SPLIT-PIN software enabling confocal and super-resolution imaging with a virtually closed pinhole.

In point-scanning microscopy, optical sectioning is achieved using a small aperture placed in front of the detector, i.e. the detection pinhole, which rejects the out-of-focus background. The maximum level of optical sectioning is theoretically obtained for the minimum size of the pinhole aperture, but this is normally prevented by the dramatic reduction of the detected signal when the pinhole is closed, leading to a compromise between axial resolution and signal-to-noise ratio. We have recently demonstrated that, instead of closing the pinhole, one can reach a similar level of optical sectioning by tuning the pinhole size in a confocal microscope and by analyzing the resulting image series. The method, consisting in the application of the separation of photons by lifetime tuning (SPLIT) algorithm to series of images acquired with tunable pinhole size, is called SPLIT-pinhole (SPLIT-PIN). Here, we share and describe a SPLIT-PIN software for the processing of series of images acquired at tunable pinhole size, which generates images with reduced out-of-focus background. The software can be used on series of at least two images acquired on available commercial microscopes equipped with a tunable pinhole, including confocal and stimulated emission depletion (STED) microscopes. We demonstrate applicability on different types of imaging modalities: (1) confocal imaging of DNA in a non-adherent cell line; (2) removal of out-of-focus background in super-resolved STED microscopy; (3) imaging of live intestinal organoids stained with a membrane dye.

A phasor-based approach to improve optical sectioning in any confocal microscope with a tunable pinhole.

Confocal fluorescence microscopy is a well-established imaging technique capable of generating thin optical sections of biological specimens. Optical sectioning in confocal microscopy is mainly determined by the size of the pinhole, a small aperture placed in front of a point detector. In principle, imaging with a closed pinhole provides the highest degree of optical sectioning. In practice, the dramatic reduction of signal-to-noise ratio (SNR) at smaller pinhole sizes makes challenging the use of pinhole sizes significantly smaller than 1 Airy Unit (AU). Here, we introduce a simple method to "virtually" perform confocal imaging at smaller pinhole sizes without the dramatic reduction of SNR. The method is based on the sequential acquisition of multiple confocal images acquired at different pinhole aperture sizes and image processing based on a phasor analysis. The implementation is conceptually similar to separation of photons by lifetime tuning (SPLIT), a technique that exploits the phasor analysis to achieve super-resolution, and for this reason we call this method SPLIT-pinhole (SPLIT-PIN). We show with simulated data that the SPLIT-PIN image can provide improved optical sectioning (i.e., virtually smaller pinhole size) but better SNR with respect to an image obtained with closed pinhole. For instance, two images acquired at 2 and 1 AU can be combined to obtain a SPLIT-PIN image with a virtual pinhole size of 0.2 AU but with better SNR. As an example of application to biological imaging, we show that SPLIT-PIN improves confocal imaging of the apical membrane in an in vitro model of the intestinal epithelium. RESEARCH HIGHLIGHTS: We describe a method to boost the optical sectioning power of any confocal microscope. The method is based on the sequential acquisition of multiple confocal images acquired at different pinhole aperture sizes. The resulting image series is analyzed using the phasor-based separation of photons by lifetime tuning (SPLIT) algorithm. The SPLIT-pinhole (SPLIT-PIN) method produces images with improved optical sectioning but preserved SNR. This is the first time that the phasor analysis and SPLIT algorithms are used to exploit the spatial information encoded in a tunable pinhole size and to improve optical sectioning of the confocal microscope.

Fourteen years' clinical experience and the first million babies protected with human live-attenuated vaccine against rotavirus disease in Italy.

Rotavirus (RV) causes up to half of hospital and community acute gastroenteritis (AGE) cases in young children in Italy. Two RV vaccines, available since 2006, are human RV (HRV) and human bovine RV (HBRV). This report looks back at the implementation of RV vaccination with HRV in Italy, and at HRV current and future perspectives. Initial regional policies led to national implementation by 2018, after scientific societies' disease awareness efforts. Following vaccination, RV hospitalizations declined significantly, and cost savings were observed. The two-dose HRV vaccine is easily administered during compulsory vaccine visits, helping increase coverage. Intussusception, a serious event in children <1 year, was reported in Italy with a rate of 33-40 per 100,000 infants. RV vaccination presents a low increased risk of intussusception after the first dose, estimated at 0.6 cases per 100,000 doses in Italy in 2019. Parents should be aware of the intussusception risk and symptoms to ensure prompt treatment. It is widely recognized that the vaccination benefits (large numbers of RV hospitalizations prevented) outweigh the risk. HRV introduction in Italy was supported by epidemiologic burden studies, healthcare provider opinions, and congress debates, which significantly contributed to implementation of RV universal routine infant vaccination in Italy.

[The importance of flu vaccination during CoViD time]. / L'importanza della vaccinazione anti influenzale ai tempi della CoViD.

In the next months, the risk of coinfection with f lu virus and Sars-CoV-2 is high. Despite the number of studies dealing with the consequences of the interaction between the two viruses, the impact of this coexistence on human health is still uncertain. However, achieving high f lu vaccination coverage would mean avoiding hospital overload due to hospitalizations for f lu complications and facilitating a timely differential diagnosis that allows a quick and appropriate treatment of CoViD-19 cases. These are two valid reasons for actively promoting f lu vaccination, particularly this year when the risk of a "twindemic" determined by f lu and CoViD-19 is high. In Italy this year, for the first time, f lu vaccination is offered free of charge also to people aged 60 to 64, expanding individuals entitled to free vaccination. Furthermore, it is strongly recommended to healthcare workers and to elderly who live in residential or long-term care facilities.

[Routine vaccinations during SARS-CoV-2 epidemics]. / Le vaccinazioni di routine ai tempi del SARS-CoV-2.

The circulation of SARS-CoV-2 required the deplacement of resources from routine preventive activities to pandemic-related interventions. The vaccination services have been adapted to the individual territorial realities on the basis of virus circulation and restrictive measures put in place. The reduction of coverage with the consequent accumulation of susceptible subjects increases the risk of vaccine-preventable diseases' epidemics. Catch-up programs and strategies to optimize sessions, such as carrying out co-administrations, are in place on the national territory in order to reduce the risk.

[Vaccines: from the identification of the microorganism to marketing. How long does it take?] / Vaccini: dall'identificazione del microrganismo alla commercializzazione. Quanto tempo ci vuole?

The development of a vaccine, particularly one that can help against the recent pandemic, is a topic that has recently attracted public opinion. More than ninety vaccines are currently being developed against Sars-Cov2 by universities and companies around the world. They are protein-based vaccines, viral vector vaccines, DNA or RNA vaccines and inactivated or attenuated viral vaccines. The development of a vaccine starts from the identification and characterization of the microorganism that causes the disease. The second step is the preclinical phase. Then, there is the phase of clinical experimentation, which allows to study the posology, efficacy and safety of the vaccine, on an increasingly larger sample. In the European Union, vaccines are authorized through two procedures (EU and national) based on the quality, safety and efficacy requirements defined by the European and international guidelines. Timing of realization, authorization and marketing of new vaccines can be shortened in cases of particular need, through an accelerated evaluation known as "Priority Medicines". In this period, it is crucial not to neglect current vaccinations. In fact, during the pandemic period, many countries postponed vaccination campaigns against many vaccine-preventable diseases, causing a marked decrease in routine immunizations in childhood.

Healthcare Workers Training Courses on Vaccinations: A Flexible Format Easily Adaptable to Different Healthcare Settings.

Since 2017, Italy has expanded the compulsory vaccination from 4 to 10 for those aged 0 to 16 years. Because of the great organizational effort required for the immunization services, minor attention was given to the vaccinations not included among the mandatory ones. This situation led to a real difficulty in harmonizing the vaccination procedures even inside a single region. In the Lazio region, the Laboratory of Vaccinology of the University of Rome Tor Vergata established a working group to create a new training model for healthcare professionals. The course program proposed an update of three vaccinations which are not mandatory but actively offered. It included the same part of scientific updating and a variable part based on local experiences. A specific anonymous questionnaire on knowledge and attitude was administered. The study aimed to propose a general format of training courses for vaccination centers adaptable to the individual local health units (ASLs) and to evaluate through questionnaires. The results show differences in knowledge and attitudes toward non-mandatory vaccinations among the ASLs of Lazio, confirming the usefulness of a support to make knowledge and procedures homogeneous. This model could be adapted to any healthcare setting and exported to other services.

[Tuberculosis: an ever present disease but difficult to prevent]. / Tubercolosi patologia sempre attuale e difficile da prevenire.

Tuberculosis (TB) is an infectious and contagious disease caused by Mycobacterium tuberculosis, commonly called Koch's bacillus. TB is spread by air and is present all over the world. Not everyone who become infected develop the disease; the immune system can cope with the infection and the bacterium can remain dormant for years. Despite advances in therapy and prevention, TB remains one of the top 10 causes of death worldwide. Over 9 million new cases and over 1 million deaths occur each year. Since 1921 BCG has been the only authorized vaccine. More than ten experimental vaccines are currently in different stages of development. Depending on the type, they are divided into vaccines consisting of: live attenuated, inactivated mycobacteria and subunits. According to their purpose they can be divided in: vaccines aimed at preventing infection, vaccines aimed at preventing disease, vaccines aimed at preventing recurrence and therapeutic vaccines. Hopefully, these numerous attempts to develop new vaccination approaches will lead to obtain products with greater immunogenicity and efficacy.

The potential public health impact of Herpes Zoster vaccination in the 65 years of age cohort in Italy.

Herpes Zoster (HZ) presents a considerable public health burden in Italy among people aged ≥50 years. This study aimed to assess the clinical and economic impact of HZ vaccination in the 65 years of age (YOA) cohort in Italy, by comparing the new Adjuvanted Recombinant Zoster Vaccine (RZV) with the currently available Zoster Vaccine Live (ZVL). A static Markov model was developed to follow all 65 YOA subjects from the year of vaccination over their lifetime by comparing three different HZ vaccination strategies: no vaccination, vaccination with ZVL and vaccination with RZV. In the base-case scenario, three 65 YOA cohorts were assumed to be vaccinated within three years, with a vaccine coverage rate of 20%, 35% and 50% at Year 1, 2 and 3 respectively, as recommended by the National Immunization Plan. The three 65 YOA Italian cohorts accounted altogether for 2,290,340 individuals. Of these, it was assumed that 564,178 subjects could be vaccinated with either RZV or ZVL in three years. The vaccination with RZV could prevent an additional total number of 35,834 HZ and 8,131 postherpetic neuralgia (PHN) cases over ZVL, leading to additional total savings of €12.4 million for the national healthcare and social systems. The introduction of RZV can be expected to have higher impact on the burden of HZ disease in the 65 YOA cohort in Italy. The avoided HZ and PHN cases can lead to an associated reduction in economic burden to the healthcare and social systems.

Seasonal influenza vaccination among health-care workers: the impact of different tailored programs in four University hospitals in Rome.

Seasonal influenza vaccination (SIV) of health-care workers (HCWs) is recommended in most countries to protect them and their patients from infection. Although SIV can reduce the risk of influenza complications among vulnerable patients, vaccination uptake is generally unsatisfactory. The present study aimed to assess the impact of different programs in promoting SIV uptake among HCWs during the season 2017/2018 in four teaching hospitals in Rome. A multicentric cross-sectional study was carried out, in order to describe the four different campaigns and to assess their impact by identifying and developing a set of indicators that provide information about the vaccination services, the percentage of invited HCWs, the vaccinators' workforce and the vaccination coverage rates.The hospitals organized different strategies: Hospital 1, 3 and 4 organized educational courses for HCWs and actively invited every single HCW through e-mail. All the hospitals organized a dedicated unit for influenza vaccination, and Hospital 1 added on-site vaccination sessions that required a large number of staff. Hospital 1 and hospital 4 registered a comparable vaccination coverage rate, 12.97% and 12.76%, respectively, while it was 6.88% in Hospital 2 and 4.23% in Hospital 3. Our indicators demonstrated to be effective and useful for analyzing the different SIV campaigns. The results suggest that the best practice to promote SIV among HCWs should include multiple approaches. Among those, an easy access to the vaccination site seems to play a key role in determining a higher vaccination coverage.

[Adverse reactions after repeated doses of anti-tetanus vaccine: scientific evidences]. / Reazioni avverse dopo dosi ravvicinate di vaccino anti-tetanico nell'adulto: una ricerca sulle evidenze scientifiche.

The possible risk of hyperimmunization after tetanus vaccination is currently discussed after the National Vaccine Prevention Plan 2017-2019 confirmed the recommendation of a booster dose every ten years. Due to the ubiquitous nature of tetanus spores and the inability to obtain herd-immunity through vaccination, efforts to reduce the incidence of tetanus aim at eliminating the disease. The only way to prevent infection is vaccination followed by recommended periodic booster doses. Between 2012 and 2016, Italy notified 45% (252/564) of all cases reported by the 26 EU Member States, most of them in the over 65 age group, generally women in the rural areas. The recommendation of the antipertussis vaccine, combined with anti-tetanus, in pregnancy and the indications for antitetanic prophylaxis by vaccination or specific immunoglobulins in emergency setting, gives rise to doubts about the risk of hyperimmunization. Studies generally agree on the safety of diphtheria-tetanus-pertussis combined vaccines during the third trimester of pregnancy, and the time elapsed since the previous tetanus vaccination seems not to be related to significant differences in the incidence of adverse events or obstetrical complications. In the emergency wards, given the relatively high incidence of tetanus in Italy, the risk/benefit ratio often leads to prefer vaccination to no-intervention. The administration of tetanus immunoglobulins in subjects not vaccinated in the last 10 years seems justified by the epidemiology of tetanus in Italy.

[Anti-pneumococcus, anti-herpes-zoster and anti-papillomavirus vaccinations: experience in programming continuing education courses in local health units in Rome]. / Vaccinazioni anti-pneumococco, anti-herpes-zoster e anti­ papillomavirus: esperienza nella programmazione di corsi di formazione nelle asl di Roma.

The National Vaccine Prevention Plan considers the recommendations for immune prophylaxis in all ages of life. However, compulsory vaccination introduced in 2017 focused the attention on improving global vaccination coverage in infants and children, giving less attention to adult/elderly vaccinations. The immunization of this population is necessary considering the change in the age structure of the population, whose average life expectancy is increasing. Aim of this work was the organization of continuing education courses about anti-Pneumococcus, anti-Herpes-Zoster and anti-Papillomavirus vaccinations to offer an update of knowledge and to discuss the attitudes of health professionals in vaccination centers of the Local Health Units in Rome.

[Anti-flu Vaccine: tailor made solutions in large-scale distribution]. / Vaccino anti-influenzale: scelte sartoriali nella grande distribuzione.

Influenza is a public health problem with a strong impact from an epidemiological, clinical and economic point of view. Many factors contribute: ubiquity and contagiousness of the disease, antigenic variability of the viruses and the possibility of serious complications. Influenza rarely causes complications in healthy subjects. On the contrary complications can occur and be severe in people with fragile conditions. More susceptible to infection are: children, elderly, pregnant women, people with chronic conditions or undergoing immunosuppressive therapies. Vaccine is an important preventive measure. It allows to protect both the vaccinated person and those at risk. Thanks to the presence of diversified vaccines, we can offer targeted prevention. Vaccine innovations also involved production methods, allowing GPs to choose a quadrivalent cell-derived in addition to the quadrivalent egg-derived and the trivalent vaccine. Age, existing pathologies and immune system reactivity are fundamental elements guiding the customized choice of GPs. Recent years innovations concerning the increased immunogenicity (adjuvated and high-dose vaccines) and the protection against a greater number of strains (quadrivalent vaccine) in addition to new production methods (cell derived vaccine) allow us to trust in further possibilities for the future.

[Old outbreaks and new vaccines: Italy facing Ebola]. / Vecchie epidemie e nuovi vaccini: l'Italia di fronte al problema Ebola.

Zaire is the name of the most deadly Ebola Virus (ZEBOV), which caused many outbreaks in past years and is the causative agent of the most recent one. In July 2019, Ebola was declared a public health emergency of international concern. After this declaration, the steps required to prequalify and approve the r VSV ZEBOV vaccine followed each other quickly. Nowadays in Europe the risk of an outbreak is low. However, secondary cases in USA and Spain have alerted public opinion. Italy is not involved except for the assistance to its citizens infected in countries at risk mainly in contexts of humanitarian aid. In epidemic areas the situation deserves close monitoring, as shown by the interest of the WHO, FDA and EMA. Last year the Italian Ministry of Health issued four circulars on this subject to update epidemic data and procedures and to remember precautions. The live attenuated vaccine in use now shows encouraging results; however, the need to evaluate further vaccines remains to guarantee efficacy, safety and adequate quantities.

Empathy in vaccination counselling: a survey on the impact of a three-day residential course.

BACKGROUND: In an era of hesitance to use vaccines, the importance of effective communication for increasing vaccine acceptance is well known. This study aimed to assess the impact of a three-day residential course concerning empathy and counselling abilities on patients' ratings of the level of empathy of physicians and nurses working in vaccination centers. METHODS: The empathy of healthcare providers was evaluated using the Adapted Consultation and Relational Empathy (CARE) Measure. The survey involved 20 healthcare workers, doctors, and nurses in three immunization services of a Local Health Unit in South Italy. Before and after attending the course, all of them administered the questionnaire to 50 consecutive parents of vaccinated children. Statistical tests were used to assess the homogeneity of pre- and post -course samples, to measure the level of empathy perceived by parents in doctors and nurses in pre- and post-course evaluations, and to compare the average CARE Measure scores among groups. RESULTS: Analysis of the questionnaires showed an increase of "excellent" scores and statistically significant differences between the pre- and post -course median values. Statistically significant differences between doctors and nurses were shown in almost all questions pre-course and in only four questions post-course. CONCLUSIONS: This study demonstrated that a residential course is effective at improving patient-rated empathy of doctors and nurses working in vaccination centers and could result in an increase of parents' adherence to vaccination programs.

[Epidemics of Hepatitis A and opportunities for vaccination: a focus on the category of men who practice sex with men (MSM)]. / Epidemie di Epatite A e opportunità della vaccinazione: un focus sulla categoria degli uomini che praticano sesso con uomini (MSM).

Hepatitis A is an infectious disease caused by a virus (HAV), which is highly contagious and widespread all over the world. In industrialized countries, Hepatitis A is commonly considered a disease with an important socio-economic impact, as the clinical disease affects mostly young adults. After the development of a specific anti-HAV vaccine, a reduction in the incidence of Hepatitis A was observed, with a subsequent change in the prevalence of HAV. HAV spreads mainly in children and risk categories, whose work or activities or medical conditions lead to a close contact with the virus. In particular, in Men who have sex with Men (MSM) many outbreaks have been reported in the last five years. Aim of this paper is an evaluation of the recent literature about HAV infection in MSM in order to update the current guidelines on HAV procedures for MSM.

[Prevention of vector-borne diseases in Italy]. / Prevenzione delle Infezioni trasmesse da vettori in Italia.

In recent years, a constant increase of vector-borne diseases has been seen in Italy, due to various conditions including climate change, trade and international travel. These factors contribute to the diffusion on the Italian territory of newly introduced carriers and pathogens. These new pathogens can be an emerging cause of epidemics, as in the case of the Chikungunya virus in 2007 and in 2017, or can establish a true endemic disease, as in the case of the West Nile virus. It is even possible that diseases that had been endemic in the past, which were subsequently eliminated in Italy, could be reintroduced, as in the case of malaria. This review aims to illustrate the recent evolution of the diffusion of vectors and of the pathogens they convey, and to investigate which emerging pathogens could be responsible in the immediate future for new epidemics / endemics in Italy. Nowadays it is necessary to keep those neglected emerging diseases in mind, in order to establish a correct diagnostic-therapeutic approach at the individual level, and to identify the measures to avoid their diffusion at the community level. Preventive interventions, such as available vaccines or individual protection from vectors, are also discussed.

[Vaccines and sport: risk perception among athletes]. / I vaccini in ambito sportivo: percezione del rischio tra gli atleti.

Infection prevention is a key issue in both general population and in categories at risk, among which the athletes. There are risk factors related to the sporting practice performed at every level, from the amateur to the elite athletes. The control of the vaccination schedule and the empowerment of the sportsman in this area should be carried out both by the General Practitioner or the Pediatrician and by the federations to which the athletes belong. There is a need for guidelines and it is necessary to inform the athletes. This study was performed to evaluate the attitude of groups of athletes regarding infectious diseases and vaccination and confirms the lack of attention towards immunization in sports. Protocols for sport medical examinations, indeed, do not require controls on the state of vaccination and the offer of specific vaccines. It is therefore important the role that scientific societies are taking on in recommending immune prophylaxis for sportsmen.

Herpes zoster in frail elderly patients: prevalence, impact, management, and preventive strategies.

Population aging is a worldwide phenomenon with significant and manifold impacts on society. Advanced age correlates with the onset of frailty. In this vulnerable state, the immune response is weakened and a higher susceptibility to infectious diseases is observed. The present narrative review aims to cover the topic of herpes zoster (HZ) and its complications in frail populations. The lifetime risk of developing HZ is estimated at about 20-30%, and the risk increases with age. In older people, HZ can lead to the inability to recover the lifestyle, the interests, and the level of activity that existed before its development. Severity of the disease at presentation and depression are the major correlates of pain burden in patients with acute HZ and postherpetic neuralgia (PHN). The frail elderly need careful assessment prior to treatment initiation and could be affected to a greater extent by treatment-related adverse events. In light of the significant burden caused by HZ and its complications in the frail elderly, the adoption of a preventive strategy appears to be promising, particularly using vaccination in appropriate age- and risk-groups. Although very few vaccine studies consider explicitly the frail elderly as their study population, there is evidence that the live, attenuated vaccine induces significant immunological responses. An adjuvanted recombinant subunit vaccine has recently been approved in Canada, in the United States, in the European Union, and in Japan, and will likely provide additional opportunities for prevention.

Cost-effectiveness of switching from trivalent to quadrivalent inactivated influenza vaccines for the at-risk population in Italy.

Seasonal influenza is caused by two subtypes of influenza A and two lineages of influenza B. Although trivalent influenza vaccines (TIVs) contain both circulating A strains, they contain only a single B-lineage strain. This can lead to mismatches between the vaccine and predominant circulating B lineages, a concern especially for at-risk populations. Quadrivalent influenza vaccines (QIVs) containing a strain from both B lineages have been developed to improve protection against influenza. Here, we used a cost-utility model to examine whether switching from TIV to QIV would be cost-effective for the at-risk population in Italy. Costs were estimated from the payer and societal perspectives. The discount rate for outcomes was 3.0%. Univariate and probabilistic sensitivity analyses were performed to examine the effects of variations in parameters. Switching from TIV to QIV in Italy was estimated to increase quality-adjusted life-years (QALYs) and produce cost savings, including €1.6 million for hospitalization and approximately €2 million in productivity. The incremental cost-effectiveness ratio was €23,426 per QALY from a payer perspective and €21,096 per QALY from a societal perspective. Switching to QIV was most cost-effective for individuals ≥ 65 years of age (€19,170 per QALY). Probabilistic sensitivity analysis showed that the switching from TIV to QIV would be cost-effective for > 91% of simulation at a maximum willingness-to-pay threshold of €40,000 per QALY gained. Although the model did not take herd protection into account, it predicted that the switch from TIV to QIV would be cost-effective for the at-risk population in Italy.