In-silico time-course studies indicate initial increase and subsequent collapse in clonal diversity. Finally, we show that radiologically evident budding structures in early-stage ccRCCs could potentially predict future evolutionary steps.Genetic intra-tumour heterogeneity (ITH) fuels cancer evolution, but our understanding of temporal evolution and the ability to predict clinically relevant evolutionary trajectories remain limited. Towards enhancing this ability, we investigated features of clonal diversification in clear cell renal cell carcinomas (ccRCCs) through combined modelling and tumour analysis. Tumour growth modes impact the extent of subclonal diversification, enabling interpretation, through the lens of temporal evolution, of static data on tumour sizes and clonal diversity in the TRACERx Renal study. Patterns of proliferation and necrosis in diverse growth models underlie the spatial features of clonal diversity and recent subclone births, with proliferation at the surface linked to highly branched evolution and attenuated disease progression.  Acquisition of genomic alterations including mutations and somatic copy-number alterations (SCNAs) drives the emergence of genetically heterogeneous subpopulations of cancer cells or subclones, resulting in intra-tumour heterogeneity (ITH). A subset of genomic alterations (termed drivers) endows subclones with increased fitness. Subclones compete for resources, including physical space, and undergo expansion or extinction according to their fitness under selective pressures imposed by the tumour microenvironment (TME) or therapeutic intervention. With advances in next-generation sequencing, clonal architecture and evolutionary features have been elucidated in a variety of tumour types. However, the ability to predict clinically relevant evolutionary trajectories remains limited.The development of cancer is an evolutionary process. Detection of ITH is sampling-dependent and accurate measurement of diversity is enhanced by sampling of multiple small tumour areas. While macrodiversity  reflects established clonal diversity within a tumour, clonal diversity at a narrow spatial scale, or microdiversity , could represent under-detected ongoing clonal evolution  uniform growth throughout the tumour volume (referred to as “Volume Growth Model”) and (ii) active proliferation restricted to the tumour surface (referred to as “Surface Growth Model”) (see . We related our model to the TRAcking Cancer Evolution through therapy (Rx) (TRACERx) Renal study, which previously evaluated the genomic profiles and spatial coordinates of 756 patient tumour regions (referred to as “PT regions” hereafter) from 66 tumours. Tumours with high clonal diversity don’t necessarily harbour metastasising clones, suggesting that the development of metastatic competence and continuing subclonal diversification may be uncoupled in the tumour as independent evolutionary processes. Through combined modelling and clinical analysis, we show how tumour growth modes determine the extent and trajectories of clonal diversity. Crucially, we explore temporal aspects of tumour evolution that would otherwise be inaccessible from single timepoint biopsies.Despite increasing understanding of ITH and macrodiversity, understanding of the temporal features of clonal evolution remains limited owing to ethical and logistical challenges in obtaining serial single or multi-regional biopsies from patientsr events . Variousel”) see . We addi. Each model unit, referred to as a “tumour voxel”, represents a tumour volume of 1 mm3. Tumour voxels stochastically undergo growth, death, and acquisition of driver events upon growth. As proliferation proceeds, some tumour voxel acquires a driver event conferring selective advantages, manifested in the current study as an increase in the growth probability (growthp). Two ways of implementing selective advantages are considered and referred to as “saturated” and “additive” driver advantage models. In the saturated driver advantage model, growt hp of a tumour voxel can be at one of the three levels s, are assumed to reflect their association with the Ki67 score in tumour regions  than SCNAs. A second mutation in the same gene is assumed to never occur in the same tumour voxel. As the majority of ccRCCs have clonal VHL inactivation events, in general and in the TRACERx Renal cohort, the founder tumour voxel is assumed to harbour VHL inactivation together with 3p loss as a clonal event. Based on data from the TRACERx Renal study, and functional evidence, mutations in PBRM1 or BAP1, are assumed to enhance the probability of SCNA acquisition. Mutations and acquisition of SCNAs are assumed to be proliferation-dependent, which imply DNA replication and chromosome mis-segregation as the main source of genomic alterations. Lastly, the selective advantage endowed by a driver is assumed to be fixed, so the variation in driver advantage dependent on changing environments is not considered in the current study.Additional assumptions were made to keep the model minimal. Individual driver gene mutations are assumed to be acquired with a greater probability   see . In both0.5) see . To furt0.5) see . Surface0.5) see -5. When 0.5) see , in keepWe next examined the spatial distribution of clonal diversity . Under Sd, depicted power law scaling (P(D ≤ d)~kd, where k is the power law exponent to be fitted). Comparably, the Surface Growth model displayed a larger k  cases from TRACERx Renal, we observe events that spanned a single region (assumed to be the most recent/youngest subclones) close to the tumour margin  and located at varying distances from the tumour margin   . Observaopically  and micropically  in the iopically . These oin-silico time-course analysis. Overall, the number of subclones remained limited in Volume Growth models but increased over time before reaching a plateau in Surface Growth models  to shape the spatial patterning of subclones.Tumour growth and clonal evolution in a spatio-temporal context have increasingly been studied with the aid of computational models that incorporate spatial elements of tumour growth and acquisition of genomic alterationsmm3, referred to as a “tumour voxel”. The full simulation lattice comprises 200 × 200 × 200 lattice sites, each of which can accommodate a single tumour voxel when a tumour grows. As a simulation proceeds, tumour voxels stochastically undergo growth, death, and acquisition of driver events upon growth , are considered in the present work . For simgrowt hp of a tumour voxel can be at one of the three levels  . In a general form, h(s) ≥ g(s) ≥ 1 reflect the growth advantages relative to the baseline. As one specific implementation, s ≤1 reflects the selective advantage. For simplicity, individual driver gene mutations are assigned with growth_kp reflects the amount of growth probability added by driver k , and the difference in ks between consecutive two drivers in their advantages, namely, Δks.  than SCNAs (0.001driverp). A second mutation in the same gene is assumed to never occur in the same tumour voxel, but multiple independent, distinct mutations in the same gene may be acquired in parallel within a simulated tumour in different tumour voxels. As the majority of ccRCCs have clonal VHL inactivation events, in general and in the TRACERx Renal cohort, the founder tumour voxel is assumed to harbour VHL inactivation together with 3p loss as a clonal event. The subpopulation of tumour voxels that only harbour these two events is referred to as the parental clone. Based on their association with a high weighted genome instability index (wGII) in the data from the TRACERx Renal study, and functional evidence, mutations in PBRM1 or BAP1, are assumed to enhance the probability of SCNA acquisition (to driverp). A range of driver acquisition probabilities have been studied to explore its impact on patterns we investigate . Mutations and acquisition of SCNAs are assumed to be proliferation-dependent, which imply DNA replication and chromosome mis-segregation as the main source of genomic alterations. Lastly, the selective advantage endowed by a driver is assumed to be fixed, so the variation in driver advantage dependent on changing environments is not considered in the current study.Upon proliferation of a parent tumour voxel, child tumour voxels inherit existing driver events harboured by the parent tumour voxel and stochastically acquire new drivers. Individual driver gene mutations are assumed to be acquired with a greater probability . During the evaluation of possible death events, for each of all tumour voxels alive, deat hp is compared to a random number generated between . If deat hp is larger, a death event occurs, resulting in the lattice site freed for accommodating a newly born tumour voxel in the future. During the evaluation of possible proliferation events, for each of all valid tumour voxels (see above for the difference between Surface Growth and Volume Growth), growt hp is determined according to the driver events harboured (see above for the difference between “saturated” and “additive” models of fitness advantage) and compared to a random number generated between . If growt hp is larger, a proliferation event occurs, resulting in a new tumour voxel created nearby. During the evaluation of possible acquisition of driver events, for each of all daughter tumour voxels just arising from proliferation and for each of the ccRCC drivers, driverp is compared to a random number generated between . If driverp is larger and that driver is not currently harboured by the tumour voxel, acquisition of the driver takes place in the given tumour voxel. In a subset of simulations, necrosis is implemented. During the evaluation of necrotic death events, for each of all tumour voxels alive, if it’s located at a distance of greater than necrosisd from the tumour surface, necrosisp is compared to a random number generated between . If necrosisp is larger, a necrotic death event occurs, resulting in the lattice site freed for accommodating a newly born tumour voxel in the future. The simulation runs until the tumour grows to at least 1 million tumour voxels after the last simulation step. The computer code is written in CUDA C++.The procedure for simulating events of death, proliferation, and acquisition of driver events is illustrated in a flow diagram . BrieflyN replicate tumours are reconstructed, each with a copy of the same evolutionary snapshot at a given time point t. Then, these replicate tumours undergo the events described above, each with a different unique random seed, and grow to the predefined stopping size. Evolutionary outcomes from these replicate tumours are evaluated and compared.The procedure for simulating evolutionary replay is illustrated in a flow diagram . Brieflyz = z0); (3) regional biopsy level, which takes into account tumour voxels within regional biopsies. A regional biopsy is defined as all tumour voxels within a region in the 2D slice. Spatially uniform sampling is performed in this study. This process is carried out by locating the centres of candidate regional biopsies in the 200mm × 200mm 2D lattice with a spacing of 20 mm and collecting all voxels within a distance of 5 mm from each biopsy centre.Analyses are conducted at three different levels : (1) whoCCF of a subclone is calculated as the number of tumour voxels that belong to a subclone divided by the total number tumour voxels in the domain of interest, depending on the level of analyses. A subclone is identified by a set of driver events, shared by a subpopulation of tumour voxels, which are accumulated within the subclone-initiating tumour voxel. A subclone-initiating tumour voxel is defined as a tumour voxel that acquires a new driver event upon birth. A subclone is considered detectable if the CCF is greater than 0.01.if is the CCF of the subclone i. All subclones are considered in this calculation.As a measure of clonal diversity, the Shannon diversity index is defined as Drivers (including both mutations and SCNAs) harboured by each tumour voxel within a tumour slice are counted at each time point. The average number of drivers among all tumour voxels is then calculated.growt hp, dependent on the set of drivers harboured by that tumour voxel. In representative cases, fitness of tumour voxels is mapped within a 2D tumour slice. To examine the spatial features of tumour fitness, the distances from every tumour voxel to the centre of a tumour slice and to the nearest point along the tumour contour are calculated, respectively. The central-most 10% of tumour voxels, with the shortest distances to the centre, and the marginal-most 10% of tumour voxels, with the shortest distances to the margin, are sampled to calculate a sample-average fitness. Additionally, another 10% of tumour voxels is randomly sampled for comparison. The ratio of the mean fitness of the central-most 10% of tumour voxels to that of the marginal-most 10% of tumour voxels, denoted as “Ratio_C2M” is calculated for each simulation.Fitness of a tumour voxel is defined as the instantaneous growth probability, mm2 region within the tumour slice. In representative cases, microdiversity is spatially mapped within a tumour slice, by sliding a 3 × 3 mm2 spatial window throughout the tumour slice. Microdiversity hotpots are defined as a subset of these small regions with 5 or more subclones. The distance from a microdiversity hotspot to the centre of a tumour slice is referred to as the distance to tumour centre (1d). The distance from a microdiversity hotspot to the nearest point along the tumour contour is referred to as the distance to tumour margin (2d). The normalised distance to tumour centre is defined as d = d1/(d1 + d2). Cumulative probability distribution, P(D ≤ d), of d is generated by combining microdiversity hotspots from repeat simulations. The power law exponent is obtained by bootstrapping 100 samples of 400 hotspots per sample and fitting a power law function to the cumulative probability distribution of d in each sample.Microdiversity is defined as the number of subclones contained in a 3 × 3 Youngest subclones are defined as the subclones that emerge closest to the end of a simulation. For the analysis of their spatial distribution, we recorded the 100 youngest subclones within a 2D tumour slice and, for each of them, measured the distance from its position to the tumour margin. The mean of the distances for these 100 subclones was calculated for comparison between different replicate simulations.x, y) position of this tumour voxel and adjacent tumour voxels. After smoothening, contour circularity is calculated as: Tumour contours are determined from 2D tumour slices. Smoothening is performed for each tumour voxel along the tumour contour by calculating the mean  images were obtained using standard-of-care imaging sequences in 91 patients and curated using a local research picture archiving and communication system (PACS) (based on the Extensible Neuroimaging Archive Toolkit). Outlines were drawn by consensus between an oncologist (S.T.C.S.) and a radiologist (D.A.) giving volumetric tumour coverage, from which image strips were prepared for rapid visualisation of all tumour slices for all patients using an in-house script written in python.(XNAT) platformTo detect presence or absence of budding structures in the contoured CT data, qualitative assessment of contoured tumours was performed by X.F. and S.T.C.S and verified by a radiologist (D.A.).Haematoxylin and eosin-stained histological sections representative cases were evaluated by two pathologists (J.I.L./C.E.S.). Each sample was qualitatively assessed for the relative amount of viable tumour cells, fibrosis and presence of necrosis.Spatial maps of regional clone diversity are created for two representative tumour sections. In these maps, regions are colour-coded based on the number of subclones. Regions that harbour at least one subclone are treated as a proxy for microdiversity hotspots defined in the model analysis. In total, there are 606 regions from 54 tumours that satisfy this criterion. In evaluation of association between microdiversity features and clinical annotations, subsets of tumours are considered. Subsets with different relapse statuses consist of 270 (“Relapse”) and 336 regions (“No relapse”), respectively. Subsets with different rates of disease progression consist of 276 (“No progression”), 265 (“Attenuated progression”), and 65 regions (“Rapid progression”), respectively. For tumour regions, the normalised distance to tumour centre is measured as described above in “Model analysis - Microdiversity”.PBRM1 and in BAP1, respectively, are created for two representative tumours. In these maps, regions are coloured differently according to different parallel mutational events. Regions that harbour more than one event are indicated with multiple colours. To study the spatial distribution of mutational events with limited clonal expansion, the maximum distance from an event spanning a single region to the tumour margin is measured.Spatial maps of parallel mutational events in stat_compare_means in R.The two-sided Wilcoxon’s rank test was performed to compare a particular measurement between different conditions. Statistical significance is annotated within box plots using random.choice in Python. The power law exponent was then determined by fitting a power law function to the cumulative probability distribution from each sample, using scipy.optimize.curve_fit in Python.Bootstrapping was performed to generate 100 random samples of 400 microdiversity hotspots per sample with replacement, using statsmodels.graphics.gofplots.qqplot in Python.The Quantile-Quantile (Q-Q) plot was generated to compare the actual distribution of microdiversity hotspots to a power law distribution with the exponent as the median of fitted values in bootstrapping, using seaborn.jointplot(kind=“kde”) in Python.Kernel density estimation was performed for simulations with respect to the size of tumour slice and the number of subclones, using R version 3.6.2 and Python version 3.7.7 are used for these analyses.Source Data for Extended Data Figure 1Source Data for Extended Data Figure 2Source Data for Extended Data Figure 4Source Data for Extended Data Figure 5Source Data for Extended Data Figure 6Source Data for Extended Data Figure 7Source Data for Extended Data Figure 8Source Data for Extended Data Figure 10Source Data for Figure 2Source Data for Figure 3Source Data for Figure 4Source Data for Figure 5Supplementary InformationSupplementary Tables


G3 Genes|Genomes|Genetics, 2021, 11(5), jkab077 https://doi.org/10.1093/g3journal/jkab077When this paper first published online, there was an error in figure 5. Figure 5 originally appeared asFigure 5 should have appeared as


The COVID‐19 crisis brought numerous challenges to work life. One of the most notable may be the acceleration of digital transformation, accompanied by an intensification of e‐mail usage and related demands such as high e‐mail workload. While research quickly started to examine the implications of these changes for employees, another important group of stakeholders has been overlooked: leaders. We focus on leaders during the COVID‐19 crisis and examine how COVID‐19‐related work intensification links to leaders' e‐mail overload appraisal and finally exhaustion and transformational leadership, a leader behaviour especially needed in times of crisis. In a 5‐day diary study in September 2020, 84 leaders responded to daily surveys on 343 days. Results of multilevel analysis showed that perceived COVID‐19‐related work intensification was positively linked to worktime spent dealing with e‐mail and appraised e‐mail overload. E‐mail overload appraisal was positively related to leaders' exhaustion, but unrelated to their transformational behaviour. Day‐specific time spent dealing with e‐mail, however, was negatively related to transformational leadership. E‐mail overload appraisal mediated the relationship between COVID‐19‐related work intensification and exhaustion. Turning the focus on leaders during the COVID‐19 crisis, our study has important implications for the design of work of leaders in times of crisis and beyond. The world of work has changed in the wake of the COVID‐19 pandemic. For example, many started to work from home what brought changes in the way employees—and their leaders—collaborate and communicate  hinders transformational leadership. We base this assumption again on the transactional stress model .From the originally registered 112 persons, 97 responded to the general survey and indicated that they are indeed in a leadership position. From those, in turn, 91 responded to a total of 350 workday surveys. To render within‐person variation in the day‐specific variables  possible, we included only data from leaders who responded to at least two daily surveys on days on which they had worked. Further, to ensure that leaders could have engaged in transformational leadership in principle, we included only days for which leaders reported that they had contact with their employees (e.g. in person or via e‐mail). This resulted in our final data set with responses from 84 leaders on 343 days (SD = 11.94) and a mean job experience of 18.00 years (SD = 11.22). Their mean weekly worktime was 41.65 hours (SD = 7.16). They had leadership responsibility for an average of 22.01 employees (SD = 43.31). The majority had completed a university degree (78.6%). With regards to job titles and occupations, our sample was diverse, representing different hierarchical levels , working in different branches including finance, health and social care, retail, and manufacturing. All leaders reported to currently use e‐mail at work to some extent, ranging from about 10 to about 90% of their worktime .Leaders in the final sample  had a mean age of 42.95 years (N = 84) did not significantly differ from those who had responded to the general survey but were not included in the final sample (N = 13) with regard to their weekly workhours . They, however, reported significantly lower perceived COVID‐19‐related work intensification . This must be kept in mind when interpreting our findings.Leaders included in the final sample  to 5 (strongly agree/completely). Table The study was conducted online. We used short measures for the daily variables to reduce participant burden .We measured COVID‐19‐related work intensification in the general survey. We adapted the original German five‐item work intensification scale by Kubicek et al.  to the pWe measured day‐specific e‐mail overload appraisal with four items, three of which we adapted from the e‐mail overload scale of the ICT demands scale by Dabbish and Kraut . We tranWe measured end‐of‐work exhaustion with the five‐item physical fatigue subscale of the Shirom‐Melamed Burnout Measure , “Today, I challenged my subordinates to think about old problems in new ways” , “Today, I encouraged my subordinates to see changing environments as situations full of opportunities” , and “Today, I behaved in a manner which was thoughtful of my subordinates' personal needs” (supportive leadership). We adapted the items from the transformational leadership scale proposed by Rafferty and Griffin  and tran0%) to 10 (100%). ICC was .76.To make sure that not sheer time spent dealing with e‐mail  = 0.9155, AIC = 11461.023, RMSEA = 0.057, CFI = 0.884, SRMRwithin = 0.060, SRMRbetween = 0.110, fit the data better than any alternative plausible model, for example, a model in which e‐mail overload appraisal and exhaustion items formed a common factor on both levels, χ2 = 694.228, df = 196, p < .001, SCF = 0.8617, AIC = 11680.400, RMSEA = 0.086, CFI = 0.727, SRMRwithin = 0.097, SRMRbetween = 0.156. We conclude that the measures capture distinct constructs.To test discriminant validity of our measures, we conducted multilevel confirmatory factor analyses in Mplus 8.5. We specified a two‐level model with three within‐level factors  and four between‐level factors . This model,We conducted two‐level path modelling , this model explained 6% of the variance in exhaustion and 3% of the variance in transformational leadership. At the person level (i.e. between person), this model explained 31% of the variance in exhaustion, 12% of the variance in transformational leadership, 16% of the variance in appraised e‐mail overload, and 7% of the variance in time spent dealing with e‐mail. Because time spent dealing with e‐mail was measured using a different response scale, we report standardised estimates  to enable easier interpretation.We tested all hypotheses simultaneously by specifying one overall two‐level path model with parallel paths at the within‐person level and at the between‐person level. Results are shown in Table p < .001, providing support for Hypothesis p = .02.Hypothesis p < .001. Thus, Hypothesis p = .88. At the between‐person level, results were comparable: E‐mail overload appraisal was positively related to exhaustion, est. = .53, p < .001, whereas time spent dealing with e‐mail was unrelated to exhaustion, est. = −.17, p = .18.Hypothesis p = .61.Hypothesis p = .52. Thus, Hypothesis p = .03. At the between‐person level, neither appraised e‐mail overload, est. = .20, p = .20, nor time spent dealing with e‐mail, est. = .04, p = .78, was related to transformational leadership.Hypothesis p = .15.Hypothesis p = .70, but was negatively related to exhaustion between persons, est. = −.24, p = .02.Transformational leadership behaviour and end‐of‐work exhaustion might not occur independent of each other but might rather be causally related: Engaging in transformational leadership is resource demanding  and those who did not. Hence, our findings might suffer from range restriction and related biased estimates  measurements and all data is self‐reported. Thus, we cannot preclude that our results suffer from same‐source bias  work intensification and high e‐mail demands are especially prevalent or problematic. Furthermore, it might be interesting to examine how leader exhaustion and transformational leadership are related to each other at the day level. In our study, transformational leadership and end‐of‐work exhaustion were not related within person. This result differs from longitudinal between‐person findings that show transformational leadership to positively predict leaders' exhaustion over time (Lin et al., The Global Leadership Forecast 2021 (Development Dimensions International, Inc., To start with, we caution organisations to not ignore the potential negative implications that the COVID‐19‐related acceleration of digital transformation Kudyba,  might haGiven that reliance on e‐mail for work communication will likely stay a central element of leaders' work, though, organisations and leaders should also adopt practices that make work e‐mail manageable (see Barber & Hu, The authors declare that they have no conflict of interest.The present research is in line with the ethical guidelines of the German Psychological Society.


Varicella zoster virus (VZV) meningitis is primarily an infection of the immuno-compromised. However, it can also affect immunocompetent individuals. Reactivation of VZV typically presents with a distinct dermatomal rash suggestive of varicella zoster, but there have also been reports of VZV meningitis presenting without a rash.We describe a case of VZV meningitis in a healthy, 30-year-old male presenting to the emergency department shortly after receiving his first coronavirus disease 2019 vaccination. He was treated with intravenous acyclovir and then discharged home on oral valacyclovir.Emergency physicians should consider aseptic meningitis in immunocompetent patients presenting with atypical headaches in this population. Varicella zoster virus (VZV) is part of the herpesvirus family. Initial infection causes varicella, also known as chickenpox. The virus becomes latent within the cranial nerve, dorsal root, and autonomic ganglia. Reactivation of the virus can occur as a result of decreased T cell-mediated immunity, typically causing a dermatomal vesicular rash.A 30-year-old, previously healthy male presented to the emergency department with a three-day history of a persistent, throbbing headache. He described the headache as worse in the morning upon awakening and exacerbated by bending over, lying down, and sneezing. The patient reported headaches in the past, but this episode had not responded to ibuprofen as usual. He denied any medical history aside from a diagnosis of Lyme disease approximately 10 years prior. This was treated with a full course of doxycycline and yielded no further complications. He had received his first COVID-19 vaccination 12 days prior to presentation, but otherwise had only taken daily ibuprofen since the onset of his headache. He denied sick contacts, recent travel, and tobacco or drug use history. Review of systems was negative for fever, changes in vision, photophobia, chest pain, shortness of breath, nausea, vomiting, diarrhea, rashes, or recent traumas.His vital signs were within normal limits. On physical exam, pupils were equal, round, and reactive to light. The patient had no focal neurologic deficits. Cranial nerves II–XII, sensation, motor strength, speech, and coordination were intact. His neck was supple with full range of motion and no rigidity. There were no rashes or skin lesions.Complete blood count and basic metabolic panel were unremarkable. A non-contrast computed tomography of the head revealed no evidence of acute intracranial hemorrhage or midline shift, mass, or intra-axial or extra-axial fluid collection. A lumbar puncture was performed and was significant for opening pressure of 29 centimeters (cm) water (reference range: 6–25 cm water). Cerebrospinal fluid was significant for 706 white blood cells/microliter (WBC/uL) (reference range 0–5 WBC/uL), with 95% lymphocytes; 4 red blood cells (RBC)/uL (0 RBC/uL); protein of 144 milligrams/deciliter (mg/dL) (15–45 mg/dL), and glucose 52 mg/dL (40–70 mg/dL). The polymerase chain reaction (PCR) meningitis/encephalitis panel detected VZV. The patient was empirically started on intravenous (IV) vancomycin, ceftriaxone, dexamethasone, and acyclovir. Further questioning revealed that the patient had chickenpox as a child.The patient was hospitalized and continued treatment with high-dose IV acyclovir (10 mg/kilogram every eight hours). Blood cultures and testing for Lyme disease and HIV returned negative. After a 48-hour hospitalization, the patient requested discharge, and the infectious disease service recommended switching the patient from IV acyclovir to a 10-day course of valacyclovir (1000 mg every eight hours). The patient’s headache had resolved prior to discharge, and he was recommended to follow-up with his primary care provider (PCP). Unfortunately, we reviewed the chart a month afterward, and the patient had not followed up with his PCP.Varicella zoster virus reactivation causes a variety of neurologic complications including acute retinal necrosis, herpes zoster ophthalmicus, and aseptic meningitis. Advanced age and immunocompromised states are considered major risk factors for the development of herpes zoster and other manifestations of VZV reactivation. The most common presentations are a dermatomal rash and neuritis. Complications of VZV include postherpetic neuralgia and aseptic meningitis.4Although rare, vaccines have also been associated with reactivation of VZV. This has been reported in patients who received vaccinations for hepatitis A, influenza, rabies, and Japanese encephalitis.7Varicella zoster virus can be diagnosed clinically with the presence of a dermatomal rash. It can also be diagnosed in the laboratory by using PCR to analyze vesicular skin material and cerebrospinal fluid.12What do we already know about this clinical entity?Reactivation of varicella zoster virus (VZV) can cause meningitis, and typically affects older and immunocompromised patients and presents with a rash.What makes this presentation of disease reportable?Our case describes an uncommon presentation of aseptic meningitis secondary to VZV in a healthy, young adult without a rash.What is the major learning point?It is important to consider aseptic meningitis on the differential when evaluating a patient who presents with an atypical headache.How might this improve emergency medicine practice?Because VZV meningitis is rarely seen in the immunocompetent population, this case report adds additional information for a better understanding of VZV.15 After an appropriate dose of IV acyclovir, valacyclovir can be a suitable option for treating minimally symptomatic patients in the outpatient setting, such as the young man in this case report.Case reports have described successfully treating patients for VZV meningitis with IV acyclovir and transitioning to oral valacyclovir.While rare, aseptic meningitis secondary to VZV can occur in young, healthy, immunocompetent patients and can present without a rash. Emergency physicians should consider this diagnosis for atypical headaches in this population. Once the diagnosis is made, it would be prudent to initiate IV acyclovir and have the inpatient team determine whether to transition to oral valacyclovir.


Salmonella enterica serovar Enteritidis. The use of lytic bacteriophages (phages) to reduce foodborne pathogens has emerged as a promising biocontrol intervention for Salmonella spp. Here, we describe and evaluate the newly isolated Salmonella phage STGO-35-1, including: (i) genomic and phenotypic characterization, (ii) an analysis of the reduction of Salmonella in chicken meat, and (iii) genome plasticity testing. Phage STGO-35-1 represents an unclassified siphovirus, with a length of 47,483 bp, a G + C content of 46.5%, a headful strategy of packaging, and a virulent lifestyle. Phage STGO-35-1 reduced S. Enteritidis counts in chicken meat by 2.5 orders of magnitude at 4 °C. We identified two receptor-binding proteins with affinity to LPS, and their encoding genes showed plasticity during an exposure assay. Phenotypic, proteomic, and genomic characteristics of STGO-35-1, as well as the Salmonella reduction in chicken meat, support the potential use of STGO-35-1 as a targeted biocontrol agent against S. Enteritidis in chicken meat. Additionally, computational analysis and a short exposure time assay allowed us to predict the plasticity of genes encoding putative receptor-binding proteins.Salmonellosis is one of the most frequently reported zoonotic foodborne diseases worldwide, and poultry is the most important reservoir of  Salmonella, can be transmitted to humans along the farm-to-fork continuum, commonly through contaminated foods of animal origin . In. In88]. S. Enteritidis. We selected three variants  using lysis plaque morphology after exposure , but win domain .Listeria monocytogenes.We selected variants using lysis plaque morphology because of evidence suggesting that the change in morphology is related to the infectivity of tail phage proteins, such as T-even phages ,90,91,92Salmonella–phage interactions in the environments in which phages will be applied.Our study found the importance of identifying RBPs and their plasticity. In the future, determining the mutation rate of these particular genes in complex systems, such as chicken meat or other environments, is necessary to better understand Siphoviridae), and has been described using an approach involving comparative genomic and phenotypic tools that have been used to identify the genomic unit of diversity inside the siphoviral morphotype. In the future, this may contribute to the classification of new similar phages. In addition, the short exposure time assay allowed us to predict the sequence plasticity of proteins that possibly function as RBPs. Finally, their successful biocontrol trial in chicken meat supports the potential use of STGO-35-1 as a biocontrol agent for targeting S. Enteritidis.The phage described in this study, STGO-35-1, belongs to the siphoviral morphotype (formerly family


In the last few years, there has been concern about the dissemination of false health information on social media. It is therefore important that we assess the influencers of this health information in the field of cardiology.Twitter represents a growing aspect of the social media experience and is a widely used tool for public education in the 21We sought to identify the top 100 Twitter influencers within cardiology, characterize them, and examine the relationship between their social media activity and academic influence.Twitter topic scores for the topic search “cardiology” were queried on May 01, 2020 using the Right Relevance application programming interface (API). Based on their scores, the top 100 influencers were identified. Among the cardiologists, their academic h-indices were acquired from Scopus and these scores were compared to the Twitter topic scores.h-index and the Twitter topic score, r = +0.32 .We found out that 88/100 (88%) of the top 100 social media influencers on Twitter were cardiologists. Of these, 63/88 (72%) were males and they practiced mostly in the United States with 50/87 (57%) practicing primarily in an academic hospital. There was a moderately positive correlation between the Our study highlights that the top ranked cardiology social media influencers on Twitter are board-certified male cardiologists practicing in academic settings in the US. The most influential on Twitter have a moderate influence in academia. Further research should evaluate the relationship between other academic indices and social media influence. It has been reported as an excellent place to discover current topics of discourse about vaccines and also to promote vaccination However, sometimes, it is unclear which individuals are influencing these interactions. Given the potential that exists for the dissemination of inaccurate health information h-index [Hirsch index—productivity in terms of number of publications and impact (number of citations) of the publication] Traditionally cardiologists are considered experts by their years of experience and their research output. This research output can be measured by different matrixes, one of which is the 2.h-index scores of the top cardiologist influencers were obtained using Scopus  on May 07, 2020, and added to the database to represent their academic influence. The median h-index of the influencers that were cardiologists was calculated and a Pearson correlation was performed between the h-indices of the cardiologists and their Twitter topic score to evaluate the relationship. Statistics and graphical representation were performed in Microsoft Excel .On May 01, 2020, similar to the method used in other studies 3.The top 100 most influential individuals in cardiology on Twitter were evaluated . Males mApproximately 63/88 (72%) of the top influencers that were cardiologists were males and 25/88 (28%) were females. Of the 88 cardiologists, 87 were actively practicing. Of the practicing cardiologists, about 50/87 (57%) of them worked primarily in an academic hospital setting, 33/87 (38%) in non-academic hospitals, 2/87 (2%) in both academic & private facilities, 1/87 (1%) in private hospitals alone, and 1/87 (1%) worked both in a non-academic hospital and as an entrepreneur. As shown in h-index of the top influencers who were cardiologists was 22 (interquartile range = 32.5) and 41.84 ± 9.89 (mean ± 95% CI) respectively. There was a moderately positive correlation between their Twitter topic score and h-index, r = +0.32 .The median and mean 4.The study aimed to assess the top individuals driving the discussions in cardiology on Twitter and to analyze if they were as influential in academia as they were on Twitter. We found out that the top 100 Twitter influencers were male cardiologists in the United States with 30% women, they work in academic hospitals and interventional cardiologists represent the largest proportion of cardiologists among the influencers. In addition, there was a moderately positive correlation between their academic and Twitter influence.Most of the top 100 cardiology Twitter influencers were US cardiologists. This made up about 85% of the total population studied. They also practice mostly in academic institutions. These individuals are currently influencing the engagements in the field of cardiology on Twitter, and it is consistent with findings from other studies. These other studies evaluated the top influencers in other medical fields on Twitter and found them to be experts in these fields We also found out that among the influencers that were cardiologists, 2 out of 3 were males. This mostly can be attributed to the small percentage of women who are currently cardiologists With regards to their location, only a few cardiologists outside the US were part of the top 100 cardiology influencers. This may be attributed to reports of anti-social media policies in some European countries In addition, we found out that most of the top cardiologist influencers practice in academic hospitals. Studies have shown a high research output from cardiologists who practice in this setting as compared to those who practice in non-academic settings h-index of the top cardiologist influencers  found in our study was higher than that of the orthopedic  and plastic surgeons  in studies done in 2018 and 2019 respectively h-index and their Twitter influence, there was a moderately positive correlation between the two. The moderate positive relationship implies that not only are these top influential cardiologists more active in research as compared to other specialties, they are also almost as influential on Twitter as they are in academia. The most active influential cardiologists may be tweeting more about breakthroughs in cardiovascular research h-index of an author With regards to the overall academic influence, the median h-index, a scoring system that shows a high correlation with other variants This study has a few strengths. First, the large sample size of this study allowed for adequate characterization of the influencers. Second, we used the Right Relevance API which has successfully been used to mine data from Twitter for other studies. Third, the academic influence was computed using the In conclusion, our study showed that when examining the influential voices in cardiology on Twitter, there is a broad range of sub-specialties represented, with interventional cardiologists being the most prominent. There was a geographical diversity as well as a flattening of the hierarch, with a mix of early career , mid-career  and advanced stage career  professionals. Thirty percent were women, which more than doubles the number of women estimated to be practicing cardiovascular medicine. This reflects the challenges that remains in closing the gender gap between men and women as influencers in cardiovascular medicine. These influencers were as influential in the academia as they are on Twitter. Future studies should exam the contents of the posts made by these influencers and also consider other indexes of academic influence like g-index, AR-index, p-index, and integrated impact indicator or academic trace as they relate to social media influence.


Background: Glioblastoma is one of the most aggressive tumours despite all advanced therapies. We aimed to investigate the correlation between qualitative markers of dynamic contrast-enhanced magnetic resonance imaging and vascularity in different tumour regions and elucidate their potential in predicting recurrence.Methods: Radiological markers of vascularity as wash-in rate, washout rate, and capillary time to peak in different single tumour regions were extracted for all glioblastoma patients before being surgically resected using preoperative dynamic contrast-enhanced MRI (DCE-MRI). Tissue samples were obtained from different intratumoral regions and peritumoral oedema and evaluated for the vascular endothelial growth factor (VEGF).Results: Two hundred sixty individuals were included in the final analysis, with 180 dead ones and 80 survivors. Radio- and chemo-therapy were received by all surviving patients and 77.8% (n= 140) of the dead ones. The mean time to peak, in seconds, was longest at the peritumoral oedema region (71.7±23.5), followed by the tumour's necrotic centre (50.0±28.5) and its periphery (2.9±1.8). The expression of VEGF at the peritumoral edema region was inversely correlated to the washout rate at the periphery  and positively correlated to peritumoral TTP .Conclusion: Using DCE-MRI, VEGF expression may be used as a non-invasive marker to estimate tumour grade for clinical diagnosis and treatment. Moreover, the risk of glioblastoma recurrence could be determined by evaluating the washout rate at the tumour's periphery. Further large-scale studies are needed to validate the results and to have concrete evidence. Glioblastoma (GBM) is considered to be the most aggressive primary brain tumour with a horrible prognosis. Recurrence after treatment is a significant problem. The survival rate for one year is about 39.7% . Ideal oOne of the most striking features of GBM is hypoxia. GBM is a highly vascularized tumour with characteristically aberrant vessels . PseudopThe maestro regulator of hypoxia-induced changes is the HIF gene . SeveralAll methods were carried out following relevant guidelines and regulations. The ethical board approved all experimental protocols used in this study. Informed consent was obtained from all subjects or a parent/legal guardian if under 18 years of age. In the case of dead patients, the legal guardian consented.Patient enrollmentThis is a prospective single-centre study. Patients with a history of newly diagnosed GBM with a maximum diameter from 3 to 5 cm as a single lesion identified by MRI were enrolled Figure . The lesDiagnosis of recurrenceIn most cases, recurrence was considered when there was either an unequivocal increase in fluid-attenuated inversion recovery (FLAIR)/T2 signal abnormality or newly detected areas of contrast enhancement on follow-up MRI requiring further surgery, radiation, or chemotherapy.Preoperative imagingPatients who fulfilled all the inclusion criteria underwent preoperative conventional gadolinium-enhanced MRI brain and DCE-MRI. We identified three regions of interest for further analysis to extract qualitative vascular markers. We divided the tumour into a necrotic centre, a peripheral thickness of the tumour, and a peritumoral oedema region Figure . All imaDCE-MRI data extractionAt first, standard preoperative non-contrasted MRI was obtained, and then, the lesion of interest was identified. Utilizing a fast 3-dimensional gradient echo, the dynamic perfusion MRI was obtained on the 1.5T Philips machine. About 0.1 mmol/kg of gadolinium particles (Omniscan) were injected over 4 seconds through an 18-gauge catheter into a prominent vein using a power injector.Semi-quantitative parameters are more manageable and more straightforward than quantitative parameters using the T1-weighted sequence after gadolinium injection. We defined specific parameters to extract in a region of interest about 5 mm in diameter. T0 was considered as the basal contrast at a duration of 0 seconds. Time to peak (TTP) was defined as the beginning of contrast injection to the highest enhancement peak. Wash-in is the time of contrast rush in the capillary from zero to peak level, while the washout rate of the contrast is the time elapsed from the peak to the return to zero level Figure .SurgerySurgery was done at a maximum of two days post-imaging. Contrasted MRI was loaded into the navigation system to target the predefined areas we specified for sampling. Sampling was done before completely resecting the lesion to avoid the volume shift from debulking and the resulting brain relaxation. The first sample was sent for frozen section pathology. According to the WHO classification 2016 ,16 and VImmunohistochemistryAll slides were examined under a 200X magnification microscope and scored by a neuropathologist blinded to the tumour, the imaging, and the patient's data. All samples retrieved were labelled according to the region from where they were obtained. All were stained with VEGF and scored from zero to four based on the proportion of cells stained.Statistical methods and data analysisAll the data were analyzed using R software version 4.0.2 and two-sided, considering P-value ≤ 0.05 was considered statistically significant for all tests. Categorical variables were represented as frequencies and percentages with Chi-square test  for testing the difference, according to their living/death status. Following the normal distribution test, continuous variables were expressed as means, and standard deviations (SDs) with independent t-test  were used for testing the difference, according to their living/death status.Pearson's correlation coefficient (r) was used to determine the relationship between different variables and their recurrence correlation. Moreover, univariable and multivariable Cox proportional hazard regression models were used to identify all possible prognostic factors affecting GBM recurrence. The adjustment in the multivariate model was made for other co-founders; TTP , washout rate (other than periphery), VEGF , radiotherapy status, and chemo-therapy status of the included patients. Regression results were expressed as hazard ratios (HRs) and 95% confidence interval (95% CI) for both regression models. Kaplan-Meier analysis in the form of survival curves was used to present the recurrence probabilities of significant predictors, and a log-rank test was used to compare their recurrence rates.Patients’ clinical dataTwo hundred sixty individuals were included in the final analysis, with 180 dead ones and 80 survivors. Radio- and chemo-therapy were received by all surviving patients, and 77.8% (n= 140) of the dead ones. In total, recurrence was confirmed in 69.2% (n= 180) of the patients; seven patients have passed away , followed by the tumour's necrotic centre (50.0±28.5) and its periphery (2.9±1.8). The same pattern was observed in the measured washout rates (seconds), where they were high at the peritumoral oedema region (70.3±24.1) and the tumour's centre (53.1±28.1) compared to its periphery (29.1±25.6)  and tumour's centre (11.8±4.8), compared with that noted in the surrounding peripheries (6.3±2.8). For all tested parameters, there were no statistically significant differences based on the survival status  and positively correlated to peritumoral TTP . Additionally, VEGF expression at the tumor’s center was positively correlated to central TTP  and the central wash out rate was inversely correlated to TTP at tumor’s periphery  Table .The risk of recurrence was solely correlated to the washout rate at the tumor’s periphery . In contrast, risk of recurrence was not correlated to central TTP , peritumoral TTP , peripheral TTP , central washout rate , peritumoral washout rate , central VEGF , peritumoral VEGF , or peripheral VEGF  , the multivariate model showed a highly significant association with recurrence risk . To test the effect of different peripheral washout rates, 20 seconds cut-off was used. The Kaplan-Meier analysis showed statistically significant results (Figure Over the last few years, the DCE-MRI has been introduced as a part of the preoperative assessment and the follow-up of brain tumours, including GBM ,18. The To date, several studies reported a comparable accuracy of DSC-MRI and DCE-MRI in tumour grading -29, withIn this study, we obtained DCE-MRI and conventional MRI for all patients with suspected GBM before surgery. We also obtained frameless navigation guided biopsies from different tumour regions as illustrated before, from the centre, tumour edge and peritumoral tissue guided by the increased T2 and FLAIR intensity surrounding the tumour. Localization was a rough estimate of these regions. There are no strict borders between these regions, resulting in a limitation to precisely sample the theoretical partitions of the GBM. Sampling was attempted before debulking surgery to avoid architectural changes resulting from fluid aspiration or volumetric changes. Each sample was analyzed for the degree of vascular hyperplasia, using VEGF staining. A group of researchers used imaging to sample different tumour regions, and found a correlation between histological markers and certain imaging markers. They observed that the semi-quantitative markers of DCE-MRI were correlated with the enhanced regions, while diffusion parameters were correlated with the non-enhanced regions .We found that specific semi-quantitative DCE-MRI markers as the washout rate and TTP were high in the peritumoral oedema area, which indicates the profuse vascularity of that area. Moreover, we found that VEGF expression is high in the peritumoral area and strongly correlates with the washout rate and TTP in the same areas. VEGF expression was higher in the peritumoral area than the centre and tumour edge Figure . At the The observed results may help us to predict vascularity in different regions of the single GBM lesion. That may guide us to target anti-vascular therapy to the highly vascular regions compared to the low vascular regions. Moreover, preoperative non-invasive determination of the microvascularity may aid in prognosis and follow-up Figure .Using DCE-MRI, VEGF expression may be used as a non-invasive marker to evaluate tumour grading for clinical prognosis and therapy. Moreover, the risk of GBM recurrence could be determined by evaluating the washout rate at the tumour's periphery. Further large-scale studies are suggested to validate the results and obtain concrete evidence.


In the Methods section, the age range of the youngest category should have appeared as “0-24.” In the key to Figure 2, the top age category should have appeared as “≤24.” This article has been corrected.1In the Research Letter titled “Trends in Drug Overdose Mortality in Ohio During the First 7 Months of the COVID-19 Pandemic,”


In “Computer-Aided Diagnosis of Gastrointestinal Ulcer and Hemorrhage Using Wireless Capsule Endoscopy: Systematic Review and Diagnostic Test Accuracy Meta-analysis” :e33267), one error was noted.In the originally published paper, the Acknowledgments section appeared as follows:This work was supported by the Technology development Program (#S2931703) funded by the Ministry of SMEs and Startups (Korea).In the corrected version of the paper, the Acknowledgments section has been revised as follows:This work was supported by the Institute of Information & Communications Technology Planning & Evaluation (IITP) grant funded by the Korean government (MSIT) (grant number 2020-0-01604).The correction will appear in the online version of the paper on the JMIR Publications website on January 11, 2022, together with the publication of this correction notice. Because this was made after submission to PubMed, PubMed Central, and other full-text repositories, the corrected article has also been resubmitted to those repositories.


Brown tumour is a rare tumour-like lesion of the bone, which is considered as an end-stage lesion of abnormal bone metabolism caused by persistently high parathyroid hormone (PTH) levels. Brown tumour can be found in any part of the skeleton; in some cases, it can occur in multiple bones and can be easily misdiagnosed as a metastatic tumour.We report the case of a 44-year-old man who presented to the Department of Oncology in our hospital with a 2-month history of local pain in his left shoulder joint. The initial diagnosis was an aneurysmal bone cyst by biopsy, for which the patient underwent tumour resection surgery. The diagnosis of a malignant tumour was made again following postoperative pathological examination. The pathological sections and all clinical data were sent to the Department of Pathology of the First Affiliated Hospital of Sun Yat-sen University; the diagnosis made there was brown tumour. His blood PTH level was 577 pg/ml (15–65 pg/ml). Colour Doppler ultrasonography of the parathyroid gland suggested a parathyroid adenoma. For further treatment, the left parathyroid adenoma was removed by axillary endoscopic resection. Postoperatively, a pathologic examination was performed, and the diagnosis of a parathyroid adenoma was confirmed. One year after the surgery, the left humerus was completely healed, and the left shoulder joint had a good range of movement.In summary, histopathological diagnosis is not sufficient for the diagnosis of brown tumours. A comprehensive analysis combining clinical symptoms with findings of imaging and laboratory tests is also required. Generally, the treatment of brown tumour includes only partial or complete resection of the parathyroid glands. However, when the tumour is large, especially when it involves the joint, surgery is indispensable. Brown tumour is a rare tumour-like lesion of the bone, which is considered as an end-stage lesion of abnormal bone metabolism caused by persistently high parathyroid hormone (PTH) levels. The incidence of primary hyperparathyroidism (PHPT) is lower (< 5%) than that of secondary hyperparathyroidism , 2. BrowGenerally, the diagnosis of brown tumour is based on medical history, clinical examination, laboratory results, and imaging findings. Pathological examination is the gold standard for diagnosis, which shows mononuclear cells mixed with multinuclear giant cells . HoweverHerein, we described a case that emphasizes the importance of considering PHPT in the differential diagnosis of patients with multiple lytic bone lesions, thus avoiding unnecessary and harmful interventions.A 44-year-old man presented to the Department of Oncology in our hospital with a 2-month history of local pain of the left shoulder joint. He initially visited a local hospital, where magnetic resonance imaging (MRI) was performed Fig. . A 70 × 2. His blood investigations revealed the following: calcaemia, 3.09 mmol/L (2.11–2.52 mmol/L); phosphoraemia, 0.55 mmol/L (0.85–1.51 mmol/L); and alkaline phosphatase level, 461 U/L (45–125 U/L). The other investigation results were normal. Standard anteroposterior and oblique radiographs of the left shoulder joint showed large osteolytic lesions involving the proximal humerus and humeral head without any joint involvement . Colour Doppler ultrasonography of the parathyroid gland showed that the left dorsal thyroid was hypoechogenic, 17 × 12 mm, with unclear boundaries and an irregular shape, which was considered as a parathyroid adenoma (Fig. 5 pg/ml. Regarding the treatment of PHPT, the patient was transferred to the Department of Otolaryngology for surgery. The left parathyroid adenoma was removed by axillary endoscopic resection. Intraoperatively, 20 × 10 mm solid yellow nodules with clear boundaries were observed at the left dorsal lobe of the thyroid gland Fig. c, d. TheOne year after the surgery Fig. , the lefPHPT is the third most common endocrine disease after diabetes and thyroid disease. Its incidence ranges from 0.025 to 0.065%, with the highest incidence in postmenopausal women –13. The Brown tumour, which is one of the pathognomonic signs of PHPT, is a focal, tumour-like bony lesion caused by osteoclastic turnover of bone. Although it is reported that musculoskeletal manifestations in patients with PHPT can reach 54.7%, the incidence rate of brown tumour ranges from 1.5 to 4.5% , 16. In To increase the diagnostic rate of brown tumours, some diagnostic methods are needed in clinical practice, including clinical examination, laboratory investigations, imaging examinations, and pathological examination . Among tControversies on the use of surgery and conservative treatment for brown tumour still exist. Generally, brown tumour does not need surgical treatment because it regresses spontaneously after partial or complete resection of the parathyroid glands . Hu et aIn summary, we described a rare case of brown tumour caused by PHPT, which was initially misdiagnosed as other bone diseases. Clinically, histopathological diagnosis is not enough for the diagnosis of brown tumours. A comprehensive analysis combining clinical symptoms, imaging, and laboratory tests is also required. Particularly, the possibility of bone metabolic diseases, especially brown tumour caused by hyperparathyroidism, should be considered when multiple osteolytic bone destruction occurs throughout the body, with extensive osteoporosis, hypercalcaemia, hypokalaemia, and high alkaline phosphatase levels. Generally, the treatment of brown tumour includes partial or complete resection of the parathyroid glands alone. However, when the tumour is large, especially when it involves the joint, surgery is indispensable.


Klebsiellae spp. arises through mutational and acquired mechanisms and is considered an “urgent threat” by the CDC. VNRX-5236 is a bicyclic boronate β-lactamase inhibitor (BLI) that combines oral bioavailability  and activity against all three Ambler classes of serine β-lactamases. VNRX-7145 is currently in development with the oral cephalosporin, ceftibuten (CTB) .Carbapenem resistance in Figure 1. Structures of VNRX-7145, VNRX-5236, and ceftibuten. The β-lactamase inhibitors are in red and the β-lactam antibiotic is in black.Klebsiellae from the Consortium on Resistance against Carbapenems in Klebsiella (CRACKLE) was assessed in this study. Among these, 193 expressed class A KPC enzymes, one expressed a class B NDM enzyme, and six expressed a class D OXA-48 or variant enzyme. Minimum inhibitory concentrations (MIC) were determined by broth microdilution (CLSI M07 Ed. 11) using the ThermoFisher Sensititre system with custom assay panels. MICs were interpreted using CLSI M100 Ed. 30, except the EUCAST breakpoint for CTB (S≤1 µg/mL) was used for CTB and was applied for comparative purposes to CTB/VNRX-5236 MICs where VNRX-5236 was fixed at 4 µg/mL. American Type Culture Collection strains were used for quality control.The activity of CTB/VNRX-5236 against 200 carbapenem-resistant 50s were in the susceptible range for CZA, MVB, and CTB/VNRX-5236; and resistant for CTB, ceftazidime (CAZ) and meropenem (MEM). MIC90s were in the susceptible range for CZA, MVB, and CTB/VNRX-5236 and resistant range for CAZ, MEM, and CTB (Table 1). One of four CZA-resistant and three of nine MVB non-susceptible strains were provisionally susceptible to CTB/VNRX-5236.92.5% of stains studied in this CRACKLE collection were provisionally susceptible to CTB/VNRX-5236. In comparison, strains were 95.5% and 98% susceptible to meropenem-vaborbactam (MVB) and ceftazidime-avibactam (CZA), respectively. MICMIC50 and MIC90 values (µg/mL) and percent susceptibility for Klebsiella pneumoniae strains (n=200). AMK, amikacin; CST, colistin; CAZ, ceftazidime; CZA, ceftazidime-avibactam; FEP, cefepime; MEM, meropenem; MVB, meropenem-vaborbactam; CTB, ceftibuten; TGC, tigecycline. * The breakpoint for CST is intermediate, as no susceptible breakpoint is available. ** The CTB breakpoint is valid only for urinary tract isolates. *** The breakpoint for CTB was provisionally used for CTB/VNRX-5236, where VNRX-5236 was fixed at 4 µg/mL.Klebsiella isolates tested, reaching a total of 92.5% susceptibility. The prodrug (VNRX-7145) allows for oral administration, making it a potential option for step-down therapy. Importantly, VNRX-5236 has a broader spectrum of activity than existing oral BLIs, opening new treatment options for resistant infections as a key addition to the existing antibiotic arsenal.The addition of VNRX-5236 enhanced the activity of CTB against the 200 Robin Patel, MD, 1928 Diagnostics (Consultant)BioFire Diagnostics (Grant/Research Support)ContraFect Corporation (Grant/Research Support)Curetis (Consultant)Hylomorph AG (Grant/Research Support)IDSA Infectious Diseases Board Review Course Mammoth Biosciences (Consultant)NBME Netflix (Consultant)Next Gen Diagnostics (Consultant)PathoQuest (Consultant)PhAST (Consultant)Qvella (Consultant)Samsung Selux Diagnostics (Consultant)Shionogi & Co., Ltd. (Grant/Research Support)Specific Technologies (Consultant)TenNor Therapeutics Limited (Grant/Research Support)Torus Biosystems (Consultant)Up-to-Date  Robin Patel, MD, BioFire  Involved: Self): Grant/Research Support; Contrafect  Involved: Self): Grant/Research Support; IDSA  Involved: Self): Editor’s stipend; NBME, Up-to-Date and the Infectious Diseases Board Review Course  Involved: Self): Honoraria; Netflix  Involved: Self): Consultant; TenNor Therapeutics Limited  Involved: Self): Grant/Research Support; to Curetis, Specific Technologies, Next Gen Diagnostics, PathoQuest, Selux Diagnostics, 1928 Diagnostics, PhAST, Torus Biosystems, Mammoth Biosciences and Qvella  Involved: Self): Consultant David van Duin, MD, PhD, Entasis (Advisor or Review Panel member)genentech (Advisor or Review Panel member)Karius (Advisor or Review Panel member)Merck Pfizer Qpex (Advisor or Review Panel member)Shionogi Utility (Advisor or Review Panel member) Vance G. Fowler, Jr., MD, MHS, Achaogen (Consultant)Advanced Liquid Logics (Grant/Research Support)Affinergy Affinium (Consultant)Akagera (Consultant)Allergan (Grant/Research Support)Amphliphi Biosciences (Consultant)Aridis (Consultant)Armata (Consultant)Basilea Bayer (Consultant)C3J (Consultant)Cerexa Contrafect Debiopharm Destiny (Consultant)Durata Genentech Green Cross Integrated Biotherapeutics (Consultant)Janssen Karius (Grant/Research Support)Locus (Grant/Research Support)Medical Biosurfaces (Grant/Research Support)Medicines Co. (Consultant)MedImmune Merck (Grant/Research Support)NIH (Grant/Research Support)Novadigm (Consultant)Novartis Pfizer (Grant/Research Support)Regeneron sepsis diagnostics Tetraphase (Consultant)Theravance Trius (Consultant)UpToDate Valanbio xBiotech (Consultant) Daniel D. Rhoads, MD, Becton, Dickinson and Company (Grant/Research Support) Michael Jacobs, MBBS, Venatorx Pharmaceuticals, Inc. (Grant/Research Support) Focco van den Akker, PhD, Venatorx Pharmaceuticals, Inc. (Grant/Research Support) David A. Six, PhD, Venatorx Pharmaceuticals, Inc. (Employee) Greg Moeck, PhD, Venatorx Pharmaceuticals, Inc. (Employee) Krisztina M. Papp-Wallace, Ph.D., Merck & Co., Inc. (Grant/Research Support)Spero Therapeutics, Inc. (Grant/Research Support)Venatorx Pharmaceuticals, Inc. (Grant/Research Support)Wockhardt Ltd.  Robert A. Bonomo, MD, entasis (Research Grant or Support)Merck (Grant/Research Support)NIH (Grant/Research Support)VA Merit Award (Grant/Research Support)VenatoRx (Grant/Research Support)


Advanced imaging of CRLM including multiparametric MRI and functional imaging techniques have the potential to provide clinically-actionable phenotypic characterisation. This includes assessment of the tumour-liver interface, internal tumour components and treatment response. Advanced analysis techniques, including radiomics and machine learning now have a growing role in assessment of imaging, providing high-dimensional imaging feature extraction which can be linked to clinical relevant tumour phenotypes, such as a the Consensus Molecular Subtypes (CMS). In this review, we outline how imaging techniques could reproducibly characterize the histopathological features of CRLM, with several matched imaging and histology examples to illustrate these features, and discuss the oncological relevance of these features. Finally, we discuss the future challenges and opportunities of CRLM imaging, with a focus on the potential value of advanced analytics including radiomics and artificial intelligence, to help inform future research in this rapidly moving field.Colorectal liver metastases (CRLM) have heterogenous histopathological and immunohistochemical phenotypes, which are associated with variable responses to treatment and outcomes. However, this information is usually only available after resection, and therefore of limited value in treatment planning. Improved techniques for  Colorectal cancer (CRC) remains the second leading cause of cancer-related death in the developed world . Liver mThere are two main therapeutic strategies for CRLM. For those patients with polymetastatic disease, palliative systemic therapy is the mainstay of treatment. In patients with ‘oligometastatic’ disease , curativafter resection, and is therefore of limited value in pretreatment prognostication, or when planning the neoadjuvant or polymetastatic treatment.The pathological literature describes the varied histopathological features of CRLM, both their internal architecture and their interface with the surrounding liver parenchyma , 10. Sevin vivo disease assessment, which can characterise the variable tumour biology, would allow clinicians to plan personalized management strategies. Imaging already plays a central role in assessing the sites and burden of metastatic cancer both before and after treatment  (Contrast enhanced CT (ceCT) is the mainstay of oncological imaging, and is the first line test for staging, surveillance and response assessment. Where there is diagnostic uncertainty, or precision about the number of metastases is crucial, mpMRI is the gold-standard technique for detecting and characterising focal liver lesions. Standard sequences include unenhanced T1- and T2-weighted sequences (including opposed-phase imaging), diffusion weighted imaging (DWI) and multiphase acquisitions following intravenous extracellular gadolinium chelate contrast media or liver-specific contrast agents. For CRLM, multiparametric liver MRI has superior per patient and per lesion sensitivity to CT , 14, and-PET/CT) . Additio-PET/CT) . 18F-flu-PET/CT) .Arguably the most clinically-relevant histopathological feature amenable to evaluation by imaging is the interface between normal liver and tumour. Interface features could influence chemotherapy selection, in particular for antiangiogenic agents , 18, infvia vascular co-option, rather than angiogenesis, which is supported by their proven poor response to anti-angiogenic agents  can be identified with hepatocellular carcinoma (HCC) as reproducible hypoperfusion of liver parenchyma surrounding the tumour due to subtle tumour infiltration . HoweverVascular invasion is a common feature of colorectal cancer and its liver metastases. Intrahepatic portal venous invasion local to resected metastases occurs in approximately a quarter of cases (reported range 10-49%) and hepatic venous invasion in approximately one tenth of cases (reported range 5-24%) . High reLymphatic invasion is a less common feature than venous invasion but has again been shown to be associated with poorer outcomes –33. LympThe internal composition of tumours differs, comprising variable proportions of tumour cells, fibrosis, necrotic material and, in some cases mucin and calcification. It is also influenced by adjuvant treatment , 35. TheViable tumour cells contrast with normal liver on T1 and T2 weighted sequences , 36–38. Defining the internal composition of metastases may help to categorise tumour biology and thus influence immunological and surgical strategy recently described Consensus Molecular Subtypes (CMS) of colorectal liver metastases , can be Pitroda et al.  have proChemotherapy and other systemic treatments, such as immunotherapy, are used in the palliative and neoadjuvant settings. Pre-treatment, proportions of viable and necrotic tumour occurs as a result of intralesional hypoxia and ischaemia as tumour growth outstrips angiogenesis or vascular co-option, and has not been found to be prognostically relevant , 57. HowThe method of histological tumour regression grading described by Rubbia-Brandt et al  is the mThere is already evidence to support using morphological features to assess response of CRLM to chemotherapy. Lesion fibrosis, demonstrated as late gadolinium enhancement, which is the principle marker of response on pathological assessment, is a feature strongly linked to improved survival outcomes Figure 4in vivo assessment of multisite disease, which is a particular advantage in the metastatic setting. In this article, we have described the basis for more complete morphological characterization of CRLM (The major benefit of imaging is the capacity to perform repeated,  of CRLM . A furth of CRLM  and func of CRLM , 55. The of CRLM , 70 and Molecular imaging techniques, in particular PET/CT, can provide further functional assessments of CRLM. FDG-PET is well-established in oncological practice, and routinely acquired in staging oligometastatic CRC, principally for detecting disease that would be beyond the scope of local therapy. However, FDG-uptake can also be used as marker of hypoxia, and as a prognostic marker and for response evaluation . The pot2. Although higher spatial resolutions can be achieved, an important feature of liver imaging is managing respiratory and, to a lesser extent, cardiac motion, which limits acquisition time and spatial resolution achievable in the upper abdomen. This issue is compounded through tissue features which can be substantially smaller than the imaging resolution, which may preclude accurate assessment of some features and place a limit on the achievable performance of imaging.However, despite the potential for multiparametric structural, functional and molecular imaging to provide a more comprehensive assessment of CRLM, there are limitations. While these techniques might be valuable, and are often integrated into trials, complex multimodality assessments are challenging in the clinical workflow, and reliably and repeatedly combining information from multiparametric imaging is difficult for human observers. A second major technical challenge to the use of imaging for assessing tissue features of CRLM is the achievable spatial resolution. For MRI, the in-plane spatial resolution is typically in the region of 1mmThese two key challenges may be overcome by developments in image acquisition and analysis techniques. MRI acquisition will increasingly use computer-assisted techniques to decrease acquisition time and improve image resolution, for example through the use of ‘super-resolution’ techniques, which may improve the potential for tissue feature assessment by imaging . SecondlThe most promising opportunity within radiology is the incorporation of machine learning in image interpretation. Analysis of tumour features has historically relied on expert radiological assessment of imaging features. However, modern radiomic image analysis can be used to extract high-dimensional data from medical imaging , and macSeveral key hurdles must be overcome before radiomics and machine learning becomes robust enough to influence patient care in daily clinical practice. Firstly, many radiomics and machine learning studies on CRLM have been conducted on relatively small datasets , 76, 78.Even assuming a robust algorithm and analysis platform is developed in the research setting, adapting them into a convenient final product for use in the routine clinical workflow is a further challenge. In addition, many radiomic techniques are time-consuming from an operator perspective with careful lesion contouring required , 81. Autin vivo tumour assessment, in particular circulating tumour biomarkers such as circulating tumour DNA (An important consideration for imaging biomarkers in cancer is the development of alternative techniques for mour DNA . Howeverin vivo characterization of clinically relevant histopathological features of CRLM. We have outlined the imaging findings of these histopathological features, focusing on the tumour-liver interface, intralesional component analysis, and morphological response assessment following systemic or liver-directed treatment. In vivo assessment of the tumour-liver interface has the potential to play and important role in defining the surgical approach and chemotherapy selection. As well as improving our characterisation of response to chemotherapy, imaging analysis of internal tumour components could play an increasingly important role as predictors of CRLM molecular subtypes. However, in the absence of studies providing robust validation of imaging techniques to report these features in practice, our assessment of CRLM by imaging is limited to documenting their size, number and location.This review has highlighted the potential for advanced imaging to provide Advanced analysis methods, such as radiomics and machine learning, will be crucial tools in developing and validating novel imaging biomarkers for CRLM. However, these rely on curated and annotated datasets of sufficient size to build reliable algorithms, which is likely to require the cross-institutional collaborations that have been achieved in pathology and molecular biology.DM was involved with literature review, and writing and editing the manuscript. MT was involved with literature review, and writing and editing the manuscript. FG was involved with writing and editing the manuscript. DB was involved with writing and editing the manuscript. RG was involved with writing and editing the manuscript. JP was involved with writing and editing the manuscript. AH was involved with writing and editing the manuscript. JF was involved with literature review, writing and editing the manuscript. All authors contributed to the article and approved the submitted version.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Universal varicella vaccination might reduce opportunities for varicella-zoster virus (VZV) exposure and protective immunological boosting, thus increasing herpes zoster incidence in latently infected adults. We assessed humoral and cell-mediated immunity (CMI), as markers of VZV exposure, in adults aged ≥50 years.We repurposed data from placebo recipients in a large multinational clinical trial (ZOE-50). Countries were clustered based on their varicella vaccination program characteristics, as having high, moderate, or low VZV circulation. Anti-VZV antibody geometric mean concentrations, median frequencies of VZV-specific CD4 T cells, and percentages of individuals with increases in VZV-specific CD4 T-cell frequencies were compared across countries and clusters. Sensitivity analyses using a variable number of time points and different thresholds were performed for CMI data.P < .0001) but not by VZV circulation. No significant differences were identified in VZV-specific CMI between participants from 2 high versus 1 low circulation country. In 3/5 sensitivity analyses, increases in CMI were more frequent in high VZV circulation countries (.03 ≤ P < .05).VZV-specific humoral immunity from 17 countries  varied significantly between countries (We found no consistent evidence of reduced VZV exposure among older adults in countries with universal varicella vaccination.NCT01165177. By repurposing humoral and cell-mediated immunogenicity data from a large multinational trial enrolling adults aged ≥50 years, we found no consistent evidence of differences in varicella-zoster virus exposure between countries with widely circulating varicella versus countries with universal vaccination. Varicella-zoster virus (VZV) is a human alphaherpesvirus that infects most individuals, usually during childhood, although its incidence may be delayed in warmer climates . PrimaryIn 1965, Hope-Simpson hypothesized that VZV reactivation is under an immunological control that is periodically boosted by exposures to varicella  and/or by prior subclinical VZV reactivation events  .Based on the observation that herpes zoster incidence was higher in adults living without children compared to those living with children (a surrogate for VZV exposure), Brisson et al  construcSeveral countries have implemented universal varicella vaccination , leadingFor this post hoc analysis, we repurposed data from a multinational randomized clinical herpes zoster vaccine trial  to deterBox 1.The varicella-zoster virus, which causes varicella in childhood, remains latent and may reactivate later in life, leading to herpes zoster disease.Protection against varicella-zoster virus reactivation and herpes zoster is expected to be boosted by 2 mechanisms: exposure to varicella (exogenous boosting) and/or by asymptomatic reactivation of the virus (endogenous boosting). To induce exogenous boosting, the varicella exposure should be intense enough to stimulate an immune response.Varicella vaccines are highly efficacious in preventing varicella disease in children and are routinely used in several countries. Many other countries have delayed implementation based on the hypothetical concern that decreased exposure to varicella (through vaccination) may lead to an increase of herpes zoster disease in older adults, due to the lack of exogenous boosting.We assessed the differences in varicella-zoster virus immune status among participants ≥ 50 years of age using samples from a clinical trail conducted in 18 countries. The objective was to seek evidence that there are more opportunities for intense varicella exposure in countries with endemic varicella (high virus circulation) compared to those with varicella vaccination programs (low virus circulation).We did not detect any differences in immunity among populations from different countries.In our analysis, we found no consistent evidence that varicella vaccination programs reduce opportunities for varicella-zoster virus exposure among older adults.The results should be carefully considered by national authorities when implementing vaccination policies.This post hoc exploratory analysis was based on data derived from placebo recipients in a large, placebo-controlled, randomized clinical trial of the adjuvanted recombinant zoster vaccine, conducted in 18 countries across North America, Latin America, Europe, Asia, and Australia, between August 2010 and July 2015   of 1067 and 119 [54.6%] of 218) and of Caucasian/European heritage (747 [70.0%] of 1067 and 128 [58.7%] of 218). Characteristics of the overall study population enrolled in the placebo arm of ZOE-50 have been previously published  versus 464.79 [SD 565.55]). A summary of CMI data is shown in IFN-γ+(+) T cells was not statistically significant (P = .56). Point estimates of the median frequency of VZV-specific CD4IFN-γ+(+) T cells/106 CD4 IFN-γ+(+) T cells counted were higher in the 2 countries with high circulation compared to the country with low circulation: 414  for Czech Republic and 448  for Japan versus 380  for the United States.CMI samples were available from 3 countries: 2 with high and 1 with low circulation. CMI status at month 0, assessed by the mean frequency of CD4P values below 5% were observed for the results of participants with data available for at least 2 time points (both for at least 2-fold and 3-fold increases) and all 5 time points (only for at least 3-fold increase). However, CIs were large and overlapping for all analyses.In the main analysis, based on participants with at least 2-fold increases and data available for the first 4 time points, no statistically significant difference was observed between countries with high and low VZV circulation . When seConcerns that universal varicella vaccination could result in increased herpes zoster incidence are based on the assumption that in varicella-endemic settings, large portions of the latently VZV-infected population experience periodic VZV exposure of sufficient intensity to stimulate the still unrecognized components of immunity that control reactivation . HoweverChanges in humoral immunity of adults following reexposure to VZV have been explored in several studies. Two studies , 23 founIFN-γ+(+) T cells of participants from countries with high versus low VZV circulation levels. This finding supports the hypothesis that VZV exposures in the general population are insufficient to elicit detectable immune responses in a significant proportion of individuals. Similar results were reported by Ogunjimi et al [IFN-γ+ T cells of grandparents with known reexposure to VZV over 1 year, or compared to controls.Age-related decreases of VZV-specific T-cell frequencies are associated with increased risks of herpes zoster, although no proven correlates of protection have been identified . Circulami et al , who fouThe kinetics of circulating VZV-specific CD4 T cells are somewhat similar during symptomatic reactivation and following reexposure to VZV, reaching a peak within the first 2 to 4 weeks and then declining by week 6 to modestly elevated and persistent levels , 23. In We observed transient CMI increases in all 3 countries with available data and although percentages were lower in the United States (a country with low VZV circulation), the differences were not statistically significant in the main analysis. In 3 of the 5 sensitivity analyses, differences between the United States and the 2 countries with high circulation (the Czech Republic and Japan) were marginally significant, although CIs were wide and overlapping for all analyses. While this could reflect reduced VZV exposure, it can also represent a country effect, considering that only 3 countries were included in this analysis. Although it is not clear whether endogenous boosting protects against development of herpes zoster , 28, it In certain settings and subpopulations, the protective effects of reexposure to varicella seem quite possible. For example, in adults, a protective effect of living with children was observed in the prevaccination era but disappeared in the years after vaccination implementation , and a mOur study has limitations. Humoral immune responses following VZV reexposure decline rapidly, with cellular immune responses being somewhat more durable ; the outIn conclusion, we assessed humoral and CMI to determine whether VZV exposures are more common in adults aged ≥50 years in countries with widely circulating varicella versus countries with universal vaccination and good varicella control but found no consistent differences. Our results suggest a credible explanation why countries that have introduced varicella vaccination programs have not experienced postulated increases in herpes zoster incidence and can inform national authorities considering implementation of universal varicella vaccination. Repurposed immunological data from completed clinical trials can provide useful information with which to address important questions relating to the dynamics of infectious diseases.The Journal of Infectious Diseases online. Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author.Supplementary materials are available at jiab500_suppl_Supplementary_DataClick here for additional data file.jiab500_suppl_Supplementary_FigureClick here for additional data file.