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Fluorescently labeled antibody and aptamer probes are used in biological studies to characterize binding interactions, measure concentrations of analytes, and sort cells. Fluorescent nanoparticle labels offer an excellent alternative to standard fluorescent labeling strategies due to their enhanced brightness, stability and multivalency; however, challenges in functionalization and characterization have impeded their use. This work introduces a straightforward approach for preparation of fluorescent nanoparticle probes using commercially available reagents and common laboratory equipment. Fluorescent polystyrene nanoparticles, Thermo Fisher Scientific FluoSpheres, were used in these proof-of-principle studies. Particle passivation was achieved by covalent attachment of amine-PEG-azide to carboxylated particles, neutralizing the surface charge from − 43 to − 15 mV. A conjugation-annealing handle and DNA aptamer probe were attached to the azide-PEG nanoparticle surface either through reaction of pre-annealed handle and probe or through a stepwise reaction of the nanoparticles with the handle followed by aptamer annealing. Nanoparticles functionalized with DNA aptamers targeting histidine tags and VEGF protein had high affinity (EC50s ranging from 3 to 12 nM) and specificity, and were more stable than conventional labels. This protocol for preparation of nanoparticle probes relies solely on commercially available reagents and common equipment, breaking down the barriers to use nanoparticles in biological experiments.

Summary Background Awareness of healthcare-associated infections (HAIs) and antimicrobial use in long-term care facilities (LTCFs) is increasing. In 2017, the third national point prevalence survey (PPS) was conducted in Italy as part of the third ‘Healthcare-Associated Infections in European Long-Term Care Facilities’ (HALT3) study. Aim To report the results of HALT3 and analyse the resident population of LTCFs, implementation of good practices, prevalence of infections and antimicrobial use. Methods The survey was designed as a PPS, carried out from April to June 2017. All residents who lived full-time in the institution were included. All facilities were asked to complete an institutional questionnaire, a ward list for all residents, and a resident questionnaire for those residents presenting with signs/symptoms of active infection and/or receiving an antimicrobial agent. Findings In total, 418 facilities took part in the study; 24,132 residents were eligible, and most were aged >85 years, disoriented and incontinent. The prevalence of HAIs was 3.9%, and 50% of the institutions reported that they had a professional trained in infection control on their staff. Only 26.4% of infections were confirmed by a microbiological sample, and 26.9% of the isolated micro-organisms were resistant to at least one antimicrobial class. In total, 1022 residents received at least one antimicrobial agent, and cephalosporins were prescribed most commonly. Conclusion The number of infection control and antimicrobial stewardship measures implemented was found to be considerably higher in this study compared with previous studies. This could lead to a reduction in the prevalence of HAIs, antimicrobial use and antimicrobial resistance. Further studies are needed to monitor these aspects.

In Italy there are no rules concerning the establishment of a hospital hygiene structure in hospitals and other healthcare settings, and the hospital organization plans vary widely. The aim of the survey, carried out by the Italian Study Group of Hospital Hygiene of the Italian Society of Hygiene, Preventive medicine and Public health, was to evaluate the presence in the hospital organization plan of a structure referred to as Hospital hygiene, or including in its denomination the words "hygiene" or "hospital hygiene", the activities carried out, the relation to other areas, like patient safety, the type and quantity of professionals involved, the strengths and the critical aspects.A semi-structured questionnaire was administered to Healthcare Trusts representing all Italian Regions through the members of the above Study Group.35 Trusts, 13 in Northern, 8 in Central, 14 in Southern Italy (including Sicily and Sardinia), completed the questionnaire. In 19 Trusts (54.3%) a structure whose denomination included the words "hospital hygiene" or "hygiene" was present. The activities related to the management of infectious risk were most represented, carried out autonomously or in collaboration, but many other activities were covered. In all hospitals the activities of the Hospital Hygiene Unit inter-linked with those of the clinical risk, with different forms of collaboration.This survey, even though on a limited sample, provided a picture of hospital hygiene at a national level, showing a considerable heterogeneity and highlighting critical issues but also strengths. It is essential to share organizational and management models that enhance and promote hospital hygiene, to ensure the appropriateness of healthcare practices offered in a safe and comfortable environment to patients, operators, and visitors.

Although hospital hygiene has a long history in Italy it is necessary to reflect about it because of the innovation in healthcare systems and because of the evolution due to European Union related activities. Different traditions exist in European countries about hospital hygiene and European Centre for Disease Prevention and Control (ECDC) adopted the term of "infection control and hospital hygiene" which includes all the engaged European healthcare professionals. We propose to modify hospital hygiene as "healthcare organisation hygiene" in order to focalise the attention to all care settings not only hospitals and to adopt the following definition: "all activities aimed to guarantee, in an appropriate, scientifically sound and efficient way, that structures and processes support healthcare practices in a safe comfortable and healthy environment both for patients, caregivers and healthcare workers". Hospital hygiene and its professionals, besides the long tradition, still remain a relevant pillar in guaranteeing quality and safety of healthcare in Italy.

In 2014, the Italian Study Group of Hospital Hygiene of the Italian Society of Hygiene, Preventive Medicine and Public Health (GISIO-SItI), in collaboration with the National Association of Medical Hospital Managers (ANMDO), conducted a survey on the availability of procedures for cleaning and disinfecting ambulances in order to assess the practices in use.An online questionnaire was prepared through the Survey Monkey® platform and a web link access was sent to a convenience sample of ANMDO doctors working in healthcare management in public and private healthcare facilities.Ninety-six questionnaires were collected (26% response rate). In 73% of cases there was a procedure for cleaning and disinfecting ambulances, which had been produced at a company level (67%) and involved various professionals. In 21% of cases the procedure had been prepared in expectation of an epidemic or following an epidemic (5%). The recommendations had been presented to the staff (90%), in 28% of cases through training events with verification of the knowledge acquired. Monitoring of the implementation of the procedure is planned in the majority of cases (88%), mainly through direct observation (92%). In 67% of cases the tender specifications for ambulance services did not include a section dedicated to cleaning and disinfection and, in the absence of a procedure, this was provided by the hospital in only 51% of case.This survey represented a first step towards the development of guidelines for standardising procedures and providing indications useful for their evaluation and monitoring their implementation.

Development of multifunctional biomaterials that sequester, isolate, and redeliver cell-secreted proteins at a specific timepoint may be required to achieve the level of temporal control needed to more fully regulate tissue regeneration and repair. In response, we fabricated core-shell heparin-poly(ethylene-glycol) (PEG) microparticles (MPs) with a degradable PEG-based shell that can temporally control delivery of protein-laden heparin MPs. Core-shell MPs were fabricated via a re-emulsification technique and the number of heparin MPs per PEG-based shell could be tuned by varying the mass of heparin MPs in the precursor PEG phase. When heparin MPs were loaded with bone morphogenetic protein-2 (BMP-2) and then encapsulated into core-shell MPs, degradable core-shell MPs initiated similar C2C12 cell alkaline phosphatase (ALP) activity as the soluble control, while non-degradable core-shell MPs initiated a significantly lower response (85 + 19% vs. 9.0 + 4.8% of the soluble control, respectively). Similarly, when degradable core-shell MPs were formed and then loaded with BMP-2, they induced a ∼7-fold higher C2C12 ALP activity than the soluble control. As C2C12 ALP activity was enhanced by BMP-2, these studies indicated that degradable core-shell MPs were able to deliver a bioactive, BMP-2-laden heparin MP core. Overall, these dynamic core-shell MPs have the potential to sequester, isolate, and then redeliver proteins attached to a heparin core to initiate a cell response, which could be of great benefit to tissue regeneration applications requiring tight temporal control over protein presentation. Statement of Significance Tissue repair requires temporally controlled presentation of potent proteins. Recently, biomaterial-mediated binding (sequestration) of cell-secreted proteins has emerged as a strategy to harness the regenerative potential of naturally produced proteins, but this strategy currently only allows immediate amplification and re-delivery of these signals. The multifunctional, dynamic core-shell heparin-PEG microparticles presented here overcome this limitation by sequestering proteins through a PEG-based shell onto a protein-protective heparin core, temporarily isolating bound proteins from the cellular microenvironment, and re-delivering proteins only after degradation of the PEG-based shell. Thus, these core-shell microparticles have potential to be a novel tool to harness and isolate proteins produced in the cellular environment and then control when proteins are re-introduced for the most effective tissue regeneration and repair.

Venezuelan equine encephalitis virus (VEEV) poses a major public health risk due to its amenability for use as a bioterrorism agent and its severe health consequences in humans. ML336 is a recently developed chemical inhibitor of VEEV, shown to effectively reduce VEEV infection in vitro and in vivo. However, its limited solubility and stability could hinder its clinical translation. To overcome these limitations, lipid-coated mesoporous silica nanoparticles (LC-MSNs) were employed. The large surface area of the MSN core promotes hydrophobic drug loading while the liposome coating retains the drug and enables enhanced circulation time and biocompatibility, providing an ideal ML336 delivery platform. LC-MSNs loaded 20 ± 3.4 μg ML336/mg LC-MSN and released 6.6 ± 1.3 μg/mg ML336 over 24 hours. ML336-loaded LC-MSNs significantly inhibited VEEV in vitro in a dose-dependent manner as compared to unloaded LC-MSNs controls. Moreover, cell-based studies suggested that additional release of ML336 occurs after endocytosis. In vivo safety studies were conducted in mice, and LC-MSNs were not toxic when dosed at 0.11 g LC-MSNs/kg/day for four days. ML336-loaded LC-MSNs showed significant reduction of brain viral titer in VEEV infected mice compared to PBS controls. Overall, these results highlight the utility of LC-MSNs as drug delivery vehicles to treat VEEV.

Healthcare-Associated Infections are a great concern for worldwide healthcare systems and represent a considerable threat to patient safety, leading to adverse clinical outcomes. A defined panel of indicators represents a key element to guide Healthcare Organizations towards identification of main gaps, implementation of effective actions and continuous improvements on Healthcare-Associated Infections prevention and control activities. A review on accreditation systems conducted by the Italian Study Group of Hospital Hygiene of the Italian Society of Hygiene Preventive Medicine and Public Health revealed a substantial heterogeneity of implemented standards and led to the development of a core set of indicators and requirements for Healthcare-Associated Infections' prevention and control within the hospital setting. The main aim of the study was to test the feasibility of the Healthcare-Associated Infections' prevention and control within the hospital setting tool to calculate performance scores on a sample of Italian Healthcare Organizations and to identify major critical issues. The potential benefits of the possibility of future implementation of the tool within Institutional Accreditation Programs is discussed.Cross sectional pilot survey.The Healthcare-Associated Infections' prevention and control within the hospital setting included 96 criteria and 20 key areas including an area for outcomes indicators. For applicable criteria, standards fulfilment was evaluated according to a 4 point Likert scale. A composite score was calculated for each Healthcare Organization and five performance levels were identified. Data were further analysed by computing performance scores at the level of each area and requirement.20 Healthcare Organizations agreed to take part in this pilot study including two rehabilitative Healthcare Organizations. Among the whole sample a mean of 12.20% of requirements resulted not fulfilled, leaving space for further improvements. Critical areas were easily identified and the instrument was able to capture substantial differences between Healthcare Organizations. Only a few number of standards resulted "Not Applicable" (Mean = 4.71%) and most of them regarded Rehabilitative Healthcare Organizations. Mean composite performance index resulted 74.06% (SD = 16.96, range 36.30 - 94.27%); area of outcome indicators obtained a mean score of 56.17.The Healthcare-Associated Infections' prevention and control within the hospital setting resulted an useful tool to assess Healthcare Organizations' performance in the field of Healthcare-Associated Infections prevention and control and to identify necessary actions for further improvements. The distribution of total scores by Healthcare Organizations showed a high heterogeneity. Implementation of the Healthcare-Associated Infections' prevention and control within the hospital setting tool as an institutional accreditation tool may help to drive the required harmonization at a national level of Healthcare-Associated Infections management and control strategies and overcome current substantial regional differences.

Recent studies have found that uncontrolled diabetes and consequential hyperglycemic conditions can lead to an increased incidence of osteoporosis. Osteoblasts, adipocytes, and mesenchymal stem cells (MSCs) are all components of the bone marrow microenvironment and thus may have an effect on diabetes-related osteoporosis. However, few studies have investigated the influence of these three cell types on each other, especially in the context of hyperglycemia. Thus, we developed a hydrogel-based 3D culture platform engineered to allow live-cell retrieval in order to investigate the interactions between MSCs, osteoblasts, and adipocytes in mono-, co-, and tri-culture configurations under hyperglycemic conditions for 7 days of culture. Gene expression, histochemical analysis of differentiation markers, and cell viability were measured for all cell types, and MSC-laden hydrogels were degraded to retrieve cells to assess their colony-forming capacity. Multivariate models of gene expression data indicated that primary discrimination was dependent on the neighboring cell type, validating the need for co-culture configurations to study conditions modeling this disease state. MSC viability and clonogenicity were reduced when mono- and co-cultured with osteoblasts at high glucose levels. In contrast, MSCs showed no reduction of viability or clonogenicity when cultured with adipocytes under high glucose conditions, and the adipogenic gene expression indicates that cross-talk between MSCs and adipocytes may occur. Thus, our unique culture platform combined with post-culture multivariate analysis provided a novel insight into cellular interactions within the MSC microenvironment and highlights the necessity of multi-cellular culture systems for further investigation of complex pathologies such as diabetes and osteoporosis.

The authors examine the effectiveness of preoperative embolization in the treatment of rhinopharyngeal angiofibroma. In addition, they evaluate the effect various embolizing substances have in the aim of establishing what is the ideal time interval between embolization and surgery. Thirteen patients with rhinopharyngeal angiofibroma (3 were recurrences) were submitted to preoperative embolization. Of these, 4 were treated with a reabsorbable microparticulate substance (Gelfoam) while the remaining 8 were treated with non reabsorbable microparticulate substances (Ivalon, ITC contour, Terbal). The time interval between embolization and surgery ranged from a minimum of 1 day with a maximum of 4 days. The authors conclude that embolization markedly reduces blood loss during surgery, so much so that the available blood supply was not required. As regards the various embolizing substances and the time lapse between embolization and surgery, the authors feel that when using reabsorbable materials the interval should not exceed 48 hours so that the supply artery can quickly be recanalized. On the other hand, for the materials which are not reabsorbed, the time interval can exceed 48 hours but must be less than 4 days because collateral circulation can arise.

Background. Healthcare-associated infections (HAIs) are an important issue in terms of quality of care. HAIs impact patient safety by contributing to higher rates of preventable mortality and prolonged hospita- lizations. In Italy, analysis of the currently available accreditation systems shows a substantial heteroge- neity of approaches for the prevention and surveillance of HAIs in hospitals. The aim of the present study is to develop and propose the use of a synthetic assessment tool that could be implemented homogenously throughout the nation. Methods. An analysis of nine international and of the 21 Italian regional accreditation systems was conducted in order to identify requirements and indicators implemented for HAI prevention and control. Two relevant reviews on this topic were further analyzed to identify additional evidence-based criteria. The project team evaluated all the requirements and indicators with consensus meeting methodology, then those applicable to the Italian context were grouped into a set of “focus areas”. Results. The analysis of international systems and Italian regional accreditation manuals led to the iden- ti cation respectively of 19 and 14 main requirements, with relevant heterogeneity in their application. Additional evidence-based criteria were included from the reviews analysis. From the consensus among the project team members all the standards were compared and 20 different thematic areas were identi ed, with a total of 96 requirements and indicators for preventing and monitoring HAIs. Conclusions. The study reveals a great heterogeneity in the de nition of accreditation criteria between the Italian regions. The introduction of a uniform, synthetic assessment instrument, based on the review of national and international standards, may serve as a self-assessment tool to evaluate the achievement of a minimum standards set for HAIs prevention and control in healthcare facilities. This may be used as an assessment tool by the Italian institutional accreditation system, also useful to reduce regional disparities.

Avoidance of corticosteroids could be beneficial after pediatric liver transplantation (LTx). To test this hypothesis, we performed a randomized prospective study to compare immunosuppression with tacrolimus (TAC) and steroids versus TAC and basiliximab (BAS) after pediatric LTx. Seventy-two patients were recruited, 36 receiving TAC and steroids and 36 TAC and BAS. The primary endpoint was the occurrence of the first rejection episode. Secondary endpoints were the cumulative incidence and severity of rejection, patient and graft survival, and incidence of adverse events. Overall 1-year patient and graft survival rates were 91.4% and 85.5% in the steroid group, and 88.6% and 80% in the BAS group (p = NS). Patients free from rejection were 87.7% in the BAS group and 67.7% in the steroid group (p = 0.036). The use of BAS was associated with a 63.6% reduction in incidence of acute rejection episodes. Overall incidence of infection was 72.3% in the steroid group and 50% in the BAS group (p = 0.035). We conclude that the combination of TAC with BAS is an alternative to TAC and steroid immunosuppression in pediatric LTx, which allows for a significant reduction in the incidence of acute rejection and infectious complications.

Alagille syndrome (AS) is a dominantly inherited, multisystem disorder involving the liver, heart, eyes, face, and skeleton. From October 1997 through July 2004, 260 pediatric orthotopic liver transplantations (OLTx) were performed in 231 patients. This report describes 21 patients of median age 1.95 years (range, 0.7-16.7) who had alagille syndrome. We present the technical features of the OLTx, incidence and type of complications, medical conditions related to the syndrome, need for retransplantation, as well as patient and graft survival rates. A split liver technique was used in 16 patients (76%) who received a left lateral segment (LLS) graft whereas 7 patients (33%) received a whole liver. Only cadaveric donors were used. The major surgical complications requiring reintervention in 11 patients (52%) included biliary problems (19%) and vascular complications (17%). One case of hepatic artery thrombosis required retransplantation. Three recipients (14%) died. All other patients are alive with an actuarial survival rate of 90% at 1 year and 80% at 5 years. The actuarial graft survival rate is 85% at 1 year and 75% at 5 years. Patients with AS, despite the associated cardiovascular anomalies, can be treated successfully by a combined approach between cardiologist, radiologist, cardiothoracic, and liver transplant surgeons. With careful planning and operative management, the results are comparable with those obtained with other more common cholestatic diseases.

In this study we analyzed the features of 12 patients who underwent liver transplantation for progressive familial intrahepatic cholestasis (Byler's disease [BD]) in view of the technical features of the OLTx, incidence and type of complications, need for retransplantation, as well as patient and graft survivals. BD was the indication in 12 patients of median age 1.32 years and median weight 10 kg. Median follow-up was 670 days. Major surgical complications requiring reintervention occurred in three patients. No thrombosis of the hepatic artery was observed. Infections with positive blood cultures were diagnosed in four patients. One patient had a biliary anastomotic stenosis successfully treated by percutaneous techniques. Four patients had episodes of acute rejection treated with steroids. Two patients were retransplanted, both of whom died in the early postoperative period due to hepatic vein thrombosis and venoenteric fistula. The actuarial patient and graft survival was 83% at 1 year and 83% at 5 years. Split-liver grafts represent an excellent organ supply for these patients, achieving good results with no mortality on the waiting list.

Early portal vein thrombosis (PVT) represents a serious complication after liver transplantation (OLTx). From October 1997 through July 2004, 260 OLTx were performed in 231 children, including 189 of left lateral segments (LLS). We retrospectively analyzed the incidence and the outcome of early PVT in this group. A daily doppler US scan was performed during the first week after transplantation. Early PVT occurred in 14 patients (8%), 10 males and four females of median age 0.77 years. The main indication for primary transplantation was biliary atresia (10), followed by Byler's disease (2), acute liver failure on cryptogenetic cirrhosis (1), and Alagille syndrome (1). Four children underwent retransplantation; three cases of thrombectomy and revision of the anastomosis, two children were treated with beta blockers, one of whom had a later failed attempt at percutaneous revascularization and eventually a meso-caval shunt. Five patients were followed with observation and no treatment. Among the four patients who died, three were in the retransplantation group and one in the thrombectomy and revision of the anastomosis group; the overall mortality was 28%. With a median follow up of 399 days, 10 patients are alive with an actuarial survival at 1 and 5 years of 72%, and graft survival rates at 1 and 5 years of 64%. PVT represents a serious complication after pediatric OLTx with LLS grafts. Prompt detection and aggressive surgical treatment in selected cases are required to reduce the mortality and graft loss.