Correlative light and electron microscopy (CLEM) combines the strengths of both light and electron imaging modalities and enables linking of biological spatiotemporal information from live-cell fluorescence light microscopy (fLM) to high-resolution cellular ultra-structures from cryo-electron microscopy and tomography (cryoEM/ET). This has been previously achieved by using fLM signals to localize the regions of interest under cryogenic conditions. The correlation process, however, is often tedious and time-consuming with low throughput and limited accuracy, because multiple correlation steps at different length scales are largely carried out manually. Here, we present an experimental workflow, AutoCLEM, which overcomes the existing limitations and improves the performance and throughput of CLEM methods, and associated software. The AutoCLEM system encompasses a high-speed confocal live-cell imaging module to acquire an automated fLM grid atlas that is linked to the cryoEM grid atlas, followed by cryofLM imaging after freezing. The fLM coordinates of the targeted areas are automatically converted to cryoEM/ET and refined using fluorescent fiducial beads. This AutoCLEM workflow significantly accelerates the correlation efficiency between live-cell fluorescence imaging and cryoEM/ET structural analysis, as demonstrated by visualizing human immunodeficiency virus type 1 (HIV-1) interacting with host cells. On one hand, fluorescence light microscopy (fLM) enables spatial localization and analyses of dynamic properties of molecules in cellular processes. Particularly, proteins, macromolecular complexes and organelles that have been labeled with fluorescence probes can be localized, tracked, and distinguished from the crowded content of the cell, however, with limited spatial resolution (dozens of nanometers at best in super-resolution mode1). On the other hand, cryo-electron tomography (cryoET) has emerged as a powerful technique for visualizing three-dimensional (3D) cellular structures at high resolution in their near-native state and natural environment2. With the recent development of subtomogram averaging techniques, cryoET is now able to generate detailed views of cellular components at sub-nanometer resolutions6. However, the information obtained from cryoET is limited to 3D snapshots of a biological event at specific time points. To achieve a time-lapse panorama and capture meaningful scenes during biological processes, such as virus entry, budding or cell signaling, combined live-cell imaging and high-resolution cryoET with subtomogram averaging using cryoCLEM, is especially desirable8.Cryogenic correlative light and electron microscopy (cryoCLEM) has become a valuable tool for investigating the high-resolution ultra-structures of specific biological entities and events 14, cryofLM probes16 and methods21 for cryofLM imaging. Recently cryofLM devices have become available from several commercial sources . Such systems have been successfully applied to image neurons, viruses, mitochondria and many other cellular components28. To improve the accuracy and efficiency of cryoCLEM, various probes (visible in both fLM and EM) have been employed to guide the correlation step; these include toner particles29, FluoroNanogold (FNG)30, quantum dots (QDs)31, 3,3′-diaminobenzidine (DAB after photoconversion)32, fluorescent dye beads33, and cathodoluminescence pointers34. Further, on the EM side, automation software, such as Leginon35, serialEM36 and ThermoFisher/FEI EPU/TOMO, have allowed users to generate grid atlas maps and to import fLM maps for alignment and registration.One challenging aspect of cryoCLEM is to image fluorescence signals at cryogenic temperature in order to keep the sample vitrified. For this purpose, several groups have developed cryo-light microscopy stages20, (ii) inefficient with low throughput due to multiple steps of manual operation and correlation, and (iii) difficult due to low signals in cryoEM that make direct correlation a challenge. To simplify the cryoCLEM procedure and to make it more robust and accurate, we developed AutoCLEM, a workflow employing fluorescent fiducial beads for automated alignment of correlative images in three sequential steps, from low-resolution fLM to low and medium magnification cryoEM, and to high resolution cryoET. We applied it to examine the early stages of infection by human immunodeficiency virus type 1 (HIV-1) and demonstrate a greatly reduced investment of time for correlation processes (10 fold) as well as improved accuracy (5 fold).However, the entire process of cryoCLEM is (i) tedious and time-consuming due to difficulties in localizing the corresponding region of interest (ROI) under cryoEMThe conventional cryoCLEM experiment usually involves four main steps: I) imaging fixed or live cells with fLM, II) plunge-freezing of cells on EM grids, III) cryofLM imaging, and IV) cryoEM/ET data collection Fig. . The key22 or a polara cartridge7 minimizes direct manual handling of EM grids, thus, greatly reduces the artifact.The first step of correlation is at the level of grid-square, indicated as ‘square’ correlation Fig. . It is nThe next step of correlation is at the fluorescent bead level, indicated as ‘bead’ correlation Fig. . This stThe last step of correlation, indicated as “virus” correlation in Fig. 37 (DIC) map (5 × 5 tiles are shown) of the entire EM grid in less than 5 minutes with auto-focusing  no recognizable or usable feature on the ‘finder’ EM grid is present to help correlate grid-squares; or 2) the grid is flipped during loading for cryoEM. To get around this manual step, we utilized a new feature of the fLM software NIS-Elements, ‘scan large map’, to automatically generate an overview at 20 × magnification, Differential Interference Contrasting Fig. . Furthering Fig.  to facilThe selected grid-squares, such as those indicated in Fig. 39 making it easier to capture intact capsids in imaging studies compared to wild-type virus particles7. To image HIV-1 and host cell interactions by cryoET, we targeted thin (<300 nm) periphery regions of the cell. High-speed time-lapse confocal fLM images were automatically recorded, with Z-sections from the fiducial beads located on the back-side carbon film to the top surface of the cell , fluorescent beads (blue) and cells (red) were recorded simultaneously at the end of time-lapse series, and these were projected in 2D, as visualized in the overlay image Fig. . The inf41 Fig. , the sam41 Fig.  were ide42. All viruses show clear envelope spikes as they are located outside of cells.After generating the coordinate transfer matrix . The 1 µm bead also was not suitable, as beads of this size are easily blotted away during plunge freezing, whereas 100 nm and 200 nm sized beads remain on the EM grid. In addition, precisely determining the positions of the 1 µm beads in cryoEM is a challenge, as they often retain a significant amount of liquid during freezing, which obscures the shape of beads in cryoEM. We found that 200 nm beads work best as fiducial markers compared to either smaller or larger beads. We have also discovered that the beads must be pre-coated on the back side (non-sample side) of the EM grid, or added instantly before plunge freezing. If added to the sample side, the beads are taken up by the cells through endocytosis, as shown in Supplementary Fig. 34. Since most of the correlations were performed manually, the procedures were very time-consuming and tedious. The autoCLEM workflow presented here comprises a near-fully automatic three-step correlation procedure; only the picking of fiducial beads in fLM image and cryoEM grid-square maps for calculating the transformation matrix is manual, and this takes less than 5 minutes. In principle, this manual aspect of the procedure can also be automated in AutoCLEM, but in practice, the variability of fluorescence signals among beads, the amount of ice contamination and variable ice thickness make this challenging. All together the grid-square correlation process (step 1) can be shortened to 10 minutes from 1 hour typically required in a manual effort, and the bead correlation and viral localization steps (steps 2–3) can be shortened to 10 minutes per square from previous 2 hours per square  or external particles  for manual correlation of grid-squares or areas of interest, followed by medium or high magnification correlation using immunogold, Quantum Dots, dye beads, holes on Quantifoil grid carbon, or even intrinsic cellular features as fiducial markers7 E45A HIV-1 was produced by transfecting 293T cells with three plasmids: pL-VSV-G (a kind gift from M. Emerman), peGFPC3-Vpr (a kind gift from T. Hope), and a proviral plasmid, pNLdE-luc, encoding HIV-1NL4-3 with deletions in env and with the luciferase gene in place of nef43. Transfections were performed in 293T cells with lipofectamine 2000  in the cell medium DMEM (10% FBS Pen/Strep/Glu) (Gibco). Virus infectivity was determined on GHOST-R3/X4/R5 cells and capsid levels were measured by p24 ELISA .As previously described2 in EGM™-2 BulletKit™. Cultures at approximately 70–85% confluence were rinsed with 5 ml room temperature HEPES Buffered Saline (HEPES-BSS) and incubated in Trypsin/EDTA solution (0.5 ml Trypsin/EDTA for 60 mm culture dishes). After about 90% of the cells were rounded, the cells were diluted in cell medium, to a density of 0.8–1 × 105 cells/ml, and plated (2 ml/dish) onto disinfected grids placed in glass-bottom culture dishes  with the cell side up. Each dish was incubated overnight.Au or Ni R2/2 Quantifoil ‘finder’ EM grids  were glow-discharged on the back-side of the grid at 20 mA for 20 seconds, coated with 1 µl 1000x diluted FluoSphere 200 nm blue beads , and left to dry. The grids were then disinfected under UV light for 2 hours and treated with 50 µg/ml fibronectin  on the front-side. Human umbilical vein endothelial cells (HUVEC) were cultured at 37 °C with 5% CO2 tissue culture conditions. The system was controlled by NIS Elements software . The EM grid was imaged in glass-bottom dish, which was centered and automatically scanned in DIC channel at 20 × magnification (20 × 0.75 NA plan apo lens), in 5 × 5 stitched tiles, covering the entire 3.05 mm EM grid  to the culture medium. After rinsing and replacement of the culture medium, the cells were infected and imaged as follows. During these processes, careful attention should be paid to not disturb the EM grids in the dishes. For fluorescence live-cell imaging, a Nikon Ti widefield microscope, Bruker Optera confocal scan head and Andor Ultra EM CCD camera was used. A Tokai hit environmental chamber was used to maintain temperature and normal 5% COrid Fig. . Grid-sq7.Immediately after fluorescence confocal imaging, 4 µl of 15 nm gold beads were applied to the EM grid, which was subsequently blotted and plunge frozen using an FEI Vitrobot  at ~100% humidity. The frozen grids were clipped into a Polara cartridge and imaged with a home-built cryo-sample stage to record cryo-fluorescence images, as described previouslyThe frozen grids were loaded to Polara G3 microscope , equipped with a Gatan US4000 4k × 4k Ultrascan CCD  and a Falcon II direct detector  camera, for cryoEM analysis and cryoET data collection. A cryoEM overview map  of the entire grid was recorded using serialEM, with which a 14 × 14 tiles LMM overview was recorded at 200 × on the Ultrascan camera. This LMM overview is correlated to the fLM tile images to find the location of the grid-squares of interest.2 on the Falcon II camera. The tilt series were aligned using 15 nm gold fiducial beads and reconstructed with weighted back-projection algorithm using the IMOD package41. The tomograms of viruses were denoised using 3D nonlinear anisotropic diffusion edge enhancing program implemented in IMOD with 6 iterations and a κ value of 0.8 to enhance visualization.After the grid-squares of interest were located, a medium magnification montage (MMM) was recorded for each square at 4,700× on Falcon II camera with 20% overlap and 300 µm defocus. Using the correlative script developed in this study, coordinates of virus particles were determined, and tilt series were collected bidirectionally, with a tilt range of −60° to 60° and 3° interval, at a nominal magnification of 39,000×, defocus values between 5–8 µm and a total dose of ~100 e/ÅSupplementary InformationMovie 1Movie 2


Arabidopsis thaliana accessions with Turnip mosaic virus (TuMV) and Cucumber mosaic virus (CMV) under high and low light intensity. Results indicated that higher light intensity increased TuMV multiplication and/or plant tolerance, which was associated with more efficient seed transmission. Conversely, higher light intensity reduced plant tolerance and CMV multiplication, and had no effect on seed transmission. This work provides novel insights on how environmental factors modulate plant virus transmission and contributes to understand the underlying processes.Increased light intensity has been predicted as a major consequence of climate change. Light intensity is a critical resource involved in many plant processes, including the interaction with viruses. A central question to plant–virus interactions is understanding the determinants of virus dispersal among plants. However, very little is known on the effect of environmental factors on virus transmission, particularly through seeds. The fitness of seed-transmitted viruses is highly dependent on host reproductive potential, and requires higher virus multiplication in reproductive organs. Thus, environmental conditions that favor reduced virus virulence without controlling its level of within-plant multiplication  may enhance seed transmission. We tested the hypothesis that light intensity conditions that enhance plant tolerance promote virus seed transmission. To do so, we challenged 18  In both light conditions, plant accessions conformed two allometric groups as previously described [Eighteen Arabidopsis accessions were used . Ten acc.05–1.06 . After iescribed , allomet® reagent , and 10 ng of total RNA were added to the Brilliant III Ultra-Fast SYBR Green qRT-PCR Master Mix  according to manufacturer’s recommendations. Specific primers were used to amplify a 70 nt fragment of the TuMV, and a 106 nt fragment of the CMV, coat protein (CP) gene, respectively [−3 ng to 2 × 10−7 ng.TuMV and CMV multiplication were quantified as viral RNA accumulation via qRT-PCR in each individual plant. Virus accumulation was quantified from three disks of 4 mm in diameter collected from different systemically infected leaves. Form these plant samples, total RNA extracts were obtained using TRIzolectively ,65. EachRW), inflorescence (IW), and seeds (SW), were obtained. RW was used as an estimate of plant resources dedicated to growth, and IW was taken as an estimate of plant resources dedicated to reproduction [iTrait/m, iTrait and m denoting infected and mock-inoculated plants, respectively). Virulence was estimated as ratio between infected and mock-inoculated plants of seed weight (iSW/mSW).Aboveground plant structures were harvested at complete senescence and dry weight was determined after maintaining plants at 65 °C until constant weight. The weights of the rosette (oduction . The effGP), as days from inoculation to the opening of the first flower; and reproductive period (RP), as days from the opening of the first flower to the shattering of the first silique. In Arabidopsis, the opening of the first flower co-occurs with the end of the rosette growth, and the shattering of the first silique co-occurs with the end of flower production [LP), as time to plant senescence, was also quantified. Three plant life-history traits were recorded: Growth period . Also, virus infection did not affect the weight of a single seed . Thus, SW similarly reflects the number of viable seeds in both mock-inoculated and infected plants.Fecundity and mortality tolerances of each Arabidopsis genotype were calculated as the slope of the linear regression of d plants ,13. Impo2). Following [ST) was then estimated using the expression reported by [p is the probability of virus transmission by a single seed, y is the number of positive samples, n is the total number of samples assayed (n = 33), and k is the number of seedlings per sample (k = 3).The efficiency of virus seed transmission was estimated as per cent of infected seeds that gave rise to infected progeny per plant. Accordingly, we measured virus seed transmission to seedlings. For each replicate, 100 seeds were washed in a 10% household bleach solution (4% active chlorine) to ensure that any viral infection that occurred was not simply the result of virus presence on the seed coat, but rather the result of embryonic infection. Seeds were kept in this solution for 5 min and washed three time in sterile distilled water. Then, seeds were placed in Petri dishes containing Murashige and Skoog medium, stratified for three days at 4 °C, and germinated in a growth chamber at 22 °C, under 16 h light . The transformed and the rest of untransformed traits were fitted to a Log-normal distribution according to Akaike’s Information Criteria (AIC), with the exception of iRP/mRP that was fitted to a Gaussian distribution . Light butions, ,71,72. To describe the relation between the effect of infection on the plant developmental schedule, growth and reproduction traits, virus accumulation, fecundity and mortality tolerance and virus seed transmission a Principal Component Analysis (PCA) was done for each virus. PCAs were performed using mean values per accession, which were scaled to zero mean and unit variance, inserted in a regression matrix and rotated using Varimax to obtain the Principal Components (PCs) using R-libraries FactorMiner and factoextra ,74. Stat


C. neoformans/C. gattii may have the potential to be more virulent according to the molecular type. This study aims to investigate the association between virulence in the G. mellonella model and genotypic diversity of Colombian clinical and environmental isolates of C. neoformans/C. gattii. A total of 33 clinical and 12 environmental isolates were selected according to their geographical origin and sequence types (STs). Pathogenicity was determined using the G. mellonella model, and the cell and capsular size before and after inoculation was determined. For C. neoformans, virulence in G. mellonella revealed that death occurred on average on day 6 (p < 0.05) and that ST5C, 6C, 25C and 71C were the most virulent. In C. gattii, death occurred at 7.3 days (p < 0.05), and ST47C, 58C, 75A and 106C were the most virulent. Capsular size increased for both species after passage in G. mellonella. In conclusion, the pathogenicity of Cryptococcus strains in the G. mellonella invertebrate model is independent of molecular type or pathogenicity factor, even within the same ST, but it is possible to find variable degrees of pathogenicity.Cryptococcosis is a potentially fatal opportunistic mycosis that affects the lungs and central nervous system. It has been suggested that certain strains of  Cryptococcus neoformans (var. grubii and var. neoformans), which is widely distributed and mainly affects immunosuppressed individuals, and Cryptococcus gattii, which can be found in tropical, subtropical, and temperate regions and affects primarily immunocompetent individuals or patients with certain predisposing risk factors [C. neoformans species complex and C. gattii species complex [Cryptococcosis is a potentially fatal opportunistic mycosis affecting the lungs and central nervous system . The inf factors ,2. Recen complex .Galleria mellonella larva due to the cost and bioethical implications of experimentation with mammalian models [G. mellonella a good model for fungal pathogenesis are hemocytes, expert phagocytes, and the large number of antimicrobial peptides in its hemolymph [C. neoformans/C. gattii [Candida albicans, C. tropicalis, Histoplasma capsulatum, and Paracoccidioides lutzii, among others [G. mellonella to infection resembles the activity exerted by neutrophil macrophages during the innate immune response in mammals [The virulence of human pathogens has been studied classically in mammals, the mouse being the most widely used model. In recent years, interest has been shown in the use of alternative non-vertebrate models such as n models ,5,6. Theemolymph . An exam. gattii ,5. Candig others ,8,9. The mammals , which iC. gattii infection in both murine and G. mellonella models [C. neoformans and C. gattii [Similar responses have been recorded during a models , suggest. gattii ,6,7,10.C. neoformans and C. gattii, previous studies have implemented a wide variety of molecular biology techniques, such as polymerase chain reaction (PCR) fingerprinting [PLB1 and URA5 genes [CAP59, GPD1, LAC1, PLB1, SOD1, URA5 and IGS1) are used [To study the epidemiology of printing , restricA5 genes ,13, and are used .C. gattii strains in Drosophila melanogaster [C. neoformans and C. gattii in the invertebrate model G. mellonella.Some studies have revealed an association between molecular type and virulence; isolates of the molecular type VGIIa recovered from the outbreak reported on Vancouver Island, British Columbia, were reported to be more virulent in a mouse model than the strains of the minor subtype, VGIIb ; in addinogaster . In thisC. neoformans and C. gattii recovered in 9 departments in Colombia between 1993 and 2014, stored in the collection of the Microbiology Group of the Instituto Nacional de Salud, were selected. Of these, 33 isolates were of clinical origin (24 C. neoformans and 9 C. gattii), and 12 isolates were from environmental sources (7 C. neoformans and 5 C. gattii). Inclusion criteria were source , geographical origin and ST. A detailed description of the C. neoformans and C. gattii strains used in the study is described in A total of 45 isolates of C. neoformans, a total of 24 clinical and 7 environmental isolates were used for the analysis. Among the clinical isolates, 79.1% were male; the mean age was 39 years, and HIV/AIDS was diagnosed in 79.1% of cases. The 7 environmental isolates came from plant material (n = 6) and bird droppings (n = 1). For C. gattii isolates, nine were of clinical origin, and five were environmental. Among the clinical isolates, 55.5% were male; the average age was 37 years, with HIV/AIDS reported in 33.3%, cases. Five environmental isolates came from three different tree species.For 7 CFU/mL with a spectrophotometer at a wavelength of 530 nm and a reading of 0.561 absorbance. A dilution of 1:1000 and 100 µL of the inoculum was inoculated onto SDA incubated at 27 °C, and 20 colonies were randomly selected. The morphological characteristics were observed by macroscopic visualization according to the texture (mucoid or non-mucoid) and aspect of the colony (wrinkled and smooth) for seven days [Each isolate was plated onto Sabouraud dextrose agar (SDA) and incubated at 27 °C for 48 h, and an inoculum was adjusted to 3.0 × 10ven days .a.DNA extraction was performed as described by Casali . Mating The larvae were obtained from a Scientia breeding facility , late-stage larvae (fifth and sixth) with weights between 250 and 330 mg and a length of approximately 2 cm were selected. A group of 20 larvae were used for each of the controls: absolute control; larvae without any manipulation, inoculation control; larvae inoculated with phosphate buffered saline (PBS) to monitor killing due to physical injury, and disinfection control; larvae disinfected with sodium hypochlorite to monitor toxicological effects of disinfections. To compare mortality, we performed three biological replicates, with 20 larvae for each strain evaluated .8 UFC/mL using a Neubauer chamber, were used to inoculate 20 larvae per isolate, each with 10 µL of inoculum by injection into the last left proto-leg using a 0.5 mL gauge insulin syringe [The strains were grown in SDA and incubated for 48 h at 27 °C. Suspensions, adjusted to 1.5 × 10C. neoformans and C. gattii used in the study is shown in After inoculation, larvae were placed in Petri dishes and incubated in darkness at 37 °C, and the number of dead larvae were recorded daily . A detaiC. neoformans or C. gattii were determined pre- and post-inoculation of the larvae with each isolate.The cellular and capsular sizes of Isolates were cultured in Sabouraud broth for 48 h at 150 rpm at 30 °C; next, a microscopic slide containing one drop of Indian ink and one drop of each strain was prepared and visualized in a Zeizz Axiophot Microscope in a 40× lens. The total cell and capsule area was measured, and the cell area was calculated according to the total area minus the area of the capsule (20 cells were measured for each isolate). A small capsule size was established as a measurement of 0.6 to 2.2 µm; intermediate, 2.3 to 3.3 µm; and large, 3.3 to 4.2 µm [G. mellonella after the survival assay. Each dead larva was macerated and homogenized in 1 mL of 1× PBS and the procedure previously described in preinoculation was performed to measure cell and capsular size [C. neoformans and C. gattii used in the study is shown in To determine these measurements after inoculation in the larvae, strains were recovered from lar size ,20. A de® program; the analysis was performed separately for C. gattii and C. neoformans. Stata software version 11.0 was used, and numerical variables were developed by means of measures of central tendency, and a Chi square test or Fisher exact test was used for categorical variables, with significance lower than 0.05% and 95% confidence. Survival analysis was performed using the Kaplan–Meier method for the invertebrate model; the analysis of the effect of capsular and cellular change before and after inoculation was performed using the McNemar statistic generated with 95% confidence [Data collection was tabulated in the Microsoft Corporation Excelnfidence .C. neoformans was 4.3 mm, with maximum values of 6.8 mm and minimum values of 3.0 mm; for C. gattii, this measure was 4.1 mm, with maximum values of 6.9 mm and minimum values of 2.3 mm  in C. neoformans and ST 25 in C. gattii ; ST5C, 6C, 25C and 71C were the most virulent, with a mean survival of 4.5 days, with respect to control strain H99 ; ST47C, 58C, 75E and 106C were the most virulent, with a survival of 5.5 days with respect to control strain CDCR-272 (H0058-I-2508)  c,d. In cC. neoformans and C. gattii, respectively, were responsible for the higher mortality of larvae in less time. C. neoformans ST77 and ST93, and C. gattii ST25, assigned to clinical and environmental isolates, had a similar degree of virulence in the invertebrate model . The mean total cell size for C. neoformans was 5.66 μm and for C. gattii was 4.28 μm. A total of 66.6% of the isolates evaluated presented medium size capsules for the two species, and it was observed that the largest capsules in pre-inoculation were presented in ST93 and ST47 for C. neoformans and C. gattii, respectively, in isolates of clinical origin with respect to the environmental ones .Observing each ST individually, it was evident that  3.42 µm , Table 2Cryptococcus spp. have diverse effects on virulence, and changes in the phenotype characteristics allow adaptation to the environment. Variation in colony morphology and principally mucoid colonies have been associated with strain virulence [Phenotype traits in irulence ,20,21,22irulence  reportedirulence ,21,22,23G. mellonella model has shown correlation in the results obtained from the pathogenicity of microorganisms in the larva with that presented in vertebrate animal models. In this work, it was observed that there is a high variability among the isolates in their pathogenic capacity and that this one is not associated with the origin of the isolate. Several studies agree that mating α is considered a factor associated with a high virulence [C. neoformans clinical and environmental isolates [C. neoformans and in most of the isolates of C. gattii.Studies of the pathogenesis of microorganisms in invertebrate hosts during the last decade have contributed to knowledge about the mechanisms of pathogenesis and host defense. The irulence  and, in isolates . Our resC. gattii before infection ranged from 5 to 12 microns and after the inoculation varied from 15 to 75 microns [In the present work, the capsular and cellular size was evaluated, pre-inoculation and post-inoculation, in the invertebrate model; significantly larger capsules were observed after the fungus passed through the larvae for both species, which is related to several pathogenicity studies such as that performed by Firacative et al. , who fou microns ; this in microns .The morphology of colonies can be smooth, mucoid, or wrinkled, with increased virulence associated with a mucoid or wrinkled phenotype ,27. We fC. neoformans and C. gattii to cause disease in mice. The studies of Sabiiti et al. [Galleria. These data reaffirm that the virulence of Cryptococcus strains varies greatly, highlighting some important differences between the various infection models.However, it was observed that three clinical isolates did not present a significant change in capsular size, which coincides with several investigations when finding small capsules or cells without capsules recovered from clinical cases. Kimura et al. , reportei et al.  showed ti et al.  found hyC. neoformans and sought relationships between the genotype, phenotype and clinical presentation of the disease. The authors conclude that clinical and phenotypic differences were detectable among genetic lineages; however, explaining the complex relationships between genetic diversity, disease presentation and outcome in a host is difficult [Among the tested sequence types of isolates from clinical cases and environmental samples, it is possible to say that only in ST77 was there an association found between mortality in the invertebrate model, even though virulence factors presented similar values between the tests; however, with this evidence, it cannot be concluded that STs are related to virulence factors. It is suggested in future investigations to expand the cohort of isolates related to each ST to be evaluated for different virulence factors, as proposed by Beale et al. , who genifficult .C. gattii VGIIa isolates had a higher virulence profile [In this study, no specific ST strains predominated with high mortality, colony morphology, or cell or capsular size. Although this molecular characterization is a great taxonomic tool, the method did not show any significant clustering or association to permit distinguishing virulent from less virulent strains from clinical and environmental origins. The study carried out by Litvintseva and Mitchell  with a g profile .C. neoformans nor C. gattii in the invertebrate model of G. mellonella is specifically associated with a specific virulence factor. Several studies have determined that the virulence of a strain is highly variable, not only between different isolates but also between cells of the same strain, demonstrating that different factors can influence virulence, as suggested by Trevijano-Contador N et al., who demonstrated the effect of different conditions on the formation of titan yeast cells [In summary, these findings demonstrated that pathogenicity of neither st cells .


The reported outcomes of the metabolic syndrome (MetS), containing atherosclerotic cardiovascular disease and diabetes, vary according to the definitions used. This study was designed to compare the performance of the Adult Treatment Panel III/American Heart Association/National Heart, Lung, and Blood Institute (ATP III/AHA/NHLBI) and International Diabetes Federation (IDF) criteria for the risks of atherosclerosis and diabetes.We sifted subjects from a self-paid Health examination program from 1999 to 2015 in this cross-sectional population-based study. On the basis of the ATP III/AHA/NHLBI and IDF criteria, the MetS diagnosis and scores were concluded. A brachial-ankle pulse wave velocity (baPWV) more than or equal to 1400 cm/s indicated more severe arterial stiffness, and a high fasting glucose level more than or equal to 6.99 mmol/L or postprandial glucose level more than or equal to 11.10 mmol/L indicated diabetic-level hyperglycemia. Comparisons of the areas under receiver operating characteristic curves (AUC–ROC) for both MetS scores to correlate with a higher baPWV and diabetic-level hyperglycemia were evaluated.p < 0.001) were classified as having MetS on the basis of the ATP III/AHA/NHLBI and IDF criteria, respectively. The AUC–ROC for the ATP III/AHA/NHLBI-MetS score were higher than those for the IDF-MetS score .In the 26,735 enrolled subjects with an average age of 55 (± 12) years, 6633 and 7388 (24.8% vs. 27.6%, To the best of our knowledge, this is the first study to demonstrate that through a holistic approach, the performance of the ATP III/AHA/NHLBI-MetS score for the risks of atherosclerosis and diabetes was superior to the IDF-MetS score for Asians. Throughout the world, the prevalence of the metabolic syndrome (MetS) is rising , 2. MetSCurrently, there are many different MetS definitions, as the MetS is a combination of different unfavorable conditions with the joint pathophysiology of abdominal obesity and insulin resistance, and not just one single disease . MoreoveAtherosclerosis-related arterial stiffness is examined by pulse wave velocity (PWV), which is highly associated with ASCVD and has been well-studied as a powerful risk factor for coronary artery disease  and all-Database from 1999 to 2015 from a Health Examination Program at one’s own expense at the Health Care Center of Taoyuan Chang Gung Memorial Hospital was gathered in the cross-sectional population-based study. In our recent report, the database and the selection criteria have been described previously to test the weight of different WC cut-off values of the MetS diagnosis for the risks of atherosclerosis and diabetes in a Chinese population . BrieflyAll subjects were required to give their written informed consent. The Ethics Committee of the Institutional Review Board of Chang Gung Memorial Hospital  approved the study, and we did on basis of the ethical principles of the Declaration of Helsinki.A baPWV more than or equal to 1400 cm/s was set as a higher level indicating more serious arterial stiffness . A high On the basis of the 2005 ATP III/AHA/NHLBI  and 2005Brachial BP measured by an appropriately sized cuff was recorded in duplicate, and values were reported as a mean of two recordings. WC was measured at the midpoint between the iliac crest and lowest rib when a subject was in a standing position with feet 15 cm apart. All subjects underwent laboratory measurements, and after fasting overnight for 12 h, venous serum samples were gathered to test in the early morning. The biochemical measurements contained tests of fasting plasma glucose, HDL-C, and triglyceride at the only laboratory of our hospital. We supplied the uniform food and drink for every subject, and tested their post-prandial plasma glucose after 2 h.An automated machine  were applied to achieve measurements of baPWV. Details were recorded in our previous report . Brieflyp value less than 0.05 was applied to determine statistical significance. We applied the Kolmogorov–Smirnov test to assess all variables for normal distribution. Moreover, the comparisons were performed using one-way analysis of variance for continuous variables and using 2-sample proportions test for the prevalence of the both MetS. We plotted receiver operator characteristic (ROC) curves for the ATP III/AHA/NHLBI- and IDF-MetS scores to correlate with a higher baPWV and diabetic-level hyperglycemia. Next we compared the areas under the ROC curves (AUC–ROC) for the performance by applying R software version 3.3.2 . The optimal cutoff points of both MetS scores were obtained by computing the equation: sensitivity − (1 − specificity), when the maximum values were reached, called the Youden index [We applied SPSS version 22.0 for Windows 7  to analyze most data which were presented as the average (± SD) or frequency in last. A en index .In all, 26,735 adult subjects were concluded from 2006 to 2015 with an average age of 55 (± 12) years (range 20–101 years), WC of 87 (± 12) cm, SBP of 135 (± 20) mmHg, DBP of 81 (± 12) mmHg, fasting glucose level of 5.55 (± 1.55) mmol/L, post-prandial glucose level of 6.44 (± 3.00) mmol/L, HDL-C level of 1.32 (± 0.36) mmol/L, triglyceride level of 1.57 (± 1.13) mmol/L, and baPWV of 1506 (± 336) cm/s. Among them, 10,514 (39.3%) were female and 2738 (10.2%) suffered from diabetic-level hyperglycemia.p < 0.001).The number of individual components of both MetS definitions included: (1) 4158 (15.6%) for WC according to the ATP III/AHA/NHLBI-MetS and 12,602 (47.1%) according to the IDF-MetS; (2) 15,713 (58.8%) for BP; (3) 8223 (30.8%) for fasting glucose; (4) 8408 (31.4%) for triglyceride; and (5) 7751 (29.0%) for HDL-C. In all 26,735 subjects, 6633  with an average age of 57 (± 12) years and 2695 (41%) female were classified as having the ATP III/AHA/NHLBI-MetS compared to 7388  with an average age of 57 (± 12) years and 3107 (42%) female as having the IDF-MetS    Fig. a. In add01) Fig. b.Fig. 2Ap for trend < 0.001)   Fig. c. In add01) Fig. d.p < 0.001)   were classified as having MetS on the basis of the ATP III/AHA/NHLBI criteria, compared to 7388 (27.6%) on the basis of the IDF criteria. The stepwise raises in the baPWV and prevalence of diabetic-level hyperglycemia corresponded significantly to both MetS scores, except for a score of 0 in the IDF-MetS, indicating no abdominal obesity. The performance of the ATP III/AHA/NHLBI-MetS score for a higher baPWV and diabetic-level hyperglycemia was superior to the IDF-MetS score. The optimal cutoff points of the ATP III/AHA/NHLBI-MetS score were more than or equal to two, with a batter sensitivity from 32.7 to 62.2% to correlate with a higher baPWV and from 62.6 to 89.4% to correlate with diabetic-level hyperglycemia.In the enrolled subjects, 24.8 and 27.6% were classified as having MetS on the basis of the ATP III/AHA/NHLBI and IDF criteria, respectively. Retrospectively, a study from Taiwan in 2002 reported a value of 14.3% with the IDF-MetS , almost We also showed that stepwise raises in both MetS scores corresponded to the baPWV and prevalence of diabetic-level hyperglycemia, as they were highly correlated with the joint pathophysiology of insulin resistance and represent groups of risk predictors of diabetes and ASCVD . In our Since these different MetS definitions were established for more than 10 years, differences in sex-specific characteristics and prevalence of the ATP III/AHA/NHLBI- and IDF-MetS among the same population have been frequently published , 25. HowIn this study, we showed that through a holistic approach, the performance of the ATP III/AHA/NHLBI-MetS score for the risks of atherosclerosis-related arterial stiffness and diabetes was superior to the IDF-MetS score for Asians. Considerable individual variation has been reported in the clinical pattern of metabolic factors in patients with insulin resistance or obesity . This meIn a cross-sectional survey, Hwang et al. reported that since the IDF-MetS criteria failed to identify non-obese subjects with hypertension, diabetes and dyslipidemia, the ATP III-MetS definition was better than the IDF-MetS definition to detect subjects at a higher risk of ASCVD . This isBy contrast, in the prospective San Antonio Heart Study with 7.4 years of follow-up, the ATP III/AHA/NHLBI and IDF criteria were both associated with an increased but similar ratio of incident CVD and diabetes . In the Interestingly, we also showed that for Asians, the presence of more than or equal to 2 of 5 components of the ATP III/AHA/NHLBI-MetS criteria yielded a better sensitivity than the current criteria. This is a novel finding and very important because increasing obesity caused by a hypernutrition and a sedentary lifestyle , 5 is prThe study has some limitations. First, some data containing smoking habits, comorbidities, and medications were missed. The information of the questionnaires including family and personal history of chronic diseases and lifestyle optionally are routinely obtained for patients seeking health check. The information may be incomplete, and has not been verified. To avoid misunderstanding we didn’t include the data in our manuscript. However, subjects were mostly asymptomatic and relatively healthy people, who needed to have their health status checked in a self-paid Health Care Center, but different from the National Health Insurance Program in Taiwan , 19, 20.Second, we were unsure as to the appropriate WC cutoff value of the ATP III/AHA/NHLBI-MetS in our Chinese population. However, in the published literature, the same WC cutoff value has been applied in other Asian nations including China  and HongThis is the first study to demonstrate that through a holistic approach, the performance of the ATP III/AHA/NHLBI-MetS score for the risks of atherosclerosis-related arterial stiffness and diabetes was superior to the IDF-MetS score for Asians. Our novel findings also supported that for Asians, the presence of at least two of five components of the ATP III/AHA/NHLBI-MetS was better than the current criteria, with an improved sensitivity. Through a holistic approach, further prospective and longitudinal randomized controlled studies are required to compare the clinical outcomes between the different MetS definitions.


Differences between country-specific guidelines for economic evaluations complicate the execution of international economic evaluations. The aim of this study was to develop cross-European recommendations for the identification, measurement and valuation of resource use and lost productivity in economic evaluations using a Delphi procedure.A comprehensive literature search was conducted to identify European guidelines on the execution of economic evaluations or costing studies as part of economic evaluations. Guideline recommendations were extracted by two independent reviewers and formed the basis for the first round of the Delphi study, which was conducted among European health economic experts. During three written rounds, consensus (agreement of 67% or higher) was sought on items concerning the identification, measurement and valuation of costs.Recommendations from 18 guidelines were extracted. Consensus among 26 panellists from 17 European countries was reached on 61 of 68 items. The recommendations from the Delphi study are to adopt a societal perspective, to use patient report for measuring resource use and lost productivity, to value both constructs with use of country-specific standardized/unit costs and to use country-specific discounting rates.This study provides consensus-based cross-European recommendations on how to measure and value resource use and lost productivity in economic evaluations. These recommendations are expected to support researchers, healthcare professionals, and policymakers in executing and appraising economic evaluations performed in international contexts.The online version of this article (10.1007/s10198-017-0947-x) contains supplementary material, which is available to authorized users. The introduction of new pharmaceuticals and technologies has caused healthcare costs to steadily rise over recent decades in Europe , 2. ThisTo ensure the comparability and quality of economic evaluations, several European national agencies developed methodological guidelines on the principles and methods for the design, execution and reporting of economic evaluations over recent decades. In many countries, for example in Belgium, Germany, Norway, Portugal, Poland, the Netherlands, the Slovakia, and Slovenia –10, it iIn recent years, the EU stimulated the development of multidisciplinary partnerships among government agencies, research institutions and health ministries across European countries by funding several health technology assessment (HTA) projects at the European level –13. AlsoThis study is part of the European Identifying Best Practices for Care-Dependent Elderly by Benchmarking Costs and Outcomes of Community Care (IBenC) project. IBenC’s primary aim is to identify best practices of community care delivery systems across Europe by comparing their costs and quality of care outcomes while also developing methods to support the accomplishment of this aim .A Delphi study with three consecutive, blinded rounds was conducted between December 2014 and January 2015 among European health economic experts. The Delphi study was conducted online to ensure anonymity of the panellists. A steering committee was appointed to force consensus on items that were not agreed on by the panel after the final Delphi round –24.A group of 110 international experts working in the field of health economics at government agencies, universities, research institutes and pharmaceutical agencies from 27 European countries was informed and invited by e-mail to participate in the Delphi study. Experts were selected on the basis of HTA-related publications in peer-reviewed journals, their participation in the development of national guidelines, their participation in other European HTA projects or their involvement with the International Society for Pharmacoeconomics and Outcomes Research (ISPOR) or EUnetHTA. Experts who were unable to participate in a Delphi round, but expressed their interest, were invited again for the subsequent round.An independent steering committee consisting of four professionals  in the field of health economics decided on items for which no consensus was reached after the third Delphi round. None of them were involved in selecting potential panellists, designing the questionnaires and analysing the results.The questionnaire used in the first round of the Delphi study was based on a review of existing European guidelines for designing and conducting economic evaluations or costing studies as part of economic evaluations. To identify existing guidelines, the website of the ISPOR was searched . ISPOR pThe recommendations extracted from the identified guidelines were used to formulate questions for the first Delphi round. In this Delphi study, the starting point was the societal perspective for identifying relevant costs in an economic evaluation, because this is the most comprehensive perspective. Other commonly used perspectives, such as the healthcare and government perspectives, can be derived from this perspective. For each item, panellists were asked to indicate the most appropriate option, or to choose “no expertise” if they felt they had insufficient expertise on a specific topic. To capture recent methodological developments that were not included in the identified guidelines, alternative response options for each question could be provided by panellists. In all rounds, panellists were asked to justify their answers to every question. Together with the questions from the second and third rounds, panellists received a feedback report with the individual and group results from the previous round.Consensus among the panel was defined a priori as an agreement of 67% or higher to include or exclude a specific item  rated with a dichotomous response option. Agreement of 67% or higher is commonly used in Delphi studies to indicate consensus . AgreemeIn the first round, we asked the Delphi panel to indicate for each of the listed perspectives, cost categories, and resource use items whether they should be included in an economic evaluation conducted from a societal perspective. Additionally, the panel was asked to indicate appropriate methods for measuring and valuing resource use and lost productivity, and whether value added taxes (VAT) should be included for all cost categories.Questions in the second round were developed on the basis of the analysis of the previous responses. Items for which no consensus was reached in the first Delphi round were included once more, accompanied by arguments for and against inclusion reported in the first Delphi round. Alternative perspectives, new resource use items and alternative valuation methods suggested by the panellists in the first Delphi round were put forward for consideration as well. In addition, panellists were asked to rank the listed methods for assessing and valuing resource use and lost productivity with regard to their relevance. Finally, the panellists were asked to indicate which of the listed discounting rates and study design they found appropriate.To construct the questionnaire for the third Delphi round, rankings on relevance given by the panellists in the second round were converted into relevance scores (a higher score indicates a more relevant method). First, for each respondent, the least relevant method was awarded one point, and the next relevant method was awarded one point more than the previous method. An exception was made for the two methods that were considered most relevant by the panellist. To discriminate better between methods ranked first and second and the other methods, the method placed in the second position received a relevance score twice that of the method in the third position. The method placed in the first position received twice the relevance score of the method in the second position. Subsequently, mean relevance scores for every method were calculated by summation of relevance scores and their division by the number of panellists. The final rankings were based on these mean relevance scores.On the basis of the relevance scores, the two methods considered most relevant per topic were presented in the third Delphi round. Panellists were asked to choose the method they found most suitable to use in a European economic evaluation from a societal perspective. Also, items for which no consensus was reached in the second Delphi round were addressed again.Finally, when no consensus was reached on an item, the steering committee was requested to make a final decision. By e-mail, the steering committee was provided with the arguments for and against a specific recommendation given by the panellists so that the members of the steering committee could weight these considerations to reach a final decision. Individual opinions from the members of the steering committee were gathered and used to make a final decision.Eighteen national guidelines were included in the study. These guidelines were published in Austria, the Baltic states , Belgium, Croatia, Denmark, Finland, France, Germany, Hungary, Ireland, Italy, the Netherlands, Norway, Poland, Portugal, Spain, Sweden and the UK , 28–40. Table Of the 110 invited experts, 26 (24%) participated in one or more rounds, six agreed to participate but did not participate (5%), 48 (44%) did not respond and the remainder  did not wish to participate mainly because of time constraints. Of the 26 invitees who participated, 11 (42%) participated in all three rounds, three (12%) participated in two rounds, and 12 (46%) participated in one round. Each round was completed by at least 16 experts. Background information on the panellists is presented in Table Table The panel reached consensus on 58 of 65 items (89%). The steering committee decided on the seven items without consensus after three rounds. These items comprised the inclusion of four cost items, two measurement methods and one valuation method.Table The societal perspective is recommended for economic evaluations in a cross-European context (88% agreement). The societal perspective helps to identify cost shifting between sectors. According to the panel, it is likely that relevant costs are missed when a narrower perspective is used because often sectors other than healthcare may incur costs or costs savings as a result of the intervention.It is recommended to take a broad perspective with regard to costs, and to include all cost categories for which differences are expected between treatments. The relevant cost categories according to the Delphi panel are healthcare service costs, intervention costs, patient and family costs, lost productivity costs and future costs.Healthcare services are services directly related to the prevention, diagnosis, treatment, rehabilitation and nursing care for a particular disorder. Assistance with  activities of daily living also falls into this category. The panellists did not reach consensus on the inclusion of costs of complementary therapies . The arguments provided by the panellists in favour of inclusion were that all related resource use should be included, and that costs of complementary therapies may be significant. A counterargument was that complementary therapists do not represent evidence-based medicine. The steering committee recommended that these costs should be included, since these costs represent resources that were used. Depending on the country’s funding system, complementary therapies may be reimbursed by health insurance and are then considered part of healthcare costs. If the costs are not reimbursed, they have to be categorized as out-of-pocket costs. An additional category that emerged from the Delphi study was the costs of e-health interventions .Intervention costs include all costs related to the implementation of a particular intervention, and should cover both personnel costs , and the costs of materials needed for implementation of the intervention, including costs of donated items  (71% agreement). On the inclusion of development and training costs of the intervention, no consensus was reached . The panellists’ arguments in favour of inclusion were that all production costs should be included, ad that training costs should be included if training is a prerequisite for the intervention to be effective. However, some argued against inclusion, because a ‘steady state’ should be assumed; the cost of development is usually covered by the price of the intervention. The steering committee recommended that a ‘steady state’ of the intervention should be assumed, implying that costs are estimated for routine implementation of the intervention in daily practice, and thus not to include development costs separately. If training of staff is a prerequisite for adequate execution of the intervention, then training costs represent a true use of resources and should be included. However, if only a one-time initial training is required, it is recommended not to include these costs. Care should be taken that intervention costs are not double counted with healthcare costs.Patient and family costs include expenses incurred by patients as a consequence of the disorder under study, and costs of informal care. The following cost categories should be taken into account: patient-out-of-pocket expenses such as costs of over-the-counter-medication and costs of assistive aids (89% agreement); patient time costs associated with seeking and receiving care for the disorder under study (78% agreement); the costs travel required by patients (84% agreement); and informal care costs (94% agreement). Informal care costs include costs related to time spent and resources used by informal caregivers that are not compensated (76% agreement) or not partially or fully compensated (94% agreement).Lost productivity costs are defined as costs related to reduced productivity from paid labour as a direct consequence of the disorder under study. The costs of both absenteeism (i.e. absence from paid and unpaid work) (100% agreement) and presenteeism (i.e. reduced efficiency when present at work) (83% agreement) should be included in an economic evaluation from a societal perspective. Lost productivity costs due to absenteeism from unpaid labour should not be included in an economic evaluation according to the panel (73% agreement). The main argument not to include these costs was the lack of standardized methods to value unpaid labour, leading to unreliable cost estimates and a risk of double counting. However, it was also argued that costs of absenteeism from unpaid labour may be an important cost category in specific patient populations such as elderly people, where the proportion of people performing unpaid labour is higher as compared with the general population.Future healthcare costs are costs for treatment of disorders occurring in life years gained as a result of the intervention under study. Part of the future healthcare costs is directly related to the intervention, whereas other costs are not (e.g. costs for dementia treatment in added years because of successful cancer treatment). Future healthcare costs related to the intervention should be included according to the panel (100% agreement). A consensus was not reached on the inclusion of healthcare costs unrelated to the intervention . Panellists in favour of inclusion argued that unrelated future healthcare costs should theoretically be included if an intervention (significantly) prolongs life and if important differences in future costs between interventions are expected. However, others argued against inclusion on the basis that the calculations are difficult as many assumptions are made. The steering committee recommended the inclusion of related and unrelated future healthcare costs if the intervention is expected to result in an extension of life, because it represent a true use of resources.A summary of relevant cost items per cost category is provided in Appendix 1 in the electronic supplementary material.The panellists did not agree on the most suitable method to assess healthcare utilization and were divided between patient-based reporting and the use of national insurance fund utilization databases (secondary-level data). Some panellists indicated that these databases are more accurate than patient-level data, whereas others argued that these databases are less precise, may not contain all relevant information and may create problems when data are linked to individual patients. The steering committee opted for patient-based reports as the most preferable method, because not all services are covered in national databases and such databases are not easily available for all countries.The recommended methods to collect patient-reported data are resource use questionnaires and interviews, activity logs and cost diaries. If patients are incapable of self-reporting, proxy reports are recommended. Patient out-of-pocket expenses are country specific as they depend on the reimbursement level and funding system. Therefore, the patient is considered the most reliable source to obtain these data. Patient time costs are also preferably measured with use of patient reports. In situations where patient reports cannot be used, these costs can also be based on standard time estimates associated with seeking and receiving care. However, researchers should be aware that patient time costs in seeking care may overlap with absenteeism from paid labour, with the risk of double counting. When travel costs are being assessed, it is recommended to use standard distances from the patient’s home to the healthcare provider over patient-reported distances, to avoid random differences between groups (100% agreement). Costs related to time spent and resources used by informal caregivers should be based on self-reports.No consensus was reached on what is considered the most suitable method to measure absenteeism from paid work (company-registered data or self-reported sick leave). The panellists in favour of using company registered data (50% of the panel) indicated that they are cheaper, easier to collect, more trustful and more credible than self-reported sick leave, whereas opponents (50%) argued that company-registered data are less precise, rarely available, and difficult to obtain in certain situations. The steering committee recommended that absenteeism from paid work should preferably be measured with self-report questionnaires, because self-reported productivity losses can be more accurately attributed to the disorder under study. Also, self-reported productivity losses may be more easily available than reports from a large number of different employers. The recommended method to collect data on presenteeism is to obtain ratings of both the quantity and the quality of work from the patient by standardized questionnaires (73% agreement).Opportunity costs are recommended to value resource use. Costs used in an economic evaluation should be representative of the country under study. Therefore, country-specific costs are preferred (100% agreement), and European average costs should not be used (71% agreement). Since country-specific costs may differ considerably, resource use rates and costs should be presented separately to facilitate the generalization of study results to other settings.The preferred proxy measure for the opportunity cost of healthcare services is country-specific standard unit costs, when available (77% agreement). Standard unit costs include all costs related to the provision of a particular service. However, standard unit cost estimates should not be used for patient out-of-pocket expenses because of large variations between patients (75% agreement). The panellists did not agree on the most suitable valuation method for the opportunity costs of patient time and informal care . The arguments in favour of the use of national averages wages were the availability and “otherwise disease affecting highly skilled or paid people have an advantage”, whereas the counterargument was that patients are not unskilled. The steering committee recommended the use of sex- and age-specific average wages of unskilled labour for the valuation of patient time and informal care, although the committee recognizes this may underestimate the true opportunity costs associated with informal care. Use of shadow prices may be more flexible since they can be adapted to the specific informal care situation. With regard to costs of traveling by public transport, tariffs should be used (86% agreement). Tariffs are expected to be closely related to market prices, and are therefore considered to resemble opportunity costs adequately. For travel by car, it is recommended to use standard costs per kilometre/mile, including costs for petrol, maintenance, depreciation, and taxes (86% agreement). Lost productivity should be valued with age- and sex-specific national average wages (93% agreement). For the valuation of absenteeism from paid work, the friction cost approach is preferred over the human capital approach because the latter is expected to lead to overestimation of productivity losses (73% agreement). With the friction cost approach, the period over which the production loss is calculated is limited to the friction period (i.e. the time that an employer needs to replace a sick employee). It should be taken into account that the length of the friction period depends on the local economic situation and that friction periods are not available for most European countries. Therefore, countries should try to determine the friction period for their country or, when this is not feasible, perform a sensitivity analysis in which productivity losses are valued with use of the human capital approach.VAT should preferably be included in the societal costs (79% agreement). VAT are indirect taxes on the domestic consumption of goods and services. Some goods are zero rated such as essential drugs and medical devices. Although VAT is a transfer from the individual to the government, it can be seen as part of the true costs of healthcare services according to the panel.Country-specific discounting rates should be used in the reference case (80% agreement). It is recommended to perform sensitivity analyses with the lowest and highest European discounting rates.The choice of a model-based approach or a trial-based approach should depend on the research question according to the panellists. The study should be model based when the intervention effects are expected in the long term, when it is not possible to include all relevant treatment alternatives in one study, or when the incidence of the clinical end point is low. In other situations, a trial-based approach suffices.This Delphi study aimed to develop cross-European recommendations for identifying, measuring and valuing resource use and productivity losses in economic evaluations. The use of a societal perspective is recommended for economic evaluations in a cross-European context because this perspective provides a broad view of the impact of healthcare interventions on society as a whole. Resource use is preferably measured by means of patient-reported measures and valued with use of country-specific standardized costs. Lost productivity is preferably measured by means of self-report and valued with use of the friction cost approach with age- and sex-specific national average wages.The Delphi design used allowed us to elicit the opinion of a heterogeneous international panel of experts with extensive expertise in economic evaluations within a relatively short time. By inviting the panel to suggest more suitable, alternative options in addition to the ones prespecified, we expected to capture recent developments in this field as well. Consensus was reached relatively easily for items regarding identification of cost categories. It was more difficult to reach consensus for items regarding assessment and valuation of resource use and lost productivity: in Delphi round 1, no consensus was reached for 21 the 52 valuation methods 40%) that were listed in the questionnaire. This is in line with previous studies that tried to harmonize existing national guidelines 0% that w.Recently, EUnetHTA developed a guideline with a framework for the method of economic evaluations based on common denominators in existing national guidelines from 25 European countries. The recommendations from our study on how to measure and value resource use and lost productivity in economic evaluations complement the EUnetHTA guideline since it does not extensively cover these topics. Also, the recommendations on topics discussed in both guidelines are in line with each other. Together, the EUnetHTA guideline and the recommendations described in this article are expected to support researchers, healthcare professionals and policymakers when they are conducting and appraising economic evaluations.Because of its comprehensiveness, the societal perspective is recommended as the reference case. However, in some countries  decision making is solely based on the healthcare perspective. To provide relevant information for these specific countries, an additional analysis from a healthcare perspective is required. If indicated, additional sensitivity analyses can be performed from other relevant perspectives [e.g. health insurance–public funds or healthcare payer(s)]. From a societal perspective, prices should reflect societal opportunity costs. However, in some countries such prices are unavailable or may be closer to costs from the healthcare perspective, whereas other countries, such as the Netherlands, attempt to approach societal opportunity costs by providing standard costs. The Delphi panel was not asked to decide between discounted and undiscounted costs. However, to facilitate the transfer of the results to other settings, we suggest that both discounted and undiscounted costs are presented.The lack of agreement among the panellists on the method to assess healthcare utilization may suggest that this is one area that it may not be possible to standardize, since national insurance fund databases differ considerably. On the basis of our Delphi study, the use of patient-based reports is recommended, but we are aware that may be infeasible in some economic evaluations, especially those that use a modelling approach.Because of the lack of standardized methods to measure absenteeism from unpaid labour, these costs should not be included in an economic evaluation according to the panel. However, in some populations this may be an important cost category (e.g. elderly people). Therefore, further research is needed to develop appropriate methods to validly assess costs due to decreased productivity in unpaid work.Among the panel and in the literature, strong differences of opinion exist with regard to the inclusion of unrelated future healthcare costs . InclusiSome limitations of the current study need to be acknowledged. First, the Delphi study had a lower response rate than anticipated; only one quarter of the invited experts participated in at least one of the Delphi rounds. This may have led to selection bias. However, since the panellists were distributed across Europe, the results likely reflect the variety in opinions and methods applied across Europe. Moreover, we considered the number of panellists to be sufficient, since the literature generally recommends 10–18 experts for a Delphi panel . Second,Other strengths are the structured and systematic approach used to identify agreement and disagreement in the methods among the panel and to generate consensus about the recommendations presented in this article. By developing recommendations based on existing guidelines and opinions of experts from all over Europe, we expect that the recommendations can be validly used across Europe. Also, the inclusion of generally accepted methods for economic evaluation greatly enhances the feasibility of complying with the recommendations formulated in this articles.We developed consensus-based recommendations for the identification, measurement and valuation of healthcare utilization and lost productivity that are applicable for European economic evaluations, despite differences between national guidelines. We think that these recommendations are generally acceptable because the recommendations were developed on the basis of a Delphi study among European experts in this field, and consensus was reached for most items within three rounds. Application of these recommendations will improve the comparability of economic evaluations conducted in Europe. Although pricing and reimbursement decisions will remain the full competence of national authorities, the recommendations will help decision makers better interpret the results of a study conducted elsewhere and facilitate the exchange of results between European countries. Moreover, it will allow countries to build on each other’s expertise and make cross-country evaluations easier to perform.Supplementary material 1 (DOCX 24 kb)Below is the link to the electronic supplementary material.


Most of the human pandemics reported to date can be classified as zoonoses. Among these, there is a long history of infectious diseases that have spread from non-human primates (NHP) to humans. For millennia, indigenous groups that depend on wildlife for their survival were exposed to the risk of NHP pathogens' transmission through animal hunting and wild meat consumption. Usually, exposure is of no consequence or is limited to mild infections. In rare situations, it can be more severe or even become a real public health concern. Since the emergence of acquired immune deficiency syndrome (AIDS), nobody can ignore that an emerging infectious diseases (EID) might spread from NHP into the human population. In large parts of Central Africa and Asia, wildlife remains the primary source of meat and income for millions of people living in rural areas. However, in the past few decades the risk of exposure to an NHP pathogen has taken on a new dimension. Unprecedented breaking down of natural barriers between NHP and humans has increased exposure to health risks for a much larger population, including people living in urban areas. There are several reasons for this: (i) due to road development and massive destruction of ecosystems for agricultural needs, wildlife and humans come into contact more frequently; (ii) due to ecological awareness, many long distance travelers are in search of wildlife discovery, with a particular fascination for African great apes; (iii) due to the attraction for ancient temples and mystical practices, others travelers visit Asian places colonized by NHP. In each case, there is a risk of pathogen transmission through a bite or another route of infection. Beside the individual risk of contracting a pathogen, there is also the possibility of starting a new pandemic. This article reviews the known cases of NHP pathogens' transmission to humans whether they are hunters, travelers, ecotourists, veterinarians, or scientists working on NHP. Although pathogen transmission is supposed to be a rare outcome, Rabies virus, Herpes B virus, Monkeypox virus, Ebola virus, or Yellow fever virus infections are of greater concern and require quick countermeasures from public health professionals. Pan troglodytes) and sooty mangabeys (Cercocebus atys) , etiological agents of AIDS that cause about 1 to 2 million annual deaths, have been linked to cross-species transmission of simian immunodeficiency virus (SIV) from chimpanzees (us atys) .Alouatta maconnelli) and the squirrel monkey (Saimiri sciureus), are allowed on the hunt but prohibited for sale , mangabeys (Cercocebus), guenons (Cercopithecus), baboons (Papio), and green monkeys (Chlorocebus). This pilot study revealed the presence of simian foamy virus and herpesviruses  in several NHP samples  from Morocco which were sold as pets in the suburbs and became aggressive during adulthood  were sold openly in North Sumatra markets despite being totally protected in Indonesia (Pongo) were rescued by the judicial services in Kalimanatan, Indonesia  are traded each year. Despite night monkey trade being regulated by the CITES convention, there is evidence for illegal trade at the Columbia, Peru and Brazil cross-border (Macaca mulatta and Nycticebus spp.) were rescued and confiscated in Chinese markets from 2000 to 2017 , with China being the largest exporter . The CITMacaca fascicularis at monkey temple in Bali was reported . For example, despite no quotas being allocated for trading of NHP as pets in Indonesia, during the last decade in North Sumatra NHP such as long-tail macaques (Macaca fascicularis) and pig-tailed macaques (Macaca nemestrina) were illegally sold at markets in Medan (Pongo abelii and Pongo pygmaeus), protected by the Indonesian law since 1931, were formally confiscated at the receivers in Indonesia  were the most affected , but other species such as the endangered golden-headed lion tamarin (Leontopithecus chrysomelas) and wooly spider monkeys (Brachyteles arachnoides) became sick, indicating that they are vulnerable to YF  occurs. Conversely, non-infected humans can be infected when they visit wild sites were infected NHP and mosquito vectors are present. Such situation has forced the Brazilian public health authorities to urgently launch a national anti-YF vaccination campaign.Another source of concern for public health resides in pathogens which can be transmitted during the blood meal of arthropods. The outbreaks of yellow fever (YF) in South American NHP are a recent example. A first YF outbreak in 2008 left more than 2,500 dead NHP in Brazil. The most recent outbreak from 2016 to 2018 killed at least 732 monkeys in Southeast Brazil. The black and brown howler monkeys , adaptation selected defense mechanisms preventing the most serious consequences of the disease. Persons who expressed sickle hemoglobin or those who present glucose-6-phosphate dehydrogenase deficiency, evaded the worst complications of malaria  and not worry about pathogens abnormally present in their livestock because of the interconnectedness of different ecosystems. Despite veterinary cares, these animals may serve as intermediate for transmission of wildlife-borne pathogens (including NHP-borne pathogens on rare occasions) to the livestock owners and thereby represent the main source of human infectious disease and pandemics . AccordiGorilla gorilla beringei) can be exposed up to 2,000 h of human visits/year  enteric pathogen virus such as poliovirus, coxsackie virus, herpes simplex virus, Hepatitis virus, or bacteria such as Salmonella spp., Shigella spp., Campylobacter spp.; and, (iii) parasites such as, Giardia lamblia or Schistosoma spp. The diversity of human pathogens found in NHP leaves no doubt as to the susceptibility of great apes to pathogens widely present in humans. In recent years, primatologists pointed out the misdeeds and risks of ecotourism and have advocated: (i) to limit the frequency and duration of visits; (ii) to reduce the number or visitors; (iii) to prohibit access to people with known diseases; (iv) to banish the ecotourists' consumption of food on wildlife site; (v) to define a minimum distance of observation or physically separate NHP and visitors in wildlife parks; and, (vi) to wear a facemask  respiratory viral pathogens such as measles virus, influenza virus, respiratory syncytial virus, rhinovirus, adenovirus, cytomegalovirus, and bacteria such as facemask .Pongo pygmaeus) , which had acute stomatitis. For exclusion of possible pathogen transmission, a sample from the marmoset's oral mucosa was tested and found positive for HHV1 and despite treatment, this NWM died 2 days later (Papio hamadryas) of the kingdom of Saudi Arabia, was reported (Papio anubis) living in the Maasai Mara Reserve of Kenya and half of the males died  . Human hys later . Human mys later . Simultays later . Evidencreported . It was reported . Howeverles died . Mycobacuminants . In 1988n origin . Attemptn origin . In Uganotourism . What companzees , 112 (Fimpanzees .Regarding pathogens that pass from humans to NHP (named reverse zoonotic disease transmission) and are capable to infect a variable diversity of hosts  –115, it Cynomolgus monkey bites had occurred over a 6 year period  was found to include Haemophilus spp., Fusobacterium cleatum, Peptostreptococcus micros, Streptococcus spp., Actinobacillus actinomycetemcomitans, Wolinella spp., Campylobacter spp., Eikenella corrodens, and spirochetes (Klebsiella pneumoniae was found in the nose and throat of NHP (Mycobacterium tuberculosis could infect back humans (Mycobacterium leprae was reported in chimpanzees and sooty mangabeys (Cercocebus atys) , bites are located on the hands, arms and legs . Dependiium spp. . A bacte animals . The subrochetes . Regardit of NHP . Tubercuk humans . Naturalus atys) . Recentlmacaques . Mycobac monkeys . As far y others . Specialematoses . An isol contact , 132. Vil Africa .Trypanosoma cruzi, a pathogen isolated in 1924 from South American squirrel monkeys (Chrysothrix sciureus), that causes anorexia, weight loss, dehydration in monkeys and Chagas disease in humans. Natural T. cruzi infection has been reported for several NHP such as marmosets, spider, cebus, rhesus monkeys, and gibbons  as a cause of severe enteric disease. It can infect humans as well, leading to dysentery. OWM such as mangabeys and great apes such as chimpanzees can carry either Schistosoma mansoni or S. haematobium , sooty mangabeys (C. atys) and baboons (Papio doguera) with diarrheal disease (Gorilla beringei) are widely infected (43%) with lymphocryptoviruses  and SIVsm from sooty mangabeys (Cercocebus atys) (Chlorocebus pygerythrus), grivet (C. aethiops), sabaeus (C. sabaeus), and tantalum  are the natural hosts of SIVagm, and do not show symptoms of immunodeficiency. SIVmac infection of macaques, is often accompanied by lymphadenopathy and immunodeficiency (Gorilla gorilla), chimpanzees (Pan troglodytes), and baboons, whereas the related STLV-II infects bonobos (Pan paniscus) and cause lymphomas in baboons (M. mulatta) and Japanese macaques (M. fuscata) colonies (M. fascicularis) in Indonesia (The risk for retroviruses transmission from NHP to humans has been deeply studied. These viruses include simian immunodeficiency virus (SIV), simian T-cell lymphotropic virus (STLV), simian type D retrovirus (SRV), and simian foamy virus (SFV). The emergence of HIV-1 and HIV-2 in humans, has been linked to cross-species transmission of SIVcpz from chimpanzees ; gibbons (Hylobates lar)  a picornavirus, induces outbreaks of fatal myocarditis in NHP. It was responsible for heart failure, renal failure and cerebral infarction causing the death of bonobos (tes lar) , Macaca sylvanus , and Papamadryas . EMCV waamadryas . EMCV waamadryas  In Peru,amadryas . This viCallithrix jacchus) was a source of human rabies in the state of Ceara (C. jacchus were registered (Cebus apella) had antibodies against rabies virus. Rabies was also reported in great apes such as chimpanzees. In India, rabies was reported in macaques (M. mulatta) (M. fascicularis) are used to being fed by tourists, and sometime bite visitors. In India, rabies is not a notifiable disease, yet there are a few reports of human rabies following exposure to NHP (Rabies is a zoonotic disease characterized by severe neurologic signs caused by rabies virus (genus Lyssavirus). This disease is responsible for almost 55,000 human deaths every year, mostly in Asia and Africa. Natural rabies was described a long time ago in laboratory monkeys . In counof Ceara . Betweengistered . Anothermulatta) . In Thaimulatta) . This obmulatta) . The dife to NHP . For exae to NHP .M. mulatta and M. fascicularis). Related viruses named SA8 and Herpes papio 2 were isolated from vervet (Cercopithecus aethiops) and baboon (Papio), respectively  in adult macaques (ectively . Asymptoectively . There aectively . Transmiectively . A womanectively . About 5ectively . It was ectively . Recentlectively . This poM. fascicularis) is a zoonotic virus endemic in Western and Central Africa. MPXV was then found in monkeys caged in zoos and primate research facilities (C. aethiops) and 6% of colobus monkeys had antibodies against MPXV  found dead with pox-like lesions in Tai National Park in Ivory Coast. In 2014, a monkeypox outbreak among chimpanzees was reported in a sanctuary in Cameroon with 6 out of 72 monkeys infected by MPXV; the sick animals lacked appetite and showed gradual appearance of vesicles and nodules on the forelimbs and the face and one died from the disease (The monkeypox virus (MPXV), isolated in 1958 from cynomolgus monkeys ; indeed, these MARV-infected animals were imported in Europe from the Lake Kyoga region of Uganda for further experimentation in animal facilities in Marburg, Germany, aimed at obtaining kidney cells required to culture the poliomyelitis vaccine. Thirty-one persons (25 staff members and 6 secondarily infected humans) developed severe hemorrhagic fever, among which 6 died from the disease (The transmission of the Marburg virus (MARV), from NHP to laboratory workers in 1967, is quite interesting to analyze regarding the spread of the pathogen via inter- and intra-species events. The source of the human outbreak was traced back to African green monkeys —, and touching wildlife with a special attraction for NHP, may still have undesirable consequences, especially that of being contaminated by a foreign pathogen infecting the NHP . Conversely, there is also a risk of introduction of new infectious pathogens in the visited ecosystem. The “One Health” concept recognizes that human and animal health are intimately connected . Implemet be >1)—, 201. Unt be >1)—, 203, ant be >1)—.in situ NHP recovery centers, such as the Pan African sanctuary Alliance  according to National Advisory Committees, and the greatest caution to those who wish to meet NHP in their natural environment . Pre- an0, or a deadly pathogen transmitted from NHP to humans via a flying blood-sucking vector insect.Post-exposure clinical survey of the patient is necessary to identify possible signs of illness . If there is evidence for a new pathogen, warning signal are needed for early detection and control of new infectious disease, and biosurveillance of humans and NHP in the area of emergence should be established to determine its evolutionary potential, its impact on health and the ability of leaders and stakeholders to control the phenomenon. The most serious risk for public health is a deadly pathogen able to spread through human-to-human transmission with high RCD, OM, HM, and DR conceived the paper. CD wrote the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Dear reader,The recent report on a veterinarian bitten by a horse seropositive to Borna Disease Virus-1 (BoDV-1) in the Netherlands  in Germany had encephalitis with similar clinical signs and died 2–4 months after onset of the clinical symptoms associated with variegated squirrel 1 Bornavirus (VSBV-1) , Vetsuisse Faculty, University of Bern, Bern, Switzerlandjohannes.vanderkolk@vetsuisse.unibe.chJ.H van der Kolk