Arthropod-borne viruses are a major cause of emerging disease with significant public health and economic impacts. However, the factors that determine their activity and seasonality are not well understood. In Australia, a network of sentinel cattle herds is used to monitor the distribution of several such viruses and to define virus-free regions. Herein, we utilize these serological data to describe the seasonality, and its drivers, of three economically important animal arboviruses: bluetongue virus, Akabane virus and bovine ephemeral fever virus. Through epidemiological time-series analyses of sero-surveillance data of 180 sentinel herds between 2004–2012, we compared seasonal parameters across latitudes, ranging from the tropical north (−10°S) to the more temperate south (−40°S). This analysis revealed marked differences in seasonality between distinct geographic regions and climates: seasonality was most pronounced in southern regions and gradually decreased as latitude decreased toward the Equator. Further, we show that both the timing of epidemics and the average number of seroconversions have a strong geographical component, which likely reflect patterns of vector abundance through co-varying climatic factors, especially temperature and rainfall. Notably, despite their differences in biology, including insect vector species, all three viruses exhibited very similar seasonality. By revealing the factors that shape spatial and temporal distributions, our study provides a more complete understanding of arbovirus seasonality that will enable better risk predictions. Arthropod-borne viruses (arboviruses) are a group of viruses that can have major impacts on public health, animal health and agricultural trade, and appear to be increasing in both number and prevalence worldwide. Despite their importance as emerging pathogens, the spatial patterns, long-term seasonal characteristics and drivers of seasonality in many arboviruses are poorly understood. The island continent of Australia provides an ideal case study for the spatial analysis of emerging arboviruses, harboring diverse climatic conditions across a wide range of latitudes. Herein we utilize long-term serological data from a nationwide network of sentinel herds in Australia to describe the seasonality of three economically important animal arboviruses: bluetongue virus, Akabane virus and bovine ephemeral fever virus. Using epidemiological time series analysis, we demonstrate that these viruses exhibit a distinct spatial pattern in both the peak timing and intensity of annual epidemic cycles, with the strongest seasonality observed in southerly geographic regions. In addition, we reveal the climatic factors that drive patterns of arbovirus distribution and, by doing so, provide a more complete understanding of arbovirus seasonality, which in turn will improve the risk assessment of these viruses. Arthropod-borne viruses (arboviruses) are one of the most important categories of emerging pathogens. They cause a wide range of diseases in humans and domestic animals, and many are increasing in global distribution as a result of climate change, urbanization and changing patterns of travel and trade Australia, which spans tropical to sub-tropical latitudes favorable to arthropod survival, experiences a wide range of arthropod-borne viruses. In addition, as an island continent with a strict quarantine policy and extensive disease surveillance systems, it presents an extremely informative study population. Human populations in Australia experience seasonal outbreaks of infection with Ross River, Barmah Forest, Murray Valley encephalitis and West Nile (Kunjin strain) viruses, as well as outbreaks of dengue fever, which is introduced sporadically by travellers. Other arboviruses affect wildlife and livestock and have the potential to severely impact the Australian economy Orbivirus, family Reoviridae . Bluetongue disease primarily affects sheep and white-tailed deer, causing acute and widespread haemorrhaging and ulceration of the oral and nasal tissue, coronitis and laminitis, and a pulmonary edema that can be fatal Bunyavirus, family Bunyaviridae , which also contains Schmallenberg virus and Aino virus. AKAV has been reported in several African countries, Israel, Turkey, Korea, Japan and Australia Ephemerovirus, family Rhabdoviridae . It occurs as a single serotype and infects wild and domestic ruminants across a vast area of Africa, the Middle East, Asia and Australia, causing ‘three-day sickness’, a highly debilitating febrile disease in cattle and water buffalo. Although long-term sequellae and mortalities can occur, the disease and the viraemia are usually of short duration (approximately three days) and primarily affect milk production Three major arboviruses affect domestic and wild ruminants in Australia: bluetongue virus (BTV), Akabane virus (AKAV) and bovine ephemeral fever virus (BEFV). BTV is regarded as a globally important emerging pathogen, with many of the 26 serotypes occurring on all continents other than Antarctica. The virus is classified in the genus Culicoides brevitarsis although other Culicoides species  have been found to play a role in BTV transmission and have different efficiencies and distributions Culicoides are found worldwide, with the exception of New Zealand and Antarctica, and, in light of the recent emergence of BTV and Schmallenberg virus in Europe, Culicoides-borne arboviruses appear to have the potential to spread rapidly in previously unaffected geographic regions Culex annulirostris, thought to be the major vector in Australia Cx. annulirostris is similar to that of C. brevitarsis, it is often more widespread Anopheles bancrojtiiCx. annulirostris in a region containing many of the sentinel herds. This suggests that mosquito species involved in BEFV transmission are subject to regional disparities, or that the virus was absent in this area during this time.The distribution of arbovirus vectors is determined by complex interactions between climate, geography and their animal hosts C. brevitarsis exploits bovid dung for breeding sites, such as those found near farmlands Cx. annulirostris breed in a variety of habitats, typically in temporary ground pools on grassland and in freshwater ponds, swamps and lakes, and its emergence in large numbers follows heavy rains. The availability of these resources and environments, as well as suitable climatic conditions, are key determinants of vector geographic distribution.In both midges and mosquitos, blood-feeding is restricted to adult females who utilize protein for egg production. Since 1969, sentinel herds have been employed in Australia to monitor for arbovirus activity Arboviruses often display well-defined seasonal peaks in temperate climates compared to tropical regions where annual patterns are not as clear The quality and quantity of the Australian national surveillance data on BTV, AKAV and BEFV, as well as its coverage across a wide range of latitudes, and hence climatic conditions, enables us to determine patterns of arbovirus distribution in time and space, thereby defining their seasonal characteristics. Using epidemiological time-series analyses, the present study draws on this data set to reveal the factors that drive the seasonality of a number of economically important arthropod-borne viruses.http://www.animalhealthaustralia.com.au, although not publicly available). The locations of sentinel herds are based on defining the transmission, surveillance and free zones for trade, which can be found in coastal and hinterland areas where viruses are prevalent, and largely absent in the central, desert region due to the inhospitableness of these areas and the absence of cows and sheep  which measures BTV-specific antibodies without detecting cross-reacting antibodies to other orbiviruses known to circulate in Australia Culicoides, spp. were sorted, identified by wing pattern and counted.Light traps for the collection of biting midges were placed near sentinel herds. These traps employ green light-emitting diodes and were deployed before dusk and remained in the field for no more than two nights. Insects were collected monthly, throughout the year in northern Australia (12 collections), from December to May in intermediate locations (6 collections) and from December to March in southern Australia (4 collections). Insects were collected into 70% ethanol and Culicoides trapped. In our analysis we have included four important Culicoides species: C. brevitarsis, C. fulvus, C. actoni and C. wadai, all of which have different efficiencies and distributions as vectors. It is important to note that in this chosen data set there is no distinction between midge sex, nor does it contain information regarding other insect vectors such as mosquitoes. In addition, all collections and isolations are undertaken by NAMP.Sentinel cattle herds and insect collections have been operating in Australia since 1969. However, we restricted our analysis to data collected between July 2004 and June 2012 because sampling over this period was more consistent across all monitoring locations compared with previous years . In the southern hemisphere the vector season begins in July and ends in the following June. Accordingly, we analyzed data over these months. Each month, the number of seroconversions to AKAV, BEFV and BTV were reported from a total of 180 sentinel herds, as well as the number of To obtain a measure of the seasonal activity for these viruses, time-series analyses were conducted using the Epipoi epidemiological software package To determine seasonal drivers of arbovirus infection in Australia we focused on climate data as seasonal predictors. Maximum and minimum monthly temperature (°C) and rainfall (mm) between July 2004 and June 2012 were obtained from the Australian Bureau of Meteorology. These data were collected from 31 weather stations in close proximity (within one degree of latitude and longitude) to at least one sentinel herd that detected positive serology within this time frame . SeasonaTo visualize seasonal changes in arbovirus activity across climatic conditions, we analyzed the number of seroconversions to BTV, AKAV and BEFV in four distinct climate regions across Australia: tropical, grasslands, arid and warm-temperate. Notably, our time-series analysis reveals a well-defined annual periodicity  of all three viruses in warm-temperate regions . GrasslaSeasonal parameters, such as the timing and amplitude of the annual primary peak, were extracted from the time series . OverallPeak amplitude, which is a measure relative of the magnitude of the average seasonal signature across all years, was obtained by dividing the wave height  by the peak value The percentage of seroconversions to all three viruses was greater in northern regions between August and October, during which time very few seroconversions occurred in the south, coinciding with low temperatures that are likely to affect arthropod blood-seeking behavior On average, the seroconversions to BTV, AKAV and BEFV increased significantly as latitude decreased towards the Equator (C. brevitarsis trapped) and geographic data (latitude) as predictors of arbovirus seasonal characteristics, using only data from herds located in close proximity to a weather station. Geographic and climate variables were often strongly associated with seasonal characteristics for all three arboviruses  and temperature (°C), along with entomological  with well-defined annual periodicity; a pattern comparable to the seasonality of BTV in California where the majority of seroconversions also occur during Autumn (September–October) Culicoides spp., AKAV has been isolated from both Culicoides and mosquitoes Culicoides spp. compared to orbiviruses. This was evident during the recent outbreak of Schmallenberg virus versus BTV in Europe, where Culicoides populations were more susceptible to the former Culicoides are very efficient vectors for the closely related AKAV Also of note was that the relative amplitude of the annual primary peak varied across latitudes for all three viruses. Peak amplitude was strongest in the south and decreased as latitude drew closer to the Equator. In contrast, the proportion of seroconversions to all three viruses increased significantly as latitude decreased. The number of seroconversions is, on average, greater in northern, tropical regions compared to southern, more temperate, regions. This pattern is consistent with conditions favorable for increased arthropod population size, as well as shorter extrinsic incubation periods of virus development. Finally, although AKAV and BTV are taxonomically assigned to different virus families with different genome architecture and modes of replication and transcription, they exhibited strongly congruent spatial and temporal patterns, reflected in their shared host range and transmission by biting midges. Nevertheless, AKAV was proportionally more dominant during warmer months than BTV. One possible explanation for this disparity is that while BTV has only ever been isolated from Culicoides spp. and Culex annulirostris, and thus their associated viruses, is strongly influenced by weather patterns. For example, C. brevitarsis larvae and pupae can survive only when winter temperatures are mild. If temperatures are too low  or too high , larval survival diminishes Cx. annulirostris, is less susceptible to climatic extremes compared to the biting midge, C. brevitarsisC. brevitarsis has a strict need for cattle dung whereas Cx. annulirostris breeds in freshwater habitats that are typically associated with heavy rainfall.As well as revealing arbovirus seasonality, we also set out to reveal its underlying causes. Climatic factors such as temperature and rainfall affect the spread of arboviruses by influencing vector behavior and survival Consistent with global climate trends, long-term climatic data in Australia show that temperature is increasing Serological surveillance of sentinel herds – which covers a vast geographical scale across Australia, including the island of Tasmania – is a powerful tool for monitoring the epidemiology of arboviruses. Herein, we have utilized these data to undertake a novel investigation of arbovirus seasonal characteristics using time-series analysis and from this determine the drivers of seasonality in three economically important animal arboviruses. Accordingly, this study has revealed important differences in arbovirus seasonality across a wide range of latitudes in Australia, covering tropical to subtropical regions, and shown how the interaction between climatic and vector abundance shapes patterns of viral seasonality and transmission. Although the current study does not account for abiotic factors that may also influence patterns of viral spread, such as the human-mediated movement of livestock within Australia, these are likely to be of negligible importance in the overall seasonal pattern. Finally, although there is an increasing availability of genome sequence data from these viruses that might ultimately reveal aspects of viral population structure and migration e.g., , includiFigure S1Plots depict the prevalence of Akabane virus (left), bovine ephemeral fever virus (center), and bluetongue virus (right). The proportion of positive seroconversions as a function of latitude is shown in the upper panel and semivariograms are shown in the lower panel. Colors in the upper panel correspond to different climate regions . Pearson's R and Spearman's rho are indicted on the plot.(TIF)Click here for additional data file.Figure S2Mean number seroconversions per month between 2004–2012 (with error bars showing the standard error of the mean) ; and the mean percentage seroconversions per month, comparing each virus in northern regions (latitudes north of −23°S) and southern regions (latitudes south of −24°S) (bottom panel – (d): AKAV and BTV; (e): AKAV and BEFV; (f): BEFV and BTV). The x-axis ranges from July through June, representing start-to-finish of the vector season.(TIF)Click here for additional data file.


Osteoporotic fractures cause a large health burden and substantial costs. This study estimated the expected fracture numbers and costs for the remaining lifetime of postmenopausal women in Germany.A discrete event simulation (DES) model which tracks changes in fracture risk due to osteoporosis, a previous fracture or institutionalization in a nursing home was developed. Expected lifetime fracture numbers and costs per capita were estimated for postmenopausal women (aged 50 and older) at average osteoporosis risk (AOR) and for those never suffering from osteoporosis. Direct and indirect costs were modeled. Deterministic univariate and probabilistic sensitivity analyses were conducted.The expected fracture numbers over the remaining lifetime of a 50 year old woman with AOR for each fracture type (% attributable to osteoporosis) were: hip 0.282 (57.9%), wrist 0.229 (18.2%), clinical vertebral 0.206 (39.2%), humerus 0.147 (43.5%), pelvis 0.105 (47.5%), and other femur 0.033 (52.1%). Expected discounted fracture lifetime costs (excess cost attributable to osteoporosis) per 50 year old woman with AOR amounted to €4,479 . Most costs were accrued in the hospital €1,743 (€751) and long-term care sectors €1,210 (€620). Univariate sensitivity analysis resulted in percentage changes between -48.4% (if fracture rates decreased by 2% per year) and +83.5% (if fracture rates increased by 2% per year) compared to base case excess costs. Costs for women with osteoporosis were about 3.3 times of those never getting osteoporosis , and were markedly increased for women with a previous fracture.The results of this study indicate that osteoporosis causes a substantial share of fracture costs in postmenopausal women, which strongly increase with age and previous fractures. With a lifetime risk of 40% to 50%, an osteoporotic fracture is one of the most likely negative health events in the remaining lifetime of a 50 year old woman. The indStudies have shown that a decrease in bone mineral density (BMD) is a strong predictor for fractures on different sites of the human body-6. AccorOsteoporotic fractures have serious consequences on the individual and the societal level. Fracture patients often have a decreased functional mobility, health Internationally, the economic burden of osteoporosis and osteoporotic fractures has frequently been investigated in the last decade,18-21,23Our study had different aims. First, a discrete event simulation model (DES) was developed to estimate the expected lifetime fracture numbers and costs of six osteoporotic fracture types for 50 year old women in Germany. Second, the same estimates for hypothetical women who never suffer from osteoporosis over their remaining lifetime were made. Third, lifetime fracture costs attributable to the risk factor osteoporosis (excess cost) were calculated which indicate the economic potential of preventing osteoporosis as the main risk factor for fractures. Finally, lifetime fracture costs were estimated for women with different risk profiles  and at different starting ages. In summary, our model should give a deeper insight into the economics of six osteoporotic fractures in Germany, and should provide new and valuable information to the international literature.A DES model-31 was dA DES is an individual based simulation technique where all individuals are simulated one by one. The maiIn order to estimate the expected lifetime fracture numbers and costs of a 50 year old woman, we conceptualized a “time driven” DES with time intervals of one year, no resource constraints and no interaction between individuals. The patient flow diagram in Figure There is evidence that fractures increase the probability of institutionalization in a NH. TherefoNot all fracture patients are treated in an inpatient setting . Therefore, the model determines on the basis of fracture specific hospitalization probabilities, whether a fracture patient is treated in a hospital or exclusively treated in an outpatient setting. If a patient is treated in a hospital, the model tracks acute hospital and post-hospital outpatient treatment costs; otherwise only outpatient costs are tracked. Also, not all patients receive inpatient rehabilitation after a hospital stay. For this reason, the same cost tracking approach, based on fracture specific post-hospital rehabilitation probabilities, was applied for rehabilitation costs. For tracking fracture-attributable long term care costs, a parallel background simulation approach was implemented. The appAll other cost categories in the model are tracked as unit costs (rewards) per fracture event, similar to the common cohort model approach. A detaiIn total, two risk groups of 200,000 hypothetical women (simulation runs) each were simulated through the model. For the first risk group it was assumed that these women were at average osteoporosis risk over their remaining lifetime , whereas the second risk group was assumed to be free of osteoporosis over their remaining lifetime. To determine osteoporosis-attributable fracture costs (excess costs), the difference in costs between the two considered risk groups was calculated. To elimAge-dependent fracture probabilities for all considered fracture types were calculated based on official German population and hospThe calculated “total” fracture probabilities pertain to the German female general population (see Table HipPreviousFxvs.NoPreviousFx) and other osteoporotic fractures (RROsteoPreviousFx vs. NoPreviousFx), comparing women with a previous fracture to those without a previous fracture, were taken from a meta-analysis[NoPreviousFx vs. GpFx) were estimated. The calculation method was adopted from Schousboe et al.[Clinical studies showed that a previous fracture is an important risk factor for a subsequent fracture, even after adjustment for BMD. The ageanalysis. Howevere et al. using thAnyPreviousFx vs. NoPreviousFx) and age-dependent prevalence rates for previous fractures (PrevPreviousFx) were both taken from the previously mentioned meta-analysis[NoPreviousFx vs. GpFx were multiplied with the fracture probabilities of the general population to obtain fracture probabilities for women without a previous fracture. If a woman suffered from a fracture in the model, further fracture probabilities were calculated by combining fracture probabilities for women without a previous fracture with RRHipPreviousFx vs. NoPreviousFx and RROsteoPreviousFx vs. NoPreviousFx depending on the fracture type . The RROST were estimated based on a method described by Kanis et al.[fx)[Fracture probabilities for women with osteoporosis were derived by multiplying the age-dependent female general fracture probabilities with age and fracture specific relative risks for women with osteoporosis compared to the female general population -6, the o al.[fx), and age al.[fx) were tak al.[fx)  of women born in 1959 was used to reflect a realistic remaining life expectancy for a 50 year old woman living in the year 2009 (start year of the model). Dependithree main categories: inpatient costs include costs due to acute hospital care, rehabilitation after hospitalization and long term care, outpatient costs include costs due to physician and physiotherapist visits, analgesics (medication) as well as home care, and family costs include informal care costs. An overview over the main direct unit costs is shown in Table Fracture related unit costs were estimated from a societal perspective for Germany. The base year of cost evaluation was 2009 and a discount rate of 3% was applied. IncludeRehabilitation costs after a hospital stay were estimated by combining the fracture type related duration of an inpatient rehabilitation treatment[Long term care costs are based on the official care statistic 2009[In Germany, hospitals are reimbursed according to a dual system: Operating costs due to direct resource uses are paid on the basis of a German version of diagnosis related groups (G-DRG) by health insurance funds (private or mandatory), whereas capital costs are compensated by federal states. Thereforeatment with a dreatment (see Addtic 2009. This sttic 2009 were addFracture related costs for physician visits (including outpatient surgeons), physiotherapeutic treatments and analgesics for patients exclusively treated in the outpatient sector, as well as outpatient cost for post-hospital treatment were estimated using German unit costs, inflateCosts for home care per fracture type were determined based on information about fracture type specific hours of home care needed and hourTo reflect the fracture related costs of informal care, fracturThe model considers two categories of productivity costs. First, loss in productivity due to the fracture related inability to perform paid work, and second, the productivity loss due to osteoporosis related premature deaths. ProductIn order to improve model transparency, all model assumptions are presented in Table A number of scenarios of direct lifetime fracture costs were analyzed for women with different start characteristics. Each considered scenario consists of a combination of four of the following (model) start characteristics: Age (50 or 75), residential status (community dwelling (C) or NH (N)), disease status , at average risk of getting osteoporosis (avO) or not at risk of getting osteoporosis (nO)) and fracture history (with a previous fracture (P) or with no previous fracture (nP)). In total we modeled 18 scenarios, and each scenario was labeled based on the combination of the four start characteristics e.g. a 50 year old woman, community dwelling with average risk of getting osteoporosis and no previous fracture was labeled as 50_C_avO_nP in the results section. All scenarios were simulated separately, direct costs are presented undiscounted and discounted by 3%.Deterministic one way and probabilistic sensitivity analyses were conducted. We performed 20 deterministic sensitivity analyses (S1-S20) to evaluate the impact on excess costs due to osteoporosis-attributable fractures:S1: We valued informal care based on the opportunity cost approach with an In order to perform a probabilistic sensitivity analysis, we simulated 10,000 hypothetical women for each risk group (simulation runs (n)) 1,500 times (simulations (m)) by varying parameters simultaneously for each of the 1,500 simulations (second-order uncertainty). Common Figure Table Table Considering discounted productivity costs due to the inability to perform paid work, we found only low excess lifetime costs per capita of around €63 applying the human capital approach and €22 applying the friction cost approach.The scenario analysis in Figure The results of the deterministic sensitivity analyses are presented in Figure internal validation of our model we compared the modeled fracture incidence rates with the expected fractures rates (input data) of the female general population. We also reported the distribution of the number of fractures per woman to support internal validity and transparency of the model estimates. Table For an The expected and modeled fracture incidence rates are very similar Table , which sexternal model validation, the modeled hip fracture incidence rates were compared to epidemiological studies from Sweden, USA and UK . For both risk groups, around 70% of costs were due to inpatient treatment, like acute hospital treatments and long term care. The fracture type with the largest proportion of direct lifetime costs were hip fractures.In a previous study, we estiA modeling study from Hiligsmann et al. conducteIn comparison to other models in the field of osteoporotic fractures, our model has some strengths. Compared to a Markov cohort model our model allows calculating event probabilities dependent on the history of a modeled individual, which overcomes the classical Markov assumption of “memorylessness”. AnotherIn terms of cost modeling, a main strength of our model is the large number of cost categories included and the way how fracture attributable long term costs are modeled. Using the parallel simulation of NH entries under real world NH mortality allowed us to estimate fracture attributable long term care costs more precisely.fx only to hip fractures, but not to non-hip fractures. If the age related patterns of RRfx for hip fractures are similar to other fracture types, the fracture risk for non-hip fractures in women with osteoporosis may be overestimated in older, and underestimated in younger age classes. In the model we applied osteoporosis prevalence rates and BMD data from a US population (NHANES III study). Applying these data was necessary, because no German study determined osteoporosis prevalence rates in a comparable design to the NHANES III study (in terms of osteoporosis definition based on BMD (T ≤ -2.5), sample size and study quality[The results of model-based studies should be interpreted against the background of the model assumptions, e.g. model type, time interval, input data. In the model, we applied age-dependent fracture specific RR quality). Furthe quality. In termIn terms of cost data, there are further limitations. We did not consider costs due to transport, medical aids and early retirement. Cost for osteoporosis specific medication was also not considered. The main reason for neglecting medication costs was based on the aim of the study to determine fracture costs attributable to the risk factor osteoporosis, and not the cost of treating osteoporosis. A study which analyzed German insurance claims data found that the share of osteoporosis medication costs was low in comparison to other fracture attributable cost categories. HoweverThe estimation of expected lifetime fracture numbers and costs is highly uncertain in terms of changes in age-adjusted fracture incidence rates (secular trends). Studies from Europe showed an inconsistent picture of changes in age-adjusted fracture incidence rates, with increasing and decreasing trends reported in the last years. Potential reasons for an increase in age-adjusted hip fracture rates may be higher survival rates in very old and fragile persons, a decrease in physical activity and deficiency in vitamin D. HoweverThe results of this study show that fractures cause relevant costs in postmenopausal women, which strongly increase with age, previous fractures and osteoporosis status. The high costs in age classes over 70, in combination with demographic change, could in addition intensify the economic burden due to fractures in Germany. Thus, tIn the future, our model can be used to perform cost-effectiveness analyses of fracture prevention interventions in Germany. Beside the described model functions, we optionally implemented an osteodensitometry module. This feature makes the modeling of different screening to treat scenarios for Germany possible. Moreover, the real-world adherence of medical anti-osteoporotic therapy can be realistically implemented in the model. As prior studies found cost-effectiveness results to be highly sensitive to medication adherence assumptions, a realistic implementation of the respective empirical evidence seems particularly important in health economic modeling of osteoporotic fractures.This is the first analysis modeling the epidemiology and costs of fractures for Germany. The analysis shows that 44.5% of all discounted direct fracture lifetime costs of a 50 year old woman are attributable to osteoporosis. For a 50 year old woman already suffering from osteoporosis, fracture lifetime costs are expected to be 3.3 fold the fracture costs of a healthy woman at the same age. The main cost drivers are hospital and long term care costs, causing around 70% of total fracture related lifetime costs. Beyond cost of illness, the model can be used to evaluate the cost-effectiveness of fracture prevention.The authors declare that they have no competing interests.FB and HHK conceived the analysis. FB developed the simulation model, conducted the analyses and drafted the manuscript. FB and AJ prepared data as input for the model. KR, CB, HHK, FB, AJ participated in study design and implementation. HHK, AJ, KR and CB critically revised the manuscript for important intellectual content. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6963/14/284/prepubFile name: Bleibler_BMCHSR_ESM.pdf . Includes all used input data of the model).Click here for file


To the Editor: Circulation of new arboviruses of the genus Flavivirus poses a major problem for differential diagnosis. Zika virus, a mosquitoborne virus of the family Flaviviridae, is closely related to other arboviruses circulating in the Americas, including dengue, yellow fever, Saint Louis encephalitis, and West Nile viruses  antigen. Gyurech et al. (Cross-reactivity in NS1 dengue tests (ELISA and immunochromatographic) using serum samples from patients with acute Zika virus infection would have medically significant consequences. We therefore conducted a retrospective analysis of the differential diagnosis for dengue and Zika virus infections since the beginning of the Zika virus outbreak in French Guiana, a department of France on the northeast coast of South America.French Guiana is subject to endemoepidemic circulation of dengue and experienced a large outbreak of chikungunya in 2014. We conducted our study from December 17, 2015 (the time of biologic confirmation of the first case of Zika virus disease in French Guiana), through March 2, 2016. During that time, the incidence of dengue virus infection in French Guiana was low, and only 1 sporadic case was confirmed. We studied clinical samples collected during this period from all patients with suspected arbovirus infection. Samples were analyzed for the differential diagnosis of dengue, chikungunya, and Zika virus disease. Zika virus diagnosis was conducted by the National Reference Centre for Arboviruses (NRC) at the Institut Pasteur of French Guiana, in Cayenne, according to the real-time reverse transcription PCR (rRT-PCR) protocol described by Lanciotti et al.  were 33.0 (± 3.4) for serum samples and 33.2 (± 3.5) for urine samples . Of the This retrospective analysis of dengue and Zika virus diagnoses indicates that no false-positive dengue NS1 test results occurred among samples with acute-phase Zika virus infection. Indeed, samples from all 65 patients with rRT-PCR–confirmed acute Zika virus infection were negative by both dengue NS1 tests. Zika virus is closely related to dengue virus, and during the acute phase of disease, Zika virus might release NS1 into patients’ serum; however, this putatively released nonstructural protein does not appear to cross-react with the dengue NS1 tests used in our study. No Zika NS1 antigen assay currently exists, and acute-phase release of Zika NS1 has not been verified. If a Zika virus NS1 test is developed, it should be evaluated for cross-reactivity with serum from patients with acute dengue infection. The false-positive result reported by Gyurech et al. (The co-circulation of Zika virus and dengue virus in the Americas is causing a health emergency. Our findings show that dengue NS1 antigen assays are still entirely appropriate for dengue surveillance, even during the epidemic circulation of Zika virus.


Our aim was to elaborate a novel method for fully controllable large-scale nanopatterning. We investigated the influence of the surface topology, i.e., a pre-pattern of hydrogen silsesquioxane (HSQ) posts, on the self-organization of polystyrene beads (PS) dispersed over a large surface. Depending on the post size and spacing, long-range ordering of self-organized polystyrene beads is observed wherein guide posts were used leading to single crystal structure. Topology assisted self-organization has proved to be one of the solutions to obtain large-scale ordering. Besides post size and spacing, the colloidal concentration and the nature of solvent were found to have a significant effect on the self-organization of the PS beads. Scanning electron microscope and associated Fourier transform analysis were used to characterize the morphology of the ordered surfaces. Finally, the production of silicon molds is demonstrated by using the beads as a template for dry etching. The development of nanoscience and nanotechnology has led to a continuous miniaturization of microelectronic components. 193 nm optical-mask lithography is the driving force behind this evolution , extremeIn this study, we concentrated on topology-assisted self-organization by using polystyrene beads (PS) dropped on a patterned silicon substrate. We optimized the self-organization of the PS beads on the silicon substrate, wherein we observed that the deposited particles (PS beads) are very sensitive to different physico-chemical parameters. Colloidal concentration and the nature of the solvent were the two important parameters which affected the self-organization of the beads. The optimisation of these parameters allows us to produce large-scale colloidal 2D monocrystals. Another problem addressed in this study was the presence of dislocations in these PS bead patterns. By using patterned silicon substrates, we were able to minimize the presence of these dislocations resulting in the formation of single crystal structures of PS beads on the entire pre-patterned area, which is scalable depending on the technique used for pre-patterning.In our experiments, only the colloidal concentration and the nature of the solvent were addressed. First, we used only water as a solvent, but the drop did not cover the entire surface due to hydrophobicity. Hence, we decided to replace water by ethanol thereby increasing the wettability of the surface. With only ethanol, we were able to cover the entire surface, but the overall structure was not ordered even within a short distance. Therefore, we used a solution of 50% water and 50% ethanol. The use of this solvent mixture resulted in well-dispersed and ordered structures with the presence of micron-sized dislocations. We further decreased the concentration of ethanol to 20%. With this lesser amount of ethanol, we were able to decrease the width of the dislocations observed with the mixture of water and ethanol in a 1:1 ratio. We also changed the initial concentration of the solution which was 0.2 to 3 wt %. It was found that when the concentration is very high, the surface occupied by a multilayered structure becomes larger. If, on the other hand, the concentration is low we notice the presence of large empty spaces between the structured areas. Moreover, in the presence of a low concentration many particles adhere to the surface of the substrate before reaching the ordered regions and form small aggregates consisting of several particles. With the concentration used (1 wt %) after optimization we had a maximum thickness of 1.2 microns, which corresponds to 3 layers of beads. Moreover, multilayered assembly of the particles occurs at the edge of the sample. However, by forming patterns at the center of the sample prevents the formation of multilayers. The development of self-organization of PS beads on silicon substrates allows us to obtain a large-scale ordering (of the order of a few centimeters), but the problem of the presence of dislocations in the structure still remains. In this work, as already mentioned, our goal was to use the pre-patterned substrates as a means to form single crystal 2D structures especially on a large scale. The SEM images in Although self-organization presents a simple structure obtained with different grain orientations, the presence of dislocations limits its applications. So the role of assisted self-organization is to obtain a structure with a single orientation based on a hexagonal lattice of HSQ posts. When we compare the structures obtained by these two techniques, it is clear that assisted self-organization occurs in the presence of the posts, and single crystal structures of the PS beads on the entire pre-patterned area is obtained. Indeed, the presence of these posts allows us to control the self-organization and get a 2D structure with a well-defined orientation and long-range order as seen in the SEM images in By varying the post diameter, we tried to understand its influence on the self-organization. In order to form a single crystal structure without defects, we used different post sizes: 195 nm, 296 nm, 390 nm and 420 nm, respectively. The effect of dry etching on the patterned silicon has also been studied. Silicon substrates with PS beads on them are directly etched –24. FiguIn summary, we demonstrated a simple, inexpensive and rapid procedure to produce large-scale colloidal 2D monocrystals. The technique is based on the topology assisted self-assembly of polystyrene beads (PS) on patterned silicon surfaces. The development of self-organization of PS beads on silicon substrates allows us to obtain a single crystal, which is 100 times larger compared to the ones on non-patterned Si surfaces. The actual size of the monocrystal is limited by the pre-patterned area. To yield well-defined structures, we optimized the parameters of the patterned posts. The best result was obtained for the sample with a post diameter identical to the beads diameter and a pitch corresponding to 5 to 7 times the latter. Finally, the 2D monolayers were used as etching masks to realize silicon mold. As a perspective, it is worth mentioning that this study of assisted self-organization of PS beads can be extended to a variety of substrates of different materials. This paves the way to large-scale patterning applications as an example of biosensitive surfaces useful for detecting proteins and cells. Patterned substrates using assisted self-organization can also be employed as templates for the selective growth of semiconducting materials.Polystyrene beads with a diameter of 400 nm suspended in an aqueous solution (1 wt %) were purchased from AGAR scientific Ltd. 20% ethanol and 80% de-ionized water were used for the dispersion of the beads. The substrates used were (100) oriented n-type silicon.2 were fabricated on a Si substrate of 2 × 2 cm2. Si substrates were cleaned with acetone and then with a piranha solution for 24 h at 80 °C. After this step, the samples were left in a beaker containing ionized water for 12 h. The samples were dried immediately after use. HSQ is a radiation sensitive spin on glass which forms silica-like materials directly upon exposure to an electron beam. Development of these samples reveals the exposed posts, without requiring further etching or processing. We tried various diameters and pitches of the posts obtained by electron beam lithography and studied their influence on the self-organization of the beads. Diameters of these posts ranged from 195 nm to 420 nm with a height of 600 nm and the pitch varied between 5 to 20 times the diameters of the beads  resist layer on a Si substrate. 20 patterns each with a size of 500 × 500 µmhe beads .PS beads were deposited on the silicon patterned substrates by a convective self-assembly technique. 6 and O2. The regions covered by the beads are not etched by RIE. In this manner, we obtained a pattern on silicon. Beads with a diameter of 400 nm were used for patterning. The total etching time utilized was 4 min.Reactive ion etching (RIE) was implemented for the dry etching of the silicon surfaces by using the beads as a mask. The gases used for RIE were SFOptical microscopy, scanning electron microscopy (SEM) , and associated Fourier transform analysis were employed to characterize the structure of the PS bead arrays on the patterned substrate.


Ciona intestinalis, commonly known as a ‘sea squirt’, has become an important model for embryological studies, offering a simple blueprint for chordate development. As a model organism, it offers the following: a small, compact genome; a free swimming larva with only about 2600 cells; and an embryogenesis that unfolds according to a predictable program of cell division. Moreover, recent phylogenies reveal that C. intestinalis occupies a privileged branch in the tree of life: it is our nearest invertebrate relative. Here, we provide an organismal perspective of C. intestinalis, highlighting aspects of its life history and habitat—from its brief journey as a larva to its radical metamorphosis into adult form—and relate these features to its utility as a laboratory model.The ascidian DOI:http://dx.doi.org/10.7554/eLife.06024.001 Ciona intestinalis spends its adult life anchored to a hard substrate, filter feeding and releasing gametes (eggs and sperm) into the surrounding sea water. For a fleeting day or two of its life, however, the larva of C. intestinalis adopts a tadpole morphology  within the molluscs, based on their adult form (The tunicate (sea squirt) rphology . This morphology . Prior tult form .10.7554/10.7554/eLife.06024.003Box 1.Ascidian—A common name for a member of the Ascidiacea, a class within the Tunicata. Ascidians include C. intestinalis and other models like Botryllus schlosseri, Phallusia mamillata and roretziHalocynthia .Biofouling—The accumulation of organisms on underwater surfaces such as pilings, boats, etc.Cephalochordate—These coastal-dwelling fish-like chordates (up to about 7 cm length) are filter feeders and lack vertebra and key head sensory apparatus. They feature somites, highlighting them as a characteristic of the common chordate ancestor, indicating their loss in tunicates.Chordate—This phylum includes tunicates, cephalochordates and vertebrates, and their features include the notochord, dorsal nerve cord, post-anal tail, and a muscular tail or somites. Some investigators consider the endostyle/thyroid to be a shared feature, while pharyngeal gill slits, although shared by all chordates, are not unique to the phylum and are found in related non-chordates as well.Hox cluster—An array of Hox genes along a single chromosome. These genes have conserved patterning functions in animal development. Their order within a chromosomal cluster reflects the anterior-posterior order in which they are expressed during development.Gastrulation—A morphological event early in embryogenesis, leading to the formation of ectoderm, mesoderm and endoderm, the precursors of all adult tissues.Larvacean—An order within the tunicates, larvaceans are so-called because of their retention of a larval-like body, most prominently the larval tail, into adult life.Neural crest cells—Multipotent migratory cells in vertebrates that form or contribute to (among other derivatives) specialized sensory and feeding structures, including the jaw and skull, the inner ear and the lateral line sensory system.Neurogenic placodes—Similar to neural crest in their contributions, these structures originate as local thickenings in the embryonic ectoderm. Their derivatives include sensory neurons of the olfactory and auditory systems, and cranial ganglia.Neurulation—The rolling up or hollowing out of tissue in chordates to create a dorsal hollow nerve cord along the anterior-posterior of the developing embryo.Operon—A cluster of genes sharing common transcriptional control, originally identified in bacteria but now known to occur in animals, plants and fungi, as well.Somites—Paired segmental blocks of mesoderm formed during embryogenesis that give rise to skeletal muscle and, in vertebrates, to other structures, including vertebrae and rib cage elements.Thaliacean—A class of free-swimming, ascidian-like tunicates that use their oral and atrial siphons for propulsion through water.Tunicate—The closest living group to vertebrates, the Tunicata subphylum of the chordates is characterized by its ability to make cellulose and by a radical metamorphosis following the larval phase.DOI:http://dx.doi.org/10.7554/eLife.06024.003C. intestinalis is the model organism of choice for many labs that aim to uncover the mechanisms that drive embryonic development and morphogenesis. In the era of recombinant DNA and genomics, many standard tools are available to the C. intestinalis researcher, including a well-annotated genome, methods to manipulate gene function and to create mutant strains and reporter lines, and techniques for expressing engineered DNA —Ascidian Network for In Situ Expression and Embryological Data; provides databases and tools for research on C. intestinalis and other ascidians.Ascidian Stock Center (www.ascidiancenter.ucsb.edu)—Provides wild type and stable transgenic C. intestinalis and C. savignyi to Ciona researchers.CIPRO (http://cipro.ibio.jp)—C. intestinalis Protein Database.Dutch ascidians homepage (ascidians.com)—Photographs of ascidian species worldwide, showing intra-specific variation.FABA (http://chordate.bpni.bio.keio.ac.jp/faba/1.4/top.html)—Four-dimensional Ascidian Body Atlas; provides high resolution images of C. intestinalis during development.Ghost Database (http://ghost.zool.kyoto-u.ac.jp/cgi-bin/gb2/gbrowse/kh/)—Gene expression, genomic and cDNA resources for C. intestinalis research.Tunicate Meeting (tunicatemeeting.info)—A biennial forum for tunicate biologists that takes place alternately in Japan, North America or Europe.Tunicate Web Portal —Provides links to tunicate databases and websites, and community information for tunicate researchers.DOI:http://dx.doi.org/10.7554/eLife.06024.004C. intestinalis rely on wild-caught specimens. The large-scale culturing of C. intestinalis is limited to only a few laboratories that have marine facilities, and attempts to establish an inbred laboratory strain of C. intestinalis for widespread use have not yet been successful. Researchers should be aware that differences between isolates , while potentially confounding, might also lead to new insights regarding regional adaptation and variation, and dispersal.Unlike researchers working on more common model organisms, those working on C. intestinalis is a member of the Tunicata (see Glossary). The Tunicata include the sessile ascidians, like C. intestinalis, as well as the free floating (pelagic) thaliaceans and larvaceans (see Glossary). Together with vertebrates and cephalochordates (see Glossary), tunicates make up the chordates. The defining feature of Chordata is the notochord, an anterior-to-posterior oriented rod of cells, which lies just below the spinal cord. In the first chordates, the notochord was likely to have conferred a biomechanical advantage to swimming larvae by providing them with stiffness and turgor, as it does for the C. intestinalis larva. Within the vertebrates, the notochord has an added role, acting as a signaling center for neighboring tissues of the developing embryo. Other morphological features uniting C. intestinalis (and other tunicates) to the chordates include a dorsal, hollow nervous system and a muscular post-anal tail .C. intestinalis in providing a window on the evolution of such traits.Chordate phylogeny has been recently reordered , with tuDespite these conserved structures that unite the chordates, tunicates have several morphological features that diverge radically from their vertebrate cousins, including the adult body form, the absence of somites (see Glossary), hermaphroditic reproduction, and, curiously, the ability to synthesize cellulose . CelluloC. intestinalis is a member of the marine biofouling (see Glossary) community, lining the hulls of boats and crowding the underwater surfaces of buoys and docks. It thrives even in the presence of the anthropogenic insults generated in a marina setting, such as effluent from boats, surface petroleum slicks and organic waste. C. intestinalis is cosmopolitan in distribution, found on both sides of the Atlantic and Pacific Oceans, and it has been reported in the oceans off Australia and South Africa ?What are the origins of cellulose synthesis, and how have tunicates integrated a functional cellulose synthase-encoding gene into their genome?C. intestinalis cannot undergo metamorphosis if cellulose synthase function is blocked   . The totC. intestinalis—are highly successful marine invertebrates. In most environments, C. intestinalis is considered to be invasive, and it can be so successful as to constitute a considerable fraction of the local biomass (C. intestinalis, together with the genetic and genomic resources available for this organism, make this a ripe area for research.The tunicates—including  biomass . Many quC. intestinalis, that of a sessile filter feeder, has served it well and has shaped its anatomy, physiology and embryology. Many of the features of C. intestinalis that at various times have been called primitive or basal, such as the simplicity of the embryo or the adult body form, are best seen as products of the animal's unique natural history. From a practical point of view, because of its inherently high levels of polymorphism and because of the many sites used to collect experimental animals worldwide, C. intestinalis researchers must remember that locally collected animals might be quite different to those used to generate the data found in C. intestinalis databases (see C. intestinalis in mind, we can expect this relatively obscure marine model organism to contribute much to our understanding of basic chordate biology and to the history of the chordate clade.The lifestyle adopted by the adult ases see . With th


Phlebotomus-borne (PhB-) viruses are distributed in large areas of the Old World and are widespread throughout the Mediterranean basin, where recent investigations have indicated that virus diversity is higher than initially suspected. Some of these viruses are causes of meningitis, encephalitis and febrile illnesses. In order to monitor the viral presence and the infection rate of PhB-viruses in a recently identified and well characterized human zoonotic leishmaniasis focus in southwestern Madrid, Spain, a sand fly collection was carried out.Phlebovirus presence both via isolation on Vero cells and via polymerase chain reaction (PCR), using degenerated primers targeting a portion of the L segment, was performed. The serological identity and phylogenetic relationships on the three genomic segments of the viral isolates were carried out.Sand fly insects were collected in four stations using CDC light traps during 2012–2013 summer seasons. Screening for Phlebovirus were obtained from fifty pools on a total of 963 P. perniciosus . Phylogenetic analysis and serological assays allowed the identification of two isolates of Toscana virus (TOSV) B genotype, three isolates strongly related to Italian Arbia virus (ARBV), and one isolate of a novel putative Phlebovirus related to the recently characterized Arrabida virus in South Portugal, tentatively named Arrabida-like virus. Positive male sand fly pools suggested that transovarial or venereal transmission could occur under natural conditions.Six viral isolates belonging to different serological complexes of the genus Phlebovirus species in the South-West area of the Madrid Autonomous Community where an outbreak of cutaneous and visceral human leishmaniasis has been recently described. The evidence of viral species never identified before in Spain, as ARBV and Arrabida-like virus, and TOSV B genotype focus stability was demonstrated. Environmental aspects such as climate change, growing urbanization, socio-economic development could have contributed to the genesis of this wide ecological niche of PhB-viruses and Leishmania  spp. The potential role of vertebrates as reservoir for the phleboviruses identified and the possibility of Phleboviruses-Leishmania co-infection in the same sand fly should be assessed. Furthermore the PhB-viruses impact on human health should be implemented.Our findings highlighted the presence of different  Leishmania spp.), bacteria (Bartonella bacilliformis) and viruses. Phlebotomus borne (PhB-) viruses belong to the genus Phlebovirus (family Bunyaviridae), Vesiculovirus (family Rhabdoviridae) and Orbivirus (family Reoviridae) are distributed in large areas of the Old World  and are widespread throughout the Mediterranean region [Phlebovirus are relevant as an emerging human health problem. Among them, Sand fly Fever Sicilian Virus (SFSV) and Sand fly Fever Naples Virus (SFNV) are the causative agents of transient febrile illness in humans, while Toscana virus (TOSV) exhibits peculiar neurotropism. Indeed, TOSV infection has been associated with aseptic meningitis or, less frequently, meningoencephalitis or encephalitis without meningitis [Phlebovirus diversity in the Mediterranean basin is higher than initially suspected and novel viruses are yearly discovered [Phlebotomine sand flies are documented vectors of human disease agents including parasitic protozoa . The first human case of TOSV infection was revealed, by serological investigation (1986–1989), in a Swedish tourist returning from Catalonia (north-east of Spain) [Phlebotomus  spp. in Granada province (southeastern Spain) during 2003, and its presence in phlebotomine sand flies was reported in subsequent years also in other areas of Spain  [Epidemiological studies in Spain have demonstrated, up to now, the circulation of two different f Spain) . The firf Spain) , 15. In f Spain) , Majorcaf Spain) , Catalonf Spain) . GRV, cltigation 86–1989, Leishmania infantum is ongoing resulting up to now in the largest reported community outbreak of leishmaniasis in Europe [http://www.madrid.org/cs/Satellite?boletin=Si&c=CM_Publicaciones_FA&cid=1142685874033&language=es&pageid=1265797458663&pagename=PortalSalud%2FCM_Publicaciones_FA%2FPTSA_publicacionServicios&site=PortalSalud&volver=Si). The epidemiological link between phleboviral and Leishmania human infections has been widely highlighted in different European countries, both in phlebotomine and invertebrates acting as possible vectors and reservoirs of these pathogens [Phlebotomus perniciosus and Phlebotomus ariasi, out of the seven Phlebotomus spp. described in the Madrid region, are proven vectors of L. infantum with P. perniciosus being the main vector [P. perniciosus was also a proven L. infantum vector in Fuenlabrada municipality [The Madrid area , investigated in the present study, has been recently characterized as a relevant focus of human zoonotic leishmaniasis. In this area, leishmaniasis is endemic in rural, periurban and suburban areas and from July 2009 to date, a cutaneous and visceral leishmaniasis outbreak by n Europe . The majathogens –26. Onlyn vector , 28. P. cipality , 30.PhB-viruses, leishmaniasis and the geographical distribution of the sand fly vectors, virological, parasitological and entomological surveillance activity represents an important tool for the control of the spread of these pathogens. In the framework of the European FP7 EDENext project for the surveillance of phlebotomines and fly-borne diseases, the objective of this work was to monitor the PhB-viruses presence and infection rate in the recent human zoonotic leishmaniasis focus of the South-West area of Madrid, Spain [Due to the closed relationship between d, Spain .P. perniciosus is the prevalent sand fly in the area at high densities (152.6 sand flies/m2) [Phlebovirus isolation [P. perniciosus. Only few specimens of a different sand fly genus, Sergentomyia minuta, were previously identified. This was a technical advantage for the present study because it required the immobilization of sandflies with CO2, specimens were then placed in a glass Petri dish over a portable chill table for the separation of both species under a stereomicroscope. Erect or recumbent abdominal setae on the posterior margins of abdominal tergites 2–6 were used for the fast identification of the genera Phlebotomus and Sergentomyia, respectively. Secondary aspects such as the general shape of the body and its color were also helpful in the identification process.The sampling of sand flies was performed in a newly constructed periurban green park of around 450 ha, surrounded by large urban densely populated areas of southwestern Madrid region. Afforestation of this park began in 2005, finishing in 2011 . The prelies/m2) , 30, 32.solation , in fourPhB-viruses were subsamples of total catches obtained during seasonal dynamics studies carried out in the same area. Sand fly monospecificity was confirmed when the taxonomic identification of the remaining specimens collected at the same stations and dates of 2012–2013 was performed. Thus, P. perniciosus was the only species of the genus Phlebotomus identified (95.3 %) while S. minuta was found in a very low proportion (4.7 %) [Pools of sand flies for the analysis of  (4.7 %) . Standar (4.7 %) , 35 wereCollected sand flies for virus isolation were pooled of ~20 specimens separated by species and sex and immediately stored at −80 °C until examination.All sand fly pools were homogenized, suspended in 1 mL of Hank’s solution containing 7.5 % bovine albumin and 1 % antibiotic-antimycotic mix  and centrifuged at 3,000 × g for 30 min . One hunPhlebovirus detection was performed amplifying Phlebovirus RNA-dependent RNA polymerase gene on a portion of L segment using degenerated primers [The RNA was extracted from supernatant aliquots by using the QIAamp viral RNA kit  according to the manufacturer’s protocol.  primers . The RT- primers . PCR pro, Waltham, MA, USA). After 1 h incubation at 37 °C in an atmosphere containing 5 % CO2, 2 mL of medium, consisting of Dulbecco’s MEM (DMEM), 2 % FBS and 1 % antibiotic-antimycotic mix , was added. The tubes were incubated at 37 °C and observed daily up to 14th day after inoculation to monitor the cytopathic effect (CPE). The culture samples showing the CPE were frozen at −80 °C. Viral stocks of the isolates were obtained by propagation on Vero cells and then stored at −80 °C in aliquots until use. The titration of virus stocks was carried out on six-well plates containing confluent monolayers of Vero cells infected with serial 10-fold dilutions of viral supernatants. Cells were incubated at 37 °C for 6–14 days under an overlay consisting of DMEM, 2 % FBS, 1 % antibiotic-antimycotic mix  and 2 % tragacant gum (Sigma Aldrich). The plaques were counted after staining with a solution of crystal violet  and the viral titre was expressed as Plaque Forming Units (PFU)/mL. The re-isolation on Vero cells of each isolate was carried out.The virus isolation was carried out as described by Verani et al. . BrieflyThe culture samples showing no CPE up to the 14th day were stored at −80 °C and then used for blind passages on Vero cells. At least three passages on Vero cells were performed.The overall virus isolation was assessed by calculating the Minimum Field Infection Rate (MFIR)/100 sand fly specimens. This value is obtained by the ratio of the number of positive pools to the total number of tested sand flies in the sample assuming that only one infected specimen is present in a positive pool .2 and stained with a crystal violet solution. A reduction of 50 and 80 % in number of plaques was selected as criterion for virus neutralizing titres. PRN test titres < 1:10 for serum were considered negative.PRN assays were performed, as previously described , using sPhlebovirus RNA [RNAs were extracted from isolates viral stocks by using the QIAamp viral RNA kit and RT-PCR amplifications of portions of L, M and S segments were performed. A partial coding region of L sequences of all isolates were obtained by amplification with the same degenerated primers used as screening in this study to detect the presence of irus RNA . The priirus RNA  and (ii)Phlebovirus sequences present in GenBank using the program ClustalW  as implemented in the Bio-Edit software version 7.2.5 [The amplicons of partial L, M and S segments were sequenced and the sequences of 438 bp, 449 bp and 258 bp respectively, were aligned with other on 7.2.5 . DAMBE sFor each of three partial segments, phylogenetic trees were constructed with MEGA software utilising the parameters of analysis indicated by ModelTest with 1,000 bootstrap reiterations. The same procedures were utilised also for the amino acid translation of each segment. Distances among sequences for each gene segment were calculated with MEGA software without any correction for different evolutionary models to better evaluate the absolute distances among sequences.The sequences of the isolates had been submitted to GenBank, and the corresponding accession numbers are given in Table P. perniciosus resulted to be the only species of the genus Phlebotomus collected at high densities (152.6 sand flies/m2) [L. infantum present. Mapping of sand fly collection stations, close to an urban park, is reported in Fig. P. perniciosus specimens were processed and tested for PhB-viruses presence, 366  for 20 pools in 2012 and 597 sand flies  for 30 pools in 2013 (Table The selected collection sites have been restricted to an area where lies/m2) , 30, 32 Phlebovirus sequences, available in the GenBank, identified: the isolates Fue-Sp45 and Fue-Sp136 as TOSV genotype B; the isolates Fue-Sp40, Fue-Sp42, and Leg-Sp49 as ARBV. The sequences of the Fue-Sp149 isolate showed the highest similarity with the sequences relative to virus named Arrabida recently characterized in Portugal  [The results obtained by generic RT-PCR plus nested-PCR amplifying a portion of the L segment and showed positivity in 4 pools of sand flies collected in 2012  and in 2 pools of sand flies collected in 2013 (Fue-Sp136 and Fue-Sp149)  was 0.27 and 0.18 for TOSV in 2012 and 2013, respectively, and 0.82 for ARBV and 0.18 for Arrabida-like virus.80 = 40) and ARBV (20 < PRN80 < 40), respectively. No serological reactions were produced using MIAFs against viruses belonging to different serocomplexes. The Arrabida-like strain showed a low positivity (PRN50 = 1:10) only with MIAFs against viruses belonging to SFNV serocomplex, as TOSV and SFNV, while no serological relationship was detected with MIAFs against viruses belonging to other serocomplexes. On the basis of PRN test results, the Arrabida-like virus seems to be strongly correlated to but clearly distinct from other tested viruses belonging to the SFNV complex.The viral isolates, molecularly characterized as TOSV, ARBV and Arrabida-like viruses, were assayed by PRN test. The results of the serological relationships with other selected Phleboviruses are reported in Table P. perniciosus in Madrid.Nucleotide and amino acid analysis of the six viral isolates were performed on all three partial sequences of the L, M and S segments. From the analysis of the phylogenetic trees of the three segments analyzed, Fue-Sp45, Fue-Sp136 and Fue-Sp149 were shown to belong to the SFNV complex as well as Fue-Sp40, Fue-Sp42 and Leg-Sp49 were related to the SALV complex Fig. , b, c. Tb, Phlebovirus groups (P distance ≥ 37 % in nucleotide composition). Analysis of the variation of nucleotide identities within members of the SALV complex indicated that these viruses were more closely related to the Italian ARBV sequences, with a similarity of 80, 79 and 91 % on L, G2 and N nucleotide sequences, respectively.The relatedness of Fue-Sp40, Fue-Sp42 and Leg-Sp49 isolates with viruses belonging to the SALV complex, in accordance with the serological assay, was demonstrated from nucleotide distance analysis   to Arrabida virus sequences recently described in Portugal [As shown in Table Portugal . The G2 PhB-pathogens epidemics. The present study was carried out in the context of the EU FP7 EDENext project (http://www.edenext.eu) with the aim to monitor the PhB-viruses presence in sand flies collected in Fuenlabrada and Leganés Municipalities, areas of Madrid known for a recent outbreak of human cutaneous and visceral leishmaniasis and where the presence of L. infantum in P. perniciosus has been also demonstrated [P. perniciosus pools collected in the same area and serological and molecular characterization showed that these Phleboviruses were belonging to two different serocomplexes of the Phlebovirus genus.Active entomological and virological surveillance is an important approach to provide early warning and predictive capacity about the risk of the nstrated , 30. FroPhlebovirus and Leishmania represent today an important public health problem in countries in which these microorganisms circulate [L. infantum and TOSV in the sand fly vector P. tobbi and L. tropica and TOSV in the sand fly vector P. sergenti were reported in Cyprus [L. infantum and TOSV and MASV were detected in P. perniciosus collected from the same trapping site in Marseille urban area [Infections by irculate . Indeed,irculate , 22. Indn Cyprus  and Moron Cyprus , respectban area .PhB-pathogens, the presence of insect vectors is an important prerequisite for transmission; however, it is not the only factor determining whether or not a pathogen can be established. In particular local environmental components  represent factors influencing the pathogens amplification and the diffusion of the diseases transmitted by them. Interestingly, in this periurban area of Madrid, following afforestation started in 2005 and finished in 2011 [P. perniciosus females [P. perniciosus sand flies, strongly suggesting they contribute to the maintenance of high sand fly populations in this epidemic area [Leishmania highlighting the infectivity of apparently healthy L. infantum infected hares (L. granatensis) to phlebotomine sand flies (P. perniciosus) [In the ecological spread of  in 2011 , a large in 2011 . The stu females  indicatemic area , 30, 44.iciosus) .P. perniciosus. However the role of vertebrates in the maintenance of the PhB-viruses transmission cycle remains unclear [Pipistrellus kuhli which was trapped in areas where P. perniciosus and P. perfiliewi were present [At present, limited informations are available about Phleboviruses seroprevalence both in lagomorphs and in other vertebrates. However all the above-mentioned evidences could suggest that the presence of this unexpected ecological niche of Phleboviruses in Madrid foci could be the result of the massive presence of the vertebrates potential reservoirs, (e.g. rabbits and hares) and  unclear . To date present , 45. Rec present .Phlebovirus serocomplexes from P. perniciosus confirmed the important role of this sand fly species as vector, being the main natural vector of the other Phleboviruses, such as GRV and TOSV, previously identified in Spain [Phlebovirus Arrabida, belonging to SFNV complex, isolated by Phlebotomine sand flies in South Portugal. The recent published data on genome sequencing suggested that this Arrabida virus from Portugal could be considered a reassortant of GRV and MASV, donors of the long and short segments, with an unknown Phlebovirus, donor of the medium segment [P distance in M segment G2 gene between MASV, GRV and Arrabida virus and Fue-Sp149 was 25, 26 and 17 % at nucleotide level, respectively. The differences in the M segment are a common distinctive characteristic among Phlebovirus and is frequently the basis for the distinction among different Phleboviruses. Indeed GRV is a natural reassortant of MASV, donor of the long and short segments, with a yet unidentified Phlebovirus, donor of the medium segment. The full FueSp-149 isolate genome characterization (in particular the M segment) will clarify the possible presence of a new reassortant Phlebovirus genetically related to Arrabida virus. Pending the results of the full genome characterization, the FueSp-149 isolate has been considered as putative novel virus tentatively named Arrabida-like virus. Due to the Arrabida-like virus, which belongs to the SFNV complex and includes many viruses able to infect humans  [The detection of several isolates belonging to different in Spain , 19. Thein Spain , 46, 47. segment . Accordi segment , Fue-Sp1In the present study, for the first time, the ARBV presence was demonstrated in Spain. The analysis of the phylogenetic trees showed some differences between the Spanish and Italian ARBVs Fig. . Indeed,Phlebovirus known to be circulating in Madrid region. In addition the MFIR of 0.21 % for TOSV, obtained from sand flies collected in Fuenlabrada in 2012–2013 summers, was higher than the infection rate of 0.05 % up to now found in Spain [The isolation of TOSV lineage B confirmed the stability of the natural focus active in Madrid community. Even if no recent epidemiological data on human population in the area of study are reported, seroprevalence analysis confirmed the TOSV circulation in human population in Spain and in Madrid area , 49. Morin Spain  but simiin Spain  where yein Spain , 49–51. Phlebovirus species in a recently identified human leishmaniasis focus in south-west of Madrid area showing the co-circulation of both pathogens. Based on our findings, an active field-based study, that combines entomological, parasitological and virological aspects, results to be a valid approach to monitor the possible appearance of vector-borne emerging diseases. A better knowledge of PhB-viruses epidemiological aspects may be important in public health to prevent epidemics and/or spreading of these viruses in geographic areas where no evidence of their circulation has been highlighted.The present study demonstrated the presence of different


S100P is a member of the S100 family of calcium-binding proteins,and it participates in pathophysiological events, such as tumor growth and invasion.Based on the striking similarities between trophoblast cells and tumor cells with regard to proliferative and invasive properties, we raised the question of whether andhow S100P expresses in trophoblast cells during development. This study aimed toinvestigate the expression pattern of S100P in the human placenta during pregnancydevelopment.In this experimental study, we collected 16 first-trimesterplacental tissues, 10 second-trimester placental tissues, and 12 term placentas. ThemRNA expression levels of S100P were detected by reverse-transcription-polymerase chain reaction (RT-PCR) and quantitative real-time PCR, the protein expressionlevels were detected by western blot, and the localization of S100P was measuredby immunohistochemical staining. The values obtained from PCR and western blotanalysis were expressed as the mean ± SD. Levene’s test was used to test equal variances, and one-way analysis of variance (ANOVA) was used to evaluate differencesbetween groups.Protein and mRNA expression of S100P could be detected in placenta during pregnancy, with minor higher levels in first-trimester (p>0.05). Immunohistochemical staining revealed that S100P protein was strongly expressed in syncytiotrophoblasts, and moderate expression was detected in villous cytotrophoblasts andcytotrophoblast columns. The S100P protein was localized to both cytoplasm andnuclei in syncytiotrophoblasts, while it only existed in the cytoplasm of cytotrophoblasts.S100P was strongly detected in human placenta during pregnancy. The specific expression and distribution of S100P in human placenta throughout gestation suggested that S100P function might vary with its location in the placenta. Patients attending the Assisted Reproduction Unit, Department of Obstetrics and Gynecology, Sir Run Run Shaw Hospital, College of Medicine, Zhejiang University, Hangzhou, China, for treatment were invited to participate in the study. The clinical characteristics of patients are stated in The inclusion criteria were 22-35 years of age, regular menstrual periods with a cycle length between 26 and 35 days, and physical health. The exclusion criterion was a history of any kind of pharmacotherapy, hypertensive disorders, and fetal growth restriction. Samples of placental tissue of the first trimester (n=16) were obtained from healthy women undergoing suction termination of pregnancy (6-9 weeks) for inevitable abortion. Placental tissue of the second trimester (n=10) was taken from healthy women undergoing induced labor as a result of inevitable abortion or fetus cheilognathopalatoschisis. Term placentas (n=12) were collected after an uncomplicated pregnancy and vaginal delivery. All of the samples were rinsed in sterile phosphate-buffered saline (PBS) to remove blood and debris, and collected in -80˚C or formalin.2, 1 μl of 10 mmol/l dNTPs, 0.2 μl of Taq DNA polymerase (5 U/μl), 1 μl of 0.25 mmol/l specific primers, 2.5 μl of 10× reaction buffer and double-distilled water to a final volume of 25 μl.Total RNA was extracted using TRIzol . Total RNA (2 μg) was used for first-strand complementary DNA (cDNA) synthesis in a 25-μl reaction volume. The cDNA was then amplified by RT-PCR. The reactions were prepared as follows: 5 μl of 5× reaction buffer, 1 μl oligo (dT), 1 μl deoxy-NTP mix (10 mmol/l each), and 1 μl Moloney murine leukemia virus reverse transcriptase. The PCR reactions were prepared containing: 2 μl of cDNA, 2 μl of 25 mmol/l MgClA 5-minute pre-cycle at 95˚C was followed by 30 cycles of 30 seconds at 95˚C, 30 seconds at 56˚C, and 30 seconds at 72˚C. After the final cycle, the samples were kept at 72˚C for 4 minutes to complete the synthesis. The PCR products were then separated by electrophoresis in a 1.0% agarose gel stained with ethidium bromide and visualized using an imaging analysis system. The intensities of bands of S100P were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which was amplified simultaneously. The primer pairs for cDNA amplification were as follows: S100P, 5΄-ATGACGGAACTAGAGACAGCCATGGGC-3΄ and 5΄- GGAATCTGTGACATCTCCAGGGCATCA-3΄; and GAPDH, 5΄-TGACTTCAACAGCGACACCCA-3΄ and 5΄-CACCCTGTTGCTGTAGCCAAA-3΄.2, 0.3 μl of probe, 1 μl of specific primers, and 26.4 μl of double-distilled water. The PCR was performed by monitoring in real time the increase of fluorescence of SYBR green I dye  with a Rotor-Gene 3000 . The relative copy number of each transcript was calculated by the concentration-CT standard curve method and normalized using the average expression of GAPDH.Thirty-eight tissues were selected for real-time PCR analysis, with GAPDH as a control. Total RNA, 2 μg, was used to generate cDNA. The PCRs contained the following: 4 μl of cDNA, 4 μl of 10× reaction buffer, 0.8 μl of 10 mmol/L dNTP, 0.3 μl of 5 U/mL Taq DNA polymerase, 3.2 μl of 25 mmol/L MgClTotal protein was separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The separation gel concentration was 15% and the voltage was 80-100 V. Then the gel was transferred to polyvinylidene fluoride membranes using an electroblotting apparatus for 1 hour. Membranes were blocked in 5% non-fat dried milk in Tris-buffered saline (TBS) with Tween 20 (TBS-T) for 1 hour at room temperature. The polyvinylidene fluoride (PVDF) membranes were incubated with rabbit anti-S100P at 1:1000  for 2 hours at room temperature. Membranes were then incubated with horseradish peroxidase and goat anti-rabbit IgG at 1:5000 for 1 hour at room temperature. Chemiluminescence was detected using an ECL Chemiluminescent Substrate Kit, according to the manufacturer’s instructions . GAPDH was used as an internal control. The protein level of S100P was normalized to that of GAPDH.Placental tissues were fixed in formalin, embedded in paraffin, and sectioned (4 μm thickness). The tissue sections were de-paraffinized in xylene, dehydrated in graded alcohols, and placed in buffered saline. Subsequently, antigen retrieval was achieved by heating the slides in a microwave oven in 10 mmol citrate buffer. After antigen retrieval, sections were incubated with an aqueous solution of 3% hydrogen peroxide followed by incubation with 5% non-fat milk, which serves as a blocking agent for nonspecific binding, for 20 minutes at 37˚C.After rinsing the slides in buffer for 5 minutes, slides were incubated with the rabbit anti-S100P , anti-cytokeratin 7 antibody , anti-vimentin  for 30 minutes at 37˚C. The anti-cytokeratin-7 antibody was positive for trophoblast cells, while the anti-vimentin antibody was positive for stromal cells. For S100P, using secondary antibody alone was applied as negative control. After washing, the secondary biotinylated antibody was then applied for 30 minutes at room temperature. After rinsing with PBS, an avidin-biotin-peroxidase complex  was added to the sections and incubated for 1 hour at room temperature. Then, 3, 3΄-diaminobenzidine (DAB) peroxidase substrate kits  were used to visualize the immunostaining and hematoxylin was used to counterstain.Each experiment was repeated at least three times. The values obtained from PCR and western blot analysis were expressed as the mean ± SD. Statistical analysis was carried out using the Statistical Package for the Social Sciences  version 11.0. Levene’s test was used to test equal variances, and one-way analysis of variance (ANOVA) was used to evaluate differences between groups. Differences were considered significant at p<0.05.This study was approved by the Ethics Committee of the College of Medicine, Zhejiang University, China. Informed consent was obtained from all patients.In the present study, we applied RT-PCR and real-time PCR to detect the expression of S100P mRNA in human placental tissue. S100P mRNA expression was detected in placental tissues from the first-trimester gestation, second trimester, and at term , and theS100P protein expression was analyzed by western blot on solubilized protein extracts from placental tissue. A band of 11 kDa was found in placental tissue . CompareIn first-trimester placenta, S100P immunopositive staining showed an intense signal in the cytoplasm and nuclei of syncytiotrophoblasts, whereas villous cytotrophoblasts  and cytoS100P can be detected in placenta , 12. HowS100P is one of the least studied members of the S100 family. It is an 11-kDa protein and was originally isolated from the placenta. S100P expression was then detected in tumor cell lines and carcinomas derived from the prostate, breast, colon, pancreas, and other tumor types, and it is associated with immortalized, malignant, hormone-independent, and chemoresistant phenotypes -15. ConvIn the present study, in first-trimester placenta, S100P was moderately expressed in the cytoplasm of villous cytotrophoblasts and cytotrophoblast columns, and was found in the cytoplasm and nuclei of syncytiotrophoblasts. Furthermore, in term placenta, the striking localization of syncytiotrophoblasts was still invariant.The villous cytotrophoblasts are proliferative and capable of fusing to form the multinucleated syncytiotrophoblast. The column cytotrophoblasts actively proliferate and tightly anchor the placenta to the uterine wall; in addition, they can further differentiate into invasive extravillous trophoblasts to remodel the uterine decidua and spiral arteries . TrophobAs the placenta developed, the cytotrophoblasts and cytotrophoblast columns gradually disappeared. Syncytiotrophoblasts became the main kind of trophoblast cells at term. Syncytiotrophoblasts were differentiated from cytotrophoblasts. The present findings, showing strong cytoplasm and nuclei-staining syncytiotrophoblasts, suggest that, upon differentiation, syncytiotrophoblasts acquire S100P synthesis and transport capabilities. The component of S100P in the nucleus might bind to S100P binding protein (S100PBP), the exact role of which remains unclear. The syncytiotrophoblast in the placenta serves as an interface for efficient maternal-fetal exchange of nutrients, metabolites, and xenobiotic compounds, and membrane transport processes play pivotal roles in the efficient uptake of certain compounds and in the exclusion of others . SyncytiThe present data show an intimate pattern of distribution of S100P in placenta, and demonstrate that the expression of S100P is strongly detected in human placenta during pregnancy. Further investigation is required to study the roles and associated molecular mechanisms of S100P in normal placenta.


Direct costs associated with an intensive care unit (ICU) admission for sepsis are approximately €30,000. However,To estimate the difference in annual health care expenditure before and after an ICU admission for sepsis.Data were derived from a prospective cohort study in two tertiary ICUs in the Netherlands in 2011 and 2012. Patients were included if they had survived one year following a sepsis episode in the ICU. Health care consumption and reimbursed costs were derived from a database of a Dutch health insurance company. The medical ethics committee of the UMC Utrecht approved the study and waived the need for informed consent (IRB-number 14-095).Of the 396 eligible patients, for 21 (5.3%) there was no information on costs available in the study period, leaving 375 subjects for analysis. An overview of reimbursed costs is given in table 1. For each stage of sepsis severity, total costs were significantly higher in the year following a sepsis admission compared to the year before . MB received research funding from the Netherlands Organization of Scientific Research (NWO Vici 918.76.611).


To provide an overview of the economic aspects of needlestick and sharps injury (NSI) management among healthcare personnel (HCP) within a Health Technology Assessment project to evaluate the impact of safety-engineered devices on health careA systematic review of economic analyses related to NSIs was performed in accordance with the PRISMA statement and by searching PubMed and Scopus databases (January 1997–February 2015). Mean costs were stratified by study approach (modeling or data driven) and type of cost (direct or indirect). Costs were evaluated using the CDC operative definition and converted to 2015 International US dollars (Int$).A total of 14 studies were retrieved: 8 data-driven studies and 6 modeling studies. Among them, 11 studies provided direct and indirect costs and 3 studies provided only direct costs. The median of the means for aggregate (direct + indirect) costs was Int$747 . The medians of the means for disaggregated costs were Int$425  for direct costs (9 studies) and Int$322  for indirect costs (6 studies). When compared with data-driven studies, modeling studies had higher disaggregated and aggregated costs, but data-driven studies showed greater variability. Indirect costs were consistent between studies, mostly referring to lost productivity, while direct costs varied widely within and between studies according to source infectivity, HCP susceptibility, and post-exposure diagnostic and prophylactic protocols. Costs of treating infections were not included, and intangible costs could equal those associated with NSI medical evaluations.NSIs generate significant direct, indirect, potential, and intangible costs, possibly increasing over time. Economic efforts directed at preventing occupational exposures and infections, including provision of safety-engineered devices, may be offset by the savings from a lower incidence of NSIs.Infect Control Hosp Epidemiol 2016;37:635–646 In the United States, the Needlestick Safety and Prevention Act was passed in 2000,–Several studies have considered the economic burden of occupational NSIs,Given the increased costs of SEDs compared to conventional devices, in 2012, a Health Technology Assessment project was launched in Italy to evaluate this new technology and its overall impact on health care, including an economic analysis of costs deriving from NSIs and savings arising from their prevention. This systematic review represents the first part of this project.A systematic review of economic analyses of occupational NSIs among HCP was performed in accordance with the PRISMA statement.The titles and abstracts of all articles were evaluated, eliminating duplicates. Articles were then assessed for eligibility through a full-text examination, and an article was included in the analysis if it provided an original estimate of either direct or indirect costs of managing a single NSI. The main stages of the systematic review are shown in Data extraction forms included author’s name, publication year, country, source of the occurrence data, study period, population studied, NSI incidence, perspective, cost estimating approach (modeling or data-driven), items considered in cost, type of cost studied (direct and/or indirect), injury management cost (range), and currency equivalent.http://data.worldbank.org/indicator/fp.cpi.totl.zg) were used to express the injury management costs in national currencies 2015. These values were then converted to 2015 International US dollars (Int$) using purchasing power parity (ppp) exchange rates (http://data.worldbank.org/indicator/pa.nus.ppp). To facilitate comparisons, all costs included in this review were converted to Int$.To compare monetary values expressed in different currencies and for different years, the national inflation rates provided by the World Bank  as well as minimum and maximum mean costs abstracted from each article. The weighted mean was used to account for the different proportion of resources used according to the type of event. The findings were stratified by study approach and type of cost (direct and/or indirect). All data used in this analysis were true cost data.Direct and indirect costs of an NSI were evaluated using the CDC operative definitions:1. Direct costs: Costs generally borne by a healthcare organization when an NSI occurs: baseline and follow-up laboratory testing, PEP, and other treatment eventually provided, including PEP side-effect management. According to which costs are borne by the organization, in certain circumstances, workers’ compensation or the management of occupationally exposed HCP may be included with direct costs.2. Indirect costs: These costs include time and wages diverted to receiving or providing exposure-related care: lost productivity associated with reporting and receiving initial and follow-up treatment for the exposure; healthcare provider time to evaluate and treat an employee; and healthcare provider time to evaluate and test the source, including obtaining informed consent for testing if applicable. For occupational health and infection control practitioners, these measures are part of their job responsibilities and therefore are not considered a diversion. Moreover, when staff absence and compensation are borne by a third-party payer, these may not be included with indirect costs.When analyzing in detail the items included within direct and indirect costs in each study, the authors originally assigned baseline and follow-up post-exposure visits differently according to the study context. However, when stratifying the study findings, the post-exposure visits, when disaggregated, were always considered as a direct cost and were assigned accordingly.The quality of each study was evaluated using Drummond’s checklist modified by La Torre et al.,,,,,–In total, 14 studies from Europe, America, Asia, and Australia published from 1997 to 2013 met all the inclusion criteria.Of the 14 studies estimating costs, 8 studies were data driven, while 6 studies used modeling ; 11 stud,,,,,,,,,Costs were analyzed mainly from a hospital or university perspective in 7 studiesDirect costs included testing the source and exposed HCP as well as post-exposure medical visits and treatment . Costs to treat an occupational infection were included in 2 studies only, which reported annual treatment costsIndirect costs included lost productivity (time off work) associated with the time required for reporting and receiving initial and follow-up treatment as well as exposure consequences , overhead, compensation, and litigation.Considering the 11 studies that provided aggregate direct + indirect costs, the overall median of the means of costs for managing an NSI was Int$747   and 4.TAThe median of the means of the 9 studies that estimated disaggregated direct costs was Int$425 , and the median of the means of the 6 studies that estimated disaggregated indirect costs was Int$322 .Considering the type of approach, modeling studies had higher disaggregated and aggregated costs than data-driven studies: direct cost ratio, 3.4:1; indirect cost ratio, 1.84:1; combined direct+indirect cost ratio, 1.13:1. Costs from data-driven studies, however, showed greater variability, with standard deviations more than twice those of modeling studies .,,,,,–,,,,,,,,,,,,When estimating direct and indirect NSI management costs, the included cost items differed among studies . Regardi,,,,,,,,,,,,,,,Regarding HCP, in 4 studies,Furthermore, in a single study, the source and HCP were also screened for syphilis, and in some cases, the source was screened for hepatitis A virus. These tests account respectively for 13.4% and 1.7% of the total tests performed in the study period.,,,The antiretroviral HIV PEP regimen was specified in 8 of 13 studies. In 6 studies conducted between 1990 and 2003, a zidovudine-based regimen was adopted alone (in 1 study),,,,,,,,,,,Finally, administration of hepatitis B immune globulin (HBIG) and HBV vaccination were included in the direct costs of an NSI in eight,,,,-Regarding indirect costs, 10 studiesThe quality of the studies is presented in Three studies presented the incremental analysis (#31), provided details of the design and results of effectiveness (#9), and used a synthesis method or meta-analysis of estimates (#10): the data-driven study by Armadans Gil, which had the highest quality score (97%),The overall aggregate direct + indirect costs for managing an NSI ranged from Int$650 to Int$750, considering the median and mean value of the mean costs borne by the hospital or NHS to manage exposures with different scenarios. These values were derived from both modeling and data-driven studies conducted across different countries and continents over a period of approximately 20 years.Indirect costs are relatively consistent between studies; they mostly refer to lost productivity, which is usually calculated in minutes spent in baseline and follow-up visits by the exposed HCP and more rarely on days of staff absence; the median and mean values of the mean indirect costs ranged between Int$175 and Int$350.Direct costs vary. The wide range of possible scenarios regarding the infectivity of the source, the susceptibility of the exposed HCP, and the post-exposure diagnostic and prophylactic protocol account for the differences in the average direct costs within and between considered studies, particularly when analyzing studies using a different approach. In modeling studies, the source and exposed HCP are tested according to an optimized protocol. Prophylaxes are provided to all susceptible HCP, and those protocols are always considered to have been completed. All exposed HCP attend follow-up visits and testing. In real life, HCP anxiety can influence the choice of baseline and follow-up testing, or the source situation may be more complicated . Acceptance, adherence, and completion rate of prophylactic treatments may be suboptimal; regimens may vary and cause adverse reactions requiring additional interventions; and compliance with follow-up protocols may differ.,Moreover, regardless of the approach, treatment costs vary between studies: the standard antiretroviral PEP regimen changed significantly over time, including different combinations and newer, more tolerable and expensive drugs in recent studies. Variations in HBIG dosage among protocols increased costs up to 4-fold, and diagnostic tests evolved over time. Two recent modeling studies considering direct and indirect costs and using newer antiretrovirals had a median cost of Int$1,187 Notwithstanding all the observed differences, comparisons between studies also identified significant similarities. Therefore, considering the wide range of possible situations, settings, and behaviors covered in selected studies, this overall amount (including direct and indirect costs) should represent a reliable estimate of the actual average cost for managing an NSI. However, the wide range of observed values should be taken into due account to avoid under- or overestimating the economic costs of managing these incidents or the potential savings resulting from their prevention.A cautious interpretation of these findings is warranted. The literature search may be incomplete; moreover, different periods, countries, and study designs were compared. Part of the heterogeneity due to the approach and type of estimated costs was controlled by stratifying the studies. Furthermore, weighted means were calculated where disaggregated costs were available. However, this was not possible for all the studies considered. The effect of this heterogeneity is evident in the high variability (SD of the mean and width of the range) of the estimates. In addition, Drummond’s checklist was designed to include all possible aspects concerning economic evaluations, but in this review, different study designs were compared, and it was not possible to apply all items to all articles. To eliminate disparities between the studies, a quality score was calculated based on the items considered appropriate for each study.Despite these issues, this review confirms that the economic impact of managing NSIs is not negligible, and our analysis provides clues about where to reduce costs.Protocols should be optimized based on the source; HCP vaccination should increase, and the response should be appropriately evaluated post-vaccination and readily available in case of injury. This recommendation is clearly demonstrated in the high-quality study by Armadans Gil et al,Additional strategies to reduce costs could include the following: using rapid HIV tests to assess the source serostatus, thus avoiding unnecessary PEP, tests, and anxiety for the exposed HCP,Even if none of the retrieved studies included HCV-RNA testing in post-exposure protocols, it is recommended as alternative strategy in the United States and is strongly supported in a cost-effectiveness analysis. HCV-RNA testing at 1 month, at an average cost of Int$242 per exposure, leads to earlier detection of HCV transmission and lowers the risk of progression to chronic hepatitis.Are these costs truly representative of this issue? The costs of treating an occupational infection were not included in the calculation of average NSI costs, nor were those of litigation and compensation. Bloodborne infections other than HBV, HCV, or HIV were not considered, though many different pathogens can be, and have been, occasionally transmitted through an NSI. Most importantly, intangible costs arising from an NSI were not evaluated. The single willingness-to-pay analysis identified in the literature reported that the high median amount HCP were willing to pay to avoid a sharps-related injury were Int$828 in the base case and Int$1,237 when exposed to HIV or HCV. This finding suggests that the costs of intangible aspects of HCP injuries, such as anxiety and distress, could equal costs associated with the medical evaluation of these injuries.In conclusion, NSIs generate significant direct, indirect, potential, and intangible costs. While the costs for their prevention may seem high in the beginning, ultimately they prove to be the opposite. The economic expenditures directed toward enhancing interventions to prevent occupational exposures and infections, including provision of SEDs, will likely decrease over time, while enhancing HCP perception of their own value and affecting the quality of the care they provide. As highlighted in the preamble of the EU Directive, “Health and safety of workers is paramount and is closely linked to the health of patients.”