HCV NS3 sequencing as a reliable and clinically useful tool for the assessment of genotype and resistance mutations for clinical samples with different HCV-RNA levels.

OBJECTIVES: This study aims to evaluate the reliability and clinical utility of NS3 sequencing in hepatitis C virus (HCV) 1-infected patients who were candidates to start a PI-containing regimen. METHODS: NS3 protease sequencing was performed by in-house-developed HCV-1 subtype-specific protocols. Phylogenetic analysis was used to test sequencing reliability and concordance with previous genotype/subtype assignment by commercial genotyping assays. RESULTS: Five hundred and sixty-seven HCV plasma samples with quantifiable HCV-RNA from 326 HCV-infected patients were collected between 2011 and 2014. Overall, the success rate of NS3 sequencing was 88.9%. The success rate between the two subtype protocols (HCV-1a/HCV-1b) was similarly high for samples with HCV-RNA >3 log IU/mL (>92% success rate), while it was slightly lower for HCV-1a samples with HCV-RNA ≤3 log IU/mL compared with HCV-1b samples. Phylogenetic analysis confirmed the genotype/subtype given by commercial genotyping assays in 92.9% (303/326) of cases analysed. In the remaining 23 cases (7.1%), 1 was HCV-1g (previously defined as subtype 1a), 1 was HCV-4d (previously defined as genotype 1b) and 1 was HCV-1b (previously defined as genotype 2a/2c). In the other cases, NS3 sequencing precisely resolved the either previous undetermined/discordant subtype 1 or double genotype/subtype assignment by commercial genotyping assays. Resistance-associated variants (RAVs) to PI were detected in 31.0% of samples. This prevalence changed according to PI experience (17.1% in PI-naive patients versus 79.2% in boceprevir/telaprevir/simeprevir-failing patients). Among 96 patients with available virological outcome following boceprevir/telaprevir treatment, a trend of association between baseline NS3 RAVs and virological failure was observed (particularly for HCV-1a-infected patients: 3/21 failing patients versus 0/22 achieving sustained virological response; P = 0.11). CONCLUSIONS: HCV-NS3 sequencing provides reliable results and at the same time gives two clinically relevant pieces of information: a correct subtype/genotype assignment and the detection of variants that may interfere with the efficacy of PI.

Hepatitis C virus genetic variability and the presence of NS5B resistance-associated mutations as natural polymorphisms in selected genotypes could affect the response to NS5B inhibitors.

Because of the extreme genetic variability of hepatitis C virus (HCV), we analyzed the NS5B polymerase genetic variability in circulating HCV genotypes/subtypes and its impact on the genetic barrier for the development of resistance to clinically relevant nucleoside inhibitors (NIs)/nonnucleoside inhibitors (NNIs). The study included 1,145 NS5B polymerase sequences retrieved from the Los Alamos HCV database and GenBank. The genetic barrier was calculated for drug resistance emergence. Prevalence and genetic barrier were calculated for 1 major NI and 32 NNI resistance variants (13 major and 19 minor) at 21 total NS5B positions. Docking calculations were used to analyze sofosbuvir affinity toward the diverse HCV genotypes. Overall, NS5B polymerase was moderately conserved among all HCV genotypes, with 313/591 amino acid residues (53.0%) showing ≤1% variability and 83/591 residues (14.0%) showing high variability (≥25.1%). Nine NNI resistance variants (2 major variants, 414L and 423I; 7 minor variants, 316N, 421V, 445F, 482L, 494A, 499A, and 556G) were found as natural polymorphisms in selected genotypes. In particular, 414L and 423I were found in HCV genotype 4 (HCV-4) (n = 14/38, 36.8%) and in all HCV-5 sequences (n = 17, 100%), respectively. Regardless of HCV genotype, the 282T major NI resistance variant and 10 major NNI resistance variants (316Y, 414L, 423I/T/V, 448H, 486V, 495L, 554D, and 559G) always required a single nucleotide substitution to be generated. Conversely, the other 3 major NNI resistance variants (414T, 419S, and 422K) were associated with a different genetic barrier score development among the six HCV genotypes. Sofosbuvir docking analysis highlighted a better ligand affinity toward HCV-2 than toward HCV-3, in agreement with the experimental observations. The genetic variability among HCV genotypes, particularly with the presence of polymorphisms at NNI resistance positions, could affect their responsiveness to NS5B inhibitors. A pretherapy HCV NS5B sequencing could help to provide patients with the full efficacy of NNI-containing regimens.

Posterior component separation with TAR: lessons learned from our first consecutive 52 cases.

Patients with complex incisional hernia (IH) is a growing and challenging category that surgeons are facing in daily practice and represent indeed a technical challenge for most of them. The posterior component separation with TAR (PCS-TAR) has become the procedure of choice to repair most complex abdominal wall defects, including those with loss of domain, subxiphoid, subcostal, parastomal or after trauma and sepsis treated initially with "open abdomen" and in those scenarios in which the fascia closure was not performed to avoid an abdominal compartment syndrome. Most recent studies showed that the PCS-TAR represents a valid procedure in recurrent IH. The purpose of our study is to evaluate the reproducibility of the PCS-TAR, describing our experience, our surgical technique and the rate of postoperative complications and recurrences in a cohort of consecutive patients. 52 consecutive patients with complex IH, who underwent PCS-TAR at "Betania Hospital and Ospedale del Mare Hospital" in Naples between May 2014 and November 2019 were identified from a prospectively maintained database and reviewed retrospectively. There were 36 males (69%) and 16 females (31%) with a mean age of 57.88 (range 39-76) and Body mass index (BMI kg/m2) of 31.2 (24-45). More than half of patients (58%) were active smokers. Mean defect width was 13.6 cm (range 6-30) and mean defect area was about 267.9 cm2. Mean operative time was 228 min. Posterior fascial closure was reached in all cases, while anterior fascial closure only in 29 cases (56%). Mean hospital stay was 5.7 days. 27% of patients developed minor complications (Clavien-Dindo grade I-II) and one case (1.9%) major complication (Clavien-Dindo III). Seroma was registered in 23% of cases. SSI was reported to be 3.8% with no deep wound infection. Recurrence rate was 1.9% in a mean follow-up of 28 months. In Univariate analysis Bio-A surface > 600 cm2 and drain removal at discharge were significantly associated with major complications, while in a multivariate analysis only Bio-A surface > 600 cm2 was related. Considering univariate analysis for recurrences, number of drains, SSO, Clavien-Dindo score > 2 and defect area were significantly associated with recurrence, while in a multivariate analysis no variables were related. PCS-TAR is an indispensable tool in managing complex ventral hernias associated with a low rate of SSO and recurrence. Tobacco use, obesity and comorbidities cannot be considered absolute contraindications to PCS-TAR. Peri and postoperative management of complications and drainages have an impact on short term outcomes. Based on these outcomes, posterior component separation with transversus abdominis release has become our method of choice for the management of patients with complex ventral hernia requiring open hernia repair in selected patients.

Phylogenetic analysis in the clinical risk management of an outbreak of hepatitis C virus infection among transfused thalassaemia patients in Italy.

BACKGROUND: Occurrence of hepatitis C virus (HCV) infection is reduced by effective risk management procedures, but patient-to-patient transmission continues to be reported in healthcare settings. AIM: To report the use of phylogenetic analysis in the clinical risk management of an HCV outbreak among 128 thalassaemia outpatients followed at a thalassaemia centre of an Italian hospital. METHODS: Epidemiological investigation and root-cause analysis were performed. All patients with acute hepatitis and known chronic infection were tested for HCV RNA, HCV genotyping, and NS3, NS5A, and NS5B HCV genomic region sequencing. To identify transmission clusters, phylogenetic trees were built for each gene employing Bayesian methods. FINDINGS: All patients with acute hepatitis were infected with HCV genotype 1b. Root-cause analysis, including a lookback procedure, excluded blood donors as the source of HCV transmission. The phylogenetic analysis, conducted on seven patients with acute infection and eight patients with chronic infection, highlighted four transmission clusters including at least one patient with chronic and one patient with acute HCV infection. All patients in the same cluster received a blood transfusion during the same day. Two patients with acute hepatitis spontaneously cleared HCV within four weeks and nine patients received ledipasvir plus sofosbuvir for six weeks, all achieving a sustained virological response. CONCLUSION: Combined use of root-cause analysis and molecular epidemiology was effective in ascertaining the origin of the HCV outbreak. Antiviral therapy avoided the chronic progression of the infection and further spread in care units and in the family environment.

[Surgical treatment and prognostic factors in colorectal cancer]. / Trattamento chirurgico e fattori prognostici nel cancro colo-rettale.

AIM: The aim of this study was to make a contribution to improve the care of patients with colorectal cancer by optimizing times and methods of the follow-up; particular attention is given to factors which may be important for the prognosis and for the quality of life in the immediate postoperative period. METHODS: The study includes all the patients with colorectal cancer who underwent laparotomic surgical treatment from 1996 to 2003. The total number of patients was 226 with an average age of 65 years; male to female ratio was 1.57:1. According to the stage of tumor, an adjuvant radiotherapeutic and/or chemiotherapeutic treatment was associated to surgery. RESULTS: On the basis of the preoperative staging and tumor localization, our patients underwent: 3 total colectomies, 57 right hemicolectomies, 137 left hemicolectomies, 6 Hartmann resections, 19 Miles resections, 4 transverse resections. CONCLUSION: The results obtained show that the 5-year overall survival is particularly influenced by the stage and the factors which directly or indirectly affect the primary tumor; thus the prognostic factors which should be considered for the survival and in the follow-up of these patients are: stage, grading and nodal involvement of the tumor.

Further evidence of benzene carcinogenicity. Results on Wistar rats and Swiss mice treated by ingestion.

Wistar rats and Swiss mice were treated by ingestion (stomach tube) with benzene in olive oil at a dose of 500 and 0 mg/kg b.w. once daily, 4-5 days weekly, for 104 weeks (rats) or for 78 weeks (mice). In Wistar rats, benzene caused Zymbal gland carcinomas, carcinomas of the oral cavity, and carcinomas of the nasal cavities, and an increase in the incidence of total malignant tumors. In Swiss mice, benzene produced Zymbal gland carcinomas and dysplasias and an increase in the incidence of mammary carcinomas (in females), lung tumors, and total malignant tumors. These experiments further confirm that benzene is a multipotential carcinogen as was shown before by long-term bioassays performed on Sprague-Dawley rats in the same Experimental Unit.

Civilian gunshot wounds of the brain.

The authors report on 42 fatal gunshot wounds to the brain in civilians. The firearms used were those commonly available to civilians, ranging from a .22 revolver to a .45 semiautomatic pistol. Missle tracks were measured and the volume computed. The relatively low-velocity missles produced by these weapons to not create the devastation that characterizes wounds from high-velocity military firearms. Instead, there is much variation in the size of the missle tracks, and they cannot be directly related to caliber. Pressure marks and contusions, impaction of bone chips, internal richochet, and cerebral edema occurred frequently. The missle passed through the brain completely in very case but was retained by the skull or soft tissues in a large percentage of cases. The mechanism of death may be acute pressure on the brain stem from the passage of the missle through the brain.

Wounds from civilian and military centerfire rifles.

This article explores the differences between rimfire and centerfire rifles and the differences in ammunition used. Permanent and temporary cavity wound tracks are explained. Radiographs, intermediary targets, and assault rifles are also discussed.

A Brownian model of glutamate diffusion in excitatory synapses of hippocampus.

We simulated the diffusion of glutamate, following the release of a single vesicle from a pre-synaptic terminal, in the synaptic cleft by using a Brownian diffusion model based on Langevin equations. The synaptic concentration time course and the time course of quantal excitatory post-synaptic current have been analyzed. The results showed that they depend on the number of receptors located at post-synaptic membrane. Their time course are dependent both on the total number of the post-synaptic receptors and on the eccentricity of the pre-synaptic glutamate vesicle.

Four deaths due to intravenous injection of cocaine.

Cocaine is a potent psychotropic drug that alters mood and behavior by stimulating the central nervous system. Deaths from abuse of cocaine are relatively rare. Most such deaths appear to be related to the intravenous injection of the drug. Acute fatal cocaine intoxication is characterized by headache, cold sweats, rapid pulse, tremors and nausea, followed by convulsions, unconsciousness and death. The prime mode of death appears to be respiratory paralysis, secondary to the effects of cocaine on the medullary portion of the brain. This paper presents four deaths due to acute cocaine intoxication following intravenous injection. Blood concentrations of cocaine were 0.11, 0.37, 0.36 and 0.75 mg/dl.

A death from fentanyl overdose.

A case of death from self-injection of fentanyl and a method for its detection in autopsy specimens are reported. Fentanyl was measured by extraction of biological samples with ethyl acetate, partitioning into 0.1N HCl, then reextracting into ethyl acetate after alkalinization of the acid phase. After reducing to a residue, the fentanyl was measured by selective ion monitoring gas chromatography/mass spectrometry. Concentrations of 4.8, 6.3, and 4.7 micrograms/L were found in blood, urine, and bile, respectively.

Allogeneic gene-modified tumour cells in metastatic kidney cancer. Preliminary report.

An allogeneic irradiated RCC cell line, engineered to produce IL-2 (ACHN-IL-2), admixed with autologous metastatic formalin-treated tumour cells, was used to vaccinate ten MRCC patients in progression of disease in spite of IL-2 immunotherapy. The cells were administered subcutaneously and/or intra-tumourally. Sixty-four MRCC patients in progressive disease, not treated by vaccination but receiving similar IL-2 immunotherapy, were considered as the control group. Patients received 4-16 injections (mean 9 +/- 4), containing an average of 10.6 x 10(7) +/- 7.7 x 10(7) ACHN-IL-2-transfected cells (a minimum of 4 x 10(7), and a maximum of 31 x 10(7)). Four patients also received intra-tumour injections. Vaccination was administered during 30-418 days, and the follow-up continued for 649 +/- 353 days (190-1342). Throughout this period, the patients continued receiving the previously set immunotherapy treatment. No adverse side effects related to the treatment were observed. One complete and one partial tumour response were observed, as well as two stable and one no-relapse disease. All but one patient died. Responding patients resumed progression in 4-11 months and died 18 and 36 months after beginning the vaccine therapy. In spite of the small number of treated patients, Wilcoxon's test showed a significant (P < 0.05) improvement of the survival in the vaccinated group compared to that of the control. The described vaccination protocol seems safe, devoid of adverse side effects and promising. It warrants further investigation.

Allogeneic gene-modified tumour cells in metastatic kidney cancer. Report II.

In a limited study, comprising only ten patients, we have previously reported that allogeneic irradiated RCC-cell-line cells, engineered to produce IL-2 (ACHN-IL-2), admixed with autologous metastatic formalin-treated tumour cells were used to vaccinate MRCC patients in progression of disease and also receiving IL-2 immunotherapy. The cells, admixed to autologous TC, were administered subcutaneously. We now report an extended study on thirty patients and one hundred thirty-one controls. Patients received 4-20 injections (mean 10 +/- 4), containing an average of 92 x 10(6) +/- 45 x 10(6) ACHN-IL-2 transfected cells (a minimum of 25 x 10(6), and a maximum of 200 x 10(6)). Autologous TC, admixed to allogeneic, were also administered by 4-16 s.c. injections (mean 7 +/- 3), i.e. a total of 12 x 10(6)-160 x 10(6) cells. Vaccination was administered during 73-1451 (307 +/- 316) days, and the follow-up continued for 1122 +/- 1240 days (106-5137). Throughout this period, the patients continued receiving the previously set immunotherapy treatment. No adverse side effects related to the treatment were noticed. One complete and four partial tumour responses were observed, as well as nine cases of stable disease. Thirteen patients died in the treated group (43%) and 63 (44%) in the control group. Responding patients resumed progression in 4-11 months and died 18 and 36 months after beginning the vaccine therapy. The Gehan Wilcoxon's test showed a significantly (P < 0.01) better survival in the vaccinated patients compared to that of the controls. Thus, we confirm, in an increased number of patients and an extensive follow-up, that our vaccination protocol is safe, devoid of adverse side effects, and promising.

Different types of noise in leaky integrate-and-fire model of neuronal dynamics with discrete periodical input.

Different variants of stochastic leaky integrate-and-fire model for the membrane depolarisation of neurons are investigated. The model is driven by a constant input and equidistant pulses of fixed amplitude. These two types of signal are considered under the influence of three types of noise: white noise, jitter on interpulse distance, and noise in the amplitude of pulses. The results of computational experiments demonstrate the enhancement of the signal by noise in subthreshold regime and deterioration of the signal if it is sufficiently strong to carry the information in absence of noise. Our study holds mainly to central neurons that process discrete pulses although an application in sensory system is also available.

Natural death as viewed by the medical examiner: a review of 1000 consecutive autopsies of individuals dying of natural disease.

A study of 1000 consecutive autopsies of individuals dying of natural disease was conducted. Cardiovascular disease was responsible for 60.9% of all deaths with coronary artery disease--not only the main cause of cardiovascular death but also the main cause of all natural deaths--accounting for 45.1% of such cases. Diseases of the central nervous and respiratory systems accounted for 8.7 and 8.6%, respectively, of the natural deaths. Seizure disorders and pneumonia were the main causes of death in these organ systems. There were 124 deaths of children less than one year in age, 91 of which were due to sudden infant death syndrome (SIDS). All of the SIDS deaths were in children less than 10 months old.

Detection of cannabinoids in homicide victims and motor vehicle fatalities.

A gas chromatographic/mass spectrometric (GC/MS) procedure is described for the detection and measurement of delta 9-tetrahydrocannabinol, 11-hydroxy-delta 9-tetrahydrocannabinol, and 11-nor-delta 9-tetrahydrocannabinol-9-carboxylic acid in blood, or 11-nor-delta 9-tetrahydrocannabinol-9-carboxylic acid in urine. About 50% of all homicide victims and motor vehicle drivers killed in Bexar County in 1985 were tested for the presence of cannabinoids. Of 130 homicides and 69 drivers tested, blood was analyzed primarily in all but 15 and 3 cases, respectively. In these latter cases, blood analyzed after urine was found to be positive. Of the homicide victims, 44 (34%), and of all drivers, 19 (28%), tested were positive for one or more of the cannabinoids. As a separate group, 16 motorcycle drivers tested had 38% positive as compared with 25% of the other vehicle drivers. Ethyl alcohol was present in 55% of the drivers, and in 63% of the homicide victims. Drugs other than alcohol or cannabinoids were found in 10% of the drivers, and in 12% of the homicide victims.

A computer program to study the area perception of squares and rectangles.

A computerized approach to study the visual perception of rectangles and squares is proposed. A program has been used for comparison of these two figures when one of them is fixed in size while the other is varied actively by a subject to match the area of the fixed one. The final errors on area and on perimeter made by the subject are computed for each pair of presented figures. The program is menu driven and thus easy to use by the investigator. Since the task procedure is very simple and non-obtrusive to the subjects, it can be used both for children and adults as well as for normal and brain impaired subjects in order to study some perceptive and cognitive functions. This program recognizes the most common graphics cards used by computers running under MS-DOS to adjust the parameters of the presented figures according to the pixel resolution of the graphics card. Quick-BASIC was used to write the program which was compiled by Microsoft Professional Basic 7.0 compiler.

Threshold effect in visual perception of geometrical figures.

Filtering of the input image has been shown to play a central role in several aspects of visual perception. In our experiments in visual perception of the area of geometrical figures the orientation in random dot patterns, and some visual illusions, we have shown that a threshold effect inferred from the filtering of the input image produces a perceptual error. This error has been explained by using the concept of Image Function. The present paper is a brief review of our experimental results and of the models we have proposed.

The Müller-Lyer illusion in interpolated figures.

The Müller-Lyer patterns formed by separate dots have been used as stimuli in an experiment on visual perception to assess the influence of the number of dots composing the figures on the magnitude of the illusion. As predicted by our model, based on the Image Function theory, an increase was noted in the magnitude of illusion when the number of dots composing the arrowheads was increased. It follows from the model that filtering of the input image plays a central role in the formation of the illusion.