Head computed tomography (CT) is a guideline recommended method to predict functional outcome after cardiac arrest (CA), but standardized criteria for evaluation are lacking. To date, no prospective trial has systematically validated methods for diagnosing hypoxic-ischaemic encephalopathy (HIE) on CT after CA. We present a protocol for validation of pre-specified radiological criteria for assessment of HIE on CT for neuroprognostication after CA.This is a prospective observational international multicentre substudy of the Targeted Hypothermia versus Targeted Normothermia after out-of-hospital cardiac arrest (TTM2) trial. Patients still unconscious 48 hours post-arrest at 13 participating hospitals were routinely examined with CT. Original images will be evaluated by examiners blinded to clinical data using a standardized protocol. Qualitative assessment will include evaluation of absence/presence of “severe HIE”. Radiodensities will be quantified in pre-specified regions of interest for calculation of grey-white matter ratios (GWR) at the basal ganglia level. Functional outcome will be dichotomized into good  and poor  at six months post-arrest. Prognostic accuracies for good and poor outcome will be presented as sensitivities and specificities with 95% confidence intervals , descriptive statistics (Area Under the Receiver Operating Characteristics Curve), inter- and intra-rater reliabilities according to STARD guidelines.The results from this prospective trial will validate a standardized approach to radiological evaluations of HIE on CT for prediction of functional outcome in comatose CA patients.ClinicalTrials.gov NCT02908308 and NCT03913065.The TTM2 trial and the TTM2 CT substudy are registered at  Post-cardiac arrest brain injury is the result of a transient ischaemia with subsequent reperfusion which may manifest as hypoxic-ischaemic encephalopathy (HIE) on neuroimaging.8To improve prognostic performance, various approaches to quantitative CT analysis have been investigated, but their clinical application is limited by the lack of consensus on which regions of interest (ROI) and cutoff values are most valid.Hypotheses of the TTM2 CT substudy are:1.A standardized qualitative assessment of head CT has a higher prognostic accuracy than non-standardized qualitative assessment (current reference standard) for poor outcome prediction2.a)Definite signs of “severe HIE” diagnosed using our SOP for qualitative assessment.b)Grey-white matter ratio (GWR) at the basal ganglia level below pre-specified cutoffs (1.10 and 1.15) determined using our SOP for quantitative assessment.c)et al.Automated, atlas-based GWR on the basal ganglia level (auto GWR) below 1.10 as described by Kenda The following findings are predictive of a poor functional outcome with 0% false positive ratio (FPR):In patients unconscious more than 48 h after CA:3.The prognostic accuracies of the CT analysis methods described above is independent from the type of CT scanner, previous use of CT contrast agent and targeted temperature management.4.No patient with “severe HIE” on head CT will have low blood levels of the brain injury marker Neurofilament light (NFL) (0% FPR).5The reliability of agreement for prediction of poor outcome using qualitative and quantitative assessment of head CT will be good (Fleisś kappa > 0.7).Clinicaltrials.gov NCT02908308) was an international, multicentre, parallel group, investigator-initiated trial which randomised 1900 adult patients with an out-of-hospital cardiac arrest to a target temperature of 33°C or to a strategy to maintain normothermia and early treatment of fever (≥37.8°C).20The TTM2-trial  is a prospective international multicentre observational study examining the prognostic accuracy of head CT for prediction of functional outcome after CA. Between 11/2017 and 01/2020, patients were recruited at 13 TTM2 sites routinely examining patients still unconscious at 48 hours post-arrest with head CT  as a “yes”/”no” answer to the question “Are there signs of global, diffuse or bilateral multifocal ischaemic injury on CT?”. The date and time for examination and the number of CT examinations performed was also reported.For this substudy, collection of the original Digital Imaging and Communications in Medicine (DICOM) images was initiated after randomisation of the last patient in the TTM2 trial. Images were collected and stored in a digital database labelled with the patient́s trial identification number. Data will be kept according to national legislation for record keeping.All types of scanners and software were permitted, and specifics for each scan will be registered and analysed. Technical prerequisites for including CTs in the analysis are: Axial slices of 4–5 mm slice thickness of the entire brain available with tube voltage 120 kV. We will extract the following technical metadata: time of CT examination, manufacturer, scanner and convolution kernel. Radiological evaluations will be started after this study protocol has been accepted for publication. Examiners will either be radiologists or neurologists, all with clinical experience in CT evaluation of CA patients.Patient outcomes will be poor functional outcome six months after randomisation, defined as modified Rankin Scale (mRS) 4 -6.Qualitative analysis will be performed according to the checklist in Image evaluation starts by applying a standard “brain window”  which is then adapted to optimize visibility of grey-white-matter differentiation.Additional characteristics of severe HIE may include “Pseudo-subarachnoid haemorrhage sign” , “reversal sign” (lower radiodensity of grey compared to white matter structures) and “white cerebellum sign” .27The main question to be answered after evaluation of the entire CT is “Are there definite signs of severe HIE?” This question should be answered with “yes”, if complete or near-complete loss of grey-white distinction is noted both in the 1) basal ganglia and 2) in the frontoparietal lobes bilaterally with additional evidence of brain swelling/sulcal effacement. Severe HIE will also be diagnosed from complete sulcal effacement when residual grey-white distinction is present.2) will be placed manually in pre-defined anatomical regions of grey and white matter bilaterally at the basal ganglia level  as previously described.The reporting of results will follow the recommendations of the Standards for Reporting Diagnostic Studies (STARD).For qualitative and quantitative evaluations, we will present sensitivities and specificities for prediction of poor functional outcome (mRS 4–6 at six months) with 95% confidence intervals (CI) calculated using Wilsońs method. Results will be reported separately for each examiner to avoid errors by averaging measurements by different investigators. Both for the manual and automated GWR evaluations we will validate cutoff values 1.10 and 1.15 for prediction of poor outcome. Several prior studies, including our own, suggest certainly poor outcome at GWR threshold below 1.10, yet at a cost of low sensitivity. To increase sensitivity we also validate the threshold 1.15.15The overall prognostic performance for good versus poor functional outcome will also be tested by the area under the receiver-operating characteristics curve (AUROC) with 95% confidence intervals. P-values will be calculated based on a test of difference in AUROC using the method of DeLong.Based on previous results, we do not expect that targeted temperature management influences the prognostic accuracies of CT.Since results from CT evaluations by local radiologists were available upon clinical decision-making, we want to gather indirect evidence whether life-sustaining therapy withdrawn for neurological reasons (in this case based on CT findings) may have been leading to self-fulfilling prophecies. We will therefore investigate whether severe HIE was diagnosed from head CT in patients with low blood levels of neurofilament light, which indicate little or no brain injury.Fleisś kappa will be calculated as a measure of the reliability of agreement for qualitative analysis between investigators  and between study investigators.A subgroup analysis will examine the group-wise difference in manually determined GWR between the different CT manufacturers, scanners, and convolution kernels.Exploratory analyses will include prediction of outcome using quantification of regional brain water uptake, in patients with two or more CTs as well as other artificial intelligence-based methods.We present the study protocol of a prospective international multicentre trial which aims to validate prognostic accuracy of standardized radiological assessments of severe HIE on CT after CA. To our knowledge, no prospective studies have examined CTs acquired in CA patients at a pre-defined time-point et al. reported a specificity of only 86% for the finding of a “poor CT” when evaluated by radiologists blinded to clinical data.Qualitative evaluation of HIE by local radiologists demonstrated a very high specificity in the TTM-trial, but since results were available to treating physicians, the risk of a self-fulfilling prophecy influencing outcome could not be excluded.Guidelines suggest “using the presence of a marked reduction of the GWR on brain CT within 72 h after ROSC in combination with other predictors for prognosticating a poor neurologic outcome in patients who are comatose after cardiac arrest and who are treated with TTM” (very low level of evidence).We previously found that a GWR below 1.1 predicted poor outcome with 100% specificity whilst a GWR below 1.15 in combination with severe HIE on the qualitative evaluation increased sensitivity and maintained this high specificity.Based on previous results, we include technical prerequisites for evaluation. Only CTs with axial slices of 4–5 mm and a tube voltage of 120kvP will be used in this study since HU are highly dependent on these parameters. We will also investigate to which extent the type of CT scanner used or whether residual contrast agent from coronary angiography may influence the reliability of our SOP.Strengths include the prospective design and the blinded assessment by several examiners. As serum NFL samples were analysed after trial completion, we can compare CT evaluation with an independent marker of HIE severity which was not available during clinical decision-making.Our study has several limitations: the TTM2 trial included adult patients with an out-of-hospital CA with a presumed cardiac origin, or with an unknown cause with a stable return to spontaneous circulation. Our results should be validated in other patient cohorts since results may vary. The results from local radiological evaluations were included when making decisions on level-of-care and the risk of a self-fulfilling prophecy cannot be excluded. We will therefore examine whether WLST for neurological reasons was performed in patients diagnosed with severe HIE on CT despite low serum levels of NFL as a surrogate marker of brain injury.The results from this prospective trial will provide a unique opportunity to validate a standardized approach to quantitative and qualitative radiological evaluations of HIE on CT for prediction of functional outcome in comatose cardiac arrest patients. We believe that our study will deliver clinically important information on an area where level of evidence is sparse.The TTM2 trial is supported by independent research grants from nonprofit or governmental agencies  and by governmental funding of clinical research within the Swedish National Health Service. In addition, the CT substudy is supported by grants from the Bundy Academy, the Segerfalk Foundation and the Elsa Schmitz Foundation. MK is supported by the Laerdal Foundation and the Berlin Institute of Health Junior Digital Clinician Scientist Program. The authors are solely responsible for the design and conduct of this study, all study analyses, the drafting and editing of the manuscript, and its final contents.CL declares institutional fees for lectures from Bard and Zoll. ND reports past lecture and travel fees for Bard and Zoll companies outside the present work. No other conflicts of interest were reported.


Identifying biomarkers associated with functional impairment is important in monitoring glaucoma patients. This retrospective cross-sectional study investigated the vasculature–function relationship in open-angle glaucoma (OAG) eyes with choroidal microvasculature dropout (CMvD) versus in OAG eyes without. Optical coherence tomography (OCT) angiography-derived circumpapillary (cpVD) and macular vessel densities (mVD) were measured in 159 early-stage OAG eyes (mean deviation >  −6 dB) in accordance with the presence or not of a CMvD. OCT-derived circumpapillary retinal nerve fibre layer thickness (cpRNFLT) and macular ganglion cell-inner plexiform layer thicknesses (mGCIPLT) were also measured as reference standards. The vasculature (cpVD and mVD)–function [24-2 visual field mean sensitivity (VFMS) and central 10° VFMS (cVFMS)] and structure (cpRNFLT and mGCIPLT)–function (24-2 VFMS and cVFMS) relationships were compared using global and sectoral maps between OAG eyes with (CMvD+) and without CMvD (CMvD−). The CMvD+ eyes showed significantly steeper cpVD-24-2 VFMS and mVD-cVFMS correlations (P < 0.05). In contrast, there were no significant differences in the cpRNFLT-24-2 VFMS and mGCIPLT-cVFMS relationships between the two groups (P > 0.05). In conclusion, OAG eyes with a CMvD have significantly stronger vasculature–function relationships than eyes without. Vessel density parameters may be useful biomarkers of disease progression in early-stage OAG patients with a CMvD. Further to this, the strength of cpVD- or mVD-VFMS association is significantly greater than that between the circumpapillary retinal nerve fibre layer (cpRNFLT)- or macular ganglion cell-inner plexiform layer thickness (mGCIPLT)-VFMS association in OAG eyes with moderate to advanced glaucoma3.Vessel density (VD) assessments using optical coherence tomography angiography (OCT-A) may be an objective method for discriminating glaucomatous patients from healthy subjects, and for monitoring disease progression, since the circumpapillary (cpVD) and/or macular VD (mVD) loss has been observed in eyes with open-angle glaucoma (OAG)5. At present, it is unclear whether CMvD is a primary event that precedes glaucomatous damage, or a by-product/epiphenomenon of nerve tissue damage in association with glaucoma. Since the parapapillary choroid is closely related to ONH perfusion due to its proximity to, and a common blood supply via the short posterior ciliary arteries8, and has extensive interconnected networks of microvasculature throughout the structure, CMvD may indicate a sign of global or generalized perfusion insufficiency to the choroid/ONH in glaucomatous eyes9. Consequently, OAG eyes with CMvD have more widespread glaucomatous damage and a poorer prognosis compared to those without CMvD at a similar stage of disease severity11.Choroidal microvasculature dropout (CMvD), which is choroidal vascular insufficiency within the β- parapapillary atrophy (β-PPA), is frequently detected in eyes with glaucomatous optic nerve head (ONH) damage10. The aim of our present study therefore was to investigate the global and sectoral correlations between the VD measurements and the corresponding VFMS data assessed by standard automated perimetry (SAP), and also to compare the vasculature–function relationships between CMvD+ and CMvD− eyes, matched for disease severity at early-stage glaucoma. We also performed global and sectoral comparisons of the structure–function relationships between the two groups as reference standards.In our present study, we hypothesized that the presence of CMvD may have a differential impact on the vasculature–function relationship in glaucomatous eyes, considering that OAG eyes with CMvD (CMvD+ eyes) exhibit greater microvasculature insufficiency in the ONH and choroid compared to OAG eyes without CMvD (CMvD− eyes)This retrospective study was approved by the Institutional Review Board (IRB) of Asan Medical Center and adhered to the tenets of the Declaration of Helsinki . The requirement for informed consent from the study subjects was waived by the IRB of Asan Medical Center due to the retrospective nature of the analyses. We consecutively reviewed medical records of patients who visited our glaucoma clinic between April 2018 and November 2021. Each had undergone a comprehensive ophthalmic examination, including best-corrected visual acuity (BCVA), an intraocular pressure (IOP), slit-lamp biomicroscopy, a refractive error determination using an autorefractor , axial length , ultrasound pachymetry  for central corneal thickness (CCT), dilated colour fundus photography , optic disc stereoscopic photography, red-free retinal nerve fibre layer (RNFL) photography (Canon), Humphrey field analyser Swedish Interactive Threshold Algorithm (SITA)-Standard 24-2 VF testing (Carl Zeiss Meditec), spectra-domain OCT , and OCT-A .12 irrespective of the IOP level; and (3) a VF mean deviation (MD) better than −6 decibels (dB) for the purpose of evaluating early-stage OAG eyes12 to avoid the confounding effects of disease severity on vasculature– and structure–function relationship and the presence of CMvD 10. To account for the learning effects with initial VF testing, a second VF test was performed within 1 week to be used in our final analysis. Subjects with any ophthalmic or neurological disease which may affect the ONH, macular structure, or VF testing were excluded. If both eyes were eligible in any subject, one eye was randomly selected for the study.The following inclusion criteria were applied to our study subjects: (1) a diagnosis of OAG with a normal anterior chamber and open angles on gonioscopy; (2) age 13: temporal , superotemporal , superonasal , nasal , inferonasal , and inferotemporal  sectors. The cpRNFLT of each sector was estimated by integrating the clock hour RNFL thickness from the Cirrus OCT14. The global mGCIPLT was measured at the macular region, centred on the fovea within an annulus with inner vertical and horizontal diameters of 1 and 1.2 mm, and outer vertical and horizontal diameters of 4 and 4.8 mm, respectively. The sectoral mGCIPLT was also measured at six sectors .SD-OCT imaging was performed using Cirrus HD SD-OCT software, version 10.0. The global (360°) cpRNFLT was measured in a 3.46 diameter circle centred on the ONH. The sectoral cpRNFLT was also measured at six sectors according to the Garway-Heath map2 scans, centred on the optic disc within the radial peripapillary capillary slab from the internal limiting membrane to the nerve fibre layer after the automated removal of large retinal vessels. The OCT-A software automatically provides the cpVD of the peripapillary regions, divided into eight 45° sectors . To obtain average VD of six sectors in accordance with the Garway-Heath map13, temporal (T) and nasal (N) sectors were estimated by averaging of the TU and TL for the T sector and NU and NL for the N sector2.OCT-A imaging of the circumpapillary and macular regions was performed using the AngioVue OCT-A system version 8.0 (Optovue Inc) to assure consistency of the data among all of the enrolled study subjects. cpVD measurements were made using images of 4.5 2 region centred on the fovea within the slabs from the internal limiting membrane to the posterior boundary of the inner plexiform layer . Eligible images were then imported into a computer program written using MATLAB software  and mVD values were calculated in the same sectors as the mGCIPLT measurements, obtained by Cirrus HD SD-OCT3. All SD-OCT and OCT-A images were reviewed by two glaucoma specialists (A.L. and J.Y.L.) for an evaluation of their quality. Images were excluded if they had (1) poor image quality with signal strength < 7; (2) motion artifacts; (3) localized weak signal intensity caused by vitreous floaters or poor clarity ; (4) images with fixation error; or (5) segmentation failure11.mVD measurements were calculated from a 6 15. CMvD was defined as a localized complete loss of the choriocapillaries and choroidal microvasculature within the 17. The margins of the optic disc, β-PPA and CMvD were evaluated by two glaucoma specialist (A.L. and J.Y.L.), while being blind to the clinical, VF, and SD-OCT information of the study patients. The presence of CMvD was confirmed regardless of its multiplicity ;1/L scale]19. The global 24-2 VFMS was calculated as the mean of VF sensitivities in 52 points of the SAP, except 2 points just above and below the blind spot and grouped into six VF sectors: SN, N, IN, IT, T, and ST  in the 12 central 10° VF points of the SAP, which corresponds topographically to the macular region within 4.8 mm of the center of the fovea20, was calculated and grouped into four central VF sectors  topographically according to previous publications  and SN cVFMS was defined as the average sensitivity of four SN VF points (paired with IT mGCIPL or IT mVD). IN cVFMS was defined as the average sensitivity of three IN VF points (paired with ST mGCIPLT or ST mVD) and IT cVFMS was defined as the average sensitivity of three IT VF points (paired with SN + SC mGCIPLT and SN + SC mVD). These six 24-2 and four central 10° VF sectors were correlated with corresponding regional cpRNFLT and cpVD , the bootstrapping method with a repetition of 1000 times with replacement was performed to examine for statistically significant differences between two correlation coefficients21. Each r value was derived from 1000 bootstrapped data and the difference between r values of the two groups was then calculated. Assuming that the bootstrapped values of the difference followed a normal distribution, a t-test was performed to test the null hypothesis that their r means between two groups were equal21. Clinical variables associated with 24-2 and central 10° VFMS in each OAG group were analysed using univariable and multivariable linear regression analysis. Variables with P values < 0.1 in the univariable analysis were included in the multivariable linear regression analyses with a backward elimination process.All statistical analyses were performed using SPSS version 21.0  and SAS version 9.4 . P values less than 0.05 were considered statistically significant and results were presented as either mean values with a standard deviation or as a frequency and percentage. The inter-observer agreement for the presence and location of the CMvD was assessed using the kappa (k) statistic. The normality of distribution was assessed using the Kolmogorov–Smirnov test. Normally distributed data were compared using an independent Student t test; otherwise, Mann–Whitney U tests were used. Categorical variables were compared using chi-square tests between the groups. The correlations of vasculature– and structure–function relationships were evaluated using linear regression analysisOf the 181 OAG eyes of 181 patients who were initially enrolled in our present study cohort, 22 eyes with unreliable VF testing and poor-quality SD-OCT/OCT-A scans were excluded. Hence, our final cohort comprised 159 eyes, consisting of 67 CMvD+ eyes and 92 CMvD− eyes. The inter-observer agreements for the determination of the presence and location of CMvD were excellent. . The demographics and baseline characteristics are summarized in Table Comparisons of vasculature– and structure–function relationships between the groups are presented in Table Table entclass1pt{minima22, which indicates the potential role of VD as an additional surrogate marker to monitor functional loss in glaucoma. As the presence of CMvD is linked to microvasculature insufficiency of the ONH/choroid and a poorer prognostic outcome15, it is plausible to hypothesize that vasculature–function relationships may differ between OAG eyes with and without CMvD. In this current study, OAG eyes with CMvD revealed stronger global and sectoral vasculature–function relationships in the 24-2 and central 10° VF maps, while there were no differences in the structure–function relationships between the two groups. These findings suggest that VD parameters, as measured by OCT-A, may be useful biomarkers for detecting and monitoring glaucomatous damage in OAG eyes, particularly in those showing signs of choroidal microvasculature insufficiency such as CMvD. To our knowledge, this present study is the first to demonstrate different impacts on the vasculature–function relationship in OAG eyes according to the presence of CMvD.VD reduction as determined by OCT-A is known to be highly associated with the loss of VFMS in glaucoma patients24. Another possibility is that as the parapapillary choroid is closely linked to ONH perfusion, insufficient blood flow within this structure in the eyes with CMvD can result in diminished blood flow to ONH, thereby leading to deep ONH structural damage, such as LC morphologic changes, which can subsequently induce axonal and vascular loss in the superficial retina25. Of note in this regard, sectoral loss of the cpVD and cpRNFLT was found to be spatially correlated with CMvD location and was mostly observed at the T and/or IT sectors in the CMvD+ eyes in the present study series (83.5%). These findings are in line with those of a previous study, which also reported an association between CMvD and reduced cpVD and RNFLT with topographic correlation5, and with prior reports that CMvDs are most often found at the IT region in the optic disc, which is the most vulnerable area to disruption of the microvasculature and ONH structural damage26.Our current analyses indicated that the global mean cpVD was significantly lower in the CMvD+ eyes but that there was no significant difference in the global cpRNFLT values between the two groups Table . In sect27, its presence can be directly related to the loss of macular structural parameters  and central VF defects28. In addition, as the density of retinal ganglion cells (RGCs) is highest in the central macula, the RGCs in the macular region require a high oxygen supply, rendering them particularly vulnerable to microvascular hypo-or non-perfusion conditions such as CMvD.In our present study series, the global and sectoral mean mVD and mGCIPLT values were significantly lower in the CMvD+ eyes, except for the ST and SC sectors of mGCIPLT Tables  and 2. TApart from the difference in the location of preferred VF damage , the CMvD+ eyes showed more widespread macular structure and VD loss compared to the CMvD− eyes in the current study. Despite that CMvD was mostly located only in the inferior hemiretina (77.6%), CMvD+ eyes showed global loss of mGCIPLT and mVD, with mean mGCIPLT and mVD values being significantly lower at the SN sector and ST, SC, and SN sectors , respectively, compared to the CMvD− eyes Table . Our fin9, thus leading to more pronounced loss of cpVD/mVD and VFMS globally and sectorally22. This in turn result in stronger vasculature–function relationships in the CMvD+ eyes based on both 24-2 and central 10° VF maps. Another explanation is that greater severity of disease in the CMvD+ eyes may be related to steeper vasculature–function relationship in these eyes, despite both CMvD+ and CMvD− eyes having similar VF MD. Considering that disruption of superficial microvasculature may be secondary to the axonal loss4, VD loss may not be profound enough to reflect functional loss in early stage of glaucoma. In contrast, more severe VD loss observed in the CMvD+ eyes may have contributed to steeper vasculature–function relationship compared to CMvD− eyes.Our comparisons of the vasculature–function relationships between the two groups demonstrated that global and sectoral vasculature–function relationship were significantly stronger in the CMvD+ eyes, according to either the 24-2 or central 10° VF maps Table . One exp29 reported that changes in RNFL thickness following crush injury lag behind the decline of RGC function, based on their histopathologic analyses. Hence, although the mechanisms of ONH injury may differ between the two studies, ONH/retinal damage induced by CMvD may first lead to microvasculature loss and functional damage, followed by a slower degeneration of the RNFL, which may explain our present findings that VD loss is better associated with the RGC dysfunction and subsequent VFMS loss than RNFL thinning. Further studies with a longitudinal design are needed to validate our hypothesis in the future.It is noteworthy that there were no significant differences in the global and sectoral structure–function relationships between the eyes with and without CMvD, according to either the 24-2 or central 10° VF maps Table . Althoug31 recently reported that glaucoma patients with vascular risk factors such as hypertension or myocardial infarction predominantly showed mGCIPLT loss, rather than a cpRNFLT reduction. These authors hypothesized that vascular dysfunction may be important in glaucomatous damage involving the macular structure. Our current findings of a significant association between the cVFMS and mGCIPLT in the CMvD+ eyes may be explained by microvasculature insufficiency present in those eyes.The linear regression analyses showed that a reduced global cpVD and mVD were independently associated with the corresponding global 24-2 and central 10° VFMS in the CMvD+ eyes, but this association was found with a reduced global cpRNFLT and mGCIPLT in the CMvD− eyes. These findings may further support our speculation that VD parameters have stronger relationship with functional loss in eyes with CMvD. Interestingly, the global cpRNFLT was not significantly associated with global 24-2 VFMS, whereas mGCIPLT was independently associated with cVFMS, in the CMvD+ eyes  or projection flow from the superficial retinal layer, can make it difficult to define the presence of CMvD and/or ONH/CMvD boundary. Nonetheless, we attempted to exclude the influence of large overlying retinal vessels and/or ODH on our scanning laser ophthalmoscopy images within the In conclusion, early-stage OAG eyes with CMvD show a significantly greater degree of global and regional macular VD loss, and stronger vasculature–function relationships, compared to eyes without CMvD. Our findings suggest that VD parameters derived from an OCT-A may be useful biomarkers of disease progression in early-stage OAG patients with choroidal microvasculature dropout.


Memory linking is the process that integrates related memories into an associative mnemonic structure of interconnected representations. Typically, memories sharing certain attributes are dynamically interlocked, such that one memory increases the likelihood of retrieval of another. Three main factors facilitate the memory linking: time, space, and perceptual/conceptual similarities.3 Mice were first exposed to a distinct context (context A), then were placed in another context (context B) after variable timeframes. Two days later, mice were again exposed to context B where they received electric footshocks. Two days after footshock, mice were finally returned to context A for a test of freezing behavior—a behavioral fear response and an indicator of memory linking between contexts A and B, since the mice received footshock only in context B. The mice with a 5-h temporal window between exposure to contexts A and B showed a significant fear response in context A, indicating effective memory linking  activation as well as neuronal excitability.4 In the current study, Shen et al. observed that mRNA levels of CCR5 and its ligand CCL5 increased until 12 h after contextual fear conditioning and fell back to baseline levels in hippocampal CA1 region  within 24 h, suggesting that CCR5 may adjust the temporal window of memory linking.To determine mechanisms that close the temporal window for memory linking to segregate temporally distinct events, Shen et al. considered the potential role of CCR5. In peripheral tissues, CCR5 is well known as an immune receptor protein in the inflammatory microenvironment and as a co-receptor for HIV infection.Shen et al. then bidirectionally manipulated the activity of CCR5 signaling to examine the effects on the temporal window for contextual memory linking. Both optical activation of a photosensitive form of CCR5 and injection of CCL5 protein compromised memory linking under specific temporal windows that would otherwise allow successful memory linking Fig. . ConversBy investigating the potential mechanisms of CCR5 in memory linking, Shen et al. found that optically activating CCR5 significantly decreased learning-induced expression of c-Fos, a characteristic marker for neuronal ensembles of memory, in the hippocampal CA1 region. CCR5 also steadily decreased the overlap between neuronal ensembles activated in both the original and linked memories of two contexts, which was consistent with the real-time calcium activity of hippocampal CA1 neurons in vivo. Collectively, CCR5 signaling decreases neuronal excitability, which prevents subsequent memories from being distributed to the initial memory ensembles, thereby reducing the overlap between the two memory ensembles and consequently diminishing memory linking. Notably, CCR5 appears to serve as a suppressor for neuronal excitability to trigger one memory retrieval via another memory and therefore closes the temporal window for memory linking.Ccr5 and Ccl5 mRNA expression levels were higher in middle-aged mice compared to their younger counterparts in the home cage. Furthermore, the middle-aged mice had a shorter temporal window than young mice, suggesting a progressing deficit in memory linking during aging. Both genetic deletion of CCR5 and pharmacological blockade of CCR5 activity with maraviroc (an FDA-approved CCR5 antagonist for HIV treatment)1 ameliorated memory linking deficits in middle-aged mice, indicating that targeting CCR5 significantly improves contextual memory linking during aging.In addition, Shen et al. observed that the hippocampal 5 Third, given that CCR5 is present in the microenvironment of memory ensembles, just as it is present in the microenvironment of peripheral inflammation, the potential roles of the broadly distributed extracellular matrix are worth considering. Targeting the neuroglia and perivascular cells within the microenvironment and their connectivity signaling cascades with neuronal ensembles may inform strategies in clinical therapies that could alter memory ensembles in mnemonic structures, thus affecting the storage, linking, and retrieval of two or more memories.In summary, this exciting work by Shen et al. revealed an unprecedented role of CCR5 in closing the temporal window for memory linking Fig. . This di


NSD1 mutations causing Sotos syndrome have been previously associated with a specific genome-wide DNA methylation (DNAm) pattern. Sotos syndrome is characterized by phenotypic overlap with other overgrowth syndromes, and a definite diagnosis might not be easily reached due to the high prevalence of variants of unknown significance (VoUS) that are identified in patients with a suggestive phenotype. Objective: we performed microarray DNAm profiling in a set of 11 individuals with a clinical suspicion of Sotos syndrome and carrying an NSD1 VoUS or previously unreported variants to solve uncertainty in defining pathogenicity of the observed variants. The impact of the training cohort size on sensitivity and prediction confidence of the classifier was assessed. Results: The Sotos syndrome-specific DNAm signature was validated in six individuals with a clinical diagnosis of Sotos syndrome and carrying bona fide pathogenic NSD1 variants. Applying this approach to the remaining 11 individuals with NSD1 variants, we succeeded in confirming pathogenicity in eight subjects and excluding the diagnosis of Sotos syndrome in three. The sensitivity and prediction confidence of the classifier based on the different sizes of the training sets did not show substantial differences, though the overall performance was improved by using a data balancing strategy. Conclusions: The present approach solved uncertainty in cases with NDS1 VoUS, further demonstrating the clinical utility of DNAm profiling.Background: Inactivating  NSD1, OMIM #606681), which encodes a histone methyltransferase that preferentially methylates lysine 36 of histone H3 and lysine 20 of histone H4, either negatively or positively influencing transcription, depending on the cellular context [NSD1 haploinsufficiency is supported by the finding of microdeletions of the 5q35 chromosomal region in a subset of patients [NSD1 pathogenic variants are point mutations causing an early termination of transcription (nonsense or frameshift variants), though changes causing amino acid substitutions within specific domains of the protein are also observed [Overgrowth syndromes are characterized by a global or regional excess of growth compared to equivalent body parts or age-related peer groups . Sotos spatients . The majNSD1 is characterized by low tolerance to LoF variants (pLI = 1). On the other hand, missense variants seem to be constrained to a lower extent (Z = 3.41), with pathogenic changes showing a clear positional clustering [https://gnomad.broadinstitute.org; accessed on 5 October 2022), 396 are classified as variant of unknown significance (VoUS) or have a conflicting interpretation in ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/ (accessed on 5 October 2022)). The pathogenic missense variants largely map within functional domains implicated in chromatin regulation at the 3′ of the gene , and predominantly occur at the consensus cysteine and histidine residues that define these domains, with few exceptions [NSD1 variants that are routinely revealed by massive parallel sequencing in the diagnostic setting is generally solved by segregation analysis, or might require dedicated functional validation, which is further more relevant in patients with atypical or incomplete presentation of the disorder. ustering ,9. Amongnt Z = 3.1, with pA large proportion of overgrowth syndromes is caused by pathogenic variants in genes that encode proteins involved in epigenetic regulation, indicating that the functional disruption of the epigenetic machinery is a recurrent mechanism causing these disorders ,10,11,12NSD1 VoUS [NSD1 VoUS and private variants identified in the routine diagnostic setting. These cases included both subjects with features suggestive of Sotos syndrome who were assessed by targeted sequencing, and individuals with a non-suggestive phenotype who had been investigated by using an agnostic approach (exome sequencing). Genome-wide DNAm profiling is becoming a powerful functional tool in clinical setting  as a secSD1 VoUS . Here, wAll subjects were originally enrolled in the context of a routine diagnostic activity. Clinical data, pictures, DNA specimens and other biological material were collected, used, and stored after signed informed consents from the participating subjects/families were collected. Permission to publish clinical pictures was obtained for all subjects reported in NSD1, either previously reported or considered bona fide as pathogenic. Their clinical and molecular characterization is summarized in NSD1 made up the testing group. Their clinical and molecular characterization is summarized in Six individuals (S1 to S6) presented with both a clinical diagnosis of Sotos syndrome and a molecularly diagnosed pathogenic variant in NSD1 variants with their functional annotation are reported in NSD1 variants annotated in ClinVar and gnomAD plotted taking into account the functional domain organization of the protein are shown in In subjects S7 to S17, all variants were identified through massive parallel sequencing using different approaches, including target panel resequencing  or trio-based exome sequencing, after the exclusion of any clinically relevant structural variant by high-resolution CMA-array. The identified NSD1 variants were analyzed, alongside 295 controls. Peripheral blood DNA was extracted using standard techniques. Bisulphite conversion was performed, and samples were analyzed using Infinium Methylation EPIC BeadChip , according to the manufacturer’s protocol. The 17 samples with ChAMP package (V.2.26.0) [p-value > 0.01 were excluded, resulting in 721,325 high-quality probes used in the subsequent analyses. Normalized methylation levels  for each sample were compared using the established DNAm signature for Sotos syndrome  [e1071 R package V.1.7 and nu-classification option. To determine the best hyperparameter and measure the accuracy of the model, the whole dataset was split into a training set (75% of samples) and a test set (25% of samples), and a 5-fold cross-validation was performed during the training process. This procedure was repeated four times to verify that each sample was used at least once for testing. Finally, a SMOTE  oversampling technique was carried out to overcome class imbalance between affected and control individuals used in the training process [NSD1 pathogenic variants.IDAT files containing methylated and unmethylated signal intensities were imported into R V.4.2.1 for subsequent analysis using the .2.26.0) . Firstly.2.26.0)  and eval of 112) , and clu process . Scores NSD1 mutations  was performed , the remaining sample with a likely pathogenic variant (S3) was validated, and the 11 tested samples were unambiguously classified  and macrocephaly (6/7), suggestive facies (8/8), and DD/ID (8/8). In particular, the cardinal facial features characterized by broad and prominent forehead (8/8), dolichocephaly (7/8), downslanted palpebral fissures (8/8), long and narrow face (8/8), and long/pointed chin (8/8) were invariably observed. On the other hand, the three individuals carrying variants reclassified as non-pathogenic for Sotos syndrome showed a phenotype that only partially fit a clinical diagnosis of the disorder, having less marked suggestive facial features, and not showing hypotonia; among these, S15 and S16 also did not show ID , which affects the PWWP1 domain, rarely occurs in the general population (rs1172667661), and has an uncertain interpretation by ClinVar (VCV000451652). Of note, conflicting annotation was also reported by MutScore, which assigned a high score for pathogenicity, though it does not map within a region with a significant PLP clustering (ustering .NSD1 variants), we performed different rounds of training, testing on the same time if prediction confidence could be further improved using a more balanced dataset by means of oversampling (see Methods). The sensitivity and prediction confidence of different classifiers were compared, showing no substantial differences among probability scores obtained from the models using the full cohort or a minimal cohort of three samples with pathogenic variants as training sets, with class imbalance correction  of the classifier, the ML-based classifier training was performed with subsets of the original training cohort. Using different sizes  has revealed an emerging problem on VoUS interpretation in either highly suggestive or nonsuggestive associated phenotypes. DNAm profiling in a subset of conditions for which a signature has been established is becoming a reflex assay for the assessment of functional significance and reclassification of VoUS. This approach has the advantage of allowing a direct assessment on the primary sample, not requiring a new sampling or a different tissue analysis. If widely applied, this strategy could overcome the “traditional” validation approaches through functional assessment in various model systems, or clinical confirmation in multiple unrelated patients/families, which are usually unavailable for routine screening due to the rarity of these conditions. In the present series, five individuals  carried previously unreported variants, while three subjects (S9-S11) were heterozygous for variants reported as VoUS or having conflicting interpretation in ClinVar. All of them were successfully reclassified as pathogenic by applying this strategy. NSD1 variant (S16) was inherited from an apparently unaffected father. S16 showed a co-occurring pathogenic homozygous variant in CFTR (p.Phe508del). She clinically presented with cystic fibrosis and relative macrocrania, which was also observed in her transmitting father. DNAm profiling allowed us to exclude a dual molecular diagnosis expressed with a blended phenotype. The application of the classifier also allowed to directly verify pathogenicity in a variant for which segregation was not available (S11) and exclude pathogenicity in a subject with a highly suggestive presentation (S17). In the latter, thanks to the negative finding, this individual is likely not to have either a deep intronic or an NSD1 intragenic deletion, avoiding further targeted analyses directed to explore other genomic events involving NSD1. Subsequent segregation analysis revealed variant transmission from the mother, who presented with macrocrania, tall stature, and mild learning difficulties, during the scholar period. Finally, S15 presented with many typical facial features of Sotos syndrome  but lacked two main features . Moreover, she did not show hypotonia and ID, thus lacking a main sign to clinically diagnose Sotos syndrome. Her variant was also reported in both ClinVar with uncertain significance and in general population as a rare allele. This variant showed a MutScore value greater than the suggested pathogenicity threshold (0.73) [NSD1 function that might underlie or contribute to the isolated macrocephaly observed in the three patients and their transmitting parents. Our findings, however, definitively exclude a pathogenic role of these variants in Sotos syndrome pathogenesis.Our classifier also managed to exclude pathogenicity of the remaining three assessed cases in the context of Sotos syndrome. One truncating d (0.73) . We cannbona fide pathogenic variants can successfully be used to effectively train a ML-based classifier based on the existing Sotos syndrome-specific DNAm signature, allowing VoUS classification. Of note, the present analyses showed that the use of even a small number (down to three) of samples with NSD1. The application of DNAm analysis in diagnostics, VoUS classification, exploration of disease pathophysiology, as well as its emerging use in biomarker identification applied to precision medicine are becoming progressively more promising and are expected to be routinely applied in the next future.In conclusion, the available episignature for Sotos syndrome has demonstrated to be a robust and reliable tool to quickly rule out or confirm a clinical suspicion of this disorder. Episignatures have the great advantage to overcome many technical limitations observed in current sequencing techniques . If properly used, this assay might be considered as a first-tier analysis in subjects with specific clinical suspicion in whom a condition caused by a gene involved in the epigenetic machinery is suspected. As exemplified in our case (S17), even in the presence of a highly suggestive Sotos syndrome phenotype, this assay could easily rule out the clinical diagnosis without proceeding with more complex targeted analyses directed to explore other genomic events involving


Liquid phase delamination of layered materials into single-orfew-layer nanosheets leads to stable nanoscale dispersions of 2D materials.The delamination of layered double hydroxide (LDH) to double hydroxidenanosheets was studied in two ionic liquids (ILs): ethylammonium nitrate(EAN) and 1-butyl-3-methylimidazolium thiocyanate (BMIMSCN).The as-prepared lamellar structure of LDH disappeared upon dispersingit in ILs due to delamination into 2D nanosheets confirmed by X-rayscattering and diffraction techniques and further evaluated by heightprofile assessment of the nanoparticles by atomic force microscopy.The results showed that both the thickness and lateral size of thedispersed particles decreased in the IL-based samples, indicatingthat cleavage of the LDH materials can be observed in addition todelamination. The findings prove the concept of delamination of layeredmaterials by ILs under ambient conditions—an excellent wayto prepare 2D double hydroxide nanosheet dispersions in one step usingnonvolatile green solvents. Most importantly, fundamentalstudies have shown that ILs can reduce the strength of attractiveinteractions between charged surfaces due to their interfacial assembly36 or partial dissociation,37 leading tothe formation of stable particle dispersions in ILs.39 Ion pair formation in ILs is an important phenomenon regarding thestabilization. In dilute aqueous solutions, ILs tend to dissociate,while the ions associate and form ion pairs as the concentration increases.41 In this way, the shielding ofattractive interactions between layers due to the repulsive solvationforces generated by the self-assembly of IL constituents on the surfaceshould also promote the collapse of stacked structures and subsequentdelamination into unilamellar nanosheets. Such a phenomenon was firstdemonstrated in IL–graphene systems.42 Among ILs, the ones with surface energies matched to the graphitesubstrate proved to be more effective, as they could exfoliate graphiteinto 2D graphene under ambient conditions, without requiring any formof external energy input.42 Moreover, delaminatedgraphene nanosheets were stabilized in IL dispersions solely by choosingthe appropriate composition of ILs.44Considering these aspects, ionic liquids (ILs)are one of the mostpromising solvent candidates for this task.48 no comprehensive studies havebeen conducted to evaluate the potential delamination of single-phaselayered LDHs into 2D double hydroxide materials with a thickness ofone or a few nanosheets. Therefore, this Letter discusses the delaminationof mesoporous LDHs under ambient conditions with minimal externalenergy input in ILs (ethylammonium nitrate (EAN) and 1-butyl-3-methylimidazoliumthiocyanate (BMIMSCN)) based on systematic characterization of theas-prepared and delaminated LDH samples by X-ray scattering and diffractionas well as by microscopy. These ILs have been widely studied, andtheir advantageous interfacial properties as well as moderate viscositieswere expected to be beneficial in delamination processes.Forthese reasons, ILs are very promising as delamination media,as they allow both one-step delamination in liquid phase and stabilizationof the resulting 2D nanosheets in dispersions. Although compositesof ILs and LDHs have been reported in the past,49 i.e., water, EAN, and BMIMSCN. The XRD result for the LDH powdersample is also shown as a black curve in 50 The average crystallite size was calculated from the half-widthof the (003) diffraction pattern using the Scherrer equation50 and estimated to be 15.4 nm.Tocheck the formation of the layers, i.e., to prove the successfulsynthesis of the mesoporous LDH powder, X-ray diffraction (XRD), small-angleX-ray scattering (SAXS), and small- and wide-angle X-ray scattering(SWAXS) measurements were performed. The results are shown in 51 it is estimated that theLDH particles in thissolid sample contain about 19 stacked layers. Comparing the powderdiffractogram (black curve) with the sharp peaks —characteristic of the layered LDH powder structure—withthe diffractogram of the aqueous LDH dispersion (green curve) in Considering the LDH basal interlayer spacing ofabout 0.8 nm,Supporting Information. The black curve in 52 To clarify the scattering contribution of the pure solvent usedto prepare the LDH dispersions, the raw SWAXS data for the liquidsamples are shown in pairs in To obtain direct evidencefor the presence of delaminated LDH nanosheetsin these IL dispersions, SWAXS and SAXS techniques were used. Theresulting experimental SWAXS and SAXS data are shown in –1), which proves the presenceof stable nanoparticles in these two dispersions. Moreover, no excessivescattering is observed in the SWAXS data of the LDH liquid dispersionin Besides, the course of the pairwise scatteringcurves for the twoIL samples in 54 (IFT) approach was used to analyzethe SAXS data shown in Figures S2a and S2b. The resulting pair distance distribution functions p(r) and the thickness pair distance distributionfunctions pt(r) are shownfor the delaminated LDH IL dispersions in To support these results, an inverseFourier transformp(r) function curves also indicates that these particlesare not spherical,which is not surprising since they are assumed to be 2D nanosheets.For plate-like (lamellar) particles that are large in two dimensions,it is possible to perform the special IFT analysis mode that usesthe cutoff at low values of q to obtain the pt(r) function. This functionis related to the thickness of the scattering nanosheets. The curvesof the pt(r) functionshown in The asymmetric course of these To further investigatethe population of these nanosheets and possiblyobtain information about their polydispersity, the atomic force microscopy(AFM) technique was used, which provided the visual results shownin Figure S3a and S4a. Namely, large platelets with lateral dimensions ofup to 400 nm can be seen. Analysis of their height profile shows thicknessesof about 15–20 nm, which is in good agreement with the thicknessof about 15.4 nm determined from the XRD data . The height/distanceprofiles in Figures S4b and S4c, also demonstrate that theaverage thickness of the particles decreased from 12 ± 6 nm inwater to 2 ± 1 and 1.7 ± 0.8 nm for EAN and BMIMSCN, respectively,while the average lateral size of crystalline LDH platelets decreasesfrom 187 ± 50 nm in water to the lateral size of nanosheets 32± 8 nm in EAN and 39 ± 8 nm in BMIMSCN. The nanosheets withlateral dimensions up to 60 nm in The morphology and thickness of thedispersed LDH nanosheets asrevealed by the AFM results are shown in 42 It was assumed that IL constituents assemble on the surface of LDHin an ordered form consisting of cation and anion layers. Such anassembly of ILs on surfaces was confirmed earlier by AFM38 and high-energy X-ray reflectivity.36 Besides, direct force measurements revealedthat the formation of IL interfacial layers leads to the developmentof repulsive oscillatory forces,55 whichovercome attractive van der Waals and electrostatic interactions acting,in our case, between the LDH layers. Therefore, delamination occursonce oscillatory forces arise due to IL assembly on the LDH surface.It is also worth mentioning that partial delamination and cleavageof aggregates were previously reported for LDH dispersed in an organicsolvent.56 The dispersions obtained inILs were stable for at least 6 months, indicating a good stabilizingeffect of ILs for LDH nanosheets.On the basis of the results presented, one canassume either thatthe LDH crystallites do not change significantly when the LDH powderis dispersed in water or that the large crystalline lamellar aggregatesform in aqueous LDH dispersion. However, in ILs, simultaneous disaggregationand delamination of LDH (accelerated by mild ultrasonication) occurred1 due to In conclusion, using XRD,SWAXS, SAXS, and AFM methods, it hasbeen shown that mesoporous LDHs can be successfully delaminated ina single step into ILs such as EAN and BMIMSCN. The disappearanceof the characteristic diffraction peaks observed for crystalline LDHpowder and its aqueous dispersion when LDH crystallites were dispersedin ILs confirmed the delamination of crystalline LDHs into doublyhydroxide nanosheets. This was also confirmed by SWAXS and SAXS results.The degree of delamination was further studied by AFM determiningthe distributions of nanosheet thickness and lateral dimensions. Thelatter two were compared with similar distributions obtained for aqueousLDH samples proving much smaller double hydroxide nanosheets in ILdispersions and indirectly the process of simultaneous delaminationand disaggregation of LDHs in ILs. The obtained 2D double hydroxidenanosheet–IL samples were stable for months without any stabilizingagents added to the samples. These results open a new way to obtainunilamellar or multilamellar nanosheets with only a few layers inliquid dispersions in one step using a green (nonvolatile) solvent.Supporting Information on pages S2–S5.67 The mesoporous nitrate-containing LDH particles studied in the presentwork were prepared using a novel colloidal approach.51 The synthesis protocol can be found in the Supporting Information. Delamination procedureswere performed by dispersing the mesoporous LDH in EAN or BMIMSCNfollowed by ultrasonication for 1 h and stirring for 48 h.Experimental details includingmaterials used for the experiments,a description of investigation methods such as AFM, XRD, SAXS, andSWAXS, and the data analysis are described in the


While personal growth has been found to be associated with multiple aspects of health in adulthood, its associations with cognitive functioning have not been fully understood. The present study aimed to assess both directions of such longitudinal associations. Using data from the second wave (T1) and third wave (T2) of the Midlife in the United States (MIDUS) study , a longitudinal measurement model containing latent variables of episodic memory and executive function was first constructed. Built on the measurement model, a structural equation model was analyzed to assess cross-lagged relationships between personal growth and the two areas of cognitive functioning, in which T1 personal growth predicted residualized changes in episodic memory and executive function, and T1 episodic memory and executive function predicted change in personal growth, controlling for covariates. The results indicated that T1 personal growth significantly predicted smaller decreases in episodic memory, whereas it did not predict change in executive function. T1 episodic memory, but not T1 executive function, significantly predicted smaller decreases in personal growth. The present findings were unique, particularly implying potential longitudinal reciprocity between personal growth and episodic memory. These findings and implications can inform future research aimed at exploring approaches to promoting personal growth and cognitive functioning among aging adults.


Whole genome sequencing of rabies lyssaviruses (RABVs) has enabled the generation of highly detailed phylogenies that reveal viral transmission patterns of disease in reservoir species. Such information is highly important for informing best practices with respect to wildlife rabies control. However, specimens available only as formalin fixed paraffin embedded (FFPE) samples have been recalcitrant to such analyses. Due to the damage inflicted by tissue processing, only relatively short amplicons can be generated by standard RT-PCR methods, making the generation of full-length genome sequences very tedious. While highly parallel shotgun sequencing of total RNA can potentially overcome these challenges, the low percentage of reads representative of the virus may be limiting. Ampliseq technology enables massively multiplex amplification of nucleic acids to produce large numbers of short PCR products. Such a strategy has been applied to the sequencing of entire viral genomes but its use for rabies virus analysis has not been reported previously. This study describes the generation of an Ampliseq for Illumina primer panel, which was designed based on the global sequence diversity of rabies viruses, and which enables efficient viral genome amplification and sequencing of rabies-positive FFPE samples. The subsequent use of such data for detailed phylogenetic analysis of the virus is demonstrated. Rabies diagnosis is normally performed by detection of the rabies virus antigen in fresh brain tissue using immunofluorescence. This technique, referred to as the direct fluorescent antibody (DFA) test, is in wide application due to the availability of specific antibodies and published protocols . AdditioHowever, tissue is occasionally formalin fixed prior to the suspicion of rabies virus infection and when a diagnosis of such a specimen is required alternative methods are necessary. After paraffin embedding and sectioning of such samples, viral antigen can be detected by immunohistochemical techniques ,11 althoThe concept of Sanger sequencing of multiple PCR products together in a single reaction was first reported in 2002 . FurtherThis report describes a protocol to generate extensive RABV sequence information using a MiSeq instrument from archived FFPE tissues infected with a variety of RABV variants. The methodology involves processing of FFPE tissue to extract RNA suitable for robust viral RNA detection by RT-qPCR, and the application of a universal RABV Ampliseq for Illumina panel to generate amplicons covering a significant portion of the genome for parallel sequencing. Use of these sequence data to infer viral type and perform detailed epidemiological analysis is demonstrated. This methodology can unlock valuable information contained in archival material relevant to the emergence and evolution of this important pathogen.From a collection of rabies-positive brain tissues, compiled at the Centre of Expertise for Rabies over many years, a cohort of 53 unfixed samples representing the seven RABV lineages that circulate world-wide was selected for proof of principle evaluation of the Ampliseq for Illumina panel. All these samples had been confirmed as rabies positive by DFA test and characterized by genetic methods of viral typing, details of which are summarized in A total of 23 rabies-positive submissions to the Centre of Expertise for Rabies which had been characterized antigenically and genetically using established inhouse methods ,25 were Total RNA was recovered from unfixed rabies-infected tissue using TRIzol  as per the manufacturer’s instructions. The final pellet was dissolved in sterile water, RNA concentration was determined using a NanoVue instrument , and samples were stored at −80 °C.Each FFPE sample tube received the following reagents, available from QIAGEN , in order: 200 µL ATL buffer, 20 µL proteinase K, and 160 µL deparaffinization solution. Immersion of the sample in the liquid was ensured using a disposable plunger as needed. Tubes were briefly vortexed and then incubated at 60 °C for 45 min followed by 80 °C for 30 min with agitation at 300 rpm in an Eppendorf Thermomixer C unit (Thermo Fisher Scientific). Samples were allowed to cool and 150 µL of the bottom phase was retrieved for RNA extraction, performed using a MagMax-96 instrument (Thermo Fisher Scientific) and an Ambion AM1830 total RNA extraction kit (Thermo Fisher Scientific) as detailed by the manufacturer. The final RNA sample was recovered in 50 µL elution buffer and RNA concentration was determined spectrophotometrically using a NanoVue instrument. Samples were brought to 6 ng/µL final concentration prior to storage at −80 °C.To establish recovery of amplifiable RNA, samples were tested using a rabies-specific RT-qPCR performed essentially as described previously  employinA collection of 190 whole genome RABV sequences were recovered from GenBank . While thttps://support.illumina.com/sequencing/sequencing_kits/ampliseq-library-plus-for-illumina/documentation.html, accessed on 12 March 2019). In brief, following a reverse transcriptase step the virus was amplified prior to partial amplicon digestion and indexing for a single primer pool using Ampliseq CD indexes set A (Illumina). The library was then purified using AMPure XP beads , amplified and recleaned prior to evaluation. Sample aliquots were used to determine (1) the DNA concentration (ng/µL) using a Qubit 2 instrument with a dsDNA HS assay (Thermo Fisher Scientific), and (2) the amplicon profile was analyzed on a QIAxcel instrument (QIAGEN) operated as per the manufacturer’s directions using a QIAxcel DNA fast analysis kit (QIAGEN). Based on these analyses, the sample’s nanomolar concentration was estimated and samples were normalized by dilution to a final concentration of 10 nM. Samples with a concentration below this value were used without further dilution. Finally, libraries, each comprised of 48 pooled samples, were run on a MiSeq sequencer (Illumina) using a 2 × 250 bp MiSeq Reagent Kit v2 (Illumina).RNA extracted from unfixed tissue was diluted first to 10 ng/µL with confirmation of the concentration using a Qubit 2 fluorimeter (Thermo Fisher Scientific) with a Qubit RNA broad range assay kit (Thermo Fisher Scientific). The concentration of the RNA recovered from FFPE samples (6 ng/µL) was also verified using the Qubit 2 fluorimeter. Finally, samples were further diluted to 2 ng/µL just prior to use. Five µL of each sample (10 ng) was then processed using an Ampliseq Library Plus for Illumina kit  together with the custom RNA panel according to chapter three of the manual “Ampliseq for Illumina custom and community panels” (available at Vulpes lagopus (GCF_018345385.1), Eptesicus fuscus (GCF_000308155.1), Bos taurus (GCF_002263795.1), Vulpes (GCF_003160815.1), Spilogale gracilis (GCA_004023965.1), as a proxy for Mephitis, and Canis lupus (GCF_014441545.1). The results of the taxonomic classification of the raw reads were summarized and visualized using R package Pavian v1.0 [The proportion of Illumina reads representing the targeted RABV and the host species of origin was explored for seven selected samples. The raw reads were analyzed using Kraken2 classifier v2.1.2  based onian v1.0  prior tohttp://www.htslib.org) was used to convert the M files to the sorted BAM files. UGENE, version 40.1, (available at https://bio.tools/ugene) was then used to inspect the mapping files and generate the assembly. The final assembled genome was recovered in FASTA format in a single contiguous sequence including gaps for those base positions with zero coverage relative to the reference sequence. An alignment of all sample assemblies with the consensus sequence was generated in MEGA, version X, software   .Lasiurus RABV variant, accession #JQ685902.1, (assembly A4) while another three samples apparently of the vampire bat or free-tailed bat RABV types were reassembled using sequence of a vampire bat variant, accession #AB519642.1, (assembly A5); three Asian samples were reanalyzed using a RABV sequence from the Philippines, accession #EU293111.1, (assembly A6); two Africa-3 samples were reassessed using an Africa-3 RABV sequence, accession #MG458308.1, (assembly A7) and the three samples from Sri Lanka were reassembled using a RABV variant from this nation, accession #AB635373.1 (assembly A8). An additional reference guided assembly was also later undertaken on a sample subset representing those of the Arctic lineage using a Canadian Arctic RABV sample NT.1993.0669AFX, accession #MN233954, as reference (assembly A9).Given the observed variation in genome coverage between samples, a second assembly (A2) was performed on the complete dataset using a sequence generated from a Canadian bat RABV variant (accession #JQ685920), a member of the American Indigenous lineage as reference. This assembly confirmed the significant impact of the reference sequence employed for read assembly on genome coverage . To bettPhylogenetic analyses were completed using MEGA version X software. Using the reference-guided assembly of each sample that resulted in optimal genome coverage, an alignment of all samples together with another 100 representative RABV genomes and an Australian bat lyssavirus (ABLV) genome as outlier recovered from the NCBI database  was comphttps://www.graphpad.com) was used for all statistical analysis. Differences in genome coverage between unfixed samples and FFPE samples were analyzed using the Mann–Whitney U test, while differences in genome coverage of unfixed or FFPE samples using different references for assemblies were calculated by the Wilcoxon matched-pairs signed rank test. A p-value < 0.05 was considered statistically significant.Graphpad Prism 7 , which we believed was due to biases introduced by the reference sequence selected, given that the FFPE sample set was extensively represented by the American Indigenous (AI) lineage. As expected, using a reference sequence from a member of the AI lineage (accession #JQ685920) for a reference-guided assembly generated significantly different genome assemblies, again with a very wide range of genome coverage from 15% for an FFPE sample of the Arctic lineage to 93% for two unfixed samples of the AI lineage (Eptesicus fuscus (EF) and Myotis (MYO) RABV variants exhibited significantly improved genome coverage while most other samples showed either little improvement or greatly reduced coverage. It was therefore apparent that the choice of reference sequence had a major impact on the extent of genome coverage that could be extracted from the sequence reads and a strategy to improve sequence recovery was clearly needed.Genome coverage varied widely when using the consensus RABV genome sequence  as the r variant . The Man lineage . In geneLasiurus and silver-haired bat types that had yielded low coverage in this assembly and yielded only short sequences (>500 bases) for the BLAST analysis while the remaining sample (V854) was a Mexican skunk sample prepared from nonfixed tissue. BLAST reassessment of these samples using the sequences generated from the A2 assembly correctly identified all the bat-associated samples as belonging to the American Indigenous lineage. Of the other 80 samples, the BLAST analysis accurately predicted the RABV variant of 71 of them. Identity values <90% or identities based on shorter sequences (<500 bases) were most prone to generate best matches that were inaccurate in terms of the viral type. Thus, while the predictive ability of this approach could not be considered highly accurate, this information identified appropriate reference sequences for additional assemblies that would optimize the sequence recovery. Accordingly, 28 samples were subject to additional reference-guided assembly as detailed in the Methods section.An alignment of the sequences of all test samples generated from assembly A1  revealedTo gauge the utility of these assembled sequence data to accurately identify the lineage and viral type of the test samples, an alignment composed of the optimized assemblies for all 86 test samples together with 100 additional reference RABV sequences and an Australian bat lyssavirus (ABLV) sequence as outlier was generated  and emplThese trees illustrate that the assembled Ampliseq reads clustered, with just one exception, within the expected lineage. The Mexican skunk sample (V854) was anomalous in that the previous N gene analysis had placed it in the CMSK variant but in this analysis, it was clustered within the Cosmopolitan lineage, typical of the South Baja California skunk (SBCSK) variant of Mexico. It remains unclear if this was a sampling error during processing or a prior misidentification. Interestingly, in this phylogeny sample V647 clustered with strong support  with this Mexican isolate. Sample V647, which originated from a cougar in California, had remained refractory to genetic typing using primers successful with the expected California skunk (CASK) viral subtype  despite Lasiurus bat type  ranged from 34 to 58% genome coverage. Despite these lower values, these samples grouped within their respective clades with good bootstrap support. Moreover, accurate variant assignment of many samples was noted in several American Indigenous clades including those of the big brown bat (Eptesicus fuscus) host. Typing of the viruses associated with this host in the USA have identified four variants: EF-E1, EF-E2, EF-W1, and EF-W2 according to their range in the eastern and western parts of the country [Myotis bat hosts all clustered within the North American Myotis bat clade and were clearly delineated as expected into two subclades MYO I and MYO II [Efforts had been made to reach a genome coverage of 60% for all samples and while many samples exceeded this value significantly, seven samples failed to reach this level, including one each of the Arctic (17-0103-116R-FFPE), Africa-3 (V039) And Indian Subcontinent (V114) lineages with coverages of 58%, 59%, and 41%, respectively. A group of four FFPE samples of the  country  which cod MYO II .Given that genome coverage and phylogenetic placement was clearly significantly impacted by the nature of the sequence used for reference-guided assembly, we explored the potential to maximize genome coverage and thereby reveal finely resolved epidemiological relationships of samples processed by the Ampliseq for Illumina approach. This was achieved for the Arctic test samples which were compared to a reference collection of previously described sequences representative of this lineage which segregates into four sub-lineages Arctic1 to Arctic4 ,36. The This phylogeny clearly shows that selecting a reference sequence belonging to the Arctic lineage to assemble the Arctic lineage test sample genomes enables their association not only with the correct RABV lineage (Arctic) and sub-lineage (Arctic1 to Arctic4) but with the correct variant of that lineage with strong support. Thus, three test samples cluster within the most common ON2 variant of the distinctive Arctic1 sub-lineage, one test sample clusters within each of the sub-lineages Arctic2 and Arctic4, while the remaining eight test samples cluster with distinct variants of the Arctic3 sub-lineage, including Arctic3-2, Arctic3-4, and two closely related variants, Arctic3-17 and Arctic3-18, which do not resolve well in this tree. Seven of these 13 test samples had been sequenced previously, and in all cases the Ampliseq for Illumina-derived sequence clustered closely with the complete genome sequence and supported a similar variant designation. The biggest discrepancy was represented by sample 17-0663-169FFPE which was somewhat distant from its corresponding sample 2017NU0663AFX but still within the same variant clade.Lasiurus group. This genomic region, or portions thereof, was employed for BLAST analysis against the Lyssavirus rabies sequence collection in the NCBI GenBank database to identify the best match for each test sample and thereby predict its viral type. Despite the conserved nature of this region, there was adequate base variation to permit differentiation of most viral types thus facilitating further read assembly using an appropriate reference sequence. Using this optimization procedure, with few exceptions, most samples in this study generated a RABV genome coverage in the range of 70–95%. However, the BLAST analysis cannot be considered completely accurate; use of shorter sequences (<500 bases) or identity matches of <90% should be interpreted with caution as many RABV variants will yield a good match with these criteria. In addition, rare variants that are poorly represented in the NCBI database will likely be misidentified in terms of their viral type. Such a situation may be responsible for the erroneous typing of the Mexican sample (V854) by the BLAST analysis. Additionally, it complicated the precise typing of the two samples from Puerto Rico for which good references were not available. Indeed, it was noted that a few samples of the Cosmopolitan lineage were not precisely typed by BLAST analysis presumably due to the limited genetic divergence within this lineage, but all samples of this lineage yielded good coverage in assembly A1 using the consensus RABV sequence as reference.This study has demonstrated the value of an Ampliseq for Illumina panel to generate amplicons covering much of the viral genome for a wide variety of RABV variants. Parallel sequencing of these amplicons provided extensive viral genome coverage in many cases, thereby enabling detailed molecular epidemiological analysis. However, it became evident during the analysis of these reads that the selection of the reference sequence used in the reference-guided assembly was critical for optimizing the subsequent sequence analysis. While the source of the sample itself can often guide selection of an appropriate reference sequence, this may not always be the case. As in this study, widely divergent RABV lineages cocirculate in the Americas, and throughout the world there is the possibility of an introduction of non-native variants. It was therefore necessary to devise a means of using preliminary sequence data to predict a likely viral type that would inform the appropriate reference sequence to be used for subsequent assembly. Accordingly, a region of the L gene corresponding to bases 7396–8603 of the consensus RABV genome was employed. This region, which corresponds to bases 7362–8570 of the PV strain, encodes amino acids 649–1051 of the polymerase corresponding to the relatively conserved domains III and IV which contain critical functional motifs . The higLasiurus group for which four FFPE samples failed to meet a threshold genome coverage of 60% and another two FFPE samples yielded coverage just above 60%. Reasons for this reduced coverage could include either low viral titer in these samples, poor match of some of the panel primers to their sequence targets, or the RNA from these samples may be of particularly low quality. It should be mentioned that these six FFPE samples originated from two submissions that were processed in triplicate during the fixation. The RT-qPCR Ct values for all six FFPE samples were in line with those of other FFPE samples (ranging from 14.39 to 17.47) arguing against a particularly low viral titer. Interestingly, two unfixed Lasiurus samples from the USA  yielded genome coverage values of 63% and 83% suggesting a rather poor correlation between viral load and genome coverage. Overall, the replicate samples included in the FFPE group generally yielded similar results. All real-time RT-qPCR Ct values were within 1.5 Ct except for one set for which the range was 2.5 Ct. Furthermore, replicate samples yielded consistent trends with respect to genome coverage using different reference sequences and they performed similarly in the phylogenetic analysis. This demonstrates the consistency of the method as currently performed.While the current panel, which was especially focused on the main variants circulating in the Americas, performed well for many variants it was less successful with some samples of the Within the unfixed sample group, evidence that higher viral loads resulted in higher genome coverage was mixed. This appeared to be the case for the two unfixed Africa-3 samples, V039 and V050, for which Ct values were 15.81 and 8.59 and genome coverage was 59% and 90%, respectively. However, of the three Indian Subcontinent samples V114 had the highest viral load  but the lowest genome coverage (41% compared to 89% and 95%). Clearly factors other than viral load impacted the final outcome of these studies. Future work to further optimize this methodology would benefit from studies to explore the effect of tweaking the primer panel to best suit the viral variants of specific regions and to determine the optimal starting amount of RNA template based on the viral load  given the variability of the viral RNA template in a sample. Furthermore, it should be acknowledged that the present study was performed with the prior knowledge of the viral type of the test samples thus potentially introducing bias in the analytical approach. Verification of this methodology would benefit from studies using double blinded panels to check for precision and accuracy of inferred viral types.While the Ampliseq for Illumina method described in this report can be applied to RNA recovered from either unfixed or FFPE tissue samples, its greatest value is in its application to the latter tissue type. To date, FFPE material has remained refractory to detailed molecular epidemiological analyses of RABVs due to challenges in generating extensive sequence information from this source. Preliminary analyses also suggest that this method may be a useful tool for the detailed analysis of material stored on solid surfaces such as Whatman FTA cards. Future iterations of this method will facilitate the analysis of archived tissue samples, including FFPE material, thereby furthering our knowledge of the historical spread of rabies.


There is a paucity of data on management strategies and clinical outcomes after recurrent venous thromboembolism (VTE). In a multicenter registry enrolling 3027 patients with acute symptomatic VTE, the current study population was divided into the following 3 groups: (1) First recurrent VTE during anticoagulation therapy (N = 110); (2) First recurrent VTE after discontinuation of anticoagulation therapy (N = 116); and (3) No recurrent VTE (N = 2801). Patients with first recurrent VTE during anticoagulation therapy more often had active cancer . Among 110 patients with first recurrent VTE during anticoagulation therapy, 84 patients (76%) received warfarin at recurrent VTE with the median prothrombin time-international normalized ratio (PT-INR) value at recurrent VTE of 1.6, although patients with active cancer had a significantly higher median PT-INR value at recurrent VTE compared with those without active cancer . Within 90 days after recurrent VTE, 23 patients (20.9%) during anticoagulation therapy and 24 patients (20.7%) after discontinuation of anticoagulation therapy died. Active cancer was a major cause of recurrent VTE during anticoagulation therapy as a patient-related factor, while sub-optimal intensity of anticoagulation therapy was a major cause of recurrent VTE during anticoagulation therapy as a treatment-related factor, particularly in patients without active cancer. VTE has a long-term risk of recurrence, and the risk of recurrence after 5 years was reported to be 20–25% in unselected cohorts of patients with VTE2. Thus, the prevention of recurrence by anticoagulation therapy is important3. The risk of recurrence is highest in the acute phase after VTE diagnosis, and it decreases rapidly after initiation of anticoagulation therapy4. Nevertheless, a certain number of patients could develop recurrent VTE beyond the acute phase irrespective of the status of anticoagulation therapy. Moreover, recurrent VTE was reported to have a considerable impact on mortality (relative risk ratio: 3.24)5. However, evidence on the optimal management strategies after recurrent VTE is scarce6. Consequently, the current VTE guidelines provide limited recommendations based on low-quality evidence for the management strategies after recurrent VTE especially during anticoagulation therapy10. Data on the current real-world management strategies and outcomes after recurrent VTE would be important for understanding the current issues and unmet needs in patients with recurrent VTE like other cardiovascular diseases. Thus, we aimed to investigate the clinical characteristics, management strategies and outcomes of recurrent VTE, using a large practice-based large observational study in Japan.Venous thromboembolism (VTE), including pulmonary embolism (PE) and deep vein thrombosis (DVT), is one of major health problems all over the world with a yearly incidence of around one case per 1000 person-years12. We searched the hospital databases for clinical diagnosis and imaging examinations, and enrolled consecutive patients who met the definition of acute symptomatic VTE diagnosed within 31 days from symptom onset during the study period13.The COMMAND VTE (COntemporary ManageMent AND outcomes in patients with Venous ThromboEmbolism) Registry is a physician-initiated, retrospective, multicenter cohort study in which consecutive patients with acute symptomatic VTE among 29 centers in Japan were included between January 2010 and August 2014. The design of the registry was previously reported in detail14.We enrolled 3027 consecutive patients with acute symptomatic VTE after screening of the consecutive 19,634 patients with suspected VTE for eligibility through chart review by the physicians at each institution. According to the occurrence of recurrent VTE during the entire follow-up period and the status of anticoagulation therapy at the time of the recurrent VTE event, the current study population was divided into the following 3 groups; (1) First recurrent VTE during anticoagulation therapy; (2) First recurrent VTE after discontinuation of anticoagulation therapy; and (3) No recurrent VTE during the follow-up period . The relevant review boards or ethics committees in all other 28 participating centers  at the time of the diagnosis. Data for prothrombin time-international normalized ratio (PT-INR) during follow-up in patients receiving warfarin were collected from the hospital charts of the centers where the index VTE was diagnosed. The presumed stable PT-INR values for warfarin users was evaluated as the first PT-INR between 1 and 6 months after VTE diagnosis. The detailed definitions of patient characteristics are described in Supplementary Appendix Detailed methodology of data collection in the COMMAND VTE Registry was previously reported11. In the current study, we evaluated the clinical outcomes at 90 days after the first recurrent VTE events, and all the clinical outcomes were evaluated irrespective of the status of anticoagulation therapy after the first recurrent VTE.Detailed methodology of clinical follow-up in the COMMAND VTE Registry was previously reported15. Death was judged to be due to PE  if it was confirmed by autopsy or if death followed a clinically severe PE after first recurrent PE events. Death was judged to be bleeding related if it followed an intracranial hemorrhage or a bleeding episode leading to hemodynamic deterioration. Death in patients with the end-stage cancer without a specific cause of death was regarded as cancer in origin. Death was judged to be due to cardiac events, if it followed acute myocardial infarction, heart failure, or ventricular arrhythmia. Final classifications for the causes of deaths were made on the basis of the full consensus of the independent clinical event committee.The primary outcome measure in the current study was all-cause death at 90 days after the first recurrent VTE. The independent clinical event committee  major bleeding, which consisted of a reduction in the hemoglobin level by at least 2 g/dL, transfusion of at least 2 units of blood or symptomatic bleeding in a critical area or organ16.The secondary outcome measures in the current study were recurrent VTE and major bleeding at 90 days after the first recurrent VTE. Recurrent VTE was defined as PE and/or DVT with symptoms accompanied by objective imaging examinations or autopsyhttps://www.jmp.com/en_gb/software/new-release/preview-jmp14.html). All reported P-values were 2-tailed, and P-values < 0.05 were considered statistically significant.In the current study, we evaluated the baseline characteristics among the 3 groups. Characteristics, management strategies, and outcomes of the first recurrent VTE were compared between the 2 groups of patients who had recurrent VTE during and after discontinuation of anticoagulation therapy. Moreover, we conducted an exploratory analysis for first recurrent VTE during anticoagulation therapy comparing patients with and without active cancer. Categorical variables are presented as numbers and percentages, and continuous variables are presented as the mean and standard deviation or the median and interquartile range (IQR) based on their distributions. Categorical variables were compared using the chi-squared test when appropriate; otherwise, Fisher’s exact test was used. Continuous variables were compared using Wilcoxon’s rank sum test between the 2 groups and one-way analysis of variance or Kruskal–Wallis test based on their distributions among the 3 groups. The 90-day clinical outcomes after the first recurrent VTE are presented as the number of events and incidences with 95% confidence intervals (CI). All statistical analyses were conducted using JMP version 14.0.0  with first recurrent VTE during anticoagulation therapy, 116 patients (3.8%) with first recurrent VTE after discontinuation of anticoagulation therapy, and 2801 patients (93%) without recurrent VTE during the follow-up period  of first recurrent VTE during anticoagulation therapy and 19% (22 patients) of first recurrent VTE after discontinuation of anticoagulation therapy occurred within 90 days after the VTE diagnosis  Table .Table 3CAmong the patients with recurrent VTE, 23 patients (20.9%) during anticoagulation therapy, and 24 patients (20.7%) after discontinuation of anticoagulation therapy died within 90 days after the first recurrent VTE events  with active cancer, and 8 patients (13.1%) without active cancer died within 90 days after the first recurrent VTE events  Patients with first recurrent VTE during anticoagulation therapy had a significantly higher prevalence of active cancer compared with those after discontinuation of anticoagulation therapy; (2) The PT-INR value for warfarin users at recurrent VTE events in patients during anticoagulation therapy was significantly higher in patients with active cancer than in patients without active cancer; (3) Approximately 20% of patients died within 90 days after the first recurrent VTE events, and fatal PE and cancer were the major causes of deaths.10. The clinical dilemma is the optimal anticoagulation strategies for recurrent VTE during anticoagulation therapy6. Recurrent VTE in the acute phase soon after initiation of anticoagulation therapy seems to be managed by more aggressive antithrombotic therapy including thrombolysis, increased intensity of anticoagulation therapy, and switching from oral anticoagulants to parenteral anticoagulants. However, currently, there have been no randomized clinical trials that have evaluated management strategies for patients with recurrent VTE during anticoagulant therapy. Potential causes of recurrent VTE during anticoagulation therapy could be divided into patient-related factors and treatment-related factors6. The investigation of the potential causes could be important to consider the appropriate management strategies after recurrent VTE during anticoagulation therapy.Generally, recurrent VTE after discontinuation of anticoagulation therapy might be managed by restart of anticoagulation therapy with indefinite duration21. Of these, the most important patient-related factor is thought to be active cancer9. A previous study reported that 31% of patients with recurrent VTE during anticoagulation therapy had active cancers17. Consistent with the previous report, the current study showed a high prevalence (45%) of patients with recurrent VTE during anticoagulation therapy. Patients with active cancer were reported to receive sub-optimal intensity of anticoagulation therapy for warfarin users due to high-bleeding risk and drug interactions of anti-cancer drug22. However, the current study showed that warfarin users with active cancer had higher PT-INR values compared with those without, suggesting the importance of active cancer as the patient-related factor of recurrent VTE on appropriate anticoagulation therapy. There might be no established anticoagulation strategies for patients with recurrent VTE during appropriate anticoagulation therapy. A current guideline weakly recommends switching from oral anticoagulants to low molecular weight heparin (LMWH) at least temporarily and increasing the dose of LMWH for LMWH users based on a low level of evidence9, although there are no recommendations for long-term anticoagulation strategies. The current study showed that a majority of patients with recurrent VTE during anticoagulation therapy were treated with parenteral anticoagulation therapy or thrombolysis in the acute phase, and continued the same anticoagulant as used at the recurrent VTE event beyond the acute phase.Several patient-related factors including cancer, thrombophilia, autoimmune disease, pregnancy, and vascular abnormalities were reported as the causes of recurrent VTE9. The recommended target PT-INR for VTE in the Japanese guidelines is 2.0  without established evidences7, which is lower than the target value  used in other Western countries10. The current study showed that there was no significant difference in the median presumed stable PT-INR values for warfarin users among the 3 groups . However, the current study also showed that the median PT-INR value at recurrent VTE events for warfarin users was 1.6 and more than half of patients had PT-INR values less than 2.0 at recurrent VTE events, and low PT-INR value at recurrent VTE events was more remarkable in patients without active cancer. These results could suggest that sub-therapeutic intensity of warfarin might be one of the causes of recurrent VTE during anticoagulation therapy as treatment-related factor especially in patients without active cancer. Clinicians should be notified for the importance of maintenance of appropriate intensity of anticoagulation therapy during the follow-up. These issues should be confirmed by further studies in the future.Treatment-related factors could include sub-therapeutic intensity of warfarin, inappropriate reduced dosage of direct oral anticoagulants (DOAC), concomitant drugs that reduce anticoagulant effect, and patient adherence to drug17. Another study reported that a considerably high risk of recurrence (8.6%) and major bleeding (1.4%) during 3 months in 70 cancer patients with recurrent VTE during anticoagulation therapy who either switched from warfarin to LMWH or had their LMWH dose increased23. The current study showed a relatively low risk of further recurrence (0.9%) and high risk of major bleeding (8.2%) at 90 days in patients with recurrent VTE during anticoagulation therapy. There results could be partly due to a relatively frequent usage of thrombolysis in the acute phase (15%) and different ethnicity between Whites and Asians24. The current study also showed that a considerably high proportion of patients (20.9%) died within 90 days after the recurrent VTE during anticoagulation therapy and fatal PE and cancer were major causes of deaths. A major cause of death was cancer among patients with active cancer, while all deaths among patients without active cancer was fatal PE. Clinicians should be careful for avoiding inappropriate anticoagulation strategies to prevent recurrent fatal PE.The clinical outcomes after recurrent VTE could also be important in considering the appropriate management strategies after recurrent VTE. A previous study reported that patients with recurrent VTE on vitamin K antagonists (warfarin) therapy have a worse prognosis with an increased risk of both further recurrences (6.8%) and major bleeding (2.3%) at 3 months compared with patients with recurrent VTE during vitamin K antagonists therapyThe current study has several limitations. First, the current study was an observational study, which can be subject to various biases inherent to observational study design. The therapeutic decision-making was left to the discretion of the attending physicians, which could have influences on clinical outcomes after recurrent VTE events. Second, detailed management strategies after recurrent VTE such as dosing of heparin and warfarin were not evaluated in the current study. Third, demographics, practice patterns as well as clinical outcomes in patients with VTE in Japan may be different from those outside Japan. Finally, the current study was conducted before introduction of DOACs for VTE in Japan. Thus, it should be interpreted with caution whether the present results could be extrapolated to patients treated with DOACs.In this real-world large registry, active cancer was a major cause of recurrent VTE during anticoagulation therapy as a patient-related factor, while sub-optimal intensity of anticoagulation therapy was a major cause of recurrent VTE during anticoagulation therapy as a treatment-related factor, particularly in patients without active cancer.Supplementary Information.


The role of the white blood cells, which form the peripheral immune system and are crucial in inflammatory processes, has been laid aside in the context of brain structural changes in schizophrenia.Determine how blood cells are associated with some brain structures volumes in first episode psychosis (FEP) and their relationship with clinical variables at baseline and 1 year follow – up.drug-naïve FEP treated between April 2013 and July 2017 at the ETEP Program at Hospital del Mar were included. Inclusion criteria were: 1) age 18-35 years; 2) fulfillment of DSM-IV-TR criteria for brief psychotic disorder, schizophreniform disorder, schizophrenia or unspecified psychosis; 3) no previous history of severe neurological medical conditions or severe traumatic brain injury; 4) presumed IQ level > 80, and 5) no substance abuse or dependence disorders except for cannabis and/or nicotine use. All patients underwent an assessment at baseline and at one-year follow-up, including sociodemographic and clinical variables . Fasting blood samples were obtained before administering any medication at baseline. Structural T1 MRI was performed at baseline and brain volumes were quantified though FreeSurfer software. SPSS program was used for statistical analyzes.Fifty Lymphocytes have a positive correlation with right and left hippocampus at baseline. Moreover, lymphocytes have a negative correlation with depressive symptoms at baseline and 1 year follow – up.Lymphocytes may have a protective effect in some brain structures in FEP patients at baseline, especially those implicated in depressive symptoms.No significant relationships.