of polyomaviruses and herpesviruses in human adrenal tumors.

The presence of polyomaviruses and herpesviruses in adrenal tumors and their role in adrenal tumorigenesis has never been investigated, even though the adrenal gland seems to be a preferential site of infection by these viruses and adrenal steroid hormones have been shown to activate their replication. We examined in a large series of normal adrenal gland tissues (n=20) and adrenal tumors (n=107) the presence of herpesviruses and polyomaviruses sequences and gene expression, which were detected in a high proportion of both normal and neoplastic adrenal samples (overall, viruses were found in 15% normal adrenals, 27.8% benign adrenal tumors and 35.3% malignant tumors). The polyomaviruses SV40 and BK virus were more frequently found in malignant adrenal tumors, whereas herpesviruses, especially Epstein-Barr virus and human cytomegalovirus, were more frequently detected in functioning benign adrenocortical tumors, often as coinfection. Moreover, tumors from patients with severe hypercortisolism frequently showed herpesvirus coinfections at high viral genome copy number. Our study suggests that the adrenal gland could be a reservoir of infection for these viruses and that hormone overproduction by the adrenal gland could represent a trigger for virus reactivation. On the other hand, these viruses could also contribute to adrenal cell proliferation and tumorigenesis.

The role of 21-hydroxylase in the pathogenesis of adrenal masses: review of the literature and focus on our own experience.

An exaggerated response of 17- hydroxyprogesterone (17-OHP) to exogenous ACTH stimulation has been found in 30 to 70% of patients with incidentally discovered adrenal tumors, supporting the concept that congenital 21- hydroxylase deficiency may be a predisposing factor for adrenocortical tumorigenesis. Decreased expression of 21-hydroxylase gene has been observed in sporadic non-functioning adrenocortical adenomas and adrenocortical carcinomas, in agreement with the reduced steroidogenic activity found in these types of tumors. Screening studies for the presence of mutations in CYP21A2 gene, encoding 21-hydroxylase, in patients with sporadic adrenocortical tumors yielded discordant results. Overall, a higher frequency of germline 21-hydroxylase mutation carriers has been found among patients with adrenal tumors, including incidentalomas, than in the general population. However, the presence of mutations did not correlate with endocrine test results and tumor mass features, suggesting that 21-hydroxylase deficiency does not represent a relevant mechanism in adrenal tumorigenesis. Mechanisms leading to reduced 21-hydroxylase expression and activity are still unknown.

Compound A, formaldehyde and methanol concentrations during low-flow sevoflurane anaesthesia: comparison of three carbon dioxide absorbers.

AIM: To determine compound A, formaldehyde and methanol concentrations in low-flow anaesthesia using different carbon dioxide absorbers. METHODS: Fifteen patients scheduled for general or urological surgery were exposed to low-flow (500 ml/min) anaesthesia with sevoflurane. The patients were randomly allocated to three groups: soda lime, DrägerSorb Free or Amsorb Plus. The concentrations of compound A, formaldehyde and methanol were sampled and analysed from the limbs of the anaesthesia circuit at T30 (30 min after the start of low-flow sevoflurane anaesthesia), T90 (90 min) and T150 (150 min). The temperatures of the absorbers were measured at the same time. RESULTS: Statistically significant differences (P < 0.05) were found in the production of compound A from soda lime (with the highest values), DrägerSorb Free and Amsorb Plus at each measurement time. Only traces of methanol (ranging from < 0.131 to 3.799 mg/m(3)) were measured, higher with Amsorb Plus (statistically significant differences were found only at T90). The formaldehyde values (ranging from < 0.1227 to 17.79 mcg/m(3) p.p.b.) were higher with soda lime, and the difference was statistically significant at T150 and, in the inspiratory limb only, at T90. The temperatures of the absorbers were higher for soda lime and lower for Amsorb Plus; the difference was statistically significant at T0 in the upper canister and at T30 in both canisters. CONCLUSION: The concentrations of harmful products in the circuit were negligible and were lower using the new-generation absorbers. Using Amsorb Plus, the temperatures in the canisters were lower than with the other two absorbers.

Evaluation of occupational exposure to high-level disinfectants in endoscopic services in an Italian hospital.

The aim of this study is the evaluation of exposure to airborne, high-level disinfectants to efficiently organize prevention of the risks due to the manipulation of these substances in the endoscopic units in medical facilities. In field and personal samples in the breathing zone of the workers were taken in 27 endoscopic units in a hospital during the replacement of high-level disinfectants in basins, tubes and lavaendoscopes. After a campaign of environmental monitoring of glutaraldehyde in nineteen endoscopic units (176 employees) of the hospital and considering the entity of glutaraldehyde problem, two substitutes, peracetic acid and electrolyzed acid water, have recently been introduced. The level of glutaraldehyde was higher than the TLV-Ceiling in 13 out of 19 working units, while the value of peracetic acid resulted higher than TLV-TWA in 1 out of 15. EAW has recently been used in two units and the chlorine environmental concentrations resulted lower than the sensibility limit of the method (<75 microg/m3). The results obtained during this study of measurements allowed to identify those operations which have the highest degree of risk for employees; this serves for taking suitable measures for prevention and individual protection as well as for evaluating current practices and decisional processes in the hospital.

Shift from Conn's syndrome to Cushing's syndrome in a recurrent adrenocortical carcinoma.

OBJECTIVE: Adrenocortical tumors may originate from the zona glomerulosa, zona fasciculata, or zona reticularis and be associated with syndromes due to overproduction of mineralocorticoids, glucocorticoids, or androgens respectively. We report an unusual case of recurrent adrenocortical carcinoma (ACC), which seems to contradict the paradigm of functional adrenal zonation. CASE REPORT: A male patient presented with severe primary aldosteronism due to an ACC, which relapsed after adrenalectomy and adjuvant mitotane therapy. After removal of the tumor recurrence and eight cycles of chemotherapy with etoposide, doxorubicin and cisplatin, the patient presented again with ACC masses, but in association with overt Cushing's syndrome and normal aldosterone levels. METHODS AND RESULTS: Extensive pathologic examination showed that this shift in steroid hormone production was paralleled by an attenuation of tumor cell atypia and polymorphism, whereas gene expression profile analysis demonstrated a change in expression of adrenal steroidogenic enzymes. Moreover, cancer progression was associated with overexpression of the inhibin-alpha subunit, which could have contributed to the phenotypic changes. CONCLUSIONS: This case of recurrent ACC demonstrates that adrenocortical cells can reverse their differentiation program during neoplastic progression and change their specific hormone synthesis, as a consequence of modifications in the expression profile of steroidogenic enzymes and cofactors. We hypothesize that this shift in steroid hormone secretion is a consequence of chromosome amplification induced by chemotherapy. These findings, besides opening new perspectives to study adrenocortical cell plasticity and potential, demonstrate how conventional clinical and pathologic evaluation can be combined with genomic analysis in order to dissect thoroughly the biology of cancer.

Determination of S-phenylmercapturic acid by GC-MS and ELISA: a comparison of the two methods.

S-phenylmercapturic acid (PMA) is a specific urinary biomarker of benzene at exposure levels lower than 1 ppm. However, measuring PMA in urine is an expensive task by either GC or HPLC due to the necessity of extensive sample pretreatment. In the present study, a commercial chemiluminescence enzyme-linked immunosorbent assay (ELISA) test for PMA and GC-MS were used for screening urine samples of 60 workers employed in petrochemical settings. The ELISA results were evaluated by comparison with the GC-MS. Overall, the ELISA test proved sensitive (limit of detection=0.1 microg l(-1)), rapid, robust and reliable, affording results in good agreement with the GC-MS (54% of measurements) and no false-negatives. On the other hand, 46% of the ELISA assays were assigned as false-positives (arbitrarily established when ELISA >5 microg l(-1), GC-MS <5 microg l(-1) and a correlation coefficient of 0.687 was calculated between the two methods. It appears that urinary PMA routine biomonitoring on large numbers of samples is carried out in a cost-effective and rapid approach by preliminary screening with the ELISA assay followed by GC-MS confirmation of concentrations exceeding the biological exposure index for PMA.

Sevoflurane low-flow anaesthesia: best strategy to reduce Compound A concentration.

BACKGROUND: To define the best strategy to reduce Compound A production in Sevoflurane low-flow anaesthesia by experiments in vitro and in vivo of different absorbers and different anaesthesia machines. METHODS: In vitro Compound A has been measured at 45 degrees C in vitro following Sevoflurane interactions with potassium hydroxide, sodium hydroxide, soda lime, Dragersorb 800 Plus and Amsorb, a new absorber that does not contain sodium or potassium hydroxide. In vivo Compound A concentration in the anaesthesia circuit (inspiratory branch) has been measured using an indirect sampling method through absorber vials (SKC) with active coal granules, during low flows (500 ml/min) general anaesthesia using soda lime, Dragersorb 800 Plus or Amsorb as absorber. Compound A was also measured during low flows (500 ml/min) general anaesthesia using as carbon dioxide absorber soda lime with different anaesthesia machines. RESULTS: In vitro at 45 degrees C Compound A concentration with soda lime and Dragersorb 800 Plus was about 10 times higher than with Amsorb. In vivo the Compound A concentrations in the inspiratory branch of the circuit were lower in the group with Amsorb. CONCLUSION: The Compound A production is minimal with Amsorb as carbon dioxide absorber.